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

VIII° MEETING NEAPOLITAN BRAIN GROUP 13 december 2018

ABSTRACT BOOK

Centro di servizio di Ateneo per le Scienze e Tecnologie per la Vita (CESTEV) Via T. De Amicis, 95, 80145

Napoli

Meeting scientific committee E. Del Giudice (UNINA), C. Lucini (UNINA),

R. Di Giaimo (UNINA), P. Sordino (SZN), A. Germanà (Università di Messina),

P. Striano (IRCCS Istituto G. Gaslini)

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Supported by

under the patronage of

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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

PROGRAMMA 08.30 – 09.00 Affissione poster 09.00 - 09.30 Saluti iniziali e Presentazione dell’Associazione NBG 09.30 - 10.00 KEYNOTE LECTURE

“Anche le cicale sanno contare” - Guido Trombetti (UNINA ) 10:00 - 10.30 KEYNOTE LECTURE

“How the lysosome tells the nucleus what to do” - Andrea Ballabio (UNINA/TIGEM) 10.30 - 11.30 PRIMA SESSIONE

Chair: A. Rubino (UNINA), S. Paladino (UNINA) 4 interventi da 10 minuti + 5 discussione

� Coppola et al. Novel chd2 and kiaa2022 mutations associated with eyelid myoclonia with absences � Nappi et al. A fluorescence-based assay to evaluate the activity of KV7.2/KV7.3 channel

modulators � Pinelli et al. The first 2 years of experience of the Telethon Undiagnosed Diseases Program � Zerillo et al. Dysregulation of autophagy has a potential role in SYNJ1-associated early-onset

parkinsonism

11.30 - 11.45 COFFEE BREAK 11.45 - 12.45 SECONDA SESSIONE

Chair: N. Brunetti Pierri (UNINA/TIGEM), M. Pinelli (UNINA/TIGEM) 4 interventi da 10 minuti+ 5 discussione

� Tunisi et al. Orexin-a prevents lps-induced neuroinflammation at the level of the gut-brain axis � Boccella et al. Ultra-micronized palmitoylethanolamide rescues the cognitive decline-associated loss

of neural plasticity in the neuropathic mouse entorhinal cortex-dentate gyrus pathway � Operto et al. Reading and writing difficulties: the role of context characteristics and teacher

judgments � Pero et al. Pathogenic role of tubulin post-translational modifications in bortezomib-induced

peripheral neuropathy (BIPN)

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13.00 - 13.30 LUNCH BREAK 13.30 - 15.00 SESSIONE POSTERS 15.00 - 15.30 KEYNOTE LECTURE

“Mechanisms regulating human neurogenesis” – Silvia Cappello (Max Planck Institute of Psychiatry, Munich, Germany)

15.30 - 16.30 TERZA SESSIONE Chair: G. Andria (UNINA), M.G. Miano (CNR-IGB) 4 interventi da 10 minuti + 5 discussione

� Indrieri et al. mir-181a and mir-181b downregulation protects from mitochondria-associated neurodegeneration by enhancing mitochondrial biogenesis and mitophagy

� Santonicola et al. Neuroprotective role of hrp-2/syncrip in a C. elegans model of spinal muscular atrophy

� Di Matteo et al. 3D human models to understand the neurogenic role of cystatin b in human epilepsy EPM1

� Punzo et al. Epigenetic landscape of D-amino acids system genes during development and in schizophrenia

16.30 - 16.45 COFFEE BREAK 16.45 - 17.45 QUARTA SESSIONE

Chair: C. Bravaccio (UNINA), G. Vitiello (UNINA/MBBM) 4 interventi da 10 minuti + 5 discussione

� Spagnuolo et al. Brain-derived neurotrophic factor modulates cholesterol homeostasis and apolipoprotein e synthesis in human models of astrocytes and neurons

� Monaco et al. The molecular tweezer CLR01 inhibits amyloid aggregation relieving lysosomal pathology and protecting against neurodegeneration in lysosomal storage diseases

� Forte et al. Chronic overweight severely impairs adult hippocampal neurogenesis and plasticity � Leggieri et al. mRNA analysis of novel genes differentially expressed during adult neurogenesis in

the short lived teleost Nothobranchius furzeri

18.00 CONCLUDING REMARKS E. Del Giudice (UNINA), G. Piccialli (CESTEV, UNINA)

Meeting scientific committee Ennio Del Giudice (UNINA), Carla Lucini (UNINA), Rossella Di Giaimo (UNINA), Paolo Sordino (SZN),

Antonino Germanà (Università di Messina), Pasquale Striano (IRCCS Istituto G. Gaslini)

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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

Il Neapolitan Brain Group Il Neapolitan Brain Group (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 essere un'occasione di incontro, in un'atmosfera informale, per tutti gli appassionati di ricerca clinica e di base nel settore delle Neuroscienze che intendano migliorare la reciproca conoscenza e, per quanto possibile, esplorare momenti di collaborazione proficua con la finalità ultima di unire in una sorta di rete culturale tutti i Centri dell’area campana in cui si fanno clinica e/o ricerca di base dedicate allo studio della fisiologia e della patologia del sistema nervoso. Recentemente l’NBG si è anche costituito come associazione senza scopo di lucro con l’obiettivo di migliorare la propria offerta culturale. Il gruppo di confronto è aperto a tutti coloro che siano interessati alle tematiche di base ed applicate delle neuroscienze e, in particolare, ai giovani in formazione delle diverse Università ed Enti di Ricerca della Campania. Fino al momento attuale l’NBG ha tenuto i seguenti meeting: 1° Meeting su invito, 4/6/2015, Dipartimento di Biotecnologie, Università di Napoli Federico II 2° Meeting su invito, 4/2/2016, Dipartimento di Biotecnologie, Università di Napoli Federico II 3° Meeting su invito, 28/4/2016, Dipartimento di Biotecnologie, Università di Napoli Federico II 4° Meeting su invito, “Meccanismi molecolari, fisiopatologici e clinici nella neuroprotezione dell’ipossia neonatale”, 9/6/2016, Dipartimento di Biotecnologie, Università di Napoli Federico II 5° Meeting su call, 15/12/2016, CEINGE 6° Meeting su call, 14/12/2017, Stazione Zoologica Anton Dohrn 7° Meeting, Corso ECM “ Le malattie del sistema nervoso: basi patogenetiche e nuovi approcci terapeutici”, 31/5/2018, CESTEV, Università di Napoli Federico II. In queste riunioni hanno presentato i loro risultati più di 150 tra ricercatori di base, medici, dottorandi, postdoc, specializzandi, tirocinanti e tesisti delle Università e degli Enti di Ricerca della Campania. L'iniziativa NBG gode del patrocinio morale dell'Università degli Studi di Napoli Federico II. La mailing list del gruppo conta ad oggi 270 membri.

Per maggiori informazioni e per essere aggiornati sul gruppo: http://www.neapolitanbraingroup.it/ https://www.facebook.com/NBG2000/

Per essere inseriti nella mailing list scrivete a: Elia Di Schiavi (elia.dischiavi@ibbr.cnr.it)

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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

LETTURE MAGISTRALI

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ANCHE LE CICALE SANNO CONTARE

Guido Trombetti

Università di Napoli Federico II, Napoli, Italia

Magicicada è il nome di una specie di cicale che vive esclusivamente in Nord America ed è in certo senso unica al mondo perché gli adulti sono perfettamente sincronizzati ed emergono tutti contemporaneamente solo una volta ogni 13 o 17 anni. Si tratta di due numeri che non possono passare inosservati, sono numeri primi. Le cicale sono solo uno dei tanti esempi affascinanti in cui i numeri primi compaiono in natura quasi magicamente. Difficile immaginare che sia solo un caso. Se una famiglia di cicale emerge ogni 13 anni ed una ogni 17, si incontreranno solo una volta ogni 221 anni. È possibile immaginare svariati motivi per cui questo risulti vantaggioso dal punto di vista evolutivo, ma qui si entra nel campo della biologia e non spetta ai matematici fornire spiegazioni. Prima ancora che l’uomo iniziasse a contare, la natura conosceva i numeri primi e li “adoperava” – se così possiamo dire – per perseguire i suoi scopi. Nel caso delle cicale, ad esempio, i numeri primi sembrano essere la chiave per la sopravvivenza di una specie, nella corsa a perdifiato della selezione naturale. I numeri primi sono i mattoni dell’aritmetica. Moltiplicandoli tra loro si ottengono tutti gli altri numeri. Sono come la tavola periodica degli elementi per un chimico. Qualsiasi essere pensante, che abiti un pianeta di un sistema solare di una qualunque galassia, se è in grado di contare si imbatte nei numeri primi. La matematica in questo senso è universale, una caratteristica che è comune solo a pochi aspetti del sapere umano. Se ad esempio volessimo comunicare a distanza con una civiltà aliena, ci troveremmo di fronte alla necessità di concordare una lingua. La matematica è una lingua ed è forse la migliore candidata. Già mezzo secolo fa, con il lancio delle prime sonde interstellari, la comunità scientifica si è posta il problema di come fare per comunicare con qualcuno di cui non si conosce nulla. Non sorprende che in molti di questi tentativi i numeri primi svolgano un ruolo fondamentale proprio perché sono universali.

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HOW THE LYSOSOME TELLS THE NUCLEUS WHAT TO DO

Andrea Ballabio

Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy In the early 50s, Christian De Duve identified a new cellular structure, the lysosome, defined as the cell’s “suicide bag”. Sixty years later, it is clear that the lysosome greatly exceeded the expectations of its discoverer. The lysosome mediates intracellular clearance by degrading and recycling a variety of substances that reach this organelle through endocytosis, phagocytosis and autophagy. Over 50 different types of lysosomal storage diseases have been identified, each due to the deficiency or malfunction of a specific lysosomal protein. In addition, an important role of the lysosome has been unveiled in several common human diseases, such as cancer, obesity, neurodegenerative diseases, and infection. We have identified a lysosomal gene network and a master gene, TFEB, that regulates lysosomal biogenesis and autophagy. TFEB and his gene network enable the lysosome to adapt to environmental cues, such as nutrient availability. The activity of TFEB is regulated by the mTORC1 kinase complex through a lysosomal signaling pathway. In addition, we found that TFEB and mTORC1 are involved in a feedback loop that is mediated by the RagD GTPase. This feedback mechanism plays an important role in the response to starvation and physical exercise and is deregulated in cancer. Overall these data reveal that the lysosome acts as a signaling hub that signals to the nucleus through the shuttling of the TFEB transcription factor to control cell homeostasis and the switch between anabolism and catabolism.

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MECHANISMS REGULATING HUMAN NEUROGENESIS Silvia Cappello

Max Planck Institute of Psychiatry, Munich, Germany

Malformations of the human neocortex are present in about 1% of the general population and represent a major cause of developmental disabilities including severe epilepsy. To date, mouse lines carrying mutations of genes so far identified in human patients with cortical malformations only partially recapitulate the expected cortical phenotypes and therefore do not provide reliable models to entirely understand the molecular and cellular mechanisms responsible for these disorders. Therefore we decided to combine the in vivo mouse model and the human derived cerebral organoids in order to better comprehend the mechanisms involved in migration of neurons during human brain development and tackle the causes of neurodevelopmental disorders. Our results show that we can model human brain development and neuronal migration disorders using human cerebral organoids and contribute to open new avenues to bridge the gap of knowledge between human brain malformations and existing mouse models.

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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

COMUNICAZIONI ORALI

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NOVEL CHD2 AND KIAA2022 MUTATIONS ASSOCIATED WITH EYELID MYOCLONIA WITH ABSENCES

A. Coppola 1, S. Krithika2,3, M. Iacomino6, S. Balestrini2,3, L. Hernandez-Hernandez2,3, S. Meletti4,5, G. Gobbi7, E. Ferlazzo8,9, L. Giordano10, S. Casellato11, V. Sofia12, P. Striano13, L. Bilo1, V. Nigro14,15, A. Torella14,15, F. Musacchia14, F. Zara6, and S.M. Sisodiya2,3

1Department of Neuroscience, Reproductive and Odontostomatological Sciences, Epilepsy Centre, Federico II University, Naples, Italy; 2Department of Clinical and Experimental Epilepsy, UCL Institute of Neurology; 3Chalfont Centre for Epilepsy, Chalfont St Peter, UK 4Department of Biomedical, Metabolic, and Neural Science, University of Modena and Reggio Emilia, Modena, Italy; 5 Neurology Unit, OCSAE Hospital, AOU Modena, Italy; 6Laboratory of Neurogenetics, Department of Neurosciences, Institute "G. Gaslini", Genoa, Italy; 7IRCCS, Institute of Neurological Sciences of Bologna, Child Neurology Unit, via Altura 3, Bologna, Italy; 8Department of Medical and Surgical Sciences, Magna Græcia University of Catanzaro, Viale Europa, Germaneto, Catanzaro, Italy; 9Regional Epilepsy Center, Great Metropolitan Hospital, Via Melacrino, Reggio Calabria, Italy; 10Child Neuropsychiatry Unit - Civile Hospital, Brescia, Italy; 11Pediatric Neuropsychiatric Division Unit, AOU Sassari, Italy; 12Department of Medical and Surgical Sciences and Advanced Technologies "G.F. Ingrassia", University of Catania, Catania, Italy; 13Pediatric Neurology and Muscular Diseases Unit, Department of Neurosciences, Rehabilitation, Ophtalmology, Genetics, Maternal and Child Health, University of Genoa, "G. Gaslini" Institute, Genova, Italy; 14Telethon Institute of Genetics and Medicine, Pozzuoli, Naples, Italy; 15Dipartimento di Biochimica, Biofisica e Patologia Generale, Università degli Studi della Campania "Luigi Vanvitelli", Naples, Italy Purpose: Eyelid myoclonia with absences (EMA) is a generalized genetic epilepsy syndrome characterised by the hallmark of rapid blinking of the eyelids and an upward deviation of the eyeballs. Understanding the genetic architecture of EMA is a challenging task as eyelid myoclonia is also seen in other epilepsy syndromes. Method: The present study is based on a cohort of individuals with either only EMA, showing eyelid myoclonia with eye closure EEG bursts and/or photosensitivity (EMA), or with EMA associated with clinically evaluated intellectual disability or psychiatric conditions (EMA+). Whole-exome sequencing (WES) was performed on probands and their parents, when available. WES data was analysed, trios were analysed for de novo and/or unique variants and the singletons for unique variants. Sanger sequencing was used to confirm variants. Results: We sequenced 112 individuals, including 19 trios, 40 singletons and 2 multiplex families. We identified 2 novel de novo CHD2 mutations in 2 EMA+ trios: a missense variant [c.4598 T>G] and a splice donor variant [c.3455+2 T>G]. We also identified a de novo frameshift deletion affecting KIAA2022 [c.2171del] in another EMA+ trio. All these variants were unique, with CADD score>20. Two singletons showed a previously reported mutation in the CHD2 gene: a frameshift variant [c.3734dupA] and a splice region variant [c.2577+7T>C]. The former variant was identified in an EMA+ case, and the latter in an EMA case. Overall, 10.5% of trios showed a de novo CHD2 variant and 5% of singletons harboured mutation in this gene. Conclusion: CHD2 gene plays a key role in cerebral cortical development. Mutations in this genehave been reported for a broad spectrum of neurodevelopmental disorders, including EMA. KIAA2022 encodes a neurite extension and migration factor. Mutations in this gene have never been associated with EMA. Our study adds growing evidence implicating novel CHD2 and KIAA2022 mutations in EMA.

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A FLUORESCENCE-BASED ASSAY TO EVALUATE THE ACTIVITY OF Kv7.2/Kv7.3 CHANNEL MODULATORS

P. Nappi1, F. Miceli1, A. Lauritano1, C. Ostacolo2, P. Campiglia3, and M. Taglialatela1

1 Dept. Neuroscience, University of Naples Federico II, Naples, Italy 2 Dept. of Pharmacy, University of Naples Federico II, Naples, Italy 3 Dept. of Pharmacy, University of Salerno, Salerno, Italy

Potassium channels encoded by the Kv7 subfamily are critical regulators of neuronal excitability and attractive pharmacological targets for several neuropsychiatric conditions. In particular, Kv7.2 and Kv7.3 subunits underlie most of the M-current (IKM), a sub-threshold K+ current controlling action potential generation at the axon initial segment. Mutations in the genes encoding for Kv7.2 and Kv7.3 are responsible for early-onset epilepsies. Moreover, the Kv7.2/Kv7.3 channel activator retigabine has been approved as a novel anticonvulsant, but has been now withdrawn because of its poor selectivity for Kv7 subtypes, short half-life, poor brain penetration and chemical instability. In this work, we have: 1. developed a fluorescence-based assay to indentify novel Kv7.2/Kv7.3 channel modulators, 2. used this fluorescence assay to evaluate the activity of a small library of retigabine derivates, and 3. compared the fluorescence results with electrophysiological data. Stable CHO cell lines expressing Kv7.2 and Kv7.3 channel subunits generated by the PiggyBac Transposon System were used in a Thallium (Tl+)-based assay with a fluorescent Tl+-sensitive dye (FluxOR Green Potassium Ion Channel Assay). Retigabine (0.3 to 100 µM) dose-dependently increased the maximal fluorescence and the initial slope of the fluorescent signal; both effects were abolished by the Kv7 blocker XE991 (10µM). The calculated EC50 for retigabine was 5.0±0.6µM, a value slightly higher than the EC50 calculated by electrophysiological techniques (1.9±0.2 µM). Several compounds able to increase the maximal fluorescence and the initial slope of the fluorescent signal, in some cases with higher potency and efficacy when compared to retigabine, were identified.

