Role Of Microglial Activation Research Articles

Effects of Tetramethylpyrazine on Microglia Activation in Spinal Cord Compression Injury of Mice

Secondary mechanisms, including inflammation and microglia activation, serve as targets for the development and application of pharmacological strategies in the management of spinal cord injury (SCI). Tetramethylpyrazine (TMP), an active ingredient of Ligusticum wallichii (chuanxiong), has shown anti-inflammatory and neuroprotective effects against SCI. However, it remains uncertain whether the inflammation-suppressive effects of TMP play a modulatory role over microglia activation in SCI. The present study investigated the effects of TMP on microglia activation and pro-inflammatory cytokines in spinal cord compression injury in mice. For a real-time PCR measurement of pro-inflammatory cytokines, SCI was induced in mice by the clip compression method (30 g force, 1 min) and TMP (15 or 30 mg/kg, i.p.) was administered once, 30 minutes before the SCI induction. For immunohistochemistry, TMP (30 mg/kg, i.p.) treatment was given three times during the first 48 hours after the SCI. 30 mg/kg of TMP treatment reduced the up-regulation of TNF-α, IL-1β and COX-2 mRNA in the spinal tissue at four hours after the SCI induction. TMP also significantly attenuated microglia activation and neutrophil infiltration at 48 hours after the SCI induction. In addition, iNOS expression in the spinal tissue was attenuated with TMP treatment. These results suggest that TMP plays a modulatory role in microglia activation and may protect the spinal cord from or potentially delay secondary spinal cord injury.

Activation of microglial cells in the trigeminal subnucleus caudalis evoked by inflammatory stimulation of the oral mucosa

To study the inflammatory hyperplasia induced by an acute noxious stimulation of oral mucosa with 5% formalin (5%FOR), we performed an immunohistochemical study on the expression of TNFá in the intermolar region of the dorsal lingual eminence (IDLE), and Iba1 and phosphorylated (phospho)- p38 MAPK involved with central nervous system microglial activation in the trigeminal subnucleus caudalis (Vc). The present study observed significantly increased expression of TNFá at either 1h or 24h of 5%FOR nociception, as well as sustained TNFá immunoreactivity in the IDLE. On the other hand, at either 1h or 24h 5%FOR nociception, Iba1- immunoreactive (IR) cells in the Vc were significantly increased after inflammatory stimulation of the IDLE; the increase was more evident at 24h 5%FOR nociception. By using the double-label immunofluorescence technique, the findings in particular demonstrated a significant increase in the number of phospho-p38 MAPK- and Iba1-IR coexpressed cells in the Vc at 24h 5%FOR nociception. The results suggest that 24h persistent microglial activation in subnuclei zonalis and gelatinosus of the Vc is evoked by 5%FOR noxious stimulation of the IDLE oral mucosa, thereby the present study indicates that the MAPK expression plays important roles in microglial activation related with central sensitization and inflammatory hyperalgesia.

Inhibitory Effects of Ginsenoside Rb1 on Neuroinflammation Following Systemic Lipopolysaccharide Treatment in Mice

Ginsenoside Rb1 (GRb1) is a major ingredient of ginseng and has a wide range of neuroprotection effects. Neuroinflammation is a feature of neurodegenerative conditions and is characterized by microglia activation and the expression of major inflammatory mediators. The present study investigated the modulatory effect of GRb1 on microglia activation, the expression of pro-inflammatory cytokines and cyclooxygenase (COX)-2 in the brain induced by systemic lipopolysaccharide (LPS) treatment in C57BL/6 mice. Systemic LPS treatment induces immediate microglia activation in the brain. Based on this information, GRb1 was administered orally, at doses of 10 and 20 mg/kg, 1 h prior to the LPS (3 mg/kg, intraperitoneally) injection. At a dose of 20 mg/kg GRb1 attenuated Iba1 protein expression and morphological activation of microglia by LPS. GRb1 significantly reduced the upregulation of tumor necrosis factor-α, interleukin (IL)-1β and IL-6 mRNA in the brain tissue at 4 h after LPS injection. In addition, the expression of COX-2 mRNA and protein in the brain tissue were also attenuated at the 20 mg/kg dose of GRb1. These results indicate that GRb1 plays a modulatory role in microglia activation and neuroinflammation. This study shows that GRb1 attenuates microglia activation in the brain using an in vivo animal model.

