6° MEETING NEAPOLITAN BRAIN GROUP 14 Dicembre 2017 -...

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6° MEETING NEAPOLITAN BRAIN GROUP

14 Dicembre 2017

LIBRO DEGLI ABSTRACT

Stazione Zoologica Anton Dohrn Villa Comunale, 80121 Napoli

Comitato scientifico E. Del Giudice (UNINA), C. Lucini (UNINA), E. Di Schiavi (IBBR, CNR),

M. Cataldi (UNINA), P. Sordino (SZN), A. Cellerino (Scuola Normale Superiore di Pisa), E.M. Valente (Università di Pavia), M.A. Hilliard (QBI, Australia).

Neapolitan Brain Group Gruppo di confronto tra ricercatori di base e clinici sulla fisiologia e la patologia del sistema nervoso con il Patrocinio morale dell'Università degli Studi di Napoli Federico II Coordinatore: Ennio Del Giudice

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Con il supporto di

con il patrocinio morale della

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6° MEETING NEAPOLITAN BRAIN GROUP

14 Dicembre 2017

PROGRAMMA


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09.00 - 09.15 Presentazione dell'SZN e saluti del Presidente Roberto Danovaro, saluti del coordinatore dell’NBG Ennio Del Giudice 09.15 - 11.00 PRIMA SESSIONE Chair: P. Sordino (SZN), G. Andria (UNINA) 09.15-9.30: Brain metabolic DNA is synthesized by reverse trascription in cytoplasmic organelles (Rutigliano, UNINA) 09.30-9.45: Nerve regeneration in the cephalopod mollusc Octopus vulgaris: from images to discoveries (Imperadore, SZN) 09.45-10.00: Characterization of smn-1 genetic interactors in a C.elegans SMA model (Santonicola, IBBR) 10.00-10.15: Study of neural stem cell biology in a mouse model of Rett syndrome (Squillaro, UNINA2) 10.15-10.30: Inhibition of monoacylglycerol lipase terminates diazepam-resistant status epilepticus in mice and its effects are potentiated by a ketogenic diet (Terrone, UNINA/M.Negri) 10.30-10.45: Orexin and endocannabinoid morphological interactions in the brain of adult zebrafish (Imperatore, UNISANNIO/ICB) 10.45-11.00: Shutting down translation of Amyloid Precursor protein in early symptomatic hAPP mutan mice rescues the Alzheimer Disease phenotype (Borreca, IBCN) 11.00 - 11.30 COFFEE BREAK e POSTERS 11.30 - 13.00 SECONDA SESSIONE Chair: M. Cataldi (UNINA), G. Fiorito (SZN) 11.30-11.45: Protocadherins and their role in brain complexity – a tale from Octopus (Styfhals, SZN) 11.45-12.00: Evidence of increased oxidative stress in Pompe disease. A new therapeutic target? (Tarallo, UNINA/TIGEM) 12.00-12.15: Regulation of trafficking and folding of cellular prion protein PrPC and its shadow Shadoo (Sarnataro, UNINA/CEINGE) 12.15-12.30: Orexin-Aenhances dopaminergic signaling in the brain of obese mice (Tunisi, ICB/UNINA) 12.30-12.45: Onecut gene function in the CNS of chordates: development and evolution (Locascio, SZN) 12.45-13.00: Targeting neuronal proteostasis to treat the CNS in lysosomal storage diseases (Monaco, TIGEM)

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13.00 - 13.30 LUNCH BREAK 13.30 - 14.00 SESSIONE POSTERS 14.00 - 15.30 TERZA SESSIONE Chair: E. Di Schiavi (IBBR), B. Franco (TIGEM) 14.00-14.15: Myoclonic Epilepsy of Unverricht and Lundborg (EPM1): understanding the role of Cystatin b in human cerebral organoids (Pipicelli, UNINA/Max Planck) 14.15-14.30: A rat model of perinatal stress: impact on gene expreession of glutamatergic postsynaptic density genes (Avagliano, UNINA) 14.30-14.45: Modulation of aging pathways as a therapeutic strategy for Parkinson’s disease (Decressac, TIGEM) 14.45-15.00: Alteration of endosomal trafficking is associated with neurodegenerative diseases (Paladino, UNINA/CEINGE) 15.00-15.15: Effects of Methylcyclopentadienyl Manganese Trycarbonil on dopaminergic neurons in zebrafish (Fasano, SZN/UNISANNIO) 15.15-15.30: Striatonigral involvement in Fabry Disease: a quantitative and volumetric MRI study (Russo, UNINA) 15.30 - 16.00 COFFEE BREAK e POSTERS 16.00 - 17.30 QUARTA SESSIONE Chair: C. Lucini (UNINA), M. Taglialatela (UNINA) 16.00-16.15: The inflammatory response following acute seizures in zebrafish brain and in chordate evolution (Nittoli, SZN) 16.15-16.30: Pragmatic abilities in multiple sclerosis: an RS-FMRI study (Cocozza, UNINA) 16.30-16.45: Default Mode Network modifications in Fabry Disease (Cocozza, UNINA) 16.45-17.00: Brain functional changes and cognitive dysfunction in Friedreich's Ataxia (Cocozza, UNINA) 17.00-17.15: Dissecting PRUNE-1/NME1 function in brain and cerebellum of Microcephaly and Peho syndrome affected patients (Zollo, UNINA/CEINGE) 17.15-17.30: De novo gain-of-function variants in the Slo2 family of Na+-activated K+ channels are responsible for developmental and epileptic encephalopathies (Manocchio, UNIMOL) 17.30 CONCLUDING REMARKS E. Del Giudice

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Il Neapolitan Brain Group L'NBG è un gruppo di confronto tra ricercatori di base e clinici dell'area napoletana (e, più in generale, campana), interessati allo studio della fisiologia e delle patologie del sistema nervoso. Il gruppo nasce nel 2015 su idea del Professor Ennio Del Giudice dell'Università degli Studi di Napoli Federico II, Dipartimento di Scienze Mediche Traslazionali (Sezione di Pediatria). Il gruppo vuole quindi essere un'occasione di incontro, in un'atmosfera informale, per tutti gli appassionati di ricerca clinica e di base che intendano migliorare la reciproca conoscenza e, per quanto possibile, esplorare momenti di collaborazione proficua. L'NBG è aperto a tutti coloro che siano interessati a queste tematiche, in particolare ai giovani in formazione, quali dottorandi, postdoc, specializzandi, studenti e tirocinanti delle Università e degli Enti di Ricerca. Il 4 giugno 2015, il 4 febbraio, il 28 aprile 2016 ed il 15 Dicembre 2016 sono stati organizzati i primi incontri con brevi comunicazioni, mentre il 9 giugno 2016 è stata organizzata una riunione monotematica di interesse generale su “Meccanismi molecolari, fisiopatologici e clinici nella neuroprotezione dell’ipossia neonatale”. In queste riunioni hanno presentato i loro risultati più di 90 tra Ricercatori, Medici, Dottorandi, Postdoc e Specializzandi delle diverse Università campane, del CNR, del TIGEM, dell'SZN e del CEINGE. Per maggiori informazioni e per essere aggiornati sul gruppo: http://www.neapolitanbraingroup.it/ https://www.facebook.com/NBG2000/ Per essere inseriti alla mailing list scrivete a: Elia Di Schiavi ([email protected]). Gli organizzatori del gruppo NBG: Ennio Del Giudice ([email protected]), Carla Lucini ([email protected]), Mauro Cataldi ([email protected]), Elia Di Schiavi ([email protected]).


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La Stazione Zoologica Anton Dohrn

Anton Dohrn, il fondatore e primo direttore della Stazione Zoologica Anton Dohrn, era nato a Stettino, in Pomerania, oggi parte della Polonia, nel 1840, da una famiglia della buona borghesia. Dohrn studiò zoologia e medicina in varie università tedesche, ma i suoi ideali cambiarono quando, nel 1862, Ernst Haeckel lo introdusse alle opere e alle teorie di Charles Darwin. Dohrn divenne un fervente difensore della teoria di Darwin della "discendenza con modificazione", la teoria dell'evoluzione per selezione naturale.

Decise allora di dedicare la sua vita alla raccolta di fatti e di idee a sostegno del darwinismo. Durante la sua carriera universitaria trascorse periodi di ricerca sulla riva del mare: a Helgoland, ad Amburgo, a Millport in Scozia e a Messina. Qui prese corpo il progetto di coprire il globo con una rete di stazioni di ricerca biologica dove gli scienziati avrebbero potuto trovare un tavolo di lavoro già pronto, con un laboratorio, servizi, prodotti chimici, riviste e libri, e le informazioni di dove e quando certe specie potevano essere trovate. La scelta di Napoli era dovuta alla grande ricchezza biologica del mar Mediterraneo ed anche alla possibilità di sviluppare un istituto di ricerca in una città a vocazione internazionale e di grandi dimensioni. Dopo una visita all'acquario di Berlino, egli aveva pensato che un acquario pubblico avrebbe potuto guadagnare abbastanza da pagare un assistente permanente per i laboratori. Mettendo insieme immaginazione, forza di volontà, abilità diplomatica ed una buona dose di fortuna, grazie al sostegno amichevole di scienziati, artisti e musicisti, Anton Dohrn superò dubbi, ignoranza ed incomprensioni e riuscì a persuadere le autorità comunali di cedergli un pezzo di terreno sulla riva del mare, nel bel Parco Reale, oggi Villa Comunale. Da parte sua promise di costruire la Stazione Zoologica a sue spese. Nel settembre 1873 il primo edificio era terminato, e l’inaugurazione ufficiale della Stazione Zoologica ebbe luogo il 14 aprile 1875. Una delle caratteristiche che era alla base del successo della istituzione era la notevole agilità e flessibilità della sua struttura. Si trattava di una istituzione internazionale per natura, fondata da un tedesco, gestita come un'impresa familiare e organizzata secondo il modello accademico tedesco, ma localizzata in Italia, con una grande apertura ai contributi finanziari e scientifici di ogni paese e istituzione. L'idea di una struttura agile, flessibile, piccola ma piena di coraggio e di spirito di iniziativa, costituisce lo "spirito" costante della Stazione, dalle sue origini ad oggi. Allo scopo di promuovere la natura internazionale della Stazione e per garantire la sua indipendenza politica ed economica, e la libertà della ricerca, Dohrn introdusse una serie di misure innovative per finanziare il suo progetto, come l'affitto di spazi di lavoro e di ricerca "senza condizioni", nel senso che i ricercatori erano completamente liberi di perseguire i propri progetti e le proprie idee, e la vendita di campioni e preparati biologici. Convinto che la disponibilità di tutte le principali fonti stampate fosse una necessità per la ricerca di punta, Anton Dohrn donò la sua importante biblioteca alla Stazione Zoologica e richiese donazioni agli editori scientifici, accademie e agli scienziati, come Darwin, Huxley, Virchow. Complessivamente, le collezioni bibliografiche della Stazione divennero rapidamente uno strumento senza pari per la ricerca bibliografica in Europa.


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COMUNICAZIONI ORALI

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BRAIN METABOLIC DNA IS SYNTHESIZED BY REVERSE TRASCRIPTION IN CYTOPLASMIC ORGANELLES B. Rutigliano, M. Prisco, J. Casalino, C. Cefaliello, A. Giuditta Biology Department, Federico II University

Brain metabolic DNA (BMD] is not involved in cell division or DNA repair but is modulated by learning, sleep memory processing, and circadian oscillations. Using routine methods of subcellular fractionation, newly synthesized BMD from male rats was shown to be localized in crude nuclear, mitochondrial, and microsomal fractions and in two fractions of purified nuclei. Sub-fractionation of the mitochondrial fraction also indicated its prevalent presence in free mitochondria, synaptosomes and myelin. Cesium density profiles of homogenate and subcellular BMD analyzed after short and longer incorporation times strongly suggested that BMD is synthesized by reverse transcription in cytoplasmic organelles before acquiring the double stranded DNA conformation and being transferred to nuclei. Kinetic analyses covering incorporation periods of several weeks provided evidence that subcellular BMD undergoes a massive turnover that depends on rat age. Recent immunofluorescence data of mouse BrdU-labeled BMD have confirmed its cytoplasmic localization and reverse transcription. Data suggest that BMD might act as a temporary store of cellular responses to environmental changes that could be used in forthcoming experiences. The view supports the concept of a somatic genome that is dynamically modulated by adaptation to the environment.

Neapolitan Brain Group Gruppo di confronto tra ricercatori di base e clinici sulla fisiologia e la patologia del sistema nervoso con il Patrocinio morale dell'Università degli Studi di Napoli Federico II

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NERVE REGENERATION IN THE CEPHALOPOD MOLLUSC OCTOPUS VULGARIS: FROM IMAGES TO DISCOVERIES P. Imperadore1,2, K. Orellana3, G. Fiorito1 1 Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, Napoli, Italy 2 Association for Cephalopod Research - CephRes, Napoli, Italy 3 Life Science Research, Leica Microsystems, Milano, Italy Pallial nerve lesion determines interruption of the circuitry connecting the central nervous system and the periphery, resulting in paralysis of Octopus vulgaris mantle muscles supporting breathing and in the loss of the central control of body patterning, at the level of the denervated area. Complete functional recovery occurs at about 30 days for normal ventilation, and about two months for reinstating the full control of body pattern. To disclose cellular and structural events occurring in the injured nerves we utilized a combination of approaches including multiphoton microscopy, that we applied to cephalopods tissues for the first time. These allow label-free ‘method’ to distinguish structures, overcoming the lack of commercial markers designed for this taxon. We identified inflammatory responses characterized by hemorragic areas and scar formation, through Coherent anti-Stokes Raman scattering (CARS) and Two-Photon Excited Fluorescence (TPEF). Hemocytes were recognized as major contributor of this response, invading all altered tissues and actively proliferating. Nerve fibers swell and fragment, giving intense endogenous fluorescence signals (TPEF), thus allowing easy recognition of degenerating fibers. Intense regeneration of fibers is observed starting in the central stump and later visible on the opposite side (CARS). The two stumps regenerate toward each other, driven by the connective tissue, as recognized using Second Harmonic Generation signal (SHG). Our results show that in octopus: i. Wallerian degeneration occurs in the pallial nerve after lesion, similar to what observed in mammals after axotomy; ii. connective tissue guides axons to re-innervate tissue targets and re-establish the lost function.


