Liya Qin

Systemic LPS causes chronic neuroinflammation and progressive neurodegeneration

Inflammation is implicated in the progressive nature of neurodegenerative diseases, such as Parkinsons disease, but the mechanisms are poorly understood. A single systemic lipopolysaccharide (LPS, 5 mg/kg, i.p.) or tumor necrosis factor alpha (TNFα, 0.25 mg/kg, i.p.) injection was administered in adult wild‐type mice and in mice lacking TNFα 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 TNFα increase that remained elevated for 10 months, while peripheral TNFα (serum and liver) had subsided by 9 h (serum) and 1 week (liver). Systemic TNFα and LPS administration activated microglia and increased expression of brain pro‐inflammatory factors (i.e., TNFα, MCP‐1, IL‐1β, and NF‐κB 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 TNFα, 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 Parkinsons disease.

Increased systemic and brain cytokine production and neuroinflammation by endotoxin following ethanol treatment

BackgroundCytokines and alcohol share a common modulation of inflammation and hormones as well as being implicated in multiple diseases, but the mechanisms are poorly understood. The purpose of this study was to investigate the interaction of liver, serum and brain cytokines as well as whether ethanol would potentiate endotoxin (Lipopolysaccharide, LPS) responses once ethanol had cleared.MethodsMale C57BL/6J mice were treated intragastrically with water (control) or ethanol (5 g/kg, i.g., 25% ethanol, w/v), with volumes matched, for 1 day or daily for 10 days. Mice were then injected intraperitoneally with saline (control) or LPS (3 mg/kg, i.p.) in saline 24 hrs after the last dose of ethanol. Gene expression and protein synthesis of proinflammatory cytokines and anti-inflammatory cytokine, oxidative enzymes, microglial activation and inhibition of neurogenesis were examined using real-time PCR, ELISA, and immunohistochemistry.ResultsLPS increased proinflammatory cytokines (TNFα, MCP-1, IL-1β) several fold in liver, brain and serum at 1 hr. Ethanol is known to increase liver cytokines and alter the risk of multiple chronic diseases. Ten daily doses of ethanol increased brain and liver TNFα, and pretreatment with ethanol potentiated LPS-induced increases in TNFα, MCP-1, IL-1βin liver, serum and brain. Proinflammatory cytokine levels in liver and serum returned to basal levels within a day, whereas brain proinflammatory cytokines remained elevated for long periods. IL-10, an anti-inflammatory cytokine, is reduced in brain by ethanol and LPS, while brain proinflammatory cytokines remain increased, whereas liver IL-10 is increased when proinflammatory cytokines have returned to control levels. Activation of brain microglia indicated by morphological changes, reduced neurogenesis and increased brain expression of COX-2 and gp91phox NADPH oxidase subunit mRNA were found in the 10 daily doses of ethanol-pretreated LPS group.ConclusionAcute increases in serum cytokines induce long lasting increases in brain proinflammatory cytokines. Ten daily doses of ethanol exposure results in persistent alterations of cytokines and significantly increases the magnitude and duration of central and peripheral proinflammatory cytokines and microglial activation. Ethanol induced differential anti-inflammatory cytokine IL-10 responses in liver and brain could cause long lasting disruption of cytokine cascades that could contribute to protection or increased risk of multiple chronic diseases.

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.

NADPH oxidase and reactive oxygen species contribute to alcohol-induced microglial activation and neurodegeneration

BackgroundActivation 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.MethodsMale 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.ResultsEthanol 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.ConclusionsEthanol 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.

