ATF4 regulates mitochondrial dysfunction and mitophagy, contributing to corneal endothelial apoptosis.

Endoplasmic reticulum (ER) stress, mitochondrial dysfunction, and mitophagy are known to contribute independently to corneal endothelial cell (CEnC) apoptosis in Fuchs’ endothelial corneal dystrophy (FECD). However, the role of a specific ER stress pathway (PERK-ATF4-CHOP) in regulating these events is unknown. This study aims to investigate the role of ATF4 in regulating mitochondrial dysfunction and mitophagy, which ultimately leads to CEnC apoptosis. Human corneal endothelial cell line (21T), Fuchs’ corneal endothelial cell line (F35T), and primary human corneal endothelial cells were treated with ER stressor tunicamycin (Tun). ATF4 siRNA was used to knock down ATF4 in 21T cell line and primary corneal endothelial cells. Mitophagy and apoptotic proteins were analyzed using Western blotting. ATF4(+/−) and ATF4 (+/+) mice were irradiated with UVA to assess ER stress and corneal endothelial apoptosis. F35T cell line had significantly increased expression of the ER stress pathway as well as caspase-mediated apoptotic molecules compared to 21T at baseline, which further increased after Tun treatment. F35T cells exhibited significantly decreased mitochondrial ATP and MMP, and increased mitochondrial fragmentation, which was further exacerbated after Tun. F35T cells also demonstrated inhibition of mitophagy, similar to 21T, after treatment with tunicamycin, despite the upregulation of mitophagy initiators. ATF4 knockdown attenuated ER and mitochondrial stress proteins, rescued mitochondrial membrane potential (MMP) loss, downregulated mitochondrial fragmentation, activated mitophagy, and prevented CEnC death under chronic ER stress. ATF4(+/−) mice had increased CE numbers, with improved cellular morphology and decreased ER stress marker CHOP expression, compared to ATF4(+/+) mice post-UVA. Pro-apoptotic ATF4 induction following ER stress disrupts mitochondrial function, leading to mitophagy inhibition and CEnC apoptosis. This study highlights the importance of ATF4 in ER-mitochondrial crosstalk and its contribution to CEnC apoptosis in FECD. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1038/s41598-026-36453-x.