Tongguang Wang

Aggregated α-synuclein activates microglia: a process leading to disease progression in Parkinson’s disease

A growing body of evidence indicates that an inflammatory process in the substantia nigra, characterized by activation of resident microglia, likely either initiates or aggravates nigral neurodegeneration in Parkinsons disease (PD). To study the mechanisms by which nigral microglia are activated in PD, the potential role of α‐synuclein (a major component of Lewy bodies that can cause neurodegeneration when aggregated) in microglial activation was investigated. The results demonstrated that in a primary mesencephalic neuron‐glia culture system, extracellular aggregated human α‐synuclein indeed activated microglia; microglial activation enhanced dopaminergic neurodegeneration induced by aggregated α‐synuclein. Furthermore, microglial enhancement of α‐synuclein‐mediated neurotoxicity depended on phagocytosis of α‐synuclein and activation of NADPH oxidase with production of reactive oxygen species. These results suggest that nigral neuronal damage, regardless of etiology, may release aggregated α‐synuclein into substantia nigra, which activates microglia with production of proinflammatory mediators, thereby leading to persistent and progressive nigral neurodegeneration in PD. Finally, NADPH oxidase could be an ideal target for potential pharmaceutical intervention, given that it plays a critical role in α‐synuclein‐mediated microglial activation and associated neurotoxicity.—Zhang, W., Wang, T., Pei, Z., Miller, D. S., Wu, X., Block, M. L., Wilson, B., Zhang, W., Zhou, Y., Hong, J. S., Zhang, J. Aggregated α‐synuclein activates microglia: a process leading to disease progression in Parkinsons disease. FASEB J. 19, 533–542 (2005)

Nanometer size diesel exhaust particles are selectively toxic to dopaminergic neurons: the role of microglia, phagocytosis, and NADPH oxidase

The contributing role of environmental factors to the development of Parkinson’s disease has become increasingly evident. We report that mesencephalic neuron‐glia cultures treated with diesel exhaust particles (DEP; 0.22 µM) (5–50 µg/ml) resulted in a dose‐dependent decrease in dopaminergic (DA) neurons, as determined by DA‐uptake assay and tyrosine‐hydroxylase immunocytochemistry (ICC). The selective toxicity of DEP for DA neurons was demonstrated by the lack of DEP effect on both GABA uptake and Neu‐N immunoreactive cell number. The critical role of microglia was demonstrated by the failure of neuron‐enriched cultures to exhibit DEP‐induced DA neurotoxicity, where DEP‐induced DA neuron death was reinstated with the addition of microglia to neuron‐enriched cultures. OX‐42 ICC staining of DEP treated neuron‐glia cultures revealed changes in microglia morphology indicative of activation. Intracellular reactive oxygen species and superoxide were produced from enriched‐microglia cultures in response to DEP. Neuron‐glia cultures from NADPH oxidase deficient (PHOX−/−) mice were insensitive to DEP neurotoxicity when compared with control mice (PHOX+/+). Cytochalasin D inhibited DEP‐induced superoxide production in enriched‐microglia cultures, implying that DEP must be phagocytized by microglia to produce superoxide. Together, these in vitro data indicate that DEP selectively damages DA neurons through the phagocytic activation of microglial NADPH oxidase and consequent oxidative insult.

Neuroprotective effect of dextromethorphan in the MPTP Parkinson’s disease model: role of NADPH oxidase

Parkinsons disease (PD) is a neurodegenerative movement disorder characterized by a progressive loss of dopaminergic neurons in the substantia nigra and depletion of the neurotransmitter dopamine in the striatum. Progress in the search for effective therapeutic strategies that can halt this degenerative process remains limited. Mechanistic studies using animal systems such as the 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) rodent PD model have revealed the involvement of the brains immune cells and free radical‐generating processes. We recently reported that dextromethorphan (DM), a widely used anti‐tussive agent, attenuated endotoxin‐induced dopaminergic neurodegeneration in vitro. In the current study, we investigated the potential neuroprotective effect of DM and the underlying mechanism of action in the MPTP rodent PD model. Mice (C57BL/6J) that received daily MPTP injections (15 mg free base/kg body weight, s.c.) for 6 consecutive days exhibited significant degeneration of the nigrostriatal dopaminergic pathway. However, the MPTP‐induced loss of nigral dopaminergic neurons was significantly attenuated in those mice receiving DM (10 mg/kg body weight, s.c.). In mesencephalic neuron‐glia cultures, DM significantly reduced the MPTP‐induced production of both extracellular superoxide free radicals and intracellular reactive oxygen species (ROS). Because NADPH oxidase is the primary source of extracellular superoxide and intracellular ROS, we investigated the involvement of NADPH oxidase in the neuroprotective effect of DM. Indeed, the neuroprotective effect of DM was only observed in the wild‐type but not in the NADPH oxidase‐deficient mice, indicating that NADPH oxidase is a critical mediator of the neuroprotective activity of DM. More importantly, due to its proven safety record of long‐term clinical use in humans, DM may be a promising agent for the treatment of degenerative neurological disorders such as PD.

