Abstract
Purpose :
Accumulation of cytosolic DNA has been found during cell senescence, a terminal cell cycle arrest and usually associated with pro-inflammatory responses. Aging is one of the highest risk factors for age-related macular degeneration (AMD) and other degenerative eye diseases. The functional significance of cytosolic DNA during retinal cell senescence is largely unexplored. Here, we are aimed to investigate the distribution, composition and pro-inflammatory effect of cytosolic DNA during RPE and photoreceptor cell senescence.
Methods :
Hydrogen peroxide (H2O2, 600 μM, 2 h) was used to induce cell senescence in human RPE cell line ARPE-19 and in mouse photoreceptor cell line 661W. β-galactosidase (β-gal) analysis and Western blot (WB) analysis confirmed cell senescence upon oxidative stress. Immunofluorescence determines DNA leakage, DNA damage and enriched chromatin marker in cytoplasm. To determine the potential pro-inflammatory of cytosolic DNA, genomic DNAs were extracted from ARPE-19 cells with or without H2O2 exposure. The obtained DNA was then transfected into ARPE-19 cells. Inflammatory factors were determined by qRT-PCR analysis at 16 h post transfection.
Results :
Oxidative stress leads to senescence of RPE and 661W cells. Leakage of nuclear DNA into cytosol and formation of micronuclei were evident in those senescent cells. The cytosolic DNA contains unrepaired fragment as evidenced by positive staining of DNA damage marker γH2AX, which is achieved by extrusion of chromatin through lamina/C-coated nuclear envelope. Transfection of genomic DNA derived from senescent cells lead to significant higher expression of IL6, IL1β and INFβ as compared to untreated normal cells.
Conclusions :
Together, our results demonstrate RPE and photoreceptor senescence is accompanied by accumulation of cytosolic DNA, which may initiate a pro-inflammatory response in age-related eye diseases.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.