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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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A model of MPP+ induced generation of two waves of ROS in dopaminergic neurons and mechanisms of blockade by losartan. MPTP is converted to MPP+ in astrocytes by monoamine oxidase B [45]. Specific uptake of MPP+ released from astrocytes into dopaminergic neurons occurs via a cell membrane dopamine transporter (DAT). After entering the cytosol, MPP+ binds and inhibits mitochondrial complex I, leading to an increase in mitochondrial ROS (First Wave), which, in turn, leads to the activation of the extramitochondrial NADPH oxidase complex to generate superoxide (Second Wave). The Second Wave of ROS can be blocked by pharmacological and genetic inhibition of NADPH oxidase, as well as inhibition of protein synthesis by cyclohexamide (c-hex), and by losartan blockade of AT1 receptor. Cell surface Nox2-generated superoxide is readily dismutated to H2O2, which can act either extracellularily or cross into the cytosol of dopaminergic neurons for propagation of the two-wave cascade. Reaction of superoxide with nitric oxide can generate highly cytotoxic peroxynitrite, which has been reported for its neurotoxicity in models of PD [74,75].
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Figure 9: A model of MPP+ induced generation of two waves of ROS in dopaminergic neurons and mechanisms of blockade by losartan. MPTP is converted to MPP+ in astrocytes by monoamine oxidase B [45]. Specific uptake of MPP+ released from astrocytes into dopaminergic neurons occurs via a cell membrane dopamine transporter (DAT). After entering the cytosol, MPP+ binds and inhibits mitochondrial complex I, leading to an increase in mitochondrial ROS (First Wave), which, in turn, leads to the activation of the extramitochondrial NADPH oxidase complex to generate superoxide (Second Wave). The Second Wave of ROS can be blocked by pharmacological and genetic inhibition of NADPH oxidase, as well as inhibition of protein synthesis by cyclohexamide (c-hex), and by losartan blockade of AT1 receptor. Cell surface Nox2-generated superoxide is readily dismutated to H2O2, which can act either extracellularily or cross into the cytosol of dopaminergic neurons for propagation of the two-wave cascade. Reaction of superoxide with nitric oxide can generate highly cytotoxic peroxynitrite, which has been reported for its neurotoxicity in models of PD [74,75].

Mentions: ROS generation is promoted by angiotensin II binding to the AT1 receptor, which induces a protein kinase C-Nox signaling cascade and leads to elaboration of superoxide from NADPH oxidase [19]. Losartan competes for binding to the AT1 receptor for suppression of angiotensin II-induced increases in ROS production (Figure 9). Although the existence of an extramitochondrial second wave is clear from our data, the specific mechanism(s) by which the mitochondria detect oxidative stress in dopaminergic cells and induce the second wave is unknown, one candidate for mitochondrial oxidative stress recognition has been proposed, viz., inactivation of mitochondrial aconitase, an iron-sulfur-containing enzyme necessary for ATP production. Increases in the release of ferrous iron from mitochondrial aconitase catalytic center suggest that iron may function as an oxidative stress biosensor [68,69]. However, it is conceivable that the Nox-induced ROS signals not only affect intracellular signaling pathways that precipitate the two-wave cascade of ROS generation and in this way may influence neighboring cells, including neurons, astrocytes, and microglia, all of which, as we show here in dopaminergic neurons, express NADPH oxidase [8,70]. In fact, CD200 ligand expressed on the surface of neurons, but not microglia, interacts with microglial CD200 receptor (CD200R) purportedly maintaining microglia in a resting state [71]. Reduced CD200/CD200R interactions between neurons and microglia may contribute to Parkinson [72] and Alzheimer pathogenesis [73] via activation of microglial 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)

A model of MPP+ induced generation of two waves of ROS in dopaminergic neurons and mechanisms of blockade by losartan. MPTP is converted to MPP+ in astrocytes by monoamine oxidase B [45]. Specific uptake of MPP+ released from astrocytes into dopaminergic neurons occurs via a cell membrane dopamine transporter (DAT). After entering the cytosol, MPP+ binds and inhibits mitochondrial complex I, leading to an increase in mitochondrial ROS (First Wave), which, in turn, leads to the activation of the extramitochondrial NADPH oxidase complex to generate superoxide (Second Wave). The Second Wave of ROS can be blocked by pharmacological and genetic inhibition of NADPH oxidase, as well as inhibition of protein synthesis by cyclohexamide (c-hex), and by losartan blockade of AT1 receptor. Cell surface Nox2-generated superoxide is readily dismutated to H2O2, which can act either extracellularily or cross into the cytosol of dopaminergic neurons for propagation of the two-wave cascade. Reaction of superoxide with nitric oxide can generate highly cytotoxic peroxynitrite, which has been reported for its neurotoxicity in models of PD [74,75].
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 9: A model of MPP+ induced generation of two waves of ROS in dopaminergic neurons and mechanisms of blockade by losartan. MPTP is converted to MPP+ in astrocytes by monoamine oxidase B [45]. Specific uptake of MPP+ released from astrocytes into dopaminergic neurons occurs via a cell membrane dopamine transporter (DAT). After entering the cytosol, MPP+ binds and inhibits mitochondrial complex I, leading to an increase in mitochondrial ROS (First Wave), which, in turn, leads to the activation of the extramitochondrial NADPH oxidase complex to generate superoxide (Second Wave). The Second Wave of ROS can be blocked by pharmacological and genetic inhibition of NADPH oxidase, as well as inhibition of protein synthesis by cyclohexamide (c-hex), and by losartan blockade of AT1 receptor. Cell surface Nox2-generated superoxide is readily dismutated to H2O2, which can act either extracellularily or cross into the cytosol of dopaminergic neurons for propagation of the two-wave cascade. Reaction of superoxide with nitric oxide can generate highly cytotoxic peroxynitrite, which has been reported for its neurotoxicity in models of PD [74,75].
Mentions: ROS generation is promoted by angiotensin II binding to the AT1 receptor, which induces a protein kinase C-Nox signaling cascade and leads to elaboration of superoxide from NADPH oxidase [19]. Losartan competes for binding to the AT1 receptor for suppression of angiotensin II-induced increases in ROS production (Figure 9). Although the existence of an extramitochondrial second wave is clear from our data, the specific mechanism(s) by which the mitochondria detect oxidative stress in dopaminergic cells and induce the second wave is unknown, one candidate for mitochondrial oxidative stress recognition has been proposed, viz., inactivation of mitochondrial aconitase, an iron-sulfur-containing enzyme necessary for ATP production. Increases in the release of ferrous iron from mitochondrial aconitase catalytic center suggest that iron may function as an oxidative stress biosensor [68,69]. However, it is conceivable that the Nox-induced ROS signals not only affect intracellular signaling pathways that precipitate the two-wave cascade of ROS generation and in this way may influence neighboring cells, including neurons, astrocytes, and microglia, all of which, as we show here in dopaminergic neurons, express NADPH oxidase [8,70]. In fact, CD200 ligand expressed on the surface of neurons, but not microglia, interacts with microglial CD200 receptor (CD200R) purportedly maintaining microglia in a resting state [71]. Reduced CD200/CD200R interactions between neurons and microglia may contribute to Parkinson [72] and Alzheimer pathogenesis [73] via activation of microglial 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