Role of the Cytokine Macrophage Migration Inhibitory Factor (MIF) in inner ear neuronal and sensory cell development by Fumi Ebisu A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Neuroscience) in the University of Michigan 2010 Doctoral Committee: Professor Kate F. Barald, Chair Professor Richard A. Altschuler Professor Peter F. Hitchcock Assistant Professor R. Keith Duncan Research Professor Emerita Margaret I. Lomax Dedication To my family, for your truly unconditional love and support. ii Acknowledgments I have many people to thank for contributing to my dissertation in various ways. First and foremost, I would like to thank my mentor, Kate Barald. It has been such an honor to be a “scientific child” of hers. She has given me an opportunity to learn the theory behind the work as well as scientific experimental approaches and techniques that I will need to be an independent scientist. The joy and enthusiasm she has for her research and teaching were always motivational for me. There is no doubt that I would not be at this position without her guidance, understanding and patience as well as financial support. Her devotion to each of us was far beyond just that of a professor. I always remember that she and her husband, Doug, picked me up at 4 o’clock in the morning on the day of my surgery and stayed with me to comfort me until the doctors brought me to the surgery room. For this and so many other things you have done for me, Kate, I thank you. My sincere thanks go to all the members of Barald lab, both past and present, for their friendship and assistance. Special thanks to my “taking walk” buddies, Yu-chi Shen, Poorna Ramamurthy and Therese Roth, who listened to my occasional worries and frustrations and finally geared me back in the right iii direction. Thank you so much to Steffi Linn for her willingness to proofread and listen to my practice talks. Her comments were always valuable and appreciated. Thank you so much to Andy Chervenak and Matt Wyatt for their help in many ways when I needed it most. Thanks so much to Dov Lerman-Sinkoff for his unstinting and always available technical help. Thanks much to John Hennessy for working with me helping me with the western blotting assays for these last several months and for sharing with me his interests in science. They all are wonderful individuals not only for working with but also being with. I hope that we will always remain in contact. I would like to thank all the members of my dissertation committee, Rick Altschuler, Keith Duncan, Peter Hitchcock and Margaret Lomax. Rick, thank you very much for your insightful comments and suggestions. Your smiling face made me feel that I was always welcome to bring my questions to you even without an appointment, and you always had the answers for me. Keith, first of all, thank you so much for becoming part of my committee last year. Your questions and suggestions during the meetings were truly valuable and helped me focus what I needed to do next. I would also like to thank you for your time and patience while my training in the basilar papilla excision technique. Peter, I would like to express my sincere thanks for your continuous encouragement and support not only as a member of my committee but also as the former director of the Neuroscience (NS) program. Your tough questions and feedback during the meetings were just amazing and helped me realize what I needed to understand to be a “scientist”. But I am ready to take that journey. Margaret, thank you so iv much for your helpful comments and suggestions. I am also thankful for your excellent example as a successful female biologist and professor. I would like to special thank my additional committee member, Beth Moore, who spent a great deal of her time teaching me the basics of Immunology. I would also like to thank my previous academic advisor and committee member, Rudy Richardson, for his time and support during the last four years. I would like to express my deep thanks to all the people in the NS program. Special thanks go to the director of the program, Steve Maren, and the associate director, Jill Becker, for their guidance and support. Thank you so much for Valerie Smith and Rachel Flaten for your unending assistance and help whenever I needed it. Thank you very much to all NS students, especially the fourth and the fifth year students who I shared a lot of my time with, for their continuous support and friendship. Thank you very much to my past mentor at Eastern Michigan University, Steve Pernecky, who accepted me as one of his students and taught me how enjoyable and challenging scientific research could be. Thank you so much to Su-Hua Sha and Fu-Quan Chen for their time and efforts to train me in the organ of Corti excision process, and