Effect of Rpe65 knockout on accumulation of lipofuscin fluorophores in the retinal pigment epithelium.

PURPOSE: In all mammalian species examined to date the retinal pigment epithelium (RPE) has been found to accumulate autofluorescent lysosomal storage bodies (lipofuscin) during senescence. Substantial evidence indicates that retinoids in the RPE-retina complex play a major role in RPE lipofuscin formation. Indeed, at least one RPE lipofuscin fluorophore is derived in part from vitamin A aldehyde. However, the precise mechanisms by which retinoids modulate RPE lipofuscin accumulation have not been elucidated. In mice without a functional Rpe65 gene, isomerization of all-trans- to 11-cis-retinol is blocked. Experiments were performed to determine whether this impairment of retinoid metabolism alters RPE lipofuscin accumulation. METHODS: RPE lipofuscin fluorophore content was compared in 12- to 13-month-old Rpe65(+/+), Rpe65(+/-), and Rpe65(-/-) mice. Lipofuscin fluorophore content was determined using quantitative fluorometric measurements. RPE lipofuscin content was also estimated with quantitative ultrastructural techniques. RESULTS: In the Rpe65(-/-) mice, RPE lipofuscin fluorophore accumulation was almost abolished. In addition, a significantly reduced accumulation of lipofuscin fluorophores was also observed in the Rpe65(+/-) animals. The inability of the RPE of)Rpe65(-/-) mice to supply 11-cis-retinal from the RPE to the retinal photoreceptors was accompanied by a massive accumulation of lipid droplets in the RPE that appeared to contain substantial amounts of retinoids. CONCLUSIONS: These findings indicate that formation of RPE lipofuscin fluorophores is almost completely dependent on a normal visual cycle. The absence of retinal (both all-trans and 11-cis) in Rpe65 knockout mice drastically reduced formation of lipofuscin fluorophores in these animals. Even an excessive accumulation of retinyl fatty acid esters in the RPE of Rpe65 knockout mice did not contribute to lipofuscin accumulation.     

Reduced phagosomal content of the retinal pigment epithelium in response to retinoid deprivation.

Previous investigations have shown that lipofuscin accumulation in the retinal pigment epithelium (RPE) is reduced greatly as a consequence of vitamin A deprivation. The mechanism by which vitamin A regulates RPE lipofuscin deposition remains to be determined. It is possible that retinoids are direct precursors of this substance. Alternatively, vitamin A deficiency may reduce the uptake and processing of other potential precursors. In retinas lacking photoreceptor cells, RPE lipofuscin accumulation is decreased substantially. This finding suggested that components of phagocytosed photoreceptor outer segments may be precursors for RPE lipofuscin. The effect of vitamin A deprivation on RPE lipofuscin content therefore could be the result of reduced outer segment phagocytosis by the RPE of vitamin A-deprived animals. To evaluate this possibility, experiments were conducted to determine whether vitamin A deprivation altered the phagosomal content of the RPE. Rats were fed diets containing or lacking retinoid precursors of 11-cis retinal. Retinoic acid was included in the diets of the vitamin A-deprived animals. After both 10 and 26 weeks, the RPE phagosomal contents were determined in animals from each dietary group. Photoreceptor cell densities also were measured in these rats. At both time points, the RPE phagosomal content was lower significantly in the retinoid-deprived animals than in those fed a vitamin A precursor of the visual pigment chromophore. This reduction was not the result of photoreceptor cell death; the density of these cells was not affected significantly by dietary vitamin A. Thus, it appears that retinoid deprivation reduces the rate of photoreceptor outer segment turnover and, consequently, outer segment phagocytosis by the RPE.(ABSTRACT TRUNCATED AT 250 WORDS)     

