ELUCIDATING SCHWANN CELL REPROGRAMMING Dr Elizabeth Byrne Cell Interactions and Cancer Group Institute of Clinical Science MRC Clinical Sciences Centre Imperial College London Thesis submitted for the degree of MPhil 1 Abstract The peripheral nervous system, unlike the central nervous system, has an exceptional capacity for regeneration following injury. This is due to the remarkable plasticity of the Schwann cells (SC), which are able to reprogramme, following injury, to a progenitor like cell which facilitates peripheral nerve repair. Current knowledge on the molecular basis of this reprogramming is incomplete and we are lacking a global overview of the transcriptional events that occur in SC following nerve injury and how these change over time. We aimed to characterise transcriptional changes in the SC, over time, following nerve injury using RNAseq. We also aimed to develop an in vitro dedifferentiation assay to use as a screening tool to asses potential key genes found using RNAseq. We developed a method of reliably extracting good quality, SC specific, RNA from the sciatic nerve of mice using fluorescence activated cell sorting. We performed RNAseq on SC from intact nerves and from the distal stump of nerves 6 days post transection. We validated this method by confirming differential expression of genes known to be up and downregulated following nerve injury, using RNAseq data. In analysing the RNAseq data we identified several potentially exciting, novel key molecular players in SC reprogramming, namely Myc and Runt-related transcription factor 1. We also developed an in vitro dedifferentiation assay to use as an initial screen for the genes identified using RNAseq. This involved the addition of neuregulin and serum to previously cyclic adenosine monophosphate differentiated SC. This assay was shown to recapitulate the changes seen in vivo, using RNAseq. 2 Table of contents ABSTRACT ........................................................................................................................................................ 2 COPYRIGHT DECLARATION............................................................................................................................... 5 DECLARATION OF ORIGINALITY ....................................................................................................................... 6 ACKNOWLEDGEMENTS .................................................................................................................................... 7 LIST OF FIGURES ............................................................................................................................................... 8 1. INTRODUCTION ..................................................................................................................................... 11 1.1 PERIPHERAL NERVOUS SYSTEM ................................................................................................................... 11 1.2 SCHWANN CELL DEVELOPMENT .................................................................................................................. 13 1.2.1 Early SC development .......................................................................................................................... 13 1.2.2 Schwann Cell Precursors ..................................................................................................................... 14 1.2.3 Immature Schwann Cells ..................................................................................................................... 15 1.2.4 Radial Sorting ...................................................................................................................................... 15 1.2.5 Schwann Cell Myelination ................................................................................................................... 16 1.3 MYELIN.................................................................................................................................................. 17 1.4 WALLERIAN DEGENERATION ...................................................................................................................... 18 1.5 REGENERATION ....................................................................................................................................... 19 1.5.1 Schwann Cell Dedifferentiation ....................................................................................................... 19 1.5.2 The Schwann Cell Repair Programme ............................................................................................. 20 1.5.3 Myelin Clearance ........................................................................................................................ 21 1.5.4 Axonal Guidance ............................................................................................................................. 22 1.5.5 Proliferation .................................................................................................................................... 23 1.6 REINNERVATION ...................................................................................................................................... 24 1.7 MOLECULAR BASIS OF SC REPROGRAMMING FOLLOWING INJURY ....................................................................... 25 1.7.1 MAPK cascades ............................................................................................................................... 25 1.7.2 Transcription factors ....................................................................................................................... 27 1.7.3 Growth Factors .............................................................................................................................. 28 1.7.4 Receptors ....................................................................................................................................... 29 1.8 CLINICAL RELEVANCE ................................................................................................................................ 30 1.8.1 Peripheral nerve injury .................................................................................................................... 30 1.8.2 Peripheral Neuropathies ................................................................................................................. 33 1.8.3 Peripheral Nerve Sheath Tumours .................................................................................................. 35 1.9 AIMS ..................................................................................................................................................... 37 2. MATERIALS AND METHODS ....................................................................................................................... 38 2.1 IN VIVO METHODS .......................................................................................................................................... 38 2.1.1 Animal housing conditions .................................................................................................................. 38 2.1.2 Mouse line ........................................................................................................................................... 38 2.1.3 Genotyping .......................................................................................................................................... 39 2.1.4 Sciatic nerve transaction ..................................................................................................................... 40 2.1.5 Harvesting the sciatic nerve ................................................................................................................ 40 2.1.6 Immunoflurescence ............................................................................................................................. 41 2.1.7 In vivo ethynyl-2’-deoxyuridine (EDU) proliferation assay .................................................................. 41 2.1.8 Digestion of the Sciatic nerve and SC extraction ................................................................................. 42 2.1.9 SC specific RNA extraction ................................................................................................................... 43 2.1.10 Bioanalyzer ........................................................................................................................................ 43 3 2.1.11 Plating and Staining SC following Sorting ......................................................................................... 44 2.1.12 RNA seq library preparation ....................................................................................................... 44 2.2 IN VITRO METHODS ......................................................................................................................................... 46 2.2.1 Isolation and culture of rat Schwann Cells .......................................................................................... 46 2.2.2 Dedifferentiation assay ....................................................................................................................... 47 2.2.3 RNA extraction .................................................................................................................................... 48 2.2.4 DNase Treatment ...............................................................................................................................