Investigating the Effects of APOE Genotype on Intracellular Cholesterol and the Endolysosomal System in the Aging Mouse Brain

Individuals who possess the ε4 allele of apolipoprotein E ( APOE ) are at a significantly increased risk for developing Alzheimer’s disease (AD). However, the precise reason for this association is not fully understood. Beyond its effects on amyloid and tau, APOE also influences fundamental cellular processes in the brain, including cholesterol trafficking between cells and within the endolysosomal system, which may be a critical component of the APOE4 -associated vulnerability to AD. Here, we examined how APOE genotype, sex, and aging alter intracellular cholesterol processing and the endolysosomal system in the mouse brain. Using the novel cholesterol-binding probe D4H*-mCherry, we quantified intracellular cholesterol levels, the levels of early endosomes (Rab5), late endosomes (CD63), and lysosomes (LAMP1), and the colocalization of cholesterol with these endolysosomal compartments. This analysis was performed in the cortex, hippocampus, and entorhinal cortex of young, middle-aged, and old APOE2 , APOE3 , and APOE4 mice. Our analysis revealed region-specific changes in intracellular cholesterol and the endolysosomal system in response to aging, sex, and APOE genotype. Notably, young APOE4 mice showed reduced cholesterol within early and late endosomes, but increased lysosomal abundance, suggesting impaired cholesterol processing. These APOE4-specific effects were less apparent in older animals. These effects were strongly modified by sex, with female APOE4 mice exhibiting elevated lysosomal cholesterol in the hippocampus and entorhinal cortex at old age, indicating sex-dependent susceptibility. Together, these results reveal that APOE genotype, age, and sex interact to influence endolysosomal cholesterol homeostasis, with female APOE4 mice showing the greatest dysregulation. These findings suggest that early and region-specific endolysosomal defects may contribute to the heightened AD risk associated with APOE4 , particularly in females.