IRBP enhances removal of 11- cis -retinaldehyde from isolated RPE membranes

Interphotoreceptor retinoid-binding protein (IRBP) greatly enhances the conversion of all- trans -retinol to 11- cis -retinal by the retinal pigment epithelium (RPE) and facilitates 11- cis -retinal release from the RPE. However, the mechanisms by which IRBP exerts these effects are not clear. Using a model system of purified bovine IRBP and isolated bovine RPE membranes, we investigated the possibility that IRBP may favor the delivery of all- trans -retinol to, or the release of 11- cis -retinal from, RPE membranes. As the interphotoreceptor space contains serum retinol-binding protein (RBP) and serum albumin in addition to IRBP, we similarly examined the exchange of retinoids between RPE membranes and human RBP or bovine serum albumin (BSA). Isolated RPE membranes were loaded with radioactive 11- cis -retinal and incubated with solutions of IRBP, RBP, BSA or with buffer alone. Membranes (pellet) and retinoid-binding protein or buffer (supernatant) were separated by centrifugation and analysed for radioactive 11- cis -retinal. Membranes incubated with buffer alone released only 4-5% of their 11- cis -retinal, while 25 microm IRBP removed 18-35%. More retinal was released as the membrane concentration was reduced. In contrast, RBP and BSA removed little retinal, even though both proteins are capable of binding this retinoid. Similar results were obtained with bovine liver membranes, consistent with the idea that the effects of IRBP do not depend on an RPE surface receptor for IRBP. IRBP was also markedly superior to RBP and BSA in removing all- trans -retinol from RPE membranes. In addition, IRBP efficiently delivered bound all- trans -retinol to membranes; however, in contrast to their differential removal of retinoids, all three binding proteins delivered comparable amounts of retinol to membranes. (This result supports the practice of using BSA as a retinoid carrier in in vitro experimental systems). We conclude that, whereas IRBP shares with other retinoid-binding proteins the ability to deliver retinol to membranes, IRBP is unique in its capacity to remove 11- cis -retinal from membranes. This may be the feature of IRBP that drives the vitamin A cycle to efficiently produce 11- cis -retinal.     

Membrane receptors for retinol-binding protein in cultured human retinal pigment epithelium

The retinal pigment epithelium (RPE) is responsible for transport of retinol from the choroidal circulation to the photoreceptors. In the intact eye, this process is mediated by membrane receptors for plasma retinol binding protein (RBP) distributed basolaterally on the RPE cells. We have shown that cultured human RPE expresses this receptor. A binding curve exhibiting saturation was generated by incubating enzymatically detached epithelial sheets with increasing concentrations of 125I-labelled RBP. 125I-RBP binding experiments also show that the receptor is expressed at a high level in first passage subcultures, suggesting de novo synthesis, and that basally oriented receptors predominate over those associated with the apical surface, reflecting the polarization characteristic of RPE in vivo. Cultured RPE can internalize 3H-retinol carried by RBP, resulting in synthesis of labelled retinyl palmitate. Production of labelled retinyl ester is competitively inhibited when incubations include an excess of holo-RBP containing non-radioactive retinol. These results indicate that RBP not only binds to the receptor specifically, but also that this interaction is functional, effecting uptake of retinol by the RPE cells. The expression of this property of differentiated RPE favors the use of cultured RPE as a model system for studying vitamin A transport and metabolism.     

Influence of dietary vitamin A on autofluorescence of leupeptin-induced inclusions in the retinal pigment epithelium.

Substantial evidence indicates that the major precursors for retinal pigment epithelial (RPE) lipofuscin are molecular components of the photoreceptor outer segments. Previous experiments have demonstrated that the retinoids required for transduction by the photoreceptors promote RPE lipofuscin fluorophore formation. Animals deficient in these retinoids accumulate lipofuscin in the RPE at greatly reduced rates. The protein components of the photoreceptor outer segments also appear to be involved in the formation of RPE lipofuscin fluorophores. When degradation of phagocytosed photoreceptor outer segment proteins by the RPE is blocked, this tissue rapidly becomes engorged with phagosome-derived inclusions that have fluorescence properties similar to lipofuscin. Experiments were conducted to determine whether the development of lipofuscin-like fluorescence in these inclusions was dependent on the availability of retinoids. Rats were fed diets containing vitamin A either in the form of retinyl palmitate (+A), which can be metabolically converted into the retinoids involved in vision, or retinoic acid (-A), which does not support visual function. After the retinas of the -A animals had been depleted of retinoids involved in vision, animals from both groups were given intraocular injections of the protease inhibitor leupeptin. Two days later, the amounts of lipofuscin-like autofluorescence from the RPEs of rats in each group were determined. Leupeptin treatment produced an increase in this fluorescence in RPEs of the +A animals, but not in the RPEs of the rats fed the -A diet, despite the fact that phagosome-like inclusions accumulated in the RPE in both dietary groups. This finding suggests that retinoids involved in the visual process are probably directly involved in RPE lipofuscin fluorophore formation.     

