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Edward Chaum, Weihong Huo, Jinggang Yin; Real-time Monitoring of Mitochondria Depletion and Rescue by Platelet Fusion Cybrids in the RPE using FlowSight Cytometry and Confocal Microscopy. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4038.
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© ARVO (1962-2015); The Authors (2016-present)
Mitochondrial activity in the RPE plays a key role in cellular energy dynamics, physiological processes, and retinal pathogenesis. Changes in mitochondria function, dynamics and gene expression may play an important mechanistic role in the pathogenesis of macular degeneration and offer potential targets for treatment. In this work we model the impact of mitochondrial dysfunction and energy depletion on the RPE by generating Rho-null (Rhoo) mitochondria-depleted ARPE19 cells followed by rescue through formation of platelet:RPE “cybrids”.
ARPE19 cells were depleted of mitochondria by culture in media containing 50ng/ml ethidium bromide (EtBr) and 50µg/ml uridine for up to six passages. At serial time points the cells were examined using confocal microscopy and FlowSight imaging cytometry. The mitochondria were stained in living cells using PicoGreen, followed by Mitotracker Red CM-H2XRos. Cell fusion of human platelets with the Rhoo cells was performed by PEG-SMEM mixture and transfer into Opti-MEM Rhoo growth media for one week. Fusion cybrids were selected by culture in media lacking uridine. Mitochondrial genes including COX4 and Tim23 expression levels were examined by Western blotting during serial passages.
ARPE19 cells were dynamically depleted of mitochondria by EtBr stress. By passage six, mitochondrial DNA could not be detected in Rhoo cells by confocal microscopy. FlowSight imaging cytometry identified 0.04% cells with detectable mitochondrial DNA. The COX4 and Tim23 protein levels decreased with mitochondria depletion. PicoGreen and Mito-Red staining was not toxic to the ARPE19 cells even during growth in mitochondrial depletion cells culture conditions.
Dynamic serial quantitative PicoGreen and Mito-Red staining methods can be used to evaluate mitochondria depletion and restoration of function using confocal microscopy and flow cytometry images analysis. These methods can be used to investigate target genes expression and modulation of mitochondrial function to explore pathogenetic mechanisms and degeneration in the RPE.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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