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THE FIRST 2 YEARS OF EXPERIENCE OF THE TELETHON UNDIAGNOSED DISEASES PROGRAMS M. Pinelli1,2*, A. Torella1,3*, G. Cappuccio1,2, R. Castello1, F. Musacchia1, M. Mutarelli1, D. Carrella1, S. Maitz4, G. Vitiello1,2,4, F. Grilli5, M. Alagia1,2, S. Fecarotta2, A. Commone6, M. Tartaglia7, B. Dallapiccola6, V. Leuzzi6, G. Parenti1,2, V. Capra5, A. Selicorni4, N. Brunetti-Pierri1,2, S. Banfi1,3, TUDP, G. Casari1,8*, and V. Nigro1,3*

1 Telethon Institute of Genetics and Medicine, Pozzuoli, Naples, Italy 2 Department of Translational Medicine, Section of Pediatrics, Federico II University, Naples, Italy 3 Department of Precision Medicine, University of Campania "Luigi Vanvitelli", Naples, Italy 4 Department of Pediatrics. ASST-Lariana. Sant'Anna Hospital, San Fermo della Battaglia (Como), Italy 5 Neurosurgery service, Giannina Gaslini Institute, Genoa, Italy 6 Genetics and Rare Diseases Research Division, Ospedale Pediatrico Bambino Gesù, IRCCS, Rome, Italy 7 Department of Human Neuroscience, "Sapienza, University of Rome", Rome, Italy 8 Vita-Salute San Raffaele University and Neurogenomics Unit, Division of Genetics and Cell Biology, San Raffaele Scientific Institute, Milan, Italy * These authors contribute equally to the work

We present the experience of the first two years of the Telethon Undiagnosed Diseases Program (TUDP). TUDP is a national program founded by Fondazione Telethon aimed at the diagnosis of children with severe and multi-organ diseases that lack a genetic diagnosis. The aim of the program is to offer an accurate clinical re-evaluation of all patients and, if indicated, a Next Generation Sequencing (NGS) test. 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. From April 2016 to June 2018, nine national pediatric centers have proposed 355 undiagnosed patients. Fifty-two have been considered as not eligible either because a diagnosis was already possible with patient data re-evaluation or the clinical manifestations were outside inclusion criteria. Of the 305 enrolled patients, 118 have completed the whole NGS analysis and results have been interpreted by comparison with medical literature and undiagnosed patient databases. Analysis of 54 patients (45%) has been positively concluded. Three patients had mutations in the same gene (DDXD3), two couples of patients in two genes (GRIN1 and ASXL3) and the rest of patients in single genes. Among these cases, 19 showed extension of the phenotypes already reported in literature and in one case a mutation in a new disease-gene was found. For unconcluded cases, 9 new candidate genes have been found with already matched second patients and follow-up studies are in progress.

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DYSREGULATION OF AUTOPHAGY HAS A POTENTIAL ROLE IN SYNJ1-ASSOCIATED EARLY-ONSET PARKINSONISM

L. Zerillo1, D. Fasano1, G. Natale2, A. De Rosa2, M. Picillo3, G. Amodio4, M.T. Pellecchia3, P. Barone3, G. De Michele2, L. Nitsch1, G.M. Pierantoni1, M. Renna1, P. Remondelli4, C. Criscuolo2, and S. Paladino1,5

1 Dept of 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 3 Center For Neurodegenerative Diseases (CEMAND), University of Salerno, Salerno, Italy 4 Dept of Medicine, Surgery and Dentistry “Scuola Medica Salernitana”, University of Salerno, Salerno, Italy 5 CEINGE Biotecnologie Avanzate scarl, Naples, Italy

Several genes responsible for some of the hereditary forms of Parkinson’s disease are implicated in distinct steps of the intracellular trafficking regulating the endo-lysosomal pathway. Among the others, we have recently reported how SYNJ1 mutations, which cause an early-onset parkinsonism (PARK20) are associated to an alteration of endosomal trafficking. In particular, we revealed a crucial role for Synaptojanin 1 (Synj1) in regulating the homeostasis and functions of early endosomal compartments. Importantly, the same alterations of early endosomal compartments and trafficking defects occur in fibroblasts derived from PARK20 patients, highlighting defective cellular pathways in PARK20 mutant cells. Furthermore, we also found that the structure of lysosomes resulted slightly altered in Synj1-deficient cells, despite any substantial difference in the levels of two lysosomal markers, Lamp-1 and cathepsin D. Because trafficking toward lysosomes is unaffected upon Synj1 silencing, the alteration of lysosomes could be due to changes in the autophagic pathway, whose activity is critical in many neurodegenerative diseases. Our results show how autophagy is indeed up-regulated in Synj1 silenced cells, as observed by measuring the levels and number of autophagosomes. The increase in autophagosome biogenesis can be, at least in part, explained by the alteration of the mTORC1 signaling, a key upstream modulator of the pathway. Nevertheless, the clearance of autophagy substrates results reduced in Sinj1 depleted cells, suggesting therefore a more complex scenario, which will need further investigations. Overall, our preliminary data corroborate the existence of a functional link between endosomal trafficking, autophagic pathway and Parkinson’s disease.

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OREXIN-A PREVENTS LPS-INDUCED NEUROINFLAMMATION AT THE LEVEL OF THE GUT-BRAIN AXIS

L. Tunisi1,2, N. Forte2, I. Mavaro1,2, R. Imperatore2,3, A.C. Fernández-Rilo2,4, R. Capasso5, N. Milic6, L. D’Angelo1,7, P. de Girolamo1, L. Cristino2, V. Di Marzo2,8, and L. Palomba2,9

1 Department of Veterinary Medicine and Animal Productions, University of Naples, Federico II, Italy 2 Endocannabinoid Research Group; Institute of Biomolecular Chemistry of CNR, Pozzuoli, Italy 3 Department of Sciences and Technologies, University of Sannio, Benevento, Italy 4 University of Campania “Luigi Vanvitelli” Naples, Italy 5 Department of Agricultural Science, University of Naples Federico II, Portici (NA) Italy 6 Department of Pharmacy, University of Novi Sad, Novi Sad, Serbia 7 Stazione Zoologica Anton Dohrn, Napoli, Italy 8 Canada Excellence Research Chair, Institut Universitaire de Cardiologie et de Pneumologie de Québec and Institut sur la Nutrition et les Aliments Fonctionnels, Université Laval, Québec, Canada 9 Department of Biomolecular Sciences, University of Urbino Carlo Bo, Urbino, Italy

Gut-brain axis, the bidirectional communication system between the central and the enteric nervous systems, is regulated also by the gut microbiota. In states of dysbiosis, the intestinal epithelial barrier (IEB) permeability is increased as consequence of disruption of the epithelial tight junction protein network, thus promoting endotoxemia, i.e. bacterial lipopolysaccharides (LPSs) translocation and systemic circulation of pro-inflammatory cytokines. Orexin-A (OX-A), a neuropeptide implicated in many physiological functions, is produced mainly in the lateral hypothalamic area but also in the myenteric plexus, where the OX-A receptor-1 (OX-1R) is located on the plasma membrane of enteric neurons. Several lines of evidence suggest a role of OX-A on the microglia activation. Here, we sought to investigate if OX-A is able to prevent the increase of IEB permeability induced by LPS and of consequent activation of microglia. In a co-culture system with intestinal epithelial Caco-2 cells in the apical side and murine primary culture of microglia in the basolateral side, we demonstrated that treatment with OX-A on Caco-2 cells preserve the expression of occludin, the main component of protein forming the tight junction network, and activation of microglia induced by LPS. These protective effects are reversed by OX-1R antagonist (SB334867). These data were confirmed by in vivo studies, showing that the i.p. injection of OX-A coupled with LPS prevented the increase of IEB permeability and microglia activation in an OX1R-dependent manner, since SB334867 treatment inhibits the OX-A beneficial effects. In conclusion, our results suggest the role of OX-A as an epithelial barrier protective factor that may prevent LPS translocation to the CNS and, consequently, neuroinflammation.

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ULTRA-MICRONIZED PALMITOYLETHANOLAMIDE RESCUES THE COGNITIVE DECLINE-ASSOCIATED LOSS OF NEURAL PLASTICITY IN THE NEUROPATHIC MOUSE ENTORHINAL CORTEX-DENTATE GYRUS PATHWAY

S. Boccellaa, C. Cristianob, R. Romanoa, M. Iannottaa, C. Belardoa, A. Farinaa, F. Guidaa, F. Piscitellic, E. Palazzoa, M. Mazzitellid, R. Imperatoree, L. Tunisic, V. de Novellisa, L. Cristinoc, V. Di Marzoc, A. Calignanob, S. Maionea, and L. Luongoa

a Department of Experimental Medicine, Pharmacology Division, University of Campania “L. Vanvitelli”, 80138 Naples, Italy b Department of Pharmacy, School of Medicine, University of Naples Federico II, Naples, Italy c Endocannabinoid Research Group, Institute of Biomolecular Chemistry, CNR, Pozzuoli, Italy d Department of Pharmacology and Neuroscience, Texas Tech University Health Sciences Center, Lubbock, TX e Department of Science and Technology, University of Sannio, Benevento, Italy

Chronic pain is associated with cognitive deficits. Palmitoylethanolamide (PEA) has been shown to ameliorate pain and pain-related cognitive impairments by restoring glutamatergic synapses functioning in the spared nerve injury (SNI) of the sciatic nerve in mice. SNI reduced mechanical and thermal threshold, spatial memory and LTP at the lateral entorhinal cortex (LEC)-dentate gyrus (DG) pathway. It decreased also postsynaptic density, volume and dendrite arborization of DG and increased the expression of metabotropic glutamate receptor 1 and 7 (mGluR1 and mGluR7), of the GluR1, GluR1s845 and GluR1s831 subunits of AMPA receptor and the levels of glutamate in the DG. The level of the endocannabinoid 2-arachidonoylglycerol (2-AG) was instead increased in the LEC. Chronic treatment with PEA, starting from when neuropathic pain was fully developed, was able to reverse mechanical allodynia and thermal hyperalgesia, memory deficit and LTP in SNI wild type, but not in PPARα null, mice. PEA also restored the level of glutamate and the expression of phosphorylated GluR1 subunits, postsynaptic density and neurogenesis. Altogether, these results suggest that neuropathic pain negatively affects cognitive behavior and related LTP, glutamatergic synapse and synaptogenesis in the DG. In these conditions PEA treatment alleviates pain and cognitive impairment by restoring LTP and synaptic maladaptative changes in the LEC-DG pathway. These outcomes open new perspectives for the use of the N-acylethanolamines, such as PEA, for the treatment of neuropathic pain and its central behavioural sequelae.

KEYWORDS: Spared nerve injury, pamitoylethanolamide, long term potentiation, PPARα, cognitive performance, synaptogenesis

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READING AND WRITING DIFFICULTIES: THE ROLE OF CONTEXT CHARACTERISTICS AND TEACHERS JUDGMENTS F. Operto, D. Esposito, C. Nicoletti, M. La Corte, R. Del Duca, A. Viggiano, G. Pastorino, and G. Coppola Department of Medicine, Surgery and Odontostomatology, Medical School of Salerno, University of Salerno, Salerno, Italy Background. In southern Italy and, specifically, in the region of Campania, many surveys show that the average of students with reading difficulties is much higher than in the northern Italy and abroad. On the other hand, specific learning disorders (SLDs) in Campania are much less certified. Aims. Since there are no etiological reasons that can explain this apparent inconsistency, an objective of this cross-sectional study was to evaluate the extent of reading/writing difficulties in students from a province of Campania and then to assess the ability of teachers to identify such difficulties in their students. Sample. Of a total of 241 enrolled students, 155 (64.31%), including 73 from primary school and 82 from secondary school, belonging to 5 schools in the province of Salerno (Italy), took part in the survey. Methods. Students’ reading and writing skills were assessed by means of standardized tests. The tests results were then compared with teachers judgments and context-related variables. Results. At the reading test, 28.7% of primary school and 13.4% of lower secondary school students fell below the 5th percentile for age. Results of the writing test were even more significant: almost half of the students of both levels of education performed below the 5th percentile. Teacher judgments showed higher agreement with standardized assessments in primary (88%, K of Cohen = 0.68) than in secondary school (78%, K = 0.23). Conclusion. Writing difficulties tend to persist to some extent in the sixth-grade classes and over, while reading skills improve with age. Further extended surveys are needed in this field.

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PATHOGENIC ROLE OF TUBULIN POST-TRANSLATIONAL MODIFICATIONS IN BORTEZOMIB-INDUCED PERIPHERAL NEUROPATHY (BIPN) M.E. Pero1,2, G. Cavaletti3, and F. Bartolini2

1 Department of Veterinary Medicine and Animal Production, University of Naples, Federico II 2 Department of Anatomy, Pathology and Cell Biology, Columbia University 3 Experimental Neurology Unit and Milan Center for Neuroscience, University of Milano-Bicocca Tubulin and microtubules (MTs) play critical roles in neuronal function and are well-established targets for certain anticancer drugs that can also induce chemotherapy induced peripheral neuropathy (CIPN) (vinca, alkaloids, taxanes). The contribution of these MT changes to the onset of CIPN is not well understood, but strongly predicted to be a determining factor. We hypothesized that seemingly unrelated CIPN-inducing drugs may share an underlying mechanism of pathogenesis based on acute alteration of one or more tubulin post-translational modifications (PTMs). To examine this, we measured the relative levels of selected tubulin PTMs in the cell bodies of dorsal root ganglia (DRG) and in sciatic nerves (SNs) isolated from rats treated with acute doses of the proteasome inhibitor Bortezomib (Bort) prior to any manifestation of neuronal injury or neuropathic behavior. Among the tubulin PTMs examined, delta-2 tubulin (D2), an irreversible tubulin PTM and marker of hyperstable MTs, was significantly increased in L4-L5 DRG and SNs of Bort treated rats, and high levels of D2 were further detected in the sural nerve from a cancer patient suffering from BIPN. We examined the pathogenic potential of D2 accumulation in dissociated adult DRG neurons and found that while induction of D2 alone was sufficient to cause axonopathy, reducing D2 accumulation significantly alleviated axonal degeneration promoted by Bort. Our results suggest that the mechanisms of CIPN drugs may converge on the acute perturbation of tubulin PTMs, and that disruption of the tubulin detyrosination cycle may play a role in the axonal injury induced by Bort.

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MIR-181a AND MIR-181b DOWNREGULATION PROTECTS FROM MITOCHONDRIA-ASSOCIATED NEURODEGENERATION BY ENHANCING MITOCHONDRIAL BIOGENESIS AND MITOPHAGY

A. Indrieri1,2, S. Carrella1,3, A. Romano1, A. Spaziano1, F. Golia1, M. Pizzo1, R. Tammaro1, E. Marrocco1, N. Giordano1,4, A. Carboncino4,5, L. Ciampi1, S. Barbato1, E. De Leonibus1,4, E.M. Surace1,3, S. Banfi1,3*, and B. Franco1,2

1 Telethon Institute of Genetics and Medicine (TIGEM), Italy 2 Department of Translational Medical Science, University of Naples “Federico II”, Italy 3 Department of Biochemistry, Biophysics and General Pathology, University of Campania “L. Vanvitelli”, Italy 4 Institute of Genetics and Biophysics (IGB), CNR, Italy 5 Department of Neuroscience, Biomedicine and Movement Science, University of Verona, Italy

Mitochondrial dysfunction underlies the pathogenesis of a variety of human neurodegenerative disorders, either directly, e.g., in the case of mitochondrial diseases, or indirectly, e.g. in the case of Parkinson’s disease. The complexity of these disorders has so far prevented the development of effective therapeutic strategies. We demonstrate that the microRNAs miR-181a and miR-181b (miR-181a/b) regulate key genes involved in mitochondrial biogenesis and function. We also show that these miRNAs are involved in the global regulation of mitochondrial turnover in the central nervous system through the simultaneous and fine tuning modulation of different gene pathways. We sought to verify whether the modulation of these miRNAs could be therapeutically exploited in neurodegenerative conditions associated to impaired mitochondrial activity. We show that miR-181a/b downregulation strongly protects neurons from cell death and significantly ameliorates the disease phenotype in in vivo models of both primary and secondary mitochondria-mediated neurodegeneration, such as Microphthalmia with Linear Skin Lesions, Leber hereditary optic neuropathy and Parkinson’s disease. Altogether our results indicate that miR-181a/b act as hubs in mitochondrial homeostasis in the central nervous system and represent novel gene-independent therapeutic targets for a wide-range of neurodegenerative diseases caused by mitochondrial dysfunction.

