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Parkinson's disease is characterized by a progressive degeneration of substantia nigra (SN) dopaminergic neurons with age. We previously found that a single systemic lipopolysaccharide (LPS, 5 mg/kg, i.p.) injection caused a slow progressive loss of tyrosine hydroxylase immunoreactive (TH+IR) neurons in SN associated with increasing motor dysfunction. In this study, we investigated the role of NADPH oxidase (NOX) in inflammation-mediated SN neurotoxicity. A comparison of control (NOX2(+/+) ) mice with NOX subunit gp91(phox) -deficient (NOX2(-/-) ) mice 10 months after LPS administration (5 mg/kg, i.p.) resulted in a 39% (P < 0.01) loss of TH+IR neurons in NOX2(+/+) mice, whereas NOX2(-/-) mice did not show a significant decrease. Microglia (Iba1+IR) showed morphological activation in NOX2(+/+) mice, but not in NOX2(-/-) mice at 1 hr. Treatment of NOX2(+/+) mice with LPS resulted in a 12-fold increase in NOX2 mRNA in midbrain and 5.5-6.5-fold increases in NOX2 protein (+IR) in SN compared with the saline controls. Brain reactive oxygen species (ROS), determined using diphenyliodonium histochemistry, was increased by LPS in SN between 1 hr and 20 months. Diphenyliodonium (DPI), an NOX inhibitor, blocked LPS-induced activation of microglia and production of ROS, TNFα, IL-1β, and MCP-1. Although LPS increased microglial activation and ROS at all ages studied, saline control NOX2(+/+) mice showed age-related increases in microglial activation, NOX, and ROS levels at 12 and 22 months of age. Together, these results suggest that NOX contributes to persistent microglial activation, ROS production, and dopaminergic neurodegeneration that persist and continue to increase with age., (Copyright © 2013 Wiley Periodicals, Inc.)

Background: Activation of microglia causes the production of proinflammatory factors and upregulation of NADPH oxidase (NOX) that form reactive oxygen species (ROS) that lead to neurodegeneration. Previously, we reported that 10 daily doses of ethanol treatment induced innate immune genes in brain. In the present study, we investigate the effects of chronic ethanol on activation of NOX and release of ROS, and their contribution to ethanol neurotoxicity., Methods: Male C57BL/6 and NF-κB enhanced GFP mice were treated intragastrically with water or ethanol (5 g/kg, i.g., 25% ethanol w/v) daily for 10 days. The effects of chronic ethanol on cell death markers (activated caspase-3 and Fluoro-Jade B), microglial morphology, NOX, ROS and NF-κB were examined using real-time PCR, immunohistochemistry and hydroethidine histochemistry. Also, Fluoro-Jade B staining and NOX gp91phox immunohistochemistry were performed in the orbitofrontal cortex (OFC) of human postmortem alcoholic brain and human moderate drinking control brain., Results: Ethanol treatment of C57BL/6 mice showed increased markers of neuronal death: activated caspase-3 and Fluoro-Jade B positive staining with Neu-N (a neuronal marker) labeling in cortex and dentate gyrus. The OFC of human post-mortem alcoholic brain also showed significantly more Fluoro-Jade B positive cells colocalized with Neu-N, a neuronal marker, compared to the OFC of human moderate drinking control brain, suggesting increased neuronal death in the OFC of human alcoholic brain. Iba1 and GFAP immunohistochemistry showed activated morphology of microglia and astrocytes in ethanol-treated mouse brain. Ethanol treatment increased NF-κB transcription and increased NOX gp91phox at 24 hr after the last ethanol treatment that remained elevated at 1 week. The OFC of human postmortem alcoholic brain also had significant increases in the number of gp91phox + immunoreactive (IR) cells that are colocalized with neuronal, microglial and astrocyte markers. In mouse brain ethanol increased gp91phox expression coincided with increased production of O2- and O2- - derived oxidants. Diphenyleneiodonium (DPI), a NOX inhibitor, reduced markers of neurodegeneration, ROS and microglial activation., Conclusions: Ethanol activation of microglia and astrocytes, induction of NOX and production of ROS contribute to chronic ethanol-induced neurotoxicity. NOX-ROS and NF-κB signaling pathways play important roles in chronic ethanol-induced neuroinflammation and neurodegeneration.

