Partial epigenetic reprogramming of RPE cells reverses age and rejuvenates mitochondrial metabolism

Abstract

Purpose : An important aspect of the aging retina is that RPE cells decrease in number, increase in cellular dysfunction, and become more susceptible to oxidative stress. This coincides with a loss of mitochondrial activity. We previously showed that “partial epigenetic reprogramming” of RPE by AAV2-OSK gene therapy (OSK: Oct4, Sox2, Klf4) reversed the age and restored visual function in old mice. However, whether partial reprogramming enhanced RPE mitochondrial activity and/or increased their resistance to oxidative stress is unknown. Complete reprogramming with OSKM (OSK+ cMyc) of somatic cells to iPSCs triggers a dramatic shift in mitochondrial activity, indicating complete reprogramming directly or indirectly alters mitochondrial activity. We hypothesize that partial epigenetic reprogramming of RPE cells reverses age, improves cellular function, and increases mitochondrial capacity.

Methods : ARPE19 cells were transduced with doxycycline (dox) inducible OSK or GFP lentiviral vectors. 24h after OSK induction, matured ARPE19 cells were challenged with oxidative agents NaIO₃ (5mM) and Ox-LDL, causing high oxidative stress. Cell viability and oxidative phosphorylation (OXPHOS) was examined by high-resolution respirometry on the Seahorse XFe96 Mito Stress Test.

Results : Epigenetic reprogramming with OSK rescued RPE cells from NaIO₃ or ox-LDL induced cell death and restored completely blunted mitochondrial metabolism. In cells that were not challenged with NaIO₃, OSK expression induced a rapid and robust upregulation in basal and maximal mitochondrial OXPHOS capacity at 24-h, with maximum upregulation at 4-days tapering down to basal levels over the 7-day treatment period compared to the GFP control and no-DOX controls.

Conclusions : Previously, we have reported that in vivo epigenetic reprogramming with OSK reversed age-induced morphological changes, restoring RPE to a youthful morphology and improved visual acuity. Mechanistically OSK induces a rapid enhancement of mitochondrial OXPHOS capacity which facilitates the rejuvenation of the metabolically active RPE. These data imply that epigenetic reprogramming can reverse the effects of aging and restore function to aging RPE and is potentially an exciting and novel therapeutic approach to treat AMD.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.