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Synergistic dopaminergic neurotoxicity of MPTP and inflammogen lipopolysaccharide: relevance to the etiology 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: F1-01, NIEHS/NIH, P.O. Box 12233, Research Triangle Park, NC 27709, USA. E-mail: gao{at}niehs.nih.gov

SPECIFIC AIMS

The specific aim of this study was to determine whether environmental toxin and neuroinflammation could act in synergy to induce a selective and progressive dopaminergic neurodegeneration, a pathological hallmark of PD. We explored the mechanism of the synergistic neurotoxicity of an inflammogen lipopolysaccharide (LPS) and a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), whose active metabolite 1-methyl-4-phenylpyridinium (MPP⁺) shares structural similarity with several environmental toxins associated with PD such as paraquat and tetrahydroisoquinolines.

PRINCIPAL FINDINGS

1. MPTP and LPS synergistically induced a selective and progressive degeneration of dopaminergic neurons
In mesencephalic neuron–glia cultures, MPTP (0.1–0.5 µM) and LPS (0.5 ng/mL) synergistically induced a progressive degeneration of dopaminergic neurons. For example, treatment with nontoxic concentrations of MPTP (0.1 µM) and LPS (0.5 ng/mL) for 7 days resulted in a significant reduction in [³H]dopamine (DA) uptake (25%), nearly threefold the sum of the reduction induced by 0.1 µM MPTP alone (4%) and by 0.5 ng/mL LPS alone (5%) (Fig. 1 A). The synergistic neurotoxicity was more prominent when lower doses of both agents were applied for a longer period (Fig. 1B ). The quantitative and morphological analysis of tyrosine hydroxylase-immunoreactive (TH-IR) neurons confirmed that MPTP and LPS caused synergistic dopaminergic death but not DA transporter (DAT) dysfunction (Fig. 1C, D ). Measurement of [³H]DA and [³H]GABA uptake and morphological and quantitative analysis of TH-IR and neuron-specific nuclear protein (Neu-N)-IR neurons indicated that the synergistic neurotoxicity was preferential to dopaminergic neurons.

View larger version (49K): [in this window] [in a new window]   Figure 1. MPTP and LPS synergistically induce dopaminergic neurodegeneration. Primary rat mesencephalic neuron–glia cultures were treated with vehicle, MPTP, and/or LPS. Three (B) or 7days (A–D) later, neurotoxicity was determined by [3H]DA uptake assay (A, B) and quantification of the TH-IR neurons (C). The results are the mean ± SE of 3 experiments performed in triplicate. *P < 0.05 and **P < 0.005 compared with the cultures treated with LPS alone, respectively. +P < 0.05 compared with the cultures treated with MPTP alone. D) Morphological analysis of TH-IR neurons. The images are from one experiment that is representative of 3 separate experiments. Scale bar, 50 µm.

2. The synergistic neurotoxicity of MPTP and LPS was observed when both agents were applied either simultaneously or in tandem
In addition to simultaneously exposing the rat mesencephalic neuron–glia cultures to MPTP and LPS (Fig. 1 ), exposure to MPTP and LPS in tandem also resulted in additive to synergistic neurotoxicity. In the first treatment scheme, cultures were first treated with one agent (0.5 µM MPTP or 0.5 ng/mL LPS); 4 days later, the other agent (0.5 ng/mL LPS or 0.5 µM MPTP) was added without a change of medium. Eight days after the initial treatment, neurotoxicity was determined by measuring DA uptake and quantifying TH-IR neurons. Neurotoxicity induced by exposure to MPTP for 8 days and LPS for the last 4 days was much greater than the sum of that induced by exposure to MPTP alone for 8 days and that induced by exposure to LPS alone for the last 4 days. Conversely, neurotoxicity induced by exposure to LPS for 8 days and MPTP for the last 4 days was also much greater than the sum of that induced by MPTP alone for the last 4 days and by LPS for 8 days. In the second treatment scheme, cultures were first treated with MPTP (0.5 µM) or LPS (0.5 ng/mL). Three days later, the spent medium was changed to fresh medium. On day 5, cultures were treated with LPS (0.5 ng/mL) or MPTP (0.5 µM). On day 7, neurotoxicity was assessed. Neurotoxicity induced by exposure first to LPS, then to MPTP, was much greater than the sum of that induced by exposure for the same length of time to either agent alone. Conversely, the neurotoxicity induced by exposure first to MPTP and then LPS was slightly greater than the sum of that induced by either agent alone.

3. Reactive free radicals are the major factors responsible for the synergistic neurotoxicity of MPTP and LPS
Analysis of the underlying mechanism of the synergistic neurotoxicity indicates that the participation of microglia is indispensable for the induction of the synergistic neurotoxicity. Specifically, in the absence of microglia (in neuron–astroglia cocultures or neuron-enriched cultures), a combination of LPS with either MPTP or MPP⁺ failed to induce additive/synergistic neurodegeneration of dopaminergic neurons, but did so quite effectively in the presence of microglia (neuron–glia cultures). Investigation of the potential toxic factors mediating the synergistic neurotoxicity illustrated that in neuron–glia cultures but not in neuron-enriched cultures, MPTP (0.5 µM) and LPS (0.5 ng/mL) synergistically stimulated the release of superoxide free radical and the production of intracellular ROS. However, in neuron–glia cultures, combined MPTP (0.5 µM) and LPS (0.5 ng/mL) appeared to cause an additive effect on NO production. The reduction in the synergistic neurotoxicity of MPTP (0.25 µM)/MPP⁺ (0.25 µM) and LPS (0.5 ng/mL) by N^G-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, supported the involvement of NO in the synergistic neurotoxicity. In sharp contrast to the involvement of reactive free radicals (NO, extracellular superoxide, and intracellular ROS), TNF did not seem to be involved in the synergistic neurotoxicity.

