Identification and cellular localization in Xenopus laevis photoreceptors of three Peripherin-2 family members, Prph2, Rom1 and Gp2l, which arose from gene duplication events in the common ancestors of jawed vertebrates.

Rod and cone photoreceptors are named for the distinct morphologies of their outer segment organelles, which are either cylindrical or conical, respectively. The morphologies of the stacked disks that comprise the rod and cone outer segments also differ: rod disks are completely sealed and are discontinuous from the plasma membrane, while cone disks remain partially open to the extracellular space. These morphological differences between photoreceptor types are more prominent in non-mammalian vertebrates, whose cones typically possess a greater proportion of open disks and are more tapered in shape. In mammals, the tetraspanin prph2 generates and maintains the highly curved disk rim regions by forming extended oligomeric structures with itself and a structurally similar paralog, rom1. Here we determined that in addition to these two proteins, there is a third Prph2 family paralog in most non-mammalian vertebrate species, including X. laevis: Glycoprotein 2-like protein or "Gp2l". A survey of multiple genome databases revealed a single invertebrate Prph2 'pro-ortholog' in Amphioxus, several echinoderms and in a diversity of protostomes indicating an ancient divergence from other tetraspanins. Based on phylogenetic analysis, duplication of the vertebrate predecessor likely gave rise to the Gp2l and Prph2/Rom1 clades, with a further duplication distinguishing the Prph2 and Rom1 clades. Mammals have lost Gp2l and their Rom1 has undergone a period of accelerated evolution such that it has lost several features that are retained in non-mammalian vertebrate Rom1. Specifically, Prph2, Gp2l and non-mammalian Rom1 encode proteins with consensus N-linked glycosylation and outer segment localization signals; mammalian rom1 lacks these motifs. We determined that X. laevis gp2l is expressed exclusively in cones and green rods, while X. laevis rom1 is expressed exclusively in rods, and prph2 is present in both rods and cones. The presence of three Prph2-related genes with distinct expression patterns as well as the rapid evolution of mammalian Rom1, may contribute to the more pronounced differences in morphology between rod and cone outer segments and rod and cone disks observed in non-mammalian versus mammalian vertebrates.

Distinct roles for prominin-1 and photoreceptor cadherin in outer segment disc morphogenesis in CRISPR-altered X. laevis.

Mutations in prominin-1 (prom1) and photoreceptor cadherin (cdhr1) are associated with inherited retinal degenerative disorders but their functions remain unknown. Here, we used CRISPR-Cas9 to generate prom1-null, cdhr1-null, and prom1 plus cdhr1 double-null Xenopuslaevis and then documented the effects of these mutations on photoreceptor structure and function. Prom1-null mutations resulted in severely dysmorphic photoreceptors comprising overgrown and disorganized disc membranes. Cone outer segments were more severely affected than rods and had an impaired electroretinogram response. Cdhr1-null photoreceptors did not appear grossly dysmorphic, but ultrastructural analysis revealed that some disc membranes were overgrown or oriented vertically within the plasma membrane. Double-null mutants did not differ significantly from prom1-null mutants. Our results indicate that neither prom1 nor cdhr1 are necessary for outer segment disc membrane evagination or the fusion event that controls disc sealing. Rather, they are necessary for the higher-order organization of the outer segment. Prom1 may align and reinforce interactions between nascent disc leading edges, a function more critical in cones for structural support. Cdhr1 may secure discs in a horizontal orientation prior to fusion and regulate cone lamellae size.This article has an associated First Person interview with the first author of the paper.

Germline CRISPR/Cas9-Mediated Gene Editing Prevents Vision Loss in a Novel Mouse Model of Aniridia.

