Mitochondrial complex-derived ROS induces lysosomal dysfunction and impairs autophagic flux in human cells carrying...

The APOE4 allele is the strongest genetic risk factor for sporadic Alzheimer's disease (sAD), yet its cell-autonomous effects remain poorly understood. While young, asymptomatic APOE4 carriers exhibit abnormal brain metabolism, the mechanistic link between mitochondrial dysfunction and lysosomal-autophagic failure remains unclear. In this study, we conducted a comprehensive analysis of primary human fibroblasts from APOE3 controls, APOE4, and sAD donors to assess mitochondrial bioenergetics, oxidative stress, autophagy, and lysosomal function. APOE4 fibroblasts displayed increased mitochondrial content-associated markers (PGC1α, mtDNA) accompanied by reduced respiratory capacity, elevated proton leak, and excessive mitochondrial ROS. In parallel, APOE4 fibroblasts showed impaired autophagic flux and reduced LC3-TOMM20 colocalization, indicating defective mitophagy. Lysosomal proteolytic activity, assessed using DQ-BSA, was significantly reduced and remained unresponsive under to starvation, in contrast to the partial recovery observed in sAD cells. Pharmacological targeting of mitochondrial ROS with site-specific inhibitors revealed that complex III-derived ROS is the predominant driver of redox stress in APOE4 fibroblasts, while complex I contributes primarily in sAD. Notably, selective inhibition of complex III-derived ROS with S3QEL restored lysosomal degradation, autophagic flux, and mitochondrial respiration in APOE4 cells. Together, these findings demonstrate that mitochondrial oxidative stress disrupts the mitochondria-lysosome axis in an APOE4-specific manner, revealing early and mechanistically distinct vulnerabilities that may precede neurodegeneration. Our results challenge the notion that APOE4 merely amplifies AD pathology and instead identity site-specific redox signaling as a promising target for allele-informed interventions.