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CHARACTERIZATION OF SMN-1 GENETIC INTERACTORS IN A C.ELEGANS SMA MODEL P. Santonicola , I. Gallotta, G. Zampi, E. Di Schiavi Institute of Bioscience and BioResources, IBBR, CNR, Naples, Italy Spinal muscular atrophy (SMA) is a genetic neuromuscular disorder caused by mutations in the evolutionary conserved Survival of Motor Neurons gene (SMN1). The loss of SMN1 induces a selective degeneration of lower motor neurons (MNs), leading to progressive muscle atrophy and death, but the pathogenesis of the disease and the function played by SMN1 in MNs are still elusive. We used C.elegans as animal model for its the powerful genetics and ease of nervous system analysis to study SMN1 in vivo function in MNs survival. We demonstrated that SMN orthologue, smn-1, is functionally conserved and its depletion in 19 ventral MNs causes an alteration in movement, an age-dependent degeneration and neuronal death (Gallotta et al., HMG 2016). We then identified several genes that genetically interacts with smn-1 for neuron survival. Interestingly some of these genes are able to prevent the apoptotic death but not the onset of degeneration, while others fully protect neuronal function, integrity and survival. By identifying new modifiers of SMN function we are elucidating the molecular mechanisms that underlie neuron degeneration. Moreover we are using a medium-thoughput drug screening to identify small synthetic and natural compounds that revert the neurodegeneration. Since the only treatment recently launched on the market has a limited uptake in many tissues, crucial targets in SMA, and presents obvious clinical challenges, the identification of new molecular targets and new small molecules will be crucial for ameliorating the symptoms of the disease.


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STUDY OF NEURAL STEM CELL BIOLOGY IN A MOUSE MODEL OF RETT SYNDROME T. Squillaro1, N. Alessio2, S. Capasso2, S. Del Gaudio2, G. Di Bernardo2, M. Cipollaro2, MAB Melone1, G. Peluso3, U. Galderisi2,4

1. Department of Medical, Surgical, Neurological, Metabolic Sciences, and Aging; Division of Neurology and InterUniversity Center for Research in Neurosciences, University of Campania "Luigi Vanvitelli", Naples, Italy. 2. Department of Experimental Medicine, University of Campania “Luigi Vanvitelli”, Naples, Italy. 3. Institute of Agro-Environmental Biology and Forestry (IBAF), CNR, Naples, Italy. 4. Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, Temple University, Philadelphia, PA, USA Factors affecting chromatin status, such as DNA methylation, histone modifications, and chromatin remodeling are key players of gene expression governance; their deregulation is connected with impairment of stem cell functions. Among the various methylated DNA binding proteins able to regulating gene expression, researchers are particularly interested in MECP2 since its mutation is associated with Rett syndrome (RTT), a severe neurological disease that affects almost exclusively girls. Nevertheless, the MECP2 role in this disease remains poorly understood. Previously, we demonstrated that mesenchymal stem cells obtained from Rett patients are prone to senescence. Thus, we decided to use a mouse model of RTT to evaluate whether residual MECP2 activity in neural stem cells (NSCs) induced senescence phenomena that could affect stem cell function. We demonstrated that Mecp2+/- NSCs showed an increase in senescence, impairment of proliferation capacity, and accumulation of unrepaired DNA foci. Moreover, mutated NSCs did not cope with genotoxic stress as it occurs in control cells. After treatment with different DNA damaging agents, Mecp2+/- NSCs accumulated more DNA damage foci (gamma-H2AX+) and were more prone to cell death with respect to controls. Senescence in mutated NSCs decreased the number of stem cells and progenitors and gave rise to a high percentage of cells that neither expressed stem/progenitor nor differentiation markers. These cells could be senescent and dysfunctional. Our results demonstrated that senescence of stem cell compartments may be associated with impaired function of MECP2 and paved the way to set up anti-aging treatments in an animal model of RTT.


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INHIBITION OF MONOACYLGLYCEROL LIPASE TERMINATES DIAZEPAM-RESISTANT STATUS EPILEPTICUS IN MICE AND ITS EFFECTS ARE POTENTIATED BY A KETOGENIC DIET G. Terrone 1,2, A. Pauletti 2, A. Salamone 2, M. Rizzi 2, BR Villa 2, L. Porcu 3, MJ Sheehan 4, E. Guilmette 4, CR. Butler 5, JR Piro 4, TA Samad 4, E. Del Giudice 1, A. Vezzani 2 1Department of Translational Medicine, Federico II University, Naples, Italy 2 Departments of Neuroscience and 3 Oncology, IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy; 3 Internal Medicine Research Unit, Pfizer Worldwide Research and Development, Cambridge, MA, USA; 4 Medicinal Chemistry, Pfizer Worldwide Research & Development, Cambridge, MA, USA Objective: Status epilepticus (SE) is a life-threatening commonly drug-refractory condition. Novel therapies are needed to rapidly terminate seizures, preventing mortality and morbidity. Monoacylglycerol lipase (MAGL) is the key enzyme responsible for the hydrolysis of the endocannabinoid 2-arachidonoylglycerol (2-AG) and a major contributor to the brain pool of arachidonic acid. Inhibiting MAGL modulates synaptic activity and neuroinflammation, two mediators of excessive neuronal activation underlying seizures. We studied the effect of a potent and selective irreversible MAGL inhibitor, CPD-4645, on diazepam-resistant SE, its neuropathological sequelae, and the mechanism underlying the drug's effects. Methods: Diazepam-resistant SE was induced in adult mice fed with standard or ketogenic diet or in CB1-receptor knock-out mice. CPD-4645 (10 mg/kg, subcutaneously) or vehicle was dosed 1 and 7 hs after SE onset in video-EEG recorded mice. At the end of SE, mice were examined in the novel object recognition test followed by neuronal cell loss analysis. Results: CPD-4645 maximal plasma and brain concentrations were attained 0.5 h post-injection (half-life=3.7 h) and elevated brain 2-AG levels by approximately 4-fold. CPD-4645 administered to standard diet-fed mice progressively reduced spike frequency during 3 h post-injection, shortening SE duration by 47%. The drug immediately abrogated SE in ketogenic diet-fed mice. CPD-4645 rescued neuronal cell loss, cognitive deficit and reduced IL-1beta and COX-2 brain expression resulting from SE. CPD-4645 effect on SE was similar in mice lacking CB1-receptors. Significance: MAGL represents a novel therapeutic target for treating SE and improving its sequelae. CPD-4645 therapeutic effects appear to be mediated by modulation of neuroinflammation.


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OREXIN AND ENDOCANNABINOID MORPHOLOGICAL INTERACTIONS IN THE BRAIN OF ADULT ZEBRAFISH R. Imperatore1,2, L. D’Angelo3, G. Annona1, , N. Forte2, L. Tunisi2,3, E. Varricchio1, P. De Girolamo3, V. Di Marzo2, L. Cristino2, M. Paolucci1

1 Department of Science and Technology, University of Sannio, Benevento, Italy; 2 Endocannabinoid Research Group, Institute of Biomolecular Chemistry, CNR, Pozzuoli, Italy, 3Department of Department of Veterinary Medicine and Animal Productions, University of Naples Federico II, Naples, Italy Orexin-A (OX-A) and Orexin-B (OX-B) are neuropeptides widely expressed among vertebrates. They act through orexin receptors (OX1R and OX2R) to regulate numerous physiological functions, such as energy homeostasis, food intake, sleep/wake cycle, learning and memory. The endocannabinoids (EC), mainly 2-arachydonoil-glycerol (2-AG), control similar biological processes by binding the cannabinoid receptor 1 (CB1). Recent studies have highlighted the potential of zebrafish to become an attractive vertebrate model to study brain function. The organization and gross architecture of many brain areas and the approximate locations of key neuromodulatory systems is similar in zebrafish and other vertebrates. Phylogenetic analyses of the zebrafish have demonstrated that OX and EC systems are structurally and functionally highly conserved with respect to the mammalian counterpart. In zebrafish, a common OX precursor gives rise to OX-A and OX-B, like in mammals, but only the OX2R has been identified showing a pattern of expression similar to CB1. We and others have found OX/EC structural and functional interactions, with OX-A being a strong enhancer of 2-AG synthesis in mammalian models. Several studies have confirmed the existence of a cross-talk between OX/EC systems in the regulation of many physiological functions. Since a similar OX2R and CB1 distribution and function has been described in homologous brain areas of zebrafish and mice, the aim of this study was to provide compelling morphological evidence of OX2R/CB1 interactions in the zebrafish brain, to outline the case for using zebrafish as a model to study these interactions and their role in the control of physiological functions such as feeding, sleep, energy homeostasis, learning and memory.


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SHUTTING DOWN TRANSLATION OF AMYLOID PRECURSOR PROTEIN IN EARLY SYMPTOMATIC hAPP MUTAN MICE RESCUES THE ALZHEIMER DISEASE PHENOTYPE Borreca A.1, Valeri F.2, De Luca M.A.2, Ernst L.3, Russo A.4, Biffo S.4, Cordella A.5,6, Mercuri N.B.6,7, Ammassari-Teule M1,6 1. CNR-IBCN Rome 2. University of Rome La Sapienza 3. Univesity of Namur 4. INGM National Institute of Molecular Genetic, Milan 5. Campus Biomedico-Rome, 6. IRCCS Fondazione Santa Lucia, Rome, Italy 7. University of Rome Tor Vergata

Overexpression of full-length Amyloid Precursor Protein (APP) associates with alterations in APP mRNA translation in Alzheimer Disease (AD) mouse models and sporadic AD patients. Whether these alterations depend on abnormalities in protein synthesis machinery is not well-understood. By performing a polysome gradient analysis in hippocampal samples from hAPPswe mutant mice, we detected a shift of APP mRNA on the polysomal fraction which indicates that the messenger are more prone to be translated before (1 month) or shortly after (3 months) mice start exhibiting AD symptoms. We observed that this shift was associated with decreased phosphorylation of the initial translation factor eIF2α We then found that pharmacological inhibition of eIF2α dephosphorylation in 3-month-old hAPP mice was able to normalize APP levels and to rescue AD hallmarks including Aβ levels, dendritic spine defects, episodic memory deficits, and lack of memory-induced hippocampal c-fos activation. By identifying an early time-window in which eIF2α phosphorylation pathway is paradoxically downregulated in hAPP mice, our findings reveal that treatments increasing eIF2α phosphorylation which shutdown APP translation within this time-window globally rescues the AD phenotype.


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PROTOCADHERINS AND THEIR ROLE IN BRAIN COMPLEXITY – A TALE FROM OCTOPUS R. Styfhals1,2, G. Ponte1, E. Seuntjens2, G. Fiorito1

1 Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, Napoli, Italy 2 Department of Biology - Research group of Developmental Neurobiology, KU Leuven, Belgium Protocadherins (PCDHs) are transmembrane adhesion molecules that have been implicated in neural wiring, synaptogenesis and neuronal diversity. The expansion of this gene family is seen as a vertebrate innovation and appears essential for creating complex brains. Drosophila melanogaster and other invertebrates have a very limited set of PCDHs. In the fruit fly the neuronal diversity is achieved through Dscam proteins (i.e. Down syndrome cell adhesion molecules). Over 168 protocadherins were found within the genome of Octopus bimaculoides, a mollusc, suggesting that it uses a vertebrate-like system to create a complex brain. We carried out an in silico analysis of the O. vulgaris transcriptome and identified putative Ov-Dscam proteins and Ov-PCDHs. By combining a Tblastn analysis with a search for conserved domains using the Pfam database, we improved the currently available annotation. In addition, construction of a phylogenetic tree allowed us to differentiate various isoforms. Subsequently, we analysed RNA-seq expression data of the identified proteins. We identified a significant number of protocadherins (>50) in O. vulgaris and were able to design specific mRNA probes for in situ hybridizations. We will present our preliminary results of the distribution of protocadherin expression within the O. vulgaris brain and will overlay it with recent available maps of the dopaminergic system in the different brain areas. Finally, we will show that only one Ov-Dscam gene is present in the O. vulgaris transcriptome, which supports the hypothesis that O. vulgaris, a species closely related to O: bimaculoides, developed the same system as vertebrates.


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EVIDENCE OF INCREASED OXIDATIVE STRESS IN POMPE DISEASE. A NEW THERAPEUTIC TARGET? A. Tarallo1,2, C. Damiano1, N. Minopoli1, M. Coletta1, C. Porto1, B. Rossi2, D. Monti3, G. Parenti1,2. 1Department of Translational Medical Sciences, Federico II University, Naples, Italy 2Telethon Institute of Genetics and Medicine, Pozzuoli, Italy 3Department of Chemical Sciences, Federico II University, Naples, Italy Pompe disease (PD), or Glycogenosis II, is a hereditary metabolic myopathy caused by the deficiency of acid alpha-glucosidase (GAA). Typical PD histologic abnormalities are the intralysosomal storage of glycogen and the accumulation of autophagic material. It is known that autophagy removes oxygen reactive species (ROS), mitohondria and damaged proteins. It might be speculated that the impairment of the autophagic pathway observed in the PD results in cellular and oxidative stress that may be deleterious for muscle and heart. To date Enzyme replacement therapy (ERT) with human recombinant GAA (rhGAA) is the only pharmacological approach for PD. We evaluated the level of oxidative stress and its effect on the uptake of rhGAA in fibroblasts of PD patients and in tissues of PD mouse model. Biochemical tests showed high levels of ROS and lipid peroxidation in PD fibroblasts and in disaese-relevant organs form the PD mouse model (heart, quadriceps, gastrocnemius and diaphragm) with respect to the controls. In the same samples we also found reduced glutathione levels and disregulation of the stress (p-ERK, pP38MAPK, Hsp27) and autophagy (LC3, Beclin1) pathways. By further increasing stress in cells (by starvation or sodium arsenite treatment) we found a reduced efficacy of ERT compared to untreated cells. By using drugs that modulate autophagy or antioxidant we obtained an attenuation of altered markers and improved internalization of rhGAA. It is reasonable to assume that these secondary abnormalities contribute to the clinical manifestations of PD and affect the efficacy of ERT; therefore, they represent potential therapeutic targets.


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REGULATION OF TRAFFICKING AND FOLDING OF CELLULAR PRION PROTEIN PRPC AND ITS SHADOW SHADOO A. Pepe1, R. Avolio1, D. S. Matassa1, F. Esposito1, L. Nitsch1, C. Zurzolo2, A. Pesapane3, N. Montuori3, A. Lavecchia4, S. Paladino1,5, D. Sarnataro1,5 1.Department of Molecular Medicine and Medical Biotechnology, University of Naples "Federico II", 80131, Naples, Italy; 2.Institut Pasteur, Unité de Trafic Membranaire et Pathogénèse, 75724 Paris CEDEX 15, France; 3.Department of Translational Medical Sciences, University of Naples "Federico II", 80131, Naples, Italy; 4.Department of Pharmacy, "Drug Discovery" Laboratory, University of Naples "Federico II", 80131, Naples, Italy; 5.Ceinge-Biotecnologie Avanzate scarl, 80145, Naples, Italy. Cellular prion protein (PrPC) is a glycosyl-phosphatidyl-inositol (GPI) anchored glycoprotein whose precise function in the brain remains elusive but may depend upon its cellular localization. PrPC is able to misfold to a pathogenic isoform PrPSc, the causative agent of neurodegenerative prion diseases. The PrPSc formation and cellular propagation has been related to the presence of the PrP receptor 37/67 kDa LR (Laminin Receptor), as well as to a member of the prion protein family called Shadoo. The misfolded PrPSc is amyloidogenic and strictly related to expression, intracellular localization and association of PrPC itself to cholesterol enriched membrane microdomains (lipid rafts). In this report we show our recent findings related to:

1. The biological effects deriving from the treatment with specific 37/67kDa LR inhibitor, NSC47924, on the trafficking and interaction between PrPC and its receptor

2. The natural tendency of Shadoo to acquire in neuronal cells “prion-like” characteristics, such as misfolding and aggregation state, and the existence of its non-translocated ER form, whose presence into mitochondria is controlled by the chaperone TRAP1.

