P4-057: ABCG1/ABCG4-mediated cellular cholesterol efflux affects β-and γ-secretase activities and β-amyloid production in N2a cells

Abstract

Aβ is a product derived from sequential proteolytic cleavage of β–amyloid precursor protein (APP) by β–and γ–secretases, while cleavage of APP by α–and γ–secretases does not yield Aβ. Since β–and γ–secretases are located in cholesterol–rich lipid rafts of cell membrane and α–secretase is located in phospholipid–rich domains of cell membrane, the activities of these secretases and APP processing may be subject to the changes of cholesterol or/and phospholipids in cellular membrane. A group of ATP–binding cassette (ABC) transporters are known to transport and regulate cholesterol and phospholipids in cells, such as ABCA1, ABCG1 and ABCG4. These transporters can mediate efflux of cholesterol and phospholipids from cell membrane to high–density lipoproteins (HDL). ABCG1 and ABCG4 are primarily expressed in the brain and can form homodimer or heterodimer to function. We have studied the roles of ABCG1/ABCG4 and cholesterol in the regulation of β–and γ–secretase activities for Aβ production and demonstrated that: 1) Cholesterol assays revealed that the level of cholesterol was significantly higher in AD than in age–matched non–demented (ND) control brain samples. 2) In vitro assays demonstrated that the activities of β–and γ–secretases were significantly higher in AD than in ND brain samples, which correspond to higher levels of cholesterol and Aβ deposition in AD brain samples. 3) ABCG1 and ABCG4 are highly expressed in normal human brain samples. However, real–time Q–PCR and Western blots showed that ABCG1 and ABCG4 were down–regulated at the protein and mRNA levels, respectively, in AD compared to ND brain samples. 4) Human ABCG1 and ABCG4 genes were cloned and their overexpression in neuroblastoma N2a cells–transfected with APP increased cellular cholesterol efflux to HDL by cholesterol efflux assays and significantly decreased cellular cholesterol levels compared to empty vector–transfected cells. 5) Reduced cellular cholesterol mediated by ABCG1/ABCG4 efflux significantly decreased β–and γ–secretase activities and Aβ production in N2a cells. These results suggest that higher cholesterol levels correlate well with higher β–and γ–secretase activities and Aβ deposition in AD compared to ND brain samples and that ABCG1/ABCG4–mediated cholesterol efflux to HDL is an important factor affecting cellular cholesterol that regulates β–and γ–secretase activities for Aβ production.