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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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Catalytic subunit of NADPH oxidase Nox2 is elevated by the MPP+ treatment. N27 cells were treated with different concentrations of MPP+ for 24 hours. (A) Survival of N27 cells as a function of MPP+ concentration. (B) Immunofluorescent detection of Nox2 in N27 cells cultured either in the absence (control, ctrl) or presence of 300 μM MPP+, a concentration of MPP+ that results in loss of 45 percent of N27 cells. Scale bar equals to 20 μm. (C) Western immunoblot illustrating the effect of treating the cultures with increasing concentrations of MPP+ on expression of Nox2 protein. β-actin served as a loading control. Data in panel A, are from 3 independent experiments with n = 6 wells per experiment and *represents p < 0.01 compared to all other concentrations examined.
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Figure 7: Catalytic subunit of NADPH oxidase Nox2 is elevated by the MPP+ treatment. N27 cells were treated with different concentrations of MPP+ for 24 hours. (A) Survival of N27 cells as a function of MPP+ concentration. (B) Immunofluorescent detection of Nox2 in N27 cells cultured either in the absence (control, ctrl) or presence of 300 μM MPP+, a concentration of MPP+ that results in loss of 45 percent of N27 cells. Scale bar equals to 20 μm. (C) Western immunoblot illustrating the effect of treating the cultures with increasing concentrations of MPP+ on expression of Nox2 protein. β-actin served as a loading control. Data in panel A, are from 3 independent experiments with n = 6 wells per experiment and *represents p < 0.01 compared to all other concentrations examined.

Mentions: Treating N27 cells for 24 hours with 300 μM MPP+ resulted in death of 45 percent of these cells by that point in time (Figure 7A). This corresponded to an increase in Nox2 protein expression in these cells as determined by immunofluorescence (Figure 7B) and by Western immunoblotting (Figure 7C). Nox2 expression, measured by Western blot, was highly sensitive to MPP+ as it was increased even at MPP+ concentration of 3 μM, which was well below the 300 μM required for cell killing (Figure 7C).


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)

Catalytic subunit of NADPH oxidase Nox2 is elevated by the MPP+ treatment. N27 cells were treated with different concentrations of MPP+ for 24 hours. (A) Survival of N27 cells as a function of MPP+ concentration. (B) Immunofluorescent detection of Nox2 in N27 cells cultured either in the absence (control, ctrl) or presence of 300 μM MPP+, a concentration of MPP+ that results in loss of 45 percent of N27 cells. Scale bar equals to 20 μm. (C) Western immunoblot illustrating the effect of treating the cultures with increasing concentrations of MPP+ on expression of Nox2 protein. β-actin served as a loading control. Data in panel A, are from 3 independent experiments with n = 6 wells per experiment and *represents p < 0.01 compared to all other concentrations examined.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Catalytic subunit of NADPH oxidase Nox2 is elevated by the MPP+ treatment. N27 cells were treated with different concentrations of MPP+ for 24 hours. (A) Survival of N27 cells as a function of MPP+ concentration. (B) Immunofluorescent detection of Nox2 in N27 cells cultured either in the absence (control, ctrl) or presence of 300 μM MPP+, a concentration of MPP+ that results in loss of 45 percent of N27 cells. Scale bar equals to 20 μm. (C) Western immunoblot illustrating the effect of treating the cultures with increasing concentrations of MPP+ on expression of Nox2 protein. β-actin served as a loading control. Data in panel A, are from 3 independent experiments with n = 6 wells per experiment and *represents p < 0.01 compared to all other concentrations examined.
Mentions: Treating N27 cells for 24 hours with 300 μM MPP+ resulted in death of 45 percent of these cells by that point in time (Figure 7A). This corresponded to an increase in Nox2 protein expression in these cells as determined by immunofluorescence (Figure 7B) and by Western immunoblotting (Figure 7C). Nox2 expression, measured by Western blot, was highly sensitive to MPP+ as it was increased even at MPP+ concentration of 3 μM, which was well below the 300 μM required for cell killing (Figure 7C).

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