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Tyro3 Modulates Mertk-Associated Retinal Degeneration.

ABSTRACT: 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.

SUBMITTER: Vollrath D

PROVIDER: S-EPMC4687644 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Tyro3 Modulates Mertk-Associated Retinal Degeneration.

Vollrath Douglas D Yasumura Douglas D Benchorin Gillie G Matthes Michael T MT Feng Wei W Nguyen Natalie M NM Sedano Cecilia D CD Calton Melissa A MA LaVail Matthew M MM

PLoS genetics 20151211 12

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 ...[more]

PMID: 26656104

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Project description:Retinitis Pigmentosa (RP) is a group of inherited retinal diseases characterized by progressive loss of rod followed by cone photoreceptors. An especially early onset form of RP with blindness in teenage years is caused by mutations in mertk, the gene encoding the clearance phagocytosis receptor Mer tyrosine kinase (MerTK). The cause for blindness in mutant MerTK-associated RP (mutMerTK-RP) is the failure of retinal pigment epithelial cells in diurnal phagocytosis of spent photoreceptor outer segment debris. However, the early onset and very fast progression of degeneration in mutMerTK-RP remains unexplained. Here, we explored the role of microglia in the Royal College of Surgeons (RCS) rat model of mutMerTK-RP. We found elevated levels of inflammatory cytokines and CD68 microglia activation marker, and more ionized calcium-binding adapter molecule 1 (Iba-1) positive microglia in RCS retina when compared to wild-type retina as early as postnatal day 14 (P14). Strikingly, renewal of photoreceptor outer segments in P14 wild-type rat retina is still immature with low levels of RPE phagocytosis implying that at this early age lack of this process in RCS rats is unlikely to distress photoreceptors. Although the total number of Iba-1 positive retinal microglia remains constant from P14 to P30, we observed increasing numbers of microglia in the outer retina from P20 implying migration to the outer retina before onset of photoreceptor cell death at ~P25. Iba-1 and CD68 levels also increase in the retina during this time period suggesting microglia activation. To determine whether microglia affect the degenerative process, we suppressed retinal microglia in vivo using tamoxifen or a combination of tamoxifen and liposomal clodronate. Treatments partly prevented elevation of Iba-1 and CD68 and relocalization of microglia. Moreover, treatments led to partial but significant retention of photoreceptor viability and photoreceptor function. We conclude that loss of the phagocytosis receptor MerTK causes microglia activation and relocalization in the retina before lack of RPE phagocytosis causes overt retinal degeneration, and that microglia activities accelerate loss of photoreceptors in mutMerTK-RP. These results suggest that therapies targeting microglia may delay onset and slow the progression of this blinding disease.

Project description:The receptor tyrosine kinase, MERTK, plays an essential role in homeostasis of the retina via efferocytosis of shed outer nuclear segments of photoreceptors. The Royal College of Surgeons rat model of retinal degeneration has been linked to loss-of-function of MERTK, and together with the MERTK knock-out mouse, phenocopy retinitis pigmentosa in humans with MERTK mutations. Given recent efforts and interest in MERTK as a potential immuno-oncology target, development of a strategy to assess ocular safety at an early pre-clinical stage is critical. We have applied a state-of-the-art, multi-modal imaging platform to assess the in vivo effects of pharmacological inhibition of MERTK in mice. This involved the application of mass spectrometry imaging (MSI) to characterize the ocular spatial distribution of our highly selective MERTK inhibitor; AZ14145845, together with histopathology and transmission electron microscopy to characterize pathological and ultra-structural change in response to MERTK inhibition. In addition, we assessed the utility of a human retinal in vitro cell model to identify perturbation of phagocytosis post MERTK inhibition. We identified high localized total compound concentrations in the retinal pigment epithelium (RPE) and retinal lesions following 28 days of treatment with AZ14145845. These lesions were present in 4 of 8 treated animals, and were characterized by a thinning of the outer nuclear layer, loss of photoreceptors (PR) and accumulation of photoreceptor outer segments at the interface of the RPE and PRs. Furthermore, the lesions were very similar to that shown in the RCS rat and MERTK knock-out mouse, suggesting a MERTK-induced mechanism of PR cell death. This was further supported by the observation of reduced phagocytosis in the human retinal cell model following treatment with AZ14145845. Our study provides a viable, translational strategy to investigate the pre-clinical toxicity of MERTK inhibitors but is equally transferrable to novel chemotypes.

Project description:PurposeOxidative injury is involved in retinal and macular degeneration. We aim to assess if retinal degeneration associated with genetic defect modulates the retinal threshold for encountering additional oxidative challenges.MethodsRetinal oxidative injury was induced in degenerating retinas (rd10) and in control mice (WT) by intravitreal injections of paraquat (PQ). Retinal function and structure was evaluated by electroretinogram (ERG) and histology, respectively. Oxidative injury was assessed by immunohistochemistry for 4-Hydroxy-2-nonenal (HNE), and by Thiobarbituric Acid Reactive Substances (TBARS) and protein carbonyl content (PCC) assays. Anti-oxidant mechanism was assessed by quantitative real time PCR (QPCR) for mRNA of antioxidant genes and genes related to iron metabolism, and by catalase activity assay.ResultsThree days following PQ injections (1 µl of 0.25, 0.75, and 2 mM) the average ERG amplitudes decreased more in the WT mice compared with the rd10 mice. For example, following 2 mM PQ injection, ERG amplitudes reduced 1.84-fold more in WT compared with rd10 mice (p = 0.02). Injection of 4 mM PQ resulted in retinal destruction. Altered retina morphology associated with PQ was substantially more severe in WT eyes compared with rd10 eyes. Oxidative injury according to HNE staining and TBARS assay increased 1.3-fold and 2.1-fold more, respectively, in WT compared with rd10 mice. At baseline, prior to PQ injection, mRNA levels of antioxidant genes (Superoxide Dismutase1, Glutathione Peroxidase1, Catalase) and of Transferrin measured by quantitative PCR were 2.1-7.8-fold higher in rd10 compared with WT mice (p<0.01 each), and catalase activity was 1.7-fold higher in rd10 (p = 0.0006).ConclusionsThis data suggests that degenerating rd10 retinas encounter a relatively lower degree of damage in response to oxidative injury compared with normal retinas. Constitutive up-regulation of the oxidative defense mechanism in degenerating retinas may confer such relative protection from oxidative injury.

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Tyro3 Modulates Mertk-Associated Retinal Degeneration.

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Tyro3 Modulates Mertk-Associated Retinal Degeneration.

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