Excessive Microglial Activation Research Articles

Lipopolysaccharide treatment arrests the cell cycle of BV-2 microglial cells in G₁ phase and protects them from UV light-induced apoptosis.

We previously reported that an optimal dose of lipopolysaccharide (LPS) markedly extends the lifespan of murine primary-cultured microglia by suppressing cell death pathways. In this study, we investigated the effects of LPS pretreatment on UV light-induced apoptosis of cells from the microglial cell line BV-2. More than half of BV-2 cells were apoptotic, and procaspase-3 was cleaved into its active form at 3h of UV irradiation. In contrast, in BV-2 cells treated with LPS for 24h, UV irradiation caused neither apoptosis nor procaspase-3 cleavage. LPS treatment arrested the cell cycle in G1 phase and upregulated cyclin-dependent kinase inhibitor p21(Waf1/Cip1) and growth arrest and DNA damage-inducible (GADD) 45α in BV-2 cells. When p21(Waf1/Cip1) and GADD45α were knocked down by small interfering RNA, procaspase-3 was cleaved into its active form to induce apoptosis. Our findings suggest that LPS inhibits UV-induced apoptosis in BV-2 cells through arrest of the cell cycle in G1 phase by upregulation of p21(Waf1/Cip1) and GADD45α. Excessive activation of microglia may play a critical role in the exacerbation of neurodegeneration, therefore, normalizing the precise regulation of apoptosis may be a new strategy to prevent the deterioration caused by neurodegenerative disorders.

Retinal microglia are activated by systemic fungal infection.

We determined whether systemic fungal infection could cause activation of retinal microglia and, therefore, could be potentially harmful for patients with retinal degenerative diseases. Activation of retinal microglia was measured in a model of sublethal invasive candidiasis in C57BL/6J mice by confocal immunofluorescence and flow cytometry analysis, using anti-CD11b, anti-Iba1, anti-MHCII, and anti-CD45 antibodies. Systemic fungal infection causes activation of retinal microglia, with phenotypic changes in morphology, surface markers expression, and microglial relocation in retinal layers. As an excessive or prolonged microglial activation may lead to chronic inflammation with severe pathological side effects, causing or worsening the course of retinal dystrophies, a systemic infection may represent a risk factor to be considered in patients with ocular neurodegenerative diseases, such as diabetic retinopathy, glaucoma, age-related macular degeneration, or retinitis pigmentosa.

Epigallocatechin gallate (EGCG) attenuates infrasound-induced neuronal impairment by inhibiting microglia-mediated inflammation

Infrasound, a kind of common environmental noise and a major contributor of vibroacoustic disease, can induce the central nervous system (CNS) damage. However, no relevant anti-infrasound drugs have been reported yet. Our recent studies have shown that infrasound resulted in excessive microglial activation rapidly and sequential inflammation, revealing a potential role of microglia in infrasound-induced CNS damage. Epigallocatechin gallate (EGCG), a major bioactive component in green tea, has the capacity of protecting against various neurodegenerative diseases via an anti-inflammatory mechanism. However, it is still unknown to date whether EGCG acts on infrasound-induced microglial activation and neuronal damage. We showed that, after 1-, 2- or 5-day exposure of rats to 16 Hz, 130 dB infrasound (2 h/day), EGCG significantly inhibited infrasound-induced microglial activation in rat hippocampal region, evidenced by reduced expressions of Iba-1 (a marker for microglia) and proinflammatory cytokines (IL-1β, IL-6, IL-18 and TNF-α). Moreover, infrasound-induced neuronal apoptosis in rat hippocampi was significantly suppressed by EGCG. EGCG also inhibited infrasound-induced activation of primary microglia in vitro and decreased the levels of proinflammatory cytokines in the supernatants of microglial culture, which were toxic to cultured neurons. Furthermore, EGCG attenuated infrasound-induced increases in nuclear NF-κB p65 and phosphorylated IκBα, and ameliorated infrasound-induced decrease in IκB in microglia. Therefore, our study provides the first evidence that EGCG acts against infrasound-induced neuronal impairment by inhibiting microglia-mediated inflammation through a potential NF-κB pathway-related mechanism, suggesting that EGCG can be used as a promising drug for the treatment of infrasound-induced CNS damage.

