Low-dose mTOR inhibitor alleviates RPE cellular senescence and pro-inflammation induced by accumulation of cytosolic nuclear DNA fragments

Abstract

Purpose : Retinal pigment epithelium (RPE) dysfunction plays an important role in the progression of age-related macular degeneration (AMD). Our previous studies have showed there is accumulation of damaged nuclear DNA (nDNA) fragments in AMD donor macular RPE cells. This accumulation induced RPE cellular senescence and pro-inflammation via STING pathway. More and more studies found long-term low dose rapamycin treatment, a mTOR inhibitor, reduced inflammation and cellular senescence without side effects. In this study, we explored whether low-dose rapamycin reduces pro-inflammatory factors secretion and cellular senescence induced by accumulation of cytosolic nDNA fragments in RPE cells.

Methods : RPE cell viability was tested by CCK-8 assay. Since lysosomal DNASE2A is essential for damaged nDNA fragments digestion, CRISPR/Cas9 was used to generate DNASE2A^-/- RPE cells to produce accumulation of cytosolic nDNA to mimic AMD donor RPE cells. AMD donor RPE cells and DNASE2A^-/- RPE cells were treated with 1nM rapamycin for 5 weeks. Cytosolic nDNA, components of mTOR and STING pathway and pro-inflammatory factors were measured by immunostaining, Western blot and ELISA. Cellular senescence was tested by SA-β-galactosidase staining. Autophagy inhibitor was used to explore cytosolic nDNA removal mechanism. nDNA secretion and LC3 conversion was measured.

Results : High-dose rapamycin (≥25μM) significantly decreased ARPE-19 and DNASE2A^-/- RPE cell viability while low dosage did not. Long-term low dose rapamycin treatment increased AMD donor macular RPE cells and DNASE2A^-/- RPE cells to secrete damaged nDNA fragments via enhancing autophagy. In this way, rapamycin reduced accumulation of cytosolic nDNA fragments in AMD donor macular RPE cells and DNASE2A^-/- RPE cells, which led to attenuate mTOR activation and cellular senescence induced by accumulation of cytosolic nDNA fragments in DNASE2A^-/- RPE cells. Furthermore, we also found STING and NFκB activation were inhibited by low-dose rapamycin treatment, thereby pro-inflammatory factors secretion (IL-1β, IL-6, IL-8) was reduced in AMD donor macular RPE cells and DNASE2A^-/- RPE cells.

Conclusions : Our studies show long-term low dose rapamycin treatment reduces RPE cellular senescence and pro-inflammation induced by accumulation of cytosolic nDNA fragments and has the potential to alleviate progressive RPE dysfunction in AMD.

This is a 2021 ARVO Annual Meeting abstract.