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The role of lipid rafts on app processing and amyloid-beta formation

The amyloid plaque formation in the brain is the major hallmark of Alzheimer’s disease (AD). Amyloid plaques are mainly composed of amyloid-β peptides (Aβ), products of the proteolytic processing of the amyloid-β precursor protein (APP). Retrospective epidemiological studies have recently shown that statin-treatment (cholesterol-lowering drugs) decreases the prevalence of AD. On the other hand, several studies have reported an association between mild hypercholesterolemia and the risk of AD. It has been observed that cholesterol accumulation in lysosomal storage disorder Niemann Pick type C (NPC) leads to increase Aβ, like in AD. Lipid rafts, a cholesterol and sphingolipid rich membrane microdomains, have been reported as a site of Aβ formation. Since cholesterol levels have shown to modulate APP processing and Aβ production, both in vitro and in vivo, we hypothesized that cholesterol-effect on Aβ may be mediated through lipid rafts. To test this we used CHO NPC1-null cells and CHOwt cells. Lipid rafts were isolated by two methods: using zwitterionic (CHAPSO) or nonionic (Triton X-100, Lubrol-WX) detergent. Briefly, the cell lysates containing 40% (w/v) sucrose were placed on the bottom of the centrifuge tube and were overlaid with 30% and 5% (w/v) sucrose. After centrifugation (3 h and 175, 000 x g) fractions were collected from the top, and protein and cholesterol levels were measured in each fraction. To detect lipid raft fractions we used a positive (flotilin 1) and a negative (transferrin-receptor) lipid raft marker. Lipid raft compartmentalization of endogenous APP was determined by western blotting and confocal microscopy. We did not observe altered lipid raft compartmentalization of APP or CTFs between CHOwt and M12 cells. In summary, our results showed that increased formation of Aβ upon cholesterol accumulation in NPC-null cells does not involve lipid rafts, suggesting that cholesterol-effect on Aβ may not be mediated through lipid rafts.

Alzheimer’s disease; amyloid-β; APP; cholesterol; lipid rafts; NPC1

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