Hui-Ming Gao

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.

Critical Role for Microglial NADPH Oxidase in Rotenone-Induced Degeneration of Dopaminergic Neurons

Increasing evidence has suggested an important role for environmental toxins such as pesticides in the pathogenesis of Parkinsons disease (PD). Chronic exposure to rotenone, a common herbicide, reproduces features of Parkinsonism in rats. Mechanistically, rotenone-induced dopaminergic neurodegeneration has been associated with both its inhibition of neuronal mitochondrial complex I and the enhancement of activated microglia. Our previous studies with NADPH oxidase inhibitors, diphenylene iodonium and apocynin, suggested that NADPH oxidase-derived superoxide might be a major factor in mediating the microglia-enhanced rotenone neurotoxicity. However, because of the relatively low specificity of these inhibitors, the exact source of superoxide induced by rotenone remains to be further determined. In this study, using primary mesencephalic cultures from NADPH oxidase—null (gp91phox-/-) or wild-type (gp91phox+/+) mice, we demonstrated a critical role for microglial NADPH oxidase in mediating microglia-enhanced rotenone neurotoxicity. In neuron—glia cultures, dopaminergic neurons from gp91phox-/- mice were more resistant to rotenone neurotoxicity than those from gp91phox+/+ mice. However, in neuron-enriched cultures, the neurotoxicity of rotenone was not different between the two types of mice. More importantly, the addition of microglia prepared from gp91phox+/+ mice but not from gp91phox-/- mice to neuron-enriched cultures markedly increased rotenone-induced degeneration of dopaminergic neurons. Furthermore, apocynin attenuated rotenone neurotoxicity only in the presence of microglia from gp91phox+/+ mice. These results indicated that the greatly enhanced neurotoxicity of rotenone was attributed to the release of NADPH oxidase-derived superoxide from activated microglia. This study also suggested that microglial NADPH oxidase may be a promising target for PD treatment.

Novel anti-inflammatory therapy for Parkinson's disease

Parkinsons disease (PD) is a movement disorder that is characterized by progressive degeneration of the nigrostriatal dopamine system. Although dopamine replacement can alleviate symptoms of the disorder, there is no proven therapy to halt the underlying progressive degeneration of dopamine-containing neurons. Recently, increasing evidence from human and animal studies has suggested that neuroinflammation is an important contributor to the neuronal loss in PD. Moreover, the pro-inflammatory agent lipopolysaccharide itself can directly initiate degeneration of dopamine-containing neurons or combine with other environmental factor(s), such as the pesticide rotenone, to exacerbate such neurodegeneration. These effects provide strong support for the involvement of inflammation in the pathogenesis of PD. Furthermore, growing experimental evidence demonstrates that inhibition of the inflammatory response can, in part, prevent degeneration of nigrostriatal dopamine-containing neurons in several animal models of PD, suggesting that inhibition of inflammation might become a promising therapeutic intervention for PD.

Critical role of microglial NADPH oxidase-derived free radicals in the in vitro MPTP model of Parkinson’s disease

1‐Methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) damages dopaminergic neurons as seen in Parkinsons disease. Although increasing evidence suggests an involvement of glia in MPTP neurotoxicity, the nature of this involvement remains unclear. Exploiting the advantage of cell culture systems, we demonstrated that microglia, but not astroglia, significantly enhanced the progression of MPTP‐induced dopaminergic neurodegeneration. Characterization of the temporal relationship between neurodegeneration and microglial activation demonstrates that reactive microgliosis resulting from MPTP‐initiated neuronal injury, but not direct activation, underlies the microglia‐enhanced MPTP neurotoxicity. Mechanistically, through the release of NADPH oxidase‐derived reactive oxygen species, microglia contribute to the progressive neuronal damage. Among the factors measured, the production of extracellular superoxide was the most prominent. NADPH oxidase inhibitor, apocynin, attenuated MPTP‐induced dopaminergic neurodegeneration only in the presence of glia. More importantly, dopaminergic neurons from mice lacking NADPH oxidase, a key enzyme for superoxide production in immune cells, are significantly more resistant to MPTP neurotoxicity than those from wild‐type controls, and microglia dictate the resistance. This study demonstrates that reactive microgliosis triggered by MPTP‐induced neuronal injury and NADPH oxidase‐mediated superoxide production in microglia constitute an integral component of MPTP neurotoxicity. This study also suggests that NADPH oxidase may be a promising target for therapeutic interventions in Parkinsons disease.

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.

Neuroinflammation and α-Synuclein Dysfunction Potentiate Each Other, Driving Chronic Progression of Neurodegeneration in a Mouse Model of Parkinson's Disease

