Early Metabolic Rewiring and Mitochondrial Remodeling in Response to Mitochondrial Impairment in a Rotenone Model of Parkinson’s Disease

Mitochondrial dysfunction is a central pathogenic mechanism in Parkinson’s disease (PD), yet early mitochondrial events linking respiratory impairment to neuronal vulnerability remain incompletely defined. Here, we investigated the impact of acute and chronic exposure to subtoxic and toxic doses of the mitochondrial Complex I inhibitor rotenone on oxidative stress, mitochondrial bioenergetics/metabolism, and mitochondrial dynamics in primary cortical neurons. Using live-cell imaging, bioenergetics and metabolomic profiling, we show that nanomolar concentrations of rotenone rapidly induce accumulation of reactive oxygen species (ROS), lipid peroxidation, and loss of mitochondrial membrane potential preceding overt neuronal death. Notably, high-content morphometric analysis revealed that even subtoxic doses of rotenone trigger an early structural remodeling characterized by mitochondrial network fragmentation into small spherical puncta (0.5–3 μm) and localized organelle swelling at both neuritic and synaptic levels. Instead, at the molecular level, whole-cell biochemical degradation of mitochondrial remodeling proteins, suppression of regulated, physiological fission–fusion cycling and accumulation of the autophagy adaptor SQSTM1/p62, indicative of defective quality control, fully manifest only at higher, toxic rotenone concentrations. Furthermore, subtoxic doses elicit widespread metabolic alterations including persistent redox imbalance, impaired glutathione (GSH) biosynthesis, and downregulation of anabolic processes such as folate/one-carbon and amino sugar metabolism. Collectively, our findings newly reveal that localized mitochondrial morphometric remodeling and metabolic pathway rewiring represent early, interconnected adaptive responses to complex I impairment, contributing to the baseline thresholds of neuronal vulnerability in PD models.