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

被引:94
作者
Anantharam, Vellareddy [1 ]
Kaul, Siddharth [1 ]
Song, Chunjuan [1 ]
Kanthasamy, Arthi [1 ]
Kanthasamy, Anumantha G. [1 ]
机构
[1] Iowa Ctr Advanced Neurotoxicol, Dept Biomed Sci, Parkinsons Disorder Res Lab, Ames, IA USA
关键词
oxidative damage; dopamine; NADPH oxidase inhibitor; neurotoxicity; neuroprotection; Parkinson's disease;
D O I
10.1016/j.neuro.2007.08.008
中图分类号
Q189 [神经科学];
学科分类号
071006 ;
摘要
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 deter-mined 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 mu M), apocynin (100-1000 mu M), and diphenylene iodonium (DPI, 3-30 mu M), to inhibit intrinsic NADPH oxidase activity in N27 cells. Flow cytometric analysis using the ROS-sensitive dye hydroethidine revealed that AEBSF blocked 300 mu M 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 It 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. (C) 2007 Elsevier Inc. All rights reserved.
引用
收藏
页码:988 / 997
页数:10
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