Hedgehog signaling activation rescues hypoxia-induced ferroptosis in human brain organoids

Fetal hypoxia disrupts neurodevelopment. In particular, the developing brain is extremely vulnerable to hypoxia injury; however, the specific vulnerable cell types and their underlying molecular mechanisms remain underexplored. In the present study, we established a human brain organoid model that reproduced the pathophysiological process of fetal hypoxia during early to mid-gestation. Through single-cell transcriptomic technology, we identified seven neural lineages in these organoids, including cortical progenitors and neurons. Further analysis revealed the specific responses to hypoxia among different types of cells regarding the mechanistic target of rapamycin complex 1 signaling pathway, fatty acid synthesis, the unfolded protein response, and the innate immune response. In terms of development, the maturation of glutamatergic and γ-aminobutyric acid-ergic neurons was significantly delayed after hypoxia exposure, whereas progenitor cells were less affected. In terms of function, we identified two subtypes of γ-aminobutyric acid-ergic neurons with different sensitivities to hypoxia. The more mature type 2 neurons were the most sensitive to hypoxia, which manifested as ferroptosis activation and impaired expression of neurite proteins (e.g., microtubule-associated protein 2). By contrast, the less mature type 1 neurons showed some tolerance to hypoxia. A mechanistic study revealed that pharmacological activation of the hedgehog pathway can inhibit ferroptosis and restore expression of the neurite protein microtubule-associated protein 2 in type 2 γ-aminobutyric acid-ergic neurons under hypoxia. Collectively, these findings delineate the lineage-specific patterns of hypoxia vulnerability and establish hedgehog pathway regulation as a potential target for neuroprotective strategies in fetal brain hypoxic injury.