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Biochemical Approaches for the Diagnosis and Treatment of Lafora Disease

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Biochemical Approaches for the Diagnosis and Treatment of Lafora Disease University of Kentucky UKnowledge Theses and Dissertations--Molecular and Cellular Biochemistry Molecular and Cellular Biochemistry 2019 BIOCHEMICAL APPROACHES FOR THE DIAGNOSIS AND TREATMENT OF LAFORA DISEASE Mary Kathryn Brewer University of Kentucky, [email protected] Author ORCID Identifier: https://orcid.org/0000-0001-5089-3570 Digital Object Identifier: https://doi.org/10.13023/etd.2019.120 Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Brewer, Mary Kathryn, "BIOCHEMICAL APPROACHES FOR THE DIAGNOSIS AND TREATMENT OF LAFORA DISEASE" (2019). Theses and Dissertations--Molecular and Cellular Biochemistry. 41. https://uknowledge.uky.edu/biochem_etds/41 This Doctoral Dissertation is brought to you for free and open access by the Molecular and Cellular Biochemistry at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Molecular and Cellular Biochemistry by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained needed written permission statement(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine) which will be submitted to UKnowledge as Additional File. I hereby grant to The University of Kentucky and its agents the irrevocable, non-exclusive, and royalty-free license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known. I agree that the document mentioned above may be made available immediately for worldwide access unless an embargo applies. I retain all other ownership rights to the copyright of my work. I also retain the right to use in future works (such as articles or books) all or part of my work. I understand that I am free to register the copyright to my work. REVIEW, APPROVAL AND ACCEPTANCE The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s thesis including all changes required by the advisory committee. The undersigned agree to abide by the statements above. Mary Kathryn Brewer, Student Dr. Matthew Shawn Gentry, Major Professor Dr. Trevor Creamer, Director of Graduate Studies BIOCHEMICAL APPROACHES FOR THE DIAGNOSIS AND TREATMENT OF LAFORA DISEASE ________________________________________ DISSERTATION ________________________________________ A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Medicine at the University of Kentucky By Mary Kathryn Brewer Lexington, Kentucky Director: Dr. Matthew S. Gentry, Professor of Molecular and Cellular Biochemistry Lexington, Kentucky 2019 Copyright © Mary Kathryn Brewer 2019 https://orcid.org/0000-0001-5089-3570 ABSTRACT OF DISSERTATION BIOCHEMICAL APPROACHES FOR THE DIAGNOSIS AND TREATMENT OF LAFORA DISEASE Glycogen is the sole carbohydrate storage molecule found in mammalian cells and plays an important role in cellular metabolism in nearly all tissues, including the brain. Defects in glycogen metabolism underlie the glycogen storage diseases (GSDs), genetic disorders with variable clinical phenotypes depending on the mutation type and affected gene(s). Lafora disease (LD) is a fatal form of progressive myoclonus epilepsy and a non-classical GSD. LD typically manifests in adolescence with tonic-clonic seizures, myoclonus, and a rapid, insidious progression. Patients experience increasingly severe and frequent epileptic episodes, loss of speech and muscular control, disinhibited dementia, and severe cognitive decline; death usually ensues in the second decade of life. LD, like one- third of all epilepsy disorders, is intractable and resistant to antiseizure drugs. A hallmark of LD is the accumulation of intracellular, insoluble carbohydrate aggregates known as Lafora bodies (LBs) in brain, muscle, and other tissues. LBs are a type of polyglucosan body, an insoluble aggregate of aberrant glycogen found in some GSDs and neurodegenerative disorders. Like most GSDs, LD is an autosomal recessive genetic disorder. Approximately 50% of LD patients carry mutations in the epilepsy, progressive myoclonus 2A (EPM2A) gene encoding laforin, a glycogen phosphatase. Remaining patients carry mutations in EPM2B, the gene that encodes malin, an E3 ubiquitin ligase. Laforin and malin play important roles in glycogen metabolism. In the absence of either enzyme, glycogen transforms into an insoluble, hyperphosphorylated and aberrantly branched polysaccharide reminiscent of plant starch. This abnormal polysaccharide precipitates to form LBs and has