THE EFFECTS OF EXCITOTOXICITY AND MICROGLIAL ACTIVATION ON OLIGODENDROCYTE SURVIVAL DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Brandon Andrew Miller, B.A. ***** The Ohio State University 2006 Dissertation Committee: Approved by Professor Phillip G. Popovich, Co-Advisor ______________________ Professor Michael S. Beattie, Co-Advisor ______________________ Professor Jacqueline C. Bresnahan, Co-Advisor ______________________ Professor Richard H. Fertel Co-Advisors, Neuroscience Graduate Studies Program Professor Chandan K. Sen ABSTRACT The nervous system transmits information over long distances by action potentials carried by neurons. In both the central and peripheral nervous system, neurons are insulated by myelin which improves the conduction of action potentials. In the peripheral nervous system, myelin is produced by Schwann cells. Oligodendrocytes (OLs) are the myelinating cells of the central nervous system (CNS). Both mature OLs and oligodendrocyte progenitor cells (OPCs) are lost in CNS disease and injury. Two contributing factors to OL and OPC cell death are inflammation and excitotoxicity. These studies were designed to better understand how OLs and OPCs respond to excitotoxicity and inflammatory stimuli. The glutamate receptor agonist kainic acid (KA) was used to induce excitotoxic cell death in OLs from optic nerve. We found that the pro- inflammatory cytokine tumor necrosis factor-alpha increased the susceptibility of optic nerve OLs to KA. Subsequent experiments tested the response of cortical OLs at different developmental stages to KA and revealed that mature cortical OLs were also vulnerable to KA-induced excitotoxicity. In the final set of experiments, we used combined culture of OLs or OPCs and microglia (MG) to study the effects of activated MG on OPC and OL survival. We found that MG activated with lipopolysaccharide induced OPC cell death, but MG, regardless of ii activation state, protected mature OLs from apoptotic cell death. We also observed that MG cell death occurs as a consequence of activation. Taken together, these studies demonstrate that both excitotoxicity and inflammatory stimuli can induce OL and OPC death but that MG can also support the survival of OLs. iii Dedicated to Rhonda and Jeff Miller iv ACKNOWLEDGMENTS I would like to acknowledge all of those who made this work possible: Rochelle Diebert, Crystal Forrider, John Komon, Jeannine Crum, Tina Van Meter, and especially Amy Tovar made invaluable technical and intellectual contributions to all of these projects. Thank you for helping me get things done right! I would like to thank Dr. Michael Beattie and Dr. Jacqueline Bresnahan for their mentorship and support for the past six years. It has been a privilege to work with and learn from you. Soon to be Dr. John Gensel – You’ve been a great friend and colleague for the past six years. It’s been an honor and a privilege to share an office with you and I wouldn’t trade you for a window any day! You will be an incredible mentor for young scientists one day soon. Dr. Adam Ferguson – Thank you for all your help in experimental design, statistics, and the occasional coffee break. You are an awesome friend and mentor. It’s been great to live just down the street from you in SF! Dr. Yvette Nout – Thank you for your advice and example for a young clinician-scientist. I know you’ll laugh (though you shouldn’t!), but one day I hope to emulate your success. Take good care of LR. v Dr. Fang Sun – Thank you for your help with immunohistochemistry and all of our discussions about oligodendrocytes. I still think of your tenacity when I feel overwhelmed. I would like to thank the members of my dissertation committee - Dr. Phillip Popovich, Dr. Richard Fertel and Dr. Chandan Sen – for their mentorship, teaching and advice. Dr. Allan Yates, Dr. Joanne Lynn, Dr. Charles Hitckcock, Dr. Mirza Baig, Dr. Abhik Chaudhury, Dr. Louis Caragine, Dr. Michael Gong, Dr. David Feldman, Dr. Howard Werman, Dr. Sandra Kostyk, Dr. David Bahner, Dr. Shirley Stiver, Dr. Guy Rosenthal, and Dr. Geoff Manley - thank you for your mentorship as I’ve ventured outside the lab during my PhD. Cheryl Yoder, Ashley Bertran, and Andrea Brown – thank you for your help with administrative issues - especially the copier! Dr. Jeff Gidday – thank you introducing me to Neuroscience, and for your patience with a college student still learning so much. I hope we will have the opportunity to work together in the future. My good friends who aren’t listed above– Nadine, Dan, Ronnie, Bethany, BT, Gitis, Kristen, Sam, Kelly, Peter, Emma, TJ, Jason, Steve, Phillip, Michelle, Amy, The