Inhibition of lysosomal degradative functions in RPE cells by a retinoid component of lipofuscin.

PURPOSE: To investigate the effect of the lipofuscin component N-retinylidene-N-retinylethanolamine (A2-E) on degradative functions of lysosomes in human retinal pigment epithelial (RPE) cells and to evaluate its mechanism of action. METHODS: A2-E was coupled to low-density lipoprotein (LDL). Human RPE cell cultures were loaded with the A2-E/LDL complex, and controls were run with medium containing LDL alone. To determine whether A2-E accumulated in lysosomes, cells were fractionated in a Percoll gradient, and protein degradation was determined by metabolic labeling and measurement of the release of low-molecular-weight radioactivity. Lysosomal degradation was distinguished from nonlysosomal degradation by inclusion of NH4Cl in the medium. The metabolism of sulfated glycosaminoglycans was studied by radiosulfate incorporation in pulse-chase experiments. Intralysosomal pH was determined using a fluorescent lysosomotropic pH indicator. RESULTS: A2-E accumulated almost exclusively in the lysosomal compartment. Lysosomal protein degradation was reduced in a dose-dependent fashion in A2-E-treated cells. The selectivity of A2-E on lysosomal function was demonstrated by its lack of effect on degradation of extralysosomal protein. Lysosomal glycosaminoglycan catabolism of RPE cells was also strongly inhibited by A2-E. Lysosomal pH was increased by A2-E. CONCLUSIONS: The findings indicate that accumulation of A2-E in RPE cells interferes with lysosomal functions as exemplified by its inhibitory effect on protein and glycosaminoglycan catabolic pathways. The quaternary amine character of the A2-E apparently causes a perturbation of the acidic intralysosomal milieu, resulting in diminished hydrolase action and consequent accumulation of undegraded material. Such mechanism could be operative in retinal diseases associated with excessive lipofuscin accumulation including age-related macular degeneration.

Lipofuscin in the retina: quantitative assay for an unprecedented autofluorescent compound (pyridinium bis-retinoid, A2-E) of ocular age pigment.

The pyridinium bis-retinoid, A2-E, has been discovered as one of the major autofluorescent components of retinal pigment epithelial lipofuscin. Due to its chemical characteristics, A2-E may contribute to cellular and molecular changes leading to age-related macular degeneration. Because A2-E is the first lipofuscin component that has been identified, purified, and its structure analysed, it represents an important marker molecule for studying lipofuscin formation under various conditions. In order to investigate the role of A2-E in ageing processes of the retinal pigment epithelium, we developed an HPLC assay for this compound using single wavelength UV-absorbance detection with continuous light emission. Standard A2-E was synthetized and purified by sequential TLC. In our assay, A2-E can be detected in amounts lower than 10 pmol. The assay has been applied to quantitative determination of A2-E amounts in albino rat eyes of different age groups. Our results demonstrate that there is a marked increase of A2-E levels in older animals. The method described is the first to allow quantification of this unusual retinoid from small amounts of biological samples.

The photochemistry of human retinal lipofuscin as studied by EPR.

Fluorescent material generated in the human retina accumulates within lipofuscin (HLF) granules of the retinal pigment epithelium (RPE) during aging. We have been investigating the possible light-induced contribution of these fluorophores to various diseases including age-related macular degeneration. Our studies have shown that some of the fluorescent components of HLF are products of the reaction of retinaldehyde with ethanolamine and that synthetic mixtures of this reaction can serve as a useful model for photophysical studies. Previous research by us has demonstrated that irradiation of either natural or synthetic lipofuscin resulted in the formation of a triplet state and possibly a free radical. Here EPR studies were performed to verify the formation of that radical. The UV irradiation of either synthetic or natural human retinal lipofuscin extracts in oxygen-free methanol led to the formation of a 5,5-dimethylpyrroline-N-oxide (DMPO) spin-trapped carbon-centered radical resulting from either hydrogen atom or electron abstraction from solvent molecules. In the presence of oxygen superoxide was formed, which was observed as a DMPO adduct. It is concluded that certain components of the chloroform-soluble fluorophores of human RPE lipofuscin granules and the fluorescent reaction products of retinaldehyde and ethanolamine are photophysically similar but not the same. Electron or hydrogen abstraction from a substrate by these fluorophores in vivo and the resulting radical products may contribute to the age-related decline of RPE function and blue light damage in the retina.

Lipofuscin fluorophore inhibits lysosomal protein degradation and may cause early stages of macular degeneration.

One of the autofluorescent compounds that accumulates within the lipofuscin granules of the human retinal pigment epithelium (RPE) has now been identified as a quaternary nitrogen-containing cationic amphiphile (the bis-retinoid pyridinium salt, A2-E). Experimental evidence suggests that it may be responsible for lipofuscinogenesis in the RPE through its ability to inhibit lysosomal proteolysis. Furthermore, it may be involved in the events that trigger the changes leading to age-related macular degeneration (AMD), the leading cause of untreatable blindness in the elderly. It is suggested that if similar weakly basic nitrogenous compounds or cationic amphiphiles arise in reactions between amines and aldehydes in other tissues, a "self-assembling lysosomotropic amine" mechanism may provide an alternative explanation for lipofuscinogenesis those cell types as well.

