ANALYSIS OF Pcp-2/L7 GENE EXPRESSION AND FUNCTION DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Yelda Serinagaoglu, B. Sc. * * * * * The Ohio State University 2007 Dissertation Committee: Dr. John Oberdick, Advisor Approved by Dr. Heithem El-Hodiri Dr. Anthony P. Young Advisor Dr. Mike Zhu Molecular, Cellular, and Developmental Biology Graduate Program ABSTRACT The mouse Purkinje cell protein-2 (Pcp-2/L7, herein called L7) gene is specifically and abundantly expressed in cerebellar Purkinje cells and in retinal bipolar neurons. Recent studies suggested that L7 functions in the tuning of P/Q type Ca2+ channels by the modulation of G-protein coupled receptors. The goals of the present studies were to extend our understanding on L7 function and control of L7 gene expression. To better understand function of the L7 protein, we carried out a variety of behavioral tests to compare wild type and L7 knock-out (L7KO) mice. We show that L7KO mice have improved performance on accelerating rotarod and display sexually dimorphic sensorimotor behavioral changes. Our results support the idea that cerebellum plays a role in sensorimotor gating, and it functions in the mediation of sensory response, which is a non-traditional role for the cerebellum. We then expanded our studies to investigate L7 gene expression in the mouse cerebellum and eye. Here we report that a third isoform of L7, which we call L7C, is the only form expressed in the eye, while two isoforms, L7A and L7B, are abundantly found in the cerebellum. To determine the molecular and genetic ii mechanisms of the Purkinje cell-specific expression of L7, we carried out in vivo analyses where we show that L7 structural gene does not have any Purkinje cell-enhancement activity, and likely contains significant repressive activity. 0.9 kb L7 proximal promoter, on the other hand, acts as an enhancer to direct expression in the cerebellar Purkinje cells. This enhancer activity requires the position-dependent action of the structural gene. In the present study, we show that retinoic acid receptor related orphan nuclear receptor-alpha (RORα) is a major activator of L7 gene expression in vivo and in vitro. Work presented here will allow us to better understand the relationship between the cellular physiology and the animal behavior. Although previous studies with RORα focused on its role during the cerebellar development, our studies show that RORα is no longer just a cerebellar developmental control molecule, but a key determinant of cerebellar physiology, as it activates L7, which in turn modulates Ca2+ channels. iii Dedicated to my family iv ACKNOWLEDGMENTS First and foremost I would like to thank my advisor Dr. John Oberdick, for his advice, support, patience, and guidance throughout my graduate study. I would like to express my gratitude for my committee members Dr. Heithem El-Hodiri, Dr. Anthony Young, and Dr. Mike Zhu for all the help and advice. I would also like to thank Dr. David Saffen for the advice and support during my candidacy exam. I am deeply indebted to Dr. Dave Bisaro for giving me the opportunity to be a member of the MCDB program. My deepest thanks go to Jan Zinaich, who always greeted me with her warm smile and has always helped me. I would like to express special thanks to the past and current staff members of the Center for Molecular Neurobiology, Keri Bantz, Scott Hines, Darlene Jackson, Juli Kanoski, Dave Long, Paula Monsma, and Laura Richter for taking care of everything for us in Rightmire Hall. I would like to thank the members of Rightmire Hall and Pressey Hall Animal Facilities for taking good care of mice. I am also grateful to Dr. Xin-An Pu for all she has done in our transgenic studies, and for taking time to answer my questions. I would especially like to thank past and current members of the Oberdick lab, Jamie Depelteau, Nichole Gebhart, Peijun Wu, Rui Zhang, and Xulun Zhang for sharing their knowledge and experience, and creating a fun and peaceful place to work at. v Special thanks go to members of Dr. Tsonwin Hai’s lab, Dr. Tony Brown’s lab, Dr. Randy Nelson’s lab, and Dr. Mike Zhu’s lab for technical support, collaborations, and scientific/non-scientific conversations. I would like to send my appreciation to my friends in Turkey and in Germany, Fulya Akyar, Arzu Baysal, Aydan Işık, İpek Kayhan, and Yasemin Tosun, for cheering me up with phone calls and e-mails from overseas. I do not know how to express my deepest appreciation for Ozan Özkuşaksız, for endlessly giving me the hope for a much better future. My sincere thanks go to my friends in the USA, Sinan Doğan, Yeşim Çapa-Aydın, Tolga Aydın, Özlem Birgül, Gülsen Çolakoğlu, Suat Gümüşsoy, and İlter Sever, for being my friends and making my life much happier. I also thank Tankut Taylan for the long conversations and for patiently helping me with computer software. I am eternally grateful to my family for their endless love, support, and encouragement. My mother Ayşe, my father Mehmet, my sister Yeşim and my brother-in-law Ahmet, words are not enough to thank you. My caring fiancé David, your love and support helped make this possible. Thank you for always believing in me, and keeping everything in perspective. Last but not least, I would like to thank Prufrock, for keeping company with me during the long and late hours. vi VITA December 26, 1977 ..................... Born – Bursa, Turkey 2001 ............................................ B.Sc. Molecular Biology and Genetics, Middle East Technical University, Ankara, Turkey 2001 – present............................. Graduate Research Associate, The Ohio State University PUBLICATIONS Yelda Serinagaoglu, Rui Zhang, Yufang Zhang, Linda Zhang, Greg Hartt, Anthony P. Young, and John Oberdick, (2007). A promoter element with enhancer properties, and the orphan nuclear receptor RORalpha, are required for Purkinje cell-specific expression of a G(i/o) modulator. Mol Cell Neurosci 34, 324-342. FIELDS OF STUDY Major Field: Molecular, Cellular, and Developmental biology vii TABLE OF CONTENTS Page ABSTRACT ........................................................................................................................ii ACKNOWLEDGMENTS.....................................................................................................v VITA ..................................................................................................................................vii LIST OF FIGURES ..........................................................................................................xiii LIST OF TABLES .............................................................................................................xv ABBREVIATIONS ...........................................................................................................xvi CHAPTER 1 INTRODUCTION.................................................................................................................1 1.1 SUMMARY ...............................................................................................................1 1.2 CEREBELLUM .........................................................................................................1 1.2.1 Functions of the cerebellum ..............................................................................2 1.2.2 Structure of the cerebellum ...............................................................................3 1.2.3 Cerebellar circuitry ............................................................................................5 1.2.4 Development of the cerebellum ........................................................................6 1.2.5 Cerebellar mutant mice: Natural mutations.......................................................7 1.3 L7 IS A CEREBELLAR PURKINJE CELL SPECIFIC GENE ...................................8 1.3.1 Discovery of L7 gene.........................................................................................9 1.3.2 Phenotype of L7KO mice ..................................................................................9 1.3.3 Structure and function of L7 protein ................................................................11 1.3.3.1 Heterotrimeric G proteins.........................................................................11 1.3.3.3 Modulation of voltage-dependent Ca2+ channels by G proteins ..............14 1.3.3.4 L7 modulates G-protein-coupled receptor-mediated inhibition of Ca2+ channels in a dose-dependent manner................................................................15 1.3.4 Structure of L7 gene........................................................................................15 1.3.5 Analysis of L7 gene expression in the mouse cerebellum ..............................17 1.3.6 Regulation of L7 gene expression...................................................................19 1.4 NUCLEAR RECEPTOR SUPERFAMILY...............................................................21 1.4.1 Orphan nuclear receptors................................................................................24 1.4.2 Retinoic acid receptor-related orphan nuclear receptors (RORs)...................25 1.4.3 RORα ..............................................................................................................27 1.4.3.1 Discovery of RORα ..................................................................................27 1.4.3.2 Genomic structure of RORα isoforms......................................................28