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
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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 ...... 99 Introduction ...... 10 Experimental Procedures...... 102 Generation of Roml targeted mice...... 102 SDS gel electmphoresis and Western Blotting ...... 103 Histology...... 104 TUNEL Assays ...... 106 Measurements and statistical analysis...... 106 ERG analysis...... 107 heparation of ROSS...... 108 Velocity Sedimentation Analysis of PeripherWRds Complexes...... 109 Immunofluorescence and confbcal microscopy ...... 109 ResuIts ...... 110 Targeting the Roml gene ...... 110 Rom-1 is required for rdphotoreceptor viability ...... 112 4- ROS disorganization in Rom1 mice becomes near-normal with age ...... 112 Loss of rom-1 increases ROS disk diameter ...... 118 4- Maximal rod photoresponses are decdin Rom1 mice ...... 121 Peripherin/rds fonns homotetramers at the ROS disk rims of ~omlmice ...... 124 Discussion ...... 125 References ...... 134
Abstract ...... 141 Introduction ...... 142 Experimenta! Proceàures...... 145 Results ...... 146 Ce11 Death Kinetics In Photoreceptor Degeneration ...... 146 Cell Death Kinetics In Non-Retinal Neurodegeneration ...... 152 Discussion ...... 154 References ...... 160
CHAFI"I'R 4 ...... 166 Abstract ...... 167 Introduction ...... 168 Experimental Procedum ...... 170 Generation of Mice ...... 170 Histology ...... 171 Measurements and statistical analysis ...... 171 Anaiysis of the kinetics of photoreceptor celi death ...... 172 Results ...... 173 Accelerated photoreceptor death in digenic mice ...... 173 ROS scnictural abnodities...... 176 Discussion ...... 179 References ...... 182
Futun Experiments ...... 186 Additional Analyses of the Roml" Phenotype ...... 186 Detemùnuig the pathogenicity of putative diseasecausing ROM1 aileles ...... 190 Determinhg the complete expression pattern of Roml : is Ruml involvecl in syndromic disease? ...... 191 Rom- 1 Associated proteins ...... 193 Evaiuation Of The "Periphecin/Rds Equivalents" Hypothesis...... 194 Identifying Photoreceptor MuRGs and MuRPs ...... 194 Extendhg the One-Hit Mode1 of Neuronal Ce11 Death ...... 196 References ...... 198
Solutions to Differential Equatioas Used in Chapter 3...... 203 1) Exponentialiy decreasing risk: ...... 203 2) Constant risk ...... 203 3) Exponentially increasing risk ...... 204
vii 4) Repeatedmeasures. non-linear regession analysis of mtabolic decline in patients with Huntington's disease ...... 204 APPENDIX 2: ...... 206 Parameter Estimates (3 significant digits) for Nonlinear Regession Analysis of Neuronal Degeneration ...... 207 Parameter Estimates for the Doubk Exponential Mode1 ...... 216
APPENDIX 3...... -220
Raw data used in analysis of metabolic decline in patients with Huntington's Disease ...... -221 List of Tables Table 1.1: The number of loci and genes involved in various photoreceptor degenerations...... 52 Table 3.1. Parameter estimates for kinetic models relating the nsk of neuronal death (p) to age...... 149
List of Figures
Figure Development of the Vertebrate Retina ...... 5 Figure Structure of the Vertebrate Retina...... -7 Figure Photoreceptor-RPE Interactions ...... 10 Figure The Enzyme Cascade in Rod Photoreceptor Photoactivation...... 14 Figure Recovery of the Rod Photoreceptor Dark State ...... 16 Figure Structure of Vertebrate Photoreceptors...... 19 Figure The Structure of the Vertebrate ROS Disk ...... 22 Figure Structural Specializations of the ROS ...... 25 Figure The Cytoskeleton Of The ROS Disk Rim ...... 30 Figure Membrane Topology of ROM- 1 and PeripheWRDS...... 35 Figure Morphogenesis of ROS disks...... 40 Figure ROS Disk Shedding and Phagocytosis...... 47
Figure 2.1. Generation of Roml mutant rnice ...... 111 Figure 2.2. Complete loss of rom-1 results in apoptotic rod photoreceptor death .... 113 Figure 2.3: The loss of rom4 leads to the development of short ROSS which exhibit a biphasic pattern of disorganization...... 116 4- Figure 2.4. ROS ultrastnictwe is abnomial in Rom1 mice...... 119 Figure 2.5: Cone outer segments (COSs) appear sûuctrirally normal in ~oml-1- mice ...... 122 Figure 2.6. Electroretinograpùic responses are abnomial in ~ml-/-mice ...... 123 Figure 2.7: Homotetramers of peripherin/rds are localized at the rims of ROS in Rom1 mutant mice ...... 126 Figure 3.1: Animai models of inheriteà photoreceptor degeneration and retinal detachment, in which the kinetics of cell death are best described by a constant risk of neuronal death...... 147 Figure 3.2: Animal models of inherited photoreceptor degeneration in which the kinetics of ceU deatb are best àescribed by an exponentialiy decreasing risk of death...... 15 1 Figure 3.3: Examples of non-retinal neuronal death which also display a constant or exponentially decreasing nsk of death...... 153 Figure 3.4: The exponentiai kinetics of ce11 death in inherited neuronal degenerations suggests the existence of a mutant steady-state (MSS)...... 157
Figure 4.1: Genotype determination of digenic mice...... 174 Figure 4.2: Digenic inheritance of a Rom1 nuil and a L 18SP Rdr mutation resuit in accelerated photoreceptor cddeath and progressive shortenhg of photoreceptor ROSS...... 175 Figure 4.3: Digenic and Ll8SP mice exhibit OS structural abnonnalities and a progressive loss of photoreceptors...... 177 Figure 4.4: Digenic mice exhibit an exponentialIy decreasing probability of photoreceptor ce11 death...... 178
Figure 5.1: Proposed mechanism of mouse rod photoreceptor outer segment morphogenesis...... 188 Figure 5.2: Introduction of putative diseasecausing alleles...... 192 Frequently Useà Abbnviations
CC...... Photoreceptor connecting cilium COS...... Cone outer segment 1s...... Inner segment MTs...... Microtubules ONL...... Outer nuclear layer OS...... Outer segment PM...... Plasma membrane RIS...... Rod photoreceptor inner segment ROS...... Rod photoreceptor outet segment RP...... Retinitis pigmentosa RPE...... Retinal pigment epithelium TLC...... Terminal loop complex Chapter 1
Introduction
Portions of the material in this chapter have been reproduced, with permission, from the publication: Clarke,G., Héon, E., and McInnes, R.R. (2000) Recent advances in the molecular basis of inherited photoreceptor degeneration. Clin. Genet. 57: 3 13-329.
O Munksgaard Press INTRODUCTION
The photoreceptor is one of the most unique nemns in the vertebrate nervous system. Its most remarkable feanire, the outer segment (OS), consists of a plasma membrane which, in primates, encases a stack of approximately 1000 membranous disks (Young, 1971). Each disk has a lumen, a central lamellar region containing rhodopsin and other proteins required for phototransduction, and a curved rim region. OSs are under a continual state of renewal: new disks are produced at the OS base and migrate distally to the OS tip, where they are phagocytoseci by the overlying pigment epitheiial celis. Elucidation of the molecular mechanisms which regulate disk morphogenesis, and which control the diurnal migration of disks to the tip of each ROS, are central problems in marnrnalian photoreceptor biology. PenpherinRds is likely the most intensively analyzed structural protein expresseci in the photoreceptor. Twenty years ago, it was demonstrated that Retirzal degeneration slow
mice (~dr")exhibit progressive photoreceptor degeneration (Sanyal et al., 1980), and never
develop OSs (Sanyal and Jansen, 198 1). Since that time, the gene responsible for the Rds phenotype has been identified (Travis et ai., 1989) and shown to encode a trammembrane protein loçalized to the rims of photoreceptor OS disks (Comell and Molday, 1990). Moreover, approximately 70 mutations in the RDS gene in humans have been associateci with inherited photoreceptor degenerations, indicating that it is critical to the maintenance of ceil viability (Kohl, 1998). Biochemical analyses have further indicated that peripherin/rds can promote membrane fusion in vitro, irnplicating it in critical developmental events regulating photoreceptor structure (Boesze-Battagha et ai., 1998). Our lab has previously identified an additional OS rim-speciîïc protein, the mi outer segment membrane protein- 1 (rom-1) and demonstrated that it is a close relative of peripheridrds (Bascom et al., 1992). Unfortunately, no naturally occuning mutation has been found in rnice, and extensive screening has failed to conclusively associate ROM1 with any monogenic photoreceptor degeneration (Bascom et al., 1995; Sakuma et aL , 1995). The only convincing ROM1 mutations so far identifrd in patients with retinitis pigmentosa (RP) cosegregate with a mutation in RDS, resulting in the bbôigenichypothesis" of RP. Whereas the function of rom4 is presently unknown, biochemical experiments indicate that it forrns a complex with peripherin/rds (Loewen and Molday, 2000), suggesting that the two function together at the disk rim. To explore the role of rom4 in the maintenance of OS sûuchue, to detemine if loss of rom4 resulr in photoreceptor degeneration, and to test the hypothesis that individuals heterozygous for both a ROM1 nul1 allele and an RDS substitution mutation exhibit RP (the "digenic hypothesis"), 1have used gene targeting to generate mice carrying a nuii mutation in the Rom1 gene. Determining answers to these question is the major focus of this thesis. In Chapter 1,I discuss the biology of the vertebrate retina with an emphasis on its structural organization. 1provide an ovetview of photoreceptor biology, and discuss the putative function of selected proteins within the ceIl. I conclude the chapter with a discussion of the most common form of photoreceptor degeneration in humans, retinitis pigmentosa, and briefly review the role of known stnictural proteins in this disease. In Chapter 2, 1demonstrate mice homozygous for a nul1 aliele exhibit progressive photoreceptor ce11 death and OS stmctural abnormalities. Chapter 3 explom the common assumption that neuronal death results from the gradua1 accumulation of cellular damage, presents a mathematical madel suggesting that neurons die due to a single random event. Chapter 4 describes histological studies of mice with the digenic genotype, and presents evidence that the digenic hypothesis of Ri? is comct. Finaliy, Chapter 5 explores future experiments that might be perfonned to further our knowledge regarding the deof rom4 in photoreceptor biology, and to extend our one-hit made1 of neuronal ce11 death. FUNCTIONAL ORGANIZATXON OF THE VERTEBRATE RETINA
The vertebrate retina is the thin layer of neural tissue lining the back of the eye that has evolved to perform two major functions (Rodieck, 1998). First, specific cells in the retina transduce light intensity into a neural signal. In otber words, the retina extracts information about light levels in a visual scene and converts it into a chernical signal that can be interpreted by other cells in our central nervous system (CNS). Second, the retina must also compate variations in neuronal activity, and has consequently evolved into a highly ordered tissue with a limited number of ceIl types which interact to perform these initial steps of image processing. For example, contrast enhancement and motion detection are visual functions that are perfomed in the retina, early in the pathway of visual information processing (Dowling, 1987). These, and other complex processes are believed to be the result of neural coding mechanisms which are common to al1 regions of the CNS. Thus, as the most accessible part of the CNS, the retina is a valuable mode1 system for elucidating both the structural and physiologicd principles underlying brain function. Furthemore, because it is non-essentiai to the viability of an organism, and at least 12 1 genes or loci have been associated with inherited retinal degeneration (Daiger et al., 20),the retina is a powerful system for understanding the pathophysiological mechanisms which result in neuronal ce11 death.
The laminated vertebrate retina
The vertebrate neuroretina is a highly ordered tissue that develops as an outgrowth of the laterai embryonic forebrain. The first morphological evidence of retinal development is the formation of two srnall indentations, termed the optic sulci, located on either side of the anterior neural plate (Saha et al., 1992; Walis, 1991) (Figure 1. la), a region of the surface ectoderm committed to a neural fate. As the cells of the neuroepithelium proliferate, the neutal crests on either side of the neural plate meet dorsaily, forming the neural tube. By the Neural Plate
v Surf' Ectoderm Neural Epitheliwn
Opac Vesicle Stage Optic Sta1 k
Neuroepithelium
Optic Cup Stage
Figure 1.l: Development of the vertebrate retina. A) The fhtmorphological evidence of the developing vertebrate eye is observed as an indentation (optic sulcus) in the antenor neural plate. As the neuroepithelial cells proliferate, the two sulci enlarge and form the optic vesicles when the neural crests meet and fuse to form the newal tube. B) Contact between the neuroepithelium of the optic vesicle and the surface ectoderm induces the formation of the optic and lens cups. The optic cup consists of two layers, an outer layer which differentiates into the tetinal pig- ment epithelium (RPE), and an inner layer which will ultimately differentiate into the cells of the adult neworetina. time of neural tube closure, the epitheliai cells of the optic sulci have pliferate., increasing the size the forebrain outgrowtbs, now termed the optic vesicles. The vesicles continue to grow and change shape until they resemble pouches connecteci to the forebrain by a short stalk. This growth continues until the vesicles contact the embryonic surface ectoderm, thereby completing the final stages of lens determination (Henry and Grainger, 1990). After lens induction is complete, the ceils which contacteci the surface ectoderm invaginate and form a bilayered structure known as the optic cup (Figure 1. lb) which consists of an outer layer destined to differentiate into the retinal pigment epithelium (RPE), and an inner layer that will proliferate fiirther before differentiating into the six neuronal cell types present in the adult neuroretina. The neurons of the adult retina are fùnctionally unique and organized into a laminateci tissue containhg three nuclear layers separated hmeach otkr by two synaptic regions (Figure 1.2). The ceIl bodies of each neuronal type are containeci within specific layers: photoreceptors in the outer nuclear layer (ONL), the bipolar, amacrine, horizontai, and interplexifonn cells ate situated within the inner nuclear layer (INL), and the ganglion celis comprise the ganglion ceIl layer (GCL). These cells interact via synapses confïned almost exclusively to the plexiform, or synaptic layers. Photoreceptor cells, the primary newons of the visual system, are speciahd for the absor&on and transduction of iight into a neuronal signai. In general, diurnal species have evolved two major types of photoreceptor cells which are classified according to functional and structural characteristics. Rod photoreceptors possess a cylindrically shaped iight sensitive region at their distal tip, or outer segment (OS) that is most sensitive to low intensity monochromatic light (scotopic vision) at a wavelength of approximately 500 nm. Most diurnd species have two types of cone cells, one maximally sensitive to short wavelengths and another with a peak absorbance in the long wavelength portion of the spectnun, which function predominantly under bright light (photopic vision) conditions Choroid (vasculature) Retinal Pigment Epitheiium
Photoreceptor Outer Segments
Outer Limiting Membrane Outer Nuclear Layer
Outer Plexiform Layer
Inner Nuclear Layer
Inner Plexifonn Layer
Ganglion Cell Layer
Nerve Fibre Layer
To Optic Nerve ,-*
Light
Figure 1.2 Structure of the vertebrate retina. The vertebrate neworetina is a laminateci structure composed of three nuclear layers, each containing specific ce11 types, that are separated by plexifom, or synaptic layers. A=amacrine cell, B=bipolar cell, C=cone photonceptor, G=ganglion cell, H=horizontal cell, I=interplexifom layer celi, M=Miiller glial cell, R=md photoreceptor. (Dowling, 1987). Humans and other old world primates are unusual in that they possess three distinct cone types: the S, M,and L cones are maximally sensitive to short (419 nm), medium (53 1 nm), and long (558 nm) wavelengths of light, respectively @artnaIl et al., 1983). Alter stimulation by light, photoreceptors transmit a chemical signal to bipolar cells in the INL, which then pass the signals to the ganglion cells. Axons of the ganglion cells converge at the optic disk and form the optic nerve which transfers visuai idonnation to higher brain centers where it undergoes further processing. During the transmission of signals through the retina, information from individual photoreceptors both converges and diverges on cells of the next layer. That is, signals hmmultiple photoreceptors converge on the synapses of a fewer number of bipolar cells, whereas the signal hman individual photoreceptor may diverge through synapses with multiple bipolar ceils (Rowe, 1991). The analysis of spatial variation in photoreceptor activity, i.e. variation in light levels across a visual scene, is gerformed by cells in the INL. Individuai horizontal cells synapse with several hundred photoreceptors and therefore receive input hma correspondingly large maof the retina. Horizontal ceils respond to this input by generating graded potentials correlated with the magnitude of the input. Axons of horizontal cells then synapse with other photoreceptors, where they inhibit the stimulation of bipolar ceils by photoreceptors. Therefore, increasing the stimulation of horizontal cells decreases the stimulation of bipolar cells by photoreceptors, resulting in an increase in visual contrast (Rodieck, 1998).
Photoreceptor-RPE Interactions
The RPE, a monolayer of cells situated between the photoreceptors and the vascular network positioned beneath the sclera of the eye (Figure 1.2), performs multiple functions that are critical to the developrnent and nonnal function of manire photoreceptors. The RPE differentiates before other cells in the retina, suggesting that it may influence the formation of the other retinal ce11 types (Sheedlo and Tumer, 1995). Retinal ceIl culture experiments support this idea and indicate that ciiffusible fabctors produceci in the RPE, such as pigment epithelial-derived factor (PEDF) (Tbmbran-Tink et al., LW 1), are necessary for the proper differentiation of photoreceptors (Sheedlo and Turner, 1995). Additionally, separaîion of the RPE from developing photoreceptors inhibits OS elongation and photoreceptor synaptogenesis in explant culnues of Xe110pu.s retinas (Holiyfîeld and Witkovsky, 1974; Stiemke et al., 1994), and completely inhibits OS formation in mice (Wdet al., 1989). Once formed, if the mature retina detached hmthe RPE, photoreceptor OSs degenerate, and the celis ultimately die (Berglin et al., 1997; Erickson et al., 1983; Guérin et al., 1993; Mac hemer, 1968). The RPE is also responsible for reguiating the movernent of nutrients and metabolites required for the maintenance of photoreceptors throughout the interphotoreceptor space. The visual cycle (Figure 1.3), the chemical reactions that regenerate the 11 -cis-retinddehyde used dunng phototransduction (see below), occurs in both the photoreceptor and the RPE. After absorption of a photon, the 114s retinaldehyde bound to opsin (rhodopsin) is converted to all-trans-retinaldehyde,which subsequently dissociates from the opsin protein. Ali-trans- retinaldehyde is then isomerized to aii-trans-retinol by retinoI dehydrogenase in the photoreceptor OS(Wa1d and Hubbard, 1949), and transported through the interphotoreceptor matrix (IPM), a glycopmtein-rich extracellular medium believed to facilitate the biochemical and physical interactions between the RPE and photoreceptors (Hageman and Johnson, 1991), to the RPE where it is enymatically converted to 11-cis-retinaldehyde and retumed to the photoreceptors (Saari, 1990). Interphotoreceptor retinol-binding protein (IRBP), a major component of the PM,was initiaily believed to be responsible for the transport of retinoids between photorezeptors and RPE (Lai et al., 1982). However, ment analysis of mice homozygous for a nuH mutation in the gene encoding IRBP has indicated that loss of IRBP does not appreciably alter visuai cycle kinetics (Palczewski et al., 1999), and the molecular mechanisrn of retinoid translocation rem- unclear. dl-tram rctind from Mood supply Glucose from Mood supply
Retinal Pigment Epithelial cell O
Foi1A Photorecepor-RPE interactions. Interactions ktween photoreceptors and the RPE are requind for the wmpletion of the visual cycle, the conversion of dl-~rm-rttinolto 1 14s-cetinal and the regeneration of photopigments within the photorecepor outer segment. The interaction is also necessaq for the generation of NADPH needcd to convert all- @ans-retinal to dl- t+u.m-retinol w ithin the photoreceptor. In addition to its diriect involvement in the conversion of ail-@culs-retinolto 1I-cis- retinaldehyde, the RPE is believed to be required for the NADPH-dependent conversion of all-tram retinal to ail-trans-retinol within the photoreceptor. Sina no transhydmgenase that can conven NADP to NADPH has been identifie-in the photoreceptor OS, the production of NADPH in the photoreceptor has been suggested to occur via the pentose phosphate pathway (PPP) (Tsacopoulos et al., 1998),. In this mode1 glucose fiom the choroidal vasculature is passed through the RPE and taken up by glucose transporters (Hsu and Molday, 1994) in the photoreceptor OS. Once within the OS, glucose is used in the PPP to produce lactate and to convert NADP to NADPH. Finally, the RPE is also critical in maintaining the health of the mature photoreceptor by phagocytosing the tips of photoreceptor OSs (see below, OS Renewal: Disk Morphogenesis and Turnover). Over time,damage caused by exposure to light can accumulate within the photoreceptor outer segments, jeopardizing the health of the al1 (Young, 1976). By phagocytosing the tips of photoreceptors, damaged components are absorbed and digested within the RPE, limiting the effects of photodamage. To compensate for this continual digestion of photoreceptor components, rod and cone cells continuaily produce new outer segment material, and therefore maintain th& overall length and structure.
Photoreceptor Topography
Since photoreceptors convert a spatially continuous signal into discrete elements, the distribution of these cells across the surface of the retina is an important determinant of how much visual information is retained during the initiai steps of visual processing (Rowe, 1991). Photoreceptor topography varies from species to species, reflecting the characteristics of the organism's behaviour or environment in which they live. In general, however, there are two classes of organization (Rodieck, 1998). The visuai streak, nomally observed in animals that evolved in Qat, open regions, consists of a horizontal area containhg a high density of cones, cone bipolar, and ganglion celis which provides these animals with a high degree of visual acuity across the visual field. In contrast, the area centralis, of which the hurnan fovea is a speciaüzed example, contains a spherical concentration of these cell types that has evolved to best discem a discrete point. Some organisms such as the cat and dog, use a combination of both organizations. The average human retina contains a non-uniform distribution of approximately 92 million rod photoreceptors and approximately 4.6 million cones (Curico et al., 1990). The fovea, the region of the retina most sensitive to detail, has the highest density of cone photoreceptors (approxirnately 200,000 mm-2);no rods are located in a central region approximately 0.35 mm in diameter (Curcio et al., 1987; Cwico et al., 1990). In many dimal species, including humans, the laminateci structure of the retina is disrupted in the fovea; al1 cells of the inner retinal layers are displaced laterally so that image degradation is minimized. With greater eccentricity, cone density declines rapiàiy, king approximately an order of magnitude lower 1 mm from the foveal center (Curico et al., 1990). In contrast, rod density increases quickly with retinal eccentricity, to a maximum of appmximateiy 175,000 mm-* in a 5 mm eliiptical ring centered on fovea (Curico et al., 1990). Rod numbers decline rapidly towards the central fovea, and slowly towards the retinal periphery. There are some species that do not exhibit patterns of retinal topography correspondhg to either an area centralis or a visual streak. For example, the retinas of rnice contain an almost unifonn distribution of both rdand cone photoreceptors (Carter-Dawson and Lavail, 1979; Sn51 et al., 1992). The mouse retina is also topographically unique in that the two cone subpopulations, the middle-wavelength (maximum sensitivity at 5 11 nm) M- cones and the short-wavelength S-cones (maximum sensitivity at 360 nm), exhibit almost rnutually exclusive distributions (Szél et al., 1992). M-cones are present in the dorsal retina at a concentration of approximately 4- 12 x 103 mm2 whiie they are almost completely absent from the ventrai hernisphere. In contrast, S-cones concentration ranges hm12- 18 x 1@ mm-2 in the ventral retina and approximately 1,000 mm-2 in the dorsai retina. Physiological measwements are consistent with these measurements; the mouse retina is more sensitive to ultraviolet light when it is directeci towards the ventral hemisphere (Calderone and Jacobs, 1995).
Phototransduction
The fmt step in the conversion of a visual sane into a neural signal is the sampling of the image itself, a function perfonned by the photoreceptor OS, a light sensitive organelle that captures photons and tramduces their energy into a chernical signai (Figure 1.4) (Jindrov5, 1998; Molday, 1998; Yau, 1994). The mokule responsible for photon capture in rod photoreceptors is rhodopsin, which accounts for more than 70% of total rod ceIl protein (Molday, 1998). Rhodopsin consists of a light sensitive chromophore bound to Lys296 of the membrane protein opsin. In dark-adapted cells, the chromophore is I l-cis- retinaldehyde, a denvative of vitamin A. Before exposure to light, Na+ and Ca*+ flow into rod photoreceptors through cGMP-gated ion channels which are kept open by the relatively high levels of intracellular cGMP present in the dark. Simiiarly, K+ ions flow out of the photoreceptor inner segment (1s) through voltage gated channels, completing the 'dark current loop'. Ionic gradients are maintained in the dark by the Na+/K+ ATPase, which removes Na+ ions from the IS, and by the removal of Ca*+ and K+ ions from the rod outer segment @OS) by the Na+/Ca2+-K+ exchanger. Tkse conditions result in a constant release of the neurotransmitter g1utamate while the ceil remains in the dark state. The initial event in phototransduction involves the absorption of a photon by 1l-cis- retinaldehyde, which is thereby converted into the dl-tram isomer (Figure 1.4). This conversion produces a conformational change in rhodopsin, generating a catalyticaily active form (R*) that interacts with transducin, a member of the family of heterotrimeric G proteins which couple environmental signals to ceiiular effactor molecules. Interaction with R* triggers the exchange of GDP for GTP on the a-subunit of transducin, pennitting the now active Ta* subunit to dissociate hmboth R* and the inhibitory T& dimer. The activation of Ta (i.e. the formation of Ta*) is greatly amplif~ed:one R* molecule can catalyze the formation of several hundred Ta* proteuis. Once formed, Ta* activates phosphdiesterase
(PDE), which consists of two active subunits, a and $, and two identical inhibitory y- subunits. Each Ta* interacts with one of the PDE inhibitory ysubunits, causing its dissociation from the activated PDEa and subunits. PDEa and B subunits both have catalytic activity and hydrolyze cGMP to 5'-GMP. Surpisingly, mice homozygous for a nul1 ailele in PDEy exhibit a higher than nonnal intracellular concentration of cGMP, suggesting that the interaction between the cataiytic and inhibitory PDE subunits is critical for the formation of a stable holoenzyme and for the enzymatic activity of the protein (T'sang et al., 1996). The decrease in intraceilular cGMP caused by the activation of PDE closes the cGMP-gated ion channels, which decreases the intraceilular Cs+concentration since this ion is still transported out of the cell by the Na+/Ca2+-K+exchanger. This efflux of positive ions hyperpolarizes the photoreceptor, closing voltage-gated Ca2+ channels in the photoreceptor synapse and reducing the release of the neurotransmitter glutamate. The retum to the dark state (Figure 1.5) is a necessary part of the phototransduction cascade, and requires both the shutting down of the enzymatic cascade and the restoration of intracellular cGMP levels (Jindrovd, 1998). R* becomes inactivated by a two step pmcess: the fmt is the ATP-dependent phosphorylation of multiple serine and threonine residues in the C-terminus of rhodopsin by rhodopsin kinase. In the dark state, rhodopsin kinase is nomdly inhibited by the Gaz+-bindiag protein recovenn (Calvert et al., 1995; Chen et al.,
1995), but the Light-sensitive decrease in intraceildar Ca2+ removes this inhibition, and results in partial inactivation of R*. Further inactivation of R* is accornpiisheû by the binding of arrestin (S-antigen) to phosphorylated rhodopsin, which hiaders tbe interaction between rhoâopsin and transducin by steric hindrance (Wilden, 1995). However, since mice homozygous for a null aiiele in the gene encoding arrestin exhibit an initial fast recovery of the dark state, followed by slower second phase (Xu et al., 1997),additional arrestin- independent mechanisms of rhodopsin inactivation may hinction in rod photoreceptors.
Activated Ta* has intrinsic GTPase activity and can therefore hydrolyze the GTP bound to itself to GDP, thereby inactivating itself. It has been observed, however, that the rate of GTP hydrolysis and Ta* inactivation is dependent upon the Imai concentration of both PDEy and membrane, indicating that other factors are involved in regdation of transducin inactivation (Arshavsb et al., 1994). Once GTP hydrolysis has accurred, Ta re-associates with Th,completing it's inactivation. The final step in the tennination of the phototransduction enzymatic cascade is the deactivation of PDE* by the reassociation of the catalytic a and $ subunits with the inhibitory PDEy. This may occur after either the phosphorylation of PDEy or after GDP-bound Ta has released PDEy by reassociating with the TPy subunits. The retum to the dark state also requks the resynthesis of intracellular cGMP. The decrease in intracellular Ca*+ after photoexcitation activates the guanylate cyclase activating proteins (GCAP 1and II), which facilitate the conversion of GTP to cGMP by the enzyme guanylate cyclase (GC). The subsequent increase in cGMP opens the ion channels that were closed upon light absorption, and the photoreceptor returns to its depolarized state. More detailed knowledge is available about the phototransduction cascade in rod than in cone photoreceptors, simply because they are easier to isolate in experimental settings. However, mom recent analysis has indicated that almost every protein dong the rod transduction pathway has a close homologue in cone photoreceptors (for a review, see (Yau, 1994)). Not swprisingly, thetefore, the mechanism of phototransduction in the two ceIl types is quite similar; known differences are mostly quantitative, in spite of the overall differences in the light sensitivity and response kinetics that are observed between the two cell types (Yau, 1994).
THESTRUCTUREOF VERTEBRATE PHOTORECEPTORS
Mature photoreceptors are stmcturally and functionaiiy polarized newons that can be subdivided into 4 compartments: the outer segment (OS), the inner segment (IS), the cell body, and the synaptic region (Figure 1.6). The synaptic region is localized within the outer plexifonn layer and transmits neural signais to the interneurons of the inner nuclear layer. The synaptic junctions, or pedicles, of cones photoreceptors are large, flat regions containing a number synaptic ribbons located at the base of prominent invaginations (Dowiing, 1965). In contrast, rod spherules normally contain one large invagination and synaptic ribbon. Photoreceptor cell bodies contain the nuclei and are found within the outer nuclear layer. Since the celi body of cones are usually a direct extension of the inner segment, they are usualiy situated directly adjacent to the outer limiting membrane. Rod ce11 bodies are located closer to the synaptic terminals and extend narrow processes which cross the region of cone cell bodies before reaching the photoreceptor inner segments. The IS contains the major metaboiic machinery of the ce11 and is typicaliy divided into two zones (Figure 1.6) (Rowe, 199l), The most proximal zone, the myoid, contains the Golgi complex and a smdnumber of mitochondria The distal portion, the ellipsoid, is the widest part of the IS and contains the major proportion of photoreceptor mitochondria and a high density of microtubules. In some species, including virtually ali birds, some reptiles, arnphibians and fish, the ellipsoid also contains an oil droplet that significantly increases the refractive index of the IS, thereby incming the light gathering efficiency of the photoreceptor. A small connecting cilium Outer Segment Disk Outer Segment (0s)
Connecting Cilium
,Mitahondria - Inner Segment as) ho@Cornpiex Endoplasmic Reticulum,
Cell Body
Rod Spherule Cone Pedicle
Synaptic Terminal uar Rod Photoreceptor Cone Photoreieptor Fi- 1.6: Structure of vertebrate photoreceptors. The two major types of photomptor can each be divided into the outer segment (OS). the imer segment (1s). the ce11 body. and the synaptic terminai. spans the junction between the IS and the higidy specialized OS, the site of iight adsorption and phototransduction.