Altogether our findings contribute i) to reveal NSC47924 inhibitor as a useful tool to regulate PrPC and 37/67 kDa LR trafficking and degradation, representing a novel small molecule to be tested against prion diseases and ii) to understand the role of molecular chaperones and of PrP-related folding intermediates in "prion-like" conversion.


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OREXIN-AENHANCES DOPAMINERGIC SIGNALING IN THE BRAIN OF OBESE MICE L.Tunisi1,2, N. Forte1, R.Imperatore1,3, L. D’Angelo2, G. Busetto4, L.Palomba5, P. De Girolamo2, V. Di Marzo1, L. Cristino1

1 Endocannabinoid Research Group, Institute of Biomolecular Chemistry, CNR of Pozzuoli, Naples; 2 Department of Veterinary Medicine and Animal Productions, University of Naples, Federico II; 3Department of Science and Technology, University of Sannio, Benevento, Italy; 4Department of Neuroscience Biomedicine and Movement, University of Verona, Italy and National Institute of Neuroscience, Verona, Italy; 5Department of Biomolecular Sciences, University of Urbino, Italy. An endocannabinoid-mediated dishinibition of orexin-A expressing neurons has been described inthe hypothalamus of leptin signaling-defective obese mice,such as ob/ob or mice fed with high fat diet, (HFD), concurrently with elevation of orexin-A trafficking and release to many target areas. In addition to its role in arousal, the orexin system also has been implicated in reward behaviors since OX-A injection in the vetrotegmental area (VTA) increases dopamine (DA) release in the prefrontal cortex and nucleus Accumbens (NAcc) triggering reward-associated behaviors. Here we sought to investigate if aberrant OX-A signaling occurs at VTA DA neurons of obese mice and if it could enhance DA synthesis and release to NAcc and promote reward-associated food seeking behaviors and hyperphagia. With this purpose, by exploiting morphological, biochemical, pharmacological and behavioral approaches we found a significant increase of OX-A release to the VTA of obese mice concomitantly with elevation of DA synthesis and release in the VTA and NAcc. The pharmacological treatment with the OX-1R antagonist SB334867 was able to lower DA levels to control values in the VTA and NAcc of both ob/ob and HFD mice. These data suggest that DA synthesis in the VTA and its release to NAcc are modulated by OX-A signaling and are associated with hyperphagia and body weight gain since SB334867 treatment of obese mice reduced body weight. These results are of special relevance since aberrant OX-A signaling during obesity could trigger the vicious circle underlying food seeking reward-associated behaviors, thus contributing to hyperphagia.


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ONECUT GENE FUNCTION IN THE CNS OF CHORDATES: DEVELOPMENT AND EVOLUTION Q.A. Vassalli1, C. Colantuono2, V. Nittoli1, A. Ferraioli1, G. Fasano1, M.L. Chiusano2, R. Kelsh3, P. Sordino1 and A. Locascio1

1. Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, Villa Comunale, Napoli, Italy. 2. Department of Soil, Plant, Environmental and Animal Production Sciences, University of Naples Federico II, Via Università 100, Portici, Italy. 3. Centre for Regenerative Medicine and Department of Biology and Biochemistry, University of Bath, London, UK.

Onecut genes (OC) have been identified in all major groups of metazoans and are expressed in

the nervous system and in some endodermal derived tissues. Their function in liver and pancreas differentiation in mammals has been quite well studied, while almost nothing is known about their function in neurogenesis and eye formation in chordates.

By using the tunicate Ciona intestinalis and vertebrate zebrafish Danio rerio as model systems, we studied the role and the genetic cascade of OC genes in photoreceptor cells and in eye formation during chordate evolution. The Ciona genome contains a single OC gene, while the analysis of the zebrafish genome revealed the presence of five OC orthologues OC1, OC2, OClike and two gene copies of OC3, named OC3a and OC3b. To acquire novel insights into the degree of OC gene functional conservation across chordates, we performed OC targeted perturbation by transgenic approach in Ciona and OC morpholino-mediated knockdown in zebrafish. By differential transcriptomic analyses on Ciona OC transgenic embryos we set the ground for the identification of OC target genes. The analysis of OC morphant phenotypes in zebrafish revealed a OC conserved role in the formation of specific eye neural structures. These data insert a new piece in the genetic cascade controlling the specification of the ocellus and eye and highlight a conserved and important role played by OC genes in the control of synaptic transmission in various regions of the central nervous system.


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TARGETING NEURONAL PROTEOSTASIS TO TREAT THE CNS IN LYSOSOMAL STORAGE DISEASES A. Monaco, Y. Ezhova, T. Giuliano, N.C. Sorrentino, A. Fraldi. Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy. Lysosomal storage disorders (LSDs) are severe childhood conditions (incidence: 1/5,000) caused by inherited defects of lysosomal function and often characterized by a neurodegenerative course. There is no cure for the central nervous system (CNS) pathology in these diseases. Understanding the cascade of events consequent to lysosomal dysfunction makes it possible to develop new therapies for LSDs. Data in our laboratory revealed a disease-relevant link between lysosomal dysfunction and defective neuronal proteostasis. Alpha-synuclein and CSP-alpha are two presynaptic chaperones involved in maintaining normal proteostasis at nerve terminals during synaptic activity. By studying mouse models of LSDs we demonstrated that lysosomal dysfunction causes accumulation of alphasynuclein in amyloid aggregates and increased proteasomal degradation of CSP-alpha. These events lead to a concurrent loss of these two chaperones at nerve terminals that disrupts presynaptic proteostasis and function, thus initiating neurodegeneration. Building upon these findings we are exploring the possibility to slow down neurodegenerative processes in LSDs reestablishing the physiological levels of CSP-alpha and alpha-synuclein at nerve terminals. Furthermore, characterization of amyloid aggregates showed that these deposits are localized in the perinuclear regions of neurons and beside alpha-synuclein contain several other aggregate-prone proteins. We are also evaluating the overall impact of amyloid aggregation on neuropathogenic cascades in LSDs in order to find new therapeutic targets to treat the CNS in these devastating disorders.


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MYOCLONIC EPILEPSY OF UNVERRICHT AND LUNDBORG (EPM1): UNDERSTANDING THE ROLE OF CYSTATIN B IN HUMAN CEREBRAL ORGANOIDS F. Pipicelli1,2, S. Cappello2, R. Di Giaimo1,2

1: Department of Biology, University of Napoli, Complesso Universitario Monte S.Angelo, via Cinthia 80126 Napoli, Italy 2: Max Planck Institute of Psychiatry, Kraepelinstraße 2-10, 80804 München, Germany EPM1 is the most common type of progressive myoclonus epilepsy due to different mutations of the Cystatin B (CSTB) gene, such as the null D68 mutation that produces a truncated protein. CSTB is a widely distributed protein which inhibits proteases of the cysteine family, most commonly Cathepsin B (CTSB). Preliminary in vivo studies show an high expression of CSTB in radial glial cells during embryonic cortex development and its importance for the maintenance of an appropriate cellular environment, suggesting a non-cell autonomous effect in regulating cell proliferation. In this study we analyze the role of CSTB in an in vitro model system of human brain development, using human cerebral organoids from induced pluripotent stem cells (IPSC). The aim is to assess if the overexpression, obtained by electroporation, of CSTB-WT and D68 is able to alter the proliferation of neigbouring cells. Proliferating cells were quantified by immunohistochemistry analysis showing that the overexpression of either CSTB and D68-CSTB causes an alteration of proliferation. Specifically, we detected a significant increase of proliferating cells in CSTB-WT electroporated organoids and a significant reduction of proliferation in CSTB-D68 electroporated organoids. These results indicate that CSTB has an important function in the niche to maintain an appropriate cellular environment regulating the proliferation of neighbouring cells in human cerebral organoids, as in mouse developing cortex.


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A RAT MODEL OF PERINATAL STRESS: IMPACT ON GENE EXPREESSION OF GLUTAMATERGIC POSTSYNAPTIC DENSITY GENES E.F. Buonaguro1, S. Morley-Fletcher2,3, C. Avagliano1, L. Vellucci1, S. Maccari2,4,3, A. de Bartolomeis1. 1University of Naples 'Federico II', Laboratory of Molecular and Translational Psychiatry- Department of Neuroscience- Reproductive Sciences and Odontostomatology, Naples, Italy. 2University Lille, Unité de Glycobiologie Structurale et Fonctionnelle, Lille, France. 3University Lille, International Associated Laboratory LIA 'Prenatal Stress and Neurodegenerative Diseases', Lille, France. 4Sapienza University of Rome, IRCCS Neuromed, Rome, Italy. Background The glutamatergic neurotransmission, particularly the scaffolding family of Homer 1 interacting with mGluR5, is associated with stress psychopathology. It has been observed that perinatal stress may increase the risk for major psychiatric diseases and that both antidepressants and antipsychotics modulate the immediate early gene Homer1a in medial prefrontal cortex. Materials and methods In perinatally restraint stressed (PRS) rats, i.e., the offspring of dams exposed to repeated episodes of restraint stress during pregnancy, we investigated: i) light-dark boxes test (LDB) measuring the time and the latency spent in the light compartment; and ii) modifications of expression of genes linked to glutmatergic signaling in different brain regions related to stress response in radioactive in situ hybridization histochemistry. Results PRS rats showed significant reduction of time spent in the light compartment compared to the control group (p<.01), expressing therefore reduced exploratory behavior. PRS rats showed reduced cortical and hippocampal levels of Homer1a and mGluR5. The mRNA levels of the constitutive isoform Homer1b were also tested, with the result of a significant gene expression reduction in the amygdala. Conclusions Perinatal stress in rats triggers alterations that make adult offspring less resilient to stress, thereby increasing vulnerability to stress-related disorders. The present results highlight an involvement of Homer1a in a perinatal epigenetic animal model of stress and supports the hypothesis of the mGluR5-Homer1 complex as a possible target for new therapeutic approaches.


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MODULATION OF AGING PATHWAYS AS A THERAPEUTIC STRATEGY FOR PARKINSON’S DISEASE F. Esposito1, Y. Colino-Sanguino2, I. Gallotta1,3, S. Coakley4, M. Faideau1, S. Montefusco1, E. Polishchuck1, A. Carissimo1, A.Bjorklund3, A. Ballabio1, M. Hilliard4, D. Medina1, E. Di Schiavi2, M. Decressac1. 1: Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy. 2: Wallenberg Neuroscience Center, Lund University, Lund, Sweden. 3: Institute of Biosciences and Bioresources (IBBR), National Research Council, Naples , Italy. 4: CJCADR, Queensland Brain Institute, The University of Queensland, Brisbane, Australia. Introduction Aging is the primary risk factors of the most common neurodegenerative diseases including Parkinson’s disease. In this line, Parkinson’s disease can be seen as a stochastic acceleration or dysfunction of cellular pathways governing cellular senescence. Consequently, a better understanding of the molecular pathways underpinning the aging process can lead to the development of novel therapeutic strategies. Methods and results Using high-content screening, we have identified an evolutionary-conserved signaling pathway that regulates aging. We demonstrate that genetic or pharmacological modulation of this pathway provides neuroprotection and regeneration in in vitro and in vivo models of Parkinson’s disease. Conclusions Aging manipulation is foremost attractive as a therapeutic approach since it simultaneously targets multiple defensive cellular mechanisms rather than on component of the proteostasis network at a time.


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ALTERATION OF ENDOSOMAL TRAFFICKING IS ASSOCIATED WITH NEURODEGENERATIVE DISEASES D. Fasano1, L. Zerillo1, G. M. Pierantoni1, A. De Rosa2, M. Picillo3, G. Amodio4, Maria T. Pellecchia3, P. Barone3, G. De Michele2, D. Saranataro1,5, L. Nitsch1, P. Remondelli4, C. Criscuolo2, S. Paladino1,5

1Dept Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Naples, Italy 2 Dept of Neuroscience, Reproductive, and Odontostomatological Sciences University of Naples Federico II, Naples, Italy 3Center For Neurodegenerative Diseases (CEMAND), University of Salerno, Salerno, Italy 4Dept of Medicine, Surgery and Dentistry “Scuola Medica Salernitana”, University of Salerno, Salerno, Italy 5CEINGE Biotecnologie Avanzate scarl, Naples, Italy Homeostasis of eukaryotic cells is largely dependent on dynamic compartmentalization of the endo-membrane system. The membrane trafficking linking different organelles is essential to maintain a proper composition of various compartments as well as to transport various molecules to appropriate compartments. Respect to other cell types nervous system is more sensitive to disturbances of the membrane trafficking. In the last years, we studied the role of endosomal trafficking in neurodegeneration focusing on two nervous system disorders, Charcot-Marie Tooth disease 4J (CMT4J) and a new form of autosomic recessive early-onset parkinsonism (PARK20), both caused by mutations of an inositol phosphatase (Fig4 and Synj1, respectively). Our recent investigations show that: i) Synj1 plays a crucial role in regulating the homeostasis and functions of early endosomal compartments in different cell types and in fibroblasts of PARK20 patients; ii) the loss of Fig4 drastically alters the whole endo-lysosome axis (lysosomes, but also late and early endosomes result enlarged and more numerous) implying its essential role for the homeostasis and function of these compartments. All together, our data provide evidence for the implication of endosomal pathway in neurodegeneration, emphasising the link between endosomal trafficking and neurodegenerative diseases.


