UNIVERSITY OF CALGARY Neuropathogenic effects of Syncytin-1 in Multiple Sclerosis by Joseph Mathew Antony A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF MEDICAL SCIENCE CALGARY, ALBERTA JUNE, 2006 © Joseph Mathew Antony 2006 iii ABSTRACT Human endogenous retroviruses (HERVs) constitute 8% of the human genome and have been implicated in both health and disease. Increased HERV gene activity occurs in activated glia although the consequences of HERV expression in the nervous system remain uncertain. Relative quantification and quantitative PCR analysis of HERV envelopes (env) revealed selectively increased abundance of HERV-W-7q encoded glycoprotein, Syncytin-1 in brains but not in blood-derived leukocytes from patients with Multiple Sclerosis (MS) relative to non-MS patients. Syncytin-1 expression in astrocytes induced the release of redox reactants, which were cytotoxic to oligodendrocytes. Increase in Syncytin-1 expression in astrocytes in the brain white matter of MS patients was accompanied by induction of the ER stress genes, OASIS, BiP, PERK, and GADD153. Expression of OASIS in astrocytes induced iNOS and thus, nitric oxide. ASCT-1, a neutral amino acid transporter and Syncytin-1 receptor, was suppressed in brain white matter astrocytes of MS patients and also in astrocytes expressing Syncytin-1 or OASIS. Nitric oxide enhanced the expression of the repressor transcription factor, Egr1, which concurrently suppressed ASCT1. Syncytin-1 mediated neuroinflammation and death of oligodendrocytes with ensuing neurobehavioral deficits were prevented by the antioxidant ferulic acid in a mouse model of MS. TNF-α implantation into a novel Syncytin-1 transgenic mice induced ER stress, loss of ASCT1 complemented by glial activation and T cell infiltration, indicating that astrocytes actively participate in MS pathogenesis. Thus, Syncytin-1’s proinflammatory properties in the nervous system demonstrate a novel pathogenic role for an endogenous retrovirus-encoded protein, which may serve as a target for future therapeutic intervention. iv PREFACE Some of the work presented in this thesis has been published previously. As required by thesis guidelines, full citations of these articles and an account of division of labor with all co-authors are listed below. Articles are listed in the order that they are published. As a general note, all work presented in this thesis was performed by Joseph Mathew Antony unless explicitly stated. 1. Antony JM, van Marle G, Opii W, Butterfield DA, Mallet F, Yong VW, Wallace JL, Deacon RM, Warren K, Power C. Human endogenous retrovirus glycoprotein-mediated induction of redox reactants causes oligodendrocyte death and demyelination. Nat Neurosci. 2004; 7 (10): 1088-95. Copyright permission has been obtained from Nature Publishing Group (Appendix F) This publication comprises all of Chapter 3. I performed the majority of the work associated with this study. Dr. Guido van Marle originally designed the SINrep5- EGFP and SINrep5-JRFL vectors that were used as controls in this study. Wycliffe Opii and Dr. Allan Butterfield measured protein carbonyls and 4-HNE. Dr. Francois Mallet provided the Syncytin-1 expressing vectors and monoclonal antibody against Syncytin-1. Dr. Wee Yong guided and provided materials for the oligodendrocyte assays. Dr. John Wallace guided and provided materials for anti-oxidant assays. Dr. Robert Deacon’s protocols were used for designing animal behavior protocols. Dr. Kenneth Warren supplied clinical samples from MS patients. All experiments were performed in the laboratory of Dr. C. Power under his supervision. v 2. Antony JM, Izad M, Bar-Or A, Warren A, Vodjgani M, Mallet F and Power C. Quantitative analysis of Human Endogenous Retrovirus-W env in neuroinflammatory diseases (AIDS Research and Human Retroviruses, in Press). This publication comprises all of Chapter 4. I did the majority of the work associated with this study. Dr. Maryam Izad assisted in the PCR assays. DNA was obtained from an Iranian cohort of MS patients provided by Dr. Mohammed Vodjgani. Dr. Amit Bar-Or (cDNA from a cohort of MS patients and controls) and Dr. Kenneth Warren (CSF and plasma) provided additional clinical samples. Dr. Francois Mallet provided the Syncytin-1 expressing vectors and monoclonal antibody against Syncytin-1. All experiments were performed in the laboratory of Dr. C. Power under his supervision. vi 3. Antony JM, Ellestad K, Shariat N, Hammond R, Imaizumi K, Mallet F and Power C. Syncytin-1 mediates Endoplasmic Reticulum Stress in a transgenic mouse model of Multiple Sclerosis (Manuscript submitted to Journal of Clinical Investigation). This publication comprises all of Chapter 5. I performed the majority of the work associated with