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Role of Cellular Prion Protein in Central Nervous System Myelination

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Role of Cellular Prion Protein in Central Nervous System Myelination Scuola Internazionale Superiore di Studi Avanzati - SISSA Trieste, Italy ROLE OF CELLULAR PRION PROTEIN IN CENTRAL NERVOUS SYSTEM MYELINATION Thesis submitted for the degree of “Philosophiae Doctor” CANDIDATE SUPERVISORS Elisa Meneghetti Prof. Giuseppe Legname, Ph.D. Dr.Federico Benetti, Ph.D. Academic year 2014/2015 2 DECLARATION The work described in this thesis has been carried out at Scuola Internazionale Superiore di Studi Avanzati (SISSA), Trieste, Italy, from November 2011 to October 2015. During my Ph.D. course I contributed to two research articles: - Gasperini, L., E. Meneghetti, B. Pastore, F. Benetti and G. Legname (2015). "Prion Protein and Copper Cooperatively Protect Neurons by Modulating NMDA Receptor Through S-nitrosylation." Antioxidants & Redox Signaling 22(9): 772-784. Included in the “APPENDIX” section. - Gasperini, L., Meneghetti, E., Legname, G. and Benetti, F. “The Absence of the Cellular Prion Protein Impairs Copper Metabolism and Copper-Dependent Ferroxidase Activity, Thus Affecting Iron Mobilization.” To be submitted. 3 4 INDEX LIST OF ABBREVIATIONS .............................................................................................................. 7 ABSTRACT ............................................................................................................................................ 11 INTRODUCTION ............................................................................................................................... 13 PRION DISEASES ............................................................................................................................... 13 PRION PARADIGM ............................................................................................................................ 15 PRION NEUROTOXICITY ................................................................................................................... 16 THE CELLULAR PRION PROTEIN ................................................................................................. 17 PrPC EXPRESSION AND SUBCELLULAR LOCALIZATION ................................................................ 19 PrP KNOCK-OUT AND TRANSGENIC MICE ..................................................................................... 21 C PHYSIOLOGICAL FUNCTION OF PrP ........................................................................................ 25 PrPC ROLE IN METAL HOMEOSTASIS .............................................................................................. 26 PrPC PROTECTIVE ROLE IN OXIDATIVE STRESS ............................................................................. 29 PrPC AND SYNAPTIC FUNCTIONALITY ............................................................................................ 32 PrPC ROLE IN MYELINATION ............................................................................................................ 36 AIM ........................................................................................................................................................... 51 MATERIALS AND METHODS .................................................................................................... 53 ANIMALS ............................................................................................................................................. 53 TISSUE HOMOGENIZATION AND MEMBRANE PURIFICATION ..................................... 53 ENZYMATIC CHOLESTEROL .......................................................................................................... 54 PROTEIN EXPRESSION ANALYSIS ............................................................................................... 54 ANTIBODIES ....................................................................................................................................... 55 RNA EXTRACTION AND TEAL TIME PCR ................................................................................. 55 ELECTRON MICROSCOPY .............................................................................................................. 58 5 ORGANOTYPIC HIPPOCAMPAL CULTURES PREPARATION ............................................. 58 ORGANOTYPIC HIPPOCAMPAL CULTURES CUPRIZONE TREATMENT ....................................... 58 MTS CELL VIABILITY ASSAY .............................................................................................................. 59 STATISTICAL ANALYSES ................................................................................................................. 59 RESULTS ................................................................................................................................................ 