Bin Xing

Microglial p38α MAPK is a key regulator of proinflammatory cytokine up-regulation induced by toll-like receptor (TLR) ligands or beta-amyloid (Aβ)

BackgroundOverproduction of proinflammatory cytokines from activated microglia has been implicated as an important contributor to pathophysiology progression in both acute and chronic neurodegenerative diseases. Therefore, it is critical to elucidate intracellular signaling pathways that are significant contributors to cytokine overproduction in microglia exposed to specific stressors, especially pathways amenable to drug interventions. The serine/threonine protein kinase p38α MAPK is a key enzyme in the parallel and convergent intracellular signaling pathways involved in stressor-induced production of IL-1β and TNFα in peripheral tissues, and is a drug development target for peripheral inflammatory diseases. However, much less is known about the quantitative importance of microglial p38α MAPK in stressor-induced cytokine overproduction, or the potential of microglial p38α MAPK to be a druggable target for CNS disorders. Therefore, we examined the contribution of microglial p38αMAPK to cytokine up-regulation, with a focus on the potential to suppress the cytokine increase by inhibition of the kinase with pharmacological or genetic approaches.MethodsThe microglial cytokine response to TLR ligands 2/3/4/7/8/9 or to Aβ1-42 was tested in the presence of a CNS-penetrant p38α MAPK inhibitor, MW01-2-069A-SRM. Primary microglia from mice genetically deficient in p38α MAPK were used to further establish a linkage between microglia p38α MAPK and cytokine overproduction. The in vivo significance was determined by p38α MAPK inhibitor treatment in a LPS-induced model of acute neuroinflammation.ResultsIncreased IL-1β and TNFα production by the BV-2 microglial cell line and by primary microglia cultures was inhibited in a concentration-dependent manner by the p38α MAPK-targeted inhibitor. Cellular target engagement was demonstrated by the accompanying decrease in the phosphorylation state of two p38α MAPK protein substrates, MK2 and MSK1. Consistent with the pharmacological findings, microglia from p38α-deficient mice showed a diminished cytokine response to LPS. Further, oral administration of the inhibitor blocked the increase of IL-1β in the cerebral cortex of mice stressed by intraperitoneal injection of LPS.ConclusionThe p38α MAPK pathway is an important contributor to the increased microglial production of proinflammatory cytokines induced by diverse stressors. The results also indicate the feasibility of targeting p38α MAPK to modulate CNS proinflammatory cytokine overproduction.

Pioglitazone inhibition of lipopolysaccharide- induced nitric oxide synthase is associated with altered activity of p38 MAP kinase and PI3K/Akt

BackgroundPrevious studies have suggested that peroxisome proliferator activated receptor-gamma (PPAR-γ)-mediated neuroprotection involves inhibition of microglial activation and decreased expression and activity of inducible nitric oxide synthase (iNOS); however, the underlying molecular mechanisms have not yet been well established. In the present study we explored: (1) the effect of the PPAR-γ agonist pioglitazone on lipopolysaccharide (LPS)-induced iNOS activity and nitric oxide (NO) generation by microglia; (2) the differential role of p38 mitogen-activated protein kinase (p38 MAPK), c-Jun NH(2)-terminal kinase (JNK), and phosphoinositide 3-kinase (PI3K) on LPS-induced NO generation; and (3) the regulation of p38 MAPK, JNK, and PI3K by pioglitazone.MethodsMesencephalic neuron-microglia mixed cultures, and microglia-enriched cultures were treated with pioglitazone and/or LPS. The protein levels of iNOS, p38 MAPK, JNK, PPAR-γ, PI3K, and protein kinase B (Akt) were measured by western blot. Different specific inhibitors of iNOS, p38MAPK, JNK, PI3K, and Akt were used in our experiment, and NO generation was measured using a nitrite oxide assay kit. Tyrosine hydroxylase (TH)-positive neurons were counted in mesencephalic neuron-microglia mixed cultures.ResultsOur results showed that pioglitazone inhibits LPS-induced iNOS expression and NO generation, and inhibition of iNOS is sufficient to protect dopaminergic neurons against LPS insult. In addition, inhibition of p38 MAPK, but not JNK, prevented LPS-induced NO generation. Further, and of interest, pioglitazone inhibited LPS-induced phosphorylation of p38 MAPK. Wortmannin, a specific PI3K inhibitor, enhanced p38 MAPK phosphorylation upon LPS stimulation of microglia. Elevations of phosphorylated PPAR-γ, PI3K, and Akt levels were observed with pioglitazone treatment, and inhibition of PI3K activity enhanced LPS-induced NO production. Furthermore, wortmannin prevented the inhibitory effect of pioglitazone on the LPS-induced NO increase.ConclusionWe demonstrate that pioglitazone protects dopaminergic neurons against LPS insult at least via inhibiting iNOS expression and NO generation, which is potentially mediated via inhibition of p38 MAPK activity. In addition, the PI3K pathway actively participates in the negative regulation of LPS-induced NO production. Our findings suggest that PPAR-γ activation may involve differential regulation of p38 MAPK and of the PI3K/Akt pathway in the regulation of the inflammatory process.