Activation of KCNN3/SK3/KCa2.3 channels attenuates enhanced calcium influx and inflammatory cytokine production in activated microglia

In neurons, small-conductance calcium-activated potassium (KCNN/SK/K(Ca)2) channels maintain calcium homeostasis after N-methyl-D-aspartate (NMDA) receptor activation, thereby preventing excitotoxic neuronal death. So far, little is known about the function of KCNN/SK/K(Ca)2 channels in non-neuronal cells, such as microglial cells. In this study, we addressed the question whether KCNN/SK/K(Ca)2 channels activation affected inflammatory responses of primary mouse microglial cells upon lipopolysaccharide (LPS) stimulation. We found that N-cyclohexyl-N-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-4-pyrimidinamine (CyPPA), a positive pharmacological activator of KCNN/SK/K(Ca)2 channels, significantly reduced LPS-stimulated activation of microglia in a concentration-dependent manner. The general KCNN/SK/K(Ca)2 channel blocker apamin reverted these effects of CyPPA on microglial proliferation. Since calcium plays a central role in microglial activation, we further addressed whether KCNN/SK/K(Ca)2 channel activation affected the changes of intracellular calcium levels, [Ca(2+)](i), in microglial cells. Our data show that LPS-induced elevation of [Ca(2+)](i) was attenuated following activation of KCNN2/3/K(Ca)2.2/K(Ca)2.3 channels by CyPPA. Furthermore, CyPPA reduced downstream events including tumor necrosis factor alpha and interleukin 6 cytokine production and nitric oxide release in activated microglia. Further, we applied specific peptide inhibitors of the KCNN/SK/K(Ca)2 channel subtypes to identify which particular channel subtype mediated the observed anti-inflammatory effects. Only inhibitory peptides targeting KCNN3/SK3/K(Ca)2.3 channels, but not KCNN2/SK2/K(Ca)2.2 channel inhibition, reversed the CyPPA-effects on LPS-induced microglial proliferation. These findings revealed that KCNN3/SK3/K(Ca)2.3 channels can modulate the LPS-induced inflammatory responses in microglial cells. Thus, KCNN3/SK3/K(Ca)2.3 channels may serve as a therapeutic target for reducing microglial activity and related inflammatory responses in the central nervous system.

A substituted 3,4-dihydropyrimidinone derivative (compound D22) prevents inflammation mediated neurotoxicity; role in microglial activation in BV-2 cells

A novel synthetic 3,4-dihydropyrimidinone derivative, compound D22 (ethyl 6-methyl-4-(3-phenoxyphenyl)-2-thioxo-3,4-dihydropyrimidine-5-carboxylate), was found to exert anti-inflammatory properties in lipopolysaccharide-stimulated microglial BV-2 cells. Compound D22 reduced the pro-inflammatory factors such as nitric oxide, prostaglandin E2, tumor necrosis factor-α and interleukin-1β. Moreover, it suppressed the expressions of inducible NO synthase and cyclooxygenase-2. Compound D22 inhibited the activation of mitogen-activated protein kinases. When compound D22-conditioned media from BV-2 cells were applied to N2a cells, neuronal cell death was inhibited via suppression of caspase-3 activation and regulation of Bcl-2 and Bax proteins expression. These results suggest that compound D22 may be useful for treating neurodegenerative diseases related with neuroinflammation.

