Probing mechanisms of photoreceptor degeneration in a new mouse model of the common form of autosomal dominant retinitis pigmentosa due to P23H opsin mutations

Rhodopsin, the visual pigment mediating vision under dim light, is composed of the apoprotein opsin and the chromophore ligand 11-cis-retinal. A P23H mutation in the opsin gene is one of the most prevalent causes of the human blinding disease, autosomal dominant retinitis pigmentosa. Although P23H cultured cell and transgenic animal models have been developed, there remains controversy over whether they fully mimic the human phenotype; and the exact mechanism by which this mutation leads to photoreceptor cell degeneration remains unknown. By generating P23H opsin knock-in mice, we found that the P23H protein was inadequately glycosylated with levels 1-10% that of wild type opsin. Moreover, the P23H protein failed to accumulate in rod photoreceptor cell endoplasmic reticulum but instead disrupted rod photoreceptor disks. Genetically engineered P23H mice lacking the chromophore showed accelerated photoreceptor cell degeneration. These results indicate that most synthesized P23H protein is degraded, and its retinal cytotoxicity is enhanced by lack of the 11-cis-retinal chromophore during rod outer segment development.     

Rhodopsin mutant P23H destabilizes rod photoreceptor disk membranes

Mutations in rhodopsin cause retinitis pigmentosa in humans and retinal degeneration in a multitude of other animals. We utilized high-resolution live imaging of the large rod photoreceptors from transgenic frogs (Xenopus) to compare the properties of fluorescently tagged rhodopsin, Rho-EGFP, and Rho(P23H)-EGFP. The mutant was abnormally distributed both in the inner and outer segments (OS), accumulating in the OS to a concentration of ～0.1% compared to endogenous opsin. Rho(P23H)-EGFP formed dense fluorescent foci, with concentrations of mutant protein up to ten times higher than other regions. Wild-type transgenic Rho-EGFP did not concentrate in OS foci when co-expressed in the same rod with Rho(P23H)-EGFP. Outer segment regions containing fluorescent foci were refractory to fluorescence recovery after photobleaching, while foci in the inner segment exhibited recovery kinetics similar to OS regions without foci and Rho-EGFP. The Rho(P23H)-EGFP foci were often in older, more distal OS disks. Electron micrographs of OS revealed abnormal disk membranes, with the regular disk bilayers broken into vesiculotubular structures. Furthermore, we observed similar OS disturbances in transgenic mice expressing Rho(P23H), suggesting such structures are a general consequence of mutant expression. Together these results show that mutant opsin disrupts OS disks, destabilizing the outer segment possibly via the formation of aggregates. This may render rods susceptible to mechanical injury or compromise OS function, contributing to photoreceptor loss.     

A diffusible factor from normal retinal cells promotes rod photoreceptor survival in an in vitro model of retinitis pigmentosa

Transgenic mice expressing a dominant mutation in the gene for the phototransduction molecule rhodopsin undergo retinal degeneration similar to that experienced by patients with the retinal degenerative disease, retinitis pigmentosa (RP). Although the mutation is thought to cause photoreceptor degeneration in a cell‐autonomous manner, the fact that rod photoreceptor degeneration is slowed in chimeric wild‐type/mutant mice suggests that cellular interactions are also important for maintaining photoreceptor survival. To more fully characterize the nature of the cellular interactions important for rod degeneration in the RP mutant mice, we have used an in vitro approach. We found that when the retinas of the transgenic mice were isolated from the pigmented epithelium and cultured as explants, the rod photoreceptors underwent selective degeneration with a similar time course to that observed in vivo. This selective rod degeneration also occurred when the cells were dissociated and cultured as monolayers. These data indicate that the mutant rod photoreceptors degenerate when removed from their normal cellular relationships and without contact with the pigmented epithelium, thus confirming the relative cell autonomy of the mutant phenotype. We next tested whether normal retinal cells could rescue the mutant photoreceptors in a coculture paradigm. Coculture of transgenic mouse with wild‐type mouse or rat retinal cells significantly enhanced transgenic rod photoreceptor survival; this survival‐promoting activity was diffusible through a filter, was heat labile, and not present in transgenic retinal cells. Several peptide growth factors known to be present in the retina were tested as the potential survival‐promoting molecule responsible for the effects of the conditioned medium; however, none of them promoted survival of the photoreceptors expressing the Pro23His mutant rhodopsin. Nevertheless, we were able to demonstrate that the mutant photoreceptors could be rescued by an antagonist to a retinoic acid receptor, suggesting that the endogeneous survival‐promoting activity may function through this pathway. These data thus confirm and extend the findings of previous work that local trophic interactions are important in regulating rod photoreceptor degeneration in retinitis pigmentosa. A diffusible factor found in normal but not transgenic retinal cells has a protective effect on the survival of rod photoreceptors from Pro23His mutant rhodopsin mice. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 475–490, 1999     

