Isolation of intact lysosomes from human RPE cells and effects of A2-E on the integrity of the lysosomal and other cellular membranes
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Background. Accumulation of lipofuscin in RPE cells occurs with age and in association with various retinal diseases. Lipofuscin and its major retinoid compound and fluorophore A2-E interfere with the cellular metabolism of RPE cells in various ways. One of these mechanisms is thought to be related to detergent properties of A2-E.
Methods. We isolated pure and intact lysosomes from RPE cell cultures and investigated detergent-like effects of the lipofuscin compound A2-E on the integrity of lysosomal membrane and other cellular membranes, using latency measurements. A postnuclear supernatant prepared from cultured human RPE cells was used to isolate intact lysosomes by fractionation of cellular organelles in two sequential gradients. Destabilization of the lysosomal membrane was tested by incubating the purified lysosomal fraction in the presence of A2-E and subsequent measurement of the latency of the lysosomal luminal marker β-hexosaminidase. In order to compare the effect of A2-E on other cellular membranes, latencies of the specific markers succinate dehydrogenase and UDP-galactosyltransferase were assessed using partially purified mitochondria and microsomes. Intactness of the plasma membrane was tested by including A2-E in the culture medium before leakage of lactate dehydrogenase into the medium was determined.
Results. A more than 100-fold purification of the lysosomal fraction was achieved. Except for a minor activity of the mitochondrial marker, no contamination with other cell fractions was observed. Intactness of the purified lysosomes was well preserved upon incubations in isotonic media providing the base for investigations on a possible detergent-like action of A2-E on lysosomal integrity. At concentrations above 2 µM A2-E, progressive leakage of the lysosomal marker was observed. In comparison, leakage of the mitochondrial marker was induced at significantly lower concentrations (1µM), whereas ER/Golgi membranes and the plasma membrane were relatively insensitive to a detergent effect of the retinoid. The described methodology to obtain highly purified and intact lysosomes from RPE cells provides a suitable tool for investigations on compounds affecting lysosomal structure. A2-E was shown to cause desintegration of the lysosomal membrane at relatively low concentrations, which may implicate an involvement of such mechanism in triggering lipofuscin-induced dysfunction of RPE in vivo. Secondary to disintegration of the lysosomal membrane, damage to mitochondria might be an additional pathogenic mechanism.
Conclusions. Our data provide evidence for surfactant-like properties of A2-E on biomembranes which might be operative in retinal diseases associated with excessive lipofuscin-accumulation, such as age-related macular degeneration.
KeywordsRetinoid Succinate Dehydrogenase Macular Degeneration Lysosomal Membrane Retinal Disease
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