Inflammation is implicated in the progressive nature of neurodegenerative diseases, such as Parkinson's disease, but the mechanisms are poorly understood. A single systemic lipopolysaccharide (LPS, 5 mg/kg, i.p.) or tumor necrosis factor alpha (TNFalpha, 0.25 mg/kg, i.p.) injection was administered in adult wild-type mice and in mice lacking TNFalpha receptors (TNF R1/R2(-/-)) to discern the mechanisms of inflammation transfer from the periphery to the brain and the neurodegenerative consequences. Systemic LPS administration resulted in rapid brain TNFalpha increase that remained elevated for 10 months, while peripheral TNFalpha (serum and liver) had subsided by 9 h (serum) and 1 week (liver). Systemic TNFalpha and LPS administration activated microglia and increased expression of brain pro-inflammatory factors (i.e., TNFalpha, MCP-1, IL-1beta, and NF-kappaB p65) in wild-type mice, but not in TNF R1/R2(-/-) mice. Further, LPS reduced the number of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra (SN) by 23% at 7-months post-treatment, which progressed to 47% at 10 months. Together, these data demonstrate that through TNFalpha, peripheral inflammation in adult animals can: (1) activate brain microglia to produce chronically elevated pro-inflammatory factors; (2) induce delayed and progressive loss of DA neurons in the SN. These findings provide valuable insight into the potential pathogenesis and self-propelling nature of Parkinson's disease., ((c) 2007 Wiley-Liss, Inc.)

Microglia are activated by lipopolysaccharide (LPS) to produce neurotoxic pro-inflammatory factors and reactive oxygen species (ROS). While a multitude of LPS receptors and corresponding pathways have been identified, the detailed mechanisms mediating the microglial response to LPS are unclear. Using mice lacking a functional toll-like receptor 4 (TLR4), we demonstrate that TLR4 and ROS work in concert to mediate microglia activation, where the contribution from each pathway is dependent on the concentration of LPS. Immunocytochemical staining of microglia in neuron-glia cultures with antibodies against F4/80 revealed that while TLR4(+/+) microglia were activated the low concentration of 1 ng/ml of LPS, TLR4(-/-) microglia exhibit activated morphology in response to LPS only at higher concentrations (100-1,000 ng/ml). Additionally, tumor necrosis factor-alpha (TNF-alpha) was only produced from higher concentrations (100-1,000 ng/ml) of LPS in TLR4(-/-) enriched microglia cultures. Diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, reduced TNF-alpha production from TLR4(-/-) microglia. The influence of TLR4 on LPS-induced superoxide production was tested in rat enriched microglia cultures, where the presence or absence of serum failed to show any effect on the superoxide production. Further, both TLR4(-/-) and TLR4(+/+) microglia showed a similar increase in extracellular superoxide production when exposed to LPS (1-1,000 ng/ml). These data indicate that LPS-induced superoxide production in microglia is independent of TLR4 and that ROS derived from the production of extracellular superoxide in microglia mediates the LPS-induced TNF-alpha response of both the TLR4-dependent and independent pathway., ((c) 2005 Wiley-Liss, Inc.)

Here, we report that leucine enkephalin (LE) is neuroprotective to dopaminergic (DA) neurons at femtomolar concentrations through anti-inflammatory properties. Mesencephalic neuron-glia cultures pretreated with femtomolar concentrations of LE (10(-15)-10(-13) M) protected DA neurons from lipopolysaccharide (LPS)-induced DA neurotoxicity, as determined by DA uptake assay and tyrosine hydroxylase (TH) immunocytochemistry (ICC). However, des-tyrosine leucine enkephalin (DTLE), an LE analogue that is missing the tyrosine residue required for binding to the kappa opioid receptor, was also neuroprotective (10(-15)-10(-13) M), as determined by DA uptake assay and TH ICC. Both LE and DTLE (10(-15)-10(-13) M) reduced LPS-induced superoxide production from microglia-enriched cultures. Further, both LE and DTLE (10(-14), 10(-13) M) reduced the LPS-induced tumor necrosis factor-alpha (TNFalpha) mRNA and TNFalpha protein from PHOX+/+ microglia, as determined by quantitative real-time RT-PCR and ELISA analysis in mesencephalic neuron-glia cultures, respectively. However, both peptides failed to inhibit TNFalpha expression in PHOX-/- cultures, which are unable to produce extracellular superoxide in response to LPS. Additionally, LE and DTLE (10(-14), 10(-13) M) failed to show any neuroprotection against LPS in PHOX-/- cultures. Together, these data indicate that LE and DTLE are neuroprotective at femtomolar concentrations through the inhibition of oxidative insult associated with microglial NADPH oxidase and the attenuation of the ROS-mediated amplification of TNFalpha gene expression in microglia.

