engleski

Cholesterol Levels Modulate Endosomal Localization of BACE1

Objectives: Dysfunction of a cholesterol transport protein NPC1 causes Niemann-Pick type C (NPC) disease. NPC disease shares similarities with Alzheimer´s disease (AD), showing increased amyloid-beta (Abeta), APP C-terminal fragment (APP-CTF) and phosphorylated tau levels. We have recently demonstrated that increased Abeta/APP-CTF levels in NPC1-null cells are due to increased cleavage of APP by beta-secretase (BACE1). In this work we analyzed trafficking of BACE1 upon cholesterol loaded/depleted conditions in NPC1-null and wt-cells. Methods: BACE1 trafficking was analyzed by immunofluorescent confocal microscopy and endosome fractionation in BACE1-GFP stably transfected cells. Cholesterol was depleted by growing the cells in the media containing a lipid-deficient serum, by statin-treatment and by methyl-beta-cyclodextrin (MbetaC)-treatment. To load cholesterol we used two different approaches: U18666A-treatment and MbetaC-cholesterol treatment. Filipin staining and Amplex Red Cholesterol assay were used to analyze free cholesterol accumulation and levels. Results: We demonstrate that cholesterol accumulation in NPC1-deficient cells alters endocytic trafficking of BACE1 causing its sequestration within enlarged vesicular TfR-positive structures in contrast to its more perinuclear and Golgi-like staining in wt-cells. Cholesterol-depletion corrects BACE1 misstrafficking in NPC1-null cells, while cholesterol loading shows altered BACE1 trafficking even in non NPC1-deficient environment. Conclusions: Our results indicate that cholesterol-dependent trafficking defect of BACE1 in NPC1-null cells could lead to increased processing of APP by beta-secretase. Our findings support previous notion that altered endocytic trafficking may be the primary defect in AD and that increased cholesterol levels, such as in NPC disease and sporadic AD, may be the upstream effector that drives these events.

BACE1; endosomes; cholesterol; Niemann-Pick type C disease

nije evidentirano