CHARACTERISATION OF THE TRANSCRIPTOMIC AND PROTEOMIC PROFILE OF ASTROCYTES IN MULTIPLE SCLEROSIS By Rachel Waller Submitted for the degree of Doctor of Philosophy (PhD) Sheffield Institute for Translational Neuroscience University of Sheffield January 2014 Volume I For Joan Ann Waller Forever in my thoughts. This is for you. i ACKNOWLEDGEMENTS First and foremost I would like to acknowledge my significant academic supervisory team; Dr. Julie Simpson, Prof. Stephen Wharton, Prof. Paul Ince, Dr. Paul Heath, Dr. Simona Francese, Prof. Nicola Woodroofe and Prof. Basil Sharrack for their scientific and at times personal guidance, support and patience throughout my PhD. My utmost gratitude goes to Julie for her continuous help, support and reassurance throughout. I am grateful to Julie for her natural ability to calm me and keep me sane with her thoughtful and encouraging words, having the knack of saying the right thing at the right time to me. But most of all I am thankful to Julie for her belief in me when at times I found it hard to. I will be eternally grateful to Nicola, you have inspired me to get where I am today. Thank you for the countless conversations over many a glass of wine, and most of all for giving me a chance from the beginning. Thank you to Biogen Idec for funding my PhD and importantly to all the donors and their families at the UK MS Society Tissue Bank. Thank you for deciding to donate your tissue to scientific research, without you this work would not have been possible. Thanks to all my work colleagues and friends both at Sheffield University and Sheffield Hallam University, for your never ending support and always being there for me to moan at and conspire with! A special thanks to Natalie and Ruth for your excellent lab assistance and Dr. Laura Cole for your understanding and patience. I will forever be grateful to Paul, my rock throughout. You have been by my side during this long journey and have always believed in me and encouraged me, helping me to realise and fulfil my ambitions and dreams. You’ve shared with me my ultimate lows always being there to pick me up, and we’ve laughed out loud together through the highs. I will be eternally grateful for your down to earth attitude towards life, words of wisdom and witty sense of humour, you’ve kept me grounded and I love you for all these things. Finally a huge thank you to my long suffering parents and sister, Rebecca. You have ALWAYS encouraged me to be the best I can be. No dream is out of reach ‘as long as you’ve tried your hardest that’s the best you can do’ (Waller et al 1988-present). Well, thanks to your love and support, the endless cups of tea, lemon barley and ginger biscuits (for the nerves!) I’ve tried my hardest and I’ve done it! ii Every accomplishment in life starts with a decision to try... iii ABSTRACT Multiple sclerosis (MS) is a chronic, neuroinflammatory demyelinating disease of the central nervous system (CNS). Typical white matter lesions (WML) in MS are surrounded by areas of non-demyelinated normal appearing white matter (NAWM) with complex subtle pathology, including blood brain barrier (BBB) dysfunction, axonal damage and glial activation. Astrocytes, the most abundant cell type within the CNS, are known to support neuronal function, maintain homeostasis within the CNS and regulate neurotransmission. Yet conversely, can promote an inflammatory response, inhibit myelin repair and support the production of autoreactive T cells in MS. This thesis aimed to investigate the transcriptomic and proteomic profile of astrocytes in MS NAWM to determine whether specific astroglial changes exist, which may contribute/prevent disease progression in MS. Initial data presented in this thesis demonstrated a change in astrocyte phenotype within different pathological regions of the CNS in MS, as observed by the distinct immunoprofile of a variety of known astrocyte markers. Being able to isolate cell types from human tissue is fundamental in beginning to define a particular cell’s role in disease pathogenesis. An immuno-laser capture microdissection (LCM) method was developed to enable the isolation of glial cells from human post mortem (PM) CNS tissue. In the current study glial fibrillary acidic protein (GFAP) positive astrocytes were isolated from MS NAWM and control WM via immuno-LCM and microarray analysis completed to compare their transcriptome. Significantly differentially expressed genes were associated with the immune response, cell signaling, cytoskeletal changes and regulation of homeostasis which relate to the distinct roles of astrocytes. Interestingly, from the top 20 significant differentially upregulated genes, six of them were related to the regulation of iron homeostasis and oxidative stress, including metallothionein I-II (MT-I+II), ferritin light chain (FTL) and transferrin (TF). Subsequent transcriptomic and proteomic investigations were carried out on candidate genes using polymerase chain reaction, immunohistochemistry, western blotting and mass spectrometry to investigate the neuroprotective role of astrocytes in regulating iron homeostasis and oxidative stress in MS NAWM. Evidence presented in this thesis demonstrates the importance of astrocytes in the pathogenesis of MS. The results indicate that further investigations into the protective roles of astrocytes in regulating iron and oxidative stress in MS NAWM are warranted. iv TABLE OF CONTENTS ♦ Dedication - i ♦ ♦ Acknowledgments - ii ♦ ♦ Quotation - iii ♦ ♦ Abstract - iv ♦ ♦ Table of contents - v ♦ ♦ List of figures - xii ♦ ♦ List of tables - xv ♦ ♦ Abbreviations - xvii ♦ CHAPTER 1 AN INTRODUCTION TO MULTIPLE SCLEROSIS ........................................................ 1 1.1 Multiple Sclerosis (MS) ..................................................................................... 2 1.2 Clinical features and disease course in MS ........................................................ 2 1.3 Pathological hallmarks of MS – lesion type ....................................................... 5 1.4 MS normal appearing white matter .................................................................... 6 1.5 Proposed mechanisms underlying the aetiology of MS ..................................... 7 1.5.1 Influence of genetics on MS ....................................................................... 7 1.5.2 Environmental factors associated with MS ................................................. 8 1.6 Proposed pathophysiology of MS – an inflammatory or degenerative disease? .. .......................................................................................................................... 10 1.6.1 The blood brain barrier (BBB) .................................................................. 11 1.6.2 BBB dysfunction in MS ............................................................................ 11 1.6.3 Inflammation in MS – the autoimmune hypothesis. ................................. 13 1.6.4 Neurodegeneration in MS ......................................................................... 15 1.6.4.1 Mitochondrial damage in MS lesions ................................................ 16 1.6.4.2 Oxidative damage in MS lesions ....................................................... 17 1.6.4.3 Glutamate excitotoxicity in MS lesions ............................................. 17 1.7 Animal models of MS ...................................................................................... 18 1.7.1 Induction of experimental autoimmune encephalomyelitis (EAE) ........... 19 1.7.2 Autoimmune hypothesis of EAE .............................................................. 19 1.7.3 Translation of EAE to MS; the pros and cons .......................................... 21 1.8 Glial Cells in MS .............................................................................................. 22 v 1.8.1 Oligodendrocytes in MS ........................................................................... 22 1.8.2 Microglia in MS ........................................................................................ 22 1.8.3 Astrocytes in MS ....................................................................................... 24 1.8.3.1 Astrocytes and the BBB in MS .......................................................... 24 1.8.3.2 Astrocytes and their role in preventing myelin repair in MS ............ 25 1.8.3.3 Reactive astrocytes associated with inflammation and gliotic scar formation ............................................................................................ 25 1.8.3.4 Astrocytes and the immune system ................................................... 26 1.8.3.5 Astrocyte cytokine actions in MS pathology ..................................... 27 1.8.3.6 Astrocyte derived chemokines in MS pathology ............................... 27 1.9 Overall aims of this thesis ................................................................................ 29 CHAPTER 2 HISTOLOGICAL CHARACTERISATION OF THE ASTROCYTE PHENOTYPE IN THE WHITE MATTER IN MULTIPLE SCLEROSIS .............................................................. 30 2.1 Introduction .................................................................................................... 31 2.2 Aims and objectives of the study ................................................................... 36 2.3 Material and methods .................................................................................... 37 2.3.1 Suppliers ...................................................................................................