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AMP Deaminase 1 Transcriptional Regulation and Knockdown Mouse Skeletal Muscle Function

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AMP Deaminase 1 Transcriptional Regulation and Knockdown Mouse Skeletal Muscle Function AMP Deaminase 1 Transcriptional Regulation and Knockdown Mouse Skeletal Muscle Function by Travis Brendan Kinder B.A. in Biochemistry, May 2011, McDaniel College A Dissertation submitted to The Faculty of The Columbian College of Arts and Sciences of The George Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy August 31, 2017 Dissertation directed by Kanneboyina Nagaraju Founding Chair and Professor of Pharmaceutical Sciences, and Adjunct Professor of Integrative Systems Biology The Columbian College of Arts and Sciences of The George Washington University certifies that Travis Brendan Kinder has passed the Final Examination for the degree of Doctor of Philosophy as of April 24, 2017. This is the final and approved form of the dissertation AMP Deaminase 1 Transcriptional Regulation and Knockdown Mouse Skeletal Muscle Function Travis Brendan Kinder Dissertation Research Committee: Kanneboyina Nagaraju, Founding Chair and Professor of Pharmaceutical Sciences, and Adjunct Professor of Integrative Systems Biology David Leitenberg, Associate Professor of Microbiology, Immunology, and Tropical Medicine and Pediatrics, Committee Member. Ljubica Caldovic, Associate Professor of Integrative Systems Biology, Committee Member. ii © Copyright 2017 by Travis Brendan Kinder. All rights reserved iii Acknowledgments This dissertation was truly a collaborative effort involving mentors from different disciplines, research conducted across multiple institutions, and work performed with the aid of scientists with diverse backgrounds and levels of education. I am thankful for the wealth of support and guidance I received from colleagues in high school, college, graduate school, post-doctoral fellows, and principal investigators. I could not have completed these projects without the generous contributions of time and effort from the following people. I would like to acknowledge the continued support, mentoring, and advice from Linda L. Werling, PhD, Kanneboyina Nagaraju, PhD, DVM, and David Leitenberg, MD, PhD, who oversaw my entire doctoral training and gave invaluable help at every step of the process. Dr Leitenberg and Jyoti K Jaiswal, PhD, provided their time and feedback at all committee meetings and guided the path of these projects as they progressed. Thank you to my committee members Ljubica Caldovic, PhD, who was a reader, and Robert Freishtat, MD, for participating in my defense committee. I am grateful to those providing materials indispensable for this work; Drs. Judith K. Davie, PhD, sent us the Ampd1 promoter used in luciferase plasmids, and Annie Colberg-Poley, PhD, provided the HEK293 cells used in high throughput screening. I am also thankful for those who contributed work to this dissertation research: William D. Coley, PhD, not only generously taught me many of the methods integral to this dissertation but also designed primers for site-directed mutagenesis and created the Ampd1-pGL4.15- immortomouse myoblasts used in Figures 3 and 10. Additionally, Dr. Coley obtained the original Ampd1 knockout mice from the Knockout Mouse Project Repository and helped plan the creation of a conditional, muscle-specific knockdown (KD) mouse described in iv Chapter 5. Sree Rayavarapu, PhD, also taught me many of the methods in this dissertation and collaborated with me in publications of primary research and review manuscripts. Chapter 3 detailing our high throughput drug screen was conducted in collaboration with the National Center for Advancing Translational Sciences principal investigator James Inglese, PhD. Working under Dr. Inglese, Patricia Dranchak, PhD, conducted the high-throughput screening assays, and Ryan MacArthur, PhD, analyzed the data and helped create graphs for Figures 4, 5, 6, and 9. Stephane P. Roche, PhD, and his undergraduate research assistant Nagalakshmi Jeedimalla at Florida Atlantic University created the aza-podophyllotoxin analogues tested and displayed in Figure 9. Kathryn White, PhM, was a co-author for the review on myositis that is the basis for part of the literature review in Chapter 1. Aditi Phadke, MS, conducted the grip strength measures and Digiscan open-field measures for the Ampd1 KD mice displayed in Figures 21 and 22. I received insightful advice and assistance with muscle cell culture and luciferase assays from post-doctoral research fellow Alyson Fiorillo, PhD. I am grateful for the deliberations and comradery with a fellow graduate student Prech (Brian) Uapinyoying and his creation of a computer program to analyze data from the voluntary running wheel displayed in Figure 23. I am indebted to my mentees from high school and