Abstract
Early endolysosomal and autophagic defects are among the earliest cellular alterations observed in Alzheimer's disease (AD). However, the molecular mechanisms linking amyloid precursor protein (APP) metabolism to vesicle trafficking dysfunction remain incompletely understood. The APP-derived fragment C99 has emerged as a potential upstream mediator of intracellular toxicity, but its impact on organelle homeostasis and its modulation by metabolic interventions remain unclear. To investigate these mechanisms, we expressed human C99 in Drosophila neurons and examined intracellular pathology using ultrastructural analysis, fluorescent reporters of autophagy and mitochondrial turnover, and proteomic interactome mapping. The effects of the ketone body β-hydroxybutyrate (BHB) were evaluated to assess the impact of metabolic intervention. Neuronal C99 expression induced pronounced vesicular abnormalities, impaired autophagic turnover, and disrupted mitochondrial quality control. Transmission electron microscopy revealed extensive accumulation of enlarged vesicular compartments, accompanied by reduced mitochondrial turnover and accumulation of aged mitochondria. BHB treatment restored autophagic cargo clearance, improved mitochondrial turnover, and normalized vesicular ultrastructure. These protective effects required neuronal ketone transport, indicating a neuron-intrinsic metabolic mechanism. Proteomic analysis of the C99-associated interactome revealed that ketone treatment remodels networks enriched for vesicle trafficking and proteostasis pathways. Network prioritization identified the retromer component VPS35 as a candidate regulatory hub. Functional analyses demonstrated that depletion of VPS35 abolished the BHB-dependent restoration of autophagy, mitochondrial turnover, and vesicle morphology. Ketone treatment restores mitochondrial quality control and autophagic homeostasis through a VPS35-dependent mechanism in C99-induced neurodegeneration. These findings provide mechanistic insight into how metabolic interventions may restore intracellular homeostasis in Alzheimer's disease.
