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Familial Cortical Myoclonus Caused by Mutation in NOL3 by Jonathan Foster Rnsseil DISSERTATION Submitted in partial satisfaction of the requirements for the degree of DOCTOR OF PHILOSOPHY in Biomedical Sciences in the Copyright 2013 by Jonathan Foster Russell ii I dedicate this dissertation to Mom and Dad, for their adamantine love and support iii No man has earned the right to intellectual ambition until he has learned to lay his course by a star which he has never seen—to dig by the divining rod for springs which he may never reach. In saying this, I point to that which will make your study heroic. For I say to you in all sadness of conviction, that to think great thoughts you must be heroes as well as idealists. Only when you have worked alone – when you have felt around you a black gulf of solitude more isolating than that which surrounds the dying man, and in hope and in despair have trusted to your own unshaken will – then only will you have achieved. Thus only can you gain the secret isolated joy of the thinker, who knows that, a hundred years after he is dead and forgotten, men who never heard of him will be moving to the measure of his thought—the subtile rapture of a postponed power, which the world knows not because it has no external trappings, but which to his prophetic vision is more real than that which commands an army. -Oliver Wendell Holmes, Jr. iv ACKNOWLEDGMENTS I am humbled by the efforts of many, many others who were essential for this work. To be provided the privilege of pursuing truth as a profession requires the stars to align. For me, they have. I wish to thank my thesis advisor, Louis J. Ptáček, M.D., for his mentorship. During the course of my interviews with about 50 MSTP faculty members across the country, the most challenging interview I had was with Louis! It didn’t scare me off, though: I appreciated the intensity of his questions and the direction of the science, and I was eager to join the lab. Louis took me under his wing as a neophyte first-year medical student, and he has provided me with priceless advice and encouragement over the last four years. I have benefitted immensely from the help of many members of the laboratory, viz.: S.Y. (Christin) Chong, Ph.D., Mary Heng, Ph.D., Lorna Kategaya, Ph.D., Sehoon Keum, Ph.D., Mary Lange, Hsien-Yang Lee, Ph.D., Emily Quinn, Jenise C. Wong, M.D., Ph.D., Linda Zhang, and Ying Zhang. Zachery Kornberg aided me with the linkage and Sanger sequencing. I could not have performed the FACS experiments without Philip Kurien, M.D. The biochemistry expertise of William (Casey) Hallows, Ph.D., was crit- ical. Perhaps more importantly, he was a fun guy to eat lunch with daily. I am especially indebted to Shen-Yi (Bruce) Howng, Ph.D., for serving as my scientific sounding board, and for his friendship. Over the past few years I spent many (most?) of my waking hours working alongside Bruce. There may never again be a time in my life when I have the freedom and environs for such frequent, deep, freewheeling discussions. I will always cherish them. v I am grateful to the many UCSF faculty members who took time out of their incredibly busy lives to generously offer their mentorship. I thank Daniel H. Lowenstein, M.D., Scott A. Oakes, M.D., and Anthony Wynshaw-Boris, M.D., Ph.D., for their service and helpful advice during my qualifying exam. Kevin M. Shannon, M.D., directed the MSTP and provided invaluable experimental suggestions and career advice during my dissertation committee meetings. Kevin also repeatedly demonstrated that he could beat me in golf. Finally, Robert L. Nussbaum, M.D., has guided my development for five years: first as my MSTP advisor, then as my BMS advisor, then as chair of my qualifying exam committee, and ultimately as chair of my dissertation committee. His scientific in- sight, ethical standards, and moral support were all far beyond the call of duty. I am willing to bet that faculty advisors underestimate the influence of a meeting that takes place each quarter and may only last an hour. More than once I entered Bob’s office deep in the throes of graduate school despondency, and yet he found a way to buoy my spirits. This was, as they say, nontrivial. His creed – to never lose sight of our patients, for they are the reason we do science – has become mine. Many others at UCSF were critical to this work, including Jana Toutolmin and Catherine Norton in the MSTP office, and Lisa Magargal et al. in the BMS office. MacKenzie A. Howard, Ph.D. and Scott C. Baraban, Ph.D., helped with electro- physiological characterization of NOL3 knockout mice. Jonathan Trinidad, Ph.D., as- sisted with mass spectrometry experiments. I was lucky to benefit from numerous collaborations outside of UCSF. Jamie L. Steckley, M.D., Angelika F.G. Hahn, M.D., and their colleagues at London Health Science University and other universities