Photoreceptor and RPE co-expression of a dominant mutant polymerase gamma subunit 1 leads to accelerated retinal degeneration in wild type mice

Abstract

Purpose : AMD patients accumulate RPE mitochondrial DNA mutations. Furthermore, mitochondrial haplotypes, (e.g., haplotype J) are linked to AMD. In mice, increased expression of a dominant mutant polymerase gamma-subunit 1 (PolG1^D1134A) causes accelerated skin aging. We have reported that this mutant PolG1 increases mitochondrial DNA mutations, decreases mitochondrial genomes copies, lowers expression of antioxidant genes in ARPE-19 cells. Herein, we define the effects of mutant PolG1 expression in photoreceptors and RPE in C57BL6/J mice.

Methods : We injected C57BL6/J mice subretinally with 10¹⁰ vector genome copies of rAAV5, delivering either GFP or PolG1^D1134A under the control of the small CBA promoter. We evaluated these mice's retina at one, two, and four months. We use electroretinography (ERG), spectral-domain optical coherence tomography (SD-OCT), and fundoscopy for each evaluation. After the final assessment, we euthanized these mice harvested their eyes. We used immunofluorescence to detect DNA damage (8-OHdG), changes in RPE65, and tight junctions (ZO-1). Finally, we used ultra-thin plastic sections to identify morphological changes within the retina.

Results : Mutant PolG1 expression in the retina decreased the a-, b-, and c-wave amplitude by 50% compared to GFP treated eyes beginning at one month after injection. The ONL of these mice was also significantly thinner when measured in SD-OCT images. Retinas cross-sections showed an abnormal distribution of RPE65 in mutant PolG1 treated mice. Flat-mounts co-stained with ZO-1 and 8-OHdG showed localized increase in oxidized DNA in animals treated with mutant PolG1. Ultra-thin retina sections of mutant PolG1 treated eyes showed accumulation of toluidine positive material under the Bruch's membrane, and death of RPE cells.

Conclusions : Our studies suggest that dominant mutant PolG1 expression in both photoreceptors and RPE cells results in accelerated retinal degeneration. This degeneration was characterized by a substantial decrease in retinal function (ERG response) and structure (SD-OCT, immunofluorescence, and ultra-thin sections). Future studies will elucidate the RPE and photoreceptors individual contribution to retinal degeneration due to mutant PolG1 expression, while also identifying changes in markers of accelerated aging in the retina.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.