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At least 973 records · Page 54Linked to original sources
PubMed2002

Role of p38 and p44/42 mitogen-activated protein kinases in microglia.

Although microglial cells are thought to play a beneficial role in the regeneration and plasticity of the central nervous system (CNS), recent studies have indicated that at least some molecules released by microglia may be harmful in acute brain insults and neurodegenerative diseases. Therefore, the pathways mediating the synthesis and release of these neurotoxic compounds are of importance. p38 and p44/42 families of mitogen-activated protein kinases (MAPKs) in microglia respond strongly to various extracellular stimuli, such as ATP, thrombin, and beta-amyloid, a peptide thought to be responsible for the neuropathology in Alzheimer's disease. In this review we describe in vivo evidence implicating that p38 and p44/42 MAPKs may play a critical role in harmful microglial activation in acute brain injury, such as stroke, and in more chronic neurodegenerative diseases, such as Alzheimer's disease. We also clarify the extracellular signals responsible for activation of p38 and p44/42 MAPK in microglia and review the responses so far reported to be mediated by these kinases.

Animals↗
PubMed2002

Microglia in human immunodeficiency virus-associated neurodegeneration.

Infection with the human immunodeficiency virus (HIV) is associated with a syndrome of cognitive and motor abnormalities that may develop in the absence of opportunistic infections. Neurons are not productively infected by HIV. Thus, one hypothesis to explain the pathophysiology of HIV-associated dementia (HAD) suggests that signals released from other infected cell types in the CNS secondarily lead to neuronal injury. Microglia are the predominant resident CNS cell type productively infected by HIV-1. Neurologic dysfunction in HAD appears to be a consequence of microglial infection and activation. Several neurotoxic immunomodulatory factors are released from infected and activated microglia, leading to altered neuronal function, synaptic and dendritic degeneration, and eventual neuronal apoptosis. This review summarizes findings from clinical/pathological studies, animal models, and in vitro models of HAD. Most of these studies support the hypothesis that altered microglial physiology is the nidus for a cascade of events leading to neuronal dysfunction and death. Several molecular mediators of neuronal injury in HAD that emanate from microglia have been identified, and strategies for altering the impact of these neurotoxins are discussed.

AIDS Dementia Complex↗
PubMed2003

Loss of microglial ramification in microglia-astrocyte cocultures: involvement of adenylate cyclase, calcium, phosphatase, and Gi-protein systems.

Reduction in microglial branching is a common feature in brain pathology and culminates in the transformation into small, rounded, microglia-derived phagocytes in the presence of neural debris. The molecular factors responsible for this transformation are unknown. Here we explored the effect of different classes of intra- and extracellular stimuli in vitro on the morphology of ramified microglia cultured on a confluent astrocyte substrate. These studies showed a strong dose-dependent effect for the Ca(2+) ionophore calcimycine/A21837 (50 microM) and for dibutyryl-cAMP (1 mM), with a loss of microglial ramification. Direct activation of the adenylate cyclase with forskolin (0.1 mM) also led to the disappearance of microglial branching. Okadaic acid (70 nM), the inhibitor of protein phosphatases 1 and 2A (PP1/PP2A), and pertussis toxin (12.5 microg/ml), a G(i)-protein inhibitor, also showed similar effects. No effect was observed for dibutyryl-cGMP or for UTP; addition of ATP had a moderate effect, but only at very high, probably nonphysiological concentrations (100 mM). Extracellular matrix components such as keratatan-sulfate, integrin receptor blockers, the disintegrins kistrin, echistatin, and flavoridin, or the serine protease thrombin all had no effect. Addition of prostaglandin D(2) (PGD(2)), a molecule produced by activated microglial cells, had a transforming effect, but at concentrations two orders of magnitude higher than that of established PGD(2) receptors. In summary, addition of agents causing intracellular elevation of Ca(2+) and cAMP or inhibition of G(i)-proteins and phosphatases to ramified microglia cultured on top of confluent astrocytes leads to a rapid loss of microglial branching. Signaling cascades controlled by these molecules may play an important role in the regulation of this common physiological process in the injured brain.