Tangeretin exerts anti-neuroinflammatory effects via NF-κB modulation in lipopolysaccharide-stimulated microglial cells

Increasing evidence suggests that tangeretin, a flavonoid from citrus fruit peels, exhibits anti-inflammatory properties and neuroprotective effects in animal disease models. However, the underlying molecular mechanisms are not clearly understood. In this study, we investigated whether tangeretin suppresses excessive microglial activation implicated in the resulting neurotoxicity following stimulation with lipopolysaccharide (LPS) in primary rat microglia and BV-2 microglial cell culture models. The results showed that tangeretin decreased the production of nitric oxide (NO), prostaglandin E₂ (PGE₂), tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6), in a dose-dependent manner. Additionally, it inhibited the LPS-induced expression of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) (examined at the protein level) as well as TNF-α, IL-1β, and IL-6 (examined at the mRNA level) in microglial cells. To explore the possible mechanisms underlying these inhibitions by tangeretin, we examined the mitogen-activated protein kinase (MAPK) protein levels and the NF-κB protein signaling pathway. Tangeretin clearly inhibited LPS-induced phosphorylation of ERK, N-terminal Kinase (JNK), and p38. In addition, tangeretin markedly reduced LPS-stimulated phosphorylation of IκB-α and IKK-β, as well as the nuclear translocation of the p65 subunit of pro-inflammatory transcription factor NF-κB. Taken together, these results support further exploration of the therapeutic potential and molecular mechanism of tangeretin in relation to neuroinflammation and neurodegenerative diseases accompanied by microglial activation.

The NAD-dependent deacetylase sirtuin 2 is a suppressor of microglial activation and brain inflammation

Deleterious sustained inflammation mediated by activated microglia is common to most of neurologic disorders. Here, we identified sirtuin 2 (SIRT2), an abundant deacetylase in the brain, as a major inhibitor of microglia-mediated inflammation and neurotoxicity. SIRT2-deficient mice (SIRT2(-/-)) showed morphological changes in microglia and an increase in pro-inflammatory cytokines upon intracortical injection of lipopolysaccharide (LPS). This response was associated with increased nitrotyrosination and neuronal cell death. Interestingly, manipulation of SIRT2 levels in microglia determined the response to Toll-like receptor (TLR) activation. SIRT2 overexpression inhibited microglia activation in a process dependent on serine 331 (S331) phosphorylation. Conversely, reduction of SIRT2 in microglia dramatically increased the expression of inflammatory markers, the production of free radicals, and neurotoxicity. Consistent with increased NF-κB-dependent transcription of inflammatory genes, NF-κB was found hyperacetylated in the absence of SIRT2, and became hypoacetylated in the presence of S331A mutant SIRT2. This finding indicates that SIRT2 functions as a 'gatekeeper', preventing excessive microglial activation through NF-κB deacetylation. Our data uncover a novel role for SIRT2 opening new perspectives for therapeutic intervention in neuroinflammatory disorders.

Understanding brain dysfunction in sepsis

Sepsis often is characterized by an acute brain dysfunction, which is associated with increased morbidity and mortality. Its pathophysiology is highly complex, resulting from both inflammatory and noninflammatory processes, which may induce significant alterations in vulnerable areas of the brain. Important mechanisms include excessive microglial activation, impaired cerebral perfusion, blood–brain-barrier dysfunction, and altered neurotransmission. Systemic insults, such as prolonged inflammation, severe hypoxemia, and persistent hyperglycemia also may contribute to aggravate sepsis-induced brain dysfunction or injury. The diagnosis of brain dysfunction in sepsis relies essentially on neurological examination and neurological tests, such as EEG and neuroimaging. A brain MRI should be considered in case of persistent brain dysfunction after control of sepsis and exclusion of major confounding factors. Recent MRI studies suggest that septic shock can be associated with acute cerebrovascular lesions and white matter abnormalities. Currently, the management of brain dysfunction mainly consists of control of sepsis and prevention of all aggravating factors, including metabolic disturbances, drug overdoses, anticholinergic medications, withdrawal syndromes, and Wernicke’s encephalopathy. Modulation of microglial activation, prevention of blood–brain-barrier alterations, and use of antioxidants represent relevant therapeutic targets that may impact significantly on neurologic outcomes. In the future, investigations in patients with sepsis should be undertaken to reduce the duration of brain dysfunction and to study the impact of this reduction on important health outcomes, including functional and cognitive status in survivors.

