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Aging, neurodegeneration and Alzheimer’s disease

  • A cell permeable ADAM10 endocytosis inhibitor promotes neuronal differentiation of adult hippocampal neural progenitors from a mouse model of Alzheimer’s disease.

    Authors:
    Valentina Bonetto (1), Valeria Bortolotto (1), Emanuele Brai (1), Giulia Boni (1), De Cicco Greta (1), Gibin Borzoni Giada (1), Pagano Corinna (1), Manfredi Marcello (2), Canonico Pier Luigi (1), Stringhi Ramona (3), D'Urso Annarita (3), La Greca Filippo (3), Scheggia Diego (3), Di Luca Monica (3), Marcello Elena (3), Grilli Mariagrazia (1)

    Organisations:
    1: Laboratory of Neuroplasticity, Department of Pharmaceutical Sciences, University of Piemonte Orientale, Novara, Italy
    2: University of Eastern Piedmont, Department of Translational Medicine (DIMET), Novara, Italy
    3: Department of Pharmacological and Biomolecular Sciences, University of Milano, Milano, Italy.

    Presenting author: Valentina Bonetto

    Exposition date: Saturday, September 16, 2023

    Exposition position: 024/2

    Alzheimer’s disease (AD) is a neurodegenerative disease with progressive memory loss and severe cognitive deficits.
    Adult hippocampus harbors neural progenitor cells (ahNPCs). It has been demonstrated that adult-born hippocampal immature neurons (DCX + cells) can be recruited into the engram but their functional role is defective in AD animal models (1). Altogether disrupted adult neurogenesis, an early defect in AD, can be regarded as a potential contributor to AD pathophysiology.
    One protein expressed in immature neurons is ADAM10, an enzyme involved in non-
    amyloidogenic Amyloid Precursor Protein (APP) processing and in dendritic spine shaping.
    Compared to healthy controls, in AD hippocampi ADAM10 membrane expression is reduced due to its increased endocytosis, mediated by AP2 interaction (2). To restore this alteration, a cell-permeable peptide (PEP3) disrupting the interaction between ADAM10 and AP2 has been developed and proven, under subchronic treatment, to rescue synaptic and cognitive impairment in 6-7 month-old APP/PS1 mice (3).
    Since immunoprecipitation confirmed ADAM10/AP2 interaction in adult hippocampal
    progenitors, cells isolated from WT and APP/PS1 mice could represent in vitro models to investigate consequences of disrupted ADAM10-dependent pathways. Under basal
    conditions, we observed a reduced ability of APP/PS1-derived ahNPC to generate neurons, compared to their WT counterpart. NPCs were treated with PEP3 or an inactive peptide for 24 h. PEP3 treatment promoted neuronal differentiation in both genotypes, but more significantly in APP/PS1 ahNPC. Preliminary data obtained in a proximity ligation assay confirmed that pro-neurogenic PEP3 concentrations reduced ADAM10/AP2 interaction in WT ahNPC. These results suggest that PEP3 may exert novel activities on ahNPC, potentially contributing to its pro-cognitive effects. Studies are ongoing to assess PEP3 in vivo effects on immature neurons and to identify ADAM10 targets involved in PEP3- mediated modulation of ahNPC neuronal fate specification. Additionally, current proteomic data analysis aims at unraveling differentially modulated signaling pathways in APP/PS1 compared to WT ahNPC.

  • An alternative Ellagic acid delivery in neurodegenerative ex-vivo model

    Authors:
    Nunzia Maisto (1,2), Dalila Mango (2), Gaetano Barbato (3), Carlotta Marianecci (1), Maria Carafa (1), Federica Rinaldi (1), Robert Nisticò (2,3)

    Organisations:
    1: Università La Sapienza di Roma, Italy
    2: Laboratory of Pharmacology of Synaptic Plasticity, EBRI Rita Levi-Montalcini Foundation, Roma,Italy,
    3: University of Rome Tor Vergata, Rome, Italy,

    Presenting author: Nunzia Maisto

    Exposition date: Saturday, September 16, 2023

    Exposition position: 004/2

    Background: Ellagic acid (EA) has been shown to have protective effects in Alzheimer's disease, but it shows poor pharmacokinetic profile. A valid approach could be delivering the drug in non-ionic surfactant vesicles (NBVs) through the nose to brain (N2B) route coupled to focused ultrasound (FUS) improving brain targeting. The EA will be loaded inside the vesicles and their neuroprotective effect will be explored using electrophysiological recordings (field and whole cell patch clamp) of hippocampal synaptic transmission and plasticity in ex-vivo Alzheimer model.

    Method: Empty and loaded NBVs were prepared and deeply characterized. In vitro FUS analysis were performed to evaluate the ability of FUS to induce a stable and prolonged release of EA. The electrophysiological experiments were conducted using C57BL6/J mice of both sexes aged between 30 and 40 days. Paired pulse facilitation was assessed at inter-stimulus intervals ranging from 50 to 500 ms. An additional recording of baseline was evaluated before inducing Long-Term Potentiation by theta-burst stimulation and responses (fEPSP) were recorded for 1h. In vitro Alzheimer model were obtained applying Aβ1-42 oligomers (200 nM). As a control, we tested the effect of Aβ1-42 with a scrambled sequence (200 nM). Empty and loaded NBVs were used in presence and not of Aβ1-42 and Aβ1-42 scrambled.

    Result: Preliminary results of empty and loaded NBVs present an optimal range of size for N2B delivery and in vitro FUS release shows a stable and controlled release profile of loaded drug. Slices incubated in presence of Aβ1-42 showed an impairment of LTP, while this effect didn’t occur after incubation of slices with Aβ1-42 scrambled. Preliminary results show that NBVs formulations didn’t compromise LTP suggesting the possibility of using them as drug carriers in Alzheimer’s disease.

    Conclusion: A better brain delivery of EA could be a valid approach in pathologies characterized by synaptic dysfunction.

  • Anti-GluA3 autoantibodies define a new sub-population of Frontotemporal dementia patients with distinct neuropathological features

    Authors:
    Maria Italia (1), Michela Salvadè (1), Filippo La Greca (1), Elisa Zianni (1), Alessio Spinola (1), Elena Ferrari (1), Silvana Archetti (2), Antonella Alberici (3), Alberto Benussi (3), Alessandro Padovani (3), Monica DiLuca (1), Diego Scheggia (1), Barbara Borroni (3), Fabrizio Gardoni (1)

    Organisations:
    1: Department of Pharmacological and Biomolecular Sciences (DiSFeB), University of Milan
    Milan, Italy
    2: Department of Laboratories, Central Laboratory of Clinical Chemistry Analysis. ASST Spedali Civili
    Brescia Italy
    3: Neurology Unit, Centre for Neurodegenerative Disorders, Department of Clinical and Experimental Sciences, University of Brescia: Brescia, Italy

    Presenting author: Maria Italia

    Exposition date: Saturday, September 16, 2023

    Exposition position: 003/2

    Over the last two decades, growing attention has been paid to the role of autoantibodies that target brain antigens in disorders of the central nervous system. Within this field, autoantibodies directed against the GluA3 subunit (anti-GluA3 hIgGs) of AMPA-type glutamate receptors (AMPARs) have been identified in 20%–25% of patients with frontotemporal dementia (FTD).

    To clarify the contribution of anti-GluA3 antibodies to central nervous system symptoms and pathology, we developed a model of autoimmunity by infusing C57BL76J mice with anti-GluA3 hIgGs isolated from patients for 1 month through an intracerebroventricular cannula. The model was used to conduct morphological and biochemical analyses and behavioural tasks. Moreover, we treated mice with a well-validated positive allosteric modulator of the AMPAR (S 47445) as a possible rescue strategy to counteract the detrimental effects mediated by anti-GluA3 hIgGs.

    Data showed that chronic anti-GluA3 hIgG administration in mice mediated the internalisation of GluA3-containing AMPARs and led to selective accumulation of the phosphorylated form of tau in the post-synaptic fraction and dendritic spine loss in the prefrontal cortex of mice. In addition, mice chronically treated with anti-GluA3 hIgGs exhibited behavioural disturbances that mostly reflected the deficits detected only in FTD patients positive for the autoantibodies, such as apathy and binge-like eating. Finally, anti-GluA3 hIgG-mediated alterations were rescued in the animal model by enhancing glutamatergic neurotransmission by S 47445 treatment.

    Overall, this study clarified the contribution of anti-GluA3 autoantibodies to central nervous system symptoms and will be instrumental in the development of a therapeutic personalised medicine strategy for patients positive for anti-GluA3 hIgGs.

  • APP and Bace1: effect of cholesterol enrichment on processing and plasma membrane mobility

    Authors:
    Claudia Capitini (1,2), Laura Maggi (3), Martino Calamai (1,2)

    Organisations:
    1: LENS - European laboratory for non-linear spectroscopy, Italy
    2: National Institute of Optics, National Research Council of Italy (CNR), Italy
    3: Department of Experimental and Clinical Medicine, University of Florence and Regenerative Medicine Unit, Careggi University Hospital, Italy

    Presenting author: Martino Calamai

    Exposition date: Saturday, September 16, 2023

    Exposition position: 005/2

    Several findings point at high cholesterol levels as a risk factor for the development of Alzheimer’s disease. Experiments investigating the role of cholesterol on the processing of the amyloid precursor protein (APP) and its proximity to the b-secretase Bace1 in cells have led to conflicting results, often relying on pharmacological approaches based on cholesterol depletion, which may alter additional important cellular features.

    Here we revealed the effect of membrane cholesterol enrichment on the proteolytic processing of APP in living human neuroblastoma cells using a previously developed fluorescence bioassay coupled with flow cytometry. A direct correlation was found between the increase in membrane cholesterol amount and the degree of APP shedding. The observed increase in shedding rate was within 10%, hardly appreciable by Western blotting. Analogue results were obtained for cells overexpressing an APP mutant that cannot be processed by a-secretase, highlighting a major influence of cholesterol enrichment on the cleavage of APP carried out by Bace1. Accordingly, higher cholesterol levels led to increased release of extracellular soluble APP b fragments relative to a fragments, and to higher amounts of intracellular C-terminal fragments.

    In addition, we have investigated the effect of higher levels of cholesterol on the plasma membrane dynamics of APP and Bace1 using single molecule tracking in living cells. No direct correlation was observed between cholesterol content and the plasma membrane dynamics of both proteins, indicating that the effect of cholesterol enrichment on APP processing by Bace1 is uncoupled from changes in their lateral diffusion, and suggesting that the localization of these two proteins inside or outside ordered microdomains is not the prominent factor governing their diffusion on the plasma membrane.

  • Bay-41 acts on sGC in microglia to reduce plaque load in Alzheimer’s disease mice independently of Clec7A

    Authors:
    Giovanni Cosco (1), Burcu Seker (2), Joshua Shrouder (2), Nikolaus Plesnila (2)

    Organisations:
    1: Università degli Studi di Padova, Italy
    2: Institute for Stroke and Dementia Research (ISD), Klinikum der Universität München, Germany

    Presenting author: Giovanni Cosco

    Exposition date: Thursday, September 14, 2023 (Satellite Events Day)

    Exposition position: 91

    Alzheimer’s Disease is characterized by a pathological accumulation of amyloid beta driving neuronal loss. cGMP is a multitarget molecule that regulates many processes in the brain. Its production is initiated by the binding of Nitric Oxide (NO) to its receptor Soluble Guanylate Cyclase (sGC). However, in the context of AD, NO can act like a double-edged sword and drive Aß plaque clearing “Disease Associated Microglia” (DAM) into a harmful proinflammatory state through reactive oxygen species (ROS) mediated damage. Therefore, a carefully tailored DAM phenotype is required to limit AD progression.

    The research focus is analyzing the state of microglia and DAM markers expression after chronic sGC stimulation in Alzheimer’s disease mice model 5xFAD.

    The brain sections (50µm) which will be used in this study were already collected from WT, 5xFAD+Vehicle, 5xFAD+sGC mice that were treated with sGC stimulator or vehicle pellets over 10 months. We evaluated the expression and the localization of Clec7a and CD68 molecule with Aß plaques and microglia cells by Free-Floating Immunohistochemistry. Imaging data was acquired by confocal microscopy and analyzed by 3D reconstruction using Imaris software.

    Here we show that Bay-41 acts downstream of NO leading to a transcriptional signature in DAM which reduces plaque load in a mice model of Alzheimer’s disease.

  • Deregulated microRNAs in Alzheimer’s and Frontotemporal Dementia patients’ extracellular vesicles

    Authors:
    Rosalinda Di Gerlando (1,2), Francesca Dragoni (1,2), Maria Garofalo (2), Maria Busacca (1), Bartolo Rizzo (1), Matteo Bordoni (2), Eveljn Scarian (2), Alfredo Costa (1,2), Giulia Perini (2), Matteo Cotta Ramusino (2), Stefano Cappa (2,3), Orietta Pansarasa (2), Stella Gagliardi (2)

    Organisations:
    1: University of Pavia, Pavia, Italy
    2: IRCCS Mondino Foundation, Italy
    3: ICoN Cognitive Neuroscience Center, Institute for Advanced Studies, IUSS, Pavia, Italy

    Presenting author: Rosalinda Di Gerlando

    Exposition date: Saturday, September 16, 2023

    Exposition position: 010/2

    Objectives: A central role of RNA metabolism and non-coding RNAs biogenesis has emerged in Alzheimer’s disease (AD) and Frontotemporal Dementia (FTD). We investigated miRNA cargo of Small Extracellular Vesicles (SEVs) derived from plasma of FTD and AD patients, and Healthy Controls (HC). The purpose was to evaluate deregulated miRNAs in patients to identify new peripheral biomarkers and to determine mRNA targets involved in FTD and AD pathogenesis.

    Materials and methods: SEVs were isolated from plasma of 13 FTD patients, 20 AD and 20 HC by differential centrifugation and characterized by Nanosight and Western Blot. MicroRNA libraries were generated using Small RNA Seq Library Prep Kit (Lexogen) and sequenced on a NextSeq 500 (Illumina). Interaction prediction was carried out on TarBase v.8 database.

    Results and discussion: We found a total of 339 Differentially Expressed (DE) microRNAs in FTD, and 291 DE microRNAs in AD compared to controls. Interestingly, 74 miRNAs were commonly deregulated: 38 had the same trend and 36 had an opposite trend. In order to find a miRNA biomarker deregulated in both FTD and AD pathology, we selected 8 DE miRNAs with the same or opposite trend based on fold changes difference and targets. These miRNAs were found to have validated mRNAs target that may be of interest for molecular study of dementia, such as APP and PTEN.

    Conclusions: In conclusion, our data highlight the importance of miRNAs cargo examination in EVs of FTD and AD patients. In fact, their potential is exploitable both for biomarkers discovery and for study of gene expression alteration in dementia pathogenesis.

  • Dipyridamole rescued cognitive deficits in a mouse model of Niemann Pick type C1 disease (NPC1).

    Authors:
    Lucia Gaddini (1), Andrea Matteucci (1), Valentina Chiodi (1), Zaira Boussadia (1), Nazzareno Di Carlo (1), Manuela Marra (2), Carla Raggi (3), Rita Di Benedetto (4), Federica Fratini (2), Loredana Le Pera (2), Patrizia Popoli (1), Antonella Ferrante (1)

    Organisations:
    1: Istituto Superiore di Sanità, National Center for drug research and evaluation, Italy
    2: Istituto Superiore di Sanità, Core facility, NGS and Proteomic Units
    3: Istituto Superiore di Sanità, National Center for control and evaluation of medicines
    4: Istituto Superiore di Sanità, Dept. of food safety, nutrition and veterinary public health

    Presenting author: Antonella Ferrante

    Exposition date: Saturday, September 16, 2023

    Exposition position: 002/2

    NPC1 is a rare, genetic disorder characterized by impaired intracellular cholesterol trafficking and accumulation of unesterified cholesterol, sphingolipids, and other lipids in the cellular endo-lysosomal compartment. Disease severity is defined by pathology of the central nervous system, which is characterized by cerebellar neurodegeneration, hippocampal atrophy, and neuroinflammation. The only drug approved in the EU is miglustat, an inhibitor of glycosphingolipid synthesis which has only limited therapeutic effects; thus, an unmet medical need exists for NPC1.

    Previously, we demonstrated that the stimulation of the adenosine A2A receptor reduced pathological hallmarks in in vitro and in vivo models of NPC1. The present study aims at verifying if repurposing the approved drug dipyridamole (DIP), a blocker of the adenosine transporter ENT1, could represent a therapeutic strategy for NPC1. DIP efficacy was tested in a mouse model of NPC1: NPC1 and WT mice were treated with vehicle or with 15 and 30mg/kg i.p. DIP for one month. The effect of the drug was evaluated on motor function (by Erasmus Ladder) and on recognition memory (by the novel object recognition test, NOR). Motor deficits displayed by NPC1 mice were not reverted by DIP; however the drug rescued the reduced motivation of NPC1 mice (N=14-16; p=0.01) to complete the task.
    In the NOR test NPC1 mice displayed a lower ability to discern the novel object with respect to the familial one, and 30mg/kg DIP rescued this deficit (N=14-16; p=0.0034). Interestingly, calbindin expression is known to correlate with recognition memory performance; we found that NPC1 mice expressed a lower amount of calbindin in the CA1 and dentate gyrus of hippocampus and that DIP at 30mg/kg restored its levels. These results indicate that increasing adenosine tone could be an effective tool to ameliorate NPC1 symptoms, and that calbindin could be involved in such an effect.

  • Gamma-oryzanol: a new drug repositioning as a neuroprotective compound

    Authors:
    Giulia Abate (1,2), Mariachiara Pucci (1), Emanuela Tirelli (1), Sara Bonini (1,2), Andrea Mastinu (1,2), Giuseppina Maccarinelli (1), Daniela Zizioli (1), Alex Pezzotta (3), Mariagrazia Grilli (4), Maurizio Memo (1,2), Daniela Uberti (1,2)

    Organisations:
    1: Università degli Studi di Brescia, Dipartimento di Medicina Molecolare e Traslazionale (DMMT), Sezione di Farmacologia, Brescia.
    2: NutriFun4Health, Center for Research and Services on Nutraceuticals and Functional Food for Health, Università degli Studi di Brescia, Brescia.
    3: Università degli Studi di Milano, Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Milano.
    4: Università degli Studi del Piemonte Orientale "Amedeo Avogadro", Dipartimento di Scienze del Farmaco, Laboratorio di Neuroplasticità, Novara.

    Presenting author: Giulia Abate

    Exposition date: Saturday, September 16, 2023

    Exposition position: 025/2

    Plant-derived functional foods can be a surprising source of bioactive phytochemicals with as yet unknown or under-explored beneficial effects. Recently, we explored the brain potential of a ferulate-enriched fraction obtained after an innovative rice bran extraction process, among which Gamma-oryzanol (ORY) was identified as the most relevant compound. In fact, ORY is a mixture of ferulic acid esters and triterpene alcohols mainly known for their antioxidant and anti-inflammatory properties. In Japan, ORY is also registered as a prescription drug for its anti-lipidemic and anti-glycemic properties. Our research group has demonstrated the action of ORY as an activator of the NRF2 transcription factor. Our work demonstrates that chronic administration of ORY in adult mice improves cognitive performance and can protect against LPS-induced neuroinflammatory damage. Furthermore, we show that ORY is able to modulate at the hippocampal level the expression of proteins involved in synaptic plasticity, neuroprotection, and mitochondrial metabolism.

    Our recently obtained data also demonstrate how ORY is able to modulate neurogenesis by addressing progenitor cells toward a neuronal phenotype. The neurogenic effect was also demonstrated in a zebrafish Tg(- 3.1neurog1:GFP). This effect is nullified in morphants in which nrf2a and nrf2b genes were silenced, suggesting that the neurogenic and neurotrophic action of ORY is mediated by NRF2.

    Therefore, our results suggest that ORY is a potential molecule of pharmacological interest with neuroprotective action as it is able to modulate BDNF gene expression and promote neurogenesis both in vitro and in vivo. Although more in-depth studies are needed, several data exploitation of ORY's mechanism of action suggested an intriguing link between ORY, BDNF, and Nrf-ARE signaling. Therefore, we suggest ORY as a potential therapeutic drug because the activation of BDNF and the Nrf2-ARE signaling system that is a candidate feature for the design of novel therapeutic agents for brain disorders.

  • Hepcidin/Ferroportin1 axis activation and astrocytic-neuronal crosstalk in brain during aging

    Authors:
    Mariarosa Mezzanotte (1), Giorgia Ammirata (2), Marina Boido (1), Antonella Roetto (3), Serena Stanga (1)

    Organisations:
    1: Neuroscience Institute Cavalieri Ottolenghi, Department of Neuroscience Rita Levi Montalcini, University of Turin, Italy
    2: Molecular Biotechnology Center Guido Tarone, University of Turin, Italy
    3: Department of Clinical and Biological Sciences, University of Turin, Italy

    Presenting author: Mariarosa Mezzanotte

    Exposition date: Saturday, September 16, 2023

    Exposition position: 006/2

    During aging the accumulation of nonheme iron occurs in specific areas of the brain vulnerable to neurodegeneration. Typical aging features including increased Brain Blood Barrier (BBB) permeability, neuroinflammation, and oxidative stress cause iron redistribution and imbalance in the cerebral cortex. Therefore, it becomes urgent to investigate the mechanisms driving iron dyshomeostasis in the context of aging, and age-related diseases.

    In this study, conducted in C57BL/6 mice during physiological aging by western blotting we measured a gradual reduction of the amount of Zonula occludens1, a tight junction protein of the BBB, indicative of a loss of BBB integrity. As consequence, the altered permeability of the BBB favours the income of iron in the brain. Indeed, in the cerebral cortex we found iron overload, demonstrated by Prussian blue Perl’s staining, accompanied by the increase of the expression of inflammatory and antioxidant genes (SAA1 and Nfr2), and reactive astrocytes and microglia verified by immunohistochemistry.

    Interestingly, we observed that Hepcidin (Hepc), the iron-regulating peptide hormone produced mainly by hepatocytes, is expressed and upregulated in the aged brain in response to iron overload. Consequently, we found also that Ferroportin1 (Fpn1), the only iron exporter and target of Hepc, is downregulated in old mice cortexes, causing iron retention within neurons. Indeed, by immunohistochemistry, we observed that the iron exporter Fpn1 is expressed mainly by astrocytes, while the iron storage protein ferritin accumulates within neurons. These observations showed that the Hepc/Fpn1-axis is also active in the brain during aging. Moreover, we demonstrated for the first time that iron accumulation within neurons is due to the activation of a selective form of autophagy mediated by NCOA4, termed “ferritinophagy”.

    Overall, our data highlight new regulators of brain iron and potential targets in the prevention of oxidative damage, stress, and neurodegeneration.

  • Identification of Sex-specific autophagy enhancers for dementia

    Authors:
    Brunella Mongiardi (1,2,3), Vittorio Loffredo (1,2,4), Mariagrazia Monaco (2), Cristina Somma (2,3), Greta Fabiani (1), Maria Sanzari (1), Giulia Torromino (1,2), Maria De Risi (1,2), Elvira De Leonibus (1,2)

    Organisations:
    1: Institute of Biochemistry and Cell Biology (IBBC), National Research Council, Monterotondo (Rome), Italy
    2: Telethon Institute of Genetics and Medicine, Telethon Foundation, Pozzuoli (NA), Italy
    3: PhD Program in Biomolecular Sciences, Luigi Vanvitelli University of Campania
    4: PhD Program in Behavioral Neuroscienc, Sapienza University of Rome, Rome, Italy

    Presenting author: Brunella Mongiardi

    Exposition date: Saturday, September 16, 2023

    Exposition position: 011/2

    Aging leads to the accumulation of misfolded proteins, such as β-amyloid and α-synuclein, which are believed to underly the onset of neurodegenerative disorders. Most of the treatments for brain disorders come from pre-clinical evidence in males, although women are more vulnerable than men to Alzheimer’ disease (AD) which is the first cause of age-related dementia. However, the mechanisms underlying these sex-differences are not known. To date, two therapeutic approaches are predominantly used to prevent and/or slow neurodegeneration; they include autophagy pharmacological enhancers and exercise training. Autophagy is one of the most relevant catabolic process whereby misfolded proteins are degraded. However, most of the preliminary observations of the efficacy of these treatments have been obtained in male mouse models, although sex-differences in genes expression and response to pharmacological treatments have been reported for non-brain pathologies. In this study, using a mouse model of early aging, we report that female mice have similar cognitive deficits to males, but they occurred earlier. In both sexes this cognitive decline is associated with the accumulation of misfolded protein and impaired autophagy-lysosomal function. However, they differently respond to the precognitive effects of exercise training and autophagy agonists. Similar results were obtained using a genetic mouse model of AD. These findings provide novel and translational relevant preclinical evidence of sex-dependent efficacy of anti-dementia treatments, filling a major knowledge gap in gender medicine for AD.

  • In vitro study of p300 role in cellular senescence

    Authors:
    Elisabetta Di Fede (1,2), Antonella Lettieri (1), Silvia Ancona (1), Esi Taci (1), Silvia Castiglioni (1), Elisa Adele Colombo (1), Paolo Grazioli (1), Clara Bernardelli (1), Elena Lesma (1), Stefania Corti (3,4), Alberto Priori (1,2,5), Cristina Gervasini (1,2), Valentina Massa (1,2)

    Organisations:
    1: Department of Health Sciences, University of Milan, Milan, Italy
    2: Aldo Ravelli Center for Neurotechnology and Experimental Brain Therapeutics, Università degli Studi di Milano, Milan, Italy
    3: Dino Ferrari Centre, Department of Pathophysiology and Transplantation, Neuroscience Section, University of Milan, Milan, Italy
    4: IRCCS Foundation Ca’ Granda Ospedale Maggiore Policlinico, Neurology Unit, Milan, Italy
    5: ASST Santi Paolo e Carlo, San Paolo University Hospital, Milan, Italy

    Presenting author: Elisabetta Di Fede

    Exposition date: Saturday, September 16, 2023

    Exposition position: 018/2

    Recently, it was reported that p300, a lysine acetyl transferase protein, could represent a driver of senescence, a process involved in both physiological and pathological process (i.e. cancer and neurodegeneration). Thus, to deepening our understanding of p300 role in senescence we performed experiments on lymphoblastoid cell lines (LCLs) obtained from Rubinstein-Taybi syndrome patients carrying EP300 mutations (EP300+/-) and healthy donor (HD). We induced a senescent-phenotype by oxidative stress in both cell lines revealing that EP300+/- cells were less responsive to senescence induction compared to HD LCLs. We validated our results, obtained with a natural model, mimicking the genetic variants using in HD LCLs a specific pharmacological inhibitor of p300, CCS1477. We confirmed in our models that mutations in EP300 or p300 inhibition impair senescence response processes paving the way to implementing new potential therapeutic target for senescence in age-related disorders, that we are currently studying using neuronal and 3D models.

  • Inhibition of fatty acid amide hydrolase reverted the amyloid-beta-induced microglia polarization by restoring autophagy activity

    Authors:
    Federica Armeli (1), Giacomo Giacovazzo (2), Beatrice Mengoni (1), Roberto Coccurello (2), Rita Businaro (1)

    Organisations:
    1: Sapienza Università di Roma, Italy
    2: IRCCS - S. Lucia Foundation , Rome, Italy

    Presenting author: Federica Armeli

    Exposition date: Saturday, September 16, 2023

    Exposition position: 007/2

    The potential of endocannabinoids (eCBs) to influence microglia homeostasis may be considered as a target for therapeutic intervention in neurodegenerative diseases, including Alzheimer's disease (AD).

    The first endocannabinoid to be characterized was anandamide (AEA), which is hydrolyzed by fatty acid amide hydrolase (FAAH).

    In AD patients, a decrease in brain levels of AEA was found to correlate with an increase in FAAH activity. Using murine BV2 microglia cells stimulated with Aβ peptide, we aimed to determine if inhibition of FAAH by administration of URB597 could affect microglia polarization and provide beneficial effects in restoring the autophagy process, which is impaired in AD. Few recent studies have evaluated the role that impaired autophagy plays on the microglial phenotype. Modulation of microglia autophagy may influence the immune response and thus neuroinflammation in aging and AD.

    In our hands, the evaluation of morphology, pro- and anti-inflammatory cytokine analysis and key markers of microglial activation (iNOS and ARG-1) showed the ability of URB597 to revert microglial activation toward the anti-inflammatory status. Moreover, URB597 administration restored autophagy activity in microglial cells as demonstrated by the increase of ATG7, Beclin1, LC3 and P62 mRNA expression. Overall, our data suggest that FAAH inhibition can drive microglial polarization toward an anti-inflammatory phenotype and promote autophagy. Thus, the modulation of ECBS signaling represents a promising therapeutic target against AD.

  • Insight into the biological properties of trodusquemine and its use as nerve marker for light-sheet and expansion microscopy

    Authors:
    Claudia Capitini (1,2), Luca Pesce (2), Giulia Fani (3), Michael Zasloff (4,5), Fabrizio Chiti (3), Francesco Saverio Pavone (1,2,6), Martino Calamai (1,2)

    Organisations:
    1: National Institute of Optics –National Research Council (CNR-INO), Sesto Fiorentino, Italy
    2: European Laboratory for Non-Linear Spectroscopy (LENS), University of Florence, Sesto Fiorentino, Italy
    3: Department of Experimental and Clinical Biomedical Sciences, Section of Biochemistry, University of Florence, Florence, Italy
    4: MedStar-Georgetown Transplant Institute, Georgetown University School of Medicine, Washington, DC, USA
    5: Enterin Inc., Philadelphia, Pennsylvania, USA
    6: Department of Physics, University of Florence, Sesto Fiorentino, Italy

    Presenting author: Claudia Capitini

    Exposition date: Saturday, September 16, 2023

    Exposition position: 014/2

    Trodusquemine is an aminosterol with a variety of biological and pharmacological functions, such as acting as an antimicrobial, stimulating body weight loss and interfering with the toxicity of Aβ and α-synuclein oligomers involved in the development of Alzheimer's and Parkinson's diseases, respectively. The mechanisms of interaction of this aminosterol with cells are, however, still largely uncharacterized. Here, by using fluorescently labeled trodusquemine (TRO-A594, TRO-ATTO565 and TRO-ATTO488), we show that it binds initially to the plasma membrane of living cells, that the binding affinity is dependent on cholesterol, and that trodusquemine is then internalized and mainly targeted to lysosomes after internalization. We also found that TRO-A594 is able to strongly and selectively bind to myelinated fibers in fixed mouse brain slices, and that it is a marker compatible with light-sheet fluorescence microscopy and expansion microscopy. In addition, ongoing experiments are aimed at deeply investigating the mechanism of action adopted by trodusquemine to lower the toxicity of Aβ oligomers in neuronal cells using a fluorescence-based approach, and evaluating the influence of membrane cholesterol and GM1. In conclusion, this work contributes to further characterize the biology of trodusquemine, with a focus on its protective role against Aβ oligomers, and provides a new tool for nerve labeling suitable for the most advanced microscopy techniques.

  • Investigating the molecular and cellular mechanisms explaining the connection between metabolic changes and the deterioration of cognitive function

    Authors:
    Silvia Penati (1,2), Chiara Adriana Elia (2), Valentina Murtaj (3), Marta Medaglia (2,4), Erica Tagliatti (2), Cecilia Gotti (5), Irene Corradini (1,2), Rosa Maria Moresco (3,6,7), Michela Matteoli (1,2,8), Maria Luisa Malosio (1,2)

    Organisations:
    1: Institute of Neuroscience, Consiglio Nazionale delle Ricerche (CNR), URTc/o IRCCS Humanitas Clinical and Research Center, Rozzano (MI), Italy
    2: Laboratory of Pharmacology and Brain Pathology, Neuro-Center Humanitas Clinical and Research Center, Rozzano (MI)
    3: Department of Nuclear Medicine, IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy
    4: University of Pavia, Pavia, Italy
    5: Institute of Neuroscience (CNR), Milan, Italy
    6: Department of Medicine and Surgery, University of Milano-Bicocca, 20900 Monza (MB), Italy
    7: Institute of Molecular Bioimaging and Physiology, CNR, 20090 Segrate (MI), Italy
    8: Humanitas University, Department of Biomedical Sciences, Pieve Emanuele (MI), Italy.

    Presenting author: Maria Luisa Malosio

    Exposition date: Saturday, September 16, 2023

    Exposition position: 023/2

    Increasing evidence suggests a link between metabolic disorders, such as insulin-resistance and type 2 diabetes, and cognitive decline as well as Alzheimer's Disease (AD). Changes in peripheral insulin sensitivity due to diet can affect brain cognitive functions. Altered glucose metabolism in the brain, along with high levels of fatty acids and chronic low-grade inflammation, could be the underlying mechanism linking type 2 diabetes and AD.

    In this study, an animal model of diet-induced glucose intolerance and insulin-resistance was established to mimic obesity and prediabetes. Mice were fed with 45% and 60% high-fat diets (HFD) for several weeks, and various tests were conducted to evaluate their weight gain, glucose tolerance, insulin tolerance, plasma leptin, and IL6 levels.

    The results showed that weight gain increased significantly after only two weeks of HFD consumption, while glucose intolerance became evident after three weeks and insulin-resistance after five weeks. At six weeks of high-fat diet, the mice showed significant changes in anxiety-like behaviors. At later time points (13 weeks), episodic memory was also affected.

    Further examination of the hippocampal tissues of mice exposed to HFD revealed altered levels of p-AKT, reduced levels of excitatory subunit receptors, reduced levels of cfos, and elevated levels of markers of endoplasmic reticulum (ER) stress.

    PET studies were performed to investigate the brain glucose metabolism.

    To investigate the molecular mechanisms responsible for insulin resistance in the brain, palmitate treatments were used on primary neuron cell cultures in vitro to mimic the metabolic conditions induced by HFD.

    Overall, the study findings suggest that even short-term exposure (8 and 14 weeks) to HFD can alter relevant brain functions, including neurotransmitter receptors, and induce anxiety and memory deficits at later time points.

  • JNK Activation Correlates with Cognitive Impairment and Alteration of the Post-Synaptic Element in the 5xFAD AD Mouse Model

    Authors:
    Arianna Giani (1,2), Erica Cecilia Priori (1,2), Clara Alice Musi (1,2), Luca Colnaghi (3,4), Ivana Milic (5), Andrew Devitt (5), Tiziana Borsello (1,2), Mariaelena Repici (5)

    Organisations:
    1: Department of Pharmacological and Biomolecular Sciences - University of Milan, Milan,Italy
    2: Mario Negri Institute for Pharmacolgical Research - IRCCS, Via Mario Negri 2, Milan,Italy
    3: Division of Neuroscience - IRCCS San Raffaele Scientific Institute, Milan,Italy
    4: School of Medicine, Vita-Salute San Raffaele University, Milan,Italy
    5: College of Health and Life Sciences, Aston University, Birmingham,UK

    Presenting author: Arianna Giani

    Exposition date: Saturday, September 16, 2023

    Exposition position: 017/2

    The c-Jun N-terminal kinases (JNKs) are a family of proteins that, once activated by stress stimuli, can alter neuronal functions and survival. The JNK cascade plays a crucial role in the post-synaptic neuronal compartment by altering its structural organization and leading, at worst, to an overall impairment of neuronal communication. Increasing evidence suggests that synaptic impairment is the first neurodegenerative event in Alzheimer’s disease (AD). To better elucidate this mechanism, we longitudinally studied 5xFAD mice at three selected time points representative of human AD symptom progression. We tested the mice cognitive performance by using the radial arm water maze (RAWM) in parallel with biochemical evaluations of post-synaptic enriched protein fraction and total cortical parenchyma. We found that 5xFAD mice presented a strong JNK activation at 3.5 months of age in the post-synaptic enriched protein fraction. This JNK activation correlates with a structural alteration of the post-synaptic density area and with memory impairment at this early stage of the disease that progressively declines to cause cell death. These findings pave the way for future studies on JNK as a key player in early neurodegeneration and as an important therapeutic target for the development of new compounds able to tackle synaptic impairment in the early phase of AD pathology.

  • Midbrain lesion triggers hippocampal inflammation by a simultaneous Dopamine-Serotonin depletion

    Authors:
    Emma Cauzzi (1,2), Livia La Barbera (2,3), Paraskevi Krashia (2,3), Annalisa Nobili (2), Elena Possemato (3), Maria Luisa De Paolis (2,3), Ilaria Paoletti (2,3), Elena Spoleti (2,3), Luana Saba (2,3), Maria Teresa Viscomi (4), Marcello D'Amelio (2,3)

    Organisations:
    1: University of Rome "Tor Vergata", Italy
    2: IRCCS Fondazione Santa Lucia, Rome
    3: University Campus Bio-Medico of Rome, Italy
    4: Università Cattolica del Sacro Cuore, Rome, Italy

    Presenting author: Emma Cauzzi

    Exposition date: Saturday, September 16, 2023

    Exposition position: 015/2

    Monoamines such as dopamine (DA) and serotonin (5HT) are neurotransmitters synthetized in the midbrain and are known to tune the functions of midbrain-innervated areas involved in neurodegenerative disorders, like the hippocampus affected in Alzheimer’s disease (AD). Incidentally, recent preclinical and clinical works highlighted structural, functional and metabolic midbrain alterations in the AD prodromal phase. Hence, we hypothesized that precocious midbrain degeneration might impair monoamine release, leading to the appearance of neurodegenerative disease hallmarks, including neuroinflammation and behavioral deficits.

    To prove this, we developed ventral midbrain-lesioned mice through a unique viral approach allowing the overexpression of the pro-apoptotic active Caspase-3 (Casp-3) under the Tyrosine Hydroxylase promoter expressed in DA midbrain neurons and in neighboring 5HT cells of the interpeduncular nucleus (IPN).

    Here, by stereology we confirmed the loss of midbrain DA neurons and of 5HT neurons in the IPN. Confocal microscopy and HPLC analysis revealed reduced DA and 5HT fiber density and monoamine levels in the hippocampus, respectively.

    Importantly, the depletion of both monoamines resulted in hippocampal neuroinflammation characterized by microglial proliferation and microglial NLRP3-inflammasome activation. Of note, Casp3-injected mice also displayed deficits in Novel-Object-Recognition, in line with the importance of these monoamines for memory functions.

    Surprisingly, microglial-mediated neuroinflammation was not present in the striatum of Casp3-injected mice, affected only by DA deprivation, nor in midbrain 6OHDA mice, suggesting that DA loss alone is not sufficient to activate neuroinflammation. Nonetheless, pharmacological treatments aimed at boosting the DA and 5HT tone were both able to tamp down NLRP3-mediated neuroinflammation in the hippocampus of Casp3-injected mice.

    Overall, these results demonstrate that the neuroinflammatory events observed early in neurodegenerative diseases such as AD, could be a direct consequence of an early midbrain damage leading to a combined DA-5HT deprivation of hippocampus, suggesting that early boost of this monoaminergic signaling can blunt neuroinflammation.

  • Morphological and mitochondrial changes in murine choroid plexus epithelial cells during healthy aging

    Authors:
    Valentina Scarpetta (1,6), Felipe Bodaleo (2), Chiara Salio (3), Marco Sassoè-Pognetto (1), Amit Agarwal (2,4), Annarita Patrizi (4,5,6)

    Organisations:
    1: Department of Neurosciences Rita Levi Montalcini, University of Turin, Turin, 10126, Italy
    2: Chica and Heinz Schaller Research Group, Institute for Anatomy and Cell Biology, Heidelberg, 69120, Germany
    3: Department of Veterinary Sciences, University of Turin, Turin, 10095, Italy
    4: Interdisciplinary Center for Neuroscience, Heidelberg University, Heidelberg, 69120, Germany
    5: Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, Heidelberg, Germany
    6: Schaller Research Group, German Cancer Research Center (DKFZ), Heidelberg, 69120, Germany

    Presenting author: Valentina Scarpetta

    Exposition date: Saturday, September 16, 2023

    Exposition position: 012/2

    Choroid plexuses (ChPs) are intraventricular structures mainly composed by specialized epithelial cells interconnected by tight junctions establishing the blood-cerebrospinal fluid (CSF) barrier. ChPs are essential to produce CSF and transport solutes from and into the brain. Changes in ChP morphology and function have been correlated to neurodegenerative disorders. Using electron microscopy, two-photon microscopy and biochemistry, we have investigated ChP epithelial cells during healthy aging, starting from young adult to two-years old mice. We identified distinct morpho-functional modifications in ChP epithelial cells in mice aged 8-12 months, which mostly remained stable up to two years. These changes include flattening of the epithelium, reduction of microvilli length, enlargement of basolateral membrane invaginations and augmentation of interrupted tight junctions. We also found a decrease in mitochondria density together with elongation of mitochondria in older mice. Two-photon imaging of whole ChP living explants revealed that morphological rearrangements of mitochondria were accompanied by increased superoxide levels, decreased membrane potential and decreased mitochondrial motility in aged mice, suggesting possible mitochondrial oxidative damage and impaired ability to synthetize ATP at baseline. Interestingly, most of the age-related changes were not accompanied by modification of protein and/or gene expression levels and aged mitochondria effectively responded to acute pharmacological stressful stimuli, raising the possibility that the morphological remodeling observed in mitochondria may represent an adaptive mechanism engaged by ChP epithelial cells to endure stressful events. Our study suggests a long-term progression of multiple morpho-functional features of the mouse choroid plexus epithelium during lifetime.

  • Neurocognitive decline during aging is associated with functional alterations in the ventral tegmental area and in the hippocampal plasticity

    Authors:
    Claudia Sagheddu (1), Shima Kouhnavardi (2,3), Ahmed M. Hussein (2,4), Marco Pistis (1,5,6), Francisco J. Monje (7), Gert Lubec (2), Jana Lubec (2)

    Organisations:
    1: University of Cagliari, Italy
    2: Paracelsus Medical University, Salzburg, Austria
    3: University of Vienna, Vienna, Austria
    4: Al-Azhar University, Assiut, Egypt
    5: National Research Council of Italy (CNR), Section of Cagliari, Italy
    6: University Hospital, Cagliari, Italy
    7: Medical University of Vienna, Vienna, Austria

    Presenting author: Claudia Sagheddu

    Exposition date: Saturday, September 16, 2023

    Exposition position: 013/2

    The process of ageing can be influenced by several factors, ranging from genetics to lifestyle. In particular, general well-being over the years can be associated with either healthy cerebral aging or functional decline, which could account as a prodromal phase towards the development of dementia, as well as neurodegenerative disorders.

    The midbrain ventral tegmental area (VTA) operates as a hub amidst affective and cognitive facets, processing sensory inputs related to motivated behaviors and hippocampal memory, among other functions. Increasing evidence show a direct dopaminergic and non-dopaminergic input form the VTA to the hippocampus. This connection could be compromised throughout the lifespan1-3, though the mechanisms are understudied.

    Here, we took advantage of a distinctive procedure from our group to separate superior- and inferior-performing rats over the course of the time4-5, as a model of healthy- versus compromised-brain aging. By combining behavioral, in vivo, and ex vivo electrophysiological recordings we investigated the functional outcomes of different neuronal populations in the VTA and the hippocampal plasticity. In aged rats, we found decreased locomotor activity, electrophysiological alterations in the VTA, and hippocampal plasticity impairment, as compared to young. In the VTA, we further evidenced cell-specific functional changes, which were associated with the superior/inferior cognitive performance in the old group.

    This study provides new insight into possible mechanisms underlying the compromised cerebral ageing. Further understanding of the brain senescence, possibly related to neurocognitive decline, will help developing social, psychological, as well as pharmacological, strategies towards preservation of high quality of life.

    1 McLachlan 2009. Can J Neurol Sci. Aug;36 Suppl 2:S84-7.

    2 Bettio et al., 2017 Neurosci Biobehav Rev. Aug;79:66-86.

    3 Penner and Mizumori 2012, Front Aging Neurosci. Aug 8;4:22.

    4 Lubec et al,. 2019 Front Aging Neurosci. Jul 31;11:198.

    5 Lubec et al,. 2021 Mol Psychiatry. Dec;26(12):7076-7090.

  • Novel approach for pre-diagnosis and follow-up of patients affected by Gaucher disease based on specific flow cytometry and fluorescent labeling

    Authors:
    Costanza Ceni (1,2), Francesca Clemente (3), Rodolfo Tonin (4), Amelia Morrone (4), Francesca Cardona (3), Martino Calamai (2,5)

    Organisations:
    1: Department of Chemistry, University of Florence, Sesto Fiorentino, Italy
    2: European Laboratory forNon-Linear Spectroscopy (LENS), University of Florence, Sesto Fiorentino, Italy
    3: Department of Chemistry "U. Schiff", University of Florence, via della Lastruccia 3-13, 50019 Sesto F.no (FI), Italy
    4: Molecular and Cell Biology Laboratory of Neurometabolic Diseases, Neuroscience Department, Meyer Children's Hospital, Florence, Italy
    5: National Institute of Optics - National Research Council (CNR-INO), Sesto Fiorentino, Italy

    Presenting author: Costanza Ceni

    Exposition date: Saturday, September 16, 2023

    Exposition position: 016/2

    Gaucher disease (GD) belongs to the lysosomal storage disorders (LSDs) and results from biallelic mutations in the gene GBA1, disrupting the function of the lysosomal glycoside hydrolase glucocerebrosidase (Gcase) and causing an accumulation of glucosylceramide (Glc-cer), as well as secondary storage materials, including ganglioside GM1. GD can be divided into three types, based on the presence and progression of neurological manifestations: while the type 1 is not related with any neuronal damage, types 2 and 3 typically cause neuronal impairments. Moreover, in patients carrying GBA1 mutations it has also been observed an increased incidence of Parkinson’s disease. Up to date, the measurement of in vitro enzymatic activity of Gcase with fluorescent substrates represent one of the most used techniques to evaluate therapeutic effects of drugs, potentially useful for the treatment of GD. However, an in vitro increase of Gcase activity might not lead to a reduction of Glc-cer accumulation in cells. In this contest, by using frozen/thawed fibroblasts, we explored the possibility of using specific fluorescent markers together with flow-cytometry and confocal microscopy in order to monitor changes in storage materials directly in fibroblasts from patients with different GD phenotypes.

    We demonstrated that with our methods it is possible to clearly distinguish the levels of accumulated Glc-cer and GM1 in fibroblasts from affected and unaffected patients, providing the basis for a rapid, sensitive and unbiased approach, applicable in routine laboratory practice, to monitor the efficacy of therapeutic strategies on reducing the storage materials directly in cells.

    Thanks to the Union European – NextGenerationEU for financial support in research project FluoPCPark and Cassa di Risparmio di Pistoia e Pescia Foundation for financial support in research project IDANEUROPARK.

  • PF-04691502, a PI3K/mTOR dual inhibitor, improves learning deficits in APP/PS1 mice.

    Authors:
    Marika Lanza, Giovanna Casili, Salvatore Oddo, Salvatore Cuzzocrea, Emanuela Esposito

    Organisations:
    University of Messina, Italy

    Presenting author: Marika Lanza

    Exposition date: Saturday, September 16, 2023

    Exposition position: 021/2

    Aging is the greatest risk factor for several neurodegenerative disorders, including Alzheimer’s disease (AD). Overwhelming evidence indicates that reducing mTOR signaling improves health span and lifespan in a multitude of organisms. PI3K is a key regulator of mTOR activity; the PI3K/mTOR signaling pathway regulates several key biological mechanisms related to cell development, cell survival, protein synthesis, autophagy, metabolism, and learning and memory. To this end, up-regulation of the PI3K/mTOR signaling contributes to AD neuropathology and causes neurodegeneration and learning and memory deficits. In this study, we sought to determine the molecular correlates of memory deficits in APP/PS1 mice, a widely used animal model AD. 18-month-old APP/PS1 and WT mice were dosed orally with 1 mg/Kg PF-04691502, an ATP-competitive PI3K/mTOR dual inhibitor, for 12 weeks. At the end of the treatment, we assessed changes in spatial learning and memory using the Morris water maze. We then processed their brains for neuropathological and biochemical assessment of amyloid-β (Aβ). We found that PF-04691502 improved learning and memory in APP/PS1 mice. Currently, we are processing the tissue to assess potential changes in brain Aβ deposits and soluble and insoluble Aβ levels. We will also assess the effects of reducing PIK3/mTOR signaling on inflammation. These results provide preclinical data indicating that PF-04691502 may be a valid therapeutic approach for AD and other neurodegenerative disorders associated with aging and mTOR hyperactivity.

  • Phenotype-specific gene expression patterns in unmutated sporadic ALS patients

    Authors:
    Francesca Dragoni (1,2), Rosalinda Di Gerlando (1,2), Maria Garofalo (2), Luca Diamanti (2), Bartolo Rizzo (1), Matteo Bordoni (2), Eveljn Scarian (2), Orietta Pansarasa (2), Stella Gagliardi (2)

    Organisations:
    1: University of Pavia, Italy
    2: IRCCS Mondino Foundation, Italy

    Presenting author: Francesca Dragoni

    Exposition date: Saturday, September 16, 2023

    Exposition position: 009/2

    Amyotrophic lateral sclerosis (ALS) is an adult neurodegenerative disorder and according to clinical criteria, sporadic ALS (sALS) patients can be classified into subgroups: classic, bulbar, pyramidal, pure lower motor neuron, flail arm, pure upper motor neuron, flail leg, and respiratory. There are no well-established biomarkers for early diagnosis, prognosis, and disease progression. The involvement of RNA-mediated toxicity is a key event in ALS, representing an opportunity to identify specific traits in ALS subgroups. Classification based on clinical phenotypes could be associated with different gene expression patterns shaped during lifespan, allowing the identification of specific ALS subtypes with homogeneous clinical, biological features. Our objective is to define the transcriptomic signatures of distinct ALS phenotypes, and to use this information for biomarker assessment and personal therapy development. We characterized n=48 sALS patients by clinical and paraclinical phenotype, and subdivided them in “Classic” (n=12), “Bulbar” (n=10), “Flail Arm” (n=7), “Flail Leg” (n=10) and “Pyramidal” (n=9). RNAs extracted from PBMCs isolated from patients and n=19 controls were sequenced. By performing a Principal Component Analysis (PCA), we observed genes expression clusterization between patients and controls, except for “Flail Arm” group. A significant number of deregulated genes have been shown to be associated with the bulbar phenotype. Only one gene (Y-RNA, a misc_RNA component of the Ro60 ribonucleoprotein involved in cellular response to interferon-alpha) was upregulated across all phenotypes, whereas the other genes appeared to be phenotype-specific. Y-RNA belongs to a conserved family of non-coding RNA which are the most abundant in plasma. There is evidence of a relationship between the ratio of the main members of the Y-RNA family and diseases progression. Hence, our future goal is to determine a 'Y-RNA signature' in plasma and a multi-parametric prognostic score that could represent a simple and fast element for early diagnosis and discrimination among sALS phenotypes.

  • rTMS effects on neuroplasticity in mild cognitive impairment (MCI)

    Authors:
    Minoo sharbafshaaer, Sabrina Esposito, Fabrizio Canale, Giulia D'Alvano, Francesca Aammora, Danilo Atripaldi, Dario Ricciardi, Gioacchino Tedeschi, Francesca Trojsi

    Organisations:
    Department of Advanced Medical and Surgical Sciences, MRI Research Center, Università degli Studi della Campania Luigi Vanvitelli, 80138 Naples, Italy

    Presenting author: Minoo sharbafshaaer

    Exposition date: Saturday, September 16, 2023

    Exposition position: 001/2

    rTMS is used as a therapeutic tool in neurodegenerative disease and has been shown to induce plasticity of excitatory synapses in the brain. It’s effective over the dorsolateral prefrontal cortex (DLPFC) in cognitive performances within mild cognitive impairment (MCI) patients. We investigated the efficacy of rTMS on brain plasticity in 9 MCI patients receiving high-frequency (10 Hz) rTMS compared to 9 patients receiving sham stimulation daily for four weeks. Six months of monitoring and ministering the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) before and after rTMS showed a significant improvement in visuospatial functions in the treated group.

  • Terpenes and alzheimer’s disease: a preliminary in vitro study

    Authors:
    Sveva Dallere (1,2), Elena Signorino (1), Gianna Pavarino (1), Clelia Ferrero (1), Marina Boido (1), Alessandro Vercelli (1)

    Organisations:
    1: Department of Neuroscience Rita Levi Montalcini, Neuroscience Institute Cavalieri Ottolenghi, University of Turin, Italy
    2: University School for Advanced Studies IUSS Pavia

    Presenting author: Sveva Dallere

    Exposition date: Saturday, September 16, 2023

    Exposition position: 008/2

    Alzheimer's disease (AD) is the leading cause of dementia during aging. Its pathological hallmarks (such as neuronal loss, amyloid beta (Aβ) plaques, neurofibrillary tangles and neuroinflammation) have been identified, while disease-modifying treatments are not yet available.  Lifestyle interventions could play an important role in the prevention and treatment of multifactorial diseases, as AD. For instance, greenness exposure is known to positively impact on our health through different and not fully understood mechanisms. In this context, we aim to clarify how molecules released by plants (terpenes) can mitigate AD, fostering neuronal survival and modulating neuroinflammation. We will focus on pinene, a terpene abundantly released by plants of Piedmont (Italy). As a first step, we have performed in vitro experiments on NE-4C cells (a murine neural cell line) that upon retinoic acid stimulation differentiate in a mixed population of neurons and astrocytes. In this model Aβ1-42 monomers administration (between 1 and 5 µM for 48h) recapitulates AD-linked neurodegeneration, decreasing cell viability (assessed by MTT assay) in both undifferentiated and differentiated cells. Preliminary results demonstrate that Pinene (100 to 500 µM), co-administered with Aβ1-42, could be able to reduce cell death (in particular 250 and 500 µM). By cell viability assays and live cells imaging we are identifying the optimal concentration of pinene to counteract neurodegeneration and glial activation, and by molecular analyses we will unravel the underlying mechanisms. These observations will be then verified also on human iPSC-derived glutamatergic neurons (in presence of Aβ1-42) and in vivo, on a well-known murine AD model (5xFAD mice). The study will clarify the mechanisms underlying the benefits derived from green exposure, thus helping in giving the scientific basis to promote the diffusion of lifestyle interventions to prevent and treat AD.

  • The cyclase-associated protein 2 controls cofilin-actin rods formation in Alzheimer’s Disease

    Authors:
    Ramona Stringhi (1), Laura D'andrea (1), Lina Vandermeulen (1), Miriam Ascagni (2), Monica Di Luca (1), Silvia Cecilia Pelucchi (1), Elena Marcello (1)

    Organisations:
    1: Università degli Studi di Milano, Department of Pharmacological and Biomolecular Science, Italy
    2: Università degli Studi di Milano, Unitech NOLIMITS, Italy

    Presenting author: Silvia Cecilia Pelucchi

    Exposition date: Saturday, September 16, 2023

    Exposition position: 019/2

    Several neurodegenerative disorders, among which Alzheimer's disease (AD), are associated with cytoskeletal abnormalities, called cofilin-actin rods. These aggregates are described as composed of cofilin and actin and their formation occurs upon exposure to different stressors, including Aβ oligomers, the main drivers of synaptic failure. We have recently demonstrated that the actin-binding protein cyclase-associated protein 2 (CAP2) is a master regulator of cofilin localization and activity, through the Cys32-dependent CAP2 dimerization. Remarkably, the mechanisms are altered in AD suggesting the involvement of CAP2/cofilin pathway in AD pathogenesis. In light of these results, the aim of the study is to investigate CAP2 role in the generation of cofilin-actin rods in AD. Taking advantage of 3D confocal analysis, we found that CAP2 accumulates within rods when specifically induced by Aβ oligomers and not by another stressor. Short-term Aβ oligomers exposure triggers the removal of CAP2 and cofilin from postsynaptic compartment. Instead, after long-term treatment, able to induce synaptic loss and actin rods formation, cofilin is still reduced while CAP2 dimerization is impaired. To prove that CAP2 is a key element in rods formation, we tested the effects of CAP2 overexpression. In hippocampal cultures, CAP2 prevents Aβ-induced rods formation and synapse loss. Besides this, we tested the same rescue strategy in an AD mouse model: the analysis of the hippocampal region confirmed that CA overexpression prevents rods formation. Overall, our data support the involvement of cofilin/CAP2 pathway in the generation of cytoskeleton abnormalities that can affect synaptic function in AD.

  • Transcriptomic Profile Across Different Brain Areas in Alzheimer’s Disease

    Authors:
    Riccardo Rocco Ferrari (2,3), Valentina Fantini (2,7), Maria Garofalo (4), Francesca Dragoni (4,8), Rosalinda Di Gerlando (4,8), Annalisa Davin (2), Xhulja Profka (1), Valentina Medici (1,6), Antonio Guaita (1,2), Stella Gagliardi (4), Tino Emanuele Poloni (1,5)

    Organisations:
    1: Department of Neurology and Neuropathology, Golgi-Cenci Foundation, 20081 Abbiategrasso, Milan, Italy
    2: Laboratory of Neurobiology and Neurogenetics, Golgi-Cenci Foundation, 20081 Abbiategrasso, Italy
    3: Department of Brain and Behavioral Sciences, University of Pavia, 27100 Pavia, Italy
    4: IRCCS Mondino Foundation, 27100 Pavia, Italy
    5: Department of Rehabilitation, ASP Golgi-Redaelli, 20081 Abbiategrasso, Milan, Italy
    6: Department of Translational Medicine, University of Eastern Piedmont, 28100 Novara, Italy
    7: Laboratory of Translational Research, Azienda USL-IRCCS of Reggio Emilia, Reggio Emilia, Italy
    8: Department of Biology and Biotechnology “L.Spallanzani”, University of Pavia, 27100 Pavia, Italy

    Presenting author: Riccardo Rocco Ferrari

    Exposition date: Saturday, September 16, 2023

    Exposition position: 020/2

    Background: Aging is often characterized by a progressive loss of cognitive abilities due to Alzheimer Disease (AD). Recently, -omic sciences have allowed us to begin to shed light on its molecular pathways in order to speculate about new and attractive hypothesis on AD pathogenesis.

    Methods: In this study, a whole transcriptome analysis and a Gene Set Enrichment Analysis (GSEA) have been carried out on brain samples from hippocampus (HI), temporal and parietal cortex (TC, PC), cingulate cortex (CG), and substantia nigra (SN) from the Abbiategrasso Brain Bank (ABB) of 6 subjects with clinical and neuropathological diagnosis of AD and 3 healthy age-matched controls in duplicate. Clinical and neuropsychological profile were obtained through evaluations made at the baseline and in the following years. The neuropathological diagnosis was based on a complete histological characterization (ABB protocol).

    Results: Transcriptomic results showed a greater number of differentially expressed genes (DEGs) in TC (1571) and CG (1210) and a least amount of DEGs in HI (206), PC (109) and SN (60). Furthermore, the GSEA showed a difference between the group of early affected brain areas (HI, TC) and the group of the subsequently involved areas (PC, CG, SN). Notably, in HI and TC there was a significant down-regulation of shared DEGs primarily involved in the synaptic transmission and in the development of the central nervous system (CDK5R1, KCNC1, KCNC2, BSN), while in PC, CG and SN there was a significant down-regulation of genes primarily involved in the proper protein folding and trafficking and inflammation (HSPA1B, HSPA1A, DNAJB1, FKBP4, CHORDC1, STIP1).

    Conclusions: The course of AD could follow a definite time- and severity-related pattern that arises from inflammatory processes and protein misfolding and leads to synaptic impairment. Therefore, we can relate neurobiological processes to clinical manifestations and outline a clearer picture of AD spreading and pathogenesis.

Applications and development of neuroimaging

  • Fluorescently labelled trodusquemine as myelin marker compatible with tissue clearing techniques .

    Authors:
    Marta Rojas-Rodriguez (1), Claudia Capitini (1,2), Alessandra Franceschini (1,3), Irene Costantini (1,4), Martino Calamai (1,2)

    Organisations:
    1: LENS-European laboratory for non linear spectroscopy, Italy
    2: National Institute of Optics –National Research Council (CNR-INO)
    3: Department of Physics and Astronomy, University of Florence
    4: Department of Biology, University of Florence

    Presenting author: Marta Rojas-Rodriguez

    Exposition date: Friday, September 15, 2023

    Exposition position: 002/1

    Trodusquemine is a naturally occurring aminosterol with a variety of biological and pharmacological functions, among which antimicrobial activity and stimulation of body weight loss. Moreover, trodusquemine was found to reduce the toxicity of proteins involved in the development of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s. Additionally, the binding affinity of this aminosterol to the cell membrane is proportional to the amount of cholesterol present in the membrane meaning that it has high affinity for cholesterol rich plasma membranes.

    We have recently reported that fluorescently labelled trodusquemine is able to strongly and selectively bind to myelinated fibers and it can be used as a marker compatible with expansion microscopy and light sheet fluorescence microscopy.

    Here, we explore the use of trodusquemine labelled with two different dyes, namely TRO-ATTO565 and TRO-ATTO488, and its compatibility with a variety of tissue clearing techniques, including CLARITY, SWITCH and iDISCO in both fixed mouse and human central nervous system tissue. A common problem with these clearing methods is the retaining of the myelin labeling after processing. So far, we have observed that iDISCO is not compatible with TRO-ATTO565 staining. By contrast, we developed a novel fixation protocol, to use in combination with CLARITY, that retains the staining in place after the clearing of the tissue. The staining efficacy of myelinated fibers was improved in terms of both signal intensity and specificity compared to common methodologies based on the use of anti-myelin basic protein antibodies.

    This discovery points at labeled trodusquemine as a promising tool that can be coupled with advanced microcopy techniques based on whole brain and spinal cord imaging to better follow the pathophysiology and treatment of demyelinating diseases, such as multiple sclerosis.

  • Radiomic Analysis to Predict Language Deficit in Glioma Patients, Studying Neuroplasticity Mechanisms of White Matter Tracts

    Authors:
    Alberto Morello (1), Francesco Carbone (2), Augusto Leone (2), Uwe Spetzger (2), Diego Garbossa (1)

    Organisations:
    1: Neurosurgery Unit, Department of Neuroscience “Rita Levi Montalcini”, “Città della Salute e della Scienza” University Hospital, University of Turin, Italy
    2: Department of Neurosurgery, Städtisches Klinikum Karlsruhe, Moltkestraße 90, 76133 Karlsruhe, Germany

    Presenting author: Alberto Morello

    Exposition date: Saturday, September 16, 2023

    Exposition position: 027/2

    Pathological brain processes induce adaptive cortical and subcortical reorganization, however, the mechanisms underlying neuroplasticity that occurs in the presence of lesions in eloquent areas are not fully explained. Diffusion tensor imaging (DTI) tractography can be used to assess white matter (WM) microstructural changes and its intra- and interhemispheric plasticity mechanisms in the post-operative in the language network. This study demonstrates the use of DTI as an anatomical and pathophysiological tool to better understand the impact of gliomas on targeted major WM pathways and their post-operative reorganization. Making a quantitative analysis on the associations between WM bundles reorganization, histopathological and molecular characterization of the tumor and the language recovery,we aim to find an algorithm to predict post surgical language deficits and functional compensatory.

    We prospectively included 27 brain tumour patients (continuously updated data) suffering from left supratentorial gliomas. Through DTI tractography, we evaluated how gliomas affect the surrounding neural fibers integrity and we analyzed DTI - derived metrics (ADC, FA, RD parameters) of the frontal aslant tract (FAT), the inferior fronto-occipital fasciculus (IFOF), the arcuate fasciculus (AF) and the superior longitudinal fasciculus (SLF), compared to the homolateral and contralateral hemisphere pre- and postoperatively. We explored, through a statistical analysis, the correlations between language deficits and phonemic functional compensatory with WM tracts microstructural changes.

    In 16 patients with greater recovery from aphasia we found FAT and IFOF microstructural alterations in the perilesional area and the contralateral hemisphere. Furthermore, patients with IDH-1 mutation and without TERT mutation, showed better cognitive performance in tests measuring general semantic fluency.

    Through the correlations between phonemic functional compensatory with DTI - derived metrics and histopathological and molecular characterization of the glioma, we developed an algorithm based on a support vector machine based features of diffusion MRI tract profiles to predict language deficits and neuroplasticity ability compensatory postoperatively.

  • Rewiring dopaminergic nigral connections in the human brain: a multi-scale and integrated study

    Authors:
    Giovanni Cirillo (1), Giuseppina Caiazzo (2), Mario Cirillo (2), Michele Papa (1), Fabrizio Esposito (2)

    Organisations:
    1: Division of Human Anatomy, Neuronal Network Morphology & Systems Biology Lab, University of Campania "Luigi Vanvitelli", Italy
    2: Department of Advanced Medical and Surgical Sciences, University of Campania "Luigi Vanvitelli", Italy

    Presenting author: Giovanni Cirillo

    Exposition date: Friday, September 15, 2023

    Exposition position: 003/1

    Background: the subcortical network of the substantia nigra pars compacta (SNc), one of the main dopaminergic nuclei of the brain, is well known and mainly represented by the nigro-striatal pathway. However, dopaminergic modulation of other brain centers, such as the thalamus, has been only investigated in non-human primates and never characterized in the humans.

    Methods: we aim to dissect the dopaminergic innervation of the human thalamus using multi-scale and integrated analyses.

    First, immunohistochemical analysis will be performed on human thalamic sections, stained for the main dopaminergic markers (TH, VMAT, DAT, AADC).

    Second, starting from high-resolution MRI segmentation of the SN (that allows the identification of the postero-medial SNc from the gabaergic anterolateral SN pars reticulata - SNr) and multi-shell high-angular resolution diffusion MRI (MS-HARDI) (that allows the tractographic reconstruction of the SNc) will be performed in a group of 10 healthy subjects.

    Finally, PET-FDOPA data coming from healthy subjects (n=20) will be evaluated for both qualitative and quantitative analysis of the thalamic region.

    Results: our preliminary MS-HARDI results performed with two previously validated diffusion MRI schemes demonstrate a reproducible structural connectivity between the SNc and the thalamus, with up to an average of ~19% of the total number of streamlines encompassing the SNc and the thalamus depending on the algorithmic constrains but with no other major subcortical structures involved (and without necessarily reaching the cortex).

    Conclusions: to our best knowledge, this is the first pilot report of a direct nigro-thalamic dopaminergic projection, with a multi-scale and integrated approach. The significance and the characterization of these connections, however, is still under investigation.

  • Volumetric changes of subcortical structures in Parkinson’s disease.

    Authors:
    Carlo Tamba, Martina Noé, Mara Cinque, Davide Marnetto, Paolo Provero, Fabrizio Pizzagalli

    Organisations:
    Department of Neuroscience ‘Rita Levi Montalcini’, University of Turin, Italy

    Presenting author: Carlo Tamba

    Exposition date: Saturday, September 16, 2023

    Exposition position: 026/2

    Parkinson's disease (PD) is a progressive neurodegenerative disorder that primarily affects movement control. Understanding the structural changes that occur in the brain of individuals with PD is crucial for advancing our knowledge of the disease and developing effective diagnostic and therapeutic strategies. Voxel-based morphometry (VBM) has emerged as a valuable neuroimaging technique that allows researchers to investigate these structural alterations in PD. Grey matter volume changes implicated in PD have been reported in the substantia nigra, striatum, thalamus, and frontal cortex [1,2,3].

    Here, we investigated volumetric changes in subcortical structures, combining a detailed brainstem atlas [4] and a large PD cohort, the Parkinson's Progression Markers Initiative (PPMI) [5].

    SPM (https://www.fil.ion.ucl.ac.uk/spm/) was used to pre-processed the MRI T1w images acquired from 317 participants, 216 PD (70 F) and 101 healthy controls (HC) (38 F), from the PPMI cohort (age: 61.16 ± 10.11). Briefly, the T1w images were segmented into grey matter (GM), white matter and cerebrospinal fluid tissues, spatially normalized in the MNI space and smoother with a 6mm isotropic gaussian kernel. The probabilistic atlas developed by [4] allowed then to extract the grey matter volumes of 86 rois.

    Analysis of variance (ANOVA) was used for the VBM comparison of GM between HC and PD. Age, diagnosis, sex, diagnosis*sex and total GM volume were used as covariates. We found a robust gender-related influence on GM changes in subcortical areas, in particular on the pontine tegmentum of the left hemisphere. This opens the way to the investigation of the underlying biological mechanisms of lateralized grey matter changes in subcortical areas, in PD.

    References:

    1. Charroud, Neuroimage Clinical, 2021

    2. Hawkes, Neuropathology and Applied Neurobiology, 2007

    3. Moro, Annals of Neurology, 2020

    4. Garcia-Gomar, Front. Neurosci. 2019

    5. Marek, Prog. Neurobiol.,2011

Artificial intelligence in neuroscience

  • A New Artificial Intelligence Drug Repurposing Approach for Fibromyalgia

    Authors:
    Sveva Bonomi, Mauro Fasano, Tiziana Alberio

    Organisations:
    Laboratory of Biochemistry and Functional Proteomics, Dept. of Science and High Technology, University of Insubria, Busto Arsizio (VA)

    Presenting author: Sveva Bonomi

    Exposition date: Friday, September 15, 2023

    Exposition position: 004/1

    Background. Fibromyalgia (FMS) is a complex syndrome with still unknown aetiopathogenesis and an intricate polysymptomatology. Consequently, there is still a tremendous lack of efficacious therapies. In the last years we implemented, with promising results, an algorithm that assigns a probability score to drug-receptor pairs. In our proof-of-concept study, we worked on another disease (anxiety) to set up the method for drug repurposing. Objective. The main objective of the present work is to implement an artificial intelligence-based drug repurposing approach applied on the proteins putatively involved in FMS. Methods. DisGeNET database was used to retrieve FMS related proteins. Networks were generated and topologically analysed by Cytoscape. The implemented algorithm to assign a probability score to drug-receptor pairs was written in Python (drugs: retrieved by DrugCentral; receptors: proteins of the FMS network, structure predicted by AlphaFold). Results. By retrieving available information about proteins related to FMS (143), we built a protein network that allowed us to visualize the interactions among proteins variously associated to the disease. Surprisingly, the network had more edges than expected (p1e-16). Moreover, we analysed the topology of the network to discriminate the most important nodes, i.e., the probable central proteins in the etiopathogenesis. ALB and BDNF resulted to have the highest betweenness centrality. Conclusion. The high connectivity of the FMS network suggests that proteins so far associated to the disease may have a role in the same biological pathways/complexes. The central nodes are the most interesting druggable targets. Pair scoring is ongoing

  • Dissecting the role of computer-based approaches in the identification of potential biomarkers and treatment for neurodegenerative diseases

    Authors:
    Letizia Messa (1,2), Federica Rey (3), Pietro Pinoli (2), Gianvincenzo Zuccotti (3,4), Stefano Ceri (2), Cristina Cereda (1), Stephana Carelli (1,3)

    Organisations:
    1: Center of Functional Genomics and Rare Diseases, Dept. of Pediatrics, Buzzi Children’s Hospital, Milan, Italy
    2: Department of Electronics, Information and Bioengineering (DEIB), Politecnico di Milano, Milan, Italy
    3: Pediatric Clinical Research Center “Fondazione Romeo ed Enrica Invernizzi”, Dept. of Biomedical and Clinical Sciences, University of Milan, Milan, Italy
    4: Department of Pediatrics, Buzzi Children’s Hospital, Milan, Italy

    Presenting author: Letizia Messa

    Exposition date: Saturday, September 16, 2023

    Exposition position: 028/2

    Parkinson’s Disease (PD) is clinically heterogeneous both in symptoms and in progression. Omics-based approaches are a useful tool to gain insights on the whole cellular dysregulations present in disease-affected patients, but most of these studies lack follow up for the identification of the mechanistic dysregulation. In this work, we aim at dissecting the use of advanced computer-based approaches, such as machine learning methods, to identify new potential innovative biomarkers in PD starting from publicly brain transcriptomic data stored in GEO repository. Raw FASTQ files were reprocessed using a common pipeline to obtain comparable data. Differential expression analysis was performed with DESeq2 R while functional enrichment analysis with g:Profiler. We highlighted a global transcriptional deregulation in whole brain as 112 genes emerged as deregulated. The functional enrichment analysis emphasized that differentially expressed genes are involved in neurodegenerative-related processes such as synaptogenesis. Since the disease pathology may affect brain differently in areas, we also considered the transcriptional dysregulation that occurs in specific brain tissues, highlighting a different degree of gene expression alteration in different PD-affected brain areas. Furthermore, we exploited advanced computer-based methods such as Non-Negative Matrix Trifactorization (NMFT) to identify the novel therapeutic agents from existing approved clinically drugs. Starting from the global transcriptional dysregulation found in the whole brain, we combined the 112 differentially expressed genes with a list of approved drugs in DrugBank, highlighting that 17 out of 112 dysregulated genes were already target of approved drugs. We predicted novel association with drugs for the remaining 95 genes by means of NMTF. Here, we highlighted a global transcriptional deregulation in the brain with differences in brain PD-affected areas. Moreover, we shed a light on the use of advanced computer-based methods to the identification of molecular mechanisms and new biomarkers towards the treatment of PD with a more personalized approach.

Autism and related disorders

  • Altered glial activation in a rat model of Fragile X Syndrome

    Authors:
    Alessandro Feo (1), Alessandro Rava (1), Emilia Carbone (1), Antonia Manduca (2), Barbara Peruzzi (3), Viviana Trezza (1)

    Organisations:
    1: Department of Science, Section of Biomedical Sciences and Technologies, University “Roma Tre”, Rome, Italy
    2: Department of Physiology and Pharmacology “V. Erspamer”, Sapienza University of Rome, Rome, Italy
    3: Bone Physiopathology Research Unit, Bambino Gesù Children’s Hospital, IRCCS, 00165 Rome, Italy.

    Presenting author: Alessandro Feo

    Exposition date: Friday, September 15, 2023

    Exposition position: 022/1

    Neuroinflammation is a complex process involving the activation of immune cells in the brain in response to injury, infection, or chronic disease. In recent years, there has been growing evidence suggesting that neuroinflammation may play a critical role in the pathophysiology of neurodevelopmental disorders, including autism spectrum disorder (ASD) and Fragile X Syndrome (FXS). Astrocytes and microglia have emerged as essential regulators of brain functions and immune defense through a complex interplay with neuronal cells. Despite alterations in the neuron-glia crosstalk have emerged as important mechanisms underlying neuropathology, how astrocyte and microglia activation occurs and affects brain function remains to be clarified. In the present study, we performed a comprehensive biochemical characterization of the inflammatory profile of the Fmr1-Δexon 8 rat model of ASD, that is also a model of FXS, both in adolescence (PND 35) and adulthood (PND 75). We first carried out a multiplex assay on plasma samples to assess the peripheric inflammatory state of these animals compared to wild-type controls, finding an altered production of pro- and anti-inflammatory cytokines in the blood of Fmr1-Δexon 8 rats. Next, we analyzed the glial activation state in the hippocampus and amygdala, as a marker of neuroinflammation, by using immunostaining and western blotting assays. Our preliminary results show the presence of astrogliosis in Fmr1-Δexon 8 rats, characterized by an upregulated GFAP expression at PND 75. These observations encouraged us to investigate the expression of inflammatory cytokines modulating astrocyte and microglia activation, including Interleukin-6 (IL-6), Interferon gamma (IFN-g) and Interleukin-1b (IL-1b). Our findings support the growing body of evidence indicating a neuroinflammatory state in ASD-related disorders and might help to enhance our understanding of the pathogenesis of these disorders, potentially favoring the development of new pharmacological treatments aimed to reduce reactive glial toxicity.

  • Autophagy and microtubules dynamics in Ubiquitin ligase E3a (UBE3A)-deficient neurons

    Authors:
    Ilaria Tonazzini (1), Laura Marchetti (2), Allegra Coppini (2), Ype Elgersma (3), Marco Cecchini (1)

    Organisations:
    1: Istituto Nanoscienze - Consiglio Nazionale delle Ricerche (CNR) @ NEST, Pisa (IT)
    2: Dip. di Farmacia, Università di Pisa (IT)
    3: Clinical Genetics, Erasmus Medical Center, Rotterdam (NL)

    Presenting author: Ilaria Tonazzini

    Exposition date: Friday, September 15, 2023

    Exposition position: 014/1

    Ubiquitin E3A ligase (UBE3A) has crucial functions in the brain and changes in its expression levels lead to neurodevelopmental disorders, to Angelman Syndrome (AS) or 15duplication-autisms. It is known that cytoskeleton and autophagy processes are connected, and of pivotal importance for neuronal homeostasis and development. The cytoskeleton has an important role in autophagy, the process by which cellular waste is isolated inside specialized vesicles called autophagosomes for recycling and degradation. We investigated neuronal guidance in vitro in UBE3A-deficient neurons, model of AS, by using micro-grooved substrates: we found deficits in topographical contact guidance in AS neurons, linked to a dysregulated focal adhesions’ sensing and cytoskeleton polarization, with an aberrant axonal branching.

    Here, we investigate the microtubules dynamics, by imaging the end-binding protein EB3, and the autophagy process in primary AS neurons, under control and stimulated conditions. The migration behaviour of wild-type and AS neurons is also studied, with the aim to clarify UBE3A role during neurodevelopment.

    We show that EB3-mediated microtubules dynamics are impaired and that the autophagy flux is dysregulated in AS murine neurons.

    Overall, cytoskeleton dynamics and autophagy process emerge as players in UBE3A-mediated pathogenesis. These results support the view that UBE3A-related deficits in early neuronal morphogenesis and homeostasis may lead to defective neuronal connectivity and plasticity. This work was supported by MSCA-IF-2017 grant Neuroguide-795948.

  • Blunted mGlu5-mediated polyphosphoinositide hydrolysis in two mouse models of autism

    Authors:
    Luisa Di Menna (1), Rosamaria Orlando (1,2), Giovanna D’Errico (1), Roxana Paula Ginerete (1), Agata Machaczka (3), Carmela Maria Bonaccorso (4), Andrea Arena (2,4), Michela Spatuzza (4,5), Roberta Celli (1), Marika Alborghetti (6), Eleonora Ciocca (2), Anna Rita Zuena (2), Maria Rosaria Scioli (1), Valeria Bruno (1,2), Giuseppe Battaglia (1,2), Ferdinando Nicoletti (1,2), Maria Vincenza Catania (5)

    Organisations:
    1: IRCCS Neuromed, Pozzilli, Italy
    2: Department of Physiology and Pharmacology, Sapienza University, Roma, Italy
    3: Department of Neurobiology, Maj Institute of Pharmacology, Polish Academy of Science, Krakow, Poland
    4: IRCCS Oasi Research Institute, Troina, Italy
    5: Institute for Biomedical Research and Innovation, The National Research Council (IRIB-CNR), Catania
    6: Department of Neuroscience, Mental Health, and Sensory Organs, Sapienza University, Italy

    Presenting author: Luisa Di Menna

    Exposition date: Friday, September 15, 2023

    Exposition position: 025/1

    Autism spectrum disorder (ASD) includes a variety of behavioral phenotypes, characterized by a defect in social interaction, behavioral stereotypies, and unusual responses to sensory inputs. The involvement of the mGlu5 receptors in the pathophysiology of several forms of monogenic autism has been supported by numerous studies following the seminal observation that mGlu5 receptor-dependent long-term depression was enhanced in the hippocampus of fmr1 knockout mice modeling the fragile-X syndrome (FXS). Surprisingly, the canonical signal transduction pathway activated by mGlu5 receptors (i.e. polyphosphoinositide – PI – hydrolysis) has never been examined in models of autism. We have developed a method for in vivo assessment of PI hydrolysis based on systemic injection of lithium chloride followed by treatment with the selective mGlu5 receptor PAM, VU0360172, and measurement of endogenous inositolmonophosphate (InsP) in brain tissue. Here, we report that mGlu5 receptor-mediated PI hydrolysis was blunted in the cerebral cortex, hippocampus, and corpus striatum of Ube3am-/p+ mice modeling Angelman syndrome (AS), and in the cerebral cortex and hippocampus of fmr1 knockout mice. In contrast, the expression of mGlu5 receptors was reduced in the cortex of FXS mice and increased in the striatum of AS mice, but it was unchanged in the other regions of both FXS and AS mice. Subtle changes in the expression of Homer, phospholipase Cbeta and Galphaq were also detected in different brain regions of both FXS and AS mice.

    This is the first evidence that the canonical transduction pathway activated by mGlu5 receptors is downregulated in brain regions of mice modeling monogenic autism, casting doubts on the use of mGlu5 antagonists in the treatments of these disorders.

  • CDKL5 regulates GABAergic innervation of oligodendrocyte progenitor cells in the cerebral cortex: myelination and behavioural consequences

    Authors:
    Sunaina Devi, Riccardo Pizzo, Anna Mellano, Debora Comai, Antonia Gurgone, Martina Lorenzati, Annalisa Buffo, Chiara Salio, Maurizio Giustetto

    Organisations:
    University of Turin, Italy

    Presenting author: Sunaina Devi

    Exposition date: Friday, September 15, 2023

    Exposition position: 020/1

    Cyclin-dependent kinase-like 5 (CDKL5) is a serine/threonine kinase highly expressed in forebrain neurons where it regulates epigenetic factors, dendritic morphology, and synaptogenesis. Lack of functional CDKL5 leads to an X-linked neurodevelopmental condition, CDKL5 deficiency disorder, characterized by early-onset drug-resistant epilepsy, intellectual disability, autistic-like features, and sensory impairments. Along with neurons, CDKL5 is also expressed by cells of the oligodendroglial lineage and CDD patients show white matter alterations. However, whether CDKL5 mutation can affect myelin organization remains unexplored. To address this issue, we assessed myelin basic protein and phospho-neurofilaments in primary sensory corticesand we found that both young and adult CDKL5-KO mice show a robust reduction of their expression.Because myelination plays a crucial role in cortical coding of somatosensory information, we next investigated behavioural correlates of defective myelination and we found that CDKL5 loss affects tactile discrimination. Cortical processing of tactile stimuli critically depends on the myelination of parvalbumin (PV+) interneurons (INs). By using 3D reconstruction of confocal microscopy images, we found that CDKL5-KO mice show a decrease in the myelin volume surrounding PV+ axons in the barrel cortex. As PV+ INs myelination is regulated by synaptic communication with oligodendrocyte progenitor cells (OPCs), we examined GABAergic connectivity onto OPCs and we revealed that CDKL5-KO mice show an increase in these contacts compared to WT animals. Finally, as GABAergic synapses control OPCs maturation into myelinating oligodendrocytes (OLs), we assessed OLs in the somatosensory cortex and found that the density of mature OLs is strongly reduced in mutant mice whereas OPCs number was unaffected. In sum, our findings reveal that CDKL5 is engaged in the regulation of cortical myelination and OPCs to OLs transition by orchestrating GABAergic connectivity onto OPCs.

  • Characterization of the transcriptional landscape of endogenous retroviruses at the fetal-maternal interface in a mouse model of Autism Spectrum Disorder

    Authors:
    Chiara Cipriani (1), Antonella Camaioni (2), Anna Maria Tartaglione (3), Martina Giudice (1), Vita Petrone (1), Claudia Matteucci (1), Gemma Calamandrei (3), Paola Sinibaldi-Vallebona (1,4), Laura Ricceri (3), Emanuela Balestrieri (1)

    Organisations:
    1: Department of Experimental Medicine, University of Rome Tor Vergata, Rome, Italy
    2: Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Ita
    3: Centre for Behavioural Sciences and Mental Health, Istituto Superiore di Sanità, Rome, Italy
    4: Institute of Translational Pharmacology, National Research Council, Rome, Italy

    Presenting author: Martina Giudice

    Exposition date: Friday, September 15, 2023

    Exposition position: 007/1

    Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder resulting from the complex interactions among genetic, environmental, and epigenetic factors. A study conducted on two mouse models of ASD, the inbred BTBR T+tf/J (BTBR) mice, and mice prenatally exposed to valproic acid, showed an altered expression of several endogenous retroviruses (ERVs) and cytokines, in whole embryos, blood and brain samples at different post-natal ages, supporting the involvement of ERVs in the ASD pathophysiology. Recently, in a mouse model of maternal immune activation, a deregulated expression of some ERVs and ERV-related genes was found in the prefrontal cortex (PFC) and hippocampus, with higher expression levels in females than in males in PFC (Cipriani et al, 2018 and 2022). ERVs are relics of ancestral germline infections by exogenous retroviruses, stably integrated into the host cellular DNA, involved in the pathogenesis of many human complex diseases. In this work we studied the expression of several ERVs and pro-inflammatory cytokines in dissected embryos and maternal tissues, in order to identify the specific areas in which early ERV overexpression and immune dysregulation occur. Whole embryos from BTBR mice were explanted at gestational day 10.5, separated from maternal tissues and dissected to obtain: cephalic and non-cephalic embryonic tissues, maternal decidua and extra-embryonic tissues. The expression of several ERV families, pro-inflammatory cytokines and Toll-like receptors, were assessed in all tissues by Real-time PCR. The results demonstrated that some ERVs, pro-inflammatory cytokines and Toll-like receptors were expressed at higher levels in BTBR mice compared to controls (i.e. the inbred C57BL6/J strain), depending of the tissue analysed, with the highest levels in embryonic cephalic tissue and maternal decidua. These preliminary results support the hypothesis that changes in the expression of ERVs and inflammatory cytokines could be related, or at least proceed in parallel, with the acquisition of the ASD phenotype.

  • Discriminative and Emotional components of Affective Touch in Autism Spectrum Disorder population

    Authors:
    Francesca Capiotto, Giulia Romano Cappi, Alessandro Mazza, Cariola Monia, Ilaria Mirlisenna, Olga Dal Monte

    Organisations:
    University of Turin, Italy

    Presenting author: Francesca Capiotto

    Exposition date: Friday, September 15, 2023

    Exposition position: 016/1

    Autism Spectrum Disorder (ASD) is a pervasive neurodevelopmental disorder that includes symptoms related to both emotional and sensory domains. Although altered tactile end emotional processing are commonly reported in ASD, no study has so far investigated whether the abnormal processing, typical of ASD patients, is related to a discriminative or emotional deficit.