Observation of A2E oxidation products in human retinal lipofuscin

Several retinal dystrophies are associated with the accumulation of lipofuscin in the retinal pigment epithelium (RPE). The only structurally characterized component of human retinal lipofuscin is the bis-retinoid pyridinium compound A2E. We report here on the observation of a monooxygenated product of A2E in the organic soluble portion of human retinal lipofuscin and in the organic extract of bovine RPE cells that have been fed A2E and irradiated. Liquid chromatography mass spectrometry confirms that the products are identical. This is the first observation of a photoproduct of A2E in human retinal lipofuscin.     

Uptake, processing and release of retinoids by cultured human retinal pigment epithelium

Upon absorption of a photon, the 11-cis retinaldehyde chromophore of rhodopsin is isomerized and reduced to all-trans retinol (vitamin A) in the photoreceptor outer segments, whereupon it leaves the photoreceptors, and moves to the retinal pigment epithelium (RPE). To clarify the function of the RPE in the regeneration of 11-cis retinaldehyde, we delivered all-trans retinol to monolayer cultures of human RPE. During delivery the retinol was associated with its putative natural carrier, interphotoreceptor retinoid binding protein (IRBP). IRBP has been proposed as a carrier protein involved in the exchange of retinoids between the photoreceptors and the retinal pigment epithelium. The retinoid composition of RPE cells and culture medium was analyzed by HPLC following several incubation periods. The RPE monolayer was found to process all-trans retinol into two distinct end-products: all-trans retinyl palmitate, which remained within the RPE monolayer: and 11-cis retinaldehyde which was released into the culture medium. These results demonstrate retinoid isomerase, retinol oxidoreductase and retinyl ester synthetase activity in human RPE cells cultured under the appropriate conditions. They show that IRBP can serve as a carrier of retinol through an aqueous medium to the RPE, and they illustrate that the visual cycle can be studied in vitro.     

A2E, a byproduct of the visual cycle

A substantial portion of the lipofuscin that accumulates with age and in some retinal disorders in retinal pigment epithelial (RPE) cells, forms as a consequence of light-related vitamin A recycling. Major constituents of RPE lipofuscin are the di-retinal conjugate A2E and its photoisomers. That the accretion of A2E has consequences for the cell, with the adverse effects of A2E being attributable to its amphiphilic structure and its photoreactivity, is consistent with evidence of an association between atrophic age-related macular degeneration (AMD) and excessive lipofuscin accumulation.     

Screening genes of the visual cycle RGR, RBP1 and RBP3 identifies rare sequence variations

The visual cycle is essential for vision and several genes encoding proteins of the cycle have been found mutated in various forms of inherited retinal dystrophy. We screened 3 genes of the visual cycle. RGR, encoding the retinal pigment epithelium (RPE) G protein-coupled receptor acting in vitro as a photoisomerase; RBP1, encoding the ubiquitous cellular retinol binding protein carrying intracellular all-trans retinoids; RBP3, encoding the interphotoreceptor retinoid binding protein, a retinal-specific protein which shuttles all-trans retinol from photoreceptors to RPE and 11-cis retinal from RPE to photoreceptors. We used denaturing high performance liquid chromatography (D-HPLC) and direct sequencing to screen 216 patients (134 with autosomal recessive or sporadic retinitis pigmentosa (RP) and 82 with other retinal dystrophies) for RBP1 and RBP3, and 331 patients for RGR (79 cases with autosomal dominant RP and 36 RP cases with undetermined inheritance were added to the 216 previous patients). Several variants were found in the 3 genes, including unique amino acid changes, but none of them showed evidence of pathogenicity. It is likely that mutations in RGR, RBP3, and possibly RBP1 occur rarely in inherited retinal dystrophies.     

