Investigative Ophthalmology & Visual Science Cover Image for Volume 65, Issue 7
June 2024
Volume 65, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2024
Role of ATF4 in ER stress-induced mitochondrial dysfunction and apoptosis in Fuchs’ endothelial corneal dystrophy
Author Affiliations & Notes
  • Saba Qureshi
    Ophthalmology, Icahn School of Medicine at Mount Sinai, New York, New York, United States
  • Stephanie Lee
    Ophthalmology, Icahn School of Medicine at Mount Sinai, New York, New York, United States
  • Lukas Ritzer
    Ophthalmology, Icahn School of Medicine at Mount Sinai, New York, New York, United States
  • Ananya Chaubal
    Herricks High School, New Hyde Park, New York, United States, New York, New York, United States
  • Varun Kumar
    Ophthalmology, Icahn School of Medicine at Mount Sinai, New York, New York, United States
    Pharmacological sciences, Icahn School of Medicine at Mount Sinai, New York, New York, United States
  • Footnotes
    Commercial Relationships   Saba Qureshi None; Stephanie Lee None; Lukas Ritzer None; Ananya Chaubal None; Varun Kumar None
  • Footnotes
    Support  NEI-4R00EY031339-04, Mount Sinai Seed Fund, New York Eye and Ear Foundation, Sarah K de Coizart Charitable Trust/Foundation, Challenge Grant from Research to Prevent Blindness
Investigative Ophthalmology & Visual Science June 2024, Vol.65, 5779. doi:
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    • Get Citation

      Saba Qureshi, Stephanie Lee, Lukas Ritzer, Ananya Chaubal, Varun Kumar; Role of ATF4 in ER stress-induced mitochondrial dysfunction and apoptosis in Fuchs’ endothelial corneal dystrophy. Invest. Ophthalmol. Vis. Sci. 2024;65(7):5779.

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      © ARVO (1962-2015); The Authors (2016-present)

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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.

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