Microglial p38α MAPK is critical for LPS-induced neuron degeneration, through a mechanism involving TNFα

BackgroundThe p38α MAPK isoform is a well-established therapeutic target in peripheral inflammatory diseases, but the importance of this kinase in pathological microglial activation and detrimental inflammation in CNS disorders is less well understood. To test the role of the p38α MAPK isoform in microglia-dependent neuron damage, we used primary microglia from wild-type (WT) or p38α MAPK conditional knockout (KO) mice in co-culture with WT cortical neurons, and measured neuron damage after LPS insult.ResultsWe found that neurons in co-culture with p38α-deficient microglia were protected against LPS-induced synaptic loss, neurite degeneration, and neuronal death. The involvement of the proinflammatory cytokine TNFα was demonstrated by the findings that p38α KO microglia produced much less TNFα in response to LPS compared to WT microglia, that adding back TNFα to KO microglia/neuron co-cultures increased the LPS-induced neuron damage, and that neutralization of TNFα in WT microglia/neuron co-cultures prevented the neuron damage. These results using cell-selective, isoform-specific KO mice demonstrate that the p38α MAPK isoform in microglia is a key mediator of LPS-induced neuronal and synaptic dysfunction. The findings also provide evidence that a major mechanism by which LPS activation of microglia p38α MAPK signaling leads to neuron damage is through up-regulation of the proinflammatory cytokine TNFα.ConclusionsThe data suggest that selective targeting of p38α MAPK signaling should be explored as a potential therapeutic strategy for CNS disorders where overproduction of proinflammatory cytokines is implicated in disease progression.

Neuroprotection with pioglitazone against LPS insult on dopaminergic neurons may be associated with its inhibition of NF-κB and JNK activation and suppression of COX-2 activity

Increasing evidence links neuroinflammation to Parkinsons disease. Microglia are mediators of neuroinflammation. Overactivation of microglia contributes to the release of cyclooxygenase 2 and prostaglandin E(2) during neuronal insults. We have previously shown that pioglitazone, a peroxisome proliferator-activated receptor gamma agonist, inhibits microglia activation, reduces proinflammatory factors, and protects dopaminergic neurons. Here, we demonstrated that pioglitazone protects dopaminergic neurons by inhibiting abnormal microglia activation, interfering with phosphorylation of jun N-terminal kinase and nuclear factor kappa-B, and by suppressing cyclooxygenase 2 expression and the subsequent prostaglandin E(2) synthesis. Therefore, the anti-inflammatory properties of pioglitazone may be useful for ameliorating the progression of Parkinsons disease.

Development of Novel In Vivo Chemical Probes to Address CNS Protein Kinase Involvement in Synaptic Dysfunction.

