[Introduction of the lipofuscin-fluorophor A2E into the lysosomal compartment of human retinal pigment epithelial cells by coupling to LDL particles. An in vitro model of retinal pigment epithelium cell aging].
OBJECTIVE
Lipofuscin accumulates with age and in association with various retinal 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 (A2-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 harvested by trypsinization and disrupted by nitrogen cavitation. After ultra-centrifugation, the postnuclear supernatant was fractionated on a self-generating gradient and fractions were analyzed by measuring marker enzyme activities of various cellular compartments.


RESULTS
A2-Eaccumulated almost exclusively in the lysosomal compartment, as indicated by the identical peaks of the marker enzyme ss-hexosaminidase and the relative fluorescence of A2-E. Only a small amount of A2-E appeared to associate with the cell membrane, as shown by a minor peak of A2-E corresponding to the distribution of phosphodiesterase activity. The lysosomal marker enzyme was not present in the cytosolic fraction.


CONCLUSIONS
The feeding of A2-E/LDL complexes to cultured RPE-cells proved to be highly effective in specific loading of the lysosomal compartment, providing a suitable in vitro cell culture model for RPE aging and the investigation of A2-E-effects on lysosomal functions in RPE cells. Such a model may contribute to the understanding of the pathogenesis of degenerative diseases of the outer retina associated with excessive lipofuscin accumulation, including age-related macular degeneration, Stargardt's disease and Best's disease.
Holz
F G
FG
Universitäts-Augenklinik Heidelberg.
Schütt
F
F
Kopitz
J
J
Völcker
H E
HE
D002478:Cells, Cultured; D016922:Cellular Senescence / Q000502:physiology*; D005456:Fluorescent Dyes; D006801:Humans; D008062:Lipofuscin / Q000378:metabolism*; D008077:Lipoproteins, LDL; D008247:Lysosomes / Q000378:metabolism* / Q000648:ultrastructure; D008856:Microscopy, Fluorescence; D010857:Pigment Epithelium of Eye / Q000166:cytology*; D012176:Retinoids / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism / Q000648:ultrastructure
D005456:Fluorescent Dyes; D008062:Lipofuscin; D008077:Lipoproteins, LDL; D012176:Retinoids