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EFFECTS OF METHYLCYCLOPENTADIENYL MANGANESE TRYCARBONIL ON DOPAMINERGIC NEURONS IN ZEBRAFISH G. Fasano1,2, R. S. Godoy3, E. Angiulli4, E. Alleva5, M. Toni4, E. Biffali, M. Ekker3, P. Sordino2, L. MT. Canzoniero1

1 Department of Sciences and Technologies, Università del Sannio, Benevento, Italy 2 Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, Naples, Italy 3 Department of Biology, University of Ottawa, Ottawa, Canada 4 Department of Biology and Biotechnology ‘‘Charles Darwin”, Sapienza University, Rome, Italy 5 Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, Rome, Italy 6 Research Infrastructures for Marine Biological Resources, Stazione Zoologica Anton Dohrn,

Naples, Italy Heavy metals are environmental factors whose role in the pathogenesis of several neurological disorders is taken increasingly into account. Particularly, alteration of brain manganese (Mn) homeostasis has been linked to neurodegenerative diseases, such as Alzheimer and Parkinson’s disease. Recently, strong concern was expressed over Methylcyclopentadienyl Manganese Trycarbonil (MMT), an organic Mn-containing gasoline additive, due to health risk from long-term exposure to Mn. Zebrafish is a useful animal model for studying effects of neurotoxicants on the nervous system, by virtue of teleost brain homologies with the mammalian one. Despite evidence of structural and functional damage induced by Mn-based chemicals on dopaminergic (DA) neurons, no in vivo studies of the effects on neuronal differentiation following chronic exposure to sub-lethal MMT have been performed. The aim of this study was to investigate transcriptional, morphological and behavioral alterations caused by sub-lethal MMT exposure during neuronal differentiation in zebrafish. In addition, adult cognitive impairments following MMT exposure during embryonic development of the brain were analyzed. Zebrafish embryos were exposed to MMT (10-300 µM) for 5-72 hours post fertilization (hpf), critical developmental stages of DA differentiation. We performed a transcriptional analysis of genes involved in differentiation of DA neurons following MMT treatment during development. Particularly, sub-lethal MMT (100 µM) altered specification (lmx1b), intermediate differentiation (otpa) and maturation (th) of DA neurons. MMT treatment (30-100 µM) was also able to alter the morphology of DA neurons increasing both the number and size of tyrosine hydroxylase-positive (Th+) cells of specific ventral diencephalic cluster 2 DA neurons, also confirmed in vivo using Tg(dat:EGFP) transgenic line. Interestingly, MMT treatment (100 µM) evoked a hyperactive behavior in embryos at 72 hpf. In adult stage, embryos treated with MMT (100 µM; 5-72 hpf), were grown up to 5 months and submitted to the Y-maze test for evaluating their behavior. The analysis revealed changes in cognitive capabilities affecting exploration, orientation and spatial memory. Collectively, these findings suggest that chronic exposure to sub-lethal MMT during neuronal differentiation can alter the development (differentiation, number, shape) of DA neurons and the short- and long-term behavioral traits controlled by the DA system.


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STRIATONIGRAL INVOLVEMENT IN FABRY DISEASE: A QUANTITATIVE AND VOLUMETRIC MRI STUDY C. Russo, G. Pontillo, A. Pisani, F. Saccà, E. Riccio, A. Macera, G. Rusconi, A. Stanzione, P. Borrelli, V. Brescia Morra, E. Tedeschi. G. Palma, S. Cocozza, A. Brunetti - Department of Advanced Biomedical Sciences, University “Federico II”, Naples, Italy - Department of Public Health, Nephrology Unit, University “Federico II”, Naples, Italy - Department of Neurosciences and Reproductive and Odontostomatological Sciences, University “Federico II”, Naples, Italy - IRCCS SDN, Naples, Italy - Institute of Biostructure and Bioimaging, National Research Council, Naples, Italy Purpose Aim of this study is to elucidate possible mechanisms of extrapyramidal involvement in Fabry Disease (FD), simultaneously assessing morphometric changes of striatonigral pathway as well as their possible correlations with clinical variables evaluating motor functions in a representative group of patients. Methods In this cross-sectional study, we investigated possible differences in terms of QSM values and volumes of different extrapyramidal relays, including striatum and substantia nigra (SN), in 30 FD patients (M/F=11/19, mean age 42.6±12.2) and 37 healthy controls (HC) of comparable age and sex (M/F=16/21, mean age 43.2±14.6). FD patients underwent a clinical examination for the study of different motor functions, and the relationship between MRI and clinical variables was tested with a nonparametric correlation analysis by using the Spearman’s coefficient. Results Compared to HC, FD patients showed an increase in susceptibility values of the SN (p<0.001) and striatum (p=0.005), while no differences emerged for the other tested extrapyramidal structures, suggesting their relative sparing. The increased susceptibility was coupled to a reduced volume of the SN (p<0.001), but not of the striatum (p=0.31). Finally, no significant correlations emerged when probing the relationship between these modifications and the clinical variables. Conclusion In FD patients, susceptibility and volumetric alterations are present throughout the extrapyramidal pathway, with the SN being particularly affected by these changes. Such results are in line with the subtle extrapyramidal involvement recently suggested in FD, and could further contribute to the understanding of the physiopathological bases of cerebral involvement in FD.


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THE INFLAMMATORY RESPONSE FOLLOWING ACUTE SEIZURES IN ZEBRAFISH BRAIN AND IN CHORDATE EVOLUTION. V.Nittoli

1, A. Gentile

2, G. Fasano

1, A. Palladino

1, M. Borra

1, P. De Girolamo

3, A. Spagnuolo

1, P.

Sordino1

1Stazione Zoologica Anton Dohrn, Napoli, Italy; 2MPI- HLR, Bad Nauheim, Germany;

3Università

degli Studi di Napoli Federico II, Italy.

Inflammation represent the natural body defense to various types of insults and is a part of a complex cascade of events, closely linked to the activation of the immune system. Although inflammation is linked to repair processes, it can be detrimental if dysregulated. Recently, clinical and experimental findings have highlighted the importance of inflammation in epilepsy, supporting an important role for inflammatory mediators in seizure activity, and suggesting the beneficial effects of anti-inflammatory medications for some refractory epilepsy forms. In this context, the discovery of new molecules targeting inflammatory pathways gives the opportunity to enrich the repertoire of drugs counteracting epileptic seizures. To this aim, we adopt the zebrafish model of seizure induced by pro-convulsive agent, the pentylenetetrazole (PTZ), to analyze the expression dynamic of inflammatory molecules and cells, and investigated the functional role of some inflammatory pathways. Moreover, to explore the degree of phylogenetic conservation of the seizure-related inflammatory mechanisms among chordates, we are investigating other emerging model systems such as the tunicate Ciona robusta. PTZ-induced seizures in zebrafish are associated with an increase of key inflammatory molecules that accompanies the activation of microglial cells and with a rapid induction of neuroprotective mechanisms. The genetic manipulation and pharmacological treatments strongly support the hypothesis of a direct role of some inflammatory pathways in the seizure activity. Our findings provide further therapeutic targets for the high-throughput usage of zebrafish in the discovery of anti-inflammatory drugs. Furthermore, preliminary data suggest the use of C. robusta to explore seizure-induced inflammatory response during evolution.


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PRAGMATIC ABILITIES IN MULTIPLE SCLEROSIS: AN RS-fMRI STUDY S. Cocozza, A. Carotenuto, M. Quarantelli, G. Arcara, V. Brescia Morra, E. Tedeschi, G. Orefice, V. Bambini, R. Iodice, A. Brunetti - Department of Advanced Biomedical Sciences, Federico II University, Naples, Italy - Department of Neurosciences, Reproductive Science and Odontostomatology, Federico II University, Naples, Italy - Institute of Biostructure and Bioimaging, National Research Council, Naples, Italy - Center for Neurocognition, Epistemology and theoretical Syntax (NEtS), Scuola Universitaria Superiore IUSS, Pavia, Italy - IRCCS Fondazione Ospedale San Camillo, Venice, Italy Purpose Cognitive functions have been largely investigated in multiple sclerosis (MS). Recent studies showed that MS patients could also experienced deficits in pragmatics. Despite the classical model of language involve a posterior temporal region (Wernicke’s area) and the inferior frontal gyrus (Broca’s area), several pieces of evidence have outlined the role of the inferior parietal lobule (Geshwind’s area) for high-level language aspects. We evaluated the relationship between pragmatic abilities and the functional connectivity of the GA in MS patients. Methods MS patients were tested for pragmatics (Assessment of Pragmatic Abilities and Cognitive Substrates) and underwent a clinical data collection. A seed based Resting-State fMRI (RS-fMRI) analysis of the functional connectivity (FC) of both the left and the right GA was performed. Results When testing for possible correlation of pragmatic data with the RS-fMRI data, a direct correlation between APACS Total score and a cluster of FC with the paracingulate cortex was found (p=0.003) when evaluating FC of the right GA seed. A similar of correlation between the APACS Total score and a cluster located at the level of the paracingulate cortex was found when testing the left GA seed (p=0.009). Conclusion We observed that the FC of right GA and paracingulate cortex is directly related to pragmatic scores, confirming that language is not simply a left hemisphere function but involves a more mazy network over both hemisphere. Furthermore, we also high-lightened that paracingulate cortex is an important hub for pragmatic abilities.


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DEFAULT MODE NETWORK MODIFICATIONS IN FABRY DISEASE S. Cocozza, G. Pontillo, M. Quarantelli, F. Saccà, E. Riccio, T. Costabile, G. Olivo, V. Brescia Morra, A. Pisani, E. Tedeschi, A. Brunetti - Department of Advanced Biomedical Sciences, University “Federico II”, Naples, Italy - Institute of Biostructure and Bioimaging, National Research Council, Naples, Italy - Department of Neurosciences and Reproductive and Odontostomatological Sciences, University “Federico II”, Naples, Italy - Department of Public Health, Nephrology Unit, University “Federico II”, Naples, Italy - Department of Neuroscience, Uppsala University, Uppsala, Sweden Purpose Aim of the study was to evaluate the presence of Default Mode Network (DMN) alterations in Fabry Disease (FD), and their possible correlations with neuropsychological scores. Methods Thirty-two FD patients with genetically confirmed diagnosis of classical FD (12 males, mean age 43.3 ± 12.2) were enrolled, along with thirty-five healthy controls (HC) of comparable age and sex (14 males, mean age 42.1 ± 14.5). Resting-State fMRI data were analyzed using a seed-based approach, with four different seeds sampling the main hubs of the DMN. Differences between patients and HC, and correlations with neuropsychological variables were probed voxelwise over the whole brain. Results Clusters of increased functional connectivity emerged in FD patients among the main DMN hubs, as well as between the DMN and the middle temporal gyri, with a reduced anticorrelation between the midline DMN hubs and the right cerebellar Crus I and II. The connectivity between right inferior frontal gyrus and precuneus was significantly correlated with the results of the Corsi span test (P=0.0001). Conclusion Widespread DMN changes are present in FD patients, and correlate with cognitive performance. Our results confirm the current view of a cerebral involvement in FD patients not simply associated to major cerebrovascular events, but also related to significant and diffuse functional changes.


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BRAIN FUNCTIONAL CHANGES AND COGNITIVE DYSFUNCTION IN FRIEDREICH'S ATAXIA S. Cocozza, T. Costabile, E. Tedeschi, F. Abate, C. Russo, A. Liguori, W. Del Vecchio, F. Paciello, M. Quarantelli, A. Filla, F. Saccà, A. Brunetti - Department of Advanced Biomedical Sciences, University “Federico II”, Naples, Italy - Department of Neurosciences and Reproductive and Odontostomatological Sciences, University “Federico II”, Naples, Italy - Institute of Biostructure and Bioimaging, National Research Council, Naples, Italy Purpose Along with the progressive motor disability, impairment of neuropsychological functions, affecting different domains, has been described in Friedreich’s Ataxia (FRDA) patients. Aim of our study was to perform the first resting-state functional MRI (RS-fMRI) analysis in FRDA patients to assess possible brain functional connectivity (FC) changes in these patients, and test their correlations with neuropsychological performances. Methods Twenty-four FRDA patients (M/F:15/9, mean age 31.3±15.0) and twenty-four healthy controls (HC; M/F:15/9, mean age 30.7±15.5) were enrolled in this cross-sectional study. All patients underwent a thorough neuropsychological battery, investigating different cognitive domains. RS-fMRI data were analyzed using a seed-based approach, probing the FC of cortical areas potentially referable to specific executive and cognitive functions compromised in FRDA. Results Compared to HC, FRDA patients showed overall worse neuropsychological scores in several domains, including global cognitive assessment, clear-thinking ability and non-verbal assessment of intelligence, spatial and verbal memory, visuo-perception and visuo-spatial functions, and executive functions. Analysis of RS-fMRI data showed an increased FC in FRDA patients compared to HC between paracingulate gyri and the medial frontal gryrus, between the superior frontal gyrus and bilateral angular gyri and between the middle temporal gyrus and the cingulate gyrus, with a decreased FC between the medial frontal gryrus and cerebellum. Conclusion In FRDA patients, widespread alterations of FC are present compared to HC. These results, in conjunction with clinical findings and neurofunctional tests, may shed new light on the pattern of supratentorial and infratentorial involvement and on dynamics of brain plasticity in this disease.


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DISSECTING PRUNE-1/NME1 FUNCTION IN BRAIN AND CEREBELLUM OF MICROCEPHALY AND PEHO SYNDROME AFFECTED PATIENTS V. Ferrucci,1-2 F. P. Pennino1-2, R. Siciliano1-2 F. Asadadeh1-2, I. Scogliamiglio1-2, Y. Ferrara1-2 , P. Pucci 2-3, A. Duilio3, A. Di Somma3, H. Houlden4, V. Salpietro3 And M. Zollo1-2

1- Dipartimento di Medicina Molecolare e Biotecnologie Mediche Università Federico II, Naples, Italy 2- CEINGE Biotecnologie Avanzate, Via G. Salvatore 486, Naples, Italy 3- Dipartimento di Scienze Chimiche, Universita` Federico II, Naples, Italy 4- Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK

PRUNE first identified in Drosophila through impairing the formation of eye pigments (drosopterins), belongs to the DHH family of phosphoesterases (Timmons et al., 1995).With a prominent exopolyphosphatase activity, PRUNE-interacts with NME1 and glycogen synthase kinase-3(GSK-3 β), a negative regulator of canonical WNT/β-catenin signalling (Diana et al., 2013; Carotenuto et al., 2014). While a PRUNE’s binding partner GSK-3β is a crucial inhibitory regulator of many neuronal functions including neurite outgrowth, synapse formation, neurogenesis and survival, which may be mediated via GSK-3 β promotion of apoptotic signalling in cultured neural precursor cells (Eom et al., 2007). In addition, neuronal overexpression of GSK-3 β has been shown to result in delayed postnatal maturation and differentiation of neurons in the mouse brain (Spittaels et al., 2002). We already have presented the identification of 15 patients mutated in Prune exons (D30N; D106N; D237W) and affected by Microcephaly and Peho syndrome (Zollo et al 2017; Salpietro et al., 2017). In the current study we will present the model of action of the PRUNE-D106N mutation together with NME1 on influencing microtubule polymerization during mitosis. This data is of importance for applications in preclinical studies in mice, in an effort to bring in clinics this new therapeutic approach.