Photoreceptor Outer Segments
The photoreceptor OS is stmchuaily specialized for the transduction of electmmagnetic energy into a chernical signal. in order to maxunize the number of rhodopsin molecules in a photoceptor, and thereby increase their sensitivity, photoreceptors have undergone a remarkable proliferation of membranes during evolution (Fein and Szuts, 1982). To accommodate the drarnatic increase in membrane volume that occurs dwing photoreceptor maturation, the outer segment develops as a stacked array of membranous disks (approximately 10disks in the primate rod OS (Young, 197 1)) oriented perpendicular to the long axis of the outer segment (Figure 1.6). This organization optUnizes photon absorption by concentrating and orienting opsin molecules dong the iight path (Liebman, 1975) and by limiting the diffusion of phototransduction molecules within the ROS (JindrovA, 1998). The OS disks are continually fomd at the OS base and are phagocytosed by the retinal pigment epitheliwn (RPE)cells. This nmewal process, whkh in primates occurs at a rate of approximately 10% of the total OS disk number per day, has been suggested to be critical for limiting the effects of photodamage on the constituent molecules of the photoreceptor (Young, 19%). Eady observations of rod and cone outer segment (ROS and COS, respectively) structure indicated that the cell types are morphologically diffemnt in two important ways. First, ROS are cylindrical in shape, while the COS of many species are pyramidal. me reason for these differences in OS geometry is uaclear, but it mybe related to alternative mechanisms of disk morphogenesis in that occur in both developing and mature cells photorecepton (see below, OS Disk Renewal and Chapter 5: Future Experiments). Second, the majority of COS disk membrane is continuous with the plasma membrane (PM) (Cohen, 1961; Cohen, 1968; Cohen, 1970; Laties et al., 1976). whereas ROS disks, except for approximaîely the 10 basai-mat disks, are separate hmthe PM and fomi individual intracellular elements (Sjostrand, 1959). This difference between the two cell types results in a dramaticaily larger COS surface area, and has been suggested to account for the difference in absolute sensitivity between rod and cone photoreceptors (Raynauld and Gagne, 1987). Since cone disks (these structures should be referred to as lameîiae since they are not separate fiom each other or fiom the PM. However, to reflect the fact that they are homologous to the disks of ROSs, 1will use the tenn cone disk hmnow on.) are continuous with the PM and open at one margin, molecules are unable to difhise freely around the entue rirn region and are therefore limited with respect to the number of molecules with which they can interact. In contrast, the components of the phototransduction cascade in ROSs are fiee to diffuse anwnd the entire nm region of a rd disk. This free diffusion of phototransduction components may amplify the light response of rods to a greater extent than in cones. However, this mechanism can not completely account for the differences in sensitivity of rods and cones, since cones of marnmals have a simcant fiaction of their disk completely enclosed within the OS plasma membrane. Additionally, a single photon is unable saturate an entire cone disk, suggesting that other factors Mtthe efficacy of the cone phototransduction cascade.
Outer Segment Disks
Outer segment disks can be divided into two structuraily distinct regions (Figure 1.7a): the flattened, centrai lamelia, and the highly curved disk nm, or terminal loop (Corless, 1986). The disk lamelle are biochemically distinct hmthe photoreceptor PM in bat the lipid composition of the two membrane partitions differ. Disks contain less cholestemi than the PM (Boesze-Battaglia et al., 1989) and are therefore more fluid. This increased fluidity is believcd to allow the fke diffusion of rhodopsin that is necessary for efficient interaction with downstream components of the visual transduction cascade Terminal Loop Terminal Loop Lammclla
Frog
Mouse Cat
Figure 1.7: The stmcture of the vertehate ROS disk. A) ROS disks can be dividcd into two structurally distinct rcgions, the terminal loop (disk rim) and the disk lamella B) ROS disks of diffmnt species Vary drarnatically in size and structure. Frog disks are much larger than mammalian disks and contain up to 30 incisures that can pemtrate close to the centcr of the disk. In conaast, mammalian disk arc small and possess few inciswes. The location of the terminal loop complex (TLC), a protein complex thought to tùnction in the maintenance of ROS structure, is indicated. (Modified from Corlcss, 1986). (Bœsze-Battaglia and Albert, 1990). Moreover, ROS disks contain appmximately four times the pbpbatidylethanolamine (PE) and twice the pbosphatidylinositol (Pi) fwnd in the PM, but only two thirds the amounts of phosphatidylse~e(PS) and phosphatidylcholine (PC) in the ceU membrane (Boesze-Battaglia and Albert, 1992). It has been suggested that this lipid partition may regulate the abundance of cholesterol in the two membranes (Boesze- Battaglia and Albert, 1992), since cholesterol wiii preferentialiy segregate out of a membrane with high levels of PE and into membranes with high PC content (Yeagle and Young, 1986). ROS disks of some species appear to develop an excess amount of rirn material which affects their shape. Although ROSS are cylindrical in shape, the disks contained within them are not simply circular. Instead, disks are divided into sectors by clefts, or incisures (Figure 1.7b), which are axially aligned dong the length of the ROS. The degree of this disk lobulation varies considerably between species (Corless, 1986; Pedler and Tilly, 1978). Disk nms have been thought to be biochemically distinct hmthe lamellae since early electron micrographs indicated that the rirn membranes appeared more dense than that of the lamellae (Sjostrand, 1949). Subsequently, biochemical analyses indicated the two regions are chernically different: disk lamellae can be extracted with tris (Falk and Fatt, 1%9), organic solvents (Borovjagin and Ivanina, 1973), or detergents menweiler, 1972) afker fixation for electron microscopy, whereas disk rims remain intact. It has been suggested that known differences in protein distributions may account for these biochemical differences. For example, rhodopsin, which comprises approximately 70% of al1 protein in rod photoreceptors (Molday, 1998), is not present in disk rims (Corless et al., 1987a; Moiday et al., 1987), whereas at lest three integral membrane proteins, rom-1, peripheridrds, and
ABCR, ate known to be located exclusively at the disk nms (Bascom et al., 1992a; Iliing et al., 1997; Molday et al., 1987; Papennaster et al., 1982; Papennaster et al., 1978; Sun and Nathans, 1997). Mamrnalian COS disks display several morphological featuries that distinguish them ftom those of ROSS. First, COS disks lack incisures and are therefore almost circular in shape (Fetter and Corless, 1987). Second, COS disk diameter decreases towards the distal tip in most species (Figure 1.6). Third, the membrane of individual COS disks is continuous both with the plasma membrane and with adjacent disks for approximateIy half of their circumference (Cohen, 1% 1; Cohen, 1968; Cohen, 1970; Laties et al., 1976). Additionally , COS disk rims adjacent to the comecting ciliwn are structuraiiy different fiom the rims on the opposite side of the COS. Rims adjacent to the cone connecting cilium join two membranes of the same disk in a structure that resembles ROS disk rims, and are refemd to as the disk closed margin ( Figure 1.6). In contrast, the disk rim opposite the cilium (the open margin) is shaped by the divergence of the membranes of an individual disk, and so is composed of the membranes of two adjacent disks (Fetter and Corless, 1987). The open margin is not cytoplasmic, but instead is smunded by the extracellular ma&.
Cytoskeletal Specializations of the Photoreceptor OS
Although phototransduction involves molecules found throughout the photoreceptor OS, additional processes occur in discrete areas of the organelle. In general, the OS can be segregated into three such compartments (Figure 1.8): the distal tip, the body, and the OS base (Roof et al., 1991). Each of these regions contains ubiquitous cytoskeletal elements, such as actin and tubulin, and other proteins that are specializeû for particular functions within the photorieceptor. For example, the OS tip of toad rod photoreceptors contains specialized cytoskeletal proteins that may be involved in the phagocytosis of OS disks (Roof et al., 199 1) (Figure 1.8). Microtubules (MTs) that arise in the IS and form the connecting cilium do not extend beyond the proximal 80% of the OS (Roof et al., 199 1). Thus, the most distal5-10 pm of the OS do not contain MT'S derived hmthe CC, but instead contain many elements that are structuraüy sirnilar to MTs. These unique molecules are not affixted Tubule "cage" surmunding disks - in distal 10pm of ROS
1
OSdiskFsnis MT Filaments connect OS disks to each other and
MT-PM A cross-linkers
F'iId: Structural Specializations of the ROS. The OS can be separateci into three regions wntaining distinct cytoskeletal specializations. The distai tip contains elements believed to function in the regulation of disk phagocytosis by the RPE. The rims of OS disks contain elements that maintain the structurai stability of the OS. Finally, the OS base contains a non-motile cilium and additional ptoieins thought to be critical for the transport and segregation of molecules within the OS and for the morphogenesis of OS disks. MT= microtubules; PM= plasma membrane; CC= connecting cilium. by MT'S dismpting agents, indicating that they are not composed of a-or ptubuiin (Roof et al., 1991). Thus, these tubules may represent a photoreceptor-specific variant of MTs, similar to the pl5 tubules of touch sensitive neurons in Caenorhabdiis elegans (Chalfie and Thomson, 1982). In contrast to proteins at the ROS distal tip, cytoskeletd proteins at the rims of mature disks are thought to be critical for the maintenance of OS structure, and those localized in the region of the CC have been suggested to be crucial for the development of OS structure
(Bascom et al., 1992b; Conneil and Molday, 1990; Corless and Fetter, 1987; Corless et al.,
1987b; Steinberg et al., 1980). Since rom-1, the subject of this thesis, has been suggested to function during one or both of these processes (Bascom et al., 1992b), 1 will focus discussion of the photoreceptor cytoskeleton on elements contained within these regions.
Comecting Cilium (CC)
The CC is the only stable connection between the IS and the OS. Thus, it is a critical structure important for the transport of material between the two compartments. Moreover, OS disk formation occurs adjacent to the distal part of the CC, implying its involvement in disk morphogenesis. Accordingly, understanding the stmctural components of the CC is critical to ow comprehension of these processes. The photoreceptor CC is structufaiiy similar to aU non-motile cilia, possessing a 9+0 arrangement of MTs (Rohlich, 1975), but lacking the spokes and dynein arms associateci with motile cilia (Dame11 et al., 1990). The MT doublets within the CC resemble those of motile cilia, with an A-fiber containing a ring of 13 protofdaments and a B-fiber with 11 protofilaments (Wen et al., 1982). These MTs associate with the overlying plasma membrane (PM) by way of membrane-spanning cross-linkers that project into the extracellular space (Figure 1.8). possibly interacting with the extracellular matrix surrounding the photoreceptor (Horst et al., 1987; Muresan and Besharse, 1994; Rohlich,
26 1975). A putative function that has been suggested for this cytoskeletal system is in maintaining the polarized distribution of membrane components in the photoreceptor. Large protein concentration gradients exist between the IS and the OS, implying that some active mechanism functions to counter random diffusion and maintain photoreceptor polarity. It has been suggested that the trammembrane cross-linkers present in the CC act as a banier to diffusion and prevent the movement of membrane molecules between the adjacent cellular compartments (Horst et al., 1987; Horst et al., 1990; Muresan and Besharse, 1994). ATP has been shown to destabilize the MT-transmembrane cross-linkers and to release proteins that associate with the CC (Muresan et al., 1997), suggesting that ATP may act as a gate controUing this transport route. The CC may also have an important role during the vectorial transport of molecules into the OS. Immunofluorescence and biochernical analyses have shown that kinesin-related proteins are associated with the CC (Eckrniiier, 1993; Eckmiller and Toman, 1997; Muresan et al., 1997). Kinesin is a ubiquitous motor protein that forms cross-bridges between adjacent MTs, and in the presence of ATP, can move dong the MT hmthe (-) to the (+) end (Dame11 et al.. 1990). Another motor protein, the unconventional myosin type VIIa (myo7a), is localized outside the ring of CC microtubules, further implicating the CC in protein transport (Liu et al., 1997a). This hypothesis is supported by the phenotype observecl in the shaker-1 mouse, which carries a missense mutation (Arg502Pro) near a putative actin binding domain in myo7a (Liu et al., 1998a). These mice exhibit a three-fold increase in rhodopsin labeling in the CC, suggesting that the mutation directly decreases the rate of rhodopsin transport to the OS (Liu et al., 1998a). The identification of motor proteins within the CC axoneme (the MT array of the CC) and their suggested role in protein transport to the OS is consistent with current models of protein targeting to photoreceptor OSs. Opsin and other OS components produced within the IS are packaged into vesicles and transportai to the penciliary ridge, a region of the apical IS surroundhg the CC (Defoe and Besharse, 1985; Peters et al., 1983; Tai et al., 1999). Once at the ndge, the vesicles fuse with the PM, their contents are incorporated into the PM and are transported to the OS. The mechanism of transport is unclear, but it must be an active process since diffusion would require a concentration gradient opposite to that which exists
(Peters et al., 1983). The CC in the region of the OS base also contains a unique arrangement of actin, myosin and other cytoskeletal elements that has been suggested to be critical for the morphogenesis of new OS disks (Figwe 1.8). Electron immunomicmscopy has shown that actin is present in the distal region of the CC in both rod and cone photoreceptors (Chaitin and Bok, 1986; Chaitin and Buniside, 1989; Chaitin et al., 1984). Similar results obtained using fluorescent phallotoxùi, a mushroom derived toxin which specifically binds filamentous actin (Vaughan and Fisher, 1987), indicates that these results reflect, at least in part, the distribution of actin microfilarnents. Conventionai myosin II and a-actinin have also been detected in the CC region of the OS base (Chaitin and Coelho, 1992; Williams et al., 1992), but the localization of myosin II does not extend beyond the CC. The presence of both actin and myosin within the CC axoneme is unusual, but is entirely consistent with the involvement of the CC in the morphogenesis of nascent OS disks (see below, Disk
Morphogenesis in the Mature ROS) (Williams et al., 1992).
The Disk Rim Cytoskeleton
At least three different structural functions can be attributed to cytoskeletal elements present at the disk rims of photoreceptor OSs: 1) linking adjacent disk rims to each other, 2) linking disk rims to the plasma membrane, and 3) providing structural support for the entire OS (Roof and Heuser, 1982) (Figure 1.8). A ubiquitous cytoskeIetal protein that may be involved in di of these functions is tubulin, an expectation supporteâ by early histological studies (for example, see (Cohen, 1965; Nilsson, 1964)) which indicated that MTs denved from the CC are major structural components in the OS. Tubulin is now known to be present within the rod OS at levels appmximately 5 to 10-fold highet than that contained within the CC (Matesic et al., 1992). Approximately 2096 of the non-axonemal tubulin is tightly associateci with the PM, wbaslittle is associated with the OS disks (Matesic et ai., 1992). Tubulin and the PM pmbably interact thugh integral membrane proteins since the PM-tubulin association is oniy dismpted by treatment with urea or aikali washes. Moreover, tubulin CO-pwifieswitâ 39 kDa, 100 kDa, 220 kDa, and 260 kDa proteins, further supporthg the association through membrane proteins (Matesic et al., 1992). More recently, an additional network of MTs has been identified in rod photoreceptors. Immunofluorescence with anti-tubulin antibodies reveals MTs within the disk incisures and extending the length of mature OS (Eckmiller, 1993; Eckmiller and Toman, 1997). Kinesin and spectrin have also been identified in the incisures and ciliary axoneme of rod photoreceptors (Eckmiller, 1993; Eckmiller and Toman, 1997), suggesting that a MT-motor protein complex may be involved in the transport of molecules within the ROS. In cones, however, kinesin labeling was detected only dong one side of the COS, corresponding to the cone CC. Proteins associated closely with the disk rim have also been identifie. and are believed to play a role in maintainhg mature OS structure. The existence of these molecules was initially suggested by early electron microscopic analysis of the structure of disk rims which idenmed a well defined, U-shaped structure at the disk terminal loop (Sjostrand, 1949). Subsequent analyses indicated that the terminal loop was composed of a lipid bilayer and a densely staining component designated the tenninal loop complex (TLC) (Corless et al., 1987a). Further microscopic studies indicated that the nCof rod photoreceptors is composed of three elements (Figure 1-9a). First, interdisk densities that span the cytoplasm between adjacent disk rims and between disk incisures of individuai disks have been obseved approximately 15-20 nm from the edge of ROS disks (Corless et al., 198%; Roof and Heuser, 1982; Usukura and Yamada, 1980; Usukura and Yamada, 1981). In transverse sections, these densities are distributeci around the perimeter of ROS disks every 16-19 nm Ciosed margin (rods, cones ) Open margin (cones)
Figure 1.9: nie cytoskeleton of the ROS disk nm. Photoreceptor disk rims contain both unique and specialized cytoskeletal proteins. A. Electmn micfoscopy reveals the presence of three types of densities at the closed margin of rod d cone disk rim. A unique arrangement of densities is obsewed at the cone disk open margin (modified hmFetter and Corkss, 1987). & Five known proteins have been shown to interact at the disk rim of ROSS. GARPl and 2 intemct with tk cGW-gated channel and with abcr. Rom-1 and peripherialrds interact with each other to fom non-covalcntly linkeâ tertamers (as indicated) and disulficb linked higher order oligomers. The higher order multimers are no< indicated kcause it is unclear if the tetramers are onented side-by-side, or if they interact across the disk lumen to link opposite sides of the disk. (Corless et ai., 1987b; Roof and Heuser, 1982) and are tilted with respect to the ROS axis. This tilt may explain the observation that ROS incisures display a helical arrangement dong the axis of frog ROSS (Corless et al., 1987b): each disk is slightly rotateci with tespect to adjacent disks and the degree of this rotation is reflected in the tilt of the interdiscal filaments. These filaments may provide mechanical force that keeps âisk in alignment with each other: it has been estimated that Brownian motion of individual disk would result in a rotation of approxirnately 900 in four hours (Roof and Heuser, 1982), a movement that has never been observed even over the life span of a disk (this amount of rotation requires -40 days in fiog). A second type of füament has been identified between the disk rims and the adjacent plasma membrane (Roof and Heuser, 1982). These molecules are less evenly spaced than the interdisk filaments, and also appear braached. These proteins may interact with the tubulin associated with the PM, thereby serving a structurai role by keeping the PM closely associated with the disk rims (Roof and Heuser, 1982). Finally, components of the TLC have also been identified within the disk rims themselves (Figure I .9a). Experiments using freeze-fracture electron microscopy to analyze disk membrane components have identified rows of transrnembrane proteins in ROS disk rims (Corless et al., 1987b; Leeson, 1971; Sjostrand and Kreman, 1978; Usukwa and Yamada, 1981). In addition, semicircular densities within the intradisk space have also been detected. These components have been postulated to be responsible for organizing the TLC since their distribution more extensively and closely resembled rim structures remaining after chernical treatment (Corless et al., 1987b; Falk and Fatt, 1969). Altematively, the membrane particles may represent proteins that link the intradisk and cytoplasmic components of the TLC. Cone photoreceptors possess unique elements at the disk rims of their open margins to aid in OS structurai maintenance. In spite of the continuous nature of the disk and plasma membranes, COS disks maintain their organized stnicnire over time, implying that structural proteins maintain COS structure, much as they are thought to do in ROS. Analysis of COS structure using electron microscopy identified extracellular globular densities located at 20 nm intervals dong the outer edge of the disk open rnargin (Figwe 1.9a), between the COS disks and the calycal processes (Figure 1.6). Additionally, axially oriented filaments were identified that appeared to connect adjacent open margin loops. The localization of these structures suggests that the densities may Epresent proteins that link COS disks and the calycai processes to maintain the organization of the disks within the mature cone photoreceptor (Fetter and Corless, 1987). ïnterestingly, COS structural integrity is ca2+ sensitive (Corless et al., 1994). an observation that led to the proposa1 that the molecule(s) that Links the COS disks to the cdycal processes may be related to the calcium sensitive "tip links" present in auditory hair cens. The proteins that make up the TLC stnictmes are not presently known. However, rnolecular cloning techniques have allowed the identification and biochemical analysis of five putative TLC proteins (Figure 1.9b). One of these proteins, ABCR (rd photoreceptor ABC transporter), is a transmembrane protein that was first identif~edas a 290kDa integral membrane protein found at the rims of fkog ROS disks, and was referred to as the 'rim protein' (Papemaster et al., 1982; Papemaster et al., 1978). The gene encoding ABCR has been cloned and found to encode a member of the ABC transporter superfamily, which includes other proteins such as the cystic fibrosis transmembrane conductance regulator. The bovine ABCR protein is a 220-250 kDa membrane protein predicted to contain two structuraily related haives (Illing et al., 1997), each containing six transmembrane domains and one ATP-binding cassette that can bind both ATP and GTP (Allikmets et aï., 1997b; Iliing et al., 1997). It was initially suggested that ABCR might represent one of the filamentous molecules obsewed to connect adjacent disks and the PM (Roof and Heuser, 1982). However, its homology to ABC transporters &es this less likely. In fact, recent analyses of the phenotype of abcr knockout mice (&cd-)have provideci iasight into the biochemical function of the protein, and have cast doubt on the importance of ABCR to OS structure (Weng et al., 1999). Mice that lack all functional abcr exhibit abnonnal levels of biochemicd intermediates involved in the visual cycle. These, and other analyses, led to the suggestion that abcr transports alî-trans-retinaldehyde hmthe OS disk interior to the photoreceptor cytoplasm, where it is then moved out of the ce11 and to the RPE (Sun et al.,
1999; Weng et al., 1999). Two different potential TLC proteins, the cytoplasmic glutamic acid-rich proteins GARPl and GARP2, have recently been show to interact with abcr at the rims of ROS disks (Khchen et al., 1999). The photoreceptor GARP proteins exist in three fonns: the cytoplasmic domain of the psubunit of the cGMP-gated channel (GARP') and hvo soluble splice isofonns (GARP1 and GARP2) that are tightly bound to ROS membranes. GARP;! also binds tubulin and min, suggesting that it may also play a structurai role at the ROS disk rim (Kthchen et al., 1999). GARP2 also interacts with PDE and guanylate cyclase (GC), suggesting a role for it in phototransduction. Interestingly, GARP2 has a higher affinity for active PDE than for the holoenzyme, and cm inhibit PDE activity in a concentration dependent manner (Korschen et al., 1999). These results have suggested the possibility that GARP proteins function in a macromolecdar phototransduction signaling complex (transducisome) at the disk rim in the vicinity of the cGMP-gated channel (Korschen et al., 1999). The specific role of the GARP's may be either to aid in the termination of PDE signaling or to localize additional signal transduction proteins to the region of the ion-gated c hannel. Two additional proteins, rom- l and peripherinirds, are homologous membrane proteins localized to the disk rims of photoreceptor outer segments, where they associate as tetramers and larger oligomers (Bascom et al., 1WZb; Kedzierski et al., 1999; Loewen and Molday, 2000). Peripherin/rds has been shown to be critical for the development and maintenance of OS structure: mice homozygous for a nul1 aiieie in peripherinlrds (R&-1- or PM-rnice*) fail to fom OSs (Sanyaî and Jansen, 1981), whereas OSs of Rds+/- rnice are disorganized and shorter than nod(Hawkins et al., 1985). Given the established importance of peripherinlrds in photoreceptor structure, the rom-1: peripâerin/& complex will be discussed in greater detail.
Rom-1 and peripherin/rds are transrnembrane proteins that interact at the rims of rod and cone OS disks, and are more related to each other than to any other known proteins
(Arikawa et al., 1992; Bascom et al., 1992a; Conne11 and Molday, 1990, Moritz and Molday, 1996). The two pmteins are of comparable predicted molecular weight (rom-1: 37 kDa, and peripheridrds: 39 kDa), and share several structwal features including four putative transmembrane domains, a large hydmphilic domain between the third and fourth membrane spanning stretch, and an extended C-temiinal cytoplasmic domain. Overali, rom-1 and peripheridrds exhibit 35% identity at the amino acid Ievel, including seven conserved cysteines in the hydrophilic domain that are required for the correct folding and interaction of peripherinlrds with rom-1 (Bascom et al., 1992b; Goldberg et al., 1998). The large hydrophilic domains share 49% identity with each other, and encompass a stretch of 16 identical amino bmken by only one ciifference. Smaller regions of identity are also scattered throughout the two molecules. These structural properties allow rom-1 and peripherin/rds to be classified as members of the tetraspaain (also known as TM4SF, 4TM, TM4SF) superfamiiy of cell- surface transmembrane proteins (Maecker et al., 1997). Al1 tetraspanin proteins contain four transmembrane domains and two extracellular loops (Figure 1.10). Whemas the physiological functions of this protein family rernains elusive, the ability of tetraspanin
' The symbol for the gene encoding penpherin/rds has been changed to Prph. However, to maintain consistency with previous literature and with the designation for the human homologue, RDS,1 wiii refer to the mouse gene as Rds for the remainder of the thesis.
34 PXXC Figure 1.10: Membrane topology of ROM-1 and PenpheridRDS. Predicted membrane topology of human ROM-1and Pcripherin/RDS indicate they are members of the TM4SF superfamily of proteins. The motifs found in the large hydmphilic domain of TM4SF family members are indicated by the dark lines beside the amino acid residues. Residues that are identical in ROM-1and Peripherin/RDS are represented by open circles, while substitutions are exhibitied by closed circles. Shaded amino acids indicate the regions of significant identity between ROM-1 and Peripherin/RDS (Modified hmBascom et. al, 1992). members to interact with each other and with other molecules has suggested that they may function as "molecular facilitators," grouping specific cell-surface proteins and thus facilitating the fornation and stabiionof functional signaling complexes (Maecker et ai., i 997). Like other members of the tetraspanin fdy,rom-1 and peripherinlrds can interact with each other to form multimenc complexes (Figure 1.9b). Initialiy, experiments utilizing two dimensional electrophoresis (non-reducing conditions, then reducing) indicated that rom- 1 and peripheridrds existed as monomers or disulfide-linked homodimers (Bascom et al., 1992b). Immunoprecipitation experiments further indicated that the two proteins were expressed in approximately equal amounts, and that they interact quantitatively with each other (Bascom et al., 1992b;Moritz and Molday, 1996). Based on these results, it was proposed that rom4 and peripherui/rds formed disulfide-lùiked homodimers that associate non-covalently at the disk rim to fonn heterotetramers (Bascom et al., 1992b; Moritz and Molday, 1996). Recently, several lines of evidence have indicated that many of the early conclusions regarding the association between rom- 1 and peripherin/rds were incorrect, and that the two proteins exist in a more complex oligomenc structure than previously thought. First, analysis of the relative abundance of OS pmteins indicates that peripherinirds accounts for approximately 2% of the total OS protein, and is approximately 2.5-fold more abundant than rom-1 (Kedzierski et al., 1999; Loewen and Molday, 2000). This result indicates that the two proteins can not exist solely in a heterotetramer composed of two disulfide-linked homodimers. Second, analyses utiiizing a combination of velocity sedimentation and immunoaffinity chromatography of reduced and non-reduced OS membranes indicated that peripheridrds and rom-1 associate non-covalently as rnultisubunit "core complex" (Loewen and Molday, 20). The core complex exhibits three distinct forms: rom-1 and peripheridrds homomeric complexes, and a heteromenc core involving both pmteins (Figure 1.9b). Densitometry perfonned on western blots inâicate that the most abundant species in the bovine core complex are the penpberidrds homomeric and betemmeric core; rom-1 homomeric core complexes constitute only about 10% of total amount of core complex in ROS (Loewen and Molday, 2000). The size of the core complex is presently uncertain. While estimates using hydrodynamic measurements have predicted that the core exists as a tetramer (Goldberg and Molday, 19%c), glutaraldehyde cross-linking of corn complexes results in the preferential formation of peripherintràs homodimers and heterodimers with rom- 1. These results suggest either that the hydrodynamic studies overestimate the size of the core complex (Loewen and Molday, 2000), or that dl of the residues involved in core complex assembly are not be available for glutaraldehyde cross-linking. Finally, velocity sedimentation of non-reduced ROS proteins indicates that disuifide bridges form between core complex subunits to produce two higher order oligomenc complexes at the disk rim (Loewen and Molday, 2000). The largest complex is composed entirely of disulfide-linked periphenn/rds. In contrast, the intermediate sized oligomer, which is believed to be an octomer based on its sedimentation coefficient, cm be separated into disulfide-linked dimers and individual monomers. The dimers are both homodimers of rom-1 and peripherin/rds and heterodimers. Since rom-1 expressed by itself in COS- 1 cells exhibits little tendency to form disulfide-linked complexes, the majority of cross-linking in the oligomers is due to interactions involving peripherin/rds (Loewen and Molday, 2000). The critical peripherinhds residue appears to be the CyslSO Iocated in the large hydrophilic loop since a CISOS peripherin/rds mutant protein fails to assemble into either higher order complex when expressed in COS-1 cells (Loewen and Molàay, 2000). The peripherinhds Cl50 residue is located within the disk lumen of ROS disks, suggesting that disulfide bonds may fonn across the lumen and aid in sustaining the flattened structure of disk membranes. While it riemains unciear if the peripheridrds: rom-1 complex represents the transmembrane particles or the sernicircular densities observed at the disk rim using fieeze- fracture EM (Corless et al., 1987b; Leeson, 197 1; Sjhtrand and Kreman, 1978; Usukwa and Yamada, 198 l), it is known that peripherinlrds is required for the dcvelopment and maintenance of OS structure. Mice homozygous for a ndi aiiele in peripherin/rds (~dr-~or
~rph2-1-mice) faü to form OSs (Sanyal et al., 1980). whereas OSs of R&+/- mice are disorganized and contain whorls of membranes (Hawkins et al., 1985). This phenotype indicates that at least the peripherin/rds component of this complex has an important structural function at the disk rim. These observations, combined with the ment elucidation of the oligomenc structure of the peripherin/rds: rom4 complex, has led to the suggestion that the formation of disulfide linked oligomers of peripheridrds is cntical for the formation and structural maintenance of the OS (Loewen and Molday, 2000). In this modei, mm4 functions by inhibithg the formation of larger sized oligomers, thereby acting as a negative modulator of peripherin/rds function.
OS Renewal: Msk Morphogenesis and Phagocytosis
Although photoreceptors contain numerous cytoskeletal proteins that maintain the structure of the mature OS, the OS is by no means a static organelle. Photoxeceptors experience continual exposure to Light that can produce a simcant amount of damage (photodamage), thereby jeoparcking the health of the cell (Young, 1976). To limit the extent of photodamage, photoreceptors have evolved a mechanism by which molecules of the mature OS are continually renewed. The existence of tbis protective process was !kt demonstrated by classic autoradiographic experiments which indicated that disks form at the ROS base. migrate distally, and are eventually phagocytosed by the RPE cells (Young, 1967; Young and Bok, 1969). These two process are normally in equilibriwn with each other, and in primates, approximately 10% of the ROS disks are renewed each day (Young, 197 1). While these experiments were unable to provide comparable evidence of COS mnewal, subsequent morphological analyses of the photoreceptor-RPE interface of a varïety of species indicated that COS disks aric also pbagocytosed by RPE cells (Anderson et al., 1978; Eckmiller, 1WO).
Disk Morphogenesis in the Mature ROS
The generally accepted view of ROS disk morphogenesis postdates that disks form by a two-step process (Steinberg et al., 1980). The fmt event involves the formation of nascent, or open disks at the base of the ROS, while the second, distinct process is the development of the disk rim and the sepadon of the nascent disk from the PM.
Early morphological studies indicated that the PM at the OS base of both rod and cone photoreceptors contained numerous infoldings (Figure 1.1 1) and therefore led to the initial hypothesis that nascent disk formation occurred by a process of plasma membrane invagination (Cohen, 1% 1; Nilsson, 1964; Sjostrand, 1959). This mode1 was ul timately rejected when it was noted that the ciliary rnargin of all nascent disks were verticaliy aligned, indicating that nascent disks were in a completely invaginated state from the outset of the morphogenesis process (Anderson et al., 1978; Steinberg et al., 1980). Accordingly, open disk formation is now speculated to occur by the evagination and growth of the ciliary PM (Figure 1.1 1). As the evaginating nascent disks increase in size, they move distally dong the length of the OS and are replaced by PM invaginations (Nilsson, 1964). This distal migration has recently been shown to be independent of the presence of actin filaments, and has been suggested to involve a MT-associated molecular motor (Kaplan, 1998). The mechanism by which membrane evagination occurs in not fidiy understd, but several studies indicate that a contractile mechanism involving several known proteins is involved. Treatment of retinas with cytochalasin D, which de-polymerizes the actin filaments surrounding the CC, results in an inhibition of nascent disk formation and an abnormal increase in the diameter of the nascent disks that fodbefore chernical treatment began (Hale et al., 1992; Hale et al., 1996; Vaughan and Fisher, 1989; Williams et al., 1988). Terminal loop 'complex WC)
Mature ROS disks
Actidmyosin dense region \
Nascent KOS disks
6) Rim closure
Cilium Lipid addition to plasma membrane
Fiin 1.11: Morphogenesis of ROS disks. ROS disk morphogenesis is a two step process. A) The first step involves the evagimtion of nascent disks (Mue) at the OS base. A plasma membrane anchor complex (green), possibly containing pripherin/rds, has been suggested to interact with the actin-myosin motor of the conneting cilium during the initiation of disk morphogenesis. B) Rim closure results in the sepaation of nascent disks hmthe PM. Closure initiates in the region of the comecting ciliwn and progresses around the disk in what has been described as a 'zippering' event. Moldesat the leading edge of the disk rim. possibly the TU: (d),act as an organizational template that aiigns adjacent disks *andparticipates in the catalysis of membrane fusion between the distal (D) and proximal (P) surfaces of adjacent open di&. Membrane fusion continues dong the disk penmeter (yellow) until the disk is entirely containcd witbin the OS. These observations indicate that while actin filaments are not required for the continued evagination of nascent disks, they are necessary for the initiation of new disks. The larger ROS disks project from the OS base and tum either towards the RPE or the ONL and maintaining an association with the photoreceptor PM (Vaughan and Fisher, 1989; W'iaxns et al., 1988). These nsults suggest that an interaction occm ktween the nascent disk surface and the PM during disk formation, and that new disks may utilize this association to organize developing disks (Williams et al., 1988). Alternatively, the larger ROS disks may not directly interact with the PM, but associate with the photoreceptor PM because of the limited extracellular space available for their expansion. A detailed analysis of the locaüzation of peripherinlrds resdted in the extension of this disk morphogenesis hypohesis. Irnmunogold labeling of rod and cone photoreceptors indicates that peripherinlrds, and possibly rom-1, is localized adjacent to the CC in the most basai nascent disks, but not at the open rnargïns of nascent disks (Arikawa et al., 1992). This restricted loçaiization suggests that one or more cytoplasmic domains of peripherin/rds may act as an anchor, linking the ciliw PM to the actin-myosin motor present in the distal CC (Arikawa et al., 1992). Since this anchor would keep portions of the ciliacy PM in proximity with the CC, the addition of components to the ciliary PM would resuit in the formation of outgrowths, or evaginations, in the PM at the OS base (Figure 1.1 1). The phenotype of mice without functional peripheriahds (~ds-1-or ~tph2-i-mice) indicates that it is essential for the morphogenesis and organization of OS disks, supporthg a role for the protein in disk morphogenesis. R&-1- rnice fail to form OSs (Sanyal et al., 1980), whereas
OSs of ~ds+/-photoreceptors are disorganized and shorter than normal (Hawkins et al..
