engleski

Increased Formation Of Amyloid-beta Peptide In CHO NPC1-null Cells Does Not Involve Lipid Rafts

Background: Lipid rafts, a cholesterol and sphingolipid rich membrane microdomains, have been reported as a site of amyloid-beta (Abeta) formation. Since cholesterol levels have shown to modulate APP processing and Abeta production, both in vitro and in vivo, we hypothesized that cholesterol-effect on Abeta may be mediated through lipid rafts. To test this we used CHO NPC1-null (M12) and, in parallel, CHOwt cells. It has been previously shown that dysfunction of NPC1 protein, that causes Niemann Pick type C disease (NPC), leads to increased cholesterol levels, increased C99/CTFbeta levels and increased levels of Abeta. Methods: The M12 and CHOwt cells were stably transfected with APPsw-6myc construct. Lipid rafts were isolated by two methods: using zwitterionic (0, 5% CHAPSO) or nonionic (0, 5% 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 (DC Protein Assay, BioRad) and cholesterol levels (AmplexRed cholesterol assay, Molecular Probes) 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 APP was monitored by western blotting, while Abeta levels were determined using ELISA Abeta assay (BioSource International, Inc.). Results: Flotilin 1 staining showed that lipid rafts were mainly isolated in fraction 4. Using either CHAPSO or Lubrol-WX detergent to isolate lipid rafts, we did not observe altered lipid raft compartmentalization of APP between CHOwt and M12 cells. APP was localized mainly in non-lipid raft fractions. In addition, we found similar levels of Abeta  peptide in lipid raft fractions of CHOwt and M12 cells. Conclusion: Our results showed that increased formation of Abeta upon cholesterol accumulation in CHO NPC1-null cells does not involve lipid rafts, suggesting that cholesterol-effect on Abeta may not be mediated through lipid rafts. This work was funded by the grants: NIH-FIRCA 1R03TW007335-01 (to A.G.) and Ministry of Science of the Republic of Croatia 098-0982522-2525 (to S.H.).

Alzheimer's disease; cholesterol; APP protein; Abeta peptide; lipid rafts

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