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DE NOVO GAIN-OF-FUNCTION VARIANTS IN THE SLO2 FAMILY OF Na+-ACTIVATED K+ CHANNELS ARE RESPONSIBLE FOR DEVELOPMENTAL AND EPILEPTIC ENCEPHALOPATHIES L. Manocchio1, I. Mosca.1, M.V. Soldovieri1, P. Ambrosino1, F. Miceli2, G. Coppola 3, A. Weisz 4, J.R. Lemke 5, and M. Taglialatela1,2

1 Department of Medicine and Health Science, University of Molise, Campobasso, Italy;

2 Department of Neuroscience, University of Naples “Federico II”, Naples, Italy; 3 Child and Adolescent Neuropsychiatry, Department of Medicine and Surgery; 4 Molecular Pathology and Medical Genomics, “SS. Giovanni di Dio e Ruggi d'Aragona - Schola Medica Salernitana” Hospital of the University of Salerno, Salerno, Italy; 5 Institute of Human Genetics, University of Leipzig Hospitals and Clinics, Leipzig, Germany Epileptic encephalopathies represent a heterogeneous group of early onset and childhood disorders characterized by severe, intractable seizures, specific electroencephalographic (EEG) signatures, and different levels of developmental delay or regression, often with a poor prognosis. Advances in genomic analysis in the last 10 years have identified novel genes and variants in a large fraction of affected children, often defining novel clinical entities. Mutations in several potassium (K+) channels have been found in a variety of developmental and epileptic encephalopathies. KCNT1 (Slo2.2 or Slack), together with KCNT2 (Slo2.1 or Slick), belongs to the SLO2 family of Na+-dependent K+ channel genes, encoding for pore-forming α-subunits variably gated by changes in voltage and/or concentrations of intracellular ions or second messengers (1). In the present work we describe the functional and pharmacological consequences of two de novo, heterozygous KCNT1 mutations (G288S and, not previously reported, M516V) in two unrelated probands affected by Malignant Migrating Partial Seizures in Infancy (MMPSI), a severe early-onset epileptic encephalopathy characterized by pharmacoresistant focal seizures migrating from one brain region or hemisphere to another (2). We also report on a recent, parallel investigation in two independent individuals with de novo variants in KCNT2 at position R190 and neurodevelopmental disorders (3). Overall, the results obtained suggest a gain-of-function pathogenetic mechanism responsible for both KCNT1- and KCNT2-related disorders, thus highlighting treatment opportunities with K+ channel blockers such as quinidine as targeted therapies in these individuals. References

1. Salkoff L, et al. Nat Rev Neurosci. 2006 Dec;7(12):921-31. 2. Rizzo F, et al., Mol Cell Neurosci. 2016 Apr;72:54-63. 3. Ambrosino P, et al. 2017 (submitted)


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POSTERS

35

SEROTONIN 5-HT7 RECEPTOR INCREASES THE DENSITY OF DENDRITIC SPINES AND FACILITATES SYNATOGENESIS IN FOREBRAIN NEURONS L. Speranzaa, F. Volpicellia, S. Pulcranoa, C. Crispinob, G.C. Bellenchia, U. di Porzioa, C. Perrone-Capanoa,c (a) Institute of Genetics and Biophysics “Adriano Buzzati Traverso”, CNR, Naples, IT; (b) Department of Biology, University of Naples Federico II, Naples, IT; (c) Department of Pharmacy, University of Bari "A. Moro", Bari, IT. (d) Department of Pharmacy, University of Naples Federico II, Naples, IT. In normal and pathological brain functions the precise control of dendritic spine density and synapse formation is critical. Therefore, how signaling pathways influence dendrite outgrowth and remodeling is still difficult to define. Here, we report that prolonged activation of the serotonin 5-HT7 receptor (5-HT7R) with a selective agonist promotes formation of dendritic spines and facilitates synaptogenesis in postnatal cortical and striatal neurons. Acute activation of 5-HT7R results in pronounced neurite elongation in postnatal striatal and cortical neurons, thus extending previous data on the morphogenic role of 5-HT7R in embryonic neurons. Critical role of 5-HT7R in neuronal morphogenesis was confirmed by analysis of neurons isolated from 5-HT7R-deficient mice and by pharmacological inactivation of the receptor. In addition, we also observed decreased number of spines when 5-HT7R was blocked pharmacologically, and in 5-HT7R-knock-out neurons, suggesting that constitutive 5-HT7R activity is critically involved in the spinogenesis. Moreover, cyclin-dependent kinase 5 and small GTPase Cdc42 were identified as important downstream effectors mediating morphogenic effects of 5-HT7R in neurons. Altogether, our data suggest that the 5-HT7R-mediated structural reorganization during the postnatal development might have a crucial role for the development and plasticity of forebrain areas such as cortex and striatum, and thereby can be implicated in regulation of the higher cognitive functions. Future analyses will be addressed to identify specific miRNAs involved in remodeling of dendritic spines and synaptogenesis. Understanding the way miRNAs are contributing to synaptic plasticity could provide clues to establish novel therapeutic strategies for several diseases of the Nervous System.


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MODULATION OF SYNAPTIC PROTEIN SYNTHESIS IN ANIMAL MODEL OF ALZHEIMER'S DISEASE C. Barbato1, C. Cefaliello1, E. Penna1, G. Di Ruberto1, C. Perrone-Capano2, M.C. Miniaci2, M. Crispino1 1Department of Biology, 2Department of Pharmacy, University of Naples Federico II, Naples, Italy A local system of protein synthesis, independent and remote from the cell body, is present in the presynaptic domains of axons. The prompt synthesis of protein on site and on demand near the synapses makes key contributions to synaptic plasticity. Nonetheless, not much has been explored whether its deregulation leads to the neurodegenerative diseases. In this study, we examine the involvement of the synaptic system of protein synthesis in the pathogenesis of Alzheimer’s disease (AD), using transgenic mice overexpressing APP as animal model (TG). To this end, we have tested if the local system of protein synthesis is modulated by contextual fear conditioning, and if such modulation is deregulated in the TG animals. We prepared synaptosomes as an in vitro model for synaptic regions. The synaptosomal fractions from cerebral cortex and cerebellum were incubated with a radio-labelled aminoacid and the pattern of newly synthetized proteins was analyzed. In the wild type animals, the newly-synthesized proteins were modulated by training. By contrast, this modulation did not occur in the TG animals, suggesting that the synaptic system of protein synthesis is impaired in AD mice. These results indicate that the synaptic protein synthesis may also be involved in the molecular mechanism leading to synaptic degeneration in AD.


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A C.ELEGANS MODEL TO STUDY LDL-RELATED PROTEINS INVOLVEMENT IN ALZHEIMER’S DISEASE C. Bertapelle1,2, A. Medoro1, F. Cocco1, E. Di Schiavi2 and C. Russo1. 1Department of Health Sciences, University of Molise, Campobasso, Italy 2Institute of Bioscience and BioResources, CNR, Naples, Italy The Alzheimer’s disease (AD) is the most common neurodegenerative disorder and its neuropathologic hallmarks are the extracellular deposits of β-amyloid (Aβ) plaques and intraneuronal tangles, resulting from Tau hyperphosphorylation. These aggregates both contribute to the loss of synapse functions and neuronal death. The major genetic risk factor in sporadic AD is represented by the E4 isoform of ApolipoproteinE (ApoE), ligand of the Low Density Lipoprotein-Related Proteins Receptor 8 (LRP8). LRPs processing is regulated by γ-secretase cleavage in a similar way to the Amyloid Precursor Protein (APP), which after cleavage by γ-secretase produces the Aβ peptide. Considering the lack of informations about LRPs involvement in AD, we propose a C.elegans model to study in vivo the correlation of human LRP8 to APP and Tau. C.elegans presents orthologs of APP, γ-secretase and Tau (apl-1, sel-12 and ptl-1), and it has been extensively used to study AD. We generated multiple transgenic lines overexpressing in all neurons the human LRP8 protein. By Western blot we verified the expression of LRP8 protein as full-length and cleaved forms, resembling the peptides observed in cell cultures and AD patients. The transgenic lines present defects in locomotion, development and lifespan in a similar way to other C.elegans AD models. In order to verify if LRP8 is cleaved by γ-secretase ortholog, we are inhibiting the protease activity using both a pharmacological and a genetic approach. We set up a new model which may help clarify the role of LRPs in AD, and its involvement in regulating APP and Tau.


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DIFFERENTIAL EFFECTS OF SUBCHRONIC MK-801 ON BEHAVIOR, AMINO ACIDS AND GLUTAMATE RECEPTOR OF THE NMDA TYPE IN THE PREFRONTAL CORTEX OF ADULT NAPLES HIGH-EXCITABILITY RATS G. Boatto1, M. Nieddu1, M. A. de Souza Silva2, C. Treno3, U. A. Gironi Carnevale3, C. Pagano4, P. Illiano4, A. Tino4, A. G. Sadile3*. 1 Dept. of Chemistry and Pharmacy, Univ. of Sassari, Sassari, Italy 2 Center for Behavioral Neuroscience, Univ. of Duesseldorf, Duesseldorf, Germany 3 Exptl. Med., Univ. of Campania "Luigi Vanvitelli", Naples, Italy 4 CNR, ISASI "E. Caianiello" Pozzuoli, Naples, Italy Introduction: The Naples High Excitability (NHE) rats model the mesocortical variant of ADHD. The aim of these studies was to investigate the subchronic effects of the non-competitive antagonist MK-801 on behavior, the amino acids L-Glutamate (L-Glu), L-Aspartate (L-Asp), L-Leucine (L-Leu) and NR1 expression in the prefrontal cortex (PFc), dorsal (DS) and ventral (VS) striatum in adult NHE rats. Methods: Young male adults (55-60 days old) rats were exposed to a spatial novelty, a Làt-maze, followed by a radial maze after subchronic daily administration for 14 days of MK-801 (0.01 mg/kg-subchronic experiment). Horizontal (HA) and vertical (VA) activities were monitored. Amino acids and NR1 subunit were detected by Liquid Cromatography Tandem Mass Spectrometry (LC/MS/MS) and western-blot and qRT-PCR, respectively, in the PFc, DS and VS. Results: (i) subchronic MK-801 increased HA in both NHE and NRB rats and decreased VA frequency in NRB and its duration in NRB and NHE (ii) the level of expression of NMDA-R1 protein was increased by 78% in PFc of NRB rats after subchronic treatment iii) there was a difference in AA content in left vs. right side in PFc and VS only in the forebrain of vehicle treated NHE rats. Conclusions: subchronic MK-801 increased NMDA-R1 expression in PFc of NRB rats only. A differential effect of MK-801 upon subcellular distribution of the measured AAs was found in the target size of mesocortical and mesolimbic dopamine systems. Finally a dissociation emerged between behavior and NMDA-R1 expression at prefronto-striatal interface of NRB rats, whereas in NHE rats this system appeared to be out of control. KEYWORDS: LOCOMOTOR ACTIVITY, NEUROTRASMITTERS, PLASTICITY, ADHD


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A FUNCTIONAL STUDY OF THE ENDOCANNABINOID SYSTEM IN ZEBRAFISH NEURODEVELOPMENT: IMPLICATIONS IN VISION AND LOCOMOTION R.M. Sepe1, R. De Paolo1, J.M. Cioni2, J. Zang3, S. Neuhauss3, W.A. Harris2, P. De Girolamo4, V. Di Marzo5, P. Sordino1

1. Biology and Evolution of Marine Organisms (BEOM), Stazione Zoologica Anton Dohrn, Naples, Italy

2. Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge CB2 3DY, UK

3. Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland 4. Dipartimento di Medicina veterinaria e Produzioni animali, University of Naples “Federico

II”, Naples, Italy 5. Endocannabinoid Research Group, Institute of Biomolecular Chemistry, Consiglio

Nazionale delle Ricerche, Pozzuoli (NA), Italy.

The endocannabinoid system (ECS) comprises neuromodulatory lipids and their receptors, capable of regulating neuronal excitability, and recently, it has been suggested that the ECS may play an important role in early neuronal development. The arachidonoylglycerol (2-AG) is synthesized at high levels in the CNS by diacylglycerol lipase α (Daglα). Our aim was to investigate the role of 2-AG and its receptors (CB1 and CB2) in the development and differentiation of neurons, and in the formation of neuronal circuits that control spontaneous locomotion and visual system, using zebrafish as model organism. Through the use of morpholino-induced transient knockdown of the zebrafish daglα and its pharmacological rescue, we suggest that synthesis of 2-AG is implicated in the control of axon formation in defined areas of the developing brain, and animals lacking Daglα display defective axonal growth and fasciculation, and abnormal physiological behavior in tests measuring stereotyped eye movement and motion perception. Furthermore, pharmacological treatments using antagonists for CB1 and CB2 highlight their role in the correct differentiation and lamination of zebrafish developing retina. Animals treated with these antagonists display also defective swimming behavior, suggesting the implication of CB1 and CB2 receptors also in the correct formation of neuronal circuits that control spontaneous locomotion. In conclusion, our results suggest an important role of endocannabinoids as mediators in axonal outgrowth with implications for the control of vision and movement.


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THE LRRK2-R1441C MUTATION DISRUPTS LONG-TERM POTENTIATION-LIKE PLASTICITY IN PARKINSON’S DISEASE PATIENTS M. Ranieri, R. Dubbioso, A. De Rosa, M. Esposito, S. Peluso, R. Iodice, G. De Michele, L. Santoro, F. Manganelli. Department of Neurosciences, Reproductive Sciences and Odontostomatology, Federico II University of Naples, Italy. Background: Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) account for up to 13% of dominant familial Parkinson’s disease (PD) cases. The clinical and pathological features of LRRK2-PD are often indistinguishable from idiopathic PD (iPD). Recent evidence pointed out that LRRK2 acts directly at the secretory and endocytic molecular machinery. Importantly, in southern Italy the R1441C mutation is the most frequent mutation reported so far. Objective: Based on the LRRK2-role on synaptic transmission, we aimed to investigate in vivo excitability and plasticity of the motor cortex by means of transcranial magnetic stimulation (TMS). Methods: Paired pulse TMS was used to investigate cortical excitability by using short intracortical inhibition and facilitation and short afferent inhibition paradigm. Intermittent theta burst stimulation (iTBS) was used to test long-term potentiation-like cortical plasticity (LTP). Eight LRRK2-PD patients harboring the R1441C mutation and 11 iPD were tested on the more affected side in ON l-dopa therapy. Results: We observed the lack of LTP-like motor cortex plasticity in R1441C-LRRK2 patients compared to healthy-subjects and with a similar profile observed for iPD patients. Motor cortex excitability profile did not exhibit any difference among the three groups. Conclusion: R1441C mutation disrupts motor cortex LTP-like plasticity in Parkinson’s disease patients.