High Mobility Group Box 1/Toll-like Receptor Danger Signaling Increases Brain Neuroimmune Activation in Alcohol Dependence

BACKGROUND Innate immune gene expression is regulated in part through high mobility group box 1 (HMGB1), an endogenous proinflammatory cytokine, that activates multiple members of the interleukin-1/Toll-like receptor (TLR) family associated with danger signaling. We investigated expression of HMGB1, TLR2, TLR3, and TLR4 in chronic ethanol-treated mouse brain, postmortem human alcoholic brain, and rat brain slice culture to test the hypothesis that neuroimmune activation in alcoholic brain involves ethanol activation of HMGB1/TLR danger signaling. METHODS Protein levels were assessed using Western blot, enzyme-linked immunosorbent assay, and immunohistochemical immunoreactivity (+IR), and messenger RNA (mRNA) levels were measured by real time polymerase chain reaction in ethanol-treated mice (5 g/kg/day, intragastric, 10 days + 24 hours), rat brain slice culture, and postmortem human alcoholic brain. RESULTS Ethanol treatment of mice increased brain mRNA and +IR protein expression of HMGB1, TLR2, TLR3, and TLR4. Postmortem human alcoholic brain also showed increased HMGB1, TLR2, TLR3, and TLR4 +IR cells that correlated with lifetime alcohol consumption, as well as each other. Ethanol treatment of brain slice culture released HMGB1 into the media and induced the proinflammatory cytokine, interleukin-1 beta (IL-1β). Neutralizing antibodies to HMGB1 and small inhibitory mRNA to HMGB1 or TLR4 blunted ethanol induction of IL-1β. CONCLUSIONS Ethanol-induced HMGB1/TLR signaling contributes to induction of the proinflammatory cytokine, IL-1β. Increased expression of HMGB1, TLR2, TLR3, and TLR4 in alcoholic brain and in mice treated with ethanol suggests that chronic alcohol-induced brain neuroimmune activation occurs through HMGB1/TLR signaling.

NADPH oxidase and aging drive microglial activation, oxidative stress and dopaminergic neurodegeneration following systemic LPS administration

Parkinsons 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 gp91phox‐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.

Chronic ethanol increases systemic TLR3 agonist-induced neuroinflammation and neurodegeneration.

BackgroundIncreasing evidence links systemic inflammation to neuroinflammation and neurodegeneration. We previously found that systemic endotoxin, a TLR4 agonist or TNFα, increased blood TNFα that entered the brain activating microglia and persistent neuroinflammation. Further, we found that models of ethanol binge drinking sensitized blood and brain proinflammatory responses. We hypothesized that blood cytokines contribute to the magnitude of neuroinflammation and that ethanol primes proinflammatory responses. Here, we investigate the effects of chronic ethanol on neuroinflammation and neurodegeneration triggered by toll-like receptor 3 (TLR3) agonist poly I:C.MethodsPolyinosine-polycytidylic acid (poly I:C) was used to induce inflammatory responses when sensitized with D-galactosamine (D-GalN). Male C57BL/6 mice were treated with water or ethanol (5 g/kg/day, i.g., 10 days) or poly I:C (250 μg/kg, i.p.) alone or sequentially 24 hours after ethanol exposure. Cytokines, chemokines, microglial morphology, NADPH oxidase (NOX), reactive oxygen species (ROS), high-mobility group box 1 (HMGB1), TLR3 and cell death markers were examined using real-time PCR, ELISA, immunohistochemistry and hydroethidine histochemistry.ResultsPoly I:C increased blood and brain TNFα that peaked at three hours. Blood levels returned within one day, whereas brain levels remained elevated for at least three days. Escalating blood and brain proinflammatory responses were found with ethanol, poly I:C, and ethanol-poly I:C treatment. Ethanol pretreatment potentiated poly I:C-induced brain TNFα (345%), IL-1β (331%), IL-6 (255%), and MCP-1(190%). Increased levels of brain cytokines coincided with increased microglial activation, NOX gp91phox, superoxide and markers of neurodegeneration (activated caspase-3 and Fluoro-Jade B). Ethanol potentiation of poly I:C was associated with ethanol-increased expression of TLR3 and endogenous agonist HMGB1 in the brain. Minocycline and naltrexone blocked microglial activation and neurodegeneration.ConclusionsChronic ethanol potentiates poly I:C blood and brain proinflammatory responses. Poly I:C neuroinflammation persists after systemic responses subside. Increases in blood TNFα, IL-1β, IL-6, and MCP-1 parallel brain responses consistent with blood cytokines contributing to the magnitude of neuroinflammation. Ethanol potentiation of TLR3 agonist responses is consistent with priming microglia-monocytes and increased NOX, ROS, HMGB1-TLR3 and markers of neurodegeneration. These studies indicate that TLR3 agonists increase blood cytokines that contribute to neurodegeneration and that ethanol binge drinking potentiates these responses.

