Neto1 and Neto2 are Auxiliary Subunits of Synaptic Kainate Receptors by Man Tang A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Molecular Genetics University of Toronto © Copyright by Man Tang 2013 Neto1 and Neto2 are Auxiliary Subunits of Synaptic Kainate Receptors Man Tang Doctor of Philosophy Graduate Department of Molecular Genetics University of Toronto 2013 Abstract Neto1 and Neto2 are CUB domain-containing transmembrane proteins that are expressed in the mammalian brain. Previous studies showed that Neto1 is a NMDAR-associated protein with important roles in synaptic plasticity and learning/memory (Ng et al., 2009). To establish the functions of Neto2, I first searched for its binding partners. Using yeast two-hybrid analysis, GST pull-down and co-immunoprecipitation studies, I found that Neto2 can bind to the PDZ domain-containing protein GRIP. In the brain, GRIP regulates the synaptic trafficking and stability of AMPA and kainate receptors (KARs) (Hirbec et al., 2003). To determine whether Neto2 is required for the synaptic expression of KARs and/or AMPARs, I examined whether Neto2 was associated with these receptors at the postsynaptic membrane. Coimmunoprecipitation studies showed that while Neto2 is a component of postsynaptic KAR protein complexes, it is not associated with AMPARs. In the cerebellum, Neto2-null mice showed a 44% (n=3;p<0.01) decrease in the abundance of postsynaptic KARs with no change in the level of total KARs, thus suggesting a specific deficit in KAR synaptic localization. Unexpectedly, loss of Neto2 had no effect on the abundance of hippocampal KARs (n=3; p>0.05), or on neurotransmission by them (n=12; p>0.05). To determine whether this normal KAR function might be due to compensation by Neto1, which also interacts with KARs, I ii examined KAR abundance in Neto1-null, and Neto1/2-double null hippocampus. Loss of Neto1 resulted in a 53% decrease in postsynaptic levels of GluK2-KARs (n=3;p<0.01). However, in double null animals, the reduction was indistinguishable from Neto1 single null mice, suggesting that Neto2 is not involved in the postsynaptic localization of hippocampal KARs. In Neto1-null mice, KAR-mediated currents showed smaller amplitude (61% of wild- type;n=14;p<0.01), and faster decay kinetics (40% of wild-type;n=14;p<0.001). Together, these findings establish both Neto1 and Neto2 as auxiliary proteins of native KARs: Neto1 regulates the synaptic abundance and kinetics of KARs in the hippocampus, while Neto2 mediates the synaptic localization of cerebellar KARs. Additionally, the results presented here, in conjunction with previous findings, reveal a unique ability of Neto1 in controlling synaptic transmission by serving as an auxiliary protein for two different classes of ionotropic glutamate receptors. iii Acknowledgements Being part of the McInnes lab has been a truly fantastic experience for me. Not only did it open my eyes to a wonderful and challenging field of science, but it allowed me to work in a collaborative environment with many intelligent, kind, and fun people. As I approach the end of my graduate journey, I would like to thank all of the people who have helped me along the way. I would not be completing my dissertation today without your guidance, support, and friendship. First, and foremost, my deepest gratitude goes to my wonderful supervisor Roderick McInnes. Rod is a fabulous mentor who has expertly guided me through my graduate education. His unwavering passion and enthusiasm for science have been an inspiration for the career I have chosen to follow. Thank you Rod for taking me on as an international student, and giving me the opportunity to work with you; for being a supportive advisor who continuously helped and challenged me to become a better scientist, writer, and public speaker; for believing in me and genuinely caring about my career development, and for always making sure I had a place to be during the holiday seasons. I would also like to thank my supervisory committee members Dr. Mike Salter, and Dr. Sabine Cordes for their advice and encouragement. I feel very fortunate to have you both in my committee!! In the last two years of my PhD, Dr. Salter has also taken on a co-supervisory role, and has provided invaluable input and established critical collaborations that made the Neto studies possible. Living away from home as a grad student, the McInnes lab has become my second family. I’d like to first thank all the past and present members of the super-awesome Neto team, Rachel Szilard, Dave Ng, Zhenya Ivakine, Jeff Gingrich, and Vivek Mahadevan. Rachel is the most helpful, patient, and knowledgeable biochemistry “lab consultant” I’ve ever met. Her iv guidance (and world’s most thorough protocols!) has been instrumental in getting the project moving during my first two years in the lab. I thank you for your continued friendship, support, and advice! Dave, the “father of Netos” has been a wonderful resource of all knowledge on Netos. I truly enjoyed the many hours we spent brainstorming ideas and discussing new avenues to take. Zhenya has been a most wonderful mentor, scientific advisor, “archenemy”, and partner in crime. Thank you for teaching me so much about science, and about life, and for giving me the confidence to pursue my dreams. I feel truly blessed to have your friendship! Vivek was the last member to join the Neto team but his immense enthusiasm for neuroscience research is unrivalled, and his perseverance and positive attitude, admirable. Thank you for bringing so much joy, and laughter into the lab, and for always cheering me on! I’d also like to thank Jeff, our lab’s “walking encyclopedia” for sharing his expertise and knowledge in neuroscience. I’m grateful to Irene Chau, Cynthia Jung, Coco Jiang, and Alexa Bramall for their friendship, to Lynda Ploder for her support on the project, and Dorothy Carlin for helping me set up all my meetings. I am also extremely grateful for my phenomenal collaborators, Dr. Ken Pelkey and Dr. Chris McBain. Ken has done a magnificent job on all the electrophysiology experiments needed for this project, and has generously devoted his time to review and provide feedback on my manuscripts. Our first paper would not have been possible without his timely contribution. Finally, I’d like to thank my family for teaching me the value of hard-work, and the importance of integrity; and for supporting me throughout this very long journey. v Table of Contents ABSTRACT……………………………………………………………………………………………………………………………………………….ii ACKNOWLEDGEMENTS……………………………………………………………………………………………………………….…………iv LIST OF TABLES………..……………………………………………………………………………….……………………………………………ix LIST OF FIGURES…………………………….………………………………………………………………………………………………………ix LIST OF APPENDICES………………………………………………………………………………………………………………………………xi FREQUENTLY USED ABBREVIATIONS………………………………………………………………………………………….………….xii Chapter 1: Introduction .............................................................................................................................1 1.1. Introduction to the mammalian central nervous system ...................................................................2 1.1.1. The hippocampus structure .......................................................................................................2 1.1.2. Basic hippocampal neural pathways .........................................................................................6 1.1.3. The cellular organization of the cerebellum ..............................................................................9 1.1.4. The basic cerebellar circuitry ................................................................................................. 12 1.2. Neuronal communication at chemical synapses ............................................................................ 15 1.2.1. Structure of a chemical synapse ............................................................................................. 16 1.2.2. Chemical synaptic transmission in the brain .......................................................................... 17 1.2.3. Ionotropic glutamate receptors ............................................................................................... 20 1.3. Kainate Receptors ......................................................................................................................... 22 1.3.1. KAR subunits: general structure and biophysical properties ................................................. 23 1.3.2. KAR pharmacology ............................................................................................................... 26 1.3.3. KAR expression, protein distribution and trafficking ............................................................ 27 1.3.4. Neuronal function of KARs ................................................................................................... 33 1.3.5. KAR interacting proteins ....................................................................................................... 38 1.3.6. KARs and disease .................................................................................................................. 40 1.4. The Neto family of transmembrane proteins ................................................................................. 41 1.4.1. Domain structure and organization ........................................................................................ 42 1.4.2. Expression of Neto1 and Neto2 in the CNS ..........................................................................