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CORPUS CALLOSUM ABNORMALITIES: NEUROIMAGING, CYTOGENETICS AND CLINICAL CHARACTERIZATION OF A VERY LARGE MULTICENTER ITALIAN SERIES G. Vitiello1, G. Terrone1 , A. Romano1, R. Genesio2, F. Imperati1, O. Zuffardi3, R. Borgatti4, L. Nitsch2, The Italian CCA Study Group and E. Del Giudice1 1 Department of Translational Medicine, Section of Pediatrics, Federico II University, Naples, Italy; 2 Neuropsychiatry and Neurorehabilitation Unit, Scientific Institute, IRCCS Eugenio Medea, Bosisio Parini, Lecco, Italy; 3 Department of Molecular Medicine and Medical Biotechnologies, Federico II University, Naples, Italy; 4 Department of Molecular Medicine, University of Pavia, Pavia, Italy; 5 Department of Advanced Biomedical Sciences, Federico II University, Naples, Italy Corpus callosum abnormalities (CCA) have an estimated prevalence ranging from 0.3% up to 0.7% in patients undergoing brain imaging. CCA can be identified incidentally, or can be part of a developmental disease. We performed a retrospective study of 551 patients, identified non-syndromic (NS) CCA and syndromic (S) CCA, reviewing clinical features, neuroradiological aspects, genetic etiology, and chromosomal microarray (CMA) results.Syndromic CCA subjects were prevalent (60%) and they showed the most severe clinical features.Cortical malformations and cerebellar anomalies were 23% of cerebral malformation associated to CCA (plus), 23% and 14% respectively in syndromic forms. A clinical and/or genetic diagnosis was obtained in 37% of syndromic CCA including chromosomal rearrangements on high-resolution karyotype (18%), microdeletion/microduplication syndromes (31%) and monogenic diseases (51 %).Non-syndromic CCA anomalies had mildest clinical features, although intellectual disability was present in 49% of cases and epilepsy in 13%. CMA diagnostic rate in our cohort of patients ranged from 11% to 23% (NS to S).A high percentage of patients (76% 422/551) remain without a diagnosis. Combined high resolution CMA studies and next-generation sequencing (NGS) strategies will increase the probability to identify new causative genes of CCA and to redefine genotype-phenotype correlation.


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PERAMPANEL AND TUBEROUS SCLEROSIS COMPLEX: A CASE REPORT. M.F. de Leva, S. Graziano,V. Lanzara, M. Pandolfi, C. Tucci, S. Buono. SC Neurologia, AORN Santobono-Pausilipon, Napoli, Italia. INTRODUCTION: TSC is a genetic disorder resulting in a variety of hamartomatous lesions that may affect every organ system. It exhibits an AD inheritance pattern, and two distinct genetic loci have been identified: TSC1 and TSC2. Perampanel is a selective noncompetitive antagonist at the AMPA receptor, highly effective in a wide range of experimental models of epilepsy. To date there are no data about perampanel efficacy in TSC. We describe the case of a TSC patient treated with perampanel as add-on therapy with a significant improvement of her seizure disorder. CASE REPORT: We report the case of a 21 year old female with TSC2 mutation realizing refractory epilepsy, severe intellectual disability and behavioral disorder. Her MRIs showed cortical tubers and subependymal nodules stable over time. Last EEG showed focal right temporal paroxysmal activity. Seizures begun in the first days of life as myoclonic, then different types of seizures appeared such as tonic, tonic-clonic, atonic. Several antiepileptic drugs such as vigabatrin, levetiracetam, ethosuximide, etc. had poor results. In the last year we added perampanel at a dose of 6 mg/day to the actual therapy with felbamate, clobazam and phenobarbital, with a significant and permanent improvement in seizures frequency and severity, and a global improvement of the behavioral disorder and social participation. CONCLUSION: Our report suggest efficacy of perampanel in TSC. The early add-on could be a significant therapeutic opportunity in the refractory various seizures of these patients.


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EPM1: UNDERSTANDING THE ROLE OF CYSTATIN B IN THE MOUSE DEVELOPING CORTEX Giordano M.1 ,Kyrousi C.1 ,Cappello S. 1,Di Giaimo R.1,2

1: Max Planck Institute of Psychiatry, Kraepelinstraße 2-10, 80804 München, Germany 2: Department of Biology, University of Napoli, Complesso Universitario Monte S.Angelo, via Cinthia80126 Napoli, Italy Progressive myoclonus epilepsy of the Unverricht–Lundborg-type (EPM1) is an autosomal recessive neurodegenerative disorder and it is the most common type of progressive myoclonus epilepsy. The physiological function of CYSTATIN-B (CSTB) in the CNS and the dysfunction caused by the mutants are still unknown but loss of-function mutations in the gene encoding CSTB are the primary genetic cause of EPM1. CSTB-KO mice show neurological disorder in mice similar to EPM1-patients. Mutations which lead to epilepsy often develop during embryogenesis, for this reason we decided to study the role of CSTB by interfering with its expression in the mouse developing cortex. To this aim, we overexpressed CSTB wild type and 2 human pathological mutants, G4R and D68, by in utero electroporation, in mouse developing cortex at E14 and we analysed the results 2 days later. Our results show different distribution of electroporated cells. Cells overexpressing wtCSTB at 2dpe migrated from VZ to IZ while cells electroporated with the 2 mutants are located only in the VZ, indicating a difference in cell migration and/or differentiation. In addition, using different markers for cell proliferation, we detected an increase of proliferation in the region surrounding the wtCSTB-electroporated cortical area. On the contrary we detected a decrease in proliferation in the cortex upon overexpression of the 2 EPM1 mutants.Our results clearly indicate a neurogenic role of CSTB during cortical development, which is altered by overexpression of the protein or by the expression of an EPM1-mutant form.


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FOUR-YEAR ELECTROPHYSIOLOGICAL AND NEUROPSYCHOLOGICAL FOLLOW-UP IN THE ADULT FORM OF NIEMANN PICK DISEASE TYPE C G. Mele, S. Tozza, R. Dubbioso, R. Iodice, A. Topa, M. Esposito, L. Ruggiero, E. Spina, A. Iovino, L. Santoro, F. Manganelli. Department of Neurosciences, Reproductive Sciences and Odontostomatology, Federico II University of Naples, Italy. Background: Niemann-Pick type C (NPC) is a recessive lysosomal lipid storage disorder, characterized by vertical supranuclear gaze palsy, ataxia, cognitive impairment, psychiatric manifestations, hearing loss, dystonia, dysarthria and dysphagia. New evidence about physiopathology of disease emerged from different electrophysiological approaches, like transcranial magnetic stimulation (TMS) protocols and multi-modal evoked potentials evaluation. Objective: The aim of our study was to describe through extensive electrophysiological and neuropsychological evaluation a four-year follow-up of two sisters affected by the adult form of NPC. Methods: Patients 1 (P1) and 2 (P2) were evaluated before starting therapy with miglustat (T0) and after 4 years (T4) of treatment. Patients underwent standard neuropsychological battery and electrophysiological assessment, consisted of nerve conduction study, motor (MEP), visual (VEP), somato-sensory (SSEP) and brainstem auditory (BAEP) evoked potentials. We evaluated the cholinergic central circuits by applying a TMS protocol, named short-latency afferent inhibition (SAI). Results: Neuropsychological evaluation demonstrated a mild cognitive impairment in P1 and mild dementia in P2 at T0 with a worsening of cognitive performances in both at T4. SAI paralleled such cognitive decline, being reduced at T0 and worsened at T4. Lastly, patients showed altered BAEP and lower limb SSEP at T0, and P1, with a poor compliance with treatment, had also abnormal lower limb MEP and VEP at T4. Conclusion: Our report strengthens the role of central cholinergic circuits impairment in the cognitive decline of NPC patients and we suggest to apply a multi-modal evoked potentials protocol in monitoring the progression of disease.


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BENEFICIAL EFFECTS OF CURCUMIN ON PERIPHERAL SYMPTOMS OF HUNTINGTON’S DISEASE E. Montano1, S. Castaldo2, F. Elifani2, V. Calabrò1, T. Angrisano1, V. Maglione2, A. Di Pardo2, A. Pollice1

1 Department of Biology, University of Naples Federico II, Naples 2 IRCCS Neuromed Centre for Neurogenetics and Rare Diseases, Pozzilli, Isernia Huntington’s disease (HD) is a genetic neurodegenerative disorder characterized by a complex pathogenic and clinical profile with neuronal dysfunction and progressive atrophy in the striatum and in the cerebral cortex. Aside defects in the nervous system, the disease is characterized by a variety of peripheral complications among which progressive weight loss that correlates with disease progression and significantly affects the quality of life of HD patients. The mechanism underlying weight loss is unknown but several evidences suggest that it may likely be attributable to structural and functional alteration of the intestinal tract. Although efforts have been made in HD research, no effective treatment to counteract any of the symptoms is currently available. Over the last years utilization of natural products like curcumin, have had large diffusion for the treatments of neurodegenerative diseases, however much remains to be explored yet. The aim of our study it to investigate the molecular mechanism underlying alterations in the intestinal tract potentially linked to the weight loss in HD and to explore the therapeutic potential of curcumin administration in R6/2 mice, which represent the best characterized and most widely used animal model of HD that recapitulate most of the features of the human pathology.


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CHARACTERISATION IN C. ELEGANS OF MUTATIONS IN DAT GENE CAUSATIVE OF DOPAMINE TRANSPORTER DEFICIENCY SYNDROME M. Paglione1; A. Lanzo1; G. Zampi1; M. Kurian2; E. Di Schiavi1 1 Institute of Biosciences and BioResources, IBBR, CNR, Naples, Italy 2 UCL Institute of Child Health, Molecular Neurosciences, Developmental Neurosciences Dopamine transporter deficiency syndrome (DTDS) is an autosomal recessive disorder caused by mutations in the human gene encoding for the dopamine transporter (hDAT), which leads to the partial or total loss of function of the protein. The DTDS phenotypic spectrum is characterized by a typical form, with early onset parkinsonism-like symptomatology, that was distinguished from atypical forms presenting milder conditions and caused by partial loss of protein function. The aim of this work is to investigate in vivo the effects of the new genomic variants found in DAT gene in patients, using Caenorhabditis elegans. hDAT is cedat-1 in C.elegans and its knockout causes a locomotion defect due to a lack of reuptake of dopamine and resistance to 6-hydroxydopamine toxic effects. We established the first animal model for DTDS and demonstrated that hDAT is able to rescue the two phenotypes observed when cedat-1 is knocked out. To examine the effects on DAT function of two mutations recently found in patients, we expressed the human mutated forms hDATG433R or hDATG467V only in dopaminergic neurons in cedat-1 KO and studied the possible rescue of the defective phenotypes. Transgenic animals expressing hDATG467V showed a partial rescue of only one of the two phenotypes, whereas the hDATG433R mutant showed an impairment in DAT function in both phenotypic paradigms. Taken together, these observations suggest that G433R mutation causes the complete loss of function in vivo, while the G467V mutation causes a partial loss of protein function, resembling other mutations causing atypical forms of DTDS.


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CYSTATIN B SECRETION IN ADULT MAMMALIAN BRAINS E. Penna1, F. Pipicelli1, A. Cerciello1, R. Di Giaimo1,2 and M. Crispino1

1: Department of Biology, University of Naples Federico II, Naples, Italy 2: Max Planck Institute of Psychiatry, Kraepelinstraße 2-10, 80804 München, Germany Cystatin B (CSTB) is a widely distributed protein expressed in most cell types and tissues where it inhibits proteases of the cysteine family. Mutation of CSTB causes Progressive Myoclonus Epilepsy (EPM1), a disease of the Central Nervous System, suggesting a key role of this protein in the physiology of the Nervous System. Previous studies of our group showed that CSTB is secreted during brain development, as CSTB was found in the cerebrospinal fluid (CSF) from the developing brain of mouse. In addition, the culture medium of primary cortical cells transfected with a plasmid expressing the CSTB-GFP fusion protein contained endogenous CSTB as well as the CSTB-GFP protein. Interestingly, transfection of a natural EPM1 mutant did not result in CSTB secretion suggesting that secretion of the protein is critical for its function. Here we detected the presence of CSTB in the synaptic regions of adult rodents brain, using synaptosomes isolated from brain cortex as in vitro model of nerve endings. Synaptosomes are an useful tool to study the function of synaptic terminals, such as secretion. After 2h incubation of synaptosomes CSTB was detected in medium, indicating an active secretion of this protein from the nerve endings of adult rodents brain. In addition, we found CSTB in a crude synaptosomal fraction prepared from human cerebral organoids. Its expression level increased with the maturation of the organoids, suggesting that CSTB is indicative of progressive synaptogenesis in organoids which can be used as an innovative tool to study neuronal plasticity in a human model.


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BRAIN DERIVED NEUROTROPHIC FACTOR (BDNF) EXPRESSION IS ASSOCIATED WITH NEURAL REPAIR OF INJURED ADULT ZEBRAFISH TELENCEPHALON Cacialli P1,2,3, D’Angelo L1, Pellegrini E2, Lucini C1

1Dpt. Veterinary Medicine and Animal Productions, University of Naples Federico II, Italy 2Inserm, UMR 1085, Research Institute in Health, Environment and Occupation, SFR Biosit, University of Rennes 1, Rennes, France 3Department of Pathology and Immunology, CMU, University of Geneva, Switzerland. The reparative ability of the central nervous system varies widely in the animal kingdom. In the mammalian brain, the regenerative mechanisms are very limited and newly formed neurons do not survive longer, probably due to a non-suitable local environment. On the opposite, fish can repair the brain after injury, with fast and complete recovery of damaged area. The brain of zebrafish, a teleost fish widely used as vertebrate model, also possesses high regenerative properties after injury. Taking advantage of this relevant model, the aim of the present study was to investigate the role of Brain-derived neurotrophic factor (BDNF) in the regenerative ability of adult brain, after stab wound telencephalic injury. BDNF is involved in many brain functions and plays key roles in the repair process after traumatic brain lesions. It has been reported that BDNF strengthens the proliferative activity of neuronal precursor cells, facilitates the neuronal migration toward injured areas, and shows survival properties due to its anti-apoptotic effects. BDNF mRNA levels, assessed by quantitative PCR and in situ hybridization at 1, 4, 7 and 15 days after the lesion, were increased in the damaged telencephalon, mostly suddenly after the lesion. Double staining using in situ hybridization and immunocytochemistry revealed that BDNF mRNA was restricted to cells identified as mature neurons. BDNF mRNA expressing neurons mostly increased in the area around the lesion, showing a peak 1 day after the lesion. Taken together, these results highlight the role of BDNF in brain repair processes and reinforce the value of zebrafish for the study of regenerative neurogenesis.