Cyclooxygenase-2-deficient mice are resistant to 1-methyl-4-phenyl1, 2, 3, 6-tetrahydropyridine-induced damage of dopaminergic neurons in the substantia nigra

Cyclooxygenases (COX), key enzymes in prostanoid biosynthesis, may represent important therapeutic targets in various neurodegenerative diseases. In the present study, we explored the role of COX in Parkinsons disease (PD) by using 1-methyl-4-phenyl1, 2, 3, 6-tetrahydropyridine (MPTP) as a tool to create a rodent Parkinsonian model. MPTP (20 mg/kg, subcutaneously) was injected daily into COX-1- and COX-2-deficient mice and wild-type (WT) controls for five consecutive days. Immunocytochemical analysis of tissues collected 7 days after the final MPTP treatment showed that MPTP significantly decreased the number of tyrosine hydroxylase-immunoreactive (TH-ir) neurons in the substantia nigra pars compacta (SNc) of WT (40% decrease) and COX-1(-/-) (45% decrease) mutants. However, a much smaller loss of TH-ir neurons in COX-2(-/-) mutants (20% decrease) was observed. Furthermore, electrochemical analysis revealed a more than 70% decrease in the levels of dopamine and its metabolites (3,4-dihydroxyphenylacetic acid and homovanillic acid) in the striatum of the WT control COX-1(-/-) and COX-2(-/-) mutant mice. These results indicate that loss of COX-2 activity reduces MPTP-induced damage to the dopaminergic neurons of the SNc, but does not alter the levels of dopamine and its metabolites in the striatum. Interestingly, MPTP caused the same degree of loss of dopaminergic neurons in both COX-2(+/-) and COX-2(-/-) mice (20% loss). The results of this study indicate an important role of COX-2 in MPTP-induced neuronal degeneration and suggest the possibility that manipulation of the COX-2 could be an important target for therapeutic interventions in PD.

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

The importance of cyclooxygenase‐2 (COX‐2) in mediating Parkinsons disease (PD) was suggested in reports, indicating that COX‐2 selective inhibitors or genetic knockout reduce 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induced dopaminergic (DA) neurotoxicity in a mouse model of PD. However, cell types and mechanisms underlying the activation of COX‐2 have not been clearly elucidated in these animal studies. Using primary neuron‐glia cultures, we aimed to determine 1) whether microglia participate in 1‐methyl‐4‐phenylpryridinium (MPP+)‐induced COX‐2 activation and 2) whether the activation of COX‐2 contributes to subsequent neurotoxicity. MPP+, in a concentration‐dependent manner, increased prostaglandin E2 (PGE2) production in mixed neuron‐microglia cultures but not in enriched neuron, microglia, or astroglia cultures nor in mixed neuron‐astroglia cultures. MPP+‐induced PGE2 increase was completely abolished by treatment with DuP697, a COX‐2 selective inhibitor. DuP697 also significantly reduced MPP+‐induced DA neurotoxicity as determined by DA uptake assay. Immunocytochemistry and confocal microscopy studies showed enhanced COX‐2 expression in both microglia and neurons after MPP+ treatment. However, neuronal increase in COX‐2 expression was not totally dependent on the production of PGE2 from microglia, since microglia deficient in COX‐2 only attenuated, but did not completely block, MPP+‐increased PGE2 production in mixed neuron‐microglia cultures, suggesting that part of PGE2 production was originated from neurons. Together, these results indicate that MPP+‐induced COX‐2 expression and subsequent PGE2 production depend on interactions between neurons and microglia. Microgliosis may also be responsible for the COX‐2 activation in neurons, leading to the enhanced DA neurotoxicity, which, in turn, reinforces microgliosis. Thus inhibition of microgliosis and COX‐2 activity may stop this vicious circle and be valuable strategies in PD therapy.

Femtomolar concentrations of dextromethorphan protect mesencephalic dopaminergic neurons from inflammatory damage.