The herbicide paraquat (PQ) has been implicated as a potential risk factor for the development of Parkinson's disease. In this study, PQ (0.5-1 microM) was shown to be selectively toxic to dopaminergic (DA) neurons through the activation of microglial NADPH oxidase and the generation of superoxide. Neuron-glia cultures exposed to PQ exhibited a decrease in DA uptake and a decline in the number of tyrosine hydroxylase-immunoreactive cells. The selectivity of PQ for DA neurons was confirmed when PQ failed to alter gamma-aminobutyric acid uptake in neuron-glia cultures. Microglia-depleted cultures exposed to 1 microM PQ failed to demonstrate a reduction in DA uptake, identifying microglia as the critical cell type mediating PQ neurotoxicity. Neuron-glia cultures treated with PQ failed to generate tumor necrosis factor-alpha and nitric oxide. However, microglia-enriched cultures exposed to PQ produced extracellular superoxide, supporting the notion that microglia are a source of PQ-derived oxidative stress. Neuron-glia cultures from NADPH oxidase-deficient (PHOX-/-) mice, which lack the functional catalytic subunit of NADPH oxidase and are unable to produce the respiratory burst, failed to show neurotoxicity in response to PQ, in contrast to PHOX+/+ mice. Here we report a novel mechanism of PQinduced oxidative stress, where at lower doses, the indirect insult generated from microglial NADPH oxidase is the essential factor mediating DA neurotoxicity.

Inflammation has been increasingly recognized to contribute to the pathogenesis of Parkinson's disease. Several compounds are neuroprotective at femtomolar concentrations through the inhibition of inflammation. However, the mechanisms mediating femtomolar-acting compounds are poorly understood. Here we show that both gly-gly-phe (GGF), a tri-peptide contained in the dynorphin opioid peptide, and naloxone are neuroprotective at femtomolar concentrations against LPS-induced dopaminergic neurotoxicity through the reduction of microglial activation. Mechanistic studies demonstrated the critical role of NADPH oxidase in the GGF and naloxone inhibition of microglial activation and associated DA neurotoxicity. Pharmacophore analysis of the neuroprotective dynorphin peptides and naloxone revealed common chemical properties (hydrogen bond acceptor, hydrogen bond donor, positive ionizable, hydrophobic) of these femtomolar-acting compounds. These results support a common high-affinity site of action for several femtomolar-acting compounds, where NADPH oxidase is the critical mechanism governing neuroprotection, suggesting a novel avenue of anti-inflammatory and neuroprotective therapy.

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.

Parkinson's disease is characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra. We have previously reported that lipopolysaccharide (LPS)-induced degeneration of dopaminergic neurons is mediated by the release of proinflammatory factors from activated microglia. Here, we report the pivotal role of NADPH oxidase in inflammation-mediated neurotoxicity, where the LPS-induced loss of nigral dopaminergic neurons in vivo was significantly less pronounced in NADPH oxidase-deficient (PHOX-/-) mice when compared with control (PHOX+/+) mice. Dopaminergic neurons in primary mensencephalic neuron-glia cultures from PHOX+/+ mice were significantly more sensitive to LPS-induced neurotoxicity in vitro when compared with PHOX-/- mice. Further, PHOX+/+ neuron-glia cultures chemically depleted of microglia failed to show dopaminergic neurotoxicity with the addition of LPS. Neuron-enriched cultures from both PHOX+/+ mice and PHOX-/- mice also failed to show any direct LPS-induced dopaminergic neurotoxicity. However, the addition of PHOX+/+ microglia to neuron-enriched cultures from either strain resulted in reinstatement of LPS-induced dopaminergic neurotoxicity, supporting the role of microglia as the primary source of NADPH oxidase-generated insult and neurotoxicity. Immunostaining for F4/80 in mensencephalic neuron-glia cultures revealed that PHOX-/- microglia failed to show activated morphology at 10 h, suggesting an important role of reactive oxygen species (ROS) generated from NADPH oxidase in the early activation of microglia. LPS also failed to elicit extracellular superoxide and produced low levels of intracellular ROS in microglia-enriched cultures from PHOX-/- mice. Gene expression and release of tumor necrosis factor alpha was much lower in PHOX-/- mice than in control PHOX+/+ mice. Together, these results demonstrate the dual neurotoxic functions of microglial NADPH oxidase: 1) the production of extracellular ROS that is toxic to dopamine neurons and 2) the amplification of proinflammatory gene expression and associated neurotoxicity.

