Coupling of the lipofuscin fluorophor (A2-E) to LDL particles allows specific loading of the lysosomal compartment in cultured human RPE cells

Purpose: Lipofuscin accumulates with age and in association with various re tinal diseases. To investigate cellular effects of lipofuscin components in an in vitro RPE cell model, specific loading of the lysosomal compartment is required. Herein a major lipofuscin fluorophor was complexed to LDL and the subsequent subcellular localization of the retinoid was examined. Methods: The lipofuscin component N-retinylidene-N-retinylethanolamine (AZ- E) was synthesized and coupled to LDL. Human RPE cell cultures were loaded with the A2-E/LDL complex over 4 weeks. Thereafter, RPE cells were harveste d by trypsinization and disrupted by nitrogen cavitation. After ultra-centr ifugation, the postnuclear supernatant was fractionated on a self-generatin g gradient and fractions were analyzed by measuring marker enzyme activitie s of various cellular compartments. Results: A2-E accumulated almost exclusively in the lysosomal compartment, as indicated by the identical peaks of the marker enzyme beta-hexosaminidas e and the relative fluorescence of AZ-E. Only a small amount of A2-E appear ed to associate with the cell membrane, as shown by a minor peak of A2-E co rresponding to the distribution of phosphodiesterase activity. The lysosoma l marker enzyme was not present in the cytosolic fraction. Conclusions: The feeding of AZ-VLDL complexes to cultured RPE-cells proved to be highly effective in specific loading of the lysosomal compartment, pr oviding a suitable in vitro cell culture model for RPE aging and the invest igation of AZ-E-effects on lysosomal functions in RPE cells. Such a model m ay contribute to the understanding of the pathogenesis of degenerative dise ases of the outer retina associated with excessive lipofuscin accumulation, including age-related macular degeneration, Stargardt's disease and Best's disease.