Imaging of microglia in patients with neurodegenerative disorders

Microglia constitute the main immune defense in the central nervous system. In response to neuronal injury, microglia become activated, acquire phagocytic properties, and release a wide range of pro-inflammatory mediators that are essential for the annihilation of the neuronal insult. Although the role of microglial activation in acute neuronal damage is well defined, the pathophysiological processes underlying destructive or protective role to neurons following chronic exposure to microglial activation is still a subject of debate. It is likely that chronic exposure induces detrimental effects by promoting neuronal death through the release of neurotoxic factors. Positron emission tomography (PET) imaging with the use of translocator protein (TSPO) radioligands provides an in vivo tool for tracking the progression and severity of neuroinflammation in neurodegenerative disease. TSPO expression is correlated to the extent of microglial activation and the measurement of TSPO uptake in vivo with PET is a useful indicator of active disease. Although understanding of the interaction between radioligands and TSPO is not completely clear, there is a wide interest in application of TSPO imaging in neurodegenerative disease. In this article, we aim to review the applications of in vivo microglia imaging in neurodegenerative disorders such as Parkinson’s disease, Huntington’s disease, Dementias, and Multiple Sclerosis.

Factor XIII Deficiency Improves Motor Function in Mice Challenged with Experimental Autoimmune Encephalomyelitis, An Established Model of Multiple Sclerosis

Abstract 858Multiple sclerosis (MS) is an inflammatory disease characterized by autoimmune demyelination of neurons and axonal damage within the central nervous system, leading to relapsing/remitting neurological deficits, including sensory and motor deficits. MS plaques are characterized by blood-brain barrier disruption leading to perivascular deposition of fibrin that correlates with areas of microglia activation and myelin damage. Consistent with multiple studies showing that hemostatic factors can serve as important modulators of the inflammatory response in vivo, fibrin(ogen) has been identified as a novel regulator of microglial activation and differentiation. Furthermore, fibrin(ogen) has been found, via the αMβ2 binding motif on the γ chain, to play a role in microglial activation in experimental autoimmune encephalomyelitis (EAE), a murine model of MS. However, previous work by our lab, as well as others, suggests that thrombin-mediated proteolysis plays a greater role in driving neuroinflammatory disease than by merely supporting fibrin formation. Our working hypothesis is that thrombin-mediated proteolysis drives autoimmune neuroinflammation through both fibrin(ogen)-dependent and fibrin(ogen)-independent pathways. To test this hypothesis, we have initiated studies of EAE in mice lacking selected thrombin substrates, including protease activated receptors (PARs) and factor XIII (fXIII). Clinical evaluation of loss of motor function in EAE-challenged cohorts of PAR-1-deficient, PAR-4-deficient and WT control mice revealed a similar outcome in all three cohorts, suggesting PARs may have either a modest or a secondary role in EAE-induced neuroinflammatory disease. However, mice with a constitutive deficiency of another thrombin substrate, the fibrin cross-linking transglutaminase fXIII, were found to have a significant amelioration in motor function loss when challenged with EAE. To further explore this phenotype, we have utilized cystamine, a transglutaminase inhibitor, as a pharmacologic complement to the fXIII knockout mice. Mice with cystamine placed in their drinking water during the EAE challenge period also experienced significantly less disease than control mice that did not receive cystamine. This data suggests that (pro)thrombin contributes neuroinflammatory disease by both supporting local fibrinogen polymerization and by activation of fXIII leading to fibrin stabilization. Furthermore, this line of research suggests that more detailed studies of fXIII may reveal novel strategies for limiting or reversing the devastating pathologies associated with multiple sclerosis. Disclosures:Mullins:Baxter: Consultancy. Palumbo:Novo Nordisk Corporation: Research Funding.

The role of microglial activation in spinal cord in a rat model of persistent postoperative pain evoked by skin/muscle incision and retraction

