A dominant mutant form of the POLG1 gene promotes the accumulation of the common mitochondrial deletion (mtDNA^del4977)

Abstract

Purpose : Deletions in the mitochondrial genome have been detected in multiple human diseases. These deletions, specifically the common deletion (mtDNA^del4977), have been detected significantly within the RPE of patients with early age-related macular degeneration (AMD). This common deletion eliminates genes required for the electron transfer chain (ETC). Thus, its accumulation is likely to result in increased oxidative stress, another characteristic of AMD. We have reported that RPE expression of a dominant mutant version of the PolG1 (POLG1^D1134A) increases mtDNA point mutations, oxidative stress and accumulation of oxidized lipids resulting in retinal degeneration when expressed in the RPE of wild-type mice. Herein we use in vitro methods to determine the effect of this mutant on the accumulation of the common deletion.

Methods : HEK293TN cells were transiently transfected with a plasmid delivering either GFP or POLG1^D1134A. Total DNA was isolated, and the relative presence of the common deletion was measured with qPCR. The presence of the common deletion was standardized to the mitochondrial gene ND1. In addition, we assess the presence of accumulated damaged lipids by staining cells with an Oil Red O stain. Finally, oxidative stress was evaluated using MitoSox staining in transfected cells.

Results : Transient expression of POLG1^D1134A caused a 4-fold increase in the presence of the mitochondrial common deletion compared to GFP transfected cells. Oil Red O stain of these cells showed increased staining compared to GFP transfected cells. Finally, the MitoSox stain showed high oxidative stress in cells transfected with the POLG1^D1134A mutant.

Conclusions : The expression of the POLG1^D1134A causes mitochondrial DNA common deletion (mtDNA^del4977) accumulation. Consequently, cells expressing this dominant mutant accumulate damaged lipids (Oil Red-O staining) and show more significant oxidative stress (MitoSox stain). These results establish a causative role for the accumulation of mtDNA^del4977 and cellular damage. Our future studies will focus on quantifying changes in the mitochondrial DNA common deletion in mice expressing the POLG1^D1134A mutant in their RPE. We will also correlate retinal pathology with differences in the presence of the mitochondrial common deletion. These studies will help us establish a new retinal degeneration model that recapitulates salient features of AMD.

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