Inflammation in the brain has increasingly been recognized to play an important role in the pathogenesis of several neurodegenerative disorders, including Parkinsons disease (PD). Progress in the search for effective therapeutic strategies that can halt this degenerative process remains limited. We previously showed that micromolar concentrations of dextromethorphan (DM), a major ingredient of widely used antitussive remedies, reduced the inflammation‐mediated degeneration of dopaminergic neurons through the inhibition of microglial activation. In this study, we report that femto‐ and micromolar concentrations of DM (both pre‐ and post‐treatment) showed equal efficacy in protecting lipopolysaccharide (LPS)‐induced dopaminergic neuron death in midbrain neuron‐glia cultures. Both concentrations of DM decreased LPS‐induced release of nitric oxide, tumor necrosis factor‐α, prostaglandin E2 and superoxide from microglia in comparable degrees. The important role of superoxide was demonstrated by DMs failure to show a neuroprotective effect in neuron‐glia cultures from NADPH oxidase‐deficient mice. These results suggest that the neuroprotective effect elicited by femtomolar concentrations of DM is mediated through the inhibition of LPS‐induced proinflammatory factors, especially superoxide. These findings suggest a novel therapeutic concept of using “ultra‐low” drug concentrations for the intervention of inflammation‐related neurodegenerative diseases.—Li, G., Cui, G., Tzeng, N.‐S., Wei, S.‐J., Wang, T., Block, M. L., Hong, J.‐S. Femtomolar concentrations of dextromethorphan protect mesencephalic dopaminergic neurons from inflammatory damage. FASEB J. 19, 489–496 (2005)

Potent regulation of microglia-derived oxidative stress and dopaminergic neuron survival: substance P vs. dynorphin

Unregulated microglial activation has been implicated as a pivotal factor contributing to Parkinsons disease. Using mesencephalic neuron‐glia cultures, we address the novel possibility that peptides endogenous to the substantia nigra (SN), substance P and dynorphin (10‐13–10‐14 M), are opposing mediators of microglial activation and consequent DA neurotoxicity. Here, we identify that substance P (10‐13–10‐14 M) is selectively toxic to DA neurons in a microglia‐dependent manner. Mechanistically, substance P (10‐13–10‐14 M) activated microglial NADPH oxidase to produce extracellular superoxide and intracellular reactive oxygen species (ROS). Neuron‐glia cultures from mice lacking a functional NADPH oxidase complex (PHOX−/−) were insensitive to substance P (10‐13–10‐14 M) ‐induced loss of DA neuron function. Mixed glia cultures from (PHOX−/−) mice failed to show a significant increase in intracellular ROS in response to substance P compared with control cultures (PHOX+/+). Further, dynorphin (10‐14 M) inhibited substance P (10‐13 M) ‐induced loss of [3H] DA uptake. Here we demonstrate a tightly regulated mechanism governing microglia‐derived oxidative stress, where the neuropeptide balance of dynorphin and substance P is critical to DA neuron survival.— Block, M. L., Li, G., Qin, L., Wu, X., Pei, Z., Wang, T., Wilson, B., Yang, J., Hong, J. S. Potent regulation of microglia‐derived oxidative stress and dopaminergic neuron survival: substance P vs. dynorphin. FASEB J. 20, 251–258 (2006)

3-Hydroxymorphinan, a metabolite of dextromethorphan, protects nigrostriatal pathway against MPTP-elicited damage both in vivo and in vitro

We investigated the neuroprotective property of analogs of dextromethorphan (DM) in lipopolysaccharide (LPS) and 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) models to identify neuroprotective drugs for Parkinsons disease (PD). In vivo studies showed that daily injections with DM analogs protected dopamine (DA) neurons in substantia nigra pars compacta and restored DA levels in striatum using two different models for PD. Of the five analogs studied, 3‐hydroxymorphinan (3‐HM), a metabolite of DM, was the most potent, and restored DA neuronal loss and DA depletion up to 90% of the controls. Behavioral studies showed an excellent correlation between potency for preventing toxin‐induced decrease in motor activities and neuroprotective effects among the DM analogs studied, of which 3‐HM was the most potent in attenuating behavioral damage. In vitro studies revealed two glia‐dependent mechanisms for the neuroprotection by 3‐HM. First, astroglia mediated the 3‐HM‐induced neurotrophic effect by increasing the gene expression of neurotrophic factors, which was associated with the increased acetylation of histone H3. Second, microglia participated in 3‐HM‐mediated neuroprotection by reducing MPTP‐elicited reactive microgliosis as evidenced by the decreased production of reactive oxygen species. In summary, we show the potent neuroprotection by 3‐HM in LPS and MPTP PD models investigated. With its high efficacy and low toxicity, 3‐HM may be a novel therapy for PD.—Zhang, W., Shin, E‐J., Wang, T., Lee, P. H., Pang, H., Wie, M. B., Kim, W‐K., Kim, S‐J., Huang, W‐H., Wang, Y., Zhang, W., Hong, J‐S., Kim, H‐C. 3‐Hydroxymorphinan, a metabolite of dextromethorphan, protects nigrostriatal pathway against MPTP‐elicited damage both in vivo and in vitro. FASEB J. 20, 2496–2511 (2006)

3-Hydroxymorphinan is neurotrophic to dopaminergic neurons and is also neuroprotective against LPS-induced neurotoxicity