Retinoids assist the cellular folding of the autosomal dominant retinitis pigmentosa opsin mutant P23H

The clinically common mutant opsin P23H, associated with autosomal dominant retinitis pigmentosa, yields low levels of rhodopsin when retinal is added following induction of the protein in stably transfected HEK-293 cells. We previously showed that P23H rhodopsin levels could be increased by providing a 7-membered ring, locked analog of 11-cis-retinal during expression of P23H opsin in vivo. Here we demonstrate that the mutant opsin is effectively rescued by 9- or 11-cis-retinal, the native chromophore. When retinal was added during expression, P23H rhodopsin levels were 5-fold (9-cis) and 6-fold (11-cis) higher than when retinal was added after opsin was expressed and cells were harvested. Levels of P23H opsin were increased approximately 3.5-fold with both compounds, but wild-type protein levels were only slightly increased. Addition of retinal during induction promoted the Golgi-specific glycosylation of P23H opsin and transport of the protein to the cell surface. P23H rhodopsins containing 9- or 11-cis-retinal had blue-shifted absorption maxima and altered photo-bleaching properties compared with the corresponding wild-type proteins. Significantly, P23H rhodopsins were more thermally unstable than the wild-type proteins and more rapidly bleached by hydroxylamine in the dark. We suggest that P23H opsin is similarly unstable and that retinal binds and stabilizes the protein early in its biogenesis to promote its cellular folding and trafficking. The implications of this study for treating retinitis pigmentosa and other protein conformational disorders are discussed.     

Inherent Instability of the Retinitis Pigmentosa P23H Mutant Opsin

The P23H opsin mutation is the most common cause of autosomal dominant retinitis pigmentosa. Even though the pathobiology of the resulting retinal degeneration has been characterized in several animal models, its complex molecular mechanism is not well understood. Here, we expressed P23H bovine rod opsin in the nervous system of Caenorhabditis elegans. Expression was low due to enhanced protein degradation. The mutant opsin was glycosylated, but the polysaccharide size differed from that of the normal protein. Although P23H opsin aggregated in the nervous system of C. elegans, the pharmacological chaperone 9-cis-retinal stabilized it during biogenesis, producing a variant of rhodopsin called P23H isorhodopsin. In vitro, P23H isorhodopsin folded correctly, formed the appropriate disulfide bond, could be photoactivated but with reduced sensitivity, and underwent Meta II decay at a rate similar to wild type isorhodopsin. In worm neurons, P23H isorhodopsin initiated phototransduction by coupling with the endogenous G_i/o signaling cascade that induced loss of locomotion. Using pharmacological interventions affecting protein synthesis and degradation, we showed that the chromophore could be incorporated either during or after mutant protein translation. However, regeneration of P23H isorhodopsin with chromophore was significantly slower than that of wild type isorhodopsin. This effect, combined with the inherent instability of P23H rhodopsin, could lead to the structural cellular changes and photoreceptor death found in autosomal dominant retinitis pigmentosa. These results also suggest that slow regeneration of P23H rhodopsin could prevent endogenous chromophore-mediated stabilization of rhodopsin in the retina.     

BiP prevents rod opsin aggregation

Mutations in rod opsin-the light-sensitive protein of rod cells-cause retinitis pigmentosa. Many rod opsin mutations lead to protein misfolding, and therefore it is important to understand the role of molecular chaperones in rod opsin biogenesis. We show that BiP (HSPA5) prevents the aggregation of rod opsin. Cleavage of BiP with the subtilase cytotoxin SubAB results in endoplasmic reticulum (ER) retention and ubiquitylation of wild-type (WT) rod opsin (WT-green fluorescent protein [GFP]) at the ER. Fluorescence recovery after photobleaching reveals that WT-GFP is usually mobile in the ER. By contrast, depletion of BiP activity by treatment with SubAB or coexpression of a BiP ATPase mutant, BiP(T37G), decreases WT-GFP mobility to below that of the misfolding P23H mutant of rod opsin (P23H-GFP), which is retained in the ER and can form cytoplasmic ubiquitylated inclusions. SubAB treatment of P23H-GFP-expressing cells decreases the mobility of the mutant protein further and leads to ubiquitylation throughout the ER. Of interest, BiP overexpression increases the mobility of P23H-GFP, suggesting that it can reduce mutant rod opsin aggregation. Therefore inhibition of BiP function results in aggregation of rod opsin in the ER, which suggests that BiP is important for maintaining the solubility of rod opsin in the ER.     