    By combining behavioral and physiological measures, we aimed to disentangle how ASD patients compared to typical controls (TC) process the discriminative and emotional components of an affective touch. First, to investigate the discriminative component, subjects received an affective touch either with a real or a fake hand and were asked which hand touched them. ASD patients showed a significantly lower accuracy than TC group in correctly discriminating the two types of hands. However, we also observed a comparable physiological activation between the two groups: both TC and ASD groups displayed a higher physiological response during an affective touch from a real hand compared to a fake hand. Second, to investigate the emotional component, subjects received either an affective touch or a non-affective touch (i.e., tapping) and were asked to evaluate how affective was the touch they received. While control subjects rated affective touch as more affective than tapping, ASD patients did not show a preference for affective touch. Also, similarly to the discriminative component, the two groups did not show differences in autonomic activation, with higher physiological responses associated to affective touch compared to tapping.

    Our results suggest a dissociation between explicit and implicit processing of discriminative and affective components of touch: while explicit processing of both the discriminative and emotional components of touch is impaired in ASD patients, autonomic responses suggest that implicit processing in ASD patients is comparable to the neurotypical population.

  • Dopaminergic alteration triggers autistic-behaviours in lysosomal storage disorders.

    Authors:
    Maria De Risi (1,2), Lorenzo Cusimano (1), Xabier Bujanda Cundin (1), Mariateresa Pizzo (1), Simona Fecarotta (3), Giancarlo Parenti (1,3), Elvira De Leonibus (1,2)

    Organisations:
    1: Telethon Institute of Genetics and Medicine, Telethon Foundation, Pozzuoli, Italy.
    2: Institute of Biochemistry and Cell Biology (IBBC), National Research Council, Rome, Italy.
    3: Department of Pediatrics "Federico II" University Hospital, Naples, Italy

    Presenting author: Maria De Risi

    Exposition date: Friday, September 15, 2023

    Exposition position: 019/1

    Mucopolysaccharidosis IIIA (MPS-IIIA) is a severe inborn errors of metabolism caused by mutations of the sulfamidase gene (SGSH), a lysosomal enzyme that participates in the metabolism of the heparan sulfate (HS). Defective sulfamidase activity leads to the lysosomal HS accumulation which results in defective autophagosomal/ lysosomal degradative capacities and build-up of primary and secondary storages, ultimately leading to neurodegeneration and dementia in children. However, dementia is preceded by severe and incapacitating autistic-like behaviours, including self-injury, stereotypic behaviours and social impairment. We recently discovered that young MPS-IIIA mice show autistic-like behaviours due to increased proliferation of mesencephalic dopamine neurons originating during embryogenesis. This hyperdopaminergia is not due to lysosomal dysfunction, but to altered HS function as co-receptor of growth factors (De Risi et al., 2021, Nature Communications). We have also shown that pharmacological inhibition of D1 dopamine receptors, but not D2, rescues autistic-like behaviours in young MPS-IIIA mice. We are therefore testing in preclinical and clinical settings FDA-approved dopaminergic drugs that might act, through different biochemical pathways, on the same dysfunctional mechanism responsible for autistic-behaviour in MPS-IIIA.

  • Early appearance of developmental brain alterations in a mouse model of CDKL5 deficiency disorder

    Authors:
    Beatrice Uguagliati, Marianna Tassinari, Stefania Trazzi, Camilla Bruna Cerchier, Ottavia Vera Cavina, Nicola Mottolese, Manuela Loi, Giula Candini, Giorgio Medici, Elisabetta Ciani

    Organisations:
    Department of Biomedical and Neuromotor Science, University of Bologna

    Presenting author: Beatrice Uguagliati

    Exposition date: Friday, September 15, 2023

    Exposition position: 006/1

    Mutations in the CDKL5 gene are the cause of CDKL5 deficiency disorder (CDD), a rare and severe neurodevelopmental condition characterized by early-onset epilepsy, motor impairment, intellectual disability, and autistic features. The Cdkl5 KO mouse, a mouse model of CDD, that recapitulates several aspects of the disease symptomatology, has helped to investigate brain alterations leading to CDD neurological defects. Studies of brain morphogenesis in adult Cdkl5 KO mice showed that the absence of CDKL5 negatively affects dendritic arborization, synaptic connectivity, neuronal survival, and microglia activation. However, at present, no studies are available regarding the early appearance of these brain alterations in Cdkl5 KO mice, and their severity in the early stages of life compared to adulthood. A deeper understanding of the development of the CDKL5 deficient brain would represent an important milestone for the further validation of the Cdkl5 KO mouse model, and for the identification of the optimum time window for treatments targeted to brain developmental defects. In sight of this, we comparatively evaluated the dendritic arborization and spines of cortical pyramidal neurons, cortical excitatory and inhibitory connectivity, microglia activation, and proliferation and survival of granule cells of the hippocampal dentate gyrus in Cdkl5 KO mice aged 7, 14, 21, and 60 days. We found that most of the structural alterations in Cdkl5 KO brains are already present in pups aged 7 days and do not worsen with age. Nonetheless, the difference in the number of excitatory and inhibitory terminals between Cdkl5 KO and wild-type mice changes with age, suggesting an age-dependent cortical excitatory/inhibitory imbalance. Moreover, confirming the precocious onset of brain defects, Cdkl5 KO pups are characterized by an impairment in neonatal sensory-motor reflexes.

  • Effects and mechanism of action of psilocybin in a genetic rat model of autism

    Authors:
    Valeria Buzzelli (1), Fabrizio Ascone (1), Emilia Carbone (1), Antonia Manduca (1), Alessandro Feo (1), Marvin Hausman (2), Viviana Trezza (1)

    Organisations:
    1: 1Department of Science, Section of Biomedical Sciences and Technologies, University “Roma Tre”, Rome, Italy
    2: Nova Mentis Life Science Corp., Vancouver BC, Canada

    Presenting author: Valeria Buzzelli

    Exposition date: Friday, September 15, 2023

    Exposition position: 005/1

    Fragile X Syndrome (FXS) is the most common form of inherited intellectual disability (ID) and the leading monogenic cause of autism spectrum disorder (ASD). Serotonergic neurotransmission has a key role in the modulation of neuronal activity during development and therefore it has been hypothesized to be involved in ASD and co-occurring conditions including FXS. As serotonin is involved in synaptic remodeling and maturation, serotonergic insufficiency during childhood may have a compounding effect on brain patterning in neurodevelopmental disorders, manifesting as behavioral and emotional symptoms. Thus, compounds that stimulate serotonergic signaling such as psilocybin may offer promise as effective early interventions for developmental disorders such as ASD and FXS. The aim of the present study was to test whether different protocols of psilocybin administration mitigate cognitive deficits displayed by the recently validated Fmr1-Δexon 8 rat model of ASD, which is also a model of FXS. Our results revealed that systemic and oral administration of psilocybin microdoses normalizes the aberrant cognitive performances displayed by adolescent Fmr1-Δexon 8 rats in the novel object recognition test – a measure of exploratory behavior, perception, and recognition. These data support the hypothesis that serotonin modulating drugs such as psilocybin may be useful to ameliorate ASD-related cognitive deficits. Ongoing the experiments are investigating the mechanism of action underlying the effect of psilocybin in fragile x syndrome . Overall, this study provides evidence of the beneficial effects of different schedules of psilocybin treatment in mitigating the cognitive deficit observed in a rat model of FXS.

  • Effects of early-life butyrate supplementation in an ASD mouse model

    Authors:
    Irene Coppola (1), Mattia De Magistris (2), Annacandida Villani (3), Maria Antonietta Ajmone-Cat (1), Valerio Pazienza (3), Roberta De Simone (1), Laura Ricceri (2), Gemma Calamandrei (2), Anna Maria Tartaglione (2)

    Organisations:
    1: National Centre for Drug Research and Evaluation, Istituto Superiore di Sanità, Rome, Italy
    2: Centre for Behavioral Sciences and Mental Health, Istituto Superiore di Sanità, Rome, Italy
    3: Gastroenterology Unit IRCCS "Casa Sollievo della Sofferenza", Hospital San Giovanni Rotondo, Foggia, Italy

    Presenting author: Irene Coppola

    Exposition date: Friday, September 15, 2023

    Exposition position: 013/1

    Autism Spectrum Disorder (ASD) is a heterogeneous neurodevelopmental condition characterized by deficits in social communication and interaction, and repetitive/stereotyped behaviors. Microbiota alterations as well as signs of neuroinflammation have been also reported in ASD. The involvement of immune activation in ASD is further supported by epidemiological studies suggesting that maternal immune activation (MIA), especially during early pregnancy, may be a risk factor for ASD. In recent years, the role of microbiota-gut-brain axis (MBGA) in ASD pathogenesis received growing attention, appearing as an attractive therapeutic target. A key mediator in MBGA is butyrate, a short-chain fatty acid produced by bacterial fermentation of dietary fibers, involved in the regulation of gut permeability and immune system, found impaired in ASD. The present study evaluated the role of early-life sodium butyrate (NaB) supplementation in the modulation of ASD-like behavioral phenotype, neuroinflammatory response, and gut microbiota alterations in the MIA mouse model.
    We modeled MIA by treating C57BL6/J female mice with a single dose of polyinosinic:polycytidylic acid (Poly I:C, 20 mg/kg, i.p.) at gestational day 12.5. After delivery, dams received NaB (200 mg/kg) administered in drinking water through the whole duration of lactation. In order to detect ASD-like behavioral deficits in offspring, we assessed their behavioral phenotype from early neonatal stage to adulthood. At the end of behavioral testing, we analyzed i) gut microbiota composition by 16S rRNA sequencing and ii) neuroinflammatory markers in prefrontal cortex, hippocampus and cerebellum by Real-Time PCR.
    Early-life NaB supplementation attenuated some of the MIA-induced behavioral alterations (i.e. familiar olfactory cues recognition, stereotyped behavior), and exerted modulatory effects on neuroinflammation and gut microbiota composition in MIA adult offspring.
    Our data support the development of therapeutic interventions targeting gut microbiota to modulate brain and behavioral changes in ASD.

  • Expanding the clinical spectrum of PPP3CA variants: alternative isoforms matter

    Authors:
    Silvia Castiglioni (1), Antonella Lettieri (1), Elisabetta Di Fede (1), Lidia Pezzani (2,3), Silvia Ancona (1), Elisa Colombo (1), Chiara Parodi (1), Paolo Grazioli (1), Daniele Bottai (4), Carlotta Pipolo (1), Laura Pezzoli (5), Donatella Milani (2), Maria Iascone (5), Valentina Massa (1,6), Cristina Gervasini (1,6)

    Organisations:
    1: Department of Health Sciences, Università degli Studi di Milano
    2: Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan.
    3: Molecular Genetics Section, Medical Genetics Laboratory, Papa Giovanni XXIII Hospital, Bergamo.
    4: Department of Pharmaceutical Sciences, Section of Pharmacology and Biosciences, Università degli Studi di Milano.
    5: Pediatrics, Papa Giovanni XXIII Hospital, Bergamo.
    6: “Aldo Ravelli” Center for Neurotechnology and Experimental Brain Therapeutics, Università degli Studi di Milano.

    Presenting author: Silvia Castiglioni

    Exposition date: Friday, September 15, 2023

    Exposition position: 008/1

    Background: PPP3CA variants cause developmental and epileptic encephalopathy-91 (DEE91). This gene encodes for a protein with two main tissue-specific isoforms (1 and 2) that differ for the presence of exon13. The cellular function is essential in intracellular signals transduction. It is ubiquitously expressed, mostly in the brain where it dephosphorylates DNM-1 protein. By Whole Exome Sequencing analysis, we identified two PPP3CA de novo variants: a 4-nucleotide duplication predicting a premature truncated protein in Pt.1 presenting the classical clinical phenotype, and a splicing mutation altering the exon13 inclusion in Pt.2 showing a mild phenotype, undescribed for this condition.

    Methods: we evaluated the isoforms 1 and 2 expression in several healthy donor tissues. Then, immortalised lymphoblastoid cell lines (LCLs) derived from the two patients were used to study the effects of the variants, mainly by assessing cell growth, DNM-1 phosphorylation levels and the two isoforms expression pattern.

    Results: transcript analyses confirmed the isoform1 prevalence in the brain conversely to all other tissues investigated. Pt.1 LCLs showed a reduced cellular growth and an increase in phosphorylated DNM-1 compared to controls. Interestingly, Pt.2 LCLs showed a growth rate comparable to controls and an absence of phosphorylated DNM-1.

    Conclusion: in this study we characterized two PPP3CA variants studying the effects on the two main isoforms. The predicted effect of variant altering the phosphatase function is in accordance with the classical Pt.1 phenotype. We hypothesize that the variant identified in Pt.2 with an unusual phenotype could perturb the isoform1 formation with deleterious effects mainly in brain function.

  • Exploring the relationship between sensory habituation, autistic traits and attentional control

    Authors:
    Alessia Santostefano (1,2,4), Vincenza Tarantino (1,4), Ilaria Tasca (1), Noemi Passarello (3), Laura Mandolesi (3), Patrizia Turriziani (1,2,4)

    Organisations:
    1: Department of Psychology, Educational Sciences and Human Movement, University of Palermo, Italy
    2: NeuroTeam Life and Science, Palermo, Italy
    3: Department of Humanities, University Federico II, Naples, Italy
    4: Neuropsychology Lab, University of Palermo

    Presenting author: Alessia Santostefano

    Exposition date: Friday, September 15, 2023

    Exposition position: 026/1

    People with autistic spectrum disorder (ASD) exhibit deficits in processing sensory information in daily life. Specifically, they show difficulty in detecting, modulating, interpreting or responding to sensory inputs, which determine profiles of hyper- or hypo-sensitivity. Also, they show a typical attentional bias toward processing local details of stimuli at the expense of global features. We hypothesized that altered habituation processes could contribute to sensory profile and attentional biases. To this end, we performed a study on people recruited from an adult general population, with no diagnosis of ASD or other developmental or acquired disorders. We measured their autistic traits by the Autistic Spectrum Quotient questionnaire and their sensory profile by the Sensory Perception Questionnaire. Habituation was assessed by a new validated questionnaire, the Sensory Habituation Questionnaire, and participants were asked to perform a Navon task. The results confirmed previous literature, showing that higher autistic traits correlated with hyper-sensitivity profiles. In addition, they revealed that higher resistance toward habituation was associated with higher autistic traits and poorer processing of global relative to local features of stimuli.

  • Gut-brain axis: effects of probiotics on behavior and function

    Authors:
    Federica Serafini (1), Tommaso Seri (1), Giusi Sabatini (2), Tamara Diamanti (1), Benedetta Citro (1), Maria Emanuela Greco (1), Laura Ricceri (3), Aldo Corsetti (2), Arianna Rinaldi (1), Natalia Battista (2), Antonella De Jaco (1)

    Organisations:
    1: Università degli Studi di Roma "La Sapienza", Italy
    2: Università degli Studi di Teramo
    3: Istituto Superiore di Sanità, Roma

    Presenting author: Federica Serafini

    Exposition date: Friday, September 15, 2023

    Exposition position: 018/1

    Fermented food microbes are gaining increasing attention for their health-promoting potential and for being genetically related to human probiotic bacteria. Among them, Lactiplantibacillus (Lpb.) plantarum represents a versatile and robust dominant specie in fermented foods, safe for humans and with anti-inflammatory and antioxidant properties, on top of its ability to modulate compromised gastrointestinal microbiota. The gut microbiota has been shown to modulate brain function and behavior. The endocannabinoid system (ECS) is one of the major modulators in the microbiota–gut–brain signaling pathways and is linked to the inhibitory GABAergic neurotransmitter system in the brain. ASDs are complex syndromes characterized by behavioral impairments in social interaction and by altered neurotransmission with an imbalance between excitatory/inhibitory pathways. We have tested the effect of Lpb. plantarum supplementation to ameliorate endophenotypes associated with ASDs in the Neuroligin3 (NLGN3) R451C mouse, a monogenic model of autism presenting highly reduced levels of post-synaptic NLGN3, social deficits, altered synaptic transmission and gastro-intestinal dysfunction. After a 5-weeks long treatment with Lpb. Plantarum, we investigated the rescue of impaired social deficits by several behavioral tests, such as three chamber and the male-female social interaction test, and found an increase in time R451C mice interacted with a co-specific WT mouse. We also found a modulation of the ECS, after the treatment. Overall, our findings point toward an effective cross-talk between functional microorganisms and ECS in ameliorating the phenotype of a mouse model of ASD. These encouraging results underline a potential implication of the host-microbe interaction in terms of alleviation of ASD-GI symptoms, opening the way to human clinical trials in order to develop functional foods that can vehicle specific probiotic strains directly isolated from fermented foods.

  • Increased expression of metabotropic glutamate receptors in infant VPA-exposed rats

    Authors:
    Simona D'Antoni (1), Sara Schiavi (2), Valeria Buzzelli (2), Samuele Giuffrida (1), Viviana Trezza (2), Maria Vincenza Catania (1)

    Organisations:
    1: Institute for Biomedical Research and Innovation, National Research Council (IRIB-CNR), via Paolo Gaifami 18, 95126, Catania, Italy
    2: Department of Science, Section of Biomedical Sciences and Technologies, University “Roma Tre", Viale G. Marconi 44600146, Rome, Italy

    Presenting author: Simona D'Antoni

    Exposition date: Friday, September 15, 2023

    Exposition position: 023/1

    Autism spectrum disorders (ASDs) are a group of clinically and genetically heterogeneous debilitating neurodevelopmental disorders commonly characterized by impaired social interaction and stereotyped behaviours. A genetic cause can be identified in at least 20% of cases and a genetic predisposition is largely the main pathogenic factor of ASD. Nevertheless, many autistic cases have an environmental origin and prenatal exposure to valproic acid (VPA), a common anti-epileptic drug, induces ASD in humans and autistic-like behaviors in rodents. Although the pathogenic mechanism of ASD is still elusive, several studies from ASD patients and animal models indicate that a dysfunction of glutamate receptors at synapses is associated with these disorders. In the VPA rat model a potential involvement of glutamatergic receptors has been suggested; however, few studies were carried out on metabotropic glutamate (mGlu) receptors.

    To this aim, we examined the protein expression levels of group I (mGlu1 and mGlu5) and group II (mGlu2/3) mGlu receptors in synaptosomes obtained from forebrain of control and prenatally VPA-exposed rats at different ages (post-natal day PND 13, 35, 90).

    We found an increase in the expression of all these receptors in the VPA rats only at PND 13. Interestingly, an acute treatment with the mGlu2/3 antagonist LY341495 (1mg/kg, one hour before test) corrected the reduced ultrasound vocalization emission, an early autism-like phenotype observed in VPA-exposed infant rats. In contrast, no effect was detected after a treatment with the mGlu5 selective antagonist MTEP (0,3 mg/Kg and 1mg/kg, one hour before test).

    Our results provide the first evidence of an up-regulation of both group I and group II mGlu receptors at synapses in infant rats prenatally exposed to VPA and offer a scientific basis for additional studies on mGlu2/3 receptors as a potential therapeutic target in ASDs and related conditions.

  • Modulation of the affect facial emotion recognition and theory of mind using a device combining prism adaptation and serious games in in Children with Autism Spectrum Disorder

    Authors:
    Angela Catania (1,3,4,5), Federica Calistro (5), Giovanna Montalto (5), Sandra Giordano (5), Massimiliano Oliveri (1,3,5), Patrizia Turriziani (2,3,5)

    Organisations:
    1: Department of Biomedicine, Neurosciences and Advanced Diagnostics, University of Palermo, Italy.
    2: Department of Psychology, Educational Sciences and Human Movement, University of Palermo, Italy
    3: Laboratory of Neuropsychology, University of Palermo, Italy
    4: International School of Advanced Studies, University of Camerino, Italy
    5: NeuroTeam Life and Science, Palermo, Italy

    Presenting author: Angela Catania

    Exposition date: Friday, September 15, 2023

    Exposition position: 024/1

    Autism Spectrum Disorder (ASD) is a neurodevelopmental condition displaying a typical neuropsychological profile in addition to the specific behavioral, communicative and social cognition patterns. In the present study, 30 ASD subjects were tested with NEPSY-II in the baseline condition and following two weeks of cognitive training with Mindlenses, a medical device combining prismatic lenses with digital cognitive training (10 sessions) through a series of serious games. Prior evidence showed that prism adaptation increases the excitability of fronto-parietal areas ipsilateral to the visual field deviation induced by prisms. Rightward or leftward digital prism adaptation was made by presenting lateralized visual stimuli on an 11” tablet. In each session, prism adaptation was followed by a battery of 8 digital serious games stimulating visuospatial and executive functions.The main results showed a significant improvement of ASD in the affect facial emotion recognition tasks with the association of rightward digital prism adaptation and serious games (p=0.003), while after leftward digital prism adaptation and serious games subjects made significative higher scores in theory of mind tasks (p=0.003).These findings, if confirmed across larger series of studies, may suggest a new training option for cognitive-affective difficulties in ASD based on non-invasive modulation of hemispheric excitability.

  • Neuronal extracellular vesicles as a novel tool to study and monitor Angelman Syndrome neurodevelopmental disorder

    Authors:
    Chiara De Cesari (1), Asia Botto (2), Laura Baroncelli (3), Liam McDonnell (2), Ilaria Tonazzini (1)

    Organisations:
    1: Nanoscience Institute - National Research Council (CNR), Pisa, Italy
    2: Fondazione Pisana per la Scienza ONLUS, San Giuliano Terme, Pisa, Italy
    3: Neuroscience Institute - National Research Council (CNR), Pisa, Italy

    Presenting author: Chiara De Cesari

    Exposition date: Friday, September 15, 2023

    Exposition position: 015/1

    Angelman syndrome (AS) is a neurodevelopmental disorder caused by mutations or deletions of the maternally inherited Ube3a gene, which encodes for the ubiquitin ligase E3A (UBE3A) in neurons. AS is characterized by severe intellectual disability, speech impairment, ataxia, epilepsy, and behavioural abnormalities. UBE3A plays a key role in neurodevelopment and, although several targets and functions were described, still little is known about its role in the pathogenesis of AS. Recent studies in the AS mouse model demonstrated the feasibility of unsilencing the paternal Ube3a gene with synthetic antisense oligonucleotides (ASO) to restore UBE3A activity, and today a few ASO clinical trials for AS are on the way.

    Small extracellular vesicles (sEVs) are crucial in several biological processes, such as cellular communication, inflammation, and neuronal plasticity. Importantly, sEVs of neural origin can cross the blood-brain barrier: they represent a potential window into brain pathological processes and can be exploited to study the brain. Nowadays there is a high need for reliable and quantitative biomarkers of brain function, for monitoring preclinical and clinical trials, particularly in AS. In fact, monitoring neurodevelopmental disorders’ progression and the effects of therapies is often complex and requires invasive procedures or behavioural observations (highly prone to subjective bias).

    Here we present a clinical assay based on the selective enrichment of neuronal sEVs from wild-type and AS neuronal models, with the aim to identify molecular biomarkers deregulated or defective in AS. The assay allows the collection of neuronal sEVs through size exclusion chromatography and immunocapture, and their subsequential proteomic and transcriptomic analysis.

    The identified biomarkers will be useful: I) to identify new potential therapeutic targets in AS, clarifying the role of UBE3A in neuronal cells; II) to monitor therapies, for the longitudinal follow-up of AS patients. This type of analysis will then be applicable to any neuropathological model.

  • Prioritization of ASD-candidate genes by a systems biology approach

    Authors:
    Veronica Remori (1,2), Heather Bondi (1), Manuel Airoldi (1), Diana Carli (3,4), Giulia Borini (3), Chiara Davico (5), Lisa Pavinato (3,6,7), Emanuela Nobile (8), Roberta Marinoni (9), Eleonora Di Gregorio (9), Roberto Keller (8), Alessandro Musso (3), Giovanni Battista Ferrero (10), Alfredo Brusco (3,9), Mauro Fasano (1,11)

    Organisations:
    1: Department of Science and High Technology (DiSAT), University of Insubria, Como
    2: Department of Information Engineering and Mathematics, University of Siena, Siena
    3: Department of Medical Sciences, University of Torino, Torino
    4: Immunogenetics and Transplant Biology, AOU Città della Salute e della Scienza, Torino
    5: Department of Public health and Pediatrics, University of Torino, Torino
    6: Institute of Oncology Research (IOR), BIOS+, Bellinzona, Switzerland
    7: Università della Svizzera Italiana, Lugano, Switzerland
    8: Adult Autism Center, DSM Local Health Unit, Torino
    9: Medical Genetics Unit, AOU Città della Salute e della Scienza, Torino
    10: Department of Clinical and Biological Sciences, University of Torino, Torino
    11: Center of Research in Neuroscience, University of Insubria, Busto Arsizio

    Presenting author: Veronica Remori

    Exposition date: Friday, September 15, 2023

    Exposition position: 010/1

    Autism spectrum disorders (ASD) are complex neurodevelopmental disorders with a strong genetic component. To unravel the genetic complexity of ASD, several large-scale genome-wide association studies, copy number variants (CNVs) testing, and genome sequencing have been performed, resulting in the identification of many candidate variants.

    In this context, computational methods that prioritize candidate genes based on additional functional information can be valuable. We hypothesized that possible candidate autism risk genes can be identified through their connections with known autism risk genes. Here, we propose a systems biology approach to prioritize candidate ASD-associated genes. From the Simons Foundation Autism Research Initiative (SFARI) Gene database, an input list of 750 non-syndromic genes associated with ASD with high confidence or strong candidate gene ratings (with SFARI score 1 or 2, respectively) was collected. We generated a protein-protein interaction (PPI) network that included first interactors of the SFARI genes, using the IMEx database (nodes = 12896, edges = 305609), and performed topological analysis.

    As a proof of concept, we mapped a list of genes with CNVs of unknown significance identified through array-Comparative Genomic Hybridization (CGH) analysis in 128 ASD patients on PPI-network. A prioritized list of genes was obtained by ranking the gene list by betweenness centrality score. We are currently performing over-representation analysis (ORA) using WebGestalt to assess whether genes in the prioritized list were significantly associated with metabolic or disease pathways. Furthermore, we are implementing our approach with an unsupervised classification to strengthen the identification of novel candidate genes.

    Although the relevance of these genes needs to be further validated in functional studies, our approach offers a promising strategy for identifying potential ASD risk genes and may contribute to our understanding of the genetic basis of ASD.

  • Sex-specific autistic-like endophenotypes in a rat model of Fragile X Syndrome: role of endocannabinoid neurotransmission

    Authors:
    Fabrizio Ascone (1), Valeria Buzzelli (1), Alessandro Rava (1), Alessandro Feo (1), Antonia Manduca (1,2), Viviana Trezza (1)

    Organisations:
    1: Department of Science, Section of Biomedical Sciences and Technologies, University “Roma Tre”, Rome, Italy
    2: Department of Physiology and Pharmacology “V. Erspamer”, Sapienza University of Rome, Rome, Italy

    Presenting author: Fabrizio Ascone

    Exposition date: Friday, September 15, 2023

    Exposition position: 011/1

    Fragile X Syndrome (FXS) is the most common inherited form of intellectual disability, caused by a mutation in the FMR1 gene. Individuals with FXS often display symptoms of Autism Spectrum Disorder (ASD), including social and communicative deficits, and repetitive behaviors. Due to the X-linked nature of the condition, FXS is more often associated with males. Furthermore, females with FXS typically have a milder presentation of symptoms compared to males. The increased severity of presentation in males and the lower prevalence in females has led to underrepresentation and often exclusion of females in research. However, females with FXS can still experience significant cognitive, behavioral and physical symptoms.

    In this study, we aimed to evaluate the presence of sex-specific ASD-like behaviors in the Fmr1-Δexon 8 rat model of FXS using a battery of behavioral tests to evaluate cognitive performance, social behavior, communication, and repetitive behaviors. Furthermore, since we have recently shown that the endocannabinoid system is involved in the behavioral deficits displayed by Fmr1-Δexon 8 male rats, our second aim was to investigate the role of the endocannabinoid system in the altered phenotype displayed by Fmr1-Δexon 8 female rats.

    Our preliminary results indicate that Fmr1-Δexon 8 female rats display deficits in the cognitive, social and communicative domain, similar to Fmr1-Δexon 8 male rats. Interestingly, we observed that Fmr1-Δexon 8 female rats display reduced anxiety-like behaviors and increased repetitive behaviors, while male Fmr1-Δexon 8 rats are spared. These findings indicate that Fmr1-Δexon 8 rat females exhibit several ASD-like symptoms. Ongoing experiments are investigating whether the anandamide hydrolysis inhibitor URB597 is able to correct the behavioral changes displayed by Fmr1-Δexon 8 female rats.

    Altogether, these findings highlight sex-specific autistic-like endophenotypes in a rat model of FXS and introduce a new therapeutic target for reversing autistic-like symptoms in both sexes.

  • Social behavior characterization during development and effects of oxytocin treatment in R451C Neuroligin3, a mouse model of autism

    Authors:
    Tamara Diamanti (1), Camilla Perfetto (1), Federica Serafini (1), Maria Egle De Stefano (1,2), Valentina Ferretti (1,2), Antonella De Jaco (1,2)

    Organisations:
    1: Dept. Biology and Biotechnology “C. Darwin”. Sapienza University of Rome, Italy
    2: Center for Research in Neurobiology “Daniel Bovet”. Sapienza University of Rome, Italy

    Presenting author: Tamara Diamanti

    Exposition date: Friday, September 15, 2023

    Exposition position: 021/1

    Alterations in social interactions represent early and distinctive features of neurodevelopmental disorders, as observed for autism spectrum disorders (ASDs). Social abilities dramatically change during adolescence along with brain structural and functional changes. Mutations in the ASD-associated synaptic molecule Neuroligin3 (Nlgn3) have been associated to aberrant social behavior in adult mice. However, the time window in which these alterations appear has not been characterized. In this work, we studied the social responses of the R451C NLGN3 mouse model (B6;129-Nlgn3tm1Sud/J) at three postnatal ages: adolescence (P28-35), young (P40-50) and adulthood (>P60), compared to age-matched wild type control mice. With the investigation of the social abilities of the NLGN3 R451C mice during development, we aim to identify possible windows for pharmacological or molecular intervention. Here, we identified alterations in both social habituation and social novelty preference since adolescence in the R451C NLGN3 mice, exposed to a nonfamiliar conspecific, while abnormal aggressive behaviors manifest uniquely in adult mutant mice. These results suggest that alterations in social interactions in this monogenic mouse model of ASDs may arise at different time points, indicating the presence of specific mechanisms and circuits underlying. To prevent or rescue the impact that the R451C NLGN3 mutation has on mice social abilities we report the effect of intranasal oxytocin treatment in young and adult mice.

  • Study of cholesterol metabolism in Rett syndrome: a new role for BET proteins

    Authors:
    Michela Varone, Noemi Martella, Mayra Colardo, Marco Segatto

    Organisations:
    Department of Biosciences and Territory, University of Molise, Contrada Fonte Lappone, 86090 Pesche, Italy

    Presenting author: Michela Varone

    Exposition date: Friday, September 15, 2023

    Exposition position: 009/1

    Rett syndrome (RTT) is a neurological disorder that affects almost exclusively female individuals. It is caused by mutations in the gene encoding methyl-CpG binding protein 2 (MeCP2), encoding for a transcriptional repressor. RTT present an important metabolic component. Indeed, there is considerable evidence of disruption of lipid metabolism in mouse models of the disease. Additionally, the plasma lipid profile of RTT patients also appears to be altered. These findings suggest that alterations of cholesterol metabolism can contribute to the pathological phenotype and can be considered a promising target for a therapeutic approach aimed at counteracting the disease physiopathology.

    Recent data describe Bromodomain and Extra-Terminal domain (BET) proteins as epigenetic factors strongly involved in the regulation of cholesterol homeostasis.

    Therefore, the aim of this project is to evaluate whether BET inhibition could restore cholesterol derangements observed in RTT. To this end, primary cultures of fibroblasts derived from both healthy and RTT donors were used.

    Our results demonstrate that cholesterol levels are higher in RTT fibroblasts. These abnormalities seem to be associated to enhanced biosynthesis and uptake, as evidenced by the increased expression of the main proteins controlling cholesterol metabolism. Notably, BET inhibition by the small molecule JQ1 attenuates the alterations observed in RTT fibroblasts, thus normalizing the intracellular cholesterol content.

    Taken together, the results suggest that cholesterol metabolism is perturbed in RTT, and that BET inhibition could represent an advantageous pharmacological approach to improve these deregulations.

  • The therapeutic potential of psychedelics on the behavioral and synaptic abnormalities of the Shank3 mouse model of Autism Spectrum Disorder

    Authors:
    Lorenzo Curti (1), Elena Montagni (2,3), Manuel Ambrosone (2), Alessandra Franceschini (2), Alessia Costa (4), Michela Amendola (2), Antonino Iurato La Rocca (1), Ludovico Silvestri (2), Anna Letizia Allegra Mascaro (2,3), Guido Mannaioni (1), Alessio Masi (1)

    Organisations:
    1: Department of Neurosciences, Psychology, Drug Research and Child Health, University of Florence, Florence, Italy
    2: European Laboratory for Non-Linear Spectroscopy, Sesto Fiorentino, Italy
    3: National Research Council, Neuroscience Institute, Pisa, Italy
    4: Department of Health Sciences, University of Florence, Florence, Italy

    Presenting author: Lorenzo Curti

    Exposition date: Friday, September 15, 2023

    Exposition position: 012/1

    Introduction: Autistic Spectrum Disorder (ASD) is a heterogeneous set of neurodevelopmental disorders defined by distinctive behavioral core symptoms, such as reduced sociability and stereotypy, and impaired communication skills. Haploinsufficiency of the SH3 and multiple ankyrin repeat domains (Shank) 3 gene is associated with Phelan-McDermid Syndrome, a rare genetic ASD, and idiopathic ASD forms as well. Shank3 is a multi-domain protein playing a crucial scaffolding role at the excitatory postsynaptic site. Shank3 KO mice exhibit social deficit and obsessive self-injurious grooming, associated with cortico-striatal synaptic defects. Evidence from this and other ASD models points to the synapse as a key element in ASD pathophysiology, therefore functional synaptic rescue could represent an effective therapeutic strategy. In this respect, a group of chemically heterogeneous compounds sharing psychedelic activity exhibit strong dendritogenic and synaptogenic activity in vitro and in vivo.

    Methods: With behavioral test, slice electrophysiology and single neuron morphometric analysis, we extended the characterization of the neurological phenotype of Shank3B +/- mice and tested the post-acute effects of a sub-anasthetic/halluginogenic single dose of ketamine and N, N-dimethyltryptamine.

    Results: In Shank3B +/- mice, social deficit was reverted 24 hours after a single, subanaesthetic dose (10 mg/kg) of racemic ketamine. Ex-vivo electrophysiological recordings indicate that intrinsic and synaptic properties of Layer V pyramidal neurons are not affected by Shank3 genotype or systemic ketamine administration yet they respond to acute application of ketamine and DMT at micromolar concentrations.

    Conclusions: We defined a panel of neuroanatomical and functional assays to characterize synaptic pathology in the Shank3 ASD mouse model. The phenotypes we investigated are closely related to the genetic alteration underlying the pathology and thus represent optimal readouts to probe the efficacy of compounds with neurogenic potential, such as ketamine, 2,5-Dimethoxy-4-iodoamphetamine, and N, N-dimethyltryptamine. Preliminary results suggest that ketamine ameliorates the defect caused by Shank3 ablation.

Cerebral ischemia

  • Effects of MRS3997, a dual adenosine A2A/A2B receptors agonist, in cerebral ischemia.

    Authors:
    Ilaria Dettori (1), Irene Bulli (1), Elisabetta Coppi (1), Federica Cherchi (1), Martina Venturini (1), Lucia Frulloni (1), Clara Santalmasi (1), Kenneth A. Jacobson (2), Hobin Lee (2), Anna Maria Pugliese (1), Felicita Pedata (1)

    Organisations:
    1: Department of Neuroscience, Psychology, Drug Research and Child Health (NEUROFARBA), Division of Pharmacology and Toxicology, University of Florence, Italy
    2: Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, US.

    Presenting author: Ilaria Dettori

    Exposition date: Saturday, September 16, 2023

    Exposition position: 035/2

    Cerebral ischemia is a multifactorial pathology characterized by different events evolving in time. Acute injury, characterized by a massive increase of extracellular glutamate levels, is followed by a secondary inflammatory damage that develops from hours to days after ischemia. Adenosine, by acting on its receptors (A1, A2A, A2B and A3), exerts a crucial role in post-ischemic damage. Previous data obtained by our laboratory demonstrated that the adenosine A2A receptor agonist CGS21680, as well as the adenosine A2B receptor agonist BAY 60-6583, chronically administered for 7 days after transient ischemia, are protective by contrasting the secondary inflammatory damage. Therefore, we hypothesize that the simultaneous activation of both A2Rs may represent a valid pharmacological approach to this pathological condition.

    On these bases, we investigated, for the first time, the effects of the newly synthetized A2A/A2B receptor dual agonist, MRS3997, chronically administered after transient (1 h) focal cerebral ischemia induced by middle cerebral artery occlusion (MCAo) in the rat.

    Chronic treatment with MRS3997 significantly reduced the neurological deficit and the body weight loss, up to 7 days after tMCAo. At this time, the dual agonist significantly reduced the volume of ischemic brain damage, microglial activation and astrogliosis in cortex and striatum. Also, MRS3997 protected from the damage to myelin in striatal perischemic area. Moreover, 2 days after ischemia the dual agonist reduced granulocytes infiltration in both cortex and striatum.

    These results show that synergic stimulation of A2A and A2B receptors reduces the ischemic brain damage and improves the neurological deficit, contrasting the secondary inflammatory damage that occurs after an ischemic insult. Interestingly, A2A and A2B receptors in most cases are co-expressed on hematic cells and might work synergistically to exert anti-inflammatory effects. Protection might be ascribed to the simultaneous activation of A2A and A2B receptors located both on brain, blood and vascular cells.

  • Impact of perinatal stroke on motor and cognitive functions

    Authors:
    Emanuela Beretta (1), Gianmarco Cuboni (1), Livia Vignozzi (1), Manuela Allegra (1,2), Gabriele Deidda (1,3)

    Organisations:
    1: Department of Biomedical Sciences, University of Padua, Padua, Italy
    2: Neuroscience Institute, National Research Council (IN-CNR), Padua, Italy
    3: Padova Neuroscience Center, Padua, Italy

    Presenting author: Emanuela Beretta

    Exposition date: Saturday, September 16, 2023

    Exposition position: 031/2

    Adult cerebral ischemia (stroke) is one of the leading causes of long-term disability and death in developed countries. However, an ischemic insult can occur also early in ontogenesis during the perinatal age thereby overlapping with the critical time windows for normal brain development. Despite the advances in adult stroke pathophysiology and recovery strategies, very little is still known about the impact on brain functions associated with perinatal stroke. Here, we provide insights into the structural and functional consequences of a focal disruption of cerebral blood flow (stroke) in young (perinatal) age at motor cognitive and functional level. We used the middle cerebral artery occlusion (MCAO) stroke model in mice at postnatal age (P) 14 to interrupt cerebral blood flow and induce a lesion in the primary and secondary somatosensory cortices; in this model the adjacent primary motor cortex represents the peri-lesional area. We then studied longitudinally during ontogenesis the impact on motor functions by means of the grid walk test, the grip test and rotarod test, and the impact on cognitive functions by means of the Y-maze test. Our results show the impact of an ischemic insult occurring at perinatal ages on brain functions.

  • In vivo characterization of putative neuronal biomarkers for recovery in a mouse model of adult stroke

    Authors:
    Livia Vignozzi (1), Stefano Varani (1), Emanuela Beretta (1), Gabriele Deidda (1,2), Manuela Allegra (2,3,4)

    Organisations:
    1: Department of Biomedical Sciences, University of Padua, Padua, Italy
    2: Padova Neuroscience Center (PNC), Padua, Italy
    3: Neuroscience Institute, National Research Council (IN-CNR), Padua, Italy
    4: Fondazione Istituto di Ricerca Pediatrica Città della Speranza (IRP), Padua, Italy

    Presenting author: Livia Vignozzi

    Exposition date: Saturday, September 16, 2023

    Exposition position: 034/2

    Stroke is a leading cause of long-term disability including impaired motor function worldwide. During a “critical period” after stroke, in the peri-infarct zone neural circuits undergo plastic changes to allow a spontaneous restoration of the neurological function. However, recovery outcomes among patients are highly variable, only partially depending on structural and functional factors related to the damage, such as the lesion location, volume and the topology of the neural connectivity within the peri-infarct zone. Currently, finding neurophysiological biomarkers to promptly predict recovery outcomes is a major challenge. To address this demand, we used a mouse model for ischemic injury (middle cerebral artery occlusion, MCAO) and investigated putative biomarkers at both behavioral and functional level. To evaluate the recovery outcomes, we characterized the motor function impairments by performing the gridwalk test in MCAO- and sham-mice at different days (D) after stroke induction (D2, D9, D16, D23, and D30) which revealed higher motor deficits in MCAO-mice compared to controls. Moreover, to investigate whether the recovery outcome of each animal could be reflected in neural connectivity, we performed in vivo electrophysiological recordings of local field potentials (LFPs) from the peri-infarct zone, i.e. the forelimb primary motor cortex. To investigate neuronal changes of inter- and intra-hemispheric connectivity, spontaneous and evoked activity were characterized at D9 and D30, the subacute and chronic (D30) phases of stroke, respectively. Our findings revealed higher amplitude responses to intra-hemispheric stimulation in stroke mice with a good recovery outcome, suggesting a possible correlation between higher neuronal plasticity and the recovery of the motor function.

  • Investigate the relationship between pirnas and lncrnas in in-vivo and in-vitro stroke model

    Authors:
    Rohan Mahesh Patil

    Organisations:
    University of Naples Federico II, Italy

    Presenting author: Rohan Mahesh Patil

    Exposition date: Saturday, September 16, 2023

    Exposition position: 032/2

    Stroke is a major cause of morbidity and mortality worldwide, and understanding the molecular mechanisms underlying this complex disease is essential for the development of effective treatments. Non-coding RNAs, notably piRNAs and lncRNAs, seem to play crucial roles in the pathophysiology of stroke, according to new research. The association between piRNAs and lncRNAs in stroke, as well as the tools utilized to examine this relationship, will be discussed in this abstract.

    piRNAs are a class of small non-coding RNAs that are typically involved in the regulation of transposable elements in germ cells. However, recent studies have shown that piRNAs are also expressed in somatic cells, including neurons, and may play important roles in neuronal function and disease. lncRNAs are a class of non-coding RNAs that are longer than 200 nucleotides in length and are involved in the regulation of gene expression. Several studies have reported that piRNAs and lncRNAs are functionally linked in stroke.

    Methology:- In-vivo TMCAO, stereotaxic and the in-vitro cell culture.

    RNAseq

    RNA pull-down

    CRISPR-Cas9 gene editing

    Chromatin immunoprecipitation (ChIP)

    Crosslinking immunoprecipitation (CLIP)

    Bioinformatics analysis

    Preliminary results

    Through computational analysis and text mining, we identified eight upregulated and eight downregulated piRNAs. Site specificity and transcriptional query length analysis revealed a correlation between the upregulated piRNAs and lncRNAs, with 85-100% similar sites of transcription, while the downregulated piRNAs demonstrated 40-60% similarity in transcription, suggesting an inter-relationship between the non-coding RNAs.

    Conclusion:-

    In conclusion, researchers have used a variety of techniques to investigate the relationship between piRNAs and lncRNAs. These techniques have enabled researchers to identify genes and pathways that are regulated by piRNAs and lncRNAs, to investigate the specific proteins or RNAs that are regulated by these non-coding RNAs, and to investigate the roles of these RNAs in gene regulation and development.

  • Montelukast, a safe anti-asthmatic drug, protects against stroke by improving anti-oxidant defence of oligodendrocyte precursor cells

    Authors:
    Majeda Muluhie (1), Juliana Helena Castro E Silva (1), Benedetta Mercuriali (1), Laura Castiglioni (1), Paolo Gelosa (1), José Maria Delgado-Garcia (2), Agnès Gruart (2), Davide Lecca (1), Mauro Cimino (3), Joanna Rzemieniec (1), Maria Pia Abbracchio (1), Luigi Sironi (1)

    Organisations:
    1: Department of Pharmaceutical Sciences, University of Milan, Italy
    2: Division of Neurosciences, Pablo de Olavide University, Seville, Spain
    3: Department of Biomolecular Sciences, University of Urbino, Italy

    Presenting author: Majeda Muluhie

    Exposition date: Saturday, September 16, 2023

    Exposition position: 030/2

    Montelukast was shown to exert neuroprotection after brain ischemia by preserving white matter integrity, but whether this could occur by influencing myelin-producing oligodendrocytes remains unexplored. Here, we assess the effect of montelukast on oligodendrocyte precursor cells (OPCs), in a model of permanent cerebral ischemia (pMCAo) in reporter florescent protein (GFP) mice, in which, upon tamoxifen treatment, OPCs expressing GPR17 become green and traceable for their entire life. Mice subjected to pMCAo were treated with montelukast (10 mg/kg/day) or vehicle for 3 days, 2 and 8 weeks. Electrophysiological analyses were performed weekly to evaluate functional outcomes. Oligodendrocyte dynamics were assessed by immunohistochemistry and the protective effects of montelukast on oligodendrocytes corroborated in vitro. Electrophysiological analyses showed that pMCAo induced a significant decrease in the amplitude of field potentials, indicating a reduction in fiber connectivity. Conversely, in montelukast-treated mice, the reduction of the amplitude of field potentials observed at week 4 and 5 was recovered and returned to baseline by week 5 and 7 after stroke, suggesting improved fibers reorganization. Immunofluorescence analyses showed that montelukast treatment increases OPCs recruitment and proliferation and promotes their differentiation to mature oligodendrocytes suggesting a link between OPCs activation, maturation and fiber connectivity. Moreover, montelukast treatment significantly increased the anti-oxidant defense of OPCs as evident from the enhanced number of GFP+-oligodendrocytes expressing heme oxygenase 1 (HO-1) 3 days after MCAo. In vitro, montelukast was able to counteract TNF-α-induced apoptosis and defective OPCs maturation. We are currently evaluating the effect of montelukast in protecting OPCs by oxidative stress. In conclusion, we demonstrated that montelukast improves long-term functional recovery after brain ischemia by enhancing the recruitment, survival and maturation of OPCs towards myelinating stage through anti-oxidant capacity. These data raise the possibility of repositioning an already marketed drug with immediate advantages for patients suffering for stroke.

  • MSC-derived-exosomes promote functional recovery in a preclinical model of stroke

    Authors:
    Chiara D'Amelio (1), Saviana Antonella Barbati (1), Chiara Feroleto (1), Federica Milella (1), Ida Nifo Sarrapochiello (1), Lucia Leone (1,2), Salvatore Fusco (1,2), Maria Vittoria Podda (1,2), Claudio Grassi (1,2)

    Organisations:
    1: Department of Neuroscience, Università Cattolica del Sacro Cuore, Rome, Italy
    2: Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy

    Presenting author: Chiara D'Amelio

    Exposition date: Saturday, September 16, 2023

    Exposition position: 033/2

    Stroke is a leading cause of death and long-term disability, making it crucial to develop strategies that promote functional recovery after stroke. Several studies have demonstrated that early treatment of ischemic stroke may reduce complications, disability, and mortality in the long term. Our study aimed at evaluating: i) the impact of a non-invasive approach, specifically intranasal (IN) administration of exosomes derived from human bone marrow mesenchymal stem cells (MSC-exo) on the recovery of forelimb motor function after stroke; ii) elucidating the underlying mechanisms. We performed experiments on mice subjected to focal ischemia by photothrombosis on the caudal forelimb primary motor cortex. The treatment started 48 hours after stroke, consisting of a single dose of exosomes that was then administered twice a week for 4 consecutive weeks. Motor function was assessed by performing a battery of tests before and 48 hours after the ischemia and then subsequently every week up to end of treatment (week 4). Our results indicated that mice treated with MSC-exo showed better performance compared to vehicle treated mice starting from the second week after stroke, as revealed by highest score in the cylinder test (% touches with impaired forelimb: 53.4% vs 33.9%; MSC-exo vs. vehicle: p=0.02) and in the grid walking test (% foot faults: 10% vs. 13%; MSC-exo vs. vehicle: p=0.009). Additionally, a preliminary histopathological assessment revealed a reduced infarct size in exosomes-treated mice (infarct volume: 0.49 mm3 vs. 1.03 mm3; MSC-exo vs. vehicle: p= 0.03), suggesting a neuroreparative effect of exosomes, favoring functional recovery.

  • Neural cortical organoids from self-assembling human iPSC as a model to investigate neurotoxicity in brain ischemia

    Authors:
    Massimiliano De Paola, Davide Comolli, Joe Kelk, Ilaria Lisi, Milica Cerovic, Stefano Fumagalli, Gianluigi Forloni, Elisa Zanier

    Organisations:
    Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Italy

    Presenting author: Davide Comolli

    Exposition date: Saturday, September 16, 2023

    Exposition position: 029/2

    Brain ischemia is a common acute injury resulting from impaired blood flow to the brain. Translation of effective drug candidates from experimental models to patients has systematically failed. The use of human induced pluripotent stem cells (iPSC) offers new opportunities to gain translational insights into diseases including brain ischemia. We used a human 3D self-assembling iPSC-derived model (human cortical organoids, hCO) to characterize the effects of ischemia caused by oxygen-glucose deprivation (OGD). hCO exposed to 2h or 8h of OGD had neuronal death and impaired neuronal network complexity, measured in whole-mounting microtubule-associated protein 2 (MAP-2) immunostaining. Neuronal vulnerability was reflected by a reduction in MAP-2 mRNA levels, and increased release of neurofilament light chain (NfL) in culture media, proportional to OGD severity. Glial fibrillary acidic protein (GFAP) gene or protein levels did not change in hCO, but their release in medium increased after prolonged OGD. In conclusion, this human 3D iPSC-based in vitro model of brain ischemic injury is characterized by marked neuronal injury reflected by the release of the translational biomarker NfL which is relevant for testing neuroprotective strategies.

Cognition and behavior

  • Audiovisual crossmodal congruency may enhance visuo-spatial long-term memory

    Authors:
    Francesca Piovesan, Irene Ronga, Pietro Sarasso, Maria-Chiara Villa, Irene Lozzi, Katiuscia Sacco

    Organisations:
    BraIn Plasticity and behavior changes (BIP) - research group. Department of Psychology, University of Turin

    Presenting author: Francesca Piovesan

    Exposition date: Friday, September 15, 2023

    Exposition position: 038/1

    Sensory modalities simultaneously operate to create a coherent representation of the world in our brain. The synchronous presentation of multimodal stimulation has been demonstrated to enhance short-term memory (i.e. crossmodal integration) in a mechanism that has long been established in literature. However, it is unclear whether crossmodal-related facilitations may occur even when multimodal inputs are presented asynchronously. By capitalizing on the well-known spatial elevation-musical pitch correspondences, here we investigate the effect of visual-auditory crossmodal facilitation when auditory sequences were followed by the presentation of congruent or incongruent visual stimulation. During the initial auditory acquisition phase, participants were asked to listen to and repeat 4 sets of arpeggiated chords. Subsequently, in the memorization phase, participants were asked to memorize 4 dot sequences, 2 of them remapping in the visual domain auditory pitch sequences. After a distraction task, participants were then asked to detect previously presented visuospatial configuration, among never-seen distractors. Memorization performance resulted to be significantly enhanced for configurations corresponding to previously presented musical chords. This result demonstrates that crossmodal facilitation effects enhance long-term memory and may be observed even for non simultaneous multimodal input. Our findings could have significance in the creation of rehabilitative protocols directed at patients diagnosed with dementia. Although these patients often suffer from visuospatial deficits, their auditory domain is often spared. Employing the common representation of sounds and spatial location in rehabilitative protocols could enhance spatial memory, at the same time supporting motivation, by exploiting the pleasure provided by harmonic music.

  • Behavioural effects of an intranasal AMPK-targeted treatment for Rett syndrome

    Authors:
    Alessia De Matteis, Chiara Urbinati, Livia Cosentino, Alessandra Monaco, Bianca De Filippis

    Organisations:
    Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Roma, Italy

    Presenting author: Alessia De Matteis

    Exposition date: Friday, September 15, 2023

    Exposition position: 044/1

    Rett syndrome (RTT) is a rare neurological disorder with a monogenic origin, mainly caused by de novo mutations in the methyl-CpG binding protein 2 (MECP2) gene. RTT principally affects females, who develop symptoms at around 1 year of age, and results in impairments at different levels by causing cognitive, motor and social defects, associated with several comorbidities. No cure is currently available. Recently, special interest has been directed toward the involvement of mitochondria dysfunction in the pathogenesis of RTT, as alterations have been described both in the brain and in the periphery. The 5’-adenosine-monophosphate-activated protein kinase (AMPK) is an energy sensor that regulates ATP levels and is essential for the control of mitochondrial function and the maintenance of energy homeostasis. AMPK is also involved in several neurological diseases, characterized by motor and cognitive dysfunction. These features make AMPK a target of potential interest for RTT. The present work evaluated whether the activation of AMPK in the brain can exert beneficial effects on the behavioural alterations of a validated mouse model of RTT (MeCP2-308 heterozygous female mice). To this aim, an 11-day-long intranasal treatment was administrated in fully symptomatic MeCP2-308 mice (12-month-old) with the selective AMPK activator GSK621 (1.5 mg/Kg), which passes the blood-brain barrier. A battery of tests focusing on the most affected behavioural domains in RTT, such as memory and motor skills, was carried out to assess treatment effects thereon. The treatment with GSK621 had a selective effect on recognition memory assessed in the novel object recognition test. No effects on the impaired motor coordination or the general health status were observed. Despite the necessity of follow-up studies to confirm the results obtained, this study lays the foundation for further investigations of the possible involvement of AMPK in the pathogenesis of RTT and the therapeutic potential of its modulation.

  • Beyond marbles buried: a comparative analysis of tests targeting restricted and repetitive behaviors in C57B6/J mouse strain

    Authors:
    Irene Coppola (1), Mattia De Magistris (2), Gemma Calamandrei (2), Laura Ricceri (2), Anna Maria Tartaglione (2)

    Organisations:
    1: National Center for Drug Research and Evaluation, Italian National Institute of Health (ISS), Rome, Italy
    2: Centre for Behavioral Sciences and Mental Health, Italian National Institute of Health (ISS), Rome, Italy

    Presenting author: Mattia De Magistris

    Exposition date: Friday, September 15, 2023

    Exposition position: 048/1

    Restricted and repetitive behaviors (RRBs), referring to set of responses showed with high frequency and for unusually long periods, are defining features of autism and other psychiatric conditions. The study of RRBs in animal models plays an important role in elucidating the mechanisms underlying these conditions and in screening treatment response. Thus, the choice of reliable behavioral tests provides the foundation of such investigations.

    One of the paradigms commonly used to analyze RRBs is the marble burying test (MBT) that allows to assess digging and burying behaviors showed by mice when they are introduced into a cage with marbles lined up on the surface of the bedding. The main outcome is the total number of marbles buried under the sawdust. However, it has not yet been established whether this parameter really reflects RRBs, or may be simply a proxy of digging behavior or anxiety-like response elicited by the presence of marbles.

    In order to disentangle different behavioral responses underlying the marble burying behavior, the present study was aimed to: i) identify potential correlations between the number of marbles buried and behavioral responses showed by mice during the MBT and ii) perform a comparative analysis between MBT and other behavioral tasks targeting RRBs (i.e., hole board, grooming test and spontaneous alternation in Y-maze test) and anxiety-like behavior (i.e., Elevated Plus Maze) in adolescent C57B6J mice.

    Our preliminary analyses revealed that the number of marbles buried did not correlate with anxiety-like behavior, whereas it was associated not only with digging in MBT, but also with number of head dipping in the Hole Board exploration and percentage of spontaneous alternation in Y-maze. These data indicate that the marble burying behavior is not simply a byproduct of digging behavior, and provides a useful tool to evaluate RRBs in murine models.

  • Brain and behavior as targets of perinatal exposure to bisphenols

    Authors:
    Brigitta Bonaldo (1,2), Antonino Casile (1,2,3), Martina Bettarelli (2), Marialaura Teresa Ostuni (2), Sofia Nasini (4), Marilena Marraudino (1,2), GianCarlo Panzica (1,2), Stefano Gotti (1,2)

    Organisations:
    1: Department of Neuroscience “Rita Levi-Montalcini”, University of Turin, Via Cherasco 15, 10126 - Turin, Italy
    2: Neuroscience Institute Cavalieri Ottolenghi (NICO), Regione Gonzole 10, 10043 - Orbassano, Turin, Italy.
    3: Department of Chemical and Pharmaceutical Sciences and Biotechnology, University of Camerino, Cavour 19/f 62032 square Camerino (MC), Italy.
    4: Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Largo Meneghetti 2, 35131 Padua (PD) – Italy

    Presenting author: Brigitta Bonaldo

    Exposition date: Friday, September 15, 2023

    Exposition position: 030/1

    Bisphenols, synthetic organic compounds used to produce plastics, are an abundant class of Endocrine Disrupting Chemicals. Bisphenol A (BPA) is the first and the most highly produced one. Thanks to its structure, BPA can act through different types of nuclear and membrane-bound hormone receptors, exerting a wide range of effects on the organism. To stem the deleterious effects of BPA, other bisphenols, such as bisphenol S (BPS), have been proposed as BPA substitutes. Unfortunately, BPS seems to display the same, or even worse, endocrine-disrupting properties as BPA. It has to be considered that i. exposure to bisphenols is still increasing, ii. while there is European legislation for BPA, it is still lacking for its analogs, iii. consequences of exposure to such compounds differ based on numerous parameters. Thus, we investigated the effects of perinatal oral exposure to low dose (4µg/kg BW/day, EFSA TDI for BPA) BPA or BPS in both male and female adult mice. In particular, we focused on the analysis of sexual and anxiety-related behaviors and, thanks to immunohistochemical techniques, on specific neuronal populations involved in the control of such behaviors. We observed alterations, different according to sex and treatment, in puberty onset, estrous cycle, and sexual and anxiety-related behaviors, together with changes in the hypothalamic kisspeptin system and in the serotonin system within the dorsal and median raphe nuclei. Our results strengthened previous data about the harmful effects of BPA and highlighted those of BPS, which cannot represent a safe alternative.

  • Differences and similiarities in the neural patterns of activity related to correct and incorrect trials in the macaques lateral prefrontal cortex: an electrophysiological study

    Authors:
    Claudio Basile, Virginia Beretta, Alfonso Gravante, Leonardo Fogassi, Stefano Rozzi

    Organisations:
    Department of Medicine and Surgery, University of Parma, via Volturno 39, 43125 Parma, Italy.

    Presenting author: Claudio Basile

    Exposition date: Friday, September 15, 2023

    Exposition position: 041/1

    The lateral prefrontal cortex (LPF) is crucially involved in the ability to flexibly select, organize and adapt behavioral responses to the continuously changing context (Tanji et al., 2008). Two main factors can lead to a failure of this process and, thus, to errors: lack of attention/motivation and incorrect implementation of contextual relevant information. Most studies investigated LPF neural activity when the latter factor occurs, showing that the discharge of neurons coding for abstract rules is modulated when incorrect decisions are taken (Genovesio et al., 2008). Little is known, however, about the neural substrate of behavioral errors in terms of population activity. Recently, Rozzi et al., (submitted) showed that, in correct trials, populations of LPF neurons encode instructions and goals in terms of their behavioral outcomes.

    To assess whether this encoding format also characterizes incorrect trials, we analyzed LPF activity recorded during a Go/NoGO task in which, based on different instructing cues, the monkeys had to act or withhold acting on different objects. We considered two types of incorrect trials: Type-I, in which the response was congruent with the behavioral rule but inaccurate (i.e., early response); Type-II, in which the response was incongruent with the behavioral rule (i.e., acting when required not to). Preliminary analyses were carried out on 120 cells recorded in at least one incorrect trial per type. We show that single neuron and population activity during early phases of the task is similar when comparing Type-II and correct trials in which behavioral rule is the opposite, while it is different when comparing Type-II and correct trials in which behavioral rule is the same. The opposite occurs when comparing Type-I and correct trials. This suggests that, starting from the initial phases of correct and incorrect trials, task-relevant information is already encoded in terms of the subsequent behavioral response.

  • Dissecting the role of Androgen receptor in the central nervous system.

    Authors:
    Emanuela Zuccaro (1,2), Elisa Bregolin (1,2), Diana Piol (1), Maria Pennuto (1,2)

    Organisations:
    1: Department of Biomedical Sciences, University of Padova, Italy
    2: Veneto Institute of Molecular Medicine (VIMM), Padova, Italy

    Presenting author: Emanuela Zuccaro

    Exposition date: Thursday, September 14, 2023 (Satellite Events Day)

    Exposition position: 92

    Androgen Receptor (AR) is a steroid hormone-activated transcription factor located in the cytoplasm in an inactive state. In response to androgen binding, AR shuttles from the cytosol to the nucleus, where it regulates the expression of specific AR target genes. AR is widely expressed throughout the brain, hippocampus, hypothalamic nuclei (like the preoptic area, POA), and amygdala.

    Of note, these brain areas hold sexually dimorphic characteristics, which are translated in sex-typed behaviour, including aggression, parental care, mating, and anxiety. AR gene is located in the X chromosome; therefore, it is present in a single copy in men. Males lacking or expressing mutant AR display female-like external sexual organs and infertility, which are recapitulated in rodent models of androgen insensitivity syndrome. Many studies have been focused on understanding the role of AR in male phenotype. However, the AR-driven molecular mechanisms leading to sexually dimorphic brain and behaviours are still largely unknown. In the nucleus, AR can regulate the expression of its target genes by recognizing and binding specific DNA sequences, termed androgen response elements (ARE), in their promoter and enhancer regions. Our data suggest that AR is able to change its subcellular localization in response to androgens and neuronal activity, ultimately modulating the expression of immediate-early and late-response downstream target genes.

    Our findings suggest a new role for AR as activity-dependent transcription factor. We are further investigating AR role in the CNS by dissecting the molecular changes driven by AR in one of the typical male behaviours known to be governed by androgens and AR, which is the male mating behaviour.

  • Dystrophic mdx mice are hypersensitive to acute and sub-chronic restraint stress: a behavioural and biochemical study

    Authors:
    Elisa Pizzichini, Valentina Ferretti, Antonella De Jaco, Maria Egle De Stefano

    Organisations:
    Sapienza University of Rome, Italy

    Presenting author: Elisa Pizzichini

    Exposition date: Friday, September 15, 2023

    Exposition position: 032/1

    Duchenne Muscular Dystrophy (DMD) is a lethal neuromuscular disease caused by the lack of full-length dystrophin (Dp427), a cytoskeletal protein expressed in skeletal muscles, as well as in selected brain regions. In patients, as in mdx mice, an elective DMD animal model, lack of Dp427 severely affects the hippocampus, a brain region target of glucocorticoids (GCs) and important relay in stress responses. As chronic treatments with GCs are a golden standard in DMD therapy, it is conceivable that their persistent administration may further compromise hippocampal physiology. Our previous in vitro and in vivo studies have demonstrated that mdx mouse hippocampal neurons are significantly more sensitive, compared to wild-type, to both acute and sub-chronic administrations of GCs, highlighting a condition of hyper-responsiveness, characteristic of chronic stress responses. Aim of this study was to verify whether hippocampal neurons of the two genotypes respond differently also to induced stress conditions. Mice were subjected to either acute or sub-chronic tube-restraint stress, and several behavioural parameters were analysed by Elevated Plus Maze (EPM) and Open Field (OF) tests. In contrast to wild-type, mdx mice behaved similarly after both stress paradigms, not differentiating between acute and sub-chronic exposures. This was revealed by equally increased frequencies of entries in the open arms of the EPM, and higher distance covered and longer time spent in the periphery rather than in the center of the OF, with respect to their control. Accordingly, protein levels of hippocampal phosphorylated and total glucocorticoid receptors ratio (pGR/GR), used as an indicator of stress response, were significantly increased only in mdx mice after both acute and sub-chronic restraint procedures, suggesting inability in preventing GR hyperactivation in adverse situations. Overall, these results confirm a peculiar susceptibility of dystrophic hippocampal neurons to stress conditions and mediators, which on the long term could further impair their physiology.

  • Effect of sustained cognitive control on error rate and event-related potentials in a go/no-go task

    Authors:
    Fabio Taddeini (1), Giulia Avvenuti (3), Alberto Arturo Vergani (1), Emiliano Ricciardi (3), Silvestro Micera (1,2), Giulio Bernardi (3), Alberto Mazzoni (1)

    Organisations:
    1: The BioRobotics Institute and Department of Excellence in Robotics and AI, Sant'Anna School of Advanced Studies, Italy
    2: Bertarelli Foundation Chair in Translational Neuroengineering, EPFL, Switzerland
    3: IMT School for Advanced Studies Lucca, Italy

    Presenting author: Fabio Taddeini

    Exposition date: Friday, September 15, 2023

    Exposition position: 033/1

    Cognitive control refers to a set of mental processes necessary for the modulation of behaviour in order to reach a specific goal. These processes include: focused attention, working-memory and inhibitory control, which are regulated by differential responses of fronto-parietal networks. However, the effects on neural activity, and consequently, on behavior, of hours-long sustained cognitive control are not completely clear. Here we studied in 24 young adults (9 males, age 30.0+/-2.8 years) the effects of fatigue induced by sustained use of cognitive control on behavioral performance during a go/no-go task. As a control condition, the same participants completed a duration-matched experimental session not involving the exertion of cognitive control. EEG data were continuously recorded from 64 scalp electrodes. We showed that cognitive fatigue leads to an increase in error rate, mainly driven by anticipated responses, during the go/no-go task. Event-related potentials (ERPs) related to these errors were observed in both frontal and posterior parietal areas, confirming the fundamental role of these regions in cognitive control. Conversely, the control experimental condition was associated with increasingly long reaction times. The ERP analysis suggested that this was likely due to a decrease in engagement. Our results show that cognitive fatigue can lead to a change in the subjects' strategy to perform the task, which is less stressful but can lead to more errors. Moreover, we observed how several components of ERPs could be indicative of a subject’s ability to exercise adequate control over his or her actions. This study can help shed light on which areas of the cortex are primarily related to errors in choosing a correct response and on what are the possible effects caused by the continuous use of cognitive control processes on behavior.

  • Effects of Galvanic Vestibular Stimulation on Actions Recognition: a single-blind study

    Authors:
    Roberto Gammeri (1), Maria-Chiara Villa (2), Giuliano Carlo Geminiani (2), Anna Berti (1), Raffaella Ricci (1)

    Organisations:
    1: SAN Lab (Space, Attention ad Action), Department of Psychology, University of Turin, Torino, Italy
    2: BIP (BraIn Plasticity and Behavior Changes) Research Group, Department of Psychology, University of Turin, Turin, Italy

    Presenting author: Roberto Gammeri

    Exposition date: Friday, September 15, 2023

    Exposition position: 046/1

    Introduction: Observing other people's actions involves the activation of corresponding motor representations in the observer's brain, which is generally known as mirror process. Therefore, familiar actions are typically recognized more quickly (i.e., faster reactions times) than unfamiliar ones, the so-called familiarity effect. Although the vestibular system has been shown to play a crucial role in the perception of self-action, its contribution to the recognition of others’ actions is still unclear. We used Galvanic Vestibular Stimulation (GVS) to explore the vestibular system’s contribution to the mirror processes underlying recognition of others’ actions.

    Methods: Eighteen healthy subjects were randomly assigned to either left-GVS or right-GVS groups (N=9 each), which consisted in 5 minutes of supra-threshold bipolar galvanic vestibular stimulation (GVS). The two groups underwent both GVS and sham condition while performing a full-body version of the action recognition task, in which participants were specifically asked to recognize familiar and unfamiliar actions that may or may not involve the vestibular system.

    Results: The familiarity effect was present in recognition of non-vestibular actions, (p < .01) and this effect was not affected by GVS. In contrast, for vestibular actions, the familiarity effect was only observed during sham and GVS increased RTs to recognize familiar actions. (p < .05). In other words, GVS selectively disrupted recognition of familiar vestibular actions, independently of its polarity.

    Conclusions: The results of the present study indicate that the vestibular system plays a specific role in the mirror process underlying recognition of others’ actions. These findings are in line with previous evidence showing that action-observation therapies are effective in improving balance performance. Furthermore, the finding that GVS can modulate action observation performance may have relevant clinical implications for the neuro-rehabilitation of patients with movement disorders.

  • Evaluation of in vivo effects of non-psychotropic phytocannabinoid mixtures

    Authors:
    Marinella Carone, Roza Anastasia Latsi, Marika Premoli, Giuseppina Maccarinelli, Maurizio Memo, Sara Anna Bonini

    Organisations:
    Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy

    Presenting author: Marinella Carone

    Exposition date: Friday, September 15, 2023

    Exposition position: 039/1

    The cannabis plant (Cannabis Sativa) produces more than 100 specific neuroactive compounds, known as cannabinoids. The major non-psychoactive cannabis constituent is cannabidiol (CBD), which has been proven to display beneficial properties in a variety of medical conditions. However, the potential therapeutic role of other phytocannabinoids, such as cannabigerol (CBG), and their use in combination with CBD has remained largely unexplored. In this study, we wanted to assess the in vivo effects of two novel non-psychotropic cannabinoid formulas, both containing relatively high percentage of CBD but differing mainly for CBG content, hereafter called CBG+ and CBG- formulas. Both formulas present other phytocannabinoids in variable proportions. We employed a battery of distinct behavioral tests to evaluate the effects of three different dosages (25 mg/kg, 50 mg/kg, 100 mg/kg via oral gavage), administered acutely and sub-chronically (2 weeks), on mice locomotor activity, anxiety-related behaviors, short-term memory and sociability. We found that the two formulas tested display unique behavioral profiles: CBG- formula produces an increase in mice locomotor activity and in mice sociability, whereas CBG+ formula displays anxiolytic properties. Both formulas improved spatial short-term memory. The results obtained suggest that different combinations of phytocannabinoids are able to determine different behavioral effects and highlight the importance of studying the effects of less known phytocannabinoids (such as CBG), which used in combination with other phytocannabinoids can change the profile of action of already characterized active compounds (such as CBD).

  • Exploring transgenerational inheritance in epigenotypes of DAT heterozygous rats: circadian anomalies and attentional vulnerability in the phenotype.

    Authors:
    Concetto Puzzo (1), Fabiana Festucci (2), Giuseppe Curcio (2), Antonella Gigantesco (3), Walter Adriani (3)

    Organisations:
    1: Università Telematica Internazionale Uninettuno, Italy
    2: Università degli Studi dell'Aquila
    3: Istituto Superiore di Sanità

    Presenting author: Concetto Puzzo

    Exposition date: Friday, September 15, 2023

    Exposition position: 029/1

    Dopamine, is a catecholamine neurotransmitter, mainly involved in locomotor activity, reward processes and maternal behaviors. Rats with KO gene coding for truncated DAT protein are in hyperdopaminergic conditions and develop stereotyped behaviors and hyperactivity. We studied transgenerational inheritance innon - classical heterozygotes [1] based on two specular lines, with putative grand-maternal vs. grand-paternal imprinting. MAT females (offspring of KO male and WT female) mated with a KO male to generate MIX offspring. Specularly, PAT females (offspring of KO female and WT male) mated with KO male to generate PIX offspring. We observed also the third generation (MYX: HET offspring of KO male and MUX female, in turn offspring of MAT male and KO female, thus with KO maternal grandmother like also for PIX).We run comparisons of circadian locomotor activity and comparisons of behavior expressed in the elevated-plus-maze (EPM) for all three generations. Locomotor hyperactivity occurs in the first generation, the opposite occurs in the second generation with combined pooling of second generation vs. third generation and control group. We were able to dismiss a role for gestation in hyperdopaminergic uterus. The MAT, MIX and MYX showed a significant vulnerability for ADHD like symptoms (duration of rearing): with seesaw profile evident in their phenotype from the first progeny it tends to be transferred to the third- generation. Our hypothesis is that the DAT gene develops transgenerational changes in heterozygous rats: for major features the similar odd generations may appear opposed to even ones; for minor specific features, the phenotype transfer may affect the progenies with a male but not a female ancestor.

  • Facial Motor Control in Monkeys and Rodents : An Anatomical Study

    Authors:
    Anna Bertucci (1,2), Dalila Biancheri (3), Elena Borra (3), Marco Lanzilotto (2)

    Organisations:
    1: Department of Life Sciences and Systems Biology, University of Turin, Italy
    2: Department of Psychology, University of Turin
    3: Department of Medicine and Surgery, University of Parma,Italy

    Presenting author: Anna Bertucci

    Exposition date: Friday, September 15, 2023

    Exposition position: 040/1

    Facial expressions play a fundamental role in social communication and interactions especially in primates. Facial displays involve rhythmic movements between different muscles and several studies have shown that different facial muscles are activated in a specific, opposite manner depending on the emotional valence of the facial expression observed or produced. Neurons within the facial motor nucleus provide the final output for controlling facial expressions. Although previous studies have focused on the anatomical characterization of this nucleus, little is known about the inhibitory circuits that may coordinate synchronous facial muscle activity. The aim of this study is to comparatively investigate in macaque and rat the presence of subpopulations of neurochemically defined neurons within the facial nucleus and facial premotor brainstem structures, which may act as orchestrators for automatic coordination of activity during muscle contractions. To identify cells distribution and circuits organization, we performed Nissl staining and immunohistochemistry for parvalbumin (PV) and calbindin (CB) and immunohistochemistry and in-situ hybridization for glutamic acid decarboxylase (GAD), to detect GABAergic interneurons and projections. We observed a subpopulation of PV+ neurons within the nucleus in both species, although the number of labelled cells was higher in the monkey. Conversely, CB+ and GAD+ cells were not located within the nucleus in either species. Interestingly, CB+ and GAD+ cells were found anteriorly and around the nucleus in facial premotor structures, and the number of these labelled cells was higher in the monkey. Finally, we found a marked difference in immunoreactivity for GAD+ fibers and terminals between species. Specifically, we observed a greater concentration of neuropil in the lateral and medio-ventral portion of the monkey facial nucleus, indicating more inhibitory afferents compared with the rat. These results pave the way for new insights into the neural mechanisms underlying facial motor coordination in primates during social interactions.

  • How the interpersonal distance varies throughout the lifespan

    Authors:
    Ilaria Mirlisenna, Greta Bonino, Francesca Capiotto, Monia Cariola, Giulia Romano Cappi, Alessandro Valvo, Olga Dal Monte

    Organisations:
    University of Turin, Italy

    Presenting author: Ilaria Mirlisenna

    Exposition date: Friday, September 15, 2023

    Exposition position: 036/1

    Interpersonal distance is defined as the physical distance that individuals maintain between themselves and other people during a social interaction. Previous studies have shown that interpersonal distance can vary according to individual characteristics such as age, gender, cultural norms, and attachment styles. Additionally, situational variables like the context and the identity of the person we are interacting with may play a role in regulating the space that individuals keep between each other during a social exchange. Despite a substantial amount of studies has investigated individual differences in interpersonal distance preferences, no studies have investigated how interpersonal space varies throughout the lifespan, nor how the identity of the other individual may influence such preferences.

    In the present study, by using an ecological setting, we collected individual preferences in interpersonal distance in a large sample of subjects ranging from 3 to 85 years old. We implemented a stop distance paradigm where subjects were asked to alternately approach and being approached by another individual until reaching a distance where they felt comfortable for interacting with the person in front of them. In order to determine to what extent the identity of the other person influences the interpersonal distance, each subject completed the procedure with both a familiar and an unfamiliar individual.

    We found that interpersonal distance preferences not only differ based on the other person identity (familiar vs unfamiliar) but, critically, the preferred physical distance, during a social interaction, changes according to the person age. Overall, the present study suggests that interpersonal distance is a complex and acquired, rather than innate, behavior that changes throughout the lifespan and varies according to individual and situational variables.

  • Improved social and object discrimination memory in Transient Receptor Potential Ankyrin 1 (TRPA1) channel-deleted mice

    Authors:
    Maria Vittoria Becagli (1), Alessia Costa (2), Daniel Souza Monteiro De Araujo (1), Romina Nassini (1), Ludovico Silvestri (3), Francesco de Logu (1), Gustavo Provensi (2)

    Organisations:
    1: Department of Health Sciences, University of Florence, Italy
    2: Department of Neurosciences, Psychology, Drug Research and Child Health (NEUROFARBA), University of Florence, Italy
    3: Department of Physics and Astronomy, University of Florence, Italy

    Presenting author: Gustavo Provensi

    Exposition date: Friday, September 15, 2023

    Exposition position: 027/1

    Transient Receptor Potential Ankyrin 1 (TRPA1) channels are non-selective cation channels expressed in peripheral and central tissues which respond to a wide range of exogenous and endogenous chemical and physical stimuli. Recently, a series of observations are shedding light on their involvement in cognition, for instance, Trpa1 genetic deletion (Trpa1-/-) resulted in improvement of animals’ performance in spatial- and fear-related tasks and in attenuation of age-induced cognitive decline. Here, we expanded these observations by investigating the expression of the TRPA1 channels in different neuronal populations and the impact of Trpa1 genetic deletion on recognition memory. The performance of Trpa1-/- male and female mice compared to wild type (Trpa1+/+) littermates were evaluated in the novel object recognition (NOR) and social discrimination (SD) paradigms. TRPA1 channels seem not play a key role on animals’ sociability, since all animals showed a clear preference in exploring a cylinder containing a social stimulus over an empty cylinder. In the test session Trpa1-/- mice recognized the previously encountered conspecific as revealed by the significantly higher percentage of time spent in exploring the novel compared to the familiar juvenile while Trpa1+/+ mice were no able to discriminate the stimulus. Similar results were obtained in the NOR task. In situ hybridization performed in brain tissue sections of Trpa1+/+ mice revealed the expression of the receptors in both GABAergic and glutammateric neurons in the hippocampus and frontal cortex. Altogether, our results confirm the hypermnestic phenotype previously described for Trpa1-/- mice by extending the observation to other types of memory. Further studies are required to fully understand the consequences of the phenotype and the specific role played by TRPA1 channels expressed in different neuronal populations.

  • Medial prefrontal cortex stimulation abolishes implicit reactions to threats and prevents the return of fear

    Authors:
    Eugenio Manassero, Giulia Concina, Maria Clarissa Chantal Caraig, Pietro Sarasso, Adriana Salatino, Raffaella Ricci, Benedetto Sacchetti

    Organisations:
    University of Turin, Italy

    Presenting author: Benedetto Sacchetti

    Exposition date: Friday, September 15, 2023

    Exposition position: 031/1

    Down-regulating emotional overreactions toward threats is fundamental for developing treatments for anxiety and post-traumatic disorders. The prefrontal cortex (PFC) is a critical brain area for downstream modulatory processes, and despite previous studies adopting repetitive Transcranial Magnetic Stimulation (rTMS) over this region provided encouraging results in enhancing extinction processes, no studies have hitherto explored the effects of stimulating the medial PFC (mPFC) on threat memory and generalization. Here we showed that rTMS applied before threat memory retrieval abolishes implicit reactions to learned and new stimuli in humans. These effects were not due to inhibition of overall electrodermal reactivity and enduringly persisted one week later in the absence of rTMS. No effects were detected on explicit recognition patterns. Critically, we observed stronger attenuation of defensive responses in subjects stimulated over the mPFC than over the dorsolateral PFC (dlPFC). Our findings uncover a prefrontal region whose modulation can permanently hamper implicit reactions to learned dangers, representing an advance to long-term deactivating overreactions to threats.

  • Neurons responsive to yawn in the monkey ventral premotor cortex

    Authors:
    Elena Ferretti (1), Francesca Lanzarini (2), Monica Maranesi (1), Luca Bonini (1)

    Organisations:
    1: University of Parma, Italy
    2: Ernst Strüngmann Institute (ESI) for Neuroscience in Cooperation with Max Planck Society, Frankfurt am Main, Germany

    Presenting author: Elena Ferretti

    Exposition date: Friday, September 15, 2023

    Exposition position: 042/1

    Yawning is an involuntary behaviour frequently regarded as a “fixed action pattern”, not influenced by sensory feedback and highly stereotyped among species. Coherently, it has been enlightened a possible role of subcortical structures (e.g. paraventricular nucleus of the hypothalamus, brainstem) in orchestrating the respiratory-motor pattern of yawning. Despite its highly stereotyped nature, yawning can vary in its duration, frequency, and form, and can be under partial voluntary control depending on autonomic, environmental, and social factors. Thus, based on the motor representation of face and mouth actions in the ventral Premotor Cortex (PMv) and the presence of neurons with sensorimotor and social functional properties in this region, we hypothesize that PMv may play a role in modulating the neural control of yawning. To date, the neurophysiological support to this hypothesis is lacking. To address this issue, we recorded single neurons from freely moving monkeys (Macaca mulatta) in a plexiglass enclosure (NeuroEthoRoom), monitored with a multiple camera system. Using a neural data logging system, we recorded PMv neurons during yawning behaviour as well as during other mouth/face behaviours (e.g. sucking, chewing, threatening). We found neurons responding specifically during yawn and not during other mouth-related behaviours. Interestingly, the discharge of some of these “yawn units” did not correlate with kinematic parameters of mouth opening. Therefore, our findings suggest that PMv neurons, differently from subcortical regions, may play a role in regulating yawning displays, possibly facilitating/inhibiting yawning for social and communicative purposes.

  • Rhythmic discrimination of languages in infants with hearing loss

    Authors:
    Gaia Lucarini (1), Caroline Nallet (1), Davide Brotto (1), Patrizia Trevisi (1), Alessandro Martini (1), Judit Gervain (1,2)

    Organisations:
    1: Università degli Studi di Padova, Padova, Italy
    2: CNRS & Université Paris Cité, Paris, France

    Presenting author: Gaia Lucarini

    Exposition date: Friday, September 15, 2023

    Exposition position: 047/1

    At birth, newborns show sensitivity to the rhythm of their native language, i.e. the language they heard prenatally, being able to discriminate it from a rhythmically different language (Pena et al. 2003, May et al., 2018). A current hypothesis (Gervain, 2018) suggests that prosody provides the basis of early speech perception and helps infants discover other linguistic units after birth, when the full-spectrum speech signal is available. Prenatal experience is thus hypothesized to be foundational for language learning. But what happens when prenatal experience is disrupted?

    To investigate this, we are testing the ability of 0-10 month-old infants with hearing loss (HL) to discriminate their native language (Italian) from a rhythmically different unfamiliar language (English). Sentences in both languages are presented forward and backward. Backward speech, with perturbed temporal features, is a standardly used non-linguistic control (Pena et al. 2003). A control group of age-matched normal-hearing (NH) infants is also tested. Infants’ brain responses are recorded using functional Near-Infrared Spectroscopy (fNIRS) covering the frontal, temporal, and parietal regions, bilaterally.

    Data collection is ongoing. Preliminary results (n=17 HL, n=18 NH) suggest that NH babies show overall larger responses than HL babies. In addition, it seems that Italian is triggering larger brain responses than English in the two groups.

    If found, a deficit in prosodic perception in infants with hearing loss could provide theoretical insights into the role of prenatal and early postnatal experience in language development, as well as important applications for screening and intervention in this population.

  • Targeting Vasopressin signaling to prevent behavioral alterations in PTSD mice

    Authors:
    Alessandra Monaco, Livia Cosentino, Chiara Urbinati, Alessia De Matteis, Bianca De Filippis

    Organisations:
    Istituto Superiore di Sanità, Italy

    Presenting author: Alessandra Monaco

    Exposition date: Friday, September 15, 2023

    Exposition position: 043/1

    Post-Traumatic Stress Disorder (PTSD) is a mental disorder that may arise after a traumatic event and it is characterized by invalidating symptoms, such as trauma-related memory impairments and persistent avoidance of trauma-associated stimuli. Stress susceptibility plays a key role in the disorder etiology and alterations in the methyl-CpG binding protein 2 (MECP2), an epigenetic regulator, are associated with an increased susceptibility to develop PTSD-like symptomatology. MECP2 plays an important role in regulating several components of the HPA axis and previous studies have demonstrated that the aberrant functionality of this protein is accompanied by an overexpression of vasopressin (AVP) which has been suggested to contribute to the onset of PTSD symptoms. In the present study, we hypothesized that modulation of AVP signaling in the early aftermath of a traumatic event may prevent the onset of PTSD-like symptomatology. To this aim, MeCP2-308 male mice (mouse model of PTSD vulnerability) and wild-type controls received two footshocks (0.8mA, 3s) to mimic the traumatic event, or no footshocks as a control, and 30 min prior to trauma exposure mice were injected with SSR149415, an antagonist of the receptor type 1b of AVP. Mice were tested for cued and contextual fear memory retrieval (1st and 8th days after the shock delivery) and persistent avoidance of trauma-related stimuli (30th day). MeCP2-308 traumatized mice showed PTSD-like fear responses when re-exposed to a non-predicting cue, but SSR149415 did not prevent the onset of this abnormal freezing response. Interestingly, MeCP2-308 traumatized mice exhibited a persistent avoidance in the active avoidance task and SSR149415 prevented this abnormal behavior, thus normalizing the PTSD-like phenotype. Present data demonstrate the potential involvement of AVP signaling in the persistence of PTSD-like avoidance behavior and provide the basis for future studies aimed at dissecting the involvement of AVP signaling in MeCP2-induced vulnerability to PTSD.