Inflammation in the brain has increasingly been recognized to play an important role in the pathogenesis of several neurodegenerative disorders, including Parkinson's disease and Alzheimer's disease. Inflammation-mediated neurodegeneration involves activation of the brain's resident immune cells, the microglia, which produce proinflammatory and neurotoxic factors, including cytokines, reactive oxygen intermediates, nitric oxide, and eicosanoids that impact on neurons to induce neurodegeneration. Hence, identification of compounds that prevent microglial activation may be highly desirable in the search for therapeutic agents for inflammation-mediated neurodegenerative diseases. In this study, we report that dextromethorphan (DM), an ingredient widely used in antitussive remedies, reduced the inflammation-mediated degeneration of dopaminergic neurons through inhibition of microglial activation. Pretreatment (30 min) of rat mesencephalic neuron-glia cultures with DM (1-10 micro M) reduced, in a dose-dependent manner, the microglia-mediated degeneration of dopaminergic neurons induced by lipopolysaccharide (LPS, 10 ng/ml). Significant neuroprotection by DM was also evident when DM was applied to cultures up to 60 min after the addition of LPS. The neuroprotective effect of DM was attributed to inhibition of LPS-stimulated microglial activation because DM significantly inhibited the LPS-induced production of tumor necrosis factor-alpha, nitric oxide, and superoxide free radicals. This conclusion was further supported by the finding that DM failed to prevent 1-methyl-4-phenylpyridinium- or beta-amyloid peptide (1-42)-induced dopaminergic neurotoxicity in neuron-enriched cultures. In addition, because LPS did not produce any significant increase in the release of excitatory amino acids from neuron-glia cultures and N-methyl-D-aspartate antagonist dizocilpine maleate failed to afford significant neuroprotection, it is unlikely that the neuroprotective effect of DM is mediated through N-methyl-D-aspartate receptors. These results suggest that DM may be a promising therapeutic agent for the treatment of Parkinson's disease.

The purpose of this study was to assess and compare the toxicity of beta-amyloid (Abeta) on primary cortical and mesencephalic neurons cultured with and without microglia in order to determine the mechanism underlying microglia-mediated Abeta-induced neurotoxicity. Incubation of cortical or mesencephalic neuron-enriched and mixed neuron-glia cultures with Abeta(1-42) over the concentration range 0.1-6.0 microm caused concentration-dependent neurotoxicity. High concentrations of Abeta (6.0 microm for cortex and 1.5-2.0 microm for mesencephalon) directly injured neurons in neuron-enriched cultures. In contrast, lower concentrations of Abeta (1.0-3.0 microm for cortex and 0.25-1.0 microm 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 Abeta, including the observations that low concentrations of Abeta activated microglia morphologically in neuron-glia cultures and that addition of microglia to cortical neuron-glia cultures enhanced Abeta-induced neurotoxicity. To search for the mechanism underlying the microglia-mediated effects, several proinflammatory factors were examined in neuron-glia cultures. Low doses of Abeta significantly increased the production of superoxide anions, but not of tumor necrosis factor-alpha, interleukin-1beta or nitric oxide. Catalase and superoxide dismutase significantly protected neurons from Abeta toxicity in the presence of microglia. Inhibition of NADPH oxidase activity by diphenyleneiodonium also prevented Abeta-induced neurotoxicity in neuron-glia mixed cultures. The role of NADPH oxidase-generated superoxide in mediating Abeta-induced neurotoxicity was further substantiated by a study which showed that Abeta 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 Abeta is via the production of reactive oxygen species.

