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MPP⁺-induced COX-2 activation and subsequent dopaminergic neurodegeneration

Tongguang Wang^*,1, Zhong Pei^*, Wei Zhang^*, Bin Liu, Robert Langenbach, Christopher Lee, Belinda Wilson^*, Jeffrey M. Reece, David S. Miller^|| and Jau-Shyong Hong^*,2

^* Neuropharmacology Section,
^>*,|| Laboratory of Pharmacology and Chemistry,
Laboratory of Molecular Carcinogenesis,
Confocal Microscopy Center, Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA; and
College of Pharmacy, University of Florida, Gainesville, Florida, USA

² Correspondence: Neuropharmacology Section, Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences/National Institutes of Health, Research Triangle Park, P.O. Box 12233, NC, USA. E-mail: hong3{at}niehs.nih.gov

SPECIFIC AIMS

The purpose of this study was to determine whether in an in vitro Parkinson’s disease (PD) model 1) microglia participate in MPP⁺-induced COX-2 activation and 2) the activation of COX-2 contributes to subsequent dopaminergic neurotoxicity. A series of experiments using mouse primary mesencephalic neuron-glia cultures and related reconstitution studies were performed to delineate the underlying mechanisms of the COX-2 activation in MPP⁺-treated neuron-glia cultures.

PRINCIPAL FINDINGS

1. Microglia are necessary for MPP⁺-induced PGE₂ production in primary neuron-glia cultures
To determine whether microglia are necessary for MPP⁺-induced PGE₂ production in neuron-glia cultures, different cultures including enriched neuron, enriched microglia, and mixed neuron-microglia cultures were treated with 0.1–0.5 µM MPP⁺ and levels of released PGE₂ were determined after 4 days. As shown in Fig. 1 A, MPP⁺ treatment significantly increased PGE₂ production in mixed neuron-microglia cultures, in a dose-dependent manner. However, neither enriched microglia nor enriched neuron cultures showed any increase in response to MPP⁺ treatment. The role of astroglia in MPP⁺-induced PGE₂ production was also investigated. MPP⁺ (0.1–0.5 µM) failed to increase PGE₂ production in neuron-astroglia or enriched astroglia cultures (Fig. 1B ). These findings indicate that the interaction between neurons and microglia, or microgliosis, is necessary for MPP⁺ to increase PGE₂ production.

View larger version (28K): [in this window] [in a new window]   Figure 1. MPP+ treatment increased PGE2 production in mixed neuron-microglia cultures. Enriched neuron, microglia, astroglia, and mixed neuron-microglia/neuron-astroglia cultures were treated with MPP+ at given doses. Four days after MPP+ treatment, media were collected and PGE2 production was determined with an ELISA kit as described in Material and Methods. Results are mean ± SE of 3 experiments performed in triplicate. *P <0.05, **P<0.01 compared with control cultures.

2. COX-2 inhibitor reduces MPP⁺-induced PGE₂ production and subsequent dopaminergic neurotoxicity in mixed neuron-microglia cultures
A selective COX-2 inhibitor, DuP697, was used to determine the role of COX-2 in mediating MPP⁺-induced PGE₂ production and dopaminergic neurodegeneration in mixed neuron-microglia cultures. DuP (10 nm) was added to the media 30 min prior to MPP⁺ addition in enriched neuron or mixed neuron-microglia cultures. PGE₂ release into the medium and dopamine (DA) uptake activity as the functional index for dopaminergic neurons were determined 4 days later. As shown in Fig. 2 A, MPP⁺ treatment significantly increased PGE₂ production in mixed neuron-microglia cultures but not in enriched neuron cultures. Pretreatment with DuP697 decreased PGE₂ levels in both control cultures and abolished MPP⁺-induced PGE₂ production in neuron-microglia cultures. These results demonstrated that COX-2 is the main enzyme responsible for MPP⁺ increased PGE₂ production in mixed neuron-microglia cultures. As shown in Fig. 2B , pretreatment with DuP697 also significantly attenuated MPP⁺-induced reduction of DA uptake in mixed neuron-microglia cultures but not in enriched neuron cultures, indicating that COX-2 activation mediates part of the MPP⁺-induced dopaminergic neurodegeneration in the neuron-microglia cultures; this COX-2 related neurodegeneration is microglia-dependent. The link between COX-2 and microglia was also supported by the observation that pretreatment with DuP697 attenuated MPP⁺-increased F4/80 (a morphological index of microglial activation) positive microglia (Fig. 2C ). These results suggest that COX-2 activation participates in MPP⁺-induced microgliosis and neurodegeneration; inhibition in COX-2 activation may result in attenuation in microgliosis and subsequent neurodegeneration.

View larger version (51K): [in this window] [in a new window]   Figure 2. Effect of COX-2 specific inhibitor DuP697 on MPP+-induced dopaminergic neurodegeneration. Neuron-enriched cultures, neuron-microglia cultures or neuron-glia cultures were treated with MPP+ with/without DuP697 pretreatment. PGE2 production in media (A) was determined and DA uptake assay (B) was performed 4 days later to determine neurotoxicity. Results are mean ± SE of at least 3 experiments performed in triplicate. *P <0.05 compared with MPP+ treated cultures. Microgliosis was observed in neuron-glia cultures 4 days later by F4/80 immunocytochemistry (C), which showed DuP697 pretreatment significantly attenuated the MPP+-induced F4/80 immunoreactivity in microglia.

