Pharmacological inhibition of neuronal NADPH oxidase protects against 1-methyl-4-phenylpyridinium (MPP +)-induced oxidative stress and apoptosis in mesencephalic dopaminergic neuronal cells

Abstract

Oxidative stress is widely recognized as a key mediator of degenerative processes in Parkinson's disease (PD). Recently, we demonstrated that the dopaminergic toxin MPP + initiates oxidative stress to cause caspase-3-dependent apoptotic cell death in mesencephalic dopaminergic neuronal (N27) cells. In this study, we determined the source of reactive oxygen species (ROS) produced during MPP +-induced apoptotic cell death. In addition to mitochondria, plasma membrane NADPH oxidase is considered a major producer of ROS inside the cell. Here, we show that N27 neuronal cells express key NADPH oxidase subunits gp91 phox and p67 phox. We used structurally diverse NADPH oxidase inhibitors, aminoethyl-benzenesulfonylfluoride (AEBSF, 100–1000 μM), apocynin (100–1000 μM), and diphenylene iodonium (DPI, 3–30 μM), to inhibit intrinsic NADPH oxidase activity in N27 cells. Flow cytometric analysis using the ROS-sensitive dye hydroethidine revealed that AEBSF blocked 300 μM MPP +-induced ROS production for over 45 min in N27 cells, in a dose-dependent manner. Further treatment with DPI, apocynin, and SOD also blocked MPP +-induced ROS production. In Sytox cell death assays, co-treatment with AEBSF, apocynin, or DPI for 24 h significantly suppressed MPP +-induced cytotoxic cell death. Similarly, co-treatment with these inhibitors also significantly attenuated MPP +-induced increases in caspase-3 enzymatic activity. Furthermore, quantitative DNA fragmentation ELISA assays revealed that AEBSF, DPI, and apocynin rescue N27 cells from MPP +-induced apoptotic cell death. Together, these results indicate for the first time that intracellular ROS generated by NAPDH oxidase are present within the mesencephalic neuronal cells, and are a key determinant of MPP +-mediated dopaminergic degeneration in in vitro models of dopaminergic degeneration. This study supports a critical role of NADPH oxidase in the oxidative damage in PD; targeting this enzyme may lead to novel therapies for PD.