  • The body knows what the mind does not: The physiology of Self-Deception

    Authors:
    Giulia Romano Cappi, Ilaria Mirlisenna, Alessandro Mazza, Olga Dal Monte

    Organisations:
    Turin University, Italy

    Presenting author: Giulia Romano Cappi

    Exposition date: Friday, September 15, 2023

    Exposition position: 037/1

    Humans are social creatures and feeling part of a group is crucial for successful survival and wellbeing. Being accepted by a group often implies showing our best qualities, and sometimes we adopt deceptive behaviors to enhance our social-self. Compared to Truth-telling, Deception provokes physical reactions such as blushing and sweating associated with increased physiological activation, and these physiological changes might lead to lie-detection by others. However, sometimes individuals can unconsciously lie to themselves about their qualities and get genuinely convinced of their deceptive ideas, a phenomenon known as Self-Deception. But what are the physiological responses associated to Self-Deception?
    To answer this question, we designed an ecologically valid paradigm capable of capturing three different experimental conditions: Deception, Self-Deception, and Truth. We first invited participants to solve several logical-mathematical tasks; then we told them to convince a confederate that their performance was among the best of all the other subjects, independently of the feedback they received. Experimental conditions were implemented to observe Deception, Self-Deception, or Truth in random order across trials. For the duration of the whole experiment, we measured skin conductance response and facial muscle activation. As expected, we found that Deception was associated to a higher physiological activation than Truth-telling, replicating previous results from the existing literature. Crucially, we also found that Self-Deception displayed a lower autonomic activation than Deception and a higher activation compared to Truth-telling.
    Our results therefore suggest that Self-Deception provokes physiological changes that lie in-between Truth-telling and intentional Deception. This is the first study to provide physiological evidence of a natural and common mechanism in humans such as Self-Deception.

  • The Impact of Cognitive Effort on Prosocial Choices: an EEG study

    Authors:
    Alessandro Valvo, Alessandro Mazza, Ilaria Mirlisenna, Marta Campagnola, Olga Dal Monte

    Organisations:
    University of Turin, Italy

    Presenting author: Alessandro Valvo

    Exposition date: Friday, September 15, 2023

    Exposition position: 035/1

    Prosocial behaviour is a fascinating and complex social construct, and the degree of prosocial choices can be influenced by several factors such as social distance between the individuals as well as empathic abilities. Specifically, real life prosocial actions always come with a certain amount of effort required to the social decision maker, but this aspect has been often neglected in most studies. Previous literature exploring this variable usually adopted money both as a reward and as a cost for being prosocial, thus very little is known about other types of effort.
    To address this gap, in our study we focused on exploring the impact of cognitive effort on prosocial choices. We developed a new social decision-making paradigm, where participants had to decide whether to make or not a cognitive effort to reward themselves or another person. The reward at stake was a discount on the time spent on the experimental activity. Together with behavioural data, we also collected EEG data to explore with high temporal resolution the neural dynamics of three phases characterizing social decision making: Options evaluation, Choice, and Feedback processing.
    Our results showed that participants significantly reduced their prosocial choices when a cognitive effort was required to reward another individual with respect to when no effort was required. At neural level, we found different processing patterns in the temporal dynamics of neural activity in each decision-making phase. Moreover, we observed frequency-specific differences in connectivity between cortical hubs when a prosocial choice was computed with or without effort. Our findings suggest that prosocial behaviours are processed across multiple brain regions and that their synchrony patterns change as a function of the effort required.

  • The interaction between pain and the perception of effort during a motor task.

    Authors:
    Ilaria Monti (1,2), Thomas Mangin (1,2), Mélysiane Marcotte (1), Stéphane Baudry (3), Pierre Rainville (1,2), Benjamin Pageaux (1,2)

    Organisations:
    1: University of Montreal, Canada
    2: Centre de Recherche de L'Institut Universitaire de Gériatrie de Montréal (CRIUGM), Montréal, Québec, Canada
    3: Laboratoire de Biologie Appliquée (LAB Neuro), Faculté des Sciences de la Motricité (FSM), Université Libre de Bruxelles, Bruxelles, Belgique

    Presenting author: Ilaria Monti

    Exposition date: Friday, September 15, 2023

    Exposition position: 028/1

    Context. Perception of effort (PE) is crucial in the regulation of human behavior. Its intensity increases with the cognitive and physical demands of a task. Pain, through its inhibitory effect on motor control and its attentional cost, increases the demand of a task. It is therefore possible that in the presence of pain, the PE to perform a motor task increases. Objective. To assess the interaction between pain and PE during the performance of an isometric force maintenance task. Methods. Twenty healthy participants (50% female) (18-35 years) participated in the study. A thermal calibration was used to identify temperatures eliciting for each participant a perception of warm (70/100, visual analog scales-VAS warm), mild pain (30/100, VAS pain) and intense pain (70/100, VAS pain). Successively, participants executed the motor task in the presence/absence of pain. The motor task consisted of producing isometric contractions at 5% or 30% of maximal force, by squeezing a dynamometer, to match a target line on a screen; or holding the dynamometer without producing a contraction (control). Effort and pain/warm perceptions were recorded after each contraction with a VAS 0-100. Performance was evaluated as force stability. Data were analyzed with repeated measures ANOVAs. Results. No effect of temperatures was found on the produced force (p=.858), which remained stable during all task duration. The intense pain condition increased the perceived effort to perform the motor task compared to the warm condition (p=.016). The 30% of maximal force condition reduced pain perception compared to the control condition (p=.037). Conclusion. Pain has no impact on motor performance. However, on one side performing the motor task in the presence of pain is perceived as more effortful. On the other side, engaging in the motor task has an analgesic effect and pain is perceived as less intense.

  • To Cooperate or to compete? Benefits and costs of performing in a social setting.

    Authors:
    Lucia De Francesco, Alessandro Mazza, Matilde Sorrenti, Virginia Murino, Edoardo Battegazzorre, Francesco Strada, Andrea Bottino, Olga Dal Monte

    Organisations:
    Università degli studi di Torino, Italy

    Presenting author: Lucia De Francesco

    Exposition date: Friday, September 15, 2023

    Exposition position: 034/1

    Social cognition developed thanks to complex exchanges between group-direct and self-direct tendencies: in this sense, cooperation and competition shape everyday human behavior, as they impact individuals’ chances of success in a variety of activities. Further, within competing and cooperating contexts, knowledge of another individual’s qualities strongly affects our predictions about our chances of success, thus influencing our motivations and behaviors. In this study, we adopted a virtual Stroop test to assess in a highly controlled manner how competing and cooperating influenced participants’ performance. We also manipulated the levels of task-related skillfulness of the other virtual participant, making them higher or lower than the experimental subject. During the whole session, we monitored changes in autonomic activation by measuring heart rate and skin conductance levels. Finally, these implicit measures were compared with subjective ratings of the perceived stress level expressed by the experimental subject across different contexts. Consistent with previous studies, we found that participants performed better in a social context than in a solo condition. Also, as expected, we found that participants’ performance significantly improved when competing with a better performer. This result confirms previous studies pointing out the relevance of the other’s skillfulness in defining the behavioral effects of competition. However, we also found a comparable performance boost when cooperating with a worse performer and, crucially, the associated autonomic activity and self-reported stress levels were significantly lower compared to competing with a better performer. These results suggest that cooperating promotes a similar increase in performance as competing, but it is not associated with such a high psycho-physiological cost as competing, and thus, it might represent a preferable way of boosting individual performance in interpersonal contexts.

  • What the dynamic between hemispheres can teach us about attention – a TMS-EEG study

    Authors:
    Selene Schintu (1,2,3), Amelia G. Stapleton (2), Sarah Shomstein (3), Eric M. Wassermann (2)

    Organisations:
    1: CIMeC, University of Trento, Rovereto (TN), Italy
    2: National Institute of Neurological Disorders and Stroke, NIH, Bethesda (MD), USA
    3: Department of Psychology, George Washington University, Washington DC (DC), USA

    Presenting author: Selene Schintu

    Exposition date: Friday, September 15, 2023

    Exposition position: 045/1

    Unilateral stroke can disrupt the equilibrium between hemispheres, which is necessary for efficient attentional deployment. Given the variability characterizing stroke patients’ cognitive profiles and the classical neuropsychological tools’ lack of sensitivity, we used a combination of behavioral and imaging techniques to identify biomarkers of interhemispheric equilibrium that predict attentional performance. Since the posterior parietal cortex (PPC) is a crucial node of the dorsal attention network and the PPC-to-PPC communication supports the interhemispheric equilibrium, we investigated whether the PPC-to-PPC communication predicts attentional performance. Given that the PPC is also a generator of alpha oscillations and alpha power correlated with attentional deployment, we tested whether interhemispheric asymmetries in alpha predict attentional performance.

    The within-subjects experiment consisted of two sessions. In the first session, healthy participants underwent resting-state fMRI that allowed us to identify individualized TMS targets. In the second session, participants underwent both Rest- and TMS-EEG recordings, 100 TMS pulses at 65% of the maximum stimulator output over the right and 100 over the left PPC individualized target. EEG recordings were preceded by a set of behavioral tasks quantifying different components of attention such as visuospatial attention, attentional orienting and reorienting, and temporal selective attention.

    Preliminary results showed that the interhemispheric signal propagation (IPS) from right to left PPC predicted participants’ performance in task quantifying spatial and temporal attention, while both the PPC and FEF alpha asymmetry index predicted participants’ search efficiency.

    This is the first fMRI-guided TMS-EEG study to investigate ISP and alpha asymmetry index as possible biomarkers for attentional performance.

Computational and theoretical neuroscience

  • Computational whole-brain model of progression to dementia accounts for event-related potentials non-linear dynamics during attention task

    Authors:
    Lorenzo Gaetano Amato (1), Alberto Arturo Vergani (1), Valentina Bessi (2), Antonello Grippo (3), Alberto Mazzoni (1)

    Organisations:
    1: Istituto di Biorobotica, Scuola Superiore Sant'Anna, Pisa
    2: Careggi University Hospital, Firenze
    3: IRCCS Fondazione Don Carlo Gnocchi, Firenze

    Presenting author: Lorenzo Gaetano Amato

    Exposition date: Saturday, September 16, 2023

    Exposition position: 036/2

    1 / 1 Attention and memory tasks and associated event-related potentials (ERPs) are gaining popularity as early biomarkers for dementia. The 3-choice vigilance task (3CVT), a test designed to estimate the level of sustained attention seems particularly promising in this respect. However, there is a lack of comprehension on the structural and neural correlates of the behavioral anomalies in these tasks observed in demented and pre-demented subjects. Here we propose a computational whole-brain model of structural (connectivity) and synaptic alterations due to prodromic Alzheimer disease, capable of reproducing the EEG dynamics observed in pre-demented subjects during the execution of a 3CVT task. We recorded EEG activity of 42 Mild Cognitive Impairment (MCI) subjects, 84 Subjective Cognitive Decline (SCD) subjects, and 18 healthy controls (HC) while they performed the 3CVT task. ERP analysis displayed significant differences in both slope and amplitude of the N1 component in the decoding phase of the task, and in the amplitude of the P2 component of the decision phase. Interestingly, all these features were not progressively decreasing with dementia severity, but displayed a larger decrease between the HC and the SCD group than between the HC and MCI group. Of note, this non-linearity was primarily due to a subset of subjects that displayed low levels of functional connectivity (FC) between electrodes. Our computational model reproduced the observed non-linearities and associated them with the degradation of white matter fibers during disease progression. Coherently with experimental results, this degradation led to a second-order phase transition between a low-FC and a high-FC zone: in the low-FC zone, ERP features did not decrease linearly with dementia severity, while they did in the high FC zone. Overall, our model of dementia progression is not only able to reproduce the EEG dynamics observed in SCD and MCI, but also to shed light on the underlying mechanisms.

  • Gamma oscillations in mouse primary visual cortex as a biomarker of pathological conditions

    Authors:
    Nicolo Meneghetti (1,3), Eleonora Vannini (2,4), Laura Restani (2), Matteo Caleo (2,5), Alberto Mazzoni (1,3)

    Organisations:
    1: The Biorobotics Institute, Sant'Anna School of Advanced Studies, 56025, Pontedera, Italy
    2: Neuroscience Institute, National Research Council (CNR), 56124, Pisa, Italy,
    3: Department of Excellence for Robotics and AI, Sant'Anna School of Advanced Studies, 56025, Pontedera, Italy
    4: Fondazione Umberto Veronesi, 20122, Milan, Italy
    5: Department of Biomedical Sciences, University of Padua, via G. Colombo 3, 35121 Padua, Italy

    Presenting author: Nicolo Meneghetti

    Exposition date: Friday, September 15, 2023

    Exposition position: 049/1

    Neural gamma oscillations are ubiquitous: they are associated with a broad range of functions in multiple cortical areas and across many animal species. Experimental and computational works have established how gamma rhythms primarily reflect timescales of the balanced and coordinated interaction between local excitation and inhibition. Coherently, gamma rhythms are strongly influenced by alterations impacting the synaptic equilibria throughout cortical circuitry. We argue therefore that these oscillations might serve as an optimal biomarker for probing the mechanism underpinning the mechanisms of cortical (dys)functions.

    In line with this reasoning, we present two works of our group that showcase the adaptability of gamma oscillations in mice primary visual cortex (V1) under two distinct pathological conditions.

    We investigated1 the interplay between etiologic synaptic alterations and network-level computations in a genetic mouse model of familial-hemiplegic-type1-migraine (FHM1). Computational modeling was capable of dissecting the impact of every synaptic alteration observed in vitro into the neuronal dynamics we recorded in awake mice. Specifically, we found that (i) the asymmetry of thalamocortical afferents, which exerted a stronger impact on interneurons, led to reduced cortical activity as observed experimentally through decreased multi-unit-activity; (ii) the enhanced intracortical glutamatergic signaling shifted gamma oscillations induced by high visual contrasts towards higher frequencies.

    We also examined2 the interlaminar alterations in visual sensory processing of a mouse model of focal epilepsy. We found a layer-unspecific increase in excitatory firing rate, which was conversely localized in infragranular layers for interneurons. Additionally, we observed a significant spreading of local field potentials gamma oscillations across cortical layers induced by visual stimuli.

    Altogether these examples show the importance of experimental and computational investigations of gamma oscillations, which could potentially serve as valuable benchmarks in clinics to probe the alterations of information processing in multiple pathological conditions.

    1.Meneghetti, N. et al.(2022) doi:10.1186/s10194-022-01495-9

    2.Panarese, A. et al.(2022) doi:10.1093/cercor/bhac335.

  • Network properties underlying remission mechanisms at early stages of psychosis (EP): bridging hierarchical levels with a whole-brain computational approach.

    Authors:
    Ludovica Mana (1), Manel Vila-Vidal (1,2), Ane López (1), Yasser Aleman (3), Philipp S. Baumann (3), Paul Klauser (3), Raoul Jenni (3), Kim Q. Do (3), Philippe Conus (3), Patric Hagmann (3), Gustavo Deco (1,4)

    Organisations:
    1: Universitat Pompeu Fabra, Italy
    2: Computational Biology and Complex Systems Group, Department of Physics, Universitat Politècnica de Catalunya, Barcelona, Spain
    3: Department of Radiology, Lausanne University Hospital (CHUV) and University of Lausanne (UNIL), Lausanne, Switzerland
    4: Institucio Catalana de la Recerca i Estudis Avancats (ICREA), Passeig Lluis Companys 23, Barcelona 08010, Spain.

    Presenting author: Ludovica Mana

    Exposition date: Friday, September 15, 2023

    Exposition position: 050/1

    Psychotic disorders are characterised by great heterogeneity of disease progression, and the neural mechanisms underlying remission and relapse after a first psychotic episode remain unclear.

    Here, we included resting-state fMRI and DTI data from 128 healthy controls and 88 patients with early psychosis, stratified based on their ability to recover after the first episode. We focused on differences between stage-III remitting-relapsing (EP3-R) and stage-III non-remitting (EP3-NR) patients. We first of all searched for stage-specific resting-state functional connectivity (FC) alterations. We then combined functional and structural empirical information of each condition into diverse generative whole-brain models to explore the interplay between the global and local dynamics underlying pathological mechanisms.

    Opposite alterations in FC could be found in different subgroups of patients as compared to controls. In EP3-NR we observed a reduction of FC, aligned with the reduced structural connectivity found in previous studies, while EP3-R shows increased FC, potentially indicating a relevant compensatory mechanism. Using a theoretical model to fit the empirical data, we showed that, in the healthy condition, a subset of areas is characterized by increased stability to filter out irrelevant stimuli and prevent over synchronisation of the network. Additionally, we proved that node stability strongly correlates with combined functional-structural properties across nodes. We then showed how such crucial property is lost in EP3 patients. Notably, this alteration is more relevant in the remitting than in non-remitting patients, and correlates with changes in empirical functional strength. Therefore, it probably indicates a compensatory response to the reduced structural conductivity (global coupling) highlighted by the model in both EP3 conditions as compared to controls. Finally, we used a mean-field model approach to prove the role of excitation/inhibition modulation underlying this compensatory mechanism.

    These results allow us to progress in understanding some of the key mechanisms in emergence and progression of psychosis.

  • Neural circuitry in the brain of Octopus vulgaris: contributing to JZ Young legacy

    Authors:
    Federica Pizzulli (1,3), Marisa Barjami (2,3), Graziano Fiorito (1,2), Giovanna Ponte (1,2)

    Organisations:
    1: Stazione Zoologica Anton Dohrn, Italy
    2: Association for Cephalopod Research (CephRes)-ETS, Italy
    3: contributed equally to this work

    Presenting author: Federica Pizzulli

    Exposition date: Friday, September 15, 2023

    Exposition position: 051/1

    J.Z. Young, or simply JZ, is a well know zoologist “who paved the way for modern studies in neurobiology”, as recognized in many circumstances. He contributed to the most authoritative description of the anatomy and organization of the nervous system of the cephalopod mollusc, Octopus vulgaris, and other cephalopods. His seminal discoveries (e.g., the giant fiber) provided great influence on the work of many others and provided a detailed analysis of the octopus learning capabilities and of the neural basis of its behavioral plasticity. The description of octopus nervous system allowed to the identification of multiple matrices, concurring for visual and chemo-tactile sensory motor processing and memory storage.

    Here, we analyzed JZ The Anatomy of the Nervous System of Octopus vulgaris (1971) with particular attention to the complex network of interconnected and interacting neuronal populations (i.e. lobes and areas in the brain) and computed O. vulgaris brain network for the first time. In this presentation we will:

    1. account our methodological approach and data collection, based on JZ data, regarding tracts, afferent and efferent fibers, etc. including octopus’ brain intra- and inter-lobes neural pathways;

    2. examine neural connectivity by adopting neural network approach, and estimated integration, segregation, centrality and modularity, neighborhood overlap, density of connections within and between lobes and masses, clustering and community structures in O. vulgaris brain;

    3. compare our results with recent connectivity maps analyzed for other octopus’ species.

    Our approach allowed a comprehensive charting of the topological organization of the octopus connectome, and to attempt a link between the structural attributes, neural network architecture and its function. Collectively, these results pave the way for a novel approach in the analysis of brain network in cephalopods, and provide a foundation for future anatomical, functional, and behavioral studies in these organisms.

Development and stem cells

  • A pilot in vitro model of Adult Neural Stem cells to study energy metabolism contribution in Alzheimer disease.

    Authors:
    Giorgia Cerqueni (1), Silvia Piccirillo (1), Valentina Terenzi (1), Alessandra Preziuso (1), Tiziano Serfilippi (1), Salvatore Amoroso (1), Vincenzo Lariccia (1), Monia Orciani (2), Simona Magi (1)

    Organisations:
    1: Department of Biomedical Sciences and Public Health, Università Politecnica delle Marche, Ancona, IT.
    2: Department of Clinical and Molecular Sciences, Università Politecnica delle Marche, Ancona, IT

    Presenting author: Simona Magi

    Exposition date: Saturday, September 16, 2023

    Exposition position: 037/2

    Through neurogenesis, adult neural progenitor cells (NPCs) can contribute to the repair processes providing new-born neurons that enhance spatial information processing and memory formation by interacting with brain network1.

    Our study aims to explore the potential differences of neuronal network morphology between NPCs from healthy and model mice of Alzheimer's disease. Furthermore, the study wants to provide a tool to investigate possible energy metabolism modifications, considering that bioenergetic failure can influence the regenerative potential of adult AD brain.  

    NPCs were isolated from adult healthy (H) and triple transgenic (AD) mice. NPCs were characterised by stem-related markers’ expression. Neuron (N) and astrocyte (A) differentiation protocols were set up. Morphological analyses were performed on H, and AD N-NPCs and the expressions of glutamate transporters and sodium/calcium exchangers (NCXs) were investigated.

    NPCs isolated from H and AD mice were strongly positive for Nestin and Sox2. NeuN and GFAP positive cells were detected in N-NPCs and A-NPCs, respectively. AD N-NPCs revealed a smaller number of neurite end-points respect H cells. Both populations expressed all the isoforms of glutamate transporters and, preliminary data revealed a significant increase of EAAC1 in AD NPCs. Interestingly, we observed that the increase in EAAC1 expression was accompanied by a concomitant increase of NCX1 in AD NPCs.

    Preliminary data suggest that the pathological features of adult neurons from AD brain can be traced back to “stem level” and continue during differentiation. The different morphological organization of neurite network observed in AD N-NPCs could be involved in the alteration of the impulse transmission and plasticity. Further studies will be necessary to clarify the consequence of an altered expression and/or activity of glutamate transporters as well the NCX1 involvement in a possible preferential use of glutamate in NPCs energy metabolism2.

    1.DOI:10.1523/JNEUROSCI.2195-06.2006

    2.DOI:10.1007/s00018-018-3002-x

  • Activation of canonical WNT pathway as a possible therapeutic approach in in vitro and in vivo models of Cornelia de Lange Syndrome

    Authors:
    Paolo Grazioli (1), Luciano Conti (2), Simona Totaro (1), Chiara Parodi (1), Antonella Lettieri (1), Elisabetta Di Fede (1), Silvia Castiglioni (1), Esi Taci (1), Thomas Vaccari (3), Cristina Gervasini (1,4), Valentina Massa (1,4)

    Organisations:
    1: Dipartimento di Scienze della Salute, Università degli Studi di Milano
    2: Dipartimento CIBIO, Università di Trento
    3: Dipartimento di Bioscienze, Università degli Studi di Milano
    4: CRC Aldo Ravelli for Neurotechnology and Experimental Brain Therapeutics, Dipartimento di Scienze della Salute, Università degli Studi di Milano

    Presenting author: Paolo Grazioli

    Exposition date: Friday, September 15, 2023

    Exposition position: 058/1

    Cornelia de Lange Syndrome (CdLS) is a rare genetic disorder affecting almost any organ including the central nervous system, inducing a variable neurodevelopmental delay. CdLS is caused by mutations in genes coding for the cohesin complex (proteins involved in the cohesion control of sister chromatids). We have previously shown that many abnormalities result from deregulation of developmental pathways, including the canonical WNT pathway, downregulated at specific times in specific tissues.

    In order to revert the phenotype, we have explored possible ameliorative effects by chemically activating the canonical WNT pathway using lithium, in a model of human induced pluripotent stem cells (hiPSCs) and in a Drosophila melanogaster model of CdLS.

    For the in vitro studies, commercial hiPSCs have been differentiated in neural precursor cells (hNPCs) and, to mimic cohesins haploinsufficiency, treated with different concentrations of the drug PCI-34051, a specific inhibitor of HDAC8, a protein known to be implicated in CdLS pathogenesis. For the in vivo studies, we exploited adult flies carrying a loss-of-function allele in a cohesins gene (Nipped-B) and reared on food supplemented with lithium as activator of canonical WNT pathway.

    Upon lithium treatment, we observed an improved differentiation capability along the neuronal lineage compared to the cells exposed to the inhibitor only. Flies’ offspring showed a significant rescue in mushroom bodies morphology, a central nervous system structure important for the olfactory learning and memory.

    These results support the hypothesis that disruption of the canonical WNT pathway, caused by cohesins mutations, plays a central role in CdLS etiopathogenesis. This concept is corroborated by the rescue induced by lithium administration in experimental models, paving the way for new possible and urgently needed therapeutic strategies.

  • Brain organoids for the study of neural maturation in physiological and pathological conditions

    Authors:
    Francesca Ciarpella (1), Giulia Pedrotti (1), Benedetta Lucidi (1), Chiara Santanatoglia (1), Eros Rossi (1), Raluca Georgiana Zamfir (1), Alessandra Campanelli (1), Elisa Ren (2), Marzia Di Chio (1), Sissi Dolci (1), Annika Ahtiainen (3), Jari Hyttinen (3), Andrea Borioli (1), Rita Bardoni (2), Davide Caron (4), Gemma Palazzolo (4), Gabriella Panuccio (4), Ludovica Sagripanti (5), Francesco Bifari (5), Giovanni Malerba (6), Giorgio Malpeli (7), Giulia Curia (2), Emanuela Bottani (1), Ilaria Decimo (1)

    Organisations:
    1: University of Verona, Dept. Diagnostics and Public Health, Pharmacology Section, Verona, Italy
    2: University of Modena and Reggio Emilia, Dept. of Biomedical, Metabolic and Neural Sciences, Modena, Italy
    3: BioMediTech, Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland
    4: Department of Neuroscience and Brain Technologies (NBT), Istituto Italiano di Tecnologia (IIT), Genova, Italy
    5: University of Milan, Dept. of Medical Biotechnology and Translational Medicine, Milan, Italy
    6: University of Verona, Dept. of Neurosciences, Biomedicine and Movement Sciences, Verona, Italy
    7: University of Verona, Dept of Surgery, Dentistry, Pediatrics and Gynecology, Verona, Italy

    Presenting author: Francesca Ciarpella

    Exposition date: Friday, September 15, 2023

    Exposition position: 052/1

    Neural Stem Cells (NSCs) are early and uncommitted cells of the nervous system with the capacity to generate different specialized cells of the central nervous system, i.e. neurons, astrocytes, and oligodendrocytes. NSCs represent the first building block for the generation of brain organoids, in vitro three-dimensional (3D) self-organized structures able to raise and recapitulate the in vivo brain complexity. In this work, we obtained in vitro culture of mature neuronal tissue, owing specific brain-regional signature (e.g. hippocampal phenotype), starting with multipotent NSCs isolated from mouse embryonic subgranular zone. Within the 3D structure, NSCs self-organize and differentiate mimicking the in vivo brain development. NSCs progressively differentiate trough defined early, intermediate, and mature stages, delineated by stem (SOX2, Vimentin), neural progenitors (DCX) and neuronal (Tubb3, MAP2) cells. As for in vivo, the external mature neuronal layer own functional properties (measured by electrophysiology, transsynaptic tracing and calcium imaging techniques) and it is able to build active synaptic connections in multiple directions. Brain organoids can also model neurodevelopmental disorders. We assess the impairment of brain organoid maturation in Allan-Herndon-Dudley syndrome (AHDS). Under pathological hypothyroidic condition, we observed a delayed brain organoid maturation and increased number of immature DCX+ immature neural cells. AHDS disease brain organoids were still able to organize into 3D structure though they showed a sustained proliferative and stemness profile over time and reactive gliosis. Overall, we highlighted the NSCs property to mature into structurally organized system resembling the in vivo brain development which can be exploited to study neural maturation in physiological and pathological conditions.

  • Cohesin and PARP inhibitors: finding new treatments for pediatric medulloblastoma

    Authors:
    Antonella Lettieri (1), Simona Totaro (2), Silvia Castiglioni (1), Silvia Ancona (1), Paolo Grazioli (1), Elisabetta Di Fede (1), Grazia Fazio (3), Stefano Rebellato (3), Cristina Gervasini (1), Thomas Vaccari (2), Valentina Massa (1)

    Organisations:
    1: Department of Health Sciences, Università degli Studi di Milano, Milano, Italy
    2: Department of Biosciences, Università degli Studi di Milano, Milano, Italy
    3: Centro Ricerca Tettamanti, Università degli Studi di Milano-Bicocca, Monza, Italy

    Presenting author: Antonella Lettieri

    Exposition date: Saturday, September 16, 2023

    Exposition position: 039/2

    Among pediatric brain cancers, medulloblastoma (MB) is the most frequent and one of the most aggressive tumors with a 5-year survival rate of 55-80%. MB is of embryonic origin, and it is likely caused by the impaired differentiation of hindbrain neural progenitors. Four main groups of MB have been originally described. Now 12 groups are recognized on the bases of transcriptomic profile; however, the genetics and predisposing factors underlying MB are still largely unknown. Because of the heterogeneity of both genetics and prognosis of pediatric MB, the urgency of specific and targeted therapies is much needed. Overall, the involvement of cohesin complex, a highly conserved multimeric structure involved in many physiological and pathological processes, including first stages of cerebellar development, and their role in gene expression regulation during embryogenesis, makes them excellent candidates for MB onset investigation. Among cohesins, Stromalin 2 (STAG2) is the most frequently mutated subunit in cancers, and it is supposed to promote tumorigenesis by altering cohesin complex functions as genome organization and transcriptional regulation. We preliminary found predicted pathogenetic variants in cohesin-encoding genes in 18% of MB patients by interrogating available databases. In addition, the pilot in vivo fly model of MB obtained by silencing the ortholog of STAG2, showed increased proliferation and defective differentiation of neuroblasts, consistent with malignant phenotype, suggesting that cohesins might concur to MB onset. Last, PARP inhibitors (PARPi), a class of drugs recently implemented in trials for glioblastoma carrying cohesin variants and experimented in vitro in NMYC expressing neuroblastoma, were tested in an in vitro model of MB generated from a hiPSC STAG2-silenced stable line. Overall, by demonstrating the interplay between STAG2 and NMYC, it will be possible to deepen the knowledge of MB pathogenesis and to set the ground for the potential therapeutic use of PARPi.

  • Description of Two Groups of Dopaminergic Cells Isolated from the Mouse Olfactory Bulb

    Authors:
    Angela Pignatelli (1), Fabio Casciano (1), Nicoletta Bianchi (1), Vittorio Vellani (2), Simona Capsoni (1)

    Organisations:
    1: University of Ferrara, Italy
    2: University of Modena and Reggio Emilia

    Presenting author: Angela Pignatelli

    Exposition date: Friday, September 15, 2023

    Exposition position: 065/1

    In the mammalian brain, the olfactory bulb (OB) is one of the two regions that undergo continuous neuronal replacement during adulthood. Among the cells added to the bulbar circuitry in adult neurogenesis a significant portion are dopaminergic (DA) interneurons located in the glomerular layer (GL). By using transgenic mice expressing eGFP under the control of the tyrosine hydroxylase (TH) promoter, we were able to identify TH-eGFP+ cells that were found not only in the GL layer but also in the mitral and external plexiform layer. We also observed a different fluorescence intensity of the TH-eGFP+ cells in the two layers. We supposed that these cells could be adult-generated neurons committed to becoming DA but not yet fully differentiated. The different levels of fluorescence could possibly reflect varying degrees of maturation of TH-eGFP+ cells. Our hypothesis comes from the electrophysiological analysis of DA neurons located in the glomerular layer (GL) that display full degree of neuronal maturation, whereas eGFP+ cells in the mitral layer (ML) exhibit characteristics of immature cells. To investigate whether this difference in maturation could be confirmed at the gene expression level, we used a fluorescence-activated cell sorting technique (FACS) on enzymatically dissociated cells from the OB. We split the cells into two groups based on their fluorescence level, possibly corresponding to immature ML cells and fully mature DA neurons from the GL. Semiquantitative real-time PCR was performed to detect the expression level of genes associated with the degree of maturation of DA neurons. We demonstrated that cells expressing low eGFP fluorescence are indeed immature neurons. Our method can be not only utilized to explore the differences between these two groups of DA neurons but also to engineer new neurons for therapeutic approaches such as transplantation into specific areas of the brain affected by Parkinson's disease.

  • Dissecting schizophrenia through patient iPSC-derived thalamic reticular nucleus neurons

    Authors:
    Marilyn Marlene Angulo Salavarria (1), Claudia Dell’Amico (1), Elena Rita Vecchi (2,7), Michela Giacich (1), Francesco Luciano Donati (2), Anna Castelnovo (3), Marta Re (4), Eleonora Maggioni (4,5), Armando D’Agostino (2,6), Luciano Conti (7), Marco Onorati (1)

    Organisations:
    1: Dep. of Biology, Unit of Cell and Develop. Biology, University of Pisa, Italy
    2: Dep. of Health Sciences, University of Milan, Milan, Italy
    3: Sleep Medicine Unit, Civic Hospital of Lugano, Switzerland
    4: Dep. of Neurosciences and Mental Health, IRCCS Ca’ Granda Foundation, Milan, Italy
    5: Dep. of Electronics, Information and Bioengineering, Politecnico di Milano, Milan, Italy
    6: Dep. of Mental Health and Addiction, ASST Santi Paolo e Carlo, Milan, Italy
    7: Dep. of Cellular, Computational, and Integrative Biology, University of Trento, Trento, Italy

    Presenting author: Marilyn Marlene Angulo Salavarria

    Exposition date: Friday, September 15, 2023

    Exposition position: 057/1

    The human brain is a complex and sophisticated organ containing several sub-types of neural populations wired together in myriad circuits. Alteration in specific network generation could lead to psychiatric disorders such as schizophrenia (SCZ). SCZ is a severe neurological disorder with a worldwide prevalence of 1% with a prominent genetic basis. Current studies have highlighted that SCZ-related genes impact neuronal differentiation and maturation which, ultimately, result in neural circuits impairment. In this regard, several studies also identified alterations in sleep spindles – generated by the interplay of the thalamic reticular nucleus (TRN) with the dorsal thalamus and then relayed to the cortex – as potential inheritable biomarkers of SCZ. Indeed, in the last decade, dysregulation of this thalamocortical connectivity has been considered at the center of the most important pathogenetic hypotheses about SCZ. Here, we generated hiPSC lines derived from SCZ patients with sleep endophenotype. We developed a protocol to mimic TRN ontogenesis and generate an in vitro model to study SCZ-related endophenotype. In particular, we optimized a diencephalic differentiation protocol by using Insulin and CHIR99021 to promote a mild caudalizing effect necessary to obtain the diencephalic fate. We also performed gene expression analysis to confirm the induced cellular identity. We compared this expression pattern to that of human RNA tissue samples from the Human Development Biology Resource (HDBR) collection – from 7 to 20 postconceptional weeks; this allowed us to generate a reference dataset and validate the expression profile of TRN neurons generated from hiPSCs.

    The application of this optimized protocol will enable us to recapitulate the main stages of human diencephalic development and generate TRN neurons. This approach will offer the possibility to investigate the sleep endophenotype closely associated with the SCZ disorder in a patient-specific manner.

  • Dysregulation of FLVCR1-dependent mitochondrial calcium handling in neural stem cells causes congenital hydrocephalus

    Authors:
    Francesca Bertino (1), Dibyanti Mukherjee (2), Massimo Bonora (3), Christoph Bagowski (4), Jeannette Nardelli (5), Nicolas Santander Grez (6), Andreas Hentschel (7), Georgia Panagiotakos (8), Pierre Gressens (5), Chiara Riganti (9), Paolo P Pinton (3), Andreas Roos (10), Thomas Arnold (2), Emanuela Tolosano (1), Deborah Chiabrando (1)

    Organisations:
    1: Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Torino, Turin, Italy
    2: Department of Pediatrics, University of California San Francisco, San Francisco, United States
    3: Department of Medical Sciences, Section of Experimental Medicine, Laboratory for Technologies of Advanced Therapies, University of Ferrara, Ferrara, Italy
    4: Prenatal Medicine Munich, Department of Molecular Genetics, Munich, Germany
    5: Université de Paris, NeuroDiderot, Inserm, 75019 Paris, France
    6: Instituto de Ciencias de la Salud, Universidad de O'Higgins, Rancagua, Chile
    7: Leibniz Institute of Analytical Sciences, ISAS, Dortmund, Germany
    8: Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143, USA.
    9: Department of Oncology, University of Torino, Italy
    10: Department of Pediatric Neurology, Developmental Neurology, and Social Pediatrics, Center for Neuromuscular Disorders in Children and Adolescents, University of Duisburg-Essen, Essen, Germany

    Presenting author: Francesca Bertino

    Exposition date: Friday, September 15, 2023

    Exposition position: 060/1

    Congenital hydrocephalus (CH), occurring in approximately 1/1000 live births, represents an important clinical challenge due to the limited knowledge of underlying molecular mechanisms. The discovery of novel CH-genes is thus essential to shed light on the intricate processes responsible for ventricular dilatation in CH. Here we identify FLVCR1 as a novel gene responsible for a severe form of CH in humans and mice. Mechanistically, our data reveal that the heme exporter FLVCR1a interacts with IP3R3-VDAC, a complex located on mitochondrial-associated membranes (MAMs) that controls mitochondrial calcium handling. Loss of Flvcr1 in mouse neural progenitor cells (NPCs) affects mitochondrial calcium levels and energy metabolism, leading to defective cortical neurogenesis and brain ventricle enlargement. These data point to defective NPC mitochondrial metabolism as one of the pathogenetic mechanisms driving CH.

  • Epigenetic features in hESC-derived neuronal cells carrying the Huntington’s Disease gene

    Authors:
    Michela Villa (1,2), Paola Conforti (1,2), Maria Rosaria Nucera (1,2), Silvia Brocchetti (1,2,5), Giulia Birolini (1,2), Laura Antonelli (4), Francesco Gregoretti (4), Gennaro Oliva (4), Chiara Lanzuolo (2,3), Elena Cattaneo (1,2)

    Organisations:
    1: Laboratory of Stem Cell Biology and Pharmacology of Neurodegenerative Diseases, Department of Biosciences, University of Milan, 20122 Milan, Italy
    2: INGM, Istituto Nazionale Genetica molecolare, Milan, Italy
    3: Institute of Biomedical Technologies (ITB)-CNR Milan, Italy
    4: Consiglio Nazionale delle Ricerche, Institute for High Performance Computing and Networking, Naples, Italy
    5: Axxam S.p.A.

    Presenting author: Michela Villa

    Exposition date: Saturday, September 16, 2023

    Exposition position: 041/2

    Huntington's Disease is a neurodegenerative disorder caused by an expansion of CAG repeats (>35) in the huntingtin (HTT) gene. This mutation results in the progressive degeneration of specific brain neurons, particularly the medium spiny neurons (MSNs) in the striatum, leading to atrophy.

    While HD symptoms typically manifest in adulthood, there is growing evidence suggesting a neurodevelopmental component in the pathology. HTT interacts with various developmental regulatory factors and plays a vital role in multiple aspects of neurodevelopment, including neuronal tube formation, maturation, and survival. One of these factors is PRC2, a chromatin-modifying factor that collaborates with Trithorax Group Proteins to maintain the pluripotent state of embryonic stem cells (ESCs).

    Based on these findings, our research focuses on characterizing the epigenetic features during in vitro differentiation of an isogenic panel of human embryonic stem cell lines carrying a gradual increase in CAG length in the HTT gene. Initially, we conducted immunocytochemistry at different time points during in vitro maturation, which revealed abnormal differentiation and persistent expression of the pluripotent marker Oct4 at later stages of differentiation in the presence of mutant HTT. These observations suggest the presence of chromatin abnormalities in the HD lines at early stages. We then performed a whole-genome analysis using ChIP-seq to investigate different epigenetic histone modifications. The aim is to identify whether there is an epigenetic signature in the HD lines and how it evolves during differentiation. Both ChIP-seq and RNA-seq analyses are currently underway, and the results will be presented.

  • Gross Anatomical Profiling of the Brain and Cerebellar Histology of the Nigerian Cattle Egret (Bubulcus ibis)

    Authors:
    Idris Ayodeji Azeez (1), Jamiu Omirinde Oyewole (1), James Olukayode Olopade (2)

    Organisations:
    1: University of Jos, Nigeria
    2: University of Ibadan, Nigeria

    Presenting author: Idris Ayodeji Azeez

    Exposition date: Friday, September 15, 2023

    Exposition position: 063/1

    The structural parts of brain are similar in all vertebrates, but they differ in their complexity and organization. The avian brain for instance, is organised differently compared to mammals, with variations existing in the relative size and location of their structures. The cattle egret (Bubulcus ibis) is a cosmopolitan avian species native to Africa. The juveniles lack coloured plumes and have black beaks/feet, while adults possess greyish-yellow feet/yellowish pointed beaks. Despite their economic importance as excellent sentinels in assessing environmental pollutants, there is a dearth of information on their neuroanatomy. Hence, we here investigated their gross morphological features and morphometric parameters, and the cerebellar histology. Twelve transcardially perfused juvenile cattle egrets were studied, with the body and brain weight and brain linear measurements considered. The brain weight constituted 0.46% of the total body mass, with a strong positive correlation recorded between the body and brain weights (r=0.9204). Morphologically, the brain was lissencephalic, with the corpus callosum absent and the olfactory bulbs rudimentary with no discernable division into the olfactory bulb, the olfactory tract and the olfactory lobe. We observed prominent sagittal eminence and obvious vallecula telencephali and fovea limbica on the dorsal and lateral surfaces of the cerebral hemispheres, respectively. The diencephalic structures were completely covered by telencephalon. Mesencephalic tectum appeared as large oval bilaterally bulging structure with prominent optic tracts and constituted bulk of the midbrain. The hypophysis was distinct, while pineal gland occupied the polygonal space between cerebrum, optic lobe and cerebellum. There was a dorsal extension of the fourth ventricle into the cerebellum known as the ventriculus cerebelli and the cerebellar histology presented a persistent outer molecular layering suggestive of a potential for adult neurogenesis. Our data has added relevant literature on the cattle egret brain and could prove useful in comparative, developmental and evolutionary avian neuroanatomy.

  • High throughput in vivo compound screen for linking genes, the habenular circuit and mental disorders

    Authors:
    Anja Bühler, Judith Habicher, Samuele Sartori, Niccolo Militello, Matthias Carl

    Organisations:
    University of Trento, Italy

    Presenting author: Matthias Carl

    Exposition date: Friday, September 15, 2023

    Exposition position: 064/1

    During brain development neurons differentiate to form neural circuits and build functional connections. The bilateral habenulae in the forebrain of vertebrates is the central component of a neurotransmitter system that relays cognitive information to the ventral mid- and hindbrain. It has been implicated in behaviors from fear and social behavior to reward responses and addiction. In addition, genes necessary for habenular neuron differentiation have been linked to pathophysiological syndromes such as major depression disorder, autism and schizophrenia. Hence, a better understanding of the genetic hierarchies underlying habenular neural circuit establishment and connectivity is a fundamental entry point for developing therapies.

    We have previously defined the temporal differentiation process of habenular neurons. This allowed us to develop a high-throughput compound screening platform to identify novel genetic pathways impacting on habenular neural circuit development. Using transgenic fish, which carry reporters for specific habenular neuron subpopulations, and the advantage of the small size of fish embryos allows for the rapid screening of hundreds of compounds in vivo.

    Our screen of about 400 compounds resulted in 10 promising candidates showing effects on habenular neurogenesis, survival and/or axonal pathfinding. Here we present a detailed analysis of the phenotypes showing altered reporter- and marker-gene expression for specific subpopulations of neurons in the habenulae. One compound in particular leads to an abolishment of habenular differentiation and continued apoptosis of a specific subset of habenular neurons even days after an initial two-hours compound treatment. Our current work aims at elucidating the mechanisms, the affected gene(s) and eventually the behavioral consequences of this specific phenotype.

  • In vitro neurotransmitters release to study developmental neurotoxicity (DNT) in hiPSCs-derived neurons

    Authors:
    Sarah Amato (1), Chiara Cervetto (1,2), Francesca Pistollato (3), Simona Baldassari (4), Federico Zara (4), Anna Bal-Price (3), Guido Maura (1), Manuela Marcoli (1,2)

    Organisations:
    1: Department of Pharmacy (DIFAR), Section of Pharmacology and Toxicology, University of Geno
    2: Interuniversity Center for the Promotion of the 3Rs Principles in Teaching and Research (Centro 3R), Pisa, Italy
    3: European Commission, Joint Research Centre (JRC), Ispra, Italy
    4: Unit of Medical Genetics, IRCCS Istituto Giannina Gaslini, Genova, Italy

    Presenting author: Sarah Amato

    Exposition date: Friday, September 15, 2023

    Exposition position: 055/1

    Human induced pluripotent stem cells (hIPSCs) and their neuronal/glia derivatives are considered a valid resource to set up an in vitro model to study developmental neurotoxicity (DNT). Within this field, an in vitro battery to assess DNT through the evaluation of neuronal specific endpoints, such as NSC proliferation and apoptosis, differentiation into neurons and glia, neuronal migration, synaptogenesis, and neuronal network formation, is proposed by the official guidelines. However, none of these tests includes the possibility to assess how neurotoxicants may hinder neurotransmitters (in particular glutamate) release and clearance, creating a gap in the biological applicability of such battery. Hence, we implemented an high-performance liquid chromatography (HPLC)-based methodology to measure glutamate and aspartate release in a previously characterized model of hIPSC-derived NSC at different stages of differentiation towards a mixed neuron/glia culture. The analytical method involves precolumn derivatization with o-phalaldehyde, followed by separation on a C18 reverse-phase chromatography column and fluorimetric detection. We found that hiPSC-derived neurons acquired Ca2+-dependent glutamate release properties as a hallmark of neuronal maturation. Then, we determined the ability of the cell culture of releasing the neurotransmitters in basal conditions or upon depolarization, as well as after repeated exposure to known neurotoxicants, alone or in three different chemical mixtures. Concurrently, the effect of the toxic molecules was determined through non-conventional electrophysiology on Multi Electrode Arrays, in order to evaluate any alteration on neuron-glia networks. Our results suggest that hIPSC-derived neurons are able to release glutamate in a vesicular manner, and that the treatment with the different chemicals induces alterations at this level. In conclusion, our methodology provides essential information about hIPSC-derived cultures in the context of DNT, helping clarifying the mechanisms underlying neurotoxicants-induced damage. Moreover, given its sensitivity, the analysis of neurotransmitters release should be considered a reliable in vitro test to define functional neuronal endpoints.

  • Indeterminate growth and regeneration: dissecting their molecular fingerprint in octopus

    Authors:
    Giulia Bergamini, Graziano Fiorito, Pamela Imperadore

    Organisations:
    Stazione Zoologica Anton Dohrn, Napoli, Italia

    Presenting author: Giulia Bergamini

    Exposition date: Friday, September 15, 2023

    Exposition position: 066/1

    Many organisms, particularly most terrestrial and some aquatic vertebrates, develop along the major axis of their body to reach a predictable size genetically determined. Indeterminate growth (IG) is the opposite phenomenon, in which the body (or part of it) keeps growing throughout life. The final body size of individuals of these species is mainly guided by environmental conditions. IG is considered as an ancestral condition, and it is recognized in different taxa especially among aquatic invertebrates. Interestingly enough, many organisms able for IG are also endowed with impressive regenerative capabilities, a phenomenon in which damaged structures regrow after injury and completely recover their functionality. While mammals exhibit limited regenerative capabilities, commonly restricted to early-life stages or to particular organs, most invertebrates retain this ability throughout adulthood. Among them, an interesting case study if offered by cephalopods, which can regenerate several complex structures, including appendages and nervous tissues. Cephalopods are also known for the continuous growth of the arms. Although the increasing interest in identifying key molecular and cellular mechanisms involved in these events, knowledge is still scanty. We investigated for the first time the molecular fingerprint involved in indetermined growth and regeneration in Octopus vulgaris arms. In silico analysis of transcriptomic data revealed that the arm tip exhibits many differentially expressed genes compared to the remaining part of the same appendage. Genes including transcription and neurogenic factors, cell fate and proliferation markers and RAGs have been identified. Through RTqPCR experiments we found that a selected set of the same genes are also upregulated during different phases of arm regeneration. Our data will aid future studies in understanding biological plasticity in O. vulgaris and other cephalopods.

  • Leukodystrophy -affected patients harboring mutations in Pol III gene present a profound transcriptional dysregulation

    Authors:
    Federica Rey (1), Letizia Esposito (1), Alessia Mauri (1), Gianvincenzo Zuccotti (1,2), Davide Tonduti (2,3), Jessica Rosati (4), Stephana Carelli (1,5), Cristina Cereda (5), Clarissa Berardo (1)

    Organisations:
    1: Pediatric Clinical Research Center "Romeo ed Enrica Invernizzi", Department of Biomedical and Clinical Sciences, University of Milan, Milan, Italy
    2: Department of Pediatrics, Buzzi Children's Hospital, Milan, Italy
    3: Department of Biomedical and Clinical Sciences, University of Milan, Milan, Italy
    4: Cellular Reprogramming Unit, Fondazione IRCCS Casa Sollievo della Sofferenza – Istituto Mendel Roma, Italy
    5: Center of Functional Genomics and Rare Diseases, Department of Pediatrics, Buzzi Children's Hospital, Milan, Italy

    Presenting author: Clarissa Berardo

    Exposition date: Friday, September 15, 2023

    Exposition position: 056/1

    Leukodystrophies (LDs) are genetic disorders which impact the white matter in the central nervous system, leading to a deficiency in myelin development. Mutations in RNA Polymerase III (PolIII) subunits cause POLR3-related LDs, such as those present in POLR3A and POLR3B genes. PolIII is involved in the transcription of small ribosomal units, tRNAs, 7SL RNA, U6 spliceosomal RNA, and more. tRNAs are implicated in translation, whilst small RNAs can perform regulatory functions on mRNA transcripts. The work hereby presented aimed to identify the transcriptional dysregulations present in primary fibroblasts of POLR3-mutated patients and to also assess alterations in the translation process. Fibroblasts were obtained from skin biopsies of 2 POLR3A and 1 POLR3B mutated patients and matched controls. RNA was extracted with Trizol reagent and Total RNA sequencing was performed with the CORALL Total RNA-Seq Library Prep Kit using Illumina NextSeq 500 Sequencing. Differential expression analysis was performed with DESeq.2 package and enrichment analyses were performed on differentially expressed genes (DEGs). Click-iT Protein Labeling approach was used to analyze newly synthesized proteins. Trascriptomic profiling highlighted a strong dysregulation in POLR3 patients, identifying 418 DEGs when comparing LDs patients to controls. Moreover, when comparing each patient to its matched control, a personalized DEGs signature was also observed, suggesting caution when grouping patients harboring different mutations in the same gene. Moreover, nascent protein synthesis analysis highlighted a down-regulation in nascent protein synthesis, suggesting an impaired translation possibly due to alterations in tRNA metabolism. In conclusion, our results highlight a profound impact on transcription and translation processes, in patients’ specific primary cells used as pre-clinical experimental model of the disease.

  • Microcephaly-Associated Protein WDR62 Shuttles from the Golgi Apparatus to the Spindle Poles in Human Neural Progenitors

    Authors:
    Claudia Dell'Amico (1), Marilyn M. Angulo Salavarria (1), Yutaka Takeo (2), Ichiko Saotome (2), Maria Teresa Dell’Anno (3), Maura Galimberti (4,5), Enrica Pellegrino (1,6), Elena Cattaneo (4,5), Angeliki Louvi (2), Marco Onorati (1)

    Organisations:
    1: Department of Biology, Unit of Cell and Developmental Biology, University of Pisa, Italy
    2: Departments of Neurosurgery and Neuroscience, Yale School of Medicine, New Haven, USA
    3: Fondazione Pisana per la Scienza ONLUS, San Giuliano Terme, Italy
    4: Dipartimento di Bioscienze, Università degli Studi di Milano, Milan, Italy
    5: INGM, Istituto Nazionale Genetica Molecolare, Milan, Italy
    6: Host-Pathogen Interactions in Tuberculosis Laboratory, The Francis Crick Institute, London, UK

    Presenting author: Claudia Dell'Amico

    Exposition date: Friday, September 15, 2023

    Exposition position: 053/1

    WDR62 is a spindle pole-associated scaffold protein with pleiotropic functions. Recessive mutations in WDR62 cause structural brain abnormalities and account for the second most common cause of autosomal recessive primary microcephaly (MCPH), indicating WDR62 as a critical hub for human brain development. Here, we investigated WDR62 function in corticogenesis through the analysis of a C-terminal truncating mutation (D955AfsX112) found in a subject with MCPH and severe malformations of neocortical development. For the present study, we employed induced pluripotent stem cells (iPSCs) derived from the patient (Mut iPSCs) and his unaffected parent (Het iPSCs), and isogenic corrected lines (Iso iPSCs) obtained from Mut iPSCs via CRISPR-Cas9 genome editing. We then generated 2D and 3D models of human neurodevelopment, including neuroepithelial stem cells, cerebro-cortical progenitors, terminally differentiated neurons, and cerebral organoids. We report that WDR62 localizes to the Golgi apparatus during interphase in Iso, Het, and Mut neural cells, and cerebral organoids, as well as in human fetal brain tissue. Additionally, we find that WDR62 translocates from the Golgi apparatus to the mitotic spindle poles in a microtubule-dependent manner in Iso and Het 2D and 3D neuroderivatives, while the mutant protein fails to localize to the spindle poles during mitosis. Moreover, we demonstrate that WDR62 mis-localization impairs mitotic progression and results in alterations of the neurogenic trajectories of Mut iPSC neuroderivatives. In summary, impairment of WDR62 localization and function results in severe neurodevelopmental abnormalities, thus delineating new mechanisms in the etiology of MCPH.

  • Oligodendrocyte-derived SEMA6A shapes GnRH neurovascular congruency and puberty onset

    Authors:
    Roberto Oleari (1), Antonella Lettieri (2), Marieke Verhagen (3), Eljo Y. van Battum (3), Marleen H. van den Munkhof (3), Carlotta Tacconi (4), Alyssa J.J. Paganoni (1), Roberta Azzarelli (5), Luca Palazzolo (1), Ivano Eberini (1), Sasha R. Howard (6,7), Alessandro Fantin (4), R. Jeroen Pasterkamp (3), Anna Cariboni (1)

    Organisations:
    1: Università degli Studi di Milano, Dept. Pharmacological and Biomolecular Sciences, Italy
    2: Università degli Studi di Milano, Dept. Health Sciences, Italy
    3: Utrecht Univeristy, Utrecht Medical Center, The Netherlands
    4: Università degli Studi di Milano, Dept. Biosciences, Italy
    5: University of Cambridge, Wellcome - Medical Research Council Cambridge Stem Cell Institute, United Kingdom
    6: Queen Mary University London, Centre for Endocrinology William Harvey Research Institute, United Kingdom
    7: Barts Health NHS Trust, Department of Paediatric Endocrinology, United Kingdom

    Presenting author: Roberto Oleari

    Exposition date: Friday, September 15, 2023

    Exposition position: 059/1

    Puberty onset and successful sexual reproduction are ensured by axon-dependent migration of gonadotropin releasing hormone (GnRH) neurons from the nose to the hypothalamus and by their innervation of the median eminence (ME), where GnRH peptide is released. Despite several studies highlighted multiple roles of semaphorins (SEMAs) during GnRH neuron development, the requirement of class 6 semaphorins has not been investigated yet. Further, molecular and cellular mechanisms underlying ME development and pubertal timing are only partially understood.

    Here, we identified an unusual role for the axon guidance cue semaphorin 6A (SEMA6A) in the control of ME vascular function, GnRH innervation and puberty onset.

    First, we found that SEMA6A was highly expressed on nasal axons during mouse embryogenesis, but its loss did not affect GnRH neuron migration, suggesting that SEMA6A is dispensable for early GnRH neuron development. Interestingly, we detected strong SEMA6A expression also on oligodendrocytes adjacent to GnRH neuron axons and fenestrated capillaries in the ME, from late embryonic development to adulthood, suggesting a possible SEMA6A role in GnRH axon patterning and neurovascular congruency. Accordingly, Sema6a-null mice exhibited reduced GnRH innervation in the ME, resulting in delayed puberty onset.

    To confirm this hypothesis, we combined tailored experimental tools including trans-endothelial electrical resistance measurements in primary brain endothelial cells and in vivo permeability assay. Specifically, we revealed an unexpected function for SEMA6A in the structural and functional rearrangements of ME fenestrated capillaries, which are required to maintain GnRH neurovascular congruency. Strongly supporting our mechanistic observations in mice, we detected novel pathogenic SEMA6A variants in patients with delayed puberty, which hampers protein stability and cellular localization by in vitro and in silico analyses.

    Collectively, these results identify an unrecognized role for oligodendrocyte-derived SEMA6A in GnRH neuron patterning by tuning ME vascular permeability and provide new insight into pathogenic mechanisms underlying delayed puberty.

  • Patients derived organoids show differences compared to mice model in DNA damage accumulations and mitotic defects leading microcephaly syndrome

    Authors:
    Gianmarco Pallavicini (1), Amanda Moccia (2), Giorgia Iegiani (1), Emily Peirent (2), Roberta Parolisi (1), Gaia Elena Berto (1), Martina Lorenzati (1), Fiorella Balzac (3), Emilia Turco (3), Enrica Boda (1), Annalisa Buffo (1), Stephanie Bielas (2), Ferdiando Di Cunto (1)

    Organisations:
    1: Neuroscience Institute Cavalieri Ottolenghi, Turin, Italy.
    2: Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI, USA
    3: Departement of Molecular Biotechnology and Health Sciences, University of Turin, Italy

    Presenting author: Gianmarco Pallavicini

    Exposition date: Friday, September 15, 2023

    Exposition position: 054/1

    In primary hereditary microcephaly (MCPH), brain volume reduction is the main clinical phenotype, associated with conserved brain architecture and mild to moderate intellectual disability. Mutations in citron (CIT), leading to loss or inactivation of the citron kinase protein (CITK), cause primary microcephaly in humans and rodents. This disorder is associated with cytokinesis failure and apoptosis in neural progenitors. It has therefore been postulated that the apoptosis observed after CITK loss is a consequence of impaired cytokinesis. However, studies performed in many different models indicate that cytokinesis failure leads more frequently to cell cycle arrest than apoptosis, suggesting that another fundamental event must occur. Using CIT ko and kinase inactive mice models compared to fore brain organoids derived from CIT mutated patients iPSCs, we found that CITK inactivation induces DNA damage accumulation and chromosomal instability in human and mouse neural progenitors. Moreover, recruitment of RAD51 to DNA damage foci is compromised by CITK loss or inactivation indicating that CITK is involved in homologous recombination. Despite same molecular lesion in ko and kinase inactive mutations in neural progenitors, different amount of damage generates syndrome with less severity compared to ko. This suggests that there is a soil of damage to induce a precise apoptosis in neural progenitors that can represent a common thread between unrelated microcephaly syndromes.

  • Regenerative approaches in spinal cord injuries by organotypic and cortico-spinal assembloid models

    Authors:
    Francesca Merighi, Sara De Vincentiis, Alessio Zanelli, Luca D'Adamo, Angela Toffano, Claudia Dell'Amico, Vittoria Raffa, Marco Onorati

    Organisations:
    Department of Biology, University of Pisa, Italy

    Presenting author: Francesca Merighi

    Exposition date: Saturday, September 16, 2023

    Exposition position: 038/2

    Spinal cord injury is a pathological condition with devastating physical and socio-psychological consequences, but effective treatments are lacking due to the complex pathophysiology. Recent investigations in the field of regenerative medicine show the therapeutic potential of human neuroepithelial stem (NES) cells to treat this type of injury, while advancement in nanotechnologies enables the development of novel nanomedical tools. Previous studies investigated the use of magnetic nanoparticles (MNPs) and magnetic fields to induce stretch-growth, i.e., the stimulation of axonal outgrowth by mechanical stimuli. Indeed, the success of stem cell-based therapies depends on the capacity to promote cell engraftment, neuronal differentiation, and axonal growth, which is necessary to reconstitute lost neural circuits. Our data concern mechanically induced stretch-growth of MNP-labelled human-derived differentiating neurons both in vitro and transplanted into an ex vivo model of mouse spinal cord (SC) organotypic slices. Here, we show the ability of our approach to promote elongation and guidance of the neurites of stretched NES cells both in vitro and in the SC tissue. Interestingly, we observed also accelerated maturation of neural precursors in vitro, when cultured long-term under mechanical stimuli. Once transplanted into the SC organotypic slice system, cells integrated diffusely throughout the tissue thickness, showing optimal survival rate. Beyond higher elongation, cells stimulated in the tissue showed a preferential orientation, suggesting the ability of the proposed approach to guide process outgrowth towards a specific direction. We are also developing an innovative platform of cortico-spinal assembloids to model the human cortico-spinal tract in vitro. Cortico-spinal assembloids will represent a further platform to test the effective regenerative potential of our approach in a human 3D cytoarchitectural context, thus offering new avenues to challenge spinal cord lesions.

  • Striatal astrocytes generate a novel type of axonless neuron that transiently integrates into damaged circuits

    Authors:
    Giulia Nato (1), Marco Fogli (1,2), Nicolas Marichal (3), Valentina Proserpio (1,4), Ilaria Ghia (1,2), Valentina Cerrato (1,5), Salvatore Oliviero (1,4), Paolo Peretto (1,2), Benedikt Berninger (3), Annalisa Buffo (1,5), Federico Luzzati (1,2)

    Organisations:
    1: Neuroscience Institute Cavalieri Ottolenghi, University of Turin, Orbassano, Italy
    2: Department of Life Sciences and Systems Biology, University of Turin, Italy.
    3: Centre for Developmental Neurobiology, King
    s College London, United Kingdom
    4: Candiolo Cancer Institute, FPO-IRCCS, Candiolo, Torino, Italy
    5: Department of Neuroscience Rita Levi-Montalcini, University of Turin, Italy.

    Presenting author: Giulia Nato

    Exposition date: Saturday, September 16, 2023

    Exposition position: 040/2

    In the adult brain, subsets of astrocytes act as neural stem cells in two neurogenic niches, the sub-ventricular zone and hippocampal dentate gyrus, where they produce olfactory bulb interneurons and dentate gyrus granule cells. Outside of these niches, neurogenesis has been occasionally observed under specific conditions, particularly in the striatum where it has been described in both pathological conditions, in rodents models of progressive and acute neuronal degeneration, and physiological conditions, in adult rabbits, humans and guinea pigs. At least in mice and guinea pigs the neurogenic progenitors are local astrocytes. However, the phenotypic commitment of these progenitors remains unclear, as their neuronal progeny have been observed to live only temporarily and do not express markers of mature or immature striatal neurons.

    To address this issue, here we performed an in depth molecular, morphological and functional characterization of immature neurons induced by excitotoxic lesion in mice. Single cell-RNA sequencing analyses of these cells indicated they belong to the class of lateral ganglionic eminence (LGE)-derived GABAergic interneurons and are distinct from striatal projection neurons (LGE-derived) or striatal interneurons (MGE-derived). Nonetheless, morphometric analysis of individually reconstructed neuroblasts reveals that these cells often attain complex morphologies with long dendrites and variable amounts of dendritic spines. Similarly to other LGE-derived interneurons they appear axonless. Electrophysiological recordings indicated that most cells receive excitatory inputs and accordingly, rabies virus-based monosynaptic tracing indicated that new neurons in the striatum integrated into neuroanatomically appropriated cortico-striatal-thalamic loops. Thus, astrocyte-generated neurons in the striatum do not replace degenerated cells but still transiently integrate into pre-existing circuitry, thereby potentially contributing to post-lesion network plasticity supporting functional recovery after damage. Fundings: Fondazione Compagnia di San Paolo, NSCR-Reconstruct.

Epilepsy and hyperexcitability

  • Changes in evoked and spontaneous brain activity following focal controlled brain lesions

    Authors:
    Chiara Maria Cassani (1,3), Flavia Maria Zauli (2,4,5), Ivana Maria Sartori (2), Maria Del Vecchio (3), Pietro Avanzini (3), Piergiorgio d'Orio (2,6)

    Organisations:
    1: Department of School of Advanced Studies, University of Camerino, Italy
    2: Centre of Epilepsy Surgery “C. Munari”, Department of Neuroscience, Niguarda Hospital, Milan, Italy
    3: Institute of Neuroscience, National Research Council of Italy, Parma, Italy
    4: Department of Philosophy ‘Piero Martinetti’, University of Milan, Milan, Italy
    5: Department of Biomedical and Clinical Sciences “L. Sacco”, University of Milan, Milan, Italy
    6: Department of Medicine and Surgery, Unit of Neuroscience, University of Parma, Parma, Italy

    Presenting author: Chiara Maria Cassani

    Exposition date: Friday, September 15, 2023

    Exposition position: 072/1

    Open surgical resection is the highest successful treatment in drug-resistant focal epilepsy in case the pathological zone can be delineated. Nowadays, less invasive techniques are being considered to reduce possible surgical complications.

    Radiofrequency-Thermocoagulation (RFTC) is a technique that produces local lesions in the pathological sites by applying radio frequency current pulses. Its main purpose is to disconnect the epileptic network, interrupting the spread of the seizure. However, quantitative estimations of how evoked and spontaneous activity changes upon thermocoagulation are still lacking, despite their potential to unravel the neural mechanisms subserving the reorganization of cortical circuits upon focal injuries.

    To address this issue, we acquired the SEEG signal of three drug-resistant epileptic patients before and after a single territory RFTC, including spontaneous activity and responses evoked by visual stimulations (i.e., flash). In the first case, we assessed the power density spectrum and its modulation induced by the RFTC, showing how lesions increase slow frequency representation. Most importantly, we evaluated the reactivity of the gamma band power (50-150 Hz), first extracting several univariate features (i.e., pre-stimulus mean power, starting time, maximal peak, duration, and area under the curve), further grouping them into four major classes: i) unresponsive, ii) responsive both pre and post RFTC, iii) responsive only before RFTC and iv) responsive only after RFTC.

    Along with the Cortico-Cortical Evoked Potentials recorded before RFTC, the data collected on this small sample suggest how brain lesions do not simply impair the local functioning of the cortex but rather instantiate and favour reserve neural processes or pathways that were not exploited before the lesions. Cascade implications regard neuroplasticity and the neurophysiology of rehabilitation.

  • Dissecting the role of HCN1 in Developmental and Epileptic Encephalopathy (DEE) by exploiting patient-specific models of cerebral cortex development

    Authors:
    Giulia Demenego (1,2), Sara Mancinelli (1), Elena Florio (1,2), Filippo Mirabella (1,2), Monica Tambalo (1), Gabriele Di Napoli (1), Carla Marini (3), Jacopo Di Francesco (4,5), Christel Depienne (6), Davide Pozzi (1), Simona Lodato (1,2)

    Organisations:
    1: Humanitas University, Pieve Emanuele, Milan, Italy
    2: Neuro Center, IRCCS Humanitas Clinical and Research Center, Rozzano-Milan, Italy
    3: Child Neurology and Psychiatric Unit, Pediatric Hospital G. Salesi, United Hospitals of Ancona, Ancona, Italy
    4: School of Medicine and Surgery, Milan Center for Neuroscience (NeuroMI), University of Milano-Bicocca, Monza
    5: Department of Neurology, ASST San Gerardo Hospital, University of Milano-Bicocca, Monza
    6: University Hospital Essen, Institute of Human Genetics, Essen, Germany

    Presenting author: Giulia Demenego

    Exposition date: Friday, September 15, 2023

    Exposition position: 071/1

    Developmental and Epileptic Encephalopathies (DEE) are a heterogeneous group of devastating conditions characterized by very early onset, unprovoked and recurrent seizures, that often show poor response to pharmacological treatment. Recent advancements in genome sequencing have expanded our knowledge on DEE, and the number of associated mutations in voltage-gated ion channels has grown exponentially. Indeed, new evidence supports the surprising role of ion channels, classically studied exclusively in mature neurons, in developing neurons, and in prenatal human brain pathology. Among others, many mutations in Hyperpolarization-activated Cyclic Nucleotide-gated (HCN1) gene have been causative of the DEE24, whose affected patients show also impaired global development, intellectual disability, and behavioral abnormalities. Here, we aim to understand how brain developmental processes are altered by the HCN1 pathogenic variants associated with DEE24. We selected 3 de novo HCN1 variants (M153I, G391D, A387S) showing association with autistic/behavioral and morphological traits and evaluated their effect on electrophysiological properties in both heterologous cell lines and neurons. We observed that the HCN1A387S mutant displays an abnormal electrophysiological profile, while HCN1G391D shows a subcellular mislocalization. By exploiting a highly reproducible human cortical organoids (hCOs) system, we generated hCOs from HCN1-DEE24 patient-specific iPSC lines. Moreover, through in utero electroporation, we generated chimeric brains harboring these variants in a temporally and spatially regulated manner. We analyzed the activity and morphology of electroporated neurons in vitro, as well as their cell-intrinsic and non-cell autonomous effects on early cortical development in vivo.

    Our preliminary results suggest that the generation of personalized models, bridging genetic data with molecular neurodevelopment, allows the identification of specific endophenotypes of distinct HCN1 variants and their effects on cortical assembly. The obtained results will open the way for future research and investigations, aiming at improving the clinical conditions of epileptic patients.

  • Functional study of a mutant α2 subunit of the neuronal nicotinic acetylcholine receptor linked to sleep-related generalized seizures with cognitive deficit

    Authors:
    Chiara Villa (1), Laura Clara Grandi (2), Valerio Conti (3), Simone Meneghini (2), Eleonora Giagnorio (1), Renzo Guerrini (3,4), Romina Combi (1), Andrea Becchetti (2)

    Organisations:
    1: Dept. of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy
    2: Dept. of Biotechnology and Bioscience, University of Milano-Bicocca, Milan, Italy
    3: Dept. of Neuroscience, Meyer Children's Hospital IRCCS, Florence, Italy
    4: Dept. NEUROFARBA, University of Florence, Florence, Italy

    Presenting author: Chiara Villa

    Exposition date: Friday, September 15, 2023

    Exposition position: 067/1

    Aims: Mutations in the CHRNA4, CHRNB2, and CHRNA2 genes, respectively encoding the α4, β2 and α2 subunits of the neuronal nicotinic receptors (nAChRs) are increasingly recognized to be linked to autosomal dominant sleep-related hypermotor epilepsy and other epileptic syndromes. The pathogenesis is uncertain, as different nAChR subunits exert complex physiological roles in the mature neocortex as well as during synaptogenesis.

    Materials and methods: We generated different F2A-based tri-cistronic constructs for equimolar expression of α2/β4 nAChR subunits, with enhanced green fluorescent protein as a reporter and carried out functional tests by patch-clamp recording on HEK 293 cells transiently transfected with mutant or wild-type (WT) constructs. Whole-cell currents were elicited by fast application of nicotine or ACh, at -70/-80 mV. Concentration-response relations were obtained by applying nicotine concentrations ranging from 0.1 to 600 µM.

    Results: a novel missense mutation in the CHRNA2 gene (c.926T>C; p.Val309Ala) was detected in heterozygosity in two members of a family displaying a peculiar phenotype and characterized by sleep-related tonic-clonic epileptic seizures accompanied by cognitive deficit. The mutation is located in a highly conserved region of the third transmembrane domain (M3), which closely interact with the pore-lining M2 segments during the gating conformational rearrangement operated by ACh binding. The mutation is described as potentially deleterious by PolyPhen-2, SIFT and MutationTaster predictors. In homo- and heterozygous condition, mutant α2 decreased the nAChR response to nicotine and ACh. The peak current density tested with 100 µM nicotine was 143 ± 11.7 pA/pF in WT and decreased by approximately 75% in both homo- and heterozygous condition. Similar effects were produced by 100 µM ACh (WT: 7.6 ± 1.25 pA/pF, n=13; homozygote: 2 ± 0.32 pA/pF, n=27; p < 0.001, Student t-test).

    Conclusions: Our data suggest that CHRNA2 loss-of-function could contribute to the generation of sleep-related tonic-clonic seizures associated with cognitive deficit.

  • Investigating activity-dependent processes during cortical neuronal assembly in development and disease

    Authors:
    Sara Mancinelli (1), Giulia Demenego (1,2), Matteo Miotto (2), Marco Erreni (2), Christel Depienne (3), Davide Pozzi (1,2), Simona Lodato (1,2)

    Organisations:
    1: Humanitas University, Pieve Emanuele, Milan, Italy
    2: IRCCS Humanitas Research Center, Rozzano-Milan, Italy
    3: University Hospital Essen, Institute of Human Genetics, Essen, Germany

    Presenting author: Sara Mancinelli

    Exposition date: Friday, September 15, 2023

    Exposition position: 070/1

    The cerebral cortex contains an extraordinary diversity of excitatory projection neurons (PN) and inhibitory interneurons (IN), wired together to form complex circuits. Spatiotemporally coordinated execution of intrinsic molecular programs defines both PN and IN subtype diversity. Spontaneous neuronal activity in the early stages of development, as well as evoked activity by external stimuli can contribute to the formation of balanced microcircuits. Alterations of these delicate processes, indeed, have often been associated to neurological/neurodevelopmental disorders. However, it is still unclear how cortical neuronal diversity influences early spontaneous activity, and whether subtype-specific molecular features (i.e. ion channel repertoire) can correlate with distinctive functional roles in the early assembly. In this study, we combined in utero genetic perturbations and pharmacological inhibition of selected ion channels (i.e HCN1Hyperpolarization. Activated Cyclic Nucleotide Gated Potassium Channel 1) with RNA-sequencing and live imaging technologies to identify the activity-regulated processes controlling the development of different cortical PN classes, their wiring and the acquisition of subtype specific features. Our preliminary results suggests that HCN1 channel, which is associated with infantile genetic epilepsy, is a possible modulator of spontaneous electrical activity in developing cerebral cortex and proposes HCN-current as a fundamental signal to drive cortical wave propagation during development. Further validation of this finding would support HCN1 as potential intervention targets for developmental epilepsies.

  • Low glycemic index diet restrains epileptogenesis in a gender specific fashion

    Authors:
    Caterina Michetti (1,2), Daniele Ferrante (1), Barbara Parisi (1), Lorenzo Ciano (2), Cosimo Prestigio (1), Silvia Casagrande (1), Sergio Martinoia (1), Enrico Millo (1), Pierluigi Valente (1,3), Silvia Giovedi (1,3), Fabio Benfenati (2,3), Pietro Baldelli (1,3)

    Organisations:
    1: Department of Experimental Medicine, University of Genova
    2: Center for Synaptic Neuroscience and Technology, Istituto Italiano di Tecnologia
    3: IRCCS, Ospedale Policlinico San Martino, Genova

    Presenting author: Caterina Michetti

    Exposition date: Friday, September 15, 2023

    Exposition position: 069/1

    Dietary restriction, such as low glycemic index diet (LGID), have been extensively and successfully used as antiepileptic approach; however, if such diet could also exert a real antiepileptogenic action is still unclear. Here, we investigated in Synapsin (Syn) II KO mice, a mouse model of hereditary temporal lobe epilepsy, whether administration of LGID during the latent period, which precedes the appearance of the first seizure, inhibits epileptogenesis.

    In these mice, seizures appear around 3 months after birth, offering a window of opportunity in which LGID may affect the epileptogenic process. Pregnant Syn II KO mice were fed either a LGID or standard diet (StD) during gestation and lactation. Both diets were maintained in weaned mice up to 5 months of age. The latency and duration of the first behavioral seizure, as well as the behavioral analyses of the seizure elements were investigated by video recordings. Notably, LGID delayed the seizure onset and induced a reduction of seizures severity only in Syn II KO female mice. Behavioral seizure analysis was followed by high-density multielectrode array recordings in acute brain slices. Coherently with the behavioral results, a reduction of the frequency, amplitude, duration, covered-area and velocity of propagation of inter-ictal events was observed in the hippocampus of LGID-fed Syn II KO females, but not in males, suggesting a possible involvement of sexual hormones in this gender-specific effect of LGID. ELISA-based analysis of plasmatic and cortical ALLO revealed did not show any gender-influence. Notably, LGID increased cortical but not plasmatic concentrations of ALLO in female, while it remained unchanged in male mice.

    These results strongly suggest that the gender-specific interference with the epileptogenic process induced by LGID finds a fundament in a gender-specific increase of cortical ALLO, a neurosteroid known for its capability to strength GABAergic inhibitory inputs, acting as an endogenous benzodiazepine.

  • SCN2A A1659V loss-of-function variant causes early infantile onset encephalopathy

    Authors:
    Lisastella Morinelli (1,2), Martina Albini (1,2), Loretta Ferrera (3,4), Alessandra Bianchi (1), Bruno Sterlini (1,2), Antonella Riva (3,4), Alessandra Ludovico (3), Giulia Lombardo (5), Francesca Madia (3), Raffaele Falsaperla (5), Federico Zara (3,4), Anna Corradi (1,2)

    Organisations:
    1: Department of Experimental Medicine, University of Genoa, Genoa, Italy
    2: Department of Neuroscience and Brain Technologies, Fondazione Istituto Italiano di Tecnologia, Genoa, Italy
    3: Unit of Medical Genetics, IRCCS Istituto Giannina Gaslini, Genoa, Italy
    4: Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health, University of Genoa, Genoa, Italy
    5: General Pediatrics and Pediatric Acute and Emergency Unit, Neonatal Intensive Care Unit and Neonatal Accompaniment Unit, Azienda Ospedaliero-Universitaria Policlinico-San Marco, San Marco Hospital, University of Catania, Catania, Italy

    Presenting author: Lisastella Morinelli

    Exposition date: Friday, September 15, 2023

    Exposition position: 068/1

    Pathogenic variants in SCN2A, gene encoding for the α-subunit type 2 of the voltage-gated Sodium channel Nav1.2, are known to cause a phenotypic spectrum including epileptic encephalopathy and intellectual disability or autism without epilepsy. Gain-of-function (GOF) mutations’ resulting phenotypes may be treated with Sodium channels blockers, while loss-of-function conditions are non-respondent.

    We focused on the effects of the pathogenic SCN2A variant c.2976C>T (p.A1659V) found, by Next Generation Sequencing, at a mosaic state in an infant affected by Developmental Epileptic Encephalopathy non respondent to carbamazepine.

    To study the functional effects of this variant, we generated by site-directed mutagenesis a plasmid encoding for the A1659V mutated SCN2A and demonstrated that this mutation does not impact on channel expression and subcellular localization in HEK293 cells by Western blot and immunofluorescence assays.

    Whole-cell patch-clamp recordings showed a reduced Na+ current in HEK293 cells with overexpression of the A1659V channel compared to the one generated by the wild-type channel. The quantitative analysis of A1659V channel activation properties showed a shift of the activation curve towards more negative potentials and a time constant slower than the one of the wild-type channel. These results explain the loss-of-function (LOF) mechanism underlying the epileptic phenotype of patients and help define the genotype-phenotype correlation with the ultimate goal of improving therapeutic strategies to treat epileptic syndromes.

Glial cells in physiology and pathology

  • Analysis of the biocompatibility and biomimeticity of a corn maltodextrin electrospinned fiber membrane used as a scaffold to promote peripheral nerve regeneration

    Authors:
    Miriam Metafune (1), Claudio Cecone (2), Marco Zanetti (2), Stefania Raimondo (1,3), Federica Fregnan (1,3)

    Organisations:
    1: Department of Clinical and Biological Sciences, University of Torino
    2: Department of Chemistry, Nis Interdepartmental Centre, University of Turin
    3: Neuroscience Institute Cavalieri Ottolenghi, University of Torino

    Presenting author: Miriam Metafune

    Exposition date: Saturday, September 16, 2023

    Exposition position: 053/2

    The objective of the present study is to evaluate biocompatibility and biomimeticity of an innovative membrane with the aim to apply it for repairing somatic and autonomic peripheral nerves in case of traumatic or iatrogenic lesions. Starch-derived (GLUCIDEX®) hyper-crosslinked polymers with suitable mechanical properties were electrospinned as membrane and tested, in vitro using immortalized Schwann Cells (RT4-D6P2T cells), for cell survival and proliferation to evaluate the biocompatibility and biomimetic nature of the scaffolds.

    RT4-D6P2T cells were cultured i) in direct contact with the membrane, to investigate the interaction with the substrate and ii) in the presence of membrane dissolution products, to test the effect on cell proliferation and organization.

    i) Concerning to the adhesion assays, the actin cytoskeleton results more organized in the control group, however, after 24 hours, the density and the area occupied by RT4-D6P2T increased.

    ii) Several analyzes were conducted using the dissolution products of Glucidex® membranes; the proliferation assay revealed that, after 1, 4 and 7 days of culture, cells maintain proliferative behavior under all conditions tested although a slight decrease, compared to the control, is observed at the first two time points. The actin cytoskeleton profile revealed that cells cultured in conditioned medium have a high organization and generate membrane protrusions, lamellipodia, correlated to cell migration, an important feature of glial cells in support of peripheral nerve regeneration.

    Investigating apoptosis and the specific cellular alterations due to Bax, pro-apoptotic protein, and Bcl-2, anti-apoptotic protein, our study revealed that the dissolution products of the membrane are not related with cell death, contrarily, they are associated with good survival. Further investigations are underway to deepen the effect of the dissolution products on expression of gene involved in the regulation of nerve regeneration by Schwann cells.

  • Astrocytes diversity across mammals: a comparative analysis on distribution and single cell morphology

    Authors:
    Caterina Ciani (1), Giulio Pistori (1), Marika Mearelli (1), Jean-marie Graic (2), Carmen Falcone (1)

    Organisations:
    1: Scuola Internazionale Superiore di Studi Avanzati (SISSA), Trieste, Italy
    2: Università di Padova, Padova, Italy

    Presenting author: Caterina Ciani

    Exposition date: Saturday, September 16, 2023

    Exposition position: 046/2

    Over the recent years, there has been an increasing need for a better understanding of the identity and the roles of astrocytes and their involvement in the cognitive abilities. Indeed, they play a crucial role in brain functions, they show primate-specific features, and they are relevant in several diseases. Investigations on astrocytes across evolution have rarely been performed. However, a comprehensive comparison of astrocytes in a diverse range of mammals is pivotal for understanding the morphology across species and the related modifications in gene expression and functions. We have recently described primate-specific features of two subtypes of astrocytes, such as the Interlaminar astrocytes (ILAs) and the Varicose Projection astrocytes (VP-As), which can be of particular interest in the context of astrocyte evolution. Our project will expand from previous data and aims to investigate astrocytes’ diversity across mammals, by characterising the distribution and the single-cell morphology of different subpopulations of astrocytes within different cortical layers across mammals. In details, we analyzed samples from prefrontal cortex of: Primates (chimpanzee, rhesus macaque, human), Carnivora (tiger, lion, leopard), Artiodactila (cow, tursiops), Rodentia (mouse) and Chiroptera (Seba’s short-tailed bat).

    We immunostained these samples with various astrocyte markers (i.e., GFAP, ALDH1L1, S100β, GLAST) in order to compare: (1) the distribution of different astrocyte subpopulations, and (2) the single-cell astrocyte morphology reconstructed with an algorithm-driven segmentation and Image-J plugin Neurotracer analyzer. Our previous results showed an increase of ILA morphological complexity and density in primates. With this project, we will unlock unprecedented details of the distribution and the single-cell morphological complexity of different astrocyte’s subtypes across different layers and different mammals, with a special focus on primates and, in particular, humans.

    Data obtained from this research have the potential to lead to new fascinating hypotheses on the role of astrocytes in primate neuroanatomical, behavioural and cognitive complexity.

  • Bay-41 acts on sGC in microglia to reduce plaque load in Alzheimer’s disease mice independently of Clec7A

    Authors:
    Giovanni Cosco (1), Joshua Shrouder (2), Burcu Seker (2), Nikolaus Plesnila (2)

    Organisations:
    1: Università degli Studi di Padova, Italy
    2: Institute for Stroke and Dementia Research (ISD), Klinikum der Universität München, Germany

    Presenting author: Giovanni Cosco

    Exposition date: Saturday, September 16, 2023

    Exposition position: 043/2

    Introduction: Alzheimer’s Disease is characterized by a pathological accumulation of amyloid beta driving neuronal loss. cGMP is a multitarget molecule that regulates many processes in the brain. Its production is initiated by the binding of Nitric Oxide (NO) to its receptor Soluble Guanylate Cyclase (sGC). However, in the context of AD, NO can act like a double-edged sword and drive Aß plaque clearing “Disease Associated Microglia” (DAM) into a harmful proinflammatory state through reactive oxygen species (ROS) mediated damage. Therefore, a carefully tailored DAM phenotype is required to limit AD progression.

    Purpose of the study: The research focus is analyzing the state of microglia and DAM markers expression after chronic sGC stimulation in Alzheimer’s disease mice model 5xFAD.

    Methods: The brain sections (50µm) which will be used in this study were already collected from WT, 5xFAD+Vehicle, 5xFAD+sGC mice that were treated with sGC stimulator or vehicle pellets over 10 months. We evaluated the expression and the localization of Clec7a and CD68 molecule with Aß plaques and microglia cells by Free-Floating Immunohistochemistry. Imaging data was acquired by confocal microscopy and analyzed by 3D reconstruction using Imaris software.

    Conclusion: Here we show that Bay-41 acts downstream of NO leading to a transcriptional signature in DAM which reduces plaque load in a mice model of Alzheimer’s disease.

    Specifically, we demonstrated using RT-PCR an increase in DAM-associated transcriptional expression of phagocytic markers CD68 and Clec7a in 5xFAD mice brain homogenates. We then used free-floating immunohistochemistry, confocal imaging and quantitative 3D structural reconstruction analysis to colocalize both molecules in Plaque-associated microglia. We observed a prominent increase in CD68 within plaque-associated microglia in the treated group, while Clec7A, although co-localized, remained unaffected by Bay-41 treatment. Therefore, Bay-41 exerts its therapeutic effect by boosting a carefully tailored Clec7A-independent phagocytotic DAM response in microglia to ameliorate AD progression.