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NEUROPROTECTIVE ROLE OF HRP-2/SYNCRIP IN A C.ELEGANS MODEL OF SPINAL MUSCULAR ATROPHY P. Santonicola1, F. Cieri1, F. Rizzo2, M. Nizzardo2, S. Corti2,3, and E. Di Schiavi1

1 Institute of Bioscience and BioResources, IBBR, CNR, Naples, Italy 2 Dino Ferrari Centre, Neuroscience Section, Department of Pathophysiology and Transplantation (DEPT), University of Milan, Italy 3 IRCCS Foundation Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy

SMN1 is the gene responsible for Spinal Muscular Atrophy (SMA), a devastating neuromuscular disease characterized by progressive and selective degeneration of lower motor neurons (MNs), leading to muscle atrophy and death of patients. SMA has been extensively studied, but the molecular mechanisms leading to MNs degeneration are still unknown. Therefore, animal SMA models are crucial to understand the pathogenesis of the disease and the functions played by SMN. In C.elegans, as in all models, SMN ortholog (smn-1) full depletion causes strong larval lethality and impaired locomotion. We overcame smn-1 KO pleiotropic effects by silencing smn-1 only in 19 MNs (Gallotta et al., HMG 2016) and we observed an age-dependent degeneration and neuronal death that mimic the key features of SMA pathology. Hence, we used our SMA model as a tool to identify new neuroprotective modifiers, genetically interacting with smn-1 in the MNs. By RNA sequencing of SMA patients-derived iPSC we identified SYNCRIP, a heterogeneous nuclear ribonucleoprotein, as a new modifier of SMN function. SYNCRIP, similarly to SMN1, is part of the spliceosome and is involved in pre-mRNA processing and transport. SYNCRIP homolog in C. elegans is hrp-2, a gene expressed in all tissues, including MNs. We demonstrated that HRP-2/SYNCRIP overexpression in neurons is able to partially rescue, in vivo, the neurodegeneration, the neuronal death and the defects in locomotion observed in our model (Rizzo et al., Brain under revision). Our results strongly suggest SYNCRIP as a new potential target for innovative combinatorial therapeutic approaches for SMA.

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3D HUMAN MODELS TO UNDERSTAND THE NEUROGENIC ROLE OF CYSTATIN B IN HUMAN EPILEPSY EPM1

F. Di Matteo1,2, C. Kyrousi1, F. Pipicelli2, L. Canafoglia3, S. Cappello2, and R. Di Giaimo1,2

1 Department of Biology, University of Napoli, Napoli, Italy 2 Max Planck Institute of Psychiatry, München, Germany 3 Carlo Besta Neurological Institute, Milan, Italy

EPM1 is an autosomal recessive neurodegenerative disorder that has the highest incidence among the progressive myoclonic epilepsies worldwide, characterized by stimulus-sensitive myoclonus and tonic-clonic epileptic seizures. Loss of function mutations in the gene encoding CYSTATIN B (CSTB) are the primary genetic cause of EPM1. CSTB is a small ubiquitous protein, identified as inhibitor of cathepsin family proteases. It is generally accepted that the loss of the antiprotease function of CSTB is the cause of EPM1 but the evidence supporting the involvement of CathepsinB is debatable. Here we model EPM1 and the role of CSTB by using patient-derived cerebral organoids, a 3D model of human brain development. To this aim, we collected blood samples from two patients with EPM1: one patient homozygous for the most common EPM1 mutation, a dodecamer amplification in the promoter of CSTB gene; and one young patient, heterozygous compound with promoter amplification and point mutation in intron 1. Starting from these 2 samples, we generated induced pluripotent stem cells (hIPSCs) that were then used to generate cerebral organoids. We found that EPM1-derived cerebral organoids show premature differentiation and alteration of progenitors proliferation related to controls. Moreover, we generated neurons derived from EPM1 and control cerebral organoids, confirming the results obtained in 3D cultures. Furthermore, the effects on progenitors proliferation are in line with previous results showed both in human cerebral organoids and in mouse brain, where CSTB or one of the pathological mutants were overexpressed, suggesting a role of CSTB in regulating early steps of human neurogenesis and development.

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EPIGENETIC LANDSCAPE OF D-AMINO ACIDS SYSTEM GENES DURING DEVELOPMENT AND IN SCHIZOPHRENIA

D. Punzo1,2, F. Errico3, T. Nuzzo4, M. Cuomo5, O. Affinito5, S. Keller5, L. Chiariotti5, and A. Usiello1,2

1 Laboratory of Behavioural Neuroscience Ceinge Biotecnologie Avanzate, 80145 Naples, Italy 2 Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, 81100 Caserta, Italy 3 Department of Agricultural Sciences, University of Naples “Federico II”, 80055, Portici, Italy 4 Medical Genetics Unit, IRCCS Casa Sollievo della Sofferenza, Mendel, Roma, Italy 5 Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, 80131 Naples, Italy

D-aspartate and D-serine modulate NMDA receptor-dependent transmission. Herein, we aimed to describe an epigenetic landscape of the genes modulating D-amino acids system evaluating the impact of their expression on D-amino acids levels during development, and in a neuropathological condition like schizophrenia. It is acknowledged that in the mammalian brain, D-aspartate content decreases after birth, as a result of the postnatal expression of the catabolizing enzyme D-aspartate oxidase (DDO). On the other hand, D-serine displays a region-specific occurrence during ontogenesis, under the regulation of the biosynthetic enzyme, serine racemase, and the catabolizing enzyme, D-amino acid oxidase (DAAO). Our studies in the mouse brain revealed that postnatal Ddo gene expression is paralleled by progressive demethylation within its putative promoter. In this regard, we revealed that embryonic cortical neurons treated with the DNA-demethylating agent, azacitidine, showed non-physiological increased Ddo mRNA levels. Then we analyzed DNA methylation state and mRNA expression of the genes regulating D-aspartate and D-serine levels at different stages of mouse development, from birth to adulthood, and in different brain regions, like the hippocampus, cortex and cerebellum. Our analysis revealed decreased Ddo gene methylation and specular increased mRNA levels in the hippocampus and cerebellum. Moreover, demethylation of Daao gene is associated with increased transcription in the cerebellum during development. Finally, we translated our studies to post-mortem brain regions of patients with schizophrenia. Epiallele classes and configuration analyses provided distinct area-specific patterns suggesting the occurrence of an orchestrated distribution of epialleles in diverse cell populations.

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BRAIN-DERIVED NEUROTROPHIC FACTOR MODULATES CHOLESTEROL HOMEOSTASIS AND APOLIPOPROTEIN E SYNTHESIS IN HUMAN MODELS OF ASTROCYTES AND NEURONS

M.S. Spagnuolo1, A. Donizetti2, L. Iannotta2, V. Aliperti2, and L. Cigliano2

1 Department of Bio-Agrofood Science,ISPAAM, CNR, Naples, Italy 2 Department of Biology, University of Naples Federico II, Naples, Italy In the central nervous system (CNS), cholesterol is critical to maintain membrane plasticity, cellular function, and synaptic integrity. A breakdown of brain cholesterol metabolism has been associated with different CNS diseases, including Alzheimer disease. Brain-derived neurotrophic factor (BDNF) was previously reported to elicit cholesterol biosynthesis and promote the accumulation of presynaptic proteins in cholesterol-rich lipid rafts, but no data are available on its ability to modulate physiological mechanisms involved in cholesterol homeostasis. Major aim of our research was to study whether BDNF influences critical events in cholesterol homeostasis, namely: a) the synthesis of Apolipoprotein E (ApoE), which is the strongest genetic risk factor for Alzheimer disease, b) the cholesterol efflux from astrocytes and c) the cholesterol incorporation into neurons. Our results show that BDNF significantly stimulates cholesterol efflux by primary astrocytes, as well as ATP binding cassette A1 (ABCA1) transporter and ApoE expression. Conversely, cholesterol uptake in neurons was downregulated by BDNF. This effect was associated with the increase of Liver X Receptor (LXR)-beta expression in neurons exposed to BDNF. Interestingly, the level of apoptosis markers was found increased in neurons treated with high cholesterol, but significantly lower when the cells were exposed to cholesterol in the presence of BDNF, thus suggesting a critical neuroprotective role of the neurotrophin, likely through its reducing effect of neuronal cholesterol uptake. Also, cholesterol treatment actually induces BDNF production by neurons. Overall, our findings evidenced a novel critical role of BDNF in the modulation of ApoE and cholesterol homeostasis in glial and neuronal cells.

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THE MOLECULAR TWEEZER CLR01 INHIBITS AMYLOID AGGREGATION RELIEVING LYSOSOMAL PATHOLOGY AND PROTECTING AGAINST NEURODEGENERATION IN LYSOSOMAL STORAGE DISEASES A. Monaco, V. Maffia, C. Sorrentino, Y. Ezhova, I. Sambri, V. Cacace, E. Nusco, E. De Leonibus, and A. Fraldi

Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy

Lysosomal storage diseases (LSDs) are inherited metabolic disorders caused by either lysosomal or non-lysosomal proteins deficiencies and often showing a severe neurodegenerative course. No cure is currently available to slow neuropathology progression in these diseases. Neurodegeneration in LSDs is driven by a global dysfunction of the lysosomal system, in particular of autophagy pathway. Such lysosomal dysfunction is associated to progressive accumulation of several types of substrates, whose nature and pathogenic relevance remain, however, still unclear. Here we found that massive amyloid deposition characterizes brain pathology in several mouse models of LSDs. Brain amyloid deposits progressively build up as neurodegeneration proceeds as shown by analyzing a mouse model of mucopolysaccharidoses type IIIA (MPS-IIIA), one of the most common and severe form of neurodegenerative LSDs. Such amyloid inclusions contain α-synuclein together with several other aggregate-prone proteins, such as prion protein, Tau and amyloid ß-peptide. A major fraction of these amyloid deposits is localized to the lysosomal compartment where they act as main determinants of lysosomal dysfunction, thus boosting the development of neurodegenerative phenotype. Indeed, inhibiting amyloid deposition in MPS-IIIA mice by CLR01, a “molecular tweezer” molecule, which efficiently hamper self-assembly of multiple amyloidogenic proteins relieves lysosomal pathology, thus re-activating lysosomal-autophagic pathway and ameliorating neuropathological signs. Together, these data put more insights on mechanisms determining amyloid aggregation toxicity and identify LSDs as a new class of amyloid disorders. Moreover, our results identify CLR01 as a potent drug candidate for the treatment of brain lesions in LSDs.

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CHRONIC OVERWEIGHT SEVERELY IMPAIRS ADULT HIPPOCAMPAL NEUROGENESIS AND PLASTICITY

N. Forte1, R. Imperatore1,2, S. Boccella3, L. Tunisi1,4, I. Mavaro1,4, A.C. Fernandez Rilo1,2, L. Palomba1,5, S. Maione2, V. Di Marzo1,6, and L. Cristino1

1 Endocannabinoid Research Group, Institute of Biomolecular Chemistry, CNR, Pozzuoli, Italy. 2 Department of Science and Technology (DST), University of Sannio, Benevento, Italy. 3 Department of Experimental Medicine, Pharmacology Division, University of Campania “L. Vanvitelli”, Naples, Italy. 4 Department of Veterinary Medicine and Animal Productions, University of Naples Federico II, Naples, Italy. 5 Department od Biomolecular Sciences, University of Urbino “Carlo Bo”, Urbino, Italy. 6 Canada Excellence Research Chair, Institut Universitaire de Cardiologie et de Pneumologie de Québec and Institut sur la Nutrition et les Aliments Fonctionnels, Université Laval, Québec, QC G1V 0A6, Canada.

Adult neurogenesis is a structural form of neural plasticity that contributes to the maintenance of normal brain function. Adult neural progenitors are present in the sub-ventricular zone of the forebrain, in the hippocampal dentate gyrus and in the hypothalamus. Within the dentate gyrus, newborn neurons are known to integrate into the pre-existing circuitry and to play a substantial role in the synaptic plasticity which underlies certain types of learning and memory. Several environmental and physiological factors, including high fat diet critically modulate almost every stage of neurogenesis. In previous studies we demonstrated that leptin signal deficiency in the brain of obese mice is coupled to high levels of orexin-A (OX-A) and endocannabinoid 2-Arachidonoylglycerol (2-AG). OX-A enhances neurogenesis, by promoting neuronal proliferation and differentiation, whereas chronic administration of leptin in adult mice increases cell proliferation in the dentate gyrus. However, the functional cross-talk between these molecular interplayers in the regulation of adult neurogenesis in not well understood. At this purpose, in the present study, we exploited leptin knockout ob/ob mice, which represent a model of obesity by mimicking the leptin inefficacy in the brain of mice made obese by a high fat diet, to investigate the effects of chronic overweight on the learning and memory by a functional evaluation of the long term potentiation (LTP) in adult dentate gyrus. By combining biochemical, morphological and functional studies with electrophysiology, in vitro and in vivo, we found an activation of orexinergic/endocannabinoid pathway which regulate the neuronal progenitor fate.

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mRNA ANALYSIS OF NOVEL GENES DIFFERENTIALLY EXPRESSED DURING ADULT NEUROGENESIS IN THE SHORT LIVED TELEOST NOTHOBRANCHIUS FURZERI

A. Leggieri1,2, L. D'Angelo1,3, M. Baumgart2, L. Castaldo1, P. de Girolamo1, C. Lucini1, A. Cellerino2,4, and L. Avallone1

1 Università degli Studi di Napoli Federico II, Napoli, Italy 2 Leibniz Institute on Aging - Fritz Lipmann Institute, Jena, Germany 3 Stazione Zoologica Anton Dohrn, Napoli, Italy 4 Scuola Normale Superiore di Pisa, Pisa, Italy

Adult neurogenesis is a process that exponentially decreases with aging in many species. In mammals it occurs only into the subventricular and subgranular zones. In teleost fish, neurogenic areas are more numerous and distributed along the entire rostro-caudal brain axis. Among teleosts, Nothobranchius furzeri is a well-established aging model. Clustering temporal profile of gene expression in N. furzeri, has shown differentially expressed genes (DEGs). We investigated the neurogenic activity of three DEGs well conserved during evolution: Inhibitor of DNA Binding 3 (ID3), Alpha Chain 1 Collagen type IV (COL4A1) and XXV (COL25A1). Quantitative Real-Time PCR was assessed in the whole encephalon of young (5 weeks post hatching, wph) and old (27 wph) animals. Data show a significant age-related upregulation of ID3, COL4A1, and COL25A1. We localized the genes by means of fluorescence in situ hybridization (FISH) experiments, confirming their presence in neurogenic areas. Then, to identify cytotype expressing gene mRNAs, double labeling of FISH and immunofluorescence was performed with anti-PCNA, anti-S100; anti-DCX; anti-HuC/D, and anti-TH. A wide co-localization is observed in most of neurogenic areas of forebrain, midbrain, and hindbrain, particularly in periventricular areas. Furthermore, we performed a statistical analysis of ID3, COL4A1, and COL25A1 co-localization with single neural marker in each brain macro-area. In proliferating cells, ID3 is more expressed in new born neurons (DCX+), whereas COL4A1 and COL25A1 are homogeneously expressed in radial glia (S100+) and differentiating neurons (HuC/D+); in mature neurons (S100+ - TH+), COL25A1 is the most represented.

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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

POSTERS

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EVOLUTION OF NEURONAL NOS GENE IN CNS OF FISH G. Annona1, J.L. Ferran2, I. Conte3, J. Postlethwait4, and S. D’Aniello1

1 Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn di Napoli, 80121, Napoli, Italy 2 Institute of Biomedical Research of Murcia (IMIB), Virgendela Arrixaca University Hospital, University of Murcia, Murcia, Spain 3 Telethon Institute of Genetics and Medicine, 80078, Pozzuoli - Napoli, Italy 4 Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA

Nitric Oxide, a gaseous signaling molecule, is probably one of the oldest bio-regulatory elements playing key roles in metazoan physiology. In tetrapods, three Nos paralogs were described based on their expression profiles: two constitutive, namely the neuronal Nos (nNos) and the endothelial Nos (eNos), and one inducible Nos (iNos) involved in immune responses. In fish, due to the evolutionary tetraploidization and re-diploidization events, the Nos repertoire results more complex. Indeed, differential loss of Nos gene duplicates have been involved in the generation of fish variability. Interestingly, the nNos gene has been maintained in a single copy and is considered the predominant source of NO in neurons. Expressed in several areas of the central and peripheral nervous systems, it participates in the elaboration of olfactory, visual and neuroendocrine stimuli. nNos promotes learning and memory and is involved in the control of adult CNS neurogenesis. Nos genes share a very similar genomic structure, but the open question is whether this conservation has also a functional meaning. In this work we investigate the nNos expression profile in three fish species that occupy key phylogenetic positions in the evolution and show a different Nos gene repertoire: Oryzias latipes (1 nNos), Danio rerio (1 nNos and 2 iNos (a/b), and Lepisosteus oculatus (1 nNos, 1 eNos and 1 iNos). Our results demonstrated that nNos expression profile increases in brain areas during the embryo development. Most remarkably, we identified homologous territories as well as specie-specific regions of nNOS expression in the CNS, suggesting a complex scenario.