The purpose of this study was to develop a novel therapy for Parkinsons disease (PD). We recently reported that dextromethorphan (DM), an active ingredient in a variety of widely used anticough remedies, protected dopaminergic neurons in rat primary mesencephalic neuron‐glia cultures against lipopolysaccharide (LPS)‐mediated degeneration and provided potent protection for dopaminergic neurons in a MPTP mouse model. The underlying mechanism for the protective effect of DM was attributed to its anti‐inflammatory activity through inhibition of microglia activation. In an effort to develop more potent compounds for the treatment of PD, we have screened a series of analogs of DM, and 3‐hydroxymorphinan (3‐HM) emerged as a promising candidate for this purpose. Our study using primary mesencephalic neuron‐glia cultures showed that 3‐HM provided more potent neuroprotection against LPS‐induced dopaminergic neurotoxicity than its parent compound. The higher potency of 3‐HM was attributed to its neurotrophic effect in addition to the anti‐inflammatory effect shared by both DM and 3‐HM. First, we showed that 3‐HM exerted potent neuroprotective and neurotrophic effects on dopaminergic neurons in rat primary mesencephalic neuron‐glia cultures treated with LPS. The neurotrophic effect of 3‐HM was glia‐dependent since 3‐HM failed to show any protective effect in the neuron‐enriched cultures. We subsequently demonstrated that it was the astroglia, not the microglia, that contributed to the neurotrophic effect of 3‐HM. This conclusion was based on the reconstitution studies, in which we added different percentages of microglia (10–20%) or astroglia (40–50%) back to the neuron‐enriched cultures and found that 3‐HM was neurotrophic after the addition of astroglia, but not microglia. Furthermore, 3‐HM‐treated astroglia‐derived conditioned media exerted a significant neurotrophic effect on dopaminergic neurons. It appeared likely that 3‐HM caused the release of neurotrophic factor(s) from astroglia, which in turn was responsible for the neurotrophic effect. Second, the anti‐inflammatory mechanism was also important for the neuroprotective activity of 3‐HM because the more microglia were added back to the neuron‐enriched cultures, the more significant neuroprotective effect was observed. The 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 nitric oxide (NO), tumor necrosis factor α (TNF‐α), prostaglandin E2 (PGE2) and reactive oxygen species (ROS). In conclusion, this study showed that 3‐HM exerted potent neuroprotection by acting on two different targets: a neurotrophic effect mediated by astroglia and an anti‐inflammatory effect mediated by the inhibition of microglial activation. 3‐HM thus possesses these two important features necessary for an effective neuroprotective agent. In view of the well‐documented very low toxicity of DM and its analogs, this report may provide an important new direction for the development of therapeutic interventions for inflammation‐related diseases such as PD.

Reactive microgliosis participates in MPP+-induced dopaminergic neurodegeneration: role of 67 kDa laminin receptor

It has been reported that extracellular matrix (ECM) molecules regulate monocyte activation by binding with a 67 kDa nonintegrin laminin receptor (LR). As microgliosis is a pivotal factor in propelling the progress of chronic neurodegeneration in the brain, we hypothesized that LR may regulate the microgliosis and subsequent neurotoxicity. Using 1‐methyl‐4‐phe‐nylpyridinium (MPP+) ‐treated C57 mice primary mesencephalic neuron‐glia cultures as an in vitro Parkinsons disease (PD) model, we observed that MPP+ treatment increased LR expression only in the mixed neuron‐glia but not in microglia‐enriched or microglia‐depleted cultures, indicating that MPP+‐induced increase of LR expression is associated with neuron‐microglia interaction. Using confocal microscopic examination, we found that LR was localized in the microglia, which were F4/80 positive. Treatment with the antibody (Ab) against LR (LR‐Ab) or YIGSR, a synthetic pentapeptide inhibitor for LR, significantly attenuated the MPP+‐increased F4/80 immunoreactivity (24 h) and dopaminergic (DA) neurotoxicity. LR‐Ab also attenuated MPP+‐increased microglial phagocytotic activity (48 h) and the superoxide production (4 days). Further study demonstrated that exogenous laminin (1–10 µg/ml) treatment induced microglial activation and DA neurotoxicity, in a dose‐dependent manner, which was partially attenuated by the LR‐Ab. We concluded that by regulating cell‐ECM interaction, LR plays important roles in mediating microgliosis and subsequent DA neurotoxicity. Laminin is a potential ligand for activating this LR receptor. This study also suggests that laminin/LR is a potential target for developing new therapeutic drugs against neurodegenerative disorders such as PD.—Wang, T., Zhang, W., Pei, Z., Block, M., Wilson, B., Reece, J. M., Miller, D. S., and Hong, J.‐S. Reactive microgliosis participates in MPP+‐induced dopaminergic neurodegeneration: role of 67 kDa laminin receptor. FASEB J. 20, 906–915 (2006)