The heat-shock response co-inducer arimoclomol protects against retinal degeneration in rhodopsin retinitis pigmentosa

Retinitis pigmentosa (RP) is a group of inherited diseases that cause blindness due to the progressive death of rod and cone photoreceptors in the retina. There are currently no effective treatments for RP. Inherited mutations in rhodopsin, the light-sensing protein of rod photoreceptor cells, are the most common cause of autosomal-dominant RP. The majority of mutations in rhodopsin, including the common P23H substitution, lead to protein misfolding, which is a feature in many neurodegenerative disorders. Previous studies have shown that upregulating molecular chaperone expression can delay disease progression in models of neurodegeneration. Here, we have explored the potential of the heat-shock protein co-inducer arimoclomol to ameliorate rhodopsin RP. In a cell model of P23H rod opsin RP, arimoclomol reduced P23H rod opsin aggregation and improved viability of mutant rhodopsin-expressing cells. In P23H rhodopsin transgenic rat models, pharmacological potentiation of the stress response with arimoclomol improved electroretinogram responses and prolonged photoreceptor survival, as assessed by measuring outer nuclear layer thickness in the retina. Furthermore, treated animal retinae showed improved photoreceptor outer segment structure and reduced rhodopsin aggregation compared with vehicle-treated controls. The heat-shock response (HSR) was activated in P23H retinae, and this was enhanced with arimoclomol treatment. Furthermore, the unfolded protein response (UPR), which is induced in P23H transgenic rats, was also enhanced in the retinae of arimoclomol-treated animals, suggesting that arimoclomol can potentiate the UPR as well as the HSR. These data suggest that pharmacological enhancement of cellular stress responses may be a potential treatment for rhodopsin RP and that arimoclomol could benefit diseases where ER stress is a factor.     

Complete rescue of photoreceptor dysplasia and degeneration in transgenic retinal degeneration slow (rds) mice.

retinal degeneration slow (rds) is a semidominant mutation of mice with the phenotype of abnormal development of rod and cone photoreceptors, followed by their slow degeneration. The rds gene has been putatively cloned and its novel protein product initially characterized biochemically. In the present study we undertook to correct in vivo the retinal phenotype of mice with the rds mutation. We assembled a transgene containing a regulatory segment of the opsin gene positioned upstream of the wild-type rds coding region. Mice from three transgenic lines, homozygous for the rds mutation, were analyzed for expression of the transgene and for their retinal phenotypes. In two high expressing lines, we observed complete reversion to wild-type retinal morphology. In a third, low expressing line, we observed a retinal phenotype intermediate between wild type and rds/rds, suggesting partial rescue of the mutation. These results constitute formal proof that we have cloned the rds gene.     

A simple polymerase chain reaction assay for genotyping the retinal degeneration mutation (Pdeb(rd1)) in FVB/N-derived transgenic mice

FVB/N mice are one of the most common inbred strains for the generation of transgenic animals. This mouse strain is preferred for transgenesis because of its fertilized oocytes, which have unique pronuclei for microinjection, and its vigorous reproductive performance along with consistently large litter sizes. However, these inbred mice carry a retinal degeneration mutation caused by a proviral insertion into the Pdeb gene, encoding the beta subunit of cGMP phosphodiesterase. This mutation (Pdeb(rd1), formerly known as rd) results in postnatal rod photoreceptor degeneration and causes severe visual impairment, which may be relevant for behavioural and vision-related research. This deficit can be overcome by crossing these mice with other mouse strains carrying the wild-type allele at the Pdeb locus. We have devised a simple polymerase chain reaction (PCR)-based method for distinguishing between the mutant and the wild-type alleles, thus allowing the efficient monitoring of the Pdeb(rd1) mutation in FVB/N-derived transgenic mice prior to experimentation where visual deficit is expected to have an influence in the phenotype.     

