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Published on: February 15, 2020
Ku80 Counters Oxidative Stress-Induced DNA Damage and Cataract Formation in the Human Lens
Andrew John Oliver Smith1, Simon Sidney Robert Ball1, Kamal Manzar2
1School of Biological Sciences University of East Anglia, Norwich Research Park, Norwich, United Kingdom.
Purpose:
Oxidative stress in the human lens leads to a wide range of damage including DNA strand breaks, which are likely to contribute to cataract formation. The protein Ku80 is a fundamental component of the nonhomologous end-joining pathway that repairs DNA double strand breaks. This study investigates the putative impact of Ku80 in cataract prevention in the human lens.
Methods:
The present study used the human lens epithelial cell line FHL124 and whole human lens organ culture. Targeted siRNA was used to deplete Ku80, with Western blot and immunocytochemistry employed to assess Ku80 expression levels. Oxidative stress was induced with hydrogen peroxide and DNA strand breaks measured by alkaline comet assay and γH2AX foci counts. Visual quality of whole human lenses was measured with image analysis software.
Results:
Expression of Ku80 was predominately found in the cell nucleus of both FHL124 cells and native human lens epithelium. Treatment of FHL124 cells and whole lens cultures with siRNA targeted against Ku80 resulted in a significant knockdown at the protein level. Application of oxidative stress (30 μM H2O2) created more DNA strand breaks when added to Ku80 knockdown cells than in scrambled siRNA control cells as determined by the alkaline comet assay and the number of γH2AX foci. In whole lens cultures, exposure to 1 mM H2O2 resulted in more lens opacity in Ku80 knockdown lenses than match-paired controls.
Conclusions:
Depletion of Ku80 in the lens through acute change or a consequence of aging is likely to increase levels of DNA strand breaks, which could negatively influence physiological function and promote lens opacity. It is therefore feasible that Ku80 plays a role in retarding cataract formation.
Insights
The protein Ku80 plays a crucial role in repairing DNA damage in the human lens. Lowering Ku80 levels increases DNA breaks and lens opacity, suggesting Ku80 may prevent cataracts.
Area of Science:
- Ophthalmology
- Molecular Biology
- Genetics
Background:
- Oxidative stress causes DNA damage in the human lens, contributing to cataract formation.
- The protein Ku80 is essential for repairing DNA double-strand breaks via nonhomologous end-joining.
Purpose of the Study:
- To investigate the role of Ku80 in preventing cataract formation in the human lens.
- To determine if Ku80 deficiency exacerbates oxidative stress-induced DNA damage in lens cells.
Main Methods:
- Used human lens epithelial cells (FHL124) and whole human lens organ culture.
- Depleted Ku80 using targeted siRNA and assessed expression via Western blot and immunocytochemistry.
- Induced oxidative stress with hydrogen peroxide and measured DNA damage using alkaline comet assay and γH2AX foci.
- Evaluated lens visual quality using image analysis software.
Main Results:
- Ku80 was primarily located in the nucleus of lens cells.
- Ku80 knockdown significantly reduced Ku80 protein levels.
- Ku80-depleted cells showed increased DNA strand breaks and γH2AX foci under oxidative stress.
- Ku80 knockdown lenses exhibited greater opacity following hydrogen peroxide exposure.
Conclusions:
- Ku80 depletion in the lens likely increases DNA strand breaks, impairing function and promoting opacity.
- Ku80 appears to play a protective role in retarding cataract development.
