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Demonstrating the Importance of Membrane Repair in Response to Disease and Injury

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Demonstrating the Importance of Membrane Repair in Response to Disease and Injury Demonstrating The Importance Of Membrane Repair In Response To Disease And Injury Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Brian J. Paleo Graduate Program in Biomedical Sciences Graduate Program The Ohio State University 2020 Dissertation Committee Dr. Noah Weisleder, Advisor Dr. Brandon Biesiadecki Dr. Anthony Brown Dr. Federica Accornero 1 Copyrighted by Brian J. Paleo 2020 2 Abstract Most cells in the human body have the capacity to reseal their cellular membranes following a disruption of the lipid bilayer. This membrane repair response involves a coordinated chain of events that are essential to maintain cellular homeostasis and prevent cell death. Membrane repair initially emerged as an important field in skeletal muscle physiology, however as the field continues to mature the importance of membrane repair has been noted in many cell types and in the context of injuries related to bacterial toxins, ischemic events, and traumatic injury. As the field begins to expand, it is important to understand the value of membrane repair in the tissue studied and if proteins known to be involved in membrane repair appear in specific tissues. Studies from our laboratory group and others demonstrated that mitsugumin 53 (MG53), a muscle-enriched tripartite motif (TRIM) family protein also known as TRIM72, is an essential component of the cell membrane repair machinery in striated muscle. In an effort to demonstrate the value of both MG53/TRIM72 and membrane repair in Duchenne muscular dystrophy (DMD), I studied the effects of genetic knock out of MG53/TRIM72 in the mdx mouse model of DMD. We observed muscle pathology consistent with the mdx mouse following the initial phase of pathology at 6 weeks of age. However, aging of the mice and resulting accumulation of repeated bouts of injury due to ii the lack of dystrophin protein led to robust fibrosis throughout skeletal and cardiac muscles. To expand the knowledge of membrane repair in tissues other than skeletal muscle we investigated if increasing membrane repair can have protective effects in the peripheral nervous system. Since many neurons are terminally differentiated, increasing cell survival following injury may minimize the impact of these injuries and provide the translational potential for the treatment of neuronal diseases. While several cell types are known to survive injury through plasma membrane repair mechanisms there has been little investigation of membrane repair in neurons and even fewer efforts to target membrane repair as a therapy in neurons. Interestingly, recombinant human MG53 (rhMG53) can be applied exogenously to increase the membrane repair capacity of various cell types both in vitro and in vivo. Thus, we assessed the therapeutic potential of rhMG53 to increase membrane repair in cultured neurons and in an in vivo mouse model of neurotrauma. We found that a robust repair response exists in various neuronal cells and that rhMG53 can increase neuronal membrane repair both in cultured cells and a mouse model of peripheral nerve injury. These findings provide direct evidence of conserved membrane repair responses in neurons and that these repair mechanisms can be targeted as a potential therapeutic approach for neuronal injury. The previous study has identified the value of membrane repair in the nervous system, but identifying potential therapeutic proteins involved in membrane repair can provide targeted treatment for diseases that involve cell membrane injuries. We have previously shown that TRIM72/MG53 can increase plasma membrane repair in skeletal iii and cardiac muscle as well as non-muscle cell types where it is not usually expressed. This observation led us to screen for novel TRIM family proteins that may be able to mediate membrane repair in neuronal cells. We found that TRIM2 transfected cells show an increased capacity for membrane resealing following multi-photon laser injury, while knock down of TRIM2 decreases membrane repair. Because TRIM2 is highly expressed in the nervous system and was previously shown to regulate neurofilaments (NFL) we tested if TRIM2 protects against membrane damage in neurons through regulation of ubiquitination of NFLs and if disruption of TRIM2 or NFL leads to compromised membrane repair or neuronal cell death. Using confocal microscopy, TRIM2 and NFL were not observed to co-localize and immunoprecipitation only revealed an interaction when the two proteins were overexpressed. Additionally, knockout of NFL in primary neurons did not affect the ability of the cells to repair their membranes. The available data suggest that TRIM2 must use an alternative mechanism to mediate membrane repair in