  • Citron-kinase loss leads to hypomyelination via cell autonomous and non-cell autonomous mechanisms

    Authors:
    Martino Bonato (1,2), Maryam Khastkhodaei Ardakani (1,2), Francesca Montarolo (1,2), Annalisa Buffo (1,2), Enrica Boda (1,2)

    Organisations:
    1: Dept. of Neuroscience, Università degli Studi di Torino, Torino, Italy
    2: Neuroscience Institute Cavalieri Ottolenghi, Orbassano (Torino), Italy

    Presenting author: Martino Bonato

    Exposition date: Saturday, September 16, 2023

    Exposition position: 048/2

    Citron kinase (Cit-K) is a serine-threonine kinase involved in DNA repair and cytoskeletal dynamics. Loss-of-function of Cit-K results in microcephaly and severe Central Nervous System (CNS) hypomyelination in both humans and mice (i.e. Cit-k KO). Former studies (Boda et al. 2022, doi: 10.1038/s41467-022-30010-6) have shown that Cit-k loss results in accumulation of DNA damage and cell-autonomously induces forebrain oligodendrocyte progenitor cells (OPCs) to undergo cell death or enter a senescent state. Yet, in Cit-k mutants, severe and diffuse hypomyelination persists even when forebrain OPC population is rescued, suggesting additional Cit-K loss -dependent mechanisms involved in myelin defect. To study these aspects, at first, we took a transplantation approach and compared the maturation potential of wild-type (WT) OPCs grafted in either Cit-k KO vs. WT postnatal mouse brain. Notably, when exposed to the Cit-k KO milieu, the fraction of grafted cells that reached a mature MBP+ phenotype was significantly lower than that in the WT brain, suggesting the presence of an inhibitory/non-supportive environment in the Cit-k KO CNS tissue. This was corroborated by in vitro assays, where Cit-k KO OPCs appeared able to progress along the lineage when isolated from the tissue and exposed to pro-myelinating factors. Yet, even in these conditions the maturation potential of Cit-k KO OPCs was lower than that of WT cells, indicating the co-existence of cell-autonomous brakes to maturation. Consistently, oligodendrocyte-specific Cit-k mutants (i.e. Sox10Cre;Cit-kfl/fl mice) showed only a partial recovery of the forebrain myelination pattern compared to the germinal Cit-k KO mouse brain. In the adult Sox10Cre;Cit-kfl/fl mice, hypomyelination persisted in the cerebral cortex –where OPC density was normal or only slightly reduced– and was associated with spatial memory defects and subtle motor deficits.
    Overall, these data indicate that Cit-k loss leads to a functionally-relevant and regionalized hypomyelination via cell autonomous and non-cell autonomous mechanisms.

  • Generation and characterization of a GALC knock-out human oligodendrocyte cell line (MO3.13) using CRISPR/Cas9 methodology for studying Krabbe disease.

    Authors:
    Miriam De Sarlo (1), Luca Scaccini (1), Laura Colagiorgio (1), Ambra Del Grosso (1), Sara Carpi (1), Ilaria Tonazzini (1), Husam B. R. Alabed (2), Roberto Maria Pellegrino (2), Carla Emiliani (2), Marco Cecchini (1)

    Organisations:
    1: NEST, Istituto Nanoscienze-CNR and Scuola Normale Superiore
    2: Department of Chemistry, Biology, and Biotechnologies, University of Perugia, Perugia Italy

    Presenting author: Miriam De Sarlo

    Exposition date: Saturday, September 16, 2023

    Exposition position: 047/2

    Krabbe leukodystrophy (KD) is a rare autosomal recessive disease caused by the defective gene GALC encoding galactosylceramidase, which is an important enzyme in lysosomal catabolism. In physiological conditions, GALC hydrolyzes the galactose ester bonds of glycolipids such as galactosylceramide and galactosylsphingosine (commonly referred to as psychosine, PSY). On the other hand, the GALC activity deficiency leads to PSY accumulation in myelinating cells - oligodendrocytes in the central nervous system and Schwann cells in the peripheral nervous system - and induces widespread demyelination followed by devasting neurodegeneration. In the scientific literature, it is known that GALC correction and PSY clearance might not be sufficient to completely restore the KD phenotype, suggesting that not yet identified molecular mechanisms could be involved in the KD pathogenesis.

    Here, we report on a new in vitro cellular model of Krabbe disease, based on the human oligodendrocyte cell line MO3.13. We took advantage of the genome editing CRISPR/Cas9 system for creating a stable GALC-deficient cell line. The experimental flow was based on several steps for reagent design, construction of single guide RNA (sgRNA)-expressing plasmids, transfection and cell line expansion and characterization. We could successfully isolate and identify an isogenic cell line with a homozygous deletion of 19 nucleotides in exon 7 of the GALC gene. Exon 7 contains the nucleophilic domain, common for all isoforms and important for GALC function. To verify whether this mutation could have altered GALC activity, we performed the quantification of galactosylceramidase activity on cellular lysates by 6-hexadecanoylamino-4-methylumbelliferyl-b-D-galactopyranoside (HMU-βGal) assay. The GALC-/- cells showed a decreased enzymatic activity compared to wild-type ones. These preliminary results suggest the successful generation of a stable model, but further experiments are ongoing to validate this line as a new tool useful to explore the hallmarks of Krabbe disease and to test new interventions against oligodendrocyte alterations.

  • Insulin sensitivity in microglial cells: the impact of neuroinflammation

    Authors:
    Enrica Marzani (1), Elisa Benetti (1), Arianna Carolina Rosa (1), Massimo Collino (2), Simona Federica Spampinato (1)

    Organisations:
    1: Dipartimento di Scienza e Tecnologia del Farmaco, Università di Torino, Italy
    2: Department of Neurosciences "Rita Levi Montalcini", Università di Torino, Italy

    Presenting author: Enrica Marzani

    Exposition date: Saturday, September 16, 2023

    Exposition position: 051/2

    Several studies have convincingly demonstrated that Insulin resistance (IR) and disglycemia can increase the susceptibility to neurodegeneration. Despite the mechanisms underlying this relationship are still unknown, a key role for a low grade inflammatory response, known as metabolic inflammation or metaflammation, has been suggested. In the brain, insulin modulates hippocampal function and memory tasks, and lack of insulin signaling causes neuronal death and glial activation. Reactive microglia, the immune resident component of the CNS, contributes to the onset of neuroinflammation, producing cytokines and ROS. Here we investigated the effects of insulin on microglia and the role of neuroinflammation in modulating microglial response to insulin. A human derived microglia cell line (HMC3) was exposed to insulin (100 nM) in the presence or absence of inflammatory cytokines (TNFα+IFNγ, TI). Our results show that short term (30 minutes) exposure to insulin evoked a significant activation of the insulin signaling pathway (AKT and GSK3β phosphorylation) in microglia, whereas long term (24h) exposure resulted in insulin resistance not reverted by intermittent pulse with insulin for 30 min. We also detected Glut-4 and Glut-5 transporters in HMC3 and demonstrated that their expression was affected by insulin exposure in a time-dependent manner. Interestingly, microglial exposure to TI affected transporters expression and inhibited insulin effects on their activation. Ongoing experiments aim to elucidate the molecular mechanisms underlying these interactions, focusing mainly on the role of the NLRP3 and NF-kB pathways, we previously documented to significantly affect insulin sensitivity in the peripheral system. In conclusion, our preliminary results indicate that insulin promotes intracellular changes in microglia and that microglial response to insulin is affected by inflammatory conditions. Overall, these findings reveal that HMC3 cell line can represent an interesting experimental model to study inflammatory insults and molecular mechanisms leading to perturbations in insulin response within the CNS.

  • Metabolic cross-talk between astrocytes and neurons: implications for Alzheimer’s disease

    Authors:
    Alessandra Preziuso, Silvia Piccirillo, Giorgia Cerqueni, Tiziano Serfilippi, Valentina Terenzi, Vincenzo Lariccia, Salvatore Amoroso, Simona Magi

    Organisations:
    Department of Biomedical Sciences and Public Health, School of Medicine, University “Politecnica delle Marche”, Via Tronto 10/A, 60126, Ancona, Italy.

    Presenting author: Alessandra Preziuso

    Exposition date: Saturday, September 16, 2023

    Exposition position: 044/2

    Neurodegenerative disorders, such as Alzheimer's disease (AD), have been associated with declining brain energetics due to impaired glucose and oxygen metabolism, mitochondrial dysfunction, and oxidative stress [1]. Astrocytes, which play a crucial role in neuronal metabolism and communication, may contribute to the development and progression of AD. By modulating the release of gliotransmitters (e.g. glutamate), they provide a fine control of neuronal metabolism and cell-to-cell communication, both of them involved in AD [2]. Impaired astrocytes metabolism may incite an inflammatory response that, in turn, negatively impacts neuronal viability. In this study, primary rat cortical astrocytes were exposed to glyceraldehyde (GA) [3] to induce metabolic stress. We evaluated cell viability, cytokine release, and the involvement of the nuclear factor-kappa B (NF-kB) pathway. We also evaluated the expression of glutamate transporters GLT-1 and GLAST and the effect of glutamate on ATP levels under physiological conditions. We explored whether damaged astrocytes could elicit neuronal death in a co-culture setting. The results indicated that exposure to GA 1 mM led to a concentration-dependent cell injury in rat cortical astrocytes, starting to cause a significant cell damage after 24 hours. GA treatment also activated the NF-kB pathway and increased release of TNFα after 48 hours. GA exposure significantly increased the levels of GLAST and GLT1 after 48 hours. Of note, when injured astrocytes were co-cultured with cortical neurons, neuronal viability significantly decreased. Under physiological conditions, glutamate exposure significantly improved intracellular ATP levels after 4 and 8 hours of treatment. These findings suggest that metabolically impaired astrocytes may play a role in damaging neighboring neurons, implicating astrocytes in the pathophysiology of AD. Therefore, astrocytes can be considered a key therapeutic and neuroprotective target for future studies.

    1. Nat Rev Neurosci 2019 Mar;20(3):148-160.
    2. Front Neurol. 2021; 12: 619626.
    3. Cells. 2021 Aug 17;10(8):2109.

  • Modelling Alzheimer’s disease-related astrocytic dysfunction in immortalized hippocampal astrocytes from 3xTg-AD mice: from loss of proteostasis to homeostatic failure

    Authors:
    Giulia Dematteis (1), Laura Tapella (1), Chunmei Gong (2), Mariagrazia Grilli (1), Armando A Genazzani (1), Marcello Manfredi (3), Laura Bonfili (2), Anna Maria Eleuteri (2), Dmitry Lim (1)

    Organisations:
    1: Department of Pharmaceutical Sciences, Università del Piemonte Orientale, Novara, Italy
    2: School of Biosciences and Veterinary Medicine, University of Camerino, Camerino, Italy
    3: Department of Translational Medicine, Center for Translational Research on Autoimmune and Allergic Diseases (CAAD), Università del Piemonte Orientale

    Presenting author: Dmitry Lim

    Exposition date: Saturday, September 16, 2023

    Exposition position: 050/2

    Continuous failure of clinical trials against Alzheimer’s disease (AD) urges for better investigation of AD pathogenesis especially in preclinical and prodromal periods which represent the only window for disease-modifying therapeutic intervention. Astrocytes, principal homeostatic cells in the CNS, change early in AD, and have been proposed to play key role in neuronal vulnerability and dysfunction. However, our understanding of early AD-related astrocytic pathophysiology is incomplete, in part linked to unavailability of easy-to-handle, cost-effective, scalable and reproducible astrocytic cellular AD model.

    Recently, we generated immortalized hippocampal astrocytes from 3xTg-AD mice (WT-iAstro and 3Tg-iAstro cells), suitable for comprehensive investigation of cellular pathophysiology. WT-iAstro express specific astrocytic markers and Kir4.1 current, while 3Tg-iAstro cells maintain transcriptional profile of primary hippocampal 3xTg-AD astrocytes and release Aβ. We further found that 3Tg-iAstro have enhanced agonist-induced cytosolic Ca2+ signals, while mitochondrial Ca2+ signals are suppressed, associated with reduced ATP- and enhanced ROS production. Mass spectrometry proteomic analysis suggested alterations of proteostasis, in fact, in 3Tg-iAstro cells, protein synthesis, proteasomal and autophagic degradation were impaired, while immunoproteasome components and interferon-gamma were strongly up-regulated. Alterations in proteostasis were accompanied by a low-grade ER-stress/UPR, phenomena, found also in 3xTg-AD mouse brain. Secretome analysis suggest reduction of adhesion and extracellular matrix molecules and neuroprotective factors in 3Tg astrocytes, functionally linked to reduced support of neuronal growth/differentiation and inability to maintain blood-brain barrier integrity. Strikingly, chemical chaperone, 4-PBA, approved by FDA and tested in AD mouse models, fully reverted alterations in 3Tg-iAstro cells at the biochemical and functional levels.

    In conclusion: i) 3Tg-iAstro cells represent versatile, accessible and reproducible model to study astrocytic AD-related cell pathophysiology; ii) dysproteostasis, caused by AD mutations and/or Aβ accumulation in astrocytes leads to loss of their homeostatic functions; iii) astrocytes and their proteostatic machinery represent potential target for development of disease-modifying therapy in AD.

  • N-acetyl cysteine rescues cortical glial cell populations and results in functional improvements in a mouse model of primary autosomal recessive microcephaly 17 (MCPH17)

    Authors:
    Maryam Khastkhodaei Ardakani (1), Cecilia Astigiano (1), Anna Incerti Tinterri (1), Francesco Ferrini (2), Chiara La Rosa (3), Serena Bovetti (3), Roberta Schellino (1), Marina Boido (1), Annalisa Buffo (1), Enrica Boda (1)

    Organisations:
    1: Dept. of Neuroscience and Neuroscience Institute Cavalieri Ottolenghi, Università degli Studi di Torino, Torino, Italy
    2: Dept. of Veterinary Sciences, Università degli Studi di Torino, Torino, Italy
    3: Dept. of Life Sciences and Systems Biology and Neuroscience Institute Cavalieri Ottolenghi, Università degli Studi di Torino, Torino, Italy

    Presenting author: Maryam Khastkhodaei Ardakani

    Exposition date: Saturday, September 16, 2023

    Exposition position: 045/2

    Primary autosomal recessive microcephaly 17 (MCPH17) is a rare neurodevelopmental disorder caused by mutations in the CIT gene, which encodes for the Citron Kinase (CIT-K), a kinase involved in DNA repair and cytoskeletal dynamics. Patients show reduced brain volume, intellectual disability, motor deficits, epilepsy, and early mortality. Cit-k KO mice recapitulate MCPH17 phenotype. In the Cit-k KO mouse brain, DNA damage and reactive oxygen species (ROS) accumulation is accompanied by neural progenitor apoptosis and glial cell alterations, including oligodendroglia and astroglia reduction, hypomyelination, and increased numbers of microglia presenting dysmorphic features and engulfed synaptic material. To identify pharmacological treatments that can reduce cellular damage accumulation and improve the functional phenotype of Cit-k KO mice, we chronically treated Cit-k KO mice during the first 2 postnatal weeks with an FDA-/EMA-approved antioxidant drug N-acetylcysteine (NAC), which can pass the blood-brain-barrier. NAC treatment reduced brain ROS levels and slightly increased Cit-k KO mouse life span. Treated mice showed motor improvement and reduction of epileptic myoclonus. Deposition of perineuronal nets around cortical parvalbumin-positive interneurons was significantly rescued by NAC treatment, suggesting a positive effect on the maturation/function of inhibitory neurons. Although NAC did not rescue Cit-k KO mouse hypomyelination, cortical oligodendrocytes and astrocytes significantly increased in numbers, while microglia density and dysmorphic features decreased. Our data suggest, in the Cit-k KO mouse cortex, NAC-induced functional improvements may be at least in part mediated by the correction of Cit-k KO glial cell dysfunctions. Ongoing analyses will unveil the functional bases of NAC effects.

  • Nerve growth factor influences microglial activity via TrkA receptors

    Authors:
    Giulia Borgonovo (1), Alexia Tiberi (1,2), Giovanna Testa (1), Paola Pacifico (1,2), Ajesh Jacob (1), Mariachiara Di Caprio (1), Mariantonietta Calvello (1), Simona Capsoni (1,3), Antonino Cattaneo (1,4)

    Organisations:
    1: Scuola Normale Superiore, Italy
    2: Inst. of Neuroscience, National Research Council, Pisa, Italy
    3: University of Ferrara, Italy
    4: European Brain Research Institute (EBRI), Rome, Italy

    Presenting author: Giulia Borgonovo

    Exposition date: Saturday, September 16, 2023

    Exposition position: 052/2

    The Nerve Growth Factor (NGF), in addition to supporting neurons, has pleiotropic actions on non-neuronal cells, especially in the immune system. Indeed, peripheral immune cells secrete and respond to NGF, by expressing TrkA receptors. Recently, our lab discovered potent immunomodulatory properties of NGF via TrkA on microglia in vitro: NGF steers them toward a neuroprotective and anti-inflammatory phenotype. Here, we provide in vivo evidence of a (neuroprotective) role for NGF signaling in brain microglia, both in (1) pathological and (2) physiological conditions. (1) We first considered Rett syndrome (RTT) as a neurodevelopmental disease that could benefit from NGF and that displays the pathogenic contribution of microglia. Thus, we tested the therapeutic effects of a mutein of the NGF, called human NGF painless (hNGFp), via intranasal delivery in a female RTT model, the MeCP2+/- mice. We reveal a deficit in microglial morphology in MeCP2+/- mice completely reversed in treated animals. We also analyzed the cytokine profile after hNGFp treatment in MeCP2+/- mice, to discover that the treatment recovered the altered expression of key neuroimmune-communication molecules. The conclusion is that hNGFp can ameliorate symptoms in the MeCP2+/- model, by exerting strong neuroprotection also via microglia. (2) Secondly, to directly assess the functional role of the microglial NGF-TrkA signaling, we generated a novel inducible transgenic mouse line, in which TrkA can be specifically deleted in microglia. We report that knocking out the microglial NGF-TrkA signaling leads to a reduction of microglial density in the primary somatosensory cortex. We also found differences in microglial morphology and phagocytosis of dendritic spines. Lastly, at a behavioral level, microglial NGF-TrkA signaling deletion affects memory and learning. Altogether, these overall data suggest that modulating the NGF-TrkA axis on microglia in vivo might be harnessed as a broad neuroprotective strategy for CNS neuronal populations that are not direct NGF-targets.

  • Novel defective pathways in astrocyte-neural progenitor cell cross-talk in a murine model of Down Syndrome

    Authors:
    Valeria Bortolotto (1), Maria Elisa Salvalai (1), Morris Losurdo (1), Marcello Manfredi (2), Pier Luigi Canonico (1), Mariagrazia Grilli (1)

    Organisations:
    1: Dept. Pharmaceutical Sciences, University of Piemonte Orientale, Italy
    2: Dept. Translational Medicine & CAAD, University of Piemonte Orientale, Italy

    Presenting author: Mariagrazia Grilli

    Exposition date: Saturday, September 16, 2023

    Exposition position: 054/2

    Down syndrome (DS) is a neurodevelopmental disorder caused by triplication of chromosome 21 and the most common genetic cause of intellectual disability. Among well characterized preclinical DS models the Ts65Dn mouse line recapitulates key disease features such as reduced proliferation of neural progenitor cells (NPC), reduced neurogenesis, with, in parallel, increased astrogliogenesis and, importantly, cognitive impairment. In recent years it has been suggested that trisomy-linked brain abnormalities, including cognitive impairment, may be pharmacologically corrected by targeting, in early stages of life, NPC defects. Interestingly, NPC dysfunctional communication with other neural cell types may also contribute to DS pathophysiology. Indeed DS astroglia also exhibit functional alterations that can affect NPC and their progeny, including defective release of signals like thrombospondin 1 (TSP-1), a key protein involved in synaptogenesis and spine formation. At present, the physiopathological role of TSP-1 in DS and other CNS disorders is underexplored. For these reasons we investigated TSP-1 signalling in Ts65Dn (Trisomic, TS) and Euploid (EU) NPC and in their cross-talk with TS/EU astrocytes. We confirmed previous findings that TS astrocytes release less TSP-1 compared to EU astrocytes, but also discovered that TS NPC are quite defective in their response to TSP-1. Among the multiple receptors mediating TSP-1 modulatory effects in NPC we focussed our interest on the α2δ1 subunit of voltage-sensitive calcium channesl and downstream signaling events in TS versus EU NPC. By using a LC-MS/MS proteomic approach we identified several novel signaling pathways which were significantly disrupted downstream TSP-1/α2δ1 interaction in trisomic NPC. Moreover we disclosed novel pathways which are involved in cell fate specification of euploid neural progenitors cells. We will discuss how that these findings hold the potential to unravel novel molecular participants involved in DS brain abnormalities but also in other developmental and neurodegenerative CNS disorders.  

  • Recovering STIMulation of astrocyte Ca2+ signal to shed light on Alzheimer’s Disease

    Authors:
    Annamaria Lia (1,2), Gabriele Sansevero (3,4), Angela Chiavegato (2), Miriana Sbrissa (2), Diana Pendin (1,2), Letizia Mariotti (1,2), Tullio Pozzan (1,2,5), Nicoletta Berardi (3,4), Giorgio Carmignoto (1,2), Cristina Fasolato (2), Micaela Zonta (1,2)

    Organisations:
    1: Neuroscience Institute - CNR, Padova, Italy
    2: Department of Biomedical Sciences - University of Padua, Italy
    3: Neuroscience Institute - CNR, Pisa, Italy
    4: Department of NEUROFARBA, University of Florence, Italy
    5: Veneto Institute of Molecular Medicine (VIMM), Padova, Italy

    Presenting author: Annamaria Lia

    Exposition date: Saturday, September 16, 2023

    Exposition position: 042/2

    Alzheimer’s disease (AD) is a chronic incurable neurodegenerative disorder characterized by progressive memory loss and cognitive dysfunctions. Brain function is governed by dynamic interactions between neurons and astrocytes. Noteworthy, calcium dynamics in astrocytes represent a fundamental signal that through gliotransmitter release regulates synaptic plasticity and behaviour. Here, by using cutting-edge techniques including 2-photon Ca2+ imaging, electrophysiology and behavioural memory tests, we present a longitudinal study in the PS2APP mouse model of AD linking astrocyte Ca2+ hypoactivity to memory loss. At the onset of plaque deposition, somatosensory cortical astrocytes of AD female mice swtich to a reactive pro-inflammatory state and exhibit a drastic reduction of Ca2+ signaling, closely associated with decreased endoplasmic reticulum Ca2+ concentration and reduced expression of the Ca2+ sensor STIM1. In parallel, astrocyte-dependent long-term synaptic plasticity declines in the somatosensory circuitry, anticipating specific tactile memory loss. Notably, we show that both astrocyte Ca2+ signaling and long-term synaptic plasticity are fully recovered by selective STIM1 overexpression in astrocytes. Our data unveil astrocyte Ca2+ hypoactivity in neocortical astrocytes as a functional hallmark of early AD stages and indicate astrocytic STIM1 as a target to rescue memory deficits.

  • Role of mitochondria-ER interaction in astrocyte cellular disfunction associated with neurodegeneration

    Authors:
    Giulia Dematteis (1), Laura Tapella (1), Fabio Cavaliere (2), Mariagrazia Grilli (1), Armando Genazzani (1), Dmitry Lim (1)

    Organisations:
    1: Department of Pharmaceutical Sciences, Università del Piemonte Orientale, Via Bovio 6, 28100, Novara, Italy
    2: Achucarro Basque Center for Neuroscience, Leioa, Spain

    Presenting author: Giulia Dematteis

    Exposition date: Saturday, September 16, 2023

    Exposition position: 049/2

    Neurodegenerative diseases are characterized by long lasting and complex pathogenesis, that dramatically impact on the determination of disease primary events, and consequently to the development of disease modifying therapy. In both Parkinson disease (PD) and Alzheimer disease (AD) bioenergetic deficit and intracellular calcium deregulation have been described. Moreover, in both these pathologies alteration mitochondria-Endoplasmic Reticulum contact sites (MERCS) has been reported in neurons, representing a possible connection point between calcium signalling and bioenergetics. Indeed, mitochondrial calcium uptake, from the ER, occurs at MERCS via IP3R-VDAC complex, and regulates mitochondrial metabolism is regulated at different level.

    Reduced mitochondrial metabolism has been described in astrocytes form AD model (AD Astro), and in hiPSC-derived astrocytes from PD patients with LRRK2(G2019S) mutation (PD Astro). Therefore, we hypothesized that bioenergetic deficit identified in AD Astro and PD Astro, could be due to altered ER-Mit interaction, and calcium signalling.

    To validate this hypothesis we measured, using genetically encoded calcium probes, calcium singling in ER and mitochondria, and we accessed Mit-ER interaction via Split-GFP contact sites sensors (SPLICS) and electron microscopy.

    Both AD and PD Astro displayed reduced mitochondrial calcium, while in AD Astro ER calcium release was increases, and in PD Astro it was reduced. Evaluating Mit-ER distances we found an opposite behaviour between AD Astro and PD ones: AD Astro presented an increase of 8-10 nm distances, while PD Astro presented an increase of 50nm distances, and both presented a reduction of interactions at 20nm, that is the optimal distance to allocate ER-Mit calcium transfer complex. We proposed that restoring interactions at 20nm may solve Mit-calcium signalling, and as a proof of principle, we overexpressed in AD Astro a 20nm ER-Mit linker, and we rescued Mit-caclium uptake. In summary, our results point out MERCS alteration as an early event in astroglial dysfunction in neurodegeneration.

  • The interaction between gut microbiota and peripheral nervous system

    Authors:
    Giulia Ronchi (1), Matilde Cescon (2), Giovanna Gambarotta (1), Sonia Calabrò (2), Davide Pellegrino (1), Chiara Cicconetti (3), Francesca Anselmi (3), Svenja Kankowski (4), Luisa Lang (4), Marijana Basic (5), Andre Bleich (5), Silvia Bolsega (5), Salvatore Oliviero (3), Stefania Raimondo (1), Dario Bizzotto (2), Kirsten Haastert-Talini (4,6)

    Organisations:
    1: Department of Clinical and Biological Sciences & Neuroscience Institute Cavalieri Ottolenghi (NICO), University of Torino, Orbassano, 10043, (Torino), Italy
    2: Department of Molecular Medicine, University of Padova, Padova, Italy
    3: Department of Life Sciences and Systems Biology, University of Torino, Torino, Italy
    4: Institute of Neuroanatomy and Cell Biology, Hannover Medical School, 30625 Hannover, Lower-Saxony, Germany
    5: Institute for Laboratory Animal Science and Central Animal Facility, Hannover Medical School, 30625 Hannover, Lower-Saxony, Germany
    6: Center for Systems Neuroscience (ZSN), Hannover, 30559 Hannover, Lower-Saxony, Germany

    Presenting author: Giulia Ronchi

    Exposition date: Thursday, September 14, 2023 (Satellite Events Day)

    Exposition position: 93

    The gut microbiota (the overall constellation of microorganisms that reside in the human gut) is responsible for essential functions in human health and it has been demonstrated that its perturbation is implicated in a growing list of neurodegenerative diseases and psychiatric disorders. Recent studies have started investigating the interactions between gut microbiota and various organs and systems besides brain, including lung, liver, bone, cardiovascular system, and others.

    To date, no studies have reported how the gut microbiota regulates the development or functions of the peripheral nervous system (PNS). In the current study, we analyzed nerves and dorsal root ganglia (DRG) of neonatal and adult mice: a) lacking all microorganisms throughout their lifetime (GF, Germ free); b) GF selectively colonized with 12 known bacteria (OMM12, Gnotobiotic); c) colonized with a normal gut microbiome (CGM, Complex Gut Microbiota).

    Stereological and morphometrical analysis mainly revealed that absence of gut microbiota impairs the development of peripheral nerves, resulting in smaller diameter and hypermyelinated axons, as well as smaller unmyelinated fibers. Accordingly, transcriptomic analysis on adult DRG and sciatic nerves highlighted a panel of differentially expressed developmental and myelination genes. Finally, we also analysed skeletal muscles and we found atrophic muscle and immature neuromuscular junctions in GF mice.

    Taken together, these results evidence a regulatory impact on proper development of the peripheral nervous system, thus establishing the existence of a novel ‘Gut Microbiota-Peripheral Nervous System-axis’

Innovative methods and technologies in neuroscience

  • Assessing the effects of transcutaneous vagus nerve stimulation on noradrenaline related cognitive function: the effects on alerting and its possible implication for Tourette syndrome

    Authors:
    Sara Boscarol (1), Elisabetta Ferrari (1), Viola Oldrati (1), Niccolo Butti (1), Cosimo Urgesi (1,2), Alessandra Finisguerra (1)

    Organisations:
    1: Scientific Institute, IRCCS E. Medea, Italy, Italy
    2: 3Laboratory of Cognitive Neuroscience, Department of Languages and Literatures, Communication, Education and Society, University of Udine, Udine, Italy

    Presenting author: Sara Boscarol

    Exposition date: Friday, September 15, 2023

    Exposition position: 081/1

    Objective. Tourette syndrome (TS) is a neurodevelopmental disorder characterized by vocal and motor tics. Even though the central neural mechanisms of tic generation are still debated, impaired cortico-striato-thalamo-cortical activity and a general elevation of the sympathetic (over the parasympathetic) tone are important signatures of TS. To increase tic control, the use of pharmacological treatments targeting the dopaminergic system, but also the GABA and Noradrenergic systems has been proposed. Previous studies have suggested that the invasive stimulation of the vagus nerve may trigger the Noradrenergic system activity and modulate noradrenaline NE transmission. As a preliminary investigation on the effectiveness of transcutaneous vagus nerve stimulation (tVNS) in TS, here we investigated the effects of tVNS in modulating alerting, which is known to be a proxy of NE concentration. Material. We applied active and sham tVNS during the execution of the Attentional Network Test (ANT). This test allows measuring the effectiveness of three cognitive and attentive components, namely the alerting, the orienting, the executive control components, which are thought to rely, respectively, over the noradrenergic, cholinergic and dopaminergic systems. Methods. 40 healthy participants underwent a two-sessions, sham-controlled, within-subjects single-blind design in which active or sham tVNS was applied during the execution of the ANT. Results. The results showed that active tVNS, with respect to sham, selectively boosted the alerting, but not the orienting and the executive control components. Discussion. Our data provides evidence for the effects of tVNS on alerting response, and this facilitation could be related to the regulation of noradrenergic activity. Conclusions. By considering the wide spectrum of effects induced by tVNS, encompassing also an infliuence on GABA neurotransmission and autonomic regulation, this finding encourages the development of future clinical studies using tVNS for addressing TS symptomatology.

  • Brain complexity in stroke recovery after bihemispheric tDCS

    Authors:
    Francesca Miraglia (1,2), Chiara Pappalettera (1,2), Maria Vittoria Podda (3,4), Saviana Barbati (3,4), Lorenzo Nucci (1,3), Alessia Cacciotti (1,2), Claudio Grassi (3,4), Paolo Maria Rossini (1), Fabrizio Vecchio (1,2)

    Organisations:
    1: Brain Connectivity Laboratory, Department of Neuroscience and Neurorehabilitation, IRCCS San Raffaele Roma, Rome, Italy
    2: Department of Theoretical and Applied Sciences, eCampus University, Novedrate, Como, Italy
    3: Department of Neuroscience, Università Cattolica del Sacro Cuore, Rome, Italy
    4: Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy

    Presenting author: Francesca Miraglia

    Exposition date: Saturday, September 16, 2023

    Exposition position: 057/2

    Stroke is one of the leading causes of disability worldwide.There are many different rehabilitation approaches aimed at improving clinical outcomes for stroke survivors. One of the latest therapeutic techniques are the Non Invasive Brain Stimulation (NIBS) tools. Among these, the transcranial direct current stimulation(tDCS) has demonstrated promising results in enhancing motor and cognitive recovery in animal models of stroke and stroke survivors. In this framework, one of the most innovative methods is the bihemispheric tDCS, based on simultaneously increasing excitability in one hemisphere and decreasing excitability in the contralateral one.As bihemispheric tDCS can create a more balanced modulation of brain activity, this approach may be particularly useful in counteracting imbalanced brain activity, such as in stroke. Giving these premises, the aim of the current study has been to explore the recovery after stroke in mice, that underwent a bihemispheric tDCS treatment by recording their electric brain activity with electroencephalography (EEG) and by measuring the behavioral outcomes of Grip Strength test. An innovative parameter that explores the complexity of signals, namely the Entropy, recently adopted to describe brain activity in physiopathological states,was evaluated based on EEG data. Results showed that stroke mice had higher values of Entropy compared to healthy mice, indicating an increase in brain complexity and signal disorder due to the stroke. Additionally, the bihemispheric tDCS reduced Entropy in both healthy and stroke mice compared to sham stimulated mice, with a greater reduction in stroke mice.Moreover, correlation analysis showed a negative correlation between Entropy and Grip Strength, indicating that higher Entropy values resulted in lower Grip Strength engagement. Concluding, the current evidence suggests that the Entropy index of brain complexity is able to characterize stroke pathology and recovery and the bihemispheric tDCS can modulate brain rhythms in animal models of stroke, providing potentially new avenues for rehabilitation in humans.

  • Comparison of Anesthetics Prior to Microwave Fixation for Preservation of Glycogen

    Authors:
    Maria Fernanda Veloz Castillo (1,2,3), Pierre J. Magistretti (1), Corrado Cali (2,3)

    Organisations:
    1: BESE Division, King Abdullah University of Science and Technology, Saudi Arabia
    2: Department of Neuroscience “Rita Levi-Montacini”, University of Turin, Italy
    3: Neuroscience Institute Cavalieri Ottolenghi, Orbassano, Italy

    Presenting author: Maria Fernanda Veloz Castillo

    Exposition date: Friday, September 15, 2023

    Exposition position: 079/1

    Glycogen is the largest cerebral energy reservoir and is localized in astrocytes under normal physiological conditions. Astrocytes possess the enzymatic machinery necessary for glycogen breakdown and its further conversion to lactate, which could be shuttled to neurons via monocarboxylate transporters to fuel their tricarboxylic acid cycle (a mechanism known as Astrocyte-Neuron Lactate shuttle, or ANLS). Therefore, one of the primary roles of glycogen is to provide a metabolic buffer during neurotransmission. Multiple studies report the role of glycogen metabolism in long-term memory formation, learning-dependent synaptic stabilization, memory consolidation, and maintenance of long-term potentiation. Nonetheless, accurately quantifying brain glycogen levels at different stages of the memory consolidation process is still a challenge. In the present work, we use a microwave fixation system as a euthanasia and fixation method to preserve glycogen levels in a way it reflects in vivo conditions. We compare the effects of six different anesthetic cocktails and three microwave fixation settings to determine the adequate condition for glycogen quantification. We found significant differences in brain anatomical preservation across the different fixation settings and clear sex-specific anesthetics that less alter brain glycogen concentration. Finally, our results support the idea that proper biochemical quantification of brain metabolites requires a special experimental setup that needs to be finely tuned.

  • CRISPR/Cas9-based approaches for Frataxin gene reactivation in Friedreich’s ataxia

    Authors:
    Elena Melacini (1,2), Sharon Muggeo (1), Mirko Luoni (1,2), Serena Giannelli (1), Margherita Rossi (1), Vania Broccoli (1,2)

    Organisations:
    1: San Raffaele Scientific Institute, Italy
    2: National council of reasearch (CNR), Milan, Italy

    Presenting author: Elena Melacini

    Exposition date: Friday, September 15, 2023

    Exposition position: 075/1

    Friedreich’s ataxia (FA) is an autosomal-recessive neurodegenerative disorder caused by the silencing of the Frataxin (FXN) gene, due to the expansion of GAA trinucleotide repeats in its first intron. Transcriptional activators constitute an innovative approach to overcome the repression of endogenous FXN. Moreover, newly available CRISPR/Cas9-associated systems represent a powerful editing platform for genome engineering. Intriguingly, the chance to selectively excise the FA pathological GAA expansion represents an intriguing opportunity for a permanent restoration of FXN expression.

    To promote FXN gene expression reactivation we compared two complementary CRISPR/Cas9 approaches based on either stimulate FXN expression or to excise the GAA pathological tract. For the former approach, FA patient fibroblasts were transduced with a lentiviral vector expressing the dCas9-VP160, containing the VP16 transactivator. Multiple sgRNAs targeting the FXN gene promoter were used to enhance the initiation of gene transcription. For the latter approach, we explored the compact and high-fidelity Neisseria meningitidis Cas9 (Nme2Cas9) in order to selectively remove the GAA pathological expansion. We produced an all-in-one AAV vector expressing Nme2Cas9 with two sgRNAs targeting the regions flanking the GAA triplet expansion.

    We demonstrated that the transcriptional activator, when combined with one or more selected sgRNAs, stimulate FXN expression by 2 to 4 fold, restoring RNA and protein levels comparable to the control condition. Simultaneously, we assessed the efficiency of the AAV-Nme2Cas9 system with two selected sgRNAs in exciding the GAA expansion in patient fibroblasts.

    Our findings indicate that dCas9-transcriptional activators can be effectively employed as tools for FXN gene reactivation in patient fibroblasts. Moreover, we confirmed that the NmeCas9 is an efficient nuclease that, giving its reduced size, is suitable for gene therapy application. These results pave the way for testing these approaches on FA mouse models to evaluate the efficiency of symptomatic rescue.

  • Effect of 3D Synthetic Microscaffold Nichoid on the Morphology of Cultured Hippocampal Neurons and Astrocytes

    Authors:
    Clara Alice Musi (1), Luca Colnaghi (2), Arianna Giani (1), Erica Cecilia Priori (1), Matteo Tironi (3), Claudio Conci (4), Giulio Cerullo (5), Roberto Osellame (5), Manuela Teresa Raimondi (4), Andrea Remuzzi (6), Tiziana Borsello (1)

    Organisations:
    1: University of Milan, Italy and Mario Negri Institute for Pharmacological Research-IRCCS Via Mario Negri, 2 20156 Milano Italy
    2: Division of Neuroscience, IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy and School of Medicine, Vita-Salute San Raffaele University, 20132 Milan, Italy
    3: Mario Negri Insitute for Pharmacolgical Research-IRCCS, Via Mario Negri, 2, 20156 Milano, Italy
    4: Department of Chemistry, Materials and Chemical Engineering "G. Natta", Politecnico di Milano, 20133 Milano, Italy
    5: Department of Physics, Istituto di Fotonica e Nanotecnologie (IFN)-CNR, Politecnico di Milano, 20133 Milano, Italy
    6: Department of Management, Information and Production Engineering, University of Bergamo, Via Marconi, 5, 24044 Dalmine, Italy

    Presenting author: Clara Alice Musi

    Exposition date: Friday, September 15, 2023

    Exposition position: 076/1

    The human brain is the most complex organ in biology. This complexity is due to the number of intricate connections of brain cells and has so far limited the development of in-vitro models for basic and applied brain research. We decided to create a new, reliable, and cost-effective in-vitro system of hippocampal neurons and astrocytes co-cultured based on the Nichoid, a 3D microscaffold microfabricated by two-photon laser polymerization technology.

    After 21 days in culture, we morphologically characterized the 3D spatial organization of the hippocampal astrocytes and neurons within the microscaffold and we compared our observations to those made using the classical 2D co-culture system. We found that the co-cultured cells colonized the entire volume of the 3D devices. Using confocal microscopy, we observed that the different cell types had well differentiated. This was further elaborated with the use of Drebrin and PSD-95 as markers for mature and differentiated dendritic spines. Drebrin and PDS95 labeled the majority of neurons both in the 2D as well as in the 3D co-cultures. Using scanning electron microscopy, we found that neurons in the 3D co-culture displayed a significantly larger amount of dendritic protrusions compared to neurons in the 2D co-culture. This latter observation indicates that neurons growing in a 3D environment may be more prone to connections than those co-cultured in 2D. The next step will be the addition of microglia to complete the scenario of cells acting in synaptic function/dysfunction.

    Our results show that the Nichoid can be a 3D device exploitable to investigate the structure and morphology of cells in-vitro as well as the complex cell-cell interactions within the brain. This model can also potentially be used as a tool to study mechanisms governing synaptic plasticity/dysfunction and to drug discovery.

  • Efficiency and robustness in three cortical areas: frontal pole cortex, dorsolateral prefrontal cortex and orbitofrontal cortex

    Authors:
    Davide Cipollini (1,2), Fabrizio Londei (3,4), Aldo Genovesio (3)

    Organisations:
    1: Bernoulli Institute for Mathematics, Computer Science and Artificial Intelligence, University of Groningen, The Netherlands
    2: Cognigron - Groningen Cognitive Systems and Materials Center, Groningen, The Netherlands
    3: Department of Physiology and Pharmacology, Sapienza University, Rome, Italy
    4: PhD Program in Behavioral Neuroscience, Sapienza University, Rome, Italy

    Presenting author: Davide Cipollini

    Exposition date: Saturday, September 16, 2023

    Exposition position: 058/2

    In this work we analyze the functional differences between three cortical areas: frontal pole cortex, dorsolateral prefrontal cortex and orbitofrontal cortex. Functional differences are investigated under the lens of the coding mechanism developed by these three areas. Namely, we estimate robustness and efficiency in the coding mechanism similarly to the work of Pryluk et al. [1]. In their work the authors developed new entropy theoretical tools to demonstrate a trade-off between efficiency and robustness across species, humans and macaques, and regions, amygdala and cingulate cortex. They provided evidence suggesting that the functional gap between species and areas due to the many evolutionary years separating them, might also arise from the different coding mechanisms. Efficiency is intended as the capability to better use the information capacity of the channels-neurons, to ultimately develop cognition. To quantify the efficiency of a single area, we use a novel measure, contrast entropy [1], defined as the entropy of the neuronal spiking activity normalized by expected theoretical maximum, thus naturally defining an efficiency. On the other hand, robustness is intended as the ability to provide reliable responses to threats from the environment. It is measured as the synchrony of activity between neurons belonging to the same area but also as the “vocabulary” similarity, that is similarity in the distribution of “words”, through the Jensen-Shannon divergence normalized to the expected theoretical maximum. We find that the frontal pole cortex shows higher efficiency, in analogy to what would be expected from an evolutionary argument as this area is present only in humans and anthropoids. No differences are observed between areas when robustness is measured.

    [1] Pryluk, R., Kfir, Y., Gelbard-Sagiv, H., Fried, I., & Paz, R. (2019). A Tradeoff in the Neural Code across Regions and Species. In Cell (Vol. 176, Issue 3, pp. 597-609.e18). Elsevier BV. https://doi.org/10.1016/j.cell.2018.12.032

  • Examination of the Brain Areas by Importance Scores Using Braingraphs

    Authors:
    Dániel Hegedűs (1,2), Vince Grolmusz (1,2)

    Organisations:
    1: Eötvös Loránd University, Hungary
    2: PIT Bioinformatics Group

    Presenting author: Dániel Hegedűs

    Exposition date: Friday, September 15, 2023

    Exposition position: 073/1

    From diffusional MRI data of 1064 subjects, braingraphs are constructed: a braingraph is a mathematical graph, containing 1015 vertices, which correspond to the different brain areas. The vertices are connected by edges, which describe the connection between the pair of areas by axonal fibers. A consensus graph is calculated from all the 1064 braingraphs, in the motivation to interpret it as an average braingraph of the 1064 human subjects. Seven different orderings are calculated on the vertices by different importance aspects, including the average length, the average thickness and the number of axonal fibers for each vertex. The correlation between these orderings is proved using the Spearman correlation coefficient: all of these orderings carry great similarity with each other. This result means that the vertices having thick axonal fibers are usually the ones, which have either long or a large number of connected fibers. Furthermore, by calculating similar orderings for edges instead of vertices, we show that the edges, which are present in many subjects, also have high thickness. Furthermore, for each vertex, two different centroids are calculated, which describe the average position of the other endpoints of its edges. An attribute of the direction from the vertex to its centroid correlates with the previously described importance orderings. Also, restricted graphs are calculated for the individuals, and for the consensus graph too: for each vertex, only its one or two thickest edges are drawn. With these graphs, the connectivity through the thickest edges is investigated. The results are proven by strict statistical analyses.

  • Extracellular matrix deregulation in spinal cord organoids of amyotrophic lateral sclerosis patients

    Authors:
    Matteo Bordoni (1), Eveljn Scarian (1), Letizia Messa (2,3), Maria Garofalo (1), Emanuela Jacchetti (2), Manuela Teresa Raimondi (2), Luca Diamanti (1), Stella Gagliardi (1), Stephana Carelli (4), Cristina Cereda (3), Orietta Pansarasa (1)

    Organisations:
    1: IRCCS Mondino Foundation, Italy
    2: Politecnico of Milan, Italy
    3: Children's Hospital "V Buzzi", Italy
    4: University of Milan

    Presenting author: Matteo Bordoni

    Exposition date: Saturday, September 16, 2023

    Exposition position: 055/2

    Amyotrophic lateral sclerosis (ALS) is a non-cell autonomous disorder as many cell types contribute to motor neurons death. The lack of effective treatments is probably due to the absence of a realistic model. Organoids are pluripotent stem cell-derived self-organizing structures that allow in vitro generation of the tissues. We developed a new method for the generation of spinal cord organoids (SCOs). Aim of the work was to characterize a 3D organoid model for the study of ALS pathogenesis. Firstly, we found that SCOs derived from sALS patients were smaller and with irregular morphology compared to healthy controls. Using the GFAP marker, we found that sALS SCOs have a thicker glial layer compared to healthy controls. We also found that healthy controls show longer neurites compared to sALS SCOs. By RNAseq, we found a ten-fold increase of deregulated gene in SCOs respect to 2D cell models. Moreover, in ALS SCOs we found an extensive deregulation of genes involved in extracellular matrix organization when compared to control. By deconvolution analysis we found that healthy controls SCOs show a higher amount of differentiated cells compared to sALS SCOs. Finally, we compared transcriptomic profile of both sALS SCOs and human spinal cord tissue, finding many similarities. Our data suggest that brain organoids represent a promising tool for the investigation of pathogenic mechanisms of ALS. In fact, we found typical pathological hallmarks of the pathology, such as the presence of gliosis, the smaller length of neurites, decreased level of mature MNs, and deregulation of genes implicated in ALS. Moreover, we found strong evidences that SCOs share many characteristics with the pathological human spinal cord. In conclusion, SCOs represent a promising tool for the investigation of pathogenic mechanisms of ALS.

  • From diamond multiarrays to nanodiamonds: new probes for neuroscience down to the nanoscale

    Authors:
    Giulia Tomagra (1,4), Federico Picollo (2,4), Marco Genovese (3), Valentina Carabelli (1,4)

    Organisations:
    1: University of Torino -Department of Drug and Science Technology
    2: University of Torino - Physics Department - INFN,
    3: Istituto Nazionale di Ricerca Metrologica (INRIM), Torino
    4: NIS Inter-departmental research centre, Torino

    Presenting author: Giulia Tomagra

    Exposition date: Friday, September 15, 2023

    Exposition position: 074/1

    Advances to understanding brain complexity relies on the construction of biosensing probes which should be targeted to optimize the multiparametric detection of neuronal activity with increasing spatial- and time-resolution. Diamond offers excellent features for biosensing that are crucial for the fabrication of integrated sensors, namely wide optical transparency, large electrochemical window, high chemical inertness, good biocompatibility, the possibility of directly writing sub-superficial graphitic microelectrodes and realizing atomic-scale defects (nitrogen-vacancy (NV) centers), pivotal for sensing at the nanoscale.

    Taking advantage of these properties, we have realized different prototypes of diamond-based multi-electrode arrays (DBM) that are suitable to combine amperometric and potentiometric recordings: once interfaced with Substantia Nigra cultured neurons. DBMs allow to monitor over time, both neuronal firing discharges and quantal dopamine release in developing networks. As such, DBM provides a reliable probe for assessing the progressive impairment and the consequent lack of dopamine release during neurodegeneration.

    Another parameter whose detection is attracting a growing interest is the intracellular temperature: localized temperature changes are strictly coupled to neuronal activity, ion fluxes through the membrane, cell metabolism, which may be altered under pathological conditions, such as neurodegeneration or cancer. By exploiting the fluorescence of NV centers in nanodiamond, combined with the optically detected magnetic resonance (ODMR) technique, we could quantify the correlation of intracellular temperature changes with variations of neuronal activity. In cultured hippocampal neurons, NV centers in NDs revealed up to 1 °C temperature increase when the spontaneous firing was potentiated by blocking GABAergic synapses with picrotoxin or 0.5 °C temperature decrease when the neuronal activity was silenced by a solution containing TTX and CdCl2 to block voltage-gated Na+ and Ca2+ channels.

    Our results prove that NV-centers in the nanodiamonds represent a potent tool for mapping neuronal spiking activity changes with high sensitivity and spatial resolution.

  • Generation and characterization of ap1s2 mutant lines in Danio rerio

    Authors:
    Elena Massardi (1), Nicola Facchinello (2), Luca Mignani (1), Barbara Gnutti (1), Dario Finazzi (1), Eugenio Monti (1), Daniela Zizioli (1), Giuseppe Borsani (1)

    Organisations:
    1: University of Brescia, Italy
    2: Neuroscience Institute, National Research Council of Italy (CNR), Padova, Italy

    Presenting author: Elena Massardi

    Exposition date: Friday, September 15, 2023

    Exposition position: 078/1

    Adaptor protein 1 (AP-1) promotes cargo sorting between trans-Golgi network and early or recycling endosomes and it is composed of four subunits (γ1, β1 μ1 and σ1; Robinson, 2015). Although deficiency for the ubiquitous subunits γ1 and μ1A is embryonic lethal (Zizioli et al., 1999; Meyer et al., 2000; Zizioli et al., 2017; Mignani et al., 2023), Ap1s2 (σ1B) knockout mice are viable, but reveal impaired tissue functions (Glyvuk et al., 2010; Baltes et al., 2014). Mutations in the human orthologue cause the Pettigrew syndrome (MIM#304340), an X-linked intellectual disability characterized by mental retardation, choreoathetosis, hydrocephalus, Dandy-Walker malformation, seizures and iron or calcium deposition in the brain (Cacciagli et al., 2014).

    Main objective of this work is the integration of previous knowledge through the generation of zebrafish CRISPR/Cas9 knockout lines for ap1s2.

    The approach led to the generation of four mutations in the coding sequence. Among these, we decided to study lines characterized by deletion of 5 and 27 nucleotides. F2 generations produced the expected ratio for -/- individuals.

    Spatio-temporal expression studies of ap1s2 by ISH on wild-type AB embryos revealed a strong signal in the CNS at 24 and 48 hpf, which is weaker in ap1s2del5/del5 embryos but not in ap1s2del27/del27, data supported by qPCR analyses.

    The expression of key neural markers in ap1s2del5/del5 embryos (pax2a, ngn1, neurod1, dlx2a) appears to be slightly altered in different CNS areas (cranial ganglia, midbrain-hindbrain-boundary and telencephalon), supporting a role of the gene during neurogenesis.

    Preliminary behavioural analysis on 5 dpf larvae, using a standard dark/light routine protocol, showed a predisposition of mutants to move slower and travel shorter distances when compared to controls.

    Further investigations will include biochemical, histological and immunofluorescence analyses as well as detailed behavioural studies to verify the presence of alterations in the locomotor system or the memory tasks.

  • Mechanical stimuli as a neuroprotective strategy in Alzheimer’s disease

    Authors:
    Alice Alessandra Galeotti, Alessandro Falconieri, Vittoria Raffa

    Organisations:
    Università di Pisa, Dipartimento di Biologia, SS12 Abetone e Brennero 4, 56127, Pisa

    Presenting author: Alice Alessandra Galeotti

    Exposition date: Saturday, September 16, 2023

    Exposition position: 059/2

    Alzheimer's disease is a neurodegenerative disorder characterized by progressive loss of memory and cognitive function. The main histopathological features are amyloid-β plaques and Tau neurofibrillary tangles, that interfere with normal neuronal function and cause microtubules destabilization.

    It has long been known that mechanical stimulation of neurons by exogenous forces can induce axon growth1. Recently, our team set up a method to mechanically stretch axons through the administration of biocompatible magnetic nanoparticles and the application of an external static magnetic field2. This protocol was found to stimulate axonal growth and neuron maturation in hippocampal neurons.3 Our findings suggest a central role of microtubules stabilization in the molecular pathways activated by mechanical stimuli4. Indeed, the hypothesis behind the present study is that the microtubule stabilization promoted by the mechanical stimulation could prevent Tau alterations and their neurotoxic effects in neurodegeneration.

    To set up an Alzheimer’s disease cell model, primary mouse hippocampal neurons were treated with 1 μM amyloid-β peptides oligomers for 48 hours. A statistically significant shortening of axons in the treated samples compared to control ones was observed, confirming the generation of a neurodegeneration model. The neuroprotective effect of mechanical stimulation was then assessed, by stretching axons for 48 hours with pico-Newton forces. Data showed that mechanical stimulation prevented neurites shortening and damages to the axonal cytoskeleton. Future experiments will aim at validating this protocol at the functional level.

    1.          Raffa, V. Force: A messenger of axon outgrowth. Semin Cell Dev Biol (2022) doi:10.1016/j.semcdb.2022.07.004.

    2.          Raffa, V. et al. Piconewton Mechanical Forces Promote Neurite Growth. Biophys J 115, 2026–2033 (2018).

    3.          de Vincentiis, S. et al. Extremely Low Forces Induce Extreme Axon Growth. Journal of Neuroscience (2020) doi:10.1523/JNEUROSCI.3075-19.2020.

    4.          Falconieri, A. et al. Axonal plasticity in response to active forces generated through magnetic nano-pulling. Cell Rep 42, 111912 (2023).

  • Monkey ventral premotor cortex generates rhythmic activity during spontaneous walking

    Authors:
    Rossella Sini (1), Davide Albertini (2), Francesca Lanzarini (2,3), Monica Maranesi (2), Luca Bonini (2)

    Organisations:
    1: CNR Institute of Neuroscience, Parma, Italy
    2: University of Parma, Department of Medicine and Surgery, Parma, Italy
    3: Ernst Strüngmann Institute (ESI) for Neuroscience in Cooperation with Max Planck Society, Frankfurt am Main, Germany

    Presenting author: Rossella Sini

    Exposition date: Friday, September 15, 2023

    Exposition position: 077/1

    Quadrupedal walking is a whole-body naturalistic behavior, involving forelimbs and hindlimbs, which begins as a voluntary action and evolves into a stereotyped and rhythmic one. Classical monkey neurophysiological studies implied to keep the animal head-fixed on a primate chair, thereby preventing the investigation of spontaneous whole-body movements. Here, we leveraged a newly developed setup to record multisite neuronal activity from ventral premotor cortex (PMv), a region thought to be primarily related to hand-and-mouth goal directed actions, of two rhesus macaques while they spontaneously moved in a large plexiglass room enriched with several stimuli and monitored by an 8-camera system. We performed an ethological analysis of the videos, and aligned the spike trains of isolated neurons to specific event of the walking pattern (e.g. hand-ground contact, hand detachment). We found several PMv neurons that fired rhythmically in correspondence with specific phases of the walking cycle, with a prevalence at the ending of the swing phase of the arm contralateral to the recorded hemisphere. Moreover, some neurons responded differently when the monkey was walking on an elevated wooden structure compared to when it was walking on the floor, suggesting that PMv neuron activity can code different postures and muscle synergies to maintain balance. Electrical microstimulation of the chronically recorded region provided causal evidence that the recorded sites were indeed primarily related to the control of hand and mouth actions. Therefore, these results suggest that rhythmic signals of whole-body movements, such as walking, can be decoded even by using neural signals primarily linked to the control of different bodily actions (e.g. reaching-grasping or hand-to-mouth actions), paving the way to the exploitation of neural redundancy for improving neuroprosthetic applications.

  • Multifunctional magnesium alginate matrix for in vitro brain models as well as for brain regenerative approaches

    Authors:
    Giulia Della Rosa (1,2), Natalia Gostynska (1), John Wesley Ephraim (1,3), Stefania Bartoletti (4), Elisa Ren (4), Beatrice Casadei Garofani (4), Federica Raimondi (4), Francesca Ciarpella (5), Alessandra Campanelli (5), Ilaria Decimo (5), Gabriella Panuccio (1), Giulia Curia (4), Gemma Palazzolo (1)

    Organisations:
    1: Istituto Italiano di Tecnologia, Enhanced Regenerative Medicine, Italy
    2: University of Pavia, Department of Molecular Medicine, Pavia, Italy
    3: University of Genova, Department of Neuroscience, Rehabilitation, Ophtalmology, Genetics, Maternal and Child Sciences, Genova, Italy
    4: University of Modena and Reggio Emilia, Department of Biomedical, Metabolic and Neural Sciences, Modena, Italy
    5: University of Verona, Department of Diagnostics and Public Health, Pharmacology Section, Verona, Italy

    Presenting author: Gemma Palazzolo

    Exposition date: Saturday, September 16, 2023

    Exposition position: 060/2

    Sodium alginate (NaAlg) is an algal biopolymer turning into hydrogel by Ca2+-mediated crosslinking. By fine-tuning the polymer and crosslinker concentration it is possible to obtain very soft alginate hydrogels providing structural support and bioactive cues for neural stem cell (NSC) differentiation and functional neuronal network maturation. However, NaAlg undergoes a rapid gelation by Ca2+ ions, limiting the hydrogel homogeneity and mechanical stability for in vitro neural cultures as well as the in vivo injectability for neuroregenerative purposes.

    Here we present the magnesium salt of alginate (MgAlg), obtained by Na+ - Mg2+ exchange. First, we investigated the physicomechanical properties of MgAlg in comparison to NaAlg. Next, we assessed their potential to protect NSCs from oxidative stress and to promote functional neuronal networks in vitro. Finally, we evaluated the in vivo injectability, anti-inflammatory and neurotrophic activity of the two salts of alginate in an epileptic rat model.

    Mg2+ conferred multiple advantages to alginate, starting from the low viscosity, slow gelation kinetics and low stiffness. These features make MgAlg ideal for generating soft yet more homogeneous and stable hydrogels, suitable for 3D neural cultures achieving extensive neuronal maturation and functionality. In addition, MgAlg exerted a greater neuroprotection against oxidative stress, preventing NSC damage and oxidant species production. Preliminary in vivo studies indicate that both alginates work well as NSC delivery system in the lesioned brain of epileptic rats and elicit an anti-inflammatory response, demonstrated by the reduction of microglia cell body size and of glial scar thickness when co-injected with NSCs. However, the delayed gelation of MgAlg offers an advantage over NaAlg for minimally-invasive intracranial injection. Overall, introducing Mg2+ as the alginate counterion provides a multifunctional bioactive matrix suitable both for generating in vitro brain tissue models and for sustaining brain tissue repair/regeneration processes.

  • Nanocarriers as vectors to up- or downregulate signal transduction pathways in peripheral nerve regeneration

    Authors:
    Marina García Bejarano (1,2), Carlos Cuestas Ayllón (2), Isabelle Perroteau (1), Jesús Martínez de la Fuente (2), Giovanna Gambarotta (1)

    Organisations:
    1: Department of Clinical and Biological Sciences (DSCB) and Neuroscience Institute Cavalieri Ottolenghi (NICO), University of Torino, 10043 Orbassano (Torino), Italy
    2: Institute of Nanoscience and Materials of Aragon (INMA), University of Zaragoza, 50009 Zaragoza (Zaragoza), Spain

    Presenting author: Marina García Bejarano

    Exposition date: Thursday, September 14, 2023 (Satellite Events Day)

    Exposition position: 94

    Peripheral nerve injury is a relatively common event that is ineffective when the nerve injury is severe and the two nerve stumps are separated, requiring a graft to bridge them. Artificial conduits are frequently employed as an advantageous alternative approach to autografts. Macrophages colonize these scaffolds, releasing vascular endothelial growth factor (VEGF-A) -stimulating angiogenesis-, while nerve fibroblasts release soluble neuregulin 1 (NRG1) -stimulating Schwann cell dedifferentiation and migration- and VEGF-A, aiding in nerve regeneration. Furthermore, nerve fibroblasts mainly express the NRG1 α isoform, while Schwann cells primarily express the β isoform, but the different roles of these isoforms have not yet been fully elucidated.

    Regeneration within conduits is inefficient when the injury repair is delayed, possibly because cells in the distal portion of the injured nerve lose the ability to release adequate amounts of the factors necessary for nerve regeneration. Nanoscience plays a crucial role in addressing these limitations, offering therapeutic advancements to overcome these deficiencies. For this reason, in this work we analysed the factors released within the conduit after immediate repair and delayed nerve repair and we proposed to enrich nerve conduits with polymeric nanocapsules (PLGA, among others) containing VEGF-A and recombinant soluble NRG1 to obtain a controlled release of these factors when their expression is not sufficient to promote nerve repair. Moreover, it is necessary to find out what the exact roles of NRG1 α and β are. Thus, silencing each of these isoforms is a strategy that would provide knowledge about essential signalling pathways in nerve regeneration. Hence, small interfering RNAs (siRNAs) bioconjugated to polyethylene glycol-coated gold nanoparticles (AuNPs) were developed and will be used to this aim.

  • Neuronal network characterization of cortico-hippocampal spontaneous activity during development

    Authors:
    Edoardo Fraviga (1,2), Genni Desiato (1), Riccardo Grassi (1,2), Davide Pozzi (1,2)

    Organisations:
    1: Humanitas IRCCS
    2: Humanitas University

    Presenting author: Edoardo Fraviga

    Exposition date: Friday, September 15, 2023

    Exposition position: 082/1

    Neuronal development represents a finely tuned process occurring during early postnatal life, that paves the way for the proper brain functioning in adulthood. Among the factors found to be crucial for this process, electrical activity represents a key aspect whose alterations may have a deep impact for the proper establishment of neuronal circuits. Multi-electrode arrays (MEAs) electrophysiology is considered a useful tool for studying the pattern of whole neural activity and network dynamics. In this study, we take advantage of high-density MEA (HD-MEA) technology to probe the dynamic changes of neural network activity along development. Cortico-hippocampal acute brain slices were established at different, postnatal (P), developmental stages (P4-P9-P15) and either spontaneous or evoked network activity was recorded. Our preliminary results indicate that network activity progressively increases during development, from P4 to P9, with a significant drop occurring at P15. We also observed that network spiking at early developmental stages is mainly driven by depolarizing GABAergic axes, as indicated by the complete ablation of spiking activity through Bumetanide. These findings provide a new tool and novel insights into the dynamic changes of network activity during development.

  • spaCe: assessing relative biological effectiveness of simulated deep space radiation in altering neural function and survival in C. elegans

    Authors:
    Giada Onorato (1,2,3), Pamela Santonicola (1), Emanuele Scifoni (4), Francesco Tommasino (4), Alessandra Bisio (5), Ferdinando Di Cunto (3,6), Elia Di Schiavi (1)

    Organisations:
    1: Institute of Biosciences and BioResources (IBBR), CNR, Naples, Italy
    2: Department of Environmental, Biological and Pharmaceutical Science and Technologies, Università degli Studi della Campania "Luigi Vanvitelli", Caserta, Italy
    3: Department of Neuroscience ‘Rita Levi Montalcini’, University of Torino, Turin, Italy
    4: Dipartimento di Fisica, Università di Trento, Trento, Italy
    5: Dipartimento di Biologia Cellulare, Computazionale e Integrata (CIBIO), Università di Trento, Trento, Italy
    6: Neuroscience Institute ‘Cavalieri Ottolenghi’, Orbassano, Italy

    Presenting author: Giada Onorato

    Exposition date: Friday, September 15, 2023

    Exposition position: 080/1

    The onset of neurodegenerative diseases is clearly determined by environmental and genetic factors. Insults external to the body, such as radiations, affect human body and mind in several ways. Thus, understanding how human mind adapts to the environment is an important challenge. Moreover, in light of future human explorations of deep space, a fundamental need is understanding how the nervous system may be affected by peculiar radiations that characterize this extreme environment and what genetic background can predispose to neuron dysfunction. We use the nematode C. elegans as a simple and convenient model, allowing to assess the effects of different space-relevant radiation beams on neurological function of selected groups of neurons. We demonstrated that gamma-rays affect selectively dopaminergic neurons, causing a dose-dependent degeneration of the dendrites and alteration in functionality. Interestingly, we observed in gamma-rays irradiated animals a reduction in dopamine content which might be the cause of dopaminergic neurons dysfunction. We compared this effect with another type of photons by performing X-rays irradiation and we observed that they affect dopaminergic neurons functionality in a similar way. We also tested the effect of a different type of ionizing radiation by performing protons irradiation and we observed that also protons affect dopaminergic neurons functionality, with a slightly milder effect than photons. The project aims also to discover genetic backgrounds that might sensitize or protect neurons from ionizing radiations and the molecular basis of ionizing radiation sensitivity. Thus, we discovered that cep-1/p53 has a neuroprotective role, with cep-1 mutants being more affected by gamma-rays irradiation. These results suggest that ionizing radiations reduce dopamine levels and alter dopaminergic neurons functions in vivo and also support C. elegans as a biological dosimeter for neuronal damage induced by peculiar environmental conditions like radiations in deep space.

  • Study of epigenetic characteristics of control and sALS motor neuron organoids

    Authors:
    Eveljn Scarian (1), Matteo Bordoni (1), Camilla Viola (2), Francesca Dragoni (1,2), Rosalinda Di Gerlando (1,2), Luca Diamanti (1), Stella Gagliardi (1), Orietta Pansarasa (1)

    Organisations:
    1: IRCCS Mondino Foundation, Italy
    2: University of Pavia, Pavia, Italy

    Presenting author: Eveljn Scarian

    Exposition date: Saturday, September 16, 2023

    Exposition position: 056/2

    Amyotrophic lateral sclerosis (ALS) is a rare neurodegenerative disease which causes the death of upper and lower motor neurons (MNs) in cortex, brainstem and spinal cord. ALS patients suffer from weakness, muscle atrophy and spasticity and the death occurs between three to five years after symptoms onset. 90% of ALS cases are classified as sporadic ALS (sALS). Till now, only symptomatic treatments are available and the principal cause is the lack of realistic models which can mimic the physiological environment in which cells grow. Organoids are pluripotent stem cell-derived self-organizing structures which can recapitulate the tissue of origin in vitro. Brain organoids are now widely used for disease modeling and for the study of cells interactions. We reprogrammed induced pluripotent stem cells from peripheral blood mononuclear cells of a healthy subject and a sALS patient, and we differentiated them in neural stem cells in 2D. Neural stem cells were then cultured for the generation of MNs progenitors organoids and finally in MNs organoids (MNOs). We performed an epigenetic characterization of organoids and 2D cultured cells at each differentiation step. Trough ELISA assay we investigated DNA methylation status on 5-methylcytosine (5-mC) and found a decreased methylation in sALS MNOs. Moreover, we tested the expression of DNA methyl-trasnferase 1 (Dnmt1) and the DNA methyl-transferase3a (Dnmt3a) by western blot, finding a significant decrease in the protein expression of Dnmt1 in both CTRL and sALS MNs progenitors organoids and MNOs when compared to the corresponding NSCs organoids, and by Real-Time qPCR, observing some differences both between organoids and 2D cultured cells and between CTRL and sALS cultures. Finally, we analyzed the methylation status of lysine 9 and 27 on histone 3, finding an increasing trend of their protein expression during the differentiation protocol.

Mechanisms of recovery after neuronal damage

  • A non-redundant role of EAAT3/EAAC1 for ATP synthesis mediated by GDH in dopaminergic neuronal cells: a new avenue for glutamate metabolism and protection in Parkinson’s disease.

    Authors:
    Alessandra Preziuso (1), Marwa Toujani (2), Tiziano Serfilippi (1), Giorgia Cerqueni (1), Valentina Terenzi (1), Gerardo Galeazzi (1), Salvatore Amoroso (1), Simona Magi (1), Vincenzo Lariccia (1), Silvia Piccirillo (1)

    Organisations:
    1: Department of Biomedical Sciences and Public Health, School of Medicine, University “Politecnica delle Marche”, Via Tronto 10/A, 60126 Ancona, Italy
    2: Department of Pediatrics, Meyer Children's University Hospital, Florence, Italy

    Presenting author: Silvia Piccirillo

    Exposition date: Saturday, September 16, 2023

    Exposition position: 061/2

    Parkinson’s disease (PD) is a devastating neurodegenerative disorder with a distinct loss of the nigrostriatal dopaminergic pathway [1]. Despite the multiplicity in etiology, alterations of affected neurons can be traced back to defects in fundamental processes that typically run under mitochondrial inputs [2]. Evidence indicates that mitochondrial activities are hierarchically integrated with the energetic performance of these organelles, so that an interesting perspective holds that interventions aimed at improving mitochondrial bioenergetics can potentially mitigate the severity of PD phenotype expression. In this mechanistic framework, approaches that facilitate the mitochondrial anaplerotic use of glutamate (Glu) [3, 4] might counteract the detrimental shift from Glu metabolism (which is typically altered in PD; [5]) to excessive Glu transmission that feed excitotoxicity and neurodegenerative spiral [6]. In this study we investigated whether the enhancement of glutamate dehydrogenase (GDH) activity, by using the GDH activator 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) [4], has neuroprotective potential against PD injury. In both retinoic acid-differentiated SH-SY5Y cells and primary rat mesencephalic neurons challenged with α-synuclein/rotenone (to mimic PD; [3]), BCH-dependent GDH activation significantly ameliorated cell viability, improved mitochondrial ATP synthesis and lessened to control levels the cellular redox burden. Strikingly, we collected evidence for the existence of a functional axis connecting GDH activity to a specific intracellular pool of the Excitatory Amino Acid Transporters (EAATs), namely the EAAT3/EAAC1. Overall, our results reveal a novel and non-redundant role of EAAT3/EAAC1 for GDH-dependent protection against PD injury, which may inspire new pharmacological approaches against PD pathology.

    1.Int J Mol Sci. 2022;23(21):13043.

    2.Int J Mol Sci. 2022;23(16):9212.

    3.Cells. 2020;9(9):2037

    4.Neuroscience. 2017;340:487-500.

    5.Biology (Basel). 2016;5(4):53

    6.J Clin Neurosci. 2021;90:178-183.

  • Analysis of the muscular fibre contribution to the early stage of nerve regeneration inside a vein graft enriched with fresh skeletal muscle

    Authors:
    Federica Zen (1,2), Alessandro Crosio (1,3), Giulia Ronchi (1,2), Debora Molinaro (1), Stefano Geuna (1,2), Giovanna Gambarotta (1,2), Stefania Raimondo (1,2)

    Organisations:
    1: Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.
    2: Neuroscience Institute Cavalieri Ottolenghi, University of Turin, Turin, Italy.
    3: UO Microchirurgia e Chirurgia della Mano, Ospedale Gaetano Pini, Piazza Andrea Ferrari 1, 20122 Milano, Italy.

    Presenting author: Federica Zen

    Exposition date: Thursday, September 14, 2023 (Satellite Events Day)

    Exposition position: 101

    Although the autograft represents the gold standard on the peripheral nerve repair, this technique raises non-negligible side effects: therefore, the muscle-in-vein (MIV) graft has gradually been employed as alternative in the clinical practice to repair severe peripheral nerve injury in human. In fact, MIV technique guarantees a lower donor site morbidity compared to the autograft and provides a protected environment in which the nerve can regrow without the economic cost of synthetic conduits, regrettably MIV technique works in gap longs no more to 6 cm.

    The fundamental role of Schwann cells in the peripheral nerve regeneration is abundantly reported in the scientific literature and, recently, we demonstrated how Schwann cells use a capillary network as pathway to colonize a hollow conduit during the peripheral nerve regeneration, providing a key role to vascularization. Moreover, we proved that fresh muscle fibres express high contents of Vascular Endothelial Growth Factor which is fundamental to vascularization.

    Against this background, our purpose is to investigate the biological processes that occur during MIV regeneration focusing on the vascularization process as potential key factor of the effectiveness of MIV technique even in longer gaps.

    In order to understand how muscle fibre presence could affect the vascularization, and Schwann cell migration as a result, we performed an injury on the median nerve of adult female rats, repaired with 10 mm vein graft filled with striatum muscle. The regenerated nerves within graft were analysed at different time points through molecular and immunofluorescent analyses.

    Our data underline that an organized capillary network is formed in the MIV graft, as previously studied in the hollow conduit, already in the early stages of regeneration and on which can be observed migrating Schwann cells. The role of endothelial cells and satellite cells naturally available in the muscle are still under investigation.

  • Fibrin-collagen hydrogel with or without adipose-derived mesenchymal stem cells enriched chitosan conduits for critical nerve gap repair

    Authors:
    Marwa El Soury (1,2), Óscar Darío García-García (3,4), Isabella Tarulli (1,5), Jesús Chato-Astrain (3,4), Isabelle Perroteau (1), Stefano Geuna (1,2), Stefania Raimondo (1,2), Giovanna Gambarotta (1,2), Víctor Carriel (3,4)

    Organisations:
    1: Dipartment of Clinical and biological sciences, University of Torino, Torino, Italy.
    2: Neuroscience Institute Cavalieri Ottolenghi, University of Torino, Torino, Italy
    3: Department of Histology, Tissue Engineering Group, University of Granada, Granada, Spain
    4: Instituto de Investigacion Biosanitaria, Ibs.GRANADA, Granada, Spain
    5: Ecole polytechnique fédérale de Lausanne, Switzerland

    Presenting author: Marwa El Soury

    Exposition date: Thursday, September 14, 2023 (Satellite Events Day)

    Exposition position: 99

    Repairing severe peripheral nerve injuries remains a great challenge for surgeons, as the regeneration outcomes are not usually satisfactory. In case of injuries accompanied by substance loss where tensionless repair is not applicable, nerve autografts are the gold standard technique applied by surgeons in order to fill the nerve gap and re-join the two transected nerve stumps. Tubulization technique is an alternative that has been developed to repair nerves and to overcome the different limitations accompanied by the use of autografts.

    Hollow conduits are found to be highly efficient just in case of repairing small gaps (up to 3 cm), while to improve the conduit efficiency in repairing long gaps intraluminal enrichment with different extracellular materials and cells could provide a better biomimicry of the natural nerve regenerating environment and is expected to ameliorate the conduit performance.

    In this study, we evaluated nerve regeneration in vivo using hollow chitosan conduits or conduits enriched with fibrin-collagen hydrogels alone or with the further addition of adipose-derived mesenchymal stem cells in a 15 mm rat sciatic nerve transection model. Unexpected changes in the hydrogel consistency and structural stability in vivo led to a failure of nerve regeneration after 15 weeks. Nevertheless, the molecular assessment in the early regeneration phase (7, 14, and 28 days) has shown an upregulation of useful regenerative genes in hydrogel enriched conduits compared with the hollow ones. Hydrogels composed of fibrin-collagen were able to upregulate the expression of soluble NRG1, a growth factor that plays an important role in Schwann cell transdifferentiation. The further enrichment with adipose-derived mesenchymal stem cells has led to the upregulation of other important genes such as ErbB2, VEGF-A, BDNF, c-Jun, and ATF3.

  • Functional circuit reconstruction of transplanted striatal neurons into a rodent model of Huntington’s Disease

    Authors:
    Marta Ribodino (1,2), Roberta Parolisi (1,2), Gabriela Berenice Gomez Gonzales (1,2), Giulia Concina (1), Malin Akerblom (3), Malin Parmar (3), Tomas Bjorklund (4), Benedetto Sacchetti (1), Annalisa Buffo (1,2)

    Organisations:
    1: Department of Neuroscience “Rita Levi-Montalcini”, University of Turin
    2: Neuroscience Institute Cavalieri Ottolenghi, University of Turin
    3: Developmental and Regenerative Neurobiology, Wallenberg Neuroscience Center, Department of Experimental Medical Science, Lund University
    4: Molecular Neuromodulation unit, Wallenberg Neuroscience Center, Department of Experimental Medical Science, Lund University

    Presenting author: Marta Ribodino

    Exposition date: Saturday, September 16, 2023

    Exposition position: 063/2

    Huntington Disease (HD) is an autosomal dominant neurodegenerative disorder characterized by the progressive degeneration of striatal GABAergic projection neurons, called medium spiny neurons (MSNs), in the early stage of the pathology. Although there is increasing knowledge about the genetics and molecular mechanisms underlying this disease, only symptomatic treatments are currently available. We showed that transplantation of both rodent and human MSN progenitor cells into a toxin-based rat model of HD leads to the survival, integration and differentiation of the graft and support a degree of recovery as measured by sensory-motor tests. However, it remains to be demonstrated to what extent these cells can form long-range striatal circuits, in particular the graft-mediated reconstruction of the direct and indirect striatal pathways, and what is the role of graft activity in the observed behavioural improvements.

    Towards these aims we are (i) screening AAV libraries to identify capsids to selectively target MSNs and optimise the investigation of the precise reconstruction of graft-derived circuitries and (ii) modulating the activity of the graft to ask whether and how this affects the observed functional rescue. Preliminary data suggest that the graft-mediated behavioural outcome can be modified through optogenetic-induced modulation supporting the causal relationship between graft electrical activity and motor rescue.

  • Investigation on carbon nanotubes-based devices for peripheral nerve injury recovery

    Authors:
    Marta Confalonieri (1), Elena Stocco (2), Silvia Barbon (2), Lucia Petrelli (2), Martina Contran (2), Ludovica Ceroni (3), Cesare Tiengo (4), Silvia Todros (1), Piero Giovanni Pavan (1), Veronica Macchi (2), Enzo Menna (3), Raffaele De Caro (2), Andrea Porzionato (2)

    Organisations:
    1: Department of Industrial Engineering, University of Padova, Italy
    2: Department of Neurosciences, Section of Human Anatomy, University of Padova, Italy
    3: Department of Chemical Sciences, University of Padova, Italy
    4: Department of Neurosciences, Clinic of Plastic Surgery, University of Padova, Italy

    Presenting author: Marta Confalonieri

    Exposition date: Thursday, September 14, 2023 (Satellite Events Day)

    Exposition position: 102

    Peripheral nerve injuries (PNI) are not uncommon in upper limb trauma and can have serious consequences on patients' quality of life. Current surgical treatments, including nerve autografts and conduits positioning are not fully satisfactory. In consideration of that, this research project has the aim of analyzing, through in vitro and preclinical studies, the effectiveness of vanguard electroconductive NC based on oxidized polyvinyl alcohol (OxPVA) with/without (+/-) carbon nanotubes (CNT). Hence, OxPVA polymer solution was preliminary mixed with a weighted amount of multi-walled CNT to prepare new nanocomposite electroconductive hydrogels (CNTs concentration of 0.1 wt%). OxPVA+/-CNT scaffolds were assayed for ultrastructure/electroconductivity/in vitro cytotoxicity/in vivo biocompatibility. Then, OxPVA+/-CNT conduits were fabricated to be implanted in animal model of disease (Sprague Dawley rat; sciatic nerve injury, gap: 5 mm). Four experimental groups were compared (reverse autograft/Reaxon/OxPVA/OxPVA+CNT) and after 6 weeks, conduits effectiveness was verified through histological, immunohistochemical analyses and morphometric study; animals’ weight was constantly monitored to assess their wellbeing. According to the in vitro data, the hybridization with CNTs conferred to OxPVA a rougher surface than CNT-free OxPVA; an increased superficial electroconductivity was detected and potential CNT-related toxicity was excluded as no cytotoxic response was evident over SH-SY5Y cells. After 14-days of subcutaneous implant, no thick fibrotic capsule was recognized in correspondence of the implants, suggesting biocompatibility. Focusing on implanted conduits, at dissection all the guides were clearly identifiable (no dislocations/neuroma); moreover, similarly to OxPVA and Reaxon, axonal regeneration was interestingly observed also within OxPVA+CNTs conduits, suggesting their potential in vivo, confirmed by morphometry. A certain atrophy of the operated-limb side gastrocnemius was evident in all groups. This study highlights NC as a valid reconstructive alternative to autografts. Furtherly, incorporation of electroconductive CNT within OxPVA, may be an appealing strategy to improve outcomes associated with tubular devices.

  • Microtophographies and phosphodiesterase inhibitors to improve the regenerative capabilities of a chitosan membrane. an in-depth study on nervous cells

    Authors:
    Federica Fregnan (1), Luisa Muratori (1), Federica Zen (1), Marwa El Soury (1), Ilaria Tonazzini (3), Luca Scaccini (3), Francesco Porpiglia (2), Matteo Manfredi (2), Cristian Fiori (2), Stefano Geuna (1), Stefania Raimondo (1)

    Organisations:
    1: Department of Clinical and Biological Sciences, and Cavalieri Ottolenghi Neuroscience Institute, University of Turin.
    2: Division of Urology, Department of Oncology, and Department of Clinical and Biological Sciences, San Luigi Gonzaga Hospital, University of Turin
    3: NEST (National Enterprise for nanoScience and nanoTechnology), Istituto Nanoscienze-CNR & Scuola Normale Superiore, Pisa

    Presenting author: Federica Fregnan

    Exposition date: Thursday, September 14, 2023 (Satellite Events Day)

    Exposition position: 96

    The treatment of localized prostate cancer is radical prostatectomy with frequent iatrogenic damages to the periprostatic neurovascular bundles (NVB) responsible of erectile dysfunctions. Our team previously demonstrated the neuro-regenerative effect of chitosan membrane on ex vivo cultures of autonomic ganglia and its anti-proliferative effect on metastatic prostatic cancer cells.

    The aim of the present study was to test in vitro and to develop a functionalized microstructured chitosan membrane to support and promote the main regenerative mechanisms, underlying nerve outgrowth and glial cell survival and proliferation for an in vivo use, to repair a lesion affecting the cavernous nerve. The chitosan was blended with 5-15% glycerol and micropatterned to obtain the well-established gratings (GR) and the asymmetric pattern with scalene triangles (SCA). Moreover, the controlled release of phosphodiesterase inhibitors (PDEI) was designed to chemically promote nerve regeneration and functional recovery.

    The results of in vitro and ex vivo direct cultures on microstructured chitosan membranes demonstrated the oriented growth of neurons, a very important step in making regeneration more effective.

    The in vitro protocol for the administration of PDEI (sildenafil-PDE5I and rolipram-PDE4I) was developed and for both stimulations an interesting gene regulation linked to the neuroprotective brain-derived neurotrophic factor (BDNF) in immortalized cultures of sensory and motor neurons was observed. In glial cell cultures, the administration of PDEI resulted in up-regulation of the transcription factor Krox20, which can positively influence the expression of myelin genes, of the proangiogenic Vascular endothelial growth factor (VEGF) and in a decrease of cell migration. Furthermore, the administration of Sildenafil and Rolipram increased the neuritic extension in neuronal populations. Further investigations are underway to deepen the study of the effect of PDEI administration on organotypic cultures of somatic and autonomic ganglia, where neuronal and glial cells co-exist, similarly to what happens in vivo.

  • Mirtazapine rescues neuronal atrophy in Rett syndrome by directly binding SOS1 and triggering the MAPK/ERK pathway

    Authors:
    Stefano Donegà (1,2), Ottavia Maria Roggero (1), Nicolò Gualandi (4,5), Vittoria Berutto (1), Andrea Colliva (1), Gianluca Masella (1), Emanuele Carosati (3), Remo Sanges (4), Gabriele Baj (1), Enrico Tongiorgi (1)

    Organisations:
    1: Cellular and Developmental Neurobiology Lab – Department of Life Sciences, University of Trieste – Trieste, Italy.
    2: Biomedical Research Center, National Institute on Aging, National Institute of Health, Baltimore, MD, USA
    3: Department of Chemical and Pharmaceutical Sciences, University of Trieste, 34127, Trieste, Italy
    4: Computational Genomics Laboratory, Scuola Internazionale Superiore di Studi Avanzati (SISSA), 34136 Trieste, Italy
    5: Department of Medicine, University of Udine, 33100 Udine, Italy

    Presenting author: Ottavia Maria Roggero

    Exposition date: Saturday, September 16, 2023

    Exposition position: 062/2

    Rett syndrome (RTT) is a genetic, progressive neurodevelopmental disorder mainly caused by sporadic mutations in the X-linked methyl-CpG binding protein 2 (MECP2) gene with an incidence of 1/10.000 newborn females worldwide. Mutations leading to MECP2 ablation cause developmental arrest, loss of speech and motor abilities, seizures, breathing abnormalities and brain atrophy characterized by more closely packed neurons and decreased dendritic complexity. In previous studies, we showed that neuronal atrophy was rescued in MeCP2y/-mice by chronic treatment with the antidepressant mirtazapine (MTZ). However, the mechanisms of action of MTZ in RTT remain unclear. Here, we tested the hypothesis that MTZ could have a neurotrophic action by activating the BDNF/TrkB pathway, which is disrupted in RTT. We found that in vitro treatments with MTZ completely restored dendritic arborization and soma size of cultured MeCP2y/-hippocampal neurons. Phosphorylation of TrkB receptor was increased after 1h treatment MTZ and not after longer treatments, but surprisingly, BDNF levels in vivo and in vitro were unaffected by MTZ treatment. In this scenario, we moved to in silico prediction by inverse docking on ligand-bound pockets from all human protein crystal structures available in PDB, using the BioGPS software. MTZ exhibited high binding affinity for a specific pocket of Son Of Sevenless Homolog 1 (SOS1) that controls the cycling of Ras-GDP to Ras-GTP. Downstream RAS-activation, phosphorylation of extracellular signal-regulated kinase (ERK) is enabled, inducing the complete rescue of dendritic atrophy in RTT neurons. In conclusion, mirtazapine is able to elicit neurotrophic effects by directly binding SOS1, triggering the MAPK/ERK pathway. These results reveal a novel mechanism of action of MTZ and support its use as a treatment for Rett syndrome.

  • Optimised hESC-derived medium spiny neurons for cell replacement: challenges and opportunities.

    Authors:
    Roberta Parolisi (1), Marta Ribodino (1), Gabriela Berenice Gomez Gonzales (1), Linda Scaramuzza (2), Dario Besusso (2), Roberta Schellino (1), Greta Galeotti (2), Elena Cattaneo (2), Annalisa Buffo (1)

    Organisations:
    1: Department of Neuroscience Rita Levi-Montalcini, University of Turin, Turin 10124, Italy and Neuroscience Institute Cavalieri Ottolenghi, University of Turin, Orbassano, 10043 Italy.
    2: Department of Biosciences, University of Milan, Milan, 20122 Italy and National Institute of Molecular Genetics “Romeo ed Enrica Invernizzi”, Milan, 20133 Italy

    Presenting author: Roberta Parolisi

    Exposition date: Saturday, September 16, 2023

    Exposition position: 064/2

    Neurotransplantation represents a promising alternative for the treatment of certain localized neurodegenerative pathology, such as Huntington disease (HD). The survival, maturation and integration of medium spiny neurons (MSN) progenitors in the lesioned striatum is a key requirement for a successful cell therapy. The toxin-based HD model, obtained with unilateral quinolinic acid (QA) injection into the striatum of athymic rats, recapitulates the cell loss of MSN seen in HD, and allows to investigate the efficacy of the graft.

