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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 13, 2004 as doi:10.1096/fj.04-1586fje. |
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,1
* Neuropharmacology Section, Laboratory of Pharmacology and Chemistry,
National Center for Toxicogenomics,
Inorganic Carcinogenesis Section, Laboratory of Comparative Carcinogenesis, NCI, National Institute of Environmental Health Sciences/National Institutes of Health, Research Triangle Park, North Carolina, USA;
Department of Neurology, First Clinical Hospital,
|| Department of Physiology, Dalian Medical University, Dalian, China;
Department of Pharmacodynamics, College of Pharmacy, University of Florida, Gainesville, Florida, USA; and
College of Pharmacy, Kangwon National University, Chunchon, Korea
1Correspondence: 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: zhang11{at}niehs.nih.gov
SPECIFIC AIMS
The purpose of this study was to explore the neuroprotective effect of a novel compound 3-Hydroxymorphinan (3-HM), an analog of dextromethorphan (DM), on dopaminergic neurons against inflammagen lipopolysaccharide (LPS) -induced neurodegeneration. A series of experiments using primary mesencephalic neuron-glia cultures and related reconstitution studies were performed to delineate the underlying mechanisms of the neuroprotective effect of 3-HM.
PRINCIPAL FINDINGS
1. 3-HM is neurotrophic to dopaminergic neurons and is also neuroprotective against LPS-induced neurotoxicity
Mesencephalic neuron-glia cultures were pretreated with vehicle or 1–5 µM 3-HM before application of 10 ng/mL LPS. Seven days later, degeneration of dopaminergic neurons was determined by functional assay of [3H]DA uptake and morphometric measurement of dopaminergic neurons after immunostaining with anti-TH antibody. As shown in Fig. 1
A, results indicated that LPS reduced 
60% of DA uptake capacity compared with the vehicle-treated cultures. 3-HM significantly attenuated LPS-induced reduction in DA uptake in a dose-dependent manner. Treatment with 3-HM alone (1–5 µM) for 7 days dose-dependently increased DA uptake capacity by 20–60% compared with vehicle-treated cultures, indicating 3-HM exerted a neurotrophic effect on dopaminergic neurons. Results from morphometric measurements revealed a pattern of changes similar to that of DA uptake studies. Results (Fig. 1B
) showed that pretreatment with 3-HM (5 µM) significantly restored LPS-induced reduction in the number of dopaminergic neurons and loss of neurite length. Pretreatment with 3-HM, dopaminergic neurons were significantly more numerous, with neurites less affected than LPS-treated cultures (Fig. 1C
).
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2. Astroglia are the contributor to the neurotrophic effect of 3-HM
To determine the target of 3-HM’s neurotrophic effect, we used neuron-enriched cultures to investigate whether 3-HM has a direct effect on dopaminergic neurons. 3-HM (0.1–5 µM) failed to show a significant increase in DA uptake capacity, indicating that the 3-HM-induced neurotrophic effect was not due to a direct effect on dopaminergic neurons (Fig. 2
A). To explore the possibility that glial cells mediated the 3-HM-induced neurotrophic effect, reconstitution experiments were performed by adding microglia or astroglia back to neuron-enriched cultures. Addition of 10% (5x104/well) (in our normal mesencephalic neuron-glia cultures, microglia constitute 10% of total cells) or 20% (1x105/well) microglia back to neuron-enriched cultures failed to enhance DA uptake capacity in 3-HM-treated cultures (Fig. 2B
). Addition of 40% or 50% of astroglia (in our normal mesencephalic neuron-glia cultures, astroglia constitute 40–50% of total cells) back to the neuron-enriched cultures increased DA uptake capacity of 3-HM by 135.8% and 158.3%. It appeared that the neurotrophic effect was positively correlated with the composition of astroglia (Fig. 2C
).
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3. Microglia are the targets of anti-inflammatory effect of 3-HM
Microglial activation and subsequent release of pro-inflammatory and neurotoxic factors may underlie LPS-induced dopaminergic neurotoxicity in mesencephalic neuron-glia cultures. To study the roles of microglia in mediating 3-HM-induced neuroprotecive effect, microglia (10% or 20% of total cells) were added to neuron-enriched cultures.Results showed that the more microglia was added, the more pronounced LPS-induced neurotoxicity (25% decrease by 10% and 52% by 20%) and the more significant neuroprotective effects exerted by 3-HM (13% increase by 10% and 30% by 20%) were seen (data not shown). These findings strongly indicated that microglia are the targets of anti-inflammatory effect of 3-HM.
