engleski

Cholesterol accumulation in NPC fibroblasts leads to lipid raft comartmentalization of APP and C99

Background: Lipid rafts, cholesterol and sphingolipid rich membrane microdomains, have been reported to be a site of amyloid-beta (Aβ) formation. The effect of cholesterol on APP processing and Aβ production was demonstrated both in vitro and in vivo as well as in Niemann Pick type C disease (NPC), in which cholesterol accumulation in late endosomal/lysosomal compartments leads to increased C99/CTFβ and Aβ levels. The goal of this work was to elucidate whether the “cholesterol-effect” on APP/C99 and Aβ in NPC disease involves increased localization in lipid rafts. To test this we used normal human skin fibroblasts (HSF) and fibroblasts from patients with NPC disease. Methods: Lipid raft compartmentalization of endogenous APP/C99 was monitored by lipid raft fractionation in a sucrose gradient and by confocal microscopy. The fibroblast lysates (in 1% Triton X-100 MBS buffer) were centrifuged in a gradient containing 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 visualization by confocal microscopy was performed using Vybrant Alexa Fluor 488 Lipid Raft Labeling kit (Invitrogen). Results: Flotilin 1 staining showed that lipid rafts were mainly localized in fraction 4, in which the highest ratio of cholesterol and protein levels was found. We did not observe altered lipid raft compartmentalization of endogenous APP/C99 in NPC fibroblasts compared to normal HSFs. APP and C99 were mainly localized in non-raft fractions in both normal and NPC fibroblasts. In addition, confocal microscopy revealed no gross overlap between APP/C99 and flotilin1 or cholera-toxin (subunit ), further suggesting that increased C99/Aβ levels in NPC disease does not involve lipid rafts. Conclusion: Our results show that increased cholesterol levels in NPC disease do not lead to increased lipid raft compartmentalization of APP/C99, indicating that the “cholesterol-effect” on APP and Aβ formation may not be mediated through lipid rafts. Funding: NIH-FIRCA 1R03TW007335-01, Ministry of Science of the Republic of Croatia 098-0982522-2525 (S.H.).

Alzheimer’s disease; APP; cholesterol; lipid rafts; Niemann Pick type C disease; NPC1

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