1985). Moreover, ROS disks of ~ds+/-mice are larger than normal (D. Bok, person communications), suggesting that the initiation of nascent disks is delayed in these mice. Experiments have indicated tbat at least two separate pst-translational protein modifications are critical for disk morphogenesis. Treatment of Xenopw retinas with hmicamycin, which inhibits the glycosylation of asparagine residues on proteins such as rhodopsin, results in an accumulation of numemus interconnected membrane vesicles in the extraceiiular space between the ROS and the RIS (Fliesler et al., 1985; Ulshafer et (II., 1986). These findings indicate that protein glycosylation is cntical for the targeting of membrane to the base of the OS where it can be incorporated into nascent disks. Alternatively, the nascent disks may be unstable in the absence of the sugar moieties, and prone to degeneration. Disuifide bond formation has also been shown to be required for normal disk formation (Wetzel et al., 1994) since treatment of Xenop retinas with the sulfhydryl reagent TCEP (tris-(2-carboxyethyl) phosphine hydrochloride) also results in the formation of interconnected vesicles at the ROS base. Since rom4 and peripheridrds form disulfide-linked oligomers at the disk rims, it is possible that the effects of TCEP on these proteins account for the observed abnonnalities. To fully mature, a nascent ROS disk must undergo a second process that resdts in its separation from the plasma membrane (Steinberg et al., 1980). This process, rim closure, is often described as a membrane pwthevent that 'Wppers' disk surfaces together while sealing the PM around OS (Corless and Fetter, 1987; Steinberg et al., 1980) (Figure 1.1 1). Like nascent disk formation, developrnent of disk rims is initiated at the ciliary margin of each nascent disk, where it begins with what appears as a srnail 'notch' in the plasma membrane. The notch forms a hairpin tum in the PM which represents the fmt evidence of a disk rim. At the point of rim formation, the PM and disk rim are continuous (Figure 1.1 1) (Steinberg et al., 1980). During nm closure, paired rim growth points progressively migrate around the circumference of maturing nascent disks. When the growth points meet opposite the CC, a membrane fusion event between the two pwthfronts separates the now mature disk from the PM (Steinberg et al., 1980). In this model, incisures are thought to represent an excess in rim fonnation since incisures are not obsemed in either the PM or in nascent disk membranes pnor to rim fonnation (Steinberg et al., 1980). Rim formation can readily be obsewed in tangentid sections of COS,whereas it is rare to observe developing rims in nascent ROS disks (Steinberg et al., 1980). Rim closure in both ce11 types, however, is thought to begin concurrently with nascent disk evagination, suggesting that COS disk rim formation progresses more slowly in cones, although it continues throughout their existence (Steinberg et al., 1980). This is consistent with the observations that COS disks remain continuous with the PM throughout their existence, but the extracellular space between adjacent disks becomes smaller as disks age and migrate distally (Anderson et al., 1978). A model of rim closure that incorporates observations of disk rim structure proposes that components of the TLC act as templates during rim developrnent (Corless and Fetter, 1987; Corless et al., 1989). In this model, individual TLC elements are monomers with cytoplasmic, transmembrane and extracellular (i.e. intradiscal) parts. The TLC monomers of mature disks are confmed to the disk rims and incisures where they form a structurai scaffold (Figure 1.1 1). Rim closure is initiated near the CC when a TLC element in a nascent disk interacts with the TLC of the mature disk imrnediately distal to it. Closure progresses when the TLC of the mature disk and the TLC elements of the nascent disk sequentially interact around the circumference of the two disks (Figure 1.1 1). The interaction between TLC elernents produces a rearrangement of disk membranes that result in the growtb of both the disk rim and the overlying PM. Thus, the TLC of closed disk rirns acts as a template, organizing the TLC of the open disks below it which subsequently induce the membrane changes that ~sultin rim closure. If this model is correct, peripherin/rds and rom4 may be critical proteins essential for rim closure. Several observation support this hypothesis. First, peripheridrds has been observed only at the leading edge of the nm closure process, supporting the existence of a developing scaffold at the ROS disk rim (Arikawa et al., 1992). Although it has not been experimentally determined, it is likely that mm4 exhibits a similar distribution at the closed margins of nascent disks. Second, the disulfide bonds required to fonn the rom4 and penpherin/rds oligomers at the disk nm, C 150 of peripherin/rds and C 153 of rom- 1, are located within the disk lumen (Goldberg et al., 1998; Loewen and Molday, 2000). It is possible that these disuifide bonds bridge the lumen of the disk rim, aiding in rim fusion by bringing the proximal and distal membtane domains in close proximity to-each other. Finally, a 15 amino acid stretch in carboxy terminus of peripheridrds has been shown to catalyze membrane fusion (Boesze-Battaglia et al., 1997; Boesze-Battaglia et al., 1998). further implicating the two proteins in the rim closure process.
COS Disk Morphogenesis and Remodeling
The mechanisms of COS disk formation are commoniy assurned to mirror those occurring in ROS. However, since cone photoreceptors are not as abundant as rods, and therefore more difficult to study during their morphogenesis, it remains uncertain if homologous mechanisms operate in rods and cones. However, it is clear that at least the rate of rim closure is different in the two ce11 types. In rod photoreceptors, this process is completed quickly and encept for a smali number of nascent disks, each mature OS disk is physicaüy separate fiom the PM. In contrast, cone photoreceptors exhibit iimited rim closure (Jindrovii, 1998). Moreover, rim closure in cones is asymrnetrical in that it does not occur to the same extent in ali disks and the disk membranes that remain continuous with the PM are not aligned (Anderson et al., 1978). Remodeling is an event unique to the morphogenesis of COS disks (Eckrniller, 1987). Disks located a specific distance hmthe COS base are invariaMy smailer than the more proximal disks. Moreover, COS maintain a constant taper even though their disks are phagocytosed by RPE cell (Anderson et al., 1978), indicating that a remodehg process continuaüy decreases disk diameter during their distal migration dong the COS. Since this reduction is inconsistent with models of disk morphogenesis denved from data relating to rod photoreceptors, different hypotheses have been suggested to explain COS disk remodeling. One of the earliest suggestions was thaî COS maintain taper by the continuai loss of membrane from distal disks (Steinberg et al., 1980). Structural analysis of developing COS disks identified disk membrane invaginations, referred to as 'distal invaginations', in the open margin of non-basal COS disks. Originally, it was suggested that each distal invagination arises hma sdindentaîion in the open margin of the COS disk. The invagination expands within the COS disk, eventually reaching the CC and dividing the disk in two (Eckmiller, 1987). Observations made on mature COS suggest that a similar process occurs in cone photoreceptors (Eckmiller, 1990).
Corless et al. have suggested an alternative model of COS disk remodeling based on their observation that as disks migrate dong the COS, the amount of closed margin, the disk rim that is separated hmthe PM (see Outer Segment Disks, above), remains relatively constant. In contrast, the length of open margin, the âisk membrane stili continuous with the PM (see Outer Segment Disks, above), decreases over tune, causing an overall reduction in disk diarneter (Corless et al., 1989). This change in the relative lengths of open and closed margins implies that the molecules at the open disk margin of cone disks must also undergo some son of remodeling process. Therefore, Corless et al (1989) proposed that open margin components are shifted fiom the margin to the PM surface and then removeci. In this model, the distal invaginations observed by Eckmiller (1987) are thought to represent the sites of resorption of the open rnargin components.
Disk Phagocytosis
The continual formation of disks at the base of rod and cone OSs is counterbaianced by shedding of disk packets at the photoreceptor distal tip, and phagocytosis of those packets by the RPE. This process is known to be critical for the maintenance of photoreceptor viability since the Royal College of Surgeons (RCS) rat, which is incapable of phagocytosing shed OS disks, exhibits photoreceptor degeneration (Strauss et al., 1998). The digestion of OS disks can be separated into îhree steps: detachment, phagocytosis, and phagosorne degradation (Beshm, 1986). In this section, emphasis will be given to disk detachment and phagocytosis. Evidence suggests that active rnechanisms function within the RPE cells during disk detachment. Structurai analysis of the RPE-photoreceptor interface identified RPE intmsions into the OS that separated packets of disks from the rest of the OS (Spitznas and Hogan, 1970). Additionally, Xempus eyecups in which shedding has been induced by exposure to Light or by the addition of L-glutamate (which enhances photoreceptor-RPE interaction
(Defoe et al., 1992; Matsumoto et al., 1987) and disk shedding (Greenberger and Besharse, 1985)) exhibit RPE prucesses whose appearance is comlated with disk detachment
(Matsumoto et al., 1987). These processes, which have also been observed in rat RPE cells (Chaitin and Hall, 1983), resemble stmctures found on macrophages, and have therefore been referred to as pseudopodia (Figure 1.12). RPE pseudopodia associate closely with the PM of the OS, and contain fdamentous actin. Treatment with cytochalasin D, which depolyrnerizes actin filaments, inhibits the formation of RPE pseudopodia and disk shedding, confinning that the RPE has an active role in disk shedding (Besharse and Dunis, 1982; Matsumoto et al., 1987). Furthemore, the enhancement of photorieceptor-RPE interaction that is observeù after incubation with L- glutamate (Matsumoto et al., 1987) was also not observed in presence of cytochalasin D, suggesting that the increased interaction is related to the presence of the pseudopodia. Several lines of evidence indicate that photoreceptors also have an active part in disk shedding. First, invaginations in the PM of the distal OS have been identifid prior to the formation of RPE pseudopodia, suggesting that initiation of disk shedding may occw in the photoreceptors (Young, 197 1). Second, vesiculation of ROS disks has been observed in areas where detachment is expected (Figure 1.12) (Cheet al., 1978), and the distal portion of ROSS stain with the fluorescent dye Lucifer Yellow (Matsumoto and Besharse, 1985). These observations indicate that stnrctural alterations of both the PM and OS disks occur in Pisment granules
Fimm 1.12: ROS Disk Sheddinn and Phanocvtosis. Stëps involved in the phagocyto& of phot&&eptor OS disks by the retind pigment epithelium (RPE). Active processes in the photorecepor result in the vesiculation of disks in the region where sheâding will occur, Pdymerization of actin the the RPE nsults in the fonnation of pseudopodia tbat invaginate into the OS. Once packets of disks have ken sheâ and ingested by the RPE, the phagosome fuses with lysosomes which enzyrnatically degrade the disks. photoreceptors during the shedding proœss. Finally, the MT-like elements that appear to form a cage mund OS tip are cleared just before disk shedding, and reforrn after disk packets detach from the OS (Roof et al., 1991). suggesting that they may function to inhibit continuai shedding. These stnicturai analyses indicate that photoreceptors are also actively involved in the control of disk turnover. These observations have led to a mechanistic mode1 of disk shedding that is similar to receptor-mediated phagocytosis by macrophages (Matsumoto et al., 1987). Once shedding is activated, RPE pseudopodia containing actin form in close association with the PM. At the same tirne, an active ROS process, possibly involving the fusion of PM and disk membranes, begins where detachment will occur. These structural alterations are then used by the RPE pseudopodia for intrusion into the OS (Matsumoto et al., 1987). The RPE pseudopodia move further into the OS, potentiaüy providing the physical rnechanism required for detachment. A final membrane fiision event wbich may be dependent upon the fusion protein rddperipherin (Boesze-Battaglia et al., 1997; Boesze-Battaglia et al., 1998), results in the complete detachment of the disk packet from the OS. Once this phagosome moves îùrther into the RPE cell, the actin cytoskeleton in the pseudopodia depolymerizes. After disk packets are shed, they are phagocytosed by the RPE ceiis. Phagocytosis of OS disks is usually thought to be specific, or receptor-mediated, although RPE cells can also exhibit non-specific uptake of carbon or latex beads. These two mechanisms are independent in RPE cells since the mutant RPE cells of the RCS rat can not phagocytose OS disks (Goldman and O'Brian, 1978), although they cm stiil ingest latex beads (Seyfried and McLaughlin, 1983). Receptor-mediated phagoc ytosis has several requirements: binding should occur with high afï5nity, should exhibit saturability, and phagocytosis must exhibit cornpetitive inhibition of receptor-ligand interaction by the putative ligand. These requirements are al1 met by the RPE-ROS interaction (McLaughlin et al., 1994). While the identity of ROS receptor is unknown, it appears to be glycosylatcd (Coliey et al., 1987; Hall et al., 1996). Unfortunately, the transient nature of RPE pseudopodia suggest that it may be difficult to identiQ the ROS receptor (Matsumoto et al., 1987). Once packets of disks bave been ingested by the RPE ceil, phagosomes fuses with lysosomes, resuiting in the rapid decrease in phagosome pH, and the concomitant degradation of opsin (Bosch et al., 1993;
Deguchi et al., 1996) (Figure 1.12).
Lightdependent Control Of OS Renewal
The timing of disk addition and shedding in most species varies according to light levels. Most ROS disk shedding and addition occurs around the tirne of light onset
(Besharse et al., 1977a; Besharse et al., 197%; LaVail, 1976). In contrast, the majority of cone shedding takes place at light offset (Young, 1978). However, exceptions to these patterns have been observed. For exarnple, in cats both rods and cone disk shedding increases on exposure to light (Fisher et al., 1983). Additionally, pigmented mice do not exhibit any appreciable light-induced patterns in disk turnover (Besharse and Holiyfield, 1979). These variations in the pattern of disk turnover indicates that the lightdependent regulation of OS disk renewal is a remarkably complex process. Disk renewal in the hghas been suggested to be directly controlled by the patterns of light and dark (Basinger et al., 1976). In these studies, frogs exposed to a cycle of 14 hours of light followed by 10 hours of darkness exhibit synchronous shedding of ROS disks in approximately 25% of their photoreceptors. When the da& phase of the cycle was extended by up to 14 hours, shedding was not observeà until exposure to light, indicating that illumination acted as a diFect signal controlling disk shedding. During prolonged exposure to darkness, some disk shedding occurs, indicating that light is not absolutely required to initiate the shedding process in frogs (Basinger et al., 1976). This conclusion is supporteci by additional studies in which the rate of disks shedding retwned to near-nod levels during prolonged exposure to constant light or darkness, suggesting that an endogenous circadian signal may also be involved in the regulation of disk twnover (Besharse et al., 1977b). Disk tumover in mammals has been show to exhibit many of the characteristics of a process under circadian control (Cahiii and Besharse, 1995). In rats, the bwst of shedding noxmaily observed approximately 30 minutes after light onset is maintained in the absence of lighting cues (Lavail, 1976; Lavail, 1980). However, like most biological oscillations under circadian control, the period of the turnover cycle lengthens slightly in the absence of environmental cues. Reserpine, a chernical which abolishes some circadiaa rhythms, can inhibit the periodic burst of disk shedding normaiiy observed in albino rats (Lavail, 1976). Finally, disk turnover can also be reentrained, or reset to a novel light cycle that is shifted from the previous rhythm (Lavail, 1980). In addition to cyclic patterns in disk turnover, other photoeceptor processes appear
to by under circadian control (for a review, see Cahill and Besharse, 1995). For example, the rate of expression of the gene encoding the red cone photopigment in chicken peaks just at the onset of darkness and declines throughout the night (Pierce et al., 1993). Similarly, c- fos expression by photoreceptors has been demonstrated to be controlled both by a circadian oscillator and directly by light (Yoshida et al., 1993). IUiythrns in the expression of rhodopsin and other phototransduction gene products have also been demonstrated in toad, fish (Korenbrot and Femald, 1989) and mouse (McGinnis et al., 1992). However, these have not been studied in the absence of environmental cues, so it is not certain that they are under circadian contrd (Cahill and Besharse, 1995). Consistent with the changes in phototransduction gene expression, visual sensitivity as measured by electroretinograms (ERGS) also exhibits circadian rhythmicity is several species (Cahill and Besharse, 1995). The source of circadian control has been demonstrated to reside within the photoreceptors themselves by studies which exarnined cyclic melatonin synthesis in cultured photoreceptors (Cahill and Besharse, 1993). However, the biochemical signals that set and modulate the rhythm are unclear. The most obvious signai that results in entrainment is light, which can met darcircadian rhythm in a manner &pendent of the patterns of exposure (Cam and Besharse, 1995). Dopamine has also been suggested to act as an entrainment signal since agonists of D2dopamine receptors exhibit an inhibitory effect on meletonin synthesis similar to that resulting from light exposure (Cahill and Besharse, 1991). However, since the receptor agonists did not measurably alter the effects of phase shifls in light exposure, its role as a photoreceptor circadian entrainment signal is unciear (Cahill and Besharse, 1995). While the mechanisrns that regdate disk turnover rernain unclear, one effector molecule may prove to be peripherin/rds. This disk rim-specific protein (saabove, PeripherinlRds: Rom- l Cornplex) bas been shown to undergo Light-dependent phosphorylation (Boesze-Battaglia et al., 1997) and to catalyze to fusion of photoreceptor disk and PM (Boesze-Battaglia et al., 1998). The phosphorylation of periphe,rin/rds may be under circadian or light-dependent control that increases its ability to promote membrane fusion required for both disk morphogenesis and shedding.
PHOTORECEPTOR DEGENERATION
The human retina is affected by a large nurnber of inherited diseases. To date, 121 disease loci have been identified and 56 genes chamcterized that are asmciated with retinal diseases (Daiger et al., 2000) (Table 1.1). Individual subtypes of photoreceptor degeneration are distinguished by their mode of inhentance, the pattern of visual loss, the appearance of the fundus (retina), and most recently, by the defective gene involved (Bird, 1998). In addition to the photoreceptor-specific diseases, syndromic disordm are known which affect photoreceptors and additional systems such as the ear, CNS,kidney, limbs or the endocrine system. In rnost progressive photoreceptor degenerations, the function of both cone and rod photoreceptors is compromised, but the degree to which these cell types are affected varies Table 1.1: The number of loci and genes involved in variom photoreceptor degenerations.
Retini tis pigmentosa
Cone or cone-rod dystrophy dMacular degeneration Congenital arnaurosis
Congenital stationary night blindness
Syndromic or systemic retinopathy
IOther retinopathy
Total between disorciers. For example, retinitis pigmentosa (RP) is characterized by a specific and predominant loss of rod function that is associated with tunnel vision and pigmentary changes in the retina (Bird, 1998). In contrast, a predominant involvement of cones is usually characterized by a loss of central vision. A considerable degree of variability is also exhibite. by each type of photomceptor degeneration in that they can exhibit locus (different mutant genes produce similar clinical phenotypes), ailetic (different mutations in the same gene cause an abnormal phenotype) and clinid heterogeneity (clinicaily different phenotypes result from mutations in the same gene).
Rethitis Pigmentosa
The most common fom of inherited photoreceptor degeneration, affecting approximately one in every 4000 individuals (Berson, 1993; Pagon, 1988), consists of a group of disorciers collectively referred to as retinitis pigmentosa (RP). RP is genetically heterogeneous and characterized by the progressive loss of photoreceptor and RPE function. The disease exhibits several modes of inheritance: autosomal dominant (ADRP, 15-25% of ail RP probands), autosomal recessive (ARRP, 2-20%), X-linked (XLRP,SIS%), and simplex (40-50%). XLRP is considered to be the most severe form of the disease, since male patients manifest considerable vision loss while stiil in kirthirties (Pagon, 1988). Obligate femaie carriers usually exhibit clinical manifestations of XLRP as well. The severity of ARRP, which exhibits onset dwing adolescence and progresses more rapidly than ADRP, is between that of XLRP and ADRP. Finally, while there are severe early onset fom, ADRP is generally considered to be a rnild form of the disorder in that central vision is ofien maintained until the sixth, seventh, or eighth decade (Pagon, 1988). Diagnosis of RP requires the dernonstration of 4 main chacacteristics: 1) bilateral involvement, 2) an early loss of peripheral vision, 3) del@ adaptation to the dark and/or the reduction of rod photoreceptor responses as measureâ by electroretinograms (ERGS), and 4) symptorns become more severe with age (Pagon, 1988). Patients typically describe nightblindness and difficulty in dark adaptation during adolescence (Berson, 1993). As the disease advances, there is a loss of the peripherai visual field resulting in tunnel vision, and finally the loss of central vision as well. Other symptorns include the narrowing of retinal blood vessels, the loss of pigment hmthe RPE, a waxy appearance to the optic nerve, and the migration of RPE cells to the inner retina with a comsponding increase in fine pigmentation present within the retina (Milam et al., 1998; Pagon, 1988). In advanced stages of the disease, large accumulations and clumps of melanin are found in the inner retina ("bone spicule" pigmentation) which partially accounts for the name given to the disorder. Abnodties in ERG recordings, which measure the electricai potential in the retina after stimulation by light, are often the earliest observable changes in patients with RP and can therefore be critical to making an early diagnosis. The ability of the ERG to icientiQ eady phototeceptor abnodtiesis such that it has been suggested that an individuai at risk for RP, but exhibiting no ERG abnodties by 20 yem of age will not develop RP later (Berson, 1976). These early ERG anomalies inclu& a reduction in the amplitude of the a- and b-wave, which represent the responses of the photoreceptors and the cells of the INL, respectively. In tirne, the a- and b-wave responses degenerate until they are no longer measurable. Histological analysis of the retins of patients with RP has revealed that the primary defects is the death of rod photoreceptors (Bunt-Milam et al., 1983; Mizuno and Nishida, 1967). The earliest observed change is the shortening of the ROS (Milam et al., 1998). but as the disease progresses, COS aiso shorten (Li et al., 1994). Rod photoreceptors also exhibit long newites that extend into the inner retina (Li et al., 1995b). These neurites bypass the horizontal and bipolar celis to which the rods nonnally synapses, and do not appear to make synaptic contacts with other retinal neurons. Instead, the newites closely associate with Müller cells. It has therefore been suggested that neurites represent a response of rod phototieceptors either to novel Müller ceIl swface markers or to growth factors that might be upregulated in response to disease (Milam et al., 1998). Altematively, newite sprouting may result from a disease-related loss of inhibitory façtors that norrnally control the growth of the roâ axons (Li et al., 199Sb). Changes to other components of the retina are also observed in RP patients, including a reduction in the size of the subretinal space, the region between photoreceptor ISs and RPE cells (Miiam et al., 1998). Additionally, RPE cells loose pigment and detach from the overiying tissue, migrating into the inner retina Within the retina, these RPE cells are localized in perivascular regions where they deposit extracellular ma& components, sometimes resulting in the complete obstruction of smaller retinal bldvessels(Milam et al., 1998).
Structural Proteins Associated with Photoreceptor Degeneration
Genetic studies of progressive photoreceptor degenerations were spearheaded by the discovery of mutations in the rhodopsin gene in families affected with retinitis pigmentosa
@ryja et al., 199 1; Dryja et al. , 1990a; Dryja et al., 1990b). In this section, 1will present a bief overview of OS proteins that have been associated with progressive photoreceptor degeneration, discussing those proteins localized to the disk rim, or those for which a structural function has been suggested.
RH0 (Rhodopsin)
Mutations in genes encoding many of the proteins involved in phototransduction have been identified in patients with photoreceptor degeneration. These include rhodopsin, the a and p subunits of phosphodiesterase, the a subunit of the cGMP-gated channel and rhodopsin kinase (for a complete list of proteins and references, see (Daiger et al., 2000)). Rhodopsin is the most abundant of these, accounting for greater than 70% of d protein in rod photoreceptors (Molday, 1998). Accordingly, normal function of rhoâopsin is criticaiiy important to the health of rod photoreceptors. Rhodopsin is lmalized to the PM and OS disks of rod photoreceptors and contains seven transmembrane domains. There have been approximately 100 RH0 mutations associated with ADRP identified to date (Van Soest et al., 1999), accounting for an estimated
25% of ail ADRP cases (Inglehearn et al., 1998). The characteristics of over 30 of these mutant proteins have been studied in cultured cells, resulting in the identification of two classes of RH0 mutations (Sung et al., 1991). Class 1mutations are usually located within the C-terminus of the protein, and include approximately 15% of all known RHO mutations
(Van Soest et al., 1999). Class 1 proteins usually bind to 1 1-cis-retinal and exhibit subcellular localization comparable to wild type protein when expressed in cultured cells. Class II mutations are generally scattered within the transmembrane or intradiscal domains of rhodopsin (or extracellular, in the case of molecules in the PM) (Rattner et al., 1999). These proteins exhibit abnormal folding and/or stability, tend to accumulate in the rough ER, and bind 1 1-cis-retinal poorly, or not at ail (Ratmer et al., 1999). The most common mutation in North American pedigrees, Pro23His, is a Class II mutation. Mutations in RHO have also ben associated with congenital stationary night blindness (CSNB)and ARRP, although far less frequently than with ADRP (Rattner et al., 1999; Van Soest et al., 1999). Rhodopsin is a signal transduction protein involved in the conversion of light into a biochemical signal (see above, Phototransduction). However, the analysis of transgenic mice carrying mutant Rho alleles has provided evidence that rhodopsin may also have a structural role in ROSS. Mice with Val20Gly, Pro23His, and Pro27Leu mutations (VPP mice) (Naash et al., 1993) are a good mode1 for RP since they express approximately equal amounts of normal and mutant rhodopsin and exhibit a slow degeneration of photoreceptors. In these mice, the basal OS disks are disorganized and vesicular, rather than flat and associated with djacent disks (Liu et ai., 1997b). This phenotype is similar to what is observed upon incubation of retinas with tunicamycin, which inhibits the glycosylation of rhodopsin and other retinal proteins (Fliesler et al., 1985). This obsemation has led to the suggestion that these N-terminal mutations interfere witb the interaction between adjacent nascent disks, which is thought to be required for normal organization of developing disks
(Fliesler et al., 1985).
ROMl (Rod outer segment membrane protein- 1)
ROM-1 is an integral membrane protein localized to the rims of photoreceptor OS disks (Bascom et al., 1992b; Moritz and Molday, 1996) (see above, Penpherin/Rds: Rom4 Complex), and has been suggested to be associated with approximately 1% of ADRP cases
(Bascom et al., 1995; Dryja et al., 1997). Evidence that ROM- 1 is essential to the photoreceptor, at least in certain genetic contexts, has been âernonstrated by the identification of patients with digenic RP. In four digenic RP families, affected individuds were found to be heterozygous for either one of two putative nuil mutations (Gly80 lbp ins, Leu1 14 1bp ins), or a base-pair substitution (Gly 1l3Glu) in ROMI,as well as heterozygous for a
Leu l8SPro mutation in the RDS gene (Dryja et al., 1997; Kajiwara et al., 1994). The effects of the Gly 113Glu ROM1 mutation on the structure and funçtion of the protein is presently unclear. However, the Gly 1 13 residue is located within the third transmembrane domain, and its substitution with a larger, charged residue may disrupt the membrane-spanning properties of this region, and alter the ability of ROM-1 to associate with Peripherin/RDS. Biochemical studies attempting to analyze subunit composition in the presence of the putative ROMl null alleles associated with digenic RP has led to the suggestion that the disorder results from an insufficiency in the number of Peripherin/Rds- containing tetramers (Goldberg and Molday, 1996a). However, this mode1 was based on the assumption that the Periphenn(RDS:Rom- 1 complex existed only in a heterotetrameric state. In light of more recent information indicating that more complex associations comprise Peripherin/Rds:Rom- 1 complex (Loewen and Molday, 2000). a more detailed analysis of subunit assembly is requhâ to completely understand the effects of these mutations. Few patients with dominant or sporadic photoreceptor degeneration have been found to cany putative ROMI diseaseausing alleles in the absence of RDS mutations. The
Leu1 14 1bp insertion mutation identified in patients with âigenic RP (Dryja et al., 1997;
Kajiwara et al., 1994) has also been identified, in the absence of RDS mutations, in two families with RP (Bascorn et al., 1995; Sakuma et al., 1995). This suggests that either alternative digenic partnefs may exist for ROM1 or that this allele has reduced penettance in the genetic background of individuals suggested to be unafTected carriers (Dryja et al., 1997; Kajiwara et aï., 1994). The mutation may encode a protein lacking the last transmembrane domain and the conserved intradiscal domain, or it may result in a reduction in the abundance of ROM1 mRNA (Bascom et al., 1995). Additional ROM1 mutations include a single ailele containhg both a Pro- and a Thrl08Met mutation that have been suggested to alter the stnictwe of ROM-1 (Bascom et al., 1995; Dryja et al., 1997). However, the identification of this allele in an unaffectecl individual cas& doubt on its pathogenicity (Martinez-Mir et al., 1997b). Finally, a Gly7SAsp substitution in the middle of the second transmembrane dornain could alter the membrane spanning properties of this domain. producing a non-functional protein (Bascom et al., 1995). Each of these putative ROM1 alleles associated with monogenic RP have been found in either sporadic cases or smali families (Bascom et al., 1995; Sakuma et al., 1995). Thus, the association between monogenic photoreceptor degeneration and ROM1 mutations is presently unclear.
RDS (PeripherWRDS)
The mouse Rds gene, which encodes the rds protein (hereafter referred to as peripheridrds), was cloned in an attempt to identify the gene responsible for the semidominant mouse phenotype retid degeneration slow (Travis et al., 1989). Mice homozygous for tbe mutation (~ds-1-1exhibit a graduai loss of d and cone photoreceptors that kgins at two or three weeks after birtb (Pl4-P2 1) and is complete by 9- 12 months of age (Sanyal et al., 1980).
