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Critical role of microglial NADPH oxidase-derived free radicals in the in vitro MPTP model of Parkinson’s disease¹

Neuropharmacology Section, Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences/National Institutes of Health, Research Triangle Park, North Carolina, USA

²Correspondence: College of Pharmacy, PO Box 100487 HSC, University of Florida, Gainesville, FL 32610, USA. E-mail: liu{at}cop.ufl.edu

SPECIFIC AIMS

The specific aim of this study was to determine the nature of glial involvement in the pathogenesis of Parkinson’s disease (PD). Exploiting the advantages of cell culture systems, we examined the contributions of glial cells to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurodegeneration and the temporal relationship between neurodegeneration and glial activation. We also determined major factors responsible for the glia-enhanced MPTP neurotoxicity using NADPH oxidase-deficient mice.

PRINCIPAL FINDINGS

1. Microglia but not astroglia render dopaminergic neurons more sensitive to MPP⁺ neurotoxicity
To investigate the role of glia in dopaminergic neurodegeneration in PD, we treated neuron–glia or neuron-enriched cultures with MPP⁺ for 7 days and assessed the neurotoxicity. In the presence of glia, dopaminergic neurons were more sensitive to MPP⁺ neurotoxicity. To further determine the type of glia mediating the enhanced MPTP/MPP⁺ neurotoxicity, we analyzed the effect of addition of microglia or astroglia to neuron-enriched cultures on MPP⁺-induced dopaminergic neurodegeneration. The addition of microglia significantly increased MPP⁺ neurotoxicity (Fig. 1 A), which was positively correlated with the number of microglia added to the neuron-enriched cultures (Fig. 1B ). In contrast, addition of astroglia (7.5x10⁴/well) did not significantly affect the sensitivity of dopaminergic neurons to MPP⁺ toxicity (Fig. 1C ); the addition of more astroglia (>15x10⁴/well), close to the composition of astroglia in neuron–glia cultures, significantly decreased the basal [³H]DA uptake but did not affect sensitivity. Similarly, in neuron–astroglia cocultures prepared by coculturing fetal midbrain neurons with a confluent monolayer of astroglia, the presence of astroglia failed to affect MPP⁺ neurotoxicity (Fig. 1D ). These results indicate that microglia, but not astroglia, play a pivotal role in the enhancement of MPP⁺-induced neurodegeneration.

View larger version (40K): [in this window] [in a new window]   Figure 1. Microglia enhance the sensitivity of dopaminergic neurons to MPP+ neurotoxicity. Neuron-enriched cultures (N) were supplemented with 7.5 x 104 microglia/well (A), 3.75–15 x 104 microglia/well (B), or 7.5 x 104 astroglia/well (C). 24 h later, the cultures were treated with the vehicle, MPP+ (A, C), or 0.1 µM MPP+ (B). [3H]DA uptake was determined 7 days after the treatment. D) The neuron–astroglia cocultures (N/A) were treated with the vehicle or indicated concentrations of MPP+ and [3H]DA uptake was determined 7 days later. The results are the mean ± SE of 3 experiments performed in triplicate. *P < 0.01, significantly different from the corresponding MPP+-treated, neuron-enriched cultures. ANOVA followed by the Bonferroni’s t test, was used for statistical analysis.

2. MPTP produces reactive microgliosis but not direct microglial activation
To examine the temporal relationship between neurodegeneration and microglial activation, we treated neuron–glia cultures with 0.5 µM MPTP, then dopaminergic neurodegeneration, morphological change of microglia, and the production of neurotoxic factors were analyzed. The degeneration of dopaminergic neurons was detected as early as 1 day after MPTP treatment, while the activation of microglia was undetectable, indicating that MPTP-induced neurodegeneration preceded microglial activation. Another line of evidence supporting this temporal relationship is that microglial-derived factors were detected only after the occurrence of substantial neuronal damage. Blockade of the conversion of MPTP to MPP⁺ by pargyline, a monoamine oxidase inhibitor, not only abolished MPTP-induced dopaminergic neuronal damage but also abrogated the activation of microglia. Stimulation of primary glial cultures or BV2 microglial cell line with either MPTP or MPP⁺ did not cause superoxide, NO, or TNF production. These results demonstrate that MPTP does not exert direct effect on microglia; rather, the activation of microglia represents a reactive gliosis process in response to the death of dopaminergic neurons.

