Exploring mechanism of stress induced accelerated aging under repetitive stress.

Abstract

Purpose : Previously, we have shown that young retinas exposed to recurrent IOP elevations show transcriptional dysregulation, chromatin remodeling, accumulation of senescent cells and an acceleration of epigenetic age and vision loss. In this study, we explore the mechanism behind observed changes and the potential of senolytic approach to protect RGCs. The goal of the study is to elucidate molecular mechanism behind stress-induced aging and to explore novel therapeutic avenues for glaucoma.

Methods : 3-month-old C57BL/J animals were exposed to unilateral IOP elevation (1h, 30mm Hg) 4 times, every two weeks. The mechanism of accelerated aging was explored through: 1. qPCR – to reveal upregulation of target genes. 2. DNA-methylation pattern – to estimate epigenetic age. 3. Pattern electroretinography (PERG) – to assess RGC function. 4. RBPMS immunostaining – for RGC counts. 5. In situ hybridization (RNAscope) – to show changes in the retina and within RGC layer. Next, after each IOP challenge, animals were treated intraperitoneally (i.p.) for 5 days, with a known senolytic agent, dasatinib. Sham-treated animals (PBS/DMSO i.p.) served as a control.

Results : Exposure of young retinas to repeated low level IOP elevation leads to significant vision loss. RGC function shows a dramatic drop after 3^rd IOP elevation episode (mean 3.67 uV vs. 12.56 uV, p<0.001). RGC loss shows a similar pattern (30% reduction in 4xIOP, p>0.0001). Gene expression analysis revealed upregulation of genes involved in senescence (e.g., Serpine, CCL2) and aging (e.g., GDF15). Specifically, robust upregulation of Timp1 was observed after 3^rd IOP elevation (fold>=200) and was further enhanced after 4^th (fold>=1000). RNAScope showed upregulation of genes in RGC layer. Interestingly, epigenetic clock analysis revealed increased epigenetic age following only the 4^th IOP. Finally, dasatinib treatment show significant protection of vision following exposure to repetitive IOP (mean 3.38 uV vs. 6.66 uV, p<0.05).

Conclusions : Our data show that repetitive low-level stress leads to a stepwise loss of function, structure, and upregulation of genes associated with inflammation, senescence, and aging. Ultimately, these processes lead to accelerated aging on molecular level. Moreover, we show a potential approach to halt the vision loss associated with age-related eye conditions.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.