Retinal light damage reduces autofluorescent pigment deposition in the retinal pigment epithelium

Lipofuscin in the retinal pigment epithelium (RPE) is thought to be derived from phagocytosed photoreceptor outer segment disc membranes. Based on this hypothesis, one would predict that the rate of lipofuscin deposition in the RPE would be proportional to the density of photoreceptor cells in the retina. In previous studies it was demonstrated that specific loss of photoreceptor cells due to a genetic defect resulted in a substantial decrease in the rate of age-related lipofuscin accumulation in the RPE. In order to confirm that this decreased RPE lipofuscin deposition was directly related to reduced photoreceptor cell density, experiments were conducted to determine whether light-induced photoreceptor cell destruction affected RPE lipofuscin content. The effects of retinal light damage on RPE autofluorescent pigment accumulation resulting from both normal aging and vitamin E deficiency were examined. Starting immediately after weaning, albino Fisher 344 rats were fed diets either containing or lacking vitamin E. All animals were maintained on a 12 hr/12 hr light/dark cycle. During the light phases of the cycles, the cage illuminance for one-half the animals in each dietary group was 750 lux, while the remaining rats were exposed to a light level of 15 lux. Illumination was provided by 40 watt cool-white fluorescent lamps. After 17 weeks, rats in both dietary groups that were maintained under the higher light intensity had substantially reduced photoreceptor cell densities relative to animals in the same dietary group maintained under dim light conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lack of fundus autofluorescence to 488 nanometers from childhood on in patients with early-onset severe retinal dystrophy associated with mutations in RPE65

PURPOSE: Fundus autofluorescence is due to accumulation of lipofuscin in the retinal pigment epithelium (RPE) resulting from incomplete digestion of N-retinylidene-phosphatidyl-ethanolamine from shed photoreceptor outer segment discs. Alteration in autofluorescence reflects changes in lipofuscin content of the RPE. Mutations on both alleles of RPE65 result in absent or largely decreased formation of rhodopsin, due to a defect in all-trans retinol isomerization in the RPE. Autofluorescence could therefore be altered. This study was conducted to evaluate fundus autofluorescence in patients with early-onset severe retinal dystrophy (EOSRD, or early-onset rod-cone dystrophy) associated with mutations on both alleles of RPE65. DESIGN: Case series. PARTICIPANTS AND CONTROLS: Ten 10- to 55-year-old patients with EOSRD and compound heterozygous or homozygous mutations in RPE65. For comparison, 6 heterozygous parents and 2 patients with other forms of EOSRD were examined. METHODS: Participants underwent, in addition to standard clinical and electrophysiological examination, autofluorescence imaging using a confocal scanning laser ophthalmoscope. Three of the patients were also examined by optical coherence tomography (OCT) to evaluate the status of retinal degeneration. Mutations in 7 patients have been reported previously; the other patients were investigated by polymerase chain reaction-single-strand conformation polymorphism and direct sequencing for mutations in RPE65 and lecithin retinol acyltransferase (LRAT). MAIN OUTCOME MEASURES: Fundus autofluorescence and OCT. RESULTS: Absent or minimal autofluorescence was found in all patients with compound heterozygous or homozygous RPE65 mutations. Autofluorescence was normal in the heterozygous parents. Autofluorescence was present in 2 children with EOSRD not associated with mutations in RPE65 or LRAT, another gene involved in retinol recycling. Optical coherence tomography in younger patients revealed an intraretinal appearance similar to that of their healthy, heterozygous parents. CONCLUSIONS: Lack of autofluorescence in patients with EOSRD associated with mutations in RPE65 is in accordance with the biochemical defect and can be used as a clinical marker of this genotype. Optical coherence tomography results in younger patients would indicate still viable photoreceptors despite the absence of autofluorescence.     

The bisretinoids of retinal pigment epithelium

The retina exhibits an inherent autofluorescence that is imaged ophthalmoscopically as fundus autofluorescence. In clinical settings, fundus autofluorescence examination aids in the diagnosis and follow-up of many retinal disorders. Fundus autofluorescence originates from the complex mixture of bisretinoid fluorophores that are amassed by retinal pigment epithelial (RPE) cells as lipofuscin. Unlike the lipofuscin found in other cell-types, this material does not form as a result of oxidative stress. Rather, the formation is attributable to non-enzymatic reactions of vitamin A aldehyde in photoreceptor cells; transfer to RPE occurs upon phagocytosis of photoreceptor outer segments. These fluorescent pigments accumulate even in healthy photoreceptor cells and are generated as a consequence of the light capturing function of the cells. Nevertheless, the formation of this material is accelerated in some retinal disorders including recessive Stargardt disease and ELOVL4-related retinal degeneration. As such, these bisretinoid side-products are implicated in the disease processes that threaten vision. In this article, we review our current understanding of the composition of RPE lipofuscin, the structural characteristics of the various bisretinoids, their related spectroscopic features and the biosynthetic pathways by which they form. We will revisit factors known to influence the extent of the accumulation and therapeutic strategies being used to limit bisretinoid formation. Given their origin from vitamin A aldehyde, an isomer of the visual pigment chromophore, it is not surprising that the bisretinoids of retina are light sensitive molecules. Accordingly, we will discuss recent findings that implicate the photodegradation of bisretinoid in the etiology of age-related macular degeneration.     

