Belinda Wilson

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)

Microglial activation-mediated delayed and progressive degeneration of rat nigral dopaminergic neurons: relevance to Parkinson's disease

The etiology of sporadic Parkinsons disease (PD) remains unknown. Increasing evidence has suggested a role for inflammation in the brain in the pathogenesis of PD. However, it has not been clearly demonstrated whether microglial activation, the most integral part of the brain inflammatory process, will result in a delayed and progressive degeneration of dopaminergic neurons in substantia nigra, a hallmark of PD. We report here that chronic infusion of an inflammagen lipopolysaccharide at 5 ng/h for 2 weeks into rat brain triggered a rapid activation of microglia that reached a plateau in 2 weeks, followed by a delayed and gradual loss of nigral dopaminergic neurons that began at between 4 and 6 weeks and reached 70% by 10 weeks. Further investigation of the underlying mechanism of action of microglia‐mediated neurotoxicity using rat mesencephalic neuron‐glia cultures demonstrated that low concentrations of lipopolysaccharide (0.1–10 ng/mL)‐induced microglial activation and production of neurotoxic factors preceded the progressive and selective degeneration of dopaminergic neurons. Among the factors produced by activated microglia, the NADPH oxidase‐mediated release of superoxide appeared to be a predominant effector of neurodegeneration, consistent with the notion that dopaminergic neurons are particularly vulnerable to oxidative insults. This is the first report that microglial activation induced by chronic exposure to inflammagen was capable of inducing a delayed and selective degeneration of nigral dopaminergic neurons and that microglia‐originated free radicals play a pivotal role in dopaminergic neurotoxicity in this inflammation‐mediated model of PD.

Microglia enhance β‐amyloid peptide‐induced toxicity in cortical and mesencephalic neurons by producing reactive oxygen species

The purpose of this study was to assess and compare the toxicity of β‐amyloid (Aβ) on primary cortical and mesencephalic neurons cultured with and without microglia in order to determine the mechanism underlying microglia‐mediated Aβ‐induced neurotoxicity. Incubation of cortical or mesencephalic neuron‐enriched and mixed neuron–glia cultures with Aβ(1–42) over the concentration range 0.1–6.0 μm caused concentration‐dependent neurotoxicity. High concentrations of Aβ (6.0 μm for cortex and 1.5–2.0 μm for mesencephalon) directly injured neurons in neuron‐enriched cultures. In contrast, lower concentrations of Aβ (1.0–3.0 μm for cortex and 0.25–1.0 μm for mesencephalon) caused significant neurotoxicity in mixed neuron–glia cultures, but not in neuron‐ enriched cultures. Several lines of evidence indicated that microglia mediated the potentiated neurotoxicity of Aβ, including the observations that low concentrations of Aβ activated microglia morphologically in neuron–glia cultures and that addition of microglia to cortical neuron–glia cultures enhanced Aβ‐induced neurotoxicity. To search for the mechanism underlying the microglia‐mediated effects, several proinflammatory factors were examined in neuron–glia cultures. Low doses of Aβ significantly increased the production of superoxide anions, but not of tumor necrosis factor‐α, interleukin‐1β or nitric oxide. Catalase and superoxide dismutase significantly protected neurons from Aβ toxicity in the presence of microglia. Inhibition of NADPH oxidase activity by diphenyleneiodonium also prevented Aβ‐induced neurotoxicity in neuron–glia mixed cultures. The role of NADPH oxidase‐generated superoxide in mediating Aβ‐induced neurotoxicity was further substantiated by a study which showed that Aβ caused less of a decrease in dopamine uptake in mesencephalic neuron–glia cultures from NADPH oxidase‐deficient mutant mice than in that from wild‐type controls. This study demonstrates that one of the mechanisms by which microglia can enhance the neurotoxicity of Aβ is via the production of reactive oxygen species.

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.

Histone deacetylase inhibitors up-regulate astrocyte GDNF and BDNF gene transcription and protect dopaminergic neurons

Parkinsons disease (PD) is characterized by the selective and progressive loss of dopaminergic (DA) neurons in the midbrain substantia nigra. Currently, available treatment is unable to alter PD progression. Previously, we demonstrated that valproic acid (VPA), a mood stabilizer, anticonvulsant and histone deacetylase (HDAC) inhibitor, increases the expression of glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) in astrocytes to protect DA neurons in midbrain neuron-glia cultures. The present study investigated whether these effects are due to HDAC inhibition and histone acetylation. Here, we show that two additional HDAC inhibitors, sodium butyrate (SB) and trichostatin A (TSA), mimic the survival-promoting and protective effects of VPA on DA neurons in neuron-glia cultures. Similar to VPA, both SB and TSA increased GDNF and BDNF transcripts in astrocytes in a time-dependent manner. Furthermore, marked increases in GDNF promoter activity and promoter-associated histone H3 acetylation were noted in astrocytes treated with all three compounds, where the time-course for acetylation was similar to that for gene transcription. Taken together, our results indicate that HDAC inhibitors up-regulate GDNF and BDNF expression in astrocytes and protect DA neurons, at least in part, through HDAC inhibition. This study indicates that astrocytes may be a critical neuroprotective mechanism of HDAC inhibitors, revealing a novel target for the treatment of psychiatric and neurodegenerative diseases.