Deficiency in Serine Protease Inhibitor Neuroserpin Exacerbates Ischemic Brain Injury by Increased Postischemic Inflammation

The only approved pharmacological treatment for ischemic stroke is intravenous administration of plasminogen activator (tPA) to re-canalize the occluded cerebral vessel. Not only reperfusion but also tPA itself can induce an inflammatory response. Microglia are the innate immune cells of the central nervous system and the first immune cells to become activated in stroke. Neuroserpin, an endogenous inhibitor of tPA, is up-regulated following cerebral ischemia. To examine neuroserpin-dependent mechanisms of neuroprotection in stroke, we studied neuroserpin deficient (Ns−/−) mice in an animal model of temporal focal ischemic stroke. Infarct size and neurological outcome were worse in neuroserpin deficient mice even though the fibrinolytic activity in the ischemic brain was increased. The increased infarct size was paralleled by a selective increase in proinflammatory microglia activation in Ns−/− mice. Our results show excessive microglial activation in Ns−/− mice mediated by an increased activity of tPA. This activation results in a worse outcome further underscoring the potential detrimental proinflammatory effects of tPA.

Neurotransmitter signaling in the pathophysiology of microglia

Microglial cells are the resident immune cells of the central nervous system. In the resting state, microglia are highly dynamic and control the environment by rapidly extending and retracting motile processes. Microglia are closely associated with astrocytes and neurons, particularly at the synapses, and more recent data indicate that neurotransmission plays a role in regulating the morphology and function of surveying/resting microglia, as they are endowed with receptors for most known neurotransmitters. In particular, microglia express receptors for ATP and glutamate, which regulate microglial motility. After local damage, the release of ATP induces microgliosis and activated microglial cells migrate to the site of injury, proliferate, and phagocytose cells, and cellular compartments. However, excessive activation of microglia could contribute to the progression of chronic neurodegenerative diseases, though the underlying mechanisms are still unclear. Microglia have the capacity to release a large number of substances that can be detrimental to the surrounding neurons, including glutamate, ATP, and reactive oxygen species. However, how altered neurotransmission following acute insults or chronic neurodegenerative conditions modulates microglial functions is still poorly understood. This review summarizes the relevant data regarding the role of neurotransmitter receptors in microglial physiology and pathology.

Microglial Activation in Young Adults With Autism Spectrum Disorder

A growing body of evidence suggests that aberrant immunologic systems underlie the pathophysiologic characteristics of autism spectrum disorder (ASD). However, to our knowledge, no information is available on the patterns of distribution of microglial activation in the brain in ASD. To identify brain regions associated with excessively activated microglia in the whole brain, and to examine similarities in the pattern of distribution of activated microglia in subjects with ASD and control subjects. Case-control study using positron emission tomography and a radiotracer for microglia--[11C](R)-(1-[2-chrorophynyl]-N-methyl-N-[1-methylpropyl]-3 isoquinoline carboxamide) ([11C](R)-PK11195). Subjects recruited from the community. Twenty men with ASD (age range, 18-31 years; mean [SD] IQ, 95.9 [16.7]) and 20 age- and IQ-matched healthy men as controls. Diagnosis of ASD was made in accordance with the Autism Diagnostic Observation Schedule and the Autism Diagnostic Interview-Revised. Regional brain [11C](R)-PK11195 binding potential as a representative measure of microglial activation. The [11C](R)-PK11195 binding potential values were significantly higher in multiple brain regions in young adults with ASD compared with those of controls (P < .05, corrected). Brain regions with increased binding potentials included the cerebellum, midbrain, pons, fusiform gyri, and the anterior cingulate and orbitofrontal cortices. The most prominent increase was observed in the cerebellum. The pattern of distribution of [11C](R)-PK11195 binding potential values in these brain regions of ASD and control subjects was similar, whereas the magnitude of the [11C](R)-PK11195 binding potential in the ASD group was greater than that of controls in all regions. Our results indicate excessive microglial activation in multiple brain regions in young adult subjects with ASD. The similar distribution pattern of regional microglial activity in the ASD and control groups may indicate augmented but not altered microglial activation in the brain in the subjects with ASD.

