Gabriel H. Travis

Rpe65 Is the Retinoid Isomerase in Bovine Retinal Pigment Epithelium

The first event in light perception is absorption of a photon by an opsin pigment, which induces isomerization of its 11-cis-retinaldehyde chromophore. Restoration of light sensitivity to the bleached opsin requires chemical regeneration of 11-cis-retinaldehyde through an enzymatic pathway called the visual cycle. The isomerase, which converts an all-trans-retinyl ester to 11-cis-retinol, has never been identified. Here, we performed an unbiased cDNA expression screen to identify this isomerase. We discovered that the isomerase is a previously characterized protein called Rpe65. We confirmed our identification of the isomerase by demonstrating catalytic activity in mammalian and insect cells that express Rpe65. Mutations in the human RPE65 gene cause a blinding disease of infancy called Leber congenital amaurosis. Rpe65 with the Leber-associated C330Y and Y368H substitutions had no isomerase activity. Identification of Rpe65 as the isomerase explains the phenotypes in rpe65-/- knockout mice and in humans with Leber congenital amaurosis.

Isomerization and Oxidation of Vitamin A in Cone-Dominant Retinas: A Novel Pathway for Visual-Pigment Regeneration in Daylight

The first step toward light perception is 11-cis to all-trans photoisomerization of the retinaldehyde chromophore in a rod or cone opsin-pigment molecule. Light sensitivity of the opsin pigment is restored through a multistep pathway called the visual cycle, which effects all-trans to 11-cis re-isomerization of the retinoid chromophore. The maximum throughput of the known visual cycle, however, is too slow to explain sustained photosensitivity in bright light. Here, we demonstrate three novel enzymatic activities in cone-dominant ground-squirrel and chicken retinas: an all-trans-retinol isomerase, an 11-cis-retinyl-ester synthase, and an 11-cis-retinol dehydrogenase. Together these activities comprise a novel pathway that regenerates opsin photopigments at a rate 20-fold faster than the known visual cycle. We suggest that this pathway is responsible for sustained daylight vision in vertebrates.

The retinal degeneration slow (rds) gene product is a photoreceptor disc membrane-associated glycoprotein

Mice homozygous for the retinal degeneration slow (rds) mutation exhibit abnormal development of photoreceptor cells, followed by their slow degeneration. We have recently cloned the rds gene and determined the structure of the wild-type rds mRNA. Here we show that the gene is expressed exclusively in photoreceptor cells. We demonstrate that it encodes a 39 kd membrane-associated glycoprotein that is restricted to photoreceptor outer segments. By electron microscopy, we show that the rds protein is distributed uniformly within outer segment discs. The developmental appearance of the rds protein coincides with outer segment disc formation. We propose that the rds protein functions as an adhesion molecule for stabilization of the outer segment discs.

Treatment with isotretinoin inhibits lipofuscin accumulation in a mouse model of recessive Stargardt's macular degeneration

Recessive Stargardts macular degeneration is an inherited blinding disease of children caused by mutations in the ABCR gene. The primary pathologic defect in Stargardts disease is accumulation of toxic lipofuscin pigments such as N-retinylidene-N-retinylethanolamine (A2E) in cells of the retinal pigment epithelium. This accumulation appears to be responsible for the photoreceptor death and severe visual loss in Stargardts patients. Here, we tested a therapeutic strategy to inhibit lipofuscin accumulation in a mouse model of recessive Stargardts disease. Isotretinoin (Accutane) has been shown to slow the synthesis of 11-cis-retinaldehyde and regeneration of rhodopsin by inhibiting 11-cis-retinol dehydrogenase in the visual cycle. Light activation of rhodopsin results in its release of all-trans-retinaldehyde, which constitutes the first reactant in A2E biosynthesis. Accordingly, we tested the effects of isotretinoin on lipofuscin accumulation in abcr−/− knockout mice. Isotretinoin blocked the formation of A2E biochemically and the accumulation of lipofuscin pigments by electron microscopy. We observed no significant visual loss in treated abcr−/− mice by electroretinography. Isotretinoin also blocked the slower, age-dependent accumulation of lipofuscin in wild-type mice. These results corroborate the proposed mechanism of A2E biogenesis. Further, they suggest that treatment with isotretinoin may inhibit lipofuscin accumulation and thus delay the onset of visual loss in Stargardts patients. Finally, the results suggest that isotretinoin may be an effective treatment for other forms of retinal or macular degeneration associated with lipofuscin accumulation.

THE PHOTORECEPTOR RIM PROTEIN IS AN ABC TRANSPORTER ENCODED BY THE GENE FOR RECESSIVE STARGARDT'S DISEASE (ABCR)

Rim protein (RmP) is a high‐M r membrane glycoprotein that has been localized to the rims of photoreceptor outer segment discs, but its molecular identity is unknown. Here, we describe the purification of RmP and present the sequence of its mRNA. RmP is a new member of the ATP‐binding cassette (ABC) transporter superfamily. We show that RmP is expressed specifically in photoreceptors and predominantly in outer segments. Further, RmP is identical to the protein recently shown to be affected in recessive Stargardts disease. RmP is the first ABC transporter observed in photoreceptors and may play a role in the photoresponse.