Retinal age pigments generated by self-assembling lysosomotropic detergents.

A universal biomarker of cellular ageing in eukaryotic postmitotic cells is the appearance over time of autofluorescent lysosomal residual bodies called age pigments or lipofuscin granules. Their role in the process of cellular ageing has been debated without resolution. Neither the identity nor mechanism of formation of the fluorophores has been definitively determined. A postmitotic cell type that accumulates large quantities of age pigments is the ocular retinal pigment epithelium. We have now identified the major orange-emitting fluorophore of these pigments using fast-atom bombardment tandem mass spectrometry with collisional activation analysis. It is an amphoteric quaternary amine that arises as a Schiff base reaction product of retinaldehyde and ethanolamine. This compound should display lysosomotropic detergent behaviour which would help explain many of the age-related changes shown in this cell. These results suggest a new role for Schiff base reaction products as lysosomotropic amines in the genesis of cellular age pigments.

The autofluorescent products of lipid peroxidation may not be lipofuscin-like.

The fluorescent molecules of cellular age pigment granules (lipofuscin) are commonly thought to be end products of membrane lipid autoxidation. Lipofuscin fluorophores of the retinal pigment epithelium (RPE) appear to be derived from photoreceptor outer segment membranes. Experiments were therefore conducted to determine whether the in vitro oxidation of retinal homogenates would generate fluorophores similar to the naturally occurring lipofuscin fluorophores of the RPE. Neural retina and RPE-choroid homogenates from young (2-3 month old) albino rats were subjected to an iron-ascorbate-air pro-oxidant reaction medium, and compared to unoxidized control samples from young age-matched animals as well as senescent (24 month old) rats. In addition, neural retina and RPE-choroid homogenates from 3 month old albino rats were subjected to a 100% oxygen atmosphere to test whether the fluorescent products of autoxidation differ substantially from those generated in the pro-oxidant medium. The chloroform-soluble fluorophores of chloroform-methanol sample extracts were analyzed by corrected fluorescence spectroscopy and thin-layer chromatography (TLC). In vitro pro-oxidation of both the neural retina and the RPE from young rats produced blue-emitting fluorophores which differed from the orange- and yellow-emitting fluorophores extracted from the RPE of senescent rats. Corrected fluorescence spectroscopy of aged tissue extracts revealed vitamin A-related fluorescence (330 nm excitation maximum; 515 nm emission maximum) and a spectrally resolvable age-related fluorescence (420 nm excitation maximum; 600 nm emission maximum). Only the vitamin A-related fluorescence could be measured in the control of young samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Failure of vitamin E to protect the retina against damage resulting from bright cyclic light exposure.

Cumulative light-mediated damage to the retina over a long time period may be involved in the development of age-related retinopathies. Light is thought to produce retinal damage by initiating autoxidative reactions among the molecular components of the retina. Experiments were therefore conducted (1) to confirm that long-term differences in cyclic light intensity affect the rate of age-related photoreceptor cell loss from the retina; and (2) to determine whether the antioxidant, vitamin E, is an effective inhibitor of damage to the retina by bright cyclic light. Albino rats were fed a basal diet either supplemented with or deficient in vitamin E. Each dietary group was divided into two light-treatment groups which were exposed to 12 hr cyclic light of either 15 lux or 750 lux. After 10 and 17 weeks of treatment, retinal photoreceptor cell densities were determined for animals in each group. Vitamin E deficiency resulted in moderate decreases in photoreceptor cell densities in the dim-light groups after both 10 and 17 weeks. Rats exposed to the bright-light condition suffered a pronounced loss of photoreceptor cells by 10 weeks, and an even greater cell loss by 17 weeks. Vitamin E deficiency did not enhance the effect of bright cyclic light in reducing photoreceptor cell densities. Thus, it appears unlikely that retinal damage by cyclic light occurs via an autoxidative mechanism.

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)

Fluorophores of the human retinal pigment epithelium: separation and spectral characterization.