beta-Carotene conversion into vitamin A in human retinal pigment epithelial cells

PURPOSE: Vitamin A is essential for vision. The key step in the vitamin A biosynthetic pathway is the oxidative cleavage of beta-carotene into retinal by the enzyme beta,beta-carotene-15,15'-monooxygenase (BCO). The purpose of the study was to investigate beta-carotene metabolism and its effects on BCO expression in the human retinal pigment epithelial (RPE) cell line D407. METHODS: BCO mRNA and protein expression were analyzed by real-time quantitative PCR and Western blot analysis, respectively. BCO activity was assayed in protein extracts isolated from D407 cells. The conversion of beta-carotene to retinoids was determined by measuring retinol levels in D407 cells on beta-carotene supplementation. RESULTS: By RT-PCR, BCO mRNA was detected in D407 cells, bovine RPE, and retina. Western blot analyses revealed the presence of BCO at the protein level in D407 cells. Exogenous beta-carotene application to D407 cells resulted in a concentration (75% at 0.5 microM and 96% at 5 microM; P < 0.05)- and time (127% at 2 hours and 97% at 4 hours in 5 microM beta-carotene, P < 0.05)-dependent upregulation of BCO mRNA expression. Application of exogenous retinoic acid downregulated BCO mRNA levels at higher concentrations (1 microM; -96%, P < 0.0005) and upregulated it at a lower concentration (0.01 microM; 399%, P < 0.005). The RAR-a-specific antagonist upregulated BCO expression by sixfold (P < 0.005). Tests for enzymatic activity demonstrated that the mRNA upregulation resulted in enzymatically active BCO protein (7.3 ng all-trans-retinal/h per milligram of protein). Furthermore, D407 cells took up beta-carotene in a time-dependent manner and converted it to retinol. CONCLUSIONS: The results suggest that BCO is expressed in the RPE and that beta-carotene can be metabolized into retinol. beta-Carotene cleavage in the RPE may be an alternative pathway that would ensure the retinoid supply of photoreceptor cells.     

Vitamin A metabolism in the retinal pigment epithelium: genes,mutations, and diseases

Mutations in the genes necessary for the metabolism of vitamin A (all-trans retinol) and cycling of retinoids between the photoreceptors and retinal pigment epithelium (RPE) (the visual cycle) have recently emerged as an important class of genetic defects responsible for retinal dystrophies and dysfunctions. Research into the causes and treatment of diseases resulting from defects in retinal vitamin A metabolism is currently the subject of intense interest, since disorders affecting the RPE are, in principle, more accessible to therapeutic intervention than those affecting the proteins of photoreceptor cells. This chapter presents an overview of the visual cycle, as well as the function of the RPE genes involved in the conversion of vitamin A to 11-cis retinal, the chromophore of the visual pigments. The identification of disease-causing mutations in this group of genes is described as well as the associated phenotypes that range from stationary night blindness to childhood-onset severe visual handicap. Consideration is also given to alternative genetic paradigms potentially relevant to defects in vitamin A metabolism, including a discussion of the relationship of this pathway to age-related macular degeneration, a non-Mendelian disease of late onset. Finally, progress and prospects for targeted therapeutic intervention in vitamin A metabolism are presented, including retinoid and gene replacement therapy. On the basis of early successes in animal models, and plans underway for Phase I/II clinical trials, it is hoped that the near future will bring effective therapies for many retinal dystrophy patients with defects in vitamin A metabolism.     

