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Synaptic Mitochondria Regulate Hair Cell Synapse

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Synaptic Mitochondria Regulate Hair Cell Synapse SYNAPTIC MITOCHONDRIA REGULATE HAIR CELL SYNAPSE SIZE AND FUNCTION by Hiu-tung Candy Wong A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland January, 2020! Abstract Mechanosensitive hair cells of the inner ear and lateral line are specialized sensory cells that are required to evoke vital behaviors such as hearing, maintaining an upright posture and evading predators. To encode these sensory signals, hair cells use specialized ribbon synapses. Mitochondrial dysfunction has been implicated in hearing loss but the role of healthy mitochondria in hair cells or at ribbon synapses is unclear. I show that mitochondrial Ca2+ couples with presynaptic activity and plays distinct roles at hair cell synapses in mature and developing cells. I show that in mature hair cells, evoked-presynaptic-Ca2+ influx initiates mitochondrial-Ca2+ uptake; block of mitochondrial-Ca2+ uptake depresses presynaptic-Ca2+ activity and long-term block of mitochondrial-Ca2+ uptake can impact synapse integrity. I show that in developing hair cells, mitochondrial-Ca2+ uptake coincides with spontaneous presynaptic rises in Ca2+. Block of spontaneous presynaptic rises in Ca2+ or mitochondrial-Ca2+ uptake in developing hair cells enlarges presynaptic ribbon structure during synapse formation. Presynaptic ribbon size is composed primarily of the self-aggregating structural protein Ribeye. My work indicates that mitochondrial-Ca2+ may impact ribbon formation by modulating Ribeye-Ribeye protein interactions via the NAD(H) binding domain on Ribeye. Spontaneous mitochondrial- Ca2+ loading lowers cellular NAD+/NADH and ultimately downregulates ribbon formation. Furthermore, I found that direct application of NAD+ or NADH can directly increase or decrease ribbon formation respectively. Our results propose that mitochondrial-Ca2+ is an important component of presynaptic function and formation. ! ii Advisory committee: Dr. Katie S. Kindt (Thesis advisor) Dr. Paul A. Fuchs (Second reader) Dr. Marnie E. Halpern (Chair) Dr. Wei Li Dr. Chen-Ming Fan! ! iii Dedication I dedicate this thesis to my parents, Yuen Han and Tak Sing. A special thank you to Dr. Daniel Cummins, who shares his life with me, and provided plentiful encouragement and deoxygenated water to support this work. To every wonderful person who have influenced me and my work; know that I am grateful for your companionship and support. ! ! iv Acknowledgments I would like to extend a heartfelt thanks to past and current lab members for their suggestions, friendship, and influence. It cannot be said enough that Dr. Katie Kindt is everything I could hope to look for in a good mentor. Alisha Beirl is a wonderful friend and dependable source of amazing immunohistochemistry and dry humor. Former postbaccalaureate researcher Suna Li is a friend and an inspiration and provided helpful knowledge on zebrafish handling when I was learning the craft. Thank you to Dr. Doris Wu for her invaluable advice, especially in times of uncertainty. A special feeling of gratitude to my thesis committee, Drs. Paul Fuchs, Marnie Halpern, Wei Li, and Chen-Ming Fan for their support for both my academic endeavors and personal growth. Much thanks to the office of the Johns Hopkins Biology department, whose support is indispensable during my thesis work. My undergraduate research training from Dr. Yehoash Raphael and his laboratory staff, especially Dr. Don Swiderski and Lisa Beyer, shaped my interest which eventually cumulated in this thesis. ! ! v Table of Contents Abstract .......................................................................................................................................................... ii Dedication ................................................................................................................................................... iv Acknowledgments ....................................................................................................................................... v List of Tables .............................................................................................................................................. vi List of Figures ............................................................................................................................................. xi Abbreviations ............................................................................................................................................ xiii Chapter 1: Introduction ............................................................................................................................. 1 Overview .................................................................................................................................................. 1 Anatomy of the mammalian inner ear ................................................................................................. 4 Hair cell ribbon synapse formation .................................................................................................... 25 Ca2+ imaging using genetically-encoded Ca2+ indicators ................................................................. 28 Mitochondrial Ca2+ uptake in hair cell physiology and pathology ................................................. 33 Zebrafish posterior lateral line ............................................................................................................ 42 Chapter 2: Synaptic mitochondria are critical for hair-cell synapse function and integrity ........... 48 Abstract .................................................................................................................................................. 49 Introduction ........................................................................................................................................... 50 Results ..................................................................................................................................................... 52 Mitochondria are located near presynaptic ribbons ............................................................................. 52 ! vi 2+ Mitochondrial-Ca uptake at ribbons is MCU and CaV1.3 dependent ............................................ 55 Mitochondrial-Ca2+ uptake occurs in cells with presynaptic-Ca2+ influx .......................................... 62 Blocking mitochondrial-Ca2+ entry impairs presynaptic Ca2+ signals in mature hair cells ............. 65 2+ Evoked mitochondrial-Ca uptake is important for mature synapse integrity and cell health .... 68 2+ MCU and CaV1.3 channel activities regulate subcellular Ca homeostasis ..................................... 76 Discussion .............................................................................................................................................. 80 Role of evoked mitochondrial-Ca2+ uptake in mature hair cells ........................................................ 80 Role of mitochondrial-Ca2+ in hair cell death and synapse integrity ................................................. 82 Materials and Methods ......................................................................................................................... 83 Zebrafish husbandry and genetics .......................................................................................................... 83 Cloning and transgenic fish production ................................................................................................. 84 Pharmacological treatment of larvae for immunohistochemistry ...................................................... 86 In vivo imaging of evoked Ca2+ signals .................................................................................................... 86 Electron microscopy ................................................................................................................................. 87 Immunofluorescence staining and Airyscan imaging .......................................................................... 88 Quantification and Statistical Analysis ............................................................................................... 89 Analysis of Ca2+ signals, processing, and quantification ..................................................................... 89 Image processing and quantification of synapse morphology ........................................................... 90 Statistics ...................................................................................................................................................... 92 Acknowledgements ................................................................................................................................... 93 ! vii Chapter 3: Spontaneous mitochondrial-Ca2+ uptake modulates ribbon synapse formation ......... 94 Abstract .................................................................................................................................................. 95 Introduction ........................................................................................................................................... 96 Results ..................................................................................................................................................... 98 Blocking mitochondrial-Ca2+ entry does not impair presynaptic-Ca2+ signals in immature hair cells .............................................................................................................................................................
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