in Canada phenotyped the patients and col- vi lected DNA samples. Giovanni Coppola, M.D., Daniel H. Geschwind, M.D., Ph.D., and others at UCLA performed targeted sequencing. Joe Quadrilatero, Ph.D., of the University of Waterloo generously shared Nol3 knockout mice. Peter Kuffa and Gabriel Nunez, Ph.D., of the University of Michigan shared human NOL3 cDNA constructs. Kinya Ishikawa, M.D., Ph.D., of Tokyo Medical and Dental University dug through his freezers and transcribed the sequences of many primer sequences for the BEAN, tk2, and FLJ27243 genes. The Nol3 knockin mouse was generated through collaborations with inGenious Targeting Laboratory, Inc., and with Caiying Guo, Ph.D., of the Howard Hughes Medical Institute Janelia Farm Research Campus. The proteomic screen was done in collaboration with Applied Biomics. It goes without saying that we are forever indebted to the patients and their families for their participation. Graduate school is just one stop of many on my educational journey, and I would be remiss to omit important teachers and mentors at prior junctions. They have influenced me greatly. They include Tas Anthony, Margaret Shullaw, and James Ruebush at Iowa City West High School; Alejandro Aballay, Ph.D., David Brady, Ph.D., Kevin DeLapp, Ph.D., Kylie A. Haskins, Ph.D., V. Louise Roth, Ph.D., and Thomas P. Witelski, Ph.D., at Duke University; and Louise Aronson, M.D., and Don Ganem, M.D., at UCSF School of Medicine. Personal financial support was provided by the UCSF MSTP (funded by NIH NIGMS grant T32GM07618 and the UCSF School of Medicine), and later by NIH NINDS grant F31NS077533. The research itself was funded by NIH grants NS044379 and U54 RR19481, by the Sandler Fund for Neurogenetics, and by the Howard Hughes Medical Institute. In the stress generated by interminable grant applications, we often vii lose sight of the fact that, historically, it is a very recent phenomenon for anyone other than a select group of aristocrats to have the wherewithal to conduct meaningful scientific research. I am grateful to be born in this age and for the resources that modern society chooses to allocate for research. That they go towards science rather than guns or butter underscores our responsibility to conduct our research nobly. Lastly, I wish to express my heartfelt dependence upon my friends and especially my family. My fiancée has offered unyielding support throughout the ecstasy and agony of graduate school. Her love, loyalty, good cheer, and unconditional kindness made it less agony, mostly ecstasy. My grandparents have loved and provided for me in in- numerable ways. My brother, sister, sister-in-law, nephews, and niece were all constant sources of joy and encouragement. Most importantly, I have always been able to count on my parents. I can be a trying child, yet they are a wellspring of emotional, financial, and moral ballast. They instilled in us a thirst for knowledge that cannot be slaked, ethical standards requiring constant vigilance, and a love for man that necessitates im- proving his lot. These principles have impelled this work. For these reasons, I dedicate this dissertation to them. viii ABSTRACT Familial Cortical Myoclonus Caused by Mutation in NOL3 Jonathan Foster Russell Many neurologic diseases cause discrete episodic impairment. Study of the genes and mechanisms underlying these diseases has informed our understanding of the nervous system. Here we describe a novel episodic neurologic disorder, which we term familial cortical myoclonus (FCM). FCM is characterized by adult onset, slowly progressive, multifocal, cortical myoclonus, inherited as an autosomal dominant trait. On the basis of clinical, electrophysiological, and genetic data, FCM is nosologically distinct. We uti- lized genome-wide single nucleotide polymorphism genotyping, microsatellite linkage, and massively parallel sequencing to identify a mutation in the gene nucleolar protein 3 (NOL3) that likely causes FCM. NOL3 is thought to bind to pro-apoptotic proteins and thereby repress apoptosis, but our extensive experimentation did not replicate these claims. In vitro, the NOL3 mutation leads to post-translational modification of NOL3 protein. We could not pinpoint the identity of the modification, but did find that this process is regulated by phosphorylation at residue T114. Finally, a proteomic screen for novel binding partners identified two candidates that modulate neuronal/astroglial differ- entiation. We hypothesize that the NOL3 mutation abrogates these interactions to cause FCM. This hypothesis will be tested with Nol3 mutant mice that we generated. In total, this work defines a novel episodic neurologic phenotype and the associated mutation, calls into question some of the published functions of NOL3, and presents an alternative mechanism that may explain the pathophysiology of FCM.

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