Objective To investigate the role of microglial activation in spinal cord in a rat model of persistent postoperative pain evoked by skin/muscle incision and retraction (SMIR) .Methods Seventy-two male SD rats weighing 200-250 g in which intrathecal (IT) catheter was successfully inserted were randomly divided into 3 groups ( n = 24 each) : group sham operation; group SMIR and group SMIR + FT minocycline (a specific microglia inhibitor) . The rat model of persistent postoperative pain evoked by SMIR was established according to the method described by Flatters. Pain behavior was assessed by paw mechanical withdrawal threshold ( MWT) to von Frey filament stimulation at 1 day before (T0,baseline) and 3, 7, 12, 22 and 32 days after operation (T1-5,) . Four animals were sacrificed at each time point in each group for detection of the expression of Iba-1 (a specific marker of microglia) in the spinal dorsal horn by immunofluorescence and the microglia was counted. Results MWT was significantly decreasedat T1-4, while the expression of Iba-1 and microglia counts in the spinal dorsal horn were significantly increased at T1, 2 by SMIR in group Ⅱ. IT minocycline significantly attenuated the hyperalgesia induced by SMIR at T1-4 and decreased Iba-1 expression and microglia counts at T1,2 in group Ⅲ. Conclusion Microglial activation in the spinal cord plays an important role in the development and maintenance of SMIR-evoked persistent postoperative pain in rats. Key words: Mictoglia; Pain,postoperative; Spinal cord

RSAD2 is a key requirement for Golgi trafficking of TNF-α in activated microglia (117.26)

Abstract Antiviral protein RSAD2 (radical S-adenosyl methionine domain-containing protein 2), also known as viperin, has been recognized as a highly inducible ER (endoplasmic reticulum) protein in response to a wide range of viruses and microbial products such as LPS and double-stranded RNA. However, the functions of RSAD2 other than its antiviral activity have remained unexplored. The present studies showed that RSAD2 mRNA and protein expression was highly induced by LPS treatment in both primary microglia and BV2 cells. To investigate the function of RSAD2 in activated microglia, BV2 cells were depleted RSAD2 by using a short hairpin RNA (shRNA) plasmid. TNF-α and IL-6 production in RSAD2 depleted cells was remarkably reduced after stimulation with LPS. However, mRNA expression of TNF-α and IL-6 did not reveal significant differences between RSAD2-shRNA transfected cells and cont-shRNA transfected cells after stimulation with LPS. The intracellular distribution and trafficking of TNF-α was demonstrated by immunofluorescence staining. Non-transfected control cells showed strong intracellular staining of TNF-α in a perinuclear Golgi complex after LPS treatment. However, depletion of RSAD2 inhibited the Golgi trafficking of TNF-α. These results suggest that RSAD2 play an important role in microglia activation, especially regulation of Golgi trafficking of secretary molecules such as TNF-α.

Do PPAR-Gamma Agonists Have a Future in Parkinson's Disease Therapy?

Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor (PPAR)-γ agonists commonly used as insulin-sensitizing drugs for the treatment of type 2 diabetes. In the last decade, PPAR-γ agonists have received increasing attention for their neuroprotective properties displayed in a variety of neurodegenerative diseases, including Parkinson's disease (PD), likely related to the anti-infammatory activity of these compounds. Recent studies indicate that neuroinflammation, specifically reactive microglia, plays important roles in PD pathogenesis. Moreover, after the discovery of infiltrating activated Limphocytes in the substantia nigra (SN) of PD patients, most recent research supports a role of immune-mediated mechanisms in the pathological process leading to chronic neuroinflammation and dopaminergic degeneration. PPAR-γ are highly expressed in cells of both central and peripheral immune systems, playing a pivotal role in microglial activation as well as in monocytes and T cells differentiation, in which they act as key regulators of immune responses. Here, we review preclinical evidences of PPAR-γ-induced neuroprotection in experimental PD models and highlight relative anti-inflammatory mechanisms involving either central or peripheral immunomodulatory activity. Specific targeting of immune functions contributing to neuroinflammation either directly (central) or indirectly (peripheral) may represent a novel therapeutic approach for disease modifying therapies in PD.

