June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
ATM-mediated prominent DNA damage response in Fuchs Endothelial Corneal Dystrophy
Author Affiliations & Notes
  • Shazia Ashraf
    Department of Ophthalmology, Schepens Eye Research Institute of Massachusetts Eye and Ear, Boston, Massachusetts, United States
  • Neha Deshpande
    Department of Ophthalmology, Schepens Eye Research Institute of Massachusetts Eye and Ear, Boston, Massachusetts, United States
  • Raymond Wong
    Department of Ophthalmology, Schepens Eye Research Institute of Massachusetts Eye and Ear, Boston, Massachusetts, United States
  • Jeffrey Boakye
    Department of Ophthalmology, Schepens Eye Research Institute of Massachusetts Eye and Ear, Boston, Massachusetts, United States
  • Mohit Nishitkumar Parekh
    Department of Ophthalmology, Schepens Eye Research Institute of Massachusetts Eye and Ear, Boston, Massachusetts, United States
  • Ula V Jurkunas
    Department of Ophthalmology, Schepens Eye Research Institute of Massachusetts Eye and Ear, Boston, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Shazia Ashraf None; Neha Deshpande None; Raymond Wong None; Jeffrey Boakye None; Mohit Parekh None; Ula Jurkunas None
  • Footnotes
    Support  NIH/NEI R01EY020581
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 1334. doi:
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      Shazia Ashraf, Neha Deshpande, Raymond Wong, Jeffrey Boakye, Mohit Nishitkumar Parekh, Ula V Jurkunas; ATM-mediated prominent DNA damage response in Fuchs Endothelial Corneal Dystrophy. Invest. Ophthalmol. Vis. Sci. 2023;64(8):1334.

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

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Abstract

Purpose : Fuchs Endothelial Corneal Dystrophy (FECD) is an oxidative stress disorder leading to accelerated corneal endothelial cell (CEnCs) loss. It is a genetically complex disorder with a higher incidence in females. Ataxia Telangiectasia Mutated (ATM) is a critical DNA damage response (DDR) kinase in post-mitotically arrested cells, whose activation leads to downstream activation of cell-cycle checkpoints, DNA repair, and apoptosis. In this study, we investigate the mechanism of ATM-mediated DDR activation in response to UVA leading to FECD.

Methods : To investigate DDR activation in response to UVA in-vitro, immortalized normal and FECD patient-derived cell lines were treated with UVA (10 /cm2)+4-OHE2 followed by recovery for 1 and 24 hours. Cells were lysed at 1-hr post-treatment to compare early DDR signaling between cell lines. The phosphorylation status and levels of γH2AX and pATM and its downstream effector, pCHK2 were evaluated by western blotting. Cell-cycle distribution was measured 24-hrs post-treatment by flow cytometry. Effect of cell cycle reentry on DNA repair efficiency was determined by sorting the treated cells into G0/G1-G2/M with MoFLo and alterations in different DNA-repair genes were quantified by qPCR. In vivo, cell-cycle activation was tested by Ki67-staining in CEnCs using whole-mount corneas (+/-UVA, 500J/cm2) of wild-type (WT) and ATM-null mice.

Results : FECD cell lines showed increased activation of pATM/ATM, and its effectors: pCHK2/CHK2, γH2AX/H2AX after treatment with UVA+4-OHE2 compared to untreated, indicating that ATM phosphorylation activated DDR. Furthermore, UVA+4-OHE2-treated CEnCs were arrested in G2/M phase of the cell cycle and showed upregulation DNA-repair genes (LIG3, NEIL2, TOP3A, XRCC1, XPC) in G2/M phase compared to G0/G1, indicating that cell cycle arrest is critical for the DNA repair process. Loss of NQO1, as seen in FECD, led to hyperphosphorylation of ATM with a 2-fold increase in pATM compared to treated normal CEnCs; however, they showed a decrease in LIG3, indicating a deficient DNA repair from oxidative stress. In vivo, UVA irradiation-induced Ki67 staining and promoted cell cycle reentry in WT but not in ATM-null mice.

Conclusions : We demonstrate increased activation of ATM-mediated DDR in response to ROS-induced DNA damage in FECD. This study provides insights into understanding the role of DDR signaling cascade in determining the DNA repair pathways in FECD.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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