Neurite outgrowth inhibitors in axoglial communication at the node of ranvier

NEURITE OUTGROWTH INHIBITORS IN AXON-GLIAL COMMUNICATION AT THE NODE OF RANVIER DU-YU NIE (M.D.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF ANATOMY NATIONAL UNIVERSITY OF SINGAPORE 2005 ACKNOWLEDGEMENTS ACKNOWLEDGEMENTS I want to particularly thank my two supervisors, Dr. XIAO Zhi-Cheng and Dr. NG Yee-Kong, who have given time and energy to helping me in my research projects. They have taught me a lot not only how to design and carry out experiments according to scientific criteria but also the skills for writing and presentation. Without them, this dissertation would never be completed and my dream would not come true. I am also grateful to Prof. LING Eng-Ang and Dr. AW Swee-Eng, the heads of Department of Anatomy, National University of Singapore and Department of Clinical Research, Singapore General Hospital, respectively. They have created excellent research environments and provided superb facilities to my work. I would like to thank all those with whom I have worked together as well as those who have helped me in preparation of manuscripts and reading the thesis: Dr. Malcolm PATERSON, Dr. TANG Bor-Luen, Dr. Sohail AHMED, Dr. Gerald UDOLPH, Dr. Narender K. DHINGRA, Dr. YE Hai-Hong, Mrs CHAN Yee-Gek (EM unit) and Dr. ZHOU Zhi-Hong, Ms MA Quan-Hong. In addition, I want to thank my co-worker Mr Timothy CHIA, for helping in some of the experiments and other laboratory administration. All your help has been much appreciated. II ACKNOWLEDGEMENTS I am truly grateful to my parents for their enduring trust and support; and I would like to dedicate this dissertation along with my love to my deceased mother. I would also like to thank my dear wife, Ms XU Gang, for her endless love and timeless support. She is my friend, my confidante, my wife, and my partner. I also want to present the appreciation to my parents-in-law, my brother and sisters for their encouragement and supportive efforts. III PUBLICATIONS Publications International Journals: Du-Yu Nie, Zhi-Hong Zhou, Beng-Ti Ang, Felicia Y.H.Teng, Gang Xu, Tao Xiang, Chao-yang Wang, Li Zeng, Yasuo Takeda, Tian-Le Xu, Yee-Kong Ng, Catherine Faivre-Sarrailh, Brian Popko, Eng-Ang Ling, Melitta Schachner, Kazutada Watanabe, Catherine J.Pallen, Bor Luen Tang, and Zhi-Cheng Xiao. (2003). Nogo-A at CNS paranodes is a ligand of Caspr: possible regulation of K+ channel localization. EMBO J. 22: 5666-5678. Du-Yu Nie, Quan-Hong Ma, Janice W. S. Law, Narender K. Dhingra, Chern-Pang Chia, Gang Xu, Yasushi Shimoda, Qing-Wen Chen, Neng Gong, Qi-Dong Hu, Pierce Chow, Alan Y. W. Lee, Yee-Kong Ng, Kazutada Watanabe, Tian-Le, Xu, Amyn Habib, Melitta Schachner, and Zhi-Cheng Xiao. Oligodendrocytes regulate formation of nodes of Ranvier via the recognition molecule OMgp. (submitted). Du-Yu Nie, Qi-dong Hu, Quan-Hong Ma, and Zhi-Cheng Xiao. Neurite outgrowth inhibitors at nodes of Ranvier. (Review) (ready for submission). Gang Xu*, Du-Yu Nie*, Ju-Tao Chen, Chao-Yang Wang, Feng-Gang Yu, Li Sun, Xue-Gang Luo, Sohail Ahmed, Samuel David, Zhi-Cheng Xiao. (2004) Recombinant DNA vaccine encoding multiple domains related to inhibition of neurite outgrowth: A potential strategy for axonal regeneration. J. Neurochem. 