Aniridia is a rare eye disorder, which is caused by mutations in the paired box 6 (PAX6) gene and results in vision loss due to the lack of a long-term vision-saving therapy. One potential approach to treating aniridia is targeted CRISPR-based genome editing. To enable the Pax6 small eye (Sey) mouse model of aniridia, which carries the same mutation found in patients, for preclinical testing of CRISPR-based therapeutic approaches, we endogenously tagged the Sey allele, allowing for the differential detection of protein from each allele. We optimized a correction strategy in vitro then tested it in vivo in the germline of our new mouse to validate the causality of the Sey mutation. The genomic manipulations were analyzed by PCR, as well as by Sanger and next-generation sequencing. The mice were studied by slit lamp imaging, immunohistochemistry, and western blot analyses. We successfully achieved both in vitro and in vivo germline correction of the Sey mutation, with the former resulting in an average 34.8% ± 4.6% SD correction, and the latter in restoration of 3xFLAG-tagged PAX6 expression and normal eyes. Hence, in this study we have created a novel mouse model for aniridia, demonstrated that germline correction of the Sey mutation alone rescues the mutant phenotype, and developed an allele-distinguishing CRISPR-based strategy for aniridia.

Autophagy Induction by HDAC Inhibitors Is Unlikely to be the Mechanism of Efficacy in Prevention of Retinal Degeneration Caused by P23H Rhodopsin.

We previously found that valproic acid (VPA) and other histone deacetylase inhibitors (HDACis) ameliorate retinal degeneration (RD) caused by P23H rhodopsin in Xenopus laevis larvae and hypothesized that this may be due to enhancement of autophagy. Here we use X. laevis expressing an autophagy marker to assess effects of HDACis on autophagy. We also assess the effects of non-HDACi activators and inducers of autophagy on RD caused by P23H rhodopsin.

Prominin-1 and Photoreceptor Cadherin Localization in Xenopus laevis: Protein-Protein Relationships and Function.

Retinal degenerative diseases are genetically diverse and rare inherited disorders that cause the death of rod and cone photoreceptors, resulting in progressive vision loss and blindness. This review will focus on two retinal degeneration-causing genes: prominin-1 (prom1) and photoreceptor cadherin (prCAD). We will discuss protein localization, potential roles in photoreceptor outer segment disc morphogenesis, and areas for future investigation.

Autophagy in Xenopus laevis rod photoreceptors is independently regulated by phototransduction and misfolded RHOP23H.

We previously reported autophagic structures in rod photoreceptors expressing a misfolding RHO (rhodopsin) mutant (RHOP23H), suggesting that autophagy may play a role in degrading the mutant RHO and/or be involved in photoreceptor cell death. To further examine autophagy in normal and diseased rods, we generated transgenic Xenopus laevis tadpoles expressing the dually fluorescent autophagy marker mRFP-eGFP-LC3 in rods, which changes from green to yellow and finally red as autophagic structures develop and mature. Using transgenic lines with constitutive and inducible expression, we determined the time-course of autophagy in rod photoreceptors: autophagosomes last for 6 to 8 hours before fusing with lysosomes, and acidified autolysosomes last for about 28 hours before being degraded. Autophagy was diurnally regulated in normal rods, with more autophagic structures generated during periods of light, and this regulation was non-circadian. We also found that more autophagosomes were produced in rods expressing the misfolding RHOP23H mutant. The RHO chromophore absorbs photons to initiate phototransduction, and is consumed in this process; it also promotes RHO folding. To determine whether increased autophagy in light-exposed normal rods is caused by increased RHO misfolding or phototransduction, we used CRISPR/Cas9 to knock out the RPE65 and GNAT1 genes, which are essential for chromophore biosynthesis and phototransduction respectively. Both knockouts suppressed light-induced autophagy, indicating that although light and misfolded rhodopsin can both induce autophagy in rods, light-induced autophagy is not due to misfolding of RHO, but rather due to phototransduction. Abbreviations: CYCS: cytochrome c; bRHOP23H: bovine RHOP23H; Cas9: CRISPR associated protein 9; dpf: days post-fertilization; eGFP: enhanced green fluorescent protein; GNAT1: guanine nucleotide-binding protein G(t) subunit alpha-1 aka rod alpha-transducin; HSPA1A/hsp70: heat shock protein of 70 kilodaltons; LAMP1: lysosomal-associated membrane protein 1; LC3: microtubule-associated protein 1A/1B light chain 3; mRFP: monomeric red fluorescent protein; RHO: rhodopsin; RP: retinitis pigmentosa; RPE65: retinal pigment epithelium-specific 65 kDa protein: sfGFP: superfolding GFP; sgRNA: single guide RNA; WGA: wheat germ agglutinin; RHOp: the Xenopus laevis RHO.2.L promoter.