    We formerly showed that first generation (fg) hESC-derived striatal grafts (1), survive up to 6 months post-transplantation in the HD model, and differentiate into key striatal cell types including relevant fractions of matured MSNs that become integrated into the host circuits (2). However, fg-grafts yielded a limited fraction of human MSN (hMSN) that matured very slowly. This prompted the development of a second-generation (sg) in vitro differentiation protocol that significantly enhanced the MSN yield and maturity (3). Here, we present first analyses on short-term grafts of sg-hMSN progenitors that were transplanted into the intact and lesioned rat striatum.

    Preliminary analyses show that sg-hMSNs display peculiar properties not found in fg-graft at corresponding time points. In detail, human graft cells revealed a pronounced migratory activity suggestive of an increased integration capability of the cells. Moreover, grafted sg-hMSNs showed a particular vulnerability to the lesion environment, which decreased the rate of graft implantation. Also, the lesion environment decreased cell proliferation and expression of mature neuronal markers in the graft. We are currently testing if extending the time distance of the graft from the lesion improves the implantation rate and investigating the lesion-derived factors decreasing efficient grafting.

    NSC-Reconstruct H2020, GA no. 875758

    1- Delli Carri et al. (2013) doi: 10.1242/dev.084608.

    2- Schellino, Besusso et al., under review.

    3- Conforti et al. (2022) doi: 10.1016/j.crmeth.2022.100367.

  • Pig decellularized peripheral nerve supports fiber regeneration.

    Authors:
    Stefania Raimondo (1), Luisa Muratori (1), Giulia Ronchi (1), Alessandro Crosio (1,2), Arianna Lovati (3)

    Organisations:
    1: Department of Clinical and Biological Sciences, Neuroscience Institute Cavalieri Ottolenghi (NICO), University of Turin, Turin, Italy
    2: Reconstructive Microsurgery and Hand surgery Unit, ASST Pini-CTO Milano, Italy
    3: Cell and Tissue Engineering Laboratory, IRCCS Istituto Ortopedico Galeazzi, Milan, Italy

    Presenting author: Stefania Raimondo

    Exposition date: Thursday, September 14, 2023 (Satellite Events Day)

    Exposition position: 95

    When a peripheral nerve injury with a large defect occurs, end to end suture is not possible and conduit is not enough to obtain good results. Allograft could be an alternative, but nerves from donors frequently cause immunogenic response. Several authors are looking for the correct way to decellularize nerves preserving both the extracellular matrix (ECM) and basal lamina to improve nerve regeneration. Over the past years, the decellularization of peripheral nerves has been used to provide a natural substrate composed of nerve ECM without the resident cells to prevent the host immune response when transplanted in patients.

    The aim of this study is to evaluate the efficacy of a new decellularization protocol, that is both easy to prepare and allow to preserve the nerve ECM, on pig nerves, specie that has attracted considerable interest, since some tissues derived from it are already used in clinical practice showing excellent biocompatibility.

    Decellularized pig nerves have been analyzed for demonstrating the effectiveness in removing cells and preserving ECM. Then, they have been implanted for repairing rat median nerves demostrating their ability to sustain peripheral nerve regeneration. One month after injury and repair of the rat median nerve, regenerating fibers have colonized the graft suggesting a promising use for repairing severe nerve lesions.

  • Role of altered vitamin B12 metabolism on the central nervous system homeostasis

    Authors:
    Aimee Rachel Mathew (1), Virve Cavallucci (2,3), Marco Fidaleo (1,4)

    Organisations:
    1: Department of Biology and Biotechnologies “Charles Darwin”, University of Rome Sapienza, 00185, Rome, Italy
    2: Fondazione Policlinico Universitario A. Gemelli IRCCS, 00168, Rome, Italy
    3: Institute of General Pathology, Università Cattolica del Sacro Cuore, 00168, Rome, Italy
    4: Research Center for Nanotechnology for Engineering of Sapienza (CNIS), University of Rome Sapienza, 00185, Rome, Italy

    Presenting author: Aimee Rachel Mathew

    Exposition date: Saturday, September 16, 2023

    Exposition position: 065/2

    Vitamin B12 (VitB12) and its direct role in the central nervous system (CNS) homeostasis is well-known; however, the detailed mechanisms are not clearly understood. To identify the possible basic mechanisms, regarding the CNS, involved in the alteration of the gene expression associated with VitB12 metabolism, a comparative RNA dataset analysis was performed. For this analysis, open-source databases were queried for RNA sequencing and microarray datasets generated from CNS tissues or cells under VitB12 deficiency mimicking conditions; and two datasets from mouse and one from rat models were selected. Despite the great diversity in the experimental models employed for the analysis, the comparison of these datasets highlighted a common alteration in the ribosomal gene expression under VitB12 deficiency mimicking conditions thereby indicating a possible basic and conserved response to VitB12 deficiency. Moreover, our analysis identified E2F1, a transcription factor, as a potential player in the aforementioned process. To further investigate this hypothesis, we used an in vitro model of differentiated SH-SY5Y human neuroblastoma cell line (exhibiting a neuron-like phenotype), followed by evaluating the roles of both the deficiency and excess of VitB12 on the modulation of E2F1 and its possible association with some mature neuronal markers. This possible link of E2F1 with the neuronal markers can lead to the identification of E2F1 as a potential target for maintaining CNS homeostasis under VitB12 deficiency and lay the foundation for future medical interventions.

  • Role of fresh skeletal muscle fibers in supporting vascularization and Schwann cell migration during peripheral nerve regeneration within a chitosan conduit

    Authors:
    Debora Molinaro (1), Federica Zen (1), Benedetta Elena Fornasari (1), Giulia Ronchi (1), Alessandro Crosio (1,2), Giovanna Gambarotta (1)

    Organisations:
    1: Department of Clinical and Biological Sciences (DSCB) and Neuroscience Institute Cavalieri Ottolenghi (NICO),University of Turin, Italy
    2: Hand Surgery and Reconstructive Microsurgery Department, CTO, ASST Gaetano Pini, Milan, Italy

    Presenting author: Debora Molinaro

    Exposition date: Thursday, September 14, 2023 (Satellite Events Day)

    Exposition position: 98

    Peripheral nerve injuries are frequently encountered clinical complications which can lead to partial or complete impairment of sensory, motor and autonomic functions.

    Several approaches have been developed to allow regeneration after nerve transection by bridging the gap between the two nerve stumps (tubulization). One of the neural guide conduits currently used in clinical practice is the chitosan tube, in which the formation of a polarized vasculature within the conduit acts as a scaffold for Schwann cell migration and axonal regrowth. Multiple pre-clinical studies have been focused on finding strategies to improve this technique; in particular, we investigated the enrichment of this conduit with fresh skeletal muscle fibers, which provide both trophic factors and physical support for Schwann cell migration.

    Molecular analysis on the hollow chitosan tube (chitosan) and the conduit enriched with fresh skeletal muscle fibers (MIT, muscle-in-tube) at 7,14 and 28 days after repair displayed an increase in the expression of genes known to be positively involved in angiogenesis in the presence of skeletal muscle fibers, with a peak at 14 days after surgery.

    Moreover, qualitative morphological analysis showed that, in the MIT, Schwann cells were associated with both blood vessels and muscle fibers, suggesting they might simply require an adequate physical substrate in order to migrate, instead of a specific protein-protein interaction.

    A relevant difference in terms of nerve regeneration was detected at 14 days post-surgery between the MIT and the chitosan, with a slower progression of both vascularization and nerve regeneration in the MIT, where the abundant muscle tissue seemed to cause a delay in the first two weeks after surgery. On the contrary, no qualitative differences were observed between the two experimental models a month after repair. Further analyses will be carried out to obtain quantitative morphological results.

  • Strategies to improve cavernous nerve regeneration after radical prostatectomy

    Authors:
    Luisa Muratori (1), Federica Fregnan (1), Alessandro Crosio (2), Federica Zen (1), Matteo Manfredi (3), Ilaria Tonazzini (4), Juliette Meziere (3), Francesco Porpiglia (3), Stefano Geuna (1), Stefania Raimondo (1)

    Organisations:
    1: Department of Clinical and Biological Sciences & Neuroscience Institute “Cavalieri Ottolenghi” (NICO), Orbassano (To), 10043, Italy
    2: UOC Chirurgia della Mano e Microchirurgia Ricostruttiva - ASST Gaetano Pini P.zza A. Ferrari 1 - 20122 Milano
    3: Department of Oncology, Division of Urology, San Luigi Gonzaga Hospital, University of Turin Orbassano (To), 10043, Italy
    4: NEST (National Enterprise for nanoScience and nanoTechnology), Istituto Nanoscienze-CNR& Scuola Normale Superiore, Piazza San Silvestro 12, 56127, Pisa, Italy

    Presenting author: Luisa Muratori

    Exposition date: Thursday, September 14, 2023 (Satellite Events Day)

    Exposition position: 100

    Prostate cancer is the most frequent cancer among men and the current treatment is radical prostatectomy (RP). Unfortunately, iatrogenic damage to the periprostatic neurovascular bundles (NVB) occurs, leading to erectile dysfunction. Recently, new strategies to improve the regeneration of the prostatic nerves are arising, among these, the application of chitosan membrane have been showed to promote nerve regeneration thanks to its useful properties. At this purpose, ex vivo experiments performed on autonomic explant ganglia have been showed the neuro-regenerative effect of a flat chitosan membrane reporting a higher neurite outgrowth. At the same time, in vitro experiments on metastatic cancer cell lines displayed a lower proliferation rate when cultured with chitosan coating and dissolution products. The safety and the ability of the flat chitosan membrane to promote nerve regeneration was also tested in clinical resulting in higher potency recovery rate in patients that have undergone RP. To improve the regenerative performance achieved by the flat membrane, nanostructured membranes with grating arrangement and a zig-zag pattern were used to repair cavernous nerve on adult male rats: 3 mm of nerve was bilaterally transected and repaired with chitosan membranes. 60 days after the surgical procedure, samples were harvested and processed for “iDISCO” technique that allowed to detect the pathway of nerve fibers on the whole membranes.These in vivo results provide the first experimental evidence supporting the ability of the chitosan membrane to allow axonal regeneration demonstrating the safety of the device for clinical use and supporting its application in urological field.

  • Study of starch-derived fibres used as a scaffold to promote peripheral nerve regeneration.

    Authors:
    Miriam Metafune (1), Claudio Cecone (2), Marco Zanetti (2), Stefania Raimondo (1,3), Federica Fregnan (1,3)

    Organisations:
    1: University of Torino, Italy
    2: Department of Chemistry, Nis Interdepartmental Centre, University of Torino
    3: Neuroscience Institute Cavalieri Ottolenghi, University of Torino

    Presenting author: Miriam Metafune

    Exposition date: Thursday, September 14, 2023 (Satellite Events Day)

    Exposition position: 97

    The objective of the present study is to evaluate biocompatibility and biomimeticity of an innovative membrane with the aim to apply it for repairing somatic and autonomic peripheral nerves in case of traumatic or iatrogenic lesions. Starch-derived (GLUCIDEX®) hyper-crosslinked polymers with suitable mechanical properties were electrospinned as membrane and tested, in vitro using immortalized Schwann Cells (RT4-D6P2T cells), for cell survival and proliferation to evaluate the biocompatibility and biomimetic nature of the scaffolds.

    RT4-D6P2T cells were cultured i) in direct contact with the membrane, to investigate the interaction with the substrate and ii) in the presence of membrane dissolution products, to test the effect on cell proliferation and organization.

    i) Concerning to the adhesion assays, the actin cytoskeleton results more organized in the control group, however, after 24 hours, the density and the area occupied by RT4-D6P2T increased.

    ii) Several analyzes were conducted using the dissolution products of Glucidex® membranes; the proliferation assay revealed that, after 1, 4 and 7 days of culture, cells maintain proliferative behavior under all conditions tested although a slight decrease, compared to the control, is observed at the first two time points. The actin cytoskeleton profile revealed that cells cultured in conditioned medium have a high organization and generate membrane protrusions, lamellipodia, correlated to cell migration, an important feature of glial cells in support of peripheral nerve regeneration.

    Investigating apoptosis and the specific cellular alterations due to Bax, pro-apoptotic protein, and Bcl-2, anti-apoptotic protein, our study revealed that the dissolution products of the membrane are not related with cell death, contrarily, they are associated with good survival. Further investigations are underway to deepen the effect of the dissolution products on expression of gene involved in the regulation of nerve regeneration by Schwann cells.

Migraine and pain

  • Antinociceptive Effects of Vitamin B-Complex in Rats: The role of No-related neuron in inflammatory chronic pain.

    Authors:
    Shahaboddin Zarei (1,2), Mina Shahriari-Khalaji (3), Ian Max Andolina (1), Gila Behzadi (2)

    Organisations:
    1: Center for Excellence in Brain Science and Intelligence Technology (Institute of Neuroscience), China, People's Republic of
    2: Department of Physiology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
    3: Microbiological Engineering and Industrial Biotechnology Group, College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, China

    Presenting author: Shahaboddin Zarei

    Exposition date: Thursday, September 14, 2023 (Satellite Events Day)

    Exposition position: 103

    B-vitamins have been evaluated as a useful adjuvant therapy to treat pain. In spite of clinical and experimental evidence indicating the analgesic effect of B-vitamins, few studies have investigated their effect on aspects of the inflammatory pain response. In the present study, we investigated the analgesic effect of chronic application of B-complex vitamins (Neurobion) using an inflammatory experimental pain model in rats. Nociceptive behavioral responses were evaluated in male Wistar rats after plantar injection of formalin, comparing groups with (TG) and without (CG) Neurobion pretreatment. In addition, neuronal activity in the central pain pathway was evaluated using c-Fos immunohistochemical reactivity and NADPH-d histochemistry. A highly significant reduction of painful behaviors such as licking and flinching were observed in TG, especially during the secondary phase of the formalin test compared to CG. Results suggest that long-term pre-treatment using Neurobion can have a beneficial effect in reducing the chronic phase of pain. In addition, we observed a downregulation of c-Fos and NADPH-d in dorsal spinal neurons, suggesting that the antinociceptive effect induced by Neurobion could be due to a suppression of nociceptive transmission at the spinal level, particularly in the afferent regions of the dorsal spinal horn, which these neurons utilizing nitric oxide at least as one of their pain neurotransmitters.

  • Importin α3 contribution to the pain transduction pathway

    Authors:
    Livia Testa (1,2), Amisha Parmar (1,2), Giada Musso (1,2), Sofia Dotta (1,2), Alessandro Vercelli (1,2,3), Letizia Marvaldi (1,2)

    Organisations:
    1: University of Turin - Dept. Neuroscience, Italy
    2: Neuroscience Institute Cavalieri Ottolenghi (NICO), Italy
    3: Istituto Nazionale di Neuroscienze, Italy

    Presenting author: Livia Testa

    Exposition date: Friday, September 15, 2023

    Exposition position: 083/1

    The recent opioid crisis in the USA has demonstrated the limitations in pain management options available at the moment. Therefore, investigating the biology of pain perception is vital to promote the development of more specific and non-addictive analgesic drugs, especially in the context of chronic pain.

    Altered skin and muscle innervation, as a result of an unbalance of internal and external growth signals in neurons, is one of the causes of neuropathic pain.

    Peripheral neurons survive and grow in response to neurotrophins, released by surrounding cells. It has been speculated that intracellular growth factors also contribute to and regulate neurotrophin signaling, but the current knowledge on this topic is severely lacking. Intriguingly, this signaling axis could be relevant in shaping nociception in the peripheral neurons.

    In a recently published article (Marvaldi et al., 2020), knockout of importin α3 in dorsal root ganglia (DRG) neurons was shown to reduce neuropathic pain perception. Importin α3 is a shuttle protein of the karyopherin family that acts as an adaptor protein of KPNB1 for the delivery of transcription factors to the nucleus. Indeed, analysis has shown that importin α3 null mice have altered gene expression upon injury.

    Our research aims to investigate which genes, found to have altered expressions in importin α3 null mice, are relevant for pain perception. In particular, we are planning to look for those genes that are differently modulated at the early and late stages of chronic pain. By comparing our findings to data collected from previously published articles, we will identify candidate proteins that may more likely be involved in the nociception pathways and then further characterize their roles through biochemical assays (both in vitro and in vivo).

    Finally, we hope to unravel novel transcriptional networks controlling nociception, which could become new therapeutic targets to manage chronic pain patients.

  • Mechanisms of initiation of cortical spreading depression in the mouse cerebral cortex

    Authors:
    Marina Vitale (1), Angelita Tottene (1), Maral Zarin Zadeh (1), Daniela Pietrobon (1,2)

    Organisations:
    1: Department of Biomedical Sciences, University of Padova, 35131 Padova
    2: Padua Neuroscience Center (PNC), University of Padova, 35131 Padova

    Presenting author: Marina Vitale

    Exposition date: Friday, September 15, 2023

    Exposition position: 084/1

    There is increasing evidence that cortical spreading depression (CSD) is the neurophysiological correlate of migraine aura and a trigger of migraine pain mechanisms. The mechanisms of initiation of CSD in the brain of migraineurs remain unknown, and the mechanisms of initiation of experimentally-induced CSD in normally metabolizing brain tissue remain incompletely understood and controversial. Here, we investigated the mechanisms of CSD initiation by focal application of high KCl in mouse cerebral cortex slices. We simultaneously recorded the changes in intrinsic optic signal and in membrane potential of layer 2/3 pyramidal neurons, located near the site of CSD induction, elicited by KCl puffs of increasing duration up to the threshold duration triggering a CSD before and after application of a specific blocker of either the glutamate NMDA receptors (NMDAR), the glutamate AMPA receptors or the voltage-gated Ca2+ (CaV) channels. If the drug blocked CSD, stimuli up to 12-15 times threshold were applied. Blocking either the NMDARs with MK801 or the CaV channels with Ni2+ completely inhibited CSD initiation by both CSD threshold and largely suprathreshold KCl stimuli. Inhibiting the AMPA receptors with NBQX was without effect on CSD threshold and velocity. The analysis of the CSD subthreshold and threshold neuronal depolarizations revealed that the mechanism underlying ignition of CSD by a threshold stimulus and not by a just subthreshold stimulus is the CaV-dependent opening of a threshold level of NMDARs, and that the delay of several seconds with which this occurs underlies the delay of CSD initiation relative to the rapid neuronal depolarization produced by KCl. Our data show that both NMDARs and CaV channels are necessary for CSD initiation, which is not determined by the level of extracellular K+ or of the neuronal depolarization per se, but requires the CaV-dependent opening of a threshold level of NMDARs.

Mitochondriopathies

  • Nr2f1 shapes mitochondria in the mouse brain unraveling new insights into the neurodevelopmental disorder BBSOAS

    Authors:
    Sara Bonzano (1), Eleonora Dallorto (1), Ivan Molineris (2), Filippo Michelon (1), Isabella Crisci (1), Giovanna Gambarotta (3), Francesco Neri (2), Salvatore Oliviero (2), Ruth Beckervordersandforth (4), Dieter Chichung Lie (4), Paolo Peretto (1), Serena Bovetti (1), Michèle Studer (5), Silvia De Marchis (1)

    Organisations:
    1: DBIOS, NICO, University of Turin, Italy
    2: DBIOS, IIGM, University of Turin, Italy
    3: DSCB, NICO, University of Turin, Italy
    4: Institut für Biochemie, Friedrich-Alexander Universität Erlangen, Germany
    5: iBV, University of Côte d’Azur, NIce, France

    Presenting author: Silvia De Marchis

    Exposition date: Friday, September 15, 2023

    Exposition position: 085/1

    The nuclear receptor Nr2f1 acts as a strong transcriptional regulator in embryonic and postnatal neural cells. In humans, mutations in the NR2F1 gene cause the Bosch-Boonstra-Schaaf Optic Atrophy-intellectual Syndrome (BBSOAS), a rare neurodevelopmental disorder characterized by multiple clinical features including vision impairments, intellectual disability and autistic traits that might be compatible with mitochondrial dysfunction in the nervous system. Interestingly, clinical investigations reported defective electron transport chain/OxPhos machinery in the muscles of two BBSOAS patients. However, whether mitochondrial dysfunction is a general mechanism in BBSOAS and how NR2F1 haploinsufficiency might lead to mitochondria-related functional defects in neurons is completely unknown. By genome-wide analyses we identified a wide set of nuclear-encoded mitochondrial genes as potential genomic targets under direct Nr2f1 transcriptional control in neurons. Notably, Nr2f1 mitochondrial putative targets form a highly interconnected network of functional and physical protein associations, suggesting that Nr2f1 may contribute to a coordinated regulation of multiple aspects of mitochondrial biogenesis and function. By combining mouse genetics, neuroanatomical and imaging approaches we demonstrated that conditional Nr2f1 loss-of-function within the adult mouse hippocampal neurogenic niche reduces mitochondrial mass and induces mitochondrial fragmentation and downregulation of key mitochondrial proteins in adult-born hippocampal newborn neurons, whose functional integration and survival are impaired. Importantly, we also found dysregulation of several nuclear-encoded mitochondrial genes and downregulation of key mitochondrial proteins in the brain of Nr2f1-heterozygous mice, a validated BBSOAS model. Notably, altered Nr2f1 expression in neural tissues has been reported in Down syndrome as well as in mouse models of Alzheimer’s and Parkinson’s diseases. Therefore, our findings open new perspectives for the future investigation on the implication of Nr2f1 in the mitochondrial dysfunction associated with the pathogenesis and progression of BBSOAS as well as multiple neurological disorders, opening new possibilities for therapeutic intervention.

  • The role of Rab proteins in mitochondrial dysfunction related to Parkinson’s disease

    Authors:
    Martina Brughera, Adeena Shafique, Francesca Martorella, Chiara Frigé, Marta Lualdi, Tiziana Alberio

    Organisations:
    Department of Science and High Technology (DiSAT), University of Insubria, Como and Center of Research in Neuroscience, University of Insubria, Busto Arsizio

    Presenting author: Martina Brughera

    Exposition date: Saturday, September 16, 2023

    Exposition position: 066/2

    Mitochondria are crucial organelles involved in the maintenance of cellular homeostasis and metabolism. They are organized in dynamic networks that undergo fusion and fission events which remodel their shape according to the cellular state and functions. Mitochondria that are damaged or dysfunctional are degraded through mitophagy. Dysregulations in these processes, collectively referred to as mitochondrial dynamics, are often associated to neurodegenerative diseases like Parkinson’s disease (PD).

    Mutations in mitophagy-related PRKN gene, which encodes the E3 ubiquitin ligase Parkin, have been linked to autosomal recessive juvenile PD. Recently, also the role of Rab proteins has been associated to PD and mitophagy: mutations in RAB32 and RAB39B genes have been identified to cause familial PD. Rabs orchestrate vesicle trafficking and are involved in the endosomal-lysosomal pathway, a crucial mechanism for the disposal of damaged mitochondria.

    In order to investigate the interplay between dysfunctional mitophagy and Rab proteins dynamics in the context of PD, we investigated the levels and localization of a subset of Rab proteins in: i) CCCP-treated SH-SY5Y cells as positive control of mitophagy, ii) dopamine-treated SH-SY5Y cells, to mimic impaired dopamine (DA) homeostasis characteristic of early steps of PD, and iii) PD patient-derived fibroblasts carrying PRKN mutations.

    As results, in CCCP-induced mitophagy Rab5 levels were up-regulated, while in DA-induced dysfunctional mitophagy Rab5 and Rab7 levels were significantly increased. In PRKN-mutated fibroblasts, Rab7 and Rab11 were up-regulated compared to controls and preliminary data from control fibroblasts suggested Rabs colocalization with the mitochondrial network.

    Thus, our results further support a possible involvement of Rabs in the regulation of mitophagy. In particular, Rabs seems to be central in both classical and non-canonical/endosome-mediated mitophagy.

Multiple sclerosis

  • Applicability of sNFL in Multiple Sclerosis as additional monitoring tool in clinical practice and implications in NEDA-3 evaluation

    Authors:
    Cecilia Irene Bava (1), Paola Valentino (1,2), Simona Malucchi (3), Serena Martire (1,4), Alessia Di Sapio (3), Marco Capobianco (5), Antonio Bertolotto (1)

    Organisations:
    1: NICO - Neuroscience Institute Cavalieri Ottolenghi, Orbassano, Italy
    2: CRESM Biobank, University Hospital San Luigi Gonzaga, Orbassano, Italy
    3: Department of Neurology and CRESM, University Hospital San Luigi Gonzaga, Orbassano, Turin
    4: Department of Neuroscience "Rita Levi Montalcini", University of Turin, Italy
    5: Department of Neurology, Santa Croce and Carle Hospital, Cuneo, Italy

    Presenting author: Cecilia Irene Bava

    Exposition date: Saturday, September 16, 2023

    Exposition position: 068/2

    BACKGROUND

    Despite the increased availability of efficient therapeutic options, effective biological tools are still mandatory for individualized clinical management in MS. Neurofilament light chain (NFL) is the most promising biomarker of disease activity and treatment efficacy. Its implementation in clinical practice is still to be addressed at individual level.

    OBJECTIVE

    To evaluate the use of sNFL in Multiple Sclerosis (MS) clinical practice, as measure of disease activity and treatment efficacy, in particular within NEDA (no-evidence-of-disease-activity) assessment.

    METHODS

    sNFL were cross-sectionally evaluated in 933 MS patients (811 relapsing remitting (RR), 50 primary progressive (PP) and 72 secondary progressive (SP)) at different disease stages (diagnostic time, before and during therapies). sNFL were correlated with radiological and clinical activity, and evaluated in a subgroup of 567 treated RR patients in NEDA-3 status.

    sNFL were measured by Simoa assay (Quanterix) and interpreted according to previously defined cut-off values. Samples and data were collected within CRESM Biobank.

    RESULTS

    1) Progressive MS patients showed a greater prevalence of pathological sNFL levels (36% in PPMS,26% in SPMS) relative to RRMS (16%). 2) All therapies lowered sNFL relative to naïve patients. 3) Pathological sNFL levels were observed in 71-72% of clinical and radiological active patients respectively, but also in 9% of NEDA-3 patients: this percentage lowered with extension of NEDA-3 status duration. These patients were classified according to their different clinical characteristics.

    CONCLUSIONS

    Our study shows that sNFL use in the everyday clinical practice is informative in the individual monitoring of MS patients and in NEDA assessment; routinary use needs that sNFL levels are analyzed at individual level according to reference values.

    sNFL showed a correlation with disease subtype, clinical activity and treatments efficacy. In particular we observed pathological sNFL levels in a subgroup of NEDA-3 patients, which could suggest subclinical disease activity and/or progression to be monitored.

  • BB-CRESM: a structured institutional biobank for quality research in Multiple Sclerosis

    Authors:
    Paola Valentino (1,2), Francesca Montarolo (1,2,3), Cecila Irene Bava (1), Lucia Giorgi (1), Antonio Bertolotto (1), Marco Alfonso Capobianco (4), Alessia Di Sapio (5)

    Organisations:
    1: Neuroscience Institute Cavalieri Ottolenghi (NICO), Orbassano, Italy
    2: CRESM Biobank, University Hospital San Luigi Gonzaga, Orbassano, Italy
    3: Department of Neuroscience "Rita Levi Montalcini", University of Turin, Italy
    4: Department of Neurology, Santa Croce and Carle Hospital, Cuneo
    5: Department of Neurology and CRESM, University Hospital San Luigi Gonzaga, Orbassano, Italy

    Presenting author: Paola Valentino

    Exposition date: Saturday, September 16, 2023

    Exposition position: 067/2

    Background. Biobanks are increasingly demonstrating a crucial role in clinical/scientific research. They are no-profit unit in which collection, storage, and distribution of biospecimens/associated data are performed according to quality standards and legal/ethical issues.The Regional Reference Multiple Sclerosis Center (CRESM, AOU San Luigi Gonzaga) has been operational in transforming its “historical collection” of biological samples into a structured biobank (BB-CRESM).

    Objectives.1)To describe the establishment/management/organization of BB-CRESM 2)To show the importance of biobanking for high quality and reproducible research studies

    Methods and Results

    1)Establishment and organization: in 2020, BB-CRESM received formal Institutional commitment into AOU San Luigi. Legal and ethical (ELSI) documentation was approved by Ethical committee and BB-CRESM governance was defined. BB-CRESM is involved in BBMRI network.

    2)Participants engagement: about 1500 patients and 150 healthy participants (HC) are currently involved according to ELSI procedures.

    3)Samples and data: about 30000 biospecimens (serum plasma, RNA, whole blood, blood cells and cerebrospinal fluid samples) are currently stored, collected from MS patients at different disease stages (diagnosis, before and during therapies and at clinical exacerbations), from patients with other neurological diseases and from HC. Each biospecimen is aliquoted in barcoded tubes, according to privacy/quality requirements. Specific GDPR-based procedures for personal data protection are applied

    4)Quality: quality controls on biological samples (RNA integrity/purity), data (completeness/consistency/relevance) and equipment were defined and applied. BB-CRESM guarantees its personnel competence through training activities/working groups.

    5)Access policy/cost-recovery: researchers can have access to BB-CRESM samples/data according to definite procedures. A cost-recovery policy was defined.

    6)Sample requests/collaborations: since 2020, more than 1000 samples/data have been distributed for external research studies; several projects were carried on at CRESM; additional collaborations are ongoing.

    Conclusions. BB-CRESM is the first italian biobank focused on MS. Its activity, founded on rigorous quality standards, represents a crucial service to boost quality research studies, in the respect of involved participants.

  • High-fat diet exacerbates inflammatory synaptic damage and disease course in clinical and experimental multiple sclerosis

    Authors:
    Sara Balletta (1), Silvia Caioli (1), Alessandra Musella (2), Diego Fresegna (2), Fortunata Carbone (3), Claudio Procaccini (3), Livia Guadalupi (4), Valentina Vanni (2), Krizia Sanna (4), Alessandro Moscatelli (4), Laura Vitiello (2), Luana Gilio (1), Monica Nencini (2), Antonio Bruno (1), Ettore Dolcetti (1), Georgia Mandolesi (2), Diego Centonze (1), Francesca De Vito (1)

    Organisations:
    1: Unit of Neurology, IRCCS Neuromed, Italy
    2: Sinaptic Immunopathology Lab, IRCCS San Raffaele Roma, Italy
    3: Unit of Neuroimmunology, IRCCS - Fondazione Santa Lucia, Rome, Italy
    4: Department of System Medicine, University of Rome Tor Vergata, Italy

    Presenting author: Sara Balletta

    Exposition date: Saturday, September 16, 2023

    Exposition position: 070/2

    Background: High-fat diet (HFD) has emerged as one of the environmental factors that influence Multiple Sclerosis (MS) condition. However, its impact on the neuro-immune crosstalk remains unexplored at synaptic level although inflammatory synaptic dysfunctions are an early hallmark of MS.

    Here, we aimed at clarifying HFD effects on synaptic damage in MS and its mouse model, the experimental autoimmune encephalomyelitis (EAE).

    Methods: We explored the influence of HFD on disease severity also linked to inflammatory synaptopathy in a large cohort of patients with MS and we completed the study in mice. C57BL/6 mice were fed either standard diet or HFD for 3 weeks before MOG35–55-EAE induction to evaluate clinical, behavioral, electrophysiological and molecular parameters. Control mice (CFA) were used to assess the synaptotoxic potential of HFD in a non-autoimmune condition.

    Results: Our clinical studies revealed that HFD directly correlated with MS severity, which appeared to be related to CSF glutamate levels in overweight and obese patients, even after one-year follow-up. In accordance, HFD worsened neuroinflammation, glutamatergic transmission and clinical manifestations in EAE mice. Notably, CFA mice fed on HFD showed all the hallmarks of synaptopathy observed in EAE SD mice. Mechanistically, HFD increased peripheral inflammation and the blood-brain-barrier permeability allowing the entrance of more activated and proliferative T cells in the CNS of CFA mice. Furthermore, all neuroinflammatory and synaptic dysfunctions induced by HFD in CFA mice were associated with a prominent gut microbiota dysbiosis, which was exacerbated in EAE animals receiving HFD.

    Conclusions: Overall, we demonstrated that HFD negatively affects MS and EAE disability by prompting synaptopathy with a possible exacerbation of neurodegenerative processes. Likely, gut microbiota dysbiosis is implicated in the increase of neuroinflammation that is responsible for HFD-linked synaptic impairments. Our results suggest that reducing dietary fat intake may provide protection against the MS occurrence and progression.

  • IL-9 regulates microglia-driven synaptotoxicity and neurodegeneration in experimental multiple sclerosis

    Authors:
    Livia Guadalupi (1,2), Valentina Vanni (2), Krizia Sanna (1), Sara Balletta (3), Silvia Caioli (3), Francesca De Vito (3), Diego Fresegna (2), Monica Nencini (2), Elisabetta Volpe (4), Georgia Mandolesi (2,5), Diego Centonze (3,1), Alessandra Musella (2,5)

    Organisations:
    1: Università degli Studi di Roma Tor Vergata, Italy
    2: Synaptic Immunopathology Lab, IRCCS San Raffaele Roma, Rome, Italy
    3: Unit of Neurology, IRCCS Neuromed, Pozzilli (Is), Italy
    4: Neuroimmunology Unit, IRCCS Fondazione Santa Lucia, Via del Fosso di Fiorano 64, 00143, Rome, Italy
    5: Department of Human Sciences and Quality of Life Promotion University of Rome San Raffaele, Italy

    Presenting author: Livia Guadalupi

    Exposition date: Saturday, September 16, 2023

    Exposition position: 069/2

    Multiple sclerosis (MS) is a neuroinflammatory and neurodegenerative disease of the central nervous system characterized by inflammation-driven synaptic abnormalities. Interleukin 9 (IL-9) is emerging as a pleiotropic cytokine involved in MS pathophysiology. Here, we demonstrated that peripheral and central administration of IL-9 significantly ameliorated experimental autoimmune encephalomyelitis (EAE) clinical disability, neuroinflammation and synaptic damage. Biochemical, immunohistochemical and electrophysiological experiments clearly indicated that the beneficial effect of IL-9 was dependent on microglia- and TNF-mediated neuronal excitotoxicity. Two main mechanisms emerged: first, IL-9 modulated microglial inflammatory and homeostatic activity, promoting the expression of triggering receptor expressed on myeloid cells-2 (TREM2) and reducing TNF release; second, IL-9 suppressed TNF signaling blocking its synaptotoxic effect. Data presented in the present work point to IL-9 as a critical modulator of inflammatory synaptopathy in EAE and MS, paving the way to new treatments against the neurodegenerative damage associated with microglial activation in neuroinflammatory disorders.

  • Re-emergence of T lymphocytes-mediated synaptopathy in progressive multiple sclerosis

    Authors:
    Sara Balletta (3), Krizia Sanna (4), Antonio Bruno (3,4), Alessandra Musella (1,2), Silvia Caioli (3), Livia Guadalupi (2,4), Francesca De Vito (3), Diego Fresegna (2), Valentina Vanni (2), Laura Vitiello (2), Monica Nencini (2), Diego Centonze (3,4), Georgia Mandolesi (1,2)

    Organisations:
    1: Università di Roma San Raffaele, Italy
    2: IRCCS-San Raffaele Roma, Italy
    3: IRCCS-Neuromed, Pozzilli (IS) Italy
    4: Università di Roma Tor Vergata, Italy

    Presenting author: Georgia Mandolesi

    Exposition date: Saturday, September 16, 2023

    Exposition position: 072/2

    Background and objectives: Secondary progressive multiple sclerosis (SPMS) is characterized by the gradual accumulation of irreversible disability in patients with a previous relapsing-remitting disease course (RRMS). Peripheral T lymphocytes of relapsing patients and of experimental autoimmune encephalomyelitis (EAE) mice exacerbate excitatory transmission of central neurons, a synaptotoxic ability absent during the remitting stages of the disease. Here, we hypothesized that the re-emergence of T lymphocyte-mediated synaptotoxicity could contribute to the progression of the disease. We also explored the possible synaptoprotective effects of siponimod, a sphingosine 1-phosphate receptor (S1PR) modulator, demonstrated to reduce the accumulation of disability and
    grey matter damage in SPMS patients.
    Methods: We enrolled a group of 6 healthy controls (HC) and 10 non-active SPMS patients (3 treated with siponimod) and performed heterologous chimeric experiments. Specifically, we recorded the spontaneous excitatory postsynaptic currents (sEPSCs) from striatal neurons of healthy C57BL/6 mice in the presence of T lymphocytes derived from the blood of SPMS patients and HCs.We executed homologous chimeric experiments with T lymphocytes isolated from the spleens of EAE mice and incubated overnight with agonists and antagonists of S1PRs, to identify the receptor involved in the siponimod-mediated synaptic recovery.
    Results: All the kinetic parameters of sEPSCs recorded in the presence of T lymphocytes from non-active SPMS patients were significantly increased compared to HCs and recovered due to siponimod treatment. Moreover, EAE T lymphocytes cultured overnight with siponimod led to the recovery of sEPSC decay time and half-width compared to vehicle treatment. The synaptoprotective effect of siponimod was mediated by modulation of S1P5Rs.
    Discussion: Transition from RRMS to SPMS is accompanied by the reappearance of T
    lymphocyte-mediated synaptotoxicity, likely contributing to neurodegeneration processes. The evidence that siponimod hampers T lymphocyte-mediated excitotoxicity in SPMS confirms the relevance of inflammatory synaptopathy in progressive MS and its potential reversibility in response to disease modifying therapies.

  • Synaptic sexual dimorphism in multiple sclerosis and experimental autoimmune encephalomyelitis mouse model

    Authors:
    Alice Tartacca (1), Sara Balletta (3), Krizia Sanna (1), Silvia Caioli (3), Fabrizio Mariani (1), Alessandra Musella (2,4), Livia Guadalupi (1), Valentina Vanni (2), Diego Fresegna (2), Francesca De Vito (3), Diego Centonze (1,3), Georgia Mandolesi (2,4)

    Organisations:
    1: Department of system medicine, Tor Vergata University, Rome, Italy
    2: Synaptic Immunopathology Lab, IRCCS San Raffaele Roma, Rome, Italy
    3: Unit of Neurology, IRCCS Neuromed, Pozzilli (Is), Italy
    4: Department of Human Sciences and Quality of Life Promotion University of Rome San Raffaele, Italy.

    Presenting author: Alice Tartacca

    Exposition date: Saturday, September 16, 2023

    Exposition position: 071/2

    Multiple sclerosis (MS) is a chronic neuroinflammatory disease characterized by demyelination and neurodegeneration of the central nervous system (CNS). Sex differences influence the susceptibility and progression of MS. Indeed, females are affected by the prevalent relapsing–remitting MS (RRMS) form, with a 3:1 preponderance. Males with RRMS accumulate disability and progress to secondary progressive MS (SPMS) significantly faster than females and show worse cognitive impairment. Recently, a diffuse inflammatory driven synaptic dysfunction and loss, namely synaptopathy, is emerging as prominent feature of MS and its experimental mouse model EAE. The release of the proinflammatory cytokine TNF by microglia and T cells has been associated to the enhancement of the glutamatergic transmission in central neurons, contributing to glutamate-mediated excitotoxicity. Interestingly, chimeric MS experiments performed by combining human T cells and murine cortico-striatal slices demonstrated that, compared to healthy subjects (HS), T cells from MS patients induced an increase of the glutamatergic kinetic properties by a TNF-mediated mechanism. Here, we explored the impact of sex on T cell-mediated excitotoxicity and observed a more pronounced enhancement of the glutamatergic transmission in slices incubated with lymphocytes isolated from male MS vs female MS patients. In parallel, we performed preclinical studies to investigate the impact of sex on the inflammatory-synaptopathy in the EAE model. We did not observe any difference in the striatal glutamatergic transmission of EAE male vs EAE female mice. Curiously, a sex difference was observed in the basal glutamatergic transmission of healthy mice. Interestingly, in vitro treatment with etanercept, a TNF receptor antagonist, of male mouse slices reduced the duration of glutamate events, suggesting the involvement of this cytokine in the increase of glutamatergic transmission observed in male animals. These preliminary data suggest that synaptic sexual dimorphism might drive susceptibility and progression of MS in patients.

Muscle and motor neuron disorders

  • A motor neuron-like spheroid 3D model for in vitro evaluation of neurodegeneration

    Authors:
    Pietro Arnaldi (1), Grazia Bellese (1), Maria Cristina Gagliani (1), Valeria Crippa (2), Patrizio Castagnola (3), Cortese Katia (1)

    Organisations:
    1: DIMES, Dipartimento di Medicina Sperimentale, Cellular Electron Microscopy Lab, Università di Genova, Genova, Italy
    2: Dipartimento di Scienze Farmacologiche e Biomolecolari "Rodolfo Paoletti", Dipartimento di Eccellenza 2018-2027, Università degli Studi di Milano, Milan, Italy
    3: IRCCS Ospedale Policlinico San Martino, Genova, Italy

    Presenting author: Pietro Arnaldi

    Exposition date: Saturday, September 16, 2023

    Exposition position: 081/2

    Three-dimensional (3D) spheroid models provide a bridge between traditional two-dimensional (2D) cultures and the complex in vivo tissue environment. These models are developed using self-clustering cells to mimic an appropriate 3D extracellular matrix (ECM). The NSC-34 cell line, created by fusing mouse spinal cord motor neurons and neuroblastoma cells, serves as a widely accepted model for studying motor neuron diseases. However, the precise behavior and features of this cell line in a 3D context have not been thoroughly investigated. In this work, we successfully generated NSC-34 spheroids using a non-adhesive agarose-based micro-wells template. Over five days, we examined the formation, stability, and morphological properties of spheroids by light and electron microscopy imaging and flow cytometry analysis. Spheroids with an initial density equal to 25000 cells showed a diameter size of 377±13 µm, a high circularity index (0.91±0.07) and a viability of ~85% after 5 days in culture. Our extensive ultrastructural analysis demonstrated that NSC-34 cells in 3D adopt and maintain a distinct round-like shape, enabling a network of short yet stable membrane connections between cells. No significant differences were detected in the ultrastructural morphology of subcellular organelles compared to 2D cultures. To showcase the potential of this system for pathological studies, we utilized the NSC-34 spheroid model to investigate the influence of 3D structure and cellular assembly on the expression of GFP-tagged full length TAR-DNA-binding protein 43 (TDP-43), a known marker of ALS pathology. NSC-34 cell line showed nuclear expression of GFP-TDP-43 in the spheroidal construct, with levels comparable to standard monolayer cultures. Our results offer valuable insights into the behavior of the NSC-34 cell line in 3D environments and emphasize the potential utility of spheroid models for exploring motor neuron diseases and associated pathologies.

    Research funded by Ministero dell’Università e della Ricerca (MIUR) – Italy (PRIN2020 - n. 2020PBS5MJ)

  • ActR-Fc-nLG3: a novel biological sustaining neuromuscular junction innervation in sarcopenia

    Authors:
    Roberta Schellino (1,2), Marina Boido (1,2), Jan W. Vrijbloed (3), Ruggero Fariello (3), Alessandro Vercelli (1,2)

    Organisations:
    1: Dept. of Neuroscience, University of Turin, 10126 Turin, Italy.
    2: Neuroscience Institute Cavalieri Ottolenghi (NICO),10043 Orbassano (Turin)
    3: PharmaFox AG, Möhlin, Aargau, Switzerland

    Presenting author: Roberta Schellino

    Exposition date: Saturday, September 16, 2023

    Exposition position: 078/2

    Sarcopenia is the primary cause of impaired motor performance in the elderly, also responsible for increased morbidity and mortality. The prevailing approach to counteract such condition is the increase in muscle mass through inhibition of the myostatin system: however, this strategy is not able to sustain per se innervation of the hypertrophic muscle and only moderately improves muscular strength, causing a progressive worsening of the motor performances. In this scenario, we have synthetized a novel biological compound, named ActR-Fc-nLG3, by combining the soluble activin receptor to the C-terminal agrin nLG3 domain: this compound has the potential of providing additional innervation to the hypertrophic muscle. Indeed, we have previously demonstrated that, after its administration, young mice are capable of remarkably enhancing the endurance of their motor tasks (rotarod), with a modest gain of muscle mass, compared to common myostatin inhibitors (as ActR-Fc). Here we extended this observation by demonstrating that also in aged (2 years-old) mice, long-term administration of ActR-Fc-nLG3 is able to increase in a sustained way the muscle strength and endurance, compared with the administration of the ActR-Fc alone and the control vehicle (PBS). Histological data show that the effects of this new biological are due to changes in neuromuscular junctions (NMJs) and to the preservation of fiber innervation, without affecting muscular fiber size. The presence of increased membrane folds in the postsynaptic site offers a possible explanation to the increased endurance as a result of improved efficiency of the neurotransmission at the NMJ level. Thus, our peptide may represent a valid option for treating disorders of the striatal muscle tissue, together with muscle dysfunction caused by altered neuronal input to the muscle, raising the hope that a therapy may be developed not only for sarcopenia but also for other neuromuscular disorders.

  • Administration of adipose-derived stem cells EVs in a murine model of Spinal Muscular Atrophy: effects of a new potential therapeutic strategy

    Authors:
    Federica Virla (1), Ermanna Turano (1), Ilaria Scambi (1), Lorenzo Schiaffino (1), Raffaella Mariotti (1), Marina Boido (2)

    Organisations:
    1: Department of Neuroscience, Biomedicine and Movement Sciences, University of Verona, Verona, Italy
    2: Neuroscience Institute Cavalieri Ottolenghi, Department of Neuroscience "Rita Levi Montalcini", University of Turin, Turin, Italy

    Presenting author: Federica Virla

    Exposition date: Saturday, September 16, 2023

    Exposition position: 095/2

    Spinal Muscular Atrophy (SMA) is an autosomal-recessive neuromuscular disease affecting children. It is caused by the mutation or deletion of the survival motor neuron 1 (SMN1) gene resulting in lower motor neurons (MNs) degeneration followed by motor impairment, progressive skeletal muscle paralysis and respiratory failure. In addition to the already existing therapies, a possible combinatorial strategy could be represented by the use of adipose-derived mesenchymal stem cells (ASCs) that can be obtained easily and in large amount from adipose tissue. Their efficacy seems to be correlated to their paracrine activity and the production of soluble factors released through extracellular vesicles (EVs). EVs are important mediators of intercellular communication with a diameter between 30 and 100 nm. Their use in other neurodegenerative disorders showed a neuroprotective effect thanks to the release of their content, especially proteins, miRNAs and mRNAs. In this study, we evaluated the effect of EVs isolated from ASCs (ASC-EVs) in the SMNΔ7 mice, a severe SMA model. With this purpose, we performed intracerebroventricular administrations of ASC-EVs in SMA pups and assessed the treatment efficacy by behavioural and histological analyses. The results showed positive effects of ASC-EVs on the disease progression, with improved motor performance and a significant delay in spinal MN degeneration of treated animals. ASC-EVs could also reduce the apoptotic activation (cleaved Caspase-3) and modulate the neuroinflammation with an observed decreased glial activation in lumbar spinal cord, while at peripheral level ASC-EVs could only partially limit the muscular atrophy and fiber denervation. Thus, our results could encourage the use of ASC-EVs as a therapeutic combinatorial treatment for SMA, bypassing the controversial use of stem cells.

  • Ambiguous effects of Hydrogen Sulphide, a study in mouse spinal cord cultures

    Authors:
    Susanna De Stefano (1,8), Francesca Ciaiola (1,8), Marta Tiberi (5), Illari Salvatori (4,9), Marco De Bardi (6), Giovanna Borsellino (6), Alberto Ferri (3,4), Cristiana Valle (3,4), Nicola Mercuri (1,7), Valerio Chiurchiù (2,5), Alida Spalloni (8), Patrizia Longone (8)

    Organisations:
    1: System Medicine Department, University of Rome Tor Vergata;
    2: Institute of Translational Pharmacology (IFT), National Research Council (CNR), Rome, Italy
    3: Institute for Cell Biology and Neurobiology, CNR, Rome, Italy
    4: Laboratory of Neurochemisrty, Fondazione S. Lucia IRCCS, Rome, Italy
    5: Laboratory of Resolution of Neuroinflammation, IRCCS Santa Lucia Foundation, Rome, Italy
    6: Neuroimmunology Unit, Santa Lucia Foundation IRCCS, Rome, ItalyUnit, Santa Lucia Foundation IRCCS, Rome, Italy
    7: Laboratory of Experimental Neuroscience, Santa Lucia Foundation IRCCS, Rome, Italy
    8: Molecular Neurobiology Unit, Fondazione Santa Lucia-IRCCS, Rome, Italy
    9: Department of Experimental Medicine, University of Roma “La Sapienza”, Rome, Italy

    Presenting author: Susanna De Stefano

    Exposition date: Saturday, September 16, 2023

    Exposition position: 077/2

    Hydrogen sulphide (H2S), a colourless gas with an unpleasant odour, considered a toxic environmental pollutant, is now recognized as an important endogenous neuromodulator (Peers et al., 2012) owning protective effects, as established in “in vivo” models of Parkinson's and Alzheimer's diseases (Giuliani et al., 2013). In Amyotrophic Lateral Sclerosis (ALS), a lethal disease characterized by the progressive degeneration of the upper and lower motor neurons (MNs), we have reported poisonous levels of H2S in the liquor of patients (Davoli et al., 2015). Furthermore, we and others have described increased neuronal death and the recruitment of death-inducing pathways (apoptosis and necrosis) (Greco et al., 2018) in the presence of H2S, even at physiological concentrations (Cheung et al., 2007). We have also demonstrated that the pharmacological inhibition of H2S production is able to increase the lifespan of the female of the SOD1G93A mice, the classical model of ALS (Spalloni et al., 2019).

    The aim of the present study is to further assess the effects of NaHS (200 micromolar), an H2S donor, in mouse mixed spinal cord culture. We will show that H2S induces cell apoptosis via the release of cytochrome C and the activation of caspase 3. Alters mitochondrial respiration, and increases the expression of connexin 43 protein, a strong indicator of astrocytic gap-junction expansion, a phenomenon that has been linked to the ALS-related MNs loss (Almad et al., Glia 2016). We also report its ability to revert microglia and astrocyte polarization from M1 to M2 and A2 to A1, respectively. The ambiguous effects of H2S on the cellular and molecular homeostasis in mixed spinal cord culture and its potential role in ALS will be discussed.

  • Analysis of possible glycinergic system alterations in Spinal Muscular Atrophy

    Authors:
    Anna Caretto (1,2), Ferdinando Di Cunto (1,2), Marina Boido (1,2), Alessandro Vercelli (1,2)

    Organisations:
    1: Neuroscience Institute Cavalieri Ottolenghi, University of Turin, Italy
    2: Department of Neurosciences "Rita Levi Montalcini", University of Turin, 10126 Turin, Italy

    Presenting author: Anna Caretto

    Exposition date: Saturday, September 16, 2023

    Exposition position: 074/2

    Spinal Muscular Atrophy (SMA) is a neurodegenerative disease due to SMN1 gene mutation causing motor neuron (MN) loss in brainstem and spinal cord and some peripheral district alterations. Although gene-based treatments have improved SMA course, the available therapies still show important limitations. Therefore, the identification of novel therapeutic strategies remains needed.

    Recently it has been observed that SMA shares mitochondria alterations with other neurodegenerative diseases (e.g. Parkinson’s Disease, Alzheimer’s Disease and Amyotrophic Lateral Sclerosis) and therefore they can be considered promising target for SMA therapy.

    In this context, we exploited an anticorrelation-based bioinformatic method to detect genes implicated in mitochondrial functions potentially relevant for drug repositioning approaches. We identified 8 mitochondrial genes significantly physiologically SMN1-anticorrelated. To investigate which of them could be also related to SMA, we performed RT-PCR on tissues obtained from SMNdelta7 mice, representative of a severe model of SMA. We found that GCSH, a glycinergic cleavage system subunit, is significantly upregulated in lumbar spinal cord of SMA pups, since an early symptomatic pathological stage (postnatal day 5, P5). Western Blot analysis confirmed the trend of upregulation and immunofluorescence staining on spinal cord slices showed a significant higher expression of GCSH in MN soma. These findings raised the possibility that the glycinergic system could be affected in SMA. In particular, we hypothesized that a reduction in MN inhibition could contribute to their loss due to hyperexcitability. To address this possibility, we focused on morphological analysis of Renshaw Cells, glycinergic inhibitory interneurons involved in spinal cord-MN recurrent inhibition. We observed a cellular shrinkage since P5 that worsens in the late stage of the disease (P12).

    On this basis, GCSH and glycinergic pathway deeper analysis could be instrumental not only for a better understanding of the mitochondrial role in SMA, but also to identify new molecules for SMA complementary therapy.

  • Assessing the face validity of a mouse model of megaconial congenital muscular dystrophy: behavioral characterization of choline kinase beta (CHKB) mutant mice

    Authors:
    Anna Maria Tartaglione (1), Giorgia Macchioni (2), Deborah Pajalunga (3), Arianna Racca (1), Andrea Casagrande (3), Marco Crescenzi (4), Gemma Calamandrei (1)

    Organisations:
    1: Centre for Behavioural Sciences and Mental Health, Istituto Superiore di Sanità, Rome, Italy
    2: Centre for Behavioural Sciences and Mental Health, Istituto Superiore di Sanità, Rome, Italy
    Dept. of Physiology and Pharmacology, Sapienza University of Rome, Rome, Italy
    3: Dept. of Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy
    4: Core Facilities, Istituto Superiore di Sanità, Rome, Italy

    Presenting author: Giorgia Macchioni

    Exposition date: Saturday, September 16, 2023

    Exposition position: 088/2

    Megaconial congenital muscular dystrophy (CMD) is a rare neuromuscular disorder caused by mutations in the gene encoding choline kinase beta (CHKB) with autosomal recessive inheritance.
    CMD is characterized by early-onset hypotonia, progressive muscle weakness with subsequent loss of ambulation, intellectual disability, and autistic behaviors.
    Two mouse models for human CMD have been generated by inactivation of the gene CHKB, displaying a total loss of the ChkB protein and of its enzymatic activity. A characteristic sign of CHKB knockout (CHKB-KO) mice is muscular dystrophy with a rostro-caudal gradient of severity. It is important to note that, unlike what has been described for pathology in humans, for CMD murine models there are no data in the literature describing their neurobehavioral phenotype.
    The present project was aimed to assess CHKB-KOflp/flp mice in comparison with wildtype (WT) mice through a comprehensive behavioral test battery assessing gross and fine motor abilities, social and repetitive behaviors, and cognitive capabilities.
    Our preliminary data support the translational potential of this murine model of CMD. Results indicate behavioral abnormalities from early neonatal stage to adulthood, showed mainly by CHKB-KO KOflp/flp. Moreover, these mice exhibited muscle weakness and deficits in motor coordination. As for locomotor activity, CHKB-KOflp/flp mice travelled distance and moved with mean velocity similar to WT mice, although they achieved a lower maximum speed. Interestingly, CHKB-KOflp/flp mice showed aberrant levels of vertical activity (i.e., rearing and jumping) and increased exploration of an unfamiliar conspecific in comparison to WT mice, suggesting a behavioral disinhibition profile. Analyses carried out so far did not reveal any cognitive deficits in adolescent mice, but the onset of cognitive impairment at later stages could not be ruled out.
    Further analyses will study the progression of pathology in this promising model to identify the underlying mechanisms and potential targets for treatment of this rare condition.

  • Can stressors affect ALS onset and progression? Preliminary observations

    Authors:
    Daniela Maria Rasà (1,2,3), Ilaria Stoppa (2), Marina Boido (1,2)

    Organisations:
    1: Department of Neuroscience Rita Levi Montalcini, University of Turin, Italy
    2: Neuroscience Institute Cavalieri Ottolenghi, University of Turin, Orbassano (TO), Italy
    3: University School for Advanced Studies IUSS Pavia, Italy

    Presenting author: Daniela Maria Rasà

    Exposition date: Saturday, September 16, 2023

    Exposition position: 079/2

    Stressors can trigger several cellular alterations that similarly characterize the pathogenesis of many neurodegenerative diseases, such as Alzheimer disease, Parkinson disease and Amyotrophic Lateral Sclerosis (ALS). ALS is a motor neuron (MN) disease characterized by progressive degeneration of both upper and lower MNs. The patients show weakness, muscle atrophy and die prematurely.

    The goal of this work is clarifying how stressors can contribute to the onset and progression of ALS, through the screening of several genes, since the correlation between ALS pathophysiological mechanisms and cellular processes activated by stressor exposure is still unclear.

    First of all, an in vitro experimental model has been set-up using naive NSC-34 cells and NSC-34 cells expressing hSOD1 gene (WT or G93A) under the control of a doxycycline-inducible promoter. The cells have been differentiated in MN-like cells with retinoic acid for 4 days, while the hSOD1 expression has been induced adding doxycycline for 24h into culture medium. To mimic a stress condition, cells underwent oxygen and glucose deprivation: CoCl2 was used as hypoxic agent. Cell damage was demonstrated evaluating protein levels of different markers: HIF1α, caspase3, UCP4 and studying the mitochondrial activity (using MitoTracker Red/MitoTraker Green). We observed an early cell damage under stress conditions in mutated cells compared to “healthy cells”.

    Then we studied several genes related to stress and ALS, using pre-designed plates by real-time PCR: genes involved in cell structure, cell integrity, apoptosis and mitochondrial metabolism resulted de-regulated in the mutated cells compared to control.

    Additional experiments are needed to confirm and extend these results, however we have set-up the conditions for the following analyses, to evaluate possible genetic/epigenetic alterations, and to clarify in vivo the stressor impact on the onset and progression of ALS.

  • Development of Growth Hormone Secretagogues as new therapeutical tools for Amyotrophic Lateral Sclerosis

    Authors:
    Ramona Meanti, Laura Rizzi, Elena Bresciani, Laura Molteni, Vittorio Locatelli, Antonio Torsello

    Organisations:
    School of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy

    Presenting author: Ramona Meanti

    Exposition date: Saturday, September 16, 2023

    Exposition position: 075/2

    GHS are a large family of synthetic compounds originally developed for their capability to stimulate the release of GH. It has been proven that GHS also exert neuroprotective effects and participate in the regulation of skeletal muscle mass, in animals and humans. Their pleiotropic role on neurons and muscle cells suggest that these compounds could be developed for the treatment of ALS. The mutation in SOD1 with the replacement of glycine 93 by alanine is a well-known pathogenetic mechanism of ALS, responsible for a conformational change that leads to a gain-of-function resulting in oxidative stress, mitochondrial alterations, and apoptosis.

    This research aims to investigate the potential protective effects of two selected GHS, hexarelin and JMV2894, against massive oxidative stress conditions. Hexarelin has important neuroprotective and cytoprotective activities, both in vitro and in vivo; JMV2894 stimulates Ca2+ mobilization, modulates mitochondria functioning and ROS production in vitro, and GH-release in vivo. To study the neuroprotective effects of hexarelin and JMV2894, human neuroblastoma cells overexpressing SOD1G93A enzyme were incubated for 24h with 150µM H2O2 or with the combination of H2O2 and 1µM hexarelin or 1µM JMV2894.

    Morphometric quantification showed that H2O2-treatment induced an apoptotic phenotype that was rescued by both GHS. The quantification of mRNA levels of the BCL-2 family and those of the effector caspase proteins suggest that GHS have anti-apoptotic effects: both GHS significantly decreased Bax/Bcl-2 ratio and hexarelin also inhibited the activation of caspase-3.

    The molecular pathways involved in GHS neuroprotection include the modulation of MAPKs and PI3K/Akt phosphorylation, probably through epigenetic mediation. Immunofluorescence visualization of γH2AX nuclear foci showed that hexarelin and JMV2894 significantly decreased the percentage of γH2AX-positive cells compared to the H2O2-treated group.

    These findings suggest the possibility of developing new GHS-based anti-oxidant and neuroprotective drugs with improved therapeutic potential that may be useful in ALS therapy.

  • Exploring the metabolic profile in ependymal stem progenitor cells from spinal cord to develop an innovative therapeutic approach to rescue neurodegeneration in amyotrophic lateral sclerosis.

    Authors:
    Marco Cattaneo (1), Marcella Bonanomi (2,3), Cristina Chirizzi (4), Eleonora Giagnorio (5), Claudia Malacarne (5), Silvia Bonanno (5), Renato Mantegazza (5), Giuseppe Lauria Pinter (6,7), Francesca Baldelli Bombelli (4), Daniela Gaglio (3,8), Stefania Marcuzzo (5)

    Organisations:
    1: Neurology 3-Neuroalgology Unit, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy.
    2: Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy.
    3: ElixirNextGenIT European Infrastructure-ISBE/SYSBIO Centre of Systems Biology, Milan, Italy.
    4: Laboratory of Supramolecular and Bio-Nanomaterials (SupraBioNano Lab), Department of Chemistry, Materials and Chemical Engineering, "Giulio Natta", Politecnico di Milano, Milan, Italy.
    5: Neurology 4-Neuroimmunology and Neuromuscular Diseases Unit, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy.
    6: Department of Clinical Neurosciences, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy.
    7: Department of Medical Biotechnology and Translational Medicine, University of Milan, Milan, Italy.
    8: Institute of Molecular Bioimaging and Physiology (IBFM), National Research Council (CNR), Segrate, Italy.

    Presenting author: Marco Cattaneo

    Exposition date: Saturday, September 16, 2023

    Exposition position: 086/2

    Amyotrophic lateral sclerosis (ALS) is a fatal disorder that affects motor neurons in motor cortex, brainstem and spinal cord. The ALS neurodegenerative process stimulates an extensive but unproductive regenerative response in the spinal cord, suggesting that therapeutic interventions aimed at enhancing spinal cord ability to induce regeneration could be successful for obtaining neurogenesis. In ALS G93A-SOD1 mice, the regeneration attempt mainly consists in a proliferative response of ependymal stem progenitor cells (epSPCs), that are normally quiescent in the spinal cord. Spinal cord SPCs from ALS patients are able to differentiate into the three neural cell lineages; thus, stimulation of epSPC differentiation may represent a strategy to counteract, or even reverse, neurodegeneration in ALS. Energy metabolism is altered in ALS. In this context, dysregulated levels of metabolites might disturb epSPC differentiation, and their restoration might favor neurogenesis.

    The aim of this study was to investigate the metabolic profile of ALS epSPCs to disclose novel therapeutic targets for disease treatment.

    By metabolomic analysis, we revealed an altered metabolic profile, underlying changes in energy and mitochondrial pathways in epSPCs isolated from ALS G93A-SOD1 compared to control mice. Specifically, we observed a reduction of tricarboxylic acid cycle metabolites, and an increase of lactate, mevalonate, glutamine and glutamate in cells from affected mice during disease progression compared to control cells. To restore metabolite levels, we treated the epSPCs with nanoparticles loaded with FM19G11, a hypoxia-inducible factor modulator. Of interest, we found that the levels of metabolites implicated in energy and mitochondrial pathways were normalized by the treatment in ALS epSPCs.

    Our overall findings suggest that modulation of metabolic pathways by FM19G11-loaded nanoparticles could represent a promising strategy to affect epSPC differentiation for limiting neurodegeneration in ALS, thus opening new research avenues towards the development of innovative metabolic therapies to counteract the disease progression.

  • Extracellular vesicles from adipose stem cells: their effect on a microglial model of ALS

    Authors:
    Sylwia Dabrowska (2), Ermanna Turano (1), Ilaria Scambi (1), Alice Nodari (1), Federica Virla (1), Mirco Galiè (1), Bruno Bonetti (3), Raffaella Mariotti (1)

    Organisations:
    1: University of Verona, Italy
    2: NeuroRepair Department, Mossakowski Medical Research Institute, Polish Academy of Sciences, Warsaw, Poland
    3: Neurology Unit, Azienda Ospedaliera Universitaria Integrata, Verona

    Presenting author: Raffaella Mariotti

    Exposition date: Saturday, September 16, 2023

    Exposition position: 096/2

    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive degeneration of upper and lower motor neurons (MNs) in the brain and spinal cord leading to paralysis and early death. Increasing evidence indicates that neuroinflammation plays an important role in ALS pathogenesis and disease progression. Neuroinflammatory responses, primarily driven by activated microglia and astrocytes, and followed by infiltrating peripheral immune cells, contribute to exacerbate MNs death. In particular, the role of microglia in ALS remains unclear, partly due to the lack of a model system that is able to completely recapitulate the complexity of ALS pathology. Here we developed and characterize a microglial cells line, SIM-A9 expressing human mutant protein Cu+/Zn+ superoxide dismutase1 (SIM-A9hSOD1(G93A)) as in vitro model mimicking microglia activity in ALS. The expression of hSOD1(G93A) in SIM-A9 cells is able to induce their metabolic activity, causing polarization into a pro-inflammatory phenotype and enhancing reactive oxygen species production, which can lead to activation of cell death processes and apoptosis. Afterwards we used our microglia cellular model as an experimental set up to investigate the therapeutic action of extracellular vesicles from adipose mesenchymal stem cells (ASC-EVs). ASC-EVs represents an innovative therapeutic treatment for ALS due to their neuroprotective and immunomodulatory properties. However, their capacity to modulate inflammatory microglia in ALS is not clear. Here we demonstrate that the treatment with ASC-EVs on microglial cells is able to modulate the inflammatory microglia reducing their metabolic activity, polarizing their phenotype toward an anti-inflammatory one, and decreasing the reactive oxygen species production.

  • First evidence of altered Gas6-Axl signaling in ALS: insights into a novel therapeutic target and monitoring biomarker

    Authors:
    Mauro Giuseppe Spatafora (1), Paolo Cabras (1), Andrea Gazzano (1), Gianluca Di Nolfi (1), Loris Bandirali (1), Agnese Dimartino (1), Davide Camazzola (1), Danilo Pellin (2), Alessandra Biffi (2), Teuta Domi (3), Angelo Quattrini (3), Koen Poesen (4), Nilo Riva (5), Marco Peviani (1)

    Organisations:
    1: Universita' degli Studi di Pavia, Italy
    2: Dana-Farber/Boston Children's Cancer and Blood Disorders Center, Boston, MA, USA
    3: Institute of Experimental Neurology, San Raffaele Hospital, Milan, Italy
    4: KU Leuven, Belgium
    5: Fondazione IRCCS Istituto Neurologico "C. Besta"

    Presenting author: Marco Peviani

    Exposition date: Saturday, September 16, 2023

    Exposition position: 089/2

    Axl belongs to TAM tyrosine kinase receptor family and is involved in neuronal survival, myelination and regulation of immune responses. Up-regulation of Axl was reported in disease-associated microglia in Alzheimer’s disease (AD), in multiple sclerosis (MS) and, more recently, also in a mouse model of TDP43 proteinopathy. Gas6 (Axl endogenous ligand) is important to modulate the extent of neuroinflammation and remyelination in the Central and Peripheral nervous system. One mechanism of regulation of Gas6/Axl pathway is the cleavage of Axl extracellular domain by sheddases ADAM10/17, which are upregulated in proinflammatory conditions. Axl shedding leads to the release of a soluble ectodomain (sAxl) or the Gas6-Axl complex and failure of the membrane-bound Axl portion to transduce intracellular signals. Given the role played by Gas6-Axl pathway in neuronal survival and neuroinflammatory responses, in this work we investigated, for the first time, whether this pathway is implicated in ALS. We analyzed Gas6-Axl pathway in mutant SOD1 and TDP43 animal models of ALS.

    Overall, we evidenced for the first time a defective Gas6-Axl pathway in ALS (reduced mRNA and protein levels of Axl and defective Axl-signaling pathway in motor neurons, upregulation of Axl in glial cells), highlighting that these alterations could account not only for the engagement of reactive gliosis, but also for defective neurotrophic signaling and MN demise. Moreover, we spotted for the first time a correlation between sAxl (measured in peripheral blood) and clinical decline in ALS patients (n=48 patients vs n=24 controls); further validation of sAxl as monitoring biomarker on a larger independent patients’ cohort is in progress.

  • Intracerebroventricular hNSC graft in ALS mice: first pre-clinical results

    Authors:
    Clelia Ferrero (1,9), Edvige Vulcano (2,9), Ivan Lombardi (2,3), Daniela Maria Rasà (1), Silvia De La Morena Saavedra (1), Rose Mary Carletti (4), Maurizio Gelati (5), Daniela Celeste Profico (5), Valentina Grespi (6), Gianmarco Muzi (6), Jessica Rosati (7), Elisa Perciballi (2), Letizia Mazzini (8), Fabiola De Marchi (8), Alessandro Vercelli (1), Angelo Vescovi (2,4), Daniela Ferrari (2), Marina Boido (1)

    Organisations:
    1: Neuroscience Institute Cavalieri Ottolenghi, Department of Neuroscience “Rita Levi Montalcini, University of Turin, Italy
    2: Department of Biotechnology and Biosciences, University of Milano-Bicocca, Italy
    3: School of Medicine and Surgery, University of Bicocca
    4: Fondazione IRCCS, Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy
    5: Fondazione IRCCS Casa Sollievo della Sofferenza, Production Unit of Advanced Therapies (UPTA), Institute for Stem-Cell Biology, Regenerative Medicine and Innovative Therapies (ISBReMIT), San Giovanni Rotondo, Italy
    6: Cell Factory and Biobank Santa Maria Hospital, Terni, Italy
    7: Cellular Reprogramming Unit, Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy
    8: Centro Regionale Esperto SLA Azienda Ospedaliero-Universitaria “Maggiore della Carità”, Novara, Italy
    9: these authors have equally contributed

    Presenting author: Clelia Ferrero

    Exposition date: Saturday, September 16, 2023

    Exposition position: 073/2

    Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder characterized by the impairment of the upper and lower motor neurons (MNs), leading to muscles atrophy and weakness, and finally death due to respiratory failure.

    The potentiality of neural stem cells (NSCs) as a therapeutic approach to improve the state of the MNs and motor performances in neurodegenerative disorders have been evaluated in pre-clinical studies. Moreover, phase I clinical trials (NCT01640067, NCT01348451) have proven the safety and feasibility of human Neural Stem Cell (hNSC) transplantation in spinal cord of ASL patients. However, this administration route presents several disadvantages.

    The aim of this project is to analyse the potentiality of a new way of administration of hNSCs in SOD1 G93A mice, a well-known ALS model. For this purpose, at postnatal day 70 (P70; presymptomatic stage) the mice received an intracerebroventricular injection of hNSCs. Following the graft, the motor performances were evaluated until sacrifice (at P110 or end-stage). To support cell survival, mice underwent a transient (15 days) or an extended (40 days) immunosuppression protocol. At sacrifice, brain, spinal cord, muscles and blood were collected: we are currently evaluating the biodistribution of the hNSCs, the rate of neuroinflammation, the number of upper and lower MNs and the muscular atrophy.

    Our preliminary results suggest that the treatment might delay the decline of motor performances. However, cell survival is not optimal (in particular with the transient immunosuppression protocol), and the reduced sample size prohibits any definitive conclusion.

    However, we are confident that this administration route could be an effective approach to increase cell dosage, to favour a broader spread of transplanted cells and released factors throughout the motor neuraxis by exploiting the CSF circulation, and to possibly target also the motor cortex.

  • Investigating the effect of rare variants of ALS2: from biology to medicinal chemistry

    Authors:
    Matteo Rossi Sebastiano (1), Fabrizia Cesca (2), Gabriele Baj (2), Giuseppe Ermondi (1), Giulia Caron (1)

    Organisations:
    1: Molecular Biotechnology and Health Sciences Dept., University of Torino, CASSMed-Chem, Torino, Italy
    2: Department of Life Sciences, University of Trieste, 34127, Trieste, Italy

    Presenting author: Matteo Rossi Sebastiano

    Exposition date: Saturday, September 16, 2023

    Exposition position: 076/2

    Infantile Ascending Hereditary Spastic Paralysis (IAHSP) is an infantile recessive ultra-rare disease originating from mutations in the ALS2 gene. The protein product is Alsin, implicated in motor neuron physiology. Mutations are also found in patients suffering from other hereditary spastic paraplegias. However, the exact mechanisms are not clear, mainly because full KO mice do not recapitulate the human phenotype. Nevertheless, in vitro ectopic models and biophysical techniques unraveled that Alsin is implicated in intracellular vesicular transport and mitochondrial fusion/fission dynamics.

    Active and functional Alsin is organized in tetramers. However, since the 3D molecular structure of Alsin is not yet resolved, all related molecular aspects are unknown and there is no broad molecular interpretation of IAHSP. Mutations span the whole gene coding sequence, with most of them resulting in early stop codons. However, missense mutations are found as well, enabling medicinal chemistry strategies. This point was proven in a previous study from our group, describing the identification of a molecule to treat a missense mutant. In our opinion, the absence of markers is the major limitation to further progress in research.

    In this work we aim to develop patient-derived cell lines and use them to search for suitable marker(s) of Alsin-related pathologies, focusing on mitochondria functionality. We used our in-silico findings to test the effectiveness of repurposed drugs and presented the first experimental 3D structure of Alsin tetramers obtained with Cryo electron microscopy, to rationalize the effect of missense mutants. Our results represent an effort to conjugate computational and experimental techniques, spanning from biophysical to cell-based assays. We believe that such approach can progress the development of personalized therapeutic strategies, having a concrete impact on the life of patients suffering from IAHSP.

  • Monitoring the glutamatergic system in a mouse model of microRNA-218 (miR-218), a motor neuron enriched micro-RNA.

    Authors:
    Francesca Ciaiola (1,2), Susanna De Stefano (1,2), Juliette Gimenez (2), Dario Tomolillo (4), Claudia Vuotto (4), Alida Spalloni (2), Gian Carlo Bellenchi (3,5), Patrizia Longone (2)

    Organisations:
    1: System Medicine Department, University of Rome Tor Vergata.
    2: Molecular Neurobiology Unit, IRCCS Fondazione Santa Lucia, Rome
    3: Institute of Genetics and Biophysics, National Research Council, Naples
    4: Microbial Biofilm Laboratory, IRCCS Fondazione Santa Lucia, Rome
    5: IRCCS Fondazione Santa Lucia, Rome

    Presenting author: Francesca Ciaiola

    Exposition date: Saturday, September 16, 2023

    Exposition position: 084/2

    Amyotrophic lateral sclerosis (ALS) is a rare, progressive, neurodegenerative disorder caused by gradual degeneration and death of motor neurons (MN). ALS can result from different causes including some environmental risk factors as well as several genes that have been identified as harbouring disease-associated variation (Hardiman et al., 2017). In the last few years altered RNA metabolism has gain momentum as one of the leading causes on the ALS-related MN degeneration.

    MicroRNAs, are small sequences of oligonucleotides, whose function is to regulate the translation of mRNAs into proteins by blocking the process or guiding the mRNA molecules to degradation (Ambros, 2004). A number of microRNAs are linked to neuronal development and the occurrence of neurodegenerative diseases. MiR-218 is greatly enriched in spinal MNs and essential for their correct functioning and differentiation.

    MiR-218 has two paralogs miR-218-1 and miR-218-2, embedded within homologous introns of the Slit2 and Slit3 genes, respectively. It has been suggested that the MN-specifying transcription factors Isl1 and Lhx3 up-regulate miR-218 by increasing transcription of Slit2 and Slit3 (Amin et al., 2015) (Amin et al., 2021) (Thiebes et al., 2015).

    Hoye et al. (Hoye et al., 2018) have reported that MNs-released miR-218 can be taken up by astrocytes and directly binds to the astrocytic glutamate transporter EAAT2, and directly regulates its expression through its interaction with the EAAT2 3′ UTR. They linked its accumulation in astrocytes to the non-cell autonomous degeneration described in ALS.

    The aim of the present study is to study the interconnection between miR-218 and the glutamatergic system in mixed spinal cord primary cultures obtained from mice harbouring a miR-218 knockout by the CrispR/cas9 technology.

  • Neuroinflammation and neurodegeneration in ALS: exploring new therapeutic approach based on Multi-Targeted Direct Ligands against TSPO and Sigma 1 receptor.

    Authors:
    Andrea Gazzano (1), Davide Camazzola (1), Mauro Giuseppe Spatafora (1), Lamacchia Alberto (1), Andrea Marsala (1), Alessandra Stanchina (1), Enrico Doria (2), Mattia Sponchioni (3), Renato Auriemma (3), Alessandro Lascialfari (4), Marta Filibian (4), Davide Moscatelli (3), Giacomo Rossino (5), Roberta Listro (5), Daniela Curti (1), Simona Collina (5), Marco Peviani (1)

    Organisations:
    1: Cellular and Molecular Neuropharmacology lab, Dpt Biology and Biotechnology “L.Spallanzani”, University of Pavia, Pavia, Italy
    2: Pharmacology lab, Dpt Biology and Biotechnology “L.Spallanzani”, University of Pavia, Pavia, Italy
    3: Department of Chemistry, Materials and Chemical Engineering "Giulio Natta", Politecnico di Milano, Milano, Italy
    4: MR and micro-CT Imaging Laboratory, University of Pavia, Pavia, Italy
    5: Department of Drug Sciences, Medicinal Chemistry and Pharmaceutical Technology Section, University of Pavia, Pavia, Italy

    Presenting author: Andrea Gazzano

    Exposition date: Saturday, September 16, 2023

    Exposition position: 093/2

    Background. Amyotrophic Lateral Sclerosis (ALS) is characterized by a heterogenous neuroinflammatory response that reflect not only the extent of neuronal demise but also variable engagement glial cells in the attempt to cope with the neuronal damage. At the same time, while targeting microglial cells with TSPO-selective ligands could exert a neuroprotective effect, pro-survival and anti-apoptotic processes in neuronal cells could represent a potential target to slow down the development of the disease. Thus, we hypotized that Multi-Targeted Direct Ligands (MTDLs) against TSPO and Sigma-1R (S1R), a promising multi-functional and ligand-operated protein, expressed at different levels by motor neurons and glial cells along the disease progression, could represent new pharmacological tools. Our goals are to achieve selective modulation of neuroprotective pathways in specific cells and identifying the best time-window, along disease progression, to deliver cell-selective therapeutics.

    Aims. In this work we aimed at: i) obtaining an in-depth analysis of TSPO and S1R distribution and correlation with disease stage in ALS animal models; ii) validating TSPO-S1R bivalent ligands as potential new drugs with combined action.

    Methods. We performed in-situ hybridization and immunohistochemistry experiments to investigate the expression and distribution of TSPO and S1r in the CNS of transgenic SOD1(G93A) rat model of ALS, which recapitulates the heterogeneous disease manifestations observed in patients. In parallel, we developed and validated MTDLs on TSPO and S1r knock-down cell lines. Aside, T2w-hr MRI protocols were implemented to characterize the disease progression to better identify a proper time-window for pharmacological treatment.

    Results, conclusions. We confirmed by ISH/IHC a clearcut upregulation of TSPO in microglia cells in the CNS areas most severely affected by the disease, while S1R protein appears to be highly expressed in neurons in non-transgenic animals and presymptomatic stage of the disease. Preliminary in-vitro studies highlighted neurotrophic effect of S1R agonist RC33 in short-term treatment.

  • Neuroprotective effect of extracellular vesicles derived from adipose stem cells diffused through an epithelium on injured neuronal cells

    Authors:
    Federica Virla (1), Marco Versuraro (1), Sylwia Dabrowska (2), Ilaria Scambi (1), Ermanna Turano (1), Raffaella Mariotti (1)

    Organisations:
    1: University of Verona, Italy
    2: NeuroRepair Department, Mossakowski Medical Research Institute, Polish Academy of Sciences, Warsaw, Poland

    Presenting author: Ermanna Turano

    Exposition date: Saturday, September 16, 2023

    Exposition position: 094/2

    Adipose mesenchymal stem cells (ASCs) represent a feasible and valid alternative to other sources of mesenchymal stem cells in the treatment of several neurological disorders. Currently, the scientific community brings a growing body of evidences indicating that ASCs exert their neuroprotective and immunomodulatory effects by a paracrine mechanism through the release of extracellular vesicles (EVs). Indeed, EVs are considered important mediators in intercellular communication as they can transfer their cargo (proteins, miRNAs and mRNAs) to nearby cells promoting nerve regeneration, neuronal protection, synaptic plasticity and remyelination in different pathophysiological contexts, recapitulating the effect of origin cells. Since ASC-EVs passage through the physiological barriers once injected can be a limiting factor in their biodistribution, the aim of this study was to set up an in vitro model to mimic the passage of ASC-EVs through an epithelial barrier. Furthermore, we wanted to evaluate their neuroprotective effect after their passage through the epithelial barrier on an oxidative stress-induced model of both motor neuron (NSC-34) and neuron (SH-SY5Y) cells. Regarding the study of the ASC-EVs biodistribution, it is also crucial to identify the mechanisms by which EVs are able to cross the epithelia and reach the central nervous system. To do that we set up a fluorescent labelling protocol to isolate and characterize labelled ASC-EVs and to evaluate their uptake by injured NSC-34 cells. The results showed that ASC-EVs neuroprotective effects observed in previous studies were maintained after their passage through the epithelium model as well, with a rescue of the neuronal cells viability after oxidative stress. Moreover, the ASC-EVs labelling protocol allowed us to isolate fluorescent EVs that will pave the way for further studies in order to clarify their passage after their administration and their capture by injured cells in the central nervous system.

  • Novel insights on the role and therapeutic potential of Glycoprotein nonmetastatic melanoma protein B (Gpnmb) in Amyotrophic Lateral Sclerosis.

    Authors:
    Mauro Giuseppe Spatafora (1), Paolo Cabras (1), Gianluca Di Nolfi (1), Andrea Gazzano (1), Teodoro Manilla (1), Loris Bandirali (1), Bruno M. Custode (1), Agnese Dimartino (1), Daniela Curti (1), Alessandra Biffi (2), Teuta Domi (3), Nilo Riva (4), Marco Peviani (1)

    Organisations:
    1: Dpt Biology and Biotechnology “L.Spallanzani”, University of Pavia, Pavia, Italy.
    2: Gene Therapy Program, Dana-Farber/Boston Children’s Cancer and Blood Disorders Center, Boston, MA, USA.
    3: INSPE, San Raffaele Scientific Institute, Milan, Italy.
    4: Carlo Besta neurological institute, Milan, Italy.

    Presenting author: Mauro Giuseppe Spatafora

    Exposition date: Saturday, September 16, 2023

    Exposition position: 092/2

    Background.
    Increased levels of a peptide derived from Gpnmb in the cerebrospinal fluid (CSF) were recently associated with a poor prognosis in patients affected by Amyotrophic Lateral Sclerosis (ALS). On the other hand, other studies highlighted that upregulation of Gpnmb could play a neuroprotective and immunomodulatory role.
    Objectives.
    In this study we engaged an in-depth characterization of Gpnmb alterations in SOD1.G93A transgenic (TG) rat model of ALS and in patients, to clarify the value of Gpnmb as prognostic biomarker and to identify a precise time-window, during the disease process, suitable for successful therapeutic intervention.
    Methods.
    We applied in-situ hybridization (ISH) and immunohistochemistry (IHC) in the central and peripheral nervous system, coupled to the assessment of Gpnmb ectodomain (sGpnmb) in the CSF and blood of TG rats. In parallel, sGpnmb was assessed in a small cohort of ALS patients.
    Results and discussion.
    Gpnmb is mainly expressed in MNs in healthy conditions. However, in TG animals there is an early decrease of Gpnmb mRNA and protein levels in MNs and upregulation in reactive microglia after symptom onset. ISH and IHC highlighted a critical role for glial cells in the synthesis and release of sGpnmb. In parallel, we spotted a significant increase of sGpnmb in the CSF and blood of TG rats, as well as in ALS patients, when the pathology is more severe. After identifying the correct dose of Gpnmb for triggering the neuroprotective pathway activation in the rat model (proved by phosphorylation of AKT), we are currently running a preclinical proof of concept study to verify the therapeutic potential of early administration of recombinant Gpnmb while monitoring sGpnmb as biomarker of target engagement.

  • Preclinical Data Support the Initiation of the ACHIEVE Trial of DYNE-101 in Individuals with Myotonic Dystrophy Type 1 (DM1)

    Authors:
    Erwan Delage, Ester Cotali, Daniel Wolf, Tyler Picariello, Lydia Schlaefke, Ryan Russo, Ann Chang, Scott Hildebrand, John Najim, Qifeng Qiu, Timothy Weeden, John W Davis II, Chris Mix, Baoguang Han, Stefano Zanotti, Oxana Beskrovnaya, Wildon Farwell

    Organisations:
    Dyne Therapeutics, United States of America

    Presenting author: Erwan Delage

    Exposition date: Saturday, September 16, 2023

    Exposition position: 080/2

    Myotonic dystrophy type 1 (DM1) is caused by expansion of CUG repeats in the 3’-untranslated region of the dystrophia myotonica protein kinase (DMPK) RNA. The expanded CUG repeats form hairpin-loop structures that sequester splicing regulators into toxic nuclear foci, leading to a spliceopathy that drives DM1 clinical manifestations. No disease-modifying treatments are approved, limiting current treatment to symptom management. We developed the FORCETM platform, which harnesses the natural expression of transferrin receptor (TfR)1 on muscle cells for targeted delivery of oligonucleotides. DYNE-101 is a TfR1-targeting antigen-binding fragment conjugated to a gapmer antisense oligonucleotide that targets nuclear DMPK RNA. In hTfR1/DMSXL mice, a novel model of DM1, DYNE-101 led to reduction of toxic human DMPK RNA and a corresponding splicing correction in cardiac and skeletal muscle that were accompanied by reduction of DMPK nuclear foci in the heart. Additionally, DYNE-101 led to a substantial suppression of WT DMPK expression in cardiac, skeletal, and smooth muscle after repeat monthly dosing in non-human primates, and had a favorable safety profile. These data supported the initiation of the ACHIEVE trial, a randomized, double-blinded, placebo-controlled, multiple ascending dose (MAD) Phase 1/2 study assessing safety, tolerability, pharmacodynamics, and pharmacokinetics of DYNE-101 administered intravenously to ~64 adults with DM1 aged 18-49 years (NCT05481879). The primary outcome is the number of participants with treatment-emergent adverse events. Change from baseline in splicing index in skeletal muscle is a secondary outcome. The ACHIEVE study will inform the potential of DYNE-101 as a treatment for DM1.