Chemical genetics of neuroinflammation: natural and synthetic compounds as microglial inhibitors

Chemical genetics is a new field of study that employs diverse small-molecule compounds to interrogate specific biological functions. The chemical genetics approach has been recently applied to microglial biology. Microglial cells are the primary immune cells of the brain, and are believed to play a major role in both host defense and tissue repair in the central nervous system. However, excessive microglial activation with overexpression of proinflammatory cytokines and oxidative stress products is linked to the progression of several neurodegenerative diseases. Therefore, suppression of microglial activation and subsequent neuroinflammation may be a potential therapeutic approach against these diseases. From microglia cell-based assays, the natural compound obovatol and several synthetic compounds were identified as inhibitors of microglial activation. They showed strong anti-neuroinflammatory activities in various in vitro and in vivo studies. Nuclear factor-κB and mitogen-activated protein kinase pathways were found to be related to the effects of these compounds on microglia. In particular, based on the affinity purification approach, peroxiredoxin 2 was identified as a microglial target of obovatol. Thus, natural and synthetic compounds are not only important biological tools for the study of microglial physiology and pathology, but they are also attractive candidates for the development of therapeutic drugs against neuroinflammatory and neurodegenerative diseases.

Systemic Inflammation Modulates Fc Receptor Expression on Microglia during Chronic Neurodegeneration

Chronic neurodegeneration is a major worldwide health problem, and it has been suggested that systemic inflammation can accelerate the onset and progression of clinical symptoms. A possible explanation is that systemic inflammation "switches" the phenotype of microglia from a relatively benign to a highly aggressive and tissue-damaging phenotype. The current study investigated the molecular mechanism underlying this microglia phenotype "switching." We show in mice with chronic neurodegeneration (ME7 prion model) that there is increased expression of receptors that have a key role in macrophage activation and associated signaling pathways, including TREM-2, Siglec-F, CD200R, and FcγRs. Systemic inflammation induced by LPS further increased protein levels of the activating FcγRIII and FcγRIV, but not of other microglial receptors, including the inhibitory FcγRII. In addition to these changes in receptor expression, IgG levels in the brain parenchyma were increased during chronic neurodegeneration, and these IgG levels further increased after systemic inflammation. γ-Chain-deficient mice show modified proinflammatory cytokine expression in the brain after systemic inflammation. We conclude that systemic inflammation during chronic neurodegeneration increases the expression levels of activating FcγR on microglia and thereby lowers the signaling threshold for Ab-mediated cell activation. At the same time, IgG influx into the brain could provide a cross-linking ligand resulting in excessive microglia activation that is detrimental to neurons already under threat by misfolded protein.

