Glial cells in health and disease by Anjali Balakrishnan A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Biochemistry University of Toronto © Copyright by Anjali Balakrishnan 2020 Glial cells in health and disease Anjali Balakrishnan Doctor of Philosophy Department of Biochemistry University of Toronto 2020 Abstract Glial cells are often considered to be ‘supporting cast’ members in the nervous system, with ancillary roles in providing nutrient and structural support to neurons. However, glial cells have many essential roles, including the myelination of nerves to allow information to be transmitted rapidly and efficiently. My thesis has largely focused on the role of myelinating glial cells in health and disease. I first studied a population of malignant oligodendrocyte-like cells that form glial tumors in the central nervous system, called oligodendroglioma. For the rest of my thesis, I focused on Schwann cells in the peripheral nervous system. In Chapter 2 of my thesis, I describe my investigation into the role of extracellular vesicles in controlling oligodendroglioma growth by mediating heterotypic and homotypic cell-cell interactions. I revealed that oligodendroglioma tumor cells secrete extracellular vesicles that carry cytotoxic cargo to induce cell death in neighboring cells. Furthermore, I implicated a gene involved in extracellular vesicle biogenesis, SMPD3, in negatively regulating oligodendroglioma growth by controlling the synthesis of extracellular vesicles (Chapter 2). I then studied the development of Schwann cells, and their transition into repair Schwann cells post nerve injury. I characterized the dynamic expression patterns of a panel of transcriptional regulators during development and in repair Schwann cells post-injury (Chapter 3). I then used this panel of markers to ask whether the ets domain transcription factor Etv5, expressed transiently in ii Schwann cell precursors, played a role in regulating Schwann cell development and in repair Schwann cells by using a hypomorphic Etv5 mutant mouse model (Chapter 4). While Etv5 mutants had no apparent defects in Schwann cell development, I describe several important caveats and future considerations. Finally, I performed the first steps towards developing a non-integrative, triple transcription factor mediated lineage conversion strategy for the generation of induced repair Schwann cells from mouse embryonic fibroblasts (Chapter 5). In conclusion, I have gained new insights on how glial cells in a healthy and diseased state are regulated. My findings have therapeutic implications for the treatment of oligodendroglioma tumors in the central nervous system, and for peripheral nerve repair. iii Acknowledgments First and foremost, I want to send out my love and gratitude to my supervisor, Dr. Carol Schuurmans. Thank you for taking my call that eventful afternoon and accepting me as a graduate student! Thank you for your patience, and for spoiling us with the time you take to train each one of us individually. Carol has been an empowering role model for women in STEM. I feel hopeful that I will be able to take her legacy forward in every role I pursue in the future. I want to thank my wonderful supervisory committee, Dr. Alain Dabdoub and Dr. Micheal Ohh for being a strong support system for me at the University of Toronto! I also want to thank Dr. Jennifer Chan, Dr. Jeff Biernaskie, and Dr. Hon Leong for their scientific counsel. I had the incredible opportunity to be a student at two wonderful universities through my graduate career, and I remain grateful for all the splendid experiences. I would like to thank Dr. Dawn Zinyk, who has helped me from the first day I entered the lab. I thank her for showing me the ropes around the lab, all the hundred cloning steps that we performed together, and for patiently answering all my doubts. I want to thank Dr. Yacine Touahri for his friendship and help with nearly everything through the last seven years. It has been a wonderful bond which I will cherish in the times to come. I want to give a shout-out to all the brilliant students I have worked with- Humna Noman, Sajeevan Sujanthan, Marielle Balanaser. I have learnt something from all of them. I miss my daily discussions with you Humna! I am blessed to have worked along with all my wonderful lab mates over the years, with a special shout-out to- Dr. Rajiv Dixit, Natasha Klenin, Nobuhiko Tachibana, Luke Ajay David, Mary Chute, Sisu Han, Dr. Vorapin Chinchalongporn, and Lakshmy Vasan. You all made graduate life so much better and memorable! A special note of thanks to my closest friends- Chinmayee and Anupama. I do not know how I would have survived in Calgary without your companionship and miss you both terribly in Toronto! I would like to thank Shakir Hasan, for just about everything ever. Thank you for bearing with all my mood swings and standing by patiently even when things got tough. I wish our journey ahead is a fabulous one! And finally, I would like to thank my family- Mom, Dad, Amit, Swati, Arun, and Arunima for being the most caring family ever. Thank you for always supporting my dreams, ambitions, and rebellions. Distance makes the heart grow fonder….and it did! I am blessed to be a part of the Balakrishnan household! I hope I make you proud! iv Dedication This thesis is dedicated to my Mom and Dad. I am so proud to be your daughter. Ever thine, Ever mine, Ever ours. v Table of Contents Acknowledgments............................................................................................................... iv List of Tables ..................................................................................................................... xii List of Figures ................................................................................................................... xiii Appendices ....................................................................................................................... xvii List of Abbreviations ...................................................................................................... xviii Dissemination of work arising from this Thesis ............................................................. xxiii Other contributions during my thesis work ..................................................................... xxv Introduction ......................................................................................................... 1 1.1 Glial cells: An overview .......................................................................................... 2 1.2 Glioma: An Introduction .......................................................................................... 2 1.2.1 General introduction on glioma subtypes .................................................... 2 1.2.2 Oligodendroglioma ...................................................................................... 2 1.3 Extracellular vesicles: An Introduction ................................................................... 4 1.3.1 A brief primer on EV classification, isolation, and biogenesis .................... 5 1.3.2 EV functions in the ‘healthy’ nervous system ............................................. 9 1.3.3 Roles of EVs in glioma .............................................................................. 13 1.4 Schwann Cells: An Introduction ............................................................................ 19 1.4.1 Glial cells of the peripheral nervous system: Schwann cells ..................... 19 1.4.2 Genes regulating Schwann cell specification and differentiation .............. 27 1.4.3 Nerve growth factor receptor and Neuregulin signaling: Role in Schwann cell myelination .......................................................................................... 31 1.4.4 Peripheral nerve injury and role of Schwann cells post injury .................. 33 1.4.5 Phenotype of a repair Schwann cell ........................................................... 35 1.4.6 Identifying an alternate source for Schwann cells: reprogramming somatic cells to generate Schwann cells .................................................................. 38 1.5 Hypothesis and specific aims of the thesis ............................................................ 44 vi SMPD3-mediated extracellular vesicle biogenesis inhibits oligodendroglioma tumor growth ................................................................................................................. 45 2.1 Abstract .................................................................................................................. 46 2.2 Introduction ............................................................................................................ 46 2.3 Methods.................................................................................................................. 47 2.3.1 Patient-derived tumor tissues and cells and study approval ...................... 47 2.3.2 The Cancer Genome Atlas (TCGA) survey ............................................... 48 2.3.3 Animals ...................................................................................................... 48 2.3.4 BT088 and BT054 cell culture ................................................................... 48 2.3.5 Small molecule inhibitors .......................................................................... 49 2.3.6 Mouse NSC isolation and culture .............................................................. 49