Electrophysiological Changes During Early Steps of Retinitis Pigmentosa.

Purpose: The rhodopsin mutation P23H is responsible for a significant portion of autosomal-dominant retinitis pigmentosa, a disorder characterized by rod photoreceptor death. The mechanisms of toxicity remain unclear; previous studies implicate destabilization of P23H rhodopsin during light exposure, causing decreased endoplasmic reticulum (ER) exit and ER stress responses. Here, we probed phototransduction in Xenopus laevis rods expressing bovine P23H rhodopsin, in which retinal degeneration is inducible by light exposure, in order to examine early physiological changes that occur during retinal degeneration. Methods: We recorded single-cell and whole-retina responses to light stimuli using electrophysiology. Moreover, we monitored morphologic changes in rods after different periods of light exposure. Results: Initially, P23H rods had almost normal photoresponses, but following a brief light exposure varying from 4 to 32 photoisomerizations per disc, photoresponses became irreversibly prolonged. In intact retinas, rods began to shed OS fragments after a rod-saturating exposure of 12 minutes, corresponding to approximately 10 to 100 times more photoisomerizations. Conclusions: Our results indicate that in P23H rods light-induced degeneration occurs in at least two stages, the first involving impairment of phototransduction and the second involving initiation of morphologic changes.

Cell Death Pathways in Mutant Rhodopsin Rat Models Identifies Genotype-Specific Targets Controlling Retinal Degeneration.

Retinitis pigmentosa (RP) is a group of inherited neurological disorders characterized by rod photoreceptor cell death, followed by secondary cone cell death leading to progressive blindness. Currently, there are no viable treatment options for RP. Due to incomplete knowledge of the molecular signaling pathways associated with RP pathogenesis, designing therapeutic strategies remains a challenge. In particular, preventing secondary cone photoreceptor cell loss is a key goal in designing potential therapies. In this study, we identified the main drivers of rod cell death and secondary cone loss in the transgenic S334ter rhodopsin rat model, tested the efficacy of specific cell death inhibitors on retinal function, and compared the effect of combining drugs to target multiple pathways in the S334ter and P23H rhodopsin rat models. The primary driver of early rod cell death in the S334ter model was a caspase-dependent process, whereas cone cell death occurred though RIP3-dependent necroptosis. In comparison, rod cell death in the P23H model was via necroptotic signaling, whereas cone cell loss occurred through inflammasome activation. Combination therapy of four drugs worked better than the individual drugs in the P23H model but not in the S334ter model. These differences imply that treatment modalities need to be tailored for each genotype. Taken together, our data demonstrate that rationally designed genotype-specific drug combinations will be an important requisite to effectively target primary rod cell loss and more importantly secondary cone survival.

Generation and Analysis of Xenopus laevis Models of Retinal Degeneration Using CRISPR/Cas9.

Xenopus laevis have proven to be a useful system for rapid generation and analysis of transgenic models of human retinal disease. However, experimental approaches in this system were limited by lack of a robust knockdown or knockout technology. Here we describe a protocol for generation of Cas9-edited X. laevis embryos. The technique introduces point mutations into the genome of X. laevis resulting in in-frame and out-of-frame insertions and deletions that allow modeling of both dominant and recessive human diseases and efficiently generates gene knockdown and knockout. Our techniques can produce high-frequency gene editing in X. laevis, permitting analysis in the F0 generation.

An interaction network between the SNARE VAMP7 and Rab GTPases within a ciliary membrane-targeting complex.

