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University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2013 Bridging The Gap: Defining the Molecular Mechanisms of Cep290 Disease Pathogenesis Theodore George Drivas University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Cell Biology Commons Recommended Citation Drivas, Theodore George, "Bridging The Gap: Defining the Molecular Mechanisms of Cep290 Disease Pathogenesis" (2013). Publicly Accessible Penn Dissertations. 851. https://repository.upenn.edu/edissertations/851 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/851 For more information, please contact [email protected]. Bridging The Gap: Defining the Molecular Mechanisms of Cep290 Disease Pathogenesis Abstract Mutations in the gene CEP290 cause an array of debilitating and phenotypically distinct human diseases, ranging in severity from the devastating blinding disease Leber congenital amaurosis (LCA) to Senior Løken Syndrome, Joubert syndrome, and the embryonically lethal Meckel-Grüber syndrome. The pathology observed in these diseases is thought to be due to CEP290's essential role in the development and maintenance of the primary cilium, but despite its critical role in biology and disease we know only little about CEP290's function. Here we identify four novel functional domains of the protein, showing that CEP290 directly binds to cellular membranes through an N-terminal domain that includes a highly conserved amphipathic helix motif, and to microtubules through a domain located within its myosin-tail homology domain. Furthermore, CEP290 activity was found to be regulated by two novel autoinhibitory domains within its N- and C-termini, both of which were also found to play critical roles in regulating ciliogenesis. Disruption of the microtubule-binding domain in the rd16 mouse LCA model was found to be sufficiento t induce significant deficits in cilium formation leadingo t retinal degeneration. Taking these findings into account, we developed a novel model that accurately predicts patient CEP290 protein levels in a mutation-specific fashion. Predicted CEP290 protein levels were found to robustly correlate with disease severity for all reported CEP290 patients. All these data implicate CEP290 as an integral structural and regulatory component of the primary cilium and provide insight into the pathological mechanisms of LCA and related ciliopathies. Our findings also suggest novel strategies for therapeutic intervention in the treatment of CEP290-based disease that, if fully realized, would be the first treatment available for the many patients suffering the devastating effects of CEP290 dysfunction. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Cell & Molecular Biology First Advisor Jean Bennett Keywords CEP290, Ciliopathy, Cilium, Leber Congenital Amaurosis Subject Categories Cell Biology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/851 BRIDGING THE GAP: DEFINING THE MOLECULAR MECHANISMS OF CEP290 DISEASE PATHOGENESIS Theodore G. Drivas A DISSERTATION in Cell and Molecular Biology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2013 Supervisor of Dissertation Signature: ____ __________________________________________ Dr. Jean Bennett F.M. Kirby Professor of Ophthalmology, Perelman School of Medicine Graduate Group Chairperson Signature: ______ _________ _______________________________ Dr. Daniel Kessler Associate Professor of Cell and Developmental Biology, Perelman School of Medicine Dissertation Committee Dr. James M. Wilson Professor of Pathology and Laboratory Medicine, Perelman School of Medicine Dr. Joshua L. Dunaief Associate Professor of Ophthalmology Perelman School of Medicine Dr. Erika L.F. Holzbaur Professor of Physiology, Perelman School of Medicine Dr. Aimee S. Payne Albert M. Klingman Assistant Professor of Dermatology, Perelman School of Medicine BRIDGING THE GAP: DEFINING THE MOLECULAR MECHANISMS OF CEP290 DISEASE PATHOGENESIS COPYRIGHT 2013 Theodore George Drivas This work is licensed under the Creative Commons Attribution- NonCommercial-ShareAlike 3.0 License To view a copy of this license, visit http://creativecommons.org/licenses/by-ny-sa/2.0/ Για τους γονείς μου, Γιώργο κ Ελένη και για τους παππούδες μου, Θοδωρή, Ιφιγένεια, Παναγιώτη, κ Σταματία οι οποίοι με εκπαίδευσαν, με διασκέδασαν, με μεγάλωσαν, με ενεπνευσαν, μου έδωσαν κουράγιο, με υποστήριξαν, και θυσίασαν τόσα πολλά για να μπορέσουν τα παιδιά τους κ τα εγγόνια τους να κάνουν κάτι καλύτερο στην ζωή τους. Αν αυτό σημαίνει κάτι, τότε ας είναι η διαθήκη για όλα όσα έχετε κάνει. iii ACKNOWLEDGMENTS The greatest difficulties sometimes spring forth from the simplest of tasks. I’ve certainly learned this lesson in the lab where the well-intentioned promise of “it’ll be done in a week” can sometimes take more than a month to fulfill. I’ve discovered this aphorism to hold