Functional Characterization of Epilepsy Associated GABRG2 Mutations By Mengnan Tian Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University in Partial Fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Pharmacology August, 2012 Nashville, Tennessee Approved: Robert L. Macdonald, M.D., Ph.D. Ronald Emeson, Ph.D. Kevin Ess, M.D., Ph.D. Katherine T. Murray, M.D. Douglas P. Mortlock, Ph.D. To my grandparents, Yunbo Tian and Zhenhua Cao. ii Acknowledgements Throughout my years pursuing Ph.D. degree, I have always been grateful for the opportunity working with an amazing complement of scientists. I would like to express my sincerest gratitude to my thesis advisor Dr. Robert Macdonald. He is by far the best mentor that I could have ever wished. He has provided me with excellent guidance and support, and given me the confidence to develop into an independent scientist. He granted me unprecedented freedom to explore new scientific fields and implement novel research strategies and techniques. I greatly appreciate all his trust in me, and it has fostered my confidence in performing researches. He has shown me, by his example, what a good scientist and mentor should be. I would like to thank my colleagues and collaborators in Macdonald lab. I have been collaborating with Xuan Huang since her rotation and after she joined our lab in 2009. We have also become good friends. She has made important contributions to many parts of my thesis studies, and provided brilliant critiques to help me improve the research plan. I am also deeply indebted to Dr. Ciria Hernandez, Ningning Hu, Wangzhen Shen, Kelienne Verdier, Dr. Emmanuel Botzolakis, Kate Gurba, Dr. Aurea Pimenta and Dr. Huajun Feng, who contributed their expertise and generated necessary reagents for my thesis studies. I would like to thank our collaborators Drs. Andre Lagrange and Martin Gallagher. Andre and Martin are always ready to share expertise or constructive iii criticism, and provided valuable help in my experiments. Andre also generously spent several months to teach me patch clamp recording technique. I would like to thank members of my thesis committee: Drs. Ronald Emeson, Douglas Mortlock, Kevin Ess, Kathy Murray and Aurelio Galli. I really appreciate the time and effort that they took to offer me helpful suggestions. Dr. Douglas Mortlock shared the BAC recombineering protocol with me and taught me this technique. I would like to thank Pharmacology department, especially Drs. Heidi Hamm and Joey Barnett, for all the help that they provided during my years in Pharmacology graduate program. I have experienced many obstacles because of my lack of experience. They spent their time talking to me and helping me to figure out the best solution. If it were not for their help, I would not have survived all these troubles and finished my thesis studies. In the end, I would like to thank my families for love and emotional support. I am greatly in debt to my grandparents, Yunbo Tian and Zhenhua Cao. They brought me up since I was less than 100 days old, at the time when they were in mid-sixties. I lived with them until I went to college. They gave me all the love that they have, provided me very good education, and passed to me our family values. They are the most important influence in my whole life. Grandma Zhenhua Cao passed away in 2001 at age of 87, and grandpa Yunbo Tian passed away last year at age of 99. It is my saddest regret that I am not able to hand this thesis to them and see how happy they would be. Finally, I would like to give my special thanks to my wife, Na Tan, for sharing happiness, sadness, and hope with me. Her continuous love, support, and encouragement are with me wherever I go and whatever I pursue. iv TABLE OF CONTENTS Page DEDICATION ............................................................................................................... ii ACKNOWLEDGEMENTS .......................................................................................... iii LIST OF FIGURES ....................................................................................................... xi Chapter 1. Introduction: GABAA Receptors and Epilepsy ......................................................1 Epilepsy ...........................................................................................................................1 Seizures ...............................................................................................................2 Etiologies of epilepsy ..........................................................................................4 International classification of epilepsies and epileptic syndromes .....................6 Idiopathic/Genetic Epilepsies ..........................................................................................8 Monogenic Epilepsies .........................................................................................9 Polygenic Epilepsies .........................................................................................10 GABAA Receptors.........................................................................................................10 Antiepileptic drugs that work on GABAA receptors .........................................12 GABAA receptor assembly and trafficking .......................................................15 GABAA receptor assembly in the endoplasmic reticulum (ER) ...........15 GABAA receptor trafficking in the Golgi apparatus .............................19 Golgi-to-plasma membrane translocation .............................................20 GABAA receptor clustering at synapses. ...............................................24 GABAA receptor endocytosis and degradation. ....................................28 GABAA receptor epilepsy channelopathies ..................................................................30 Overview ...........................................................................................................30 GABRG2 gene mutations. .................................................................................32 GABRG2 gene expression pattern and γ2 subunit function...................32 v GABRG2 gene missense mutations. ......................................................38 GABRG2 gene nonsense mutations. ......................................................44 GABRG2 gene intron splice donor site mutation. .................................47 GABRA1 gene mutations. ..................................................................................48 GABRA1 gene expression pattern and α1 subunit function. ..................48 GABRA1 gene missense mutations. .......................................................51 GABRA1 gene deletion mutations. ........................................................53 GABRB3 gene mutations. ..................................................................................54 GABRB3 gene expression pattern and β3 subunit function. ..................54 GABRB3 gene mutations. ......................................................................57 GABRD variants. ...............................................................................................59 GABRD gene expression pattern and δ subunits function. ....................59 GABRD gene variants. ...........................................................................60 2. The Intronic GABRG2 Mutation, IVS6+2TG, Associated with CAE Altered Subunit mRNA Intron Splicing, Activated Nonsense-Mediated Decay and Produced a Stable Truncated γ2 Subunit ..........................................................62 Abstract .........................................................................................................................63 Introduction ...................................................................................................................64 Materials and Methods ..................................................................................................67 Expression vectors with GABAA receptor subunits ..........................................67 Cell culture, transfection and RNAi ..................................................................69 RNA extraction, RT-PCR and Taqman real-time qPCR ..................................70 Generation and maintenance of hGABRG2 BAC transgenic mice ...................71 Transgenic mouse genotyping PCR ..................................................................72 Immunohistochemistry ......................................................................................73 Immunocytochemistry and confocal microscopy .............................................74 Flow cytometry .................................................................................................76 Immunoblotting .................................................................................................77 Glycosidase Digestion .......................................................................................78 Immunoprecipitation .........................................................................................79 Electrophysiology .............................................................................................79 vi Statistical analysis .............................................................................................80 Results ...........................................................................................................................81 The GABRG2(IVS6+2TG) mutation generated a mutant hGABRG2(IVS6+2TG)