91:1018-1023. (*equal contribution authors) Gang Xu, Du-Yu Nie, Wen-Zu Wang, Pei-Hua Zhang, Jie Shen, Beng-Ti Ang, Guo-Hua Liu, Xue-Gang Luo, Nan-Liang Chen, and Zhi-Cheng Xiao. (2004). Optic nerve regeneration in polyglycolic acid-chitosan conduits coated with recombinant L1-Fc. NeuroReport. 15(14):2167-2172. Xiao-Ying Cui, Qi-Dong Hu, Meriem Tekaya, Yasushi Shimoda, Beng-Ti Ang, Du-Yu Nie, Li Sun, Wei-Ping Hu, Meliha Karsak, Tanya Duka, Yasuo Takeda, Lian-Yun Ou, Gavin S. Dawe, Feng-Gang Yu, Sohail Ahmed, Lian-Hong Jin, Melitta Schachner, Kazutada Watanabe, Yvan Arsenijevic, and Zhi-Cheng Xiao. (2004). NB-3/Notch1 pathway via Deltex1 promotes neural progenitor cell differentiation into oligodendrocytes. J Biol Chem. 279(24):25858-65. IV PUBLICATIONS Conference Abstracts: D.Y. Nie, B.T. Ang, F.Y.H. Teng, T. Xiang, G. Xu, C.J. Pallen, B.L. Tang, and Z.C. Xiao. Nogo-A at CNS paranodes is a ligand of Caspr/Paranodin: A molecular interaction that may regulate K+ channel localization. Australian Neuroscience Society Inc 24th Annual meeting. 2004, Melbourne, Australia. Z.C. Xiao, G. Xu, D.Y. Nie, S. Ahmed. A DNA vaccine enconding inhibitory domains of MAG, Tenascin-R and Nogo-A promotes axonal regeneration after spinal cord injury. Scientific Committee of the II International Congress on Neuroregeneration. 2004, Brazil. G. Xu, D.Y. Nie, J. Shen, W.Z. Wang, P.H. Zhang, N.L. Chen, G.H. Liu and Z.C. Xiao. A polymer filaments conduit coated with L1 promotes guided optic nerve regeneration. Australian Neuroscience Society Inc 24th Annual meeting. 2004, Melbourne, Australia. V TABLE OF CONTENTS TABLE OF CONTENTS TITLE PAGE………………………………………………………………… I ACKNOWLEDGEMENT………………………………………………… . II PUBLICATIONS………………………………………………… .……… . IV TABLE OF CONTENTS…………………………………………… . VI ABBREVIATIONS……………………………………… .………………… XII SUMMARY……………………………………… ……… .………………… XV LIST OF TABLES…………………………………………… .…………… . XVIII LIST OF FIGURES…………………………………………… …………… XIX CHAPTER INTRODUCTION…………………………………………… 1. Neuronal polarity and axonal initial segment…………………………… . 2. Myelination and axonal polarity………………………… .…………… 2.1 Myelinating glia initially recognizes internodes……………………… 2.2 Potassium channels aggregate within juxtaparanodes………………… 2.3 The paranode plays a central role in domain organization……… . 12 2.3.1 Cis-complex of Caspr/paranodin and contactin/F3…………… 13 2.3.2 Neurofascin 155 (Nf155)……………………………………… 15 2.4 The formation of node of Ranvier (NOR)………………… ……… 16 2.5 Sodium (Nav) channel at NORs and the action potential…………… . 19 2.5.1 Clustering of Nav channels………… … . 19 VI TABLE OF CONTENTS 3. 2.5.2 Developmental transition of Nav channel isoforms at NOR…… 21 2.5.3 Cis-interactions with axonal cell adhesion molecules (CAMs) . 23 2.5.4 Trans-interactions with extra-axonal molecules……………… . 24 Signaling pathways underlying myelination and axonal domain formation………………………………………………………………… . 4. 25 The inhibitory hypothesis and regeneration failure in the central nervous system (CNS)……………………………………………………………… 31 4.1 Myelin components regulate axonal sprouting……………………… 32 4.2 Tenascins (TNs) produce repulsive substrates.………………………… 37 4.3 Chondroitin sulphate proteoglycans (CSPGs) arrest the advance of 5. growth cones………………………………………………………… 39 4.4 NgR/p75 signaling complex………………………………………… . 40 Roles of neurite outgrowth inhibitors (NOIs) at the NOR…………… 43 5.1 TN-R and TN-C modulate Nav channels’ functions………… 43 5.2 Most of CSPGs are enriched at NORs…………………………………. 