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APPROPRIATENESS OF NEUROPSYCOLOGICAL TESTING REFERRAL IN CAMPANIA: A SINGLE CENTER EXPERIENCE M. Bellofatto, F. Lauro, M. Tedeschi, V. Brescia Morra, R. Lanzillo, and C. Criscuolo

Department of Neurosciences, Reproductive and Odonostomatologic Science – Federico II University - Naples

OBJECTIVES: To describe health care outcomes of patients referred for neuropsychological evaluation (NE) to our Dementia Center from March to April 2018. MATERIALS: Neuropsychological tests were performed by three different neuropsychologists and included Mini-Mental State Examination (MMSE, Italian version), spatial and verbal span, Rey fifteen-item memory test, visuo-spatial examination, frontal assessment battery and fluencies. RESULTS: 156 patients (75 males, 81 females) underwent NE. Mean age±SD was 67±11.1 years. Average years of education±SD were 8±4.8 (9.3±4.8 in males; 7±4.7 in females). Most patients were referred by outside physicians: 49 (31%) by neurologists, 13 (9%) by general practitioners, and 49 (31%) by other specialists. 106 patients had already CT or MRI before NE; 28 patients also had FDG-PET studies (12 requested by our center, 7 by other neurologists, 8 by other specialists). NE revealed 96 (61%) patients with a negative MMSE, however 62 of them (64%) presented at least one domain impaired. The most affected domains were executive functions (58%), long-term (57%) and short- term memory (44%). Psychiatric symptoms were reported by 104 patients. The most common were depression and anxiety (54% and 50% respectively; 45% females vs 34% males); only 30% of these patients were treated. DISCUSSION: Our data shows that NE is part of the standard diagnostic approach to dementia not only by neurologists, but also by other specialists. FDG-PET scans are most often ordered by neurologists, particularly by tertiary dementia centers. We speculate that complex cases are referred to neurologists who utilize FDG-PET in challenging cases. No differences were observed between males and females except for lower education and higher incidence of psychiatric disorders in women. Psychiatric disorders were a frequent feature, but patients were seldom treated. Considering the deep impact of these diseases on test scoring, better management by the referral physician is warranted.

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UNRAVELING THE ROLE OF LOW-DENSITY LIPOPROTEIN RECEPTOR-RELATED PROTEIN 8 IN ALZHEIMER’S DISEASE USING A NEW C. ELEGANS MODEL C. Bertapelle1,2, A. Medoro1, D. Mignogna1, C. Russo1, and E. Di Schiavi2

1 Department of Health Sciences, University of Molise, Campobasso, Italy 2 Institute of Bioscience and BioResources, CNR, Naples, Italy Alzheimer’s disease (AD) is the most common neurodegenerative disorder. Familial AD is caused by mutations in APP, PSEN1 and PSEN2, while individuals carrying the ε4 allele of Apolipoprotein E (ApoE) are at increased risk of AD. One of the neuropathologic feature of AD is represented by the extracellular deposition of β-amyloid (Aβ), deriving from the proteolysis of the Amyloid Precursor Peptide (APP) by presenilins (encoded by PSEN1 and PSEN2), the catalytic core of the γ-secretase complex. Aβ accumulation seems to be responsible for the pathogenesis, although lately this hypothesis is debated. Recent data suggest the involvement of the ApoE receptor, the Low-Density Lipoprotein Receptor-Related Protein 8 (LRP8) in AD genesis. LRP8 is involved in neuronal migration, cell proliferation and memory and, like APP, is cleaved by γ–secretase. We hypothesized that the expression levels and relative proteolytic processing of LRP8 may modulate and influence the AD phenotype, and the occurrence of neurodegeneration. To test our hypothesis, we used C.elegans to study in vivo the correlation among LRP8, PSEN and APP. We generated transgenics overexpressing in neurons human LRP8, as full-length or as fragmented proteins. These lines present a dose-dependent defect in development, locomotion and lifespan. Moreover, we showed PSEN involvement in LRP8 function, using a pharmacological and a genetic approach. The role of the different domains of hLRP8 in these phenotypes is under analysis and the impact on learning and APP proteolysis will be shown.

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NEURO-BEHÇET DISEASE WITH BENIGN INTRACRANIAL HYPERTENSION: HOW CAN WE BETTER TREAT IT? REPORT OF TWO CASES S. Bruscella, T. Somma, M. Barbato, C. Peca, and O. de Divitiis Dipartimento di Neuroscienze e Scienze Riproduttive e Odontostomatologiche, Divisione di Neurochirurgia- Università degli Studi di Napoli Federico II- Azienda Ospedaliera Universitaria Federico II, Napoli Behçet disease is a multisystemic chronic disease of unknown etiology, characterized by an inflammatory perivasculitis that can arise in almost any tissue. It was first described in 1937 with the classic clinic triad: recurrent oral and genital ulcers with uveitis. Neurological involvement is one of the most serious causes of long-term morbidity and mortality. Its prevalence is described as highly variable, ranging from 1-3% to 59% and it is reported as 2-8 times more frequent in men than in women, with age of onset usually around 20–40 years. Behçet disease’s involvement of the Central Nervous System can be categorized as parenchymal and non-parenchymal with the second one typically characterized by vascular complications such as the presence of intracranial aneurysms and thrombosis within the large vessels (usually veins). This condition may lead to a syndrome of intracranial hypertension and permanent visual loss. Clinical management of intracranial hypertension in Behcet patients resistant to medical treatment, is complex and not fully clarified. We describe two cases of Neuro-Behçet disease with benign intracranial hypertension, treated in our Department, in which lumboperitoneal shunt gave a benefit, even if not long-lasting, with several complications resulting from surgery. Our two cases highlight that if diagnosis of Behçet disease is correct, and treatment proper and timely, clinical results are more favorable. The disease’s course may not be entirely benign: further researches on its pathophysiology are required and cooperation between different specialists is mandatory, because it may lead to a better management of this complex clinical entity.

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A SURVEY ON PEDIATRIC EPILEPSY IN THE ISCHIA ISLAND: EPIDEMIOLOGIC AND GENETIC FINDINGS

V. Buono1, A. Coppola1, V. D’Ambrosio2, G. Giussani3, S. Buono4, F. Zara5, E. Beghi3, and P. Striano6

1 Department of Neuroscience, Reproductive and Odontostomatological Sciences, Federico II University, Naples, Italy 2 Family pediatrician, Ischia, Italy 3 Laboratory of Neurological Diseases, IRCCS “Mario Negri”, Milan, Italy 4 Department of Neurology, AORN “Santobono-Pausilipon”, Naples, Italy 5 Laboratory of Neurogenetics and Neuroscience, IRCCS "G. Gaslini" Institute, Genova, Italy 6 Pediatric Neurology and Muscular Diseases Unit, DINOGMI-Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health, "G. Gaslini" Institute, Genova, Italy

There are only few studies on pediatric epilepsy in geographic isolates, we investigated the frequency and characteristics of pediatric epilepsy in a typical geographic isolate, with a twofold purpose: 1. To calculate incidence and prevalence of the disease; 2. To investigate the genetic component through family history, medical history and gene mapping, to be obtained from probands and relatives. The total population of island of Ischia is 61,086, including 8,381 children and adolescents aged 0-14 years. Only children and adolescents aged 1 through 14 years in the calendar year 2017 were eligible for investigation. Epilepsy was diagnosed according to the International League Against Epilepsy’s classification. A total of 36 children and adolescents were found with confirmed epilepsy. There were 16 girls and 20 boys. Included were 18 patients with epilepsy due to structural/metabolic diseases and 18 patients with idiopathic epilepsy. Perinatal complications were the predominant cause in patients with structural/metabolic diseases. The prevalence was 4.3 per 1,000. Only one patient was diagnosed prior to 2004, leaving 35 incident cases. The incidence in general pediatric population, calculated during the 14-year period, was 33.5 per 100,000/year (29 in female pediatric population e 39,1 in male pediatric population). In contrast with other reports from geographic isolates, our findings do not confirm a higher than expected prevalence of epilepsy. The prevalence of active epilepsy in children in our study was even lower than in the Eolian islands that included in the diagnosis only patients with two or more unprovoked seizures.

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NERVE GROWTH FACTOR IS EXPRESSED AND STORED IN BRAIN NEURONS OF ADULT ZEBRAFISH

P. Cacialli1,2, C. Gatta1, L. D’Angelo1,3, A. Leggieri1, A. Palladino4, P. de Girolamo1, E. Pellegrini2, and C. Lucini1

1 Dip Medicina Veterinaria e produzioni animali, Università di Napoli Federico II, Napoli, Italy 2 Environment and Occupation, SFR Biosit, University of Rennes 1, Rennes, France 3 Stazione Zoologica Anton Dohrn, Napoli, Italy 4 Centro Ricerche Interdipartimentali sui Biomateriali, Università di Napoli Federico II, Naples, Italy

Nerve Growth Factor (NGF), a member of the neurotrophin family, was initially described as neuronal survival and growth factor, but successively has emerged as active mediator in the central nervous system of mammals. NGF is synthesized as precursor pro-NGF and is either secreted outside the cells or cleaved intracellularly into mature NGF. Despite the vast literature present in mammals, studies devoted to NGF in the brain of other animal models are scarce. Zebrafish is a teleost fish emerging as model for translational neuroscience research. Ngf organization is highly similar in zebrafish and mouse. Besides to mature NGF protein, two precursors are known in zebrafish. NGF mRNA was visualized by in situ hybridization on whole brains. NGF protein distribution was assessed on microtomic sections by using an antiserum against NGF which recognizes proNGF. To characterize NGF positive cells, anti NGF was employed on aromatase B transgenic zebrafish slides (where radial glial cells appeared fluorescent) and by means of double immunolabelling against NGF/PCNA (proliferation marker) and NGF/MAP2 (mature neuronal marker). NGF mRNA and protein were widely distributed in the brain of adult zebrafish and their distribution pattern was quite overlapping, both in males and females. MAP2 immunoreactivity was present in the majority of NGF positive cells, throughout the zebrafish brain. PCNA and aromatase B cells were not positive to NGF, but they were closely intermingled with NGF cells. In conclusion, our study demonstrated that mature neurons in the zebrafish brain express NGF mRNA and store proNGF.

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CYTOTOXIC EFFECTS OF CADMIUM CHLORIDE IN THE ZEBRAFISH BRAIN

T. Capriello1, A. Monaco2, M.C. Grimaldi1, and I. Ferrandino1

1 Department of Biology, University of Naples Federico II, Naples, Italy 2 Telethon Institute of Genetics and Medicine, Pozzuoli, Italy

Cadmium is indicated as a possible etiological factor in neurodegenerative diseases. In this study we have analysed the effects of 1mg/L cadmium chloride (CdCl2), a concentration found in industrial polluted water, on brain of adult zebrafish exposed to metal for 16 days. The brains were analysed at 2, 7 and 16 days from treatment. We evaluated the neurodegeneration by the Fluoro-Jade B stain and the myelin integrity by methasol fast blue staining. The expression of glial fibrillary acidic protein (GFAP), astroglial marker, and the accumulation levels of beta-Amyloid1-42 peptide were tested by ABC technique and western blotting. Collected data showed neurodegenerative effects time-dependent and a sensible reduction of affinity to the dye in neurons axons after 7 and 16 days of exposure respect the control. Moreover, a decrease in the levels of GFAP was observed after 2 days in the order of 40% compared with the control. This decrease arrived to 50% after 16 days. In particular, a reduction of GFAP positive fibers was revealed in midbrain and in cerebellum, in medulla oblongata and after 16 days in telencephalon. Instead the levels of increased as time passed, in telencephalon, cerebellum and medulla oblongata. These data confirm the toxicity of cadmium on neuroglia cells and represent a point for discovering possible correlations between cadmium and neurological disorders as Alzheimer’s disease for the data on beta-Amyloid1-42 peptide.

35

NOMOPHOBIA RELATED PHENOMENA IN YOUNG PEOPLE AND ASSOCIATION WITH PSYCHOPATHOLOGICAL RISK

G. Catone, S. Gritti, K. Russo, V.P. Senese, and A. Gritti

Università degli Studi Suor Orsola Benincasa Università della Campania “Luigi Vanvitelli”

Nowadays people routinely use new technologies (computers, smartphone). In particular, young people use internet for information and for social activities. Currently internet access is mainly via the smartphone. The Net children Go Mobile project indicated that, among children aged 9 to 16 years, 46% owned a smartphone. Although new technologies have brought enormous benefits to the community, we are starting to look at some negative aspects; among these nomophobia is of particular interest. Nomophobia is the sense of anxiety related to the impossibility of using the mobile phone. It includes the smartphone addiction (SA), Ringxiety, behaviour of continuously check the smartphone (RA) and the phantom phone signals, the illusion of hearing phone ringtone (PPS). We aim to describe nomophobia in a sample of 2959 students of the metropolitan city of Naples and to test their potential association with psychopathological risk. SA was endorsed by 2042 (69%) subjects. 1343 (45.4%) students indicated RA. PPS was present in 1709 (57.8%) subjects. Female had higher nomophobia phenomena and all variables significantly increase with the progressive shift to higher-grade classes. SA demonstrated positive and significant correlation with emotional problems (0.130), conduct problems (0.242), hyperactivity (0.208) and total problems (0.208). RA showed positive and significant correlation with emotional problems (0.136), conduct problems (0.252), hyperactivity (0.206) and total problems (0.219). PPS had positive and significant correlation with emotional problems (0.193), conduct problems (0.222), hyperactivity (0.187), peer problems (0.091), and total problems (0.240). In conclusion it is necessary to evaluate these phenomena among young people.

36

FISH OIL AND CONJUGATED LINOLEIC ACID SUPPLEMENTATION ALLEVIATES

STRUCTURAL AND FUNCTIONAL SIGNS OF DEPRESSION IN AGED MRL/MPJ-FASLPR MICE. L. Ciglianoa, S. Spagnuoloa, L. Treppiccioneb, E. Coccac, L. Iannottaa, and P. Bergamob

a Università degli Studi di Napoli “Federico II” - Napoli b Istituto di Scienze dell’Alimentazione - CNR (ISA) - Avellino c Istituto di Bioscienze e BioRisorse - CNR (IBBR) - Napoli Purpose Inflammation and oxidative stress play an important role in the pathogenesis of depressive disorders and Nuclear erythroid related factor 2 (Nrf2), a master regulator of RedOx homeostasis, is a promising target for depression prevention and treatment. As fish oil (FO, n-3 PUFA) and Conjugated Linoleic Acid (CLA) - composed by 1:1 amount of cis9, trans11 and trans10, cis12 C18:2 isomers - are Nr2 activator, their preventive ability in relieving functional/structural signs of depression was comparatively evaluated in a murine model of neuro-psychiatric lupus (MRL/MpJ-Faslpr). Four groups of mice (n=8 each) were used. Three composed by Old mice (17-weeks old) and one by Young animals (8-10 weeks). Two Old groups were supplemented (po) for 5 weeks with human equivalent doses of FO or CLA . Young or untreated Old mice were used as negative or diseased/positive control, respectively. At the end of the treatment, structural (brain Fatty Acids) and functional decline (oxidative stress, inflammation, neuroplasticity signalling) was evaluated to examine anti-depressive activity of the different supplements. Disrupted redox homeostasis in Old mice associated with compensatory hyperactivation of Nrf2, deterioration of brain Fatty Acids profile, increased pro-inflammatory cytokines and lower synaptic plasticity markers as compared to Young mice. FO and CLA ameliorated all the pathophysiological hallmarks at a level comparable to Young mice. This is the first time that CLA (n-6 and n-7 PUFA mixture) was shown to exhibit anti-depressive effects comparable to that of n-3 PUFAand involvement of Nrf2-mediated mechanisms in anti-depressive effect resulting from FO or CLA intake was hypothesized.

37

JOINING THE DOTS… USE OF SULFORAPHANE TO RELIEVE BOTH BDNF PRODUCTION AND NRF2-DEPENDENT ANTIOXIDANT POTENTIAL IN CDKL5 DEFICIENCY DISEASE: A CASE STUDY

A. Colarusso, M. Calvanese, C. Lauro, E. Parrilli, and M.L. Tutino

Dipartimento di Scienze Chimiche, Università Federico II, Napoli

Mutations in X-linked CDKL5 gene have been associated to an ultra-rare human disease, now called CDKL5-Deficiency Disorder (CDD). CDD is a severe neurodevelopmental disorder, which affects in majority girls and is almost always associated to early-onset epilepsy and a severe mental retardation. Amongst many other functions, CDKL5 mutations were associated with the nuclear accumulation of HDAC4, which results in the silencing of BDNF expression [Trazzi et al. 2016] potentially explaining several neurodevelopmental alterations associated with CDD. Furthermore, fibroblasts derived from CDD patients displayed an aberrant localization of Nrf2 (a master regulator of antioxidant genes expression) and were uncapable of facing even moderate oxidative stresses [Pecorelli et al., 2015]. A critical review of the recent literature highlighted the ability of sulforaphane (SFN, an isothiocyanate derivative present in broccoli sprouts at high concentration) to epigenetically enhance BDNF production in both wild type and 3 x Tg-AD primary neurons, thus activating the TrkB dependent signaling pathways [Kim et al. 2017]. It was demonstrated that this action was related to a clear inhibition of total histone deacetylase activity (HDACs). Moreover, BDNF was recently demonstrated to induce the hippocampal nuclear translocation of Nrf2, which ends up in neurons better suited to face oxidative stress related to ROS production [Bruna et al., 2018]. On this background, we elaborated a working hypothesis on the benefit of using SNF treatment in CDD patients. The outcomes of SNF treatment on one 8 years old CDKL5 girl will be presented.

