“FEDERICO II” University of Naples PhD PROGRAM IN NEUROSCIENCE XXIX CYCLE PhD Thesis: “D-Aspartate treatment stimulates differentiation of oligodendrocyte precursors, prevents demyelination and accelerates remyelination in the cuprizone mouse model of Multiple Sclerosis” Coordinator Prof. Lucio Annunziato Tutor Candidate Dr. Francesca Boscia Dr. Valeria de Rosa Academic Year 2016/2017 INDEX Abbreviations ............................................................................................................................ 4 SUMMARY ............................................................................................................................... 6 I. INTRODUCTION ..................................................................................................... 9 1. MULTIPLE SCLEROSIS ................................................................................................. 10 1.1 Epidemiology .............................................................................................................................. 11 1.2 Histological hallmarks................................................................................................................. 12 1.3 Pathological mechanisms ............................................................................................................ 13 1.4 Classification and histological heterogeneity of MS lesions ....................................................... 14 1.5 Clinical course ............................................................................................................................. 16 1.5.1 Clinical symptoms .................................................................................................................... 17 1.5.2 Diagnosis .................................................................................................................................. 17 1.6 Biomarkers .................................................................................................................................. 19 1.7 Therapy ....................................................................................................................................... 20 2. OLIGODENDROCYTES .................................................................................................. 22 2.1 Morphology ................................................................................................................................. 22 2.2 Origin .......................................................................................................................................... 24 2.3 Function ....................................................................................................................................... 25 2.4 Oligodendrocyte lineage ............................................................................................................. 25 3. MYELIN .............................................................................................................................. 27 3.1 Myelin composition .................................................................................................................... 27 3.2 Myelination ................................................................................................................................. 32 3.3 Remyelination ............................................................................................................................. 33 4. IN VIVO MODELS TO STUDY DEMYELINATION AND REMYELINATION ..... 35 4.1 EAE ............................................................................................................................................. 35 4.2 Viral infections ............................................................................................................................ 35 4.3 Toxic models ............................................................................................................................... 36 5. REGULATION OF OLIGODENDROCYTE DEVELOPMENT ................................ 41 5.1 Sonic hedgehog, bone morphogenetic proteins and Wnt signaling ............................................ 41 5.2 Transcription factors ................................................................................................................... 42 5.3 Growth factors signaling ............................................................................................................. 43 5.4 Ionic signaling through voltage-dependent ion channels ............................................................ 44 5.5 Glutamate receptors signaling ..................................................................................................... 45 5.6 Glutamatergic input role on oligodendrocyte lineage ................................................................. 50 1 5.6.1 OPC migration...................................................................................................................... 50 5.6.2 OPC proliferation ................................................................................................................. 51 5.6.3 OPC differentiation and myelination ................................................................................... 52 5.7 The Na+/Ca2+ exchanger .............................................................................................................. 53 5.7.1 Structure and function .............................................................................................................. 53 5.7.2 Bidirectional operation mode ................................................................................................... 55 5.7.3 Role of NCX in oligodendrocytes ............................................................................................ 55 7. D-AMINOACIDS ............................................................................................................... 56 7.1 D-Aspartic acid ........................................................................................................................... 56 7.1.1 Distribution........................................................................................................................... 57 7.1.2 Biosynthesis ......................................................................................................................... 58 7.1.3 Uptake, transport, degradation and release .......................................................................... 58 7.1.4 Function ................................................................................................................................ 59 AIM OF THE STUDY ........................................................................................................... 61 II. MATERIALS AND METHODS .................................................................. 64 1. Animals ......................................................................................................................................... 64 2. Cell Cultures .................................................................................................................................. 64 3. Rat and mouse primary OPC cultures and D-Asp exposure ......................................................... 64 4. Quantitative Real-Time PCR ........................................................................................................ 65 5. Western blotting ............................................................................................................................ 65 2+ 6. Microfluorimetric [Ca ]i measurement ........................................................................................ 66 7. Electrophysiology ......................................................................................................................... 66 8. Silencing ........................................................................................................................................ 67 9. Cuprizone-induced demyelination/remyelination and D-Asp treatment protocols ....................... 67 10. Behavioral testing ........................................................................................................................ 69 11. Confocal microscopy................................................................................................................... 69 12. Quantitative data analysis ............................................................................................................ 70 13. Electron microscopy .................................................................................................................... 70 14. G-ratio ......................................................................................................................................... 70 15. Hormonal dosage ........................................................................................................................ 71 16. Statistical analysis ....................................................................................................................... 71 III. RESULTS ............................................................................................................... 73 1. D-Aspartate exposure stimulates oligodendrocyte differentiation and upregulates hormone levels in OPC cells ........................................................................................... 73 2 2. Blocking the NMDA receptor and the Na+/Ca2+ calcium exchanger NCX3