Abstract
Purpose :
Retinal pigment epithelial cell (RPE) plays a vital role in the pathogenesis of age-related macular degeneration (AMD). Our lab has shown that RPE cellular senescence contributed to the pathophysiology of experimental AMD, and SASP members are involved in this process (PMID: 34986369). Recently, we presented confirmatory evidence to earlier GWAS studies that tumor necrosis factor receptor superfamily 10A (TNFRSF10A) dysregulation leads to AMD development and is linked to RPE dysfunction (PMID: 35103281). This study aims to investigate the contribution of RPE senescence to AMD pathophysiology using TNFRSF10A silenced human RPE (hRPE) cells and Tnfrsf10 KO mice.
Methods :
Subconfluent primary hRPE cells and TNFRSF10A silenced hRPE were exposed to stress-induced premature senescence with H2O2 (500 μM) for 48h, and senescence-associated markers (βgal, p16, p21, cGAS, and STING) were analyzed by RT-PCR and WB analysis. The effect of H2O2-induced senescence in non-silenced and silenced hRPE on OXPHOS and glycolysis was determined using Seahorse XF96 analyzer. 12-month-old male C57BL/6J Tnfrsf10 KO (Tnfrsf10-/-) mice were used to examine the regulation of senescence by TNFRSF10A in vivo. Expression of p16 and p21 in control and KO mice were determined by RT-PCR, WB, and immunostaining analysis.
Results :
The senescence-associated p16 and p21 showed a significant (p < 0.01) upregulation with H2O2 induction at the gene (1.8- and 3-fold) and protein (3.2- and 4-fold) in hRPE cells. The protein expression of p16, p21, cGAS, and STING was more significantly increased by co-treatment with siRNA (p < 0.05 vs. H2O2). Mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) (pmol/min/total DNA) increased with senescence induction by H2O2 for 48h in control RPE, and knockdown of TNFRSF10A caused a further increase in OCR and ECAR. Similarly, in vivo studies showed upregulation of senescence-related markers (p16 and p21) by RT-PCR, WB, and immunostaining analysis in RPE/choroid of Tnfrsf10 KO mice.
Conclusions :
Our findings suggest that TNRSF10A participates in RPE senescence. Further work on elucidating senescence pathways will facilitate the development of new therapeutic targets for AMD.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.