Quantitative proteomics links mitochondrial dysfunction to metabolic changes and epithelial differentiation defects in hyperoxia-exposed neonatal airway cells

Newborn infants, especially those born preterm, often require supplemental oxygen (O₂) therapy; however, exposure to supraphysiological oxygen (hyperoxia) can disrupt normal lung development and contribute to neonatal lung injury, including bronchopulmonary dysplasia (BPD). Mitochondrial dysfunction is increasingly recognized as a contributor to oxidative lung diseases, including BPD. However, the effects of hyperoxia on mitochondrial function and mucociliary differentiation in the developing airway epithelium remain poorly understood. This study tested the hypothesis that hyperoxia impairs neonatal airway mucociliary differentiation by disrupting mitochondrial bioenergetic function. Neonatal tracheal airway epithelial cells from term infants (<i>n</i> = 5) were cultured in a three-dimensional air-liquid interface (ALI) model and exposed to 60% O₂ during the mid-phase of differentiation (ALI <i>days 7</i>-<i>14</i>). Cellular phenotype was assessed using immunofluorescence staining and gene expression analyses. Mitochondrial function was evaluated through Seahorse metabolic flux analysis, and global protein changes were characterized by quantitative proteomics. Hyperoxia significantly impaired terminal differentiation with reduced ciliated and goblet cells. Seahorse assay revealed a decrease in baseline oxygen consumption and mitochondrial ATP production, accompanied by a compensatory increase in glycolytic ATP production. Quantitative proteomics identified disruption of mitochondrial complex I as a central feature of the hyperoxic response. Downstream proteomic pathway analyses further confirmed the metabolic shift from mitochondrial to glycolytic ATP production and demonstrated altered epithelial differentiation pathways, including NOTCH and TGF-β signaling. These findings reveal that hyperoxia impairs mitochondrial bioenergetics and alters metabolic programming, leading to disrupted mucociliary differentiation. Future studies should evaluate mitochondrial oxidative fitness as a therapeutic target in neonatal lung disease.<b>NEW & NOTEWORTHY</b> We report that moderate hyperoxia during a critical window of mucociliary differentiation disrupts terminal maturation in neonatal airway epithelial cells cultured in a three-dimensional model. Hyperoxia induces mitochondrial bioenergetic dysfunction and metabolic reprogramming, with proteomic analysis identifying complex I disruption as a key driver of impaired differentiation. Overall, these findings reveal a previously underrecognized link between mitochondrial bioenergetics and airway epithelial development, positioning metabolic dysfunction as an early trigger of hyperoxia-induced neonatal airway injury.