  • Reactive phenotype and oxidative stress in i-Astrocytes from patients affected by Amyotrophic Lateral Sclerosis: how the mGluR5 negative allosteric modulator CTEP affects these pathological features

    Authors:
    Sara Tessitore (1), Carola Torazza (1), Mandeep Kumar (1), Silvia Ravera (2), Ilaria Musante (3,4), Tiziana Bonifacino (1), Elena Angeli (5), Allan Shaw (6), Pamela J Shaw (6), Laura Ferraiuolo (6), Giambattista Bonanno (1), Marco Milanese (1,7)

    Organisations:
    1: Department of Pharmacy, Pharmacology and Toxicology Unit, University of Genoa, Genoa, Italy
    2: Department of Experimental Medicine, University of Genoa, Genoa, Italy
    3: Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health (DINOGMI), University of Genoa, Genoa, Italy
    4: Unit of Medical Genetics, IRCCS Istituto Giannina Gaslini, Genoa, Italy
    5: DIFILAB, Department of Physics, University of Genoa, Genoa, Italy
    6: Sheffield Institute of Translational Neuroscience (SITraN), University of Sheffield, Sheffield, United Kingdom
    7: IRCCS, Ospedale Policlinico San Martino, Genoa, Italy

    Presenting author: Sara Tessitore

    Exposition date: Saturday, September 16, 2023

    Exposition position: 082/2

    BACKGROUND. Amyotrophic Lateral Sclerosis (ALS) is a multifactorial non-cell-autonomous neurodegenerative disease, characterized by motor neurons (MNs) death. The metabotropic glutamate receptor type 5 (mGluR5) plays a key role in modulating astrocyte reactivity and glutamate excitotoxicity as major cause of MNs loss. We provided in-vitro and in-vivo evidence showing that modulation of the mGluR5 by the selective negative allosteric modulator CTEP positively affects the reactive phenotype and neurotoxicity of ALS astrocytes and significantly improved the life span and disease progression in SOD1G93A ALS mice. Here we investigated the impact of in-vitro mGluR5 modulation by CTEP on the phenotype of i-Astrocytesdifferentiated from inducible neural progenitor cells (iNPCs) of ALS patients and control subjects.

    RESULTS. In-vitro pharmacological modulation with CTEP did not alter the mGluR5 total expression. Confocal microscopy, immunohistochemical experiments and RT-qPCR analyses showed that 5 days in-vitro exposure to 100nM CTEP reduced the over-expression of reactive astrocytes and neuroinflammatory markers (GFAP, S100β, C3, NLRP3), in i-Astrocytes carrying the C9orf72 and SODA4V ALS-mutations vs. untreated cells and control i-Astrocytes from healthy donors. Of note, the improved phenotype following mGluR5 negative modulation, was accompanied by increased Nrf2 nuclear translocation, amelioration of the antioxidant enzymes activity (glutathione reductase, glutathione peroxidase, glucose-6-phosphate dehydrogenase, catalase), reduced ROS and malondialdehyde accumulation, compared to untreated and control cells. Preliminary results also showed that CTEP hampers the excessive intracellular calcium mobilization in ALS i-Astrocytes vs. untreated and control cells.

    CONCLUSIONS. We here show that the in-vitro pharmacological negative modulation of the mGluR5 by CTEP positively affects the reactive phenotype of human-derived i-Astrocytes from C9orf72 and SODA4V ALS patients, mainly by ameliorating the oxidative stress response of these cells. These data extend our previous results in SOD1G93A in-vitro and in-vivo models, thus further encouraging a potential translational application of mGluR5 modulators in clinical trials.

  • Repositioned SMN-independent drugs for the treatment of Spinal Muscular Atrophy

    Authors:
    Ersilia Nicorvo (1), Daniela Maria Rasà (1), Serena Stanga (1), Pamela Santonicola (2), Sandro Montefusco (3), Diego Medina (3), Alessandro Vercelli (1), Elia Di Schiavi (2), Marina Boido (1)

    Organisations:
    1: Department of Neuroscience “Rita Levi Montalcini”, Neuroscience Institute Cavalieri Ottolenghi, University of Turin, Italy
    2: Institute of Biosciences and BioResources, National Research Council, Naples 80131, Italy.
    3: TIGEM, Pozzuoli, Naples, Italy

    Presenting author: Ersilia Nicorvo

    Exposition date: Saturday, September 16, 2023

    Exposition position: 085/2

    Spinal Muscular Atrophy (SMA) is one of the most common genetic causes of infant mortality. SMA is caused by mutation/deletion of survival motor neuron 1 (Smn1) gene, determining the lack of SMN protein, and is characterized by the progressive degeneration of lower motor neurons (MNs).

    FDA and EMA have approved three revolutionary SMN-dependent treatments that are focused on increasing the production of the SMN protein. However, these approaches present some limitations: therefore, the identification of alternative/synergistic therapeutic strategies is strongly needed.

    To this aim, we used an invertebrate SMA animal model, Caenorhabditis elegans, that allowed us performing a High Content screening of thousands of natural/synthetic compounds. With this approach we identified new FDA-approved drugs that rescued the neurodegeneration.

    The most effective molecules were also tested in vitro on primary cortical neurons from SMA delta7 mice (a severe SMA model). At day in vitro 7, different assays were performed to assess cell viability and morphology. Compared to untreated cells, most of the compounds exerted positive effects, by significantly improving cell viability, increasing cell body area and length/branching of neuronal processes, and influencing the synaptic vesicle distribution. Finally, by WB, we also analysed their ability to increase SMN protein levels: since no differences were observed among groups, we suggest a SMN-independent mechanism of action.

    Taking advantage of the results, we are now further evaluating the mechanisms and time of action of the identified drugs and performing extensive tests using in vitro live-cell analysis.

    Although additional analysis are necessary, our results suggest that the new drugs identified could be combined with SMN-dependent treatments to develop synergistic pharmacological strategies and improve efficacy.

  • Robust preclinical data support development of DYNE-251 as a potential treatment for individuals with DMD mutations amenable to exon 51 skipping

    Authors:
    Erwan Delage, Ester Cotali, Maria L. Naylor, Cody A. Desjardins, Reshmii Venkatesan, John Hall, Ryan Russo, Kim Tang, John W. Davis II, Timothy Weeden, Stefano Zanotti, Chris Mix, Baoguang Han, Oxana Beskrovnaya, Ashish Dugar

    Organisations:
    Dyne Therapeutics, United States of America

    Presenting author: Ester Cotali

    Exposition date: Saturday, September 16, 2023

    Exposition position: 087/2

    Duchenne muscular dystrophy (DMD) is characterized by progressive loss of muscle function leading to premature death. Current therapeutic approaches use exon skipping phosphorodiamidate morpholino oligomers (PMOs) that enable the translation of a shortened, functional dystrophin protein, but their success has been hampered by poor muscle delivery. We developed the FORCETM platform, which harnesses the natural expression of transferrin receptor (TfR)1 on muscle cells for targeted delivery of oligonucleotides. DYNE-251, an investigational therapeutic for the treatment of DMD, consists of an exon 51 skipping PMO conjugated to an antigen-binding fragment (Fab) targeting TfR1. In non-human primates, DYNE-251 led to pronounced exon skipping in cardiac and skeletal muscle and demonstrated a favorable safety profile. We also demonstrated that a single dose of FORCE-M23D, a mouse-specific Fab-PMO conjugate designed to skip exon 23 of the murine Dmd pre-mRNA, led to robust and durable exon skipping and dystrophin restoration, and improved functional outcomes in mdx mice, compared to unconjugated PMO. These preclinical data supported the initiation of DELIVER, a randomized, double-blind, placebo-controlled, multiple ascending dose (MAD) study of DYNE-251 administered intravenously to ~46 ambulant and non-ambulant males (4-16 years) with exon 51 skip-amenable mutations (NCT05524883). The purpose of the study is to evaluate the safety, tolerability, and dystrophin levels in muscle following multiple doses of DYNE-251. Primary outcomes are the number of participants with treatment-emergent adverse events and the change from baseline in dystrophin levels in muscle at Week 25. The DELIVER study will inform the potential of DYNE-251 as a treatment for DMD.

  • Sensorimotor peripheral innervation in Duchenne muscular Dystrophy: spotlights on the sciatic nerve of dystrophic mdx mice

    Authors:
    Silvia Di Nuzzo (1), Francesca Mastrostefano (1), Marzia Soligo (2), Valerio Magnaghi (3), Valentina Ferretti (1), Maria Egle De Stefano (1)

    Organisations:
    1: Sapienza University of Rome, Italy
    2: Inst. of Traslational Pharmacology, CNR, Rome, Italy
    3: Università degli Studi di Milano, Milan, Italy.

    Presenting author: Silvia Di Nuzzo

    Exposition date: Thursday, September 14, 2023 (Satellite Events Day)

    Exposition position: 104

    Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disease, characterized by the lack of dystrophin (Dp427), a cytoskeletal protein expressed in skeletal muscles and several other cell types. Along or in substitution of Dp427, different tissues other than muscles may also express shorter dystrophin isoforms, as in the peripheral nervous system (PNS), where myelinating Schwann cells (SCs) contain the Dp116 isoform. By binding to a large multiprotein complex (DGC), described as similar in muscle and organized around the alpha and beta-dystroglycan (DG) dymer, Dp116 contributes to myelin integrity and nerve fiber conduction. Structural and functional integrity of nerve fibers rely on axon-SC intense cross-talk, a well-known mediator of which is the neurotransmitter GABA. To unveil the effects that DMD has on peripheral sensory-motor nerves, we analyzed in the sciatic nerve of mdx mice, a model of DMD, expression and localization of: myelin proteins (e.g. MBP, P0, PMP22, MAG), Dp116, proteins of the DGC (dystrobrevin, beta-DG), as well as inotropic GABAA (α4, β3, δ) and metabotropicGABAB-1b receptor subunits. Compared to wild-type, in mdx mice, level, distribution and intensity of immunolabeling of all proteins analyzed, except for Dp116, were significantly reduced, indicating alteration of myelin integrity and SCs-axons crosstalk. This was also confirmed by parallel electrophysiological studies showing a significant reduction in Abeta- mechanoreceptor fiber excitability. Furthermore, gel zymography and Western immunoblot demonstrated activation of the pro-inflammatory extracellular matrix metalloproteinase (MMP) 2, which targets both alpha and beta-DG, suggesting that cleavage of DG within the myelin sheet, induced by retrograde inflammation from muscle to nerve, could be an upstream determinant for Dp116-DGC degradation and consequent deregulation of SC-axon signaling. Altogether, these findings support the hypothesis of significant alterations of the neuron-SC cross-talk in peripheral nerves of dystrophic subject, an aspect to be considered for supporting DMD therapeutic strategies.

  • Size-Based Effects of Anthropogenic Ultrafine Particles on Lysosomal TRPML1 Channel and Autophagy in Motoneuron-like Cells as a putative mechanism of ALS

    Authors:
    Silvia Sapienza (1), Valentina Tedeschi (1), Barbara Apicella (2), Francesco Palestra (3,4), Carmela Russo (2), Ilaria Piccialli (1), Anna Pannaccione (1), Stefania Loffredo (3,4), Agnese Secondo (1)

    Organisations:
    1: Department of Neuroscience, Reproductive and Odontostomatological Sciences, University of Naples Federico II, 80131 Naples, Italy.
    2: Istituto di Scienze e Tecnologie per l'Energia e la Mobilità Sostenibili (STEMS)-CNR, 80125 Naples, Italy
    3: Department of Translational Medical Sciences, Center for Basic and Clinical Immunology Research (CISI), WAO Center of Excellence, University of Naples Federico II, 80131 Naples, Italy.
    4: Institute of Experimental Endocrinology and Oncology (IEOS), National Research Council, 80131 Naples, Italy

    Presenting author: Silvia Sapienza

    Exposition date: Saturday, September 16, 2023

    Exposition position: 091/2

    Air pollution is a major threat to human health. Atmospheric particulate matter of anthropogenic origin (PM) is recognized as the major air pollutant component, mostly ascribed to diesel exhaust and it is known for causing respiratory and cardiovascular diseases. Moreover, emerging body of evidence suggests an association between PM at ultrafine size and neurodegeneration; since it may pass BBB and negatively impact central nervous system. However, the molecular mechanisms of neurotoxicity remains unknown.

    Our work focused on the dimension-related effect exerted by PM on NSC-34 motor neuron- like cell line in order to understand the putative impact of air pollution on amyotrophic lateral sclerosis (ALS). At this end, ultrafine PM particles with a diameter < 0.1μm (PM0.1) and nanoparticles, with a diameter < 20nm (NP-20) were sampled in a lab-scale combustion system.

    NPs-20, but not PM0.1 particles, reduced NSC-34 cell movement in the space studied by time-lapse and high-content microscopy. Furthermore, NPs-20 were able to shift membrane potential of motor neurons toward more depolarizing values as measured by current-clamp electrophysiology. Both PM0.1 and NPs-20 exerted mitochondrial toxicity, inducing ROS production and ATP depletion. Then, ROS may produce an hyperactivation of the lysosomal channel TRPML1 activity, as measured by Fura2-video imaging, with a consequent overexpression of both LC3-II and p62 protein and AMPK phosphorylation. This pattern of expression suggests an engulfment of autophagy occurring in NSC-34 motor neuron- like cell line exposed to NPs-20 or PM0.1. Of note, the detrimental accumulation of autophagosomes is often found in neurodegenerative diseases like ALS, suggesting that ultrafine particles may trigger neurodegenerative processes in motor neurons through the organellar Ca2+ dyshomeostasis and autophagy dysfunction.

  • STIP1 Homology And U-Box Containing Protein 1 (STUB1/CHIP) mutants as a key factor on TATA-box binding protein (TBP) behaviour in digenic spinocerebellar ataxia type 17 (SCA17-DI)

    Authors:
    Paola Pramaggiore (1), Stefania Magri (2), Marta Chierichetti (1), Paola Rusmini (1), Veronica Ferrari (1), Barbara Tedesco (1), Marta Cozzi (1), Elena Casarotto (1), Valeria Crippa (1), Mariarita Galbiati (1), Laura Cornaggia (1), Guglielmo Patelli (1), Daniela Di Bella (2), Franco Taroni (2), Angelo Poletti (1), Riccardo Cristofani (1)

    Organisations:
    1: Dipartimento di Scienze Farmacologiche e Biomolecolari ”Rodolfo Paoletti”, Università degli Studi di Milano, Italy
    2: Unit of Medical Genetics and Neurogenetics, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy

    Presenting author: Paola Pramaggiore

    Exposition date: Saturday, September 16, 2023

    Exposition position: 090/2

    Spinocerebellar ataxias (SCAs) are hereditary, progressive and fatal, very heterogenous neurodegenerative diseases (NDs). SCA type 17 (SCA17) is characterized by the presence of expanded CAG nucleotide repeats of the TATA-box binding (TBP) gene that codes for an abnormally long polyglutamine (polyQ) tract in the N-terminal of the protein. This leads to reduced solubility and accumulation of mutated TBP in neurons. Strikingly, TBP forms containing an intermediate polyQ tract (41-47 Qs) show incomplete penetrance (SCA17-DI), which seems to be correlated to the presence of mutations in STIP1 Homology And U-Box Containing Protein 1 (STUB1/CHIP). STUB1 is an E3 ubiquitin-ligase which has a key role in the protein quality control (PQC) system. Given the hypothesis that both TBP and STUB1 may be involved in SCA17-DI, we investigated their behaviour and interplay for a deeper understanding of the underlying molecular mechanisms in the disease.

    Our data show a punctate distribution and insoluble protein accumulation of overexpressed elongated polyQ TBP (TBP-Q54) that it is not present in the wild type (TBP-WT) or intermediate polyQ TBP (TBP-Q43) expressing neurons. Interestingly, TBP accumulation is reverted by STUB1 over-expression suggesting that TBP degradation is mediated by STUB1. Since STUB1 plays a role in both ubiquitin proteasome system (UPS) and autophagy, we alternatively inhibited these pathways to study TBP behaviour. Our preliminary findings suggest that different pathways are responsible for STUB1-mediated TBP-Qs removal based on the different sizes of the polyQ tract. Moreover, other analyses show that STUB1 SCA17-DI-linked mutations are characterized by a reduced activity on TBP clearance.

    Collectively, our data demonstrate that STUB1 mutations affect TBP biochemical behaviour in SCA17-DI. Therefore, our goal is to further investigate TBP and STUB1 interplay in order to better understand their pathological role in this form of ataxia, leading to a deeper knowledge of the disease.

    GRANTS: Fondazione Cariplo (2021-1544)

  • The counter effect of DMD in peripheral innervation: alterations of the dystrophin-dystroglycan complex in the sciatic nerve of dystrophic mdx mice

    Authors:
    Silvia Di Nuzzo (1), Marzia Soligo (2), Valentina Ferretti (1), Maria Egle De Stefano (1)

    Organisations:
    1: Sapienza University of Rome, Italy
    2: Inst. of Traslational Pharmacology, CNR, Rome, Italy

    Presenting author: Silvia Di Nuzzo

    Exposition date: Saturday, September 16, 2023

    Exposition position: 083/2

    Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disease, characterized by the lack of the large cytoskeletal protein dystrophin (Dp427) in skeletal muscles and other cell types. Shorter dystrophin isoforms, also exist, expressed in a tissue-specific manner and often substituting Dp427. In the peripheral nervous system (PNS), Schwann cells (SCs), which are responsible for axon myelination, express the Dp116 isoform, which binds to the same large glycoprotein complex that in muscles connects actin, Dp427 and extracellular matrix. Central core of this complex are alpha- and beta-dystroglycans (DGs). In SCs, the Dp116-DG complex (DGC) confers myelin integrity and preservation of nerve fiber conduction properties. To date, little is known on the effects that DMD has on peripheral sensory-motor nerves. Our previous studies on the sciatic nerves of wild-type and mdx mice, a model of DMD lacking the sole Dp427, highlighted a significant reduction in the GABAergic signaling ruling SCs-axons cross-talk in dystrophic mice, compared to wild-type, as well as in Abeta- mechanoreceptor fiber excitability. Therefore, considering the role of Dp116-DGC in myelin, we analyzed whether protein levels and immunolocalization of three major complex components: alpha- and beta-DGs, and dystrobrevin, were affected. Levels, distribution and intensity of immunolabeling of all three proteins were significantly reduced in mdx mouse sciatic nerves compared to wild-type, confirming alteration of myelin integrity. Since the expression of Dp116 was not equally altered, we hypothesized as a possible cause activation of proinflammatory extracellular matrix metalloproteinases (MMP) 2 and 9, both having alpha and beta-DGs among their targets. Indeed, gel zymography and Western immunoblot revealed a significant induction of MMP2 activity in mdx mouse sciatic nerves compared to wild-type. This suggests that a DMD-induced DGs cleavage within the myelin sheets could be the upstream cause of the Dp116-DGC degradation and of the SC-axon cross talk deregulation.

Nanoscopic, microscopic, and macroscopic neuroimaging technologies

Neurobiology of psychiatric disorders

  • Attachment bond alteration and intergenerational transmission of psychopathology vulnerability

    Authors:
    Lucy Babicola (1,2), Valeria Petroni (1), Matteo Di Segni (2), Francesca Romana D'Amato (3), Rossella Ventura (1,4)

    Organisations:
    1: Dept. of Psychology and Center "Daniel Bovet", Sapienza University, Rome, Italy
    2: IRCCS Santa Lucia Foundation, Rome, Italy
    3: Biochemistry and Cell Biology Institute, National Research Council, Monterotondo Scalo, Rome, Italy
    4: IRCCS San Raffaele, Rome, Italy

    Presenting author: Lucy Babicola

    Exposition date: Friday, September 15, 2023

    Exposition position: 098/1

    The attachment figure is the most important early social stimulus for all altricial mammals. Attachment bond plays a critical role in brain development and experiences affecting this bond can drive psychopathological outcomes acting though complex epigenetic mechanism. However, whether attachment bond alteration can be also transmitted to offspring is currently puzzling. Here we investigate whether the increased vulnerability to psychopathological phenotype induced by mother-pups bond interference in C57BL/6J (B6) inbred mice is transmitted to offspring. We have previously demonstrated that exposure to an early-life manipulation interfering with mother-pups bond (Repeated Cross Fostering, RCF) induces in B6 male mice increased vulnerability to depression-like phenotype, consisting in increased passive coping strategy and reduced saccharine preference, while the opposite phenotype was observed in females. Based on this evidence, Control and RCF B6 adult mice were mated to generate groups bearing only maternal (RC), only paternal (CR) or bi-parental (RR) RCF experience. Both females and males RC, CR and RR mice were tested for attachment bond and adult behavioral phenotype. Offspring from RCF male mice (CR, RR) show alteration of mother-pups bond and depression-like phenotype regardless of maternal experience, highlighting the critical role of paternal contribution in attachment bond formation and adult psychopathological vulnerability. Our data suggest the epigenetic intergenerational transmission of phenotype.

  • Attachment bond interference and psychopathology: the positive role of earned-attachment

    Authors:
    Camilla Mancini (1), Lucy Babicola (2,3), Gilda Chilà (4), Alice Passeri (2), Diana Municchi (2,3), Elena Spoleti (4,5), Matteo Di Segni (3,2), Massimiliano Renzi (4), Carlo Cifani (1), Rossella Ventura (2,6)

    Organisations:
    1: School of Pharmacy, Pharmacology Unit, University of Camerino, Camerino, Italy
    2: Dept. of Psychology and Center "Daniel Bovet", Sapienza University, Rome, Italy
    3: IRCCS Santa Lucia Foundation, Rome, Italy
    4: Dept. of Physiology and Pharmacology, Sapienza University, Rome, Italy
    5: Dept. of Medicine and Surgery, Campus Bio-Medico University of Rome, Rome, Italy
    6: IRCCS San Raffaele, Rome, Italy

    Presenting author: Camilla Mancini

    Exposition date: Friday, September 15, 2023

    Exposition position: 100/1

    Early-life experiences affecting attachment bond can have long-lasting consequences resulting in negative outcomes, such as depression, in adult life. However, the formation of an earned-secure attachment with an alternative caregiver can act as a protective factor against attachment disruption with the primary caregiver. The Dopaminergic (DA) system of the Ventral Tegmental Area (VTA), involved in both attachment bond and depression, is particularly sensitive to early-life manipulation. VTA DA neurons are characterized by an inward regulatory mechanism, the hyperpolarized-activated cation current (Ih current) and, interestingly, this mechanism was found altered in animal model of depression. We have previously reported that C57BL/6J female mice exposed to Repeated Cross Fostering (RCF), a procedure affecting attachment bond during a sensitive time window, showed resilience to depression and decreased Ih current in VTA DA neurons. However, females by a different inbred strain, DBA/2J (DBA), exposed to RCF, shows increased vulnerability to depressive-like behavior in adulthood.
    Here, we hypothesized that the opposite phenotype observed in DBA females is related to an opposite alteration of Ih current and that by introducing a stable attachment caregiver, during RCF, we could prevent both behavioral and electrophysiological alterations. First, we analyzed the Ih current density in VTA DA neurons of adult RCF and Control females thought patch-clamp recording. Then, we performed RCF procedure introducing a Stable Attachment Caregiver (SAC) that remains with the pups from PND1 to PND4. After assessing pups’ attachment behavior, we tested mice in adulthood to evaluate the rescue effect of SAC on depressive-like phenotype. Finally, we investigated VTA DA neuron Ih current in RCF+SAC adult females. Our results indicated that depressive-like behavior induced by RCF is linked to an increased Ih current density and that rescuing the attachment bond alteration, by providing pups with an alternative stable caregiver is sufficient to prevent these alterations.

  • Cannabidiol prosocial effect in mice: crucial role of its cross-talk with the oxytocin pathway mediated by TRPV2

    Authors:
    Marika Premoli, Marinella Carone, Giuseppina Maccarinelli, Maurizio Memo, Sara Anna Bonini

    Organisations:
    University of Brescia, Italy

    Presenting author: Marika Premoli

    Exposition date: Friday, September 15, 2023

    Exposition position: 094/1

    Cannabis sativa is the most used recreational drug worldwide. In recent years, there has been a growing interest in the potential therapeutic benefits of medicinal cannabis to treat different psychiatric and neurological conditions. In particular, cannabidiol (CBD), a non-psychoactive cannabis constituent, has been investigated for its potential prosocial effects on behavior, although the molecular mechanisms underlying this effect are still largely unknown. The aim of this study was to further investigate the effect of CBD on social behavior by studying the possible cross-talk between CBD and oxytocin; our hypothesis is that this cross-talk does exists and is mediated by TRPV2 activation. We found that a chronic treatment with a CBD-rich oil on mice exerted a prosocial effect associated with an increase of ultrasonic communication. These effects were prevented by pre-treating mice with an oxytocin receptor antagonist. At the molecular level, we found that CBD oil treatment caused a significant increase in both oxytocin and the endocannabinoid receptors expression levels in the brain hypothalamus; on the contrary, TRPV2 and FAAH expression was decreased. In vitro experiments on two cell lines demonstrated the crucial role of the TRPV2 in the CBD-oxytocin cross-talk. Our results suggest that CBD could be used to promote social behavior by acting on oxytocin pathway through the TRPV2; this effect may cause also an increase in the endocannabinoid signaling tone.

  • Dissecting UBE3A-dependent regulation of synapse development

    Authors:
    Martina Biagioni (1), Federica Baronchelli (3,4), Martina Di Nunzio (1), Alessandra Folci (3), Marco Erreni (2), Matteo Fossati (1,3)

    Organisations:
    1: Humanitas Clinical and Research Center-IRCCS, Italy
    2: Unit of Advanced Optical Microscopy, Humanitas Clinical and Research Center - IRCCS, Rozzano (MI),
    3: CNR-Institute of Neuroscience, Rozzano (MI), Italy
    4: Humanitas University, Pieve Emanuele (MI), Italy;

    Presenting author: Martina Biagioni

    Exposition date: Friday, September 15, 2023

    Exposition position: 091/1

    The UBE3A gene codes for an E3 ubiquitin ligase and is critical to ensure a proper brain function. Indeed, perturbations of UBE3A dosage or function result in pathological phenotypes. Loss of UBE3A causes the Angelman Syndrome, a severe neurodevelopmental disorder characterized by intellectual disability, motor delay and seizures, while increased UBE3A copy number or activity are strongly associated with Autism. Importantly, the molecular underpinnings of UBE3A-associated pathogenic mechanisms of neurodevelopmental disorders are still poorly understood. In this project, we study the effects of changes in UBE3A dosage, mimicking the genetic alterations of the Angelman syndrome (or autism) on the regulation of synaptic development at single cell level in vivo. To this aim, we combine cortex-directed in utero electroporation to inactivate UBE3A in sparse layer 2/3 pyramidal neurons with high-resolution optical microscopy to investigate the consequences of UBE3A loss on the functional organization of excitatory and inhibitory postsynaptic compartments. As already suggested by other groups, our results indicate that UBE3A critically regulates the formation of excitatory synapses. Strikingly, our data also suggests that UBE3A controls the assembly and the maturation of specific subtypes of inhibitory synapses, namely those located in the perisomatic region and in the axon initial segment. Together, our preliminary results suggest for the first time that the UBE3A gene may be critical to set the number of excitatory and inhibitory synaptic connections at the single-cell level, thus contributing to regulate the ratio between excitation and inhibition through cell-autonomous mechanisms.

    Funding by Ministry of Health - RICERCA FINALIZZATA 2018 (GR-2018-12366478), Telethon Foundation, General grant 2020 (GP20127)

  • Electrophysiological correlates of reward anticipation in subjects with schizophrenia: an ERP microstate study

    Authors:
    Pasquale Pezzella (1), Andrea Perrottelli (1), Giulia Maria Giordano (1), Thomas Koenig (2), Edoardo Caporusso (1), Luigi Giuliani (1), Paola Bucci (1), Armida Mucci (1), Silvana Galderisi (1)

    Organisations:
    1: University of Campania "Luigi Vanvitelli!", Italy
    2: University Hospital of Psychiatry and Psychotherapy, Translational Research Center, University of Bern, Switzerland.

    Presenting author: Pasquale Pezzella

    Exposition date: Friday, September 15, 2023

    Exposition position: 086/1

    Negative symptoms are a key aspect of schizophrenia due to their remarkable impact on real-life functioning in subjects suffering from this disorder and their low responsiveness to the available pharmacological treatments. However, the neurobiological mechanisms underlying such symptoms remain uncertain. The present study aimed to investigate alterations of event-related potentials (ERPs) microstate during reward anticipation in subjects with schizophrenia (SCZ), and their association with hedonic experience and negative symptoms.
    EEG data were recorded in thirty SCZ and twenty-three healthy controls (HC) during the Monetary Incentive Delay task in which reward, loss and neutral cues were presented. Microstate analysis and standardized low-resolution electromagnetic tomography (sLORETA) were applied to EEG data and analyses correlating topographic ERPs scores with scales assessing hedonic experience and negative symptoms were performed.
    We found alterations in the first (125.0 - 187.5 ms) and second (261.7 - 414.1 ms) anticipatory cue-related microstates. Reward cues were associated to shorter duration and earlier offset of the first microstate class as compared to the neutral condition in SCZ. Furthermore, in the second microstate class, the area under the curve was smaller for both reward and loss anticipation cues in SCZ as compared to HC. sLORETA analysis showed that hypo-activation of the cingulate cortex, insula, orbitofrontal and parietal cortex was detected in SCZ as compared to HC. Finally, significant correlations between ERPs scores and the anticipation of pleasure scores were detected, while no significant association was found with the severity of negative symptoms.
    In conclusion, the current study shows that in subjects with schizophrenia, abnormalities in ERPs may be detected even during the early stages of reward processing suggesting the presence of different impairments in the effective evaluation of incoming pleasant experiences. However, negative symptoms and anhedonia are partially independent results.

  • Electrophysiological Properties and Excitatory/inhibitory Balance of Parvalbumin and Somatostatin-positive Interneurons Are Altered in the Hippocampus of the Ts65Dn mouse model of Down Syndrome

    Authors:
    Giulia Colombo (1), Alberto Potenzieri (1,2), Andrea Contestabile (1), Laura Cancedda (1)

    Organisations:
    1: Istituto Italiano di Tecnologia, Genova, Italy
    2: 2Università degli Studi di Genova, Genova, Italy

    Presenting author: Giulia Colombo

    Exposition date: Friday, September 15, 2023

    Exposition position: 087/1

    Dysfunction of GABAergic interneurons during sensitive time windows plays a crucial role in the etiopathogenesis of neurodevelopmental disorders. For example, GABAergic transmission through Cl-permeable GABAA receptors (GABAAR) is depolarizing in pyramidal neurons of the CA1 region of the hippocampus of the Ts65Dn mouse model of Down syndrome. This depends on the upregulation of the Cl-importer NKCC1 and leads to an impairment of synaptic plasticity and memory performance in Ts65Dn adult mice. Blocking GABAergic transmission by blocking the GABAAR or restoring the reversal potential of GABAAR-mediated currents by NKCC1 antagonism both rescued synaptic plasticity and memory dysfunction in Ts65Dn adult mice. However, the contribution of specific GABAergic interneurons in the hippocampal plasticity and cognitive impairment observed in Ts65Dn mice is largely unknown.

    Here, we characterized parvalbumin (PV) and somatostatin (SOM)-positive interneurons in the hippocampus of 3-months-old Ts65Dn mice versus control littermates by immunohistochemistry and electrophysiological recordings on brain slices. Adult Ts65Dn mice showed a reduced number of PV interneurons and a lower expression of their perineuronal nets in the stratum pyramidale of CA1, CA3 and dentate gyrus of the hippocampus. This is suggestive of an aberrant developmental trajectory of PV neurons in Ts65Dn mice. Accordingly, Ts65Dn PV neurons showed a higher membrane time constant, wider action potentials and a lower maximal action potential frequency, typical of immature neurons. Moreover, the excitatory/inhibitory balance of those neurons was shifted towards inhibition (smaller EPSCs together with larger IPSCs, and lower frequency of mEPSCs than in controls). On the other hand, Ts65Dn SOM interneurons showed a lower membrane time constant, a higher action potential threshold, a higher action potential frequency, and lower EPSC/IPSC frequency ratio.

    Altogether, our preliminary results suggest that PV neurons have a defective neurogenesis and maturation, whereas SOM neurons are hyperexcitable. This may concur to the defective hippocampal information-processing of Ts65Dn mice.

  • Elucidating the interplay between early-life adversities and genetic predisposition in the emergence of transdiagnostic psychiatric hallmarks

    Authors:
    Gabriele Chelini, Tommaso Fortunato-Asquini, Benedetta Catena, Ginevra Matilde Dall'O', Enrica Cerilli, Luca Pangrazzi, Yuri Bozzi

    Organisations:
    University of Trento, Trento, Italy

    Presenting author: Gabriele Chelini

    Exposition date: Friday, September 15, 2023

    Exposition position: 099/1

    The etiological complexity of psychiatric disorders arises from the dynamic interplay between genetic and environmental vulnerabilities. Among the environmental factors, early-life adversities (ELA) play a major role in the exacerbation of psychiatric symptoms. Yet, the mechanistic interaction between ELA and genetic vulnerability contributing to psychopathology is poorly understood. To fill this gap, we took advantage of the ideally controlled conditions of a pre-clinical approach. In this study we raised a mouse model with genetic vulnerability for autism and schizophrenia, the Cntnap2+/- mouse, with limited bedding and nesting (LBN), a well-established paradigm to induce early-life stress in rodents. These mice were compared to LBN-raised wild-type (WT) littermates, as well as parallel groups of WT and Cntnap2+/- raised in standard conditions. Using a specifically designed battery for behavioral testing, we discovered that LBN-raised Cntnap2+/- mice selectively showed a perseverative and purposeless risk-taking behavior assessed in the elevated plus maze and that this behavior was correlated with an altered social interaction pattern in the 3-chamber test. This phenotype was accompanied by a significant decrease in the expression the calcium-binding protein parvalbumin (PV) in the basolateral nucleus of the amygdala (BLA). These findings suggest that Cntnap2 heterozygous mutations confer a state of vulnerability to BLA-PV interneurons that exacerbates in a loss of function phenotype as a consequence of ELA, resulting in altered anxiety regulation and abnormal social interaction. This study shows that early-life experience shapes long-term phenotypic development based on genetic predisposition, highlighting the importance of an etiology-driven stratification of the mental health spectrum to improve prevention and treatment of neurodiverse people.

  • Fgf14 deletion confers resilience to basal and stress-induced depressive-like behaviour

    Authors:
    Francesca Montarolo, Linda Masante, Anita Maria Rominto, Luna Berrino, Filippo Tempia, Eriola Hoxha

    Organisations:
    Department of Neuroscience "Rita Levi Montalcini" University of Turin, and Neuroscience Institute Cavalieri Ottolenghi (NICO), Italy

    Presenting author: Francesca Montarolo

    Exposition date: Friday, September 15, 2023

    Exposition position: 092/1

    Major depressive disorder (MDD) is a debilitating neuropsychiatric disease that represents a main social problem. MDD shows a higher prevalence in women relative to men, and its core symptoms include depressed mood, loss of interest/pleasure (anhedonia), and lack of motivation.

    The fibroblast growth factor 14 (Fgf14) is an intracellular accessory protein of voltage-gated sodium channels that controls neuronal excitability and synaptic transmission. The Fgf14 gene has been indicated by linkage and genome-wide association studies to be a putative risk factor for neuropsychiatric diseases including MDD.

    Here, we investigated the consequences of targeted deletion of the Fgf14 gene in the expression of depressive-like behaviour in female Fgf14-/- mice compared to wild-type littermates. Under basal conditions, we tested the level of anhedonia/neophobia in the sucrose preference test, demonstrating a higher level of hedonia and lower neophobia in Fgf14-/- relative to wild-type mice.

    Next, we exploited the tail suspension test (TST) to induce acute stress, which is known to be associated with depressive-like behaviour. We measured the time of immobility as a marker of helplessness and despair. In the TST, Fgf14-/- mice showed less immobility than wild-types. These results confirm that Fgf14-/- mice exhibit lower levels of depressive-like behaviour compared to wild-type mice.

    To understand the neural mechanisms of such resilience to depression of Fgf14-/- mice, we investigated neuronal activation following TST-induced acute stress, by the evaluation of cFos staining in the medial prefrontal cortex (mPFC). Wild-type mice subjected to TST showed a significantly higher number of cFos positive (cFos+) cells compared to unstressed controls, suggesting increased neuronal activity in the mPFC. Conversely, unstressed Fgf14-/- mice showed a high number of cFos+ cells, that did not increase further after TST. The different activity of the mPFC suggests that this area might be critical for the behavioural resilience to depression observed in Fgf14-/- mice.

  • Oxidative stress underlies sex-dependent effects of prenatal stress in a mouse model

    Authors:
    Marianna Samà, Chiara Musillo, Alessandra Berry, Barbara Collacchi, Francesca Cirulli

    Organisations:
    Center for Behavioral Sciences and Mental Health, Istituto Superiore di Sanità, Roma, Italy

    Presenting author: Marianna Samà

    Exposition date: Friday, September 15, 2023

    Exposition position: 102/1

    Maternal stress during pregnancy may re-direct foetal brain development, increasing the susceptibility to psychopathology in the offspring, although the underlying biological mechanisms still need to be studied in detail. We hypothesized that prenatal stress (PNS) might disrupt foetal programming by acting through oxidative stress (OS)-mediated mechanisms, ultimately resulting in long-lasting effects on the offspring brain development and behaviour.

    To test this hypothesis, we used a mouse model of maternal stress (repeated restraint sessions during the last week of pregnancy), and we tested the preventive action of the antioxidant N-acetyl cysteine (NAC, 1g/kg) administered to the dams for eight weeks, until delivery. The emotional profile was assessed in male and female adult offspring through the elevated plus-maze (EPM) and the neuroendocrine activity was assessed by measuring plasma corticosterone levels under basal conditions and in response to the forced swim test (FST).

    Results showed an increase in risk-assessment behaviours, in the EPM, and in the active coping strategy to face the FST in PNS female offspring. This latter effect was associated to reduced levels of basal corticosterone, followed by an hyperactivation of the HPA axis after stress exposure, overall suggesting that PNS increases anxiety-like behaviours and emotional reactivity in female offspring. Prenatal NAC administration in PNS females was able to prevent the emotional arousal observed in the EPM and to re-establish the same coping strategy of controls. As for males, effects of PNS and NAC were less pronounced and did not associate to changes in the coping strategies.

    Overall, our results clearly highlight detrimental sex-dependent effects of PNS on behaviour and neuroendocrine function with a greater impact on female offspring. Moreover, the preventive effects of NAC, which restores the physiological redox balance, point to OS as a possible mechanism underlying PNS and shed light on antioxidant compounds as promising preventive strategies.

    ERANET-NEURON-JTC-2018-EMBED

  • Oxytocin’s role in depression-like phenotype induced by attachment bond interference

    Authors:
    Alice Passeri (1), Diana Municchi (1,2), Lucy Babicola (1,2), Camilla Mancini (3), Sebastian Luca D'Addario (1,2), Marta Tiberi (2), Matteo Di Segni (2,1), Luisa Lo Iacono (1,2), Diego Andolina (1,2), Carlo Cifani (3), Valerio Chiurchiu (2), Rossella Ventura (1,4)

    Organisations:
    1: Dept. of Psychology and Center "Daniel Bovet", Sapienza University, Rome, Italy
    2: IRCCS Fondazione Santa Lucia, Rome, Italy
    3: University of Camerino School of Pharmacy, Pharmacology Unit, Camerino, Italy
    4: IRCCS San Raffaele, Rome, Italy

    Presenting author: Alice Passeri

    Exposition date: Friday, September 15, 2023

    Exposition position: 097/1

    The formation of a stable bond with the attachment figure is one of the most important early life experiences and alterations of this bond are associated with increased vulnerability to several psychopathologies, such as depression, in adulthood. The main neuropeptide involved in social behaviors and attachment bond formation is the Oxytocin (Oxt) and Oxt alterations are reported in depressed patients as well as in animal models of depression.

    We have previously demonstrated that Repeated Cross-Fostering (RCF) affects the formation of mother-pup bond and increases vulnerability to depression-like phenotype in C57BL/6J male mice. RCF is an early life manipulation where pups change the mother every 24 hours from PND 1 to PND4.

    Here we hypothesized that RCF-induced depression-like phenotype is mediated by a dysregulation of Oxt system.

    To test this hypothesis, we analyzed two of the most important targets of the Oxt system: the Oxt Receptor (OxtR) expression in the mesocorticolimbic circuit and the Receptors for Advanced Glycation End-Products (RAGE), an Oxt transporter, across the blood-brain barrier (BBB) of adult RCF and Control mice.

    Our results indicate a significant reduction of the OxtR selectively in the Nucleus Accumbens (NAc) and altered levels of RAGE in different BBB cell populations of RCF compared to Control mice.

    Finally, to confirm the involvement of the Oxytocinergic system, we precociously treated pups with subcutaneous injections of Oxt during the four days of RCF. This treatment prevented the alteration of attachment bond in pups and the depression-like phenotype in adulthood. In progress experiment are currently investigating the effects of Oxt treatment on OxtR and RAGE.

    These data strongly suggest the protective role of Oxt system in mediating appropriate brain and behavioral development.

  • Post-traumatic stress disorder (PTSD) vulnerability and resilience: behavioral and transcriptomic characterization of two unique PTSD rat lines

    Authors:
    Giulia Federica Mancini (1,2), Maria Morena (1,3), Benedetta Di Cesare (1,3), Fausto Masi (1), Rachele Tofani (1), Onno Meijer (2), Patrizia Campolongo (1,3)

    Organisations:
    1: Dept. of Physiology and Pharmacology, Sapienza University of Rome, Rome, Italy
    2: Dept. of Endocrinology, Leiden University Medical Center, Leiden, The Netherlands
    3: Neurobiology of Behavior Laboratory, Santa Lucia Foundation, Rome, Italy

    Presenting author: Giulia Federica Mancini

    Exposition date: Friday, September 15, 2023

    Exposition position: 101/1

    After experiencing a traumatic event, stress coping mechanisms may become dysfunctional leading to the development of psychiatric disorders, such as post-traumatic stress disorder (PTSD). Interestingly, only a subset of subjects develops PTSD (susceptible), whilst most of the trauma victims fully recovers after the first acute physiological response (resilient). However, the etiopathogenesis of PTSD and the neurobiological mechanisms underlying the emergence of susceptible and resilient PTSD phenotypes remain largely unknown. Therefore, the aim of this study was to identify molecular mechanisms underlying PTSD vulnerability and resilience. By selective breeding of Sprague-Dawley rats, screened based on post-trauma behavioral indicators of PTSD-like vulnerability (or resilience) (Colucci et al. 2020 Transl Psychiatry 10:243), we generated two distinct lines of PTSD-like susceptible (SUS) and resilient (RES) rats. Data collected so far, on the 11th generations (F11), indicated a marked distinction between the two phenotypes in terms of behavioral indices of traumatic memory (i.e., increased traumatic memory consolidation and recall, and reduced extinction in SUS vs RES) and post-trauma sociability (i.e., reduced sociability in SUS vs RES) in males and females. Furthermore, transcriptome analysis (RNA sequencing) within the amygdala showed differentially expressed genes between naïve SUS and RES rats of both sexes never exposed to a traumatic experience. The availability of such SUS and RES lines of rats represents an unprecedented tool to determine mechanisms of PTSD vulnerability or resilience and identify new targets for prophylactic and/or therapeutic interventions for the treatment of trauma-related disorders.

  • Resting state mri functional connectivity and negative symptoms in subjects with schizophrenia

    Authors:
    Edoardo Caporusso (1), Andrea Perrottelli (1), Giulia Maria Giordano (1), Leonardo Fazio (2), Giuseppe Blasi (3), Giulio Pergola (3), Marina Sangiuliano (3), Linda Antonucci (3), Pasquale Pezzella (1), Armida Mucci (1), Silvana Galderisi (1), Alessandro Bertolino (3), Mario Maj (1)

    Organisations:
    1: University of Campania "Luigi Vanvitelli", Napoli, Italy
    2: LUM Jean Monnet, Libera Università Mediterranea di Bari, Bari, Italy
    3: Dipartimento Di Scienze Mediche Di Base, Neuroscienze E Organi Di Senso, Bari, Italy.

    Presenting author: Edoardo Caporusso

    Exposition date: Friday, September 15, 2023

    Exposition position: 088/1

    Introduction: Due to their substantial impact on real-life functioning in individuals with schizophrenia, negative symptoms (NS) are a major component of this condition. These symptoms have been linked to brain circuit dysfunctions that result from poor connectivity within and among neural networks. Therefore, the purpose of this study is to ascertain if functional connectivity impairments and NS in individuals with schizophrenia (SCZs) are related.Therefore, this study aims to investigate the association between functional connectivity abnormalities and NS in subjects with schizophrenia (SCZs).

    Methods: Functional magnetic resonance imaging (fMRI) data were acquired from 79 SCZs and 61 healthy controls (HC), recruited within the Italian Network for Research on Psychoses. Resting-state functional connectivity (RS-FC) was extracted from 23 regions of interests within 7 predefined functional networks. Data analysis included comparisons between SCZs and HC of the RS-FC and correlations between RS-FC and NS in SCZs.

    Results: SCZs, compared to HC, showed a significant decrease of the RS-FC in the left superior temporal gyrus region (limbic network) (F= 9.61, p= 0.002). Significant correlations were found between the RS-FC of the left superior parietal lobule region (dorsal attention network) and the total score of NS (r= 0.39, p< 0.001), as well as with the two NS subdomains, the Avolition-apathy (r= 0.35, p< 0.01) and the Expressive deficit (r= 0.42, p< 0.001).

    Conclusions: The findings seem to suggest that schizophrenia is characterized by a reduction in functional connectivity within the limbic network, which is involved in the development of emotions, auditory and language processing, as well as social cognitive processes. Additionally, NS are associated with hyperconnectivity within the dorsal attention network, which is devoted to early sensory processing.

  • Serotoninergic antidepressants are effective when administered in a supportive environment

    Authors:
    Silvia Poggini (1), Naomi Ciano Albanese (1), Caterina Barezzi (1), Claudia Delli Colli (1,2), Igor Branchi (1)

    Organisations:
    1: Istituto Superiore di Sanità, Rome, Italy
    2: PhD program in Pharmacology and Toxicology, Sapienza University of Rome, Rome, Italy

    Presenting author: Silvia Poggini

    Exposition date: Friday, September 15, 2023

    Exposition position: 103/1

    Adolescence is a sensitive phase where adverse experiences, such as a lack of appropriate social interactions, increase the vulnerability to depressive episodes and to the onset of treatment-resistant major depressive disorder in adulthood. The first-line treatment for depression is selective serotonin reuptake inhibitors (SSRIs), but their efficacy is incomplete. Recently, to explain such variable effects, it has been proposed that SSRIs do not affect mood per se, but, by enhancing neural plasticity, amplify the influences of the living environment on mood. Therefore, we hypothesized that a supportive environment is critical for recovery and antidepressant treatment can further enhance such beneficial action.

    To test this hypothesis, we compared the efficacy of the exposure to an enriched environment, the SSRI fluoxetine administration, and their combination in depressed-like adult mice. From weaning to adulthood, C57BL/6 male mice have been exposed to either standard social housing or social isolation to induce a depressive-like phenotype. In adulthood, socially housed mice received vehicle while exposed to an enriched environment (control group). By contrast, isolated subjects received either fluoxetine or vehicle while exposed to a standard or enriched environment. We assessed the effects of the different treatment strategies on the cognitive and emotional domains, and stress hormone levels.

    Our results showed that adolescent social isolation induced both depressive- and anxiety-like behaviors. Environmental enrichment, but not fluoxetine alone, counterbalanced the detrimental effects of social isolation, as indicated by the normalization of floating behavior in the forced swim test, locomotor activity in the open field test, and time spent in the open arms in the elevated plus-maze test. It is worth noting that fluoxetine effects were determined by the environment.

    These findings outline the relevance of environmental interventions alone or in combination with serotoninergic antidepressant to treat adolescence-onset depression.

  • Study of molecular mechanisms underlying Treatment-Resistant Depression using iPSCs-derived neurons

    Authors:
    Zaira Tomasoni (1), Federica Bono (1), Giulia Sbrini (1), Jessica Mingardi (2), Laura Musazzi (3), Veronica Mutti (4), Adele Guglielmi (1), Alessandra Minelli (2,4), Massimo Gennarelli (2,4), Cristina Missale (1), Alessandro Barbon (2), Chiara Fiorentini (1)

    Organisations:
    1: Division of Pharmacology, Department of Molecular and Translational Medicine, University of Brescia, 25123 Brescia, Italy
    2: Division of Biology and Genetics, Department of Molecular and Translational Medicine, University of Brescia, 25123 Brescia, Italy
    3: Department of Medicine and Surgery, University of Milano-Bicocca, 20900 Monza, Italy
    4: Genetic Unit, IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, Brescia, Italy

    Presenting author: Zaira Tomasoni

    Exposition date: Friday, September 15, 2023

    Exposition position: 089/1

    Monoaminergic systems represent the main target of traditional anti-depressant drugs (Villas Boas et al., 2019). Between them, selective serotonin reuptake inhibitors (SSRIs), such as fluoxetine, are among the most prescribed ones. SSRIs block the activity of serotonin transporter (SERT), increasing the synaptic availability of this neurotransmitter (Feighner, 1999). While the increased availability of serotonin at synaptic level occurs in hours, therapeutic effect requires several weeks (Racagni and Popoli, 2008). On this basis, it has been hypothesized that anti-depressant efficacy could be related to the ability of traditional anti-depressant drugs to enhance neuroplasticity (Duman, 2009), thus restoring cellular and structural brain defects frequently observed in patients with major depressive disorder (MDD) (Lorenzetti et al., 2009) and stress-based animal models of depression (Duman, 2009). However, traditional anti-depressant drugs failed to induce a therapeutic effect in 30-40% of MDD patients, defined as having Treatment-Resistant Depression (TRD) (Al-Harbi et al., 2012). The molecular mechanisms underlying TRD are almost unknown. On these bases, induced pluripotent stem cell (iPSCs) technology has been chosen as “in vitro” model for investigating molecular and cellular bases of TRD. In particular, iPSCs from two TRD patients and one healthy subject, previously generated and characterized in our laboratory (Bono et al., 2018; 2020; 2021), were differentiated into a mixed neuronal populations and characterized. By using immunofluorescence analysis, we found that glutamatergic and GABAergic neurons presented patient-specific alterations both in number and in morphology, suggesting an imbalance between inhibitory and excitatory pathways, consistent with MDD (Villas-Boas et al., 2019). Therefore, iPSC technology could be a useful approach for identifying specific molecular abnormalities for each patient likely contributing to complex mechanisms that lead to resistance to antidepressant drugs. Using this model, the ability of the SSRI fluoxetine or the N-methyl-D-aspartate (NMDA) receptor antagonist ketamine to rescue the morphological defects was also analyzed.

  • Sumoylation of OPHN1 controls spine density and architecture by tuning actin dynamics

    Authors:
    Cristina Guglielmetti (1), Alessia Seccia (1), Marco Erreni (2), Marta Busnelli (1), Marie Pronot (3), Marta Prieto (3), Fabrizia Guarnieri (1,4), Francesca D'Autilia (2), Gwenola Poupon (3), Silvia Bassani (1), Maria Passafaro (1), Matteo Fossati (1,5), Davide Pozzi (6), Federico Forneris (7), Stéphane Martin (8), Michela Matteoli (1,6), Alessandra Folci (1,5)

    Organisations:
    1: CNR-Institute of Neuroscience, Italy
    2: Unit of Advanced Optical Microscopy, Istituto Clinico Humanitas, Italy
    3: Université Côte d'Azur, CNRS, IPMC, Valbonne, France
    4: Division of Neuroscience, IRCCS San Raffaele Scientific Institute, Milan, Italy
    5: Istituto Clinico Humanitas, Rozzano, Italy;
    6: Humanitas University, Rozzano, Italy
    7: University of Pavia, Italy
    8: Université Côte d'Azur, INSERM, CNRS, IPMC, Valbonne, France

    Presenting author: Alessandra Folci

    Exposition date: Friday, September 15, 2023

    Exposition position: 093/1

    Ophn1 gene encodes Oligophrenin-1 (OPHN1), a Rho-GAP protein highly expressed in neurons. In humans, all Ophn1 mutations cause the loss of function of OPHN1 leading to syndromic intellectual disability (ID) characterized by cerebellum abnormalities. In neurons, OPHN1 regulates dendritic spine density and architecture, actin dynamics and AMPA receptor (AMPAR) trafficking. Interestingly, we identified for the first time OPHN1 as a novel target of sumoylation. Sumoylation is a post-translational modification essential to the modulation of several neuronal functions, including neurotransmitter release and synaptic plasticity. Altered sumoylation has been associated with neurological disorders. Here, we combined molecular biology with live imaging and super resolution microscopy to address the role of sumoylation in controlling OPHN1 function in hippocampal neurons. Excitingly, we demonstrated that sumoylation controls the activation state of OPHN1. By this mechanism, sumoylation controls the actin dynamics which in turn impact dendritic spine density and architecture. Furthermore, since the sumoylation site is located close to the novel missense mutation (G412D) identified in ID patients, we explored thrilling hypothesis that compromised sumoylation may lead to synaptic dysfunction associated to the ID phenotype.
    Altogether, our results clearly demonstrate that sumoylation is a novel regulatory mechanism tuning OPHN1 activity. Furthermore, since the ID-linked G412D mutation impacts OPHN1 sumoylation and affects spine density and morphology, AMPAR surface expression as well as actin polymerization rate to a similar extent as the non-sumoylatable OPHN1 mutant, our data support the hypothesis that impaired OPHN1 sumoylation may participate to the etiology of ID in patients carrying the G412D mutation.

  • Telomere length and mitochondrial DNA copy number as markers of accelerated aging in bipolar disorder and response to lithium treatment

    Authors:
    Anna Meloni (1), Raffaella Ardau (2), Caterina Chillotti (2), Bernardo Carpiniello (3,4), Donatella Congiu (1), Maria Del Zompo (1), Pasquale Paribello (3,4), Federica Pinna (3,4), Marco Pinna (3,4), Claudia Pisanu (1), Giovanni Severino (1), Mirko Manchia (3,4,5), Alessio Squassina (1,6)

    Organisations:
    1: Department of Biomedical Sciences, Section of Neuroscience and Clinical Pharmacology, University of Cagliari, Cagliari, Italy
    2: Unit of Clinical Pharmacology, University Hospital Agency of Cagliari, Cagliari, Italy
    3: Unit of Psychiatry, Department of Public Health, Clinical and Molecular Medicine, University of Cagliari, Cagliari, Italy.
    4: Unit of Clinical Psychiatry, University Hospital Agency of Cagliari, Cagliari, Italy.
    5: Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada.
    6: Department of Psychiatry, Faculty of Medicine, Dalhousie University, Halifax, NS, Canada

    Presenting author: Anna Meloni

    Exposition date: Friday, September 15, 2023

    Exposition position: 090/1

    Bipolar disorder (BD) is a psychiatric disease affecting 1-3% of the general population, with severe consequences on quality of life. While the pathophysiological mechanisms involved are still not completely understood, it has been strongly suggested that BD is associated with cellular aging. In this study, we aim to investigate different markers of cellular aging (telomere length (TL) and mitochondrial DNA copy number (mtDNAcn)) in a sample of BD patients under lithium treatment (n=47) and a sample of healthy controls (n=36). Lithium response was characterized with the Alda scale: 23 patients were responders (R) and 24 non-responders (NR). TL and mtDNAcn were measured with qPCR in DNA extracted from peripheral blood. Statistical analyses were run using general linear models and IBM SPSS Statistics as software.

    TL was inversely correlated with age and BMI and was shorter in BD patients compared to controls (model corrected, p=< 0.001; effect of diagnosis p=<0,001). No differences were found between R and NR. mtDNAcn was not correlated with any of the tested variables and did not differ between BD and controls or R and NR. mtDNAcn and TL did not correlate in the entire sample and in the single groups, even if some studies suggest common regulatory mechanism of the two tested markers.

    Our study confirms previous findings showing that telomeres are shorter in BD patients compared to controls, suggesting accelerated cellular aging in BD. However, considering that some studies also reported longer telomeres in BD patients treated with lithium, further investigations including patients treated with mood stabilizers other than lithium would be important to evidence possible treatment-related differences. Moreover, more studies on mtDNAcn in psychiatric patients will be useful to clarify the inconsistency of the results in the literature.

  • The biological role of “green-therapy”: a study on depressed patients

    Authors:
    Gianna Pavarino (1,2), Claudio Brasso (1,3), Roberta Schellino (1,2), Anna Carluccio (1,3), Stefania Baire (1,3), Elena Signorino (1,2), Marina Boido (1,2), Paola Rocca (1,3), Alessandro Vercelli (1,2)

    Organisations:
    1: Department of Neuroscience “Rita Levi Montalcini”, University of Turin, 10126 Turin, Italy.
    2: Neuroscience Institute Cavalieri Ottolenghi, 10043 Orbassano (TO), Italy.
    3: Struttura Complessa di Psichiatria Universitaria, Dipartimento di Neuroscienze e Salute Mentale, Azienda Ospedaliero-Universitaria "Città della Salute e della Scienza di Torino", 10126 Turin, Italy.

    Presenting author: Gianna Pavarino

    Exposition date: Friday, September 15, 2023

    Exposition position: 096/1

    In the last few years, human-nature interactions are becoming a fundamental issue. Indeed, spending time in nature and urban green spaces reduces stress and promotes mental well-being; however, the underlying biological mechanisms of action (MoA) are still elusive. Major depressive disorder (MDD) is one of the most prevalent and debilitating psychiatric diseases characterized by severe symptoms that negatively influence health perception. These symptoms also occur in other psychiatric disorders like bipolar disorder (BD). Unfortunately, even after antidepressant treatments, a high percentage of patients do not achieve a personal full recovery from MDD. Therefore, our study aims to i) evaluate the biological, molecular and epigenetic impact of spending time in the greenery on depressed patients, and ii) highlight the importance of the urban “green-therapy” on depressive symptoms. For this purpose, we are enrolling MDD and BD patients (who are recommended to walk regularly in urban parks, at least 40 minutes 3/4 times per week, for 6 months) and healthy control subjects. Then, we are evaluating serological markers of inflammation (e.g. cortisol, CRP, IL-6) and epigenetic markers (e.g. miRNAs and post-translationally modified histone proteins) that correlate with these disorders. Our preliminary results in depressed patients after the “green-therapy” show a clear trend to the decrease of IL-6 level in time (from baseline to 6 months) returning to healthy control levels. Moreover, the expression of the so far tested miRNAs (as miRNA-124-3p, miRNA-132-3p) seems to be completely restored in peripheral blood mononuclear cells. From this study, we provide new scientific evidence about beneficial effects of green environment on depressive symptoms, and propose greenness-related activities as a potential side treatment to reduce pharmacological administration and achieve a personal full recovery.

  • The psychotherapeutic use of lucid dreaming: a critical review

    Authors:
    Marina Baroni (1,3), Sara Baldini (2,3)

    Organisations:
    1: Department of Surgical, Medical and Molecular Pathology and Critical Care Medicine, University of Pisa, via Savi, 10, 56126, Pisa, Italy
    2: Neurology Unit, Department of Medicine, Surgery and Health Sciences, Cattinara University Hospital ASUGI, University of Trieste, Trieste, Italy
    3: Scuola di Psicoterapia Integrativa Interdisciplinare, Via Pratese 13, 50145, Firenze

    Presenting author: Marina Baroni

    Exposition date: Friday, September 15, 2023

    Exposition position: 104/1

    Introduction: lucid dreaming refers to the experience of being aware that you are dreaming; to date, several authors highlighted the potential usefulness of lucid dream induction in treating several psychological conditions. The present work aims at reviewing the literature research on the use of lucid dreaming in psychotherapy.

    Methods: a critical review using PubMed and Scopus® databases was carried out by identifying specific keywords to conduct searches for the pertinent full-text articles. In April 2023, the final database check was performed. In the qualitative synthesis, a total of 19 records were included.

    Results: The majority of the included studies investigated the use of lucid dreaming in the treatment of nightmares also in terms of PTSD symptoms (N=15), while others observed the use of this phenomenon in other conditions such as pain (N=2), insomnia (N=1), and depression (N=1). Moreover, most reviewed studies used lucid dreaming induction combined with other psychotherapeutic techniques. In addition, literature findings pointed out significant results concerning the effectiveness of lucid dreaming in treating the conditions above. Lastly, data also highlighted interesting neural circuitry activation results during lucid dream experiences.

    Conclusion: Despite the significant data that emerged, future studies still need to better comprehend people's emotional experiences during lucid dreaming. From a psychotherapy point of view, lucid dreaming could be considered a ground-breaking phenomenon, but to use with caution. However, exploring the default mode network and frontoparietal network activation in such a peculiar condition could be regarded as an interesting parameter concerning the functioning of these two neural circuits.

  • Unrevealing the role of the neuropeptide oxytocin in anorexia nervosa: a preclinical investigation

    Authors:
    Barbara Eramo (1), Adele Romano (1), Silvana Gaetani (1), Ida Nilsson (2)

    Organisations:
    1: Dept. of Physiology and Pharmacology “V. Erspamer”, Sapienza University of Rome, Rome, Italy
    2: Center for Molecular Medicine, Karolinska University Hospital, and Dept. of Molecular Medicine and Surgery, Karolinska Institutet, 171 76 Stockholm, Sweden.

    Presenting author: Barbara Eramo

    Exposition date: Friday, September 15, 2023

    Exposition position: 095/1

    Anorexia nervosa (AN) is a common, poorly understood metabo-psychiatric disorder with a high relapse rate and mortality. The hypothesis that anorexia nervosa results from a deranged hypothalamic neuropeptidergic signalling is well documented. Among the hypothalamic peptides, oxytocin (OXY) is rising in interest due to its role in the coordination of energy balance at the crossroad between homeostatic and non-homeostatic mechanism. Although still sparse, both pre-clinical and clinical evidence suggest that OXY might participate in the pathophysiology of AN. However, to date, there are no studies on eventual derangement of brain areas that modulate or are modulated by OXY system in AN condition.

    Therefore, the aim of the present study is to investigate the possible role of the central oxytocinergic system in the neurobiology of AN. To reach our aim, we used anx/anx mice as preclinical model of AN, and wild-type sibling mice as a control group. The anx/anx mouse shows impaired hypothalamic neuropeptidergic systems involved in regulating food intake and metabolism. These mice share several characteristics with AN patient, making it a precious resource in research on the (neuro)biology of AN. Although the anx/anx model has been well-characterised concerning the hypothalamic neuropeptide profile, the oxytocinergic system has not yet been investigated.

    By performing immunofluorescence analysis, we evaluated the expression levels of OXY, c-fos (marker of neuronal activation) and OXY-receptor in brain areas partaking in the oxytocinergic system. Pituitary OXY immunoreactivity was also evaluated. This gland releases oxytocin into the blood system, and thus influence plasma levels of OXY.

    Although it is not clear if an increase or a decrease of the oxytocinergic activity would be desirable to counteract AN, our study contributes to improve knowledge of the complex mechanisms of AN neurobiology, with novel therapeutic potential for the treatment of one of the most prevalent psychiatric conditions in our society.

Neurocovid

Neuroendocrinology and neuroimmunology

  • Genistein and predisposition to obesity in cd1 mice: from neural circuits to estrogen receptors

    Authors:
    Marilena Marraudino (1,2), Ornella Vranaraj (1,3), Roberta Maggio (1), Antonino Casile (1,2,4), Brigitta Bonaldo (1,2), Stefano Gotti (1,2)

    Organisations:
    1: Neuroscience Institute Cavalieri Ottolenghi (NICO), University of Turin, Regione Gonzole, 10, 10043, Orbassano (TO), Italy.
    2: Department of Neuroscience ‘Rita Levi-Montalcini’, University of Turin, Via Cherasco 15, 10126 Turin, Italy.
    3: Department of Life Sciences and Systems Biology, University of Turin, via Accademia Albertina 13, 10123 Turin, Italy.
    4: School of Pharmacy, Pharmacology Unit, University of Camerino, Via Madonna delle Carceri 9, 62032 Camerino, Italy.

    Presenting author: Ornella Vranaraj

    Exposition date: Friday, September 15, 2023

    Exposition position: 106/1

    Genistein (GEN) is a phytoestrogen, a metabolic endocrine disruptor capable of interfering
    in the regulation of various estrogen-controlled hypothalamic metabolic circuits, binding
    estrogen receptors (ERs). In our diet, the main sources of GEN are soybean and soybean-
    derived foods. In a previous study we showed that early postnatal exposure to GEN, in a
    dose comparable to exposure level in babies fed with soy-based formula, determines an
    obesogenic phenotype only in adult mice. Here, we investigated the sexually dimorphic
    predisposition to obesity in adult caused by the exposure to GEN at an early stage of life
    and the role of different ERs.
    CD1 mice of both sexes were treated in early postnatal period (PDN5-PDN12) with GEN
    alone or in combination with different ERs antagonists individually (MPP, ERα antagonist;
    PHTPP, ERβ antagonist; G15, GPR30 antagonist) and all together (mix group). Once
    adults CD1 mice were analyzed both for the different physiological parameters of feeding
    behavior and for the hypothalamic circuits controlling food-intake: Pro-opiomelanocortin
    (POMC) and Neuropeptide Y (NPY) immunoreactive systems.
    Only male mice treated with GEN and GEN+PHTPP significantly increased body weight.
    The effects of treatment were also observed in the analysis of leptin levels by ELISA
    assay, where GEN-treated males showed a significant increase in leptin expression. In the
    hypothalamus, POMC-ir analysis showed a significant change in cell size in the arcuate
    nucleus, but no major changes in their cell number, while the NPY system was most
    altered by postnatal treatment in a sexually differentiated manner in the different nuclei
    analyzed.
    In conclusion, early postnatal exposure of CD1 mice leads to a state of obesity in GEN-
    treated adulthood male and to marked dimorphic alterations in hypothalamic systems
    regulating food-intake. In addition, the ERβ receptor appears to play a crucial role in the
    regulation of feeding behavior in males only.

  • Maternal exposure to genistein in combination with low-protein diet during pregnancy influences maternal behavior and alters the stress axis in offspring

    Authors:
    Mariateresa Bellantoni (1), Marialaura Teresa Ostuni (1), Elena Signorino (1,2), Marilena Marraudino (1,2)

    Organisations:
    1: Laboratory of Neuroendocrinology, Neuroscience Institute Cavalieri Ottolenghi (NICO), Regione Gonzole 10, Orbassano, Torino, Italy.
    2: Department of Neuroscience ‘Rita Levi Montalcini’, University of Torino, Via Cherasco 15, Torino, Italy.

    Presenting author: Mariateresa Bellantoni

    Exposition date: Friday, September 15, 2023

    Exposition position: 108/1

    Offspring health can be affected by maternal nutrition in pregnancy, such as a low-protein diet that may present a greater risk to the development, physical and neurological development of the prole. Therefore, dietary supplements such as legumes and soybeans, that are rich in phytoestrogens, in particular Genistein (GEN), are recommended for these incorrect diets in pregnancy. GEN is recognized as an endocrine disruptor for its ability to bind estrogen receptors, affecting various estrogen-sensitive neural systems, such as the stress axis. The aim of this work is to study the effects of early exposure to GEN as nutritional substitute in a low-protein diet during pregnancy on both the maternal and feeding behavior of mothers and the stress axis of the offspring.

    Female Spague Dawly rats were used and kept on a low-protein diet with or without GEN from two months before mating. We assessed mothers' feeding and maternal behavior, as well as the number, sex ratio, and weight of pups at birth and during development. In addition, at pups birth we performed molecular analyses of plasma corticosteroid levels by ELISA assay and for glucocorticoid receptor expression in the brain by RT-PCR.

    The results showed that feeding and maternal behavior were affected by low-protein diet in combination with or without GEN. Moreover, from birth the pups born to such mothers had significantly lower body weights, a difference that was maintained during development. In addition, the ELISA assay showed lower corticosteroid levels in pups born to mothers on diets compared with controls, particularly in low protein GEN-females. Finally, the RT-PCR results showed an increase in glucocorticoid receptor expression in both treated groups compared with controls. In conclusion, these data indicate that maternal diet is critical for maintaining the balance of the stress axis in the offspring, but also for maternal nutrition and behavior.

  • Postnatal phytoestrogen diet impacts on emotional behaviors and serotoninergic system in male and female mice

    Authors:
    Marilena Marraudino (1,2), Elena Ricci (1,3), Roberta Maggio (1), Antonino Casile (1,2,4), Brigitta Bonaldo (1,2), Stefano Gotti (1,2)

    Organisations:
    1: Neuroscience Institute Cavalieri Ottolenghi (NICO), Regione Gonzole, 10, 10043 Orbassano (TO), University of Turin, Turin, Italy.
    2: Department of Neuroscience ‘Rita Levi-Montalcini’, University of Turin, Turin, Via Cherasco 15, 10126 Turin (TO), Italy.
    3: Department of Biology and Biotechnology ‘L.Spallanzani, University of Pavia, Pavia, Via Adolfo Ferrata 9, 27100 Pavia (PV), Italy.
    4: School of Pharmacy, Pharmacology Unit, University of Camerino, Via Madonna delle Carceri, 9, Camerino, 62032, Italy.

    Presenting author: Elena Ricci

    Exposition date: Friday, September 15, 2023

    Exposition position: 107/1

    Genistein (GEN) is a phytoestrogen found in leguminous plants, particularly soy, which is used in postnatal weaning of infants, as soy milk. GEN’s molecular structure similar, like that of estrogens, allows binding to their receptors (ERs) altering their estrogenic functions. Previous studies have shown that serotoninergic (5-HT) system, which is strongly regulated by estrogen, is affected by postnatal GEN exposure, inducing an anxiolytic effect in males and anxiogenic in female animal models.

    The aims of this work are dual: to understand which receptor is involved in altering the organizational effect of GEN in the postnatal period and to evaluate how such exposure may affect the 5-HT system.

    CD1 male and female mice were treated daily from PND5 to PND12 with corn oil (control group), GEN and GEN associated separately (MPP, ERα antagonist; PHTPP, ERβ antagonist, and G15, GPR30 antagonist), or all together (mix group) with each of ERs antagonist. In adulthood the animals were tested with different behavioral tests to assess anxiety and stress state. Finally, they were sacrificed at PND90 to analyze the 5-HT system within the dorsal and median Raphe nucleus.

    The behavioral tests showed an anxiogenic behavior in GEN and GEN + PHTPP only in males and a high locomotor activity along with anxiolytic behavior in all treated females. In 5-HT analyses, no significant differences were observed between males and females, but emerged that it ERα receptors was most affected by postnatal GEN treatment playing a crucial role in the pathways that regulate mood. The results show that the 5-HT system was most altered by treatment in the rostral part of the Raphe, suggesting that in that region the system is particularly sensitive to estrogen regulation.

    Thus, data obtained confirmed that postnatal GEN’s administration has effects on estrogen receptors and on 5-HT system, altering the mood pathways.

Neuroinflammation

  • Anti-inflammatory and neuroprotective potential of oleoylethanolamide in rat model of diet induced obesity

    Authors:
    Nisha Zahid, Marzia Friuli, Barbara Eramo, Silvana Gaetani, Adele Romano

    Organisations:
    Sapienza University of Rome, Italy

    Presenting author: Nisha Zahid

    Exposition date: Saturday, September 16, 2023

    Exposition position: 108/2

    Background:A well-described feature of obesity is chronic, unresolved tissue inflammation,neuroinflammation and modification in the integrity and functionality of the blood brain barrier (BBB)[1]. In this scenario, oleoylethanolamde (OEA), a naturally occurring bioactive lipid received great attention for its biological properties. It exerts many protective effects including anti-obesity, anti-inflammatory, antioxidant properties thus supporting its potential use for the treatment of obesity and eating-related disorders [2–4]. Hence, by using a rat model of diet-induced obesity (DIO), with the aim to evaluate whether rats with an obese phenotype showed neuroinflammation and modification in the integrity and functionality of the BBB and subsequently whether the chronic peripheral administration of OEA (10 mg/kg) might ameliorate such alterations.

    Methods: We used a rat model of DIO based on exposure for 11 weeks to a HFD (60% of the kcal from fats); the control group received LFD(10% of the kcal from fats). From the 12th week, all the animals were chronically treated for 2 weeks with VEH or OEA (10 mg/kg i.p.). Brains collected from all animals groups were used in immunohistochemistry and immunofluorescence experiments to evaluate: 1) GFAP expression levels as a marker of neuroinflammation; 2)VIM (ZO)-1 expression levels as proteins of the BBB; 3) DCX as a marker of neurogenesis.

    Results:Our results revealed that, in HFD-fed animals, OEA administration completely reverted the expression of GFAP as in VEH-treated animals. Moreover, OEA treatment modulated the expression of VIM and ZO-1, key proteins involved in the integrity and functionality of BBB. Finally, OEA treatment increased the number of DCX-positive neurons in both lean and obese rats

    Conclusion:Taken together, our data demonstrated that OEA is reducing food intake and body weight gain accompanied by decreased neuroinflammation and improved neuroprotection. Hence, OEA has strong potential to be used as a pharmacological target to treat obesity and its related alterations.

  • APOE4 impairs microglia-astrocyte response in Alzheimer’s disease by inducing TGFβ-mediated checkpoints

    Authors:
    Oleg Butovsky, Zhuoran Yin, Neta Rosenzweig, Kilian Kleemann, Wesley Brandão, Milica Margeta, Jen-Li Barry, Michael Aronchik, Dean Sheppard, Bart Eggen, Rudolph Tanzi, Charlotte Madore, Thomas Arnold, David Holtzman

    Organisations:
    Harvard Medical School, United States of America

    Presenting author: Oleg Butovsky

    Exposition date: Saturday, September 16, 2023

    Exposition position: 097/2

    Background

    APOE ε4 is the strongest genetic risk factor for late-onset Alzheimer’s disease (AD). The contribution of microglial APOE4 to AD pathogenesis is unknown, although APOE has the most enriched gene expression in neurodegenerative microglia (MGnD).

    Methods

    Generation of CX3CR1-CREERT2 mice crossed to APOE-KI(E3 and E4)fl/fl:APP/PS1 and APOE-KI(E3 and E4)fl/fl:P301S mice. Microglia-astrocyte crosstalk was determined using scRNAseq and NichenetR database and validated in the brain of AD donors carrying the APOE e3/3 and e3/4 alleles.

    Results

    Here we show, in mice and in humans, a negative role of microglial APOE4 in the induction of MGnD response to neurodegeneration. Deletion of microglial APOE4 restores MGnD phenotype, associated with neuroprotection in P301S tau transgenic mice and decreases pathology in APP/PS1 mice. MGnD-astrocyte crosstalk associated with β-amyloid (Aβ) plaque encapsulation and clearance are mediated via Lgals3 signaling following microglial APOE4deletion. In the brain of AD donors carrying the APOE ε4 allele, we found a sex-dependent reciprocal induction of AD risk factors associated with suppression of MGnD genes in females, including LGALS3, as compared to APOE ε3/3 carriers. Mechanistically, APOE4-mediated induction of ITGB8-TGFb signaling impairs MGnD response via upregulation of TGFβ-mediated microglial homeostatic checkpoints, including INPP5D. Microglial deletion of Inpp5drestores MGnD-astrocyte crosstalk and facilitates plaque clearance in APP/PS1 mice. Genetic and pharmacological blocking of ITGB8-TGFβ signaling enhanced MGnD response associated with increased plaque clearance in AD mice.

    Conclusions

    We identify the microglial APOE4-ITGB8-TGFβ pathway as a negative regulator of microglia-astrocyte response to AD pathology, and restoring MGnD phenotype via blocking ITGB8-TGFβ signaling may provide new molecular targets to modulate and restore functional microglia in AD.

  • Complement in the cortex of EAE mice: its relevance to synaptopathy.

    Authors:
    Alessandra Roggeri (1), Alice Taddeucci (1), Veronica Torre (1), Guendalina Olivero (1), Hanna Trebesova (1), Massimo Grilli (1), Anna Pittaluga (2)

    Organisations:
    1: Department of Pharmacy, University of Genoa, Genoa, Italy
    2: Department of Pharmacy, University of Genoa and IRCCS Ospedale Policlinico San Martino, Genoa, Italy

    Presenting author: Anna Pittaluga

    Exposition date: Saturday, September 16, 2023

    Exposition position: 105/2

    Recently, the complement system has emerged as a major actor in white and grey matter lesions in experimental autoimmune encephalomyelitis (EAE) mice, an animal model of multiple sclerosis (MS). In the cortical homogenate of these mice, we did not observe synaptic derangements (measured as SNAP25 and PSD95 density) but extensive microgliosis (quantify as CD11b density) and astrocytosis (quantified as GFAP density) in parallel with a significant increase of C1q and C3 proteins. Since MS has recently been recognized as a synaptopathy, we wanted to investigate whether complement components might play a role in the modulation of glutamate transmission in EAE mice.

    To this aim, purified nerve endings (synaptosomes) and astrocytic processes (gliosomes) were isolated from the cortex of EAE mice at the acute stage of the disease (21 ± 1 day post immunization) and used as experimental model. Then, the particles were analysed for glutamate release efficiency [measured as release of preloaded [3H]D-aspartate], C1q and C3 expression, and the viability/apoptosis levels were assessed by flow cytometry.

    In healthy mice, complement induces [3H]D-aspartate release from gliosomes more efficiently than from synaptosomes. The effect occurs in a dilution-dependent manner and involves the reversal of glutamate transporters. Differently, in EAE mice, the complement-induced release activity is significantly reduced in cortical synaptosomes, but largely amplified in cortical gliosomes. Interestingly, Western blot analysis confirmed that the adaptations paralleled the reduced expression of EAAT2 in synaptosomes and the increased density of EAAT1 in gliosomes. Similarly, both C1q and C3 proteins expression was increased in EAE cortical synaptosomes and gliosomes, although no signs of ongoing apoptosis or altered viability were detectable in both preparations. Overall, the results propose novel mechanisms of actions of the complement in the CNS of EAE mice that are relevant for disease progression and central synaptopathy, suggesting new therapeutic targets for MS management.

  • Effect of Heated Tobacco Products on oxidative status and inflammatory processes in the rat prefrontal cortex

    Authors:
    Camilla Morosini (1), Laura Rullo (1), Loredana Maria Losapio (1), Antonio Lacorte (1), Fabio Vivarelli (1), Donatella Canistro (1), Paola Franchi (2), Marco Lucarini (2), Moreno Paolini (1), Sanzio Candeletti (1), Patrizia Romualdi (1)

    Organisations:
    1: Department of Pharmacy and Biotechnology, Alma Mater Studiorum, University of Bologna, Via Irnerio 48, 40126 Bologna, Italy.
    2: Department of Chemistry "G. Ciamician", Alma Mater Studiorum-University of Bologna, Via S. Giacomo 11, 40126, Bologna, Italy.

    Presenting author: Camilla Morosini

    Exposition date: Saturday, September 16, 2023

    Exposition position: 098/2

    Perceived as a safer alternative to conventional cigarettes, “Heated Tobacco Products” (HTPs) reached high popularity in recent years, especially among never-smokers and young people, becoming an emergent public health problem. The HTPs products are equipped with technology that heats tobacco without combustion to produce a nicotine aerosol. The overall health effects of employing these products haven’t been assessed. In fact, recent data suggest that the use of HTPs produces toxic effects similar to the ones triggered by conventional cigarettes. Based on this evidence, the aim of this study was to investigate the HTPs impact on oxidative stress and antioxidant enzymatic machinery, as well as on key signalling pathways involved in DNA damage, in the rat CNS.

    To this end, male Sprague Dawley rats were exposed to HTPs aerosol using a whole-body mode. After four weeks, animals were sacrificed, the prefrontal cortex (PFCx) was collected, and the measure of reactive oxygen species (ROS) amount was performed using electron paramagnetic resonance. The mRNA and protein levels of pro-inflammatory cytokines, antioxidant enzymes and some markers of neuroinflammation, as well as of DNA damage were assessed by quantitative RT-PCR and Western Blot assay.

    Results showed the HTPs exposure enhanced ROS production and the protein level of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) as well as oxidative injury markers (e.g., OGG1 and PARP1). The exposure to HTPs aerosol induced an increase of the expression of antioxidant enzymes, like SOD1 and CAT, and altered the gene expression of PPARs receptors and KDM6A enzyme involved in the neuroinflammation response.

    This study highlighted the ability of HTPs to promote neuroinflammation in the rat PFCx. Indeed, these results showed that aerosol generated from HTPs devices may induce inflammatory and oxidative processes, as indicated by alterations in pro and anti-inflammatory genes, as well as antioxidant enzymes and DNA damage markers.

  • Exploring Toll-like receptor 4 as a target for opioids in microglia

    Authors:
    Chiara Chemello, Laura Facci, Morena Zusso

    Organisations:
    University of Padova, Italy

    Presenting author: Morena Zusso

    Exposition date: Saturday, September 16, 2023

    Exposition position: 103/2

    Opioids are the most widely used class of analgesics for acute and chronic pain; however, their chronic use is accompanied by side effects and complications. Although the mechanisms underlying these phenomena are not fully understood, neuroinflammation is likely to contribute to analgesic tolerance and opioid-induced hyperalgesia. Indeed, opioids have been shown to induce neuroinflammation and microglia activation, leading to the production and release of numerous pain and inflammatory agents. Furthermore, given the central role of Toll-like receptor 4 (TLR4) in the activation of innate immune cells, including microglia, this receptor has been proposed as an off-target site for opioid action.

    With the aim to validate TLR4 as an opioid target, we studied the effect of fentanyl on LPS-induced TLR4 activation in primary microglia cells. The production and release of different proinflammatory mediators were investigated in microglia by real-time PCR and ELISA. Fentanyl increased mRNA levels of IL-1β, IL-6, TNF-α, iNOS, and MCP1/CCL2, as well as the LPS-induced secretion of IL-1β and TNF-α. Next, we analyzed the role of TLR4 in mediating the fentanyl effect using ciprofloxacin and CLI-095, two inhibitors of TLR4 signaling, previously identified by our group and others. Ciprofloxacin and CLI-095 completely reduced the release of proinflammatory cytokines by microglia stimulated with LPS and fentanyl, suggesting that TLR4 could be the target of the proinflammatory activity of fentanyl.

    These results provide new insight into the mechanism of the proinflammatory activity of fentanyl, which involves the activation of TLR4 signaling. Our findings might facilitate the development of new molecules active in modulating opioid-mediated TLR4 signaling, for future development of effective and safe pharmacological agents for pain management.

  • Impact of a a new formulation of Pomegranate-Peel Extract on spinal astrocytosis and microglia activation in an animal model of multiple sclerosis

    Authors:
    Veronica Torre (1), Alessandra Roggeri (1), Alice Taddeucci (1), Guendalina Olivero (1), Federica Turrini (1), Raffaella Boggia (1), Anna Pittaluga (2,3)

    Organisations:
    1: Department of Pharmacy (DiFar), University of Genoa
    2: Department of Pharmacy (DiFar), Center of Excellence for Biomedical Research, 3Rs Center, University of Genoa
    3: IRCCS Ospedale Policlinico San Martino

    Presenting author: Veronica Torre

    Exposition date: Saturday, September 16, 2023

    Exposition position: 106/2

    Multiple Sclerosis (MS) is a demyelinating progressive disease that affects the central nervous system (CNS) with a highly variable and unpredictable clinical presentation. Special dietary regimens are attracting the attention of researchers, indeed, promising anti-inflammatory and neuroprotective effects have been recently obtained with pomegranate fruits and their bioactive compounds, such as ellagic acid (EA) and ellagitannins, in animal models of MS. We propose a new formulation of a pomegranate-peel extract (PEm) obtained by PUAE (Pulsed Ultrasound-Assisted Extraction), and we tested its potential healthy properties in an animal model of the non- remitting form of MS: the Experimental Autoimmune Encephalomyelitis (EAE). PEm effects were compared to those elicited by a formulation containing EA (EAm). EAm and PEm were chronically administered to Control and EAE mice, dissolved in the drinking water, starting from the day 10 post-immunization (d.p.i.), with a "therapeutic" protocol to deliver daily 50 mg/kg of EA. We analyzed the impact of the treatment on in vivo and ex vivo parameters related to the pathological progression of the EAE. Treated EAE mice did not showed changes in body weight, nor they limit their daily access to the beverage, but displayed an amelioration of the severity of the clinical symptoms, which clearly emerged in the PEm-treated EAE mice. Ex vivo histochemical analysis showed that spinal cord demyelination and inflammation in PEm or EAm treated EAE mice at 23 ± 1 d.p.i. were comparable to those in the untreated EAE animals. However, microglia activation (measured as Iba1 staining) and astrocytosis (quantified as GFAP immunopositivity) were significantly reduced by the treatments, particularly in the grey matter. It is concluded that in EAE mice both treatments, EAm and PEm, were comparably efficient in modulating the spinal pathological cellular hallmarks. This would support their use as dietary supplementation in MS patients.

  • Impact of perinatal exposure to Chlorpyriphos on neuroinflammatory responses and hypnic phenotype in mice

    Authors:
    Loredana Maria Losapio (1), Laura Rullo (1), Camilla Morosini (1), Antonio Lacorte (1), Sara Alvente (2), Viviana Lo Martire (2), Chiara Berteotti (2), Stefano Bastianini (2), Gabriele Matteoli (2), Elena Miglioranza (2), Alessandro Silvani (2), Giovanna Zoccoli (2), Sanzio Candeletti (1), Patrizia Romualdi (1)

    Organisations:
    1: Department of Pharmacy and Biotechnology, Alma Mater Studiorum, University of Bologna, Via Irnerio 48, 40126 Bologna, Italy.
    2: Department of Biomedical and NeuroMotor Sciences, Alma Mater Studiorum, University of Bologna, P.zza P.ta S. Donato 2, I-40126 Bologna, Italy.