true in the writing of my dissertation as well. This section of my thesis, seemingly the simplest, has been perhaps the most challenging to write – not because it is terribly long (I have surely forgotten to thank many people), nor because it required any amount of hard work (in fact I should have worked harder to thank, in person, the many people listed here), but because it is impossible to fully express the gratitude one feels for all the mentors, supporters, and friends that one gains along the journey from science-obsessed child to fledgling scientist. However, in the lab I also learned that the most difficult of problems are often the most rewarding to resolve. This maxim holds true here too – reflecting back on the many dozens of people who encouraged, supported, and challenged me throughout my life has been orders of magnitude more rewarding and gratifying than any number of picture-perfect western blots or journal acceptances. Each of these people shares a part in the work I present here for shaping me into the person I have become and the scientist I will grow up to be. Innumerable lab and life lessons have been imparted to me by many people, but a few mentors stand out as the ones who truly inspired me and introduced me to science and the beauty of the natural world. I have been extremely lucky to have had many such mentors during every stage of my life, beginning at day one with my father. Not every child is given a microscope and chemistry set in elementary school, nor does every father take his infant son on hour-long walks through the salt marshes and natural forests of northern Manhattan, pointing out trees by name and imparting those pieces of knowledge that impressionable young children never forget – the five needle cluster of the white pine; the fresh lemony scent of a sassafras leaf rubbed between one’s hands; the cat-faced leaves and long, straight trunk of the tulip tree, used by native Americans to make dugout canoes. These walks grew longer as I grew old enough to walk, stopping every few feet to collect insects and plants. It’s a true testament to my mother’s patience that she allowed so many collected creatures into her home. iv Eventually I graduated from my father’s classroom and was enrolled in New York City’s public school system. Here I was introduced to a number of people to whom I am forever indebted for their encouragement and support. Each of these individuals went above and beyond any expectations of what a teacher is expected to do. Every single teacher I met along the way deserves a full page to acknowledge all that they’ve done for me, but in particular I must name Ms. White, Ms. Alister, Ms. Shapiro, and Ms. Williams (as they will always be known to me, no matter how often they now tell me to use their first names) for helping me grow so much as both a student and as a person. In New York’s public schools I was also introduced to two scientific mentors who left indelible marks on my personality and ambitions. These mentors were Mr. Peter Hannon in elementary school and Dr. John Utting in high school, both of whom picked up where my father left off in fostering my interest in science. I was exceptionally lucky to have Mr. Hannon as a dedicated science teacher with a dedicated science lab – even in 1 st and 2 nd grade the time I looked forward to most was the time spent in Mr. Hannon’s classroom dissecting owl pellets (my mother still has a reconstructed vole skeleton hanging somewhere in the house), building electromagnets, and growing plants in the back garden of the school. It was probably in Mr. Hannon’s class that I first seriously considered “doing science” as a career. If there was any question about my future ambitions, however, my mind was firmly made up after meeting Dr. John Utting at Stuyvesant High School. Dr. Utting was a fantastic teacher, both in the numerous courses he taught me during the school day throughout my four years at Stuy and after school when he spent multiple hours each week teaching me and a handful of other classmates a college level cell biology course. I am, however, most indebted to Dr. Utting for the time, energy, and emotion he put into helping me complete an independent research project at the Rockefeller University, taking extra time out of his day to review the scientific literature with me, plan experiments, and review my writing. I will never forget how genuinely passionate Dr. Utting was for the subject that he taught and for the students that he cared for so deeply. While I was incredibly lucky to have had so many fantastic people push me into the sciences, I was equally lucky to have been given hands-on lab experience from a very young age. Again, my father began this task, bringing me to the lab as a child while he was a graduate student and post-doctoral fellow.

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