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Proteostasis of Glial Intermediate Filaments: Disease Models, Tools, and Mechanisms

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Proteostasis of Glial Intermediate Filaments: Disease Models, Tools, and Mechanisms PROTEOSTASIS OF GLIAL INTERMEDIATE FILAMENTS: DISEASE MODELS, TOOLS, AND MECHANISMS Rachel Anne Battaglia A dissertation submitted to the faculty at the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Cell Biology and Physiology in the School of Medicine. Chapel Hill 2021 Approved by: Natasha T. Snider Carol Otey Keith Burridge Douglas Cyr Mohanish Deshmukh Damaris Lorenzo i © 2021 Rachel Anne Battaglia ALL RIGHTS RESERVED ii ABSTRACT Rachel Anne Battaglia: Proteostasis of Glial Intermediate Filaments: Disease Models, Tools, and Mechanisms (Under the direction of Natasha T. Snider) Astrocytes are a major glial cell type that is crucial for the health and maintenance of the Central Nervous System (CNS). They fulfill diverse functions, including synapse formation, neurogenesis, ion homeostasis, and blood brain barrier formation. Intermediate filaments (IFs) are components of the astrocyte cytoskeleton that support many of these functions in healthy individuals. However, upon cellular stress or genetic mutations, IF proteins are prone to accumulation and aggregation. These processes are thought to contribute to disease pathogenesis of different tissue-specific disorders, but therapeutic targeting of IFs is hindered by a lack of pharmacological tools to modulate their assembly and disassembly states. Moreover, the mechanisms that govern the formation and dissolution of IF aggregates are poorly defined. In this dissertation, I investigate IF aggregates called Rosenthal fibers (RFs), which form in astrocytes of patients with two pediatric neurodegenerative diseases, Alexander disease (AxD) and Giant Axonal Neuropathy (GAN). My aim was to gain a better understanding of the mechanisms of how astrocyte IF protein aggregates form and interrogate the role of post- translational modifications (PTMs) in this process. In Chapter 1, I introduce fundamental information about astrocyte biology, IF proteins and their functions, and specific roles of astrocyte IFs in neurodegenerative diseases. In Chapter 2, I present a method to simultaneously isolate mammalian IFs from several tissues in order to examine in vivo-relevant PTMs. In Chapter 3, I demonstrate the utility of an image-based small molecule screen to identify IF- iii selective compounds. In Chapter 4, I reveal mechanistic information in AxD and identify a specific PTM as a new marker of AxD severity. In Chapter 5, I describe IF proteostasis in neural progenitor cells and astrocytes from GAN patients. The work in Chapters 4 and 5 involves development of clinically relevant tools for these diseases using induced pluripotent stem cells (iPSCs) and CRISPR/Cas9 gene editing technology. In Chapter 6, I summarize and contextualize the results of this work, acknowledge the limitations and remaining gaps, and highlight key future directions. In summary, these studies provide new methods, tools, disease models, and mechanisms to understand and target IF cytoskeleton abnormalities in human diseases. iv To my parents, who are my rock, my role models, and my eternal cheerleaders. v ACKNOWLEDGEMENTS First and foremost, I would like to thank my advisor, Natasha Snider. Thank you for giving me the opportunity to be a part of your lab and for being part of my journey for the past five years. From day one, there has never ever been a dull moment in the Snider lab. I have enjoyed the excitement and fast-paced energy of the lab and the privilege to learn from my PI side-by-side at the bench. Thank you for giving me the freedom to follow my curiosity and the wisdom to not get spread too thin or pulled too far down the rabbit hole. Natasha, your dedication, passion, creativity, resilience, and heart is an inspiration to me. I am so thankful for your guidance, support, enthusiasm, and encouragement throughout this work and period of growth. I also thank my research and career mentors outside of the lab. Many thanks to my dedicated thesis committee members: Carol Otey, Keith Burridge, Mohanish Deshmukh, Doug Cyr, and Damaris Lorenzo for graciously donating their time, providing insights about my project, and supporting my career plans. Adriana Beltran, thank you for being my fearless partner in iPSC gene editing and for all of your encouragement and advice about science, career, and life. Thank you also to Felix Olivares-Quintero for generous technical assistance with gene editing. Diane Armao, I thank you for devoting your time and zealous energy to my education in the methods and interpretations of neuropathology and for our spirited conversations over the years. Virginia Godfrey, our collaborative animal projects did not make it into this dissertation, but I am grateful for your significant