DEFINING METABOLIC CHANGES IN THE RPE DUE TO THE ACCUMULATION OF MITOCHONDRIAL DNA DAMAGE USING AN IN VITRO MODEL

Abstract

Purpose : The accumulation of damaged Mitochondrial DNA (mtDNA) in the RPE positively correlates with the age-related macular degeneration (AMD) disease stage. However, the mechanism by which the accumulation of damaged mtDNA impairs RPE function remains poorly defined. We hypothesize that the accumulation of damaged mtDNA causes a glycolytic switch in the RPE, leading to decreased glucose transfer towards photoreceptors. We have previously shown that expression of the PolG1^D1134A mutant gene increases both mtDNA mutations and the mtDNA^d4977 common deletion. This work aims to define the RPE metabolic changes due to an accumulation of damaged mtDNA using an in vitro model of primary human RPE cells in combination with the PolG1^D1134A gene.

Methods : We electroporated primary human RPE (hRPE) cells with a plasmid expressing either GFP or the PolG1^D1134A dominant mutant. The effect on barrier formation was studied using transepithelial resistance. The impact of mtDNA damage on the localization of GLUT1 transporter was investigated using immunofluorescence staining. [RB1] Similarly, we measured changes in glycolytic genes using RT-qPCR. Finally, to observe changes in lipid metabolism we stained ARPE-19 cells with Oil O Red.

Results : mtDNA damage caused by expression of PolG1D1134A did not significantly impact hRPE transepithelial resistance compared to the control plasmid. The expression of this mutant did cause a mis-localization of the GLUT1 transporter. hRPE cells with mtDNA damage had significant changes in the expression of glycolytic genes (e.g., a 4-fold increase in PFKM1, a 60% decrease in PKM, and an 83% decrease in HK1) 48 hrs after transfection. Finally, mtDNA damage increased the accumulation of lipid droplets positive for Oil O Red.

Conclusions : Damage to the mtDNA can alter the RPE metabolic state. Cells with damaged mitochondria appear to switch from an oxidative phosphorylation metabolism to a glycolytic metabolism based on gene expression. However, this does not appear to compromise the barrier activity of the RPE, thus suggesting a potential change towards a senescence phenotype and not cell demise. Future studies will determine whether mtDNA damage stimulates markers of senescence in the RPE and how mtDNA damage regulates the expression of nuclear genes. This work will help identify ways to control RPE damage in aging diseases like AMD.

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