college who have contributed immensely to this work; Brittany Brookner and Shayna M. Glassberg helped genotype, conducted behavioral testing, and dissect mice for Chapters 4 and 5. I would like to thank the entire Research Center for Genetic Medicine at Children’s National Medical Center for lab meeting and seminar discussions that raised valuable questions and provided insight into future research directions. Finally, thank you to the Institute for Biomedical Sciences at the George Washington University for the v opportunity to undertake this research and earn a doctorate degree. Funding for this research was provided by the following grants: R01AR050478 and F31AR065362 through the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health, and W81WXH-11-1-0809 through the US Department of Defense/The Myositis Association. vi Abstract of Dissertation AMP Deaminase 1 Transcriptional Regulation and Knockdown Mouse Skeletal Muscle Function The group of idiopathic inflammatory myopathies, collectively known as myositis, have traditionally been diagnosed and treated as autoimmune diseases. They involve cytotoxic T cells and antibodies attacking host tissue, usually respond to immunosuppressive treatment, and are associated with both environmental exposures as well as inherited alleles of immune genes like MHC Class I. Patients with myositis may experience muscle weakness, fatigue, or pain and often have systemic pathologies of the skin, lungs, or gastrointestinal tract. The standard therapy is administration of glucocorticoids and immunosuppressive drugs, but there currently are no treatments specifically for myositis. Although the current drugs can eliminate inflammatory cells within the muscle, most patients do not fully regain strength. Many recent studies have shown that there is no correlation between the amount of muscle inflammatory infiltration and weakness. Therefore, we sought other explanations for refractory muscle weakness in myositis. We and others have observed an acquired deficiency of the muscle-specific, metabolic enzyme AMP deaminase 1 (AMPD1) in both patients and a mouse model of myositis. AMPD1 catalyzes the rate-limiting step of the purine nucleotide cycle, and a deficiency in this enzyme has been hypothesized to cause muscle weakness, fatigue, or cramping. Our hypothesis was that an acquired AMPD1 deficiency is at least partially responsible for muscle weakness in myositis. In this dissertation, our three aims were to identify drugs that could increase the amount of AMPD1 in muscle, uncover mechanisms of how an AMPD1 deficiency could vii be acquired in myositis, and create a genetically engineered mouse in which Ampd1 is turned off to assess the gene’s effect on muscle strength and fatigue. We used the cutting- edge technique of quantitative high throughput screening to test over 4,000 compounds at more than 7 concentrations each and found that microtubule inhibitor drugs such as podophyllotoxin can increase the expression of AMPD1 in cell culture. To investigate how an AMPD1 deficiency could be acquired, we subjected both cultured muscle cells as well as mice to factors associated with myositis pathology and found that TLR7 stimulation, ER stress, and MHC Class I over-expression can decrease Ampd1 expression. Finally, we created a conditional, skeletal muscle-specific, Ampd1 knockdown (KD) mouse and performed standardized and rigorous protocols to measure muscle strength, fatigue, and recovery from fatigue. Compared to normal wild-type mice, the KD mice were not weak and did not fatigue quicker, but unexpectedly, their isolated extensor digitorum longus muscles had an improved recovery from ex vivo fatigue protocols. Although Ampd1 KD did not cause weakness nor fatigue by our measures in mice, a deficiency might still cause these symptoms in otherwise normal humans, in the presence of a disease, or in the context of other genes. Our drug screening efforts have identified the first known compounds to increase the expression of AMPD1, namely the microtubule inhibiting drugs like podophyllotoxin. These drugs were very potent and a custom synthetic library of aza-podophyllotoxin analogues is currently being explored for an improved therapeutic profile. Our studies on the regulation of Ampd1 expression have provided insight to up-stream causes of AMPD1 deficiency occurring in myositis. We found that MHC class I over-expression is likely the cause of ER stress as well as Ampd1 viii down-regulation. The finding that Ampd1 KD muscle recovers more force after resting from fatigue requires further research and may be related to altered metabolite levels or improved blood flow observed by others’ in humans deficient for AMPD1. Although an acquired AMPD1 deficiency may not be the cause of muscle weakness in myositis, we have discovered some of the mechanisms that control
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