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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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NADPH oxidase inhibitors and an inhibitor of de novo protein synthesis, cyclohexamide, attenuate ROS, but only at later time points after initiation of the MPP+ treatment. (A) N27 cells were treated with 300 μM MPP+ in the absence or presence of 10 nM PAO. Intracellular ROS levels were measured using carboxy-H2-DCFDA and flow cytometry at 3, 6, or 24 hours after initiation of the MPP+ treatment. * represents p < 0.001 compared to 3 hours MPP+ and # represents p < 0.001 compared to 24 hours MPP+. (B) N27 cells were treated with MPP+ for 3 or 6 hours in the absence or presence of the protein synthesis inhibitor cyclohexamide (c-hex). Intracellular H2O2 levels were measured as described above. Treatments are plotted as a percent of the 3-hour MPP+ treatment. * represents p < 0.001 compared to 3 hours MPP+ and # represents p < 0.05 compared to 6 hours MPP+. Data are from 3 independent experiments with n = 6 wells per experiment.
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Figure 6: NADPH oxidase inhibitors and an inhibitor of de novo protein synthesis, cyclohexamide, attenuate ROS, but only at later time points after initiation of the MPP+ treatment. (A) N27 cells were treated with 300 μM MPP+ in the absence or presence of 10 nM PAO. Intracellular ROS levels were measured using carboxy-H2-DCFDA and flow cytometry at 3, 6, or 24 hours after initiation of the MPP+ treatment. * represents p < 0.001 compared to 3 hours MPP+ and # represents p < 0.001 compared to 24 hours MPP+. (B) N27 cells were treated with MPP+ for 3 or 6 hours in the absence or presence of the protein synthesis inhibitor cyclohexamide (c-hex). Intracellular H2O2 levels were measured as described above. Treatments are plotted as a percent of the 3-hour MPP+ treatment. * represents p < 0.001 compared to 3 hours MPP+ and # represents p < 0.05 compared to 6 hours MPP+. Data are from 3 independent experiments with n = 6 wells per experiment.

Mentions: As MPP+ treatment led to an accumulation of ROS in a time-dependent manner (Figure 3B), N27 cell cultures were treated with MPP+ for three, 6, or 24 hours in the presence or absence of the NADPH oxidase inhibitor PAO. In the presence of PAO, increases in ROS levels were not suppressed by PAO until treatment times were prolonged to 24 hours, and this increase was not fully suppressed by PAO treatment (Figure 6A). This suggests that ROS production in response to MPP+ treatment occurs in two waves; the first detectable as early as 3 hours can be accounted for by MPP+ binding to mitochondrial complex I, a well known source of oxyradicals [51,52], followed, hours later, by the second wave arising from NADPH oxidase activation. The fact that NADPH oxidase inhibitors selectively suppressed ROS production is consistent with the idea that this second wave of ROS is mediated by extramitochondrial NADPH oxidase.


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)

NADPH oxidase inhibitors and an inhibitor of de novo protein synthesis, cyclohexamide, attenuate ROS, but only at later time points after initiation of the MPP+ treatment. (A) N27 cells were treated with 300 μM MPP+ in the absence or presence of 10 nM PAO. Intracellular ROS levels were measured using carboxy-H2-DCFDA and flow cytometry at 3, 6, or 24 hours after initiation of the MPP+ treatment. * represents p < 0.001 compared to 3 hours MPP+ and # represents p < 0.001 compared to 24 hours MPP+. (B) N27 cells were treated with MPP+ for 3 or 6 hours in the absence or presence of the protein synthesis inhibitor cyclohexamide (c-hex). Intracellular H2O2 levels were measured as described above. Treatments are plotted as a percent of the 3-hour MPP+ treatment. * represents p < 0.001 compared to 3 hours MPP+ and # represents p < 0.05 compared to 6 hours MPP+. Data are from 3 independent experiments with n = 6 wells per experiment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 6: NADPH oxidase inhibitors and an inhibitor of de novo protein synthesis, cyclohexamide, attenuate ROS, but only at later time points after initiation of the MPP+ treatment. (A) N27 cells were treated with 300 μM MPP+ in the absence or presence of 10 nM PAO. Intracellular ROS levels were measured using carboxy-H2-DCFDA and flow cytometry at 3, 6, or 24 hours after initiation of the MPP+ treatment. * represents p < 0.001 compared to 3 hours MPP+ and # represents p < 0.001 compared to 24 hours MPP+. (B) N27 cells were treated with MPP+ for 3 or 6 hours in the absence or presence of the protein synthesis inhibitor cyclohexamide (c-hex). Intracellular H2O2 levels were measured as described above. Treatments are plotted as a percent of the 3-hour MPP+ treatment. * represents p < 0.001 compared to 3 hours MPP+ and # represents p < 0.05 compared to 6 hours MPP+. Data are from 3 independent experiments with n = 6 wells per experiment.
Mentions: As MPP+ treatment led to an accumulation of ROS in a time-dependent manner (Figure 3B), N27 cell cultures were treated with MPP+ for three, 6, or 24 hours in the presence or absence of the NADPH oxidase inhibitor PAO. In the presence of PAO, increases in ROS levels were not suppressed by PAO until treatment times were prolonged to 24 hours, and this increase was not fully suppressed by PAO treatment (Figure 6A). This suggests that ROS production in response to MPP+ treatment occurs in two waves; the first detectable as early as 3 hours can be accounted for by MPP+ binding to mitochondrial complex I, a well known source of oxyradicals [51,52], followed, hours later, by the second wave arising from NADPH oxidase activation. The fact that NADPH oxidase inhibitors selectively suppressed ROS production is consistent with the idea that this second wave of ROS is mediated by extramitochondrial NADPH oxidase.

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