Abstract
Purpose :
Endoplasmic reticulum (ER) and mitochondrial stress are independently associated with corneal endothelial cell (CEnC) loss in Fuchs endothelial corneal dystrophy (FECD). However, the role of ER stress in mitochondrial dysfunction contributing to CEnC apoptosis is unknown. The purpose of this study is to explore the crosstalk between ER and mitochondrial stress in CEnCs for FECD.
Methods :
Human corneal endothelial cell line (HCEnC-21T) was treated with ER stressor tunicamycin (1,5,10,20 mg/ml) and ER stress-reducing chemical 4-phenyl butyric acid (4-PBA;2.5 mM) for 6 and 24 hours. ER stress proteins (GRP78/BiP, PERK, p-eIf2, CHOP, IRE1, XBP1, ATF6), apoptotic proteins (cleaved caspase 3 and PARP), and mitochondrial-mediated intrinsic apoptotic stress proteins (cleaved caspase 9, Bcl2, Bax, cytochrome c) and mitochondrial fragmentation (Fis1, p-Drp1) and fusion (OPA1, Mfn2) proteins were determined using immunoblotting and immunohistochemistry. Cell viability was measured using MTT assay. Mitochondrial bioenergetics was analyzed using Seahorse XFe96 analyzer at 2 hours post tunicamycin (10mg/ml). Mitochondrial dynamic changes (shape, area, perimeter, fragmentation) were analyzed using Transmission electron microscopy (TEM) and immunohistochemistry at 6 and 24 hours after tunicamycin treatment (1,10 mg/ml).
Results :
Treatment of HCEnC-21T cell line with tunicamycin activated three ER stress pathways (PERK-eIf2-CHOP, IRE1-XBP1 and ATF6), induced apoptotic proteins (cleaved caspase 3, PARP), reduced cell viability, upregulated mitochondrial intrinsic apoptotic molecules (cleaved caspase 9, Bcl2, Bax, cytochrome c), increased mitochondrial respiratory capacity, causes mitochondrial swelling and fragmentation with increased expression of mitochondrial fission proteins (Fis1 and pDRP1). 4-PBA rescued cell loss and reduced cleaved caspase 3 and 9, thereby rescuing tunicamycin-induced cell death.
Conclusions :
Tunicamycin-induced ER stress disrupts mitochondrial bioenergetics and dynamics and contributes to the loss of CEnC viability. This novel study highlights the importance of ER-mitochondria crosstalk and its contribution to CEnC apoptosis, as seen in FECD.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.