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

Zawada WM, Banninger GP, Thornton J, Marriott B, Cantu D, Rachubinski AL, Das M, Griffin WS, Jones SM - J Neuroinflammation (2011)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Donald W, Reynolds Department of Geriatrics, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA. wzawada@uams.edu

ABSTRACT

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.

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Dopamine neurons in the adult female C57BL/6J mouse substantia nigra express Nox2, p47phox and p67phox subunits of the NADPH oxidase. Immunofluorescent histochemical staining revealed the presence of Nox2, p47phox and p67phox (green) in the neurons positive for TH (red). Merged images indicate co-labeling (yellow). Occasionally cells positive for a subunit were not dopaminergic neurons (TH-cells). To illustrate such an example, arrows in the top row point to a Nox2+/TH- cell and arrowheads in the middle row indicate a cell that is p47phox+/TH-. Scale bars equal 20 μm.
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Figure 1: Dopamine neurons in the adult female C57BL/6J mouse substantia nigra express Nox2, p47phox and p67phox subunits of the NADPH oxidase. Immunofluorescent histochemical staining revealed the presence of Nox2, p47phox and p67phox (green) in the neurons positive for TH (red). Merged images indicate co-labeling (yellow). Occasionally cells positive for a subunit were not dopaminergic neurons (TH-cells). To illustrate such an example, arrows in the top row point to a Nox2+/TH- cell and arrowheads in the middle row indicate a cell that is p47phox+/TH-. Scale bars equal 20 μm.

Mentions: NADPH oxidase is present in microglia [11], astrocytes [46], and in certain types of neurons in hippocampus and cortex [46,47]. Here, in adult mice, we show that dopaminergic TH immunoreactive neurons in substantia nigra coexpress three of the NADPH oxidase subunits, viz., Nox2, the catalytic subunit responsible for superoxide generation, as well as the two subunits, p47phox and p67phox, necessary for Nox2 activation (Figure 1). The expression was predominantly cytoplasmic, no nuclear localization was observed, and negative control staining by omitting the primary antibody did not produce detectable immunoreactivity (data not shown). While all TH-immunoreactive neurons expressed these subunits, occasionally cells lacking TH expressed some subunits, suggesting that dopaminergic neurons are not the only cell type in substantia nigra capable of assembling the NADPH oxidase enzyme. Non-TH immunoreactive cell candidates that express NADPH oxidase may include other neuronal cell types, or more likely microglia and astrocytes [48], which when activated are known to express high levels of this enzyme [11,49].


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

Zawada WM, Banninger GP, Thornton J, Marriott B, Cantu D, Rachubinski AL, Das M, Griffin WS, Jones SM - J Neuroinflammation (2011)

Dopamine neurons in the adult female C57BL/6J mouse substantia nigra express Nox2, p47phox and p67phox subunits of the NADPH oxidase. Immunofluorescent histochemical staining revealed the presence of Nox2, p47phox and p67phox (green) in the neurons positive for TH (red). Merged images indicate co-labeling (yellow). Occasionally cells positive for a subunit were not dopaminergic neurons (TH-cells). To illustrate such an example, arrows in the top row point to a Nox2+/TH- cell and arrowheads in the middle row indicate a cell that is p47phox+/TH-. Scale bars equal 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198931&req=5

Figure 1: Dopamine neurons in the adult female C57BL/6J mouse substantia nigra express Nox2, p47phox and p67phox subunits of the NADPH oxidase. Immunofluorescent histochemical staining revealed the presence of Nox2, p47phox and p67phox (green) in the neurons positive for TH (red). Merged images indicate co-labeling (yellow). Occasionally cells positive for a subunit were not dopaminergic neurons (TH-cells). To illustrate such an example, arrows in the top row point to a Nox2+/TH- cell and arrowheads in the middle row indicate a cell that is p47phox+/TH-. Scale bars equal 20 μm.
Mentions: NADPH oxidase is present in microglia [11], astrocytes [46], and in certain types of neurons in hippocampus and cortex [46,47]. Here, in adult mice, we show that dopaminergic TH immunoreactive neurons in substantia nigra coexpress three of the NADPH oxidase subunits, viz., Nox2, the catalytic subunit responsible for superoxide generation, as well as the two subunits, p47phox and p67phox, necessary for Nox2 activation (Figure 1). The expression was predominantly cytoplasmic, no nuclear localization was observed, and negative control staining by omitting the primary antibody did not produce detectable immunoreactivity (data not shown). While all TH-immunoreactive neurons expressed these subunits, occasionally cells lacking TH expressed some subunits, suggesting that dopaminergic neurons are not the only cell type in substantia nigra capable of assembling the NADPH oxidase enzyme. Non-TH immunoreactive cell candidates that express NADPH oxidase may include other neuronal cell types, or more likely microglia and astrocytes [48], which when activated are known to express high levels of this enzyme [11,49].

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Donald W, Reynolds Department of Geriatrics, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA. wzawada@uams.edu

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus