Generation of reactive oxygen species in 1-methyl-4-phenylpyridinium (MPP+) treated dopaminergic neurons occurs as an NADPH oxidase-dependent two-wave cascade

BACKGROUND: Reactive oxygen species (ROS), superoxide and hydrogen peroxide (H(2)O(2)), 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 H(2)O(2)-induced carboxy-H(2)-DCFDA fluorescence. Cells were treated with MPP+ (MPTP metabolite) following siRNA silencing of the Nox2-stabilizing subunit p22(phox), 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 p22(phox), or losartan. A two-wave ROS cascade was identified: 1) as a first wave, mitochondrial H(2)O(2 )production was first noted at three hours of MPP+ treatment; and 2) as a second wave, H(2)O(2 )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.