engleski

The cholesterol-effect on amyloid-β formation upon NCP1 dysfunction involves altered APP localization

Cholesterol accumulation in lysosomal storage disorder Niemann Pick type C (NPC) leads to increased levels of amyloid-β (Aβ) peptide, a causative factor of Alzheimer’ s disease. To elucidate the mechanism(s) of increased Aβ upon NPC1 dysfunction, we monitored amyloid precursor protein (APP) processing between CHO NPC1-null (M12) and CHOwt cells. As previously reported, we observed increased levels of total cholesterol, increased C-terminal APP fragment C99 (CTFβ ) and increased levels of Aβ in M12 compared to CHOwt cells. Although levels of the γ -secretase dependent formation of Aβ were significantly increased in M12 vs. CHOwt cells (by 4-fold, p<0, 01), we observed similar levels of the γ -secretase dependent formation of AICD between these cells. When C99, a direct γ -secretase substrate, was transfected into the cells a similar increase of Aβ was observed in M12 compared to CHOwt cells, indicating that the effect on Aβ upon NPC1 loss may be dependent on increased γ -secretase activity and not on high C99 levels. However, in vitro γ -secretase assay and pulse chase assay showed that γ -secretase activity is not altered in M12 cells. To further test whether the effect on Aβ in M12 cells is related to cholesterol levels we monitored Aβ production under cholesterol starved/fed conditions. Our results show that neither cholesterol depletion in M12 cells nor cholesterol overload in CHOwt cells results in substantially decreased/increased Aβ levels, respectively, indicating that the effect on Aβ upon NPC1 dysfunction is not directly related to high cholesterol levels. Moreover, we observed a markedly decreased level of APP on the cell surface in M12 vs. CHOwt cells, suggesting that less APP could be processed through the α -secretase pathway in M12 cells. Indeed, we confirmed this finding by showing that the levels of the APP cleavage products by α -secretase (sAPPα and C83) were substantially lowered in CHO NPC1-null cells. Overall, our results show that increased levels of Aβ upon cholesterol accumulation in NPC1-deficient cells are not directly related to cholesterol levels. We assume that upon NPC1 loss cholesterol may have an indirect effect on Aβ by altering APP localization and shifting APP processing through the β -secretase pathway.

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

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