engleski

Increased Abeta Formation In CHO NPC1-null Cells Is Not Directly Related To Cholesterol Levels

Background: Cholesterol accumulation in Niemann Pick type C disease (NPC) leads to increased levels of amyloid-beta (Abeta) peptide, a causative factor for Alzheimer&#8217; s disease. To elucidate the mechanism(s) of increased Abeta upon NPC1 loss of function, we monitored APP processing in CHO NPC1-null (M12) and CHOwt cells. Methods: The cells were transiently transfected with APPsw-6myc construct. APP processing was monitored by western blotting while Abeta levels were determined by ELISA assay. To deplete cholesterol the cells were grown in medium containing 10% lipid-deficient serum (LPDS) instead of 10% FBS. To raise cholesterol the cells were grown in LPDS media supplemented with LDL. gamma-Secretase activity in M12 and CHOwt cells was monitored by in vitro gamma-secretase and pulse chase assays, while fluorescence based assays were used to measure the activities of alpha- and beta-secretases. Results: We observed increased levels of cholesterol, increased C99/CTFbeta, decreased C83/CTFalpha and increased levels of Abeta40 and Abeta42 (by 4-fold, p<0, 01) in M12 cells compared to CHOwt. Surprisingly, the levels of AICD were similar between these cells. When C99, a direct gamma-secretase substrate, was transfected a similar increase of Abeta was observed in M12 compared to CHOwt cells, indicating that the effect on Abeta upon NPC1 dysfunction is not dependent on high levels of C99. We also determined that in vitro activities of all three secretases (alpha-, beta- and gamma-) are similar between M12 and CHOwt cells. Cholesterol depletion in M12 cells lowered cholesterol to the levels seen in untreated CHOwt, however, the levels of Abeta remained significantly higher than in wt cells. When cholesterol in CHOwt cells was raised to the levels seen in M12 cells, the Abeta levels were still significantly higher in M12 cells than in CHOwt. Conclusion: Our results suggest that increased levels of Abeta upon cholesterol accumulation in M12 cells are not directly related to cholesterol levels. We assume that in M12 cells the differences in Abeta levels may be due to disturbed endosoma/lysosomal trafficking in these cells.

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

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