Dean Bok

Rpe65 is necessary for production of 11- cis-vitamin A in the retinal visual cycle

Mutation of RPE65 can cause severe blindness from birth or early childhood, and RPE65 protein is associated with retinal pigment epithelium (RPE) vitamin A metabolism. Here, we show that Rpe65-deficient mice exhibit changes in retinal physiology and biochemistry. Outer segment discs of rod photoreceptors in Rpe65–/– mice are disorganized compared with those of Rpe65+/+ and Rpe65+/– mice. Rod function, as measured by electroretinography, is abolished in Rpe65–/– mice, although cone function remains. Rpe65–/– mice lack rhodopsin, but not opsin apoprotein. Furthermore, all-trans-retinyl esters over-accumulate in the RPE of Rpe65–/– mice, whereas 11-cis-retinyl esters are absent. Disruption of the RPE-based metabolism of all-trans-retinyl esters to 11-cis-retinal thus appears to underlie the Rpe65-/- phenotype, although cone pigment regeneration may be dependent on a separate pathway.

The Pivotal Role of the Complement System in Aging and Age-related Macular Degeneration: Hypothesis Re-visited

During the past ten years, dramatic advances have been made in unraveling the biological bases of age-related macular degeneration (AMD), the most common cause of irreversible blindness in western populations. In that timeframe, two distinct lines of evidence emerged which implicated chronic local inflammation and activation of the complement cascade in AMD pathogenesis. First, a number of complement system proteins, complement activators, and complement regulatory proteins were identified as molecular constituents of drusen, the hallmark extracellular deposits associated with early AMD. Subsequently, genetic studies revealed highly significant statistical associations between AMD and variants of several complement pathway-associated genes including: Complement factor H (CFH), complement factor H-related 1 and 3 (CFHR1 and CFHR3), complement factor B (CFB), complement component 2 (C2), and complement component 3 (C3). In this article, we revisit our original hypothesis that chronic local inflammatory and immune-mediated events at the level of Bruchs membrane play critical roles in drusen biogenesis and, by extension, in the pathobiology of AMD. Secondly, we report the results of a new screening for additional AMD-associated polymorphisms in a battery of 63 complement-related genes. Third, we identify and characterize the local complement system in the RPE-choroid complex - thus adding a new dimension of biological complexity to the role of the complement system in ocular aging and AMD. Finally, we evaluate the most salient, recent evidence that bears directly on the role of complement in AMD pathogenesis and progression. Collectively, these recent findings strongly re-affirm the importance of the complement system in AMD. They lay the groundwork for further studies that may lead to the identification of a transcriptional disease signature of AMD, and hasten the development of new therapeutic approaches that will restore the complement-modulating activity that appears to be compromised in genetically susceptible individuals.

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.

Molecular and biochemical characterization of lecithin retinol acyltransferase.

The enzyme responsible for conversion of all-trans-retinol into retinyl esters, the lecithin retinol acyltransferase (LRAT) has been characterized at the molecular level. The cDNA coding for this protein was cloned and its amino acid sequence deduced. LRAT is composed of a polypeptide of 230 amino acid residues with a calculated mass of 25.3 kDa. Tissue distribution analysis by Northern blot showed expression of a 5.0-kilobase transcript in the human retinal pigment epithelium as well as in other tissues that are known for their high LRAT activity and vitamin A processing. Affinity labeling experiments using specific compounds with high affinity for LRAT and monospecific polyclonal antibodies raised in rabbits against two peptide sequences for LRAT confirmed the molecular mass of LRAT as a 25-kDa protein. High performance liquid chromatography analysis of the reaction product formed by HEK-293 cells transfected with LRAT cDNA confirmed the ability of the transfected cells to convert [3H]all-trans-retinol into authentic [3H]all-trans-retinyl palmitate as chemically determined.

Biosynthesis and assembly of the rod outer segment membrane system. Formation and fate of visual pigment in the frog retina

Abstract When a mixture of tritiated leucine and phenylalanine is injected into adult frogs, the amino acids are rapidly incorporated into newly synthesized protein in the retinal rod inner segment. Electron microscope autoradiography shows that these radioactive proteins are assembled into the basal membranous discs of the outer segment following a two-hour period of migration within the inner segment. This assembly into disc structure is coincident with a sudden rise in the specific activity of visual pigment 500 (rhodopsin) extracted from isolated, washed outer segments and purified by gel filtration. 80 to 85% of the radioactivity in washed outer segments is recovered in the visual pigment fraction. Maximum labeling of visual pigment occurs by six hours post injection, after which the specific activity remains constant for the next 8.5 weeks. During this period, autoradiograms show a successive displacement of labeled discs towards the apex of the outer segment. During this time, disc labeling neither increases nor decreases in its intensity. These findings indicate that visual pigment apoprotein does not undergo turnover during the period that it forms a structural part of the membranous disc. After 8.5 weeks there is a gradual drop in the specific activity of visual pigment. At the same time, the labeled discs disappear from the apex of the outer segment and appear as membranous inclusions undergoing degradation in the cytoplasm of the pigment epithelium. These experiments show that one structural component of the rod outer segment disc membranes, the visual pigment apoprotein, is constantly renewed. Once assembled into the disc structure, the apoprotein of visual pigment remains stable until it is destroyed by the pigment epithelium. On the basis of the experimental results, a model for the synthesis and assembly of rod outer segment disc membranes is proposed.

