EXPRESSION PATTERN AND FUNCTIONS OF DIHYDROPYRIMIDINASE LIKE-3 IN THE RODENT MICROGLIA JANANI MANIVANNAN NATIONAL UNIVERSITY OF SINGAPORE 2013 EXPRESSION PATTERN AND FUNCTIONS OF DIHYDROPYRIMIDINASE LIKE-3 IN THE RODENT MICROGLIA JANANI MANIVANNAN, M.Sc A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF ANATOMY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2013 DECLARATION I hereby declare that the thesis is my original work and it has been written by me in its entirety I have duly acknowledged all the sources of information which have been used in the thesis This thesis has also not been submitted for any degree in any university previously _ Janani Manivannan i ACKNOWLEDGEMENTS First, I would like to express my deepest appreciation to my supervisor Associate Professor S.Thameem Dheen, Department of Anatomy, National University of Singapore for his unstinted guidance and persistent support throughout my work He has been instrumental in both professional and scientific development of my research career I would like to extend my warm thanks to Professor Bay Boon Huat, Head, Department of Anatomy, for providing me an opportunity to pursue research at this Department and also for his expert advice and suggestions during my study I am greatly indebted to Emeritus Professor Ling Eng-Ang, former Head, and Associate Professor Samuel S.W Tay, Deputy Head, Department of Anatomy, National University of Singapore for their valuable advice and suggestions to my research during the candidature I would like to thank Mrs Ng Geok Lan, Mrs Yong Eng Siang and Ms Chan Yee Gek for their technical assistance, Mdm Ang Carolyne Lye Geck, Ms Teo Li Ching Violet and Mdm Diljit Kaur for their secretarial assistance I must thank my labmates, Mr Parakalan Rangarajan, Ms Nimmi Baby, Ms Sukanya Shyamasundar and Ms Shweta Jadhav for their help and valuable suggestions during lab meetings I had an opportunity to train Ms Lina Farhana (Honors student, NUS) for her final year project experiments and would like to thank her for some contribution to the preliminary analysis of this project I would ii also like to take this opportunity to thank Late Mr Dhayaparan Devaraj for his support and friendship I wish to thank the Yong Loo Lin School of Medicine, National University of Singapore for the financial support by means of Research Scholarship Research grant support from National Medical Research Council (NMRC/EDG/1039/2011; R-181-000-139-275) to A/P Dheen to carry out this present study, is gratefully acknowledged Lastly, and most importantly, I thank my family (my parents, my brother and my husband) for their love, care and support iii Dedicated to my beloved family iv TABLE OF CONTENTS DECLARATION i ACKNOWLEDGEMENTS ii TABLE OF CONTENTS v PUBLICATIONS xiii SUMMARY xv LIST OF TABLES xviii LIST OF ILLUSTRATION/TEXT FIGURES xix LIST OF SYMBOLS/ABBREVIATION xx Chapter 1: Introduction 1.1 Central nervous system and its cell types 1.2 Microglia 1.3 Morphology of microglia 1.3.1 Amoeboid microglia 1.3.2 Ramified microglia 1.3.3 Activated microglia 1.4 Functions of microglia 1.4.1 Chemotaxis and migration of microglia 1.4.2 Phagocytosis 1.4.3 Proliferation v 1.4.4 Release of cytokines and reactive oxygen intermediates 1.5 Activation of microglia 11 1.5.1 Lipopolysaccharide (LPS) 11 1.5.2 Beta –Amyloid (Aβ) 12 1.6 Signaling pathways involved in microglial activation 12 1.6.1 Nuclear factor-κB pathway (NF-κB) 12 1.6.2 Mitogen-activated protein kinase pathways (MAPKs) 13 1.6.3 Rho family of guanosine triphosphatases GTPases (Rho GTPases) 13 1.7 Cytoskeleton organization in microglia 14 1.8 Microglial activation in neuropathologies 16 1.8.1 Microglial activation in Alzheimer’s disease 16 1.8.2 Microglial activation in Parkinson’s disease 17 1.8.3 Microglial activation in traumatic brain injury 17 1.9 Current approaches for controlling microglia activation 18 1.10 BV-2 microglial cells for in vitro experimental study 18 1.11 Global gene expression profiling of microglia 19 1.12 Collapsin Response Mediator Proteins (CRMPs) 20 1.12.1 Dihydropyrimidinase like-3 (Dpysl3) or Collapsin response mediator protein-4 (CRMP4) - Gene Ontology predictions 21 1.13 Expression of Dpysl3 in different cell types 21 vi 1.13.1 Dpysl3 in the developing nervous system 21 1.13.2 Dpysl3 in adult nervous system 22 1.13.3 Dpysl3 in the peripheral nervous system 22 1.14 Function of CRMPs 22 1.14.1 CRMPs in neuronal development 22 1.14.2 CRMPs in pathological conditions 23 1.15 Signaling pathways involving Dpysl3 24 1.15.1 F-actin cytoskeletal bundling 24 1.15.2 Rho GTPase Regulators 25 1.16 Role of Dpysl3 in nerve regeneration 26 1.16 Aim of the study 27 1.16.1 To investigate the expression pattern and function of Dpysl3 in the normal or resting and activated microglia 27 1.16.2 To examine the role of Dpysl3 in microglial migration, phagocytosis and proliferation 28 1.16.3 To examine the role of Dpysl3 in cytoskeleton organization of activated microglia 28 1.16.4 To investigate the role of Dpysl3 in Rho GTPases cytoskeletal pathway 29 Chapter 2: Materials and Methods 30 2.1 Animals 31 vii 2.1.1 Injection of LPS 31 2.2 Perfusion 32 2.2.1 Materials 32 2.2.2 Procedure 32 2.2.3 Preparation of frozen sections 33 2.3 Cell culture 33 2.3.1 Primary microglial culture 33 2.3.2 BV-2 microglial cell culture 35 2.3.3 Treatment of BV-2 cells 36 2.4 siRNA mediated gene knockdown 37 2.4.1 Principle 37 2.4.2 Materials 37 2.4.3 Procedure 38 2.5 RNA isolation and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) 39 2.5.1 Principle 39 2.5.2 Materials 40 2.5.3 RNA extraction 41 2.5.4 cDNA Synthesis 42 2.5.5 Quantitative real-time -PCR 43 viii Figures and Figure legends Fig 16.Knockdown of Dpysl3 inhibits the phagocytosis by activated BV-2 microglia A-D) Confocal images of BV-2 microglial cells transfected with Dpysl3siRNA or negative control and treated with or without LPS are shown Only a small percentage of unstimulated BV-2 microglial cells transfected either with scrambled or Dpysl3 siRNA, ingested with latex beads E) The quantitative analysis shows the number of phagocytic cells laden with latex beads increased significantly after 1h LPS stimulation, while knockdown of Dpysl3 appears to inhibit this increase significantly Data are presented as mean ± SD (n=4), **p