The Arf4-rhodopsin complex (mediated by the VxPx motif in rhodopsin) initiates expansion of vertebrate rod photoreceptor cilia-derived light-sensing organelles through stepwise assembly of a conserved trafficking network. Here, we examine its role in the sorting of VAMP7 (also known as TI-VAMP) - an R-SNARE possessing a regulatory longin domain (LD) - into rhodopsin transport carriers (RTCs). During RTC formation and trafficking, VAMP7 colocalizes with the ciliary cargo rhodopsin and interacts with the Rab11-Rabin8-Rab8 trafficking module. Rab11 and Rab8 bind the VAMP7 LD, whereas Rabin8 (also known as RAB3IP) interacts with the SNARE domain. The Arf/Rab11 effector FIP3 (also known as RAB11FIP3) regulates VAMP7 access to Rab11. At the ciliary base, VAMP7 forms a complex with the cognate SNAREs syntaxin 3 and SNAP-25. When expressed in transgenic animals, a GFP-VAMP7ΔLD fusion protein and a Y45E phosphomimetic mutant colocalize with endogenous VAMP7. The GFP-VAMP7-R150E mutant displays considerable localization defects that imply an important role of the R-SNARE motif in intracellular trafficking, rather than cognate SNARE pairing. Our study defines the link between VAMP7 and the ciliary targeting nexus that is conserved across diverse cell types, and contributes to general understanding of how functional Arf and Rab networks assemble SNAREs in membrane trafficking.

Modeling Dominant and Recessive Forms of Retinitis Pigmentosa by Editing Three Rhodopsin-Encoding Genes in Xenopus Laevis Using Crispr/Cas9.

The utility of Xenopus laevis, a common research subject for developmental biology, retinal physiology, cell biology, and other investigations, has been limited by lack of a robust gene knockout or knock-down technology. Here we describe manipulation of the X. laevis genome using CRISPR/Cas9 to model the human disorder retinitis pigmentosa, and to introduce point mutations or exogenous DNA sequences. We introduced and characterized in-frame and out-of-frame insertions and deletions in three genes encoding rhodopsin by co-injection of Cas9 mRNA, eGFP mRNA, and single guide RNAs into fertilized eggs. Deletions were characterized by direct sequencing and cloning; phenotypes were assessed by assays of rod opsin in retinal extracts, and confocal microscopy of cryosectioned and immunolabeled contralateral eyes. We obtained germline transmission of editing to F1 offspring. In-frame deletions frequently caused dominant retinal degeneration associated with rhodopsin biosynthesis defects, while frameshift phenotypes were consistent with knockout. We inserted eGFP or point mutations into rhodopsin genes by co-injection of repair fragments with homology to the Cas9 target sites. Our techniques can produce high frequency gene editing in X. laevis, permitting analysis in the F0 generation, and advancing the utility of X. laevis as a subject for biological research and disease modeling.

Opposing Effects of Valproic Acid Treatment Mediated by Histone Deacetylase Inhibitor Activity in Four Transgenic X. laevis Models of Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is an inherited retinal degeneration (RD) that leads to blindness for which no treatment is available. RP is frequently caused by mutations in Rhodopsin; in some animal models, RD is exacerbated by light. Valproic acid (VPA) is a proposed treatment for RP and other neurodegenerative disorders, with a phase II trial for RP under way. However, the therapeutic mechanism is unclear, with minimal research supporting its use in RP. We investigated the effects of VPA on Xenopus laevis models of RP expressing human P23H, T17M, T4K, and Q344ter rhodopsins, which are associated with RP in humans. VPA ameliorated RD associated with P23H rhodopsin and promoted clearing of mutant rhodopsin from photoreceptors. The effect was equal to that of dark rearing, with no additive effect observed. Rescue of visual function was confirmed by electroretinography. In contrast, VPA exacerbated RD caused by T17M rhodopsin in light, but had no effect in darkness. Effects in T4K and Q344ter rhodopsin models were also negative. These effects of VPA were paralleled by treatment with three additional histone deacetylase (HDAC) inhibitors, but not other antipsychotics, chemical chaperones, or VPA structural analogues. In WT retinas, VPA treatment increased histone H3 acetylation. In addition, electron microscopy showed increased autophagosomes in rod inner segments with HDAC inhibitor (HDACi) treatment, potentially linking the therapeutic effects in P23H rhodopsin animals and negative effects in other models with autophagy. Our results suggest that the success or failure of VPA treatment is dependent on genotype and that HDACi treatment is contraindicated for some RP cases.SIGNIFICANCE STATEMENT Retinitis pigmentosa (RP) is an inherited, degenerative retinal disease that leads to blindness for which no therapy is available. We determined that valproic acid (VPA), currently undergoing a phase II trial for RP, has both beneficial and detrimental effects in animal models of RP depending on the underlying disease mechanism and that both effects are due to histone deacetylase (HDAC) inhibition possibly linked to autophagy regulation. Off-label use of VPA and other HDAC inhibitors for the treatment of RP should be limited to the research setting until this effect is understood and can be predicted. Our study suggests that, unless genotype is accounted for, clinical trials for RP treatments may give negative results due to multiple disease mechanisms with differential responses to therapeutic interventions.

