Abstract
Purpose :
7-Ketocholesterol (7KC) is a form of oxidized cholesterol present in high concentrations in drusen. While the role of 7KC in the pathogenesis of Age Related Macular Degeneration (AMD) is not well understood, there is emerging evidence of its role in altering mitochondrial function in retinal pigmented epithelial cells (RPE). Since significant changes in the mitochondria and bioenergetics of RPE have been reported in AMD pathogenesis, we sought to identify the effect of prolonged exposure to non-lethal low doses of 7KC on RPE bioenergetics.
Methods :
ARPE-19 cells were treated with 0, 1, 2 and 5µg/ml of 7KC for 7 days. Post treatment, Oxygen Consumption Rates (OCR), Extracellular Acidification Rates (ECAR) and total ATP levels were measured using an Agilent Seahorse ATP rate assay. 2-NBDG glucose assay was used to measure glucose uptake. Immunostaining and western blotting were used to measure changes in morphology and protein expression respectively.
Results :
Seven days post 7KC exposure, ECAR, total ATP production and the percentage of ATP production from glycolysis were significantly upregulated in ARPE-19 cells in a dose-dependent manner, while OCR was not significantly changed. Elevated ECAR in ARPE-19 was associated with enhanced glucose uptake and metabolism. We found an increase in TOMM20 levels as well as an increase in proteins involved in oxidative phosphorylation, NDUFS1 (Complex I), SDH (Complex II), UQCRC2 (Complex III), MTCO2 (Complex IV) and ATPB (ATP Synthase). Furthermore we found elevated expression of mitochondrial fission proteins (DRP1, FIS1 and p-DRP1Ser616) and their regulators (PINK1 and TFAM) indicating an increase in mitochondrial numbers due to fission. Additional observations in ARPE-19 cells treated with low 7KC concentrations were alterations in cell morphology with loss of epithelial markers, similar to RPE cells in AMD patients.
Conclusions :
Low doses of 7KC did not reduce mitochondrial function, but affected mitochondrial morphology and content, increased glycolysis and altered cell morphology. Increased mitochondrial numbers through fission might be an adaptive mechanism to remove damaged mitochondria and maintain mitochondrial function. Changes in morphology and bioenergetics/metabolism are critical aspects of AMD RPE pathology and suggest a possible role of 7KC accumulation in AMD pathogenesis.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.