Aithough the satureof photoreceptors in Rdr/- mice is normal at birth, there is no evidence of OS formation at any time (Sanyai and Jansen, 1981). Infiequently, cytoplasmic masses containing disorganized membranes can be observed in the apical IS or at the distal tips of the CC (Cohen, 1983; Jansen and Sanyai, 1984). These structures have been suggested to represent aborted attempts to form OSs in ~ds-1-mice (Cohen, 1983). In contrast to these photoreceptor abnormalities, the other layers of the retina appear unaffected. Heterozygous rnice (~ds+/-)also exhibit a slow photoreceptor degeneration that affects rods more severeiy than cones (Cheng et al.,
1997); by 18 months of age, photoreceptor number has been reduced by half (Hawkins et al.,
1985). Uniilce ?Us-/- mice, photoreceptors of heterozygotes form OS. However, these OSs are abnormal in that their development is delayeci, they are approximately half of the wild type OS length, and their disks associate in irregular whorls and remain disorganized throughout Life
(Hawkins et al., 1985). Using a differential hybridization strategy designeci to isolate phototeceptor-specific cDNAs, the mutated gene responsible for this phenotype was identifïed (Travis et al., 1989). One candidate hmthe screen was mapped to mouse chromosome 17, previously shown to harbour the Rdr gene (Van Nie et al., 1978). Northem blots indicated that the clone encoded an mRNA that was less abundant in R&' mice and approximately 9 kb larger than in wild type controls (Travis et al., 1989). It has since been shown that these transcnpt abnormalities result from the insertion of a 9.2 kb repetitive element into exon two of the Rdk gene (Ma et al., 1995). Proof that this was the gene tesponsible for the Rds phenotype was provided by phenotypic rescue using a transgene encoding the putative Rds gene under control of the mouse rhoâopsin gene regulatory elements (Travis et al., 1992). In contrast to ROMI, the association between RDS and monogenic photoreceptor degeneration is weU established. Appmximaly 70 potcntially pathogenic mutations have been associated with RDS ((Kohl et al., 1998). and references therein) since it was first localized to chromosome 6p 12 in 199 1 (Travis et al., 1991 a). Overall, these mutations account for approximately 3% of ADRP and 7-10% of central retina dystrophies (Kohl et al., 1998). Interestingly, al1 mutations identifieci to date have exbibited either dominant or digenic inheritance; no mutations in RDS have been documented in patients with recessive retinal degeneration (Kohl et al., 1998; Rattner et al., 1999; Van Soest et al., 1999). Of the known RDS mutations, approximately two thirds are base pair substitutions, a smali rninority of which create premature stops in the coding region (Kohl et al., 1998). Sixteen of the rernaining mutations, six insertions and 10 deletions, result in fhneshift changes that dramatically alter the structure of PeripheridRDS. Although the mutations are distributed throughout the protein, a cluster within the large hydrophilic dornain (the second intradiscal loop which contains 40% of Peripherin/RDS amino acids) contains over 70% of all the known mutations. Only 12 known changes have been detected N-temiinal to this region, and eight have been identified in the carboxy tail of the protein. The various RDS mutations produce remarkable clinical variability, with over 10 different phenotypes documented (Kohl et al., 1998; Rattner et al., 1999; Van Soest et al., 1999). Approximately 40% of the mutations result in ADRP, which is charaçterized by the primary loss of rod photoreceptors and an early decline in peripheral vision. Most of these mutations are missense substitutions located within the large hydrophilic bop, emphasizing the importance of this domain to photoreceptor viab'ity. This importance may be related to the interaction between ROM-1 and PeripheridRDS (Bascom et al., 1992h Goldberg and Molday, 1996b;Goldberg and Molday, 19%~;Goldberg et al.. 1995; Laewen and Molday, 2000), a possibility supporteci by in vitro experiments analyzing the role of the seven cysteines in the large hydrophilic loop that are conserved between ROM-1 and
PenpheridRDS (Goldberg et al., 1998). Six of these ~lesiduesare critical for the proper folding of Peripherin/RDS and for its assembly into the noncovdentiy associateâ homo- and heterotetrameric complexes. The seventh residue, Cys 150, is necessary for the formation of disulfide bonds between PeripherinIRDS and ROM4 subunits that result in the formation of higher order oligorners. Almost haif of RDS mutations are associated with diseases of the centrai retina, including Stargardt's âisease (Meins et al., 1993), Butteffly-shaped macula dystrophy
(Nichols et al., 1993a; Nichols et al., 1993b), and viteWorm maculat dystrophy (Wells et al., 1993). These àisorders are distinguished by the loss of cone photoreceptors, and result prirnarily from frameshift (insertion and deletion) and nonsense substitutions. Why these types of changes to Peripherin/RDS produce such a striking clifference in clinical outcome compared with those resulting from substitution mutations is unclear. It has been suggested that haploinsufficiency due to tnincation or nul1 allees affects the centrai retina whereas a dominant-negative effect resulting from substitution mutations preferentialiy affects rod photoreceptors (Kedzierski et ai., 1997). Altematively, the distribution of mutations throughout PeripherinIRDS suggests that different regions of the protein may have subtly different functions, each with distinct relevance to either rods or cones. An increased understanding of the fwiction of various domains in ROM-1 and Peripherin/RDS should aid our understanding of the clinical heterogeneity that results from changes in PeripherinlRDS.
MYO7A (Unconventional Myosin VIIa)
Myosin VIIA is an unconventional myosin localized to the apical processes of RPE cells (Hasson et al., 1995). the comating cilium of photoreceptors (Liu et al., 1997a), and in human embryonic cochlea and vestibular neuroepithelia It has knestirnated that mutations in MY07A can account for approximately 75% of patients with Usher syndrome subtype lb (USH1B) (Adato et al., 1997; Weil et al., 1995). Usher syndrome is a geneticaliy heterogeneous group of recessive disorclers characterized by sensorineurai hearing loss and retinal degeneration (Daiger et al., 2000). Additionaiiy, andysis of MY07A in a less severe fom of the disease known as USH3-like has identified two missense mutations, indicating that genetic background is important in coniroiîing the severity of tbe Usher phenotype (Liu et al., 1998k Weston et al., 1998). Mice with a missense mutation in the Myo7A gene (shaker4 mice) have provided insight into the possible role of this protein in rietinal biology. The mutant myo7A protein in shaker-1 mice is targeted comtly in the RPE, but localization of melanosornes in the apical processes of RPE ceUs was abrogated (Liu et al., 1998a). Thus, in the RPE, myosin VIIa may have a function similar to that of myosin V, another large unconventional myosin which is necessary for melanosorne locakation in the dendrites of melanocytes. Given the putative motor properties of myosin Wa,it is plausible that meianosomes may be transported dong the RPE apical processes as cargo of myosin VIIa (Hasson et al., 1995; Weil et al., 1996). Additionally, rhodopsin accumulation is abnonnally high in the photoreceptor comecting cilium of shaker-1 mice (Liu et al., 1998a), suggesting that this myosin is required for the transport of opsin dong the connecting cilium to the OS. This later observation provides indirect support for the idea that opsin is transported though the cilium before king incorporated into the outer segment. Moreover, it suggests that the visud loss exhibited by patients with some forms of Usher syndrome may be caused by abnonnal rhodopsin transport to the OS, leading to the destab'iation of the organelle.
ABCR (rod photoreceptor NCtransporter)
The ABCR gene encodes an ATP binding transmembrane protein (similar to CFTR, the CF protein) localized to the rims of rod OS disks, and its involvement in photoreceptor degeneration has been well established. Mutations in ABCR were initidy linked to Stargardt disease (STGD) (AlMcmets et al., 1997b. Azatian and Travis, 1997; Illing et al., 1997), a juvenile macular dystrophy (Kaplan et af., 1993) characterized by the accumulation of lipofuscin-like debris (yellow flecks) in the RPE and central vision Ioss in earîy adulthood (Stargardt, 1909). Subsequently, it was shown that mutations in ABCR could also result in ARRP. A locus for ARRP had previously been found in tbe same region on chromosome 1 (Martinez-
Mir et al., 1997a), and since ABCR is expressed in rod photoreceptors (Sun and Nathans, 1997), it became a good candidate gene for RP. Accordingly, ABCR mutations have been identified in small families with ARRP (Cremers et al., 1998; Martinez-Mir et al., 1998). Mutations in ABCR bave also been associated with recessive cone-rod dystrophy (Cremers et al., 1998) and fundus flavimacuiatus (FFM) (Rozet et al., 1998); a mild form of STGD. Interestingly, mutations tmncating the ABCR protein result in the more severe STGD while missense mutations affecthg uncharged amino acids are found only in patients with FFM (Rozet et al., 1998). The degree of phenotypic variation resulting hmdifferent ABCR mutations is unclear. Some obligate carriers of ABCR mutations appear to present with a late-onset type of macular dystrophy, and ABCR sequence changes have been identified in patients with age-related rnacular degeneration (AMD) (Allikrnets et al., 1997a; Dryja et al., 1998). Whether or not these changes are associated with a dominant susceptibility locus for AMD remains a subject of controversy (Dryja et al., 1998; Stone et al., 1998). Nevertheless, mutationai anaiysis of ABCR indicates that this gene is associated with a wider spectrum of disease phenotypes than previously suspected (Cremers et al., 1998).
AW~ mice have provided insight into the biochemicai fimction of the transporter in rod OSs, and into the mechanism of photoreceptor ce11 death (Weng et al., 1999). Anaiysis of the abundance of biochemical intemiediates involved in the visual cycle suggests that the mouse abcr protein transports ali-tram-retinaldehyde hmthe OS disk interior to the photoreceptor cytoplasm, where it is then moved out of the ce11 and to the RPE. In the absence of this transport, intermediates accumulate and are partiaiiy "digested" by the RPE cells, where their by-products accumulate as lipofuscin and kill the ceiis. In this model, photoreceptor Qegeneration is considered a secondary effet to the loss of RPE fwiction
(Weng et al., 1999).
Mechanism of Photoreceptor Cell Death
Little is known about the pathophysiological mechanisms of photoreceptor ceU death. Analysis of the biochemical mechanism involved has been hampered, until now, by a scarcity of biological reagents. However, with the advent of technologies aüowing for the creation of animal models carrying specific gene defects, we now enter an era in which it is possible to dissect, on a molecular level, the cellular abnonnalities leading to progressive photoreceptor degeneration. Analysis of such animd models, and of those occming naturally, has led to the realization that a common feahm of photoreceptor ce11 death appears to be the initiation of a cascade which ultimately leads to apoptosis (Chang et uL, 1993; Portera-Cailliau et al., 1994). Histological analysis demonstrates that apoptosis occurs in a variety of photoreceptor degenerations, including retinal detachment (Berglin et al., 1997), RP (Li and Milam, 1995), exposure to iight (Rem6 et al., 1995). and in ~ds-l-,~d-~, and transgenic Mce expressing mutant alleles of the Rho gene (for a more comprehensive list, see (Remé et al., 1998)). Apoptotic cells generally exhibit PM blebbing followed by chromatin and cytoplasmic condensation. During this process, nuclear DNA is often endonucleolytically cleaved into fragments that are multimers of 180 bp. Dying cells subsequently shrink, fragment, and the resulting apoptotic bodies are disposeci of by macrophages or adjacent cells (Remé et aL, 1998). These attributes deviate hmthose observeci during necrosis which include cellular swelling and lysis, the releaçe of lysosomal enzymes, inflammation and the death of surrounding cells. The molecular charactterization of apoptosis continues to proceed at a dizzying rate: numerous proteins involved in various apoptotic steps have been identified. Unfortunately, the molecular mechanisms by which mutations and physical injury ldto the initiation of apoptosis in photoreceptors are unclear.
The Cellular Congestion Hypothesis
Several models have been suggested over the pst decade to explain photoreceptor death that results fkom mutations in specific genes. For example, the "cellular congestion hypothesis" has been invoked to explain photoreceptor degeneration due to RHO mutations
(Li et al., 1996). Class I mutations are those with mutations in the C-terminus of rhodopsin (see above, Structural Proteins Associateci with Photoreceptor Degeneration). In vitro experiments indicate that the carboxy tail of rhodopsin is required for transport out of the Golgi complex (Deretic et al., 1996), and transgenic mice expressing a rhodopsin protein lacking 1stfive amino acids (Q344ter) exhibit an abnormal accumulation of mutant protein in
PM, consistent with the idea of protein t6cking gone awry (Sung et al., 1994). The class II rhodopsin mutant Ro23His also accumulates abnormally, initially in the synaptic PM, and then in the endoplasmic reticulun (Roof et ai., 1994). The early accumulation in synapses may represent active mislocalization of protein by the cell, while the subsequent accumulation in the ceil body occurs only after photoreceptors have lost their OSs and are unable to transport the abnormd protein elsewhere (Roof et al., 1994). The production of, and the need to degrade these abnormal proteins has ken suggested to produce a lethal metabolic stress on the photoreceptor (Sung et al., 1994). If so, this delrnay also be applicable to other disorders where the OS is lost.
The Equivalent to Light Hypothesis
Alterations in rhodopsin structure may lead to the constitutive activation of the phototransduction cascade and photoreceptor death, Le. the "quivalent to light*' hypothesis (Fain and Lisman, 1993; Fain and Lisman, 1999; Lisman and Fain, 1995). This concept is based on the observation that long-tenn expure to light can produce photoreceptor degeneration simila to RP. Momver, the action spectnun of light damage mirrom the absorption spectrum of rhodopsin, suggesting that the protein is part of the iight damage mechanism (Williams and Howeil, 1983). Confurnation of this bas recentiy been obtained through the analysis of R~~OS' mice, which lack funclionai rhodopsin (Grimm et al.,
2000). In contrast to wild type mice, ~pc65-/-mice do not show any evidence of photoreceptor death after exposure to constant light, indicating that light damage occurs through a rhodopsin-dependent mechanism. Some mutant rhodopsin proteins, such as Lys296Glu, are able to constitutively activate transducin in vitro (Rao et al., 1994; Robinson et al., 1992). The "equivalent to light" hypothesis proposes that these mutant pmteins would cause photoreceptor death by a mechanism similar to that resulting hmconstant light. Potential links between light darnage and ce11 death may include the inhibition of circadian-controlled processes, such as disk renewal (Fain and Lisman, 1993), or a reduction in photoreceptor ~a2+levels capable of triggering apoptosis (Fain and Lisman, 1999). This hypothesis may also explain ce11 death caused by mutations in other genes involved in regulating photoreceptor ion flow such as the cGMP gated channel (Dryja et al.,
1995) or retinal guanylate cyclase- 1 (Kelsell et al., 1998; Semple-Rowland et al., 1998). Moreover, photoreceptors of arrestin knockout mice undergo apoptosis when exposed to light too dim to kiU wild type celis, but the mutant photoreceptors do not die if kept in darkness (Chen et al., 1999). Data also exists which casts doubt on the devance of the equivalent to light hypothesis. Photoreceptors from transgenic rnice expressing different mutant rhodopsin alleles exhibit physiological behaviour inconsistent with this hypothesis (Goto et al., 1995; Li et al., 1996). Furthemore, the rhodopsin Lys296Glu mutant protein can bind arrestin and is phosphorylated in vivo (Li et al., 199Sa), suggesting that it cannot constitutively activate phototransduction in photoreceptors. These msuits indicate the similarities between photoreceptor death resulting light damage and fiom RH0 mutations are naas clear as was fmt suspected.
The Oxygen Toxicity Hypothesis
To accommodate the demands of disk renewal and the need to continually pump ions out of the cell (see above, Phototransduction), photoreceptors have extremely high metaboiic rates. This energy demand is reflected in the high oxygen consumption exhibited by these celis: photoreceptors use 34fold more oxygen than any other neuron (Braun et al., 1995), and are therefore particularly vulnerable to environmental and genetic insults that disrupt cellular metabolism (Stone et al., 1999; Travis, 1998). The glucose and oxygen utilized by photoreceptors diffuse fiom the choroid, a highly vascularized tissue located immediately extemal to the RPE (see Figure 1.2), and across the RPE to the photoreceptors. This arrangement results in a remarkably steep oxygen gradient between the RPE and the photoreceptor ce11 bodies (Travis, 1998). Increases in the local concentration of oxygen in the photoreceptor environment may be important causes of retinal degeneration. For example, photoreceptor death due to constant light and ''quivalent to light" mutations may be caused by an unloading of the
Na+/K+ ATPase transporter that pumps ions out of photoreceptors (Travis, 1998). During constant exposure to light, cGW-gated channels are closed and ion flux into the photoreceptor is decreased. Consequently, the rate of ion transport out of the cell required to maintain ionic balance declines, reducing the energy requirements, and therefore the oxygen consumption of the photoreceptor. However, because the supply of oxygen to the retina via the choroid is relatively constant (Stone et al., lm),the amount of oxygen delivered to the constitutively active photoreceptors exceeds their demand and the oxygen in the photoreceptor environment increases to toxic levels, killing the cells (Travis, 1998). Oxygen toxicity may dso be involved in other forms of photoreceptor degeneration.
For example, in R&' and ~ds+/-mice, OS shortenhg moves photoreceptor nuclei closer to the source of oxygen (the choroid) and may decrease the number of functional ion channels contained within the OS PM (Travis et al., 1991b). Loss of ion channels decrease the metabolic load on the dl,and the rnovement of nuclei towards the choroid increases the oxygen gradient, combining to produce toxic oxygen leveb. Since OS shortenhg is a common phenotype obsewed in many examples of phototoceptor degeneration, this model may be applicable to many forms of photoreceptor loss (Travis, 1998).
The Two-Stage Mode1 of Photoreceptor Degeneration
Developmentally regulated photoreceptor death, the death of "extra" photoreceptors that occurs as the cells are differentiaîing (Stone et al., 1999), has been suggested to be due to the competition for energy during the differentiation process (Maslim et al., 1997). Only a limited amount of nutrients are available to photoreceptors during this critical pend of development, so those photoreceptors unable to obtain sdcient energy undergo apoptosis
(Stone et al., 1999). According to the two stage model, this competition for nutrients occurs in the adult retinas as well, and provides a common mechanism explaining both inherited and acquired photoreceptor degeneration (Stone et al., 1999). The fust step in this model is a genetically or environmentaiiy caused depletion of photoreceptor number that occurs during the critical developmental period. These early insults incriease the metabolic stress on the photoreceptors and accelerate the rate of ce11 death nonnally observed during that time. The increased photoreceptor depletion decreases oxygen consumption in the retina, leading to toxic hyperoxia. At this point, the retina enters a state of positive feedback in which loss of ceils leads to -ter stress, and the subsequent loss of remaining photoreceptors. At any point in time, damaged cells that survive the toxic levels of oxygen upmgulate expression of protective fators, such as bFGF or CNTF (Valter et al.,
1998; Wen et al., 1995). decreasing the overaü rate of ce11 death. This mode1 is supported by observations made on several animal models of photoreceptor degeneration (Stone et al., 1999). For example, the RCS (Royal College of Surgeons) rat exhibits an accumulation of debris in the subretinal space between the photoreceptors and the RPE. Dwing the critical developmental period, the debris may inhibit oxygen diffusion and lead to hypoxic photoreceptor death (Valter et al., 1998). After the critical pend however, hypoxia is protective: retinas of RCS rats exhibit decreased cell death, the degeneration of OS is slowed and the decline in photoreceptor function is reduced
(Stone et al., 1999; Valter et al., 1998). Similar observations have been made during analyses of photoreceptor death caused either by light damage or by the ~d-1-mutation in mice (Stone et al., 1999), suggesting that normal oxygen levels are critical for photoreceptor surnival. Both the oxygen toxicity and the two-step mode1 of photoreceptor death can also explain observations that the equivalent-to-light hypothesis cm not: namely that photoreceptor ceIl death in several examples appears non-autonomous. For example, several forms of RP are caused by genes expressed solely within the rod photoreceptor, yet during the later stages of the disease, cones also die (Travis, 1998). Furthennore, in chimeric mice with retinas containing both nonnal photoreceptors and those expressing a mutant version of rhodopsin, a similar degree of photoreceptor loss is observed for both the wild type and the transgenic cells (Huang et al., 1993). These results indicated that the death of individual photoreceptors cm be induced by the deatth of swrounding cells, either by the production of toxic factors or by the withdrawal of trophic substances (Travis, 1998).
Rernarkable advances have been made over the past several decades in our understanding of the molecular basis of visual function. Similarly, during the past 10 years, Our knowledge of the genetic basis of photoreceptor degeneration has progressed at an astonishing rate. Unfortunately, the molecular mechanisms of photoreceptor death, and neuronal death in general, remain unclear at this time. Whiie the oxygen toxicity and two- step models are compelling, the hypotheses have not been tested rigorously. No doubt a better undemtanding of the biochemical foundations of normal photoreccptor stmcture and function, and pater insight into the molecular mechanisms of photoreceptor death wiU be required before we will be able to routinely treat visual disorders. REFERENCES:
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Rom-1 Is Required For RdPhotoreceptor ViabiJity And The Regulation Of DSsk Morphogenesis
Work in this chapter formed the basis for the publication:
Clarke,G., Go1dberg.A.F.X.. Vidgen,D.. Collins,L., Schwan,L., P10der.L.~ Molday,L.L., Rossant,J., Molday,R.S., Birch,D.G., and McInnes,R.R. (2000). Rom-1 is required for rod photoreceptor viability and the regdation of disk morphogenesis. Nature Genet. 25: 67-73.
While the majority of the work in this chapter is my own, collaborators assisted in generating the data contained in the foiiowing figures:
1) Figure 5: Sectioning, irnmunogold labeling, and imaging was performed under my supervision by Leslie Collins 2) Figure 6: ERG analysis was performed by Dr. David Birch 3) Figure 7a.b: western blots and sedimentation analysis were performed by Dr. Andy Goldberg 4) Figure 7c: Confocal rnicroscopy was perfonned under my supervision by Danka Vidgen
Reprinted by permission from Nature Genetics 25: 67-73. 0 NamAmerica Inc. (2000)
98 Abstract
Rom-1 and peripheridrâs are homologous membrane proteins localized to the àisk rims of photoreceptor outer segments (06s). wkre they associate as tetramers and larger oligomers. Disk rims are thought to be critical for disk morphogenesis, OS renewal, and the maintenance of OS structure, but the molecules which regulate these processes are unknown. Although pripherinlrds 4- is known to be required for OS formation (since lùh mice do not fonn OSs), and mutations in the RDS gene cause retinal degeneration, the relationship of rom-1 to these processes is uncertain.
-1- Here we show that Rom1 mice form OSs in which peripheridrds homotetramers are Iocalized to the disk tims, indicating that peripherinlrds alone is suscient for both disk and OS -1- morphogenesis. However, the disks produced in Rom1 mice were markedly enlarged, rdOSs were highly disorganized (a phenotype which largely nodzedwith age), and rod -1- photoreceptors died slowly by apoptosis. Furthemiore, the maximal photoresponse of Rom1 rod photoreceptors was significantly reduced. We conclude that rom-1 is requïred for the regulation of disk morphogenesis and the viability of mammalian rod photoreceptors. and that mutations in human ROM1 may cause recessive photoreceptor degeneration. Introduction
The photoreceptor is one of the most unique neurons in the vertebrate nervous system. Its most remarkable feature, the outer segment (OS), consists of a plasma membrane which, in primates, encases a stack of agproximately 100membranous disks (Young, 1971). Each disk has a lumen, a centrai larneliar region containhg rhodopsin and other proteins required for phototransduction, and a curved rim region. Biochernical analysis has indicated that the two regions are distinct; in contrast to the rims, disk lamellae are disruptted by treatment with osmium tetroxide (Falk and Fatt, 1969) and organic sotvents (Borovjagin and Ivanina, 1973). In primates, the disks of rod photoreceptors are renewed at a rate of approximately 10% per &y, by a process of disk morphogenesis occurring at the base of each rod outer segment (ROS). The newly formed disks move distaiiy dong the ROS at the same rate, until they are phagocytosed by the retinal pigment epithelium (RPE)(Young, 1971). This continual replacement of ROS disks has presumably evolved to circwnvent the problem of cumulative photodamage which would ultimately disrupt the function of ROS macromolecules (Young, 1976). Elucidation of the molecular mechanisms which regulate disk morphogenesis, and which control the diurnal migration of disks to the tip of each ROS,are central problems in IllSunrnalian photoreceptor biology. ROS disk morphogenesis has been postulated to occur by a two step process; open disk formation (disk evagination) followed by rim closure (Steinberg et al., 1980). Open disk formation begins as an outpwth of the plasma membrane at the base of the ROS and proceeds until nascent disk diameter equals that of the mature ROS. Rim closwe, the pmcess by which newly formed disks separate fiom the plasma membrane and becorne encloseci within the ROS, begiins swn after disk evagination begins, and is thought to depend critically upon a pmtein complex localized to the disk rims (Corless and Fetter, 1987; Corless et al., 1987; Steinberg et al., 1980). This pmtein complex may also facilitate the linking of adjacent disks to one another and to the plasma membrane by filamentous pmteins (Roof and Heuser, 1982). Five disk rim proteins have been characterized. One of these, ABCR, also referred to as rim protein or RmP, is an ATP-binding transporter (Illing et al., 1997) which has been suggested to be involved in the transport and recycling of ~tinoidsrequired for the phototransduction cascade (Sun et al., 1999, Weng et al., 1999). The soluble gluîamic-acid-nch proteins GARPl and GARF2 have recently been localized to the disk rim where they are believed to organize a protein complex involved in cGMP turnover (Kihchen et al., 1999). The two other disk rim proteins. rom-1 (Bascom et al., 1992) and peripherin/rds (Comell et al., 1991) are homologous integral membrane proteins of the 4TM superfamily (Wright and Tomlinson, 1994) which associate noncovalently as heterotetramers and larger disuIfide-linked oligomers (Keûzierski et al., 1999; Loewen and Molday, 2000). The locaiization of these prottins to the disk rim and the effects of a loss of pexipherin/rds on photoreceptor stmcture and viability has suggested that the rom-1 :peripherin/rds complex is required for both disk morphogenesis and the maintenance of OS structure. Mice homozygous for 4- an Rds nul1 allele (Rds mice) fail to fonn disks or OSs (Cohen, 1983; Jansen and Sanyal, 1984; +/- Sanyal et al., 1980; Sanyal and Jansen, 198 l), while Rds animal~have disorganized ROSS
(Hawkins et al., 1985). Photoreceptors of Rds mutant animals of both genotypes degenerate
(Cohen, 1983; Hawkins et al., 1985; Sanyal et al., 1980). Similady, several autosomal dominant human monogenic retinal degenerations have been associatecl with RDS mutations, including retinitis pigmentosa (RP), macular dystrophy, and pattern dystrophy (reviewed in(Koh1 et al., 1998)). Interestingly, however, mutations in RDS have never been documented in patients with recessive retinal degeneration. In contrast, no information exists on the rquirement for rom1 in disk morphogenesis or ROS structure, and the relationship of ROMI mutations to human photoreceptor degenerations is unclesu. Evidence that rom4 is essential to the photoreceptor, at least in certain genetic contexts, has been clearly dernonstrate.by the identification of patients with digenic RP (Dryja et al., 1997; Kajiwara et al., 1994). In four âigenic RP families, affected individuals were found to be heterozygous for either one of two putative nul1 mutations (Gly80 lbp ins, Leu1 14 lbp ins), or a base-pair substitution (Gly 1l3Glu) in ROMl, as weU as heterozygous for a L 185P mutation in the RDS gene. In contrast, individuals with any one of these mutations do not exhibit any obvious clinical abnormalities. On the other hanci, no convincing association between mutations in ROM2 and monogenic retinal degeneration has been obtained. Al1 of the putative ROM2 RP-associated deles have ôeen found in either sporadic cases or sdfamilies (Bascom et al., 1995; Sakuma et al., 1995). Consequently, it remains to be established whether a loss of ROM1 function aione causes retinal degeneration. To detennine whether rom- 1, like peripherin/ràs, is required for disk morphogenesis, for the formation and maintenance of the OS, or for photoreceeptor viability, we generated a nul1 allele in the Roml gene by gene targeting. The structure of cone photoreceptors was unaffkcted by the loss of rom- 1 function, and cone viability was normal. in contrast, rod photoreceptors lacking rom-1 were capable of forming ROSs, but these ROSs, and the disks within hem, were stnicturally abnormal. Moreover, rod photoreceptors lacking rom- 1 die, indicating that this protein, like peripherin/rds, is essentiai for the maintenance of the mammalian photoreceptor.
Experimental Procedures
Generation of Rom1 targeted mice.
An 18.9-kb Rom1 genornic clone was isolateci from a h. DASH II (Stratagene) phage library containing mouse strain 129Sv genomic DNA by screening with a 4.5-kb genornic fragment of Balbk mouse Rom1 gene (Bascom et al., 1993). A 1.5-kb fragrnent containing the fmt exon was removed, producing 4.6-kb upstream fragment that was ligated 5' of the Neomycin resistance gene of the pPNT targeting vectof (Tybulewicz et al., 1991). A 4.4-kb downstream fragment containing exons 2 and 3 was iigated 3' of the Neo gene. 250 pg of linearized vector 7 was electmporated into 2.8~10 RI ES cells (Nagy et al., 1993) and after 48 hours, colonies were
incubated in the presence of 250 pg/mL Geneticin (Gibco) and 2 mM gancyclovir to select for targeted clones. 332 doubly resistant ce11 lines were isolated and genomic DNA hm104 hes were hybridized (Sambrook et al., 1989) to flanking and intemal probes (Figure 1. la, b) to ident@ correctly targeted lines. Two targeted ceIl lines, A8 1 and P70, were used for momla aggregation (Nagy and Rossant, 1993) with CD1 embryos. The resulting male chimeras hmthe A8 1 ceIl line transmitted the targeted allele to appmximately half of their offspring, whereas no genn line transmission was observed from chimeras of the P70 ce11 line. Chirneric A8 1 males were bred with CD1 females to produce outbred rnice that were heterozygous for the targeted allele. Genotypes were assigned by hybridizing (Sambmok et al., 1989) genomic DNA to the flanking probe and detecting the expected band (Figure 1. lb). Heterozygotes were then bred to +/+ +/- -1- produce wild type (Roml ), heterozygous (Roml ), and homozygous (Roml ) animals. Al1 animals were treated in accordance with guideliaes established at the institutions in which the experiments were perfonned.
SDS gel electrophoresis and Western Blotting.
To analyze rom-1 abundance, retinas were dissected hmlight adapted eyes of eight week +/+ +/- -1- old Rom1 ,Rom1 and Roml mice and immersed in 30 pL of ice cold 20 mM Tris, pH 7.2, and vortexed for two minutes at top speed in a microcentrifuge. Tissues were solubilized by the addition of 30 pL of 2x loading buffer (Sambrook et al., 1989), vortexed briefly, boiled for two minutes, vortexed for 1 minute, and boiled a further two minutes. Sarnples were centrifuged for 5
O O minutes at 4 C and the supernatant collected and stored at -20 C. Three pg of protein extract was electrophoresed and transferred to Hybond-C extra membrane (Amersham) according to established protocols (Sambrook et al., 1989). Membranes were blocked with 5% BS A in TBST (10 mM Tris pH 8.0, 150 mM NaCl, 0.1% Tween ) for 1 hour at mmtemperature and incubated ovemight at 4'~with an anti-mouse rom-1 antibody diluted 1: 1000 in l%BSA in TBST.
Membranes were then incubated for 1 hour at room temperature with a 1:2500 dilution of goat anti-rabbit secondary antibody conjugated to horseradish peroxidase. Membraws were immersed in 10 mL of Lwninol (O. 1M Tris pH 8.6,0.05% 5-amino-2,3 dihydro-1,4 phthalazinedione (Sigma) in 0.03% H&) containing 1% enhancer solution (0.22% Chydroxy cinnamic acid in
MO)and immediately exposed to Reflection film (Dupont). Relative levels of rom4 were calculated from scanned images using MH Image 1.60.
For analysis of rhodopsin and peripherin/rds abundance, ROS preparations (see below) were solubilized with SDS loading buffer containing 2-mercaptoethanol, subjected to SDS gel electrophoresis on a 10% polyacrylamide gel, and transferred to Immobilon-P membranes
(Millipore) for western blotting as previously described (Goldberg et ai., 1995). PeripheWrds was detected using either the 5H2 (Connell et al., 1991) or RDH-E6 monoclonal antibody, and rhodopsin was detected with the rholD4 monoclonal antibody (Molday and MacKenzie, 1983). Western blotting was carried out by blocking the electroblotted Immobilon-P membranes in PBS containing 0.05% Tweed20 (PBST)for 30 minutes at room temperature followed by binding of
O the primary antibody in 1:20 diluted hybridoma tissue culture fluid ovemight at 4 C. Foliowing 3 x 15 minutes washes in PBST, the blots were relabelled with a secondary antibody conjugated to horseradish peroxidase for detection by enhanced cherniluminescence (Goldberg et al., 1995).
Histology . Major organs (brain, kidney, liver, lung, heart, stomach, large and srnall intestine, spleen, testes, ovary, and skeletal muscle) were dissected fiom two 6 month old mice of each genotype
O and fixed in 4% parafomaidehyde in lx PBS, ovemight at 4 C. Tissues were dehydrated, embedded in paraffin, and 7 pm sections were stained with hematoxylin and eosin, and observed on a Leitz Aristoplan microscope. Eyes were enucleated from mice of each genotype at 1,2,4,6,9, 12, and 18 months, and the superior hemisphere marked by notching the cornea. Eyecups fixed in 2.5% glutaraldehyde and 4% parafomaldehyde in O. 1 M sodium cacodylate buffer, pH 7.4, for at least 4 hours.
