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SEX-LINKED NEUROLIGINS AND THEIR ROLES AT SYNAPSES A Dissertation submitted to the Faculty of the Graduate School of Arts and Sciences of Georgetown University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Pharmacology By Thien Anh Nguyen, B.S. Washington, D.C. March 3, 2020 Copyright 2020 by Thien A. Nguyen All Rights Reserved ii SEX-LINKED NEUROLIGINS AND THEIR ROLES AT SYNAPSES Thien A Nguyen, B.S. Thesis Advisor: Katherine W. Roche, PhD ABSTRACT Autism spectrum disorder (ASD) is a neurodevelopmental disorder that results in social- communication impairments, restricted and repetitive behaviors, and is more prevalent in males. Although the underlying etiology of ASD is generally unknown, a cell adhesion molecule, Neuroligin 4X (NLGN4X) located on the X chromosome, has been specifically linked with this disorder. The male-specific NLGN4Y, the NLGN4X homolog, is thought to be functionally redundant, but this assumption has not been experimentally tested. In this dissertation, we report NLGN4X and NLGN4Y are functionally different. NLGN4Y does not effectively traffic to the surface or induce synapses. We show that a critical amino acid difference in the extracellular domain (ECD) between NLGN4X (P93) and NLGN4Y (S93) leads to this differential function. In addition, we show that NLGN4X is robustly phosphorylated by protein kinase A and protein kinase C, whereas NLGN4Y cannot. The differential phosphorylation is due to one amino acid difference in the intracellular domain (ICD) between NLGN4X (R710) and NLGN4Y (H710). Together, we demonstrate that NLGN4X and NLGN4Y do not share the same function. Interestingly, many ASD-associated variants have been identified in NLGN4X in the region surrounding P93, and there is a decrease in normal population variation in this region. In addition, we describe two new variants found in probands with ASD and intellectual disability (ID), NLGN4X R101Q and V109L. Intriguingly, several of the ASD-associated mutations in iii NLGN4X are found in males with mothers who are carriers. Furthermore, an ASD-associated mutation in the ICD of NLGN4X disrupt both PKC and PKA phosphorylation. Taken together, our results reveal that NLGN4Y cannot compensate for NLGN4X function due to a defect in trafficking to the cell surface and phosphorylation, revealing potential pathogenic mechanisms for male bias in NLGN4X-associated ASD cases. iv ACKNOWLEDGEMENTS First and foremost, I would like to express my gratitude to my mentor and thesis advisor, Dr. Katherine Roche. For the last five years, Dr. Roche had provided me with her expertise and guidance in the design and construction of well-controlled experiments. With her valuable experience and insight, I developed creative approaches in the exploration of synapse functions and critically analyzed the results. In addition to her research and experience in academia, Dr. Roche’s dedication and enthusiasm for collaboration has demonstrated how cooperation can facilitate progress and accelerate scientific discoveries. Through her encouragement, I had the incredible opportunity to meet and collaborated with many extraordinary scientists. In addition to being my scientific mentor, she understands the need for a healthy work-life balance, and allows me to visit my late grandmother yearly, and for that, I am eternally grateful. I could not have asked for a better mentor; Dr. Roche has had a profound impact upon my academic and professional development I would like to thank all of the members of Roche Lab for their contribution to my research, but above all else, I am grateful for their friendship. In particular, Drs. Michael Bemben and Sehoon Won who have trained and advised me in biochemical techniques; John D Badger II who has helped with neuronal and animal experiments; Drs. Richa Lomash and Jaehoon (Jwood) Jeong for their help with immunocytochemistry experiments; my summer student Lex Lehr for his help with biochemical experiments; and especially to the Bay of Pigs members, Drs. Marta Vieira and Jeremiah Paskus, for their advice on experimental designs and many discussions about science, life and everything else in between. I would also like to thank the many collaborators whom I have encountered to complete this thesis research. I would like to thank Drs. Kunwei Wu and Saurabh Pandey for providing electrophysiology data; Drs. David Wang and Kareem Zaghloul for providing human differentiated neurons and human brain samples; Dr. Mahim Jain and Dr. Audrey Thurm for their help in analyzing human genetic data; and I would like to thank the families that reached out to us and allowing us to study their genetic variants. I would like to express my appreciation to the faculty members Drs. Barry Wolfe, Daniel Pak, Robert Yasuda, Katherine Conant, and Wei Lu at Georgetown