neurons. Overall, these studies demonstrate the importance of membrane repair in both skeletal muscle, cardiac muscle, and nervous tissue. Future studies of membrane repair will increase the understanding of this essential process and establish that increasing membrane repair is a valuable target for therapeutic treatment. iv Dedication This document is dedicated to my wife, family and friends for their patience and understanding. v Acknowledgments This document would not have been completed without the mentorship and support of my advisor Dr. Noah Weisleder. I am extremely grateful for the time and effort he has put into helping me get through my time in graduate school. Working in his lab has been an extremely enjoyable experience, and he has taught me how to think and examine problems like a scientist. I would also like to thank the current and former members of my committee Dr. Brandon Biesiadecki, Dr. Anthony Brown, Dr. Federica Accornero, and Dr. Jessica Lerch. I appreciate the guidance you have given me, and the aid the members of your lab have given me when I needed help with my projects. Lastly, I want to thank all the current and former members of the Weisleder lab. Eric Beck has been crucial for the completion of my dissertation both through experimental help and editing of the document. I also want to acknowledge the help of my fellow graduate students Dr. Alisa Blazek, Dr. Kevin McElhanon, and Dr. Tom Kwiatkowski have been by helping me troubleshoot experimental issues. Finally, this work would not have been completed without the aid of the hard-working undergraduate research assistants that have worked with me: Kassidy Banford, Alex Carsel, Allison Miller, and Francesca Veon. This work was aided by the Nishikawara fund and the Center For Muscle Health vi and Neuromuscular Disorders. vii Vita 2011………………………………………… B.S. Biology, California State University, Long Beach 2012-2013……………………………………Discovery Post-baccalaureate Research Education Program, The Ohio State University 2013-present ………………………………...Graduate Research Associate, The Ohio State University Publications Blazek, A. D.*, Paleo, B. J.* & Weisleder, N. Plasma Membrane Repair: A Central Process for Maintaining Cellular Homeostasis. Physiology (Bethesda) 30, 438-448, doi:10.1152/physiol.00019.2015 (2015). * Equal Contribution Talbert E.E., Cuitiño M.C. Ladner K.J., Rajasekerea P. V., Siebert M., Shakya R., Leone G.W., Ostrowski M.C., Paleo B., Weisleder N,. Reiser P.J.,Webb A., Timmers C.D., Eiferman D.S., Evans D.C., Dillhoff M.E., Schmidt C.R., Guttridge D.C. Modeling viii Human Cancer-induced Cachexia. Cell reports 28, doi:10.1016/j.celrep.2019.07.016 (2019). Paleo B.J., Madalena K.M., Mital R. McElhanon K.E., Kwiatkowski T.A. Rose A.L., Lerch J.K. Weisleder N; Enhancing Membrane Repair Increases Regeneration in a Sciatic Injury Model. PloS one 15, doi:10.1371/journal.pone.0231194 (2020). Fields of Study Major Field: Biomedical Sciences Graduate Program ix Table of Contents Abstract ............................................................................................................................... ii Dedication ........................................................................................................................... v Acknowledgments.............................................................................................................. vi Vita ................................................................................................................................... viii List of Figures .................................................................................................................... xi Chapter 1. Introduction ....................................................................................................... 1 Chapter 2. MG53 Deficiency Exacerbates Skeletal Muscle Pathology in the mdx Mouse ........................................................................................................................................... 27 Chapter 3. Enhancing Membrane Repair Increases Regeneration In A Sciatic Injury Model ................................................................................................................................ 61 Chapter 4 Targeting Neuronal Membrane Repair Through TRIM2 Modulation ............. 87 Chapter 5 Summary, Significance, and Future Work ..................................................... 103 Bibliography ................................................................................................................... 115 x List of Figures Figure 1: Models of the plasma membrane repair process. .............................................. 22 Figure 2: Major membrane repair proteins and their hypothesized roles in the repair process..............................................................................................................................
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