Abstract
Purpose :
Our lab reported that tumor necrosis factor receptor superfamily 10A dysregulation leads to AMD progression and RPE dysfunction (PMID:35103281). Recently, we further showed a marked upregulation of senescence in TNFRS10A knockdown in RPE and in RPE/choroid of Tnfrsf10 KO mice (ARVO 2023). This study intends to investigate the age-related changes in senescence development in Tnfrs10 KO retina and attempts to identify potential senolytic agent(s) using TNFRS10A-silenced hRPE.
Methods :
Subconfluent primary hRPE cells and TNFRSF10A silenced hRPE were exposed to H2O2 (500 μM) alone or co-treated with a PKC activator, phorbol 12-myristate 13-acetate (PMA) for 48h. The effect of PMA treatment on senescence was quantified using immunostaining, RT-PCR, and WB analysis of senescence-associated markers (beta-gal, p16, p21, cGAS, and STING). Mitochondrial metabolic parameters in the control and treated groups were determined using a seahorse XF96e analyzer. In in vivo studies, 3, 6, and 12-month-old male Tnfrsf10 KO (Tnfrsf10-/-) mice were used to examine the effect of aging on senescence. Expression p16 and p21 in KO mice was determined by RT-PCR, WB, and immunostaining analysis.
Results :
The senescence biomarkers p16 and p21 showed a significant downregulation with PMA treatment at the gene (p < 0.05) and protein (p < 0.01) levels in hRPE cells. Mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) (pmol/min/total protein) increased with senescence induction and/or knockdown of TNFRSF10, and PMA treatment decreased both OCR and ECAR. Similarly, mitochondria-associated markers (Fis1, DRP1, PGC1-α, and mtTFA) showed a similar trend. Age-dependent upregulation of p16 and p21 was observed in the RPE/choroid of Tnfrsf10 KO mice.
Conclusions :
Our studies reveal an important age-dependent role of TNFRSF10A in senescence. Our results further suggest the potential that PKC activator could serve as a senolytic agent.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.