Synergistic neurotoxic effects of combined treatments with cytokines in murine primary mixed neuron/glia cultures

Activation of brain glial cells with the bacterial endotoxin lipopolysaccharide (LPS), the HIV-1 coat protein gp120, or beta-amyloid-derived peptides, stimulates the expression of several cytokines, including tumor necrosis factor-alpha (TNFalpha), interleukin-1 (IL-1) and IL-6. and nitric oxide (NO) which have been proposed as causes of neurodegeneration in the brain. In the present study, the neurotoxic effects of several cytokines, alone or in various combinations, and the correlations of the release of lactate dehydrogenase, the loss of neurons, and the secretion of NO in brain neuronal cell injury were investigated in murine primary mixed neuronal/glial cell cultures. A specific combination of cytokines, i.e., IL-1 (1 ng/ml)+ TNFalpha (10 ng/ml)/interferon-gamma (IFNgamma) (200 u/ml), induced a dramatic neuronal cell injury in the neuron/glia cultures, and its cytotoxic profile was very similar to that seen with the LPS/IFNgamma-induced neuron injury. This indicates that among the many toxic immune mediators secreted in response to LPS, IL-1 and TNFalpha can mimic LPS as the triggering signals and primary mediators for glia-mediated neuron injury in the presence of IFNgamma. This study provides new insights about the cytotoxic mechanism(s) for cytokine-mediated neuron injury.

Role of reactive oxygen species in LPS‐induced production of prostaglandin E2 in microglia

We determined the roles of reactive oxygen species (ROS) in the expression of cyclooxygenase‐2 (COX‐2) and the production of prostaglandin E2 (PGE2) in lipopolysaccharide (LPS)‐activated microglia. LPS treatment increased intracellular ROS in rat microglia dose‐dependently. Pre‐treatment with superoxide dismutase (SOD)/catalase, or SOD/catalase mimetics that can scavenge intracellular ROS, significantly attenuated LPS‐induced release in PGE2. Diphenylene iodonium (DPI), a non‐specific NADPH oxidase inhibitor, decreased LPS‐induced PGE2 production. In addition, microglia from NADPH oxidase‐deficient mice produced less PGE2 than those from wild‐type mice following LPS treatment. Furthermore, LPS‐stimulated expression of COX‐2 (determined by RT‐PCR analysis of COX‐2 mRNA and western blot for its protein) was significantly reduced by pre‐treatment with SOD/catalase or SOD/catalase mimetics. SOD/catalase mimetics were more potent than SOD/catalase in reducing COX‐2 expression and PGE2 production. As a comparison, scavenging ROS had no effect on LPS‐induced nitric oxide production in microglia. These results suggest that ROS play a regulatory role in the expression of COX‐2 and the subsequent production of PGE2 during the activation process of microglia. Thus, inhibiting NADPH oxidase activity and subsequent ROS generation in microglia can reduce COX‐2 expression and PGE2 production. These findings suggest a potential therapeutic intervention strategy for the treatment of inflammation‐mediated neurodegenerative diseases.

HMGB1 Acts on Microglia Mac1 to Mediate Chronic Neuroinflammation That Drives Progressive Neurodegeneration

What drives the gradual degeneration of dopamine neurons in Parkinsons disease (PD), the second most common neurodegenerative disease, remains elusive. Here, we demonstrated, for the first time, that persistent neuroinflammation was indispensible for such a neurodegenerative process. 1-Methyl-4-phenylpyridinium, lipopolysaccharide (LPS), and rotenone, three toxins often used to create PD models, produced acute but nonprogressive neurotoxicity in neuron-enriched cultures. In the presence of microglia (brain immune cells), these toxins induced progressive dopaminergic neurodegeneration. More importantly, such neurodegeneration was prevented by removing activated microglia. Collectively, chronic neuroinflammation may be a driving force of progressive dopaminergic neurodegeneration. Conversely, ongoing neurodegeneration sustained microglial activation. Microglial activation persisted only in the presence of neuronal damage in LPS-treated neuron–glia cultures but not in LPS-treated mixed-glia cultures. Thus, activated microglia and damaged neurons formed a vicious cycle mediating chronic, progressive neurodegeneration. Mechanistic studies indicated that HMGB1 (high-mobility group box 1), released from inflamed microglia and/or degenerating neurons, bound to microglial Mac1 (macrophage antigen complex 1) and activated nuclear factor-κB pathway and NADPH oxidase to stimulate production of multiple inflammatory and neurotoxic factors. The treatment of microglia with HMGB1 led to membrane translocation of p47phox (a cytosolic subunit of NADPH oxidase) and consequent superoxide release, which required the presence of Mac1. Neutralization of HMGB1 and genetic ablation of Mac1 and gp91phox (the catalytic submit of NADPH oxidase) blocked the progressive neurodegeneration. Our findings indicated that HMGB1–Mac1–NADPH oxidase signaling axis bridged chronic neuroinflammation and progressive dopaminergic neurodegeneration, thus identifying a mechanistic basis for chronic PD progression.

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.