Regulation of Schwann Cell Differentiation and Peripheral Myelination by Src-like Kinases, p38 MAPKs and Rho GTPases Shireen Hossain Department of Pharmacology and Therapeutics McGill University, Montreal, Canada June, 2010 A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy. © Shireen Hossain, 2010 1 TABLE OF CONTENTS LIST OF FIGURES ................................................................................................................. 7 LIST OF TABLES ................................................................................................................... 9 ABSTRACT ......................................................................................................................... 10 RÉSUMÉ ............................................................................................................................ 12 PREFACE ............................................................................................................................ 14 ORIGINAL CONTRIBUTION TO KNOWLEDGE ................................................................... 17 ACKNOWLEDGEMENTS .................................................................................................... 20 LIST OF ABBREVIATIONS .................................................................................................. 22 CHAPTER 1: INTRODUCTION TO SCHWANN CELLS AND PERIPHERAL MYELINATION ... 29 1.1 HISTORICAL PERSPECTIVE ........................................................................................... 30 1.2 TYPES OF SCs ............................................................................................................... 31 1.3 PERIPERAL MYELIN ...................................................................................................... 32 1.3.1 Dorsal root ganglion neurons ............................................................................ 33 1.3.2 Properties of myelin .......................................................................................... 35 1.3.3 Function of myelin ............................................................................................. 36 1.3.4 Architecture of a myelinated internode ............................................................ 36 1.3.4.1 Node of Ranvier ......................................................................................... 39 1.3.4.2 Paranode ................................................................................................... 41 1.3.4.3 Juxtaparanode ........................................................................................... 43 1.3.4.4 Internode ................................................................................................... 44 1.3.5 Lipids of myelin sheath ...................................................................................... 46 1.3.6 Proteins of Myelin Sheath ................................................................................. 47 1.3.6.1 Myelin associated glycoprotein (MAG) ..................................................... 48 1.3.6.2 Myelin basic protein (MBP) ....................................................................... 49 1.3.6.3 Protein zero (P0) ........................................................................................ 50 1.3.6.4 Periaxin ...................................................................................................... 52 1.3.6.5 Peripheral myelin protein 22 kDa (PMP22) ............................................... 53 1.3.6.6 Peripheral myelin protein‐2 (PMP‐2) ........................................................ 55 1.3.6.7 Connexins .................................................................................................. 55 1.3.6.8 Proteolipid protein (PLP)/DM20 ................................................................ 57 1.3.6.9 2’, 3’‐cyclic nucleotide 3’phosphodiesterase (CNP) .................................. 59 2 1.4 SC DEVELOPMENT AND DIFFERENTIATION ................................................................ 59 1.4.1 Origin and development of SCs ......................................................................... 59 1.4.2 SC differentiation and myelination ................................................................... 64 1.4.2.1 Cell cycle exit ............................................................................................. 64 1.4.2.2 Cell adhesion molecules ............................................................................ 66 1.4.2.3 Transcription factors ................................................................................. 68 1.5 REGULATION OF SC MYELINATION: POSITIVE AND NEGATIVE REGULATORS ............ 77 1.5.1 Positive regulators of differentiation ................................................................ 77 1.5.1.1 Axon‐Glia interactions: cell contact and secreted factors ......................... 78 1.5.1.2. Autocrine Growth factors ......................................................................... 80 1.5.1.3 Basal lamina assembly ............................................................................... 80 1.5.2 Negative regulators of differentiation .............................................................. 81 1.6 BASAL LAMINA: COLLAGEN AND LAMININ ................................................................. 82 1.6.1 Collagen ............................................................................................................. 82 1.6.1.2 Receptors: .................................................................................................. 84 1.6.2 Laminins ............................................................................................................. 85 1.6.2 Receptors ...................................................................................................... 87 1.7 INTEGRIN SIGNALING: Focal adhesion complex formation ........................................ 91 1.7.1 Protein Kinases .................................................................................................. 92 1.7.1.1 Focal adhesion kinase (FAK) ...................................................................... 92 1.7.1.2 Src‐Like Kinases (SLK) ................................................................................. 93 1.7.2 Phosphatases ..................................................................................................... 95 1.7.3 Rho GTPases ...................................................................................................... 96 1.8 SIGNALING PATHWAYS INVOLVED IN PERIPHERAL MYELINATION .......................... 100 1.8.1 Neuregulins/ErbB ............................................................................................ 100 1.8.2 Phosphoinositide 3‐kinase/Akt ....................................................................... 103 1.8.3 Mitogen‐activated protein kinases ................................................................. 106 1.8.3.1 Extracellular regulated kinase (ERK)‐1/2 ................................................. 107 1.8.3.2 Jun amino‐terminal kinases (JNK)............................................................ 108 1.8.3.3 p38 MAPK ................................................................................................ 109 1.8.3.4 Kinase targets of MAPKs: MAPK‐activated protein kinases (MK) ........... 111 1.9 DEMYELINATING DISEASES ....................................................................................... 114 3 1.9.1 Charcot‐Marie‐Tooth (CMT) disease ............................................................... 114 1.9.2 Guillain‐Barre‐Strohl (GBS) Syndrome ............................................................ 116 1.9.3 Leprosy ............................................................................................................ 118 CHAPTER 2: RATIONALE AND OBJECTIVES .................................................................... 120 2.1 RATIONALE ................................................................................................................ 121 2.2 HYPOTHESIS .............................................................................................................. 124 2.3 SPECIFIC OBJECTIVES ................................................................................................ 125 2.3.1 Specific Objective 1: Characterize the role of the SLKs in peripheral myelination125 2.3.2 Specific Objective 2: Determine whether p38 MAPK regulates transcription factors expression in SCs to ultimately modulate expression of myelin genes associated with SC terminal differentiation and peripheral myelination ................ 125 2.3.3 Specific Objective 3: Characterize the role of Rho GTPases in peripheral myelination involving activation of p38 MAPK ........................................................ 126 CHAPTER 3: MATERIALS AND METHODS .................................................................. 127 3.1 MATERIALS ................................................................................................................ 128 3.1.1 Cell culture reagents........................................................................................ 128 3.1.2 Inhibitors ......................................................................................................... 128 3.1.3 siRNA transfection reagents ............................................................................ 128 3.1.4 Adenoviruses