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Marisol del Valle Cano, Sayantan Datta, Lei Wang, Tongyun Liu, Sonny Dike, James T Handa; Nrf2 Protects Mitochondrial Function from Cigarette Smoke (CS) Independent of its Mitochondrial Antioxidant Capability.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5367.
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© ARVO (1962-2015); The Authors (2016-present)
Oxidative stress is hypothesized to contribute to RPE apoptosis in Age-related macular degeneration (AMD). CS is the strongest environmental risk factor for AMD, yet how smoking affects the RPE is poorly understood. Nrf2 is a cytoprotective transcriptional factor that controls a cascade of antioxidant genes in the RPE. Nrf2 decreases with age and chronic oxidative stress. The purpose herein is to determine how CS and Nrf2 deficiency affects RPE mitochondrial function.
Confluent ARPE-19 cells were treated with 100-500 µg/ml CS extract for 24h, after treating cells with Nrf2 or scrambled siRNA. Viability was determined by MTS assay. Protein was extracted and used for western blot. ATP was determined by Cell titer glow, cellular and mitochondrial superoxide by DHE and Mitosox, respectively, NAD/NADH and Lactate using commercial kits.
Cells exposed to 100-500 µg/ml CS for 24h remained viable. CS induced an increase in cellular and mitochondrial superoxide (p≤0.01). Nrf2 knockdown (KD) further increased cellular, but not mitochondrial derived superoxide. CS greatly reduced ATP levels (p≤0.01) without cytochrome C release as an indicator of apoptosis. Nrf2KD maximized this reduction in ATP to render the cell susceptible to apoptosis with cytochrome c release and caspase 3 cleavage. The mitochondrial impairment, as suggested by the decreased ATP levels, prompted us to evaluate mechanisms other than oxidative stress that can affect ATP production. Nrf2KD causes impairment of oxidative phosphorylation due to a lack of NAD substrate. Indeed, NAD was reduced after Nrf2KD compared to controls (p≤0.02). To compensate, the cells increased glycolysis, with increased lactate levels after Nrf2KD compared to controls (p≤0.0001). CS exposure (4 hrs) decreased glycolysis in both control cells (p≤0.01) and after Nrf2KD (p≤0.0004). However, glycolysis in Nrf2KD cells remained above Nrf2 competent cells for the same CS concentration (p≤0.01). After 24 hrs of CS exposure, glycolysis was impaired, with decreased lactate levels after Nrf2KD compared to controls (p≤0.009). The lack of glycolysis as a source of ATP in cells already with mitochondrial dysfunction, rendered cells susceptible to apoptosis.
Nrf2 deficiency does not exacerbate mitochondrial derived oxidative stress, but does impair mitochondrial derived metabolic balance that is exacerbated by CS.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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