    Presenting author: Loredana Maria Losapio

    Exposition date: Saturday, September 16, 2023

    Exposition position: 099/2

    Early-life exposure to stressors can predispose to pathologies’ development later in life. Exposure to pesticides may have a negative impact on brain maturation and on the vulnerability to developing diseases. In this frame, we focused on Chlorpyriphos (CPF) exposure during pregnancy. CPF is a potent acetylcholinesterase inhibitor whose exposure has been previously associated with the development of neuroinflammation processes that, in turn, have been claimed to be involved in the mechanisms underlying sleep disorders.

    Based on this evidence, we investigated if perinatal CPF exposure affects sleep and respiratory phenotype in adulthood. Moreover, we assessed the long-term effect of perinatal exposure to CPF on pro-inflammatory cytokine expression levels and on peroxisome proliferator-activated receptors (PPARs), modulators of the anti-inflammatory response. Furthermore, the expression of histone demethylase enzyme KDM6A, involved in the epigenetic control of the inflammatory response, has been evaluated.

    To this end, C57BL6/J pregnant mice received oral CPF or vehicle, from 2 weeks prior to mating until weaning of the pups. Twenty weeks after birth, male and female adult mice were subjected to the assessment of the hypnic and respiratory phenotype during sleep. Subsequently, the hippocampus of animals was collected for gene expression analysis of IL-6, IL-1β e TNF-α, PPARa, PPARg, and KDM6A.

    Data revealed no significant effect of treatment on sleep macrostructure, while sleep apnea occurrence rate was increased in mice born from CPF-treated dams with respect to control mice, and was higher in females than in males. Molecular data showed that perinatal CPF exposure caused an upregulation of pro-inflammatory cytokines mRNA levels and a decrease in PPARs gene expression in female mice. Interestingly, females display higher basal levels of these genes than males.  

    These data indicate that perinatal CPF exposure induces changes in the respiratory pattern of sleeping mice and alterations of the inflammatory response in a sex-dependent fashion.

  • Impaired immune response in the deep cervical lymph nodes in a mouse model of Alzheimer’s disease

    Authors:
    Giulia Castellani (1), Sarah Phoebeluc Colaiuta (1), Javier María Peralta Ramos (1), Tommaso Croese (1), Gil Stelzer (2,3), Miguel Ángel Abellanas (1), Afroditi Tsitsou-Kampeli (1), Serena Riccitelli (1), Liora Cahalon (1), Paola Antonello (1,4), Chiara Burgaletto (1), Tomer Meir Salame (2), Merav Kedmi (2,3), Hadas Keren-Shaul (2,3), Igor Smirnov (5,6), Jonathan Kipnis (5,6), Michal Schwartz (1)

    Organisations:
    1: Department of Brain Sciences, Weizmann Institute of Science, Rehovot, Israel
    2: Department of Life Sciences Core Facility, Weizmann Institute of Science, Rehovot, Israel
    3: The Nancy & Stephen Grand Israel National Center for Personalized Medicine (G-INCPM), Weizmann Institute of Science, Rehovot, Israel
    4: Department of Systems Immunology, Weizmann Institute of Science, Rehovot, Israel
    5: Center for Brain Immunology and Glia (BIG), Washington University in St Louis, MO, USA
    6: Department of Pathology and Immunology, School of Medicine, Washington University in St Louis, MO, USA.

    Presenting author: Giulia Castellani

    Exposition date: Saturday, September 16, 2023

    Exposition position: 100/2

    Immune dysfunction has been implicated in Alzheimer’s disease (AD) progression, offering novel therapeutic targets that could be adopted for disease modification. Here, we show that both T follicular helper and B cells driving germinal center (GC) reaction are reduced in the deep cervical lymph nodes (dCLNs) draining the brain of AD mice (5xFAD), compared to wild types (WTs). Using T- and B-cell receptor single-cell RNA sequencing we show a reduced clonal expansion in the dCLNs of 5xFAD animals. Importantly, these alterations were not observed in lymph nodes draining other sites. Since our data suggested a dysfunctional spontaneous follicular reaction, we next tested the dCLNs response following an immune challenge ex vivo and in vivo. Unexpectedly, upon intra-cisterna magna administration of a foreign antigen, we detected an enhanced adaptive immune response in 5xFAD mice, reaching the levels of WTs. This study implies a defective response to self-brain antigens in the dCLNs of AD mice, potentially leading to a reduced immunosurveillance. The administration of a foreign antigen might therefore represent a promising tool to enhance GC reaction, to be further exploited towards developing therapeutic interventions for AD.

  • Increased levels of glucosyl-sterols induce intestinal inflammation possibly causing neurodegeneration

    Authors:
    Francesca Terrin (1), Sofia Faggin (1), Giuseppe Borsato (2), Fabrizio Fabris (2), Alessandro Scarso (2), Stefano Cagnin (1), Maria Cecilia Giron (1), Nicoletta Plotegher (1), Luisa Dalla Valle (1)

    Organisations:
    1: University of Padua, Padua, Italy
    2: Ca’ Foscari University of Venice, Venice, Italy

    Presenting author: Francesca Terrin

    Exposition date: Thursday, September 14, 2023 (Satellite Events Day)

    Exposition position: 105

    Glucosyl-sterols are lipids composed of a sterol-derived molecule and a glucose moiety. They can be either produced endogenously or introduced into the organism by pathogens and even with the diet, as it happens with the plant-derived β-glucosyl-sitosterol. The cellular function of these molecules is still unknown, but an alteration of their homeostasis has been associated with the occurrence of neurodegenerative diseases. Exposure to dietary β-glucosyl-sitosterol, indeed, has been considered the main risk factor for the onset of a neurodegenerative disorder called Amyotrophic Lateral Sclerosis-Parkinsonism Dementia Complex (ALS-PDC).

    Mice fed with β-glucosyl-sitosterol develop a marked intestinal inflammatory phenotype evidenced by the up-regulation of genes involved in the inflammatory response, as emerged with RNA sequencing.  Ultrastructural analysis revealed that microvilli, a fundamental cell surface structure of the enterocytes, are significantly shorter in treated mice than controls. Moreover, qPCR analysis and Western Blots performed on intestinal tissue show an altered expression of genes and proteins linked with microglia activation.

    According to the gut-brain-axis model, this inflammation initially localized in the gut may lead to a more systemic inflammatory state, finally affecting the brain. RNAseq on mice brain tissues evidenced the dysregulation of genes involved in the occurrence of neurodegenerative diseases, while Western Blots further confirmed an increased expression of proteins usually prone to form toxic aggregates in damaged neurons.

    Interestingly, chronic treatment with β-glucosyl-sitosterol impacts on intestinal morphology in our zebrafish model as well. Ongoing inflammation is evidenced by the up-regulation of genes associated with inflammatory response and in addition we observed the dyshomeostasis of other genes usually linked with neurodegeneration. Altered gut functionality was also assessed ex vivo, thus indicating an impairment of the enteric nervous system.

    This evidence suggests that an increase in the dietary glucosyl-sterols may trigger bowel inflammation that could further contribute to neurodegeneration through the well-known gut-brain axis.

  • Investigating microglial activation in the Activity-based anorexia (ABA) rats.

    Authors:
    Carlotta Siddi (1), Chiara Camoglio (1), Sabrina D’Amelio (2), Vittoria Spero (2), Raffaella Molteni (2), Maria Scherma (1), Simona Dedoni (1), Ida Nilsson (3,4), Paola Fadda (1,5)

    Organisations:
    1: Department of Biomedical Sciences, Division of Neuroscience and Clinical Pharmacology, University of Cagliari
    2: Department of Medical Biotechnology and Translational Medicine, University of Milan, Milan, Italy
    3: Department of Molecular Medicine and Surgery, Karolinska Institute, Stockholm, Sweden
    4: Center for Molecular Medicine, Karolinska University Hospital, Stockholm, Sweden
    5: Institute of Neuroscience-Cagliari, National Research Council, Rome, Italy

    Presenting author: Carlotta Siddi

    Exposition date: Friday, September 15, 2023

    Exposition position: 105/1

    The pubertal transition is a crucial period for the risk of the development of eating disorders (EDs). Anorexia nervosa (AN) is a serious eating disorder prevalently widespread in female subjects (Klump et al., 2013), diagnosed on the base of the presence of several characteristic symptoms that include: restriction of energy intake, low body weight, fear of becoming fat or of gaining weight, distortion in the body perception, and amenorrhea (DSM5). Nowadays strongly connection between inflammatory processes and AN have been demonstrated, (Dalton et al., 2018; Solmi et al., 2015), as well as the involvement of some immunoinflammatory pathways in others EDs (Breton et al., 2022). Rodents model of AN such as anx/anx exhibits an increase in macrophage infiltration (Lindfors et al., 2015), moreover, Dehydration-induced anorexia (DIA) young female rats, shown a hippocampal increase in the microglial density (Ragu-Varman et al., 2019). However, the possible connection between the inflammation of the central nervous system (CNS) and AN remains enigmatic. To clarify this potential link, we investigated the expression of microglia in several key brain regions involved in AN, using Activity-based anorexia (ABA) female rodents (Scherma et al., 2017). The aim of this study was to elucidate the in-situ expression of ionized calcium-binding adapter molecule 1 (Iba1), a marker of microglial activation in the paraventricular nucleus, nucleus accumbens, dorsal and ventral hippocampus, arcuate nucleus, cingulate cortex, caudate putamen, and amygdala. Our preliminary results showed an overall reduction of microglial density after the ABA paradigm.

  • Neuroprotective effect of bFGF mimetic, SUN11602, on tissue regeneration and glial cells activation in an in vivo model of spinal cord injury

    Authors:
    Irene Paterniti, Valentina Bova, Alessia Filippone, Alessio Ardizzone, Alberto Repici, Salvatore Cuzzocrea, Emanuela Esposito

    Organisations:
    University of Messina, Italy

    Presenting author: Irene Paterniti

    Exposition date: Saturday, September 16, 2023

    Exposition position: 107/2

    Spinal cord injury (SCI) represents a debilitating traumatic event to the spinal cord that usually triggers permanent changes in motor and sensory functions. The knowledge of important functions exerted on central nervous system (CNS) make basic fibroblast growth factor (bFGF) a good therapeutic approach for long-term consequences of CNS traumatic event, although its therapeutic use is limited, due to the undesirable effects. The synthetic compound SUN11602, mimetic of bFGF, shows neuroprotective activities similar to those of bFGF, but with greater safety. Therefore, the aim of our study was investigate the neuroprotective effects of SUN11602 in a chronic mouse model of SCI. SCI was produced in mice by extradural compression of the spinal cord at T6 to T7 using an aneurysm clip with a closing force of 24 g for 1min. SUN11602 treatments (doses of 1, 2.5 and 5 mg/kg) were administered orally, daily, from day 1 to day 3. After 3 days, spinal cord tissues were collected and used for biochemical and histological analysis. By performing Basso Motor Score (BMS), we found that SUN11602 treatments significantly restored motor impairments 72 h after SCI. Oral treatments with SUN11602, especially at the higher dose of 5 mg/kg, reduced the inflammatory state and neutrophils infiltration, significantly lowering MPO activity; this data was confirmed by mast cells detection in spinal cord tissues. Treatments with SUN11602 restored neurotrophin 3 (NT-3), brain-derived neurotrophic factor (BDNF) and glial cell derived neurotrophic factor (GDNF) expression. Also, SUN11602 treatments were able to modulate NF-kB and MAPK pathways, reducing the astrogliosis and microgliosis response SCI-induced. Finally, FGFR1 activation by SUN11602, restored intracellular Ca2+ homeostasis, modulating Ca2+-binding proteins, such as Calbind-D28k, S-100β and Calmodulin. Therefore, based on the results, SUN11602 could represent a promising therapeutic and pharmacologic candidate for the attenuation of CNS traumas related consequences.

  • Role of pentraxin 3 in neurodevelopmental disorders

    Authors:
    Giuliana Fossati (2), Cecilia Zen (2), Mariassunta De Luca (1), Anna Chiara Pacentra (2), Sonia Valentino (2), Eliana Lauranzano (2), Angelo Iannielli (1), Rebecca Fry (3), Barbara Bottazzi (2), Cecilia Garlanda (2), Vania Broccoli (1), Michela Matteoli (1,2), Elisabetta Menna (1,2)

    Organisations:
    1: CNR Neuroscience Institute, Milano, Italy
    2: IRCCS Humanitas, via Manzoni 56, 20089 Rozzano, Italy
    3: University of North Carolina, Chapel Hill, USA

    Presenting author: Giuliana Fossati

    Exposition date: Saturday, September 16, 2023

    Exposition position: 102/2

    Control of synapse number and function is critical to the formation of neural circuits. We recently demonstrated that the innate immune molecule, pentraxin3 (PTX3), released by astrocytes, promotes the formation of functional excitatory synapses. PTX3 interacts with thrombospondin1 (TSP1), an astrocyte-derived factor which controls the formation of silent synapses. PTX3:TSP1 interaction exerts a negative regulation of PTX3 itself suggesting that the relative amount of these proteins and their complex are crucial to set the balance between synaptic growth and synapse maturation. Prenatal inflammation and maternal immune activation (MIA) are recognized risk factors for neurodevelopmental diseases. Preclinical studies established a causal link between MIA and the disruption of the proper neurodevelopmental trajectory in the offspring. Of note, PTX3 is stimulated by inflammatory insults and, as a consequence, PTX3 and TSP1 may change their levels upon immune challenge during brain development.

    Starting from these premises, we aim to investigate how prenatal immune stimulation affects PTX3 and TSP1 levels and how this impacts synaptogenesis and brain circuit development.

    Using a mouse model of MIA we demonstrated that prenatal inflammation disrupts the physiological developmental pattern of expression of PTX3 and alters the PTX3:TSP1 ratio in in the cerebral cortex during the postnatal period of synaptogenesis. Moreover, we observed morphological and functional alterations in astrocytes, which are the main source of PTX3. We are currently investigating the consequences at neuronal and synaptic level. Furthermore, we are investigating the involvement of PTX3 alteration in a human cohort of premature neonates (ELGAN study) characterized by enhanced frequency of autism spectrum disorders and the regulation of PTX3 inflammatory conditions in human iPSC-derived glial cells.

  • Suppressing the microglia-mediated neuroinflammation in Alzheimer’s disease with Leishmania infantum parasites

    Authors:
    Estefania Calvo-Alvarez (1), Giovanna Pepe (2), Chiara Sfogliarini (2), Marina Saresella (3), Elisabetta Vegeto (2), Donatella Taramelli (1), Mario Clerici (3,4), Nicoletta Basilico (5)

    Organisations:
    1: Department of Pharmacological and Biomolecular Sciences, University of Milan, Milan (Italy)
    2: Department of Pharmaceutical Sciences, University of Milan, Milan (Italy)
    3: IRCCS Fondazione Don Carlo Gnocchi, Milan (Italy)
    4: Department of Physiopathology and Transplants, University of Milan, Milan (Italy)
    5: Department of Biomedical, Surgical and Dental Sciences, University of Milan, Milan (Italy)

    Presenting author: Estefania Calvo-Alvarez

    Exposition date: Saturday, September 16, 2023

    Exposition position: 104/2

    Alzheimer’s disease is a progressive, irreversible and yet uncurable brain disorder. Neuroinflammation, mainly mediated by the release of pro-inflammatory mediators by overactivated microglia (the brain’s resident macrophages, MG), might be a vital component in the initiation and progression of AD pathology. Indeed, modulating neuroinflammation through MG-targeted approaches is considered an innovative strategy for AD. In distant human immunopathologies such as visceral leishmaniasis infection, ancient Leishmania infantum parasites (Li) have co-evolved sophisticated mechanisms to survive within macrophages (Mϕ) by inactivating myriads of inflammatory responses that threaten parasite persistence. Among these, our group has demonstrated that Li reduces the activation of the NLRP3 inflammasome (an innate immune sensor) in amyloid-β-treated Mϕ, resulting in the generation of an anti-inflammatory milieu. Being MG functionally similar to Mϕ, we hypothesize that Leishmania subversion tactics against the host immune response might be conserved and exploited to suppress the dysfunctional pro-inflammatory properties of MG in AD. Here, by using immortalized and primary murine MG we found that Li infection did not induce the release of pro-inflammatory mediators, and that the expression of Iba-1 (a marker of AD MG), resulted to be greatly reduced only in Li-dwelling cells. Additionally, we observed that both Li and Aβ can be concomitantly phagocytosed by MG, suggesting enhanced phagocytic activities by infected MG in the presence of Aβ. Finally, we further show that the pro-inflammatory NF-kB/NLRP3 axis is impaired in infected and Aβ-stimulated MG, with the upregulation of the NF-kB-negative regulator A20 as the main mechanistic explanation. Overall, Leishmania infection results in a wide anti-inflammatory phenotype in MG, even under Aβ stimulation, which holds promise for suppressing aberrant MG-mediated inflammatory responses. These results may represent an unprecedented bioinspired strategy against neuroinflammation in AD, with the potential of discovering new molecular targets for therapeutic intervention.

Neuronal circuits, neurophysiology, and optogenetic approaches

  • Dissecting the role of the dorsal hippocampus in observational learning of social decisions

    Authors:
    Filippo La Greca, Giulia Coccia, Elisa Zianni, Davide Maggioni, Fabrizio Gardoni, Monica DiLuca, Diego Scheggia

    Organisations:
    Department of Pharmacological and Biomolecular Sciences, University of Milan, Italy

    Presenting author: Filippo La Greca

    Exposition date: Friday, September 15, 2023

    Exposition position: 113/1

    Humans are social animals that rarely live in isolation, rather they are continuously surrounded and influenced by others' actions. In this context, humans often learn from others and take advantage of their previous observation. This ability is called observational learning and gives the possibility to learn movements, emotions, and social behaviors without the need of direct experience, the latter being often inconvenient from an evolutionary perspective. Literature shows nonhuman animals are also capable of learning from others. In particular, rodents can acquire environmental and social information from prior observation of their conspecifics. However, it remains unclear whether rodents can learn to make social decisions through a shared social experience. Here, we used a social decision-making task (SDM) in which an observer mouse can learn prosocial behaviors from a demonstrator that decides whether to share a reward. We revealed that observer mice were able to perform the SDM without training, compared to their demonstrator at start. Observational learning was similar in males and females. Observer mice did not imitate their models rather they developed a typical strategy or preference. At the neural level, we found that the dorsal CA1 area of the hippocampus (dCA1) was fundamental for the acquisition by observational learning. Indeed, silencing of the dCA1, but not ventral CA1, considerably slowed the establishment of a clear social preference despite previous observation. Furthermore, dCA1 silencing during the retrieval phase did not affect the social preference of the observer mice. Finally, we analyzed synaptic function and in vivo neural activity in dCA1 during observational learning to elucidate the role of this area in the formation of a social behavior through others’ observation. By clarifying the neural circuitry, this work might be of relevance for those brain diseases where observational learning is compromised with dramatic consequences on social cognition and social life.

  • Guiding synaptic plasticity toward specific features of visual functions

    Authors:
    Daniele Cangi (1,2), Giulia Palla (2,3), Matteo Caleo (2), Laura Restani (2), Claudia Alia (2)

    Organisations:
    1: Università di Firenze, Italy
    2: CNR, Pisa, Italy
    3: Scuola Normale Superiore, Pisa, Italy

    Presenting author: Daniele Cangi

    Exposition date: Friday, September 15, 2023

    Exposition position: 112/1

    This abstract is dedicated to the memory of Prof. Matteo Caleo

    After a focal damage in the cerebral cortex, areas in the territory of the lesion undergo a sudden loss of function which impacts dramatically on the physiology of these regions. Restoring functional connections implies a fine synaptic weights modulation of the network, but currently, there are no ways to modulate connection strength among specific populations of neurons.
    The aim of this work is to exploit spike-timing-dependent plasticity (STDP) rule to functionally guide the rearrangement of perilesional neural connections after a stab wound lesion in the primary visual cortex. We applied this strategy in head-fixed awake injured mice by coupling a specifically oriented visual stimulation with cortical optogenetic activation of V1. Mice were previously injected with an adeno-associated virus encoding for channelrhodopsin2 and were subjected to a pairing protocol for 2 weeks after the lesion. The protocol consisted of a daily visual stimulation of abrupt reversal square gratings, in which one hemicycle was coupled with optogenetic stimulation of cortical neurons, in phase with the physiological response of V1 neurons. During the same training session, visual stimulations for the orthogonal orientation were presented without optogenetic stimulation, as internal control.

    Evaluation of visual evoked potential (VEP) amplitudes after two weeks of training revealed a significant orientation preference toward the paired orientation that is exclusive for the hemicycle that was coupled with optogenetics. Longitudinal recordings confirmed long-lasting orientation preference up to 2 weeks after the ending of the training protocol, even at the level of multi-unit activity.

    In summary, this study provides a proof of principle that using STDP as a tool to guide synaptic plasticity is a potential way to restore lost function after a brain lesion.

  • Large-scale investigation of cell assembly coordination and network motifs in the mouse brain

    Authors:
    Fabrizio Londei (1,2), Giulia Arena (1,2), Francesco Ceccarelli (1), Lorenzo Ferrucci (1), Aldo Genovesio (1)

    Organisations:
    1: Department of Physiology and Pharmacology, Sapienza University, Rome, Italy
    2: PhD Program in Behavioral Neuroscience, Sapienza University, Rome, Italy

    Presenting author: Fabrizio Londei

    Exposition date: Friday, September 15, 2023

    Exposition position: 118/1

    Although first conceptualized more than 70 years ago, the idea of cell assemblies as functional units of the brain continues to attract research attention. Defined as sets of neurons that transiently and repeatedly organize into a register of temporally patterned activity, they are thought to support brain computations that represent cognitive and perceptual entities. In our study, we used Russo and Durstewitz's (2019) Cell Assembly Detection (CAD) algorithm to interrogate a publicly available dataset of neural recordings collected from more than 70 mouse brain regions (Steinmetz et al., 2018) to detail the coordinative nature of these areas. Specifically, we first focused on assemblies of two neurons, each belonging to a single area, that were also characterized by a delay in the activation of the member units, which could be considered a signature of directional information flow across areas. Such conditions, when applied to every possible 2-element combination of the available areas, revealed strong and interesting asymmetries in the ability to form assemblies with different directionality, suggesting heterogeneous information processing mechanisms. To explore network motifs, defined by Milo et al. (2002) as recurrent patterns of interaction between elements constituting different biochemical, ecological and neurobiological systems, we then examined assemblies composed of three neurons distributed over two areas, focusing on the "loop-like" motifs characterized by a reverberant coordination between the two areas involved in such interactions. Captivating results emerged by this analysis: for example, the endopiriform nucleus is capable of coordinating its activity in terms of pairs with several structures, but only when observed at the level of loop-like assemblies reveals a strong reverberant coordination that only interests the piriform cortex, which is reminiscent of the bidirectional connections it shares with this structure in the context of the olfactory system.

  • Memory-driven coding of objects in the monkey parieto-frontal grasping network

    Authors:
    Anna Mitola (1), Monica Maranesi (1), Luca Bonini (1), Marco Lanzilotto (2)

    Organisations:
    1: University of Parma, Italy
    2: University of Turin, Italy

    Presenting author: Anna Mitola

    Exposition date: Friday, September 15, 2023

    Exposition position: 116/1

    Parieto-frontal networks are thought to play a crucial role in coordinating the visuomotor transformations required to interact with objects and other subjects in the environment. In particular, several parietal and frontal areas have been shown to constitute an “extended grasping network” for transforming the physical features of graspable objects into the kinematic parameters of the hand and arm for reaching-grasping actions, which are continuously updated based on visual feedback. However, objects with known features can also be grasped in the dark, relying on memory- rather than sensory-motor transformation. Previous evidence suggests a role for posterior parietal cortex in memory-guided hand manipulation, but the relative contribution of distinct cell types within each area and of different areas within the network is still unknown.

    To address these issues, here we recorded the activity of single neurons from a set of interconnected parieto-frontal areas (AIP, F5, F2 and F6) from three macaques trained to perform a visuomotor grasping task in both light and dark conditions. In the grasping-in-the-dark condition, the three-dimensional features of the observed objects had to be temporarily stored in memory in order to shape the hand appropriately and to perform the grasping task correctly.

    By applying population decoding analyses and examining the stability of object coding over time in specific subpopulations of neurons within each area, we found that area AIP exhibits a clear and persistent tuning for the object across the different phases of memory-driven trials, area F6 exhibits a more sensory-driven and temporally specific coding, whereas F5 and F2 display intermediate tuning features.

    These results suggest that local and long-range neuronal mechanisms cooperate in exploiting the sensory- and memory-driven transformations of object physical properties in the appropriate motor plans for manual actions in primates.

  • Molecular changes underlying decay of sensory responses and enhanced seizure propensity in peritumoral neurons

    Authors:
    Marta Scalera (1), Elena Tantillo (1,2), Elisa De Santis (1), Nicolò Meneghetti (3,4), Chiara Cerri (5,6), Michele Menicagli (2), Alberto Mazzoni (3,4), Mario Costa (1,8,9), Chiara Maria Mazzanti (2), Eleonora Vannini (1,5,8), Matteo Caleo (1,7)

    Organisations:
    1: CNR, Neuroscience Institute, via G Moruzzi 1, 56124 Pisa
    2: Fondazione Pisana per la Scienza Onlus (FPS) via F. Giovannini 13, San Giuliano Terme, 56017 Pisa, Italy
    3: Department of Excellence for Robotics and AI, Scuola Superiore Sant’Anna, 56025 Pisa, Italy
    4: The Biorobotics Institute, Scuola Superiore Sant’Anna, 56025 Pisa, Italy
    5: Fondazione Umberto Veronesi, 20122 Milan, Italy
    6: Department of Pharmacy, University of Pisa, Pisa 56126, Italy
    7: Department of Biomedical Sciences, University of Padua, via G. Colombo 3, 35121 Padua
    8: Centro Pisano ricerca e implementazione clinica Flash Radiotherapy “CPFR@CISUP”, “S. Chiara” Hospital, Pisa, Italy
    9: Laboratory of Biology BIO@SNS, Scuola Normale Superiore, Piazza di Cavalieri 7, Pisa, 56124, Italy

    Presenting author: Marta Scalera

    Exposition date: Friday, September 15, 2023

    Exposition position: 110/1

    In recent years, the interaction between glioma and brain cells has emerged as one important regulator of tumor progression. In order to investigate the molecular and physiologic alterations of the peritumoral neurons induced by glioma growth, we used two well-established glioma syngeneic cell lines: GL261 and CT-2A. We performed in vivo electrophysiological recordings of visual evoked potentials (VEPs) to longitudinally monitor the neural dysfunctions along with tumor progression. With respect to controls, glioma-bearing (GBM) mice showed a decay of visual responses that started from day 14 after tumor induction. At this stage, we microdissected layer II-III pyramidal neurons and performed RNA-sequencing of a panel of genes involved in synaptic transmission. We found that only gabra1 and SNAP25 were significantly reduced in peritumoral neurons of glioma-bearing mice. These data were also confirmed by immunohistochemical analyses that showed a decrease in the level of gabra1 and SNAP25 proteins. Next, we performed LFPs recordings in freely moving control and GBM mice before and after the treatment with a subconvulsive dose of DMCM (an inverse benzodiazepine agonist that inhibits GABA currents by binding to the α subunits of the GABA-A receptor). We found that DMCM triggered epileptiform activity in both glioma murine models but not in controls, suggesting an involvement of the GABA-A receptor in seizures’ susceptibility. These data were also confirmed by the Racine Scale, where behavioural observations revealed the presence of seizures in GBM mice.

    In summary, these findings reveal novel molecular players in pyramidal neurons which may be targeted for preventing neuronal dysfunction and hyperexcitability in order to ameliorate patients’ quality of life.

  • Neuronal signals for self and other’s action in the monkey putamen

    Authors:
    Matilde Reni (1), Cristina Rotunno (1), Carolina Giulia Ferroni (2), Matteo Di Volo (3), Luca Bonini (1), Monica Maranesi (1)

    Organisations:
    1: Department of Medicine and Surgery, University of Parma, Parma, Italy
    2: Zardi-Gori Foundation and Vita-Salute University and San Raffaele Scientific Institute, Milan, Italy
    3: Stem Cell and Brain Institute, INSERM U846M, Lyon 1 University, Lyon, France

    Presenting author: Matilde Reni

    Exposition date: Friday, September 15, 2023

    Exposition position: 114/1

    It is widely accepted that an extended cortical Action Observation Network (AON) underlying the coding of other’s actions largely overlaps with areas devoted to action planning and execution. Recent anatomical data demonstrated that most of the cortical areas belonging to the AON send convergent projections to overlapping territories of the putamen in the basal ganglia, suggesting that it may play a role in the processing of other’s actions, in addition to one’s own.

    To address this issue, we recorded neuronal activity from the macaque putamen during a reaching-grasping task in which the animal grasped or observed the experimenter grasping a multi-affordance object in a shared operational space (Social condition).

    Some of the recorded neurons responded only during action execution (Self type) and some only during action observation (Other type), whereas the majority during both (Self-Other type). Facilitated activity prevailed during task execution while suppressed discharge balanced the facilitated one during task observation.

    Introducing a barrier that alternatively prevented one of the agents from interacting with the object, we observed an overall decrease in modulation amplitude with respect to the Social condition, but with similar percentages of facilitated and suppressed units.

    Local field potential (LFP) recorded together with spiking activity revealed an event-related desynchronization of the alpha/beta and the high beta/low gamma bands during action execution and observation, coupled with a synchronization of the delta/theta and high-gamma bands.

    Our findings support the existence of input and output neuronal signals related to others’ manual actions in the putamen, providing functional evidence to the hypothesized involvement of the putamen in the AON.

  • Shaping the spikes: oxytocinergic modulation of action potentials in the CA1 hippocampal region of mice

    Authors:
    Antonio Nicolas Castagno (1), Paolo Spaiardi (1,2), Arianna Trucco (1), Claudia Maniezzi (3), Francesca Raffin (1), Maria Mancini (4), Alessandro Nicois (5), Jessica Cazzola (1), Paola del Papa (1), Matilda Pedrinazzi (1), Antonio Pisani (4,6), Francesca Talpo (1), Gerardo Rosario Biella (1,2)

    Organisations:
    1: Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, Italy
    2: Istituto Nazionale di Fisica Nucleare, Sezione di Pavia, Pavia, Italy
    3: Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy
    4: Department of Brain and Behavioral Sciences, University of Pavia, Pavia, Italy
    5: Institute of Biomolecular Chemistry (ICB), National Research Council (CNR), Pozzuoli, Italy
    6: Neurological Institute Foundation Casimiro Mondino (IRCCS), Pavia, Italy

    Presenting author: Antonio Nicolas Castagno

    Exposition date: Friday, September 15, 2023

    Exposition position: 117/1

    Oxytocin (OT) is a small hypothalamic neuropeptide that in recent years has emerged as a crucial modulator of social-related behaviour and cognition in the central nervous system. While its behavioural effects have been widely investigated, the OT neuromodulation exerted on neurons remains still not completely clarified as its effects could be remarkably different depending on the brain area and neuronal type on which OT acts. It has been demonstrated that the neurons of each subregion of the hippocampus could be differently modulated by OT, thus presumably regulating the information flow within the hippocampus. For example, in hippocampal CA1 field OT can directly depolarize the GABAergic interneurons (INs) and thus indirectly inhibit the projecting pyramidal neurons (PYRs). Here we demonstrate that TGOT, a selective agonist for the OT receptor, could affects not only the membrane potential and firing frequency but it can also modify the properties of action potentials (APs) of the INs (but not of the PYRs) in CA1 hippocampal region of mice. To find out the functional meaning of these modifications, we built linear mixed-effects models. This method allows to highlight the influences of the TGOT on the different parameters of AP and how they could affect the firing frequency variation observed during drug perfusion. These data provide evidence that the OT action on neurons is not restricted to changes of the membrane potential but causes significant changes in the shape of spikes. Altogether these data suggest that the oxytocinergic modulation of the hippocampal circuit could affect the processing of the information in the different hippocampal subregions and the signal progression to the downstream targets.

  • The Case of Octopus vulgaris: exploring neural hallmarks for consciousness

    Authors:
    Marianna De Luca (1,2), Joachim Schmidt (3), Cinzia Chiandetti (2), David B. Edelman (1), Giovanna Ponte (2,4), Graziano Fiorito (2,4)

    Organisations:
    1: Association for Cephalopod Research (CephRes)-ETS, Italy
    2: Dipartimento Scienze della Vita, Università degli studi di Trieste, Italy
    3: Institut für Zoologie, Universität zu Köln, Cologne, Germany
    4: Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, Naples, Italy

    Presenting author: Marianna De Luca

    Exposition date: Friday, September 15, 2023

    Exposition position: 119/1

    Is there sufficient evidence to make a strong case for a form of sensory consciousness in cephalopod molluscs? Over the past two decades, such a case has been made on a number of occasions by researchers across a variety of disciplines. Here we will survey evidence drawn from current developmental, anatomical, physiological, and behavioral studies and attempt to build the most systematic and substantive case yet for cephalopod consciousness.

    Recent discoveries suggest that cephalopod nervous system includes some of the same genetic innovations that characterized the rise of complex brains in the vertebrates (e.g., expansion of the protocadherin gene family, large number of orphan genes, existence of active retrotransposons). These and other genomic and morphological novelties, as well as recent revelations regarding cognitive abilities in cephalopods – mirroring those of higher vertebrates - further bolster the argument we make here: namely that cephalopod molluscs are fully capable of conscious experience, albeit a variety that is markedly different from the sort of subjectivity experienced by humans and other vertebrates.

    We will also review recent technical improvements in the analysis of neural correlates of brain activity in the octopus, and will present our recent findings in assessing spontaneous brain activity in O. vulgaris in the search of the neural hallmarks of consciousness in these animals.

  • Whole-brain functional network topology for representing imprinted odor cues

    Authors:
    Alessandra Stella (1,2), Stefano Zucca (1,2), Fabrizio Pizzagalli (3), Paolo Peretto (1,2), Serena Bovetti (1,2)

    Organisations:
    1: Dept. of Life Sciences and System Biology, University of Turin, Turin, Italy
    2: Lab. of Adult Neurogenesis, Neuroscience Institute Cavalieri Ottolenghi, University of Turin, Orbassano, Italy
    3: Dep. Of Neuroscience "Rita Levi Montalcini", University of Turin, Turin, Italy

    Presenting author: Alessandra Stella

    Exposition date: Friday, September 15, 2023

    Exposition position: 115/1

    Sexual imprinting is a form of early-life learning that allows individuals to acquire memories of sensory cues, which are then used to guide mate choice in adulthood1-2. However, the neural circuits underlying sexual imprinting remain poorly understood, particularly in complex networks such as the mouse brain. To investigate this issue, we used an interdisciplinary approach to investigate how imprinted (familiar) and novel (unfamiliar) sensory cues are represented in the mouse brain and whether resulting functional networks exhibit hubs, small-world, or rich club topology3.

    Using a combination of iDISCO tissue clearing and light-sheet fluorescence microscopy, we imaged cFos-stained neurons and evaluated the number of cFos-positive cells and their fluorescence intensity in all brain areas using the ClearMap software4,5. We interpreted the number of activated cells as a proxy for the degree of activation of a given area during odor presentation, and we defined a second measure, called energy6, which weighted the number of activated cells by their average fluorescence intensity.

    We calculated correlations across areas and assessed differences between conditions and mouse strains. We identified functional subnetworks by constructing undirected graphs of brain areas that were differentially activated across experimental groups, and analyzed the graph topology to identify areas coordinating as hubs in response to the olfactory cues.

    Our results suggest that amygdalar and hypothalamic areas may be crucial in the neural circuits involved in sexual imprinting in the mouse brain. Our interdisciplinary approach, combining tissue clearing, advanced imaging techniques, and network analysis, offers a promising avenue for future studies of complex neural systems.

    References

    1. Yamazaki, K. et al. J., Exp. Med. 1976.

    2. Asaba, A. et al. T., Front. Neurosci. 2014.

    3. Watts and Strogatz, Nature. 1998.

    4. Renier, N. et al., Cell, 2014.

    5. Renier, N. et al., Cell, 2016.

    6. Lupori, L. et al., biorXiv, 2023.

Neuronal excitability

  • Neuronal hippocampal hyperexcitability and striatal synaptic dysfunctions in a mouse model of Lafora disease

    Authors:
    Alessandro Tozzi (1), Laura Bellingacci (1), Jacopo Canonichesi (1), Alessia Russo (1), Martina Geusa (1), Lorenzo Fedrizzi (1), Giulia Di Giorgio (1), Massimiliano Di Filippo (1), Miriam Sciaccaluga (1,2), Cinzia Costa (1)

    Organisations:
    1: Department of Medicine and Surgery, University of Perugia, Italy
    2: Fondazione Malattie Rare Mauro Baschirotto BIRD Onlus, Longare (VI), Italy

    Presenting author: Alessandro Tozzi

    Exposition date: Friday, September 15, 2023

    Exposition position: 120/1

    Lafora disease (LD) is a rare, fatal, recessive form of progressive myoclonic epilepsy caused by mutations in the EPM2A or EPM2B genes, which encode for laforin or malin, causing the formation of aberrant polyglucosane aggregates (Lafora bodies) in the brain and other tissues, which appear to be the main cause of disease progression. The pathophysiological mechanisms underlying LD are still poorly understood.

    In this work, we performed an electrophysiological characterization of a new genetic model of LD, a knock-in (KI) model for a mutation in the gene encoding for laforin, present in some patients (Epm2aR240X). Through ex-vivo electrophysiological recordings, we aimed at identifying possible alterations of intrinsic membrane properties, neuronal excitability, and synaptic transmission and plasticity in two different brain regions particularly involved in the mechanisms of epileptogenesis and synaptic plasticity, the hippocampus ad the nucleus striatum.

    Our results show that Epm2aR240X KI mice exhibit hyperexcitability and synaptic dysfunction in a subregion of the hippocampus, as well as impaired cortico-striatal transmission and plasticity.

    Based on these data, we suggest that the Epm2aR240X KI mouse model may be suitable to explore the pathogenetic relevance of neuronal hyperexcitability and of abnormal synaptic plasticity for Lafora disease. Subsequent experiments will focus on understanding the mechanisms underlying these alterations and will include pharmacological treatments exploring the role of glutamatergic, dopaminergic and endocannabinoid receptors.

Neurotransmission and signal transduction

  • A study of chemical neuroanatomy on the neurotensinergic system in the human cerebellar cortex

    Authors:
    Paolo Flace (1,2), Diana Galletta (3), Demetrio Milardi (4), Vincenzo Coviello (2), Giulio Fata (2), Rosanna Mallamaci (5), Andrea Marzullo (6), Antonella Bizzoca (7), Gianfranco Gennarini (7), Angelo Quartarone (8)

    Organisations:
    1: Medical School University of Bari 'Aldo Moro', Italy
    Hospital Structures of Universo Salute Opera Don Uva, Bisceglie, Italy
    2: Hospital Structures of Universo Salute Opera Don Uva, Bisceglie, Italy
    3: Unit of Psychiatry and Psychology, Federico II University Hospital, Naples, Italy
    4: Department of Biomedical, Dental Sciences and Morphological and Functional Images, University of Messina, Messina, Italy
    5: Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari ‘Aldo Moro’, Bari, Italy
    6: Section of Molecular Pathology, Department of Precision and Regenerative Medicine and Ionian Area (DiMePRe-J), University of Bari ‘Aldo Moro’, Bari, Italy
    7: Department of Translational Biomedicine and Neuroscience “DiBraiN”, University of Bari ‘Aldo Moro’, Bari, Italy
    8: IRCCS Centro Neurolesi ‘Bonino Pulejo’, Messina, Italy

    Presenting author: Paolo Flace

    Exposition date: Friday, September 15, 2023

    Exposition position: 121/1

    Neurotensin (NT) is a neuropeptide largely distributed in the mammals nervous systems. NT together dopamine (DA) play a considerable role in neuromodulation mechanisms. Studies indicate a role of NT in DA-related disorders. In the cerebellum no data are available on the presence of intrinsic neurotensinergic neurons, and only the neurotensin receptor subtypes NTR2 and NTR3 has been detected. Therefore, the aim of this study, was to evaluate in the human cerebellar cortex, the presence of an intrinsic neuronal neurotensinergic system. The study was carried out on fragments of postmortem human cerebellar cortex 36-48h after death. Each fragment were fixed in an picric acid and aldehyde solution, embedded in paraffin, cut into 5 μm sections, and subjected to light microscopy immunohistochemical procedures using rabbit and goat polyclonal antibodies respectively against NT and NTR1. For positive controls fragments of rat intestine subjected to the same experimental procedure were used. In the human cerebellar cortex, NT and NTR1 immunoreactivity were observed in the molecular layer, in subpopulations of stellate and basket neurons; in the Purkinje neuron layer, in a subpopulation of Purkinje neurons; in the granular layer, in a subpopulation of granules and Golgi neurons and in some non-traditional large neurons (i.e. candelabrum, synarmotic, perivascular neurons). NTR1 immunoreactivity was also observed in form of fine ‘puncta’ (putative axon terminals) in the neuropil and in close relationship to the wall of microvessels. This study demonstrate in the human cerebellar cortex the presence of a neurotensinergic neuronal system, may involved in neuromodulation of associative and projective circuits, in microvascular innervation. Moreover, we plan to carry out further studies to evaluate the co-expression level of the corticocerebellar neurotensiergic system with the cerebellar dopaminergic system and its possible role in DA-related disorders such as Parkinson's disease and schizophrenia.

  • Peculiar D2 dopamine receptor localization in pancreatic β-cells

    Authors:
    Marco Carli (1), Edoardo Ferrero (1), Letizia Biso (1), Michela Novelli (1), Vincenzo De Tata (1,2), Marco Scarselli (1)

    Organisations:
    1: Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Pisa, Italy
    2: CIME (Centro Interdipartimentale di Microscopia Elettronica), University of Pisa, Pisa, Italy.

    Presenting author: Marco Carli

    Exposition date: Friday, September 15, 2023

    Exposition position: 122/1

    Pancreatic β-cells respond to dopamine and are equipped with a complete dopaminergic apparatus, such as dopamine receptors (D1-D5) and enzymes for dopamine production (tyrosine hydroxylase and aromatic amino-acid decarboxylase) and degradation. In β-cells, dopamine is stored in insulin vesicles via vesicular monoamine transporter 2 (VMAT2) and co-released with insulin. β-cells also express transporters for dopamine (DAT) and its amino-acids precursors (LAT1 and 2). In a pancreatic β-cell line (INS 832/13), we confirmed the expression and function of D2 receptor, while no D1 was detected. D2 receptors stimulated with dopamine and dopamine agonists activate Gi proteins and inhibit the release of insulin stimulated by glucose (GSIS) and other secretagogues (IBMX and KCl). The inhibition of insulin secretion by L-DOPA demonstrates that L-DOPA is taken by LAT and decarboxylated to dopamine, which is in turn loaded into the insulin granules via VMAT2 and then secreted. The inhibition of VMAT2 with tetrabenazine generated a significant increase in ROS production and oxidized glutathione levels, demonstrating that cytosolic permanence of dopamine results in oxidative stress. By means of immunofluorescence and immunogold labeling, we demonstrated that D2 receptors are located within the membrane of insulin granules while they are not detectable at the plasma membrane level. D2 dopamine binding sites face the lumen of the granule, because when the granule fuses with the plasma membrane, D2 receptors are transiently exposed to the extracellular space and then activated. The unique of its kind D2-VMAT2 co-localization within insulin granule membranes poses some questions on the physiology and regulation of the activity of this system. We aim to understand how dopamine can be stored in granules expressing its receptors and if D2 receptors are active or inactive intracellularly, and if such organization can be found in other cell lines producing dopamine.

  • Presynaptic 5-HT2A-mGlu2/3 Receptor-Receptor functional interaction in the rat Prefrontal cortex: Metamodulation of Glutamate Exocytosis.

    Authors:
    Alice Taddeucci (1), Guendalina Olivero (1), Alessandra Roggeri (1), Veronica Torre (1), Claudio Milanese (2), Beatrice Garrone (2), Anna Pittaluga (1,3)

    Organisations:
    1: Department of Pharmacy, DiFAR, Pharmacology and Toxicology Section, University of Genoa, 16148, Genova, Italy
    2: Angelini Pharma S.p.A., Rome, Italy 16148, Genoa, Italy
    3: IRCCS Ospedale Policlinico San Martino, 16145, Genova, Italy

    Presenting author: Alice Taddeucci

    Exposition date: Friday, September 15, 2023

    Exposition position: 123/1

    The glutamatergic nerve terminals of a rat prefrontal cortex (PFc) have presynaptic 5-HT2A heteroreceptors and mGlu2/3 autoreceptors. These receptors have been shown to functionally interact, resulting in an integrated 'metamodulation' of neuronal transmission at the synaptic level. It has been demonstrated that mGlu2/3 and 5-HT2A receptors are also physically associated at the presynaptic level in isolated nerve terminals (synaptosomes) of rat spinal cord, where they functionally interact in an antagonistic fashion to modulate glutamate exocytosis. Because alterations in this receptor-receptor interaction have been associated with the development of psychosis and/or altered responsiveness of schizophrenic patients to antipsychotics, our study aims to verify the existence and their reciprocal role of 5HT2A and mGlu2/3 receptors in in synaptosomes isolated from rat cortex. Synaptosomes were preloaded with [3H]D-aspartate ([3H]D-asp) to monitor glutamate exocytosis elicited by 15 mM KCl-containing medium. These release experiments were carried out to verify the impact of 5HT2A and mGlu2/3 ligands on this event. (±) DOI (0.1-30 µM) as well as LY379268 (0.1-1 µM) concentration-dependently inhibited the 15 mM KCl-evoked [3H]D-asp exocytosis; the former effect was abolished by the 5HT2A antagonist MDL11,939 (1 µM). When concomitantly activated, however, a significant reduction of the tritium exocytosis was not observed. Conversely, blockade of the 5HT2A receptors with MDL11,939 amplified the LY379268-mediated inhibition of tritium exocytosis. The presence of the two receptors in PFcx synaptosomes was confirmed with WB analysis. Later, synaptosomal lysates were immunoprecipitated with anti-5HT2A antibodies: the immunoprecipitates were immune-positive for the 5HT2AR but not for the mGlu2/3 receptors. Similarly, mGlu2/3R proteins were immunoprecipitated with an anti-mGlu2/3R antibody: the immuno-precipitates were positive for mGlu2/3 but not for 5HT2A. Experiments carried out suggest that the 5HT2A and the mGlu2/3 receptors colocalize, but do not physically associate, in PFc glutamatergic terminals, where they functionally interact in an antagonist-like fashion to control glutamate exocytosis.

Parkinson’s disease and other movement disorders

  • A transgenic zebrafish line expressing human alpha-synuclein to study the biological basis of synucleinopathies

    Authors:
    Silvia Zini, Alessia Muscò, Francesca Longhena, Gaia Faustini, Giuseppe Borsani, Daniela Zizioli, Arianna Bellucci

    Organisations:
    University of Brescia, Brescia, Italy

    Presenting author: Silvia Zini

    Exposition date: Saturday, September 16, 2023

    Exposition position: 113/2

    Parkinson’s disease (PD), Lewy body dementia (LBD) and Multiple System Atrophy (MSA) are characterized by the progressive accumulation of alpha-synuclein (αSyn) insoluble aggregates affecting either neuronal or glial cells, and are thus defined as synucleinopathies (doi: 10.1093/brain/aww230). Compelling evidence supports that the deposition of αSyn aggregates plays a causative role in the onset of neuronal degeneration in these disorders (doi: 10.1111/nan.12297, doi:10.3390/genes8120377). Even though a plethora of experimental models reproducing the different features of human synucleinopathies have been developed the physiological and pathological role of αSyn still need to be fully disclosed (doi: 10.1002/mds.28387). In recent years, zebrafish arose as a valuable complementary model to study disorders of the nervous system, including PD (doi: 10.3389/fphar.2022.835827, doi: 10.1016/j.nbd.2021.105291).

    Here we describe the successful generation of a viable transgenic zebrafish line, ubiquitously expressing full length human αSyn with an N-terminal mCherry tag in the nervous system under the guidance of zebrafish HuC promoter by exploiting the Tol2 transgenesis system Tg(HuC:mCherry/hSNCA). The transgenic animals reached adulthood and as expected their progeny exhibited mCherry fluorescence signal in central and peripheral neurons from early developmental stages. The co-expression and co-localization of mCherry and αSyn was verified through PCR, western blot and immunofluorescence studies. White light routine behavioral test on the mCherry-positive larvae at 5 days post fertilization (5dpf) did not evidence major alterations when compared to non-fluorescent littermates at this developmental stage.

    These results hint that the novel Tg(HuC:mCherry/hSNCA) zebrafish represents a promising complementary model to study the physiological and pathological role by in vivo imaging. Moreover, this novel zebrafish line may open the possibility to test the efficacy of new αSyn-targeted therapies by large screening analysis.

  • Bioenergetic and lysosomal defects in blood-derived GBA-Parkinson’s disease macrophages

    Authors:
    Gerardo Ongari (1), Cristina Ghezzi (1), Micol Avenali (2,4), Antonio Pisani (3,4), Fabio Blandini (5), Silvia Cerri (1)

    Organisations:
    1: IRCCS Mondino Foundation, Unit of cellular and molecular neurobiology, Pavia, Italy
    2: IRCCS Mondino Foundation, Neurorehab ilitation Unit, IRCCS Mondino Foundation, Pavia, Pavia, Italy
    3: IRCCS Mondino Foundation, Movement disorders, Pavia, Italy
    4: University of Pavia, Department of Brain and Behavioural Sciences, Pavia, Italy
    5: IRCCS Policlinico Maggiore Hospital, Scientific Director, Milano, Italy

    Presenting author: Silvia Cerri

    Exposition date: Saturday, September 16, 2023

    Exposition position: 123/2

    Glucocerebrosidase 1 (GBA1) mutations are the most important risk factor for Parkinson’s disease (PD), which primarily affect macrophages due to their intrinsic features and vulnerability for disturbances in ceramide metabolism. While it is known that GBA-associated lysosomal dysfunctions can trigger the inflammatory response in macrophages, no studies explored the effects of GBA1 mutations on mitochondrial metabolism and oxidative stress in these cells.

    We then investigated mitochondrial dysfunction in macrophages of patients with GBA mutations, in association with lysosomal alterations and α-synuclein accumulation.

    Thirty-four patients were recruited in the study: 10 controls (HC), 10 patients with sporadic PD (iPD), 10 PD patients with GBA mutations (PD-GBA+) and 4 healthy subjects carrying GBA mutations (HC-GBA+). Macrophages were differentiated from monocytes and stimulated to M1 (pro-inflammatory) and M2 (anti-inflammatory) phenotypes. Glucocerebrosidase and β-hexosaminidase activities were quantified using fluorometric assays. Metabolic characterization was performed with a Seahorse Analyzer. A-synuclein levels were quantified using an ELISA.

    As expected, glucocerebrosidase activity was halved in PD-GBA+ and HC-GBA+ subjects compared with HC and iPD, whereas β-hexosaminidase activity was reduced in PD-GBA+ compared to HC. Lysosomal alterations in PD-GBA+ is associated with increased α-synuclein levels, compared with iPD and HC macrophages. HC-GBA+ subjects showed α-synuclein levels similar to PD-GBA+.

    M2 stimulated PD-GBA+ macrophages displayed an overall impairment in the oxygen consumption compared to iPD, while no differences have been found between HC and iPD. Conversely, M1 macrophages of all PD groups showed a decrease of respiratory parameters compared to HC, reaching the statistical significance in PD-GBA+. Very preliminary results in HC-GBA+ indicated higher respiration values both in M1 and M2 macrophages compared to PD-GBA+.

    In conclusion, this study pointed out specific bioenergetic alterations in macrophages from PD subjects with GBA1 mutations suggesting their potential contribution in disease pathogenesis.

  • Blocking the aerobic/energy source in neurons via SGLTs inhibits /delaying Parkinson’s disease biomarker α-synuclein transmission/ propagation

    Authors:
    Ahmed Tanvir

    Organisations:
    Center for Convergence Research of Neurological Disorders, Ajou University, School of of Medicine, Korea, Republic of (South Korea)

    Presenting author: Ahmed Tanvir

    Exposition date: Saturday, September 16, 2023

    Exposition position: 111/2

    Progressive loss of dopaminergic neurons in the substantia nigra (SN) pars compacta (SNpc) and the formation of cytoplasmic inclusions containing misfolded α-synuclein (α-syn), Lewy bodies (LBs) like characteristics are two major neuropathological hallmarks of Parkinson Disease (PD) Which is the second most common neurodegenerative disease. PD and Diabetic mellitus (DM) share potential contributing factors and have overlapping pathology. Studies have unveiled a crucial connection between PD and DM. DM, distinguish by impaired glucose metabolism and subsequent hyperglycemia. The elevated risk of developing cognitive abnormalities in individuals with impaired glucose metabolism has been well documented. Hyperglycemia, a common pathogenesis in T2DM, has been shown to contribute to the onset of α-syn pathology in neurons and oligodendrocytes. Phosphorylated α-syn inclusions have been found in pancreatic β cells of T2DM subjects, indicating the existence of PD-related peripheral pathology in DM. But due to the complicated effect of glucose transporter on PD induced dopaminergic degeneration still not well understood. Here, we demonstrate that A53T mutated cell and α-syn fibrils induce active glucose energy transporter SGLTs activation in neurons which increased the uptake of α-syn into neurons. Furthermore, the inhibition of SGLTs inhibits the α-syn uptake and the formation of lewy body-like inclusions. These results demonstrated a new therapeutic approach for PD.

  • Changes in PD neuromuscular control of locomotion after deep brain stimulation

    Authors:
    Marco Ghislieri (1,2), Michele Lanotte (3,4), Marco Knaflitz (1,2), Laura Rizzi (3,4), Valentina Agostini (1,2)

    Organisations:
    1: Department of Electronics and Telecommunications, Politecnico di Torino, 10129, Turin, Italy
    2: PolitoBIOMed Lab, Politecnico di Torino, 10129, Turin, Italy
    3: Department of Neuroscience “Rita Levi Montalcini”, University of Turin, 10126, Turin, Italy
    4: AOU Città della Salute e della Scienza di Torino, 10126, Turin, Italy

    Presenting author: Marco Ghislieri

    Exposition date: Saturday, September 16, 2023

    Exposition position: 124/2

    High-frequency Deep Brain Stimulation (DBS) of the SubThalamic Nucleus (STN) has been demonstrated as an effective treatment for advanced PD patients, leading to a good control of all the PD cardinal symptoms and the drug-induced motor complications. To date, no studies have investigated how PD patients’ neuromuscular control can be possibly improved after STN-DBS. In this perspective, the aim of this study is to quantitatively and non-invasively assess PD neuromuscular control changes after bilateral STN-DBS, based on a novel muscle synergy evaluation approach. A group of 20 PD patients evaluated at baseline (before surgery, T0), at 3 months (T1), and at 12 months (T2) after STN-DBS surgery, as well as a group of 20 age- and gender-matched healthy control subjects, underwent an instrumented gait analysis, including surface electromyography (sEMG) recordings from 12 lower-limb and trunk muscles. Muscle synergies were extracted from the sEMG signals by applying the Non-Negative Matrix Factorization (NNMFA) algorithm. A smaller number of muscle synergies was found in PD patients (4 muscle synergies, at each time point) compared to control subjects (5 muscle synergies), suggesting an overall reduction in the complexity of the motor control system often associated with poorer motor performance. The neuromuscular robustness of PD patients - that at T0 was smaller with respect to controls (PD T0: 69.3±2.2% vs. Controls: 77.6±1.8%, p=0.004) - increased at T1 (75.8±1.8%), becoming not different from that of controls at T2 (77.5±1.9%). From the clinical point of view, an enhanced robustness (less variable motor control along the walking task) can be hypothesized to have an impact in decreasing the fall risk of PD patients. The muscle synergies analysis may offer clinicians new knowledge on the neuromuscular structure underlying PD motor types of behavior and how they can improve after electroceutical STN-DBS therapy.

  • Early gut microbiota dysbiosis in c-Rel deficient mice, a model of Parkinson’s disease

    Authors:
    Edoardo Parrella (1,2), Sara Roversi (3), Erika Scaltriti (4), Mattia Domenichini (3), Vanessa Porrini (1), Michele Mario Gennari (1), Marina Benarese (1), Simona Fiorentini (3), Marina Pizzi (1)

    Organisations:
    1: Division of Pharmacology, Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy
    2: Dipartimento di Ingegneria per la Medicina di Innovazione, University of Verona, Verona, Italy
    3: Division of Microbiology, Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy
    4: Risk Analysis and Genomic Epidemiology Unit, Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia-Romagna, Parma, Italy

    Presenting author: Edoardo Parrella

    Exposition date: Saturday, September 16, 2023

    Exposition position: 120/2

    Parkinson's disease (PD), the most common neurodegenerative movement disorder, is characterized by dopaminergic nigrostriatal neuron loss and α-synuclein accumulation. Besides classical motor symptoms, PD patients can manifest a broad spectrum of non-motor symptoms, including constipation, that usually occur years before the onset of motor symptoms. In accordance with the early onset of gastrointestinal dysfunction, the enteric nervous system (ENS) is among the structures earliest and most frequently affected by α-synuclein pathology. Recent research has shown that gut microbiota is altered in PD patients and could be involved in the disease onset and progression interacting with ENS.

    We previously showed that male mice lacking the NF-kB/c-Rel subunit (c-rel-/- male mice) reproduce a pattern of ascending α-synuclein deposition in the olfactory bulbs and brainstem nuclei. The pathology progression is accompanied by the appearance of both non-motor and motor symptoms, including hyposmia at 2 months, anxiety- and depressive-like behaviour at 12 months, and motor deficits at 18 months. Of note, c-rel-/- male mice display early and progressive intestinal α-synuclein accumulation and inflammation starting at 2 months, paralleled by reduced colon motility.

    Here we determined whether gut dysfunction and pathology in c-rel-/- male mice were coupled with an altered microbiota composition. Stool pellets collected from wt and c-rel-/- male mice at early (0.5 and 1 months) and later (6-8 and 18-20 months) phases of the pathology were subjected to culturomics and Next Generation Sequencing (NGS) analyses. The complementary culturomics and metagenomic approaches indicated a remarkable difference between wt and c-rel-/- male mice particularly at 0.5 months. Indeed, young c-rel-/- male mice displayed a pro-inflammatory microbiota characterized by the reduction of Verrucomicrobiacae and Enterobacteriaceae bacteria families, and the increase of Prevotellacae. This microbiota profile, deriving from young c-rel-/- mice in an extremely early phase of the pathology, may be involved in the pathogenesis of the disease.

  • Evaluation of novel pharmacological chaperones targeting α-synuclein-Glucocerebrosidase interplay in Parkinson’s disease

    Authors:
    Antonino Iurato La Rocca (1), Elisabetta Gerace (1), Giuseppe Ranieri (1), Lorenzo Curti (1), Valentina Ferrara (1), Francesca Clemente (2), Camilla Matassini (2), Francesca Cardona (2), Andrea Goti (2), Rodolfo Tonin (1,3), Amelia Morrone (1,3), Guido Mannaioni (1), Alessio Masi (1)

    Organisations:
    1: Department of Neuroscience, Psychology, Drug Research and Children's Health (NEUROFARBA), University of Florence
    2: Department of Chemistry "Ugo Schiff," University of Florence
    3: Molecular and cellular biology laboratory, pediatric neurology unit and laboratories, Meyer Children's Hospital, Florence, Italy.

    Presenting author: Antonino Iurato La Rocca

    Exposition date: Saturday, September 16, 2023

    Exposition position: 114/2

    Background: Parkinson's disease (PD) is characterized by the formation of proteinaceous aggregates of α-synuclein (α-syn) in cell bodies and neurites of Substantia Nigra pars compacta (SNpc) dopaminergic neurons, eventually causing cell death. Interestingly, defective activity or mutations in GBA1, the gene encoding the lysosomal enzyme Glucocerebrosidase (GCase), are the strongest risk factor to develop PD. Based on this evidence, reverting the cytological and molecular abnormalities caused by GCase mutations or enhancing the activity of the wild type enzyme may represent a feasible therapeutic strategy to oppose α-syn accumulation in PD.

    Aim: To evaluate the safety and pharmacokinetic profile of the GCase enhancer CF30 in vivo and assess its ability to: (1) enhance GCase activity, (2) prevent/oppose the accumulation α-syn aggregates in cortico-striatal organotypic slices.

    Methods: Chaperone-induced GCase enhancement and protective efficacy was assayed with biochemical and histological analyses on cortico-striatal organotypic slices treated with oligomers of α-syn.

    Results: CF30 is well tolerated at effective concentration/dosage in cell lines and in vivo and exhibits favourable pharmacokinetic profile with good oral availability and brain penetrance. GCase activity is enhanced by 40 and 18% in vitro and in vivo, respectively. On cortico-striatal organotypic slices we have evaluated the protective action of GCase enhancement against the neurotoxic effects of oligomers.

  • Intensive exercise training counteracts nigrostriatal degeneration and striatal structural changes in an alpha-synuclein based experimental model of Parkinson’s disease

    Authors:
    Federica Servillo (1), Maria De Carluccio (1,2), Gioia Marino (1), Federica Campanelli (1), Giuseppina Natale (1), Veronica Ghiglieri (1,3), Maria Teresa Viscomi (1,4), Paolo Calabresi (1,4)

    Organisations:
    1: Facoltà di Medicina e Chirurgia, Università Cattolica del Sacro Cuore, Rome, Italy
    2: IRCCS San Raffaele Roma, Roma, Italy
    3: Department of Human Sciences and Quality of Life Promotion, Università Telematica San Raffaele, Rome, Italy
    4: Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Rome, Italy.

    Presenting author: Federica Servillo

    Exposition date: Saturday, September 16, 2023

    Exposition position: 126/2

    Parkinson's disease (PD) is characterized by the formation of Lewy body aggregates, and by dopaminergic neuronal loss. Clinically, PD is associated with motor slowing, rigidity, and tremor. Intensive physical exercise has beneficial effects on PD patients at early stages, but the underlying mechanisms are not completely understood. In this study, we investigated whether an intensive treadmill training program, started at an early phase, counteracts nigrostriatal neurodegeneration in intrastriatal alpha-synuclein (α-syn) preformed fibrils (PFFs)-injected rats. To evaluate the effects of treadmill training on nigrostriatal morpho-functional changes, we assessed the survival of dopaminergic neurons and the functional integrity of their terminals in the striatum, by counting tyrosine hydroxylase (TH)-positive neurons of the SNpc and the expression level of the dopamine active transporter (DAT), respectively. Furthermore, we analyzed the structural plasticity of the postsynaptic compartments, namely the dendritic spine density of the striatal spiny projection neurons. Comparisons were made between sedentary and active α-syn-PFFs-injected animals in comparison to active control animals. Interestingly, in active parkinsonian animals we found an increase in the number of SNpc neurons coupled to a higher density of DAT+ terminal fibers in the dorsolateral striatum, compared with the sedentary α-syn-PFF-injected rats. Furthermore, active animals showed increased spine density in the striatal projection neurons with a greater proportion of young immature spines. These structural changes were also associated with a functional outcome as animals subjected to exercise training displayed better performances in motor coordination and visuospatial learning tests for which an intact striatal function is required. In conclusion, we demonstrate that intensive exercise training in parkinsonian animals, started at the presymptomatic stages of the disease, has effects in counteracting neurodegeneration of the nigrostriatal pathway. As a result, physical activity induces striatal adaptive responses to the α-syn-PFFs intoxication, associated with improved motor and cognitive performances compared to the parkinsonian sedentary group.

  • Loss of midbrain dopaminergic neurons elevates serine enantiomers levels in the caudate putamen and cerebrospinal fluid of Parkinson’s Disease patients.

    Authors:
    Anna Di Maio (1,2), Tommaso Nuzzo (1,2), Luana Gilio (3), Marcello Serra (4), Fabio Buttari (3), Francesco Errico (1,5), Arianna De Rosa (1,2), Mario Stampanoni Bassi (3), Micaela Morelli (4), Jumpei Sasabe (6), David Sulzer (7), Manolo Carta (4), Diego Centonze (3,8), Alessandro Usiello (1,2)

    Organisations:
    1: Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Università degli Studi della Campania "Luigi Vanvitelli", Caserta, Italy
    2: Laboratory of Translational Neuroscience, Ceinge Biotecnologie Avanzate Franco Salvatore, Naples, Italy
    3: Unit of Neurology, IRCCS Neuromed, Pozzilli (IS), Italy
    4: Department of Biomedical Sciences, University of Cagliari, Monserrato, Italy
    5: Department of Agricultural Sciences, University of Naples “Federico II”, Portici, Italy
    6: Department of Pharmacology, Keio University School of Medicine, Tokyo, Japan
    7: Departments of Psychiatry, Neurology, Pharmacology, Columbia University Irving Medical Center, Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, NY, USA
    8: Department of Systems Medicine, University of Rome Tor Vergata, Rome, Italy

    Presenting author: Tommaso Nuzzo

    Exposition date: Saturday, September 16, 2023

    Exposition position: 118/2

    Parkinson’s disease (PD) is traditionally characterized as a condition mainly affecting the dopaminergic system. However, a growing body of evidence suggests the involvement of other neurotransmitter systems, among which is the glutamatergic system. In particular, research conducted on experimental models and PD patients has highlighted profound alterations in the glutamatergic ionotropic N-methyl-D-aspartate (NMDA) receptors (NMDAR) signalling within the PD brain. In the mammalian forebrain, D-serine plays a critical role in regulating NMDAR activity by serving as a major NMDAR co-agonist. High levels of D-serine have been associated with either beneficial or detrimental effects depending on the neuropathological context; nonetheless, its involvement in PD remains unclear. To address this gap, in the present work we measured the levels of D-serine, of its precursor, L-serine, and of other amino acids involved in NMDAR function, namely L-glutamate, L-aspartate, D-aspartate, and glycine, in the post-mortem caudate putamen (CPu) and superior frontal gyrus (SFG) of PD patients and control subjects. Additionally, cerebrospinal fluid (CSF) samples from drug-free PD patients, Alzheimer's disease (AD) patients, amyotrophic lateral sclerosis (ALS) patients, and control subjects with other neurological disorders (OND) were examined. We found greater D-serine and L-serine levels in the CPu of PD patients but not in the SFG, a cerebral region that, in contrast to the CPu, is not innervated by nigral dopaminergic terminals. Moreover, we found a significant reduction in the striatal protein expression of the alanine serine cysteine transporter 1 (ASCT1), an astrocytic transporter that is involved in the shuttle of serine enantiomers between neurons and astrocytes. Notably, our results also highlighted a significant elevation of both serine enantiomers in the CSF of PD but not of AD and ALS patients, compared with OND controls. In conclusion, our findings identify D-serine and L-serine increases as disease-specific signatures associated with nigrostriatal dopaminergic degeneration in PD.

  • Lysosomal and alpha-synuclein alterations: predictive biomarkers in irbd-associated parkinson’s disease?

    Authors:
    Deborah Di Martino (1), Gerardo Ongari (1), Cristina Ghezzi (1), Laura Castrignanò (1), Micol Avenali (2,5), Michele Terzaghi (3), Antonio Pisani (4,5), Fabio Blandini (6), Silvia Cerri (1)

    Organisations:
    1: IRCCS Mondino Foundation, Unit of Cellular and Molecular Neurobiology, Pavia,Italy
    2: IRCCS Mondino Foundation, Neurorehabilitation Unit, Pavia, Italy
    3: IRCCS Mondino Foundation, Unit of Sleep Medicine and Epilepsy, Pavia,Italy
    4: IRCCS Mondino Foundation, Movement Disorders Research Center, Pavia, Italy
    5: University of Pavia, Department of Brain and Behavioural Sciences, Pavia, Italy
    6: IRCCS Policlinico Maggiore Hospital, Scientific Director, Milano, Italy

    Presenting author: Deborah Di Martino

    Exposition date: Saturday, September 16, 2023

    Exposition position: 121/2

    Idiopathic REM sleep behaviour disorder (iRBD) is one of the most important prodromal markers for Parkinson’s Disease (PD). Recent studies highlighted an increased incidence of iRBD in subjects with lysosomal disorders, e.g. subjects carrying GBA mutations. We demonstrated that lysosomal-associated alterations in peripheral blood mononuclear cells (PBMCs) provide a unique profile distinguishing PD patient with GBA mutations from non‐mutated PD and that GBA mutations, as well as sleep deprivation, are associated to an increased release of extracellular vesicles (EVs).

    In this project, we investigated the presence of lysosomal dysfunctions in subjects with RBD. Moreover, we quantified intracellular, plasmatic, and EV-associated levels of α-synuclein, which can accumulate as a consequence of lysosomal impairment, to identify potential features defining the risk to develop PD.

    We recruited 21 healthy controls, 19 iRBD subjects and 16 PD patients with RBD (PD-RBD). Plasma small EVs (or exosomes) were isolated by differential centrifugations. Lysosomal proteins’ levels (glucocerebrosidase, saposin C, LIMP-2 and cathepsin D) and glucocerebrosidase activity were evaluated in PBMCs through western blot and fluorimetric tests, respectively. Α-synuclein levels were quantified using ELISA assays.

    The results showed a significant reduction of LIMP-2 and saposin C levels in PD-RBD patients, but not in subjects with iRBD. Lysosomal alterations in PD-RBD group are accompanied by a slight accumulation of intracellular α-synuclein. Compared to controls, both PD-RBD and iRBD subjects showed an increased plasma exosome concentration associated with higher content of exosomal α-synuclein. Interestingly, a significant increase of free circulating α-synuclein is observed in plasma of iRBD subjects.

    This study provides preliminary evidence concerning lysosome and α-synuclein alterations associated with RBD, paving the way to the identification of biomarkers predicting the risk of conversion to PD and new targets for possible neuroprotective strategies.

  • Mitochondrial alterations in idiopathic REM-sleep disorder could help to predict Parkinson’s disease onset

    Authors:
    Gerardo Ongari (1), Cristina Ghezzi (1), Micol Avenali (2,5), Michele Terzaghi (3), Antonio Pisani (4,5), Fabio Blandini (6), Silvia Cerri (1)

    Organisations:
    1: IRCCS Mondino Foundation, Unit of cellular and molecular neurobiology, Pavia, Italy
    2: IRCCS Mondino Foundation, Neurorehab ilitation Unit, IRCCS Mondino Foundation, Pavia, Pavia, Italy
    3: IRCCS Mondino Foundation, Unit of Sleep Medicine and Epilepsy, Pavia, Italy
    4: IRCCS Mondino Foundation, Movement disorders, Pavia, Italy
    5: University of Pavia, Department of Brain and Behavioural Sciences, Pavia, Italy
    6: IRCCS Policlinico Maggiore Hospital, Scientific Director, Milano, Italy

    Presenting author: Gerardo Ongari

    Exposition date: Saturday, September 16, 2023

    Exposition position: 122/2

    Idiopathic REM sleep disorder (iRBD) represents the most important prodromal marker of Parkinson’s disease (PD). Nevertheless, only few studies investigated the mechanisms involved in the development of PD in iRBD subjects. Since the presence of mitochondrial dysfunctions have been linked to sleep disturbances in PD (Smith et al., 2018; Milanese et al., 2019), we explored mitochondrial alterations in fibroblasts of iRBD subjects, in order to characterize a biochemical predictive profile.

    The project involved 36 subjects divided in four groups: 10 healthy subjects, 10 subjects with iRBD, 8 iRBD subjects subsequently converted to PD (RBD-PD) and 8 patients who developed RBD after the diagnosis of PD (PD-RBD). Expression levels of mitochondrial proteins were evaluated by western blotting. The rate of mitochondrial respiration was evaluated using an XFe24 Seahorse Analyzer.

    The evaluation of the mitochondrial metabolism revealed a reduction in maximal and spare respiration in all groups with RBD, with and without PD. Furthermore, the RBD-PD subjects showed a decrease of ATP production. The impairment of mitochondrial function in RBD-PD and PD-RBD groups was associated with a decrease in the expression levels of mitochondrial complex I, for PD-RBD subjects, and III and V for RBD-PD. These alterations were associated with mitochondrial fragmentation. iRBD subjects showed similar but less severe alterations.

    These findings suggest that mitochondrial alterations observed in the fibroblasts of iRBD subjects may predispose to the worsening of the bioenergetic profile observed in RBD-PDs, indicating a potential mechanism underlying the progression of PD in iRBD patients.

  • Morphological and functional effects of a pro-resolving mediator in an α-synuclein rat model of Parkinson’s disease

    Authors:
    Francesca Cossa (1,2), Mariangela Massaro Cenere (1,2), Beatrice Zarrilli (1,2), Cecilia Giacomet (1,2), Mauro Federici (1), Vladimiro Batocchi (3), Ada Ledonne (1,2), Ezia Guatteo (1,4), Nicola Berretta (1), Valerio Chiurchiù (3,5), Nicola Biagio Mercuri (1,2)

    Organisations:
    1: Department of Experimental Neurology, IRCCS Santa Lucia Foundation, Rome, Italy
    2: Department of Systems Medicine, University of Rome Tor Vergata, Rome, Italy
    3: Laboratory of Resolution of Neuroinflammation, IRCCS Santa Lucia Foundation, Rome, Italy
    4: Department of Motor Science and Wellness, Parthenope University, Naples, Italy
    5: Institute of Translational Pharmacology, CNR, Rome, Italy

    Presenting author: Francesca Cossa

    Exposition date: Saturday, September 16, 2023

    Exposition position: 116/2

    Accumulation of α-synuclein (α-syn) in Lewy bodies and Lewy neurites is one of the main pathological signatures of Parkinson’s disease (PD).

    We have previously characterized the progressive functional and morphological neuronal alterations in a transgenic PD rodent model overexpressing the full-length human α-synuclein (SNCA+/+) compared to wild-type (WT) rats, at 4 and 12-13 months of age. Altered physiological properties of substantia nigra pars compacta (SNpc) dopamine (DA) neurons and neuroinflammation were observed in this rodent model. In particular, in the aged animals, the number of SNpc DA neurons was reduced, and the cells displayed hyperexcitability in response to depolarizing stimuli.

    To test whether these changes were associated with a neuroinflammatory condition, we investigated morphological and electrophysiological features in the same model chronically treated (twice a week for ten months) with Resolvin D1 (RvD1), a pro-resolving molecule for inflammation.

    RvD1 prevented SNpc TH-positive neuronal loss and increased soma size in 12-13 month-old SNCA+/+ rats.

    These findings prompted us to investigate the electrophysiological properties of DA neurons.

    In all experimental groups, only a few spontaneously firing cells were detected by multielectrode array (MEA) recordings in the SNpc, possibly because of the old age of the animals. No evident difference in basal firing was observed among the experimental groups.

    In patch-clamp recordings, although we did not observe any differences in Ih current and passive membrane properties, the neuronal hyperexcitability was reduced in the RvD1-treated SNCA+/+ rats compared to the vehicle-treated ones.

    In conclusion, RvD1 chronic treatment attenuated functional and morphological alterations in 12-13 month-old SNCA+/+ rats.

  • MT-POM: neuroprotective effect in a Drosophila model of Parkinson’s disease

    Authors:
    Maria Antonietta Casu (1), Ignazia Mocci (1), Dong Seok Kim (2), Nigel H. Greig (3), Maria Dolores Setzu (4), Anna Rosa Carta (4)

    Organisations:
    1: CNR-Institute of Translational Pharmacology, Cagliari, Italy
    2: Aevis Bio Inc., Daejeon, Republic of Korea
    3: Intramural Research Program, National Institute On Aging, NIH, MD, USA
    4: Department of Biomedical Sciences, University of Cagliari, Italy

    Presenting author: Maria Antonietta Casu

    Exposition date: Saturday, September 16, 2023

    Exposition position: 125/2

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by the selective loss of dopaminergic neurons and motor impairment. The pathogenesis of PD may involve both genetic susceptibility and environmental factors. Mutations in Leucine-rich repeat kinase 2 (LRRK2) locus contributes to genetic forms of PD1. The common fruit fly Drosophila melanogaster (Dm) carrying the mutation LRRK2 loss-of-function in the WD40 domain (LRRK2-Dm), is an in vivo model of PD that develops motor impairment, dopaminergic neuronal loss and damaged mitochondria2. Therefore, this model can be considered a valid tool to first test novel therapeutic approaches to the disease.

    Recent studies have identified several immunomodulatory agents with a neuroprotective effect in neurodegenerative disorders2-3. Here, in the LRRK2-Dm, we tested the novel pomalidomide derivative 3-monothiopomalidomide (MT-POM) that displayed increased potency in lowering TNF-alpha levels in in vitro tests. Changes in motor performance, number of brain dopaminergic neurons and mitochondria integrity were analyzed. Moreover, we studied the survival rate of LRRK2-Dm and wild type flies under life-long drug treatment.

    Mutant and wild type flies received 3 increasing MT-POM doses via diet from day 1 post eclosion, and efficacy was evaluated after 21 days. MT-POM significantly and dose-dependently improved climbing behavior respect to controls, prevented the loss of dopaminergic neurons and fully rescued damaged mitochondria with dilated cristae observed in non-treated LRRK2-Dm. Moreover, MT-POM significantly extended the survival of LRRK2-Dm.

    Our data indicate a neuroprotective effect of MT-POM in the LRRK2-Dm PD model, prompting further investigation of this drug in mammalian models of PD.

    Refs:

    1 West AB, et al., 2005

    2 Casu et al., 2020

    3 Martinez B, et al., 2019

  • NF-κB/c-Rel DNA-binding deficit as a potential biomarker for Parkinson’s disease

    Authors:
    Michele Mario Gennari (1), Vanessa Porrini (1), Andrea Pilotto (2), Marika Vezzoli (1), Edoardo Parrella (1), Marina Benarese (1), Gaia Faustini (1), Francesca Longhena (1), Arianna Bellucci (1), Angelo Antonini (3), Alessandro Padovani (2), Marina Pizzi (1)

    Organisations:
    1: Department of Molecular and Translational Medicine, University of Brescia
    2: Neurology Unit, Department of Clinical and Experimental Sciences, University of Brescia
    3: Parkinson and Movement Disorders Unit, Study Centre for Neurodegeneration (CESNE), Department of Neuroscience, University of Padua

    Presenting author: Michele Mario Gennari

    Exposition date: Saturday, September 16, 2023

    Exposition position: 115/2

    Despite the significant increase in the prevalence of Parkinson’s disease (PD) over the past three decades, no trustworthy biomarkers or disease-modifying therapies are available. Moreover, the molecular mechanisms underlying its onset are still largely unknown. In order to provide a better clinical management of PD patients, there is an urgent need to develop specific and sensitive biomarkers. Nuclear factor-κB (NF-κB) transcription factors family is a cardinal transcriptional regulator of inflammation and apoptosis that can contribute to the pathological processes associated with neurodegeneration. We previously reported that NF-κB/c-Rel deficient mice develop a late-onset parkinsonism, encompassing nigrostriatal degeneration, caudal-rostral α-synuclein deposition and accumulation of pro-apoptotic NF-κB/RelA acetylated at the lysine 310 (Ac-RelA(lys310)). These findings suggest that dysregulation in this transcription factors may be implicated in PD pathogenesis. We demonstrated that NF-κB/c-Rel DNA-binding activity is reduced in both post-mortem substantia nigra (SN) and peripheral blood mononuclear cells (PBMCs) of PD patients when compared with healthy controls (HC) ones, although no differences in NF-κB/c-Rel protein level were observed. The decrease of NF-κB/c-Rel activity in PBMCs from followed-up PD patients appeared to be independent from disease progression or dopaminergic medication, as it was evident even in early stage, drug-naïve PD patients. Furthermore, we found an inverse correlation between NF-κB/c-Rel DNA-binding and Ac-RelA(lys310) content in post-mortem SN of sporadic PD cases. Finally, immunoprecipitation assay revealed reduced levels of O-linked-β-N-acetylglucosamine-glycosylated NF-κB/c-Rel in PBMCs from followed-up PD patients, which is a fundamental post-translational modification for the activation of NF-κB/c-Rel transcriptional function. Taken together, these data suggest that NF-κB/c-Rel dysregulation is implicated in the pathophysiology of PD, while deficiency of NF-κB/c-Rel DNA-binding activity in PBMCs could represent a potential biomarker for PD.

  • Peripheral immunophenotype and inflammatory changes in Parkinson’s disease patients

    Authors:
    Maria Francesca Palmas (1), Ana Florencia Vega Benedetti (1), Clara Porcedda (1), Tommaso Ercoli (2), Chiara Burgaletto (3), Giovanni Defazio (2), Alfonso De Simone (4), Giuseppina Cantarella (3), Valeria Sogos (1), Anna R Carta (1)

    Organisations:
    1: Department of Biomedical Sciences, University of Cagliari
    2: Department of Medical Sciences and Public Health, University of Cagliari
    3: Department of Biomedical and Biotechnological Sciences, University of Catania
    4: Department of Pharmacy, University of Naples Federico II

    Presenting author: Maria Francesca Palmas

    Exposition date: Saturday, September 16, 2023

    Exposition position: 119/2

    Parkinson’s disease (PD) is characterized by early peripheral events such as inflammation and misfolding of α‐synuclein (αSyn), which may damage brain barriers resulting in chronic CNS infiltration of peripheral immunocytes and neuroinflammation. αSyn misfolding harbors the formation of toxic aggregates, among which small soluble oligomers (αSynO) induce neurotoxicity and inflammatory responses. We hypothesized that αSyn oligomers in peripheral circulation elicit inflammatory processes and alter brain barrier functions, thereby impacting CNS infiltration. In human PBMC from PD patients we investigated changes in cellular subpopulations as compared to PBMC from healthy donors, and cytokines expression after in vitro exposure to αSynOs. PD patients enrolled at the Cagliari University hospital were evaluated with classical motor (HY and UPDRS) and non-motor (MoCA and NMSS) scales. Fresh PBMCs isolated from healthy and PD donors were immunophenotyped by flow cytometry and exposed to αSynO (0.5 μM) for 24hs for multiplex ELISA (mELISA) analysis. Our results showed that peripheral immune cells displayed an altered phenotype in PD. Among PBMC subpopulations, the NK cell phenotype was mostly affected by the disease state, showing a highly significant increase of mature NK cells (CD16+/CD56-) and a dramatic decrease of immature NK cells (CD16+/CD56+), although a subpopulation of PD patients was refractory to such change of NK phenotype. The correlation analysis suggested that changes in NK phenotype correlated with the non-motor symptoms scale score. Cytokine analysis by mELISA showed that untreated PBMC from PD patients produced a highly dysregulated cytokine profile, that remained mostly unaltered after exposure to αSynOs. In contrast, PBMC from healthy donors drastically changed their cytokine profile in response to αSynOs. Changes in the PBMC phenotype may support the clinical diagnosis of PD. The increased inflammatory profile of PD-PBMC supports a role of peripheral inflammation in BBB function defects which may impact on disease progression.

  • Phenotypic and functional characterization of Astrocytes in Parkinson’s disease mouse model

    Authors:
    Michele Tufano (1), Giulia De Riso (2), Maria Josè Sisalli (1), Elena D'Apolito (1), Rossana Sirabella (1), Valeria Valsecchi (1), Antonella Casamassa (1), Giuseppe Pignataro (1), Sergio Cocozza (2), Davide Viggiano (3), Antonella Scorziello (1)

    Organisations:
    1: Division of Pharmacology, Department of Neuroscience, School of Medicine, Federico II University of Naples, Italy.
    2: Department of Molecular Medicine and Medical Biotechnology, University of Naples "Federico II", Naples, Italy.
    3: Department of Translational Medical Sciences, University of Campania "Luigi Vanvitelli", Naples, Italy.

    Presenting author: Michele Tufano

    Exposition date: Saturday, September 16, 2023

    Exposition position: 117/2

    Although reactive Astrocytes in Parkinson’s disease (PD) represents an important hallmark of the ilness, it is still unclear whether astrocyte activation is a fundamental event or a consequence of neurodegeneration. Indeed, the role of glial cells in the different stages of PD is debatable since it seems that these cells contribute to neuroinflammation as well as the neuroprotection of dopaminergic neurons. In this study, we are exploring the hypothesis that:

    1) Astrocytes might have a different phenotype depending on the brain region.

    2) The difference in Astrocytes phenotype might be associated to the progression of the disease.

    3) The changes in the Astrocytes phenotype might lead to dopaminergic dysfunction in those brain areas mainly involved in motor and non-motor symptoms of PD.

    To address these hypotheses an unbiased technique guided by pre-existing single nucleus RNA-Seq database was adopted on mice with the human mutation A53T of α-synuclein. We evaluated the difference in Astrocytes gene expression between Midbrain and Striatum and we identified: 393 genes expressed by striatal astrocytes; 80 genes expressed by mesencephalic astrocytes and 266 commonly expressed. Interestingly, the Gprofiler website’s Gene Ontology analysis revealed a gene enrichment in the pathways involved in: Oxidative Phosphorylation, Proton motive force-driven mitochondrial ATP production, ATP biosynthetic process, more pronounced in the striatal than in mesencephalic astrocytes. Focusing on the 266 common genes, we observed that 14 genes displayed a different expression between Midbrain and Striatum. Further experiments are in progress to confirm these data and clarify the pathways involved in these effects.

