Abstract
BackgroundReactive 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.MethodsNADPH 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.ResultsNigral 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.ConclusionsA 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.
Highlights
Reactive oxygen species (ROS), superoxide and hydrogen peroxide (H2O2), are necessary for appropriate responses to immune challenges
We found that: i) N27 cells express all the components of NADPH oxidase that are required for its activation; ii) that treatment of these cells with NADPH oxidase inhibitors, or with an angiotensin II type 1 receptor blocker leads to an attenuation of MPP+ induced generation of hydrogen peroxide (H2O2, a product of superoxide dismutation); iii) MPP+ treatment induced a biphasic generation of H2O2; and iv) NADPH oxidase inhibitors blocked selectively only the second wave of H2O2 production
NADPH oxidase subunits are expressed in both dopaminergic neurons in the mouse substantia nigra pars compacta and in the rat dopaminergic cell line N27 NADPH oxidase is present in microglia [11], astrocytes [46], and in certain types of neurons in hippocampus and cortex [46,47]
Summary
Reactive oxygen species (ROS), superoxide and hydrogen peroxide (H2O2), are necessary for appropriate responses to immune challenges. Reactive oxygen species (ROS) contribute to cellular signaling, affecting most aspects of cellular function including gene expression, proliferation, differentiation, and migration [1,2]. An alternative and potentially sounder approach to suppression of NADPH oxidase-generated superoxide utilizes angiotensin II type 1 (AT1) receptor blockers, exemplified by the original compound in this class, losartan [19,20]. This is possible because generation of superoxide from NADPH oxidase is promoted by angiotensin II binding to the AT1 receptor, leading to induction of protein kinase C-induced Nox signaling [19]. Antagonists of the AT1 receptor such as candesartan and losartan suppress angiotensin II-induced increases in superoxide production and Nox expression [21]
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