Serine-threonine protein kinases are critical to CNS function, yet there is a dearth of highly selective, CNS-active kinase inhibitors for in vivo investigations. Further, prevailing assumptions raise concerns about whether single kinase inhibitors can show in vivo efficacy for CNS pathologies, and debates over viable approaches to the development of safe and efficacious kinase inhibitors are unsettled. It is critical, therefore, that these scientific challenges be addressed in order to test hypotheses about protein kinases in neuropathology progression and the potential for in vivo modulation of their catalytic activity. Identification of molecular targets whose in vivo modulation can attenuate synaptic dysfunction would provide a foundation for future disease-modifying therapeutic development as well as insight into cellular mechanisms. Clinical and preclinical studies suggest a critical link between synaptic dysfunction in neurodegenerative disorders and the activation of p38αMAPK mediated signaling cascades. Activation in both neurons and glia also offers the unusual potential to generate enhanced responses through targeting a single kinase in two distinct cell types involved in pathology progression. However, target validation has been limited by lack of highly selective inhibitors amenable to in vivo use in the CNS. Therefore, we employed high-resolution co-crystallography and pharmacoinformatics to design and develop a novel synthetic, active site targeted, CNS-active, p38αMAPK inhibitor (MW108). Selectivity was demonstrated by large-scale kinome screens, functional GPCR agonist and antagonist analyses of off-target potential, and evaluation of cellular target engagement. In vitro and in vivo assays demonstrated that MW108 ameliorates beta-amyloid induced synaptic and cognitive dysfunction. A serendipitous discovery during co-crystallographic analyses revised prevailing models about active site targeting of inhibitors, providing insights that will facilitate future kinase inhibitor design. Overall, our studies deliver highly selective in vivo probes appropriate for CNS investigations and demonstrate that modulation of p38αMAPK activity can attenuate synaptic dysfunction.

Glial cell line-derived neurotrophic factor protects midbrain dopaminergic neurons against lipopolysaccharide neurotoxicity

Aberrant microglia activation causes dopaminergic neuronal loss and nitric oxide produced by microglia plays a critical role in dopaminergic neuronal degeneration. However, no study has determined if GDNF protects dopaminergic neurons via inhibiting nitric oxide generation in Parkinsons disease animal model. We report that GDNF not only reduces lipopolysaccharide-induced degeneration of dopaminergic neurons, suppresses microglia activation and nitric oxide generation, but also reverses the inhibition of phosphoinositide 3-kinase (PI3K) in dopaminergic neurons and microglia. It suggests that the neuroprotective effect of GDNF on dopaminergic neurons may be related to its suppression of microglia activation-mediated nitric oxide via releasing the inhibition of PI3K in both neurons and microglia.

Inhibition of Neuronal p38α, but not p38β MAPK, Provides Neuroprotection Against Three Different Neurotoxic Insults

The p38 mitogen-activated protein kinase (MAPK) pathway plays a key role in pathological glial activation and neuroinflammatory responses. Our previous studies demonstrated that microglial p38α and not the p38β isoform is an important contributor to stressor-induced proinflammatory cytokine upregulation and glia-dependent neurotoxicity. However, the contribution of neuronal p38α and p38β isoforms in responses to neurotoxic agents is less well understood. In the current study, we used cortical neurons from wild-type or p38β knockout mice, and wild-type neurons treated with two highly selective inhibitors of p38α MAPK. Neurons were treated with one of three neurotoxic insults (L-glutamate, sodium nitroprusside, and oxygen-glucose deprivation), and neurotoxicity was assessed. All three stimuli led to neuronal death and neurite degeneration, and the degree of neurotoxicity induced in wild-type and p38β knockout neurons was not significantly different. In contrast, selective inhibition of neuronal p38α was neuroprotective. Our results show that neuronal p38β is not required for neurotoxicity induced by multiple toxic insults, but that p38α in the neuron contributes quantitatively to the neuronal dysfunction responses. These data are consistent with our previous findings of the critical importance of microglia p38α compared to p38β, and continue to support selective targeting of the p38α isoform as a potential therapeutic strategy.

Deficiency in p38β MAPK fails to inhibit cytokine production or protect neurons against inflammatory insult in in vitro and in vivo mouse models.