Molecular basis for photoreceptor outer segment architecture.

To serve vision, vertebrate rod and cone photoreceptors must detect photons, convert the light stimuli into cellular signals, and then convey the encoded information to downstream neurons. Rods and cones are sensory neurons that each rely on specialized ciliary organelles to detect light. These organelles, called outer segments, possess elaborate architectures that include many hundreds of light-sensitive membranous disks arrayed one atop another in precise register. These stacked disks capture light and initiate the chain of molecular and cellular events that underlie normal vision. Outer segment organization is challenged by an inherently dynamic nature; these organelles are subject to a renewal process that replaces a significant fraction of their disks (up to ∼10%) on a daily basis. In addition, a broad range of environmental and genetic insults can disrupt outer segment morphology to impair photoreceptor function and viability. In this chapter, we survey the major progress that has been made for understanding the molecular basis of outer segment architecture. We also discuss key aspects of organelle lipid and protein composition, and highlight distributions, interactions, and potential structural functions of key OS-resident molecules, including: kinesin-2, actin, RP1, prominin-1, protocadherin 21, peripherin-2/rds, rom-1, glutamic acid-rich proteins, and rhodopsin. Finally, we identify key knowledge gaps and challenges that remain for understanding how normal outer segment architecture is established and maintained.

NLRP3 inflammasome activation drives bystander cone photoreceptor cell death in a P23H rhodopsin model of retinal degeneration.

The molecular signaling leading to cell death in hereditary neurological diseases such as retinal degeneration is incompletely understood. Previous neuroprotective studies have focused on apoptotic pathways; however, incomplete suppression of cell death with apoptosis inhibitors suggests that other mechanisms are at play. Here, we report that different signaling pathways are activated in rod and cone photoreceptors in the P23H rhodopsin mutant rat, a model representing one of the commonest forms of retinal degeneration. Up-regulation of the RIP1/RIP3/DRP1 axis and markedly improved survival with necrostatin-1 treatment highlighted necroptosis as a major cell-death pathway in degenerating rod photoreceptors. Conversely, up-regulation of NLRP3 and caspase-1, expression of mature IL-1ß and IL-18 and improved cell survival with N-acetylcysteine treatment suggested that inflammasome activation and pyroptosis was the major cause of cone cell death. This was confirmed by generation of the P23H mutation on an Nlrp3-deficient background, which preserved cone viability. Furthermore, Brilliant Blue G treatment inhibited inflammasome activation, indicating that the 'bystander cell death' phenomenon was mediated through the P2RX7 cell-surface receptor. Here, we identify a new pathway in cones for bystander cell death, a phenomenon important in development and disease in many biological systems. In other retinal degeneration models different cell-death pathways are activated, which suggests that the particular pathways that are triggered are to some extent genotype-specific. This also implies that neuroprotective strategies to limit retinal degeneration need to be customized; thus, different combinations of inhibitors will be needed to target the specific pathways in any given disease.

Photoreceptors at a glance.

Retinal photoreceptor cells contain a specialized outer segment (OS) compartment that functions in the capture of light and its conversion into electrical signals in a process known as phototransduction. In rods, photoisomerization of 11-cis to all-trans retinal within rhodopsin triggers a biochemical cascade culminating in the closure of cGMP-gated channels and hyperpolarization of the cell. Biochemical reactions return the cell to its 'dark state' and the visual cycle converts all-trans retinal back to 11-cis retinal for rhodopsin regeneration. OS are continuously renewed, with aged membrane removed at the distal end by phagocytosis and new membrane added at the proximal end through OS disk morphogenesis linked to protein trafficking. The molecular basis for disk morphogenesis remains to be defined in detail although several models have been proposed, and molecular mechanisms underlying protein trafficking are under active investigation. The aim of this Cell Science at a Glance article and the accompanying poster is to highlight our current understanding of photoreceptor structure, phototransduction, the visual cycle, OS renewal, protein trafficking and retinal degenerative diseases.