4. Microglial NADPH oxidase-derived superoxide plays a pivotal role in the synergistic neurotoxicity of MPTP and LPS
NADPH oxidase inhibitor apocynin (0.25 mM) significantly attenuated the synergistic neurotoxicity of MPTP (0.25 µM) and LPS (0.5 ng/mL), suggesting that the dramatically increased release of superoxide might be catalyzed by NADPH oxidase, an enzyme that appears to be a major source of extracellular superoxide production in microglia. Moreover, in the neuron–glia cultures from wild-type C57BL/6J (gp91^phox+/+) mice, but not from NADPH oxidase-deficient (gp91^phox-/-) mice, the combination of LPS (0.5 ng/mL) with either MPTP (0.1, 0.25, or 0.5 µM) or MPP⁺ (0.1 µM) induced synergistic superoxide production and synergistic neurotoxicity (Fig. 2 ). The resistance of gp91^phox-/- cultures to the synergistic neurotoxicity of MPTP and LPS indicated that the production of superoxide was a key event in the induction of synergistic neurotoxicity.

View larger version (36K): [in this window] [in a new window]   Figure 2. Dopaminergic neurons from gp91phox-/- mice are more resistant to the synergistic neurotoxicity of MPTP/MPP+ and LPS than those from gp91phox+/+ mice. Neuron–glia cultures from gp91phox+/+ or gp91phox-/- mice were treated with vehicle, 0.5 ng/mL LPS, and/or indicated concentrations of MPTP/MPP+. Seven days later, neurotoxicity was determined by [3H]DA uptake assay (A) and quantification of TH-IR neurons (B). Results are the mean ± SE of 3 experiments performed in triplicate. *P < 0.05, **P < 0.005 compared with the LPS-treated cultures. +P < 0.05, compared with the MPTP-treated or MPP+-treated cultures.

CONCLUSIONS AND SIGNIFICANCE

Although there is substantial evidence supporting the involvement of multiple environmental factors in the pathogenesis of PD, single environmental toxin etiology remains elusive. Few animal studies have demonstrated additive/synergistic neurotoxicity of environmental toxins. However, the underlying mechanism of these additive/synergistic effects was insufficiently explored due to the limitations of in vivo study. In our current study, using primary mesencephalic neuron–glia cultures as an in vitro model of PD, we illustrated that MPTP (a neurotoxin) and LPS (an inflammogen) synergistically induced a selective and progressive degeneration of dopaminergic neurons. The synergistic neurotoxicity was observed when the two agents were applied either simultaneously or in tandem. More important, taking advantage of cell culture systems, we explored the mechanism of the synergistic neurotoxicity of MPTP and LPS. Microglial NADPH oxidase-mediated generation of superoxide played a pivotal role in the induction of the synergistic neurotoxicity, as evidenced by the observations that MPTP and LPS synergistically induced NADPH oxidase-derived superoxide production and that pharmacological inhibition and genetic inactivation of NADPH oxidase eliminated the synergistic effect of MPTP and LPS. NO might also be involved in the synergistic neurotoxicity.

This study, combined with our recent demonstration of the synergistic dopaminergic neurodegeneration induced by pesticide rotenone and LPS, lends strong support to the notion that development of PD may be a result of the interaction of environmental toxins and other risk factor(s) such as neuroinflammation. Findings from this study will help uncover the pathogenesis of PD and may also provide clues to developing effective therapeutic strategies for treatment. Confirmation of these in vitro observations in animal studies and in multi-factorial epidemiological studies should shed significant light on the etiology of PD.

View larger version (24K): [in this window] [in a new window]   Figure 3. Schematic diagram depicting the microglia-dependent synergistic dopaminergic neurodegeneration induced by combined LPS and MPTP/MPP+. MPTP is converted into MPP+ by MAO (monoamine oxidase) -B in astroglia. MPP+ is then taken up and concentrated in dopaminergic neurons, where it inhibits the mitochondrial complex I resulting in dopaminergic neuronal death. Subsequently, neuronal death triggers a reactive microgliosis. In contrast, LPS can directly induce microglial activation. Activated microglia, either due to LPS stimulation or as a result of MPP+-induced neuronal death, release NADPH oxidase-derived superoxide and produce NO, which initiates or exacerbate neuronal death. Overall, immune stimulation (LPS) and neurotoxin (MPTP/MPP+) amplify each other, inducing a synergistic dopaminergic neurodegeneration; microglial NADPH oxidase-derived superoxide is a major mediator in this process.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0203fje; doi: 10.1096/fj.03-0203fje

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