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Emily Brown, Casey Keuthan, John Ash; Measuring mitochondrial flux in RPE under conditions of oxidative stress. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6553.
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The goal of this study is to examine mitochondrial dynamics during conditions of oxidative stress using mitochondrial reporter tools. It has been demonstrated that there are changes in mitochondrial morphology, as well as an increase in oxidative stress in patients with age-related macular degeneration (AMD). Mitochondrial reporter tools allow us to examine mitochondrial flux in cell culture and in animal models under various conditions. This provides a mechanism to potentially identify whether an increase in mitochondrial flux provides protection against oxidative stress, and subsequently test therapeutic targets that are possibly capable of increasing mitochondrial flux.
ARPE-19 cells were transiently transfected with plasmids containing either YFP-tagged mitofusin 2 (Mfn2), mCherry-tagged Dynamin-related protein (Drp1), or mitochondrial-targeted pMito-Timer, with a dsRed mutant fluorophore that switches from green to red once oxidized. Confocal microscopy was used to image cells over time. Cells were treated with various concentrations of paraquat to induce oxidative stress.
Mitochondrial reporter tools demonstrate that there is a shift in mitochondrial flux during oxidative stress in ARPE-19 cells. Our data using pMito-Timer show that oxidative stress increased both oxidation in mitochondria, as well as synthesis of new mitochondrial proteins in these cells. We show that under oxidative stress Mfn-2 and Drp-1 levels are altered as well.
Mitochondrial reporter tools can be utilized to examine mitochondrial flux in ARPE-19 cells. During oxidative stress in ARPE-19 cells, levels of mitochondrial protein oxidation and synthesis are elevated. We hypothesize that an increase in mitochondrial flux can be protective against oxidative damage. Our future goal is to use these tools in animal models of oxidative stress in the RPE to determine if mitochondrial flux is altered under these conditions. We also aim to identify whether an increase in mitochondrial flux is protective and if treatment with agonists, such as metformin, results in a shift in mitochondrial dynamics in these models.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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