THE NEUREGULIN GROWTH FACTORS AND THEIR RECEPTOR ERBB4 IN THE DEVELOPING BRAIN: DELINEATION OF NEUREGULIN-3 EXPRESSION AND NEURITOGENESIS Afrida Rahman Submitted to the faculty of the University Graduate School in partial fulfillment of the requirements for the degree Doctor of Philosophy in the Department of Psychological & Brain Sciences and Program in Neuroscience, Indiana University June 2019 Accepted by the Graduate Faculty, Indiana University, in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Doctoral Committee ____________________________________ Anne L. Prieto, Ph.D. ____________________________________ Andrea Hohmann, Ph.D. ____________________________________ Cary Lai, Ph.D. ___________________________________ Kenneth Mackie, M.D. April 17, 2019 ii Dedicated to my parents, Nasima and Asirur Rahman, Who left everything in Bangladesh to come to America for a better life. Your courage has taught me to always be fearless. Thank you. iii Acknowledgements I would first and foremost like to express my heartfelt gratitude to Dr. Anne Prieto for giving me the opportunity to work in her laboratory and for offering her continuous mentorship. Her patience in teaching and constructive assessments of my science helped shape my critical thinking skills, my aptitude to do research, and my ability to teach. Her constant support and push also gave me the motivation and confidence to continue and finish my Ph.D. I would also like to thank my committee members who have dedicated their time to serve on my committee: Dr. Cary Lai, for essentially being another mentor to me and always providing me with Oreos and chocolate; Dr. Andrea Hohmann, for all her great experimental suggestions and for always supporting my immunofluorescence artwork; and Dr. Ken Mackie, for giving me countless troubleshooting advice and introducing me to calcium phosphate transfection. Furthermore, I would like to thank the Department of Psychological and Brain Sciences (PBS) and Program in Neuroscience (PNS) for years of financial support, travel grants, and opportunities to further my teaching aspirations. I would also like to thank the administrative, business, and technical support staff in PBS and PNS, especially Patricia Crouch, JeanneMarie Heeb, Amy Holtzworth-Monroe, Misti Bennett, LeAnna Faubion, Misty Theodore, Susanne Kindred, and Faye Caylor, for their willingness to help me whenever they could. Additionally, I would like to express gratitude to the Office of the Provost and Center of Excellence for Women in Technology (CEWiT) for their travel support and opportunities they have presented to me and other women in science. I am also fortunate to have made several close friends and a solid support group during my graduate career including, Erika Perez, Dr. Richard Slivicki, Dr. Justin Bollinger, Lawrence Carey IV, and Nathan Berbesque. The countless shenanigans that we’ve been a part of are some of my favorite memories and their friendships have meant absolutely everything to me. I iv would also like to thank past and current members of my lab, especially Natalie Wallace, Sarah Huber, Jeff Yu, Grace Shariat Panahi, Sarah Hocevar, and Ashwin Biju for always being entertaining lab mates and providing me with a great deal of assistance with my experiments. A special “thank you” to Meredith Krodel for not only being an amazing role model and teacher, but for also becoming my friend in the process. Lastly, I would like to thank several people that I love, especially Mom, Dad, Aftab, and Micah. Each one of you has played such a crucial role in my life through loving me, taking care of me, overprotecting me, and supporting me. You have all gotten me though some of my roughest days and have also been there to celebrate all the good ones. I will always be grateful that I have you in my life and know that I support each one of you in everything you do. v Afrida Rahman THE NEUREGULIN GROWTH FACTORS AND THEIR RECEPTOR ERBB4 IN THE DEVELOPING BRAIN: DELINEATION OF NEUREGULIN-3 EXPRESSION AND NEURITOGENESIS The developing brain is a highly dynamic structure, making it repeatedly vulnerable to possible developmental abnormalities that can endure into adulthood. The neuregulin (Nrg) family of growth factors (Nrg1-4) and their receptor, ErbB4, have been linked to mental illnesses considered to emerge as a result of abnormal neural development, such as schizophrenia, bipolar disorder, and depression. In the normal central nervous system (CNS), Nrg-ErbB ligand- receptor pairs regulate several cellular functions integral to neural development, including the enhancement of neurite extension and roles in polarity-driven events such as neuronal migration and the establishment of radial glial morphology. Due to the roles of Nrg-ErbB pairs in the healthy