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Haijiang Lin, Bo Tian, Ahmad Al Moujahed, Joan W Miller, Demetrios G. Vavvas; Accumulation of damaged nDNA promotes RPE cellular senescence and pro-inflammation. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5235.
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There is increasing evidence to support a role for chronic inflammation in the pathogenesis of age-related macular degeneration (AMD). Dysfunction of Retinal pigment epithelium (RPE) cell is a critical event in molecular pathways leading to clinical relevant AMD changes. We have reported that mitochondrial DNA (mtDNA) induces human RPE cell line to secrete inflammatory cytokines IL-6 and IL-8, which have been associated with AMD onset and progression. To study the effect of accumulation of damaged DNA on RPE cell senescence and pro-inflammation, we established an in vitro RPE cell model by knocking out DNase 2a, which codes a lysosomal enzyme responsible for degrading damaged double stranded DNA.
ARPE-19 cells were infected with lentiviral vector co-expressing Cas9 nuclease and sgRNA targeting the DNA sequence in exon 4 of Dnase 2a gene or control lentiviral vector co-expressing scamble sgRNA. Validation of Dnase 2a deletion was performed by western blot. Damaged nDNA accumulation was detected by immumofluorescence staining using both anti-dsDNA and anti-γH2AX antibody. Senescence was analyzed via SA-β-Gal staining as well as Western blot analysis of senescence pathway-associated gene expressions p16, pRB and Apo-J. Expression level of pro-inflammatory cytokines IL-6, IL-8 and IP-10 were assessed by either qPCR or western blot. Finally, levels of pro-IL-1β and pro-caspase 1 were evaluated by western blot.
DNase 2a deletion resulted in accumulation of endogenous damaged nDNA. RPE cells with accumulation of nDNA showed larger size, higher levels of positive SA-β-Gal staining, higher expression levels of the p16, APO-J, and lower expression levels of pRb compared to scramble sgRNA control cells. This indicates that accumulation of nDNA promotes RPE cellular senescence. Futhermore, multiple cytokines and chemokines such as IL-6, IL-8 and interferon gamma-induced protein 10 (IP-10) expression levels are increased in Dnase 2a -/- cells. DNase 2a deletion increased levels of pro-IL1β and pro-caspase 1, indicating that the inflammasome is primed in these cells.
Our studies demonstrated that accumulation of endogenous damaged nDNA promotes RPE cellular senescence and pro-inflammation. This study contributes to our understanding of the molecular mechanism in the development of AMD
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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