3. Microglia are necessary for MPP⁺-induced COX-2 activation
To investigate the role of microglial COX-2 activation in MPP⁺-increased PGE₂ production, microglia that are COX-2 deficient or their wild-type controls were added back to wild-type enriched neuron cultures. The mixed neuron-microglia cultures were then treated with 0.1–0.5 µM MPP⁺ for 4 days and COX-2 immunoreactivity and PGE₂ production were determined. Cocultures containing wild-type microglia showed enhanced PGE₂ production in response to increasing concentrations of MPP⁺. Even at the lowest concentration tested, PGE₂ production was significantly increased. In contrast, cocultures containing microglia from COX-2 KO mice showed no significant elevation in PGE₂ production, with 0.1 and 0.2 µM MPP⁺ and a significantly reduced increase (compared with wild-type cocultures) with 0.5 µM MPP⁺. These results indicate that microglial COX-2 was partially responsible for the MPP⁺-induced increase in PGE₂ production and that neuronal COX-2 activation secondary to microgliosis may have contributed to the remaining MPP⁺-induced increase in PGE₂ production. This notion was further supported by the immunocytochemical findings that MPP⁺ (0.5 µM) treatment increased the density of COX-2-immunoreactive neurons compared with control cultures even in cocultures with COX-2 deficient microglia. Thus, microglia are necessary for MPP⁺-induced COX-2 activation in two steps: first, COX-2 activation in microglia produces most of the MPP⁺-induced PGE₂ production; second, microglia-neuron interaction is necessary for neuronal COX-2 activation, which may be responsible for the remaining PGE₂ production.

CONCLUSION AND SIGNIFICANCE

In vitro studies from our laboratory have revealed that microglia render dopaminergic neurons more vulnerable to MPP⁺-induced neurotoxicity through reactive microgliosis. In this study, using different types of primary mesencephalic cultures, we demonstrate that 1) microglia-neuron interaction is necessary for MPP⁺-induced COX-2 activation and PGE₂ production; 2) the increase in COX-2 expression is mainly seen in microglia, and to a lesser degree in neurons; 3) inhibition of COX-2 activity partially attenuates MPP⁺-induced microgliosis and protects dopaminergic neurons against subsequent neurotoxicity.

Unique features of the pathogenesis of PD are the delayed and progressive degeneration of dopaminergic neurons in substantia nigra pars compacta. Mechanisms underlying this progressive nature are still not fully explained. However, a self-propelling mechanism has been postulated that is operative continuously and becomes progressive and exaggerated as time goes by. Many recent reports have implicated chronic neuro-inflammation in PD, suggesting it may underlie the progressive aspect of the disease. COX-2 is an important enzyme for the production of prostaglandins, which play important roles in inflammation. Specific COX-2 inhibitions have been reported to protect dopaminergic neurons against MPTP-induced neurotoxicity in mice. Therefore, COX-2 plays an important role in mediating MPTP-induced neurotoxicity. Although microglia were reported to participate in LPS-induced COX-2 expression and PGE₂ production, it is unclear whether microglia participate in MPTP-induced COX-2 expression and subsequent neurotoxicity.

Our findings that reactive microgliosis participated in MPP⁺-induced COX-2 and PGE₂ production in neuron-glia cultures and subsequent neurotoxicity provide another mechanism for COX-2-mediated neurotoxicity in MPTP PD model besides the neuronal COX-2 activation and direct cytotoxicity reported. Our study clearly demonstrated that microglia-neuron interaction, possibly through microgliosis, underlies COX-2 expression and PGE₂ production in MPP⁺-induced dopaminergic neurotoxicity, especially in treatment with lower doses of MPP⁺(0.1 and 0.2 µM). This microgliosis is also necessary for higher dose of MPP⁺ (0.5 µM) to induce COX-2 expression and subsequent PGE₂ production in dopaminergic neurons. The resulting neurotoxicity from neuronal COX-2 activation may further enhance the microgliosis and propel the pathological progression of PD (Fig. 3 ). Therefore, it is reasonable to conclude that by inhibiting COX-2 activity and microgliosis, we may attenuate neuroinflammation and delay the progression of neurodegenerative diseases such as PD.

View larger version (16K): [in this window] [in a new window]   Figure 3. Schematic flow chart depicting the possible mechanism for MPP+-induced COX-2 expression in neuron-glia cultures. MPP+ treatment exerts direct dopaminergic neurotoxicity. The neuronal damage causes reactive microgliosis, resulting in increase in the expression of proinflammatory genes, including COX-2. Release of excessive proinflammatory factors, including NO, ROS, cytokines, and PGE2, will enhance the expression of neuronal COX-2 activity. The increase in neuronal COX-2 activity, along with PGE2 production plus the other proinflammatory factors would lead to a second wave of neuronal damage, which in turn would reinforce the microgliosis process. Thus, a positive feedback circle between neuronal death and microgliosis occurs. This vicious cycle may partially explain the self-propelling nature of PD. It is hypothesized that blockade of COX-2 activity would attenuate the circle at two points: first, decreased COX-2 activity in microglia attenuates microgliosis; second, decreased COX-2 activity in neurons may also attenuate neurotoxicity.

FOOTNOTES

¹ Current address: Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2457fje; doi: 10.1096/fj.04-2457fje

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