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Thesis Template Molecular and Synaptic Alterations in the Striatum of GluN1-Deficient Mice by Yuxiao Chen A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Pharmacology and Toxicology University of Toronto © Copyright by Yuxiao Chen 2018 Molecular and Synaptic Alterations in the Striatum of GluN1-Deficient Mice Yuxiao Chen Doctor of Philosophy Department of Pharmacology and Toxicology University of Toronto 2018 Abstract N-methyl-D-aspartate receptors (NMDARs) are essential for proper neurodevelopment and cognitive function. NMDAR dysfunction contributes to many neurological disorders, with striatal changes linked to psychosis and cognitive impairment. GluN1 knockdown (GluN1KD) mice have low NMDAR expression, striatal synaptic deficits, and cognitive behavioural abnormalities. How these phenotypes functionally relate to one another is unclear. We studied molecular and synaptic changes in the GluN1KD striatum to understand the molecular underpinnings of GluN1KD behaviours. From a screen of Rho GTPase pathway proteins, which regulate synapses and cognition, Ras-related C3 botulinum toxin substrate 1 (Rac1) signaling proteins were specifically identified. GluN1KD mice at 6 and 12 weeks of age consistently had less Rac1 and its downstream effector Wiskott-Aldrich syndrome protein-family verprolin homologous protein 1 (WAVE-1). These deficits were developmentally aligned with medium spiny neuron (MSN) spine density deficits and previously published behavioural abnormalities. To assess the biological and behavioural significance of these findings, we restored WAVE-1 levels by intercross-breeding GluN1KD mice and WAVE-1 overexpressing (WAVE-Tg) mice. GluN1KD-WAVE-Tg F1 hybrids had improved WAVE-1 levels and spine density at the striatum, as well as better Y-maze and 8-arm radial maze performance. These phenotypic recoveries were not mirrored by WAVE-1 levels in the hippocampus nor by neuronal dendritic ii spines in cornu ammonis 1. From a broader RNAseq analysis of the GluN1KD striatal transcriptome, both neurodegeneration-related and cell-growth signaling pathways were distinguished as affected by NMDAR knockdown. These transcript changes were greatly sex- dependent, potentially mediated by aberrations of estrogen receptor α signaling. Our data suggest that different molecular changes in male and female GluN1KD mice contribute to similar behavioural abnormalities. Restoring WAVE-1 at the striatum improved some of these phenotypes, pointing to a nuanced relationship between its function, striatal synaptic plasticity, and cognition. Specifically, striatal WAVE-1 and MSN spine density may be important for goal- directed maze exploration in mice. iii Acknowledgments I would like to thank all my friends and family who have supported me in various ways, professors who have guided me throughout my PhD program, and labmates who worked and commiserated with me side-by-side through all the highs and lows. For all my friends and family outside the lab, I have already (I hope, by the time this is submitted) said my thanks for all of your emotional and moral support and for putting up with me. That must not have been easy. Of the professors that I must thank for their guidance over these years, my supervisor Dr. Amy J. Ramsey is first and foremost, of course. I am very thankful to you for the opportunity to conduct research in your lab. Thank you for all of your lessons and advice. You are a wonderful teacher and mentor and I attribute a lot of my development as a scientist and academic to your efforts. Similarly, I would like to thank Dr. Ali Salahpour for day-to-day support and for my overall amazing working experience in the Ramsey-Salahpour lab. I also want to express gratitude to all of my PhD supervisory committee members, past and present: Drs. José N. Nobrega, David S. Riddick, Zhengping Jia, and Jason Matthews. Thank you all for your constructive feedback throughout my PhD program. I know that your comments have helped me tremendously in progressing my research. While you all offered various forms of guidance, I particularly wanted to thank Dr. Nobrega for statistical help, Dr. Jia for in-depth insight into my research topics, and Drs. Riddick and Matthews for grounding my perspective outside of neuroscience research. Finally, I wanted to double-thank Dr. Riddick for being my early thesis reader. I swear I tried to get the best possible draft to you. Finally, I want to thank the labmates who have worked with me in the trenches (and benches) all these years. In particular, I want to show appreciation for all the work done by Marija Milenkovic and Wendy Horsfall in support of my research. Thank you Marija for all of your help with behavioural experiments presented in this thesis, not to mention your previous Rho GTPase work that laid the foundations for my research. Thank you, Wendy, for all the animal husbandry work you do every day, as well as the various other bits you do to support and teach all the students. I also want to recognize all the previous Ramsey-Salahpour graduate students who have been mentors to me in their own right: Kasia Mielnik, Kristel Bermejo, Shababa T. Masoud, Laura M. Vecchio, Vincent Lam, and Pieter Beerepoot. You guys were the best colleagues I could have asked for. iv Table of Contents Acknowledgments.......................................................................................................................... iv Table of Contents .............................................................................................................................v List of Tables ................................................................................................................................. ix List of Figures ..................................................................................................................................x List of Appendices ........................................................................................................................ xii List of Abbreviations ................................................................................................................... xiii Chapter 1 Introduction .....................................................................................................................1 Introduction .................................................................................................................................1 1.1 The N-methyl-D-aspartate Receptor ....................................................................................1 1.1.1 N-methyl-D-aspartate Receptor Characterization and Organization .......................1 1.1.2 NMDAR Activation and Subsequent Downstream Signaling .................................6 1.1.3 Physiological Roles of NMDARs ..........................................................................13 1.1.4 NMDARs in Health and Disease; Links to Neurological Disorders .....................18 1.2 Mouse Model of NMDAR Deficiency by GluN1 Knockdown .........................................25 1.2.1 Background and Overview of GluN1 Knockdown Mice ......................................25 1.2.2 GluN1KD Behavioural Abnormalities and Their Pharmacological Treatments ...28 1.2.3 Cellular and Molecular Changes in GluN1KD Mice .............................................40 1.3 Study Overview .................................................................................................................49 1.3.1 GluN1KD Model Validity, Utility and Research Impact ......................................49 1.3.2 Rationale and Purpose of the Study .......................................................................51 1.3.3 Specific Research Objectives .................................................................................56 Chapter 2 Methods and Materials ..................................................................................................58 Methods and Materials ..............................................................................................................58 2.1 Animals Used in the Study ................................................................................................58 2.1.1 GluN1KD Mice ......................................................................................................58 v 2.1.2 WAVE-Tg Mice.....................................................................................................58 2.1.3 Animal Handling and Ethics ..................................................................................59 2.2 Dendritic Spine Analyses ...................................................................................................59 2.2.1 Dendritic Spine Density Analysis of WT and GluN1KD Mice at Three, Six, and Twelve Weeks of Age .....................................................................................59 2.2.2 Dendritic Spine Density and Morphology Analyses of WT, WAVE-Tg, GluN1KD, and GluN1KD-WAVE Mice ...............................................................60 2.3 Western Blotting ................................................................................................................60 2.4 Fluorescent in situ hybridization via RNAscope ...............................................................61 2.5 Behavioural Tests of Cognition in Mice ............................................................................62 2.5.1 Y-Maze Spontaneous Alternation Test ..................................................................62 2.5.2
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