Alterations in Transcription and Connectivity in Stem Cell- Derived Neurons of Children with Neurodevelopmental Disorders by Kirill Zaslavsky A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Molecular Genetics University of Toronto © Copyright by Kirill Zaslavsky 2017 Alterations in Transcription and Connectivity in Stem Cell-Derived Neurons of Children with Neurodevelopmental Disorders Kirill Zaslavsky Doctor of Philosophy Department of Molecular Genetics University of Toronto 2017 Abstract Many neurodevelopmental disorders (NDD) have a genetic etiology. Some arise from mutations at a specific locus whereas others are polygenic. For example, Williams-Beuren Syndrome (WBS) is caused by hemizygous deletions at 7q11.23 that result in intellectual disability and increased socialization. In contrast, autism spectrum disorder (ASD) is common, results in impaired socialization, and has a polygenic, sex-dependent pattern of inheritance. While these NDDs involve genes in transcription and synapse function, it has been challenging to discover associated neuronal phenotypes owing to a lack of suitable human tissue. The recent development of human induced pluripotent stem cells (IPSC) allows to bridge this gap by providing a renewable source of neurons specific to individuals with NDDs. To examine transcription, I used IPSCs from a child with WBS and two children with ASD with mutations at the PTCHD1/PTCHD1AS locus, which encodes a protein and long non-coding RNA (lncRNA) of unknown function. In WBS neurons, I found an overall reduction in expression of synapse, voltage-gated potassium channel and extracellular matrix genes. In ASD neurons, I found a reduction in genes involved in RNA splicing, export, cellular respiration, and an increase in ii extracellular matrix genes. The large range of transcriptional alterations suggests that mechanisms of disease in these NDDs are complex and varied. To assess synaptic connectivity in ASD, I compared IPSC-derived neurons from two cases with mutations in SHANK2, a synaptic scaffolding gene, and engineered SHANK2 knockouts to parental and isogenic controls. Because heterogeneity among IPSC-derived neurons can compromise connectivity comparisons, I developed a Sparse coculture for Connectivity (SparCon) assay that increases statistical power by sparsely plating differentially labeled control and mutant neurons onto a lawn of unlabeled control neurons. Using SparCon, I found that dendrite complexity and total synapse number are increased in SHANK2 mutant cells, which was paralleled by increased excitatory synaptic activity. These findings are the first example of excitatory hyperconnectivity in IPSC-derived neurons from subjects with ASD and provide evidence that SHANK2 functions in early neuronal development as a suppressor of dendrite branching. In summary, IPSC-derived neurons are beginning to reveal transcriptional and synaptic neuronal phenotypes in vitro that are associated with NDDs. As the model system matures, methodological refinements like SparCon will further increase the ability of this system to discover disease phenotypes. Investigating alterations in transcription of synaptic genes in WBS, RNA splicing in PTCHD1/PTCHD1AS ASD, and hyperconnectivity in SHANK2 ASD will yield further insight into the pathophysiology of these NDDs. iii Acknowledgments The PhD is a physically and mentally draining process. I would not have been able to complete it without the support of my laboratory, my supervisor, my dear friends, and relatives. In particular, I want to thank my supervisor James Ellis for giving me the opportunity to work in his lab and for allowing me to pursue my ideas. I am indebted to Joel Ross for inspiring me to work in this field. I am lucky I have found a great friend in labmate Deivid Rodrigues, who helped me keep Fresh Start in business by dutifully buying coffee every day and lending a patient ear. He has helped me shape my ideas and has been crucial source of support. I would also like to thank Eric Deneault, for I have not seen anybody that works harder. My work would also not have been possible without the great support of the lab technicians who help us feed and differentiate our cells – namely Alina Piekna, Wei Wei, and Asli Romm. Peter Pasceri, our lab manager, made the lab a welcoming and warm place to work. I also thank Rebecca Mok for helpful discussions on the project. My friends have been an unwavering source of support. I’d like to thank Ilya Mukovozov, who has been by my side for the past 7 years going through MD and PhD together. Richard Wu has been the most amazing workout buddy and has kept me focused. I would be remiss if I did not acknowledge others that have helped me through it all: Rob Vanner for being a great mentor, Brian Ballios for always being an inspiration, Amanda Khan for her unwavering positivity and support, Priya Dhir for listening to me rant about things that almost always turned out better than I expected, and Richard Gao for being my one of my best friends since grade 8. Lastly, this would not have been possible without the support of my parents. I thank my mother, Elena, who is the living embodiment of the American Dream, an immigrant who arrived with nothing and achieved so much. I am also thankful to my father, Ilya, for helpful discussion iv and support. I am constantly inspired by my little brother, Maxim, who is the smartest person I know and at the age of 20 is set to begin grad school. I cannot wait to see what you will accomplish. v Table of Contents Acknowledgments ........................................................................................................................ iv Table of Contents ......................................................................................................................... vi List of Tables ................................................................................................................................. x List of Figures ............................................................................................................................... xi Chapter 1 Introduction ................................................................................................................ 1 1.1 Overview of NDDs ......................................................................................................................1 1.2 Overview of WBS .......................................................................................................................3 1.2.1 Brief History of WBS ..............................................................................................................3 1.2.2 Neurodevelopmental Alterations in WBS ................................................................................4 1.2.3 Genetic Architecture of WBS ..................................................................................................5 1.2.4 Structural brain changes in WBS .............................................................................................7 1.2.5 Transcriptional and connectivity alterations in WBS ..............................................................8 1.3 1.3 Overview of ASD ................................................................................................................10 1.3.1 Brief history of ASD ..............................................................................................................10 1.3.2 Diagnosis and Clinical Presentation ......................................................................................12 1.3.3 Genetic Architecture of ASD .................................................................................................14 1.3.4 Transcriptional and morphological alterations in post-mortem ASD brains .........................20 1.3.5 Genetic evidence for PTCHD1/PTCHD1AS ..........................................................................22 1.3.6 Genetic evidence for SHANK2 ...............................................................................................23 1.4 SHANK proteins are critical for development of synaptic connectivity. ............................24 1.4.1 SHANKs coordinate synapse development and function ......................................................25 1.4.2 SHANK1, SHANK2, and SHANK3 have overlapping and distinct functions. ....................27 1.4.3 Non-synaptic roles of SHANK ..............................................................................................29 1.4.4 Mouse models of SHANK2-associated ASD exhibit inconsistent phenotypes ......................30 1.5 IPSC in NDD modeling & Drug Discovery ............................................................................32 1.5.1 Gene editing in human iPSC ..................................................................................................33 1.5.2 Three Broad Types of Neuronal Differentiation Protocols ....................................................36 1.5.3 Recent Progress in IPSC models of NDDs ............................................................................41 1.5.4 Challenges in IPSC models of NDDs. ...................................................................................43 1.6 1.6 Rationale ..............................................................................................................................46 vi Chapter 2 Transcriptional Alterations in WBS and ASD ....................................................... 49 ....................................................................................................................................................