BACKGROUND:Reactive oxygen species (ROS), superoxide and hydrogen peroxide (H2O2), are necessary for appropriate responses to immune challenges. In the brain, excess superoxide production predicts neuronal cell loss, suggesting that Parkinson's disease (PD) with its wholesale death of dopaminergic neurons in substantia nigra pars compacta (nigra) may be a case in point. Although microglial NADPH oxidase-produced superoxide contributes to dopaminergic neuron death in an MPTP mouse model of PD, this is secondary to an initial die off of such neurons, suggesting that the initial MPTP-induced death of neurons may be via activation of NADPH oxidase in neurons themselves, thus providing an early therapeutic target.METHODS:NADPH oxidase subunits were visualized in adult mouse nigra neurons and in N27 rat dopaminergic cells by immunofluorescence. NADPH oxidase subunits in N27 cell cultures were detected by immunoblots and RT-PCR. Superoxide was measured by flow cytometric detection of H2O2-induced carboxy-H2-DCFDA fluorescence. Cells were treated with MPP+ (MPTP metabolite) following siRNA silencing of the Nox2-stabilizing subunit p22phox, or simultaneously with NADPH oxidase pharmacological inhibitors or with losartan to antagonize angiotensin II type 1 receptor-induced NADPH oxidase activation.RESULTS:Nigral dopaminergic neurons in situ expressed three subunits necessary for NADPH oxidase activation, and these as well as several other NADPH oxidase subunits and their encoding mRNAs were detected in unstimulated N27 cells. Overnight MPP+ treatment of N27 cells induced Nox2 protein and superoxide generation, which was counteracted by NADPH oxidase inhibitors, by siRNA silencing of p22phox, or losartan. A two-wave ROS cascade was identified: 1) as a first wave, mitochondrial H2O2 production was first noted at three hours of MPP+ treatment; and 2) as a second wave, H2O2 levels were further increased by 24 hours. This second wave was eliminated by pharmacological inhibitors and a blocker of protein synthesis.CONCLUSIONS:A two-wave cascade of ROS production is active in nigral dopaminergic neurons in response to neurotoxicity-induced superoxide. Our findings allow us to conclude that superoxide generated by NADPH oxidase present in nigral neurons contributes to the loss of such neurons in PD. Losartan suppression of nigral-cell superoxide production suggests that angiotensin receptor blockers have potential as PD preventatives.

Oxidative stress is a key contributor to the pathogenesis of Parkinson's disease. NADPH oxidase is a membrane enzyme responsible for the oxidative burst in activated phagocytes. NADPH oxidase also exists in many types of non‐phagocytic cells including vascular cells and neurons, with a wide distribution throughout the brain. Although the microglial NADPH oxidase is known to participate in dopaminergic neurotoxicity, whether NADPH oxidase also exists in the dopamine neurons in the substantia nigra pars compacta (SNpc) and whether it contributes to the ROS generation has not been explored. We have examined the role of NADPH oxidase in MPP+ induced oxyradical production in immortalized dopaminergic cell line (N27). MPP+ dose dependently led to upregulation of ROS in N27 cells, which was attenuated by 40% with treatment with NADPH oxidase inhibitors. N27 cells were observed to express mRNA for all the necessary components of the NADPH oxidase, Nox2, p22, p40, p47 and p67, as well as Nox1 and Nox4. SiRNA‐mediated knockdown of p22, a critical stabilizing subunit of the NADPH oxidase, reduced the amount of ROS generated. Immunocytochemical examination of mouse dopaminergic neurons in sections of the SNpc revealed that these neurons express Nox2, p47 and p67. While our results suggest that NADPH oxidase is a contributor to ROS generation in dopaminergic neurons, the functional role of this process remains to be defined. Supported by NIH AA016654 and The Neuroscience Program at the UCD.