Background Mechanisms whereby gene–environment interactions mediate chronic, progressive neurodegenerative processes in Parkinson’s disease (PD)—the second most common neurodegenerative disease—remain elusive. Objective We created a two-hit [neuroinflammation and mutant α-synuclein (α-syn) overexpression] animal model to investigate mechanisms through which mutant α-syn and inflammation work in concert to mediate chronic PD neurodegeneration. Methods We used an intraperitoneal injection of the inflammogen lipopolysaccharide (LPS; 3 × 106 EU/kg) to initiate systemic and brain inflammation in wild-type (WT) mice and transgenic (Tg) mice overexpressing human A53T mutant α-syn. We then evaluated nigral dopaminergic neurodegeneration, α-syn pathology, and neuroinflammation. Results After LPS injection, both WT and Tg mice initially displayed indistinguishable acute neuroinflammation; however, only Tg mice developed persistent neuroinflammation, chronic progressive degeneration of the nigrostriatal dopamine pathway, accumulation of aggregated, nitrated α-syn, and formation of Lewy body-like inclusions in nigral neurons. Further mechanistic studies indicated that 4-week infusion of two inhibitors of inducible nitric oxide synthase and NADPH oxidase, major free radical–generating enzymes in activated microglia, blocked nigral α-syn pathology and neurodegeneration in LPS-injected Tg mice. Conclusions Microglia-derived oxidative stress bridged neuroinflammation and α-syn pathogenic alteration in mediating chronic PD progression. Our two-hit animal model involving both a genetic lesion and an environmental trigger reproduced key features of PD and demonstrated synergistic effects of genetic predisposition and environmental exposures in the development of PD. The chronic progressive nature of dopaminergic neurodegeneration, which is absent in most existing PD models, makes this new model invaluable for the study of mechanisms of PD progression.

Synergistic dopaminergic neurotoxicity of MPTP and inflammogen lipopolysaccharide: relevance to the etiology of Parkinson’s disease

Parkinsons disease (PD) is a profound movement disorder resulting from progressive degeneration of the nigrostriatal dopaminergic pathway. Although its etiology remains unknown, increasing evidence suggests the involvement of multiple factors such as environmental toxins and genetic susceptibilities in the pathogenesis of PD. In this study using mesencephalic neuronglia cultures as an in vitro PD model, we demonstrated that the neurotoxin 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP, 0.1–0.5 µM) and an inflammogen lipopolysaccharide (LPS, 0.5 ng/ml) synergistically induced a progressive and selective degeneration of dopaminergic neurons. The synergistic neurotoxicity was observed when both agents were applied either simultaneously or in tandem. The synergistic neurotoxicity was more prominent when lower doses of both agents were applied for a longer period of time. Mechanistically, microglial NADPH oxidase‐mediated generation of reactive oxygen species played a pivotal role in the synergistic neurotoxicity: MPTP and LPS synergistically stimulated the NADPH oxidase‐mediated release of superoxide free radical; pharmacological inhibition and genetic inactivation of NADPH oxidase prevented superoxide production and the synergistic neurotoxicity. Additionally, inhibition of nitric oxide synthase afforded significant neuroprotection, suggesting the involvement of nitric oxide in the synergistic neurotoxicity. This study lends strong support for a multifactorial etiology of PD and provides clues for therapeutic interventions.

NADPH oxidases: novel therapeutic targets for neurodegenerative diseases

Oxidative stress is a key pathologic factor in neurodegenerative diseases such as Alzheimer and Parkinson diseases (AD, PD). The failure of free-radical-scavenging antioxidants in clinical trials pinpoints an urgent need to identify and to block major sources of oxidative stress in neurodegenerative diseases. As a major superoxide-producing enzyme complex in activated phagocytes, phagocyte NADPH oxidase (PHOX) is essential for host defense. However, recent preclinical evidence has underscored a pivotal role of overactivated PHOX in chronic neuroinflammation and progressive neurodegeneration. Deficiency in PHOX subunits mitigates neuronal damage induced by diverse insults/stresses relevant to neurodegenerative diseases. More importantly, suppression of PHOX activity correlates with reduced neuronal impairment in models of neurodegenerative diseases. The discovery of PHOX and non-phagocyte NADPH oxidases in astroglia and neurons further reinforces the crucial role of NADPH oxidases in oxidative stress-mediated chronic neurodegeneration. Thus, proper modulation of NADPH oxidase activity might hold therapeutic potential for currently incurable neurodegenerative diseases.

Resveratrol protects dopamine neurons against lipopolysaccharide-induced neurotoxicity through its anti-inflammatory actions.

Parkinsons disease (PD) is the second most common neurodegenerative disease characterized by a progressive loss of dopamine (DA) neurons in the substantia nigra. Accumulating evidence indicates that inhibition of microglia-mediated neuroinflammation may become a reliable protective strategy for PD. Resveratrol, a nonflavonoid polyphenol naturally found in red wine and grapes, has been known to possess antioxidant, anticancer, and anti-inflammatory properties. Although recent studies have shown that resveratrol provided neuroprotective effects against ischemia, seizure, and neurodegenerative disorders, the mechanisms underlying its beneficial effects on dopaminergic neurodegeneration are poorly defined. In this study, rat primary midbrain neuron-glia cultures were used to elucidate the molecular mechanisms underlying resveratrol-mediated neuroprotection. The results clearly demonstrated that resveratrol protected DA neurons against lipopolysaccharide (LPS)-induced neurotoxicity in concentration- and time-dependent manners through the inhibition of microglial activation and the subsequent reduction of proinflammatory factor release. Mechanistically, resveratrol-mediated neuroprotection was attributed to the inhibition of NADPH oxidase. This conclusion is supported by the following observations. First, resveratrol reduced NADPH oxidase-mediated generation of reactive oxygen species. Second, LPS-induced translocation of NADPH oxidase cytosolic subunit p47 to the cell membrane was significantly attenuated by resveratrol. Third and most importantly, resveratrol failed to exhibit neuroprotection in cultures from NADPH oxidase-deficient mice. Furthermore, this neuroprotection was also related to an attenuation of the activation of mitogen-activated protein kinases and nuclear factor-κB signaling pathways in microglia. These findings suggest that resveratrol exerts neuroprotection against LPS-induced dopaminergic neurodegeneration, and NADPH oxidase may be a major player in resveratrol-mediated neuroprotection.