this study. Mr. Kristofor Ellestad designed and optimized siRNA molecules against Syncytin-1. Ms. Neda Shariat performed immunohistochemistry for ER stress proteins from MS patients’ brain tissue provided by Dr. Robert Hammond. Dr. Kazunori Imaizumi provided the OASIS construct and antibody. Dr. Kenneth Warren provided clinical samples (CSF and plasma). Dr. Francois Mallet provided the Syncytin-1 expressing vectors and monoclonal antibody against Syncytin-1. All experiments were performed in the laboratory of Dr. C. Power under his supervision. vii ACKNOWLEDGEMENTS I would like to extend my deepest appreciation and gratitude to my supervisor, Dr. Christopher Power for his support and encouragement throughout the course of my graduate study at the University of Calgary and later, at the University of Alberta. His confidence and trust enabled me to transcend from one country and area of expertise to another. Also I would like to thank members of my supervisory committee, Dr. Wee Yong, Dr. Daniel Muruve and Dr. Robert Bell, who are a part of my career process and will continue to be. In addition, a number of faculty members from various Departments made my study exciting, for which I am very appreciative. In particular, I would like to thank Dr. Fabrizio Guilliani, Dr. Peter Dickie, Dr. Suzanne Grant (University of Alberta), Dr. Guido van Marle, Dr. John Wallace, Dr. Joseph Goren and Dr. Kamala Patel (University of Calgary). Also, I would like to thank my course coordinators, Dr. Julie Deans and Dr. David Severson for explaining to me the vagaries of grant and assignment writing. Laboratory life in Calgary was the most happiest one in my life and I owe this mainly to Claudia Silva, Shigeki Tsutsui, Shuhong Liu, Gareth Jones, Andrea Sullivan, Robyn Flynn, Qing Tang, Yu Zhu, Guido van Marle, Julie Ethier, Scot Henry, Farshid Noorbakhsh, Neda Shariat, David Vergote, Aundria Hood (the Calgary gang); Amir Afkhami, Ramin Sarrami, Kris Ellestad, Nicola Barsby and Martine Ooms (the BrainPowerLab at Edmonton), members of the Wee Lab (Tiffany, Tammy, Jennifer Wells, Viktor, Rowena, Lorraine, Angelika, Yan Fan), Zochodne Lab (Cory Toth & James Kennedy), Patel Lab (Subhadeep, Manprit, Cory, Evelyn, Vicky, Keith & Kamala) and numerous other friends at the Health Sciences Center whose names are not viii mentioned here. Graduate life would not have been a smooth without the excellent help and advise from Belinda Ibrahim, Sherry Sweeney, Rosalie Kolstad, Dr Francine Smith, Dr. Stephen Robbins and Christine Szefer for which I am forever grateful. My stay in Calgary and Edmonton was indeed a joyous one-my thanks to the family of friends- Jasprit, Jose Martinez, Elena Silva, Valentine, George and Annie, Dr. Rajan George and Deepa. Lastly, I would like to thank all the funding agencies for the financial support and encouragement received-Alberta Heritage Foundation for Medical Research, Multiple Sclerosis Society of Canada and Canadian Institutes for Health Research (CIHR)- Integrated Health Research Team (IHRT). ix DEDICATION To my wife, Smitha, for what she is to me, AND To my parents, for their unfailing commitment to my education x Table of Contents Approval Page ii Abstract iii Preface v Acknowledgements viii Dedication x Table of Contents xi List of Tables xvi List of Figures xvii Appendices xx List of Abbreviations xxi Epigraph xxii CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW 1 I.1. Multiple Sclerosis (MS) Pathogenesis 2 I.1.1. Clinical and demographic features of MS 2 I.1.2. Pathophysiology of MS 5 I.1.3. Neuroinflammation 8 I.1.3.1. Cellular components of neuroinflammation 9 I.1.3.1.1.Leukocytes 13 I.1.3.1.1.1. CD4+ T cells 13 I.1.3.1.1.2. CD8+ T cells 14 I.1.3.1.1.3. B cells 14 I.1.3.1.1.4. Mast cells 14 I.1.3.1.1.5. Neutrophils 15 I.1.3.1.1.6. Dendritic cells 16 xi I.1.3.1.2. Immunoregulatory cells in MS 17 I.1.3.1.3. Resident neural cells 18 I.1.3.1.3.1. Astrocytes 18 I.1.3.1.3.2. Oligodendrocytes 20 I.1.3.1.3.3. Neurons 20 I.1.3.1.3.4. Microglia 21 I.1.3.1.4. T cell-glia interaction 22 I.1.3.2. Molecular components of neuroinflammation 24 I.1.3.2.1. Cytokines 25 I.1.3.2.2. Chemokines 27 I.1.3.2.3. Neurotrophic factors and brain repair 28 I.1.3.2.4. Proteases 31 I.1.3.2.5. Oxidative stress 33 I.1.3.2.5. Nitric oxide (NO) 34 I.1.3.2.7. ER stress 37 I.1.4. Drawbacks of EAE as a model of MS 41 I.1.5. Genetics of MS 42 I.1.6. Environmental Factors in MS 45 I.1.6.1. Infectious agents in MS pathogenesis 45 I.1.6.2. Demyelinating viruses 46 I.1.6.3. Retroviruses and MS pathogenesis 47 I.2. Retroviruses: Introduction 51 I.2.1. Genomic and structural organization 52 I.2.2. Classification 53 I.2.3. Retroviral Biology 57 I.2.4. Retroviral pathogenesis in the nervous system 61 I.2.4.1.