Shonka, Bryce1; Skeie, Jessica1; Eggleston, Tim1; Shevalye, Hanna1; Greiner, Mark 1,2
1Iowa Lions Eye Bank, Iowa City, IA; 2University of Iowa, Department of Ophthalmology and Visual Sciences
Purpose: Oxidative damage, mitochondrial dysfunction, and UV sensitivity have been implicated in the pathogenesis of Fuch’s endothelial corneal dystrophy (FECD). Manganese superoxide dismutase (MnSOD), a mitochondrial enzyme encoded by SOD2, is an important antioxidant for CEC function and is deficient in FECD. Ferroptosis is a mechanism of nonapoptotic oxidative cell death due to iron-mediated lipid peroxidation and has been demonstrated to be upregulated in FECD surgical tissue and cell culture models. This is of interest, as SOD2, in the presence of increased iron availability, forms FeSOD2, a prooxidant peroxidase, causing mitochondrial damage, free radical formation, and increased susceptibility to oxidative stress. This has yet to be investigated in FECD.
Methods: Two SOD2-KD CEC lines, G1 and G2, were developed from B4G12 corneal endothelial cells (CECs) via CRISPR/Cas9. Knockdown was confirmed by quantifying SOD2 via microfluidic western blotting. Superoxide levels were evaluated with a MitoROS-580 assay. Mitochondrial bioenergetics, including basal respiration, ATP-associated oxygen consumption, proton leak, maximal respiration, spare respiratory capacity, and nonmitochondrial respiration were assayed via Seahorse XF96 extracellular flux analysis. All data were normalized to cell count and analyzed using a one-way ANOVA test. Cultured human patient derived TCF4 trinucleotide repeat expansion CECs, along with controls, were treated with 1 J/cm2 UVA radiation. Protein was harvested from cell lysates, and SOD2 was purified using anti-SOD2 ELISA. SOD2 isolates treated with HNO3 and H2O2, and analyzed via ICP-MS for iron concentration. Statistics were performed using an unpaired t-test.
Results: We found significant changes in superoxide levels and various markers of mitochondrial bioenergetics in SOD2-KD CECs compared to controls. Mean MitoROS levels of 0.5391 and 0.5348 for G1 and G2, respectively, were significantly higher (p<0.0001) than the levels measured for B4G12, which had a mean of 0.2978. Assessing mitochondrial bioenergetics, basal respiration was significantly decreased in both the G1 and G2 population (p<0.0001 and p<0.05), as was ATP production-coupled (p<0.0001 and p<0.05). Significant decreases in maximal respiration (p<0.0001) and spare respiratory capacity (p=0.0002) were only seen in the G1 population. Comparing UV-treated FECD cells to controls, respective means of iron-bound SOD2 were 784.6 ppm and 590.6 ppm (P=0.2889).
Conclusions: Reduced SOD2 expression in CECs results in increased levels of superoxide along with decreased levels of mitochondrial basal respiration, maximal respiration, spare respiratory capacity, and ATP production-coupled respiration in CECs. Iron-bound SOD2 protein in FECD CECs following UVA exposure is also increased compared to controls, albeit non-statistically significant due to high variability between samples. These results preliminarily indicate UV-mediated iron availability may increase transformation of SOD2 protein into prooxidant FeSOD2 in FECD CECs. Together, findings are consistent with the mitochondrial dysfunction and oxidative damage that has been noted in FECD, supporting the hypothesis that reduced or dysfunctional SOD2 may contribute to FECD pathogenesis.
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