Fcγ receptors are required for NF-κB signaling, microglial activation and dopaminergic neurodegeneration in an AAV-synuclein mouse model of Parkinson's disease

Overexpression of alpha-synuclein (α-SYN), a protein which plays an important role in the pathogenesis of Parkinson's disease (PD), triggers microglial activation and adaptive immune responses, and leads to neurodegeneration of dopaminergic (DA) neurons. We hypothesized a link between the humoral adaptive immune response and microglial activation in α-SYN induced neurodegeneration. To test this hypothesis, we employed adeno-associated virus serotype 2 (AAV2) to selectively over-express human α-SYN in the substantia nigra (SN) of wild-type mice and FcγR-/- mice, which lack high-affinity receptors for IgG. We found that in wild-type mice, α-SYN induced the expression of NF-κB p65 and pro-inflammatory molecules. In FcγR-/- mice, NF-κB activation was blocked and pro-inflammatory signaling was reduced. Microglial activation was examined using immunohistochemistry for gp91PHOX. At four weeks, microglia were strongly activated in wild-type mice, while microglial activation was attenuated in FcγR-/- mice. Dopaminergic neurodegeneration was examined using immunohistochemistry for tyrosine hydroxylase (TH) and unbiased stereology. α-SYN overexpression led to the appearance of dysmorphic neurites, and a loss of DA neurons in the SN in wild-type animals, while FcγR-/- mice did not exhibit neuritic change and were protected from α-SYN-induced neurodegeneration 24 weeks after injection. Our results suggest that the humoral adaptive immune response triggered by excess α-SYN plays a causative role in microglial activation through IgG-FcγR interaction. This involves NF-κB signaling, and leads to DA neurodegeneration. Therefore, blocking either FcγR signaling or specific intracellular signal transduction events downstream of FcγR-IgG interaction, such as NF-κB activation, may be viable therapeutic strategies in PD.

Chronic upregulation of activated microglia immunoreactive for galectin-3/Mac-2 and nerve growth factor following diffuse axonal injury

BackgroundDiffuse axonal injury in patients with traumatic brain injury (TBI) can be associated with morbidity ranging from cognitive difficulties to coma. Magnetic resonance imaging scans now allow early detection of axonal injury following TBI, and have linked cognitive disability in these patients to white matter signal changes. However, little is known about the pathophysiology of this white matter injury, and the role of microglial activation in this process. It is increasingly recognized that microglia constitute a heterogeneous population with diverse roles following injury. In the present studies, we tested the hypothesis that following diffuse axonal injury involving the corpus callosum, there is upregulation of a subpopulation of microglia that express the lectin galectin-3/Mac-2 and are involved in myelin phagocytosis.MethodsAdult mice were subject to midline closed skull injury or sham operation and were sacrificed 1, 8, 14 or 28 days later. Immunohistochemistry and immunofluorescence techniques were used to analyze patterns of labelling within the corpus callosum qualitatively and quantitatively.ResultsActivated microglia immunoreactive for galectin-3/Mac-2 were most abundant 1 day following injury. Their levels were attenuated at later time points after TBI but still were significantly elevated compared to sham animals. Furthermore, the majority of galectin-3/Mac-2+ microglia were immunoreactive for nerve growth factor in both sham and injured animals.ConclusionsOur results suggest that galectin-3/Mac-2+ microglia play an important role in the pathogenesis of diffuse axonal injury both acutely and chronically and that they mediate their effects, at least in part by releasing nerve growth factor.

Microglial MyD88 signaling regulates acute neuronal toxicity of LPS-stimulated microglia in vitro

Although the role of microglial activation in neural injury remains controversial, there is increasing evidence for a detrimental effect in the immature brain, which may occur in response to release of neurotoxic substances including pro-inflammatory cytokines. However, the signaling mechanisms involved in microglial-induced neuronal cell death are unclear. Microglia isolated from the brains of wild-type (WT) or MyD88 knockout (KO) mice were exposed to PBS or the TLR4-ligand LPS (100 ng/mL) for 2, 6, 14, or 24 h, and the microglia-conditioned medium (MCM) collected. Detection of multiple inflammatory molecules in MCM was performed using a mouse 22-plex cytokine microbead array kit. Primary neuronal cultures were supplemented with the 14 or 24 h MCM, and the degree of neuronal apoptosis examined after exposure for 24 h. Results showed a rapid and sustained elevation in multiple inflammatory mediators in the MCM of WT microglia exposed to LPS, which was largely inhibited in MyD88 KO microglia. There was a significant increase in apoptotic death measured at 24 h in cultured neurons exposed to CM from either 14 or 24 h LPS-stimulated WT microglia ( p < .05 vs. WT control). By contrast, there was no increase in apoptotic death in cultured neurons exposed to CM from 14 or 24 h LPS-stimulated MyD88 KO microglia ( p = .15 vs. MyD88 KO control). These data suggest that MyD88-dependent activation of microglia by LPS causes release of factors directly toxic to neurons.