Mechanisms of Cell Death in the Inherited Retinal Degenerations

The human retina possesses both extraordinary sensitivity and a huge adaptive range. A light flash containing only a few photons is sufficient to be consciously perceived in humans. We also can see over a range of nearly nine orders of magnitude in background illumination. The vertebrate visual system has been pushed, by evolutionary pressures, to approach its thermodynamic limitations. This exquisite tuning, however, comes at a cost. Photoreceptors are forced to carry large metabolic burdens, to renew continuously their light-sensitive outer segments and to exist under conditions of very high pO2. Modest changes in the biochemical environment or cellular structure of photoreceptors, caused by genetic lesions or environmental insults, are sufficient to induce cell death through apoptosis. For the inherited retinal degenerations, understanding the impact of each molecular defect on the biology of photoreceptors is important, in addition to studying how these effects converge upon the apoptotic pathway. Collectively, these studies should lead to the development of rational therapies that may slow or even reverse the progression of this devastating disease process.

The human retinal degeneration slow (RDS) gene: Chromosome assignment and structure of the mRNA ☆

Retinal degeneration slow (rds) is a mouse neurological mutation that is characterized phenotypically by abnormal development of rod and cone photoreceptors followed by their slow degeneration. This phenotype resembles the pathologic abnormalities seen in retinitis pigmentosa. The mouse rds gene has recently been cloned. Here we present the sequence of a full-length cDNA clone of the human RDS mRNA. We show that in human retina there are two RDS transcripts of 3.0 and 5.5 kb. By analysis of DNA from a panel of human X hamster somatic cell hybrids, and by direct in situ hybridization, we show that the RDS gene is located on the proximal short arm of human chromosome 6. Finally, we present information on the frequency of several observed restriction fragment length polymorphisms using the RDS cDNA. This information is of potential value for testing linkage of the RDS gene to the disease phenotype in families with retinitis pigmentosa.

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.

Accelerated Accumulation of Lipofuscin Pigments in the RPE of a Mouse Model for ABCA4-Mediated Retinal Dystrophies following Vitamin A Supplementation

PURPOSE Dietary supplementation with vitamin A is sometimes prescribed as a treatment for retinitis pigmentosa, a group of inherited retinal degenerations that cause progressive blindness. Loss-of-function mutations in the ABCA4 gene are responsible for a subset of recessive retinitis pigmentosa. Other mutant alleles of ABCA4 cause the related diseases, recessive cone-rod dystrophy, and recessive Stargardt macular degeneration. Mice with a knockout mutation in the abca4 gene massively accumulate toxic lipofuscin pigments in the retinal pigment epithelium. Treatment of these mice with fenretinide, an inhibitor of vitamin A delivery to the eye, blocks formation of these toxic pigments. Here the authors tested the hypothesis that dietary supplementation with vitamin A may accelerate lipofuscin pigment formation in abca4(-/-) mice. METHODS Wild-type and abca4(-/-) mice were fed normal or vitamin A-supplemented diets. Tissues from these mice were analyzed biochemically for retinoids and lipofuscin pigments. Eyes from these mice were analyzed morphologically for lipofuscin in the retinal pigment epithelium and for degeneration of photoreceptors. Visual function in these mice was analyzed by electroretinography. RESULTS Mice that received vitamin A supplementation had dramatically higher levels of retinyl esters in the liver and retinal pigment epithelium. Lipofuscin pigments were significantly increased by biochemical and morphologic analysis in wild-type and abca4(-/-) mice fed the vitamin A-supplemented diet. Photoreceptor degeneration was observed in 11-month-old albino, but not pigmented, abca4(-/-) mice on both diets. CONCLUSIONS Vitamin A supplementation should be avoided in patients with ABCA4 mutations or other retinal or macular dystrophies associated with lipofuscin accumulation in the retinal pigment epithelium.

Rpe65 Is a Retinyl Ester Binding Protein That Presents Insoluble Substrate to the Isomerase in Retinal Pigment Epithelial Cells

Photon capture by a rhodopsin pigment molecule induces 11-cis to all-trans isomerization of its retinaldehyde chromophore. To restore light sensitivity, the all-trans-retinaldehyde must be chemically re-isomerized by an enzyme pathway called the visual cycle. Rpe65, an abundant protein in retinal pigment epithelial (RPE) cells and a homolog of β-carotene dioxygenase, appears to play a role in this pathway. Rpe65-/- knockout mice massively accumulate all-trans-retinyl esters but lack 11-cis-retinoids and rhodopsin visual pigment in their retinas. Mutations in the human RPE65 gene cause a severe recessive blinding disease called Lebers congenital amaurosis. The function of Rpe65, however, is unknown. Here we show that Rpe65 specifically binds all-trans-retinyl palmitate but not 11-cis-retinyl palmitate by a spectral-shift assay, by co-elution during gel filtration, and by co-immunoprecipitation. Using a novel fluorescent resonance energy transfer (FRET) binding assay in liposomes, we demonstrate that Rpe65 extracts all-trans-retinyl esters from phospholipid membranes. Assays of isomerase activity reveal that Rpe65 strongly stimulates the enzymatic conversion of all-trans-retinyl palmitate to 11-cis-retinol in microsomes from bovine RPE cells. Moreover, we show that addition of Rpe65 to membranes from rpe65-/- mice, which possess no detectable isomerase activity, restores isomerase activity to wild-type levels. Rpe65 by itself, however, has no intrinsic isomerase activity. These observations suggest that Rpe65 presents retinyl esters as substrate to the isomerase for synthesis of visual chromophore. This proposed function explains the phenotype in mice and humans lacking Rpe65.