Kinesin family 17 (osmotic avoidance abnormal-3) is dispensable for photoreceptor morphology and function.

In Caenorhabditis elegans, homodimeric [kinesin family (KIF) 17, osmotic avoidance abnormal-3 (OSM-3)] and heterotrimeric (KIF3) kinesin-2 motors are required to establish sensory cilia by intraflagellar transport (IFT) where KIF3 and KIF17 cooperate to build the axoneme core and KIF17 builds the distal segments. However, the function of KIF17 in vertebrates is unresolved. We expressed full-length and motorless KIF17 constructs in mouse rod photoreceptors using adeno-associated virus in Xenopus laevis rod photoreceptors using a transgene and in ciliated IMCD3 cells. We found that tagged KIF17 localized along the rod outer segment axoneme when expressed in mouse and X. laevis photoreceptors, whereas KIF3A was restricted to the proximal axoneme. Motorless KIF3A and KIF17 mutants caused photoreceptor degeneration, likely through dominant negative effects on IFT. KIF17 mutant lacking the motor domain translocated to nuclei after exposure of a C-terminal nuclear localization signal. Germ-line deletion of Kif17 in mouse did not affect photoreceptor function. A rod-specific Kif3/Kif17 double knockout mouse demonstrated that KIF17 and KIF3 do not act synergistically and did not prevent rhodopsin trafficking to rod outer segments. In summary, the nematode model of KIF3/KIF17 cooperation apparently does not apply to mouse photoreceptors in which the photosensory cilium is built exclusively by KIF3.

Preparation of Xenopus laevis retinal cryosections for electron microscopy.

Transmission electron microscopy is the gold standard for examination of photoreceptor outer segment morphology and photoreceptor outer segment abnormalities in transgenic animal models of retinal disease. Small vertebrates such as zebrafish and Xenopus laevis tadpoles have been used to generate retinal disease models and to study outer segment processes such as protein trafficking, and their breeding capabilities facilitate experiments involving large numbers of animals and conditions. However, electron microscopy processing and analysis of these very small eyes can be challenging. Here we present a methodology that facilitates processing of X. laevis tadpole eyes for electron microscopy by introducing an intermediate cryosectioning step. This method reproducibly provides a well-oriented tissue block that can be sectioned with minimal effort by a non-expert, and also allows retroactive analysis of samples collected on slides for light microscopy.

Light Induces Ultrastructural Changes in Rod Outer and Inner Segments, Including Autophagy, in a Transgenic Xenopus laevis P23H Rhodopsin Model of Retinitis Pigmentosa.

PURPOSE: We previously reported a transgenic Xenopus laevis model of retinitis pigmentosa in which tadpoles express the bovine form of P23H rhodopsin (bP23H) in rod photoreceptors. In this model, retinal degeneration was dependent on light exposure. Here, we investigated ultrastructural changes that occurred in the rod photoreceptors of these retinas when exposed to light. METHODS: Tadpoles expressing bP23H in rods were transferred from constant darkness to a 12-hour light:12-hour dark (12L:12D) regimen. For comparison, transgenic tadpoles expressing an inducible form of caspase 9 (iCasp9) were reared in a 12L:12D regimen, and retinal degeneration was induced by administration of the drug AP20187. Tadpoles were euthanized at various time points, and eyes were processed for confocal light and transmission electron microscopy. RESULTS: We observed defects in outer and inner segments of rods expressing bP23H that were aggravated by light exposure. Rod outer segments exhibited vesiculations throughout and were rapidly phagocytosed by the retinal pigment epithelium. In rod inner segments, we observed autophagic compartments adjacent to the endoplasmic reticulum and extensive vesiculation at later time points. These defects were not found in rods expressing iCasp9, which completely degenerated within 36 hours after drug administration. CONCLUSIONS: Our results indicate that ultrastructural defects in outer and inner segment membranes of bP23H expressing rods differ from those observed in drug-induced apoptosis. We suggest that light-induced retinal degeneration caused by P23H rhodopsin occurs via cell death with autophagy, which may represent an attempt to eliminate the mutant rhodopsin and/or damaged cellular compartments from the secretory pathway.