Superior and inferior hemispheres were isolated, trisected radiaily through the optic nerve, and pst-fued in 1% OsO, in buffer for 90 minutes. Samples were dehydrated in EtOH and propylene oxide, embedded in TAAB 8 12-Araldite 502 =sin (Merivac), and polyrnerized at 60°c for 48 hours. For light microscopy, 0.4 psections hmcentral (within 1 mm of the optic nerve) and peripheral (within 1 mm of the ora serata) regions of the superior and inferior hemispheres were obtained using a Reichart-Jung Ultracut microtome, stained with toluidine blue (1% toluidine blue, 1% sodium borate), and imaged with a Leitz Aristoplan and Kodak Ektachrome 64T fdm. For electron microscopy, silver-gold sections (-60-80 nm) were stained with saturated uranyl acetate and 1% lead citrate and observed on a Hitachi H-7000electron microscope. For irnmunogold labeling, eyes were fixed in 4% padormaidehyde and 2% glutaraldehyde in O. 1 M sodium cacodylate buffer, washed for 90 minutes, dehydrated in an
O ethanol series, embedded in LR White resin and polymerized ovemight at 55 C. Antibody labeling was performed as previously described (Bascom et al., 1992) using polyclonal antibodies against rom-1 (Bascom et al., 1992), mouse blue cone photopigment (Rohlich and Sdl, 1993), and the LD4 monoclonal antibody against rhodopsin (Molàay and MacKenzie, 1983). Secondary antibodies were goat anti-rabbit conjugated to 10 nm gold particles or goat anti-moue conjugated to 5 nm gold particles diluted to 150in O. 1 M tris and 0.1 % BSA. TUNEL Assays.
The Apoptag Plus apoptosis detection kit (Oncor) was used according to the manufacturer's suggested protocol except for the following changes. A positive control sample was generated by incubating normal mouse retina sections with 1 mg/mL DNase 1for 10 minutes at room temperature, followed by a 5 minute wash in PBS, prior to incubation with the equilibration buffer. Sections were incubated in equilibration bder for 15 minutes and immediately incubated with TdT enzyme in reaction buffer for 5 hours in a humidifhi chamber at
370C. After the enzymatic reaction was terminateci, sections were incubated with anti-
O digoxigenin-peroxidase in a humidified chamber for 4 hours at 37 C foliowed by incubation with the DAB colour substrate solution for 7 minutes at rom temperature. Images were obtained with a Leitz Aristoplan and Kodak Ektachrome 64T film.
Measurernents and statistical anaiysis.
Measurements of ONL thickness and ROS lengh were made with an ocular micrometer on a Leitz Wetzlar microscope at 1,250x magnification. ROS length was measured dong the long axis of ROSS between the inner segments and the RPE. ONL thickness was considered to be the distance between the outer limiting membrane and the outer plexiform layer, and was measured in sections where columns of rod nuclei were apparent, to ensure that sections were not oblique. For each sample, five individual measurements were averaged and measurements from at least three mice of each genotype at each tirne point. These data were analyzed using the Mann-Whitney U test (Statistica 4.1, StatSoft) to detennine if the genotype, age or location in the retina affected either ONL thickness, ROS length, nuclear density, or celi number. For analysis of disk length, a 5x5 grid was superimposed on electmn micrographs and a ROS was selected at random hmeach subdivision. Disk diameter was measured along the length of disk cross sections which could be traced along their entire diameter. Since most sections through the ROS were oblique, each disk was selected from the center of the ROS to ensure that measurements were made through the middle of ROS disks. Fifty ROS disks wexe measureâ in tach of three individuai mice, for each genotype, at 2 and 18 montbs of age. Analysis of variance (ANOVA, Newman-Keuls pst-hoc analysis module of the Statistica 4.1, Statsoft) was useâ to compare disk diameter. Ail measurements are reported as mean Is.d.
ERG analysis. 4- +/- ERGS were obtained from Rom1 mice (n=25) at 2,4,6,9, 12, and 18 months, Rom +/+ mice (n= 15) at 6, 12, and 18 months, and Rom1 (wild type) mice (n=20) to provided age- matched normal values for each parameter. Full-field ERGS were obtained as previously described (Kedzierski et al., 1997). The leading edge of the rod a-wave was fit by a computational model of the activation phase of transduction based on the Lamb and hghmodel of transduction (Lamb and Pugh, 1992):
p3 = [l - e<-s('-'d P ]RmP3 for t > td (Equation 2.1) where P3 is the sum of the responses of individual rods, i is the flash intensity, t is time after flash onset, S is a sensitivity parameter that scales i, and RmP3 is the maximum response. The value of td, a brief delay, was held constant at 3.2 msec. To quanti@ b-wave amplitudes in each mouse, we determineci V, and log k using Naka-Ruston analysis. A quantitative measure of the rod- mediateci b-wave response was provided by fitting V - log 1 fwictions with the Michaelis-Menton relationship:
(Equation 2.2) to determine the parameters of sensitivity (k) and maximum b-wave amplitude (V-). Differences among groups were analyzed as a function of age with two-way analyses of variance (Sigma-Stat; SPSS inc., Richmond CA). Significant differences among groups were fwther analyzed with phisemultiple cornparison procedures (Tukey Tests).
Preparation of ROSs. +/+ +/- Retinas were dissected from light adapted eyes of 6 week old Roml ,Rom1 or
4- Rom1 mice and immersed in 20 pl of icecold homogenization buffer (20 pi44 Tris-acetate, pH
7.2,0.25 mM MgClr 8 mM taurine, 8 mM D-glucose, 0.4 mghl Pefabloc SC protease inhibitor
(Boehringer Mannheim)) containing 20% (w/v) sucrose. Samples were vortexed (3 x 30 seconds) and the ROSs collected as the supernatant fraction afker centrifiigation at 3,000 rpm for 1 minute (3,000g). The pellet was resuspended in homogenization buffer containing 20% sucrose (40
~Yretina),vortexed, and centrifugeci as above. Supernatant fractions fkom the two extractions were combined, layered onto a discontinuous sucrose gradient consisting of 0.8 ml of 25%
O sucrose (wlv) and 0.8 ml of 4û% sucrose in homogenization buffer at 4 C and centrifuged at
O 30,000 rpm (60,000g) for 30 minutes at 4 C. The pink layer of ROS was coîlected at the 25%-
40% sucrose interface in a total volume of 0.2 ml, diluted with three volwnes of homogenization
O buffer (without sucrose) and centrifuged at 40,000 rpm (90,000g) for 40 minutes at 4 C. The
ROS pellet was then resuspended in homogenization buffer containing 20% (w/v) sucrose (10
O CLyretina) and stored at -70 C. This procedure typically yielded about 10 pg proteidtetina as measured with a bicinchoninic acid (BCA) assay kit (Pierce, Rockforci, IL). Velocity Stdimentation Analysis of Peripherin/Rds Complexes.
ROS were hypotonically lysecl and washed once with 5 mM Na-phosphate buffet (pH 7.3, 1 mM EDTA and 1 mM DTT. Washed ROS membranes were resuspended at protein concentration of 4.7 mg/d in phosphate-buffered saline (PBS), pH 7.5 and solubilized by the addition of an equd volume of ice-cold detergent solution (2xPBS, 2% Triton X-100,2 rnM Dm, pH 7.5), with gentle vortexing. Afker 20 minutes on ice, the solution was centrifugeci in a
Beclamm TLA-45 rotor at 40,000 rpm (90,000g) for 30 minutes at 4'~to remove any unsolubilized material. Sucrose density gradient sedimentation of the Triton X- 100 solub'i ROS and detection and quantification of peripherin/rds by western blotting and laser densitomitry were perfonned as describai previously (Goldberg et ai., 1995). Penpherin/rds was detected using either the 5H2 (Conne11 et ai., 1991) or RDH-E6 monoclonal antibody. Rom-1 was detected using either the anti-rom-1 C-2 polyclonal antibody or romlD5 monoclonal antibody
(Moritz and Molday, 1996) and rhodopsin was detected with the rfiolD4 monoclonal antibody
(Molday and MaciKenzie, 1983). Sedimentation coefficients (SZ0,J were estimated using a partial specific volume of v=0.83 mVg as determined for the perïpherin/rds:rom- 1 complex from bovine
ROS membranes (Goldberg and Molàay, 1996) and dimensions of the TLS-55 rotor as previously reported (Goldberg et al., 1998).
Immunofluorescence and confocal microscopy.
Eyes were enucleated hm8 week old mice of each genotype fixed in 4% paraformaldehyde in O. 1 M sodium cacodylate buffer (pH 7.4) for at lest two hours. After rinsing (3x 30 minutes) in ice-cold lx PBS (pH 7.4), eyecups were irnmersed (2x 30 minutes) in
O ice-cold 15% sucrose in PBS, followed by 30% sucrose in PBS for 16 hours at 4 C. Tissue was blotted dry, embedded in ûCï (TissueTek), and frozen in liquid nitrogencooled 2-methylbutane. We allowed the sections (7 pm) to air dry for 30 minutes, and stodthem in air-tight containers.
Sections were rehydrated by immersion in PBS (pH 7.4), and blocked for 3x 20 minutes using
10% goat semm (Sigma) in PBS. Primary antibodies to mouse rom-1 @ascorn et al., 1992) and to the central hydrophiiic domain of peripherin/rds @2P4 (Kedzierski et al., 1999)) were diluted
O in 1% Triton X-100 and 1% BSA (Sigma) and incubated for 16 hours at 4 C. After rinsing in
PBS (3x 10 minutes) and blocking with 10% goat semm for 15 minutes, sections were incubateci with goat-antirnouse Ig secondary antibody conjugated to FITC for 1 hour at rwm temperature. Sections were then washes in PBS and distilied water, mounted using ImmunoFluor (ICN Biomedicals, Inc.), and imaged on either a Leitz Aristoplan or a Zeiss Axiovert 100 microscope using a Zeiss Micmsystems laser scanning confocal microscope system 3.80.
Results
Targeting the Roml gene
To create a Roml nul1 dele, a targeting vector was designed in which the fmt exon was replaced with a bacterial neomycin resistance gene (Figure 2. la). A targeted ES ce11 line was identified (Figure 2. lb) and used to generate germ-line chimeras. Crosses of +/- Roml mice produced litters containing appmximately equal nwnbers of male and female
+/+ +/- -1- offspring, and Roml , ,and mice were obtained in the expected rnendeiian ratios.
The targeted allele was shown to be a tme nul1 by demonstrating that retinas of homozygous -1- targeted animals containeci no rom-1 protein (Figure 2. lc). The Rom1 mice were grossly normal, and no abnormalities were detected by histological analysis of major organs. Fi= 2.1: Generation of Roml mutant mice. a, Restriction maps of the endogenous Roml gene, the targeting vector, and the Rom1 targeted allele. The bacterial Neomycin resistance gene replaced the endogenous exon 1, which encodes 56% of the entire protein, and approximately 500 bp of Roml upstream sequences including the transcriptional start site. The position of the flanking and intemal probes used to identiQ targeted ceIl lines by Southem analysis are indicated. B=BamHI; H3=HindllI; X=XhoI. b, Southem blots of genomic DNA hmelectroporated ES clone A81, indicating band shifts characteristic of a homologous targeting event. Roml +/- mice showed identical restriction patterns to the A8 1 line, while blots of DNA from Roml-l- mice exhibited only the lower bands (&a not shown). c, Western blot analysis of cmde retinal protein extracts hm8 week old rnice pmbed with antibodies to mm- 1. Retinas of heterozygous (Roml +/-) rnice containecl 54.7* 14.1 % (n4) the wild type levels of rom- 1, whereas no rom-1 was detected in the retinas of homozygous mutant mice (Roml-/-), confirming that the targeted mutation is a nul1 allele. Rom- 1 is mquired for rcxi photoreceptor viability
To detennine whether the loss of rom4 function leads to photomeptor ce11 kath, we measured outer nuclear layer (ONL) thickness, an accurate measure of photoreceptor number +/- -1- (Michon et al., 1991), in wild type, Rom1 and Roml mice between 1-18 months. No significant difference was observed in ONL thickness between any genotype up to two months of age (Figure 2.2a). However, the retinas of ~oml-/-mice became progressively thinner until at
-1- least 18 months of age when the ONL of Roml rnice was 42% thinner than in wild type mice.
-1- The density of photoreceptor nuclei in the ONL of Rom1 retinas was not significantly different from controls at any age (M.05;data not shown), indicating that the observed reduction in ONL thickness was not due to a decrease in the size of the photoreceptor ce11 Mesin mutant mice. At no age was the density or thickness of the ONL in ~ornl+/-mice significantly diffemnt from that of wild type mice (Figure 2.2a). As with other photoreceptor degenerations (Travis, 1998), rom-1 deficient cells died by apoptosis (Figure 2.2b-d). Although rom-1 is expressed in cone photoreceptors in bovine (Moritz and Molday, 1996), mouse (Bascom and McInnes, unpublisheâ), and human (Moritz and
Molday, unpublished) we found no difference in cone numbers between ~oml+/+and ~oml-/- +/+ mice at 18 months of age (mean Is.d.; Roml : 9.66I1.53 cones per 150 pn section (n=3) vs.
-1- Roml : 10.7513.86 cones (n=3); P=0.860, Mann-Whitney U-test). Thus, rom-1 is essential for the maintenance of rod, but not cone photoreceptors.
-1- ROS disorganization in Roml mice becomes near-normal with age
To detennine whether the rom- 1 protein is required to generate and maintain ROSs, we +/- 4- examined retinal histology of wild type, Rom1 and hlrnice. In contrast to the ROSs of Figure 2.2: CompIete loss of rom4 results in apoptotic rod photomceptor death. a. ONL thickness in ~ornl-'-mice (triangle) was significaatly less (*; P4.05,Mann-Whitney U test) than that of wild type retinas (circles) after 4 months of age. By 18 months of age, the ONL +/+ of ~onrl"mice was 42% thinner than in wiid type mice (Rornl : 39.e1 S6 pm (n=3) vs. +/- ~~ml":22.8+3.28 ~III(n=3); P4.01). In contrast, the rainas of Rom1 rnice (squares) did not exhibit a significant decrease in ONL thickness. Each symbol represents results from an individual mouse. b, TUNEL assays indicate that apopttotic photoreceptor death was undetectable in retinas of one month old normal mice. Staining in the RPE and the overlying choroid arises from pigment granules normally contained within these cells, and is not indicative of apoptosis. c, Phase contrast image of the same field of view as panel b, indicating the location of the ONL,
INL, and RPE. d, TUNU-positive cells (arrows) in the ONL of one month old ~ontl-~mice indicate that the photoreceptors are dying by apoptosis. e, Phase contrast image of the same section as in panel d. RPE, retinal pigment epithelium; ROS,rod photoreceptor ROSS; ONL, outer nuclear layer; INL, inner nuclear layer of retina; PL,imer plexiform (synaptic) layer. 40 wiid type Rom 1 30 Rom 1 -/- (w) 20 +/- 4- wild type (Figwe 2.3a) and Rom1 mice, those of one month old Roml mutants exhibited moderate disorganization (Figure 2.3b); they were of variable length in longitudinal sections, and were not straight. The disorganization increased at two months of age (Figure 2.3c), when mutant ROSs exhibited an almost coqlete loss of the sûaight and parallel arrangement observeû in controls. Irregular gaps were often apparent between and within ROSs, and amorphous material had accumulateci between many ROSs. Unexpectedly, we found that ROS stmchire became near-normal between four and 18 rnonths of age (Figure 2.3d; n=3 mice per genotype at each age). Although scattered pockets of disorganization and amorphous deposits and gaps were still were still observed between adjacent ROSs, it became possible to trace individual ROSs from the inner segments to the RPE. Phenotypic variability prevented me hmdetennining the exact -1- age at which this irnprovement began. The correction of ROS disorganization in Rom1 mice represents a rare example of phenotypic improvement of a neurogenetic disease with age. OSs have been shown to shorten progmssively in several models of photoreceptor degeneration in which this issue has ken examined (for example, Hawkins et al., 1985), indicating that the rate of disk phagocytosis by RPE ce11 exceeds the rate of new disk production (Young, 1976). Loss of rom-1 also resulted in a decrease in ROS length that was apparent after two months of age (Figure 2.3e). At one month, ROS length was not significantly different (PM.05)between the genotypicaily different mice. Between one and two months, the length of +/- wild type and Rom1 ROSS increased significantly by 11% (Pe0.02)and 12% (P4.01), respectively, and maintained the 2 month length thereafter (Figure 2.3e). In contrast, the ROSs of -1- Ruml mice failed to lengthen between one and two months, with a maturie length 23%
-1- (P4.001,Mann-Whitney U-test) less than controls. Photoreceptors in Rom1 mice are unique in that they retain their maximum ROS length for the temainder of their lives, indicating that the equilibrium between disk production and phagocytosis is rnaintained. Figure 2.3: The loss of rom4 leads to the development of short ROSs which exhibit a biphasic pattern of disorganization. a, ROSs of one month old wild type rnice appear as straight, parailel cylinders that extend dwctly from the photoreceptor inner segments US) to the RPE. b, In contrast, ROSs of one month old
~oml"mice are ddlydisorganized. A typical accumulation of amorphous material (*) within the ROS layer is shown. c, The disorganization of ROSS had increased by 2 months of age. d,
ROS organization is near-nonnal by 12 months of age in ~oml-~mice, but the ROSs are not as closely packed as in the wild type retina and membranous accumulations are still apparent (*). e, +/- ROSS in wild type and Roml mice increase in mean length between 1-2 months of age (1 month +/+ +/+ Rom2 : 24.6I0.635 pm (n=3), vs. 2-18 months Roml : 27.3H.868 pm (n=9); P4.02; 1 +/- +/- month Rom1 : 24.6M.653 (n=3) vs. 2-18 months Roml : 27.5I0.924 (n=18); P<0.01,
Mann-Whitney U-test). In contrast, ROSS of homozygous mutant mice are not significantly different ftom each other at any age: mature ~ornl"ROSS are 23% shorter than those of wild type +/+ mice (Roml : 27.3I0.87 pm (n=18) vs. Roml": 20.9G.54 pm (n=18), P<0.001, Mann-
Whitney U test). Tirnepoints at which the length of ~oml"ROSs are significantly different
(Mann-Whitney U test, P4.05) from wild type are indicated (*). Bar = 10 p.
Loss of rom-1 increases ROS disk diameter
To determine the cause of tbe ROS structural abnodties, we examined the ultrastructure of wild type and mutant ROSs. ROS disks were weii organized wiîhin wild type photoreceptors (Figure 2.4a,c.e): regular stacks of flat disks weE closely associated with each otkr and with the plasma membrane, each disk king roughly perpendicular to the long axis of the ROS. In 4- contrast, the ultrastructure of ROSs in Roml mice exhibited three predominant abnodties.
First, the degree of disorder varied greatly between individual ROSs, with relatively normal- looking ROSs juxtaposed to ROSs whose structure was grossly distorted by membranous whorls (Figure 2.4b). Second, many rddisks were of grossly increased diameter, and were contained in packets of similarly enlarged disks (Figure 2.4, f). Third, the size of the interdisk space was +/- often increased (Figure 2.4b). Infrequently, individual photoreceptors in Rom1 mice exhibited a similar, but milder disruption of ROS structure, with enlarged, curvilinear disks, indicating that haploinsufficiency of rom- 1 produces a mild phenotype (Figure 2.4g). -1- The ROSs of Rom1 mice often contain packets of disks with grossly increased diameters
(Figure 2.4, f), demonstrating that the loss of rom4 randomly produces sever disruptions in the regdation of disk size. To detennine whether the dysregdation of disk size was a general response to the loss of rom-1, and not confined to the packets, we measured the diameter of randomly chosen disks in two and 18 month old mice (Figure 2.4h). The mean disk diameters of +/- +/+ +/- wild type and Roml mice (Roml : 1.36M.18 pm; n=300 vs. Rom1 : 1.45M.25 p; n=300) were in agreement with previous measurements of 1.4 pn (Carter-Dawson and Lavail, 1979), were not significantly different from each other (Pd.276,ANOVA), and did not Vary with
-1- age. In contrast, the mean ROS disk diameter of Roml mice was approximately 39% larger
(1.95M.51p1, n=300, Pd.001) than that of controls at both two and 18 months. Since the size Fi2.4: ROS ultrastructure is abnormal in ~oml-/-Mce. a, Wild type ROSs âisks are packed tightly within the ROS and oriented perpendicular to the long anis of the ROS. Bar = 10 pm. b, ROS disks from two month old ~ornl-/- mice are disorganized: the interdisk space is occasional1y greater than normal (open arrowhead) and the disks often appear enlargecl (arrowhead). ROS to ROS variation in the severity of the phenotype is apparent: some ROSs contain large membranous whorls (arrow). while others appear stnicnirally normal (*). Bar = 10 pm e, Except for the developing disks at the base of ROSs (arrowhead), disks of wild type photoreceptors are of uniforni sim. Bar = 1 Pm. d, The distal disks (top of panel) of a ~ornl-1-ROS are of normal size, while the packet of disk near the ROS base (arrowhead) are approximately 50% larger than an average wild type ROS disk. Bar = 1 Fm. RPE, retinal pigment epithelium; ROS, photoreceptor ROSs; IS, photoreceptor imer segments; CC, connecting cilium linking the outer and imer segments. Number d dis
Disk biameter (pm)
Figure 24 (contbiied): ROS ultrastructure is abnocmal in a on il-/- mice. e, ROSs of 18 month old wild type mice are indistinguishable from those of younger wild type Mce. Bar = 1 Pm. f, ROS disks of 18 month old ~ml-1-mice are larger than normal, but are relatively well organized within the ROS. Note that membranous whorls are still present. Bar = 1 Pm. g, Photoreceptors in hl+/-mice occasionaily exhi bit structural abnormalities similar to those in J?oml-/- mice. Bar = 1 Fm. h, Mean ROS disk diameter is incdin !?onil-/-mice. Fquency distri butions of ROS disk length measurements indicate that the increase in mean disk diameter results hmthe increased variability in ROS disk size and not hmsampling bias. Curves represent the predicted nomal distribution of ROS disk measurements based on the mean and stan- dard deviation of the data sets. RPE, retinal pigment epithelium; ROS, photoreceptor ROSs; IS, photoreceptor imer segments; CC, connecting cilium linking the outer and imer segments. of ROS disks varid dramatiçally in ~urni'-mice, we plotted fiequency distributions of disk
diameter measmments to establish that the increase in mean disk diameter was not an artifact of biased sampling in which the larger disks were over-represented (Figure 2.4h). Additionally, the distributions of disk length diameters for al1 genotypes were slightly skewed towards smaller disks, indicating that a bias towards larger disks was not present in the sample. The fact that the disks were enlarged to the same extent throughout the Life of the Rom< animais indicates that the
-1- nonnalization of Roml ROS structure observai afier four months of age cannot be explained by an age-related reduction in disk size. Furthermore, the increase in diameter does not occur gradually as new disks fom. -1- Although cone survival was not reduced in Rom1 animals, we examined the ultrastmcture of cone outer segments (COSs) to detennine if the loss of rom-1 altered COS
-/- morphology. Cone OSs of wild type (data not shown) and Rom1 mice (Figure 2.5) were indistinguishable, with flat and well organized disks. Moreover, the mean diameter of COS disks -1- +/+ of two month old wild type and Rom1 mice were not significantly different (Roml :
-1- 0.94I0.15 pm; n=2 mice, 19 cones per mouse vs. Rom1 : 0.91M.15 pm ;n=2 mice, 19 cones per mouse; Pa.626 (ANOVA)), indicating that rom-1 is not critical for either cone structure or
-1- Maximal rod photoresponses are decreased in Rom2 rnice
To evaluate the effect of a loss of rom-1 on photoreceptor function, we measured the kinetics and magnitude of the electroretinogram (ERG) a-wave in mutant and control mice (Figure 2.6a,b). Mean values for the gain of phototransduction (log S) were not significantly different among control and mutant animals, demonstrating that mm- 1 does not Figwe 2& Cone outer segments (COSs) appear structwally normal in Rornl-/- mice. a, A COS in a two month old RomP/- mouse, similar to those in wild type mice, is located among the imer segments (1s) of rod photoreceptors. A disrupted ROS is present (arrowhead) in this tow magnification micrograph. Bar = lpm. b, Higher magnification of the boxed region in panel a shows a COS labeled with antibodies specific for rnammalian cone short-wavelength photopigment (Rohlich and Szél. 1993). The COS is well organized; disks are oriented perpendicular to the COS long axis, and are of normal size. Bar = 1 Fm a-waw d b-wave . 12monîhrmdtype
- - time (ms)
a-waw 12 month ~oml-~
5 O 10 20 O 100 200 --time (ms) time (ms)
Figure 2.6: Electroretinographic responses are abnormai in ~ornl-/-mice. a,. Initial 20 mec of photoresponses to intense (1.8 to 3.4 log SC td-s) flashes in a representative wild type mouse. The dashed cwes represent fits to equation (1). b, Photoresponses from a representative 12 month old Roml-/- mouse are defreased in amplitude. c. Summary values (means f 1 s.d.) of maximum photoresponse amplitude (-3) for each group. Simcant differences (*; Pcû.05) between wild type and ~oml-1-rnice were observed at 9 months (Roml+/+: 2.33I0.10; n=2 vs. Rornl-/-: 2.03M.08; nd), 12 months (Rornl+/+: 2.33f0.11; n=2 vs. ~oml-1-: 1.84I0.07; n=3), and 18 months (~ornl+/+:2.28kO.20; n=2 vs. Rornl-1-: 1.9m.22; n=4) d, Rod b-waves to flashes ranging from 0.3 to 1.3 log SC td-s (0.3 log unit steps) in a representative wild type mouse. e, b-wave amplitudes are reduced in ~oml-~mice at 12 months. f, Su- values (means f 1 s.d.) of maximum b-wave amplitude (Vmax) for each group. Vman was significantly lower (*) in Rornl-/- mice only ai 12 months of age (~ornl+/+:2.48 f O. 19 log mV, n=3 vs. hml -/: 2.14 f O. 16 log mV, n=3). Vmax of wild type and ~omI -/-mice at 18 months (~oml+/+:2.45 f 0.16 log mV, n=2 vs. Roml-/-: 2.28 f 0.24 log mV, n4) were not significantly different. participate in phototransduction. However, a significant Merence between the maximal rod photoreceptor response (log RmP3) was observed between 9 months old wild type and
-1- Rom1 ,mice when the maximal rcsponse of the ~ornl-/-mice was 5OaD lower than that of
wild type mice (Figure 2.6~).Maximal rod photoresponses did not show further significant reductions after 9 months of age. No significant differences were observed between log +/+ +/- RmP3 of Roml and Rom1 mice at any age.
To examine the responses of secondary retind neurons, we rneasuted Vmax (maximum b-wave amplitude) and log k (sensitivity at half-saturation) in mice of each genotype. Whereas no significant differences in log k were observed between wild type and 4- mutant mice at any age, the V,, of 12 months old Rom1 mice was 55% (pd.05) lower +/+ than in age-matched Roml mice (Figure 6d, e). Surprisingly, V,, of 18 months old
-1- Roml mice showed an improvement; the difference between responses of wild type and
-1- Rom1 mice was not significant, although they differed by 33%.
-1- Peripherinhds forms homotetramers at the ROS disk rims of Rom1 mice
Peripherin/rds and rom- 1 nodyassemble as tetrameric and multimeric complexes
at the photoreceptor rims (Loewen and Molday, 2000). Because peripherin/rds can also assemble into a hornotetrarnenc complex when expressed in COS-1 cells in the absence of rom-l(Go1dberg et al., 1995), we reasoned that such homotetramers might form at the disk 4- rims of Roml mice. To examine this possibility, the relative abundance, the localization,
4- and the multimeric structure of peripherinlrds in Roml mice were analyzed. The overail
+/- 4- protein content of wild type, Roml and Rom1 mouse ROSS was indistinguishable by SDS gel electrophoresis, and the abundance of rhodopsin and peripherin/rds did not differ arnong the genotypically Merent mice (Figure 2.7a). Additionaily, the sedinientation profdes and coefficients of peripherin/rds are comparable in wiM type and ~ornl~mice
(Figure 2.7~).Findy, confocal microscopy indicated that both peripheridrds and the Abcr protein (ILliag et al.. 1997) are nonnally situateci at the disk rims of Jtontl4 mice (Fi-
2.7~-f). These fmding indicate that the signals which direct peripherin/rds and Abcr to the disk rim, and maintain hem there, act inàependently of rom- 1.
Discussion
The characterization of the retinal abnormaüties in mice with a complete loss of Roml funçtion demonstrates that the rom-1 protein is required for the normal morphogenesis of ROS disks. Moreover, the fmdings of this study demonstrate a role for rom-1 in disk morphogenesis which is consistent with the principal function of the rom- 1:peripherin/rds disk rim complex king stmctural (Bascorn et al., 1992; Molday, 1998). The normal phototransduction kinetics in the
-1- Rom1 mice appear to eliminate mm4 hmhaving a significant function in the visual cascade and rom-1 has no informative domains or motifs that suggest a non-structurai activity. However, other activities cannot be entirely excluded. The locaiization of the ABCR and GARP proteins to the disk rim, where they rn thought to be involved in retinoid transport (Sun et al., 1999; Weng et al., 1999) and cGMP turnover (Korschen et aL, 1999) respectively, illustrates that disk rim proteins may have non-structural bctions. 4- The increased diameter of rod disks in Rom1 mice demonstrates that rom-1 is critical for the regdation of membrane lipid flow into nascent disks. The molecular mechanisms which control disk morphogenesis have not been elucidated, but in addition to rom-1, at least two other molecules, peripheridrds and actin, are known to be essential for this process. Photoreceptors in
R&'- mice, which are homozygous for an Rdr nuii dele, fail to form OSs and make aborted Figure 2.7: Homotetramers of peripherin/ds are localized at the rims of ROS in Rom1 mutant mice. a, ROS protein preparations from control and mutant mice were not visibly different as uidicated Coomassie Blue stained SDS electrophoresis gels (left), and the abundance of rhodopsin (center) and peripherin/rds (right) were not appreciably altered in the absence of Rom1 expression. a, witd +/- J- type mice; b, Rom1 ; c, Rom1 . b, Velocity sedimentation analysis indicates that peripherin/rds exists in a tetrameric complex in the ROSs of 6 week oid wild type (4.92I0.11S
Immunohistochemistry indicates th& rom-1 is present exclusively at the periphery of photoreceptor OSs (inset), consistent with its localization to the disk rim (Bascom et al., 1992). Bar = 5 W. d, Antiboàies recognizing peripheridrds specifically staia the periphery of OSs (inset) in wild type mice, consistent with the colocalization of rom-1 and peripherinJtds (Bascom et al., 1992). Bar = 4- 1ûpm. e, Xn Rom1 mice, the majority of staining with peripherinirds antibodies is detected within the photoreceptor OSs, and is confined to their periphery (inset). Bar = Iûp. f,
-1- Sunilarly, the ABCR rim protein is localized to the periphery (inset) of ROSs in Rom1 mice.