61 ANALYSIS OF PERIPHERAL NERVOUS SYSTEM MYELINATION IN Prnp+/+ AND 0/0 Prnp MICE ....................................................................................................................................... 61 SCIATIC NERVE CHOLESTEROL CONTENT ...................................................................................... 61 MYELIN PROTEIN EXPRESSION ANALYSIS IN SCIATIC NERVES ................................................... 62 TRANSCRIPTIONAL ANALYSIS OF MYELIN GENES IN THE PNS ................................................... 65 ANALYSIS OF CENTRAL NERVOUS SYSTEM MYELINATION IN Prnp+/+ AND Prnp0/0 MICE ...................................................................................................................................................... 66 BRAIN CHOLESTEROL MEASUREMENT .......................................................................................... 66 MYELIN PROTEIN EXPRESSION ANALYSIS IN THE BRAIN ............................................................ 67 TRANSCRIPTIONAL ANALYSIS OF GENES INVOLVED IN CNS MYELINATION ............................. 71 ELECTRON MICROSCOPY ANALYSIS OF CNS MYELIN................................................................... 74 ORGANOTYPIC HIPPOCAMPAL SLICE CULTURES AS AN EX VIVO MODEL FOR MYELINATION ................................................................................................................................... 76 MYELIN PROTEIN EXPRESSION ANALYSIS IN OHC ........................................................................ 76 TRANSCRIPTIONAL ANALYSIS OF MYELIN GENES IN OHC .......................................................... 78 OHC TREATMENT WITH CUPRIZONE ............................................................................................. 80 DISCUSSION ........................................................................................................................................ 83 CONCLUSIONS AND PERSPECTIVES ................................................................................... 92 ACKNOWLEDGEMENTS .............................................................................................................. 95 BIBLIOGRAPHY ................................................................................................................................. 97 APPENDIX .......................................................................................................................................... 117 6 LIST OF ABBREVIATIONS AD: Alzheimer’s disease AMPA: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate ATP: adenosine triphosphate Atp7a: ATPase 1 BACE1: β-secretase 1 BCA: bicinchoninic acid BrdU: 5’-bromo-2’-deoxyuridine BSA: bovine serum albumin BSE: bovine spongiform encephalopathy CaM: calmodulin CaMKII: Ca2+/calmodulin-dependent protein kinase II CC: charged cluster CD: central domain CDP: chronic demyelinating polyneuropathy CJD: Creutzfeldt-Jakob disease CNS: central nervous system CTR1: copper transporter 1 CWD: chronic wasting disease CZ: cuprizone DAPI: 4',6-diamidino-2-phenylindole DIV: days in vitro Dpl: Doppel EAE: experimental allergic encephalitis 7 ER: endoplasmic reticulum fALS: familial amyotrophic lateral sclerosis fALS: familial amyotrophic lateral sclerosis FFI: fatal familial insomnia Gapdh: glyceraldehyde 3-phosphate dehydrogenase GluN(1, 2A-D, 3A-B): NMDAR subunit (1, 2A-D, 3A-B) GPI: glycosylphosphatidylinositol GSS: Gerstmann, Sträussler and Scheinker syndrome HC: hydrophobic core HRP: horseradish peroxidase KO: knock-out L-MAG: long myelin-associated glycoprotein L-MPZ: large-myelin protein zero LPC: lysolecithin, lysophosphatidylcholine MAG: myelin-associated glycoprotein MBP: myelin basic protein MOG: myelin oligodendrocyte glycoprotein MPZ: myelin protein zero MS: multiple sclerosis MTS: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium NeuN: neuronal nuclei NG2: neural/glial antigen 2 NMDA: N-methyl-D-aspartate NMDAR: N-methyl-D-aspartate receptor nNOS: neuronal nitric oxide synthase 8 NO: nitric oxide NRG: neuregulin NTC: non-template control OHC: organotypic hippocampal cultures OPC: oligodendrocyte precursor cells OR: octapeptide repeat P: postnatal day PBS: phosphate-buffered saline PD: Parkinson’s disease PES: phenazine ethosulfate PFA: paraformaldehyde PKA: protein kinase A PKC: protein kinase C PLP: proteolipid protein PNS: periferal nervous system PrPC: cellular prion protein PrPSc: scrapie prion protein ROS: reactive oxygen species RT: room temperature S-MAG: short myelin-associated glycoprotein SN: sciatic nerve SNO: S-nitrosylation SOD: superoxide dismutase Sox10: (sex determining region Y)-box 10 TBST: Tris-Buffered Saline plus Tween 20 9 TGN: trans-Golgi network TSE: transmissible spongiform encephalopathies Tubb3: βIII-tubulin WB: Western
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