Previously we reported that naloxone stereoisomers, in an opioid receptor-independent manner, attenuated the inflammation-mediated degeneration of dopaminergic neurons by inhibition of the activation of microglia, the resident immune cells in the brain. Recently we discovered that beta-amyloid peptide Abeta (1-42) exhibited enhanced neurotoxicity toward both cortical and mesencephalic neurons through the activation of microglia and production of superoxide. The purpose of this study was to determine whether naloxone isomers had any effect on Abeta (1-42)-induced neurodegeneration. Pretreatment of either cortical or mesencephalic neuron-glia cultures with 1 to 10 microM (-)-naloxone, prior to treatment for up to 11 days with 0.1 to 3 microM Abeta (1-42), afforded significant neuroprotection as judged by neurotransmitter uptake, immunocytochemical analysis, and cell counting. More importantly, (+)-naloxone, the ineffective enantiomer of (-)-naloxone in binding opioid receptors, was equally effective in affording neuroprotection. Mechanistically, inhibition of Abeta (1-42)-induced production of superoxide in microglia underlay the neuroprotective effect of naloxone stereoisomers. Moreover, neuroprotection and inhibition of Abeta (1-42)-induced superoxide production was also achieved with naloxone methiodide, a charged analog with quaternary amine, suggesting that the site of action for naloxone isomers is at the cell surface of microglia. These results demonstrated that naloxone isomers, through mechanisms unrelated to the opioid receptors, were capable of inhibiting Abeta (1-42)-induced microglial activation and degeneration of both cortical and mesencephalic neurons. Combined with our previous observations with inflammagen-induced neurodegeneration, naloxone analogs, especially (+)-naloxone, may have potential therapeutic efficacy for the treatment of Alzheimer's and Parkinson's disease.

Induction of neuroimmune genes by binge drinking increases neuronal excitability and oxidative stress, contributing to the neurobiology of alcohol dependence and causing neurodegeneration. Ethanol exposure activates signaling pathways featuring high-mobility group box 1 and Toll-like receptor 4 (TLR4), resulting in induction of the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells, which regulates expression of several cytokine genes involved in innate immunity, and its target genes. This leads to persistent neuroimmune responses to ethanol that stimulate TLRs and/or certain glutamate receptors (i.e., N-methyl-D-aspartate receptors). Alcohol also alters stress responses, causing elevation of peripheral cytokines, which further sensitize neuroimmune responses to ethanol. Neuroimmune signaling and glutamate excitotoxicity are linked to alcoholic neurodegeneration. Models of alcohol abuse have identified significant frontal cortical degeneration and loss of hippocampal neurogenesis, consistent with neuroimmune activation pathology contributing to these alcohol-induced, long-lasting changes in the brain. These alcohol- induced long-lasting increases in brain neuroimmune-gene expression also may contribute to the neurobiology of alcohol use disorder. INSET: Adolescence and Persistent Neuroimmune Expression in the Brain. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) produced by activated microglia are deleterious to neurons. In this study, we studied the effect of Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), a superoxide dismutase/catalase mimetic, on the lipopolysaccharide (LPS)-induced degeneration of dopaminergic neurons in rat mesencephalic neuroglia cultures. MnTMPyP exhibited a significantly protective effect against LPS (5 ng/ml)-induced neurotoxicity as determined by [3H]dopamine uptake and immunocytochemical analysis. MnTMPyP significantly attenuated LPS-induced production of superoxide free radical and prostaglandin E2 (PGE2) in microglia. These results indicate that MnTMPyP may potentially be used for the treatment of inflammation-related degenerative neurological disorders. [Copyright &y& Elsevier]

Abstract: Addiction occurs through repeated abuse of drugs that progressively reduce behavioral control and cognitive flexibility while increasing limbic negative emotion. Recent discoveries indicate neuroimmune signaling underlies addiction and co-morbid depression. Low threshold microglia undergo progressive stages of innate immune activation involving astrocytes and neurons with repeated drug abuse, stress, and/or cell damage signals. Increased brain NF-κB transcription of proinflammatory chemokines, cytokines, oxidases, proteases, TLR and other genes create loops amplifying NF-κB transcription and innate immune target gene expression. Human post-mortem alcoholic brain has increased NF-κB and NF-κB target gene message, increased microglial markers and chemokine-MCP1. Polymorphisms of human NF-κB1 and other innate immune genes contribute to genetic risk for alcoholism. Animal transgenic and genetic studies link NF-κB innate immune gene expression to alcohol drinking. Human drug addicts show deficits in behavioral flexibility modeled pre-clinically using reversal learning. Binge alcohol, chronic cocaine, and lesions link addiction neurobiology to frontal cortex, neuroimmune signaling and loss of behavioral flexibility. Addiction also involves increasing limbic negative emotion and depression-like behavior that is reflected in hippocampal neurogenesis. Innate immune activation parallels loss of neurogenesis and increased depression-like behavior. Protection against loss of neurogenesis and negative affect by anti-oxidant, anti-inflammatory, anti-depressant, opiate antagonist and abstinence from ethanol dependence link limbic affect to changes in innate immune signaling. The hypothesis that innate immune gene induction underlies addiction and affective disorders creates new targets for therapy. [Copyright &y& Elsevier]