Photoactivation-induced instability of rhodopsin mutants T4K and T17M in rod outer segments underlies retinal degeneration in X. laevis transgenic models of retinitis pigmentosa.

Retinitis pigmentosa (RP) is an inherited neurodegenerative disease involving progressive vision loss, and is often linked to mutations in the rhodopsin gene. Mutations that abolish N-terminal glycosylation of rhodopsin (T4K and T17M) cause sector RP in which the inferior retina preferentially degenerates, possibly due to greater light exposure of this region. Transgenic animal models expressing rhodopsin glycosylation mutants also exhibit light exacerbated retinal degeneration (RD). In this study, we used transgenic Xenopus laevis to investigate the pathogenic mechanism connecting light exposure and RD in photoreceptors expressing T4K or T17M rhodopsin. We demonstrate that increasing the thermal stability of these rhodopsins via a novel disulfide bond resulted in significantly less RD. Furthermore, T4K or T17M rhodopsins that were constitutively inactive (due to lack of the chromophore-binding site or dietary deprivation of the chromophore precursor vitamin A) induced less toxicity. In contrast, variants in the active conformation accumulated in the ER and caused RD even in the absence of light. In vitro, T4K and T17M rhodopsins showed reduced ability to regenerate pigment after light exposure. Finally, although multiple amino acid substitutions of T4 abolished glycosylation at N2 but were not toxic, similar substitutions of T17 were not tolerated, suggesting that the carbohydrate moiety at N15 is critical for cell viability. Our results identify a novel pathogenic mechanism in which the glycosylation-deficient rhodopsins are destabilized by light activation. These results have important implications for proposed RP therapies, such as vitamin A supplementation, which may be ineffective or even detrimental for certain RP genotypes.

Mutant ELOVL4 that causes autosomal dominant stargardt-3 macular dystrophy is misrouted to rod outer segment disks.

PURPOSE: Autosomal dominant Stargardt macular dystrophy caused by mutations in the Elongation of Very Long Chain fatty acids (ELOVL4) gene results in macular degeneration, leading to early childhood blindness. Transgenic mice and pigs expressing mutant ELOVL4 develop progressive photoreceptor degeneration. The mechanism by which these mutations cause macular degeneration remains unclear, but have been hypothesized to involve the loss of an ER-retention dilysine motif located in the extreme C-terminus. Dominant negative mechanisms and reduction in retinal polyunsaturated fatty acids also have been suggested. To understand the molecular mechanisms involved in disease progression in vivo, we addressed the hypothesis that the disease-linked C-terminal truncation mutant of ELOVL4 exerts a dominant negative effect on wild-type (WT) ELOVL4, altering its subcellular localization and function, which subsequently induces retinal degeneration and loss of vision. METHODS: We generated transgenic Xenopus laevis that overexpress HA-tagged murine ELOVL4 variants in rod photoreceptors. RESULTS: Tagged or untagged WT ELOVL4 localized primarily to inner segments. However, the mutant protein lacking the dilysine motif was mislocalized to post-Golgi compartments and outer segment disks. Coexpression of mutant and WT ELOVL4 in rods did not result in mislocalization of the WT protein to outer segments or in the formation of aggregates. Full-length HA-tagged ELOVL4 lacking the dilysine motif (K308R/K310R) necessary for targeting the WT ELOVL4 protein to the endoplasmic reticulum was similarly mislocalized to outer segments. CONCLUSIONS: We propose that expression and outer segment mislocalization of the disease-linked 5-base-pair deletion mutant ELOVL4 protein alters photoreceptor structure and function, which subsequently results in retinal degeneration, and suggest three possible mechanisms by which mutant ELOVL4 may induce retinal degeneration in STGD3.