The Role And Mechanisms Of A2E Induce Telomere Dysfunction And Accelerate RPE Cell Senescence In The Development Of AMD

Background and Purpose: Age-related macular degeneration is the leading cause of irreversible vision loss in developed country.However,the mechanism of age-related macular degeneration is not fully understood.Advanced age is the most important risk factor.It has demonstrated that cellular senescence contributes to aging and age-related disease.Recent studies showed that retinal pigment epithelium cell senescence contributes to the pathology of dry age-related macular degeneration,which is accompanied with lipofuscin accumulation.In addition,N-retinylidene-N-retinylethanolamine(named A2E),the main fluorophore of lipofuscin,has been shown to play a detrimental role in retinal pigment epithelium normal functions by a variety of mechanisms,the most important of which is induction of DNA lesion.Interestingly,telomeres are fragile DNA sites due to enrichment of guanines.In vitro study,it has found that telomere shorting resulted from cell division could provoke RPE cell senescence.Consequently,cellular senescence could trigger the senescence-associated secretory phenotype,which may contribute to age-related pathology.Therefore,we hypothesized that A2 E may induce telomere dysfunction,thereby accelerating RPE senescence,triggering the senescence-associated secretory phenotype,which indicate that to defer senescence including telomere-protecting and controlling the senescence-associated secretory phenotype may be potential therapy for age-related macular degeneration,especially early-stage.Methods: We used ARPE19 cell line in our study.First,we estimated the level of DNA damage by western-blot and comet assay under photosensitization of A2 E in RPE cells.Then,we detected the telomere DNA damage by immunofluorescence and Q-FISH in the metaphase spreads.Second,we monitored SA-β-galactosidase staining as a marker of cellular senescence.We measured the rate of senescent cells under photosensitization of A2 E.Later,we detect the rate of senescent cells under photosensitization of A2 E when ectopic expression of the catalytic subunit of telomerase(TERT).Next,we add antioxidant NAC and flavonoid Chrysin to the medium to see whether anti-oxidative stress and telomere protection could alleviate A2E-induce DNA damage and senescence.Finally,RNA-deep sequencing was taken to detect the senescence-associated secretory phenotype.Results: First,we found that photosensitization of A2 E induces DNA damage,triggers telomere dysfunction and cell senescence in RPE cells.Antioxidant NAC treatment alleviates DNA damage and cellular senescence due to A2 E photosensitization.However,the fact that telomere loss was not significantly rescued and that the TIF was only partially rescued under the treatment of NAC suggested that the antioxidant NAC could not specifically protect the A2 E photosensitization induced telomere damage.Then,we found that flavonoid Chrysin could alleviated both telomere deprotection and cell senescence due to photosensitization of A2 E.Next,transfection TERT could alleviate RPE cell senescence caused by photosensitization of A2 E.Finally,our data showed that RPE senescence caused by photosensitization of A2 E triggers the expression of SASP factors.Conclusions: In this work,we identified photosensitization of A2 E induced DNA damage,especially telomere deprotection and deletion,and accelerated RPE cell senescence.Ectopic expression of the catalytic subunit of telomerase(TERT)alleviated RPE cell senescence,indicating that a large proportion of A2E-mediated senescence was due to telomere shortening.Antioxidant NAC alleviated DNA damage,consequently rescued cell senescence.Flavonoid Chrysin alleviated DNA damage and telomere deprotection,consequently rescued RPE cell senescence.Ultimately,photosensitization of A2 E triggers SASP gene expression in RPE cells,which may affect the microenvironment of retina and the surrounding cells,consequently contributing to age-related retina degeneration diseases,like AMD.Our study pinpoints the role of telomere dysfunction in RPE senescence and suggests that protecting telomere is a valuable strategy for treating retina degeneration diseases,such as AMD.