Minocycline reduces neuronal death and attenuates microglial response after pediatric asphyxial cardiac arrest.

The mechanisms leading to delayed neuronal death after asphyxial cardiac arrest (ACA) in the developing brain are unknown. This study aimed at investigating the possible role of microglial activation in neuronal death in developing brain after ACA. Postnatal day-17 rats were subjected to 9 mins of ACA followed by resuscitation. Rats were randomized to treatment with minocycline, (90 mg/kg, intraperitoneally (i.p.)) or vehicle (saline, i.p.) at 1 h after return of spontaneous circulation. Thereafter, minocycline (22.5 mg/kg, i.p.) was administrated every 12 h until sacrifice. Microglial activation (evaluated by immunohistochemistry using ionized calcium-binding adapter molecule-1 (Iba1) antibody) coincided with DNA fragmentation and neurodegeneration in CA1 hippocampus and cortex (assessed by deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL), Fluoro-Jade-B and Nissl stain). Minocycline significantly decreased both the microglial response and neuronal degeneration compared with the vehicle. Asphyxial CA significantly enhanced proinflammatory cytokine and chemokine levels in hippocampus versus control (assessed by multiplex bead array assay), specifically tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-1alpha (MIP-1alpha), regulated upon activation, normal T-cell expressed and secreted (RANTES), and growth-related oncogene (GRO-KC) (P<0.05). Minocycline attenuated ACA-induced increases in MIP-1alpha and RANTES (P<0.05). These data show that microglial activation and cytokine production are increased in immature brain after ACA. The beneficial effect of minocycline suggests an important role for microglia in selective neuronal death after pediatric ACA, and a possible therapeutic target.

The Ca2+ release-activated Ca2+ current (ICRAC) mediates store-operated Ca2+ entry in rat microglia

Ca2+ signaling plays a central role in microglial activation, and several studies have demonstrated a store-operated Ca2+ entry (SOCE) pathway to supply this ion. Due to the rapid pace of discovery of novel Ca2+ permeable channels, and limited electrophysiological analyses of Ca2+ currents in microglia, characterization of the SOCE channels remains incomplete. At present, the prime candidates are ‘transient receptor potential’ (TRP) channels and the recently cloned Orai1, which produces a Ca2+-release-activated Ca2+ (CRAC) current. We used cultured rat microglia and real-time RT-PCR to compare expression levels of Orai1, Orai2, Orai3, TRPM2, TRPM7, TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6 and TRPC7 channel genes. Next, we used Fura-2 imaging to identify a store-operated Ca2+ entry (SOCE) pathway that was reduced by depolarization and blocked by Gd3+, SKF-96365, diethylstilbestrol (DES), and a high concentration of 2-aminoethoxydiphenyl borate (50 μM 2-APB). The Fura-2 signal was increased by hyperpolarization, and by a low concentration of 2-APB (5 μM), and exhibited Ca2+-dependent potentiation. These properties are entirely consistent with Orai1/CRAC, rather than any known TRP channel and this conclusion was supported by patch-clamp electrophysiological analysis. We identified a store-operated Ca2+ current with the same properties, including high selectivity for Ca2+ over monovalent cations, pronounced inward rectification and a very positive reversal potential, Ca2+-dependent current potentiation, and block by SKF-96365, DES and 50 μM 2-APB. Determining the contribution of Orai1/CRAC in different cell types is crucial to future mechanistic and therapeutic studies; this comprehensive multi-strategy analysis demonstrates that Orai1/CRAC channels are responsible for SOCE in primary microglia.

