beta Hydroxybutyrate remodels the C99 interactome and coincides with restored organelle homeostasis in a Drosophila Alzheimers model

Abstract

Early endolysosomal and autophagic defects are among the earliest cellular alterations observed in Alzheimers disease (AD), yet the molecular drivers 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. Here, we show that neuronal expression of human C99 in Drosophila induces profound vesicular abnormalities, impaired autophagic turnover, and disrupted mitochondrial quality control. Ultrastructural analysis revealed extensive accumulation of enlarged vesicular compartments, accompanied by reduced mitochondrial turnover and accumulation of aged mitochondria. Treatment with the ketone body beta-hydroxybutyrate (BHB) 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 mapping 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. Together, these findings suggest that ketone treatment restores mitochondrial quality control through a VPS35-dependent mechanism in C99 induced neurodegeneration, providing mechanistic insight into how metabolic interventions may restore intracellular homeostasis in Alzheimers disease.