The etiology of sporadic Parkinson's 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.

Increasing evidence has suggested an important role for environmental factors such as exposure to pesticides in the pathogenesis of Parkinson's disease. In experimental animals the exposure to a common herbicide, rotenone, induces features of parkinsonism; mechanistically, rotenone-induced destruction of dopaminergic neurons has been attributed to its inhibition of the activity of neuronal mitochondrial complex I. However, the role of microglia, the resident brain immune cells in rotenoneinduced neurodegeneration, has not been reported. Using primary neuron-enriched and neuron/glia cultures from the rat mesencephalon, we discovered an extraordinary feature for rotenone-induced degeneration of cultured dopaminergic neurons. Although little neurotoxicity was detected in neuronenriched cultures after treatment for 8 d with up to 20 nM rotenone, significant and selective dopaminergic neurodegeneration was observed in neuron/glia cultures 2 d after treatment with 20 nM rotenone or 8 d after treatment with 1 nM rotenone. The greatly enhanced neurodegenerative ability of rotenone was attributed to the presence of glia, especially microglia, because the addition of microglia to neuron-enriched cultures markedly increased their susceptibility to rotenone. Mechanistically, rotenone stimulated the release of superoxide from microglia that was attenuated by inhibitors of NADPH oxidase. Furthermore, inhibition of NADPH oxidase or scavenging of superoxide significantly reduced the rotenone-induced neurotoxicity. This is the first report demonstrating that microglia play a pivotal role in rotenone-induced degeneration of dopaminergic neurons. The results of this study should advance our understanding of the mechanism of action for pesticides in the pathogenesis of Parkinson's disease.

Increasing evidence has suggested that inflammation in the brain is closely associated with the pathogenesis of several degenerative neurologic disorders, including Parkinson's disease, Alzheimer's diseases, multiple sclerosis, amyotrophic lateral sclerosis, and AIDS dementia. The hallmark of brain inflammation is the activation of glial cells, especially that of microglia that produce a variety of proinflammatory and neurotoxic factors, including cytokines, fatty acid metabolites, free radicals--such as nitric oxide (NO) and superoxide. Excessive production of NO, as a consequence of nitric oxide synthase induction in activated glia, has been attributed to participate in neurodegeneration. Using primary mixed neuron-glia cultures and glia-enriched cultures prepared from embryonic rodent brain tissues, we have systemically studied the relationship between the production of NO and neurodegeneration in response to stimulation by the inflammagen lipopolysaccharide. This review summarizes our recent findings on the kinetics of NO generation, the relative contribution of microglia and astrocytes to NO accumulation, the relationship between NO production and neurodegeneration, and points of intervention along the pathways associated with NO generation to achieve neuroprotection. We also describe our results relating to the effect of several opioid-related agents on microglial activation and neuroprotection. Among these agents, the opioid receptor antagonist naloxone, especially its non-opioid enantiomer (+)-naloxone, promises to be of potential therapeutic value for the treatment of inflammation-related diseases.

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) damages dopaminergic neurons as seen in Parkinson's 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 Parkinson's disease.

