Itaconic acid dissolves TFEB liquid-liquid phase separation to alleviate hyperoxia-induced bronchopulmonary dysplasia.

Supplemental oxygen in premature neonates risks bronchopulmonary dysplasia (BPD). Although the metabolite itaconic acid (ITA) promotes transcription factor EB (TFEB) nuclear translocation to alleviate BPD, the upstream mechanism driving hyperoxia-induced TFEB sequestration remains unclear. Here, we investigated whether hyperoxia exploits liquid-liquid phase separation (LLPS) to trap cytoplasmic TFEB, and if ITA directly dissolves these pathological condensates. Using hyperoxia-induced BPD mice (85 % O2) and MLE12 cells, we analyzed TFEB LLPS via FRAP, fusion, and in vitro phase separation assays. ITA-TFEB binding was evaluated by surface plasmon resonance (SPR). Autophagic/mitophagic flux and apoptosis were assessed, and the necessity of TFEB was validated using shRNA knockdown in vivo and in 3D lung organoids. We found that hyperoxia promoted TFEB accumulation into cytoplasmic condensates with liquid-like properties, which was associated with the blockage of its nuclear import. ITA directly bound TFEB with high affinity (confirmed by SPR) and effectively disrupted these pathological condensates without altering basal expression. Consequently, ITA restored TFEB nuclear translocation, reactivated mitophagy, cleared dysfunctional mitochondria, and reduced AT2 cell apoptosis in BPD lungs. These protective effects of ITA were abolished by TFEB knockdown in vivo and in lung organoids. Collectively, our findings suggest that hyperoxia-triggered TFEB phase-separation-like behavior represents a novel biophysical mechanism of autophagic arrest in BPD. By acting as an endogenous metabolite capable of disrupting biomolecular condensates, ITA alleviates pathological TFEB condensates, restoring mitophagy and alveolar development, thereby proposing a paradigm of targeting aberrant macromolecular condensation for neonatal lung injury.