Ion Channels on Microglia: Therapeutic Targets for Neuroprotection

Under pathological conditions microglia (resident CNS immune cells) become activated, and produce reactive oxygen and nitrogen species and pro-inflammatory cytokines: molecules that can contribute to axon demyelination and neuron death. Because some microglia functions can exacerbate CNS disorders, including stroke, traumatic brain injury, progressive neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and multiple sclerosis, and several retinal diseases, controlling their activation might ameliorate immune-mediated CNS disorders. A growing body of evidence now points to ion channels on microglia as contributing to the above neuropathologies. For example, the ATP-gated P2X7 purinergic receptor cation channel is up-regulated around amyloid β-peptide plaques in transgenic mouse models of Alzheimer's disease and co-localizes to microglia and astrocytes. Upregulation of the P2X7 receptor subtype on microglia occurs also following spinal cord injury and after ischemia in the cerebral cortex of rats, while P2X7 receptor-like immunoreactivity is increased in activated microglial cells of multiple sclerosis and amyotrophic lateral sclerosis spinal cord. Utilizing neuron/microglia co-cultures as an in vitro model for neuroinflammation, P2X7 receptor activation on microglia appears necessary for microglial cell-mediated injury of neurons. A second example can be found in the chloride intracellular channel 1 (CLIC1), whose expression is related to macrophage activation, undergoes translocation from the cytosol to the plasma membrane (activation) of microglia exposed to amyloid β-peptide, and participates in amyloid β-peptide-induced neurotoxicity through the generation of reactive oxygen species. A final example is the small-conductance Ca2+/calmodulin-activated K+ channel KCNN4/KCa3.1/SK4/IK1, which is highly expressed in rat microglia. Lipopolysaccharide-activated microglia are capable of killing adjacent neurons in co-culture, and show markedly reduced toxicity when treated with an inhibitor of KCa3.1 channels. Moreover, blocking KCa3.1 channels mitigated the neurotoxicity of amyloid β-peptide-stimulated microglia. Excessive microglial cell activation and production of potentially neurotoxic molecules, mediated by ion channels, may thus constitute viable targets for the discovery and development of neurodegenerative disease therapeutics. This chapter will review recent data that reflect the prevailing approaches targeting neuroinflammation as a pathophysiological process contributing to the onset or progression of neurodegenerative diseases, with a focus on microglial ion channels and their neuroprotective potential.

Inhibitors of Microglial Neurotoxicity: Focus on Natural Products

Microglial cells play a dual role in the central nervous system as they have both neurotoxic and neuroprotective effects. Uncontrolled and excessive activation of microglia often contributes to inflammation-mediated neurodegeneration. Recently, much attention has been paid to therapeutic strategies aimed at inhibiting neurotoxic microglial activation. Pharmacological inhibitors of microglial activation are emerging as a result of such endeavors. In this review, natural products-based inhibitors of microglial activation will be reviewed. Potential neuroprotective activity of these compounds will also be discussed. Future works should focus on the discovery of novel drug targets that specifically mediate microglial neurotoxicity rather than neuroprotection. Development of new drugs based on these targets may require a better understanding of microglial biology and neuroinflammation at the molecular, cellular, and systems levels.

Effects of agmatine on hypoxic microglia and activity of nitric oxide synthase

Microglia are the resident macrophages of CNS and play a crucial role in maintaining homeostasis against various neuronal injuries. However, excessive activation of microglia may destroy healthy neurons as well as damaged neurons. We investigated neuroprotective effects of amgatine on hypoxic microglia using in vitro and in vivo models for transient hypoxia. For in vitro study, BV2 immortalized murine microglia were incubated with or without 100μM of agmatine in a closed anaerobic chamber for 2h. After recovery in normoxic condition for 20h, cell viability and the amount of nitrite generation were determined. For in vivo study, 100mg/kg of agmatine or equivalent volume of saline was intraperitoneally administered, and the left middle cerebral artery of adult male Sprague–Dawley rats was occluded for 90min. After 24h from occlusion, the cortex and striatum of the forebrains was evaluated to check the immunoreactivity with a microglial marker, ionized calcium binding adaptor molecule 1 (Iba1), and inducible nitric oxide synthase (iNOS). Results showed that agmatine attenuated hypoxia-induced cytotoxicity and nitrite production by BV2 microglia. Agmatine also decreased the activities of microglia and NOS induced by transient middle cerebral artery occlusion. Finally, our findings reveal that agmatine may reduce microglial damages caused by transient hypoxia and suggest that agmatine may lead to a novel therapeutic strategy for hypoxic neuronal injuries.

