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 gp91phox and 
p67phox. We used structurally diverse NADPH oxidase inhibitors, 
aminoethyl-benzenesulfonylfluoride (AEBSF, 100-1000microM), apocynin 
(100-1000microM), and diphenylene iodonium (DPI, 3-30microM), to inhibit 
intrinsic NADPH oxidase activity in N27 cells. Flow cytometric analysis using 
the ROS-sensitive dye hydroethidine revealed that AEBSF blocked 300microM 
MPP+-induced ROS production for over 45min 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 24h 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.