IS, photoreceptor inner segments, ONL,outer nuclear layer; INL, inner nuclear layer, GCL, ganglion celï layer. -u abc abc
INL attempts at disk morphogenesis, fonning large nmbers of membmos vesicles occasionaily accompanied by multilayered membranes (Cohen, 1983; Jansen and Sanyal, 1984). +/- Photoreceptors in Ra% mice degenerate afkr developing short, disorganized ROSS (Hawkins et al., 1985) that contain enlarged disks 0.Bok, personal communication), illustrating that normal amounts of peripheridrds are also required for ROS disk rnorphogenesis. The participation of actin and myosin in the initiation of disk morphogenesis has been documenteci both by their localization to the base of the ROS where nascent disk formation begins (Chaitin et al., 1984;
Witliams et al., 1992). and by the generation of a large-disk phenotype, comparable to that -1- observed in Rom1 mice, in Xenopus and rabbit retinas treated with the actin-depolymerizing agent cytochalasin D (Vaughan and Fisher, 1989; Williams et al., 1988). The large-disk phenotype resulting from the loss of function of rom4 or actin, or from a haploinsufficiency of peripherinlrds demonstrates that each of these pmteins are required for the normal regdation of disk diameter, possibly as components of a multiprotein cornplex. On the basis of current knowledge, the regdatory events may occur at one or both of two sites in the most basal ROS disks. Because actin, myosin (Chaitin et al., 1984;
Williams et al., 1992), and peripheridrds (Arikawa et al., 1992) (and probably, therefore, also rom-1) are al1 fomd near the connecting ciliwn in the most basal disks, the initiation of new disks may be regulated at this site by a mechanism dependent on these three proteins. The loss of function of these proteins may therefore compromise the efficiency of disk initiation, and the continual addition of membrane to the ROS therefore riesults in an increase in disk diameter. Alternatively, disk size may be solely or additionally detennined by the efficiency of ri.closure. Since the presence of peripherin/rds at the leading edge of developing rims has suggested that it induces or is at least quired for rim closure (Arikawa et al., 1992), rom- 1 is also likely to participate in this process. Indeed, peripherinlrds has been shown to catalyze fusion between disk and ROS plasma membranes (Boesze-Battaglia et al., 1998). In the absence of rom- 1, the efficiency wiîh which the peripheridrds-containing wmplex mediates disk closure may be compromiseci, and nascent disks remain continuous with the plasma membrane for a greater period of the. Addition of membrane to the ROS during this period may therefore allow excess growth of nascent disks, increasing their diameter. This possibility, however, is unlikely since rim closure does not appear to be required for the regdation of disk diameter in other systems. For example, dwing the first eight hows afier light onset, the number of open disks increases by approximately three fold, but the maximum diameter of these disks remains unchanged (Besharse et al., 1977). That average disk diameter remains fixed while membrane addition to nascent disks increases indicates that the rate of rim closwe is not a limiting factor in determining ROS disk diameter. 4- +/- The phenotypic differences between both Roml and hlmice and mice with the corresponding Rdr genotype (see above) demonstrates that rom-1 is not essential for ROS morphogenesis, and that peripherin/rds is a more critical component of the mammalian -1- photoreceptor than rom-1. Furthemore, the formation of OSs in Roml mice indicates that peripheridrds can maintain at least partial function in the absence of rom- 1. The assembly of peripheridrds into a homotetrameric complex that is correctly localized to the disk rim suggests that the phenotypes of ~omFmice may k due to one of three alternative models: 1) the loss of rom- 1 per se, which assumes that rom-1 makes unique contributions to the photoreceptor; 2) a relative insufficiency of peripherin/rds "equivalents", which assumes that rom-2 has no unique functions distinct hmperipheridrds; or, 3) to some combination of these two alternatives. The proposal that rom-1 makes unique contributions to the photoreceptor suggests that the
-1- peripherin/rds homotetramers present in Roml photoreceptors are less effective in regulating disk morphogenesis, perhaps because the peripherin/rds homotetramer associates less efficiently or stably with other components of the disk morphogenesis machinesr. The evolutionary conservation of rom-1 in mammals supports the concept that rom-1 has unique functions which cannot be complemented by peripherinlrds. Mouse mm-1 is 87% and 85% identical to bovine and human rom- 1 respectively (Bascom et d-,1993; Mo& and Molday, 1996). This degree of identity between mammalian mm-1 proteins would nd be expected if rom-1 did not have some unique function(s) necessary for photoreceptor structure or viability. Additionally, peripheridrds is approximately 2.5-fold more abundant in the ROS than rom4 (Kedziemki et al., 1999). 4- Consequently, the total protein abundance in tk peripherin/rds-containing complex of hland +/- +/- Rds mice is comparable whereas the phenotype of Rrls mice is more severe, suggesting that the nivo proteins have distinct, but related functions (KedWerski et al., 1999). These considerations, taken together with the fmdings on disk morphogenesis discussed above, suggest that a major role of rom-1 in disk morphogenesis is to increase the stability of the periphedrds- containing complex to a degree greater than that achieved by peripheridds alone. Alternatively, the peripheridrds homotetramers may be able to substitute completely for the 4- wild type rom- l :peripherin/rds heterotetramer in the Roml mouse. This hypothesis implies that the retinal abnormalities in the ~ornl-~mouse are the result of insufficient peripherinlrds
"equivalents", and reflects the fact that the abundance of penpherinfrds does not increase in the
-/- Rom I retina (Figure 2.7a) to compensate for the loss of rom- 1. Since the abundance of the peripherin/rds homotetramer would be appruximately 70% of the normal levels of the mm4:peripherin/rds heterotetramer, the fwiction of the resulting peripherin/rds complex may be reduced below a threshold required for normal disk morphogenesis. These alternative models of
-1- the Roml photoreceptor phenotype can be examined directly by increasing the level of
4- peripherin/rds expression in Rml mice using peripheridrds transgenes. Correction of the
~oml-/-retinal phenotype would provide direct support for the pripherin/rds equivalent hypothesis. Our studies clearly estabLish that the rom-1 protein is essentiai for the viabiiity of tk 4- mammalian rod photoreceptor. The rate of apoptotic rod cell death in Roml mice is the slowest of any of the 11 animal models of inherited retinal degeneration in which we have examineci the kinetics of celi death (see Chapter 3). In fact, only the mouse model of gyrate atrophy, which is -1- thought to affect pnmarily the RPE, displays slower degeneration than Rom1 mice (Wang et al.,
19%). Consequently, the rate of apoptotic rod celi death in ~oml-1-mice is the slowest of any animai model of photoreceptor degeneration pduced by a mutation in a photoreceptor-specific gene. In particular, the rate of photoreceptor loss in hl-/-mice was slower than that observed in both the ~dr+/-and R& mice (Hawkins et al., 1985), indicating that a complete loss of Rom1 fùnction is less detrimental to photoreceptor viability than a loss of 50% of peripherin/rds. As a model of slowly progressive rdphotoreceptor degeneration, this mutant will be particularly valuable for the evaluation of potential therapies with subtle but definite effects, such as Vitamin A
(Berson et al., 1993). which may not be evident in models with more rapid degeneration. 4- Additionaily, the gradual photoreceptor death in the Roml mutant will be advantageous in pathophysiologicai analyses of mutant photoreceptors, since the influence of secondary effects will accrue more slowly than in other mutants. The lack of any significant phenotype in mice carrying one Rom2 nuli ailele is a major distinction in the gene dosage effect of mutations in Roml and Rh, since both mice and human heterozygous for mutations in the RDS gene manifest retinal degeneration (Hawkins et al., 1985;
Kohl et al., 1998); notably, only dominant hwnan RDS mutations have been identified to date. +/- The virtuai norxuaicy of the retina of Roml mice suggests why few patients with dominant or sporadic retinal degeneration have been found to carry putative ROM1 disease-causing alleles, of which only 4 have been identifieci to date (Bascom et al., 1995; Dryja et al., 1997; Martinez-Mu et al., 1997; Sakuma et al., 1995) (apart from those identified in patients with digenic RP). Thus, 4- since only hlrnice had retinal degeneration, it seems probable that only individuais with dominant negative ROMl deles, or two loss-of-function ROM1 alleles, will have photoreceptor disease. Although Roml is expresseci in mouse (Bascorn and Mches, unpublished observations), hwnan (Moritz and Molday, unpublished), and bovine cones (Moritz and Molday, 4- 1996), no evident effect on cone stmcture or viability was detected in Rom1 mice.
Consequently, in conttast to mutations in the RDS gene, mutations in human ROM seem unlikely to be associated with cone degeneration. As with virtually al1 other examples of photoreceptor degeneration, the mechanism of rod -1- photoreceptor death in Rom1 mice is unclear. The eniarged disks of the mutant mice may be responsible for the initiation of cell death, although it is -cuit to envisage how an increase in disk size would impair photoreceptor homeostasis sufficiently to initiate apoptosis. It is also 4- difficuît to attribute the ROS disorganization of Roml mice to photoreceptor death, particularly since the celis continue to die between 9 and 18 months, a period during when ROS disorganization became minimal. However, our analysis of the kinetics of photoreceptor death in
~oml-1-mice (see Chapter 3) demonstrates that the risk of cell death may decrease and approach zero after about 18 months of age in these animals. Without detennining the rate of ceN death after 20 months, it is not possible to determine whether ceil death actualiy ceases. If ce11 death does stop in these animals, the acquisition of nonnal ROS structure may be directly and causally related to the decrease or arrest of photoreceptor ceii death observed. If celt death does not eventually 4- stop in Roml mice, then other biochemical abnormalities must be responsible for the loss of photoreceptors in these animals. In surnmary, our results indicate that a nul1 mutation in Roml alone is sufficient to cause slow rod photoreceptor degeneration in mice and that sirniiar mutations in hurnans are Likely to result in late-onset forms of recessive rod photoreceptor degeneration. Elucidation of the biochemical basis for the different effects of loss of function of Roml vs. Rdr on disk morphogenesis, rod photoreceptor OS stmcture, and rod and cone viability will depend on deterarining whether mm-1 and peripheridrds function solely as structural proteins, adif so, on identification of the molecules with which they interact during disk morphogenesis and in the disk lumen and cytoplasm of the mature photoreceptor. Reterences
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A One-hit Mode1 of CeU Death in lnberited Neuronal Degenecations
Work in this chapter formed the basis for the publication:
Clarke,G., Collins,R. A., Leavitt,B .ReTAndrews,D.F., Hayden,M.R., Lumsden,C.J., and McInnes,R.R. (20).A one-hit mode1 of cell death in inherited neuronal degenerations. Nature 406: 195-199.
The majority of the work presented in this chapter was is my own, with the following exceptions:
1) Figure 3e: Repeated-measures, non-lin- regression was performed by Dr. David Andrews on data gathered by Drs. Blair Leavitt and Michael Hayden
Reptinted by permission fiom Nature 406: 195-199. @ Macmillan Magazines Ltd. (2000) Abstract
In genetic disorciers associated with premature neuronal death, symptoms may not be evident for years or decades. This delay in clinical onset is of'ten assurned to reflect the occurrence of age-dependent cumulative darnage (Alves-Rodrigues et al., 1998; Cassarino and Bemett Jr., 1999; CoyIe and Puttfarken, 1993; Cummings et al., 1998; Dunnett and Bjorklund, 1999; Heales et al., 1999; Schapira, 1999; Sekoe, 1999). A prediction of the cumulative damage hypothesis is that the pmbability of cell death will Uicrease over time. in contrast, we show here that the kinetics of photoreceptor cell death in 12 animal models of retinal degeneration arie exponential and better explained by mathematical models in which the risk of cell death remauis constant or decreases exponentialiy with age. Thus, the kinetics argue against neuronal death being a consequence of cumulative damage; instead, the time of death of any neuron is random. we also demonstrate that patients with Parkinson's (Feamley and Lees, 1991) and Huntington's disease exhibit a constant risk of ce11 death, as do the cerebeilar granule cells of pcd/'d mice (Triarhou, 1998). Tbese fmdings are most simply accommodatecl by a "one-bit" biochemical model in which mutation imposes a mutant steady-state (MSS) on the neuron and a single random event initiates cell death. This model of neuronal death appears to be cornmon to many, if not A,neurodegenerations. Introduction
Although much has been leamed about the morphologiccal and biochemical characteristics of neuronal cell death, the earliest events which initiate the degeneration pathway in these cells are poorly unclerstood. For example, patients suffering hma variety of inùerited diseases manifest premature photoreceptor loss that eventually leads to blindness. Whereas the genetic basis for a large number of these diseases is now known (for a detailed List, see Daiger et al., 2000), we have little clue as to the mechanisms that initiate photoreceptor cell death in these disorciers. Several meçhanisms have been suggested to explain the initial steps in photoreceptor death. For example, the "cellular congestion hypothesis" has been invoked to explain photoreceptor degeneration due to some mutations in the gene encoding the visual pigment Rhodopsin (the RHO gene) (Li et al., 1996). Mutations in the carboxy tail of the protein have been shown to result in abnormal accumulations of the mutant protein the ER (Roof et al., 1994) or the PM (Sung et al., 1994), suggesting that abnormal protein crafflcking causes an increase in metabolic stress that results in photoreceptor death (Sung et ai., 1994). Altematively, the constitutive activation of the phototransduction cascade has been suggested to cause photoreceptor ce11 death in a manner simiiar to that exbibited by photoreceptors exposed to constant illumination (Fain and Lisrnan, 1993; Lisrnan and Fain, 1995; Fain and Lisman, 1999). Unfortunately, how constant light initiates ceil death is unclear, although suggestions have rangeci fiom an inhibition of circadian-controlled processes, such as disk renewal (Fain and Lisrnan, 1993), or a reduction in photoreceptor ~a2+levels capable of triggering apoptosis (Fain and Lisman, 1999). A more recent idea is that toxic levels of oxygen can lead to photoreceptor degeneration (Travis, 1998). When OSs shorten, photoreceptor nuclei are effectively moved up the oxygen gradient that exists between ihem and the choroid, the highly vascularized tissue that pmvides most of the nutrients to the cells. As photoreceptors move up the concentration gradient, the oxygen levels pm&~essivelyinmase to toxic levels, initiating cell
death. Similady, OS shortening has been suggesteâ to decrease photoreceptor energy requirements by lowering the number of ion channels in the PM (Travis et al., 1991). This decmase in energy demand. and themfoce in oxygen consumption, may conspire with the movement of photoreceptors up the oxygen concentration gradient to fiirther increase the likelibood of cell death. Since OS shortening is a common phenotype observed in many examples of photoreceptor degeneration, this model may be applicable to many forms of visual loss. A related model suggests that photoreceptor death is a two stage process (Stone et al., 1999). Experirnents have suggested that developmentally regulated photoreceptor death, the death of "extra" photoreceptors that occurs as the cells are differentiating, is due to the competition for energy and nutrients durhg the dflerentiation pmcess (Maslim et al., 1997).
However, resowce availability is limited during this critical period of development, so those photoreceptors unable to obtain sufficient energy undergo apoptosis. Environmental or genetic insults that occur during this time can increase the level of metabolic stress and accelerate this normal loss of photoreceptors. This abnormal decrease in photoreceptor number results in increased oxygen concentration in the photoreceptor environment, a toxic situation that triggers the death of the remaining cells. Three general rnechanisms, that may act independently or cooperatively, have been suggested to cause premature neuronal death in disorders affecthg the central nervous system. The fmt, metabolic stress, primarily affects mitochondrïa and can lead to a depletion of cellular ATP ceserves and a general dysregulation of Mtochondnal function (Alexi et al., 2000). Decreased ATP concentration can ducethe activity of plasma membrane ion purnps, resulting in progressively higher intraceliular C? ievels and other ionic imbalances. The second mechanism. excitotoxicity produced by amino acid neurotransmitters, has also been suggesteâ to lead to increased levels of ca2+through the elevated activation of NMDA receptors (Alexi et ai., 2000). These two mecbanisms can, in hm, activate a third pathway leading to neuronal de&: oxiàative stress. Oxidative stress, defined as an imbalance between the production of reactive oxygen species (fiee radicals) and ceilular antioxidant mechanisms, has been widely implicated as a general mechanism of neuronal death in all regions of the nervous system (Cassarino and Bennett Jr., 1999; Coyle and Puttfarken, 1993). In fat, it has kensuggested that the discovery of the role of oxidative stress and fiee raâical generation in degenerative disease is as important as the elucidation of the role of rnicmorganisms in infectious disease (Bray, 1999). The brain is particularly Milnerable to the effects of increased free radical production for three reasons: 1) almost al1 of its energy is derived hmaerobic metabolism (Coyle and Puttfarken, 1993). 2) it is enriched in highly peroxidizable bpi& and 3) it has a relative paucity of antioxidant defense mechanisms in comparison to other tissues (Fioyd, 1999).
Oxygen free radicals, which include the superoxide anion (-0;)and the hydroxyl radical (-OH), are produced during a variety of other cellular processes (Betteridge, 2000;
Coyle and Puttfaen, 1993; Floyd, 1999) inctuding aerobic metabolism. Once produced, free radicals can react with proteins, lipids, and DNA to disrupt ce11 structure and function (Coyle and Puttfarken, 1993). In fact, a single molecule cmset off a fiee radical chah reaction within ce11 membranes that ultirnately result in the dismption of membrane integrity and damage to membrane proteins (Betteridge, 2000). Such mechanisms have been implicated in numerous neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, amyotrophie lateral sclerosis, and Huntington's disease (Cassarino and Bennett Jr., 1999; Coyle and Puttfarken, 1993; Dunnett and Bjorklund, 1999; Schapira, 1999). A common feature of these photoreceptor-specific and more generai neuronal ce11 death mechanisms is that over tirne, there is an accumulation of cellular àamage that eventualiy leads to celJ death (the "cumulative damage hypothesis"). This concept most likely amse h9m the observation that neurodegenerative disorciers usually exhibit a long delay in clinical onset during which neurons survive and fwiction relatively norrnaiiy. Unfortunately, whereas ample biochemical eviàence indicates that damage does accumulate within injured newons, it remallis unclear if the accumulations are ditiectly responsible for the ceil death observed in neurodegeneraîive diseases. One prediction of the cumulative damage hypohesis, however, is that the initial probability of newonal death is low but that as mutant neurons age, increasing celiuIar damage causes a comsponding increases in the nsk that a ce11 will die. To &termine the validity of the cumulative damage hypothesis, we generated a mathematical mode1 which enabled us to asses the probability of neuronal deaîh as a function of the. Here we show that the kinetics of ceIl death in 16 examples of neurodegeneration are better explained by mathematical models in which the risk of celi death remains constant or decreases exponentially with age. Thus, the kinetics argue against neuronal deah king a consequence of cumulative damage; instead, the time of death of any newon is random.
Experimental Procedures
The kinetics of photoreceptor degeneration was examined in animal models in which cell loss had been reported quantitatively over at lem one year, or until the majority of photoreceptors had died. The kinetics of ce11 loss was analyzed by fitting either the outer nuclear layer (ONL)thickness or ce11 number &ta to solutions of the differential equation (for solutions to differentiai equations, see Appendix 1):
(Equation 3.1) where p(t) represents the risk of ceil death at age t. Functions for p(t) were substituted as follows:
exponentially decreasing risk: P(t) = Poe-A(I-~CW (Equation 3.2) constant nsk: lm) = CLo (Equation 3.3)
exponentially increasing risk: ~(t)= Poe ~(t-dciay) (Equation 3.4) where W)represents the initial probability of cell àeath and delay repmsents the time before neuronal death begins. Equations for ~(t)were chosen based on their ability to yield exponential and sigmoidal curves. Data fitting was performeû using nonlinear regression analysis (quasi-Newton methods), a method of modeling the relationship ôetween variables, utilizing the functions containeâ within the Mathematica 3.0 (Wolfram Research) software package (Ratkowsky, 1983). Models were rejecteâ if parameter estimates did not differ significantly (P (Equation 3.5) in order to provide an estimate of A. AMcorresponds to increasing risk, A4to decreasing risk, and Adto constant risk. Estimates of A for each subject were then averaged, and a Student's t-test was used to detennine if the mean value was significantly different ftom zero (i.e.PcO.05). Results Ce11 Death Kinetics In Photoreceptor Degeneration A prediction of the cumulative damage hypohesis is that îhinitial pmbability of neuronal death is low but that as mutant neurons age, the increase in damage correspondingly increases the risk that a ceU wili die, resulting in a sigmoidal decline in ceU number. In contrast, a constant or a decreasing nsk of neuronal death over time WUbe associated with an exponential decline in cell numbet (Figure 3. la). To distinguish between these alternative kinetics of neuronal death, we fmt used riegression analysis to analyze photonxeptor neuron death in 11 animal models of inherited retinal degeneraîion. These models include animals with mutations in genes encoding proteins with a broad range of physiological functions, ranging fiom the photosensitive pigment, rhodopsin (Lavail, unpublished observations), to the enzyme cGMP phosphodiesterase(Parshail et al., 1991; Sanyal and Jansen, 1981 ; Schmidt and Aguhe, 1985) and the structural proteins rom-1 (see Chapter 2) and peripherixdrds (Hawkins et al., 1985; Sanyal and Hawkins, 1981). Additionally, three mutants were analyzed in which the affected gene is unknown. In four of these examples (Figure 3.1, Table 3. l), the data fit only to a mathematical mode1 in which the probability of photoreceptor kath remained constant with age. In six others (Figure 3.2, Table 3. l), the kinetics fit equally weli to models of constant or exponentially decreasing risk of death. Thenfore, with the exception of the ~d/' mouse (~24.980;P<0.001 for exponentiaily incrrssing risk), the increasing nsk of ce11 death predicted by the cumulative damage hypothesis can be firmly excluded. In agreement with these direct measwements of the kinetics of photoreceptor death in animai models, clinicai assessrnent of the rate of decline of photoreceptor function in patients with retinitis pigmentosa and cone-rod dystrophy has demonstrated that the decay of both visual field loss (Massof et al., 1990) and of the maximum electroretinograrn (ERG) responses of photoreceptors also observe exponential kinetics (Berson et al., 1985; Birch et al., 1999) . Thus, the exponential ce11 âeath kinetics we have identified in animal models appear to be shared by most, if not dl, examples of inherited retinal degeneration. Moreover, our fmding of similar kinetics of photoreceptor death due to retinal detachment (Figure 3. ld, Table 3. l), based on data hma cat mode1 (Erickson et al., 1983), indicates that exponentiai kinetics are also characteristic of photoceceptor neurons âamageâ by at lest some acquired insults. statting cell number c % initial % initial rnean rnean ONL ONL thickness trlii e % initial mean ONL thickiess Figure 3.1: Animal models of inherited photoreceptor degeneration and retinal detachment, in which the kinetics of ceIl death are best described by a constant risk of neuronal death (see equations 3.1 and 3.3 in Experimental Procedures). a, Constant and incteasing nsk of neuronal death will manifest as an exponen- tial or sigrnoidal decline in ce11 number, respctively. b, Retinal degeneration slow heterozygous (~ds+/-;crosses) (Hawkins et al., 1985) and homozygous rnice (Rdd-;circles) (Hawkins et al., 1985) with a nul1 mutation in the gene encoding peripheridrds. c, Nervous homozygous mice (nr/nr;crosses) (Lavai1 et ai., 1993). and purkhje ce11 degeneration mice @cd/pcd;circles) (Lavail et al., 1982). d, Mice homozygous for a nul1 mutation in the gene encoding the phototransduction enzyme rod cGMP p-phosphodiesterase (Rd-)(Sanyal and Hawkins, 198 1). e, Experimental retinal detachment in the cat (Erickson et al., 1983). Table 3.1: Panmeter estimates for Wnetie modelr rehting the risk of neuronal death (p) to agev. Animal Mode1 I Constant p: 1 Exponentially decreasing p: 1 ~ds+~mice (Hawkins cr 49.4 pl I ul., 1985) I Rds"- mice (Hawkins et 40.5 pm NIA I al., 1985) I 50.1 pin N/A NIA 10.6 nuclei NIA NIA pced mice (Lavail et al., 1982) 1 :ml-'- Nce (sa Chapter I 41.9 pm 12.1 nuclei 13.1 nuclei Schnauzer) (Parshall et aL. Mice (Sanyal and Hawkins, 1981) rcd-1 (Irish Setter) 10.6 nuclei 1 1.8 nuclei (Parshall er al.. 199 1; Schmidt and Aguirre, 1985) and Hawkins, 198 1) expressing transgenic rat (Lavail, unpublished observations) V~2values reflect the pmpoction of data variability that is explaineci by the model. All reported R~ values were statistically significant (P<0.001).See Appendix 2 for the parameter estimates for each model. * represents repssions perfonned on mean values reported in literature; t parameter estimates wem not significantiy diffmnt hmtao. Table 3.1 (continued): Parameter estimates for Unetic models relathg the risk of neuronaI death (p) to agev. Animal Model I Constant p: I ExponentiaHy decreasing p: 1 CO R&- mice (Sanyal and NIA NIA Hawkins, 198 1 ) NIA Cultured hippocarnpal 0.0773 100% of NA NA neurons (Dubinsky et al.. normal Parkinson's disease 0.0858 NIA NIA (Fearnley and Lees. 1991) 5999.52 6000 cells degeneration in cells pcd/pcd mice (Triarhou. 1998) 100% of rat model of normal Parkinson's disease (Sauer %RZ values reflect the proportion of daîa variability that is crplained by the model. Al1 reporteü R* values were sîatistically significant (PcO.001). See Appendix 2 for the parameter estimates for each model. * represents regressions performed on mean values reported in iiterature; parameter estimates were not significantly different from zero. 2 4 6 8 1012 Age (rnonths) b % initial mean Afbino ONL Mice Age (months) Figure 3.2: Animal models of inhented photoreceptor degeneration in which the kinetics of ce11 death are best descdbed by an exponentially decreasing risk of death (see equations 3.1 and 3.2 in Experimentai Procedures) a, Wiid type (triangles) and ~oml-~mice (circles) (set Chapter 2), and photomeptor dysplasia in miniature schnauzers (pi(@;crosses) (Parshall et al., 1991). b, Albino mice (Sanyal and Hawkins, 1981) (BdWcHeA) (crosses), and rod ce11 degeneration in Irish Setters (rcd-1;circles) (Parshall et al., 1991; Schmidt and Ag+, 1985) due to a null mutation in the rod cGW p-phosphodiesterase ene. c, Mice homozygous for null mutations in both the gene encoding rod cGMP B-phosphodicsterase and in the gene encoding peripheridrds (~d-/-;~ds-&)(Sanyal and Hawkins, 1981). CeU Death Kinetics In Non-Retinal Newodegeneration To determine whether a constant or exponentialiy deneashg risk of neuronal death are general phenornena which also occur in neurons outside the mtina, we examined the kinetics of death in four other diseases or experimental models involving other classes of neurons. Neuronal loss in the substantia nigra in Parkinson's disease (Feamiey and Lees, 1991), in the excitotoxic death of cultured hippocampal neurons (Dubinsky, 1995; Dubinsky et al., 1995)' and in the loss of cerebella granule celis in pcd (Purkinje cell degeneration) mice (Triarhou, 1998) have previously been shown to exhibit an exponential decline in neuronal number with tirne. Our regression analyses demonstrate that only a constant risk describes the kinetics of celi death in the fmt two examples (Table 3.1, Figure 3.3a,b). On the other hand. the loss of cerebella granule cells which occurs subsequent to the geneticalîy determined loss of their target Purkinje cells in pcct/pcd mice (Triarhou, 1998) (Figure 3.3c), as weli as the neuronal death in a chemicaliy induced rat mode1 of Parkinson's disease (Sauer and Oertel, 1994) (Figure 3.3d), can both be described by either a constant or an exponentially decreasing risk of cell death (Table 3.1). We also measured 18~- deoxyglucose uptake in the caudate nucleus of patients with Huntington's disease as an indirect measure of neuronal loss. Because each of these patients was repeatedly sarnpled at various times after clinical onset, the glucose uptake data, predictably, is highly variable in the whole population of patients (see Appendix 3). Consequently, analysis of neuronal degeneration kinetics in this heterogeneous population necessitateci the use of repeated- measures regression for each individual (see Experimental Procedures). The results of this analysis demonstrate that neurodegeneration in the caudate nucleus of Huntington's disease patients is also best described by a constant nsk of ce11 death Figure 3.3e). % of cell Parùinson's diseese number (constant risk) O Tirne in culture (hm) O % of nomal cell number 111-.--11 ' 2 6 10 14 18 lime after chernical injection (weeks) Valueof A 10 for indidual patients 0 (~10") -10 Figure 3.3: Examples of non-retinal neunnial death which also display a constant or exponentially decreasing risk of death. a, The percentage of normal neuron number in the substantia nigra of patients with Parkinson's disease as a huiction of symptom duration (Feamley and Lees, 1991) is best described by a constant probability of neuronal death. b, Cultured hippocampal neurons undergoing excitotoxic ceil death after incubation with glutamate exhibit a constant risk of ce11 death (Dubinsky et al., 1995). e, In contrast, the secondas, loss of cerebellar granule cells (Triarhou, 1998) is described equally well by either a constant or an exponentially decreasing risk of cell death. The indicated cwerepresents a constant risk. d, The percentage of substantia nigra nemns present in rats after injection with the neurotoxin 6-hydroxydopamine (Sauer and Ocrtel, 1994). a chemicaliy-induced animal mode1 of Parkinson's disease, is best fit by an exponentially decreasing risk of neuronal death. e. Analysis of patients with Huntington's disease indicates that the values of A (in the exponent of equation 3.5, see methods) do not differ significantly hmzero (P4.495,Student's t-test), indicating the Liaetics of metabolic decline are best fit by a constant nsk of ce11 loss. Each point repnsents the estimated A for an individual patient. Solid line: mean A (mean f s.d.: -0.01~10'3i 5.3 x10-3 mm, n=38 patients) across patient population; dashed lines: 95% confidence interval for the mean value of A (-1.7 x 10~~.1.7 x 10-~). 153 Discussion Taken together, the above finding of constant or decreasing nsk of ce11 kath in anunal models of photoreceptor degeneration, and in four ohrexamples of neuronal death reported here, suggest the increasing risk of ce11 death predicted by the cumulative damage hypothesis can be firrniy excludeci. Thus, even if age-dependent cumulative daaage does occur in these mutant neurons, it is not associated with an increase in the probability that the photoreceptor will die. Moreover, the identification of similar ce11 death kinetics in five different types of newons suggests that a constant or decreasing nsk of cd&ah is likely to be shared by many, if not al1 forms of neuronal degeneration. Examination of this possibility will require analysis of the cell death kinetics of other neurodegenerative disorders. Although neuronal death has been found to be mediated by apoptosis in atl photoreceptor degenerations examineci (Chang et al.