assistance and guidance with multiple animal experiments vi and for our discussions on Alexander disease and beyond. I look forward to seeing our work come to fruition and to our future collaborations. Ashalla Freeman and Jessica Harrell, thank you for welcoming me into the IMSD family and for your support through the first year in BBSP and advice in navigating rotations. I will never forget the kindness you showed me and invaluable service you provided for myself and my cohort. Adrienne Cox and Folami Ideraabdullah, thank you for your support in the later years of my PhD journey. Finally, Anna O’Connell, you have been an advisor, a confidant, a role model, and a dear friend to me through the years. You have challenged the way I think about communication and life, and I am so thankful for the time and advice you have given to me through the years. I really cannot put into words how thankful I am for your encouragement or how much I look up to you. I do not know where you find the time or the strength to do all of the wonderful things you do in service to your community and your friends, but I am forever thankful that you have always made time for me. Next, I am very thankful to all of my labmates, both past and present. Thank you to the McKim lab for welcoming me into the research world and carefully nurturing my passion for science. A special thank you to my previous PI, Kim McKim, for giving me the opportunity to work in his lab and to my dedicated bench mentor, Sarah Radford. I also thank my unofficial mentors and friends, Arunika Das, Victoria Wagner, Allysa Tuite, and Mercedes Enners for their support. Helen Willcockson, thank you for going above and beyond to help set the Snider lab up for success. I learned so much from you in my first few years in the lab: the importance of organization, managing a mouse colony, how to make friends and influence people across campus, and of course, the sweet tones of the Moody Blues. Parijat Kabiraj and Kathryn Trogden, thank you for paving the way in the PTM and simvastatin projects. Thank you to our vii fabulous undergraduates for their help to keep the lab running: Deekshita Ramanarayanan, Megan Dew, Chloe Eaton, Samed Delic, Seyoung Jung, and Morgan Rouse. Jasmine Robinson, thank you for technical assistance and your diligent and careful attention to details, and of course, thank you for our varied and candid discussions. With your adaptability and determination, I am excited to see what you will achieve. Cassie Phillips, thank you for your careful review of the GAN chapter and for being such an enthusiastic mentee. I am happy that you have chosen to join the Snider lab family and look forward to hearing about your future successes. Dr. Maryam Faridounnia, thank you for being a part of team filament. I have enjoyed being your teammate, rock climbing companion, and friend. I am so proud of the effort you have poured into confidently carving out the brave new direction of RNA work and iPSC modeling in microfluidic devices. You are by far the fanciest member of the Snider lab, and I look forward to reading your published work. Last but never the least, Dr. Kaye Alcedo, you have been in the trenches with me almost my entire tenure in the Snider lab. Thank you for being my lab sister, providing the same support, care, communication, and encouragement as a true sister. I admire how dependable you are, how you are always up for a challenge, how you engage boldly with other scientists, and your absolute thirst for knowledge. I know that you will accomplish anything you set your energy towards, and I look forward to seeing you excel at Harvard and beyond. I was also lucky to have a strong community outside of the lab. A virtual toast to the Breakfast Club who brightened my Saturdays before lab and filled my weekends with concerts, game nights, and Cheezits. To The Lids (Neil, Lex, Phil, Emma, Julia, and Maggie), thank you for providing a musical release, unforgettable performance experiences, family dinners, and so many laughs. I’ll be your biggest fan in Boston! Garrett Sharpe, thank you for being my friend viii and much-needed nature guide – both physically by bringing me along for hikes near and far and verbally by sharing your beautiful poems. Never let the iguana-ridden people in this world get you down. Drs. Adelaide Tovar and Yael Escobar, you are my best friends, and I am so grateful that you have been with me from day one at UNC. Thank you for always being there for me through the ups and downs to listen, to make me laugh, and to dole out the occasional hug when I allow it. You are two of the most whimsical, talented, trustworthy, and genuine women I have ever known. I will always treasure our memories together and look forward to making more. Last, I thank my family. Cassie and Katie, thank you for always cheering me on and being willing to hear about my research and life in the lab.
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