Abstract
Purpose :
We have previously discovered a global loss of histone expression and an epigenetic signature of specific histone marks in the aging mouse retinal pigment epithelium (RPE). This study utilizes in vitro models of chronological and replicative aging to identify novel histone expression profiles and post-translational modifications (PTMs) in primary human RPE (hRPE) cells.
Methods :
To study chronological aging, early passage (P3-5) primary hRPE cells (n=3 isolates, fetal and adult) were seeded on fibronectin (2μg/cm2) coated transwell plates (1x105 cells /cm2) in RPE medium (Lonza, 1% FBS). Cultures were evaluated by TEER, ZO-1, and pigmentation at several time points (14/21/28d) throughout their maintenance period of 3m. To induce replicative aging, hRPE cells were grown to 75-80% confluence, serially passaged by repeated trypsinization, and subcultured at a 1:4 ratio, until the cells ceased to divide. Population doublings (PDLs) were estimated as two PDLs per passage. Expression of senescence-associated markers (SASP) and SA-β-Gal activity was used to confirm cellular senescence. To study the effects of histone depletion in hRPE cells, we performed siRNA-mediated knockdown of HINFP, a key histone transcription regulator (vs. scrambled siRNA control, 50pmol, Lipofectamine 3000). Histone gene expression profiling and histone PTMs were measured via qPCR, IF, and Western blot.
Results :
In this study, we observed a significant decrease in global histone expression in both chronological and replicative aging models in hRPE cells. Aged hRPE cultures demonstrated significantly increased SA-β-Gal staining and SASP factors, including proinflammatory cytokines, interleukins, and extracellular matrix components. Global histone deficiency was accompanied by histone PTM signatures including specific acetyl- and methyl-histone marks. HINFP knockdown resulted in significantly decreased canonical histone expression (range 40-80%). Cultures exhibited reduced proliferation along with increased SASP markers and SA-β-Gal staining.
Conclusions :
These data provide highly innovative insights into epigenetic mechanisms of aging and senescence in the human RPE. Deciphering the cellular molecular responses to global histone deficiency and associated PTMs will be critical to our modern scientific understanding of biological aging and age-related macular degeneration.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.