Abstract
Purpose :
Endoplasmic reticulum (ER) stress and mitochondrial dysfunction contribute 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 and apoptosis is unknown. The purpose of this study is to explore the role of ATF4 in regulating mitochondrial dysfunction and CEnC apoptosis in FECD.
Methods :
Human 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 mg/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 (0.1, 1, 10 mg/mL tunicamycin for 24 hours). Mitochondrial bioenergetics was analyzed by measuring mitochondria membrane potential (MMP) loss using TMRE assay and ATP production using ATP synthase enzyme activity at 48 hours post tunicamycin. Mitochondrial-mediated intrinsic apoptotic pathway proteins were analyzed using immunoblotting (10 mg/mL tunicamycin for 24 hours).
Results :
F35T cell line had a significantly increased expression of ER stress pathway molecules (eIF2α, ATF4, CHOP) and mitochondrial-mediated intrinsic apoptotic molecules (cytochrome c, Bcl2, cleaved 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 decreased apoptosis, ER, and mitochondrial stress pro-apoptotic molecules, rescued MMP loss, attenuated mitochondrial fragmentation, and modulated autophagy pathway (Akt/mTOR) in 21T cell line and primary corneal endothelial cells.
Conclusions :
Pro-apoptotic ATF4 induction under tunicamycin-induced ER stress disrupts mitochondrial bioenergetics and dynamics, leading to CEnC apoptosis. This study highlights the importance of ATF4 in ER-mitochondrial crosstalk and its contribution to CEnC apoptosis in FECD.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.