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DISEASE’ MECHANISMS UNDERLYING THE NEUROPATHOLOGICAL PROGRESSION IN MUCOPOLYSACCHARIDOSIS IIIA M. De Risi1, M. Tufano1, F.G. Alvino1, S. Pulcrano2, G.C. Bellenchi2, A. Fraldi1, M. Caiazzo3, and E. De Leonibus1,4 1 Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli (Naples), Italy 2 Institute of Genetics and Biophysics, CNR, Naples, Italy 3 Utrecht Institute for Pharmaceutical Sciences (UIPS), Universiteitsweg, Utrecht, The Netherlands 4 Institute of Genetics and Biophysics (IGB), Naples and Institute of Cellular Biology and Neurobiology (IBCN), Monterotondo (Rome), National Research Council, Italy Mucopolysaccharidosis type IIIA (MPS-IIIA) is a neurodegenerative lysosomal storage disorder characterized by the deficiency of the enzyme sulfamidase. The pathology is characterized by different stages, each defined by a specific set of symptoms. The neurobiological mechanisms leading to these symptoms are still unknown. In the early phase, children with MPS-IIIA manifests with behavioural symptoms (BSs), including stereotypic and social behaviour dysfunctions; these symptoms are progressively substituted by the onset of dementia and motor impairment. Using an animal model of MPS-IIIA, we have identified endophenotypes of early and late stages of MPS-IIIA pathology, which are associated to dynamic changes in tyrosine hydroxylase (TH) expression, the dopamine (DA) synthesis-rating enzyme. This dynamic changes in TH expression are recapitulated in a cellular model of the pathology. These findings are important to define specific antipsychotic therapy for behavioral symptoms and to understand the disease’ mechanisms leading to DA dysfunctions in MPS-IIIA.

39

MAPPING NOCICEPTORS IN OCTOPUS VULGARIS ARM: DISTRIBUTION AND COMPLEXITY

G. Di Cristina, P. Imperadore, G. Ponte, and G. Fiorito

Dipartimento Biologia ed Evoluzione Organismi Marini, Stazione Zoologica Anton Dohrn

Limited morphological and behavioral evidence, supporting the view that cephalopods are able to experience pain, was used as the basis for the recommendation by EFSA Panel to the European Commission for the inclusion of these invertebrate animals in the Directive 2010/63. It is only recently that the presence of nociceptors has been postulated in the squid Doryteuthis (Loligo) paeleii, and in the octopus Abdopus aculeatus. These putative nociceptors are high threshold mechanoreceptors that undergo sensitization after the application of a potentially noxious stimulus. The central projections of these nociceptors are still not known. In the common octopus, Octopus vulgaris, the existence of a “pain circuit” has been postulated on the basis of behavioral responses to putative negative stimuli. Here we present our recent data based on the search of pain related genes and nociceptor-markers in O. vulgaris. Transcriptomes and genome sequencing allowed to estimate for the first time the relative expression of pain-related-molecules in octopus nervous systems and in the arms. Relative expression of transcripts coding for transient receptor potential channels (e.g., TRPA1, TRPV1), peptides (e.g., Substance P, CGRP), G-protein coupled receptors (e.g. ADORA2a, CALCRL) and other proteins involved in nociceptive pathways will be presented. We also provided a mapping of putative nociceptors in the octopus arm and suckers by localizing fibers and cellular structures expressing proteins involved in nociceptive pathways, e.g., Substance, P IB4 and CGRP. Our data contribute in providing evidence of the existence of a nociceptive system in Octopus vulgaris, for the first time.

40

INHIBITION OF S1P DEGRADATION IS BENEFICIAL IN THE TRANSGENIC R6/2 MOUSE MODEL OF HUNTINGTON DISEASE

A. Di Pardo, E. Amico, S. Castaldo, L. Capocci, G. Pepe, and V. Maglione

Centre for Neurogenetics and Rare Diseases, IRCCS Neuromed, Pozzilli (IS), Italy Background. Huntington’s disease (HD) is the most common neurodegenerative disorder with no effective cure currently available. Over the past few years our research has shown that alterations in sphingolipid metabolism represent a critical determinant in the pathogenesis of the disease. In particular, we have provided the first evidence of aberrant metabolism of sphingosine-1-phosphate (S1P) in multiple disease settings including human post-mortem brains from HD patients. Importantly, we have also demonstrated that pharmacological interventions aimed at reducing S1P degradation, by inhibition S1P-Lyase (SGPL1), resulted beneficial in an HD cell model. Aim. In this study, we aimed to investigate whether inhibition of SGPL1 may exert therapeutic action in-vivo in the R6/2 HD mouse model. Results. Our results indicate that chronic administration of 0.1 mg/kg THI is safe and well tolerated in manifest R6/2 HD mice. The compound significantly slowed down the progressive mouse motor deficit associated with the worsening of the disease. Immunoblotting analysis reveals a significant increase in the levels of cortical post synaptic density protein PSD-95 in HD treated animals. Further analyses are in progress for establishing any other disease-modifying properties of the compound. Conclusion. Our preliminary data indicate that the beneficial effect of THI treatment may be likely associated with an increased synaptic activity. These findings further support the idea that modulation of S1P metabolism may represent a promising therapeutic approach for the treatment of the disease.

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OBESITY-DRIVEN NEURODEGENERATIVE DISEASES: NEW INSIGHTS FOR NEW MOLECULAR INTERPLAYERS AND THERAPEUTIC TARGETS

A.C. Fernández-Rilo1,2, L. Tunisi1,3, I. Mavaro1,3, N. Forte1, R. Imperatore4, R. Capasso5, L. Palomba6, V. Di Marzo1, and L. Cristino1

1 Endocannabinoid Research Group -Institute of Biomolecular Chemistry, CNR, Pozzuoli, Naples, Italy 2 University of Campania “Luigi Vanvitelli” Naples, Italy 3 Department of Veterinary Medicine and Animal Productions, University of Naples Federico II, Italy 4 Department of Sciences and Technologies, University of Sannio, Benevento, Italy 5 Department of Agricultural Sciences, University of Naples Federico II, Portici, Italy 6 Department of Biomolecular Sciences, University of Urbino “ Carlo Bo” Italy 7 Canada Excellence Research Chair, Institut Universitaire de Cardiologie et de Pneumologie de Québec and Institut sur la Nutrition et les Aliments Fonctionnels, Université Laval, Québec, Canada

Neurodegenerative diseases are one of the main causes of worldwide disability and decreased quality of life. A link between neurodegeneration and obesity is opening novel research avenues in the search for therapeutic approaches. Unfortunately, current available treatments for both pathologies remain ineffective. Therefore, it is important to generate appropriate models to evaluate the relationship between obesity and neurodegeneration. The metabolic changes caused by obesity are able to modulate the central nervous system by altering the synaptic plasticity. In this study, we have investigated the changes in synaptic plasticity at areas implicated in cognitive function, such as the hippocampus and prefrontal cortex, in order to further understand the molecular mechanisms that control phosphorylation of Tau. GSK3β is a key enzyme responsible for the fine balance between the phosphorylated and not phosphorylated forms of Tau, modulating synaptic plasticity in these areas. Different interplayers, such as leptin hormone, orexin-A (OX-A) neuropeptide or components of the endocannabinoid (ECs) system are able to interact and modify the activity of GSK3β. By molecular, biochemical and morphological studies, we found remarkable changes in the expression of phosphorylated tau form in hippocampus and prefrontal cortex of leptin knockout ob/ob mice which represent a model of obesity by mimicking the leptin inefficacy in the brain of mice made obese by a high fat diet. Furthermore, we found elevation of 2-AG and OX-A content in the hippocampus of ob/ob confirming that OX-A and 2-AG (2-arachidonoylglycerol) are able interplayers to increase or decrease, respectively, the phosphorylation of tau in opposite way. Unraveling the functional crosstalk between ECs and OX system in the regulation of Tau phosphorylation of in areas of the brain involved in cognitive function could reveal novel molecular players and pathways that might result in drug able targets.

42

LOCOMOTION ALTERATIONS IN BDNF ZEBRAFISH MUTANTS: FIRST INSIGHTS INTO ITS GENETIC PATHWAY

E. Gagliardi, F. Salatiello, F. Berruto, Y. D’Agostino, S. D’Aniello, and A. Locascio

Stazione Zoologica “Anton Dohrn”

Background: GDP mannose pyrophosphorylase B (GMPPB) deficiency is one of several dystroglycanopathies, a heterogeneous group of neuromuscular disorders characterized by progressive myopathy, brain and eye abnormalities, and intellectual disability. GMPPB catalyzes the formation of GDP-mannose, required for mannosylation and glycosylation of alpha-dystroglycan (ADG). Patient and methods: In a patient with GMPPB deficiency due to GMPPB gene mutations (p.Q234X/p.T153I), presenting with hypotonia, elevated serum creatine kinase, psychomotor delay, seizures, and bilateral cataracts, we found a profound deficiency of the lysosomal acid alpha-glucosidase (GAA). Results: GAA activity in fibroblasts was 2.61 nmol/mg prot/h (NV 64.4 ± 22.9). The molecular analysis of the GAA gene was negative. A western blot analysis showed impaired processing of GAA in fibroblasts. GAA was mostly detectable as the 110 kDa precursor isoform, with near-complete deficiency of the mature 70 and 76 kDa peptides. Conversely, exogenous recombinant human GAA (Myozyme) was normally internalized and processed into the mature GAA. Morever, PAS staining showed glycogen accumulation, which disappeared in cells treated with Myozyme. In addition, we found reduced activities of beta-galactosidase, alpha-galactosidase, alpha-mannosidase, beta-glucosidase and alpha-fucosidase, suggesting an impairment of multiple lysosomal enzymes. Conclusions: Our results suggest aberrant processing of endogenous GAA in our patient’s fibroblasts, possibly due to defective glycosylation and mannose-6-phosphate generation. We speculate that defective GMPPB function impacts not only on ADG, but also on glycosylation of other glycoproteins, including lysosomal enzymes. It is possible that secondary GAA deficiency contributes to the pathophysiology of muscle disease in GMPPB deficiency.

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DEFICIENCY OF LYSOSOMAL ACID ALPHA-GLUCOSIDASE IN A PATIENT WITH DYSTROGLYCANOPATHY DUE TO GMPPB DEFICIENCY V. Gragnaniello1, S. Fecarotta1, A. Tarallo1,2, C. Damiano1,2, N. Minopoli1, R. Tuzzi1, R. Della Casa1, D. Garozzo3, L. Sturiale3, R. Barone3, 4, and G. Parenti1, 2

1 Department of Translational Medical Sciences, Federico II University, Naples, Italy 2 Telethon Institute of Genetics and Medicine, Pozzuoli, Italy 3 CNR, Institute for Polymers, Composites and Biomaterials, Catania, Italy 4 Pediatric Neurology Unit, Department of Pediatrics, University of Catania, Catania, Italy

Background: GDP mannose pyrophosphorylase B (GMPPB) deficiency is one of several dystroglycanopathies, a heterogeneous group of neuromuscular disorders characterized by progressive myopathy, brain and eye abnormalities, and intellectual disability. GMPPB catalyzes the formation of GDP-mannose, required for mannosylation and glycosylation of alpha-dystroglycan (ADG). Patient and methods: In a patient with GMPPB deficiency due to GMPPB gene mutations (p.Q234X/p.T153I), presenting with hypotonia, elevated serum creatine kinase, psychomotor delay, seizures, and bilateral cataracts, we found a profound deficiency of the lysosomal acid alpha-glucosidase (GAA). Results: GAA activity in fibroblasts was 2.61 nmol/mg prot/h (NV 64.4 ± 22.9). The molecular analysis of the GAA gene was negative. A western blot analysis showed impaired processing of GAA in fibroblasts. GAA was mostly detectable as the 110 kDa precursor isoform, with near-complete deficiency of the mature 70 and 76 kDa peptides. Conversely, exogenous recombinant human GAA (Myozyme) was normally internalized and processed into the mature GAA. Morever, PAS staining showed glycogen accumulation, which disappeared in cells treated with Myozyme. In addition, we found reduced activities of beta-galactosidase, alpha-galactosidase, alpha-mannosidase, beta-glucosidase and alpha-fucosidase, suggesting an impairment of multiple lysosomal enzymes. Conclusions: Our results suggest aberrant processing of endogenous GAA in our patient’s fibroblasts, possibly due to defective glycosylation and mannose-6-phosphate generation. We speculate that defective GMPPB function impacts not only on ADG, but also on glycosylation of other glycoproteins, including lysosomal enzymes. It is possible that secondary GAA deficiency contributes to the pathophysiology of muscle disease in GMPPB deficiency.

44

IMPACT OF A SHORT TERM FRUCTOSE DIET ON REDOX HOMEOSTASIS, AUTOPHAGY, AND SYNAPTIC FUNCTION MARKERS IN FRONTAL CORTEX OF YOUNG AND ADULT RATS: AN INTRIGUING MENAGE A TROIS

L. Iannotta1, P. Bergamo2, A. D’Aria1, R. Crescenzo1, S. Iossa1, L. Cigliano1, and M.S. Spagnuolo3 1 Dept of Biology, University of Naples Federico II, Naples, Italy 2 Dept of Bio-Agrofood Science, Institute of Food Sciences, National Research Council (CNR- ISA), Avellino, Italy 3 ISPAAM, CNR, Naples, Italy

The use of fructose as sweetener for beverages and processed food is strongly increased in the last decade. A high consumption of this sugar has been related to the onset of obesity, metabolic diseases and reduced synaptic plasticity. Since adolescents make a widespread consumption of added sugars, we decided to investigate the early risks of a short-term fructose-rich in frontal cortex of both young and adult rats, focusing on the effects on redox homeostasis, autophagy and synaptic function. We focused on this region, as its maturation continues until late adolescence, and includes areas critically implicated in higher-order cognitive functions. Short term fructose feeding was associated with an imbalance of redox homeostasis, as lower amount of Nuclear factor (erythroid derived 2)-like 2, lower activity of Glucose 6-phosphate dehydrogenase and Glutathione reductase as well as lower Glutathione/Oxidized Glutathione ratio were found. Furthermore, the activation of autophagy and a decrease of synaptic function markers were evidenced in fructose-fed young and adult rats. Interestingly, two key markers of brain functioning, BDNF signaling and Acetylcholinesterase, were affected by fructose diet in age dependent manner. Intriguingly, an increase of Acetylcholinesterase activity, which was previously associated with impaired learning and memory functions, was found only in young treated rats, suggesting a more detrimental effect of fructose feeding in young animals. Overall, our findings suggest that even young animals may severely suffer from the deleterious influence of fructose on brain health as the adults and suggest the need of targeted nutritional strategies to reduce its amount in foods.

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THE ZEBRAFISH AS A MODEL TO STUDY THE ROLE OF 3,5-DIIODO-L-THYRONINE AT PERIPHERAL AND CENTRAL LEVEL

R. Imperatore1,2, E. Coccia1, and M. Paolucci1

1 Department of Science and Technology (DST), University of Sannio, Benevento, Italy, 2 Endocannabinoid Research Group, Institute of Biomolecular Chemistry, Pozzuoli, Italy

The 3,5-diiodo-L-thyronine (3,5-T2) is an endogenous metabolite of thyroid hormones which can improve adiposity and associated disorders. The 3,5-T2 administration to rodents feeding a high-fat diet (HFD) prevents body weight increase and reverts the HFD-associated expression pattern of proinflammatory factors. Given that several brain and gut functions and immune genes are conserved between zebrafish (Danio rerio) and mammals, the obese adult zebrafish has been recently used as an experimental model, with pathophysiological pathways similar to mammalian obesity, to investigate fundamental processes underlying central and peripheral inflammation. Here, we aimed to determine, for the first time, whether 3,5-T2 regulates the gut morphology and the central and peripheral inflammation in the Diet Induced Obesity (DIO) zebrafish model. For this purpose, we supplemented the fish water with two different concentration of 3,5-T2, 1 nM and 10 nM. The DIO adult zebrafish showed an increase of intestinal inflammation confirmed by the damaged intestinal barrier and the increase of goblet cells, accompanied by the over-expression of several inflammatory markers. Interestingly, 3,5-T2 treatment increased the impairment of intestine morphology, as well as the expression of proinflammatory factors in the gut of both control and DIO adult zebrafish. The alterations found in the intestine were accompanied by brain inflammation as indicated by the increase of microglia activation. Our findings reveal the zebrafish as animal model to study the aversive effect of 3,5-T2, both at peripheral and central level.

46

GUT-BRAIN AXIS: BUTYRATE EFFECTS IN ANTIBIOTIC-INDUCED INTESTINAL INJURY ASSOCIATED TO PARKINSON'S DISEASE IN MICE

A. Lamaa, C. Avaglianoa, C. De Caroa, C. Cristianoa, R. Russoa, M.P. Mollicab, R. Melia, A. Calignanoa, and G. Mattace Rasoa

a Department of Pharmacy, University of Naples Federico II, via D. Montesano 49, 80131 Napoli, Italy b Department of Biology, University of Naples Federico II, via D. Montesano 49, 80131 Napoli, Italy

Early involvement of gut is observed in Parkinson’s Disease (PD) and symptoms, such as gastrointestinal dysmotility, and digestive disorders, may precede motor ones.[1] A recent study demonstrated the link between gut microbiota alteration in PD patients and a reduction of bacteria producing short chain fatty acids (SCFAs).[2] Among these, butyrate has been shown to improve motor functions when administered in PD animals; moreover, it exerts beneficial effects in the gut, reducing inflammation, improving gut integrity, visceral sensitivity and intestinal motility.

