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Investigations Into Neuronal Cilia Utilizing Mouse Models

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Investigations Into Neuronal Cilia Utilizing Mouse Models INVESTIGATIONS INTO NEURONAL CILIA UTILIZING MOUSE MODELS OF BARDET-BIEDL SYNDROME Dissertation Presented In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Nicolas F. Berbari, BS ***** The Ohio State University 2008 Dissertation Committee: Approved by: Kirk Mykytyn, PhD, Adviser Virginia Sanders, PhD __________________________________________ Georgia Bishop, PhD Adviser Michael Robinson, PhD Integrated Biomedical Sciences Graduate Program ABSTRACT Cilia are hair-like microtubule based cellular appendages that extend 5-30 microns from the surface of most vertebrate cells. Since their initial discovery over a hundred years ago, cilia have been of interest to microbiologists and others studying the dynamics and physiological relevance of their motility. The more recent realization that immotile or primary cilia dysfunction is the basis of several human genetic disorders and diseases has brought the efforts of the biomedical research establishment to bear on this long overlooked and underappreciated organelle. Several human genetic disorders caused by cilia defects have been identified, and include Bardet-Biedl syndrome, Joubert syndrome, Meckel-Gruber syndrome, Alstrom syndrome and orofaciodigital syndrome. One theme of these disorders is their multitude of clinical features such as blindness, cystic kidneys, cognitive deficits and obesity. The fact that many of these cilia disorders present with several features may be due to the ubiquitous nature of the primary cilium and their unrecognized roles in most tissues and cell types. The lack of known function for most primary cilia is no more apparent than in the central nervous system. While it has been known for some time that neurons throughout the brain have primary cilia, their functions remain unknown. Research on neuronal cilia has suffered from a paucity of tools to study them. To this end, some of this work ii describes the development of the first in vitro neuronal culture system where primary cilia are present (Chapter 2). It is apparent that the functions of cilia, at least in part, are defined by the specific proteins that localize within and on the organelle. Indeed access of proteins and signaling modules into and out of the ciliary compartment appears to be tightly regulated. In order to better understand the physiological roles of cilia throughout the body, an understanding of the signaling proteins that localize to the cilium and the mechanisms behind cilia localization is needed. To these ends, some of this work describes novel ciliary signaling proteins such as the localization of adenylyl cyclase III to primary cilia throughout the adult mouse brain, and the localization of the G protein-coupled receptor (GPCR) melanin concentrating hormone receptor 1 to certain neuronal cilia (Chapter 3 and 4). We also have identified a specific GPCR sequence involved in the localization of GPCRs to the ciliary compartment (Chapter 4). This sequence will aid in determining the mechanisms that target proteins to the cilium. The development of these tools has allowed for the opportunity to test the hypothesis that certain clinical features of the ciliary disorder Bardet-Biedl Syndrome (BBS) are due to neuronal cilia dysfunction. Indeed, we have shown that mouse models of BBS fail to localize specific receptors to their neuronal cilia (Chapter 5). These data have set the ground work for more extensive investigations into neuronal cilia function. Further, this work suggests neuronal cilia dysfunction may contribute not only to the cognitive defects associated with ciliary disorders, but may also underlie the obesity observed in these disorders. iii DEDICATION Dedicated to My Wife iv ACKNOWLEDGMENTS This work would not be possible without the help and guidance of my advisor and mentor, Dr. Kirk Mykytyn. I would like to thank you for your scientific integrity, optimism and endless patience in educating. This experience is one that I will remember forever. I would like to thank each of my committee members, Dr. Michael Robinson, Dr. Candice Askwith, and Dr. Georgia Bishop, who have been extremely generous with their time and have provided invaluable advice. To my lab mates past and present, thank you for putting up with me. I would especially like to express my gratitude to Jackie Lewis for all of her help and patience both in and out of the lab. To Dr. David Cunningham, thank you for some of the most interesting conversations. I would like to thank Tom Sherwood and Dr. Brian Davy for reminding me that we do this because it is fun. I am also grateful to the Raymond E. Mason Foundation for providing me a 2 year fellowship and The Ohio State University for the Presidential Fellowship this last year. I would like to thank all of my friends and family for all of their support this year. Finally, I would like to thank my wife who has been supportive in every possible way that one could support another. v VITA November 6, 1979 Born – Iowa City, Iowa, USA 2002 B.S. Biology, Indiana University PUBLICATIONS 1. Lemon W.J., Swinton C.H., Wang M., Berbari N., Wang Y., You M. Single nucleotide polymorphism (SNP) analysis of mouse pulmonary adenoma susceptibility loci 1-4 for identification of candidate genes. Journal of Medical Genetics. 