Mikes, Liz, Chad, Eric and Jen – your support has made this possible and your friendship continues to make life great! Most importantly, to my family: Rhonda and Jeff – thank you for dauntlessly supporting me all these years. Laura and Mike – thanks for so many vi fun times in Delaware and Philly. My grandparents and great aunt – thank you for the value you’ve placed on education and hard work. Jared – your hard work and courage will always be an inspiration to me. vii VITA 12/17/1977 . Born – Cleveland, OH 5/8/2000 . B.A. Biology with College Honors Washington University in St. Louis 2000 – 2002 . Medical School Preclinical Curriculum The Ohio State University 2002 – 2006 . Graduate Research Associate The Ohio State University 2006-present . NRSA Dual Degree Fellow National Institute of Neurological Disorders and Stroke, and The Ohio State University PUBLICATIONS 1. Miller BA, Baig M, Hayes J, Elton S. (2006) Injury Outcomes in Children Following Automobile, Motorcycle and All-Terrain Vehicle Accidents: an Institutional Review. The Journal of Neurosurgery: Pediatrics 105:182-186 2. Miller BA, Sun F, Christensen RN, Ferguson AR, Bresnahan JC, Beattie MS. (2005) A Sublethal Dose of TNF α Potentiates Kainate-Induced Excitotoxicity in Optic Nerve Oligodendrocytes. Neurochemical Research 30: 867-875 3. Miller BA, Perez RS, Shah AR, Gonzales ER, Park TS, Gidday JM. (2001) Cerebral Protection by Hypoxic Preconditioning in a Murine Model of Focal Ischemia-Reperfusion. NeuroReport 12:1663-1669 FIELDS OF STUDY Major Field: Neuroscience viii TABLE OF CONTENTS P a g e Abstract. .. ii Dedication. iv Acknowledgments . .. v Vita . viii List of Figures . xi Chapters: Chapter 1: Introduction . .1 Chapter 2: A sublethal dose of TNF α Increases Excitotoxic Cell Death in Optic Nerve Oligodendrocytes . 18 Introduction . 18 Materials and Methods . 23 Results . 27 Discussion . 33 Chapter 3: Characterization of the Response of Cortical Oligodendrocytes to Kainic Acid Excitotoxicity . 40 Introduction . 40 Materials and Methods . 44 Results . 52 Discussion . 62 Chapter 4: The Effects of LPS-Activated Microglia on Oligodendrocyte Progenitor Cells and Mature Oligodendrocytes. 73 Introduction . 73 ix Materials and Methods . 75 Results . 78 Discussion . 90 Chapter 5: Microglial Cell Death in Response to LPS-Activation . 95 Introduction . 95 Materials and Methods . 97 Results . 98 Discussion . 102 Chapter 6: Summary, Conclusions and Future Directions . 107 References . 114 x LIST OF FIGURES Figure Page 1.1 Schematic of OPC and OL morphology . 3 1.2 Pathways of OL cell death after CNS trauma. .. 15 2.1 Optic nerve OL immunocytochemistry . 28 2.2 Optic nerve OLs labeled for GluR4 . 29 2.3 Effect of short term KA exposure on optic nerve OL survival . 30 2.4 Effect of long term KA exposure on optic nerve OL survival . 32 2.5 Caspase activation in optic nerve OLs . 33 2.6 Effect of TNF α on KA excitotoxicity . 34 3.1 Cortical OL shakeoff procedure . 47 3.2 Comparison of live and dead OLs and OPCs . 51 3.3 Cortical OL response to KA . 54 3.4 Immunocytochemistry of OLs with and without TH . 55 3.5 Response of OLs with and without TH to KA . 56 3.6 OPC response to KA – cell counts . 57 3.7 OPC response to KA – LDH assay . 58 3.8 OL caspase labeling with caspase kit . 59 3.9 Caspase activation in response to time and KA . 60 3.10 OLs with and without MG . 62 3.11 Response of OLs with and without MG to KA . 63 xi 4.1 MG TNF α release in response to KA and LPS . 78 4.2 Immunocytochemistry of OPCs . 80 4.3 Immunocytochemistry of OPCs and OLs with MG . 81 4.4 OPC survival in response to MG and LPS . 82 4.5 OPCs labeled with BrdU . 83 4.6 OPC BrdU incorporation quantification . 84 4.7 OPCs labeled with caspase kit . 84 4.8 OPC apoptosis in response to MG and LPS . 85 4.9 Immunocytochemistry of OLs . .87 4.10 OL Survival in response to MG and LPS . 88 4.11 OL apoptosis in response to MG and LPS . 89 5.1 TNF α production by MG in different media . 99 5.2 MG cell death in the presence of OPCs and OLs . 100 5.3 MG cell death in the absence of OPCs and OLs . 101 5.4 LDH assay for MG cell death . 103 6.1 OL labeling with Carboxy-H2DCFDA . 109 6.2 Schematic of results in Chapters 4 and 5 . 113 xii CHAPTER 1 INTRODUCTION Oligodendrocytes in Health and Disease Myelination allows neuronal axons to quickly conduct action potentials over long distances. Oligodendrocytes (OLs) are the myelin-producing cells of the central nervous system (CNS). Each OL contributes to the myelination of about 15 CNS axons, allowing neurons to.