Abstract
The Nrf2 (NF-E2 related factor 2)-ARE (antioxidant response element) pathway controls a powerful array of endogenous cellular antioxidant systems and is an important pathway in the detoxification of reactive oxygen species (ROS) in the brain. Using a combination of quantitative proteomics and siRNA screening, we have identified novel protective mechanisms of the Nrf2-ARE pathway against oxidative stress in astrocytes. Studies from our lab and others have shown Nrf2 overexpression protects astrocytes from oxidative stress. However, the exact mechanisms by which Nrf2 elicits these effects are unknown. In this study, we show that induction of Nrf2 reduces levels of reactive oxygen species (ROS) produced by various oxidative stressors and results in robust cytoprotection. To identify the enzymes responsible for these effects, we used stable isotope labeling by amino acids in cell culture (SILAC) and quantitative shotgun proteomics to identify 72 Nrf2-regulated proteins in astrocytes. We hypothesized a subset of these proteins might play a critical role in Nrf2 protection. In order to identify these critical proteins, we used bioinformatics to narrow our target list of proteins and then systematically screened each candidate with siRNA to assess the role of each in Nrf2 protection. We screened each target against H2O2, tert-butyl hydroperoxide, and 4-hydroxynonenal and subsequently identified three enzymes–catalase, prostaglandin reductase-1, and peroxiredoxin-6–that are critical for Nrf2-mediated protection in astrocytes.
Highlights
Oxidative stress has been implicated as a causative agent in a wide spectrum of neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis [1,2,3]
NF-E2related factor-2 (Nrf2) is regulated by its binding partner Kelch ECH associating protein 1 (Keap1) [14,15,16] and under normal conditions is sequestered by Keap1 in the cytoplasm; under conditions of oxidative stress, Nrf2 is released from Keap1 and translocates to the nucleus where it binds the antioxidant response element (ARE) and drives gene expression [17]
Nrf2 induction with tert-butyl hydroquinone (tBHQ) confers robust protection against H2O2 toxicity (Figure 1A). This protection is reversed in Nrf2 knockout astrocytes, demonstrating that Nrf2 is required for tBHQ protection (Figure 1B)
Summary
Oxidative stress has been implicated as a causative agent in a wide spectrum of neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis [1,2,3]. Genes under the putatiave control of the Nrf2-ARE pathway are direct antioxidants, including glutathione [18,19]; enzymes that inactivate ROS, including superoxide dismutase and catalase [20,21]; reductive co-factors, including NADPH, [22]; and enzymes involved in protein turnover and homeostasis [23]. Astrocytic-specific Nrf activation confers protection against ROS to co-cultured neurons [6,7,9]. Astrocyte-specific overexpression in vivo has been shown to mitigate disease pathogenesis in animal models of Huntington’s disease, amyotrophic lateral sclerosis, Parkinson’s disease, and Alexander’s disease [9,24,25,26]
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