pathological consequences in the brain. Since a definitive LD diagnosis requires genetic testing, whole exome sequencing has been increasingly used to diagnose LD. As a result, numerous cases of more slowly progressing or late-onset LD have been discovered that are associated with missense mutations in EPM2A or EPM2B. Over 50 EPM2A missense mutations have been described. These mutations map to many regions of the laforin X-ray crystal structure, suggesting they produce a spectrum of effects on laforin function. In the present work, a biochemical pipeline was developed to characterize laforin patient mutations. The mutations fall into distinct classes with mild, moderate or severe effects on laforin function, providing a biochemical explanation for less severe forms of LD. LBs drive LD pathology. As a result, LBs and glycogen metabolism have become therapeutic targets. Since LBs are starch-like, and starch is degraded by amylases, these enzymes are potential therapeutics for reducing LB loads in vivo. However, amylases are normally secreted enzymes. Degradation of intracellular LBs requires a cell-penetrating delivery platform. Herein, an antibody-enzyme fusion (AEF) technology was developed to degrade LBs in vitro, in situ in cell culture, and in vivo in LD mouse models. AEFs are a now putative precision therapy for LD, potentially the first therapeutic to provide a significant clinical benefit. Prior to this work, LD was considered a homogenous disorder and treatments were only palliative. The data herein support a spectrum of clinical progression, a potential therapy for LD, and mechanistic insights into LD pathophysiology. This work illustrates how personalized medicine, both in diagnosis and treatment, can be achieved through basic biochemical approaches to human disease. KEYWORDS: Lafora disease, glycogen, epilepsy, neurodegenerative disease, phosphatase, personalized medicine. Mary Kathryn Brewer April 22, 2019 Date BIOCHEMICAL APPROACHES FOR THE DIAGNOSIS AND TREATMENT OF LAFORA DISEASE By Mary Kathryn Brewer Dr. Matthew Shawn Gentry Director of Dissertation Dr. Trevor Creamer Director of Graduate Studies April 22, 2019 Date DEDICATION For my little brother Ben ACKNOWLEDGMENTS I must first and foremost acknowledge my extraordinary mentor, Dr. Matthew Gentry. I will never be able to thank him sufficiently for his kindness, grace and guidance throughout my undergraduate and graduate career. Not only has he done everything in his power to enable me and my colleagues to succeed as scientists, the goodness of his character has profoundly impacted my life. I am forever grateful for the lessons that I have learned from him. I know he will go on to train many more outstanding scientists like himself. I am also thankful for his wife Sandy, who cheers on the whole lab and helps to hold things together. I would like to thank my dissertation committee members and outside examiner for their valuable time, questions, guidance and feedback. Dr. Craig Vander Kooi has always challenged me and encouraged me to think more deeply. He is an indispensable collaborator and an unending source of knowledge and discussion, whom I could approach with technical or scientific questions at any time. I am always thankful for the big-picture perspective of Dr. Jim Geddes, and for a very successful rotation in his lab at the beginning of graduate school. I am also very thankful for Dr. Tianyan Gao, for her intelligence and gentle demeanor, and for mentoring me during my first departmental seminar. Finally, I am very grateful to my outside examiner Dr. Jim Pauly, whom I am honored to have as a part of my defense. It was very fortuitous for us to cross paths and I deeply value his skills, knowledge, and collaboration. I am deeply grateful for the friendship and mentorship of Satrio Husodo and Dr. Madushi Raththagala during my early years in the Gentry lab. Both are talented scientists and I am glad to have known and worked with them. I am also very thankful for Dr. Ramon Sun, whose vibrancy, energy, skills and brilliance are unmatched. Ramon has been an enormous asset to the Gentry lab. The lab continues to grow and flourish over the years and I am thankful for each and every individual who passes through. I am grateful for the foundational work of Amanda Sherwood and Dave Meekins, which paved the way for mine. I deeply appreciate the hard work, help and patience of Annette Uittenbogaard and Grant Austin. I want to thank Corey
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