3. Microglial NADPH oxidase-derived reactive oxygen species (ROS) play an important role in MPP⁺/MPTP neurotoxicity
Among the factors we measured, the production of extracellular superoxide was the most prominent. Since NADPH oxidase, also called phagocyte oxidase (PHOX), appears to be a major source of extracellular superoxide production in microglia, we speculated that microglial NADPH oxidase played an important role in microglia-enhanced MPTP neurotoxicity. Indeed, in neuron–glia cultures from PHOX^+/+ mice but not from PHOX^-/- (gp91^phox-deficient) mice, treatment with MPTP (0.5 µM) for 4 days caused significant superoxide production. The NADPH oxidase inhibitor apocynin attenuated MPTP/MPP⁺ neurotoxicity in neuron–glia cultures from PHOX^+/+ mice but not from PHOX^-/- mice. More important, in neuron–glia cultures, dopaminergic neurons from PHOX^-/- mice were more resistant to MPTP neurotoxicity than those from PHOX^+/+ mice (Fig. 2 A, C). Similarly, dopaminergic neurons from PHOX^+/+ mice were more sensitive to MPP⁺ neurotoxicity than those from PHOX^-/- mice (Fig. 2B, C ), suggesting that the resistance of dopaminergic neurons from PHOX^-/- mice to MPTP neurotoxicity was not due to alterations in the formation of MPP⁺. These results indicate that microglial NADPH oxidase-derived superoxide plays a major role in mediating the microglia-enhanced MPTP neurotoxicity.

View larger version (16K): [in this window] [in a new window]   Figure 2. Differential sensitivity of dopaminergic neurons in neuron–glia cultures from PHOX-/- and PHOX+/+ mice. 7 days after treatment, [3H]DA uptake analysis (A, B) and quantification of TH-IR neurons (C) showed that in neuron–glia cultures, dopaminergic neurons from PHOX-/- mice were more resistant to MPTP/MPP+ toxicity than those from PHOX+/+ mice. Data represent mean ± SE (n=4). *P < 0.01, compared with the corresponding control. ANOVA, followed by the Bonferroni’s t test, was used for statistical analysis.

4. Neuronal NADPH oxidase does not contribute to MPP⁺/MPTP neurotoxicity
Although NADPH oxidase is mainly expressed in phagocytic cells, increasing evidence indicates that various subunits of NADPH oxidase are expressed in nonphagocytic cells such as sympathetic ganglion neurons and cortical neurons, which suggests that neuronal NADPH oxidase might also be involved in superoxide production. However, our results demonstrated that microglia, but not neurons, mediated the differential sensitivity of dopaminergic neurons to MPTP/MPP⁺ neurotoxicity between PHOX^-/- and PHOX^+/+ mice. This conclusion is based on the following observations. First, in neuron-enriched cultures, there is no difference in the MPP⁺ neurotoxicity between PHOX^-/- and PHOX^+/+ mice. Second, the MPP⁺-induced neurodegeneration in neuron-enriched cultures from both types of mice was equally increased by the addition of microglia from PHOX^+/+ mice. Third, neuroprotection of apocynin against MPP⁺ neurotoxicity was observed only in the presence of glia.

CONCLUSIONS AND SIGNIFICANCE

We demonstrated that the activation of microglia, but not astroglia, significantly enhanced the MPTP-induced dopaminergic neurodegeneration in neuron–glia cultures. Characterization of the temporal relationship between microglial activation and neurodegeneration indicates that reactive microgliosis resulting from MPTP-elicited neuronal injury, but not a direct effect of MPTP on microglia, underlies the microglia-enhanced dopaminergic neurodegeneration. Our data also indicate that via generation of superoxide catalyzed by NADPH oxidase and production of nitric oxide, microglia enhanced MPTP-induced dopaminergic neurodegeneration. However, the damage signals from dying neurons, responsible for reactive gliosis, remain unknown.

Although increasing evidence suggests an involvement of glia in PD, the nature of this involvement remains unclear. Recent reports indicate that glia participate in MPTP-induced dopaminergic neurodegeneration in animal models. However, in these in vivo studies, due to the rapid neuronal death and glial activation after dosing the animals with MPTP, precise determination of the nature of glial involvement was difficult. Specifically, the exact temporal relationship between glial activation and neurodegeneration, the type of glia that play a pivotal role, and the molecular pathways involved in this process have not yet been resolved. In this study, we took the advantages of cell culture systems to explore the mechanism of glial involvement. We found that reactive microgliosis, triggered by MPTP-induced neuronal injury, and NADPH oxidase-mediated superoxide production in microglia constitute an integral component of MPTP neurotoxicity. This study also suggests that inhibition of microglial NADPH oxidase would be a promising strategy to retard the progression of PD.

View larger version (27K): [in this window] [in a new window]   Figure 3. Schematic diagram depicting a pivotal role of reactive microgliosis in the progression of MPTP-induced neurodegeneration and the neuroprotection afforded by pharmacological inhibition and genetic inactivation of NADPH oxidase. MPTP is converted into MPP+ by MAO-B. MPP+ is then taken up by the DAT and concentrated in dopamine neurons, where it blocks the mitochondrial respiration leading to dopaminergic neuronal death. Subsequently, neuronal death triggers a reactive microgliosis, which in turn exacerbates neuronal death. Neuronal death and reactive microgliosis amplify each other in a vicious cycle of toxicity leading to neuronal dysfunction, atrophy, and finally cell death.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0109fje; doi: 10.1096/fj.03-0109fje

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