Subretinally-deposited amyloid beta (A beta) is a main contributor of developing age-related macular degeneration (AMD). However, the mechanism causing A beta deposition in AMD eyes is unknown. Hypercholesterolemia is a significant risk for developing AMD. Thus, we investigated the effects of cholesterol on A beta production in retinal pigment epithelial (RPE) cells in vitro and in the mouse retina in vivo. RPE cells isolated from senescent (12-month-old) C57BL/6 mice were treated with 10 mu g/ml cholesterol for 48 h. A beta amounts in culture supernatants were measured by ELISA. Activity and expression of enzymes and proteins that regulate A beta production were examined by activity assay and real time PCR. The retina of mice fed cholesterol-enriched diet was examined by transmission electron microscopy. Cholesterol significantly increased A beta production in cultured RPE cells. Activities of A beta degradation enzyme; neprilysin (NEP) and anti-amyloidogenic secretase; alpha-secretase were significantly decreased in cell lysates of cholesterol-treated RPE cells compared to non-treated cells, but there was no change in the activities of beta- or gamma-secretase. mRNA levels of NEP and alpha-secretase (ADAM 10 and ADAM17) were significantly lower in cholesterol-treated RPE cells than non-treated cells. Senescent (12-month-old) mice fed cholesterol-enriched chow developed subRPE deposits containing A beta, whereas age-matched mice fed standard rodent chow diet did not. Activities and mRNA levels of NEP and alpha-secretase were significantly lower in native RPE cells freshly isolated from cholesterol-enriched chow fed mice compared to standard rodent chow fed mice. These findings suggest that cholesterol enhances subretinal A beta accumulation by modulating the activities of enzymes degrading and processing A beta in RPE cells in senescent subjects. Crown Copyright (c) 2012 Published by Elsevier Inc. All rights reserved.