  • Plasma Ser129 phosphorylated alpha-synuclein / total alpha-synuclein ratio discriminates patients affected by Parkinson’s disease from controls

    Authors:
    Francesca Longhena (1), Vanessa Porrini (1), Andrea Pilotto (2), Cristina Gussago (1), Lucia Paolini (1), Paolo Bergese (1), Marika Vezzoli (1), Emirena Garrafa (3), Fiorella Guadagni (4,5), Alessandro Padovani (2), Marina Pizzi (1), Arianna Bellucci (1)

    Organisations:
    1: Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy
    2: Neurology Unit, Department of Clinical and Experimental Sciences, University of Brescia, Brescia, Italy
    3: Laboratory of Clinical Chemistry, Department of Molecular and Translational Medicine, ASST Spedali Civili and University of Brescia, Brescia, Italy
    4: Interinstitutional Multidisciplinary Biobank (BioBIM), IRCCS San Raffaele Roma, Rome, Italy
    5: Department of Radiology, IRCCS San Raffaele Roma, Rome, Italy

    Presenting author: Francesca Longhena

    Exposition date: Saturday, September 16, 2023

    Exposition position: 112/2

    Parkinson’s disease (PD) is the most common neurodegenerative disorder characterized by motor symptoms. The deposition of fibrillary aggregated alpha-synuclein (aSyn) in Lewy bodies (LB) and Lewy neurites (LN) is considered a main neurotoxic event in the brain of patients affected by PD. Of note, several lines of evidence support that mature aSyn aggregates contain elevated levels of Ser129-phosphorylated aSyn (pSer129-aSyn), supporting that this post-translationally modified form of the protein may serve as a biomarker for PD. Indeed, pSer-129-aSyn can be detected both in the cerebrospinal fluid, serum, plasma and erythrocytes.

    We recently developed a novel enzyme-linked immunosorbent assay (ELISA) to evaluate the relative amount of pSer-aSyn in plasma. By using this assay coupled with a standard ELISA protocol for the detection of total aSyn (taSyn), we found that the ratio p-Ser129-aSyn/taSyn was significantly higher in PD patients when compared with DLB and age-matched healthy controls (HC). Of note, we observed that the p-Ser129-aSyn/taSyn also enabled to differentiate DLB patients form HC when patients with Hoehn & Yahr > 2 were selected. Consistently, the p-Ser129-aSyn/taSyn ratio of PD and DLB patients at Hoehn & Yahr > 2 positively correlated with the UPDRS III score. Moreover, P-aSyn/a-Syn ratio was also found to positively correlate with the DNA binding of NF-kB c-Rel in peripheral blood mononuclear cells from both PD patients and healthy controls, supporting that the implementation of novel panels of peripheral biomarkers may enable to define novel key signatures in PD patients. Taken together, these results advocate that plasma p-Ser129-aSyn/taSyn ratio may enable to stratify patients affected by PD and DLB.

  • Salivary alpha-synuclein species as diagnostic biomarker for Parkinson’s disease

    Authors:
    Ignazia Mocci (1), Fabrizio Angius (2), Tommaso Ercoli (3), Francesco Loy (2), Maria Francesca Palmas (2), Giada Cannas (2), Aldo Manzin (2), Giovanni Defazio (3,4), Anna Rosa Carta (2)

    Organisations:
    1: Institute of Translational Pharmacology, National Research Council of Italy, Italy
    2: Department of Biomedical Sciences, University of Cagliari, Cagliari, Italy
    3: Department of Medical Sciences and Public Health, University of Cagliari, Cagliari, Italy
    4: Department of Translational Biomedicine and Neuroscience, University of Bari, Bari, Italy

    Presenting author: Ignazia Mocci

    Exposition date: Saturday, September 16, 2023

    Exposition position: 127/2

    Parkinson’s disease (PD) diagnosis is still vulnerable to bias, and a definitive diagnosis often relies on postmortem neuropathological diagnosis. In this regard, alpha-synuclein (αsyn)-specific in vivo biomarkers remain a critical unmet need, based on its relevance in the neuropathology. Specifically, content changes in αsyn species such as total, oligomeric and phosphorylated within the cerebrospinal fluid (CSF) and peripheral fluids (i.e., blood and saliva) have been proposed as PD biomarkers possibly reflecting the neuropathological outcome.

    Here, we measured for the first time the p-αsyn levels in the saliva from 15 PD patients along with tot-αsyn, o-αsyn and their ratios, and compared the results with those from 23 healthy subjects (HS), matched per age and sex. We also calculated the optimal cut-off values for different αsyn species to provide information about their capability to discriminate PD from HS. We found that p-αsyn was the most abundant alpha-synuclein species in the saliva. While p-αsyn concentration did not differ between PD and HS when adjusted for total salivary proteins, the ratio p-αsyn /tot-αsyn was largely lower in PD patients than in HS. Moreover, the concentration of o-αsyn was increased in the saliva of PD patients, and tot-αsyn did not differ between PD and HS.

    The ROC curves indicated that no single αsyn form or ratio could provide an accurate diagnosis of PD. On the other hand, the ratio of different items, namely p-αsyn/tot-αsyn and o-αsyn, yielded more satisfactory diagnostic accuracy, suggesting that the combined measure of different species in the saliva may show more promises as a diagnostic means for PD.

Proteinopathies in neurodegenerative diseases

  • Different chronic stress paradigms converge on endogenous TDP43 cleavage and aggregation

    Authors:
    Niccolò Candelise (1,2,3), Daniela Caissutti (2), Henri Zenuni (4), Valentina Nesci (3,4), Silvia Scaricamazza (3), Illari Salvatori (3), Zaira Spinello (2), Vincenzo Mattei (5), Tina Garofalo (2), Alberto Ferri (3,6), Cristiana Valle (3,6), Roberta Misasi (2)

    Organisations:
    1: Istituto Superiore di Sanità, Italy, National Center for Drug Research and Evaluation
    2: Department of Experimental Medicine, "Sapienza" University of Rome
    3: IRCCS Fondazione Santa Lucia
    4: Department of Systems Medicine, “Tor Vergata” University of Rome
    5: Biomedicine and Advanced Technologies Rieti Center, Sabina Universitas
    6: Institute of Translational Pharmacology (IFT), Consiglio Nazionale delle Ricerche (CNR)

    Presenting author: Niccolò Candelise

    Exposition date: Saturday, September 16, 2023

    Exposition position: 128/2

    The TAR-DNA binding protein (TDP43) is a nuclear protein whose cytoplasmic inclusions are hallmarks of Amyotrophic Lateral Sclerosis (ALS). Acute stress in cells causes TDP43 mobilization to the cytoplasm and its aggregation through different routes. Although acute stress elicits a strong phenotype, is far from recapitulating the years-long aggregation process. We applied different chronic stress protocols and described TDP43 aggregation in a human neuroblastoma cell line by combining solubility assays, thioflavin-based microscopy and flow cytometry. This approach allowed us to detect, for the first time to our knowledge in vitro, the formation of 25 kDa C-terminal fragment of TDP43, a pathogenic hallmark of ALS. Our results indicate that chronic stress, compared to the more common acute stress paradigm, better recapitulates the cell biology of TDP43 proteinopathies. Moreover, we optimized a protocol for the detection of bona fide prions in living cells, suggesting that TDP43 may form amyloids as a stress response.

  • Shared behaviours of KIF5A frameshift mutants in neurodevelopment and neurodegeneration

    Authors:
    Marta Cozzi (1), Barbara Tedesco (1), Guglielmo Patelli (1), Veronica Ferrari (1), Elena Casarotto (1), Marta Chierichetti (1), Paola Pramaggiore (1), Laura Cornaggia (1), Margherita Piccolella (1), Mariarita Galbiati (1), Paola Rusmini (1), Valeria Crippa (1), Cinzia Gellera (2), Daniela Di Bella (2), Stefania Magri (2), Riccardo Cristofani (1), Franco Taroni (2), Angelo Poletti (1)

    Organisations:
    1: Dipartimento di Scienze Farmacologiche e Biomolecolari “Rodolfo Paoletti” (Dipartimento di Eccellenza 2018-2027), Università degli Studi di Milano, Milan, Italy
    2: UOC Genetica Medica – Neurogenetica, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy

    Presenting author: Marta Cozzi

    Exposition date: Saturday, September 16, 2023

    Exposition position: 129/2

    KIF5A is a neuron-specific kinesin driving anterograde axonal transport. While missense mutations in KIF5A motor and stalk domains cause spastic paraplegia and Charcot-Marie-Tooth disease, translational frameshifts in KIF5A tail domain are linked to amyotrophic lateral sclerosis (ALS) and to a neurodevelopmental condition named neonatal intractable myoclonus (NEIMY). Despite the evident phenotypic differences between ALS and NEIMY, the underpinning KIF5A-related mutations share abnormal reading frame and stop codon, producing elongated KIF5A variants with 40 mutated residues in common. Based on these premises, we compared the biochemical behaviour of two representative ALS-KIF5A (p.N999VfsX39) and NEIMY-KIF5A (p.C975VfsX73) mutants to identify unique and shared features between them.

    Bioinformatic analysis of ALS- and NEIMY-KIF5A C-terminal sequences revealed that their common aberrant tail is composed of poorly soluble amino acids, differently from wild-type (WT) KIF5A. Of note, an additional low-solubility sequence was present in the NEIMY-KIF5A mutant but absent in the ALS-KIF5A one. Consistently, upon overexpression both mutants displayed low detergent solubility, but NEIMY-KIF5A accumulated into larger inclusions compared to ALS-KIF5A. The increased tendency to form condensates of the NEIMY-KIF5A mutant was also accompanied by higher WT KIF5A sequestration with respect to the ALS-KIF5A mutant, hinting at a stronger dominant-negative effect. Moreover, both ALS- and NEIMY-KIF5A mutants puncta co-localised with the ubiquitin-binding protein SQSTM1/p62, but the autophagy receptor was only found at the rim of NEIMY-KIF5A inclusions. Interestingly, both ALS- and NEIMY-KIF5A mutants accumulated upon proteasomal blockage, while no autophagy involvement was found in mutant KIF5A degradation. Finally, both mutants poorly co-localised with mitochondria, a well-established KIF5A cargo.

    Together, our observations indicate that most biochemical behaviours characterising ALS-KIF5A appear exacerbated for NEIMY-KIF5A, consistently with a more severe phenotype, and that a combination of gain- and loss-of-function mechanisms may be at the basis of both KIF5A-linked conditions.

    Acknowledgements: Italian Ministry of Health (grant RF-2018-12367768)

Reward systems and drug abuse

  • Adolescent nicotine exposure: effects on alcohol drinking behavior

    Authors:
    Cristina Cadoni (1), Augusta Pisanu (1), Alessandra Tiziana Peana (2)

    Organisations:
    1: National Research Council of Italy - Institute of Neuroscience, Italy
    2: University of Sassari - Dept. of Medicine, Surgery and Pharmacy

    Presenting author: Cristina Cadoni

    Exposition date: Saturday, September 16, 2023

    Exposition position: 140/2

    Nicotine consumption, via tobacco and increasingly electronic delivery systems, typically begins during adolescence. Early initiation with nicotine use is considered a risk factor to develop Alcohol Use Disorders and thus to function as a gateway toward alcohol abuse. Although several clinical and pre-clinical findings seem to support this relationship, causation is still far to be demonstrated. One of the reasons for this might be the role played by the individual genetic background. The use of an animal model might help to clarify this issue. Thus, the aims of the present study were to investigate a likely gateway effect of adolescent nicotine exposure to alcohol drinking behavior and to unveil the contribution of genetic background in individual vulnerability to the postulated gateway effect of adolescent nicotine.

    To this end we used two inbred rat strains, Lewis and Fischer344, as an animal model of genetic vulnerability to addiction, and compared them with the outbred rat strain Sprague-Dawley. Adolescent male rats were exposed to nicotine (0.4 mg/kg s.c.) once a day for 5 days. The following week they were trained in an intermittent alcohol access (20 % v/v) two-bottle choice paradigm for 7 weeks before undergoing alcohol operant self-administration. After six 30 min sessions at FR1, the operational requirement to gain the alcohol reward (100 ml/reward) was increased to FR3, FR5, FR10, and then to a progressive ratio (PR) schedule to test motivation to drink.

    Preliminary results of the study do not suggest a gateway effect of nicotine to drinking behavior in the F344 strain, while in SD and LEW strains different effects were observed depending on operant schedule. Moreover, adolescent alcohol exposure in Lewis rats seems to have impaired incentive learning while nicotine appears to have improved instrumental learning at high ratios with the results of increased gain of alcohol rewards.

  • Cebranopadol, a novel pan-opioid agonist with low abuse liability, attenuates heroin self-administration and seeking in the rat

    Authors:
    Caitlin Crook (1), Qianwei Shen (1), Hongwu Li (1,2), Friedbert Weiss (3), Nazzareno Cannella (1), Roberto Ciccocioppo (1)

    Organisations:
    1: School of Pharmacy, Pharmacology Unit, University of Camerino, Camerino, Italy
    2: School of Chemical Engineering, Changchun University of Technology, Changchun, 130012, China
    3: Department of Neuroscience, The Scripps Research Institute, La Jolla, California, USA

    Presenting author: Caitlin Crook

    Exposition date: Saturday, September 16, 2023

    Exposition position: 139/2

    Introduction: Gold standard pharmacological treatment for opioid use disorder (OUD) consists of maintenance therapy with long-acting opioid agonists such as buprenorphine and methadone. While these compounds have demonstrated substantial efficacy, a significant cohort of patients does not show optimal responses. Moreover, the abuse liability of these medications remains a major concern. Cebranopadol is a new, long-acting, pan-opioid agonist that differs from current therapeutic options in its additional activation of the nociceptin opioid peptide (NOP) receptor. Methods: Here, we conducted a series of experiments in the rat to evaluate the abuse potential of cebranopadol and its therapeutic potential for OUD.

    Results: Under a fixed ratio 1 (FR1) schedule of self-administration, rats maintained responding for both cebranopadol (0.3, 1.0, 6.0 μg/inf) and heroin (7, 20, 60 μg/inf). When the animals switched to a progressive ratio (PR) schedule of reinforcement, responding decreased drastically and the breakpoint (BP) reached was very low for each cebranopadol dose (0.3, 1.0, 6.0 μg/inf). Conversely, heroin-administering rats maintained PR responding at a high level at all doses tested (7, 20, 60 μg/inf). We also demonstrated that oral administration of cebranopadol (0, 25, 50 μg/kg) significantly attenuated heroin drug self-administration independently of heroin concentration (7, 20, 60 μg/inf). Furthermore, in heroin (20 μg/inf) extinction/reinstatement paradigms, pretreatment with cebranopadol significantly attenuated yohimbine stress-induced reinstatement of drug seeking. Together, these data indicate that cebranopadol has significantly limited abuse liability compared to heroin, and is highly efficacious in attenuating opioid self-administration and stress-induced reinstatement.

    Conclusions: Results indicate that cebranopadol is a potential new treatment for OUD characterized by its low abuse liability.

    Key Words: Opioid Addiction, Drug Dependence, Relapse, Nociceptin, Opioid Receptors

  • Chronic exposure to cannabinoids induces synaptic alterations in immature and mature organotypic hippocampal slice cultures.

    Authors:
    Costanza Mazzantini, Domenico E. Pellegrini-Giampietro, Elisa Landucci

    Organisations:
    University of Florence, Italy

    Presenting author: Costanza Mazzantini

    Exposition date: Saturday, September 16, 2023

    Exposition position: 132/2

    In recent years, growing evidence suggests that cannabinoids have promising medicinal and pharmacological effects, but the chronic use and abuse has risen medical concerns. The novel progress of cellular and molecular neurobiology have contributed to understand the mechanisms by which cannabinoids induce long-lasting functional changes in the brain. However, little is still known about the functional and molecular mechanisms underlying these alterations.

    Rat organotypic hippocampal slice cultures were exposed chronically for 7 days to cannabidiol (CBD) or Δ9-tetrahydrocannabinol (THC) after 2 days (immature) or 10 days (mature) of culture in vitro. The cell death in different subregions of slices was quantified by propidium iodide (PI) fluorescence, pre-synaptic and post-synaptic marker proteins were analized by Western blotting and mitochondria and ER-stress markers by RT-PCR.

    Chronically CBD or THC incubation for 7 days did not induce significant cell death in organotypic hippocampal slices as observed by PI fluorescence, but caused modifications in the expression of presynaptic and postsynaptic proteins with a significantly decrease following chronic exposure to THC in both models. RT-PCR experiments showed alterations of mitochondria and ER-stress markers with different effects in immature and mature slices.

    Our results suggest that chronic exposure of CBD or THC induced alterations in the hippocampus. For that reason, the right medicinal use of cannabinoids with chronic treatment should be contemplated with more consciousness.

  • Chronic exposure to MDMA can alter neuroinflammatory processes in rats

    Authors:
    Antonio Lacorte (1), Laura Rullo (1), Camilla Morosini (1), Loredana Maria Losapio (1), Ana Domi (2), Roberto Ciccocioppo (2), Sanzio Candeletti (1), Patrizia Romualdi (1)

    Organisations:
    1: Department of Pharmacy and Biotechnology, Alma Mater Studiorum, University of Bologna, Via Irnerio 48, 40126 Bologna, Italy.
    2: Pharmacology Unit, School of Pharmacy, University of Camerino, Camerino, Italy.

    Presenting author: Antonio Lacorte

    Exposition date: Saturday, September 16, 2023

    Exposition position: 131/2

    MDMA (3,4-methylenedioxymethamphetamine), also known as ecstasy, is an abused psychostimulant drug. Due to its world-wide increasing abuse, especially among adolescents and young adults, studies aimed to better elucidate its long-term effects on Central Nervous System (CNS) functions are mandatory. Recent studies suggested a relationship between chronic exposure to drugs of abuse and the development of neuroinflammatory processes. In particular, the dysregulation of the epigenetic histone demethylase KDM6A and KDM6B, associated with the modulation of many inflammatory processes, has been reported in alcohol dependence. In addition, a role for the peroxisome proliferator-activated receptors (PPARS) has been indicated either in alcohol intake and in nicotine withdrawal.

    On this basis, here we investigated possible gene expression alterations for the just mentioned epigenetic enzymes and receptors involved in inflammatory processes, caused by the prolonged exposure to MDMA, in some rat CNS areas typically involved in addictive behavior.

    Adult male Wistar rats were administered with MDMA (8 mg/kg, i.p.) twice a day for seven days; after treatment, the prefrontal cortex (PFC), the ventral tegmental area (VTA) and the nucleus accumbens (NAc) of animals were collected for gene expression analysis (Real-time quantitative RT-PCR) of KDM6A, KDM6B, PPARα and PPARγ. In the PFC, chronic MDMA caused a significant reduction in PPARα and KDM6B mRNA levels whereas no significant alterations in KDM6A and PPARγ levels between control and treated rats were observed. In either VTA and NAc areas MDMA caused a similar pattern of alterations: in particular, a significant downregulation of both PPARα and PPARg gene expression was detected together with a decrease of KDM6B mRNA levels. Although further studies are required, these preliminary results show that MDMA may produce alterations of molecular targets associated with neuroinflammatory and neurotoxic processes.

  • Environmental enrichment modulates mesolimbic neural activity in Sardinian alcohol-preferring rats

    Authors:
    Michele Santoni (1), Paola Maccioni (2), Marco Pistis (1,2,3), Giancarlo Colombo (2), Anna Lisa Muntoni (2)

    Organisations:
    1: Division of Neuroscience and Clinical Pharmacology, Department of Biomedical Sciences, University of Cagliari, 09042-Monserrato, Italy
    2: Neuroscience Institute, Section of Cagliari, National Research Council of Italy (CNR), 09042-Monserrato
    3: Unit of Clinical Pharmacology, University Hospital, Cagliari, Italy

    Presenting author: Anna Lisa Muntoni

    Exposition date: Saturday, September 16, 2023

    Exposition position: 133/2

    Living in an enriched environment (EE) induces structural and functional alterations of the brain which result in changes in behavior, including those motivated by drugs of abuse.

    However, while several studies suggest a beneficial effect of EE against addiction to mostly psychostimulants, the scenario appears more complex when referring to alcohol. To further disentangle the relationship between EE and alcohol-motivated behaviors, we assessed the effect of EE in a validated model of alcohol use disorder, the Sardinian alcohol-preferring (sP) rats.

    Here, we investigated the influence of EE exposure on the activity of the mesolimbic dopamine system, key in the mechanisms underlying alcohol consumption and dependence, by assessing the properties of the midbrain dopamine neurons in vivo and their response to cumulative doses of alcohol.

    Starting from postnatal day 21 (PND21), male sP rats were housed in 3 different conditions: impoverished environment (IE; single housing and no environmental enrichment), standard environment (SE; small colony and no environmental enrichment), and EE (large colony and multiple elements of environmental enrichment). From PND60, we carried out extracellular electrophysiological recordings from ventral tegmental area (VTA) dopamine neurons in anesthetized rats belonging to the different experimental groups.

    The analysis of the electrophysiological properties of VTA dopamine neurons revealed a higher number of cells/track, coupled with a higher mean spikes/burst, in EE rats compared to SE and IE rats (EE>SE>IE), while two-way ANOVA revealed an effect of environment on the cumulative doses of alcohol.

    Altogether our results suggest that living in enriched environments may reduce the reinforcing and motivational properties of alcohol in sP rats. Importantly, the tendency toward an increase in mesolimbic dopamine neuron functionality might provide a mechanism for better understanding the behavioral readout.

  • Impact of the drinking in the dark (DID) model of alcohol use disorder on locomotion and dopaminergic neurotransmission in mice

    Authors:
    Carolina Giulia Ferroni (1,2), Noemi Nicosia (1), Maria Regoni (1), Guendalina Bastioli (1), Margherita Spadaccini (1), Veronica Di Febo (1), Atea Shkodra (1), Ceren Ozcan (1), Stefano Comai (1,3), Flavia Valtorta (1), Danilo De Gregorio (1)

    Organisations:
    1: Vita-Salute San Raffaele University and Division of Neuroscience, San Raffaele Scientific Institute, Milan
    2: Zardi-Gori Foundation, Via Curtatone 25, 20122 Milan, Italy
    3: Department of Pharmaceutical and Pharmacological Sciences University of Padua, Italy

    Presenting author: Carolina Giulia Ferroni

    Exposition date: Saturday, September 16, 2023

    Exposition position: 137/2

    Background: Alcohol Use Disorder (AUD) is a major cause of health and social problems. The Drinking In the Dark (DID) paradigm has gained interest to study in animal models the neurophysiological and behavioral effects of AUD, since it resembles human binge drinking. However, the impact of DID on locomotion and on the dopaminergic (DA) activity in the ventral tegmental area (VTA), a brain region playing a pivotal role in addictive behavior, remains elusive. Employing a behavioral and electrophysiological approach, in this preliminary study we assessed the impact of the DID paradigm on locomotion and DA transmission in mice.

    Methods: C57BL6/J 8-week-old male mice underwent 6 cycles of DID. Each cycle consists in: one experimental group (AUD mice) in which the water bottles were removed from all cages and replaced with bottles containing 20% ethanol solution for 2 hours per day, for 4 consecutive days; another group of mice (control, CTL) in which the water bottles were not replaced with ethanol. After 6 cycles of DID, both groups of mice underwent alcohol consumption assessment with the 40 days two-bottle choice test. Twenty-four hours from the last drinking session, locomotion was assessed employing the open field test (OFT) and the rotarod test. Finally, in vivo single unit extracellular recordings of VTA DA neurons were performed.

    Results: AUD mice did not show difference in locomotion during the OFT, compared to CTL (p=0.012). However, AUD mice displayed decreased latency to fall (p<0.01) in the rotarod test. These effects were coupled to an increased DA VTA firing rate activity (p<0.05) in AUD mice compared to CTL.

    Conclusion: These data show that chronic alcohol consumption in the DID paradigm produces partial locomotor impairments similar to those observed in human binge drinking. The observed alterations in DA neurotransmission may underlie the development of the observed addictive behavior.

  • Increased MOP expression in the Ventral Tegmental Area mediates higher heroin self-administration and motivation expressed by marchigian sardinian alcohol preferring rats compared to non-preferring Wistars

    Authors:
    Hela Mrizak (1), Veronica Lunerti (1), Laura Rullo (2), Esi Domi (1), Patrizia Romualdi (2), Roberto Ciccocioppo (1), Amy Lasek (3), Nazzareno Cannella (1)

    Organisations:
    1: School of Pharmacy, Pharmacology Unit, Center for Neuroscience, University of Camerino,Camerino, Italy
    2: Department of Pharmacy and Biotechnology, Alma Mater Studiorum University, Bologna, Italy
    3: Department of Pharmacology and Toxicology, Virginia Commonwealth University, USA

    Presenting author: Hela Mrizak

    Exposition date: Saturday, September 16, 2023

    Exposition position: 136/2

    Marchigian Sardinian alcohol-preferring (msP) rat is an animal model genetically selected for high alcohol preference from outbred Wistars. MsPs show an innate dysregulation of Mu opioid receptors (MOP) in the amygdala and the nucleus accumbens. This prompted us to hypothesize that, similar to alcohol, msP rats may show a higher preference also for heroin compared to their non-preferring Wistar controls.

    We set out by comparing the dose/response of heroin (1, 7, 20, 60 µg/infusion) self-administration between msP and Wistar rats under fixed ratio 1 (FR1) contingency. Independently from sex, msPs self-administered higher number of heroin infusions than Wistars at the two intermediate doses. Next, we evaluated the motivation for the four doses of heroin measuring the break point reached under progressive ratio contingency. Male msP rats reached higher break-point than Wistars at the two intermediate heroin doses, whereas female msP rats showed higher break-point than Wistars at all heroin doses except the lowest. These data demonstrated that msP showed an increased preference for the primary reinforcing effect of heroin. Thus, we compared MOP expression in the ventral tegmental area (VTA) between heroin naïve msPs and Wistars finding that msPs show increased MOP expression in this area. Therefore, we next toned down the expression of MOP in the VTA of msPs by local viral transfection of a MOP short hairpin RNA (MOPshRNA) plasmid. Heroin (7 and 20 µg/infusion) self-administration and motivation of MOPshRNA rats were compared to that of scrambled virus-infected rats. As expected, the MOPshRNA group showed lower heroin self-administration and motivation regardless of the heroin dose.

    Altogether, these findings demonstrate that msP rats have a higher preference for heroin compared to Wistar controls, which is mediated by a higher expression of MOP in the VTA.

  • Inhibition of phosphodiesterase 7 enzymes reduces alcohol drinking and relapse in animal models of alcohol use disorders

    Authors:
    Adana Keshishian (1), Esi Domi (1), Gregory Demopulos (2), George Gaitanaris (2), Roberto Ciccocioppo (1)

    Organisations:
    1: University of Camerino, Italy
    2: OMEROS Corporation, USA

    Presenting author: Adana Keshishian

    Exposition date: Saturday, September 16, 2023

    Exposition position: 134/2

    Alcohol use disorder (AUD) is a major cause of disabilities and, despite a few medications having been developed, still represents a largely unmet medical need. We have recently developed a new series of brain-penetrant molecules that selectively inhibit phosphodiesterase-7 (PDE7) enzymes. PDEscontrol the rate of degradation of cAMP and cGMP, and their inhibition leads to intracellular increase of these two second messengers. The PDE7 isoform encompasses two members, PDE7A and PDE7B. Both are cAMP-specific enzymes highly expressed in brain regions known to play a role in the modulation of alcohol reinforcement and abuse.

    Here we tested the effect of OMSPDE79, a selective PDE7 inhibitor, on multiple well-validated models of AUD in the rat.First, we evaluated the effects of intraperitoneal (i.p.) administration of OMSPDE79 (0, 5, 7, 9 mg/kg) on alcohol self-administration and on cue and yohimbine-induced reinstatement of alcohol seeking in outbred Wistar rats. Next, we tested the effects of OMSPDE79 on the alcohol deprivation (ADE) model of relapse in Wistar rats with a long history of drinking. Finally, we assessed the effect of OMSPDE79 on home-cage drinking in Marchigian Sardinian alcohol-preferring (msP) rats, a genetic model of innate preference for alcohol that mimics some features of more advanced stages of AUD.

    We found that OMSPDE79 dose-dependently decreased alcohol self-administration as well as cue and yohimbine-induced reinstatement of alcohol seeking. OMSPDE79 also prevented ADE after protracted abstinence in Wistars and reduced alcohol drinking in msP rats. These effects were specific to alcohol as OMSPDE79 altered neither food nor water intake.

    Taken together, our findings strongly support the potential of OMSPDE79 as a novel therapeutic candidate in AUD.

  • Oxytocinergic system alteration induced by attachment bond disruption mediates the vulnerability to cocaine effects in adulthood

    Authors:
    Diana Municchi (1,2), Camilla Mancini (3), Sebastian Luca D'Addario (2), Alice Passeri (1,2), Marta Tiberi (2), Lucy Babicola (1), Matteo Di Segni (2), Luisa Lo Iacono (1,2), Diego Andolina (1,2), Carlo Cifani (3), Valerio Chiurchiu (2), Rossella Ventura (1)

    Organisations:
    1: La Sapienza Università di Roma, Italy
    2: IRCCS Fondazione Santa Lucia, Roma, Italy
    3: University of Camerino School of Pharmacy, Camerino, Italy

    Presenting author: Diana Municchi

    Exposition date: Saturday, September 16, 2023

    Exposition position: 138/2

    Early life experiences have the potential role to alter both brain development and adult behavior.

    Particularly, the formation of the attachment bond is crucial for well-being and mental health, and its disruption is related to expression of several psychopathologies, such as drug addiction. Interestingly, oxytocin (Oxt) plays an important role in the formation of the attachment bond and dopaminergic (DA) circuits development, and alterations of both Oxt and DA systems are involved in drug addiction. We have recently showed that an early life manipulation (Repeated Cross Fostering, RCF) able to alter the mother-pups bond induces in C57 female mice a behavioral phenotype characterized by increased vulnerability to cocaine effects in adulthood affecting DA mesocorticolimbic system. Here, we hypothesize that RCF manipulation impacts Oxt system development increasing the vulnerability to cocaine effects in adult life. To test this hypothesis, we investigated the main targets of the Oxt system: Oxt levels, Oxt receptor (OxtR) within the mesocorticolimbic system and the Receptors for Advanced Glycation End-Products (RAGE) (a recently identified Oxt transporter), across the blood-brain barrier (BBB).

    We found that RCF manipulation induced a reduction in both Oxt and OxtR levels in the brain of female C57 mice at postnatal day (PND) 5.

    Moreover, in adult RCF compared to Control females, altered OxtR levels and Oxt release in the nucleus accumbens (NAc) induced by acute cocaine injection were also observed. Finally, we also reported reduced RAGE expression. Restoring brain oxytocin levels through subcutaneous injection of oxytocin (20ul, 0.2 ng/ul) from PND1 to PND4, was sufficient to reduce increased sensitivity to cocaine effects in adult RCF females (as evaluated by cocaine-induced Conditioned Place Preference) and normalize both Oxt and OxtR levels in adulthood. Altogether, our results demonstrate the critical role for Oxt system in RCF-induced vulnerability to cocaine effects reported in adult C57 females.

  • Selective inhibition of Phosphodiesterase 7 enzyme reduces cue- and stress- induced reinstatement of cocaine seeking in the rat

    Authors:
    Sofia Vellere (1), Esi Domi (1), Adana Keshishian (1), Gregory Demopulos (2), George Gaitanaris (2), Roberto Ciccocioppo (1)

    Organisations:
    1: School of Pharmacy, Pharmacology Unit, University of Camerino, Via Madonna delle Carceri, 62032 Camerino, Italy
    2: Omeros Corporation, 201 Elliot Avenue West, Seattle, WA 98119, USA

    Presenting author: Sofia Vellere

    Exposition date: Saturday, September 16, 2023

    Exposition position: 135/2

    Cocaine use disorder (CUD) has a large societal impact, causing severe disabilities and deaths. There is no approved treatment for CUD. One of the challenges in treating this condition is the high rate of recidivism. Frequently, cocaine users try to stop abusing the drug but, in most cases, they are unable to maintain long-term abstinence, and relapse occurs.

    Previous studies on nicotine, have shown that administration of OMSPDE71 and OMSPDE79, two selective inhibitors of the phosphodiesterases7 (PDE7), reduced nicotine intake as well as nicotine relapse elicited by both cues and yohimbine stress. PDE7 inhibition (PDE7i) appears to facilitate Dopamine receptor 1 (D1)-mediated transmission in the nucleus accumbens. Considering the important role of dopaminergic transmission in cocaine effects, we decided to test PDE7i in cocaine self-administration and relapse.

    Following acquisition of stable daily cocaine self-administration (0.25 mg/kg/inf) under fixed-ratio 5 (FR5) reinforcement schedule, Wistar rats were treated intraperitoneally (i.p.) with OMSPDE71 (0, 1, 3, 9 mg/kg) and cocaine intake was evaluated. A separate group of rats was trained to FR1 cocaine self-administration followed by daily 1-hour extinction sessions during which lever pressing did not result in drug delivery. These rats were administered OMSPDE79 (0, 1, 3, 9 mg/kg) i.p. Reinstatement was then evaluated in this second group by returning the cues (house light and a bit tone) previously paired with cocaine or by treatment with yohimbine (1.25 mg/kg).

    Results showed efficacy of OMSPDE71 in attenuating cocaine self-administration. Similarly, in the reinstatement tests, OMSPDE79 significantly reduced relapse evoked by both cues and yohimbine. This effect was specific to cocaine relapse as OMSPDE79 did not modify the responses at the inactive control lever.

    In conclusion, our findings support the clinical investigation of PDE7i for the treatment of CUD.

  • The Neuropeptide S Reduces Alcohol Self-administration and Impulsivity in Rats

    Authors:
    Min LI, Sara De Carlo, Nazzareno Cannella

    Organisations:
    School of Pharmacy, Pharmacology Unit, Center for Neuroscience, University of Camerino, Camerino, Italy

    Presenting author: Min LI

    Exposition date: Saturday, September 16, 2023

    Exposition position: 130/2

    Alcohol use disorder (AUD) is a psychiatric condition often comorbid with impulsivity. The neuropeptide S (NPS) is a neurotransmitter characterized by a pro-arousal and anxiolytic physio-pharmacological. A variant of the NPS receptor has been associated with maladaptive impulsivity and alcoholism. We previously demonstrated that NPS reduces alcohol self-administration (ASA) in male marchigian sardinian alcohol preferring (msP) rats. Here we verified the effect of NPS on ASA in female msP rats and tested the hypothesis that NPS would reduce impulsive behavior in a go/no-go task.

    For ASA study, we trained 20 female and 20 male msP rats to self-administer 0.1 ml of 10% alcohol under fixed-ratio 1 contingency and tested the effect of intracerebroventricular (ICV) NPS (0.1, 1.0 and 2.0 nmole/icv) on ASA.

    To model impulsivity, we developed a go/no-go paradigm in which following a pre-trial period, during which rats had to wait for instruction, rats experienced either a go or a no-go trial. Pressing the lever during go-trials and refraining from it during no-go trials, was reinforced with 0.1 ml of 10% sucrose. This model yielded three measures of impulsivity, premature responses (pressing during pre-trials), % of correct go-trials and % of correct no-go trials. The model was validated in 12 Wistar rats by atomoxetine pretreatment (0, 1, 5 mg/kg). Next, the effect of NPS (0.1, 1.0 and 2.0 nmole/icv) was tested in 12 male Wistar rats.

    We confirmed that NPS reduced ASA in male msP rats and expanded this finding to females. In the go/no-go study, atomoxetine reduced the number of premature responses and increased the % of correct no-go trials, providing predictive validity to our model of impulsivity. A similar effect was obtained following NPS pretreatment, indicating that this neurotransmitter has anti-impulsive effects.

    Our data indicate that NPS is a potential target to treat AUD and co-morbid impulsivity.

Sensory systems

  • A comparison of young and aged neuronal growth in importin alpha3 deficient mice

    Authors:
    Sofia Dotta (1), Giada Musso (1), Amisha Parmar (1), Livia Testa (1), Franziska Rother (2,3), Micheal Baden (2,3,4), Letizia Marvaldi (1)

    Organisations:
    1: Neuroscience Institute Cavalieri Ottolenghi, Italy
    2: Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany
    3: Center for Structural and Cellular Biology in Medicine, Institute of Biology, University of Lübeck, 23538 Lübeck, Germany
    4: Charité - Universitätsmedizin Berlin, 10117 Berlin, Germany.

    Presenting author: Sofia Dotta

    Exposition date: Friday, September 15, 2023

    Exposition position: 129/1

    Neurotrophins are extrinsic factors that have several neuronal functions, mainly responsible for growth and survival during development and grow only in adult animals. Are there any differences in neurotrophins response in aged and young neurons? Our results show that in importin alpha3 ko mice females do not have a neurotrophins response compared to the wt mice. This is very intriguing and our effort will shed light on how neurotrophins signalling communicate with the intrinsic factors. Specifically, we would like to investigate how genes are modulated in sensory neurons by neurotrophins stimulation. Knowing the different signaling pathways and the gene's network will allow us to genetically modify the expression of age state from aged to young sensory neurons.

  • A comparison of young and aged neuronal growth in importin 3 deficient mice

    Authors:
    Sofia Dotta (1), Giada Musso (1), Amisha Parmar (1), Livia Testa (1), Franziska Rother (2,3), Michael Bader (2,3,4), Letizia Marvaldi (1)

    Organisations:
    1: Neuroscience Institute Cavalieri Ottolenghi, Italy
    2: Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany
    3: Center for Structural and Cellular Biology in Medicine, Institute of Biology, University of Lübeck, 23538 Lübeck, Germany
    4: Charité - Universitätsmedizin Berlin, 10117 Berlin, Germany

    Presenting author: Sofia Dotta

    Exposition date: Thursday, September 14, 2023 (Satellite Events Day)

    Exposition position: 108

    Neurotrophins are extrinsic factors that have several neuronal functions, mainly responsible for growth and survival during development and grow only in adult animals. Are there any differences in neurotrophins response in aged and young neurons? Our results show that in importin a3 ko mice females do not have a neurotrophins response compared to the wt mice. This is very intriguing and our effort will shed light on how neurotrophins signalling communicate with the intrinsic factors. Specifically, we would like to investigate how genes are modulated in sensory neurons by neurotrophins stimulation. Knowing the different signaling pathways and the gene's network will allow us to genetically modify the expression of age state from aged to young sensory neurons.

  • A comprehensive evaluation of the polygenic architecture of hearing problems in adults.

    Authors:
    Flavio De Angelis (1,2), Govanni Cirillo (2), Ciro De Luca (2), Assunta Virtuoso (2), Michele Papa (2), Renato Polimanti (1)

    Organisations:
    1: Yale University
    2: University of Campania "L Vanvitelli"

    Presenting author: Flavio De Angelis

    Exposition date: Friday, September 15, 2023

    Exposition position: 133/1

    Hearing problems (HP) in adults are common and associated with several comorbid conditions impacting human health. The complex nature of HP is reflected by an increasing prevalence with age, which echoes the cumulative effect of environmental factors and genetic predisposition. Although several risk loci have already been identified, large-scale genetic studies still under-investigated HP biology and epidemiology. Leveraging several large cohorts, we conducted a comprehensive genome-wide association study of HP in more than 700,000 participants. The GWAS findings were used to characterize HP polygenic architecture, exploring sex differences, polygenic risk across ancestries, tissue-specific transcriptomic regulation, cause-effect relationships with genetically correlated traits, and gene interactions with HP environmental risk factors. We identified 54 risk loci and demonstrated that HP polygenic risk is partially shared across ancestries. The transcriptomic regulation analyses underlined the role of the central nervous system in HP pathogenesis. Additional associations related to peripheral hormonally regulated tissues were identified, suggesting a potential role of estrogen metabolism in hearing function. The genetically informed causal inference analysis showed that HP is linked to many genetically correlated physical and mental health outcomes. The multivariate interaction analysis shows how genes impacting brain development interact with sex, noise pollution, and tobacco smoking with their HP associations.The results provide many novel insights into the biology and epidemiology of HP in adults, even identifying sensitive molecular pathways for drug development or repurposing. The potential causal relationships identified may also support novel preventive screening programs to identify individuals at risk.

  • Activation of human visual area V6 during egocentric navigation with and without visual experience

    Authors:
    Elena Aggius Vella (1), Daniel-Robert Chebat (2,3), Shachar Maidenbaum (4,5), Amir Amedi (1)

    Organisations:
    1: The Baruch Ivcher Institute for Brain, Cognition & Technology, Reichman University, Herzliya, Israel.
    2: Visual and Cognitive Neuroscience Laboratory (VCN Lab), Department of Psychology, Faculty of Social Sciences and Humanities, Ariel University, Ariel, Israel.
    3: Navigation and Accessibility Research Center of Ariel University (NARCA), Ariel, Israel.
    4: Biomedical Engineering, Ben-Gurion University of the Negev, Beersheba, Israel
    5: Zlotowski center for neuroscience, Ben Gurion University, Beersheba, Israel

    Presenting author: Elena Aggius Vella

    Exposition date: Friday, September 15, 2023

    Exposition position: 130/1

    Introduction: V6 is a retinotopic area located in the dorsal visual stream that integrates eye movements with retinal and visuo-motor signal. Despite the known involvement of V6 in visual motion, it is still unknown whether it is involved in navigation and how sensory experiences shape its functional properties. We explored the involvement of V6 in egocentric navigation in sighted and in congenitally blind (CB) participants navigating via an in-house distance-to-sound sensory substitution device (SSD), the EyeCane.

    Method: We performed two fMRI experiments on two independent datasets. In the first experiment, CB and sighted participants navigated the same mazes. The sighted performed the mazes via vision, while the CB via audition. The CB performed the mazes before and after a training session using the EyeCane SSD. In a second experiment a group of sighted people performed a motor topography task.

    Results: Results show that right V6 (rhV6) is selectively involved in egocentric navigation independently by the sensory modality used. Indeed, after training rhV6 of CB is selectively recruited for auditory navigation like rhV6 of the sighted. Moreover, we found activation for body movement in area V6, that can putatively contribute to its involvement in egocentric navigation. Taken together, our findings suggest that area rhV6 is a unique hub that transforms spatially relevant sensory information into an egocentric representation for navigation. While vision is clearly the dominant modality, rhV6 is in fact a supramodal area that can develop its selectivity for navigation in the absence of visual experience during the critical period.

  • Adjusting to a mirrored world: a study on visual adaptation

    Authors:
    Cristina Solinas (1), Matteo Diano (2), Alessio Borriero (2), Marco Lanzilotto (2), Marco Tamietto (2,3)

    Organisations:
    1: University of Parma, Parma, Italy
    2: University of Torino, Torino, Italy
    3: Tilburg University, the Netherlands

    Presenting author: Cristina Solinas

    Exposition date: Friday, September 15, 2023

    Exposition position: 132/1

    Unveiling the importance of neural plasticity in the visual system may highlight the remarkable adaptability of human perception and its potential impact on rehabilitation and technological progress. Two seminal research papers have shown that the visual system of both healthy humans and nonhuman primates can successfully adapt to an optically inverted environment byusing left-right reversal prismatic glasses. However, such an experimental procedure had a significant impact on individuals' daily lives as participants wore the prism for ~40 days, making it a costly approach to study visual adaptation, and it failed to systematically quantify behavioral responses.

    The main goal of this study was to investigate short-term adaptation to inverted vision by exploring human behavior. Six healthy participants donned left-right reversing glasses and were tested before and after a 36-hours waking adaptation period. The Poffenberger paradigm was used to assess interhemispheric information transfer. Two conditions were established: the uncrossed condition, in which stimuli were presented in the same hemifield as the responding hand, and the crossed condition, in which stimuli appeared in the contralateral hemifield. The difference in reaction times between conditions (CUD) served as a measure of interhemispheric transfer time (IHTT).

    Our analysis of the CUD results showed a significant difference between conditions with longer reaction times in the crossed condition before adaptation, consistent with the existing literature. Intriguingly, this effect disappeared after 36 hours of wearing prismatic glasses. In addition, overall reaction times were faster post-adaptation. These results suggest that prismatic adaptation involved the ipsilateral hemisphere thereby neutralizing the IHTT delay in the crossed condition and shortening participants' reaction times. This suggests that a short adaptation period is sufficient to produce measurable behavioral effects, possibly suggesting bilateral hemisphere involvement. Neuroimaging could provide more robust evidence of the mechanisms involved and allow structural and functional mapping of the affected areas.

  • Cross-talk between intrinsic and extrinsic factors in importin a3 mutant mice

    Authors:
    Giada Musso (1), Sofia Dotta (1), Amisha Parmar (1), Livia Testa (1), Franziska Rother (2,3), Michael Bader (2,3,4), Letizia Marvaldi (1)

    Organisations:
    1: Neuroscience Institute Cavalieri Ottolenghi, Italy
    2: Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany
    3: Center for Structural and Cellular Biology in Medicine, Institute of Biology, University of Lübeck, 23538 Lübeck, Germany
    4: Charité - Universitätsmedizin Berlin, 10117 Berlin, Germany.

    Presenting author: Giada Musso

    Exposition date: Thursday, September 14, 2023 (Satellite Events Day)

    Exposition position: 106

    During development there are several cues that determine the growth-rate of a neuron and the axonal determination of path-finding. Previous work in Marvaldi et al., 2020 showed that importin a3 is responsible for reducing pain upon injury and the mechanism of action is dependent of c-FOS protein. RNAseq data from embryonic DRG tissue (E13.5) followed by informatic analysis showed that c-FOS transcription factor has been enriched in our dataset. Our aim is to validate this data and understand the role of c-FOS during development in axonal path-finding. Do importin a3 and c-FOS inhibition act similarly on the phenotypical changes in peripheral cells (sensory neurons and Schwann cells)? Particularly, Inhibition of importin a3 or c-FOS makes changes in the nerve endings of the E13.5 paw. Why this is the case? Using neuronal-glia co-culture in E13.5 will highlight the grow and survival in both cell type with importin a3 and c-FOS inhibition. Finally, we will unravel if modulating intrinsic factors (importin a3 or c-FOS) or extrinsic factors (neurotrophins) will make changes in growth and survival of neurons and glia cells.

  • Cross-talk between intrinsic and extrinsic factors in importin a3 mutant mice

    Authors:
    Giada Musso (1), Sofia Dotta (1), Amisha Parmar (1), Livia Testa (1), Franziska Rother (2,3), Michael Bader (2,3,4), Letizia Marvaldi (1)

    Organisations:
    1: Neuroscience Institute Cavalieri Ottolenghi, Italy
    2: Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany
    3: Center for Structural and Cellular Biology in Medicine, Institute of Biology, University of Lübeck, 23538 Lübeck, Germany
    4: Charité - Universitätsmedizin Berlin, 10117 Berlin, Germany

    Presenting author: Giada Musso

    Exposition date: Friday, September 15, 2023

    Exposition position: 125/1

    During development there are several cues that determine the growth-rate of a neuron and the axonal determination of path-finding. Previous work in Marvaldi et al., 2020 showed that importin a3 is responsible for reducing pain upon injury and the mechanism of action is dependent of c-FOS protein. RNAseq data from embryonic DRG tissue (E13.5) followed by informatic analysis showed that c-FOS transcription factor has been enriched in our dataset. Our aim is to validate this data and understand the role of c-FOS during development in axonal path-finding. Do importin a3 and c-FOS inhibition act similarly on the phenotypical changes in peripheral cells (sensory neurons and Schwann cells)? Particularly, Inhibition of importin a3 or c-FOS makes changes in the nerve endings of the E13.5 paw. Why this is the case? Using neuronal-glia co-culture in E13.5 will highlight the grow and survival in both cell type with importin a3 and c-FOS inhibition. Finally, we will unravel if modulating intrinsic factors (importin a3 or c-FOS) or extrinsic factors (neurotrophins) will make changes in growth and survival of neurons and glia cells.

  • Dissecting the role of the mouse olfactory dopaminergic cells in sexual odor processing

    Authors:
    Ilaria Ghia, Maddalena Pieroni, Stefano Zucca, Nicole Mancin, Paolo Marcello Peretto, Silvia De Marchis, Serena Bovetti

    Organisations:
    Dept. of Life Sciences and Systems Biology and Neuroscience Institute Cavalieri Ottolenghi, University of Turin, Italy

    Presenting author: Ilaria Ghia

    Exposition date: Friday, September 15, 2023

    Exposition position: 126/1

    Olfaction is one of the most conserved senses in animal evolution and many species heavily rely on it for their behavior, including mate choice and reproduction. In mice, the main olfactory bulb (OB) hosts a large dopaminergic (DA) population, which can be identified by the expression of tyrosine hydroxylase (TH) enzyme. TH+ OB cells are inhibitory interneurons, mostly located in the glomerular layer and classified into two major categories (i.e., small and large cells) based on their morphology, generation time and biophysical properties. While midbrain DA neurons are known to be activated by salient stimuli, whether OB DA cells are involved in processing ethologically relevant odorants, and particularly sexual odors, is still completely unknown. Here, we investigated the recruitment of olfactory DA cells in response to exposure to sexual odors contained in urine of the opposite sex. By whole-brain labelling of the immediate early gene c-fos and light-sheet microscopy, we show that, in female mice, short and acute exposure to male urine increases neuronal activation mainly in the most dorsal-posterior area of the OB, where we detected a selective and strong recruitment of olfactory DA cells. Interestingly, large cells seemed to display a higher basal excitability compared to small ones even in absence of stimulation. To further analyze the in vivo physiological response of DA interneurons to opposite sex odors, we validated a Cre-lox based method to specifically express the calcium indicator GCaMP targeting preferentially large or small OB DA cells. We are currently performing 2-photon in vivo functional calcium imaging to dissect the contribution of these two subpopulations in processing opposite sex-odors and unravel the role of glomerular dopamine in the context of reproduction.

  • Dual Pathways in Visual Processing: Insights from Machine Learning approach

    Authors:
    Matteo Diano (1), Petra Vetter (2), Marco Tamietto (1,3), Patrik Vuilleumier (4)

    Organisations:
    1: University of Turin, Italy
    2: University of Fribourg, Switzerland
    3: Tilburg University, the Netherlands
    4: University of Geneva, Campus Biotech, Geneva, Switzerland

    Presenting author: Matteo Diano

    Exposition date: Friday, September 15, 2023

    Exposition position: 131/1

    The visual system has distinct neural pathways for processing visual stimuli, divided primarily between "subcortical" and "cortical" routes. The subcortical route, which includes superior colliculus (SC), and pulvinar, is involved in early-stage visual processing and focuses on basic properties (low spatial frequency, non-purple color, unconscious processing). The cortical route handles more complex processing (high spatial frequency, colors, conscious stimuli) and consists mainly of the lateral geniculate nucleus (LGN), primary visual cortex (V1), and ventral visual stream.

    To investigate these pathways, an fMRI study was conducted with 15 subjects using dog images varying in spatial frequency, color, un/conscious via continuous flash suppression, and un/conditioned modulation. A machine learning model (XGBoost) was trained to categorize neural activity in the SC (subcortical) and LGN (cortical), achieving 66% accuracy. SHAP analysis revealed that low spatial frequency, unconscious, conditioning, and grey conditions were the most significant factors for model accuracy. The trained model was then used to classify other brain areas against a null distribution. Our results, for example, showed a classification gradient within the V1, with the foveal region classified as part of the cortical route and the peripheral region as part of the subcortical route. This supports the idea that foveal vision is dominated by retino-geniculate pathways, while extra-foveal event detection for gaze shifts is managed by the SC. In conclusion, the study demonstrated the distinct roles of subcortical and cortical pathways in processing visual information, with low spatial frequency, unconscious, and grey conditions being significant factors for differentiation. The observed classification gradient in V1 further supports the division of labor between these two neural routes.

  • Inferring brain network organization from connectomic reconstructions: a network analysis approach applied in d. melanogaster and d. rerio

    Authors:
    Karan Manjunatha (1), Matteo Bruzzone (2,3,4), Giorgio Nicoletti (1), Samir Suweis (1,3), Marco Dal Maschio (2,3)

    Organisations:
    1: Department of Physics, University of Padua, IT
    2: Department of Biomedical Sciences, University of Padua, IT
    3: Padua Neuroscience Center – PNC, University of Padua, IT
    4: Department of Neuroscience, University of Padua, IT

    Presenting author: Matteo Bruzzone

    Exposition date: Friday, September 15, 2023

    Exposition position: 062/1

    The development of every-day more powerful techniques enables the generation of large and dense reconstructions of neuronal anatomy across a whole organism brain with a degree of resolution that can reach the synaptic detail. However, these reconstructions are still far from revealing the general connectivity patterns of the neuronal circuits across the brain and their functional organization in networks. Indeed, most frequently these datasets are analyzed following a bottom-up approach, starting from a “seed” neuron and then reconstructing its synaptic partners one after the other in order to reveal the organization of a single circuit component of interest. Even when the synaptic input/output relationship are known for all the neurons, there are not yet methods established to identify highly- connected neuronal communities and connectivity patterns. Here we describe a top-down approach that, combining graph theory methods with the information about the density of synaptic connections, allows to infer the most relevant neuronal connectivity patterns and with these the main functional networks within an organism brain. We applied this approach on two different datasets, the mesoscale light microscopy-based cell resolution atlas of the larval zebrafish brain and the electron microscopy reconstruction of the adult drosophila hemibrain, revealing architectural connectivity motifs aligned with their functional roles.

  • Retinal ganglion cells encode the direction of motion outside their classical receptive field

    Authors:
    Serena Riccitelli, Hadar Yaakov, Alina S. Heukamp, Lea Ankri, Michal Rivlin-Etzion

    Organisations:
    Weizmann Instituttet of Science, Israel

    Presenting author: Serena Riccitelli

    Exposition date: Friday, September 15, 2023

    Exposition position: 124/1

    Retinal ganglion cells (RGCs) typically respond to light stimulation over their spatially restricted receptive field. Using large-scale recordings in the mouse retina, we find that a subset of non-direction selective (DS) RGCs exhibit DS responses when motion stimuli are presented in an area far beyond their classical receptive field. The extraclassical DS response arises via inputs from an asymmetric distal zone and is enhanced by desensitization mechanisms. Pharmacological manipulations revealed the necessity of glycinergic amacrine cells and gap junctions for this response. Using in-vivo recordings in anesthetized mice, we identified similar extraclassical DS responses in lateral geniculate nucleus neurons, suggesting such non-conventional DS information is transferred to downstream structures. Our study reveals a novel mechanism for encoding direction selectivity in the extraclassical receptive field of non-DS cells, which could facilitate computation by compensating for phototransduction and other synaptic delays in retinal processing.

  • The role of importin α3 in sensory neurons upon in vitro injury

    Authors:
    Amisha Parmar (1), Livia Testa (1), Sofia Dotta (1), Giada Musso (1), Ferdinando Di Cunto (1), Franziska Rother (2,3), Michael Bader (2,3,4), Letizia Marvaldi (1)

    Organisations:
    1: Department of Neuroscience "Rita Levi Montalcini", Neuroscience Institute Cavalieri Ottolenghi, University of Turin, 10043 Orbassano (TO), Italy
    2: Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany
    3: Center for Structural and Cellular Biology in Medicine, Institute of Biology, University of Lübeck, 23538 Lübeck, Germany
    4: Charité – Universitätsmedizin Berlin, 10117 Berlin, Germany

    Presenting author: Amisha Parmar

    Exposition date: Thursday, September 14, 2023 (Satellite Events Day)

    Exposition position: 107

    Dorsal root ganglia (DRG) neurons can grow up to a meter long in the adult human. Upon injury, peripheral axons regenerate but mostly functional recovery is poor due to a slower growth rate and misrouting target, this as a consequence provokes neuropathic pain. How can we modulate growth and thus avoid aberrant regeneration and inaccurate target reinnervation? Previously, we observed that importin ɑ3 protein modulates the pain response via c-FOS transcription factor. So far, it is unknown if the reduction of importin ɑ3 and/or influence of neurotrophins (Neurotrophin-3, Nerve Growth Factor) modulate axonal growth and survival after injury. Here, we are investigating how the injury impacts axonal growth and neuronal survival using in vitro injury model in wildtype and importin ɑ3 mutant sensory neurons with or without neurotrophic factor treatment. Our high-throughput imaging and blinded quantification approach allowed us to obtain unbiased results. Future prospects are to provide insights on signaling mechanisms of regulation between axonal growth dynamics and pain perception in vivo.

  • The role of importin α3 in sensory neurons upon in vitro injury

    Authors:
    Amisha Parmar (1), Livia Testa (1), Sofia Dotta (1), Giada Musso (1), Ferdinando Di Cunto (1), Franziska Rother (2,3), Michael Bader (2,3,4), Letizia Marvaldi (1)

    Organisations:
    1: Department of Neuroscience "Rita Levi Montalcini", Neuroscience Institute Cavalieri Ottolenghi, University of Turin, 10043 Orbassano (TO), Italy
    2: Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany
    3: Center for Structural and Cellular Biology in Medicine, Institute of Biology, University of Lübeck, 23538 Lübeck, Germany
    4: Charité – Universitätsmedizin Berlin, 10117 Berlin, Germany

    Presenting author: Amisha Parmar

    Exposition date: Friday, September 15, 2023

    Exposition position: 128/1

    Dorsal root ganglia (DRG) neurons can grow up to a meter long in the adult human. Upon injury, peripheral axons regenerate but mostly functional recovery is poor due to a slower growth rate and misrouting target, this as a consequence provokes neuropathic pain. How can we modulate growth and thus avoid aberrant regeneration and inaccurate target reinnervation? Previously, we observed that importin α3 protein modulates the pain response via c-FOS transcription factor. So far, it is unknown if the reduction of importin α3 and/or influence of neurotrophins (Neurotrophin-3, Nerve Growth Factor) modulate axonal growth and survival after injury. Here, we are investigating how the injury impacts sensory neuronal growth and survival using in vitro injury model in wildtype and importin alpha3 mutant sensory neurons with or without neurotrophic factor treatment. Our high-throughput imaging and blinded quantification approach allowed us to obtain unbiased results. Future prospects are to provide insights on signaling mechanisms of regulation between axonal growth dynamic and pain perception in vivo.

Sociality and emotions

  • Hugging synchronizes the rhythm of two hearts

    Authors:
    Monia Cariola, Ilaria Mirlisenna, Alessandro Mazza, Olga Dal Monte

    Organisations:
    Department of Psychology, University of Turin, Torino, Italy

    Presenting author: Monia Cariola

    Exposition date: Friday, September 15, 2023

    Exposition position: 136/1

    Both in animals and humans, skin-to-skin contact represents a safety signaling mechanism which communicates proximity and affection, improves stress coping and decreases anxiety. In human social interactions, emotional support and affection is often expressed through physical touch, such as stroking, hand-holding, or hugging. It has been shown that hugging increases happiness and health through both mental and physical benefits; it also reduces blood pressure, heart rate, and cortisol levels while promoting oxytocin release.

    While several studies have investigated the beneficial effects of hugging in a single individual, the affective and physiological experience in both individuals remains largely unknown. Here, we investigated the autonomic responses (heart rate) in romantic couples when they were invited to hugging and holding hands. We also examined whether and how heart rate synchrony between romantic dyads would emerge during the act of hugging.

    Overall, participants showed a greater decrease in heart rate during hug with respect to hand-holding, suggesting a greater calming effect of hugs. We also found that both hugging and holding hands induced heart rate synchronization between romantic partners, but the physiological coupling was more pronounced during hugging conditions. These findings show for the first time that hugging a beloved one not only decreases heart rate of single individuals forming the dyad, but it also facilitates synchronization between two independent autonomic nervous systems. The strong physiological synchronization that emerges when two individuals are hugging might represent a key factor in promoting emotional engagement, and possibly contributes to the beneficial effects resulting from this social behavior.

  • Overgeneralization of implicit defensive reactions in obesity

    Authors:
    Federica Scarpina (1,2), Eugenio Manassero (1), Sofia Tagini (1,2), Giulia Concina (1), Massimo Scacchi (2), Alessandro Mauro (1,2), Benedetto Sacchetti (1)

    Organisations:
    1: University of Turin, Italy
    2: IRCCS Istituto Auxologico Italiano, Ospedale San Giuseppe, Piancavallo, Italy

    Presenting author: Eugenio Manassero

    Exposition date: Friday, September 15, 2023

    Exposition position: 135/1

    The phenomenological underpinning of anxiety is the primary emotion of fear. Because of this emotion, organisms activate defensive responses when facing dangers. Generalizing defensive responses to new events reminding learned threats is an adaptive process within an ever-changing environment. However, from a clinical perspective, evaluation mechanisms excessively biased toward generalization (i.e., overgeneralization) may underlie anxiety disorders and trauma. In the context of obesity – a clinical condition characterized by a higher prevalence of anxiety symptoms-, fear memory and generalization processes have never been investigated.

    In this research, we enrolled a sample of individuals affected by obesity and a sample of healthy individuals. We tested them at a two-days fear conditioning paradigm, in which participants learned to associate a standard tone (CS) with an unconditioned stimulus (US). Twenty-four hours later, we tested the ability to recognize the encoded stimuli as well as the reactions to novel stimuli. As experimental outcomes to verify fear overgeneralization, we assessed the skin conductance response, which is the implicit one, and the participant’s recognition of stimuli paired with the USs during the learning phase as threatening (i.e., explicit outcome).

    About the implicit outcome, participants with obesity had comparable skin conductance responses to a learned fearful stimulus, but crucially enhanced responses to new stimuli, when compared with the normal-weight participants. Instead, about the explicit outcome, no differences between groups were observed

    Our results provide the first evidence that obesity may widen fear generalization patterns, in relation to the level of awareness the process involved requires, through a fear conditioning paradigm. From a clinical perspective, this evidence supports the hypothesis according to emotional dysregulations, especially about fear, may underly clinical anxiety-related symptoms in obesity. Moreover, our results may support future translational research about fear overgeneralization in obesity.

  • Spatial frequency sensitivities for emotional body stimuli

    Authors:
    Maria-Chiara Villa (1), Alessio Borriero (1), Matteo Diano (1), Beatrice de Gelder (2), Marco Tamietto (1,3)

    Organisations:
    1: University of Turin, Italy
    2: Maastricht University
    3: Tilburg University

    Presenting author: Maria-Chiara Villa

    Exposition date: Friday, September 15, 2023

    Exposition position: 137/1

    Most of our current knowledge concerning the neuronal underpinnings of emotion perception draws on studies using facial expressions. Prior evidence suggests that partly segregated pathways engage in the processing of different spatial frequencies, with low spatial frequency signals (LSF) preferentially processed by dorsal stream areas, and high spatial frequencies (HSF) by the ventral stream. Bodily expressions are biologically meaningful signals to convey emotional information. While previous neuroimaging assays have investigated neural responses to bodily expressions, there is no evidence on the impact of spatial frequency filtering on the encoding of bodily expressions. To address this issue, we performed an fMRI study in which twenty healthy participants viewed emotional fearful and neutral bodily expressions filtered for different spatial frequencies (broadband, HSF, LSF). We used the searchlight approach, a machine learning-based multivoxel pattern analysis method (MVPA), to classify brain activations associated with the processing of fearful compared to neutral expressions according to the three different frequencies. After discounting activity associated with neutral bodily actions, we found that areas known to process emotional information and motor planning were capable of discriminating between spatial frequencies. Dorsal and ventral stream regions were selectively involved in analyzing LSF and HSF fearful expressions, respectively. Moreover, the premotor cortex was equally involved in fearful expressions in both frequencies, suggesting a role of this region in the processing of whole-body expressions regardless of frequency information. Overall, this study contributes to our understanding of how different spatial frequencies are combined to encode emotional value conveyed by bodily postures.

  • Union is strength: a novel paradigm to study social facilitation in mice.

    Authors:
    Giulia Palla (1,2), Nadia Giordano (1,2), Lorenzo Fruzzetti (3,4), Raffaele Mazziotti (2), Tommaso Pizzorusso (1,2), Luca Bonini (5), Matteo Caleo (6), Claudia Alia (2)

    Organisations:
    1: LABORATORIO DI BIOLOGIA, SCUOLA NORMALE SUPERIORE, PISA, ITALY
    2: NEUROSCIENCE INSTITUTE (IN), NATIONAL RESEARCH COUNCIL, PISA, ITALY.
    3: THE BIOROBOTICS INSTITUTE, SCUOLA SUPERIORE SANT'ANNA, PONTEDERA (PISA), ITALY.
    4: DEPARTMENT OF EXCELLENCE IN ROBOTICS AND AI, SCUOLA SUPERIORE SANT'ANNA, PISA, ITALY.
    5: DEPARTMENT OF MEDICINE AND SURGERY, UNIVERSITY OF PARMA, PARMA, ITALY.
    6: EPARTMENT OF BIOMEDICAL SCIENCES, UNIVERSITY OF PADUA, PADUA, ITALY.

    Presenting author: Giulia Palla

    Exposition date: Friday, September 15, 2023

    Exposition position: 134/1

    This abstract is dedicated to the memory of Prof. Matteo Caleo

    Action observation among conspecifics can lead, in some cases, to the repetition of the same action. This process, called social facilitation, seems to be at the basis of social interaction and communication within the members of the same group or species. It has been demonstrated that this process occurs mainly in species that live in groups such as birds, primates, and rodents.

    In the present study, we implemented a novel behavioural paradigm to investigate the social facilitation process in mice, focusing on how the presence of a drinking conspecific is able to modulate motivation and behaviour in the observer.

    We demonstrated that the observation of a highly motivated drinking mouse strongly facilitated drinking behaviour in a head-fixed observer, both if the demonstrator had free access to water or performed a foraging task. Specifically, the time spent drinking, was significantly higher when a drinking conspecific was observed compared to the observation of a non-drinking conspecific or the mere presentation of the drinking bottle. Moreover, we found that the observation of a drinking subject directly triggered the beginning of drinking by the observer. For each session, we considered the temporal distribution of the actor’s and observer’s drinking intervals, and we found a higher probability that the observer began to drink when the actor was already drinking.

    These results suggest that feeding behaviour in mice could be influenced, in particular enhanced, by the observation of feeding conspecifics. In an evolutionary perspective, our data provide information on the possibility of the existence in mice of a system involved in understanding behaviours with important social meaning.

    To support our hypothesis, we are currently studying neurophysiological mechanisms (e.g., pupil diameter, electrophysiological activity) underlying social facilitation in our murine model.

Synapses and plasticity

  • ADAM10 (dys)function at the Huntington’s Disease cortico-striatal synapse

    Authors:
    Andrea Scolz (1,2), Allegra Lenci (1,2), Elena Cattaneo (1,2), Chiara Zuccato (1,2)

    Organisations:
    1: Department of Biosciences, University of Milan, 20122, Milan, Italy
    2: Istituto Nazionale di Genetica Molecolare (INGM) Romeo ed Enrica invernizzi, 20122, Milan, Italy

    Presenting author: Andrea Scolz

    Exposition date: Saturday, September 16, 2023

    Exposition position: 143/2

    Huntington’s Disease (HD) is caused by a CAG repeat expansion in the Exon1 of the Huntingtin (HTT) gene. The striatum is the most vulnerable region, but prominent pathological alterations are visible also in the cortex. Indeed, multiple lines of evidence point to cortical dysfunctions and impaired cortico-striatal connection as early events in HD. As a result, presynaptic cortical dysfunction represents a very attractive site of action for developing new therapeutic approaches.

    Recent studies from our group have demonstrated that a novel HTT interactor, A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), is implicated in HD synaptic dysfunction. Indeed, wild-type HTT regulates glutamatergic synapse remodeling by binding to ADAM10 and inhibiting its proteolytic activity on N-Cadherin (N-CAD). Mutant HTT binds less to active ADAM10, which accumulates at the synapse weakening synaptic communication via excessive N-CAD proteolysis leading to cognitive decline in HD mice. Intriguingly, inhibiting ADAM10 rescues N-CAD proteolysis and prevents synaptic and cognitive defects (Vezzoli et al., 2019).

    Recently, we revealed that ADAM10’s interactome shares presynaptic binding partners with HTT, including vesicle transport/release regulators. These findings suggested an unexpected role of ADAM10 in orchestrating vesicle transport/release at the presynaptic district. We discovered that the level of active ADAM10 increases in the HD cortex’s presynaptic fractions, resulting in reduced synaptic vesicles. Notably, synaptic vesicle storage was restored by normalizing ADAM10 activity (Cozzolino et al., 2021).

    Based on this novel role of ADAM10 at the presynapse, we propose that cortico-striatal circuitry defects, and striatal pathology, could mostly depend on mutant HTT detrimental impact on presynaptic ADAM10 in cortical afferents. To this aim, we reconstructed the cortico-striatal circuitry on-a-chip exploiting a microfluidic-based platform that allows to inhibit ADAM10 separately in the pre- (cortical) and post-synaptic (striatal) compartments. The ADAM10 inhibitor GI254023X was administered to cortical and striatal neurons independently, and cortico-striatal functionality was assessed.

  • Brain histone beta-hydroxybutyrylation couples metabolism with gene expression

    Authors:
    Sara Cornuti (1), Siwei Chen (2), Leonardo Lupori (1), Francesco Finamore (3), Fabrizia Carli (3), Muntaha Samad (2), Simona Fenizia (3), Matteo Caldarelli (4), Francesca Damiani (1), Francesco Raimondi (1), Raffaele Mazziotti (5,6), Christophe Magnan (2), Silvia Rocchiccioli (3), Amalia Gastaldelli (3), Pierre Baldi (2), Paola Tognini (1,4)

    Organisations:
    1: Bio@SNS Lab, Scuola Normale Superiore, Pisa, Italy
    2: Institute for Genomics and Bioinformatics, School of Information and Computer Sciences, University of California, Irvine, CA, USA
    3: Institute of Clinical Physiology, National Research Council, Pisa, Italy
    4: Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Pisa, Italy
    5: Institute of Neuroscience, National Research Council, Pisa, Italy
    6: Department of Developmental Neuroscience, IRCCS Stella Maris Foundation, Calambrone, Pisa, Italy.

    Presenting author: Sara Cornuti

    Exposition date: Friday, September 15, 2023

    Exposition position: 141/1

    Little is known about the impact of metabolic stimuli on brain tissue at a molecular level. The ketone body beta-hydroxybutyrate (BHB) can be a signaling molecule regulating gene transcription. Thus, we assessed lysine beta-hydroxybutyrylation (K-bhb) levels in proteins extracted from the cerebral cortex of mice undergoing a ketogenic metabolic challenge (48 h fasting). We found that fasting enhanced K-bhb in a variety of proteins including histone H3. ChIP-seq experiments showed that K9 beta-hydroxybutyrylation of H3 (H3K9-bhb) was significantly enriched by fasting on more than 8000 DNA loci. Transcriptomic analysis showed that H3K9-bhb on enhancers and promoters correlated with active gene expression. One of the most enriched functional annotations both at the epigenetic and transcriptional level was “circadian rhythms''. Indeed, we found that the diurnal oscillation of specific transcripts was modulated by fasting at distinct zeitgeber times both in the cortex and suprachiasmatic nucleus. Moreover, specific changes in locomotor activity daily features were observed during re-feeding after 48-h fasting. Thus, our results suggest that fasting remarkably impinges on the cerebral cortex transcriptional and epigenetic landscape, and BHB acts as a powerful epigenetic molecule in the brain through direct and specific histone marks remodeling in neural tissue cells.

  • Cortical rewiring after peripheral injection of botulinum neurotoxin type A

    Authors:
    Alexia Tiberi (1,2), Verediana Massa (1), Elena Montagni (3), Marco Pirazzini (4), Ornella Rossetto (4), Anna Letizia Allegra Mascaro (1,3), Matteo Caleo (1), Laura Restani (1)

    Organisations:
    1: Neuroscience Institute, National Research Council (CNR), Pisa, Italy
    2: Scuola Normale Superiore, Pisa, Italy
    3: European Laboratory for Non-Linear Spectroscopy, University of Florence, Florence, Italy
    4: Department of Biomedical Sciences, University of Padua, Padua, Italy.

    Presenting author: Alexia Tiberi

    Exposition date: Saturday, September 16, 2023

    Exposition position: 142/2

    Botulinum neurotoxin type A1 (BoNT/A1) is a highly effective treatment for neurological conditions characterized by neuronal hyperactivity, such as dystonia. By specifically and reversibly blocking synaptic vesicle fusion at the neuromuscular junction (NMJ), this bacterial metalloprotease is indeed able to alleviate symptoms. However, evidence suggests that a fraction of BoNT/A1 can also undergo long-distance axonal transport, potentially affecting central circuits. Moreover, fMRI studies on human patients have demonstrated broad and long-lasting changes in cortical activity after BoNT/A1 injection, at timescales that are not compatible with the peripheral blockade at the level of the NMJ. In this study, we investigated whether and how BoNT/A1 peripheral injections can influence cortical areas. While BoNT/A1 injection into the whisker pad led to an evident paralysis lasting around 10 days, we found a decrease in spine density in cortical motor areas 30 days after the injection. To understand the mechanism underlying this decrease, we measured spine dynamics longitudinally in awake mice using two-photon microscopy, revealing an increase in spine elimination. Notably, BoNT/A1-injected mice also exhibited deficits in a whisker-dependent learning paradigm at the peak of paralysis, as expected because of the lack of sensory input, but also well after its disappearance, suggesting a rearrangement of central circuits. In fact, analysis of c-Fos expression showed decreased activation in different cortical and subcortical areas in these animals. Furthermore, wide-field calcium imaging uncovered profound changes in intra- and inter-hemispheric connectivity up to 30 days after injection. Taken together, our data indicate that BoNT/A1 injections can result in persistent cortical changes, which may contribute to its therapeutic efficacy in neuropathologies and the long-lasting benefits observed in patients.

  • Impaired cerebellar development/function in a mouse model of Niemann-Pick C1

    Authors:
    Greta Massa (1,2), Serena Camuso (1,2), Jessica Tiberi (1,2), Roberta Stefanelli (1), Piergiorgio La Rosa (1), Maria Teresa Fiorenza (1), Sonia Canterini (1)

    Organisations:
    1: University of Rome "Sapienza", Department of Psychology, Division of Neuroscience, Rome, Italy
    2: Ph.D Program in Behavioral Neuroscience, University of Rome “Sapienza”, Rome, Italy

    Presenting author: Greta Massa

    Exposition date: Friday, September 15, 2023

    Exposition position: 139/1

    The cerebellum is a multifunctional brain region that controls various motor and non-motor behaviors. Thus, impairments in cerebellar architecture and circuitry lead to a wide range of neurodevelopmental/neuropsychiatric disorders.

    During postnatal development, the cerebellum undergoes changes in its cellular organization, controlled by neurotrophic factors, such as BDNF-TrkB signaling that is involved in the proper development, synaptogenesis, and maintenance of cerebellar circuitry.

    In Niemann-Pick C1 (NPC1) disease, a rare lipid-storage disorder, we have previously demonstrated that a reduction in the Shh and BDNF expression in the first weeks of postnatal development disturbs the proliferation and migration of cerebellar granule cells (GCs) influencing the final cerebellar cytoarchitecture. As for the GCs, their complete differentiation into the inner granular layer by connecting, via their dendrites, with mossy fiber axons and establishing the major synaptic complex, known as the glomerulus.

    In Npc1 mice, by immunohistochemistry and Neurolucida analysis, we observed a significant reduction in the number, area and tortuosity of glomeruli at different stages of early postnatal life. These results led us to investigate the presence of functional defects at the level of mature glutamatergic synapses. Therefore, by subcellular protein fractionation, we investigated the expression levels of specific pre-synaptic (Syntaxin 1A, VAMP2, SNAP-25) and postsynaptic (Drebrin, Shank3) proteins during different stages of postnatal cerebellar development and revealed a general SNAP-25 deficiency in Npc1 mice versus wt.

    Furthermore, by Golgi-Cox staining analysis, we also characterized the density/morphology of GC dendritic spines in the IGL, both in wt and mutant mice, to identify defects in synapse maturation and pruning processes during the key steps of cerebellar development.

    Finally, male Npc1 mice showed no preference for social/nonsocial stimulus in a typical task exploited to study autistic-like behaviour, consistent with studies demonstrating reduced levels of cerebellar BDNF in autistic patients.

  • Investigating the effects of subtoxic dosage of glyphosate on primary neuronal and astrocytic cultures.

    Authors:
    Debora Comai (1), Giuseppe Chiantia (1), Antonia Gurgone (1), Vita Cardinale (1), Andrea Marcantoni (2), Luca Munaron (3), Maurizio Zibetti (1,4), Maurizio Giustetto (1)

    Organisations:
    1: Department of Neuroscience “Rita Levi-Montalcini”, University of Turin, Turin (Italy)
    2: Department of Drug Science, Laboratory of Cell Physiology and Molecular Neuroscience, N.I.S. Centre, University of Turin, Turin (Italy)
    3: Department of Life Sciences and System Biology, University of Turin, Turin (Italy)
    4: SC Neurologia 2U AOU Città della Salute e della Scienza, Turin (Italy)

    Presenting author: Debora Comai

    Exposition date: Friday, September 15, 2023

    Exposition position: 142/1

    Glyphosate (Gly)-based compounds are the most widely used herbicides worldwide that act by inhibiting the enolpyruvylshikimate-3-phosphate synthase of the shikimate pathway, an enzyme expressed in plants but not in mammals. The safety of Gly is under scrutiny since several years: beside being indicated as probably carcinogenic to humans, recent studies revealed a positive correlation between the rise of Gly application in agriculture and increased incidence of neurological disorders such as autism, Parkinson’s and Alzheimer’s diseases. Nevertheless, very little is known on the effects of Gly on brain cells and few studies tested the neurotoxicity of the acceptable daily intake (ADI) dose. To fill this gap, we investigated the consequences of an acute Gly (3µM) treatment on primary neuronal cultures using both patch-clamp recordings and immunofluorescence. Evoked excitatory and inhibitory postsynaptic currents analyses revealed that Gly tampers with inhibitory GABAergic, but not glutamatergic neurotransmission by reducing its amplitude. Accordingly, both amplitude and frequency of miniature inhibitory post-synaptic currents were affected by Gly administration, indicating that both pre- and post-synaptic GABAergic compartments are influenced. Consistently, morphological analyses supported the functional data. The density of both pre- (vGAT-positive) and post-synaptic (gephyrin-positive) inhibitory compartments was decreased in Gly-treated neurons while excitatory synapse structures seemed unaffected.

    Moreover, we are assessing the ADI dosage of Gly on astrocyte cultures as these cells play a primary role in the formation of the blood-brain-barrier. Using quantitative immunofluorescence analyses and confocal microscopy both cell viability and reactive status of these cells are being assessed. Furthermore, to verify if cell-to-cell communication mechanisms are involved in Gly-treatment consequences, we are studying the impact on neurons of astrocyte-derived extracellular vesicles on synaptic formation and transmission.

    Our study disclosed novel cellular and synaptic defects produced by subtoxic dosage of Gly and prompts further analysis to understand the underlying molecular mechanisms.

  • Investigating the molecular diversity of COPII-dependent transport in cortical neurons

    Authors:
    Federica Baronchelli (1,2), Martina Biagioni (3), Giuseppe Martano (1,3), Sara Francesca Colombo (1), Matteo Fossati (1,3)

    Organisations:
    1: CNR Neuroscience Institute, Milano, Italy
    2: Humanitas University, Pieve Emanuele (MI), Italy
    3: Humanitas Research Hospital, Rozzano (MI), Italy

    Presenting author: Federica Baronchelli

    Exposition date: Friday, September 15, 2023

    Exposition position: 140/1

    Neurons are the most highly compartmentalized and morphologically complex cells of the body. In particular, synapses are dynamic nanomachines composed by a unique repertoire of molecules arranged in multimolecular complexes. For this reason, their correct development and function require sophisticated mechanisms to target proteins and lipids to their site of action in the right amount at the right time. However, how this daunting task is achieved is largely unknown. Here, we focus on the role of the first stations of the secretory pathway, namely the Endoplasmic Reticulum (ER) and the Golgi Apparatus (GA), in the transport of newly synthetized neuronal proteins to their final destination. In particular, we investigate the role of the molecular diversity of SEC24, a component of the inner coat of COPII vesicles involved in cargo selection. To this aim, we developed a proteomic screen based on proximity-dependent biotinylation to identify the protein interaction networks of distinct SEC24 isoforms. Preliminary data obtained from heterologous cells and primary cultures of cortical neurons indicate that SEC24 proteins fused to the biotin ligase are enriched at the ER exit sites, interact with protein cargoes and, upon biotin administration, efficiently biotinylate proteins in an isoform-specific manner. Second, we are also investigating the subcellular localization of SEC24 isoforms in neuronal compartments to assess their potential role in directing cargoes to specific transport pathways. Together, our approaches may shed the light on the contribution of the early secretory pathway to neurodevelopment and unravel novel mechanisms underlying the formation and function of synaptic connections.

  • Modulation of Cofilin 1 phosphorylation affects structural plasticity in the adult mouse visual cortex.

    Authors:
    Agustina Dapueto (1), Bruno Pannunzio (2), Francesco Mattia Rossi (3)

    Organisations:
    1: Lab. Mecanismos de Neurodegeneración y Neuroprotección, Dept. Neuroquímica, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay
    2: Dept. Histología y Embriología, Facultad de Medicina, UdelaR / Lab. Neuroinflamación y Terapia Génica, Institut Pasteur, Montevideo, Uruguay
    3: Lab. Neurociencias "Unidad de Neuroplasticidad", Facultad de Ciencias, UdelaR, Montevideo, Uruguay

    Presenting author: Francesco Mattia Rossi

    Exposition date: Friday, September 15, 2023

    Exposition position: 138/1

    Cofilin 1 is an actin depolymerizing protein important for spine morphology, receptor trafficking and synaptic plasticity. We recently identified cofilin 1 as a potential candidate controlling plasticity levels in the mouse visual cortex. Here we analyze the effects of modulating cofilin 1 activity on structural plasticity processes.

    Spine density was estimated on layer II-III and V pyramidal neurons in the visual cortex of Golgi-like stained brains analyzing 40x images with Fiji software. Monocular deprivation (MD) was performed by suturing the eyelids of the right eye during three days during the critical period (CP, P28-P31) or in adults (AD, P57-P60). Cofilin 1 activity modulation was obtained by acute intravenous administration of the phospho-cofilin peptide (TAT-pS3, 15pmol/g) or a peptide control (TAT, 15pmol/g).

    We first showed that spine density decreases 55% from critical period to adulthood (CP: 176.0±4.4, n=24, vs. AD: 79.4±3.3, n=32). We then analyzed the effects of MD on structural plasticity aspects. We found that during the critical period, MD reduces spine density in the contralateral vs. ipsilateral visual cortex of approximately 35% (contral.: 113.9±1.5 vs. ipsil.: 176.0±4.4, n=24), while no effects were observed in adult mice (contral.: 88.1±3.6 vs. ipsil.: 92.4±5.5, n=21).

    Finally, we analyzed if modulation of cofilin activity affects structural plasticity processes in adult mice. One day following peptide treatment, mice were monocularly deprived for three days and sacrificed. We found that MD reduces spine density in the contralateral vs. ipsilateral visual cortex in peptide-treated mice of approximately 41% (contral.: 49.6±3.5 vs. ipsil.: 83.9±8.7, n=6), but not in adult mice treated with the control peptide (contral.: 82.3±5.0 vs. ipsil.: 76.5±4.5, n=16).

    The present data suggest that cofilin 1 is a relevant player in the control of experience-plasticity in the mouse visual cortex and that its modulation favors reopening of high plasticity levels at adult stages.

  • Role of adenosine A2A/A2B receptors on CA1 synaptic plasticity or during an ischemic-like insult

    Authors:
    Martina Venturini (1), Clara Santalmasi (1), Federica Cherchi (1), Lucia Frulloni (1), Ilaria Dettori (1), Daniela Catarzi (2), Felicita Pedata (1), Vittoria Colotta (2), Flavia Varano (2), Elisabetta Coppi (1), Anna Maria Pugliese (1)

    Organisations:
    1: Department of Neuroscience, Psychology, Drug Research and Child Health, NEUROFARBA, Section of Pharmacology and Toxicology, University of Florence, Italy
    2: Department of Neuroscience, Psychology, Drug Research and Child Health, NEUROFARBA, Section of Pharmaceutical and Nutraceutical Sciences, University of Florence, Italy

    Presenting author: Martina Venturini

    Exposition date: Saturday, September 16, 2023

    Exposition position: 141/2

    In recent years the use of multi-target compounds has become an increasingly pursued strategy to treat complex pathologies, included cerebral ischemia, considering their several advantages (i.e. eliminate the risk of drug-drug interactions, reduction of possible side effects, pharmacokinetics and metabolism). Adenosine is a neuromodulator exerting its functions via four receptor subtypes, A1, A2A, A2B, and A3 that are known to play a crucial role on hippocampal synaptic transmission, either in normoxic or ischemic-like conditions. Adenosine A2A and A2B receptors (A2AARs, A2BARs) are both coupled to Gs-protein and expressed at hippocampal level. Their stimulation by using respective selective agonists, CGS21680 and BAY60-6583, it is known to inhibit paired-pulse facilitation (PPF), an electrophysiological paradigm whose reduction reflects an increase in presynaptic glutamate release at the hippocampal level. Firstly, we investigated the effect/s of the newly synthetized dual antagonist P626, able to simultaneously block A2AAR/A2BAR, by extracellular recordings of synaptic potentials in the CA1 region of rat hippocampal slices under normoxic conditions. We demonstrated that P626 prevented PPF reduction induced by CGS21680 (50 nM) or BAY60-6583 (200 nM). Furthermore, previous data demonstrated that the selective antagonism of A2AAR or A2BAR delays anoxic depolarization (AD) appearance, an unequivocal sign of neuronal injury induced by a severe oxygen-glucose deprivation (OGD) insult in the rat hippocampus. Therefore, in a next experimental series, we investigated the effects of P626, applied before, during and after a severe OGD insult (30-min long) on CA1 neurotransmission by extracellular recordings. The dual compound was able to significantly delay the AD appearance in a concentration dependent manner without affecting AD amplitude. P626 may represent a putative neuroprotective compound for stroke treatment with the possible translational advantage to reduce side effects and to bypass differences in pharmacokinetics due to a combined treatment.