Ten fluorescent fractions originating from the chloroform extracts of retinal pigment epithelial (RPE) cells of human donor eyes (ages 52-98 yr) have been separated and characterized by UV-vis absorbance and corrected fluorescence spectroscopy. The semipurified fluorophores fall into four categories based upon their spectral properties: green-emitting fluorophores, a golden yellow-emitting fluorophore, yellow-green-emitting fluorophores and orange-red-emitting fluorophores. All share common absorbance peaks around 280- and 330 nm, and the orange-red-emitting fluorophores also exhibit a strong absorbance peak at 420 nm. No significant visible-emitting fluorophores were detected in the methanol-water phase of these extracts. While these fluorophores are abundant in extracts from adult-derived RPE, most of the fluorophores occur in much lower amounts in RPE extracts from human donors under 10 yr of age. Eyes from child human donors also have much less RPE lipofuscin than those from adult donors, suggesting that most of the fluorophores are lipofuscin derived. This interpretation is supported by the previous finding that all of the fluorophores from whole RPE are also present in extracts of purified lipofuscin granules. Characterization of the chromatographic and spectral properties of the chloroform-soluble fluorescent components from the human RPE provides an important tool for determining the mechanism of RPE lipofuscin fluorophore formation. The absorbance properties defined here are of significance to investigations into the photobiology of the RPE and to those using laser therapy in treatment of age-related retinal diseases.

Characterization of disease-specific brain fluorophores in ceroid-lipofuscinosis.

By isolating and identifying the molecular components of the storage material in the ceroid-lipofuscinoses, it should be possible to elucidate the metabolic basis for these diseases. Using brains of English setter dogs afflicted with a form of this disorder, the autofluorescent storage granules have been isolated and subjected to extraction with chloroform-methanol. A significant amount of autofluorescent material was solubilized by this procedure. Spectral analysis of the extracts indicated that the disease-specific fluorophores have corrected fluorescence excitation maxima at 335-340 nm and emission maxima at 530 nm. Analysis of the extracts with thin layer chromatography showed the presence of several disease-related fluorophores. In addition, the amounts of several nonfluorescent lipids appeared to be enhanced as a result of ceroid-lipofuscinosis. A cerebral cortex sample from a human subject who died with the late infantile form of ceroid-lipofuscinosis was subjected to the same analytical procedures. The chloroform-methanol extract from the human brain had a fluorescence spectrum very similar to the extracts from the brains of affected dogs. TLC analysis showed a single fluorophore from the human brain sample. This fluorophore had the same retention time as the most prominent fluorophore present in the samples from affected dogs. These findings are consistent with the possibility that the defects underlying the human and canine ceroid-lipofuscinoses are similar. The analytical procedures established in these experiments are useful in evaluating the various models of ceroid-lipofuscinosis and may ultimately serve as the basis for distinguishing among different forms of these disorders.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipofuscin autofluorescence: evidence for vitamin A involvement in the retina.

A lipofuscin-like autofluorescence develops in the degenerating photoreceptor cells of the RCS rat, a strain with inherited retinal dystrophy. In animals with normal retinas, age-related lipofuscin accumulation in the eye is restricted to the retinal pigment epithelium (RPE). Previous investigations have established that RPE lipofuscin accumulation in the normal rat retina can be reduced by dietary vitamin A deficiency. In order to determine whether the photoreceptor-derived fluorescence in the RCS rat retina is related to RPE lipofuscin fluorescence, the influence of dietary vitamin A on the fluorophore content of the RCS rat retina was studied. Vitamin A deficiency substantially reduced the autofluorescence associated with degenerating photoreceptor cells of the RCS rat retina. A specific vitamin A-dependent fluorophore was isolated from these retinas using thin-layer chromatography (TLC). The mobility of this fluorophore on TLC differs from that of the major age-dependent fluorophore isolated from the RPE of normal rats. Thus, if the vitamin A-dependent fluorophores of the photoreceptors and RPE are related, it appears that the fluorophore generated in the photoreceptor cells must undergo chemical modification once it has been taken up by the RPE. The fact that both the RPE- and photoreceptor-associated fluorophores are vitamin A-dependent suggests that such a relationship between them is likely. These experiments indicate that the RPE is somewhat different from other lipofuscin-accumulating tissues in that a major precursor of RPE lipofuscin fluorophores originates in another cell type and enters the RPE via phagocytosis.

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.

The fate of the phagosome: conversion to 'age pigment' and impact in human retinal pigment epithelium.

Phagosomes are converted to phagolysosomes and then to residual bodies (also known as lipofuscin granules or age pigment). Lipofuscin granules of retinal pigment epithelial (RPE) cells of single human eyes were isolated and analysed for enzyme content and fluorescence spectra. The granules are low in lysosomal enzymes and they fluoresce yellow-gold. Thin layer chromatography reveals several separable constituents. Lipofuscin granules accumulate with age, the largest increase occurring after the first decade of life in humans. Macular RPE cells accumulate granules more than non-macular cells.

Lipofuscin: resolution of discrepant fluorescence data.

Lipofuscin granules (age pigments) emit yellow light under ultraviolet excitation in the fluorescence microscope. The reported blue emission maximum of extracts of lipofuscin-laden cells may result from instrumental bias. The major fluorescent components that accumulate with age in these lysosomal residual bodies of human retinal pigment epithelium are yellow-emitting fluorophores. Different age-related fluorophores, which do emit blue light, are derived from other intracellular sources. A reevaluation of the connection between blue-emitting lipid peroxidation products and the age-related lipofuscin granules of classical pathology is necessary.