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A. Lakkaraju, S.C. Finnemann, E. Rodriguez–Boulan; Altered Cholesterol Homeostasis in the Retinal Pigment Epithelium in the Presence of the Lipofuscin Fluorophore A2E . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5885.
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Accumulating evidence suggests that cholesterol and lipid metabolism in the RPE is important for photoreceptor health and function. Further, histopathological studies of retinas from donors affected with age–related macular degeneration (AMD) indicate that alterations in RPE cholesterol homeostasis may contribute to disease progression. Here, we investigated the regulation of cholesterol trafficking in the RPE in the presence or absence of the lipofuscin component A2E.
Determinants of cholesteryl ester (CE) metabolism and cholesterol efflux were studied in human ARPE–19 cells and in primary cultures of bovine RPE. Bodipy–labeled cholesteryl ester was used as a probe to monitor the kinetics of CE degradation. Filipin staining and thin–layer chromatography were used to assess intracellular and total cholesterol pools, respectively.
The presence of A2E in RPE endolysosomes inhibited CE metabolism. As exogenous cholesteryl esters are hydrolyzed by acid lipase in the low pH of late endosomes/lysosomes, we first asked whether A2E affected either of these two determinants. Amazingly, both late endosomal/lysosomal pH and acid lipase activity were normal in A2E–loaded cells. We then studied cholesterol trafficking in the RPE because cholesterol efflux from late endosomes is crucial for driving further CE hydrolysis in these organelles. Three independent lines of evidence showed that cholesterol transport out of late endosomes is essential for CE metabolism in RPE cells: (i) pharmacological inhibition of cholesterol efflux from late endosomes inhibited CE hydrolysis; (ii) activation of peroxisome proliferator activator gamma (which increases cholesterol efflux via increased expression of the ABC cholesterol transporters) increased CE degradation in A2E–loaded cells; and (iii) acid lipase activity was inversely proportional to the amount of free cholesterol (product) in the vicinity of the cholesteryl ester (substrate). Finally, we investigated the distribution of endogenous cholesterol in RPE cells by filipin staining and found that A2E–loaded cells had an enlarged intracellular cholesterol pool compared to control cells. Studies are now underway to characterize the expression and function of cholesterol transporters in RPE.
We show that the major lipofuscin fluorophore A2E blocks late endosomal cholesterol efflux in RPE cells and thus leads to an accumulation of cholesteryl esters. Further, our results suggest that cholesterol homeostasis is tightly regulated in the RPE and may play a role in the pathogenesis of AMD.
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