Cholesterol depletion inhibits the generation of beta-amyloid in hippocampal neurons

Abstract

The amyloid precursor protein (APP) plays a crucial role in the pathogenesis of Alzheimer's disease. During intracellular transport APP undergoes a series of proteolytic cleavages that lead to the release either of an amyloidogenic fragment called beta-amyloid (Abeta) or of a nonamyloidogenic secreted form consisting of the ectodomain of APP (APPsec). It is Abeta that accumulates in the brain lesions that are thought to cause the disease. By reducing the cellular cholesterol level of living hippocampal neurons by 70% with lovastatin and methyl-beta-cyclodextrin, we show that the formation of Abeta is completely inhibited while the generation of APPsec is unperturbed. This inhibition of Abeta formation is accompanied by increased solubility in the detergent Triton X-100 and is fully reversible by the readdition of cholesterol to previously depleted cells. Our results show that cholesterol is required for Abeta formation to occur and imply a link between cholesterol, Abeta, and Alzheimer's disease.

Figure 1

Staining of cholesterol-depleted hippocampal neurons with filipin. (A) Untreated neurons stained with filipin. (B) Neurons stained with filipin after treatment for 4 days with lovastatin/mevalonate and for 20 min with 5 mM methyl-β-cyclodextrin.

Figure 2

Cholesterol depletion does not change APP biosynthesis and secretion. Hippocampal neurons were grown for 4 days in the presence (+) or absence (−) of lovastatin/mevalonate. After infection with SFV/APP, cells were treated (+) or not (−) for 20 min with 5 mM methyl-β-cyclodextrin, and metabolically labeled for 2.5 hr. Cellular and secreted APP were recovered by immunoprecipitation.

Figure 3

Cholesterol depletion inhibits production and secretion of Aβ. Hippocampal neurons were grown for 4 days in the presence (+) or absence (−) of lovastatin/mevalonate and after infection with SFV/APP were treated with 5 mM methyl-β-cyclodextrin for the indicated times (min) and metabolically labeled for 2.5 hr. Cholesterol was added back for the indicated times (min) as a cholesterol-methyl-β-cyclodextrin inclusion complex (CD-cholesterol). (A) Aβ secreted from control and cholesterol-depleted cells (immunoprecipitated using antibody B7/6). (B) Aβ secreted from cholesterol-depleted/repleted cells. (C) Aβ secretion in cholesterol-depleted cells as compared with untreated control cells (mean of 3–11 experiments). (D) Immunoprecipitation of APP and its proteolytic fragments from cell homogenates using antibody B12/4. Fragments generated by α- and β-cleavage are indicated.

Figure 4

Cholesterol depletion reduces association of APP with detergent-insoluble low-density DIGs. Hippocampal neurons treated or not for 4 days with lovastatin/mevalonate and 20 min with 5 mM methyl-β-cyclodextrin were extracted on ice with 1% Triton X-100 immediately after pulse labeling for 20 min or after an additional chase for 100 min. After flotation in an OptiPrep step-gradient, APP was immunoprecipitated from the collected fractions.