Here, we investigated whether antibiotic-induced gut alterations are involved in worsening PD symptoms and the effects of sodium butyrate (BuNa) in modulating brain and gut bidirectional interplay. In order to induce gut microbiota dysbiosis, mice were treated with ceftriaxone (CFX, 8 g/kg, per os) for 5 days; [3] afterwards mice were injected with 6-hydroxydopamine (6-OHDA, 4 µg/2µl) in the right striatum.[4] Mice challenged with 6-OHDA or with CFX and 6-OHDA were treated with BuNa (100 mg/kg os) once daily for 14 days. Here, we demonstrated the worsening effects of CFX administration in PD pattern and BuNa capability in improving motor deficit in both 6-OHDA and 6-OHDA+CFX mice. These behavioural motor effects were related to BuNa-induced reduction of inflammatory, oxidative and apoptotic parameters at striatal level. Moreover, we demonstrated that BuNa improved colonic inflammation and integrity altered by both CFX and/or 6-OHDA, which mirrored the reduction of serum inflammatory mediators. These findings addressed gut alterations as risk factor for PD and BuNa therapeutic potential in limiting PD progression. References [1] Mulak and Bonaz 2015 [2] Unger et al. 2016 [3] Ling et al. 2015 [4] Avagliano et al. 2016

47

OX-A-INDUCED ENHANCEMENT OF 2-AG LEVELS IN DIFFERENT BRAIN AREAS OF OBESE ob/ob MICE

I. Mavaro1,2, R. Imperatore2,3, N. Forte2, L. Tunisi1,2, A.C. Fernández-Rilo2,4, C. Lucini1, L. Avallone1, M. Paolucci3, L. Palomba5, V. Di Marzo2,6, and L. Cristino2

1 Department of Veterinary Medicine and Animal Productions, University of Naples, Federico II, Italy 2 Endocannabinoid Research Group; Institute of Biomolecular Chemistry of CNR, Pozzuoli, Italy 3 Department of Sciences and Technologies, University of Sannio, Benevento, Italy 4 University of Campania “Luigi Vanvitelli” Naples, Italy 5 Department of Biomolecular Sciences, University of Urbino Carlo Bo, Urbino, Italy 6 Canada Excellence Research Chair, Institut Universitaire de Cardiologie et de Pneumologie de Québec and Institut sur la Nutrition et les Aliments Fonctionnels, Université Laval, Québec, Canada

Orexin-A (OX-A) is a neuropeptide expressed by a small number of neurons of the lateral hypothalamus (LH), a key regulatory center of feeding and sleep-wake functions in the brain. According to the involvement of OX-A in the control of arousal, stress- and reward-related behaviors, OX-A projections are widely distributed throughout the central nervous system including hypothalamic nuclei [arcuate (ARC), supra-chiasmatic (SCN), ventro-medial, (VMN), dorsomedial (DMN) and periventricular (PVN)], cortex, nucleus accumbens, hippocampus, ventrotegmental area (VTA) and different locus in the brainstem. An endocannabinoid-mediated disinhibition of OX-A expressing neurons occurs in the brain of leptin signaling-defective obese ob/ob mice, concurrently with elevation of OX-A trafficking and release to the different LH target areas. By binding OX-1R receptors, OX-A has been found to promote the synthesis of 2-arachidonoylglycerol (2-AG), the main endocannabinoid regulating synaptic transmission in a retrograde manner by inhibiting the release of GABA or glutamate at presynaptic cleft. Here we provide morphological and anatomical evidence showing enhancement of OX-A trafficking into fibers projecting to many different LH target areas in concurrence with elevation of 2-AG content by biochemical LC-MS quantification of endocannabinoid levels in obese ob/ob mice compared to wild-type mice. These effects result in a change of excitatory/inhibitory balance in different brain regions of obese mice, with functional outcomes on the synaptic plasticity of neuronal network regulating stress-, reward-, sleep-wake- and arousal-related behaviours, which were prevented by i.p. injection of leptin and reversed by antagonism of OX-1R with SB334867 in ob/ob mice.

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INCREASED DOSAGE OF THE BIFUNCTIONAL TRANSCRIPTION FACTOR ARX DISTURBS ITS TUNEABLE ACTIVITY AND MAY CAUSE NEURONAL DEFECTS

A. Padula1, D. Drongitis1, M. Malacarne2, M. Piccione3, M. Mallardo1, G. Cervicato1, D. Coviello2, L. Poeta1, and M.G. Miano1

1 Institute of Genetics and Biophysics “Adriano Buzzati-Traverso”, CNR, Naples, Italy 2 Laboratory of Human Genetics, E.O. Ospedali Galliera, Genova, Italy 3 University of Palermo, Palermo, Italy Defective activity of dosage-sensitive transcription factors (TFs) has been established to change gene expression and cause a phenotypic effect during brain development. Aristaless-related homeobox protein (ARX) is a bifunctional transcription factor capable of activating or repressing gene transcription, whose mutations have been found in a wide spectrum of neurodevelopmental disorders including severe cortical malformations, refractory epileptic encephalopathy and Intellectual Disability (ID). Dosage sensitivity of ARX locus is an emerging question since it has been recently demonstrated that breakpoints in enhancer elements of ARX can alter its tissue-specific expression in the developing brain. On the other hand, the Xp21 region, where ARX maps, undergoes frequent genomic rearrangements detected in neurobehavioral phenotypes. Here we report that ARX is a dosage-sensitive gene whose protein acts as a concentration-dependent TF in the regulation of direct and indirect disease targets whose functioning is abolished by loss-of-function mutations in ARX. We further show that in an ID male patient carrying an extra-copy of ARX and its ultra-conserved enhancers, a strong increase in ARX dosage causes a deregulation in effector genes. In murine GABA-ergic oriented neurons, real-time PCR and western blot analysis reveals that under increased dosage conditions ARX causes a deregulation in key controllers of neuronal activity. Our results suggest that not only a reduction but also an extra dosage of ARX may disturb the tuneable bifunctional activity and thus produce cognition disorders.

49

UNDERSTANDING THE CELL DIVERSITY OF THE SEA URCHIN LARVA: EMPHASIS ON THE NERVOUS SYSTEM & NOVEL NEURONAL SUBTYPES

P. Paganos, and M.I. Arnone

Stazione Zoologica Anton Dohrn, Naples, Italy

The nervous system of the sea urchin larva consists of a central ganglion of serotonergic neurons located in the apical organ (central nervous system) and sensory and motor neurons associated with the ciliary band and the gut (peripheral nervous system). Signals from these neurons enable the larvae to swim and feed. Although this system seems quite simple, recent findings suggest that is far more complex than previously described as neurons of the same type are expressing different toolkits of genes and secret different combination of neurotransmitters and neuropeptides. In addition, some of these neurons bear a pancreatic like molecular signature making this neuronal diversity even greater. The aim of our project is to dissect the gene regulatory networks (GRNs) that guide the differentiation of the neuropeptide expressing pancreatic neurons as well as to clarify their function in echinoderms. Moreover, we aim to investigate the cell diversity of the sea urchin larva with emphasis on the nervous system. To accomplish these, known gene markers of these neurons were knocked-down via specific morpholino injection and gene relations were drawn. To further investigate the GRNs of these neurons and their evolution, we are applying high throughput assays such as single cell RNA sequencing (scRNAseq) in the two different sea urchin species Strongylocentrotus purpuratus and Paracentrotus lividus. Using bioinformatics, we aim to recognize the factors that are pancreatic specific and key factors will be knocked down. The effect of each knockdown will be estimated by in situ hybridization and immunofluorescence of specific probes and protein markers respectfully. In this way will be able to provide insight, for the first time, into the molecular fingerprint of these cells and clarify how ‘similar’ or ‘different’ the nervous system of the two species is.

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BAFE AND BRIEF-P FOR THE ASSESSMENT OF THE EXECUTIVE FUNCTIONS IN PRESCHOOL AGE

G. Pastorino, P. Vitale, V. De Simone, E. Amadori, D. Esposito, S. Aiello, F. Operto, and G. Coppola Child and Adolescent Neuropsychiatry, Department of Medicine, Surgery and Odontoiatry, Medical School of Salerno, University of Salerno, Italy

Introduction. The Executive Functions (EFs), namely inhibition, working memory and cognitive flexibility, are fundamental cognitive processes for human adaptation. This study aimed to early detect EFs’ disorders in a preschool pediatric population by means of BAFE and BRIEF-P tests, as well as to verify the convergence between these two diagnostic tools in order to highlight the importance of an early intervention in children at risk of EFs’ disorders.

Methods: In a cross-sectional, observational study carried out in four public nursery schools in the city of Salerno (Italy), 212 children (106 males), aged between 36 and 72 months, were evaluated by means of BAFE battery (Battery for the Assessment of Executive Functions), which consisted of four tests (Card Sort, Stroop-like day-night task, Pattern-making, Spin the pots), and, the BRIEF-P (Behavior Rating Inventory of Executive Function - Preschool Version), which is an indirect test that consists of a questionnaire including 63 items to be filled in by parents/caregivers.

Results: BAFE test showed pathological scores in about 16.5% of children, at least in one task. Executive functions were worse in males, and in children with a history of “speech therapy” and “complicated pregnancy”. Only 1.4% of children were detected as pathological at BRIEF-P questionnaire. No significant correlation was found between pathological scores in BAFE and both parents and teachers BRIEF-P tests.

Conclusions: In preschool non selected children, the direct test (BAFE) detected more executive function deficits than the indirect questionnaire (BRIEF-P) by parents and teachers.

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EXPLORATION OF THE RELATIONSHIP BETWEEN LIPOPHILICITY AND RESIDENCE TIME IN 5-HT7 RECEPTOR LIGANDS

E. Penna1,2, M. Niso3, E. Lacivita3, M. Crispino1, and M. Leopoldo2,3

1 Department of Biology, University of Naples Federico II, via Cinthia, 80126, Naples, Italy 2 Biofordrug srl. via Orabona 4, 70125, Bari, Italy 3 Department of Pharmacy-Drug Sciences, University of Bari Aldo Moro, via Orabona 4, 70125, Bari, Italy

The 5-HT7 receptor is a G protein-coupled receptor (GPCR), activated by the neurotransmitter serotonin (5-hydroxytryptamine, 5-HT). It is involved in many physiological events such as learning and memory, but also in neurological and neurodevelopmental disorders such as Fragile X syndrome, the most common form of inherited intellectual disability, and autistic spectrum disorder. The molecular bases of these diseases are unknown, so it is important to study the pharmacology of 5-HT7 through the development of selective drugs, such as N-(4-cyanophenylmethyl)-4-(2-diphenyl)-1-piperazinehexanamide (LP-211), a brain penetrant and selective 5-HT7receptor agonist. Previous studies showed that, when LP-211 was pre-bound to the 5-HT7 receptor, it remains strongly bound to it. The same experiment, performed using an analogue of LP-211, showed a different result, suggesting that the residence time of these 5-HT7 receptor ligands is structure-dependent. Based on preliminary results, we hypothesized that the residence time of this class of compounds can be related to the lipophilicity of the molecule. To test this hypothesis, we have studied the residence time of a series of 5-HT7 ligands characterized by different lipophilic properties (clogP).

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FOLDING PROPERTIES AND MITOCHONDRIAL TARGETING OF THE PRION-LIKE PROTEIN SHADOO IN NEURONAL HYPOTHALAMIC CELLS

A. Pepe1, R. Avolio1, D.S. Matassa1, F. Esposito1, L. Nitsch1, C. Zurzolo3, S. Paladino1,2, and D. Sarnataro1,2 1 Department of Molecular Medicine and Medical Biotechnology, University of Naples “Federico II”, Via Pansini 5-80131, Naples, Italy 2 Ceinge-Biotecnologie avanzate, s.c.a r.l., Via G. Salvatore, 486-80145, Naples, Italy 3 Unité de Trafic Membranaire et Pathogenese, Institut Pasteur, 25-28 Rue du Docteur Roux, 75724, Paris, CEDEX 15, France.

Shadoo (Sho), a member of prion protein family, has been shown to prevent embryonic lethality in Prnp 0/0 mice and to be reduced in the brains of rodents with terminal prion diseases. Sho can also affect PrP structural dynamics and can increase the prion conversion into its misfolded isoform (PrPSc), which is amyloidogenic and strictly related to expression, intracellular localization and association of PrPC to lipid rafts. We reasoned that if Sho possesses a natural tendency to convert to amyloid-like forms in vitro, it should be able to exhibit “prion-like” properties, such as PK-resistance and aggregation state, also in live cells. We tested this hypothesis, by different approaches in neuronal cells, finding that Sho shows folding properties partially dependent on lipid rafts integrity whose alteration, as well as proteasomal block, regulated generation of intermediate Sho isoforms and exacerbated its misfolding. Moreover, a 18 kDa isoform of Sho, likely bearing the signal peptide, was targeted to mitochondria by interacting with the molecular chaperone TRAP1 which, in turn controlled Sho dual targeting to ER or mitochondria. Our studies contribute to understand the role of molecular chaperones and of PrP-related folding intermediates in “prion-like” conversion.

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METABOLISM OF SIALO-GLYCO-CONJUGATES IS DEFECTIVE IN HUNTINGTON’S DISEASE

G. Pepe1, S. Castaldo1, E. Amico1, T.J. Boltje2, A. Di Pardo1, and V. Maglione1

1 Centre for Neurogenetics and Rare Diseases, IRCCS Neuromed, Pozzilli (IS), Italy 2 Institute for Molecules and Materials, Synthetic Organic Chemistry, Radboud University, Nijmegen, The Netherlands

Introduction. Huntington’s disease (HD) is a rare neurodegenerative disorder with no cure available. Among all the deregulated molecular mechanisms characterizing the disease, aberrant metabolism of gangliosides, sialic-acid containing glycosphingolipids, represents a critical determinant in its pathogenesis and a potential therapeutic target. Although still under investigation, such dysfunction seems to be, at least in part, attributable to a significant reduction in the expression of some of the sialyltransferases, classically involved in the synthesis of both gangliosides and glycoproteins. Hypothesis. Considering the role of sialyltransferases in the synthesis of glycoproteins, the main hypothesis of this study is that the aberrant ganglioside metabolism in HD may represent the result of an overall perturbed metabolism of sialo-conjugates. Aim. Here, we evaluated whether an overall impairment of brain sialo-conjugate metabolism really occurs in HD and may represent a potential novel therapeutic target. Results. Our data indicate that levels of Polysialic Acid (PSA), a sugar polymer exclusively linked to proteins and normally synthesized by two different sialyltransferases -ST8SIA2 and ST8SIA4-, is perturbed in brain tissues from a HD mouse model at different stage of the disease. The aberrant PSA content seems to depend on deregulated expression of ST8SIA2 and ST8SIA4. In line with our expectation, although still not conclusive, mRNA levels of these enzymes are clearly deregulated in brain tissues from HD mice. Conclusions. Our results support the idea that metabolism of sialo-conjugates may be globally impaired in HD, thus its modulation may likely represent a potential novel and alternative therapeutic strategy to treat the disease.

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THE SMALLEST 7Q11.23 DUPLICATION ENCOMPASSING GTF2I AND GTF2IRD1 GENES IN AN INDIVIDUAL WITH INTELLECTUAL DISABILITY . M. Pinelli1,2, G. Terrone1, G. Cappuccio1,2, F. Imperati1, P. Pignataro3, G. Merla4, R. Genesio3, E. Del Giudice1, and N. Brunetti-Pierri1,2 1 Department of Translational Medicine, Section of Pediatrics, Federico II University, Naples, Italy 2 Telethon Institute of Genetics and Medicine, Pozzuoli, Naples, Italy 3 Department of Molecular Medicine and Medical Biotechnologies, University of Naples Federico II, Napoli, Italy 4 Division of Medical Genetics, Poliambulatorio “Giovanni Paolo II”, IRCCS-Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy Microduplication of 7q11.23 region has been recognized as a syndrome presenting with speech delay, cognitive impairment, and behavioral abnormalities. It typically involves 1.5 Mb region including approximately 26 genes that is deleted in Williams-Beuren Syndome (WBS) deletion. The majority of reported patients have recurrent size and breakpoints because the duplication is mediated by nonallelic homologous recombination between low copy repeats flanking the critical region. GTF2I and GTF2IRD1 have been proposed as responsible for the neurobehavioral phenotype. However, to date there have been no reported cases with duplications involving only these two genes. Here we present a 9-yeat-old boy with language delay, cognitive impairment, obesity and dysmorphic facial features. Array-CGH identified a maternally inherited 184-kb duplication of the 7q11.23 region including GTF2I and GTF2IRD1 genes. The evaluation of intellectual functioning by WISC-IV resulted in a Full-Scale QI of 61, with homogeneous scores for subcategories. The adaptive behavior tested by VABS was in line with intellectual functioning. No evident behavioral and emotional problems were highlighted by CBCL 6-18. His facial dysmorphisms were not specific. Her mother had learning difficulty and is presently unemployed. Neither the patient nor the mother showed behavioral abnormalities consistent with autism spectrum disorder. GTF2I and GTF2IRD1 encode for the ubiquitous TFII-I transcription factor and for a DNA-binding protein, respectively and have been proposed as responsible for the distinctive neurobehavioral phenotypes of both 7q11.23 deletion and duplication syndromes. The individual we reported is the first case supporting the involvement of GTF2I and GTF2IRD1 in intellectual disability.