44 5.3 Myelin-associted glycoprotein (MAG) is prone to clustering in myelinated axons…………………………………… ……………… 45 Research aims and objectives…… .…………………………………… . 46 CHAPTER MATERIALS AND METHODS…………… .…………… . 50 Animals…………………………………………………………………… 51 2. Antibodies……………………………………………………………… 51 6. VII TABLE OF CONTENTS 3. Peptides and recombinant proteins……………………………….…………. 55 4. Experimental autoimmune encephalomyelitis (EAE) Model…….…………. 58 5. Oligodendrocyte myelin glycoprotein (OMgp) antisense transgenic (tg) mice…………………………………………………………………………. 59 6. Other genetically modified mouse models………………………………… . 61 7. Cell culture and transfection……………………………………………… 61 8. Retinal ganglion cells (RGCs) purification and Nav1.2 clustering study 63 9. Western blot analysis………………………………… 65 10. Fluoresecent immunohistochemistry (IF) studies…………… ………… 66 11. Conventional and immuno-electron microscopy……… .……….……… 67 12. Immunoprecipitation (IP) assay……………………… ………… ………. 68 13. Glutathione S-transferase (GST) pull-down assays…………… …………. 69 14. Cell adhesion/repulsion assay……………… …………………… …… 69 15. Phosphatidylinositol-specific phospholipase C (PI-PLC) treatment of cells. 71 16. In vivo conduction velocity recording…… ……………………………… 71 17. Morphometric quantitation and statistics……… .… ……… .… . 72 CHAPTER RESULTS……………………………………………………… 74 1. Nogo-A –Caspr interaction is involved in the paranodal axon-glial junction…………………………………………………………………… 75 VIII TABLE OF CONTENTS 1.1 NgR is uniformly distributed along the myelinated axons…….…… 75 1.2 Nogo-A has a confined distribution in the CNS paranodes……… 77 1.3 Paranodal Nogo-A is predominantly derived from oligodendroglia . 81 1.4 Nogo-A interacts with the paranodal Caspr/F3 complex….… …… . 85 1.5 The extracellular Nogo-66 trans-interacts directly with Caspr………. 86 1.6 Nogo-A and Caspr share a similar temporo-spatial relation with Kv1.1 along myelinated axons during development….… ………… 89 1.7 Nogo-A/Caspr complex interacts with Kv1 channels….…… .… . 94 1.8 Nogo-66 interacts indirectly with Kv1 channels via Caspr………… 95 1.9 Nogo-A/Caspr may assist in regulating Kv1.1 location……………… 95 2. Oligodendrocytes, via OMgp rather than TN-R, regulate the formation of NORs………………………….………… 99 2.1 OMgp deposits in the NORs of both the CNS and PNS………….… 99 2.2 Nodal OMgp is derived from oligodendrocytic lineages in the CNS . 104 2.3 OMgp could exist in secreted or soluble form……… .…… .……… 106 2.4 Nodal OMgp is preferably related to large axons in the CNS .… …. 2.5 OMgp clustering at NORs correlates with nodal maturation… …… 108 2.6 OMgp co-accumulates with TN-R and NG2 in NORs of the CNS… . 109 2.7 OMgp associates with extracellular matrix (ECM) and neuronal 107 proteins ……………………………………………… . 110 IX TABLE OF CONTENTS 2.8 OMgp expression is down-regulated in spinal cord of the antisense transgenic (tg) mice………………………….…………… ………… 112 2.9 Large spinal axons in the OMgp tg mice are hypo-myelinated……… 113 2.10 Sciatic nerves are hyper-myelinated in response to OMgp down-expression……………………………………………………… 117 2.11 Nodal gap is narrowed in large axons of the OMgp tg mice ……… . 120 2.12 Both nodal distance and OMgp expression are normal in the TN-R -/mice……………………… . 122 2.13 Lateral glial loops are disorganized in NORs of the OMgp tg mice…. 122 2.14 Normal organization of NORs in sciatic nerves of the OMgp tg mice………….………………………………………………………. 