Transport of retinol from the blood to the retina: an autoradiographic study of the pigment epithelial cell surface receptor for plasma retinol-binding protein.

Abstract Weanling rats were injected with 125 I-labeled retinol binding protein and their ocular tissues were examined by light and electron microscope autoradiography at 10 min and 1 hr after injection. Combined with earlier work on isolated cells, the results show that receptors for retinol binding protein are present on the basal and lateral plasma membrane of retinal pigment epithelial cells. The pigment epithelium of the pars plana and ciliary body also contain plasma membrane receptors for retinol binding protein. In appears as though the entire plasma membrane of the ciliary pigment epithelium contains receptors for the binding protein.

Neurotrophins enhance retinal pigment epithelial cell survival through neuroprotectin D1 signaling

Integrity of retinal pigment epithelial cells is necessary for photoreceptor survival and vision. The essential omega-3 fatty acid, docosahexaenoic acid, attains its highest concentration in the human body in photoreceptors and is assumed to be a target for lipid peroxidation during cell damage. We have previously shown, in contrast, that docosahexaenoic acid is also the precursor of neuroprotectin D1 (NPD1), which now we demonstrate, acts against apoptosis mediated by A2E, a byproduct of phototransduction that becomes toxic when it accumulates in aging retinal pigment epithelial (RPE) cells and in some inherited retinal degenerations. Furthermore, we show that neurotrophins, particularly pigment epithelium-derived factor, induce NPD1 synthesis and its polarized apical secretion. Moreover, docosahexaenoic acid (DHA) elicits a concentration-dependent and selective potentiation of pigment epithelial-derived factor-stimulated NPD1 synthesis and release through the apical RPE cell surface. The bioactivity of signaling activated by pigment epithelium-derived factor and DHA uncovered synergistic cytoprotection with concomitant NPD1 synthesis when cells were challenged with oxidative stress. Also, DHA and pigment epithelium-derived factor synergistically modify the expression of Bcl-2 family members, activating antiapoptotic proteins and decreasing proapoptotic proteins, and by attenuating caspase 3 activation during oxidative stress. Thus, our findings demonstrate that DHA-derived NPD1 protects against RPE cell damage mediated by aging/disease-induced A2E accumulation. Also, our results identify neurotrophins as regulators of NPD1 and of its polarized apical efflux from RPE cells. Taken together, these findings imply NPD1 may elicit autocrine actions on RPE cells and paracrine bioactivity in cells located in the proximity of the interphotoreceptor matrix.

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.

mTOR-mediated dedifferentiation of the retinal pigment epithelium initiates photoreceptor degeneration in mice

Retinal pigment epithelial (RPE) cell dysfunction plays a central role in various retinal degenerative diseases, but knowledge is limited regarding the pathways responsible for adult RPE stress responses in vivo. RPE mitochondrial dysfunction has been implicated in the pathogenesis of several forms of retinal degeneration. Here we have shown that postnatal ablation of RPE mitochondrial oxidative phosphorylation in mice triggers gradual epithelium dedifferentiation, typified by reduction of RPE-characteristic proteins and cellular hypertrophy. The electrical response of the retina to light decreased and photoreceptors eventually degenerated. Abnormal RPE cell behavior was associated with increased glycolysis and activation of, and dependence upon, the hepatocyte growth factor/met proto-oncogene pathway. RPE dedifferentiation and hypertrophy arose through stimulation of the AKT/mammalian target of rapamycin (AKT/mTOR) pathway. Administration of an oxidant to wild-type mice also caused RPE dedifferentiation and mTOR activation. Importantly, treatment with the mTOR inhibitor rapamycin blunted key aspects of dedifferentiation and preserved photoreceptor function for both insults. These results reveal an in vivo response of the mature RPE to diverse stressors that prolongs RPE cell survival at the expense of epithelial attributes and photoreceptor function. Our findings provide a rationale for mTOR pathway inhibition as a therapeutic strategy for retinal degenerative diseases involving RPE stress.

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.