RELN AS A CANDIDATE GENE FOR AUTISM SPECTRUM DISORDER (ASD) Item Type Dissertation Authors Lammert, Dawn Rights Attribution-NonCommercial-NoDerivatives 4.0 International Download date 08/10/2021 04:24:02 Item License http://creativecommons.org/licenses/by-nc-nd/4.0/ Link to Item http://hdl.handle.net/20.500.12648/1754 RELN AS A CANDIDATE GENE FOR AUTISM SPECTRUM DISORDER (ASD) Dawn Brianna Lammert A Dissertation in the Department of Neuroscience and Physiology Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Graduate Studies of the State University of New York, Upstate Medical University. Approved ______________________________ (Sponsor’s signature) Date__________________________________ i ACKNOWLEDGEMENTS First and foremost I would like to thank my parents Rose and Dennis for supporting me and encouraging me to follow my interests and passions. They have put countless hours and more hard work than could ever be described in the pages of this dissertation into one of the most difficult experiments: raising not just one, but three daughters to question the natural world around them and work to use the knowledge gained from those questions to better humanity. “Nature Dude” taught me to observe the biological wonders that surround us, while my mom showed me that all people, including those with disabilities, are important to our community. It is truly because of them that I have pursued such an ambitious career that combines a love for science, an unrelenting desire for discovery, and a need to share those findings with others in a way that improves the lives of people around me. There are not adequate words to express my thanks to my husband Greg Sevik for accompanying me on this journey to becoming a clinician-scientist. His own doctoral journey and perseverance have been nothing short of inspirational. No prose could adequately convey my gratitude, and I will leave the poetics to the professional. Thank you to all the people I have met on this journey. A PhD is truly a collaborative effort, and I am indebted to many for their guidance and support both personally and professionally: Michele Kyle, Dr. Mary Lou Vallano, Dr. Barry Knox, Karen Gentile, Sharon Longo, Dr. Sarah Reks, Dr. Peter Calvert, Dr. Klaus Werner, Dr. James Traver, Nancy Leotta, and Wendi Burnette. I would be remiss not to thank the DLAR staff, without whom the bulk of this work could not happen. Thank you to the many friends ii and future colleagues: Kimberly Wong, Krysten O’Hara, Dr. Malvina Prapa, Lina Marcela Barrera, Dr. Joel Wilmore, Dr. Darin Dolezal, Dr. Annie Vogel-Cierna, and my Syracuse Track Club family. Thank you to the NINDS for funding this work, and to my program officer Dr. Laura Mamounas for seeing my potential and supporting me early in the project. Thank you to my committee members, who have not only read this work, but have been there over the years for experimental guidance: Drs. Rick Matthews, Eric Olson, Andrea Viczian, and Stewart Loh. I have been fortunate in that a question has never been met with “no” for an answer, but instead mutual interest in solving and exploring any problem. Thank you to Dr. Gabriella D’Arcangelo for agreeing to serve as my outside examiner and more generally for being an exemplary role model for women in science. And finally, but most importantly, thank you to Brian, who truly embodies the archetypal figure of Mentor. My time in lab has been an epic journey, and without his unwavering support, patience, guidance, and wisdom, I would not be the scientist I am today. iii ABSTRACT RELN AS A CANDIDATE GENE FOR AUTISM SPECTRUM DISORDER (ASD) Dawn Brianna Lammert Dissertation Sponsor: Brian W. Howell Autism spectrum disorder (ASD) affects approximately 1 in 45 people, and is characterized by deficits in social communication and repetitive behaviors. Sequencing advancements have enabled the identification of numerous candidate genes, but precisely how these genes contribute to ASD remains largely unknown. RELN is consistently implicated as a candidate gene for autism. The encoded secreted glycoprotein, Reelin is important for proper brain developmental and postnatal synapse function. Here we examine the molecular and cellular consequences of the de novo RELN mutation R2290C. This mutation falls in a conserved arginine-amino acid-arginine (RXR) motif that is found within the Reelin subrepeat structure. Several other ASD patient mutations fall with in this consensus and all examined reduce Reelin secretion. Based on this we tested two hypothesis: (1) that the mutations reduce Reelin signaling and (2) that they have a gain-of-function consequence, such as ER stress. Using an engineered cell line with a heterozygous RELN R2290C mutation and the RELN Orleans (Orl) mouse line