Elevated cholesterol in APOE4 astrocytes drives mitochondrial cristae collapse and ATP synthase dysfunction

Cholesterol imbalance is a hallmark of major human diseases, including atherosclerosis and Alzheimer’s disease, both of which are also associated with mitochondrial dysfunction, yet the mechanistic links between cholesterol and mitochondria remain poorly understood. Here we show that elevated intracellular cholesterol in murine astrocytes expressing the Alzheimer’s disease risk variant APOE4 disrupts the inner mitochondrial membrane, manifesting as sparse, truncated cristae alongside an excess of cristae junction complexes. These structural abnormalities are accompanied by loss of respiratory chain complexes I and IV, and reduced respiration; nevertheless, low proton leak and reverse ATP synthase activity combine to generate an elevated mitochondrial membrane potential. Strikingly, APOE4 astrocytes are hypersensitive to the ATP synthase inhibitor oligomycin, demonstrating a profound dysfunction of the enzyme, and cholesterol sequestration with methyl-β-cyclodextrin abrogates this toxicity, establishing elevated cholesterol as its proximate cause. Another method of targeting ATP synthase, epicatechin, which prevents the enzyme operating in reverse, attenuated both cholesterol- and respiratory chain inhibitor-induced cell death. Reciprocally, reducing intracellular cholesterol via nutrient restriction restored cristae architecture and partially rescued respiratory chain complex abundance. These findings identify mitochondrial cholesterol as a critical determinant of cristae architecture and ATP synthase function and suggest that cholesterol-driven mitochondrial dysfunction may be a unifying feature of cholesterol-related disorders from neurodegeneration to atherosclerosis.