Retinal function declines with age as evidenced clinically by a variety of age-related retinopathies. One of the most obvious age- related changes in the retina is the accumulation of lipofuscin granules within the retinal pigment epithelium (RPE). These granules appear to be lysosomal storage granules filled with autofluorescent compounds. None of these compounds have yet been identified as to their chemical structure or source of origin. It is the purpose of the present proposal to identify these fluorescent compounds and to study their possible involvement in vitamin A metabolism in the RPE. Three approaches will be used: in vitro, analytical and cell biological. Unphagocytosed photoreceptor outer segments, (POS) develop lipofuscin-like fluorescence in the RCS rat. This fluorescence is affected by dietary vitamin A, as is RPE lipofuscin fluorescence in normal rats. In vitro conditions for generation of this vitamin-A related fluorescence in POS will be identified and optimized. This in vitro system will then be refined and systematically analyzed to identify the chemical reactants and mechanisms involved. Thin- layer chromatography and absorbance and corrected fluorescence spectroscopy will be used to monitor the fluorophores. RPE tissue cultures will then be used to study the conversion of the POS fluorophores into RPE lipofuscin fluorophores. To verify the chemistry deduced by these experiments, new techniques for the mass spectral analysis of thermally labile and nonvolatile compounds will be applied to the structural identification of the fluorescent molecules which have been purified. Upon identification, quantitative assays for the individual fluorophores will be established. These assays will be used to study lipofuscin composition in different regions of the retina. Immunogold labelling will be used to localize specific fluorophores in the POS, RPE lipofuscin, and any other subcellular site where these compounds may be having an influence on vitamin A metabolism. Finally, experiments into the ability of lipofuscin to act as an exchangeable reservoir for various vitamin A derivatives will be conducted utilizing radiolabelled retinoids as tracers. These studies promise to yield for the first time an unequivocal identification of the fluorophores and direct evidence on the mechanisms of lipofuscin granule accumulation in the RPE. In addition, information on the influence of lipofuscin on vitamin A metabolism in the aging retina, with possible direct correlations to clinically verified cases of age-related macular degenerations will be gained.
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