The p38 MAPK pathway plays a key role in regulating the production of proinflammatory cytokines, such as TNFα and IL-1β, in peripheral inflammatory disorders. There are four major isoforms of p38 MAPK (p38α, β, δ, γ), with p38α and p38β the targets of most p38 MAPK inhibitor drugs. Our previous studies demonstrated that the p38α MAPK isoform is an important contributor to stressor-induced proinflammatory cytokine up-regulation and neurotoxicity in the brain. However, the potential role of the p38β MAPK isoform in CNS proinflammatory cytokine overproduction and neurotoxicity is poorly understood. In the current studies, we used primary microglia from wild type (WT) and p38β knockout (KO) mice in co-culture with WT neurons, and measured proinflammatory cytokines and neuron death after LPS insult. We also measured neuroinflammatory responses in vivo in WT and p38β KO mice after administration of LPS by intraperitoneal or intracerebroventricular injection. WT and p38β KO microglia/neuron co-cultures showed similar levels of TNFα and IL-1β production in response to LPS treatment, and no differences in LPS-induced neurotoxicity. The in vitro results were confirmed in vivo, where levels of TNFα and IL-1β in the CNS were not significantly different between WT or p38β KO mice after LPS insult. Our results suggest that, similar to peripheral inflammation, p38α is critical but p38β MAPK is dispensable in the brain in regards to proinflammatory cytokine production and neurotoxicity induced by LPS inflammatory insult.

MW151 Inhibited IL-1β Levels after Traumatic Brain Injury with No Effect on Microglia Physiological Responses

A prevailing neuroinflammation hypothesis is that increased production of proinflammatory cytokines contributes to progressive neuropathology, secondary to the primary damage caused by a traumatic brain injury (TBI). In support of the hypothesis, post-injury interventions that inhibit the proinflammatory cytokine surge can attenuate the progressive pathology. However, other post-injury neuroinflammatory responses are key to endogenous recovery responses. Therefore, it is critical that pharmacological attenuation of detrimental or dysregulated neuroinflammatory processes avoid pan-suppression of inflammation. MW151 is a CNS-penetrant, small molecule experimental therapeutic that restores injury- or disease-induced overproduction of proinflammatory cytokines towards homeostasis without immunosuppression. Post-injury administration of MW151 in a closed head injury model of mild TBI suppressed acute cytokine up-regulation and downstream cognitive impairment. Here, we report results from a diffuse brain injury model in mice using midline fluid percussion. Low dose (0.5–5.0 mg/kg) administration of MW151 suppresses interleukin-1 beta (IL-1β) levels in the cortex while sparing reactive microglia and astrocyte responses. To probe molecular mechanisms, we used live cell imaging of the BV-2 microglia cell line to demonstrate that MW151 does not affect proliferation, migration, or phagocytosis of the cells. Our results provide insight into the roles of glial responses to brain injury and indicate the feasibility of using appropriate dosing for selective therapeutic modulation of injurious IL-1β increases while sparing other glial responses to injury.

The p38alpha mitogen-activated protein kinase limits the CNS proinflammatory cytokine response to systemic lipopolysaccharide, potentially through an IL-10 dependent mechanism

BackgroundThe p38α mitogen-activated protein kinase (MAPK) is a well-characterized intracellular kinase involved in the overproduction of proinflammatory cytokines from glia. As such, p38α appears to be a promising therapeutic target for neurodegenerative diseases associated with neuroinflammation. However, the in vivo role of p38α in cytokine production in the CNS is poorly defined, and prior work suggests that p38α may be affecting a yet to be identified negative feedback mechanism that limits the acute, injury-induced proinflammatory cytokine surge in the CNS.MethodsTo attempt to define this negative feedback mechanism, we used two in vitro and two in vivo models of neuroinflammation in a mouse where p38α is deficient in cells of the myeloid lineage.ResultsWe found that p38α in myeloid cells has an important role in limiting amplitude of the acute proinflammatory cytokine response to a systemic inflammatory challenge. Moreover, we identified IL-10 as a potential negative feedback mechanism regulated by p38α.ConclusionsOur data suggest that p38α regulates a proper balance between the pro- and anti-inflammatory cytokine responses to systemic inflammation, and that if circulating IL-10 levels are not elevated to counter-balance the increased systemic proinflammatory responses, the spread of the inflammatory response from the periphery to the CNS is exaggerated.