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University of Florida Thesis Or Dissertation Formatting Template THERAPEUTIC POTENTIALS OF TARGETING AMPK SIGNALING IN THE RETINA By LEI XU A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2018 © 2018 Lei Xu To my family ACKNOWLEDGMENTS I sincerely thank my mentor, Dr. John Ash. I met him back to 2009 (wow! It has been that long!), without any background knowledge about the eye and did my first PCR in his lab. Over the years, he gave me the greatest support that I could ever ask for; he was always standing on my back to hold on me and not give up on me. It was challenging to move and live in another country. English was a big problem (and still a problem). I remember the first time he talked to me about success; my face looked puzzled “what is that mean?” Then he tried to explain to me “success…success is a good word”. And another time he told me “I am running late to the lab since I have roofers working on my house,” and I delivered the message as “Dr. Ash has roosters in his house” to all my lab mates, and they were actually all convinced that he did have a rooster in his house. We had many good and fun times together, and I can picture that we talk about this 50 years later. I also had a hard time back to the first year of my graduate school with balancing all the exams, lab work, and my family. He studied together with me, practiced, gave me all the advice and helped me to stay positive, confident and be strong. The word “thank” is not enough to tell how much I appreciate all he has done for me. He trained me to be a scientist and better person throughout the years. He is the best mentor in the world and a family friend of lifelong. I also want to thank my supervisory committee members, Dr. Alfred Lewin (who has always asked me “where is your paper”), Dr. Christiaan Leeuwenburg, and Dr. Shannon Boye for taking the time to give me feedback and guidance. I would also acknowledge my lab members to make up this great Ash Family. They are Marcus Hooper, Clayton Santiago, Casey Keuthan, Emily Brown, and 4 previous lab members Ana Chucair-Elliott, Yumi Ueki, Gary Jiangang Wang, Annise Saddi, Jing Zhang, Huiming Xia, Caroline Abrahan, LaGina Shine, Xiang Ren, Haijun Liu. I would also acknowledge my undergraduate students Maria Abdo, Abraham Bell, Lena Phu Linda Qian and Eric Nayman who have been working with me and be masked observes of many of my experiments. I would like to thank my family, my parents-in-law who are the best in-laws in the world, my parents, my brother, sister-in-law, and friends for their constant love, support, and confidence in me. Foremost I would like to acknowledge my husband Bo Dong for his patients who drove me to the lab every weekend to inject metformin to the mice; for the understanding and continued supports throughout my graduate studies; for the inspiration of each day is better and happier during this nine years marriage and many more years to go. I would like to acknowledge my children Dorothy Dong and Timothy Dong who brighten my days and my life. They both love science, and they consider mom is the best scientist. I would like to thank Interdisciplinary Program (IDP) in Biomedical Sciences for offering me the wonderful opportunity and support for my graduate training. Special thanks go to Ms. Kris Minkoff for all her assistance throughout the years. In the end, I would like to mention all the mice that I have worked with who sacrificed themselves for science and allow me to explore and answer my scientific questions. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 9 LIST OF FIGURES ........................................................................................................ 10 LIST OF ABBREVIATIONS ........................................................................................... 12 ABSTRACT ................................................................................................................... 14 CHAPTER 1 RESEARCH BACKGROUND ................................................................................. 17 Visual System ......................................................................................................... 17 The Eye ............................................................................................................ 17 Visual Signal Transduction ............................................................................... 17 Photoreceptors and RPE .................................................................................. 18 Energy Metabolism in the Healthy Retina ............................................................... 19 Metabolic Dysregulation in the Diseased Retina ..................................................... 22 Retinitis Pigmentosa ......................................................................................... 22 AMD ................................................................................................................. 23 AMPK Signaling Cascade ....................................................................................... 25 AMPK as an Energy Sensor (83) ..................................................................... 26 Regulation of Mitochondrial Biogenesis by AMPK ............................................ 28 Regulation of mTOR Pathway by AMPK .......................................................... 29 Regulation of Inflammation Response by AMPK .............................................. 30 Activation of AMPK Signaling by Metformin ............................................................ 31 History of Metformin ......................................................................................... 31 Metformin Usages and its Side Effect .............................................................. 31 Mechanisms of Metformin’s Action ................................................................... 33 Metformin as a Tool to Target Neurodegeneration, Aging, and Cancer ........... 33 Concluding Remarks .............................................................................................. 34 2 METHODS AND MATERIALS ................................................................................ 36 Animals ................................................................................................................... 36 BALB/cj Mice .................................................................................................... 36 PDE6Brd10 Mice ................................................................................................ 36 Cre Loxp System to Induce Retinal Specific Knockout Mice ............................ 36 Intravitreal and Subcutaneous Injections ................................................................ 37 Sodium Iodate Induced RPE Damage .................................................................... 38 Light Damage ......................................................................................................... 39 Electroretinograms (ERGs) ..................................................................................... 39 Spectral Domain Optical Coherence Tomography (SD-OCT) ................................. 40 6 Morphometric Analysis ............................................................................................ 41 Western Blots ......................................................................................................... 42 Real-time PCR ........................................................................................................ 43 Immunohistochemistry ............................................................................................ 43 ATP and NADH Content ......................................................................................... 44 Metformin Concentration by LC-MS ........................................................................ 45 Complex I Activity ................................................................................................... 45 Metabolomic Analysis ............................................................................................. 46 Statistics ................................................................................................................. 46 3 STIMULATION OF AMPK PREVENTS DEGENERATION IN THE PHOTORECEPTORS AND RETINAL PIGMENTED EPITHELIAL ......................... 48 Introduction ............................................................................................................. 48 Results .................................................................................................................... 50 Metformin Protected Retinal Photoreceptors from Light Damage Induced Retinal Degeneration .................................................................................... 50 Metformin-induced Protection is Mediated by Local Signaling and is Acting through AMPK α2 Subunit ............................................................................ 55 A Minimum of 4 days of Daily Metformin Injection is Required to Induce Protection from Light Damage ....................................................................... 61 Metformin Induces Changes in Mitochondrial Gene Expression following Light Damage ...............................................................................................
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