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Sujoy Bhattacharya, Jinggang Yin, Weihong Huo, Edward Chaum; Modeling of mitochondrial bioenergetics and autophagy impairment in MELAS-mutant iPSC-derived retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 2022;63(7):800 – F0359.
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© ARVO (1962-2015); The Authors (2016-present)
Mitochondrial dysfunction and mitochondrial DNA (mtDNA) damage in the retinal pigment epithelium (RPE) have been implicated in the pathogenesis of macular degeneration. Elimination of dysfunctional mitochondria through mitochondrial autophagy (mitophagy) is an important quality control mechanism in the RPE. In this study, we model the impact of a prototypical systemic mitochondrial defect, mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), in RPE health and homeostasis as a model for impaired mitochondrial bioenergetics.
We used iPSCs derived from skin biopsies of MELAS patients (m.3243A>G tRNA leu mutation) with different levels of mtDNA heteroplasmy and differentiated them into RPE cells. Mitochondrial depletion of ARPE-19 cells (p0 cells) was also performed using 50ng/mL ethidium bromide (EtBr) and 50mg/ml uridine. Cell fusion of the human platelets with the p0 cells performed using PEG-SMEM mixture to generate platelet:RPE “cybrids”. Confocal microscopy, FLowSight Imaging cytometry, and Seahorse XF Mito Stress test were used to analyze mitochondrial function. Western blotting was used to analyze expression of autophagy and mitophagy proteins.
We found that MELAS iPSC-derived RPE cells exhibited key characteristics of native RPE. We observed heteroplasmy-dependent impairment of mitochondrial bioenergetics and reliance on glycolysis for generating energy in the MELAS iPSC-derived RPE. The degree of heteroplasmy was directly associated with increased STAT3 activation, reduced AMPKα activation, and decreased autophagic activity. In addition, impaired autophagy was associated with aberrant lysosomal function, and failure of mitochondrial recycling. The mitochondria-depleted p0 cells replicated the effects on autophagy impairment and aberrant STAT3/AMPKα signaling and showed reduced mitochondrial respiration, demonstrating phenotypic similarities between p0 and MELAS iPSC-derived RPE cells.
Our studies demonstrate that the iPSC-derived RPE lines from MELAS patients are useful tools for identifying molecular, cellular, and functional features that contribute to age-related RPE degeneration. We propose that heteroplasmic mitochondrial disease models can be used for the assessment of mitochondrial and cellular dynamics and provide a new experimental model for the RPE in aging and macular degeneration.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.
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