Macrophage activation and infiltration into resident tissues is known to mediate local inflammation and is a hallmark feature of metabolic syndrome. Members of the sirtuin family of proteins regulate numerous physiological processes, including those involved in nutrient regulation and the promotion of longevity. However, the important role that SIRT1, the leading sirtuin family member, plays in immune response remains unclear. In this study, we demonstrate that SIRT1 modulates the acetylation status of the RelA/p65 subunit of NF-B and thus plays a pivotal role in regulating the inflammatory, immune, and apoptotic responses in mammals. Using a myeloid cell-specific SIRT1 knockout (Mac-SIRT1 KO) mouse model, we show that ablation of SIRT1 in macrophages renders NF-B hyperacetylated, resulting in increased transcriptional activation of proinflammatory target genes. Consistent with increased proinflammatory gene expression, Mac-SIRT1 KO mice challenged with a high-fat diet display high levels of activated macrophages in liver and adipose tissue, predisposing the animals to development of systemic insulin resistance and metabolic derangement. In summary, we report that SIRT1, in macrophages, functions to inhibit NF-B-mediated transcription, implying that myeloid cell-specific modulation of this sirtuin may be beneficial in the treatment of inflammation and its associated diseases.Chronic inflammation is increasingly recognized as a causal factor leading to the development of obesity, insulin resistance, and type 2 diabetes (15, 31). This low-grade inflammatory state is in part mediated by macrophages, key sentinels of the innate immune system. Macrophages quiescently monitor the tissue milieu for signs of infection or damage (13,25). Upon stimulation, macrophages infiltrate resident tissue, perpetuating local inflammation and contributing to the development of insulin resistance and metabolic derangements (17,37,43). The nuclear factor kappa B (NF-B) transcription factor signaling pathway is a key mediator of immune response in macrophages (5, 7). NF-B is composed of a heterodimer of p50 and RelA/ p65 subunits. In unstimulated cells, NF-B resides in the cytoplasm bound to its inhibitory proteins, which are members of the inhibitor of B (IB) family. Stimulation of cells by environmental factors, including dietary fatty acids, liberates NF-B, allowing it to translocate to the nucleus, where it mediates gene transcription (12). Under environmental stresses, such as those surrounding obesity-like conditions, this chain of events is believed to ultimately lead to insulin resistance, setting in motion the vicious cycle of the metabolic syndrome.Sirtuins are highly conserved NAD ϩ -dependent deacetylases that target histones, transcription factors, coregulators, and other key regulators to adapt gene expression and metabolism to the cellular energy state (16,22,32). SIRT1, the leading family member, has been reported to promote longevity in species ranging from yeast to flies (1)(2)(3)6). It is believed that these life-exte...

␣-Synuclein (SYN) is the major component of Lewy bodies, the neuropathological hallmarks of Parkinson's disease (PD).Missense mutations and multiplications of the SYN gene cause autosomal dominant inherited PD. Thus, SYN is implicated in the pathogenesis of PD. However, the mechanism whereby SYN promotes neurodegeneration remains unclear. Familial PD with SYN gene mutations are rare because the majority of PD is sporadic and emerging evidence indicates that sporadic PD may result from genetic and environmental risk factors including neuroinflammation. Hence, we examined the relationship between SYN dysfunction and neuroinflammation in mediating dopaminergic neurodegeneration in mice and dopaminergic neuronal cultures derived from wild-type SYN and mutant A53T SYN transgenic mice in a murine SYN-null (SYNKO) background (M7KO and M83KO, respectively). Stereotaxic injection of an inflammagen, lipopolysaccharide, into substantia nigra of these SYN genetically engineered mice induced similar inflammatory reactions. In M7KO and M83KO, but not in SYNKO mice, the neuroinflammation was associated with dopaminergic neuronal death and the accumulation of insoluble aggregated SYN as cytoplasmic inclusions in nigral neurons. Nitrated/oxidized SYN was detected in these inclusions and abatement of microglia-derived nitric oxide and superoxide provided significant neuroprotection in neuron-glia cultures from M7KO mice. These data suggest that nitric oxide and superoxide released by activated microglia may be mediators that link inflammation and abnormal SYN in mechanisms of PD neurodegeneration. This study advances understanding of the role of neuroinflammation and abnormal SYN in the pathogenesis of PD and opens new avenues for the discovery of more effective therapies for PD.

Increasing evidence has suggested an important role for environmental toxins such as pesticides in the pathogenesis of Parkinson's 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.

BackgroundMechanisms whereby gene–environment interactions mediate chronic, progressive neurodegenerative processes in Parkinson’s disease (PD)—the second most common neurodegenerative disease—remain elusive.ObjectiveWe 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.MethodsWe 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.ResultsAfter 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.ConclusionsMicroglia-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.