Z-ligustilide attenuates lipopolysaccharide-induced proinflammatory response via inhibiting NF-κB pathway in primary rat microglia

To investigate the anti-inflammatory effect of Z-ligustilide (LIG) on lipopolysaccharide (LPS)-activated primary rat microglia. Microglia were pretreated with LIG 1 h prior to stimulation with LPS (1 microg/mL). After 24 h, cell viability was tested with MTT, nitric oxide (NO) production was assayed with Griess reagent, and the content of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and monocyte chemoattractant protein (MCP-1) was measured with ELISA. Protein expression of the nuclear factor-kappaB (NF-kappaB) p65 subunit, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) was detected with immunocytochemistry 1 h or 24 h after LPS treatment. LIG showed a concentration-dependent anti-inflammatory effect in LPS-activated microglia, without causing cytotoxicity. Pretreatment with LIG at 2.5, 5, 10, and 20 micromol/L decreased LPS-induced NO production to 75.9%, 54.4%, 43.1%, and 47.6% (P<0.05 or P< 0.01), TNF-alpha content to 86.2%, 68.3%, 40.1%, and 39.9% (P<0.01, with the exception of 86.2% for 2.5 micromol/L LIG), IL-1beta content to 31.5%, 27.7%, 0.6%, and 0% (P<0.01), and MCP-1 content to 84.4%, 50.3%, 45.1%, and 42.2% (P<0.05 or P<0.01), respectively, compared with LPS treatment alone. LIG (10 micromol/L) significantly inhibited LPS-stimulated immunoreactivity of activated NF-kappaB, COX-2, and iNOS (P<0.01 vs LPS group). LIG exerted a potent anti-inflammatory effect on microglia through inhibition of NF-kappaB pathway. The data provide direct evidence of the neuroprotective effects of LIG and the potential application of LIG for the treatment of the neuroinflammatory diseases characterized by excessive microglial activation.

Anti-neuroinflammatory and anti-oxidative activities of the ethanol extract from Citrus unshiu MARC.

Citrus peel extract has shown anti-inflammatory and anti-oxidative effects in several tissues. Therefore ，we investigated whether the ethanol extract from Fruit peel of Citrus unshiu MARC. suppresses brain insult-induced excessive microglial activation and neuronal oxidative stress. Releases of NO，the major inflammatory mediator in microglia，and iNOS mRNA expressions were markedly inhibited in a dose-dependent manner following treatment of the ethanol extract from Citrus unshiu MARC. in LPS-stimulated BV-2 microglia cells. In addition, the extract significantly suppressed LPS-induced mRNA expression of pro-inflammatory cytokine TNF-α IL-lβ and IL-6. On the other hand, the thanol extract exerted intracellular ROS scavenging activities in HT-22 neurons in a dose dependent manner (50-400 ug/ml)， suggesting the potent anti-oxidative stress effects in neurons. Taken together，these results suggest that Citrus unshiu MARC. peel extract has therapeutic potential against neuroinflammation and neurodegeneration via suppression of brain insult-induced microglial activation and neuronal oxidative stresses.

Celastrol Suppresses Morphological and Transcriptional Responses in Microglial Cells Upon Stimulation with Double-Stranded RNA

ABSTRACTDespite the pivotal role of microglia in the immune system of the brain, a growing body of evidence suggests that excessive microglial activation provokes neuronal and glial damage, leading to neurodegenerative and neuroinflammatory disorders. Celastrol, a triterpene, is a potent anti-inflammatory and antioxidant compound derived from perennial creeping plants belonging to the Celastraceae family. In the current study, we have analyzed the effect of celastrol on morphological and transcriptional responses in microglial MG6 cells upon stimulation with double-stranded RNA, a strong activator of innate immune cells. In the presence of celastrol, morphological changes were inhibited in double-stranded RNA-stimulated microglia. It was also found that the treatment of microglia with celastrol led to a significant decrease in the double-stranded RNA-induced expression of proinflammatory cytokines and chemokines. These data demonstrate that celastrol inhibits morphological and transcriptional responses during microglial activation.