University and the National Institutes of Health that contributed to this dissertation are. I am grateful to have had each of them on my thesis committee and for their guidance throughout my thesis research Nineteen years ago, when I moved to the USA from Vietnam, I never thought I would be here today. I would like to thank all of the wonderful teachers and the mentors for their patience, their support, their kindness, and for everything they have taught me. I want to thank Dr. Lee Graves for giving the very first opportunity to work in a research lab, and thank you to Dr. Silvia Kreda, for allowing me to grow as a scientist and encouraged me to pursue graduate school. I want to thank all my friends, especially Tai Nguyen and Kang He, for their continuing encouragement and support. v Finally, I would not be where I am today without the love and support of my family. I am deeply grateful for my parents, Quan and Dang Nguyen, for their love, support, and the countless sacrifices they had made and the hardship they endured to grant me the freedom and the opportunity to pursue what I love. To my grandparents, Nam Nguyen and Phuong Dien, and my late grandmother, Hieu Nguyen, I am thankful for their unconditional love. Lastly, I would like to thank many members of my family, especially Nhut Nguyen, Lien Nguyen and Ann Nguyen, for continuing to be my biggest advocates. Con rất muốn cảm ơn ba mẹ đã cho con rất nhiều niềm thương, giúp đỡ, và đã dậy dỗ con nên người. Con rất quý trọng những hy sinh ba mẹ đã phải trãi qua để con có được sự tự do theo đuổi những gì con yêu thích. Con cũng rất chân trọng sự giúp đở và tình thương của ông bà nội và bà ngoại dành cho con. Con muốn cảm ơn sự giúp đở do cô dượng ba nói riêng, và cả gia đình nói chung dành cho con để con có thể theo đuổi sự nghiệp của mình. Con không có thể nên người nếu không nhờ sự hỗ trợ của mọi người. Many thanks, Thien Anh Nguyen vi TABLE OF CONTENTS Chapter I: INTRODUCTION .......................................................................................... 1 SYNAPSES AND THE POSTSYNAPTIC DENSITY ............................................... 1 COMPONENTS OF EXCITATORY SYNAPSES ..................................................... 2 NEUREXINS ............................................................................................................... 7 NEUROLIGINS AT SYNAPSES ............................................................................... 8 HUMAN AND RODENT NEUROLIGIN-4 ............................................................. 13 AUTISM SPECTRUM DISORDER AND INTELLECTUAL DISABILITY ......... 16 NEUROLIGINS AND ASD/ID ................................................................................. 18 NEUROLIGIN 4Y AND ITS FUNCTION ............................................................... 23 Chapter II: MATERIALS AND METHODS ................................................................ 30 PLASMIDS AND ANTIBODIES ............................................................................. 30 CELL CULTURE AND TRANSFECTION ............................................................. 33 GENE EXPRESSION ................................................................................................ 34 DISSOCIATED NEURONAL CULTURES ............................................................. 34 IMMUNOFLUORESCENCE MICROSCROPY ...................................................... 35 NEURONAL DIFFERENTIATION FROM IPSCS ................................................. 36 HUMAN BRAIN SYNAPTOSOMES ...................................................................... 37 IMMUNOBLOTTING .............................................................................................. 38 CO-IMMUNOPRECIPITATION .............................................................................. 38 BIOTINYLATION ASSAY ...................................................................................... 39 COCULTURE ASSAY ............................................................................................. 40 LABEL-FREE MASS SPECTROMETRY ............................................................... 40 ELECTROPHYSIOLOGY ........................................................................................ 42 NOVEL MUTATION IDENTIFICATIONS ............................................................ 42 STATISTICAL ANALYSIS ..................................................................................... 43 GST-FUSION PROTEIN PRODUCTION AND IN VITRO PHOSPHORYLATION ............................................................................................. 43 Chapter III: FUNTIONAL DIFFERENCES BETWEEN NLGN4X AND NLGN4Y .. 45 INTRODUCTION ....................................................................................................
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