Exploring Nsr100/SRRM4 As a Therapeutic Target for Autism Spectrum Disorder in Mice
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Exploring nSR100/SRRM4 as a therapeutic target for autism spectrum disorder in mice by Juli Wang A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Molecular Genetics University of Toronto © Copyright by Juli Wang, 2019 I Exploring nSR100/SRRM4 as a Therapeutic Target for ASD in Mice Juli Wang Master of Science Department of Molecular Genetics University of Toronto 2019 Abstract Misregulation of nSR100 and its target microexons are common in a large proportion of ASD patients and cause ASD-associated features in mice. This thesis explores nSR100 and its target splicing program as a potential therapeutic target using a conditional knockout allele, nSR100GT. I show that nSR100 protein is effectively depleted in the cortical regions of nSR100GT mutant mice at E17.5, E18.5, and P2 stages, which correlates with phenotypes overlapping with all core behavioral domains of ASD. I show that tamoxifen-mediated rescue in prenatal nSR100GT animals restores nSR100 protein and microexon inclusion levels comparable to those observed in wildtype mice. Collectively my thesis research shows that the nSR100GT mouse strain holds the promise for examining phenotypic effects of nSR100 reactivation in ASD-like mice at different developmental stages, and complimentary models are also to be considered for investigating the therapeutic potential of targeting nSR100 in the context of ASD. II Acknowledgments I wholeheartedly thank for the tremendous support and educational experiences I have received from my mentors, Dr. Sabine Cordes and Dr. Benjamin Blencowe, as well as my committee members, Dr. Julie Lefebvre and Dr. John Calarco. Collectively, they have helped me forging realistic ideas about how research is like as a career, which further strengthens my belief that pursuing research will bring the best out of me – that is, providing meaningful and logical answers and, more importantly, inspirations for future endeavors to follow so that the world of biology is not only to be appreciated with awes but to be predicted and/or manipulated by mortal beings with some humble but growing certainties. I sincerely thank for members from the labs and colleagues in the Department of Molecular Genetics who have given me hands-on and/or hands-off guidance with sincerity and generosity. Specifically, I thank Dr. Mathieu Quesnel Vallières, a highly inspiring and visionary scientist, for providing me a great amount of knowledge, encouragement, and supervisions. Not only did he offer me valuable trainings on numerous mouse-related experimental skills, he has been kind to remind me the importance of learning how to navigate between different colleagues, designing experiments with a clear vision of what questions are to be addressed, and most importantly, listening to my passion instead of my fear so as to never let any adversity to get the best of me. I also thank Dr. Serge Gueroussov and Dr. Thomas Gonatopoulos Pournatzis, two highly prolific and knowledgeable scientists who were always open to share their own experiences during our conversations throughout my four years of research experience that initiated at Dr. Blencowe’s lab, which not only helped me troubleshoot my experiments but also realize what it means to keep moving forward against all odds. Furthermore, I have benefited greatly from many stimulating scientific discussions with Dr. Ulrich Braunschweig, Dr. Robert Weatheritt, Eesha Sharma, and Mingkun Wu, four highly intellectual and erudite scientists from whom I have learned the merit of being the most critical person when it comes to judging my own data as well as evaluating the validity of other people’s arguments. Finally, I feel truly grateful to Sabine and Ben who not only try their best to provide me an inspiring research environment but also faithfully believe that everyone has his/her unique talent that deserves a chance to III develop. Collectively, all these educational supports will keep me company throughout my scientific career path, which I will strive to pay forward at my every step forward and hence contribute very humbly but faithfully to the propagation of such noble spirit. I feel fortunate and grateful for being a member of my family, where everyone has his/her unique temperaments but converges on one of the characteristics that unify us as a cohesive unit – that is, to enjoy the challenge of never taking the easy way out, especially with regard to finding our ways to contribute to scientific discoveries with what we are gifted for. Furthermore, I would like to thank my grandmother who has been providing me the strongest spiritual support and helping guard my moral compass so I can practice virtuousness and righteousness to my best ability when coping with inevitable cultural differences. Last but not least, I salute to the billions of neurons and trillions of synapses, as well as all species of molecules in my body that work and collaborate with each other not only diligently but voluntarily. Not only do they strive to be always up and running in their autonomous and disciplined manners, they also share my daily endeavors and philosophies of making our existence worthwhile. I wholeheartedly wish to pay forward this favor by learning in depth about their microscopic but powerful “daily rituals” throughout my career life. IV Table of Contents ABSTRACT.................................................................................................................................................. II ACKNOWLEDGMENTS......................................................................................................................... III TABLE OF CONTENTS............................................................................................................................ V LIST OF TABLES.................................................................................................................................... VII LIST OF FIGURES..................................................................................................................................VIII LIST OF ABBREVIATIONS…................................................................................................................ IX CHAPTER 1 INTRODUCTION ............................................................................................................ 1 1.1 AUTISM SPECTRUM DISORDERS ……………………………………………………………………........................... 2 1.1.1 Clinical overviews .................................................................................................................................... 2 1.1.2 Formulating the mode of inheritance of ASD..............................................................................3 1.1.3 Neurological features in ASD brain s............................................................................................... 6 1.1.4 Reversal of ASD-associated phenotypes in adults.....................................................................11 1.2 TRANSCRIPTOMIC FEATURES IN POST-MORTEM ASD BRAINS…..............................................................12 1.2.1 Diverse RNA species implicated in ASD ........................................................................................12 1.2.2 Spatiotemporal dynamics of the transcriptome rooted in the brain and implicated in ASD.................................................................................................................................................................................13 1.2.3 Alternative splicing in the nervous system....................................................................................16 1.2.4 Misregulation of microexon splicing and nSR100/SRRM4 in ASD..................................21 1.2.5 NSR100/SRRM4 as a molecular hub for ASD-associated abnormalities...................... 22 1.3 THESIS OBJECTIVES: EXPLORING NSR100 AS A THERAPEUTIC TARGET IN MICE....................................23 1.3.1 ASD-associated features observed in nSR100+/D7-8 mice...................................................... 23 1.3.2 The nSR100D7-8 allele and tHe nSR100 knockout-first allele.................................................23 1.3.3 Exploration of nSR100 as a therapeutic target for ASD using mice that carry the nSR100 knockout-first allele (nSR100lox or nSR100GT)........................................................................25 CHAPTER 2 CHARACTERIZATION OF NSR100GT MUTANT MICE AND TAMOXIFEN- MEDIATED REACTIVATION OF NSR100.................................................................28 2.1 MATERIAL AND METHODS .............................................................................................................................29 2.1.1 Mouse strain and genotyping.............................................................................................................29 2.1.2 RT-PCR and RT-qPCR.............................................................................................................................30 2.1.3 Western blotting....................................................................................................................................30 V 2.1.4 Tamoxifen-mediated prenatal and neonatal restoration of nSR100.................................. 30 2.1.5 Behavior tests..........................................................................................................................................31 2.1.6 Open field..................................................................................................................................................32