127 2.15 Nodal disorganization is not observed in the TN-R -/- mice…… … 129 2.16 Conduction velocity in spinal cord of the OMgp tg mice is decreased. 131 2.17 Distinct roles of OMgp and TN-R in the NORs……… .………… . 132 3. 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(1996) The transcription factors SCIP and Krox-20 mark distinct stages and cell fates in Schwann cell differentiation. Mol Cell Neurosci. 8:129-45. 204 APPENDICES APPENDICES 205 APPENDICES Appendix 1: Solutions and Buffers Regeneration Buffer: M NaCl; 0.1 M borate acid pH8.5. Elution buffer: 7.5mM glutathione (MW: 307.3); 50mM Tris/HCl pH8.0. Denaturation buffer: 100 mM Tris/HCl pH8.0; M Urea; mM EDTA; 300 mM DTE (Dithioerythritol). Renaturation Buffer: 0.1 M Tris/HCl pH8.0; 200 mM L-arginine; 10 mM Cystamine; mM EDTA (ethylenediaminetetraacetic acid). Cell lysis buffer: 20 mM Tris-HCl pH7.5; mM EGTA; 25 mM β-glycerophosphate; mM dithiothreitol (DTT); mM sodium orthovanadate; mM phenylmethylsulfonyl fluoride (PMSF); 206 APPENDICES 1% protease inhibitors and 0.1% NP-40. Tissue lysis buffer-1 50 mM Tris-HCl pH7.5; mM EDTA; 1% Triton X-100; proteases inhibitors. Tissue homogenizing buffer 320 mM sucrose; 10 mM Tris-HCl pH 7.4; mM NaHCO3 pH 7.4; mM MgCl2. Tissue lysis buffer-2 10 mM Tris-HCl pH9; 150 mM NaCl; 0.5% Triton X-100; 1% sodium deoxycholate (DOC); 0.5% SDS; mM EDTA; 1% protease inhibitor cocktail. Solutions used for western blotting: 1x TBS: (1 Liter) Tris base 2.42 g; NaCl 0.8 g; add ddH20 up to liter; adjusted to pH7.6 with HCl. 1x TBST: (1 Liter) 1x TBS liter; 207 APPENDICES 0.1% Tween 20. Ringer’s Solution ddH2O 10 liter; NaCl KCl CaCl2 85 g; 2.5 g; 3g; NaHCO3 2g. Binding Buffer (for GST pull-down assay) NaCl 250 mM; HEPES pH7.9 50 mM; EDTA 0.5 mM; NP-40 0.1%; DTE mM. 0.1 M phosphate buffer (PB) NaH2PO4. H2O 2.76 g; Na2HPO4. 2H2O 14.24 g (or Na2HPO4 11.38 g); Add ddH2O up to liter. Or prepare stock solution: A NaH2PO4.2H2O B Na2HPO4. 2H2O 78 g/L; 89 g/L. To make 0.1 M PB, pH7.4 (100ml): A 3.8 ml; B 16.2 ml; ddH2O 80 ml. 0.5 M cacodylate buffer Sodium Cacodylate (CH3)2AsOONa.H2O 107.015 g dissolved in ddH2O (up to liter), filtered. 208 [...]... axons, the regularly interrupted regions that remain uncovered by myelin sheathes, are named the node of Ranviers (NORs) From node to node, rapid “saltatory” conduction is accomplished Next to NORs, a myelinated axon is further organized into several other continuous domains which are easily differentiated by using electron microscopy (EM) They are the internode, the juxtaparanode and the paranode (Fig... through the Notch pathway (Hu et al., 2003) In myelinated axons, a particular role of contactin/F3 is to demarcate different subcellular domains Association with contactin/F3 potentiates Caspr (contactin-associated protein) delivery to the paranodes (Faivre-Sarrailh et al., 2000) Interestingly, the portion of Contactin/F3 that is not associated with Caspr is expressed at the NORs rather than at paranodes... (Fig 1) Of particular note is that the diameter of a myelinated axon is not uniform Instead, nodes and paranodes are slightly reduced in the diameter compared to those of juxtaparanodes and internodes along the same fiber (Salzer, 2003) In evolutionary light, all these features are of great advantages Firstly, the myelin layers, the large internodal distance, and the constricted diameters in the node. .. generated in excited neurons propagate much more rapidly along the shaft of