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

Pharmacological inhibitors of NADPH oxidase and silencing p22phox using siRNA attenuate MPP+ induced ROS. N27 cells were treated for 18 hours with 300 μM MPP+ and increasing concentrations of either apocynin (A) or phenylarsine oxide (PAO) (B). H2O2 levels were measured using caboxy-H2-DCFDA and flow cytometry. ROS levels are represented as percent of MPP+ induced ROS. * represents p < 0.05 compared to cells receiving no inhibitor. (C) N27 cells were transfected with a non-targeting control (NTC) siRNA or a SmartPool siRNA targeting p22phox. Total RNA was collected and reverse transcribed to cDNA. Primers complimentary to rat p22phox were used to amplify the cDNA. An image of a single representative ethidium bromide-stained agarose gel is shown from one knockdown experiment out of three that produced an average knockdown of 40%. (D) N27 cells were transfected with NTC or p22phox siRNA Smartpool and the intracellular H2O2 was measured with flow cytometry as described above. * represents p < 0.05 compared to NTC siRNA-treated cells. 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 5: Pharmacological inhibitors of NADPH oxidase and silencing p22phox using siRNA attenuate MPP+ induced ROS. N27 cells were treated for 18 hours with 300 μM MPP+ and increasing concentrations of either apocynin (A) or phenylarsine oxide (PAO) (B). H2O2 levels were measured using caboxy-H2-DCFDA and flow cytometry. ROS levels are represented as percent of MPP+ induced ROS. * represents p < 0.05 compared to cells receiving no inhibitor. (C) N27 cells were transfected with a non-targeting control (NTC) siRNA or a SmartPool siRNA targeting p22phox. Total RNA was collected and reverse transcribed to cDNA. Primers complimentary to rat p22phox were used to amplify the cDNA. An image of a single representative ethidium bromide-stained agarose gel is shown from one knockdown experiment out of three that produced an average knockdown of 40%. (D) N27 cells were transfected with NTC or p22phox siRNA Smartpool and the intracellular H2O2 was measured with flow cytometry as described above. * represents p < 0.05 compared to NTC siRNA-treated cells. Data are from 3 independent experiments with n = 6 wells per experiment.

Mentions: The role of NADPH oxidase in the response of N27 cells to MPP+ treatment was examined by treatment of cells for 18 hours with 300 μM MPP+ in the absence or presence of increasing concentrations of NADPH oxidase inhibitors phenylarsine oxide (PAO) or apocynin. Both NADPH oxidase inhibitors led to a reduction in NADPH oxidase activity in a dose dependent manner, with a maximum of 40% attenuation of the MPP+ induced NADPH oxidase-mediated ROS effect at doses of 10 μM apocynin (Figure 5A) and 100 nM PAO (Figure 5B). To further validate the role of the NADPH oxidase in MPP+ driven generation of ROS, we genetically suppressed the expression of the p22phox subunit, which is necessary for the activity of all the Nox isoforms of NADPH oxidase. Such silencing of p22phox mRNA resulted in a 40 percent reduction in p22phox expression compared to a non-targeting control (NTC) siRNA (Figure 5C). Although the amount of p22phox silencing in these cells is restricted by the limited transfection efficiency, the examination of ROS levels in p22phox versus NTC siRNA-transfected cells revealed a significant 20% attenuation of MPP+ induced ROS production (Figure 5D). This gene silencing approach yielded a decrease in ROS production that was similar to that achieved by maximally effective concentrations of NADPH oxidase inhibitors PAO and apocynin.


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)

Pharmacological inhibitors of NADPH oxidase and silencing p22phox using siRNA attenuate MPP+ induced ROS. N27 cells were treated for 18 hours with 300 μM MPP+ and increasing concentrations of either apocynin (A) or phenylarsine oxide (PAO) (B). H2O2 levels were measured using caboxy-H2-DCFDA and flow cytometry. ROS levels are represented as percent of MPP+ induced ROS. * represents p < 0.05 compared to cells receiving no inhibitor. (C) N27 cells were transfected with a non-targeting control (NTC) siRNA or a SmartPool siRNA targeting p22phox. Total RNA was collected and reverse transcribed to cDNA. Primers complimentary to rat p22phox were used to amplify the cDNA. An image of a single representative ethidium bromide-stained agarose gel is shown from one knockdown experiment out of three that produced an average knockdown of 40%. (D) N27 cells were transfected with NTC or p22phox siRNA Smartpool and the intracellular H2O2 was measured with flow cytometry as described above. * represents p < 0.05 compared to NTC siRNA-treated cells. 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 5: Pharmacological inhibitors of NADPH oxidase and silencing p22phox using siRNA attenuate MPP+ induced ROS. N27 cells were treated for 18 hours with 300 μM MPP+ and increasing concentrations of either apocynin (A) or phenylarsine oxide (PAO) (B). H2O2 levels were measured using caboxy-H2-DCFDA and flow cytometry. ROS levels are represented as percent of MPP+ induced ROS. * represents p < 0.05 compared to cells receiving no inhibitor. (C) N27 cells were transfected with a non-targeting control (NTC) siRNA or a SmartPool siRNA targeting p22phox. Total RNA was collected and reverse transcribed to cDNA. Primers complimentary to rat p22phox were used to amplify the cDNA. An image of a single representative ethidium bromide-stained agarose gel is shown from one knockdown experiment out of three that produced an average knockdown of 40%. (D) N27 cells were transfected with NTC or p22phox siRNA Smartpool and the intracellular H2O2 was measured with flow cytometry as described above. * represents p < 0.05 compared to NTC siRNA-treated cells. Data are from 3 independent experiments with n = 6 wells per experiment.
Mentions: The role of NADPH oxidase in the response of N27 cells to MPP+ treatment was examined by treatment of cells for 18 hours with 300 μM MPP+ in the absence or presence of increasing concentrations of NADPH oxidase inhibitors phenylarsine oxide (PAO) or apocynin. Both NADPH oxidase inhibitors led to a reduction in NADPH oxidase activity in a dose dependent manner, with a maximum of 40% attenuation of the MPP+ induced NADPH oxidase-mediated ROS effect at doses of 10 μM apocynin (Figure 5A) and 100 nM PAO (Figure 5B). To further validate the role of the NADPH oxidase in MPP+ driven generation of ROS, we genetically suppressed the expression of the p22phox subunit, which is necessary for the activity of all the Nox isoforms of NADPH oxidase. Such silencing of p22phox mRNA resulted in a 40 percent reduction in p22phox expression compared to a non-targeting control (NTC) siRNA (Figure 5C). Although the amount of p22phox silencing in these cells is restricted by the limited transfection efficiency, the examination of ROS levels in p22phox versus NTC siRNA-transfected cells revealed a significant 20% attenuation of MPP+ induced ROS production (Figure 5D). This gene silencing approach yielded a decrease in ROS production that was similar to that achieved by maximally effective concentrations of NADPH oxidase inhibitors PAO and apocynin.

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