APOE4 differentially alters lipid composition in extracellular vesicles from hiPSC-derived neurons of Alzheimer's disease patients.

Background: The Apolipoprotein E4 (APOE4) is the most significant genetic risk factor for late-onset Alzheimer's disease (AD), but the cellular mechanisms by which it increases vulnerability remain poorly understood. Lipids play a crucial role in membrane organization, vesicle trafficking, and inflammatory signaling in the brain. Increasing evidence suggests that changes in lipid metabolism might serve as a connection between genetic risk and neurodegeneration. Extracellular vesicles (EVs) reflect the lipidomic composition of their parent cell offering a window into lipidomic alterations that may be tracked in living humans over time. Given high variability in human samples, we sought to define APOE4-related lipidomic EV signatures in a controlled human cellular system. Methods: We studied EVs secreted by four replicates of human-induced pluripotent stem cell (iPSC)-derived glutamatergic neurons from two healthy individuals (APOE2/E3 and APOE4/4) and two patients with AD (APOE3/3 and APOE4/4). Targeted lipidomics enabled quantitative profiling of hundreds of individual lipid species across major lipid classes. Univariate and multivariate analyses were applied to identify lipid alterations associated with APOE genotype and AD status. Results: APOE4 was associated with differences in lipid composition in neuronal EVs from both healthy and AD donors, suggesting that APOE4 influences EV lipid composition regardless of AD. Additionally, APOE4/4 carriers with AD exhibited clear, species-specific lipid imbalances. Lower levels of ganglioside GM3 and higher of phosphatidylserine emerged as consistent contributors to the APOE4-associated AD lipid signature, alongside alterations in dihydrosphingomyelin and phosphatidylinositol species linked to membrane integrity, endolysosomal function, and inflammatory pathways. These lipid species were among the strongest variables distinguishing healthy and AD APOE4/4 samples in multivariate analyses. Conclusion: These findings specify lipidomic changes in AD related to the APOE4 genotype, and support a model by which APOE4 creates a lipid profile that becomes selectively dysregulated in AD. By identifying specific lipid species associated with APOE4 and AD, this work provides further evidence that distinct lipidomic signatures are associated with both APOE4 and AD. These findings may contribute to the identification of lipid-based biomarkers and support future investigations into genotype-informed therapeutic strategies, although validation in larger, independent cohorts is required.