* 1993; Li and Milam, 1995), as well as in retinal detachment (Berglin et al., 1997), it rernains to be detennined whether a constant or exponentiaily decreasing risk of death will be found invariably to accompany the neuronal apoptosis. Although the available data does not allow us to distinguish conclusively between a mode1 with a constant risk of ce11 bath versus one in which the risk is exponentiaily decreasing, both models indicate that the tirne of death of an individuai neuron is random. Nevertheless, the two models have quite different implications about the pathophysiological rnechanisms that lead to neuronal death. In a process involving constant risk, the time of death of an individual neuron is not only random, it is also independent of the death of any other neuron. In this case, the khetics of neuronal degeneration are comparable to the simple exponential decay exhibiteci by radioactive compounds, and the Merent rates of photoreceptor degeneration (Figure 3.1) are determined largely or solely by the mutant genotype. In con- exponentialiy decreasing nsk ïndicates that the chance of ceU death decreases in direct proportion to the nwnber of cernaining ceUs. Such kinetics could msult hman increase in the concentration of a survival fxtor, such as bFGF or CNTF (Wen et al., 1995), or from a decrease in the arnount of a toxic faoras the popuiation of ceils declines. Consistent with the latter possibity, dying retinal progenitor cells have been shown to produce an apoptosis-inducing agent (Seigai and Liu, 1997). Analysis of chimeric mice has also alluded to the presence of such a toxic factor. In chimeric mice with retinas containing both normal photomeptors and those expressing a mutant version of rhodopsin, a uniform pattern of ceil death is obseved, regardless of photoreceptor genotype (Huang et al., 1993). One interpretation of this observation is that normal photoreceptors receive a 'death signal' from the surroundhg mutant cells. As more photoreceptors die in these chimenc retinas, less of this toxic factor would be produced and the nsk of death to the remaining ceUs would be lowered. The exponential kinetics we have identifieci in ali 11 photoreceptor mutants, including the rats expressing mutant P23H rhodopsin (Table 3. l), suggest that similar kinetics also apply to the photoreceptors of chimeric mice. An alternative interpretation for the exponential decline in nsk is that the decrease is an artifact resulting fiom the presence of two photoreceptor populations, each with different but constant nsks of ce11 death. We can fulnly exclude the possibility that this artifact arises from a differential rate of rod and cone ceîl de&, since cones comprise only 3% of the total number of photoreceptor ceils (Carter-Dawson and Lavail, 1979); this fraction is too smail to have an observable effect on the overall kinetics of photoreceptor hath. In addition, we have also determined that the data hmeach rietinal model, from human and rat Parkinson's disease, from cultured hippocampal cells, and hmthe cerebellar degeneration in pcd/pcd mice cannot be fit to a simple equation incorporating two exponentiai functions (see Appendix 2), suggesting that the presence of two differentialiy affected cell populations is not responsible for the exponentially decreasing risk of ceil death. Any model of the mechanisms underlying inherited neuronal degenerations must account for the major features of cell kath in these disorders. These features are i) the constant or decreasing nsk of neuronal death descnbed above, ii) the genotype-dependent nature of the nsk. iii) the random tirne of death of any ceil (iiiusttated by îhe random distribution of apoptotic photoreceptors seen in the retins of animals and humans with inherited retinal degenerations (Chang et al., 1993; Li and Milam, 1995)). and iv) the paradoXical situation in which the great majonty of neurons in anirnals or patients with inhented neurodegenerations survive and function nonnally for moaths, years, or decades, while a few genetically identical celis in the population are dying randomly. We propose that these features can be explained if the mutant neurons are in an abnormal homeostatic state, the mutant steady state (MSS). The MSS differs little hmthe nomal neufonal steady-state (since most of the mutant cells are dive and hctioning normally), except that the MSS is associated with an increased nsk of celi death. We suggest that the MSS is a response to mutation characterized by subtle, but critical changes in the expression or function of a relatively few "mutant response" genes or proteins (MuRGs or MuRPs, respectively) which mediate critical pre-death reactions. If a MuRP is an enzyme, for example, it may change the relative concentrations of "pre-lethai" molecules (Figure 3.4). Exit from the MSS and cornmitment to ce11 death wouid then occur when random fluctuations in the concentrations of pre-lethal molecules - for example, compound B in Figure 3.4 - exceed a threshold beyond which neuronal death is initiated. Different mutations would shift the steady state to varying degrees, so that mutations producing a transition to a MSS cioser to the ce11 death threshold have a greater chance of exceeding that threshold, and therefore a higher probability of ce11 death. Thus, we propose a 'one-hit' model in which the death of an individual neuron is initiated randornly by a single rare catastrophic event. A similar one hit kinetic model was proposed and rejected by Dubinsky et al. as an explanation for the exponential celi deah kinetics exhibiteâ by cultured hippocampal neurms cell death 1 Figure 3.4: The exponential kinetics of celi death in inhented neuronal degenerations suggests the existence of a mutant steady-state (MSS) in which the risk of ce11 death is increased. In wildtype neurons, a reaction, catalyzed for example by the enzyme MuRPl, is associated with a concentration of compound B of 3 units/d. In a mutant newon in the MSS, the MuRPl activity changes in response to the mutation, so that the concentration of compound B is increased to 5 units/ml. Random increaws in the concentration of compound B to 7 unitdm1 will trigger ce11 death. exposed transiently to excitotoxic amino acids (Dubinsky, 1995; Dubinsky et al., 1995). The one hit mode1 was rejected in favour of a more complex mechanisms involving a multistep biochemical cascade in which the overall death rate is detennined by the specific rate constants for each of an unknown number of transitions within the cascade. We suggest, however, that some environmental insults place neurons in an abnomai steady-state which, like the MSS, is associated with a constant nsk of ceU death. This environrnentally- induced abnormal steady-state is exemplified by the effect of excitotoxic amino acids in initiating the exponential death of hippocampal newons (Dubinsky, 1995; Dubinsky et al., 1995) Figure 3.3b), and by the effect of retinal detachment in leading to photoreceptor death (Erickson et al., 1983) (Figure 3.14, Table 3.1). Our fmdings have important implications for the understanding and treatment of retinal and possibly other types of neuronal àegeneration. First, biochemical mechanisrns proposed to underiie neuronal deah must be re-examined in light of the constant or exponentially decreasing risk of ce11 deaîh we have identifiai, and their implication that newons do not die kmcumulative damage. Many of the biochemicai mechanisms which have been suggested to mediate neuronal death, such as oxiâative stress (Cassarino and Bennett Jr., 1999; Coyle and Puttfatken, 1993; Schapira, 1999; Travis, 1998), involve a progressive increase in the severity of injury, causing a higher proportion of ceUs to enter the death cascade in older animals. Our observations indicate that the importance of this and comparable biochemicai hypotheses must be re-evaluated. Second, identification of the MuRGs and MuRPs in different mutant neurons will indicate whether mutations impose MSS's which share common MuRGs and MuRPs, or whether each MSS is uniquely or partly defined by the specific mutant gene or mutation. In mutant neurons in which the risk of death is constant throughout life, the MSS should be the same in young and old cells with the same genotype, although additionai secondary changes in gene or protein expression may occur as a consequence of ceii &ah. Altematively, in models in which the risk of death decreases exponentially, cddeath rnay be associated with a changing pattern of MuRGs and MuRPs. In eitber case, identification of MuRGs and MuRPs in different mutants is iikely to provide insight into the pathogenic events thaî increase the risk of celi death in the MSS. Pharmacologie intemention to shift the activity of MuRGs and MuRPs towards normal levels should slow or prevent initiation of the biochemical cascade leading to ceil &ath. Fmally, the absence of cwnulative damage means that the 1ikelihOOd that a mutant neuron can be rescued by treatment is not diminished by age, although fewer celis WUbe available to rescue. 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A rhodopsin gene mutation responsible for autosomal dominant retinitis pigmentosa results in a protein that is defective in localization to the photoreceptor outer segment. Jowal of Neuroscience 14: 5818-5833. Travis, G.H. (1998). Mechanisms of ce11 death in the inherited retinal degenerations. Am. J. Hum. Genet. 62: 503-508. Travis, G.H., Sutcliff, J.G. and Bok, D. (1991). The retinal degeneration slow (rds) gene product is a photoreceptor disk membrane-associateci glycoprotein. Neuron 6: 6 1-70. Triarhou, L.C.(1998). Rate of neuronal fallout in a transsynaptic cerebellar model. Brain Res. Bull. 47: 2 19-222. Wen, R., Song, Y.C., T., Matthes, M.T., Yasumura, D., Lavail, M.M. and Steinberg, R.H. (1995). Injury-induced upregulation of bFGF and CNTF mRNAs in the rat retina. J. Neurosci. 15: 7377-7385. Chapter 4 Pigenic inheritance of photoreceptor degeneration in mice with mutations in the genes for botb Rds and Roml. Abstnct Whereas the involvement of RDS in inherited photoreceptor degeneration has ken well established, convincing disease-related mutations in ROM1 have only been identified in association with RDS mutations. In four families with digenic retinitis pigmentosa, affected individuals are hetetozygous for a Leu l8SRo mutation in the RDS gene and were heterozygous for either one of two putative null mutations (Gly80 lbp ins, Leu1 14 lbp ins), or a base-pair substitution (Gly 1l3Glu) in W.In contrast, individuals carrying only one of these mutations are asymptomatic Oryja et al., 1997; Kajiwara et al., 1994). While evidence strongly supports the association between these mutations and RP, it remains formally possible that other genes are responsible for the photorecepfor degeneration observed in these families. Therefore, to test the ROMI-RDS digenic hypothesis of RP :i.e. that CO-inheritanceof these particular mutant alleles results in RP, we reconstnicted the +/- +/-, rgL185P +/-, tgLl85P digenic RP genotype(Rom 2 ,Rds ) in transgenic mice. Rds mice differed from ~anl+/-mice (see Chapter 2) in that they manifested OS disorganization and +/- +/-. rgL18SP slowly progressive photoreceptor degeneration. However, Rom1 ,Rds mice +/O, rgL185P (digenic) displayed more severe OS disorganization than the Rds mice, and an accelerated rate of photoreceptor cell death. These resuits support biochemicaî experiments indicating that ROM and PenpherWRDS interact at the OS disk rims. Furthexmore, they indicate that the RDS:ROMl digenic hypothesis of RP is correct. Rom-1 and peripherin/rds are transmembrane proteins that are localïzeâ to the rims of vextebrate rdand cone OS disks (Arikawa et al., 1992; Bascom et al., 1992a; Conne11 and Molday, 1990, Moritz and Molday, 1996), and have been show biochemically to fonn tetramers and higher order complexes (Loewen and Molday, 2000). Ample evidence exists to indicate that peripherinlrds is critical for the development of the OS and for photo~eptor viabiiity. Not only do ~ds'-photoreceptors die after making abor(ed attempts at OS formation, but mutations in RDS are associated with a varïety of photorreceptor degenerations in humans (Kohl et al., 1997; Kohl et al., 1998) (also see Chapter 1). However, little support for the association of ROMI with monogenic inherited retinopathies is available. Demonstration that ROM1 is associated with human photoreceptor degeneration bas been provided by the identification of patients with digenic RP (DSrja et al., 1997; Kajiwara et al., 1994). Analysis of families in which retinitis pigmentosa (RP) was thought to be caused by a dominant L185P RDS mutation (Kajiwara et al., 1994) distinguisbed three characteristics inconsistent with dominant inhentance: 1) the disease occurred in offspring of unaffécted individuals, 2) the disease was transmitted hmaffected individuals to only -25% of their offspring, and 3) some people camying the L l8SP ailele appeareù clinically unaffected. It was subsequently discovered that affected famiiy members were also heterozygous for a null allele in ROM2 (Kajiwara et al., 1994). To date, four families have been identified with digenic RP associated with mutations in RDS and ROM. In all of these families, afkcted individuals were heterozygous for either one of two putative nuil mutations (Gly80 lbp ins, Leu 114 lbp ins), or a base-pair substitution (Gly 113Glu) in ROMI, as well as heterozygous for a Leul85Pro mutation in the RDS gene (Dryja et al., 1997; Kajiwara et al., 1994). Biochemical snidies attempting to analyze subunit assembly invoiving the L185P peripherin/râs protein and rom-1 have suggested that digenic RP results hman insufficiency in the number of peripherin/rds=containingtetramers (Goldberg and Molday, 1996a). In contrast to the wild type protein, îhe mutant L185P peripheridrds protein is unable to assemble into homotetramers when expressed in COS-1 cells. hstead, abnormal migration during SDS-PAGE suggests that it forms aggregates and may assemble as atypical dimers. In contrast, when L l8SP peripherin/rds is co-expressed with wild type rom- 1, it is able to fonn tetramers. This has led to the suggestion that the basis of digenic RP is an inability of L l8SP peripheridrds to self associate (Goldberg and Molday, 1996a). According to this model, individuals that inherit both digenic mutant alleles would have lower levels of functional Peripherin/RDS-containing complexes. This would result in OS instability and photoreceptor death (Goldberg and Molday, 1996a). In contrast, ROMI individuais, which carry one ROM1 nul1 alkle, are normal sincc wild type levels of PeripherinlRDS can compensate for the reduced abundance of ROM-1. Similarly, RDS+n18sP individuals are asymptomatic because the normal levels of rom-1 can reçruit the mutant Peripherin/RDS into functional complexes. Although segregation of RP in these four families @ryja et al., 1997; Kajiwara et al., 1994) provides convincing evidence in favour of the "digenic inheritance" hypothesis, it remains a fodpossibility that mutations in unknown genes are actudy responsible for the disease. To detennine whether photoreceptor degeneration can result hmthe inheritance of these particular mutations in ROM1 and RDS, we crossed ~oml"mice with transgenic mice expressing the L185P RDS 4-;rgLIBSP allele on an ~d/background (Rds ) and determined if they exhibited OS structural abnormalities or photoreceptor cell death. We found that the digenic rnice exhibited OS disorganization, progressive OS shortening, and an acceleration of the photoreceptor degeneration in cornparison to control mice, phenotype consistent with the digenic hypothesis of RP. Experimentai Procedures ~s"*mice that were homozygous for the insertion of a transgene expressing a 4;tgLI8SP mutant L185P Rds cDNA dercontrol of the mouse Opsin promoter (Rds ) were obtained hmDr Gabriel Travis and cmssed with wild type and ~oml*'*mice to generate +/-;tgLI%SP* +/-;tgLl85P. L 185P "heterozygous" mice (Rds ,~ornl+'+)and digenic (Rh ,~onl ") mice, nspectively. Determination of the Roml genotype was performed as previously described (Chapm 2, Experimntal Procedures). Rds genotype was detemiined using PCR according to previously established pmtocols (Kedzierski and Travis, manuscript in preparation). Bnefly, primers were designed to amplify the following fragments of genomic DNA: L I 8SP-expressing transgene: Op3: 5'-CCTGGAG'ITGCGCI'GT-3 ' MR5: 5'-GTCTI'L'ITCATGAAGCACC-3' Mutant Rrls allele: B9A (795-809): 5'-CGCATCAAGAGCAAC-3' R24-2: 5'-CACATTACI'CITAAGGCC-3' Wiid type Rds dele: MA-1-0: 5 '4GCTGTTTCTCTTCC-3 ' B9A (1004-990): 5'-GGAATTCATGAGGCT-3' Each amplification reaction contained 0.5wof each primer, lx Taq polymerase buffer (Boehringer Mannheim), 1 unit Taq polymerase (Boehringer Mannheim), 2OqiM of each dNTP, 4p.L genomic DNA (prepared hm1 cm tail clips and resuspended in 0.5 mL Tris- EDTA buffer), and H20to 2% final voluxne. Samples were denanired at 94'~for 1 minute, and amplified with 30 cycles of 94k for 30 seconds, 55Oc for 30 seconds, and 72'~for 1 minute. Amplification products were then electrophoresed on 1.25% agarose gels in lx TAE buffer according to standard protocols (Sambrook et al., 1989). Histology . +/-;tgLIlUP Eyes were enucleated from mice of each genotype (wild type. Rds 9 +/-;tgLlBSP ~ornl+'-,and the digenic Rds ;~oml+~mice) at ages between one and 18 rnonths, and the superior hemisphere marked by notching the comea Eyecups nxed in 2.5% glutaraldehyde and 48padormaldehyde in O. 1 M sodium cacdylate buffer, pH 7.4, for at least 4 hours. Superior and inferior hemispheres were isolated, trisected radially through the optic nerve, and pst-fixed in 1% OsO, in buffer for 90 minutes. Samples were dehydrated in EtOH and propylene oxide, embedded in TAAB 81ZAraldite 502 resin (Merivac), and O polymerized at 6û C for 48 hours. Sections (0.4 p)hm the central (within 1 mm of the optic nerve) regions of the superior and inferior hemispheres were obtained using a Reichart- Jung Ultracut microtome, stained with toluidine blue (1% toluidine blue, 1% sodium borate), and imaged with a Leitz Aristoplan and Kodak Ektachrome 64T film. Measurements and statistical analysis. Measurements of ONL thickness, an accwate gauge of photoreceptor number (Michon et al., 199 l), and ROS length were made with an ocular micrometer on a Leitz Wetzlar microscope at 1,250x magnification. ROS length was measund dong the long axis of ROSS between the inner segments and the RPE. ONL thickness was considered to be the distance betweem the outer Limiting membrane and the outer plexiforni layer, and was measured in sedions where columas of rod nuclei were apparent. to ensure that sections were not oblique. For each sample, five individual measurements were averaged and measurements fiom at least thme mice of each genotype at each tirne point. These data were analyzed usbg the Mann-Whitney U test (Statistica 4.1, StatSoft) to determine if the genotype, age or location in the mina aff'ected either ONL thickriess, ROS length, nuclear density, or ce11 nurnber. Al1 measurements are reported as mean t s.d. Analysis of the kinetics of photoneceptor cell death The kinetics of photorezeptor degeneration was examined in digenic mice by fitting either the outer nuclear layer (ONL)thickness &ta to solutions of the differential equation (for solutions to differential equations, see Appendix 1): (Equation 4.1) where p(t) represents the risk of cell death at age t. Functions for p(t) were substituted as follows: exponentially âecreasing risk: p(t) = pae-" (Equation 4.2) constant risk: Mt)= CL0 (Equation 4.3) exponentially increasing risk: P(t) = ri&" (Equation 4.4) where po represents the initial pmbability of ceil death and delay represents the time before neuronal death begins. Equations for p(t) were chosen based on their ability to yield exponentiai and sigrnoidal curves. Data fitting was perfonned using nonlinear regression analysis (quasi-Newton methods), a method of modeling the relationship between variables, utilizing the functions contained within the Mathematica 3.0 (Wolfram Research) software package (Ratkowsky, 1983). Models were rejected if parameter estirnates did not differ significantly (P Reconstruction of the digenic genotype in mice was accomplished by crosshg wildtype and ~oml"mice with R&~-mice thai w«e homozygous for a transgem composeci of an RdF L185P mutant cDNA under control of the mouse Opsin promoter (Kedzierski et al., manuscript in preparation). Southern blotting and PCR was used to genotype the offspnng of these crosses (Figure 4.1). Litters of normal size (-8- 12 pups) were obtained hmal1 crosses, indicating that the digenic genotype was not lethal, and ali mice appeared grossly normai, both physicaüy and behaviordy. Accelerated photofeceptor dath in digenic mice +/-;tgLIBSP. +/-;tgLIBSP. To determine whether Rds ,Rornl+/- micc (digenic mice) or Rds , ~oml+'+mice (heceafter refend to as '2l8SP mice") exhibited photoreceptor degeneration, +/- we compared outer nuclear layer (ONL)thickness of wild type, Rom1 ,digenic, and L 1 85P mice between 1 - 18 months. No difference in ONL thickness as observed between genotypicaliy different mice at one month of age (Figure 4.2a) and the abundance of rod and cone photoreceptors was comparable arnong al1 genotypicaily different mice. However, the retins of digenic and L185P mice became pgressively thinner with age (Figure 4.2% Figure 4.3b-d). The ONL of digenic mice was significantly thinner hmwild type mice by four months of age (19% thimer, P<0.05), whereas the ONL of L185P mice was significantly reduced by six months (15% thinner, P<0.05). The rate of cell &ath in the L185P mice was slower than that observed in ~oml"mice, which exhibited significant photoreceptor loss by four months of age (see Chapter 2). Importantly, the ONL of six month old digenic mice was significautly redud in comparison to that of age-matched ~ornl+/-(digenic: 30.8 t 0.97 pm (n=3 mice) vs. ~aml+'-: 37.0 * 0.635 pm (n=3 mice)) Foire4.1: Genotype cietennination of representative digenic mice. a) Southem Mot analysis of genomic DNA hma digenic mouse probed with a fragment of genomic DNA flanking the Rom1 mutation. Expected fragments corresponding to the wild type and the mutant allele are indicated by the arrows. b) The Rds genotype was detemiined by PCR amplification of fragments hmthe wild type. mutant and transge~c Rdr genes. Redicted fragment sizes are indicauxi on the right of the figure, and size standards are on the left. Figure 4.2: Digenic inheritance of a Rom1 nuIl and a L 185P Ra3 mutation result in ac~eleratedphoto-keptor ceIl death and progressive shortening of photoreceptor ROSS. a, Although the density of nuclei within the ONL did not change (data not shown), the ONL of digenic and L185P mice was significanttly reduced (P4.05, Mann-Whitney U test) compared to wild type and ml mice by four and six months of age, respectively. By 18 months of age, the ONL of digenic mice was 47% thinner rhan in wild type mice (wild type: 39.6 I1 .S6 Fm (n=3) vs. Digenic: 2 1.3 * 1.27 pm (n=3), P4.05). Each symbol represents results hman individual mouse. Timepoints at which the ONLs of digenic and L185P mice are significantiy different from wild type are indicated (* or t, respectively). b, Digenic and L185P mice exhibit a progressive decline in ROS length. At one month, ROSS in digenic mice are 11% shorter than in wild type mice (wild type: 24.6 I0.635 pm, (n=3) vs. digenic 22.0 I1.27 pm (n=3), Pd.05). By 18 months, this diflerence has increased to 47% (2-18 month wild type: 27.3 I0.868 pm (n=9) vs. 14.7 + 0.635 pm (n=3), M.005). L185P mutants exhibit a slower decline in OS length and are significanly shorter than normal only after four months. By 6 months of age, L18SP OSs are 22% shorter than wild type OSs (wild type: 28.2 I1.27 pm (n=3) vs. L185P: 22.0 * 1.10 pm (n=3), P4.05). Timepoints at which the OSs of digenic and L185P mice are significantly different hmwild type are indicated (* or t, respectively). and L l8SP mice (digenic: 30.8 I0.97 pm (n=3 mice) vs. L l8SP: 33.0 0.635 pm (n=3 mice)), indicating that photoreceptor ce11 death is accelerated by the ceinheritance of both mutations. Rom-1 and peripherdrds are expressed in cone photoreceptors in bovine (Moritz and Molday, 1996), mouse (Bascom and McInnes, unpubished), and human (Mtzand Molday, unpublished) retinas. Correspondingly, there was a significant decrease in cone +/+ abundance in the digenic mice by 18 months of age ( Roml : 9.a1.53 cones per 150 pm section (n=3) vs. digenic: 5.33 I0.577 cones per 150 pn section (n=3), Pe0.05, Mann- Whitney U-test), indicating that both cone and rod photoreceptor viability is affecteci by co- inheritance of these mutations. No significant decrease in cone abundance was obsented in the L185P mice. To cletennine if cumulative damage is a cause of photoreceptor death in digenic mice, we fit the ONL thickness to mathematical models representing a constant, an exponentially decreasing, and an exponentiaüy increasing nsk of ce11 death with tirne (see Chapter 3). Sirnilar to the models of photoreceptor degeneration studied in Chapter 3, the kinetics of ce11 loss in digenic mice could be fit equaily well to either a constant or an exponentiaily decreasing risk of ce11 death (Figure 4.4). Therefore, cumulative cellular damage can be excluded as a cause of photoreceptor cell death in these mice. ROS structural abnormaiities To determine whether the digenic and L l8SP mice manifest photoreceptor structural +/- abnonnalities, we compared retinal histology of wild type, Roml ,L185P, and digenic +/- mice. Whereas ROSSof one month old wild type (Figure 4.3a) and Roml mice (data not shown), were straight and paraliel, those of age-matched digenic mice were disorganized: digenic ROSS appeared to have abnodylarge diameters, were shorter than in controls, Figure 4.3: Digenic and L185P mice exhibit OS structural abnormalities and a progressive loss of photoreceptors. a, ROSs of one month old wild type mice appear a9 straight, parailel cylindcrs that extend directly hmthe phdoreceptor imer segments (1s) to the RPE. Both rod (arrowhead) and cone (arrow) photoreceptors are apparent in this micrograph b, In contrast, ROSs of one month old digenic rnice are short and disorganizeû. ROSs also appear to be of greater diameter than normal, and contain occasional membranous whorls (*). However, the ONL is of normal thickness and contains both rod (arrowhead) and cone nuclei (mw). c, The ONL is thinner in 18 month old digenic mice, and their ROSs are disorganized and shorter than at one month of age. Both rod (arrow) and cone (arrowhead) nuclei remain visible in the ONL. d, Retinas of L185P mice also exhibit a decrease in photoreceptor numôer, aithough both rod (anowhead) and cone (arrow) remain present. Similar to the digenic rnice, OSs of L185P mice are shorter than normal and are disorganized. Bar = 10 Fm. 40 Digenic mice ( R~,,, +/- ; -+/-;tg L 185P) 30 ONL thickness (iim) 20 exponentially decreasing risk 10 2 4 6 8 10 12 14 16 18 Age (months) Figure 4.4: Digenic mice exhibit an exponentially decceasing probability of photoreceptor ce11 death. This is inconsistent with a role for cumulative damage in the death of these cells. and sporadicdy contained membranous whorls (Figun 4.3b). However, unlike ~0ml~- mice (see Chapter 2), the degree of ROS disorganhîion in older digenic remaineci unchanged in cornparison to that observed at one month (Figure 4.3~). ROSs in L185P mice were short, disorganized, and of greater diameter than normal (Figure 4.3d), although these phenotypes appeared less severe than in the digenic mice. ROSs have been shown to shorten progressively in several animal models of photoreceptor degeneration (for exampIe, Hawkins et al. (1985) and Humphries et al. (1997)), indicating that the rate of disk phagoçytosis by RPE ce11 exceeds the rate of new disk production (Young, 1976). To determine if ROSs in digenic and L l8SP mice exhibit similar dysregulation of OS renewal, we measured ROS length over tirne. ROSs of one month old digenic mice were shorter than in age-matfhed wild type mice, and become progressively shorter with age (Figure 4.2b). At one month, digenic ROSs were 11% shorter than wild type ROSs, while this Merence had increaseû to 47% by 18 months. ROSs of L l8SP mice were also shorter than normal, although it remains unclear if the phenotype is progressive in these mutants. Discussion The ROS structurai abnodties and accelerated photoreceptor degeneration resulting from the heterozygous inheritance of a Rom1 nul1 allele and a L18SP Rds allele support the hypothesis of digenic RP in humans. The severity of the various Rom1 and Rds mutations, based on the rate of photoreceptor degeneration, also supports clinical phenotypes observed in patients with mutations corresponding to those of the digenic and control mice. No convincing dominant ROM1 mutations have been associated with photoreceptor degeneration, compatible with the extremely mild phenotype observed in ~oml+/-mice (see Chapter 2). The pbenotype of the L185P mice is consistent with a mild clinid phnotype. Although carriers of this mutation have been describeci as asymptomatic, carriers have sometimes exhibited electroretinogram (ERG) responses that are slightly below normal (Kajiwara et al., 1994). This has been suggested to indicate that this mutation is recessive, but an alternative interpretation is that the L185P Penpherin/RDS protein has a mild dominant-negative effect (Goldberg and Molday, 1996a). This hypothesis is consistent with the ROS structural abnormaiities and the slow photoeceptor degeneration observed in the L l8SP mutant mice. The rate of photoreceptor loss in L185P mice is slower than that observed in ~ml" mice, suggesting that patients homozygous for ROMl nul1 alleles would present with symptoms more severe than exhibited by the L 185P carriers. That the rate of photoreceptor loss in digenic and Rom14 mice is comparable Mersuggests that iadividuals with recessive ROM1 nul1 alleles would display symptoms similar to those observed in patients with digenic RP. One difference between phenotypes might be a relative sparing of cone photoreceptors and central vision in patients homozygous for ROMl mutations, since cones were uaaffaited in ~oml~mice. These predictions. however, rernain to be conf-d since no individuals homozygous for ROMl nuli alleles have been identified thus far. The presence of more severe structural abnorrnalities in the photoreceptors of digenic mice than in those of either L18SP or oni il+^ rnice supports the biochemical evidence that the two proteins interact at the rirns of OS disks (Bascorn et al., 1992b; Goldberg and Molday, 19%b; Goldberg and Molday, 1996~;Goldberg et al., 1995; Loewen and Molday, 2000). Furthemore, this observation indicates that both molecules perfom some critical function at the disk rim. According to one delput forth as molecular expianation for digenic RP, peripheidrds plays a dominant role in maintainhg OS structure, and rom-1 functions by negatively regulating the abundance of higher-order peripherinkds-containing complexes (Goldberg and Molday, lm, Loewen and Molday, 2000). If this model is tme, thtn we would expect contrasting pbenotypes in Rd4-and R&+~mice. For cxample, the larger disk observeci in ~onzl-~mice would wggest that R&'- mice should exhibit a decrease in ROS disk diameter. This is in contrast to the elecuon rnicroscopic observation of larger disks in the Rh+/-mice @. Bok, persona1 communication). Additionally, given the elabrate subunit structure of the peripherin/rds:rom- 1 complex (Loewen and Molday, 20ûû), it is possible chat the different multimers perfonn discrete functions within the OS. Further experiments designed to determine the relative abundance of these complexes within mutant photoreceptors should shed Ught on the function of both proteins in photoreceptor biology . References Arikawa, K., Molday, L.L., Molday, R.S. and Williams, D.S. (1992). Localization of penpheridrds in disk membranes of cone and rod photoreceptors: Relationship to disk membrane morphogenesis and retinal degeneration. J. CeIl Biol. 116: 659- 667. Bascom, R.A., Garcia-Heras, J., Hsieh, C.-L.,Gerhard, D.S., Jones, C., Francke, U., Willard, H.F., Ledbetter, D.H. and McInnes, R.R. (1992a). Localization of the photoreceptor gene ROM1 to human chromosome 1 I and mouse chromosome 19: Sublocalization to human 1 lq13 between PGA and PYGM. Am. J. Hum. Genet. 51: 1028-1035. Bascom, R.A., Manara, S., Collins, L., Molday, R.S., Kalnins, V.I. and Mchnes, R.R. (1 992b). Cloning of the cDNA for a novel photoreceptor membrane pmtein (rom-1 ) identifies a disk rim protein family implicated in human retinopathies. Neuron 8: 1171-1184. ConneU, G. and Molday, R.S. (1990). Molecular cloning, primary structure, and orientation of the vertebrate photoreceptor ce11 protein peripherin in the rod outer segment disk membrane. Biochemistry 29: 469 1-4698. Dryja, T.P.,Hahn, L.B., Kajiwara, K. and Berson, E.L. (1997). Dominant and digenic mutations in the PeriphenMWS and ROM2 genes in retinitis pigmentosa Invest. Ophthalmol. Vis.Sci. 38(10): 1972-1982. Goldberg, A.F.X. and Molday, R.S. (1996a). Defective subunit assembly underlies a digenic form of retinitis pigmentosa linked to mutations in penpherin/ds and rom-1. Proceedings of the National Academy of Science, USA 93: 13726- 13730. Goldberg, A.F.X. and Molday, R.S. (1996b). Rom-1 dependent subunit assembly of a peripherin/RDS mutant linked to a digenically inherited form of retinitis pigmentosa. Invest. Ophthalmol. Vis. Sci. 37: S806. Goldberg, A.F.X. and Molday, R.S. (19%~).Subunit composition of the peripherinlrds- rom-l disk rùn complex hmrod photonxeptors: hydrodynamic evidence for a tetramenc quartemary structure. Biochemistry 35: 6 144-6 149. Goldberg, A.F.X., Mortiz, O.L. and Molday, R.S. (1995). Heterologous expression of photoreceptor peripheridrds and rom-1 in COS- 1 cells: assembly, interactions, and locaiization of multisubunit complexes. Biochemistry 34: 14213- 142 19. Hawkins, R.K., Jaiisen, H.G. and Sanyal, S. (1985). Development and degeneration of retina in rds mutant mice: photoreceptor abnorrnalities in the heterozygotes. Exp. Eye Res. 41: 701-720. Humphries, M.M., Rancourt, D., Farrar, G.J., Kenna, P., Hazel, M., Bush, R.A., Sieving, P.A., Sheils, D.M., McNally, N., Creighton, P., Erven, A., Boros, A., Gulya, K., Capecchi, M.R. and Humphries, P. (1997). Retinopathy induced in mice by targeted dismption of the rhodopsin gene. Nature Genet. lS(2): 216-219. Kajiwara, K., Berson, E.L. and Dryja, T.P. (1994). Digenic retinitis pigmentosa due to mutations at the unlinked periphen'n/lPDS and ROM2 loci. Science 264: 1604-1608. Kohl, S., Christ-Adler, M., Apfelstedt-Sylla, E., Kellner, U., Eckstein, A., Zremer, E. and Wissinger, B. (1997). RDUperipherin gene mutations are frequent causes of central retinal dystrophies. J. Med. Genet. 