Abstract

Objective Mitochondrial complex I sustains oxidative phosphorylation, respiratory adaptation, and mitochondrial homeostasis during osteogenic differentiation. NADH:ubiquinone oxidoreductase core subunit (NDUFS5) is a nuclear-encoded complex I subunit required for complex I assembly and function, but its role in osteoblast differentiation is unknown. This study examined whether NDUFS5 deficiency suppresses osteogenic differentiation through mitochondrial dysfunction and gamma-aminobutyric acid (GABA) shunt-related metabolic disruption. Methods NDUFS5 expression was examined in mouse tibial sections and MC3T3-E1 cells during osteogenic induction. Osteogenic differentiation was evaluated by osteogenic marker expression, alkaline phosphatase (ALP) staining, and ALP activity. Mitochondrial morphology and function were evaluated using fluorescence imaging, transmission electron microscopy, and assays of membrane potential, reactive oxygen species, and oxygen consumption. Integrated transcriptomic and metabolomic profiling was performed, and exogenous GABA supplementation was used to test GABA shunt involvement. Results NDUFS5 expression increased during osteogenic differentiation and was enriched at sites of active bone formation. Ndufs5 deficiency reduced osteogenic marker expression and ALP activity. It also caused mitochondrial swelling, cristae disruption, reduced membrane potential, increased reactive oxygen species, and impaired respiratory function. Integrated analyses indicated GABA shunt disruption, accompanied by reduced intracellular GABA. Exogenous GABA partially restored osteogenic marker expression and ALP activity in Ndufs5 -deficient cells. Conclusion NDUFS5 contributes to osteogenic differentiation by maintaining mitochondrial integrity, respiratory function, and GABA shunt-related metabolic homeostasis. These findings identify NDUFS5 as a regulator of metabolic adaptation during osteoblast maturation and suggest partial rescue through GABA replenishm