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DISRUPTION OF GABA(A)-MEDIATED INTRACORTICAL INHIBITION IN PATIENTS WITH CHOREA-ACANTHOCYTOSIS G. Ciccarelli, R. Dubbioso, M. Esposito, S. Peluso, R. Iodice, G. De Michele, L. Santoro, F. Manganelli. Department of Neurosciences, Reproductive Sciences and Odontostomatology, Federico II University of Naples, Italy. Background: Chorea-acanthocytosis (Ch-Ac) is an autosomal recessive neurodegenerative disorder characterized by adult-onset chorea, acanthocytes in the peripheral blood, and Huntington’s disease-like neuropsychiatric symptoms. Ch-Ac is caused by loss-of-function mutations in the Vacuolar Protein Sorting 13 Homolog A (VPS13A) gene, encoding for chorein. Mouse model carrying such mutations and pathological studies in humans have shown dysregulation of gamma-aminobutyric acid (GABA)ergic interneurons in its pathophysiology. Objective: Based on these findings we aimed to investigate in vivo (GABA)ergic inhibitory circuits of the motor cortex by means of transcranial magnetic stimulation (TMS). Methods: Different TMS protocols were applied in two patients with Ch-Ac to probe intracortical inhibitory circuits mainly underpinned by GABA-A mediated circuit (short interval intracortical inhibition, SICI; short-latency afferent inhibition, SAI; intracortical facilitation, ICF) and GABA-B mediated networks (cortical silent period, CSP). Results: We found that Ch-Ac patients showed a striking impairment of GABA(A)-mediated intracortical inhibition, demonstrated by means of abnormal SICI and SAI findings and an exaggerated response to ICF protocol. On the other hand, we did not find any abnormality regarding the cortical GABA(B) inhibitory network explored by CSP. Conclusion: Our findings suggest that the motor cortex of Ch-Ac patients is disinhibited and hyperexcitable due to an impaired GABA(A)ergic neurotransmission.


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GO-OPSIN EXPRESSING CELLS IN SEA URCHIN LARVAE PRODUCE A TRH NEUROPEPTIDE, WHICH INFLUENCES ARM GROWTH M. Cocurullo1, N. Wood2, P. Oliveri2, M.I. Arnone1

1Stazione Zoologica Anton Dohrn, Napoli, Italy; 2University College London, London, UK In mammals, the Thyrotropin-Releasing Hormone (TRH) has a central role on regulation of metabolism and growth by stimulating the secretion of the TSH from the pituitary gland. In non-mammalian vertebrates, e.g. amphibians and fish, TRH also regulate growth by stimulating the release of Growth hormone and prolactin from the pituitary gland, while it has little or no effect on the secretion of TSH. The role of TRH so far remains poorly investigated in invertebrates. Recently, Van Sinary et al. (Proc Natl Acad Sci U S A. 2017 114: E4065-E4074) identified a TRH-like peptide in C. elegans and investigated its role in this protostome worm. This study shows that TRH originated before the divergence of protostomes and deuterostomes and suggests that the ancestral role of this neuropeptide is on the control of postembryonic growth and reproduction. Combining in situ hybridisation, whole-mouth immunostaining and knock-down experiments we investigated the role of a sea urchin TRH neuropeptide (QYPGamide) its precursor (SpTRH) and potential receptor (SpTRHR) in Strongylocentrotus purpuratus and Paracentrotus lividus larvae. In sea urchins, SpTRH is produced by two go-opsin expressing cells bilaterally distributed at each the side of the larval apical organ. Our data shows that TRH protein production is regulated by light/dark cycle and feeding/starving conditions. Furthermore, knock-down experiments of the SpTRH precursor and of the potential receptor SpTRHR inhibit the post embryonic arm growth. In conclusion, our results describe for the first time the role of TRH in a non-chordate deuterostomes and validate the hypothesis of an ancestral role of TRH on postembryonic growth regulation.


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AUTISM WITH BENZODIAZEPINE-RESPONSIVE ELECTRICAL STATUS EPILEPTICUS IN SLEEP (ESES) CAUSED BY KCNQ3 GAIN-OF-FUNCTION VARIANTS Anna Lauritano1, Tristan T. Sands2, Gaetan Lesca3, Maria Roberta Cilio4, Piera Nappi1, MariaVirginia Soldovieri5, Francesco Miceli1, and Maurizio Taglialatela1

1 Dept. Neuoscience, Univ. of Naples Federico II, Naples, Italy; 2 Columbia University, New York, USA; 3 Claude Bernard University, Lyon, France; 4 UCSF, San Francisco, California; 5 Department of Medicine and Health Science, University of Molise, Campobasso, Italy KCNQ2 and KCNQ3 encode voltage-gated ion channel subunits mediating a subthreshold potassium current important in limiting neuronal excitability. Loss-of-function variants in KCNQ3 are known to cause Benign Neonatal Epilepsy characterized by seizures in newborns with normal subsequent development (1), although rare families with more severe phenotypes have also been described. In the present study, we have investigated the in vitro consequences of de novo variants in KCNQ3 found in heterozygosity in three patients suffering from autism, motor delays with hypotonia and electrical status epilepticus of sleep (ESES) responsive to benzodiazepine therapy. In all patients, symptoms appeared around 2-3 years of age, without previous history of neonatal seizures. Whole exome sequencing (WES) revealed a KCNQ3 R230H de novo variant in two patients, and a de novo R230S variant in the third. Patch clamp analysis of R230H and R230S - variants neutralizing the second arginine within the S4 voltage sensor, similarly to the previously described R230C variant (2) demonstrated a gain-of-function effect characterized by increased potassium current density and loss of voltage-dependence. Our findings add to growing evidence for unique gain-of-function phenotypes imparted by KCNQ2 and KCNQ3 gain-of-function variants (3). Particularly, gain-of-function affecting R230 of KCNQ3 can cause autism and neurodevelopmental disability even in the absence of seizures. We suggest that KCNQ3 gain-of-function is a monogenic form of autism associated with ESES susceptible to personalized treatment with benzodiazepines. 1. Sands TT, et al. Epilepsia. 2016;57(12):2019-2030. 2. Miceli F, et al. J Neurosci. 2015;35(9):3782-3793. 3. Mulkey SB, et al. Epilepsia. 2017;58(3):436-445.


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POLYSOMNOGRAPHIC FINDINGS IN CHILDREN WITH MILD TRAUMATIC BRAIN INJURY Ilaria Bitetti, Francesco Precenzano, Valentina Lanzara, Roberta Ida Ferrentino, E. Merolla, Marco Carotenuto UOC di Neuropsichiatria Infantile- Università degli Studi della Campania “Luigi Vanvitelli”, Napoli BACKGROUND Traumatic Brain Injury (TBI) may be considered as one of the main causes for childhood morbidity and mortality. TBI increases the risk of psycho-social problems, intellectual disability and cognitive impairment, but the most common consequences of TBI are sleep troubles. Aim of study is assessing polysomnographic findings in pediatric patients with TBI. MATERIAL AND METHODS 37 Children with TBI (mean age 9,69+/-1,81, 20 Males and 17 Females). The control group was composed by 37 children (Mean age 9, 34 +/-1,78, 19 M and 18 F). Exclusion criteria were: psychiatric and neurological disorders, intellectual disability (IQ≤70) and psychoactive drug administration. All children in both group filled out a full nocturnal polysomnogrfapy (PSG) Statistical Analysis t-Student’s analysis was used to verify the mean differences between groups for PSG parameters. Statistical software package (STATISTICA 8.0, StatSoft Inc.) was used. P value <0.05 were considered as statistically significant. RESULTS Children with TIB showed a statistically significant (p<0,001) reduction for TIB (Time In Bed) min, SPT (Sleep Period Time) min, TST (Total Sleep Time)-min, SE (Sleep Efficiency) % , N2 min, N3 min, REM min, N3 spt, REM spt , N2 tst, N3 tst, REM spt and a statistically significant increasing in parameters such as SOL (Sleep Latency) min, SS-h (stage shifts/h), AWN (awekenings)-h, WASO (wakefulness after sleep onset) min, N1 min, N1 spt, N1 tst (Table 1). Our study shows that pediatric TBI may alter sleep architecture consistently, although further studies are needed.


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Table 1

TBI (N=37) Controls (N=37) Mean Std.Dev. Mean Std.Dev. t-value p Age 9.692 1.806 9.341 1.640 0.876 0.3839 TIB-min 380.446 74.794 589.189 86.071 -11.135 0.0000 SPT-min 335.091 54.125 555.473 75.341 -14.451 0.0000 TST-min 258.449 49.810 529.878 70.227 -19.176 0.0000 SOL-min 45.355 43.355 24.676 18.747 2.663 0.0095 FRL-min 118.857 33.666 130.041 54.274 -1.065 0.2904 SS-h 9.995 2.745 7.524 3.411 3.432 0.0010 AWN-h 8.643 2.648 1.649 1.865 13.136 0.0000 SE% 68.532 8.426 90.251 5.601 -13.058 0.0000 WASO-min 76.643 27.561 25.595 26.475 8.125 0.0000 N1-min 55.016 20.490 17.351 21.956 7.629 0.0000 N2-min 78.962 30.447 231.595 46.381 -16.734 0.0000 N3-min 63.378 29.777 162.162 69.836 -7.915 0.0000 REM-min 61.092 24.109 118.689 32.431 -8.670 0.0000 WASO-spt 22.966 7.269 4.454 4.446 13.215 0.0000 N1-spt 16.587 6.152 3.173 3.932 11.176 0.0000 N2-spt 23.471 8.295 42.100 7.815 -9.944 0.0000 N3-spt 18.741 7.850 28.695 9.684 -4.857 0.0000 REM-spt 18.236 6.988 21.559 5.895 -2.211 0.0302 N1-tst 21.874 8.835 3.419 4.382 11.383 0.0000 N2-tst 30.172 9.125 44.043 7.744 -7.050 0.0000 N3-tst 24.418 10.109 30.043 10.017 -2.404 0.0188 REM-tst 23.536 8.503 22.511 5.834 0.605 0.5474

Tab 1. Comparison of full polynomographic parameters between subjects with TIB and healthy controls for t-Student’s test. Subjects with TIB show statistically significant differences (p <0.005) in almost all of the examined parameters.

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THE EXPERIENCE OF THE “FEDERICO II” UNIT OF THE TELETHON UNDIAGNOSED DISEASES PROGRAM M. Pinelli1,2, G. Cappuccio1,2, M. Alagia1,2, A. Torella1,3, V. Nigro1,3, N. Brunetti-Pierri1,2 and TUDP

1. Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italia 2. Dipartimento di Scienze Mediche Traslazionali, Università degli Studi di Napoli Federico

II, Napoli, Italia 3. Dipartimento di Biochimica Biofisica e Patologia Generale, Università della Campania

'Luigi Vanvitelli', Napoli, Italia

We present the experience of the “Federico II” unit of the Telethon Undiagnosed Diseases Program (TUDP). TUDP is aimed at the diagnosis of children with rare and severe diseases that lack a genetic diagnosis. TUDP is a national program founded by Fondazione Telethon and is open to all patients, and to clinicians following patients, with undiagnosed diseases. The general principle of the study is to offer an accurate clinical re-evaluation of all patients and, if indicated, a Next Generation Sequencing (NGS) test. Ours is the referring clinical center of TUDP for the southern and central Italy. From March 2016 to September 2017, we have enrolled 51 pediatric patients with severe, multisystem and undiagnosed diseases. Most of those patients were already been visited by multiple centers and subject to invasive and non-invasive procedures. All were already tested with array-CGH. Conclusive genetic diagnosis was reached for 11 cases, candidate-gene mutations for 7 cases, and no potential explanation for 4 cases. Out the remaining, 13 cases were considered low-priority and, thus, not addressed to NGS and 16 are still in progress. Among the concluded cases: one had a mutation in a novel disease-causing gene and its pathogenetic role has been confirmed in other cases; two had phenotypes more complex and severe than those described in literature; most of the remaining cases had clinical presentations too aspecific to prompt a specific genetic test and their diagnosis would have unlikely been reached without an NGS approach.


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VARIABLES AFFECTING PAIN THRESHOLD EVALUATION IN AUTOALGOMETRY L. Lorusso, A. Viggiano UNIVERSITA’ DEGLI STUDI DI SALERNO, DIP. MEDICINA, CHIRURGIA E ODONTOIATRIA “SCUOLA MEDICA SALERNITANA” [email protected] [email protected] Pain threshold is the measure of the intensity of a physical stimulus that evokes pain. To avoid the influence of the “tester”, a new method has been recently proposed, named “autoalgometry”, in which the subject being evaluated applies and controls by himself the force against the autoalgometer tip. The aim of the present work was to evaluate the effects of stimulation rate, gender and site of stimulation on the pain threshold evaluated in healthy subjects. Fifty healthy volunteers (21 males, 29 females, age 18-29 y) were evaluated for the pain threshold using the autoalgometric procedure by applying fast- or slow-increasing stimulation on a computerized autoalgometer tip with their fingers and reaching a minimal or maximal pain intensity. There was a positive correlation between test speed and pain threshold measures. Male participants reached higher speeds compared to female participants when asked to execute fast and showed higher pain thresholds (both for the minimal and the maximal pain intensity) compared to female participants in the fast tests. When the tests were executed slowly, the minimal pain threshold did not differ between males and females, but the maximal pain threshold was still higher in males compared to females. These results demonstrate that it is mandatory to record the rate of stimulation in a pressure pain-threshold evaluation and support the use of the autoalgometric procedure for this purpose.


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MOLECULAR AND MORPHOLOGICAL STUDY OF ADULT NEUROGENESIS IN THE SHORT LIVED FISH Nothobranchius furzeri A. Leggieri1,2, L. D’Angelo1, L. Castaldo1, C. Lucini1, P. de Girolamo1, A. Cellerino2,3, L. Avallone1

1. Department of Veterinary Medicine and Animal Productions, University of Naples “Federico II” – Napoli, Italy 2. Leibniz Institute on Aging, Fritz Lipmann Institute – Jena, Germany 3. Scuola Normale Superiore di Pisa – Pisa, Italy Adult neurogenesis is a dynamic and highly regulated process, influenced by a large number of stimuli (environment, drugs, pathology onset) and modulated by different factors (grow factors, mRNAs). Neurogenic events during adulthood are different in vertebrates: in mammals, they are spatially restricted into two specific neurogenic regions; in teleost fish extensive adult neurogenesis has been detected in sixteen regions, mostly associated with the telencephalic ventricular system, the midbrain and the cerebellum valvula and corpus. Among teleost fish, Nothobranchius furzeri is a well consolidated model organism for ageing research. It is particularly suitable for neurobiological studies for its very short lifespan strictly related to rapid growth, accelerated sexual maturation and expression of age-related changes. We have investigated the presence and assessed the localization, by mRNA expression studies, of three genes associated with neuronal stem cells niches: Inhibitor of DNA binding 3 (ID3), Collagen type IV - α 1 subunit (COL4A1) and Collagen type XXV - α 1 subunit (COL25A1). mRNA expression was assessed by fluorescence in situ hybridization (FISH), using uracil-digoxigenin-labeled RNA probes, on cryosections of old (27 weeks) animals, MZM – 04 /10 strain. Apart from a specific expression pattern, ID3, COL4A1 and COL25A1 share a common localization in some forebrain and midbrain regions: the dorso-medial region and in the internal cellular layer of telencephalon; the habenular and anterior thalamic nucleus of diencephalon; the tori and the periglomerular gray zone of both diencephalon and mesencephalon. ID3 and COL4A1 also share a common localization in the corpus of cerebellum.


