A prominent feature of aging and of some inherited retinal degenerations is the accumulation of autofluorescent granules of lipofuscin in retinal pigmented epithelial (RPE) cells. Circumstantial evidence exists for an association between age-related macular degeneration (AMD) and RPE lipofuscin; however, the impact of lipofuscin accumulation on the RPE cell has not been directly tested. Since one of the constituents of RPE lipofuscin, the fluorophore A2E, exhibits structural and photodynamic properties that could be detrimental to cells, it is hypothesized that the accumulation of this fluorophore in RPE cells plays a role in the pathogenesis of AMD.
The aim of this work is to understand the mechanisms by which A2E forms in the RPE cell and to determine the impact of its accumulation on the RPE cell. By high performance liquid chromatography (HPLC) analysis, the quantities of A2E in RPE cells isolated from human eyes will be correlated with the age, race and gender of the donors. A2E in eyes from AMD donors will also be quantified. To develop a cell culture model of A2E-containing cells, cultured RPE lacking endogenous A2E will be presented with synthetic A2E in the culture media. Subsequently, the internalization of A2E by the cells will be characterized, as will the intracellular compartmentalization of A2E. To test the hypothesis that A2E, when present at critical concentrations, can have adverse effects on RPE cells, the propensity of intracellular A2E to (a) exhibit detergent-like activity, (b) damage cells as a photosensitizing agent and (c) disrupt lysosomal function, will be evaluated. It will also be determined whether A2E-mediated phototoxicity involves the generation of reactive oxidant species and an apoptotic form of RPE cell death. The ability of melanin pigment to protect against A2E-mediated phototoxicity will also be tested. Finally, in studies related to the biogenesis of A2E, we will obtain evidence for the formation of an intermediate compound (A2-PE) during A2E biosynthesis. We will also address the issue of whether A2-PE/A2E is formed in the photoreceptor outer segment membrane as opposed to the lysosomal compartment of the RPE cell. The long-term goal of these studies is to define conditions that accelerate the formation of A2E in vivo and to evaluate the role of A2E in the causation of AMD. If it can be demonstrated that the amassing of synthetic A2E by RPE cells is significant in terms of RPE cell function, treatments could be aimed at preventing its formation or destroying the formed molecule within the RPE cells.
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