Limits...
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.

Show MeSH

Related in: MedlinePlus

MPP+ induces a dose- and time-dependent increase in intracellular ROS in the N27 dopaminergic cells. (A) N27 cells were treated with increasing concentrations of MPP+ (up to 1000 μM) for 18 hours and H2O2 (ROS) levels were measured using carboxy-H2-DCFDA fluorescence and flow cytometry. ROS levels are reported as fold increase above values observed in cells that received no MPP+, control. * p < 0.01 compared to 0 μM MPP+ control. (B) N27 cells were treated with 300 μM MPP+ and ROS levels were detected at different times after treatment. # represents p < 0.05 compared to 0 hour and * represents p < 0.01 compared to 0 hour. Data are from 3 independent experiments with n = 6 wells per experiment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3198931&req=5

Figure 3: MPP+ induces a dose- and time-dependent increase in intracellular ROS in the N27 dopaminergic cells. (A) N27 cells were treated with increasing concentrations of MPP+ (up to 1000 μM) for 18 hours and H2O2 (ROS) levels were measured using carboxy-H2-DCFDA fluorescence and flow cytometry. ROS levels are reported as fold increase above values observed in cells that received no MPP+, control. * p < 0.01 compared to 0 μM MPP+ control. (B) N27 cells were treated with 300 μM MPP+ and ROS levels were detected at different times after treatment. # represents p < 0.05 compared to 0 hour and * represents p < 0.01 compared to 0 hour. Data are from 3 independent experiments with n = 6 wells per experiment.

Mentions: MPP+ treatment of the dopaminergic N27 cell line served here as a surrogate in vitro model of the in vivo MPTP-treatment model of PD. In accordance with neurons in normal substantia nigra (Figure 1), N27 dopaminergic neurons have all the subunits necessary to produce superoxide via Nox pathways. Treating N27 cells with increasing amounts of MPP+, the active metabolite of MPTP, corresponded to a dose-dependent increase in the production of superoxide, which peaked at a 20-fold increase (Figure 3A). In addition, exposure to MPP+ at a constant level (300 μM) led to a time-dependent increase in H2O2 accumulation that was first detected at three hours and plateaued at 21 hours (Figure 3B).


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)

MPP+ induces a dose- and time-dependent increase in intracellular ROS in the N27 dopaminergic cells. (A) N27 cells were treated with increasing concentrations of MPP+ (up to 1000 μM) for 18 hours and H2O2 (ROS) levels were measured using carboxy-H2-DCFDA fluorescence and flow cytometry. ROS levels are reported as fold increase above values observed in cells that received no MPP+, control. * p < 0.01 compared to 0 μM MPP+ control. (B) N27 cells were treated with 300 μM MPP+ and ROS levels were detected at different times after treatment. # represents p < 0.05 compared to 0 hour and * represents p < 0.01 compared to 0 hour. Data are from 3 independent experiments with n = 6 wells per experiment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: MPP+ induces a dose- and time-dependent increase in intracellular ROS in the N27 dopaminergic cells. (A) N27 cells were treated with increasing concentrations of MPP+ (up to 1000 μM) for 18 hours and H2O2 (ROS) levels were measured using carboxy-H2-DCFDA fluorescence and flow cytometry. ROS levels are reported as fold increase above values observed in cells that received no MPP+, control. * p < 0.01 compared to 0 μM MPP+ control. (B) N27 cells were treated with 300 μM MPP+ and ROS levels were detected at different times after treatment. # represents p < 0.05 compared to 0 hour and * represents p < 0.01 compared to 0 hour. Data are from 3 independent experiments with n = 6 wells per experiment.
Mentions: MPP+ treatment of the dopaminergic N27 cell line served here as a surrogate in vitro model of the in vivo MPTP-treatment model of PD. In accordance with neurons in normal substantia nigra (Figure 1), N27 dopaminergic neurons have all the subunits necessary to produce superoxide via Nox pathways. Treating N27 cells with increasing amounts of MPP+, the active metabolite of MPTP, corresponded to a dose-dependent increase in the production of superoxide, which peaked at a 20-fold increase (Figure 3A). In addition, exposure to MPP+ at a constant level (300 μM) led to a time-dependent increase in H2O2 accumulation that was first detected at three hours and plateaued at 21 hours (Figure 3B).

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