Blue light-absorbing intraocular lens and retinal pigment epithelium protection in vitro

PURPOSE: To compare the Alcon AcrySof Natural (SN60AT) and AcrySof (SA60AT), the AMO Sensar (AR40e) and ClariFlex, and the Pfizer CeeOn Edge 911A intraocular lenses (IOLs) as to their ability to protect retinal pigment epithelial (RPE) cells from light damage mediated by the lipofuscin fluorophore A2E. SETTING: Department of Ophthalmology, Columbia University, New York, New York, USA. METHODS: Cultured human RPE cells (ARPE-19 cell line) that had accumulated A2E were exposed to blue (430 nm +/- 30), green (550 +/- 10 nm), or white (390 to 750 nm) light with and without an IOL in the light path. RESULTS: The blue light-absorbing AcrySof Natural IOL was associated with significant reduction (78% to 82%; P<.01) in the death of A2E-laden RPE that were exposed to blue, white, and green light. The decrease in the incidence of cell death was greater in magnitude than would be expected from the amount of light that was absorbed by the IOL. The considerably smaller declines in cell death observed with the AcrySof, Sensar, ClariFlex, and CeeOn Edge IOLs were likely due to nonspecific reductions in light transmittance. CONCLUSIONS: By absorbing blue light, the AcrySof Natural IOL shields RPE cells that have accumulated the aging lipofuscin fluorophore A2E from the damaging effects of light. A long-term population-based clinical trial would determine whether a blue light-absorbing IOL can reduce the risk for or progression of age-related macular degeneration.     

Mechanism of the inhibition of lysosomal functions in the retinal pigment epithelium by lipofuscin retinoid component A2-E

BACKGROUND: Excessive accumulation of lipofuscin in the retinal pigment epithelium with age and in various hereditary and degenerative retinal diseases, is of pathogenetic significance. We have shown that the major lipofuscin fluorophor A2-E (N-retinylidene-N-retinylethanolamine) affects the lysosomal degradation of human RPE cells and damages the cellular metabolism by phototoxic properties. Herein we sought to determine mechanisms for the inhibitory effect on lysosomal function apart from pH elevation. METHODS: Potter-Elvejem homogenates of RPE cells were used to measure the activity of 24 lysosomal enzymes before and after incubation with A2-E. RESULTS: This is the first time that RPE cells have been screened for a large spectrum of lysosomal hydrolases including proteases, lipidases, glycosidases, nucleases, sulfatases and phosphatases. The activities of these hydrolases were readily detectable in cultured RPE cells. Incubation of RPE cell homogenates even with high A2-E concentrations (up to 10 microM) did not affect the activity of isolated lysosomal enzymes. CONCLUSIONS: The results suggest that a direct inhibition of lysosomal enzyme activity would not explain the inhibitory effect on lysosomal degradation. A2-E increases the acidic intralysosomal pH thereby probably hindering pH-dependent lysosomal enzymatic activities. The understanding of the inhibitory effects of A2-E on RPE cell metabolism may contribute to new approaches for treatment of retinal diseases with excessive lipofuscin accumulation such as ARMD or M. Stargardt.     

Screening genes of the visual cycle RGR,RBP1 and RBP3 identifies rare sequence variations

The visual cycle is essential for vision and several genes encoding proteins of the cycle have been found mutated in various forms of inherited retinal dystrophy. We screened 3 genes of the visual cycle. RGR, encoding the retinal pigment epithelium (RPE) G protein-coupled receptor acting in vitro as a photoisomerase; RBP1, encoding the ubiquitous cellular retinol binding protein carrying intracellular all-trans retinoids; RBP3, encoding the interphotoreceptor retinoid binding protein, a retinal-specific protein which shuttles all-trans retinol from photoreceptors to RPE and 11-cis retinal from RPE to photoreceptors. We used denaturing high performance liquid chromatography (D-HPLC) and direct sequencing to screen 216 patients (134 with autosomal recessive or sporadic retinitis pigmentosa (RP) and 82 with other retinal dystrophies) for RBP1 and RBP3, and 331 patients for RGR (79 cases with autosomal dominant RP and 36 RP cases with undetermined inheritance were added to the 216 previous patients). Several variants were found in the 3 genes, including unique amino acid changes, but none of them showed evidence of pathogenicity. It is likely that mutations in RGR, RBP3, and possibly RBP1 occur rarely in inherited retinal dystrophies.     

