THE ROLE OF ROM-1 IN MAPNTAINING PHOTORECEPTOR STRUCTURE AM) VUBILITY, AND A MATHEMATICAL MODEL EXPLOIUNG THE ICINETiCS OF NEURONAL DEGENERATION Geoffrey Alïan Clarke A thesis submittd in cdormity with the requirements foi the degree of Worof Philosophy, Graduate Department of Mobdar and Medical Genetics, in the University of Toronto O Copyri@ by Geofffey AUan Clarke 2ûûû The author has gnmted a non- L'auteur a accordé une licence non exclusive licence ailowing the exclusive pennettaat à la National Library of Canada to Bibliothèque nationale du Canada de reprduce, 10- distnïute or sel reproduire, prêter, distribuer ou copies of this thesis in microfonn, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/nlm, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantiaî extracts firom it Ni la thèse ni des extraits substantiels may be printed or otherwise de ceiîe-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. Cana The Rok Of Rom-1 In MainWning Photoreceptor Structure and ViabUity, and a Matbernatical Mode1 Explorlng the ainetics of Neuronal Degeneration Geoffrey Ailan Clarke Department of Molecular and Medical Genetics University of Toronto Doctor of Philosophy,2000 Abstract Rom-1 and peripherinhds are homoIogous membrane proteins localized to the disk rims of photoreceptor outer segments (OSs), where they are postulated to be critical for disk -1- morphogenesis, OS renewal, and the maintenance of OS structure. Rds rnice (homozygous for a null mutation in the gene encoding peripherin/rds) do not fonn OSs, Uidicating that peripheridrds is essential for OS formation. Futhermore, mutations in RDS cause retinal degeneration, indicating that PeripheridRds is necessary for photoreceptor viabiiity. To determine if rom- 1 is also critical for maintaining photoreceptor structure and viabiiity, we gentxated ~ornl" mice using gene targeting . ~oml"mice exhibit a graduai apoptotic death of rd, but not cone, photoreceptors, so that by 18 months of age, 42% of al1 rods had died. Electroretinogram analysis indicated that although rom-1 is not involved in phototransduction, nine month Rml" mice displayed a signifiant decline in photoreceptor fiinction. These results indicate that, like peripheridrds, rom1 is required for the maintenance of photoreceptor viability. Rd" mutants form rod outer segments (ROSs), indicating that expression of peripherinfrds alone is suficient for disk and OS morphogenesis. Mutant ROSs were disorganized: they were not straight and parallel as in control mice. We suggest that the disorganization results from the production of markedly enlarged ROS disks. To test the hypothesis that the accumulation of cellular &mage causes neuronal death, we analyzed the kinetics of cell &th in 12 animal -1s of photoreceptor degeneration, and in four examples of neurodegeneration occurring in other parts of the nervous system. We found that the kinetics of cell loss in aü exarnples were best fit by mathematical models in which the risk of cell death decreased or rernained constant. These results are inconsistent with the increasing nsk predicted by cumulative damage, and instead indicate that the tirne of death of an individual neuron is random. Finaiiy, we demonstrate that the digenic hypothesis of retinitis pigmentosa (RP) is correct: mice inheriting one Roml nul1 ailele in combination with one L 18SP Rhsubstitution exhibit an accelerated rate of photoreceptor death in cornparison to mice inheriting only one of these alleles. Acknowledgemeats Many individuals have conaibuted to the work in this thesis in numerous ways, and my degree couid not have been completed without their influence. First of all, 1would like to thank my thesis advisor, Rod McInnes, for his ongoing support and friendship throughout my stay in his lab. He offered this support continually, whether 1deserved it or not. And when 1didn't, he was always there to provide a nudge in the nght direction. Most of dl, however, 1would like to thank Rod for his most important contribution to my growth over the past several years; namely inboducing me to the wonderful world of red wines. 1wouid also like to thank all members of the McInnes lab, both past and present, for their involvement in many discussions and collaborations that helped shape this thesis. Moreover, their fiiendship made working in the lab more rewardùig than 1would ever have thought. Members of my supervisory committee, Derek van der Kooy, Janet Rossant, and Vincent Giguere, were also instrumental in molding a young and inexperienced graduate student into somebody able to perfonn biomedical research with at least some semblance of proficiency. David Birch, Robert Molday, Andy Goldberg, and Gabriel Travis provided coiiaborations that ailowed me to extend the reach of my research beyond that which 1could have accomplished myseif. 1am also extremely grateful for the financiai support provided to me by the University of Toronto (Open FeIIowship) and the RP Eye Research Foundation of Canada (studentship). Of course, my parents, family and friends were ail important in helping me mach this point in my life. They provided unconditional love and support, even when 1decided to switch undergraduate prograrns for the nh time. To all of you, I'mfinnly finished, so lock your doors 'cause now 1 should actually be able to fmd time to corne and visit. Most of all, 1wish to express my utmost thanks and respect to my wife Mandy - my love, my life, my sugar mamrna You have been with me throughout my internrnent in university and have never wavered in the tolerance you showed to my insane working hows, in the support you gave me, and in the confidence you expressed in ne. If it hadn't been for you, 1 would never have been able to complete this work. This, and everything aftenvards, is for you. Table of Contents Acknowledgements.......................................................................... iv Table of Contents............................................................................. v List of Tables ................................................................................. ix List of Figures................................................................................ ix Frequently Useâ Abbreviations......................................................... xi CHAPTER 1....................................................................................... 1 INTRODUCTION................................................................................ 2 FUNCTIONAL ORGANIZATION OF THE VERTEBRATE RETINA ......... 4 The laminated vertebrate retina .......................................................... 4 Photoreceptor-ROE Interactions ......................................................... 8 Pbotoreceptor Topography .............................................................. 11 Pbototransduction .......................................................................... 13 THE STRUCTURE OF VERTEBRATE PHOTORECEPTORS ................. 18 Photonceptor Outer Segments......................................................... 20 Outer Segment Disks ........................................................................ 21 Cytoskeletai Specializations of the Photoreceptor OS ......................... 24 Comecting Cilium (CC) .................................................................... 26 The Disk Rim Cytoskeleton ................................................................ 28 PeripheridRds: Rom1 Complex ..................................................... 34 OS Renewal : Disk Morphogenesis and Phagocytosis.......................... 38 Disk Moiphogenesis in the Mature ROS .................................................. 39 COS Disk Morphogenesis and Remodehg .............................................. 44 Disk Phagocytosis........................................................................... 45 Light-dependent Control Of OS Renewal................................................. 49 PHOTORECEPTOR DEGENERATION ................................................ 51 Retinitis Pigmentosa....................................................................... 53 Structural Proteins Associated with Photoreceptor Degeneration ......... 55 RHO (Rhodopsin) ........................................................................... 55 ROM1 (Rd outer segment membrane protein- 1) ....................................... 57 RDS (Penpherin/RDS)...................................................................... 58 MY07A (Unconventional Myosin VIIa) .................................................. 61 ABCR (rod photoreceptor 88Ç transpoaeç) ............................................. 62 Mecbanhm of Pbotomeptor Cell Death ...........................................64 The ceiiuiar Congestion Hypothesis ......................................................65 The Equivalent to Light Hypothesis .......................................................65 The Oxygen Toxicity Hypothesis.......................................................... 67 The TweStage Mode1 of Photorieceptor Degeneration.................................. 68 REFERENCES. ................................................................................. 71 CHAPTER 2 ..................................................................................... 98 Abstract .......................................................................................