A functional rhodopsin-green fluorescent protein fusion protein localizes correctly in transgenic Xenopus laevis retinal rods and is expressed in a time-dependent pattern

To study rhodopsin biosynthesis and transport in vivo, we engineered a fusion protein (rho-GFP) of bovine rhodopsin (rho) and green fluorescent protein (GFP). rho-GFP expressed in COS-1 cells bound 11-cis retinal, generating a pigment with spectral properties of rhodopsin (A(max) at 500 nm) and GFP (A(max) at 488 nm). rho-GFP activated transducin at 50% of the wild-type activity, whereas phosphorylation of rho-GFP by rhodopsin kinase was 10% of wild-type levels. We expressed rho-GFP in the rod photoreceptors of Xenopus laevis using the X. laevis principal opsin promoter. Like rhodopsin, rho-GFP localized to rod outer segments, indicating that rho-GFP was recognized by membrane transport mechanisms. In contrast, a rho-GFP variant lacking the C-terminal outer segment localization signal distributed to both outer and inner segment membranes. Confocal microscopy of transgenic retinas revealed that transgene expression levels varied between cells, an effect that is probably analogous to position-effect variegation. Furthermore, rho-GFP concentrations varied along the length of individual rods, indicating that expression levels varied within single cells on a daily or hourly basis. These results have implications for transgenic models of retinal degeneration and mechanisms of position-effect variegation and demonstrate the utility of rho-GFP as a probe for rhodopsin transport and temporal regulation of promoter function.     

Autosomal recessive retinitis pigmentosa and E150K mutation in the opsin gene

Retinitis pigmentosa (RP) is a heterogeneous group of hereditary disorders of the retina caused by mutation in genes of the photoreceptor proteins with an autosomal dominant (adRP), autosomal recessive (arRP), or X-linked pattern of inheritance. Although there are over 100 identified mutations in the opsin gene associated with RP, only a few of them are inherited with the arRP pattern. E150K is the first reported missense mutation associated with arRP. This opsin mutation is located in the second cytoplasmic loop of this G protein-coupled receptor. E150K opsin expressed in HEK293 cells and reconstituted with 11-cis-retinal displayed an absorption spectrum similar to the wild type (WT) counterpart and activated G protein transducin slightly faster than WT receptor. However, the majority of E150K opsin showed a higher apparent molecular mass in SDS-PAGE and was resistant to endoglycosidase H deglycosidase. Instead of being transported to the plasma membrane, E150K opsin is partially colocalized with the cis/medial Golgi compartment markers such as GM130 and Vti1b but not with the trans-Golgi network. In contrast to the endoplasmic reticulum-retained adRP mutant, P23H opsin, Golgi-retained E150K opsin did not influence the proper transport of the WT opsin when coexpressed in HEK293 cells. This result is consistent with the recessive pattern of inheritance of this mutation. Thus, our study reveals a novel molecular mechanism for retinal degeneration that results from deficient export of opsin from the Golgi apparatus.     

Cone cells fail to develop normally in transgenic mice showing ablation of rod photoreceptor cells

Transgenic mice were derived containing the cytotoxic dt-α gene driven by opsin promoter sequences. Mice expressing this construct showed progressive degeneration of rod photoreceptor cells commencing at birth, with obvious depletion of such cells by postnatal day 7. Ablation of rod photoreceptor cells in the transgenic retina was accompanied by the failure of developing cone cells to elaborate outer segments, although all other aspects of cone cell cytodifferentiation appeared normal. The results suggest that the 1.0-kb opsin promoter segment contains rod cell type specificity and that cone cells require maturation of rod cells to complete the late stages of their terminal differentiation and for their maintenance and cellular integrity.     

Visualization of rod photoreceptor development using GFP-transgenic zebrafish

Zebrafish retina contains five morphologically distinct classes of photoreceptors, each expressing a distinct type of opsin gene. Molecular mechanisms underlying specification of opsin expression and differentiation among the cell types are largely unknown. This is partly because mutants affected with expression of a particular class of opsin gene are difficult to find. In this study we established the transgenic lines of zebrafish carrying green fluorescent protein (GFP) gene under the 1.1-kb and 3.7-kb upstream regions of the rod-opsin gene. In transgenic fish, GFP expression initiated and proceeded in the same spatiotemporal pattern with rod-opsin gene. The retinal section from adult transgenic fish showed GFP expression throughout the rod cell layer. These results indicate that the proximal 1.1-kb region is sufficient to drive gene expression in all rod photoreceptor cells. These transgenic fish should facilitate screening of mutants affected specifically with rod-opsin expression or rod cell development by visualization of rod cells by GFP.     

