Considering the fact that NADPH oxidase is the main

Considering the fact that NADPH oxidase is the main source for paraquat and maneb-induced oxidative stress, we hypothesized that NADPH oxidase activation might contribute to dopaminergic neurodegeneration induced by paraquat and maneb through ferroptosis. To test our hypothesis, we investigated the regulatory effects and underlying mechanisms of NADPH oxidase on ferroptosis by using paraquat and maneb (referred to subsequently as P + M) -induced in vitro and in vivo dopaminergic neurodegeneration models.
Materials and methods
Results
Discussion In the present study, we demonstrated that ferroptosis was involved in NADPH oxidase-regulated dopaminergic neurotoxicity in P + M-induced in vitro and in vivo dopaminergic neurodegeneration models. The salient features of our study are: 1) P + M induced ferroptosis in SHSY5Y cells, which was inhibited by NADPH oxidase inhibitors, but exacerbated when the RNA was stimulated by PMA or supplemented with H2O2; 2) Inhibition of NADPH oxidase abrogated P + M-induced lipid peroxidation and reduction of GPX4; 3) NADPH oxidase inhibitor apocynin suppressed the ferroptotic features in P + M-intoxicated mice, which was associated with dopaminergic neuroprotection and motor improvement. The inhibition of mitochondrial complex I activity has long been considered as a leading mechanism for P + M-induced dopaminergic neurotoxicity. However, the loss of mitochondrial complex I activity by genetic deletion of Ndufs4, a gene encoding a subunit required for complete assembly and function of complex I, fails to interfere with dopaminergic neurodegeneation induced by both paraquat and maneb (Choi et al., 2008; Choi and Xia, 2014). Furthermore, a recent study revealed no association between complex I deficiency and dopaminergic neurodegeneration in patients with PD (Flones et al., 2018), suggesting that alternative mechanisms underlay P + M-induced dopaminergic neurotoxicity. Accumulating evidence suggested a critical role of NADPH oxidase in mediating P + M-induced dopaminergic neurotoxicity in both in vivo and in vitro. Consistently, we recently found that inhibition of NADPH oxidase markedly mitigated P + M-induced neurotoxicity in mice (Hou et al., 2017a). In this study, we extended previous findings and showed that NADPH oxidase activation contributed to P + M-induced ferroptosis of dopaminergic neurons. Inhibition of NADPH oxidase mitigated, whereas NADPH oxidase activation exacerbated P + M-induced ferroptosis in vitro. Furthermore, the ferroptotic features, including elevated iron content, lipid peroxidation and neuroinflammation as well as reduced GPX4 expression, which in combination can separate ferroptotic cell death from apoptotic cell death (Xie et al., 2016), in P + M-treated mice were recovered by NADPH oxidase inhibitor apocynin. Similar to our findings, inhibition of NADPH oxidase also blocks ferroptotic cell death induced by deprivation of cystine in human mammary epithelial cells (Poursaitidis et al., 2017). Mechanistically, the most critical question to address is how NADPH oxidase regulates ferroptosis. NADPH oxidase is a membrane-bound enzyme complex responsible for the respiratory burst. ROS generated by NADPH oxidase can react with the polyunsaturated fatty acids (PUFAs) of lipid membranes and induce lipid peroxidation (Hernandes and Britto, 2012; Ma et al., 2017). Genetic deletion or pharmacological inhibition of NADPH oxidase not only blocks ROS production but also lipid peroxidation in multiple pathological conditions (Zhang et al., 2014a), seizure (Kim et al., 2013; Pecorelli et al., 2015). An amount of evidence suggested that oxidative stress derived by NADPH oxidase is a central mechanism of chronic neurodegeneration in various neurodegenerative disorders, including PD (Chong et al. 2005; Potashkin and Meredith, 2006). In this study, we found that inhibition of NADPH oxidase attenuated, whereas stimulating activation of NADPH oxidase exacerbated lipid peroxidation induced by P + M, which might be underlay the regulatory effects of NADPH oxidase on ferroptosis. Consistent with our findings, the canonical NADPH oxidase inhibitor DPI and the NADPH oxidase 1/4-specific inhibitor GKT137831 also inhibited erastin-induced ferroptosis in Calu-1 and HT1080 cells (Dixon et al., 2012). Additionally, recovered expressions of GPX4, a key negative regulator of ferroptosis, by NADPH oxidase inhibitor apocynin might be another potential reason. Although the mechanisms remain unclear, similar findings were observed in Pan’s study, in which a uremic toxin, para-cresol (p-cresol) inhibits NADPH oxidase-derived ROS that is associated with increased expressions of GPX4 in human endothelial progenitor cells (Pan et al., 2017).