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REDUCED INTRACRANIAL VOLUME IN FABRY DISEASE: EVIDENCE OF ABNORMAL NEURODEVELOPMENT? G. Pontillo, S. Cocozza, V. Brescia Morra, E. Riccio, C. Russo, F. Saccà, E. Tedeschi, A. Pisani, M. Quarantelli, A. Brunetti - Department of Advanced Biomedical Sciences, University “Federico II”, Naples, Italy - Department of Neurosciences and Reproductive and Odontostomatological Sciences, University “Federico II”, Naples, Italy - Department of Public Health, Nephrology Unit, University “Federico II”, Naples, Italy - Institute of Biostructure and Bioimaging, National Research Council, Naples, Italy Purpose Lysosomal storage disorders (LSD) are often characterized by a reduction of intracranial volume (ICV), as a reflection of an abnormal brain development; the possibility that in Fabry Disease (FD), similarly to other LSD, this abnormal development could occur has never been investigated. Methods Forty-two FD patients (15 males, mean age 43.3±13.0) were enrolled along with thirty-eight healthy controls (HC) of comparable age and sex. Volumetric MRI data were processed using SPM12 to obtain ICV values, as well as normalized global brain parenchyma and gray matter (GM) volumes, in order to investigate if associated parenchymal loss, independent from a possible ICV reduction, was also present. Results Mean ICV of FD patients was 8.1% smaller compared to the control group (p < 5•10-5), while no significant differences emerged between the two groups when comparing normalized global brain parenchyma and GM volumes, thus suggesting the presence of a harmonious volumetric reduction of intracranial structures, rather than a primary neurodegenerative phenomenon. Conclusion The present results suggest that in FD patients an abnormality of brain development could be present, expanding the current knowledge about central nervous system involvement in FD, and further emphasizing the importance of an early diagnosis.


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INFLAMMASOME AND AUTOPHAGY CROSS-TALK IN BOVINE BRAINS: PRELIMINARY OBSERVATIONS D. De Biase1, C. Pirozzi2, G. Piegari1, T.B. Pagano1, F. Prisco1, G. Mattace Raso1, S. Papparella1 and O. Paciello1

1Department of Veterinary Medicine and Animal Production, University of Naples Federico II via Delpino, 1, 80137 Naples, Italy 2Department of Pharmacy, University of Naples Federico II, Via Montesano 49, 80131 Naples, Italy.

Inflammaging occurs in the aged brain both as microglia senescence and as an increased production of pro-inflammatory cytokines and protein complexes known as inflammasomes. Recently, it has been suggested that autophagy acts as a regulator of NLRP3 inflammasome activation. Here, we describe our findings concerning the expression of MHC II as a marker of microglia senescence and NLRP3 inflammasome in bovine brains and the cross-talk between inflammasome, autophagy and ROS production. Samples of hippocampus were collected from 42 Podolica cattle. Animals were divided in three groups: group A (aged 15 to 24 years), group B (aged 5 to 14 years) and group C (aged up to 5 years). Double color immunofluorescence and immunohistochemistry were performed to evaluate 1) the relationship between NLRP3, SOD1 and autophagy marker Beclin 1. and 2) the expression of MHC II and NLRP3. Western blot was performed to determine the expression levels of NLRP3. Double color immunofluorescence showed a positive relationship between inflammasome, Beclin 1 and SOD1. Moreover, immunoistochemistry revealed a higher expression of MHC II and NLRP3 in older animals. Western blot confirmed the higher expression of NLRP3 in aged bovine compared to younger. These data demonstrate that MHC II and NLRP3 are up-regulated in the brain of aged cattle. We suggest that the age-related overexpression of SOD1 indicates an excessive production of ROS resulting in deleterious peroxidative reactions and ultimately in neuroinflammation. Moreover, we propose that autophagy may protect the cell by removing inflammasome components from the cell, thus maintaining cellular homeostasis.


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MOTOR PERFORMANCE DETERIORATION ACCELERATES AFTER 50 YEARS OF AGE IN CHARCOT-MARIE-TOOTH TYPE 1A PATIENTS D. Severi, S. Tozza, R. Iodice, M. Esposito, R. Dubbioso, L. Ruggiero, A. Topa, E. Spina, A. Iovino, L. Santoro, F. Manganelli.

Department of Neuroscience, Reproductive Sciences and Odontostomatology, University of Naples "Federico II", Naples, Italy Objective The aim of our study was to describe, by a case-control and cross-sectional design, the correlation between clinical impairment and age in CMT1A patients. Methods We enrolled seventy CMT1A patients and seventy sex- and age-matched healthy controls. Motor performance was assessed through 10-Meter Walk Test, 6-Minute Walk Test and 9-Hole Peg Test (9HPT) of dominant and non-dominant side, and muscle strength was measured by using the Medical Research Council Score. In CMT1A group, disability and quality of life were evaluated using the CMT Neuropathy Score (CMTNS) and the Short Form-36 (SF-36) questionnaire. We analyzed cross-sectional relationships between age and all clinical measures and calculated differences in the slopes between cases and controls. We explored the occurrence of a structural change in the age-related progression of clinical measures. Results The deterioration of motor performance correlated with age in both groups with a greater slope in CMT1A patients than controls. The deterioration of CMTNS and SF-36 correlated with age in CMT1A group. The deterioration of all clinical measures with the exception of SF-36 questionnaire showed a structural change at the 50th year of age. The rate of deterioration was not different between patients and controls until 50 years of age, whereupon it became significantly greater in CMT1A patients. Conclusion Our study supports that the disease progression in CMT1A patients is an age-related process and the 50th year of age represents a critical moment after which the clinical decline becomes faster.


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Indice degli autori A Abate, F. 31 Alagia, M. 54 Alessio, N. 12 Alleva, E. 26 Ambrosino, P. 33 Ammassari-Teule, M. 15 Amodio, G. 25 Angiulli, E. 26 Angrisano, T. 45 Annona, G. 14 Arcara, G. 29 Arnone, M.I. 50 Asadadeh, F. 32 Avagliano, C. 23 Avallone, L. 56 Avolio, R. 18 B Ballabio, A. 24 Bambini, V. 29 Barbato, C. 36 Barone, P. 25 Bellenchi, G.C. 35 Bertapelle, C. 37 Biffali, E. 26 Biffo, S. 15 Bitetti, I. 52 Bjorklund, A. 24 Boatto, G. 38 Borgatti, R. 41 Borra, M. 28 Borreca, A. 15 Borrelli, P. 27 Brescia Morra, V. 27,29,30,57 Brunetti, A. 27,29,30,31 Brunetti-Pierri, N. 54 Buonaguro, E.F. 23 Buono, S. 42 Busetto, G. 19 Butler, C.R. 13 C Cacialli, P. 48 Calabrò, V. 45

Canzoniero, L.M.T. 36 Capasso, S. 12 Cappello, S. 22,43 Cappuccio, G. 54 Carissimo, A. 24 Carotenuto, A. 29 Carotenuto, M. 52 Casalino, J. 9 Castaldo, L. 56 Castaldo, S. 45 Cefaliello, C. 9 Cellerino, A. 56 Cerciello, A. 47 Chiusano, M.L. 20 Ciccarelli, G. 49 Cilio, M.R. 51 Cioni, J.M. 39 Cipollaro, M. 12 Coakley, S. 24 Cocco, F. 37 Cocozza, S. 27,29,30,31,57 Cocurullo, M. 50 Colantuono, C. 20 Coletta, M. 17 Colino-Sanguino, Y. 24 Coppola, G. 33 Cordella, A. 15 Costabile, T. 30,31 Criscuolo, C. 25 Crispino, C. 35 Crispino, M. 36,47 Cristino, L. 14,19 D Damiano, C. 17 D’Angelo, L. 14,19,48,56 de Bartolomeis, A. 23 De Biase, D. 58 Decressac M. 24 De Girolamo, P. 14,19,28,39,56 de Leva, M.F. 42 Del Gaudio, S. 12 Del Giudice, E. 13,41 De Luca, M.A. 15 Del Vecchio, W. 31

De Michele, G. 25,40,49 De Paolo, R. 39 De Rosa, A. 40 de Souza Silva, M.A. 38 Di Bernardo, G. 12 Di Giaimo, R. 22,43,47 Di Marzo, V. 14,19,39 Di Pardo, A. 45 di Porzio, U. 35 Di Ruberto, G. 36 Di Schiavi, E. 11,24,37,46 Di Somma, A. 32 Dubbioso, R. 40,44,49,59 Duilio, A. 32 E Ekker, M. 26 Elifani, F. 45 Ernst, L. 15 Esposito, F. 18,24 Esposito, M. 40,44,49,59 Ezhova, Y. 21 F Faideau, M. 24 Fasano, D. 25 Fasano, G. 20,26,28 Ferraioli, A. 20 Ferrara, Y. 32 Ferrentino, R.I. 52 Ferrucci, V. 32 Filla, A. 31 Fiorito, G. 10,16 Forte, N. 14,19 Fraldi, A. 21 G Galderisi, U. 12 Gallotta, I. 11,24 Genesio, R. 41 Gentile, A. 28 Giordano, M. 43 Gironi Carnevale, U.A. 38 Giuditta, A. 9 Giuliano, T. 21


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Godoy, R.S. 26 Graziano, S. 42 Guilmette, E. 13 H Harris, W.A. 39 Hilliard, M. 24 Houlden, H. 32 I Illiano, P. 38 Imperadore, P. 10 Imperati, F. 41 Imperatore, R. 14,19 Iodice, R. 29,40,44,49,59 Iovino, A. 44 Italian CCA Study Group 41 K Kelsh, R. 20 Kurian, M. 46 Kyrousi, C. 43 L Lanzara, V. 42,52 Lanzo, A. 46 Lauritano, A. 51 Lavecchia, A. 18 Leggieri, A. 56 Lemke, J.R. 33 Lesca, G. 51 Liguori, A. 31 Locascio, A. 20 Lorusso, L. 55 Lucini, C. 48,56 M Maccari, S. 23 Macera, A. 27 Maglione, V. 45 Manganelli, F. 40,44,49,59 Manocchio, L. 33 Matassa, D.S. 18 Mattace Raso, G. 58 Medina, D. 24 Medoro, A. 37 Mele, G. 44 Melone, M.A.B. 12 Mercuri, N.B. 15 Merolla, E. 52 Miceli, F. 33,51 Miniaci, M.C. 36 Minopoli, N. 17 Monaco, A. 21 Montano, E. 45 Montefusco, S. 24 Monti, D. 17 Montuori, N. 18 Morley-Fletcher, S. 23 Mosca, I. 33 N

Nappi, P. 51 Neuhauss, S.C.F. 39 Nieddu, M. 38 Nigro, V. 54 Nitsch, L. 18,25,41 Nittoli, V. 20,28 O Oliveri, P. 50 Olivo, G. 30 Orefice, G. 29 Orellana, K. 10 P Paciello, F. 31 Paciello, O. 58 Pagano, C. 38 Pagano, T.B. 58 Paglione, M. 46 Paladino, S. 18,25 Palladino, A. 28 Palomba, L. 19 Palma, G. 27 Pandolfi, M. 42 Paolucci, M. 14 Papparella, S. 58 Parenti, G. 17 Pauletti, A. 13 Pellegrini, E. 48 Peluso, G. 12 Peluso, S. 40,49 Pennino, F.P. 32 Pepe, A. 18 Pellecchia, M.T. 25 Penna, E. 36,47 Perrone-Capano, C. 35,36 Pesapane, A. 18 Picillo, M. 25 Piegari, G. 58 Pierantoni, G.M. 25 Pinelli, M. 54 Pipicelli, F. 22,47 Piro, J.R. 13 Pirozzi, C. 58 Pisani, A. 27,30,57 Polishchuck, E. 24 Pollice, A. 45 Ponte, G. 16 Pontillo, G. 27,30,57 Porcu, L. 13 Porto, C. 17 Precenzano, F. 52 Prisco, F. 58 Prisco, M. 9 Pucci, P. 32 Pulcrano, S. 35 Q Quarantelli, M. 29,30,31,57 R Ranieri, M. 40

Remondelli, P. 25 Riccio, E. 27,30,57 Rizzi, M. 13 Romano, A. 41 Rossi, B. 17 Ruggiero, L. 44, 59 Rusconi, G. 27 Russo, A. 15 Russo, C. 27,31,37,57 Rutigliano, B. 9 S Saccà, F. 27,30,31,57 Sadile, A.G. 38 Salamone, A. 13 Salpietro, V. 32 Samad, T.A. 13 Sands, T.T. 51 Santonicola, P. 11 Santoro, L. 40,44,49,59 Sarnataro, D. 18 Scogliamiglio, I. 32 Sepe, R.M. 39 Seuntjens, E. 16 Severi, D. 59 Sheehan, M.J. 13 Siciliano, R. 32 Soldovieri, M.V. 33,51 Sordino, P. 20,26,28,39 Sorrentino, N.C. 21 Spagnuolo, A. 28 Speranza, L. 35 Spina, E. 44,59 Squillaro, T. 12 Stanzione, A. 27 Styfhals, R. 16 T Taglialatela, M. 33,51 Tarallo, A. 17 Tedeschi. E. 27,29,30,31,57 Terrone, G. 13,41 Tino, A. 38 Toni, M. 26 Topa, A. 44,59 Torella, A. 54 Tozza, S. 44,59 Treno, C. 38 Tucci, C. 42 TUDP, 54 Tunisi, L. 14,19 V Valeri, F. 15 Varricchio, E. 14 Vassalli, Q.A. 20 Vellucci, L. 23 Vezzani, A. 13 Viggiano, A. 55 Villa, B.R. 13 Vitiello, G. 41 Volpicelli, F. 35

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W Weisz, A. 33 Wood, N. 50

Z Zampi, G. 11,46 Zang, J. 39 Zerillo, L. 25

Zollo, M. 32 Zuffardi, O. 41 Zurzolo, C. 18

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