Influence of early photoreceptor degeneration on lipofuscin in the retinal pigment epithelium

Experiments were conducted to evaluate the role played by photoreceptor cells in the accumulation of age pigment, or lipofuscin, in the retinal pigment epithelium (RPE). The age-related accumulation of RPE lipofuscin was compared between rats with hereditary photoreceptor degeneration (RDY) and congenic rats with normal retinas. In the RDY animals, the age-related increase in RPE lipofuscin content was substantially less than in normal controls. This suggests that the photoreceptor cells play a significant role in RPE lipofuscin deposition, although they may not be the sole contributors to RPE lipofuscin formation. Evidence that outer-segment components may be converted into lipofuscin fluorophores was provided by the discovery that in young RDY rats, fragments of outer segments from degenerating photoreceptor cells had fluorescence properties similar to those of RPE lipofuscin. Chloroform-methanol extraction of retina-RPE tissue from young normal and dystrophic rats, and analysis of the chloroform fractions by thin-layer chromatography, revealed three distinct fluorescent components associated with the lipofuscin-like fluorescence of the outer-segment fragments in the RDY rats.     

A2E, a lipofuscin fluorophore, in human retinal pigmented epithelial cells in culture.

PURPOSE: To study A2E, a component of retinal pigmented epithelial (RPE) cell lipofuscin, after its internalization by cultured human RPE cells. METHODS: A2E was synthesized and incubated with an adult RPE cell line devoid of native lipofuscin. To investigate the cellular compartmentalization of A2E, cells were incubated simultaneously with A2E and a fluorescent acidotropic probe, (Lysotracker Red DND-99; Molecular Probes, Eugene, OR). Plasma membrane integrity was evaluated by assaying for leakage of the cytoplasmic enzyme lactate dehydrogenase (LDH), by fluorescence nuclear staining with a membrane-impermeant dye and by morphologic criteria. The emission spectrum of internalized A2E was also determined. The levels of A2E accumulated by the cultured cells were quantified by high-performance liquid chromatography and compared with amounts present in RPE isolated from human eyes. RESULTS: Internalization of A2E by the RPE cells was evidenced by the acquisition of intracellular granules detectable by fluorescence confocal imaging. Internalized A2E had an emission maxima of 565 to 570 nm. The levels of A2E accumulating in cells incubated with 10 to 25 microM A2E were comparable to the amounts of A2E present in equal numbers of RPE cells harvested from human eyes. Colocalization of A2E and the Lysotracker probe revealed a preferential accumulation in acidic organelles. The elevated LDH levels that were measured after exposure to 50 and 100 microM A2E were attributable to membrane damage in a subpopulation of the A2E-accumulating cells, determined by fluorescence nuclear labeling. CONCLUSIONS: Internalized A2E has an affinity for acidic organelles. The membrane damage exhibited by A2E-accumulating RPE is dependent on the concentration of A2E and reflects the ability of this amphiphilic compound to exert detergent-like effects.     

Impact of lipofuscin on the retinal pigment epithelium: electroretinographic evaluation of a protease inhibition model

With aging, the retinal pigment epithelium (RPE) becomes increasingly congested with residual debris called lipofuscin. Little is known about the impact of lipofuscin on retinal function, and this was addressed in the present study by examining the influence of RPE debris on electroretinographic (ERG) parameters utilizing an experimental model of lipofuscin accumulation. Pigmented rats were injected intravitreally with the protease inhibitor leupeptin, and examined 1 week later by electroretinogram (ERG) recording and light and electron microscopy. Relative to vehicle-injected controls, leupeptin-treated retinas showed abundant accumulation throughout the RPE cytoplasm of inclusions that resembled lipofuscin. RPE cells filled with this debris showed a marked increase in height and a displacement of melanin from their apical border. Morphological changes in the RPE had no influence on retinal function since ERG threshold, a- and b-wave maximum amplitude, latency and implicit time were not significantly different between leupeptin-treated eyes and controls. Furthermore, leupeptin-induced RPE inclusions did not alter either the rate or extent of ERG dark adaptation. These findings suggest that filling of the RPE cytoplasm with residual debris is not in itself likely to be the cause of functional alterations in the aging eye.Supported in part by the National Institutes of Health grants EY-04554 and EY-02520, and Research to Prevent Blindness, Inc.