34: 620-626. Kohl, S., Giddings, L, Besch, D., Apfelstedt-Sylla, E., Zrenner, E. and Wissinger, B. (1998). The role of the peripherinllPDS gene in retinal dystrophies. Acta Anat. 162: 75-84. Loewen, C.J.R. and Molday, R.S. (20).Disulfide-mediated oligomerization of peripherin/rds and rom-1 in photoreceptor disk membranes. Implications for photoreceptor outer segment morphogenesis and degeneration. J. Biol. Chem. 275: 5370-5378. Michon, J.J., Li, 2.-L., Shioura, N., Anderson, R.J. and Tso, M.O.M.(1991). A comparative study of methods of photoreceptor morphometry . Invest. Ophthalmol. Vis.Sci. 32(2): 280-284. Moritz, O.L. and Molday, R.S. (19%). MolecuJar cloning, membrane topology, and localization of bovine rom-1 in rod and cone plmtoreceptor cells. Invest. Ophtbalm01. Vis. Sci. 37(2): 352-362. Ratkowsky, D.A. (1983). No- Model&AUnitKd Rggiçal APeLPBfh. New York, Marcel Decker. Sarnbrook, J., Fritsch, E.F. and Maniatis, T. (1989). lratm Manual. New York, Cold Spring Harbor Laboratory Press. Young, R.W. (1976). Visual ceils and the concept of nnewal. Invest. Ophthaùwl. Vis. Sci. lS(9): 700-725. Chapter 5 Future Directions Vertebrate photoreceptors perfonn the biochemicai reactions by which light is converteci into a chemical signal that can processed and interprieted by the rest of the nervous system. As such, the heaith of photoreceptors is critical to visual function. In the work presenied in this thesis, we demonstrated that rom-1 is critical for rod photoreceptor viability and for regulating the morphogenesis of ROS disks. Mice homozygous for a &ml nul1 allele exhibit an exponential decline in photoreceptor number, so that by 18 rnontbs, approximately half of al1 rod photoreceptors have died. Furthermore, we detennined that ROSS in RO~~rnice exhibit a biphasic pattern of disorgauization, and suggest that this disorganization results fiom the production of ROS disks with an abnormally large diameter. Finaiiy, we were able to demonstrate that the digenic hypothesis of RP is correct: mice inheriting one Rom1 ndi allele in combination with one L185P Rdr substitution exhibit an accelerated rate of photoreceptor death in cornparison to mice inheriting only one of these aiieles. The conclusions drawn hmwork presented in this thesis lead directly to severai new Lines of research directed at increasing our undemtanding of the role of rom- 1 and peripherinlrds in photoreceptor biology. No doubt these additional experirnents will also increase ow understanding of photoreceptor biology in general, and of the pathophysiological mechanisms involved in diseases the prernature death of these cells. Future Experiments Additional Analyses of the ~ml"Phenotype Analysis of the abnormalities in ROS structure exhibiteci by ~oml" rnice has so fa. focused on photoreceptors between one and 18 months of age. By one month, the ROS is more or less mature: the oniy obvious change occwring after that age is a slight elongation of the ROS. Consequently, our anaiysis has concentrated on structural changes which mfltct the role of rom4 in ROS disk renewai . There is, however, some evidence to suggest that ROS morphogenesis, the initial development of the organelle as an outgrowth of the photoreceptor connecting cilium, occurs by a altogether different mechanism. Between postnatal day 5-7 in mice, nuxnerous rhodopsin-containing membraae vesicles can be observd scattered within the swoilen apical region of the rdcomecting ciiium (Obata and Usukura, 1992; Usukura and Obata, 1995). Historically, these vesicles have been assumed to represent an artifact of the chemical fmation techniques used to prepare the tissues, and it was conjecnired that the fmt ROS disks develop by a PM invagination similar to those observed in mature photoreceptors. However, more ment anaîysis using rapid freezing and electron microscopy indicated thaî small endocytotic structures are present in the apical plasma membrane of differentiating photoreceptors (Obata and Usukura, 1992). Furtheme, structures were observed that suggested that the endocytotic vesicles fuse and flatten within the cytoplasm (Obata and Usukura, 1992), and ate then arranged into a stacked array similar to that observed in mature ROSs (Figure 5.1). Since the youngest rnice examined were one month old, it is unclear if the ROS structural abnonnalities observeci at that age are caused by alterations in ROS morphogenesis, or if they are due to insuffrciencies in disk morphogenesis. Therefore, to determine the effect of the loss of rom4 function on ROS morphogenesis, the structure of ROSs should be examined in ~ml"mice between the ages of one and three weeks. By using bth rapid freezing (Obata and Usukura, 1992) and chemical futation techniques in combination with electron xnicroscopy, it will be possible to study the formation of wild type and mutant ROSs as they differentiate. Furthemore, it will be possible to evaiuate whether ROS development is delayed in Roml mutant mice. ROS Nascent ROS disks Actin filaments Connecting Cilium Figure 5.1: Roposed mechanism of mouse rcxi photoreceptor outer segment morphogenesis. An increase rate of membrane production during ROS mor- phogenesis is accompanied by the formation of endocytotic vesicles. The vesi- cles are subsequently organized and flattened by an actin-mediated pmcess. The fmt disks formed are onented parallel to the ROS long axis. However, once the ROS becomes filled disks, new disks becorne oriented perpendicuiar to this axis. Modified hmObata and Usukura (1992). Another outstanding issue regarding the stnictwe of oni il" photoreceptors is whether or not the mutation results in abnodties in rim closure. It has been suggested that rom1 is involved in this pmcess since it is localized exclusively to the rims of OS disks (Bascorn et al., 1992b). Moreover, the observation that peripheridrds is locdized exclusively to the closed margin of OS disks (Arikawa et al., 1992). and the fmding that it cm promote membrane fusion in culture (Boesze-Battaglia et al., 1997; Bœsze-Battaglia et al., 1998) support the hypothesis that rom- 1 is also involved in rim closure. In a proposed mode1 of disk mophoogenesis, the perimeter of a ROS disk, and therefore the length of the disk rim, is determineci by the number of teminal lwp complexes (TLC) in that disk (Corless, 1986) (see Chapter 1). Therefore, it is pmiicted that the loss of rom-1 decreases the amount of TLC in a disk, and results in a coniesponding decrease in rim closure. To detennine if this is indeed the case, we can use established techniques to assess whether ROS disks are isolated from the PM in ~omlmice. For example, when the dye profion yellow is injected into the subretinal space (region between photoreceptor OSs and the RPE), it difises into the region between adjacent open disks (Laties et al., 1976). Consequently, COS and any ROS disks thaî are open at the time of injected becorne stained. Therefore, if the loss of rom- 1 function resuits in abnormalities in rim closure, ROS should be stained after this procedure. Our structural studies suggest that the increased ROS disk diameter observed in ~ml"mice may result from a decrease in the rate of rim closure (see Chapter 2). Using an in vitrio assay system ,it has been shown that peripherin/rds can stimulate membrane fusion in a rom-1-independent manner (Boesze-Battagiia et al., 1997; Boesze-Battaglia et al., 1998). In these experiments, recombinant periphenn/rds was reconstituted in membrane vesicles and fùsed with PM labeled with octadecylrhodamine B chlonde (Ris) . Membrane fusion was then assayed by measuring the dilution of label after adding peripherin/rds- containing vesicles. By using aff'inity purified peripherinlrds:rom- 1 complexes (or those isolated by hydroxylapatite chromatography (Boesze-Battaglia et al., 1997)) in similar experiments, we cm therefore detenaine whether the presence of rom-1 increases the efticiency and kinetics of penpheridrds-mediated membrane fusion. Detennining the pathogenicity of putative diseasetausing ROMl alleles Although the association between RDS mutations and photoreceptor degeneration has been welî documented (Kohl et al., 1998), the same cm not be said of ROMI. Only a small number of indiviiduals with dominant or spotadic photoreceptor degeneration have been found to cary putative ROMl disease-causing aileles in the absence of RDS mutations. These include a Leu 114- 1bp insertion mutation in two fdeswith RP (Bascom et al., 1995; Sakuma et al., 1995). a Gly75Asp substitution in the middle of the second transmembrane domain (Bascom et al., 1995), and a single aliele containing both a Ro60Thr and a Thrl08Met mutation (Bascom et al., 1995; Dryja et al., 1997). However, this last mutation has also been identified in an unaffected individual, casting doubt on its pathogenicity (Martinez-Mir et al., 1997). To detennine whether these mutations are responsible for photoreceptor degeneration, these mutant alkles can be expressed in transgenic rnice on a ~ornl+~ background. First, a transgene will be constructed that expresses a wildtype human ROM1 gene under control of a photoreceptor-specific promoter. In the ideal situation, these regulatory elements would be from the endogenous mouse Rom1 gene. However, since these have not been isolated, the rod-specific promoter from the mouse opsin gene (Lem et al., 199 1 ;Travis et al., 1992) could also be utilized. Transgenic mice carrying this constnict couid then be crossed to yield Roml 4-;tgROMI mice, thereby establishing whether the human protein could hinctionaily compensate for the loss of the mouse Rom1 gene. Additionaliy, if phenotypic rescue is observed, this expriment would confm that the phenotype observed in the ~ornl~mouse is due to the targeted mutation, not caused by an unknown mutation in a closely linked gene. Once rescue is achieveà, transgenes expressing mutant ROM1 genes cm be used to generate mice that are heterozygous for each putative mutation (Figure 5.2). Histological analysis of tbe retinas of these mice will then indicaîe whether these aileles are capable of causing photoreceptor degeneration. This appmach wiU afso be beneficial in atternpting to understand the specific cellular defects resulting hmROM2 mutant alleles, Determining the complete expression pattern of Roml: is Roml involved in syndromic disease? A possible explmation for the low fiequency of ROMI mutations is that the gene rnay be expressed in a wider array of tissues than has so far been reported. A cornparison between the rate of cell loss in digenic and mice suggests that the comsponding phenotypes in humans should be similar. However, no individuals homozygous ROM1 nul1 alleles have been identified, whereas several families have been identified with digenic RP. Support for the suggestion of a broder ROMI expression pattern was provided by preliminary rtPCR experiments which indicated that Rom1 cDNA was expressed in several celi types in addition to photoreceptors (Bascorn and McInnes, unpublished observations). Additionally, cDNA libraries derived from mouse inner ear contain both Rom2 and Rds cDNAs (Ploder and McInnes, unpublished observations), and mouse cochlear hair cells stain with polyclonal antibodies to ROM- 1 (Vidgen and McInnes, unpublished observations). However, this staining is also present in f?om14 mice, suggesting that the mtibodies may recognize a related molecule, that the knockout allele is leaky, or that unidentifieci 5' exons of the Rom1 gene are expressed expression in the ear. To distinguish between these alternatives, additional antibodies that bhd to alternative rom-1 epitopes may be used to detennine whether immunofluorescent staining is due to the expression of rom- 1 or a structurally related protein. If functional mm-1 pmtein is expressed in cochlear hair celis, it eon I II 111 1 3' LI14(9 1 bp) 5.W 6.5 kb 2.5 kb Mouse RomllRho Upstream Elements Human ROMlGene Figure 53: Introduction of putative disease-causing aiieles. The mouse Rom1 or Rh0 (the gene encdng the rhodopsin protein) upstream elements will be used to express human ROM1 in the mutant mice. After phenotypic rescue with a wild type ROM1 ailele, the putative mutant alleles (G75D and L114(V 1bp)will be introduced into the ~oml+/-background in a similar manner in order to detennine if they cause retinal degeneration. WUbe important to screen DNA samples hmpatients with photoreceptor degeneration and syndromic blindness for mutations in the ROM1 gene using previously established techniques (Bascom et al., 1995). Potential candidate diseases that map to chromosome 11 q 13 (Bascorn et al., 1992a) include Usher's synàrome type 1B (USH 1B) (OMIM,2000). Additional tissues may also express Roml. To detennine the complete expression pattern of Roml,we WUuse in situ hybridization on sections of late-stage embryo and postnatal mouse tissues, focussing our analysis of tissues known be involved in syndromic biindness, such as Bardet-Biedl syndrome type 1 (OMIM,2ûûû). Expression of Rom1 in any tissue will then be confiiusing immunohistochemistry. Rom- 1 Associated proteins To fuliy understand the functioa of rom-1, it is important to identifvthe molecules with which it interacts. Locaiization to the ROS disk nm places rom-1 in a cntical region of the photoreceptor involved in many processes including the attachent of disks to the cytoskeleton, to the PM, and to each other (Corless and Fetter, 1987; Corless et al., 1987; Roof and Heuser, 1982). Only four additional proteins are known to locaiize to the ROS disk rim: peripherin/rds, the rod photoreceptor ABC transporter (ABCR), and the glutamic acid-rich proteins GARP 1 and GARE (see Chapter 1). The ody one of these proteins known to associate with rom-1 is peripheridrds (Bascom et al., 1992b). GARPl and 2 are not believed to interact with rom- 1 (K6rschen et al., 1999) based on indirect evidence, whereas the association of ABCR and rom-1 has not been rigorously exarnined. To detennine if proteins other than peripherin/rds associate with rom-1, a two hybrid screen can be utilized to identify proteins that interact with specific domains of Rom-1. Of particular interest are the regions within the central hydrophilic loop thac are conserveci arnong the tetraspanin superfamiiy of membrane proteins, and the carbxy tail which exhibits limited homology to other known molecules. Evaluation Of The bbPenpherin/RdsEquivalents" Hypotbesis The large-disk phenotype observeci in ~ml"mice has been suggested to result from either the loss of rom- 1 per se, or hma decrease in the concentration of "peripherin/rds equivalents" (se Chapter 2). Since peripherhlrds is approximately 2.5 fold more abundant than rom- 1. this latter hypothesis predicts that ~ornf-mice expressing appmximately 150% of the normal levels of peripherin/rds would exhibit no ROS abnormalities and no photoreceptor degeneration. To directly test this preâiction, we will cross ~onzl-~mice with transgenic mice expressing an Rds cDNA under control of the Rho regulatory elements (Travis et al., 1992). Two lines of such mice that are hemizygous for the transgene on a Ra%-'* background have been shown to express appmximately half of the wiid type levels of peripherin/rds (Travis et aL, 1992). Since peripherin/rds is approximately 2.5 fold more abundant than rom-1, ~oml"mice that are also hemiygous for the transgene (but are ~ds+'+)would produce roughly normal levels of "peripherin/rds equivalents". If peripherinfrds and rom-1 have no unique func tions, such ~ornl-&;Rds +/+;gRds mice should exhibit no abnormal characteristics. In contrast, any abnormaiities observe-would reflect a unique deof rom-l within the ROS. Identifying Photoreceptor MuRGs and MuRPs The one-hit mode1 of neuronal celi death makes the prediction that neurodegeneration is associated with a change in the expression of genes (MuRGs) that shifts a neuron into an abnormal homeostatic state, the MSS. Since we propose that these genes are the critical elements whose random fluctuations are responsible for the cornmitment to cell death, it is a great importance to isolate these genes and detennine how their expression changes due to various mutations. One approaich to identifvingsuch MuRGs is the use of DNA microarrays to screen large numbers of candidate genes to idemtij. those whose expression changes in the mutant retinas. The microarrays consist of fkgments of specific genes that are bound to glas slides. The slides are then hybridized to cDNA obtained hmthe tissue under scrutiny, in this case wild type and mutant retinas. Gene fragments that hybriàize differentially to the cDNA and therefore are either up- or dom-regulated in the ~ml"retinas, become candidate MuRGs. This type of experiment wili no doubt identify a wide varîety of genes with altered expression levels. It will therefore becorne important to have an assay that can isolate the tme MuRGs hmthose representing secondary effects. A MuRG must exhibit altered expression levels in ~ornl"mice that are significantly different from wild type. To obtain an accurate measure of expression levels, it will therefore be necessary to measure expression levels in individual mice, using techniques such as quantitative PCR. Additionaiiy, it will be necessary to show that altered levels of MuRG expression correlate with the probability of ce11 death. For example, if the expression level of a candidate MuRG is increased in Ronrl-'- mice, increasiag its expression to a larger degree shouid result in a corresponding increase in the risk of photoreceptor ce11 death. To prove this will require that we aiter expression levels of candidate MuRGs in transgenic animals, or in cultured photoreceptors. Roml" mice are of great value in his type of cxperiment since the rate of ceii loss is slower than any known photoreceptor degeneration caused by a gene expressed specifidy in those cells. Therefore, the likelihood that any particular ce11 will reside within the MSS at the time of an experiment is greater than for any other animal mode1 of photoreceptor degeneration. In contrast, the photoreceptors of R& mice, in which aimost aü photoreceptors die by 2 months of age, are likely to exit the MSS relatively quicicly. Consequently, it would be far more diff~cultto isolate MuRGs and MuRPs fiom these animals. Extending the One-Hit Mode1 of Neuronal CeU Death As is, the one-hit mode1 of cell death relates to the temporal pattern of cell loss in examples of uiherited photoreceptor and neuronal degeneration. However, in numemus examples of neurodegenerative diseases, cell death does not occur unifonnly throughout the tissue. For example, the fact that patients with RP experience a progressive restriction in their visual field that eventualiy leads to complete blindness indicates that 41s at the edge of the retina die before those in the central retina (Pagon, 1988). In diseases such as macular degeneration, the cone photoreceptors in the cenaai retina are the fîrst to die (Reichel and Sandberg, 1994). Finally, rods in the central retina are preferentially lost during the aging process (Curico et al., 1993), suggesting that the two cell types may possess naturaiiy different probabilities of dying. in this 1stexample, the celis are exposed to same light and are located within the same microenvironment, so the reason for the differentiai rates of ce11 loss is unclear, especially in light of the random loss of cells we have identifieci. To attempt to determine what factors are involved in generating spatial patterns of cell loss across the retinal surface, we wiil attempt to extend the one-hit mode1 to include a spatial dimension. One possible method of accomplishing this task would be to generate an array of "virtual photoreceptors'" that interact through specific mles. For example, one ce11 type might mlease trophic factors into its environment that maintain the viabiIity of the surrounàing cells. Conversely, mutant cells in such an array might release toxic factors that induce the swounding ceus to enter the MSS. Experimental evidence for such cell-cell interactions has been provided by studies of chimeric mice expressing mutant transgenes (Huang et ai., 1993). In such chimeric mice, photoreceptors expressing mutant versions of rhodopsin can induce the death of neighbouring, wild type photoreceptors. An array of virtual photoreceptors could be described by mathematicai entities known as cellular automata (CA'S) (Wolfram, 1984). Briefly, a CA would model ce11 death across the retina by subdividing it into discrete elements (i.e. ''virtual photoreceptors") that interact via specific rules that represent how photoreceptors might interact. When elements "die", the signals that nonnally are passed to neighbouring elements are no longer transmitted. Likewise, a cell in the MSS may transmit abnod"toxic" messages to adjacent cells, thereby causing those othenuise nodcells to enter the MSS. The random death demonstrated by the exponential loss of photoreceptors could then be incorporated into the model using Monte CarIo simulation techniques. These statistical methods cm most simply k described as a numerical stochastic process, or a sequence of random events (Kalos and Whitlock, 1986), and could thus be used to determine which virtual photoreceptors in the array would âie in any given tirne increment. By such computational approches, it may be possible to test vatious assumptions about the ce11 interactions in the retina that rn responsible for produce the spatial patterns of photoreceptor death that are observed in animal models of retinopathies and in hwnans with RP. Reterences Arikawa, K., Molday, L.L., Molday, R.S. and Williams, D.S. (1992). Localization of peipherin/ràs in disk membranes of cone and roà photoreceptors: Relationship to disk membrane morphogenesis and retinal degeneration. J. Ce11 Biol. 116: 659- 667. Bascom, R.A., Garcia-Heras, J., Hsieh, C.-L., Gerhard, D.S.,Jones, C., Francke, U., Willard, H.F.,Ledbetter, D.H. and McInnes, R.R. (1992a). Localization of the photoreceptor gene ROM1 to human chromosome 11 and mouse chromosome 19: Sublocalization to hwnan 1 lq13 between PGA and PYGM. Am. J, Hum. Genet. 51: 1028- 1035. Bascom, R.A., Liu, L., Heckenlively, J.R., Stone, E. and McInnes, R.R. (1995). Mutation analysis of the ROM1 gene in retinitis pigmentosa. Hum. Mol. Genet. 4: 1895-1902. Bascom, R.A., Manara, S., Collins, L., Molday, R.S., Kalnins, V.I. and McInnes, R.R. (1992b). Cloning of the cDNA for a novel photoreceptor membrane protein (mm-1) identifies a disk rim protein family implicated in human retinopathies. Neuron 8: 1171-1 184. Boesze-Battaglia, K., Kong, F., Lamba, O.P., Stefano, F.P. and Williams, D.S. (1997). Purification and light-dependent phosphorylation of a candidate fusion protein, the photoreceptor ce11 Peripheridrds. Biochemistry 36: 6835. Boesze-Battaglia, K., Lamba, O.P., Napoli Jr., A.A., Sinha, S. and Guo, Y. (1998). Fusion between retinal rod outer segment membranes and mode1 membranes: a role for photoreceptor peripheridrds. Biochemisty 37: 9477-9487. Corless, LM.(1986). A minimum diameter limit for retinal rod outer segment disks. mve1 svstem,- Hilfer, S.R. and Sheffield, J.B. New York, Springer Verlag. 5: 127- 142. Corless, J.M.and Fetter, R.D. (1987). Structural features of the terminal loop region of hgretinal rod outer segment disk membranes: m.Implications of the tenninal loop complex for disk morphogenesis, membrane fusion, and cell surface interactions. J. Cornp. Neurol. 257: 24-38. Corless, J.M.,Fetter, R.D., Zarnpighi, O.B., Costello, M.J. and Wall-Buford, D.L. (1987). Structural features of the terminal loop region of frog retinal rod outer segment disk membranes: II. Organization of the terminal loop complex. J. Comp. Neurol. 257: 9-23. Cwico, C.A., Millican, C.L., Allen, K.A. and Kalina, R.E. (1993). Aging of the human photoreceptor mosaic: Eviâence for selective vulnerability of rods in the central retina. Invest. Ophthalmol. Vis. Sci. 34: 3278-3296. Dryja, T.P., Hahn, L.B., Kajiwara, K. and Berson, E.L. (1997). Dominant and digenic mutations in the PeripheridWS and ROM1 genes in retinitis pigmentosa. Invest.Ophthalmo1.Vis.Sci. 38(10): 1972- 1982. Huang, P., Gaitan, A., Hao, Y., R.M., P. and Wong, F. (1993). Cellular interactions implicated in the mechanism of widespread photoreceptor âegeneration in transgenic mice expressing a mutant rhodopsin gene. Proc. Natl. Acad. Sci. U.S.A. 90: 8484- 8488. Kalos, M.H.and Whitlock, P. A. (1986). Monte car10 metha. New York, John Wiley &Sons. Kohl, S., Giddings, 1.. Besch, D., Apfelstedt-Sylla, E., Zrenner, E. and Wissinger, B. (1998). The role of the peripheri-s gene in retinal dystrophies. Acta Anat. 162: 75-84. Korschen, H.G., Beyennaun, M., Müller, F., Heck, M., Vantler, M., Koch, K.-W., Kellner, R., Wolfmm, U., Bode, C., Hofmann, K.P. and Kaupp, U.B. (1999). Interaction of glutamic-acid-rich proteins with the cGMP signaliing pathway in rod photoreceptors. Nature 000: 76 1-766. Laties, A.M., Bok, D. and Liebman, P. (1976). Procion Yellow: A marker dye for outer segment disc patency and rod renewal. Exp. Eye Res. 23: 139-148. Lem, J., Applebwy, ML.,Falk, J.D., Fiannery, J.G. and Simon, M.I. (1991). Tissue- specific and developmental regulation of rod opsin chïmeric genes in transgenic mice. Neuron 6: 20 1-2 10. Martinez-Mir, A., Vilela, C., Bayes, M., Vdverde, O., Dain, L., Beneyto, M., Marco, M., Baiget, M., Gnnberg, D., Baiceils, S., Gonzàlez-Duarte, R. and Vilageliu, L. (1997). Putative association of a mutant ROM1 aliele with ~tinitispigmentosa Hum-Genet. 99: 827-830. Obata, S. and Usukura, J. (1992). Morphogenesis of the photoreceptor outer segment during postnatal development in the muse (BALWc) retina. Cell Tissue Res. 269: 39-48. OMIM (2000). Online Mendelian Inhentance of Man. http://www3.ncbi.nlm.nih.gov/Omim Pagon, R.A. (1988). Retinitis pigmentosa. Surv. Ophthaimol. 33: 137-177. Reichel, E. and Sandberg, M.A. (1994). Hereditary macular degenerations. -le a tice of OD-.- Albert, D.M.and Jakobiec, F.A. Toronto, W.B. Saunders Company. 2: 124%1262. Roof, D.J. and Heuser, J.E. (1982). Surface of rod photoreceptor disk membranes: Integral membrane components. J. Cell Biol. 95: 487-500. Sakuma, H., Inana, G., Murakami, A., Yajima, T., Weleber, R.G.,Murphey, W.H., Gass, J.D.M., Hotta, Y., Hayakawa, M., Fujiki, K., Gao, Y.Q., Danciger, M., Farber, D.B ., Cideciyan, A.V. and Jacobsen, S.G. ( 1995). A heterozygous putative nul1 mutation in ROM1 without a mutation in penpherin4ZDS in a family with retinitis pigmentosa. Genomics 27: 384-386. Travis, G.H., Groshan, K.R.,Lloyd, M. and Bok, D. (1992). Complete rescue of photoreceptor dysplasia and degeneration in transgenic retinal degeneration slow (rds) rnice. Neuron 9: 1 13- 119. Usukura, J. and Obata, S. (1995). Morphogenesis of photoreceptor outer segments in retinal development. Prog. Ret. Eye Res. 15: 113- 125. Wolfram, S. (1984). Cellular automata as models of compkxity. Nature 311: 419. Appendix 1 Solutions To Dwerentiai Equations Used In Chapter 3 Sohtions to DiRerentiaJ Equations Useà in Cbapter 3 Basic equation: In other words, the rate of change in celi nwnber, etc.(left side of equation), is proportional to the number of ceils present at any given time. wewanted to detennine if p(t) increased, decreased, or remained constant during the degeneration process. This was dont by making the foliowing substitutions and solving the resulting differential equations. 1) Exponentially decreasing risk: p(t) = p,,e-"' Substitute, and use separation of variables to solve: Shce to= time when cell death begins 4, 2) Constant risk CLW = CL, Substitute, and use separation of variables to solve: There fore: 3) Exponentially increasing risk p(t) = PC" Substitute, and use separation of variables to solve: Following 1) above: 4) Repeated-measures, non-linear repssion analysis of metabolic decline in patients with Huntington's disease We now wish to &termine if AM(exponentialiy increasing nsk), Ad(constant risk, or if A4(exponentially decreasing risk). To solve the differential equatim, integrate boîh sides with respect to t: right side: ro left side: Now let t-td. Since A is srna11 (we know this because the metabolic rate does not rapidly 'crash' to zero in any of these patients), we can approximate eA(r-ro) - eM using the Taylor series: so, dd2 log[ONL(t) J = log[ONut0)J + aà + - 2 We can make the foliowing replacement to yield: This is a simple quadratic equation. Data from each patient was fit to this equation to obtain an estirnate of the parameters a and A. Snrdents t-test was then applied across al1 patient samples to determine whether A was significantly different from zero, and if so, whether it was positive (increasing risk) or negative (kreasing risk). Appendix 2: Supplementary lafornation For The Anslysis Of The Kinetics Of Neuronal CeU Death Parameter Estimates (3 significant digits) for Nonlinear Regression Analysis of Neuronal Degeneration Note: for tegressions involving a delay before the onset of ceIl death, th &lay parameter was subtracted hmt, the age of the animal. Modeb with significant regression results are indicated in bold. Al1 parameter estimates must be significanrly >O to accept a mode1 as appropriate. kp-1) exponentialiy decreasing risk: ONL(t)= ONL(tJe * constant risk: ONL(t)= ONL(t,)e*ot -!!!qp -,) exponentially increasing risk: ONL(t)= ONL(t,)e ~ornl-~mice: de- de- delay = 0.368, p= 0.482 ONL(O)= 40.947 pe0.00 l ONL(O)= 39.8. p ONL(O)= 39.8 pcO.001 OWO)= 40.0 p ONL(O)= 11.1 p ONL(O)= 9.93 p Albino mice: risk + debv: ONL(O)= 49.1 p ONL(O)= 5 1.1 p ON'i,(O)=1 1.8 @.O01 ONL(O)= 12.0 ~0.01 R= 0.086 ~0.01 R= 0.0356 p= 0.422 po= 0.0483 pc0.001 po= 0.0 174 p= 0.54 1 delay = 23.2 pc0.001 k + drlpv: OwO)= 10.6 p ONL(O)= 1 1.6 pe0.001 ONL(O)= 12.0 p ~d-A;~ds-1- mice: ONL(O)= 45.3 pcO.001 ONL(O)= 55.0 p ONL(O)= 48.0 p ONL(O)= 45.0 pcO.001 ONL(O)= 55.0 p ~ds+/-rnice: ONL(O)= 49.7 pc0.001 ONL(O)= 50.0 p CO-: ONL(O)= 48.3 ~0.001 ONL(O)= 49.4 p nrhr mice: e-: e-: . . ONL(O)= 54.7 p ONL(O)= 9.36 p ONL(O)= 9.37 p ONL(0)s 43.2 p<0.001 ONL(O)= 0.0 p + delav: constantnsk: ONL(O)= 42.2 p ONL(O)= 43.2 pc0.001 Omo)= 45.0 pdl.001 R= 0.968 pc0.001 R= 0.738 p= 0.653 w)= 0.0364 p<0.001 delay = 11.0 pc0.00 1 Retinai detachment in cat: . * âelav: e- ONL(O)= 250 @.O01 ONL(O)= 250 pcO.00 l R= 0.0181 p= 0.153 R= 0.0 185 p= 0.0872 w)= 0.0310 pc0.05 pp0.03 12 pc0.05 delay = 1.08 pc 0.809 + delav: . ONL(O)= 2 17 p<0.001 Omo)= 220 pco.001 p= 0.160 ~0.05 p= 0.0147 p R= 0.00234 p=0.8 16 R= -0.00637 w.4 13 po= 0.0708 p Parkinson's disease: (ce11 number(O)=10096 by definition) v : R= 0.03 10 p + delav: p= 0.0863 p R= -0.03 17 p=0.522 R= -0.008 17 w.730 po= 0.124 pdI.0709 CY)= 0.0914 p p= 0.264 pc0.05 p= 0.207 p Best fit equations and parameter estimates (3 signifiant digits) for Nonlincar Regrcssion Analysis of Neuronal Degeneration assuming two ceIl populations Note: for cegressions involving a delay before the onset of ceIl death, the delay parameter was subtmcted hmt, the age of the animai. Models with significant regression results are indicad in bold. All parameters must be >O to accept a model as appropriate. equation: Ob&(t) = ONL, (0)ëR8'+ ONL~ (o)ëRbt wke: Orna@) and Ra represent the initial number of cells and the rate constant for degeneration of population A ONLb(0) and Rb tepresent the initiai number of cells and the rate constant for degeneration of population B When the parameter estimates are identical, this model reduces to an equation witb a single exponent. This corresponds to the constant risk of ceIl death model. ~om+ mice: ONLa(0)= 18.9 p Ra=0.03 16 p Albino mice: 0NLa(0)=24.6 p Ra4.00917 p4.001 Rbd.009 17 -duces to single exponential equation rcd-l in Insh Settet: 0&(0&5.52 @.O01 ONi&O)=S.52 Rs=0.03 19 p4.001 Rb=0.03 19 &lay =24.8 p4.001 -duces to single exponential quation ~d-1-;~ds-/- mice: 0NLa(0)=22.8 @.O01 ONLb(O)=22.8 Rad. 122 p P23H rhodopsinexpressing rats: 0&(0)=23.4 p p ~ds-y-mice: 0NLa(0)=20.2 p 0NLa(0)=2999.76 p Rd"- mice: 0&(0)=2 1.9 p Retinal detachment in cat: ONLa(0)= 109.8 p<0.001 ONLb(O)= 109.8 p4.001 Ras0147 p4.007 Rb4.0147 @.O07 -ceduces to single exponentiai equation Cuitured hippocampal newons: (ce11 number(û)=lOO%by de finition) 0&(0)=48.3 p Ra=0.073 1 p Parkinson' s disease: (ce11 number(0)=1009b by definition) 0NLa(0)=50.3 pd.001 OI+iLt,(0)=50.3 Rad.0863 pd.001 Rb=0.0863 -duces to single exponential cquation Chemicaiiy induced rat mode1 of Parkinson's disease: (ceU number(O)= 10096 by definition) 0-(Ob57.6 p=û. 187 OW0)=57.6 Ra=0.264 p=û.3 14 bO.264 -duces to single exponential equation Appendix 3: Patient Data Used For The Analysis Of CeU Death Kiaetics Exhibited By Huntington's Disease Raw data useâ ln andysis of metabolic deciine in patients wïth Huntington's Disease {Log Glucose uptake (mM/hr)} 1 -, 1 +Patientl 0.8 ; -*. -. ----patient3 0.6 -4- Patient7 0.4 Patient9 0.2 0-0- Patientl {Time) 500 1000 1500 2000 2500 3000 {Log Glucose uptake (mM/hr)} Patientl --Patientl Patientl -+Patientl +- Patient2 {Log Glucose uptake (mM/hr) ) {Log Glucose uptake (mM/hr)) 1 - 0.8 O. 6 0.4 , 0.2 , >------ 500 ;O&I - &O - ' 2000 2500 - ;&&Ti'='e1 {~ogGlucose uptake (mM/hr) 1 - 0.8 1 -a. 1 )-km% 0.6 , * -'\+)A 0.4 '\ \ & 0.2 r- - fi----.----=------1 500 1000 1500 2000 2500 {Log Glucose uptake (mM/hr)) I r 0.8 P .* ,/.-*" - . P 0.4 - 1 0.2 - I {Tirne) 500 1000 1500 2000 2500 3000 {Log Glucose uptake (mM/hr)} 1 0.8 0.6 \Y.-+ 0.4 7:- , b 0.2 , , 500 1000 1500