ATF4 regulates mitochondrial dysfunction, mitophagy, and autophagy, contributing to corneal endothelial apoptosis under chronic ER stress in Fuchs’ dystrophy
Authors: Qureshi Saba , Lee Stephanie , Ritzer Lukas , Kim Stefan Y. , Steidl William , Krest Gayle Joliet , Kasi Anisha , Kumar Varun 
Published: November 2024

AbstractPurposeEndoplasmic reticulum (ER) stress, mitochondrial dysfunction, mitophagy/autophagy are known to contribute independently to corneal endothelial (CE) apoptosis in Fuchs’ endothelial corneal dystrophy (FECD). However, the role of a well-studied specific ER stress pathway (PERK-ATF4-CHOP) in regulating mitochondrial dysfunction, mitophagy/autophagy, and apoptosis is unknown. The purpose of this study is to explore the role of ATF4 in regulating mitochondrial dysfunction and mitophagy/autophagy, leading to CEnC apoptosis in FECD.MethodsHuman corneal endothelial cell line (HCEnC-21T), Fuchs’ corneal endothelial cell line (F35T), and primary human corneal endothelial cells were treated with ER stressor tunicamycin (0.01, 0.1, 1, 10 μg/mL) for 24 and/or 48 hours. ATF4 siRNA was used to knock down ATF4 in 21T cell line and primary corneal endothelial cells. Cell viability was measured using an MTT assay (10 μg/mL tunicamycin for 24 hours). Mitochondrial bioenergetics was analyzed by measuring mitochondria membrane potential (MMP) loss using TMRE assay and ATP production using mitochondrial complex V assay kit at 48 hours post tunicamycin. Mitochondrial-mediated intrinsic apoptotic pathway proteins, mitophagy, and autophagy marker proteins were analyzed using Western blotting (10 μg/mL tunicamycin for 24 hours). ATF4 +/- and ATF4 +/+ mice were irradiated with UVA to assess pro-apoptotic ER stress and corneal endothelial cell deathin vivo.ResultsF35T cell line had a significantly increased expression of ER stress pathway molecules (eIF2α, ATF4, CHOP) and mitochondrial-mediated intrinsic apoptotic molecules (cleaved PARP, caspase 9, caspase 3) along with mitochondrial fragmentation compared to 21T cells at the baseline, which further increased after treatment with tunicamycin. Mitochondrial membrane potential also significantly decreased in F35T compared to 21T after tunicamycin. ATF4 knockdown after tunicamycin significantly attenuated pro-apoptotic ER and mitochondrial stress molecules, rescued MMP loss, and reduced mitochondrial fragmentation in the 21T cell line and primary corneal endothelial cells. ATF4 knockdown post tunicamycin treatment also downregulated altered/excessive Parkin-mediated mitophagy and Akt/mTOR-mediated autophagy pathway with reduction of caspases, leading to increased cellular viability. ATF4+/-mice had significantly increased CE numbers with improved cellular morphology and decreased CHOP expression compared to ATF4+/+ post-UVA.ConclusionsPro-apoptotic ATF4 induction under tunicamycin-induced ER stress disrupts mitochondrial bioenergetics and dynamics, leading to activation of excessive autophagy/mitophagy. ATF4-induced activation of CHOP plays a key role in switching excessive autophagy to CEnC apoptosis. This study highlights the importance of ATF4 in ER-mitochondrial crosstalk and its contribution to CEnC apoptosis in FECD.