2003. 40(4):E36. 2. Kelly L.E., Davy B.E., Berbari N.F., Robinson M.L., El-Hodiri H.M. Recombineered Xenopus tropicalis BAC expresses a GFP reporter under the control of Arx transcriptional regulatory elements in transgenic Xenopus laevis embryos. Genesis. 2005. 41(4):185-91. 3. Doggett N.A., Xie G., Meincke L.J., Sutherland R.D., Mundt M.O., Berbari N.F., Davy B.E., Robinson M.L., Rudd M.K., Weber J.L., Stallings R.L., Han C. A 360-kb interchromosomal duplication of the human HYDIN locus. Genomics. 2006. Dec;88(6):762-71. 4. Berbari N.F., Bishop G.A., Askwith C.C., Lewis J.S., Mykytyn K. Hippocampal neurons develop primary cilia in culture. Journal of Neuroscience Research. 2007. April; 85(5):1095-100. 5. Bishop G.A., Berbari N.F., Lewis J.S., Mykytyn K. Type III adenylyl cyclase localizes to primary cilia throughout the adult mouse brain. Journal of Comparative Neurology. 2007. Oct 9; 505(5): 562-71. 6. Berbari N.F., Askwith C.C., Lewis J.S., Mykytyn K. Identification of Ciliary Localization Sequences within the Third Intracellular Loop of G Protein-Coupled Receptors. Molecular Biology of the Cell [epub ahead of print Feb 2008]. 7. Berbari N.F., Bishop G.A., Lewis J.S., Mykytyn K. Bardet-Biedl Syndrome Proteins are Required for G Protein-Coupled Receptor Localization to Neuronal Cilia. PNAS. vi FIELDS OF STUDY Major Field: Integrated Biomedical Science vii TABLE OF CONTENTS Page ABSTRACT ....................................................................................................................... ii DEDICATION ................................................................................................................... iv ACKNOWLEDGMENTS.................................................................................................... v VITA ................................................................................................................................. vi LIST OF TABLES............................................................................................................. xi LIST OF FIGURES.......................................................................................................... xii LIST OF ABBREVIATIONS............................................................................................. xv CHAPTER 1: INTRODUCTION TO CILIA.........................................................................1 Summary .................................................................................................................1 Cilia Structure, Function, And Classification............................................................2 Ciliogenesis And Intraflagellar Transport.................................................................3 Specialized Sensory Cilia and Disease ...................................................................4 Cilia Localization Sequences...................................................................................4 Cilia Disorders and Clinical Features.......................................................................6 viii The Cilia Disorder Bardet-Biedl Syndrome..............................................................7 Neuronal Cilia ..........................................................................................................9 Hypothesis and Chapter 2-5 Overview ..................................................................10 CHAPTER 2: HIPPOCAMPAL NEURONS POSSESS CILIA IN CULTURE ..................17 Summary ...............................................................................................................17 Background............................................................................................................18 Results...................................................................................................................19 Discussion .............................................................................................................23 Materials and Methods ..........................................................................................25 CHAPTER 3: TYPE III ADENYLYL CYCLASE LOCALIZES TO CILIA THROUGHOUT THE ADULT MOUSE BRAIN ..........................................................................................33
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