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TRANSPOSABLE ELEMENTS IN OCTOPUS NEURAL TRANSCRIPTOME

G. Ponte1, G. Petrosino1, M. Volpe1, R. Sanges1,2, and G. Fiorito1

1 Dipartimento Biologia ed Evoluzione Organismi Marini, Stazione Zoologica Anton Dohrn, Napoli 2 Computational Genomics Laboratory, International School for Advanced Studies (SISSA), Trieste

By sequencing Octopus vulgaris transcriptome we found high frequency of retroelements embedded in transcripts, similar to what reported in mammals. In particular, short interspersed elements (SINEs) are more abundant in transcribed long non coding RNAs (lncRNAs) than in protein coding genes. Both lncRNAs and SINEs are enriched in transcripts expressed in the octopus brain. We also found a long interspersed element (LINE), fully competent for retrotransposition and expressed in nervous tissues. In situ hybridization provided evidence of a diffuse expression of LINE mRNAs in the great majority of amacrine cells constituting the posterior buccal, frontal- and vertical lobes of the octopus brain (supra-esophageal mass) and in some large motor neurons in the sub-esophageal mass and arm nerve cord. By using a custom antibody designed for the O. vulgaris LINE, we identified a discrete number of neural cells immunoreactive to the LINE-Ab (e.g., large cells of the vertical lobe) and fibers positive to O. vulgaris LINE in selected areas of the octopus brain. Transposable elements are known to generate germinal and somatic genomic heterogeneity in mammals, and contribute to neural mosaicism in mammalian brain such as in the hippocampus and the cortex. Our findings suggest that a convergent evolutionary process, driven by transposable elements, has led to the evolution of mammalian-like molecular traits in the nervous system of the cephalopod mollusk Octopus vulgaris, possibly contributing to develop its neural plasticity and cognitive abilities.

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GLUCOCORTICOID-INDUCED CHANGES IN TRANSCRIPTION AND LYMPHOCYTES FUNCTIONALITY IN ATAXIA-TELANGIECTASIA.

M.R. Prencipe1, E. Cirillo1, G. Giardino1, V. Gallo1, L. Palamaro1, G. Scalia2, L. Del Vecchio2, E. Del Giudice1, M. Menotta3, M. Magnani3, and C. Pignata1

1 Department of Translational Medical Sciences-Section of Pediatrics, Federico II University, Naples, Italy 2 Department of Biochemistry and Medical Biotechnology–CEINGE, Federico II University, Naples, Italy 3 Department of Biomolecular Sciences, University of Urbino “Carlo Bo”, Urbino, Italy Ataxia-Telangiectasia (A-T) is an incurable and rare hereditary syndrome. Treatment with glucocorticoids (GCs) leads to a variable improvement of the neurological and immunological phenotype. We wondered whether there was a correlation between the behavior of lymphocytes functionality and the neurological response to the GC administration. We found that the proliferative response to PHA or IL-7 showed a increase only in 2 of the 4 A-T patients, referred at a single Center. A correlation between the proliferative response to IL-7 and the percentage of CD3+CD127+ cells was found. A significant inverse relationship between the proliferative response to IL-7 and the behavior of the SARA score was found. Subcellular localization studies revealed that, only in the responders, IL-7Rα was localized into the early endosome (EEA1) vesicles and recycled back to the cell surface through the late endosomes (Rab-7). Using microarray analysis we have identified 4304 differently expressed genes (DE-genes), in PBMCs from the only A-T responder patient studied before or after GC treatment. Of these, 2933 transcripts decreased and 1371 transcripts exhibited an increased transcription. Interestingly, some DE-genes are implicated in pathways relevant for A-T pathogenesis, such as cell proliferation, regeneration and differentiation, endosomal/lysosomal trafficking and inflammation. In conclusion, our data indicate a tight correlation in A-T between the in vivo neurological response to GC and the in vitro lymphocyte behavior, and that in-vitro studies on lymphocyte functionality are an excellent cellular model to achieve a better understanding of the disease pathogenesis and of the mechanism of action of GC.

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SHEDDING LIGHT ON THE ENDOCANNABINOID SYSTEM: EFFECT OF LIGHTING CONDITIONS IN THE AQUATIC ENVIRONMENT R. M. Sepe1, R. De Paolo1, F. Corato2, P. De Luca3, A. Rabbito1, F. Piscitelli4, V. Di Marzo4,5, C. Brunet2, and P. Sordino1 1 Biology and Evolution of Marine Organisms (BEOM), Stazione Zoologica Anton Dohrn – Naples – Italy 2 Integrative Marine Ecology (EMI), Stazione Zoologica Anton Dohrn – Naples – Italy 3 Research Infrastructures for marine biological resources - The Molecular Biology and Bioinformatic Unit (RIMAR- MB&B), Stazione Zoologica Anton Dohrn – Naples – Italy 4 Endocannabinoid Research Group, Institute of Biomolecular Chemistry - Consiglio Nazionale delle Ricerche (ICB-CNR) – Pozzuoli (NA) – Italy 5 Canada Excellence Research Chair, Institut Universitaire de Cardiologie et de Pneumologie de Québec and Institut sur la Nutrition et les Aliments Fonctionnels, Université Laval, Québec, Canada The aquatic habitat poses particular challenges for photoreceptive mechanisms involving the circadian clock, because light intensity and spectrum change with depth and water quality. The endocannabinoid system shows tissue-specific diurnal changes, and is involved in circadian physiological processes (e.g. sleep, body temperature, endocrine secretions, food intake, learning, memory, locomotion). We investigated the role of light spectrum on the expression of endocannabinoid system genes in zebrafish development. RT-qPCR revealed that the expression of cnr1 and pparg receptors was reduced in green light, while that of the gpr55a receptor was significantly lower in blue, green and red. Significant reduction was found in the expression of the abhd4 synthesis enzyme and ptgs2b degradation enzyme under green light. The expression of abhd6a catabolic enzyme was reduced in green and red, while that of ptgs2a was lower in blue and green. Most changes in gene expression occur under green light. These data are integrated with mass spectrometry analysis of endocannabinoids. Green light has a negative effect on zebrafish development, causing low feeding activity, poor growth, malformations, and low survival. Larvae adapt their visual system to photic environment by altering number of cones and opsin expression in the retina, which is rich in UV and blue cones in larvae, and in green- and red-sensitive opsins in juveniles. So, green light treatment could induce lower gene expression probably because of the reduced number of red and green cones in the larval retina. Here, lighting conditions clearly influence expression levels of endocannabinoid system genes during zebrafish development.

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IDENTIFICATION OF NEW BIOMARKERS TO ENABLE LIQUID BIOPSY FOR POMPE DISEASE

A. Tarallo1,2, A. Carissimo2,3, C. Damiano1,2, L. Santoro4, T. Mongini5, F. Deodato6, A. Dardis7, B. Bembi7, O. Musumeci8, A. Toscano8, A. Amoresano9, M. Ruoppolo10, W.W.M.P. Pijnappel11, A.T. van der Ploeg11, and G. Parenti1,2

1 Department of Translational Medical Sciences, Federico II University, Naples, Italy 2 Telethon Institute of Genetics and Medicine, Pozzuoli, Italy 3 Istituto per le Applicazioni del Calcolo “Mauro Picone”, Naples, Italy 4 Department of Neurosciences, Federico II University, Naples, Italy 5 Department of Neurosciences, University of Torino, Italy 6 Ospedale Pediatrico Bambino Gesù, Rome, Italy 7 Azienda Ospedaliera Universitaria "Santa Maria della Misericordia", Udine, Italy 8 Department of Neurosciences, University of Messina, Italy 9 Department of Chemical Science, Federico II University, Naples, Italy 10 Department of Molecolar Medicine and Medical Biotechnology, Federico II University, Naples, Italy 11 Center for Lysosomal and Metabolic Diseases, Erasmus MC University Medical Center, Rotterdam, the Netherlands Evaluation of patient's condition and monitoring the efficacy of Enzyme Replacement Therapy (ERT), is a critic issue for Pompe Disease (PD), a metabolic, hereditary disease, due to deficiency of lysosomal acid alpha glucosidase. In this study we are looking for a set of measurable biomarkers in plasma/serum of PD patients, based on the analysis of micro-RNA (miRNA) and oxylipins. MiRNAs are small non-coding RNAs that regulate gene expression. Oxylipins are an emerging group of metabolites, generated by oxygenation of polyunsaturated fatty acids. Both are molecoles involved in numerous biological functions and their content in circulation can alter in response to pathological conditions. We are evaluating circulating miRNAs and serum oxylipins in samples of patients (N = 52) and of mouse model. Our approach combines Next Generation Sequencing techniques, tandem mass spectrometry and bioinformatics analysis. In previous work we stated that miR-133a is upregulated in plasma of PD patients (Tarallo et al, 2018). We have evaluated others two circulating microRNAs, miR-1 and miR-206. Both are upregulated in the plasma of PD patients and correlate with the severity of the phenotype and the response to ERT. Preliminary data also suggest that the profile of serum oxylibins in PD mice is altered, compared to age matched wild type mice. The identification of multiple biomarkers measurable in blood would allow for development of so-called “liquid biopsies” in the management of PD, as tool to support the diagnosis, monitor patient's condition and evaluate the effecacy of ERT.

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VACUOLATED PAS-POSITIVE LYMPHOCYTES: A SCREENING TEST FOR POMPE DISEASE AND OTHER AUTOPHAGIC MYOPATHIES

C. Terracciano, F. Napolitano, A. Pascarella, O. Farina, T. Esposito, G. Franzese, G. Di Iorio, M.A.B. Melone, and S. Sampaolo

Department of Medicine, Surgery, Neurology, Metabolic and Aging Science, Reference Center for Neurological and Neuromuscular Rare Disease, University of Campania “Luigi Vanvitelli”, Naples, Italy

Pompe disease is an autosomal recessive lysosomal disorder caused by mutations of the acid-α-glucosidase (GAA) gene. Deficiency of GAA enzyme leads to glycogen accumulation and autophagy impairment in cardiac and skeletal muscles, but also in lymphocytes. Since an effective therapy is available, a rapid, sensitive and specific test is crucial to early identify affected subjects. Number of lymphocytes containing Periodic-acid-Schiff (PAS)-positive vacuoles were evaluated on blood smears from 78 adult patients with hyperckemia and/or muscle weakness, 13 Late-onset-Pompe-disease (LOPD) and 13 symptomatic LOPD offspring. More than 4 PAS-positive lymphocytes were found in all LOPD patients and in all LOPD offspring resulted to have only a single GAA mutation but low GAA levels. PAS-positive lymphocytes were also found in 17 out of the 78 patients: 6 new diagnosis of LOPD, 3 different glycogen storage myopathies, 1 glucose-6-phosphate-dehydrogenase deficiency, 1 caveolinopathy and 6 myopathies with glycogen deposition and autophagic vacuoles in muscles biopsies, in which mutations for Pompe disease was subsequently ruled out. Immunostaining with the autophagy marker LC3 confirmed the autophagic nature of vacuoles in all positive lymphocytes. ROC curve assessment of PAS-positive lymphocytes disclosed a 100% sensitivity and a 90% specificity in recognizing both homozygous and heterozygous GAA mutations carriers. The other myopathies with more than 4 PAS-positive lymphocytes appeared to be all related to impaired autophagy, that seems to be responsible of PAS-positive vacuolated lymphocytes formation. Quantification of PAS-positive lymphocytes in blood smears should be routinely used as first level test for Pompe disease and other autophagic vacuolar myopathies.

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A CASE OF DEVELOPMENTAL ENCEPHALOPATHY CAUSED BY A DE NOVO KCNB1 GENE MUTATION

G. Terrone1, A. Romano1, E. Gennaro2, M. Taglialatela3, and E. Del Giudice1

1 Department of Translational Medical Sciences, University of Naples “Federico II,” Naples, Italy 2 Laboratory of Genetics, E.O. Ospedali Galliera, Genoa, Italy 3 Department of Neuroscience, University of Naples "Federico II, Naples, Italy

Background: De novo KCNB1 mutations have been identified in patients with early onset epileptic encephalopathies (EE) ranging from West to Lennox-Gastaut syndrome. KCNB1 gene encodes the Kv2.1 voltage-gated potassium channels, critical for membrane repolarization during repetitive stimulation. Here we describe a 17-month-old girl with global developmental delay, post-natal microcephaly and pharmaco-resistant epilepsy caused by de novo mutation in KCNB1. Case description: the patient is the first daughter of healthy non-consanguineous parents. At 9 months of life, clinical examination revealed an axial hypotonia and she presented daily episodes of motor arrest followed by spasms in flexion, occurring especially upon awakening. Electroencephalogram (EEG) revealed normal background activity with focal (left temporal) or rare multifocal spikes, without a pattern of hypsarrhythmia. She was immediately treated with ACTH, but spasms relapsed at the end of therapy. Seizures were resistant to valproic acid and levetiracetam. A therapy with topiramate reached a partial control of seizures. Brain magnetic resonance, fundus oculi, auditory and visual evoked potentials, metabolic screening, CGH-array were all normal. Next generation sequencing panel for EE revealed a novel de novo mutation (c.1045G>7-p.Val349Phe) in the S5 domain of the KCNB1 gene. At her last evaluation at 16 months, she did not reach the upright position, persisting a truncal hypotonia, she had a poor eye contact and reduced response to social interactions. A post-natal microcephaly was also evident. Conclusion: This case expands the phenotypic spectrum of epilepsies associated with KCNB1 variants. Functional characterization of mutant channels will be crucial for target therapy.

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CHEMOGENETIC MANIPULATION OF BRAIN NEURONAL PATHWAYS RESCUES SEX-DIFFERENCES IN MEMORY CAPACITY

G. Torromino1,2, V. Loffredo1,3, F. Esposito2, M. Colucci2, M. De Risi1,2, and E. De Leonibus1,2

1 Institute of Cell Biology and Neurobiology, National Research Council (IBCN-CNR), Rome, Italy 2 Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy 3 PhD Program in Behavioral Neuroscience (XXXII cycle), Sapienza University of Rome, Italy

Memory capacity (MC) is the number of information that can be retained in memory for a short (working memory capacity – WMC) or a long (long-term memory capacity – LTMC) time interval. Its decline has been observed in many psychiatric and neurodegenerative diseases, and in physiological ageing. Although gender differences in MC are reported, less is known about the biological mechanisms at the basis. Using the Different/Identical Object Recognition Task (DOT/IOT), we found that adult male mice have a WMC of about six objects, like humans, and that only in high memory load conditions the hippocampus (HP) is recruited to solve the task. Female subjects have the same WMC as males, but they are impaired in consolidating information when challenged with high memory load conditions. This impairment in memory consolidation correlates with recruitment of different brain circuitries in the two sexes, with the HP hypoactivated in female compared to male mice. Using a chemogenetic approach, we demonstrated that reverting HP hypoactivation rescues the memory consolidation impairment in females. In parallel, we characterized MC decline during ageing in males and females at 6 and 12 months. We found that female mice have a load- and delay-dependent earlier decay of MC compared to males. These findings might be relevant in understanding the higher impact of dementia in women population compared to men, and we propose the different circuitry recruitment as a mechanism responsible for female’s higher vulnerability to age-dependent memory decay.

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CONSUMPTION OF FAT RICH DIET AFFECTS MITOCHONDRIAL FUNCTIONS IN BRAIN CORTEX AND CORTICAL SYNAPTIC REGION OF MICE

G. Trinchese, G. Cavaliere, E. Penna, A. Catapano, F. Cimmino, A. Cerciello, G. Di Ruberto, A. Speranza, M.P. Mollica, and M. Crispino

Department of Biology, University of Naples Federico II, via Cinthia, 80126 Naples, Italy

The consumption of high-fat diet (HFD), widely diffused in industrialized countries, is implicated in several metabolic pathologies, which can be risk factors for the development of neurological and neurodegenerative diseases. Therefore, it is particularly interesting to investigate the molecular mechanism underlying the HFD effect on altered neuronal plasticity. Synaptic-located mitochondria (synaptosomal mitochondria) play a key role in providing energy to support synaptic functions and plasticity. Thus, the impairment of their functions, following HFD consumption, may contribute to neurodegenerative diseases. Our project aims to analyse the dysfunctions of brain cortex mitochondria and synaptosomal mitochondria in a mouse model of diet induced obesity. Male C57Bl/6 mice were divided into two groups fed a standard diet or HFD for 12 weeks, and it was observed that HFD induces inflammation, oxidative stress and mitochondrial dysfunctions not only in brain cortex, but also in synaptosomal fraction. In conclusion, our results indicate that HFD, negatively affecting mitochondrial activity at synaptic level, alters the synaptic energy supply, leading to synaptic failures that could be an early event in neurodegenerative diseases.

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POST SYNAPTIC DENSITY AND ITS MODULATION BY ANTIPSYCOTICS, PSYCHIATRIC DISORDERS AND PRENATAL STRESS. A SOFIST MACHINE: FOCUS ON HOMER GENES. L. Vellucci, C. Avagliano, E.F. Buonaguro, F. Iasevoli, and A. de Bartolomeis Laboratory of Molecular and Translational Psychiatry, Department of Neuroscience, Reproductive Sciences and Odontostomatology, University “Federico II” of Naples, Italy

The post-synaptic density (PSD) is an ultra-specialized structure near the post synaptic membrane of glutamatergic synapses which contains receptors, signaling and scaffolding proteins organizing signal-transduction pathways [1]. This structure takes part to many processes involved in the development of nervous system [2], synaptic plasticity [3] and regulation of transductional pathways [4]. Psychiatric disorders have been widely related to PSD disfunctions. In this work we studied alterations of PSD molecules and their modulation by antipsychotic treatments and environmental conditions. In particular we evaluated Homer 1a expression in schizophrenia and stress-related murine models and Homer 1a expression after acute administration of the typical antipsychotic Haloperidol. The expression of Homer 1a was found to be altered in different brain areas related to psychiatric disorders. Moreover, acute administration of Haloperidol produced alterations in functional connectivity among brain regions relevant to schizophrenia pathophysiology, thus inducing variations in cortico-subcortical brain networks compared to control group. PSD molecules are strongly related to psychiatric disorders and are promising molecular targets of future therapeutic approaches. Keywords: Post-synaptic density, Homer, Haloperidol, Schizophrenia, stress-related disorder, network [1] Yamauchi T. Molecular constituents and phosphorylation-dependent regulation of the post-synaptic density. Mass Spectrom Rev. 2002 Jul-Aug;21(4):266-86. [2] Foa L, Rajan I, Haas K, Wu GY, Brakeman P, Worley P, Cline H. The scaffold protein, Homer1b/c, regulates axon pathfinding in the central nervous system in vivo. Nat Neurosci. 2001 May;4(5):499-506. [3] Sheng M, Kim MJ. Postsynaptic signaling and plasticity mechanisms. Science. 2002 Oct 25;298(5594):776-80. [4] Ehlers MD, Mammen AL, Lau LF, Huganir RL. Synaptic targeting of glutamate receptors. Curr Opin Cell Biol. 1996 Aug;8(4):484-9.