that produces nearly full length Reelin that is defective for secretion, we found evidence for both increased Dab1 and increased PDIA1 expression. Since, like most genes implicated in ASD RELN likely acts in a multifactorial manner, we investigated whether second site mutations might contribute to ASD-related iv behaviors. Towards this end we crossed the heterozygous Orl and Shank3b mice to model two hits that are present in at least one ASD proband. We found that the resulting double heterozygous mice had impaired socialization and altered ultrasonic vocalizations. Furthermore, forebrain and cerebellar lysates showed increased PSD-95, identifying a potentially common mechanism and therapeutic target for ASD. These studies are the first to investigate the biological relevance of RELN coding mutations in ASD. v TABLE OF CONTENTS Title Page i Acknowledgements ii Abstract iv Table of Contents vi List of Figures and Tables ix List of Abbreviations xii RELN AS A CANDIDATE GENE FOR AUTISM SPECTRUM DISORDER (ASD) Chapter 1. Introduction: A brief history of RELN gene candidacy in ASD............001 1.1 Defining Autism Spectrum Disorder……………………………………..…………002 1.2 Reelin and neurodevelopment……………………………………………..………..003 1.3 Brain architecture and ASD………………………………………………..………004 1.4 Canonical Reelin signaling and brain development………………………..………006 1.5 Reelin and Dendritogenesis………………………………………………….……..008 1.6 Reelin at the Synapse………………………………………………………….……008 1.7 Autism Network Analysis……………………………………………………….…..010 1.8 Reelin expression in ASD patients……………………………………………….....012 1.9 Reelin methylation………………………………………………………………….012 1.10 Reelin Missense Mutations in ASD…………………………………………….....012 1.11 Experimental Aims of this Dissertation…………………………………………...017 1.12 References………………………………………………………………………....018 Figures and Tables...……………………………………………………………………025 vi Chapter 2. The de novo Autism Spectrum Disorder RELN R2290C missense mutation reduces reelin signaling and increases protein disulfide isomerase expression……………………………………………………………………………....030 Acknowledgement of Authorship………………………………………………………031 Abstract…………………………………………………………………………………032 2.1 Introduction…………………………………………………………………………033 2.2 Materials and Methods……………………………………………………………..035 2.3 Results……………………………………………………………………………....043 2.4 Discussion……………………………………………………………………….….054 2.5 References…………………………………………………………………………..055 Figures and Tables……………………………………………………………………...061 Chapter 3. Heterozygous RELN and SHANK3 mutations cooperate to drive abnormal vocalization and socialization behaviors in mice………….……………..076 Abstract…………………………………………………………………………………077 3.1 Introduction………...……………………………………………………………….078 3.2 Materials and Methods…………..…………………………………………………081 3.3 Results…………………………………...………………………………………….086 3.4 Discussion………………………………..…………………………………………095 3.5 References………………………………………………………….……………….099 Figures and Tables……………………………………………………………………...103 Chapter 4. General Discussion 129 4.1 The importance of understanding ASD genetics……………………………………130 4.2 RELN as a candidate gene for ASD – Reelin as a candidate protein for ASD……..131 4.3 Future directions toward understanding of PDIA1 overexpression………………..133 4.4 Reelin, the synapse, and postnatal cerebellar expression….………………………135 4.5 RELN and the “two-hit” ASD hypothesis: synaptic disruption…………………….135 vii 4.6 Social communication and language deficits in mouse models of ASD……………137 4.7 Social motivation deficits in ASD…………………………………………………..139 4.8 Increase PSD-95 in the Orl+/-;Shank3b+/- mouse and the PSD-95 impaired turnover theory…………………………………………………………………………………...141 4.9 The future direction of ASD research………………………………………………144 4.10 References…………………………………………………………………………146 Appendix I: Supplemental RNAseq data……………………………………………152 Appendix II: P19 neurosphere characterization……………..……………………...166 Appendix III: RELNnull+/-;Shank3b+/- preliminary data…………………………..…169 Appendix IV: Increased PSD-95 may correlate with Purkinje cell dysfunction….176 Appendix V: Exploring the possibility of targeting Reelin secretion with small molecule therapeutics…………………………………………………………………179 viii LIST OF FIGURES AND TABLES FIGURES Chapter 1. 1.1 Reelin signaling and ASD-associated proteins at the synapse……………………...025 1.2 RELN missense mutations identified in ASD……………………………………...028 Chapter 2. 2.1 The RELN R2290C and other ASD mutations reside in a conserved RXR consensus and impair Reelin secretion. …………………………………………………………...061 2.2 Decreased extracellular Reelin expression by R2290C+/- compared to wild-type neurospheres……………………………………………………………………………064 2.3 Reelin repeat fusion proteins (RRfps) with ASD RXR mutations are less abundant in the media as compared with their wild-type counterparts……………………………...067 2.4