SDF-1alpha up-regulates interleukin-6 through CXCR4, PI3K/Akt, ERK, and NF-kappaB-dependent pathway in microglia

Stromal cell-derived factor-1 (SDF-1), also known as CXCL12, and its receptor CXC chemokine receptor 4 (CXCR4) express in various kinds of cells in central nervous system. The SDF-1/CXCR4 signaling pathway is regulated by diverse biological effects. SDF-1 is up-regulated in the ischemic penumbra following stroke and has been known to be associated with the homing of bone marrow cells to injury. However, the effect of SDF-1α/CXCR4 on cytokine production in microglia is mostly unknown. Here, we demonstrated that SDF-1α enhanced IL-6 production in both primary cultured microglia and BV-2 microglia. We further investigated the signaling pathway involved in IL-6 production stimulated by SDF-1α in microglia. SDF-1α increased IL-6 production in both protein and mRNA levels. These effects were attenuated by ERK, phosphatidylinositol 3-kinase (PI3K), NF-κB inhibitors, and IκB protease inhibitor. Stimulation of microglia with SDF-1α also increased Akt and ERK1/2 phosphorylation. In addition, SDF-1α treatment also increased IκB kinase α/β (IKK α/β) phosphorylation, IκBα phosphorylation, IκBα degradation, p65 phosphorylation at Ser 276, translocation of p65 and p50 from cytosol to nucleus and κB-luciferase activity. Moreover, SDF-1α-mediated increase of κB-luciferase activity was inhibited by pre-transfection of DN-p85, DN-Akt or DN-ERK2. Increase of IKK α/β phosphorylation and binding of p65 and p50 to the NF-κB element were both antagonized by PI3K and ERK inhibitors. Our results demonstrate a mechanism linking SDF-1α and IL-6, and provide additional support for the notion that SDF-1α plays a regulatory role in microglia activation.

Kv1.1 expression in microglia regulates production and release of proinflammatory cytokines, endothelins and nitric oxide

Potassium channels play an important role in microglial activation but their involvement in main functions of microglia including secretion of proinflammatory cytokines has remained uncertain. This study has revealed the specific expression of Kv1.1 in microglia both in vivo and in vitro. Kv1.1 immunoreactivity was localized in the amoeboid microglia in the rat brain between postnatal (P) day 1 (P1) and day 10 (P10); it was, however, progressively reduced with age and was hardly detected at P14 and P21 in ramified microglia, a derivative cell of amoeboid microglia. Following hypoxic exposure, Kv1.1 expression in amoeboid microglia was enhanced or induced in ramified microglia in more mature brain at P21 when compared with their matching controls. RT-PCR and Western blot analysis confirmed Kv1.1 mRNA and protein expression in murine BV-2 cells which was up-regulated by hypoxia or lipopolysaccharide (LPS) treatment; it was reduced significantly by dexamethasone. Neutralization with Kv1.1 antibody suppressed the expression and release of tumor necrosis factor-α, interleukin-1β, endothelins and nitric oxide (NO) in LPS-activated BV-2 cells. It is concluded that Kv1.1, constitutively expressed by microglia, is elicited by hypoxia and LPS and this may be linked to production of proinflammatory cytokines, endothelins and NO.