Increased receptor for advanced glycation end product expression in the human alcoholic prefrontal cortex is linked to adolescent drinking.

Adolescence is characterized behaviorally by increased impulsivity and risk-taking that declines in parallel with maturation of the prefrontal cortex and executive function. In the brain, the receptor for advanced glycation end products (RAGE) is critically involved in neurodevelopment and neuropathology. In humans, the risk of alcoholism is greatly increased in those who begin drinking between 13 and 15years of age, and adolescents binge drink more than any other age group. We have previously found that alcoholism is associated with increased expression of neuroimmune genes. This manuscript tested the hypothesis that adolescent binge drinking upregulates RAGE and Toll-like receptor (TLR) 4 as well as their endogenous agonist, high-mobility group box 1 (HMGB1). Immunohistochemistry, Western blot, and mRNA analyses found that RAGE expression was increased in the human post-mortem alcoholic orbitofrontal cortex (OFC). Further, an earlier age of drinking onset correlated with increased expression of RAGE, TLR4, and HMGB1. To determine if alcohol contributed to these changes, we used an adolescent binge ethanol model in rats (5.0g/kg, i.g., 2-day on/2-day off from postnatal day [P] 25 to P55) and assessed neuroimmune gene expression. We found an age-associated decline of RAGE expression from late adolescence (P56) to young adulthood (P80). Adolescent intermittent ethanol exposure did not alter RAGE expression at P56, but increased RAGE in the young adult PFC (P80). Adolescent intermittent ethanol exposure also increased TLR4 and HMGB1 expression at P56 that persisted into young adulthood (P80). Assessment of young adult frontal cortex mRNA (RT-PCR) found increased expression of proinflammatory cytokines, oxidases, and neuroimmune agonists at P80, 25days after ethanol treatment. Together, these human and animal data support the hypothesis that an early age of drinking onset upregulates RAGE/TLR4-HMGB1 and other neuroimmune genes that persist into young adulthood and could contribute to risk of alcoholism or other brain diseases associated with neuroinflammation.

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.

Endotoxin induces a delayed loss of TH-IR neurons in substantia nigra and motor behavioral deficits

We have previously reported that a single injection of endotoxin, lipopolysaccharide (LPS, 5mg/kg, i.p.), causes a delayed and progressive loss of TH-IR neurons in the substantia nigra (SN) in C57BL/six male mice. In this study, we determined sex differences and behavioral deficits accompanying the loss of TH-IR neurons in response to peripheral LPS injection. A single injection of LPS (5mg/kg, i.p.) failed to produce any loss of TH-IR neurons in the SN of female mice over a 12-month period. To determine if multiple-injections were required, female mice received five injections of LPS (5mg/kg, i.p.) at either weekly or monthly intervals. Behavioral motor ability and TH-IR neuronal loss were determined after the first injection of LPS. We found significant differences in both behavioral activities and neuronal loss between these two injection paradigms. Between 7 and 20 months after the first injection of LPS, progressive behavioral changes, measured by rotor-rod and open-field activities, and neuronal loss in SN were observed in monthly injected, but not in weekly injected mice. In addition, reduced rotor-rod ability in monthly injected mice were restored following treatment of l-dopa/carbidopa (30 mg/3mg/kg), i.p.). Approximately 40 and 50% loss of TH-IR neurons at 9 and 20 months, respectively, was observed after exposure to LPS, suggesting that the behavioral deficit is related to loss of dopamine function in the nigra-striatal pathway. More intense immuno-staining of alpha-synuclein and inflammatory markers were detected in brain sections exposed to LPS. In conclusion, these results show that multi-LPS monthly injections can induce a delayed and progressive loss of TH-IR neurons and motor deficits which resemble the progressive nature of Parkinsons disease. Further, the present study reveals a clear sex difference: female mice are more resistant to LPS than male mice. Repeated monthly LPS injections are required to cause both motor behavioral deficits and DA neuronal loss in female mice.