and malignant brain, the aims of this thesis were to address fundamental gaps in knowledge and to expand on the roles of the Nrgs and ErbB4 in neural development. Towards these goals, we 1) characterized the spatiotemporal localization of Nrg3, an understudied Nrg, in the rat brain, 2) defined and characterized the effects of Nrgs1-3 and ErbB4 in neurite outgrowth of GABAergic interneurons, and 3) studied the potential role of Nrg-ErbB4 signaling in neuronal polarization through its interaction with the Par polarity complex. The results from this thesis revealed that the Nrgs and ErbB4 play important roles in the development of the CNS, specifically in the differentiation of GABAergic interneurons. Nrg3 was found to be the most widely expressed Nrg in CNS neurons, including GABAergic interneurons, and likely plays non- overlapping functional roles with the other Nrgs. Our studies also revealed that Nrgs1-3 do play redundant roles in neurite development of early GABAergic interneurons by increasing the number of neurites and dramatically enhancing neurite length, including axonal elongation. These effects were driven by the activation of ErbB4. Lastly, using biochemical and vi immunocytochemical techniques, we uncovered a novel Nrg-dependent association of ErbB4 with members of the Par polarity complex (Par6, Par3, aPKC, and Cdc42), a group of proteins important for radial glial morphology, neuronal migration, and axonal specification. Collectively, the findings of this thesis highlight biological processes of the Nrgs and ErbB4 in the developing CNS, including roles in neuronal differentiation. In addition, these findings may contribute to the development of promising therapies addressing ErbB4-driven psychiatric disorders, such as depression, bipolar disorder, and schizophrenia. ____________________________________ Anne L. Prieto, Ph.D. __________________________________ Andrea Hohmann, Ph.D. ____________________________________ Cary Lai, Ph.D. ___________________________________ Kenneth Mackie, M.D. vii Table of Contents Table of Contents....................................................................................................................... viii List of Figures and Tables ......................................................................................................... xv List of Abbreviations ................................................................................................................. xix Chapter 1: Introduction .................................................................................................................1 1.1 Neural Development.........................................................................................................1 1.1.1 Cortical Interneuron Subtypes ................................................................................3 1.1.2 Stages of Neuronal Development in Culture .........................................................4 1.1.3 Neurite Outgrowth and Axon Formation ................................................................7 1.1.4 Neuronal Polarity and the Par Polarity Complex ................................................. 12 1.2 The Neuregulin (Nrg) Family of Growth Factors........................................................... 20 1.2.1 Discovery and Isolation of the Nrgs ..................................................................... 21 1.2.2 Molecular Structures of Nrg1-3 and Their Isoforms ............................................ 22 1.2.2.1 Molecular Architecture and Alternative Splicing .................................... 22 1.2.2.2 Proteolytic Cleavage ............................................................................... 23 1.2.2.3 Glycosylation .......................................................................................... 24 1.3 The ErbB Receptor Tyrosine Kinase Family ................................................................ 26 1.3.1 ErbB4 Alternative Spliced Variants ...................................................................... 27 1.4 Functions of Nrg-ErbB Signaling Outside of the CNS .................................................. 32 1.4.1 Heart Development .............................................................................................. 32 viii 1.4.2 Breast Development ............................................................................................. 33 1.4.3 Schwann Cell Development and Myelination ...................................................... 36 1.5 Functions of Nrg-ErbB Signaling in the CNS ................................................................ 39 1.5.1 Synaptic Formation .............................................................................................. 39 1.5.2 Neurotransmission