Anti-neuroinflammatory Activity of Nobiletin on Suppression of Microglial Activation

A growing body of evidence suggests that nobiletin (5,6,7,8,3',4'-hexamethoxy flavone) from the peel of citrus fruits, enhances the damaged cognitive function in disease animal models. However, the neuroprotective mechanism has not been clearly elucidated. Since nobiletin has shown anti-inflammatory effects in several tissues, we investigated whether nobiletin suppresses excessive microglial activation implicated in neurotoxicity in lipopolysaccharide (LPS)-stimulated BV-2 microglia cell culture models. Release of nitric oxide (NO), the major inflammatory mediator in microglia, was markedly suppressed in a dose-dependent manner following nobiletin treatment (1-50 µM) in LPS-stimulated BV-2 microglia cells. The inhibitory effect of nobiletin was similar to that of minocycline, a well-known microglial inactivator. Nobiletin significantly inhibited the release of the pro-inflammatory cytokine tumor necrosis factor (TNF-α) and interleukin-1β (IL-1β). LPS-induced phosphorylations of extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), and p38 mitogen-activated protein kinases (MAPKs) were also significantly inhibited by nobiletin treatment. In addition, nobiletin markedly inhibited the LPS-induced pro-inflammatory transcription factor nuclear factor κB (NF-κB) signaling pathway by suppressing nuclear NF-κB translocation from the cytoplasm and subsequent expression of NF-κB in the nucleus. Taken together, these results may contribute to further exploration of the therapeutic potential and molecular mechanism of nobiletin in relation to neuroinflammation and neurodegenerative diseases.

A novel anti-neuroinflammatory pyridylimidazole compound KR-31360

Excessive microglial activation with overexpression of proinflammatory cytokines and oxidative stress products is linked to the progression of several neurodegenerative diseases; therefore, suppression of microglial activation is a potential therapeutic approach against these diseases. Since nitric oxide (NO) is one of the major inflammatory mediators that are produced by activated microglia, inhibitory effects of novel synthetic compounds on microglial NO production were investigated. From the mouse microglia cell-based assays, an imidazo [4,5-b] pyridine compound KR-31360 was identified as an inhibitor of microglial NO production with an IC 50 value of 2 μM. Structure–activity relationship study indicated that 5-position of imidazo [4,5-b] pyridine ring is critical for the activity. KR-31360 also inhibited lipopolysaccharide (LPS)-induced secretion of tumor necrosis factor alpha (TNF-α) and transcription of TNF-α, interleukin-1 beta, and inducible nitric oxide synthase as well as activation of nuclear factor kappa B and mitogen-activated protein kinases. KR-31360 was neuroprotective by suppressing microglial neurotoxicity in a microglia–neuron coculture. The neuroprotective activity of the compound was most effective when microglia were pretreated with the compound prior to LPS challenge. The inhibitory effect of KR-31360 on microglial activation was further demonstrated in a mouse neuroinflammation model in vivo: compared to vehicle-injected animals, KR-31360 injection attenuated LPS-induced microglial activation as evidenced by isolectin B4 staining and proinflammatory gene expression of brain sections. DNA microarray analysis supported that KR-31360 targeted Toll-like receptor 4 pathways. In addition to being a new drug candidate against neuroinflammatory diseases, the compound may be a powerful tool for the better understanding of microglia biology and neuroinflammation.

Chloride channel blockers inhibit iNOS expression and NO production in IFNγ-stimulated microglial BV2 cells

Microglial cells play an important role during neuroinflammation in the central nervous system. Among other factors, activated microglia produce nitric oxide (NO), which is toxic to neurons and excessive microglial activation and NO production contribute to the pathology of neurodegenerative diseases. Chloride channels have previously been shown to participate in microglial activation. Here we investigate the effects of established chloride channel blockers with different chemical structures on interferon-γ (IFNγ)-induced activation of the murine microglial cell line, BV2. IFNγ-induced NO production was effectively reduced by NPPB, IAA-94, tamoxifen, NS3728 and NS1652, with NS1652 being the most potent. In contrast, DIDS reduced NO production only at cytotoxic concentrations. Furthermore, NS1652 reduced the protein and mRNA levels of inducible nitric oxide synthase (iNOS), without altering STAT1 phosphorylation. These observations suggest a microglial chloride conductance as a critical permissive factor downstream in the IFNγ-induced iNOS cascade. The nature of the underlying channel is unknown, but the pharmacological profile appears incompatible with the involvement of the volume activated anion conductance (VRAC).