myelinated axons than in unmyelinated axons The term myelination describes myelin wrapping around axons Oligodendrocytes in the CNS and Schwann cells in the PNS are myelin producing glia Myelin lamillae function as an insulator to confer high resistance and low capacitance to the axon membrane Along the entire length of myelinated... likely, the development of longitudinal axonal domain organization and circumferential myelin polarity are mutually dependent Myelination occurring in the CNS differs from that in the PNS in several ways A single oligodendrocyte ensheathes multiple axons simultaneously while in the PNS each Schwann cell myelinates only one axon so that they have an approximately 1:1 relationship Secondly, the outside of. .. Schmidt-Lanterman incisures (Cajal, 1928) It appears most likely that these cytoplasmic channels provide conduits communicating the inner and outer cytoplasmic compartments At the very beginning of myelinogenesis, the internode is recognized as the initiating site for oligodendrocytes or Schwann cells to lay down their myelin membrane They subsequently spread longitudinally away from the beginning sites towards the. .. whether OMgp involves myelination and nodal formation/maintenance, an antisense OMgp transgenic (tg) mouse was generated to selectively down-regulate OMgp’s expression in myelin-producing glial cells Unexpectedly, analyses showed that in these mice, myelination was inversely affected in the CNS and PNS: hypo-myelination in the spinal cord but hyper-myelination in the sciatic nerve Nevertheless, transverse... regulation of nerve conduction velocity in myelinated axons may involve at least four parameters: axon diameter, internodal distance, alteration in node size and architecture, and potentially, ion channel expression (Salzer, 2003) Figure 1 A schematic drawing depicts the myelinated axon and related axon-glial interactions The node of Ranvier (N) is the region uncovered by myelin sheaths (Ms) In the. .. internode refers to the segment extending from node to node, which is covered by compact myelin sheath Therefore, the internode constitutes the longest portion of the axon which is roughly 100 times the axon diameter (Hess and Young, 1952) One structural feature of internodes, much apparent in the PNS, is that at certain sites, the myelin laminae is not so consolidated but remains as funnel-like enlargements... domains (Arroyo et al., 1999) In this view, not only do myelin sheaths function to insulate and nourish the axon, but also differentiate the axonal membrane into distinct cytoplasmic domains in terms of structural compartmentation and protein expression Establishment of subcellular domains in myelinated axons, herein referred to as axonal polarization, depends heavily upon at least two distinct types of . channels 1 the leucine-rich repeats and Ig domain-containing, Nogo receptor-interacting protein myelin-associated glycoprotein mitogen-activated protein kinase microtubule-associated protein 2. developmental studies further demonstrated that the expression and clustering of OMgp correlated with myelin maturation and nodal formation at late phase. To examine role of OMgp in clustering Nav channels,. in myelin-producing glial cells. Unexpectedly, analyses showed that in these mice, myelination was inversely affected in the CNS and PNS: hypo-myelination in the spinal cord but hyper-myelination