Neurotoxic Activation of Microglia Is Promoted by a Nox1-Dependent NADPH Oxidase

Reactive oxygen species (ROS) modulate intracellular signaling but are also responsible for neuronal damage in pathological states. Microglia, the resident CNS macrophages, are prominent sources of ROS through expression of the phagocyte oxidase which catalytic subunit Nox2 generates superoxide ion (O2(.-)). Here we show that microglia also express Nox1 and other components of nonphagocyte NADPH oxidases, including p22(phox), NOXO1, NOXA1, and Rac1/2. The subcellular distribution and functions of Nox1 were determined by blocking Nox activity with diphenylene iodonium or apocynin, and by silencing the Nox1 gene in microglia purified from wild-type (WT) or Nox2-KO mice. [Nox1-p22(phox)] dimers localized in intracellular compartments are recruited to phagosome membranes during microglial phagocytosis of zymosan, and Nox1 produces O2(.-) in zymosan-loaded phagosomes. In microglia activated with lipopolysaccharide (LPS), Nox1 produces O2(.-), which enhances cell expression of inducible nitric oxide synthase and secretion of interleukin-1beta. Comparisons of microglia purified from WT, Nox2-KO, or Nox1-KO mice indicate that both Nox1 and Nox2 are required to optimize microglial production of nitric oxide. By injecting LPS in the striatum of WT and Nox1-KO mice, we show that Nox1 also enhances microglial production of cytotoxic nitrite species and promotes loss of presynaptic proteins in striatal neurons. These results demonstrate the functional expression of Nox1 in resident CNS phagocytes, which can promote production of neurotoxic compounds during neuroinflammation. Our study also shows that Nox1- and Nox2-dependent oxidases play distinct roles in microglial activation and that Nox1 is a possible target for the treatment of neuroinflammatory states.

Astrocytes play a key role in activation of microglia by persistent Borna disease virus infection

Neonatal Borna disease virus (BDV) infection of the rat brain is associated with microglial activation and damage to certain neuronal populations. Since persistent BDV infection of neurons is nonlytic in vitro, activated microglia have been suggested to be responsible for neuronal cell death in vivo. However, the mechanisms of activation of microglia in neonatally BDV-infected rat brains remain unclear. Our previous studies have shown that activation of microglia by BDV in culture requires the presence of astrocytes as neither the virus nor BDV-infected neurons alone activate microglia. Here, we evaluated the mechanisms whereby astrocytes can contribute to activation of microglia in neuron-glia-microglia mixed cultures. We found that persistent infection of neuronal cells leads to activation of uninfected astrocytes as measured by elevated expression of RANTES. Activation of astrocytes then produces activation of microglia as evidenced by increased formation of round-shaped, MHCI-, MHCII- and IL-6-positive microglia cells. Our analysis of possible molecular mechanisms of activation of astrocytes and/or microglia in culture indicates that the mediators of activation may be soluble heat-resistant, low molecular weight factors. The findings indicate that astrocytes may mediate activation of microglia by BDV-infected neurons. The data are consistent with the hypothesis that microglia activation in the absence of neuronal damage may represent initial steps in the gradual neurodegeneration observed in brains of neonatally BDV-infected rats.

Fibrillar Amyloid-β Peptides Activate Microglia via TLR2: Implications for Alzheimer’s Disease

Microglial activation is an important pathological component in brains of patients with Alzheimer's disease (AD), and fibrillar amyloid-beta (Abeta) peptides play an important role in microglial activation in AD. However, mechanisms by which Abeta peptides induce the activation of microglia are poorly understood. The present study underlines the importance of TLR2 in mediating Abeta peptide-induced activation of microglia. Fibrillar Abeta1-42 peptides induced the expression of inducible NO synthase, proinflammatory cytokines (TNF-alpha, IL-1beta, and IL-6), and integrin markers (CD11b, CD11c, and CD68) in mouse primary microglia and BV-2 microglial cells. However, either antisense knockdown of TLR2 or functional blocking Abs against TLR2 suppressed Abeta1-42-induced expression of proinflammatory molecules and integrin markers in microglia. Abeta1-42 peptides were also unable to induce the expression of proinflammatory molecules and increase the expression of CD11b in microglia isolated from TLR2(-/-) mice. Finally, the inability of Abeta1-42 peptides to induce the expression of inducible NO synthase and to stimulate the expression of CD11b in vivo in the cortex of TLR2(-/-) mice highlights the importance of TLR2 in Abeta-induced microglial activation. In addition, ligation of TLR2 alone was also sufficient to induce microglial activation. Consistent to the importance of MyD88 in mediating the function of various TLRs, antisense knockdown of MyD88 also inhibited Abeta1-42 peptide-induced expression of proinflammatory molecules. Taken together, these studies delineate a novel role of TLR2 signaling pathway in mediating fibrillar Abeta peptide-induced activation of microglia.