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ANALYSIS OF CORTICAL GENE EXPRESSION VARIABILITY IN A MOUSE MODEL OF X-LINKED INFANTILE SPAMS SYNDROME

L. Verrillo1,2, M. Tuccillo1, D. Drongitis1, E. Mangano3, C. Franco4, G. Terrone5, L.M.T. Canzoniero4, E. Del Giudice5, R. Bordoni3, L. Poeta1, and M.G. Miano1

1 Institute of Genetics and Biophysics “Adriano Buzzati-Traverso”, CNR, Naples, Italy 2 University of Campania “Luigi Vanvitelli”, Caserta, Italy 3 Institute of Biomedical Technologies, CNR, Segrate (MI), Italy 4 Department of Science and Technology, University of Sannio, Benevento, Italy 5 Department of Translational Medicine, University of Naples Federico II, Naples, Italy

Background: Infantile Spasms Syndrome (ISS) is a early-onset epileptic encephalopathy characterized by epileptic spasms during early infancy and severe global developmental delay. A GCG triplet repeat expansion in X-linked Aristaless-related homeobox gene (ARX) is the most commonly inherited error found in patients with X-linked ISS. Although a growing number of studies have been done on the identification of the complete subset of ARX targets, the effect of repeat instability remains unknown. Objectives: Here we describe a single-cell RNA sequencing study design aimed to identify transcriptome landscapes in the epileptogenic cortex of the Arx(GCG)7/Y mouse compared to the wild type one. The Arx(GCG)7/Y model develops severe tonic-clonic seizures in a phenotype that well recapitulates the chronic epilepsy associated to the c.304ins(GCG)7 mutation in ISS children. We have isolated cortical Arx(GCG)7/Y and wild type neurons from the developing brains and set up the experimental conditions to proceed with the scRNA-seq protocol. Our main objective is to define how the activity of the expanded-polyalanine ARX TF perturbs directly or indirectly transcriptome profiles in distinct inhibitory and excitatory sub-types. The generation of these datasets will constitute a valuable resource to probe cellular composition and molecular features of the epileptogenic cortex associated to ARX defects. Understanding all these aspects may help us to identify cell-specific epileptic-biomarkers linked to the disease-response that could be used as druggable targets in anti-epileptic drug discovery research.

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CORPUS CALLOSUM ABNORMALITIES AND AUTISM SPECTRUM DISORDERS

G. Vitiello1, F. Imperati1, G. Terrone1, R. Romaniello2, A. D’Amico3, R. Genesio4, M. Severino5; The Italian CCA Study Group; L. Nitsch4, R. Borgatti2, M.P. Riccio1, C. Bravaccio1, 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 Advanced Biomedical Sciences, Federico II University, Naples, Italy 4 Department of Molecular Medicine and Medical Biotechnologies, Federico II University, Naples, Italy 5 Neuroradiology Unit, Istituto Giannina Gaslini, Genoa, Italy

The corpus callosum (CC) represents the most important portion of white matter in the human brain. Corpus callosum abnormalities (CCA) have an estimated prevalence ranging from 0.3% up to 0.7% in patients undergoing brain imaging. Furthermore, alterations of the white matter are associated with dysfunctional cognitive and social abilities. Recent studies suggest the hypothesis of a correlation between CCA and autism spectrum disorders (ASD). We reviewed MRI data, clinical and genetics findings of patients with ASD recruited in the series of the Italian CCA Study Group. We found 26/579 individuals with ASD (10%), 8 females and 18 males. Complete CC agenesis was present in 3/26 cases (12%), partial agenesis in 9/26 (35%), thin CC in 4/26 (15%), thick CC in 10/26 (38%). Non-syndromic CCA ASD subjects were 18/26 (69%). Intellectual disability was referred in 25/26 patients (96%) with prevalent mild-moderate degree (62%). Chromosomal micro array (CMA) data were available in 22/26 (87%). CMA was negative in 15/22 (68%) and variants of unknown significance were reported in 7/22 (32%). Whole exome sequencing (WES) is in progress in a few cases. These data support the hypothesis that CCA constitutes a major risk factor for developing ASD with low cognitive functions. Individuals with CCA should be screened for ASD and CCA should be considered in autism diagnostic evaluations as well. Combined CMA and Next-generation sequencing (NGS) strategies will increase the probability to identify new causative genes for CCA and ASD. Further studies are necessary to define the role of CCA in ASD.

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THE MICRO RNA-29A MODULATES 5-HTR7 EXPRESSION AND ITS MORPHOGENIC EFFECTS IN HIPPOCAMPAL NEURONS.

F. Volpicellia,b, L. Speranzaa, S. Pulcranoa, M. Crispinoc, G.C. Bellenchia, U. di Porzioa, and C. Perrone-Capanoa,b

a Institute of Genetics and Biophysics “Adriano Buzzati Traverso”, CNR, Naples, IT b Department of Pharmacy, University of Naples Federico II, Naples, IT c Department of Biology, University of Naples Federico II, Naples, IT

miRNAs are master regulators of gene expression in diverse biological processes, including neuronal differentiation, dendritic arborization, synaptic plasticity, neuronal survival and regeneration. The identification of their physiological target genes remains one of the outstanding challenges. Recently, we demonstrated that the activation of serotonin receptor 7 (5-HT7R) plays a key role in regulating the neuronal cytoarchitecture, synaptogenesis and synaptic plasticity during embryonic and early postnatal development of the CNS. However, the possible interplay between miRNAs and 5-HT7R has not yet been studied. Starting from a computational prediction of microRNAs in 5-HT7R mouse gene, we identify miR-29-3p as an important regulator of 5-HT7R gene expression. Using a luciferase reporter system we demonstrate that miR-29a binds to 3’UTR of 5-HT7R mRNA and concomitantly downregulates its expression. On the contrary. the expression of other serotonin receptors, such as 5-HT1AR and 5-HT6R, is not affected by miR-29a. Using primary neuronal cultures from the postnatal hippocampus we show that over-expression of miR-29a impairs neurite outgrowth induced by 5-HT7R stimulation, as well as downstream signalling transduction pathways. In addition, we observed that the upregulation of miR-29a in the late postnatal stages (P20) of hippocampal development parallels with the downregulation of 5-HT7R. Our data support the idea that this miRNA could be a physiological modulator of 5-HT7R, involved in shaping hippocampal neural circuits during development. Understanding the way miR29a is contributing to the regulation of 5-HT7R-dependent structural plasticity in the CNS may provide clues to establish novel therapeutic strategies for neurodevelopmental diseases associated with altered brain connectivity.

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Author list

A

Affinito, O. 22 Aiello, S. 50 Alagia, M. 13 Aliperti, V. 23 Alvino, F.G. 38 Amadori, E. 50 Amico, E. 40, 53 Amodio, G. 14 Amoresano, A. 58 Annona, G. 28 Arnone, M.I. 49 Avagliano, C. 46, 63 Avallone, L. 26, 47 Avolio, R. 52 B Balestrini, S. 11 Ballabio, A. 9 Banfi, S. 12, 19 Barbato, M 31 Barbato, S. 19 Barone, P. 14 Barone, R. 43 Bartolini, F. 18 Baumgart, M. 26 Beghi, E. 32 Bellenchi, G.C. 38, 66 Bellofatto, M. 29 Belardo, C. 16 Bembi, B. 58 Bergamo, P. 36, 44 Berruto, F. 42 Bertapelle, C. 30 Bilo, L. 11 Boccella, S. 16, 25 Boltje, T.J. 53 Bordoni, R. 64 Borgatti, R. 65 Bravaccio, C. 65 Brescia Morra, V. 29

Brunet, C. 57 Brunetti-Pierri, N. 13, 54 Bruscella, S. 31 Buonaguro, E.F. 63 Buono, S. 32 Buono, V. 32 C Cacace, V. 24 Cacialli, P. 33 Caiazzo, M. 38 Calignano, A. 16, 46 Calvanese, M. 37 Campiglia, P. 12 Canafoglia, L. 21 Canzoniero, L.M.T. 64 Capasso, R. 15, 41 Capocci, L. 40 Cappello, S. 10, 21 Cappuccio, G. 13, 54 Capra, V. 13 Capriello, T. 34 Carboncino, A. 19 Carissimo, A. 58 Carrella, D. 13 Carrella, S. 19 Casari, G. 13 Casellato, S. 11 Castaldo, L. 26 Castaldo, S. 40, 53 Castello, R. 13 Catapano, A. 62 Catone, G. 35 Cavaletti, G. 18 Cavaliere, G. 62 Cellerino, A. 26 Cerciello, A. 62 Cervicato, G. 48 Chiarotti, L. 22 Ciampi, L. 19 Cieri, F. 20 Cigliano, L. 23, 36, 44 Cimmino, F. 62

Cirillo, E. 56 Cocca, E. 36 Coccia, E. 45 Colarusso, A. 37 Colucci, M. 61 Commone, A. 13 Conte, I. 28 Coppola, A. 11, 32 Coppola, G. 17, 50 Corato, F. 57 Corti, S. 20 Coviello, D. 48 Crescenzo, R. 44 Criscuolo, C. 14, 29 Cristiano, C. 16, 46 Crispino, M. 51, 62, 66 Cristino, L. 15, 16, 25, 41, 47 Cuomo, M. 22 D D’Agostino, Y. 42 Dallapiccola, B. 13 D’Ambrosio, V. 32 Damiano, C. 43, 58 D’Amico, A. 65 D’Angelo, L. 15, 26, 33 D’Aniello, S. 28, 42 Dardis, A. 58 D’Aria, A. 44 de Bartolomeis, A. 63 De Caro, C. 46 de Divitiis, O. 31 de Girolamo, P. 15, 26, 33 Del Duca, R. 17 De Leonibus, E. 19, 24, 38, 61 Del Giudice, E. 54, 56, 60, 64, 65 Della Casa, R. 43 De Luca, P. 57 Del Vecchio, L. 56 De Michele, G. 14 de Novellis, V. 16

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Deodato, F. 58 De Paolo, R. 57 De Risi, M. 38, 61 De Rosa, A. 14 De Simone, V. 50 Di Cristina, G. 39 Di Giaimo, R. 21 Di Iorio, G. 59 Di Marzo, V. 15, 16, 25,41, 47, 57 Di Matteo, F. 21 Di Pardo, A. 40, 53 di Porzio, U. 66 Di Ruberto, G. 62 Di Schiavi, E. 20, 30 Donizetti, A. 23 Drongitis, D. 48, 64 E Errico, F. 22 Esposito, D. 17, 50 Esposito, F. 52, 61 Esposito, T. 59 Ezhova, Y. 24 F Farina, A. 16 Farina, O. 59 Fasano, D. 14 Fecarotta, S. 13, 43 Ferlazzo, E. 11 Fernández-Rilo, A.C. 15, 25, 41, 47 Ferran, J.L. 28 Ferrandino, I. 34 Fiorito, G. 39, 55 Forte, N. 15, 25, 41, 47 Fraldi, A. 24, 38 Franco, B. 19 Franco, C. 64 Franzese, G. 59 G Gallo, V. 56

Gagliardi, E. 42 Garozzo, D. 43 Gatta, C. 33 Genesio, R. 54, 65 Gennaro, E. 60 Giardino, G. 56 Giordano, L. 11 Giordano, N. 19 Giussani, G. 32 Gobbi, G. 11 Golia, F. 19 Gragnaniello, V. 43 Grilli, F. 13 Gritti, A. 35 Gritti, S. 35 Grimaldi, M.C. 34 Guida, F. 16 H Hernandez-Hernandez, L. 11 I Iacomino, M. 11 Iannotta, L. 23, 36, 44 Iannotta, M. 16 Iasevoli, F. 63 Imperadore, P. 39 Imperati, F. 54, 65 Imperatore, R. 15, 16, 25, 41, 45, 47 Indrieri, A. 19 Iossa, S. 44 Italian CCA Study Group 65 K Keller, S. 22 Krithika, R. 11 Kyrousi, C. 21 L Lacivita, E. 51 La Corte, M. 17

Lama, A. 46 Lanzillo, R. 29 Lauritano, A. 12 Lauro, C. 37 Lauro, F. 29 Leggieri, A. 26 Leopoldo, M. 51 Leuzzi, V. 13 Locascio, A. 42 Loffredo, V. 61 Lucini, C. 26, 33, 47 Luongo, L. 16 M Maione, S. 16, 25 Maffia, V. 24 Maglione, V. 40, 53 Magnani, M. 56 Maitz, S. 13 Malacarne, M. 48 Mallardo, M. 48 Mangano, E. 64 Marrocco, E. 19 Matassa, D.S. 52 Mattace Raso, G. 46 Mavaro, I. 15, 25, 41, 47 Mazzitelli, M. 16 Medoro, A. 30 Meletti, S. 11 Meli, R. 46 Melone, M.A.B. 59 Menotta, M. 56 Merla, G. 54 Miano, M.G. 48, 64 Miceli, F. 11 Mignogna, D. 30 Milic, N. 15 Minopoli, N. 43 Mollica, M.P. 46, 62 Monaco, A. 24, 34 Mongini, T. 58 Musacchia, F. 11, 13 Musumeci, O. 58 Mutarelli, M. 13 N

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Napolitano, F. 59 Nappi, P. 12 Natale, G. 14 Nicoletti, C. 17 Nigro, V. 11, 13 Niso, M. 51 Nitsch, L. 14, 52, 65 Nizzardo, M. 20 Nusco, E. 24 Nuzzo, T. 22 O Operto, F. 17, 50 Ostacolo, C. 12 P Padula, A. 48 Paganos, P. 49 Paladino, S. 14, 52 Palamaro, L. 56 Palazzo, E. 16 Palladino, A. 53 Palomba, L. 15, 25, 41, 47 Paolucci, M. 45, 47 Parenti, G. 13, 43, 58 Parrilli, E. 37 Pascarella, A. 59 Pastorino, G. 17, 50 Pellegrini, E. 33 Peca, C. 31 Pellecchia, M.T. 14 Penna, E. 51, 62 Pepe, A. 52 Pepe, G. 40, 53 Pero, M.E. 18 Perrone-Capano, C. 66 Petrosino, G. 55 Pierantoni, G.M. 14 Pignata, C. 56 Piccione, M. 48 Picillo, M. 14 Pignataro, P. 54 Pijnappel, W.W.M.P. 58 Pinelli, M. 13, 54 Pipicelli, F. 21 Piscitelli, F. 16, 57

Pizzo, M. 19 Poeta, L. 48, 64 Ponte, G. 39, 55 Postlethwaite, J. 27 Prencipe, M.R. 56 Pulcrano, S. 38, 66 Punzo, D. 22 R Rabbito, A. 57 Remondelli, P. 14 Renna, M. 14 Riccio, M.P. 65 Rizzo, F. 20 Romaniello, R. 65 Romano, A. 19, 60 Romano, R. 16 Ruoppolo, M. 58 Russo, C. 30 Russo, K. 35 Russo, R. 46 S Salatiello, F. 42 Sambri, I. 24 Sampaolo, S. 59 Sanges, R. 55 Santonicola, P.20 Santoro, L. 58 Sarnataro, D. 52 Scalia, G. 56 Selicorni, A. 13 Senese, V.P. 35 Sepe, R.M. 57 Severino, M. 65 Sisodiya, S.M. 11 Sofia, V. 11 Somma, T. 31 Sordino, P. 57 Sorrentino, C. 24 Spagnuolo, M.S. 23, 44 Spagnuolo, S. 36 Spaziano, A. 19 Speranza, A. 62 Speranza, L. 66 Striano, P. 11, 32

Sturiale, L. 43 Surace, E.M. 19 T Taglialatela, M. 12, 60 Tammaro, R. 19 Tarallo, A. 43, 58 Tartaglia, M. 13 Tedeschi. M. 29 Terracciano, C. 59 Terrone, G. 54, 60, 64, 65 Torella, A. 11, 13 Torromino, G. 61 Toscano, A. 58 Treppiccione, L. 36 Trinchese, G. 62 Trombetti, G. 8 Tuccillo, M. 64 Tufano, M. 38 Tunisi, L. 15, 16, 25, 41, 47 Tutino, M.L. 37 Tuzzi, R. 43 U Usiello, A. 22 V Van der Ploeg, A.T. 58 Vellucci, L. 63 Verrillo, L. 64 Viggiano, A. 17 Vitale, P. 50 Vitiello, G. 13, 65 Volpicelli, F. 66 Volpe, M. 55 Z Zara, F. 11, 32 Zerillo, L. 14 Zurzolo, C. 52

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