Nrf2 deficiency decreases NADPH from impaired IDH shuttle and pentose phosphate pathway in retinal pigmented epithelial cells to magnify oxidative stress‐induced mitochondrial dysfunction

• Published in: Aging cell 2021-08, Vol.20 (8), p.e13444-n/a
• Main Authors: Cano, Marisol, Datta, Sayantan, Wang, Lei, Liu, Tongyun, Flores‐Bellver, Miguel, Sachdeva, Mira, Sinha, Debasish, Handa, James T.
• Format: Article
• Language: English
• Subjects: Aging; Animals; Antioxidants; Apoptosis; Blindness; Cell death; Cigarette smoke; Cigarettes; Dehydrogenases; Disease; Electron transport chain; Enzymes; Epithelial cells; Humans; Hydrogen peroxide; Immunohistochemistry; Induced Pluripotent Stem Cells; Isocitrate Dehydrogenase - metabolism; Macular degeneration; Mice; Mitochondria; Mitochondria - metabolism; NADP - metabolism; NF-E2-Related Factor 2 - deficiency; Original; Original Paper; Oxidative stress; Oxidative Stress - physiology; Pentose Phosphate Pathway; Photoreceptors; Proteins; Reactive oxygen species; Reactive Oxygen Species - metabolism; Retina; Retinal Pigment Epithelium - metabolism; Smoking
• ISSN: 1474-9718; 1474-9726
• DOI: 10.1111/acel.13444

The nuclear factor‐erythroid 2‐related factor‐2 (Nrf2), a major antioxidant transcription factor, is decreased in several age‐related diseases including age‐related macular degeneration (AMD), the most common cause of blindness among the elderly in western society. Since Nrf2’s mito‐protective response is understudied, we investigated its antioxidant response on mitochondria. Control and Nrf2‐deficient retinal pigmented epithelial (RPE) cells were compared after treating with cigarette smoke extract (CSE). Mitochondrial antioxidant abundance and reactive oxygen species (ROS) were quantified. Mitochondrial function was assessed by TMRM assay, NADPH, electron transport chain activity, and Seahorse. Results were corroborated in Nrf2−/− mice and relevance to AMD was provided by immunohistochemistry of human globes. CSE induced mitochondrial ROS to impair mitochondrial function. H2O2 increase in particular, was magnified by Nrf2 deficiency, and corresponded with exaggerated mitochondrial dysfunction. While Nrf2 did not affect mitochondrial antioxidant abundance, oxidized PRX3 was magnified by Nrf2 deficiency due to decreased NADPH from decreased expression of IDH2 and pentose phosphate pathway (PPP) genes. With severe CSE stress, intrinsic apoptosis was activated to increase cell death. PPP component TALDO1 immunolabeling was decreased in dysmorphic RPE of human AMD globes. Despite limited regulation of mitochondrial antioxidant expression, Nrf2 influences PPP and IDH shuttle activity that indirectly supplies NADPH for the TRX2 system. These results provide insight into how Nrf2 deficiency impacts the mitochondrial antioxidant response, and its role in AMD pathobiology. The antioxidant transcription factor Nrf2 does not regulate mitochondrial antioxidant gene expression, but instead genes in the pentose phosphate pathway and the IDH shuttle, which together provide NADPH to the mitochondria for the TRX2 system to neutralize H2O2. With Nrf2 deficiency, which occurs in age‐related macular degeneration, mitochondria become damaged by inadequately neutralized H2O2 after smoking.