Rescue of photoreceptor degeneration by curcumin in transgenic rats with P23H rhodopsin mutation

The P23H mutation in the rhodopsin gene causes rhodopsin misfolding, altered trafficking and formation of insoluble aggregates leading to photoreceptor degeneration and autosomal dominant retinitis pigmentosa (RP). There are no effective therapies to treat this condition. Compounds that enhance dissociation of protein aggregates may be of value in developing new treatments for such diseases. Anti-protein aggregating activity of curcumin has been reported earlier. In this study we present that treatment of COS-7 cells expressing mutant rhodopsin with curcumin results in dissociation of mutant protein aggregates and decreases endoplasmic reticulum stress. Furthermore we demonstrate that administration of curcumin to P23H-rhodopsin transgenic rats improves retinal morphology, physiology, gene expression and localization of rhodopsin. Our findings indicate that supplementation of curcumin improves retinal structure and function in P23H-rhodopsin transgenic rats. This data also suggest that curcumin may serve as a potential therapeutic agent in treating RP due to the P23H rhodopsin mutation and perhaps other degenerative diseases caused by protein trafficking defects.     

Immunoassay of rod visual pigment (opsin) in the eyes of rds mutant mice lacking receptor outer segments

In 020/A mice, homozygous for the retinal degeneration slow (rds) gene, the photoreceptor cells fail to develop outer segments, and in the absorption spectra of retinal extracts the rhodopsin peak is lacking. Application of an enzyme-linked immunoassay using antisera against bovine opsin shows, however, that opsin is present in the homozygous mutant retina (0.010 nmol/eye) at 3% of the level of the normal retina (0.38 nmol/eye) of Balb/c mice. In the retina of heterozygous mice the opsin level (0.19 nmol/eye) is about half of the normal. Detection of opsin in the rds mutant retina demonstrates the functional basis for the reported electroretinographic response and light-mediated reduction in cyclic nucleotide levels in this mutant.     

Q344ter Mutation Causes Mislocalization of Rhodopsin Molecules That Are Catalytically Active: A Mouse Model of Q344ter-Induced Retinal Degeneration

Q344ter is a naturally occurring rhodopsin mutation in humans that causes autosomal dominant retinal degeneration through mechanisms that are not fully understood, but are thought to involve an early termination that removed the trafficking signal, QVAPA, leading to its mislocalization in the rod photoreceptor cell. To better understand the disease mechanism(s), transgenic mice that express Q344ter were generated and crossed with rhodopsin knockout mice. Dark-reared Q344ter^rho+/− mice exhibited retinal degeneration, demonstrating that rhodopsin mislocalization caused photoreceptor cell death. This degeneration is exacerbated by light-exposure and is correlated with the activation of transducin as well as other G-protein signaling pathways. We observed numerous sub-micrometer sized vesicles in the inter-photoreceptor space of Q344ter^rho+/− and Q344ter^rho−/− retinas, similar to that seen in another rhodopsin mutant, P347S. Whereas light microscopy failed to reveal outer segment structures in Q344ter^rho−/− rods, shortened and disorganized rod outer segment structures were visible using electron microscopy. Thus, some Q344ter molecules trafficked to the outer segment and formed disc structures, albeit inefficiently, in the absence of full length wildtype rhodopsin. These findings helped to establish the in vivo role of the QVAPA domain as well as the pathways leading to Q344ter-induced retinal degeneration.     

A comparison of the efficiency of G protein activation by ligand-free and light-activated forms of rhodopsin

Activation of the photoreceptor G protein transducin (Gt) by opsin, the ligand-free form of rhodopsin, was measured using rod outer segment membranes with densities of opsin and Gt similar to those found in rod cells. When GTPgammaS was used as the activating nucleotide, opsin catalyzed transducin activation with an exponential time course with a rate constant k(act) on the order of 2 x 10(-3)s(-1). Comparison under these conditions to activation by flash-generated metarhodopsin II (MII) revealed that opsin- and R*-catalyzed activation showed similar kinetics when MII was present at a surface density approximately 10(-6) lower than that of opsin. Thus, in contrast to some previous reports, we find that the catalytic potency of opsin is only approximately 10(-6) that of MII. In the presence of residual retinaldehyde-derived species present in membranes treated with hydroxylamine after bleaching, the apparent k(act) observed was much higher than that for opsin, suggesting a possible explanation for previous reports of more efficient activation by opsin. These results are important for considering the possible role of opsin in the diverse phenomena in which it has been suggested to play a key role, such as bleaching desensitization and retinal degeneration induced by continuous light or vitamin A deprivation.     