CONCLUSION AND SIGNIFICANCE
In this study, we demonstrated 3-HM, an analog of DM, exerted potent neuroprotection on mesencephalic dopaminergic neurons in inflammation LPS in vitro PD model by acting on two different targets: a neurotrophic effect mediated by astroglia and an anti-inflammatory effect mediated by inhibition of microglial activation. After finding that 3-HM exerted a potent neuroprotective and neurotrophic effect on dopaminergic neurons in rat primary mesencephalic neuron-glia cultures treated with LPS, we demonstrated that the neurotrophic effect of 3-HM was glia-dependent since 3-HM failed to show any protective effect in neuron-enriched cultures. We verified that astroglia were contributors to the neurotrophic effect of 3-HM, as 3-HM was neurotrophic after addition of astroglia, but not microglia, back to the neuron-enriched cultures. 3-HM-treated astroglia-derived conditioned media presented a significant neurotrophic effect on dopaminergic neurons. An anti-inflammatory mechanism was also important for the neuroprotective activity of 3-HM because the more microglia was added back to neuron-enriched cultures, the more significant neuroprotective was the effect observed. Anti-inflammatory mechanism of 3-HM was attributed to its inhibition of LPS-induced production of an array of pro-inflammatory and neurotoxic factors, including NO, TNF-
, PGE2, and reactive oxygen species (ROS), such as extracellular superoxide and intracellular ROS.
Parkinson’s disease (PD) is a chronic, progressive, and devastating neurodegenerative disorder characterized by selective loss of dopaminergic neurons in substantia nigra pars compacta (SNpc) and subsequent depletion of dopamine in striatum. So far, the most effective therapeutic approaches for PD rely on alleviating symptoms and cannot halt progression or reverse the neurodegenerative changes occurring in SNpc. Therefore, there is an urgent need to develop compounds that not only prevent but also curtail the degenerative process. Current research on the development of potential neuroprotective drug(s) focuses on two fronts. 1) Neurotrophic effect: this can be achieved by administering neurotrophic factors directly to the brain or indirectly stimulating the release of neurotrophic factors from glial cells to promote the neuronal survival. 2) Anti-inflammmatory effect: due to the important role of inflammation in the pathogenesis of PD, anti-inflammmatory drugs have potentially significant implications for PD therapy. We have reported that DM, an active ingredient in a variety of widely used anti-cough remedies, was neuroprotective against LPS or MPTP-induced dopaminergic neurotoxicity in primary mesencephalic neuron-glia cultures and animal studies. The neuroprotective effects observed were attributed to DM’s anti-inflammatory ability by inhibiting overactivation of microglia. By extension, we have examined a variety of analogs of DM. In this study, we found that 3-HM, an analog of DM, showed higher potency in protecting dopaminergic neurons against inflammation-induced damage than its parent compound. The more potent neuroprotection of 3-HM was attributed to its additional neurotrophic effect in addition to an anti-inflammatory effect shared by both DM and 3-HM. Because it possesses these two features necessary for effective neuroprotective agents and in view of the documented low toxicity for DM or its analogs, 3-HM may provide important insights into developing therapeutic interventions for inflammation-related diseases such as PD.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1586fje;
This article has been cited by other articles:
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  L. Qian, K. S. Tan, S.-J. Wei, H.-M. Wu, Z. Xu, B. Wilson, R.-B. Lu, J.-S. Hong, and P. M. Flood Microglia-Mediated Neurotoxicity Is Inhibited by Morphine through an Opioid Receptor-Independent Reduction of NADPH Oxidase Activity J. Immunol., July 15, 2007; 179(2): 1198 - 1209. [Abstract] [Full Text] [PDF]
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 W. Zhang, E.-J. Shin, T. Wang, P. H. Lee, H. Pang, M.-B. Wie, W.-K. Kim, S.-J. Kim, W.-H. Huang, Y. Wang, et al. 3-Hydroxymorphinan, a metabolite of dextromethorphan, protects nigrostriatal pathway against MPTP-elicited damage both in vivo and in vitro FASEB J, December 1, 2006; 20(14): 2496 - 2511. [Abstract] [Full Text] [PDF]
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B pathway. 3-HM inhibited LPS-induced microglial activation and the production of a large amount of pro-inflammatory and neurotoxic factors, including ROS (extracellular superoxide and intracellular ROS), TNF-