more importantly for their friendship. Thank you to Mary Caponite Hurley at the Michigan Proteome Consortium for performing proteomic analyses for our studies and helping us interpret them. Thank you so much to the stuff of the CDB department, Karen Lang, Karen v Meeks, Lori Longeway, Kristen Hug and Ryan Schell, for their assistance. Thank you to all the people at the MIL and Morph Cores in CDB, particularly Chris Edwards. Thanks so much to all my friends for their support and friendship. Very special thanks go to Beth Valka, who was formerly my Bible teacher and is now my best friend, for her unending support, patience and encouragement. More importantly, thank you so much for just being with me when I needed someone most to listen to my concerns. Another special thanks to Naoko Murakami, who cooked Japanese food for me when I was sick or too busy to cook. Very many thanks go to Tomiyo Wemert, Noriko Yi, Satoko Motohara, Ayako Hayami, Christie Kim, Becky (Heekyong) Bae, Harsha jayatillake and Hemali Amunugama for their companionship. Finally and most heartfelt, I thank my family: my parents, Yukifumi Ebisu and Harumi Ebisu, for their unconditional love and endless support and encouragement to pursue my interests, and my brother, Kazuyuki Ebisu, for being close to our parents during my time in the U.S. and for believing in me vi Table of contents Dedication……………………………………………………………………….……….ii Acknowledgements……………………………………………………………………..iii List of Figures………………………………………...………………………………….x List of Tables…………………………………………………………………………...xii List of Abbreviations…………………………………………………………………..xiii Abstract………………………………………………………………………….…….xviii Chapter I. Introduction ODF is not one of the previously identified neural growth factors……..2 The neuroimmunoaxis……………………………………………………...5 Physiological basis of hearing and balance in the vertebrate ear……..6 Mammalian inner ear development……………………..……………….10 Organogenesis…………………………………………………………10 Neurogenesis…………………………………………………………..11 Spiral ganglion neurons……….…………………………………………..12 Sensorineural hearing loss (SNHL)…………………………………...…15 How hair cell loss leads secondarily to the loss of sensory neurons in the inner ear. Why this causes deafness……………………………….16 vii Why sensory neurons last longer than hair dells in the inner ear?......18 How a cochlear implant works……………………………………………20 Trophic factors on which each of the stages of neurons depends…...22 Otocyst derived factor(s) (ODF) enhance(s) directional neurite outgrowth and neuronal survival of the early stage Statoacoustic ganglion (SAG) neurons…………………………………………..………28 Proteomic studies demonstrate that the major components of ODF are immune system cytokines………………………………………………...32 Development of immortalized mouse inner ear cell lines to characterize ODF…………………………………………………………..…….............32 Macrophage migration inhibitory factor (MIF), an inflammatory cytokine, plays the role of a "neurotrophin" in inner ear development…………35 Enzymatic activity of MIF/MIF as a therapeutic target ……...........35 The pathogenesis and progression of acute inflammation in the body……………………………………………………………………..40 The pathogenesis and progression of chronic inflammatory diseases (Autoimmune diseases)……………………………………42 Inflammation and cancer………….…………………………………..47 Role of MIF in development……..……………………………………51 Role of MIF in neuronal regeneration……………………………..…56 Mechanisms of MIF……………………………………………………..…62 References…………………………………………………………………67 II. The cytokine, macrophage migration inhibitory factor (MIF) acts as a neurotrophin for neurons in the developing inner ear………………….85 viii Abstract……………………………………………………………………..85 Introduction…………………………………………………………….…...86 Materials and experimental methods……………………………………88 Results…………………………………………………………...………..102 Discussion…………………………………………….………………….128 References……………………………………………………………..…136 III. Conclusion and future work…………………………………………………143 References………………………………………………………………..159 Appendix………………………………………………………………………………163 Abstract……………………………………………………………………163 Introduction…………………………………………………………….….164 Materials and experimental methods…………………………………..168 Results…………………………………………………………...………..175 Discussion…………………………………………….…………………..183 References……………………………………………………………..…188 ix List of Figures Chapter I Figure 1.1: Schematic representations of the human ear ..................................... 9 Figure 1.2: Schematic view of the cochlea and spiral ganglion neurons ............ 14 Figure 1.3: Schematic view of a cochlear implant system………………………...21 Figure 1.4: Three-dimensional ribbon diagram of human MIF ............................ 38 Figure 1.5: MIF mRNA expression in the embryonic mouse and Xenopus ........ 54 Figure 1.6: Zebrafish mif expression and role in inner ear ................................