Misfolded rhodopsin mutants display variable aggregation properties

The largest class of rhodopsin mutations causing autosomal dominant retinitis pigmentosa (adRP) is mutations that lead to misfolding and aggregation of the receptor. The misfolding mutants have been characterized biochemically, and categorized as either partial or complete misfolding mutants. This classification is incomplete and does not provide sufficient information to fully understand the disease pathogenesis and evaluate therapeutic strategies. A Förster resonance energy transfer (FRET) method was utilized to directly assess the aggregation properties of misfolding rhodopsin mutants within the cell. Partial (P23H and P267L) and complete (G188R, H211P, and P267R) misfolding mutants were characterized to reveal variability in aggregation properties. The complete misfolding mutants all behaved similarly, forming aggregates when expressed alone, minimally interacting with the wild-type receptor when coexpressed, and were unresponsive to treatment with the pharmacological chaperone 9-cis retinal. In contrast, variability was observed between the partial misfolding mutants. In the opsin form, the P23H mutant behaved similarly as the complete misfolding mutants. In contrast, the opsin form of the P267L mutant existed as both aggregates and oligomers when expressed alone and formed mostly oligomers with the wild-type receptor when coexpressed. The partial misfolding mutants both reacted similarly to the pharmacological chaperone 9-cis retinal, displaying improved folding and oligomerization when expressed alone but aggregating with wild-type receptor when coexpressed. The observed differences in aggregation properties and effect of 9-cis retinal predict different outcomes in disease pathophysiology and suggest that retinoid-based chaperones will be ineffective or even detrimental.     

Rhodopsin activation causes retinal degeneration in Drosophila rdgC mutant

Drosophila rdgC (retinal degeneration-C) mutants show normal retinal morphology and photoreceptor physiology at young ages. Dark-reared rdgC flies retain this wild-type phenotype, but light-reared mutants undergo retinal degeneration. rdgC photoreceptors with low levels of rhodopsin as a result of vitamin A deprivation or a mutant rhodopsin (ninaE) gene fail to show rdgC-induced degeneration even after prolonged light treatment, demonstrating that degeneration occurs as a result of light stimulation of rhodopsin. Analysis of norpA; rdgC flies shows that the norpA-encoded phospholipase C, the target enzyme of the G protein activated by rhodopsin, is not required for rdgC-induced degeneration. Thus the rdgC+ gene product is required to prevent retinal degeneration that results from a previously unrecognized consequence of rhodopsin stimulation.     

Tyro3 Modulates Mertk-Associated Retinal Degeneration

Inherited photoreceptor degenerations (IPDs) are the most genetically heterogeneous of Mendelian diseases. Many IPDs exhibit substantial phenotypic variability, but the basis is usually unknown. Mutations in MERTK cause recessive IPD phenotypes associated with the RP38 locus. We have identified a murine genetic modifier of Mertk-associated photoreceptor degeneration, the C57BL/6 (B6) allele of which acts as a suppressor. Photoreceptors degenerate rapidly in Mertk-deficient animals homozygous for the 129P2/Ola (129) modifier allele, whereas animals heterozygous for B6 and 129 modifier alleles exhibit an unusual intermixing of degenerating and preserved retinal regions, with females more severely affected than males. Mertk-deficient mice homozygous for the B6 modifier allele display degeneration only in the far periphery, even at 8 months of age, and have improved retinal function compared to animals homozygous for the 129 allele. We genetically mapped the modifier to an approximately 2-megabase critical interval that includes Tyro3, a paralog of Mertk. Tyro3 expression in the outer retina varies with modifier genotype in a manner characteristic of a cis-acting expression quantitative trait locus (eQTL), with the B6 allele conferring an approximately three-fold higher expression level. Loss of Tyro3 function accelerates the pace of photoreceptor degeneration in Mertk knockout mice, and TYRO3 protein is more abundant in the retinal pigment epithelium (RPE) adjacent to preserved central retinal regions of Mertk knockout mice homozygous for the B6 modifier allele. Endogenous human TYRO3 protein co-localizes with nascent photoreceptor outer segment (POS) phagosomes in a primary RPE cell culture assay, and expression of murine Tyro3 in cultured cells stimulates phagocytic ingestion of POS. Our findings demonstrate that Tyro3 gene dosage modulates Mertk-associated retinal degeneration, provide strong evidence for a direct role for TYRO3 in RPE phagocytosis, and suggest that an eQTL can modify a recessive IPD.