Adenosine Triphosphate↗
PubMed2003

Differential activation of subtype purinergic receptors modulates Ca(2+) mobilization and COX-2 in human microglia.

We have studied modulation of purinergic receptors (P(2Y) and P(2X) subtypes) on changes in intracellular Ca(2+) [Ca(2+)](i) and expression and production of COX-2 in human microglia. Measurements using Ca(2+)-sensitive spectrofluorometry showed adenosine triphosphate (ATP) to cause rapid transient increases in [Ca(2+)](i). Application of ATP plus the P(2X) antagonist, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), or treatment with adenosine diphosphate-beta-S (ADP-beta-S), a selective P(2Y) agonist, led to a considerable prolongation in [Ca(2+)](i) responses compared with ATP. The prolonged time courses were consistent with sustained activation of store-operated channels (SOC) since SKF96365, an inhibitor of SOC, blocked this component of the response. RT-PCR data showed that microglia expressed no COX-2 either constitutively or following treatment of cells with ATP (100 microM for 8 h). However, treatment using ATP plus PPADS or with ADP-beta-S led to marked expression of COX-2. The enhanced COX-2 with ATP plus PPADS treatment was absent in the presence of SKF96365 or using Ca(2+)-free solution. Immunocytochemistry, using a specific anti-COX-2 antibody, also revealed a pattern of purinergic modulation whereby lack of P(2X) activation enhanced the production of COX-2 protein. These results suggest that modulation of subtypes of purinergic receptors regulates COX-2 in human microglia with a link involving SOC-mediated influx of Ca(2+).

Adenosine Diphosphate↗
PubMed2003

High-yield isolation of murine microglia by mild trypsinization.

Microglia can be isolated with high purity but low yield by shaking off loosely adherent cells from mixed glial cultures. Here we describe a new technique for isolating microglia with an average yield close to 2,000,000 microglial cells/mouse pup, more than five times higher than that of the shaking method. Confluent mixed glial cultures are subjected to mild trypsinization (0.05-0.12%) in the presence of 0.2-0.5 mM EDTA and 0.5-0.8 mM Ca2+. This results in the detachment of an intact layer of cells containing virtually all the astrocytes, leaving undisturbed a population of firmly attached cells identified as >98% microglia. These almost pure microglial preparations can be kept in culture for weeks and show proliferation and phagocytosis. Treatment with macrophage colony-stimulating factor and lipopolysaccharide, alone or in the presence of interferon gamma, induces typical microglial responses in terms of proliferation, morphological changes, nuclear factor-kappaB translocation, NO, and tumor necrosis alpha release and phagocytosis. This method allows for the preparation of highly enriched mouse or rat microglial cultures with ease and reproducibility. Because of its high yield, it can be especially convenient when high amounts of microglial protein/mRNA are required or in cases in which the starting material is limited, such as microglial cultures from transgenic animals.

Animals↗
PubMed2003

Selective expression of Gi/o-coupled ATP receptor P2Y12 in microglia in rat brain.

Extracellular nucleotides, including ATP, have been demonstrated to transmit important physiological signals in the brain through either G-protein-coupled P2Y receptors or P2X receptors, which are ligand-gated ion channels. In this study, we performed a detailed analysis of the expression of the Gi/o-coupled receptor P2Y12 in the brain. Northern blot analysis demonstrated that P2Y12 is expressed predominantly in the brain, and to a lesser extent in the spleen. The cellular localization of P2Y12 was investigated by in situ hybridization, and P2Y12 mRNA was detected in small cells distributed throughout the brain, including the hippocampus. Expression of P2Y12 was also observed in naive and axotomized facial nuclei, and the number of P2Y12-expressing cells increased following facial nerve axotomy. Selective expression of P2Y12 mRNA in microglia was confirmed by double-label in situ hybridization and immunohistochemistry with antibodies against NeuN and Iba1 as an immunohistochemical marker for neurons and microglia, respectively. Hardly any P2Y12 mRNA was detected in macrophages obtained from the spleen and abdominal cavity, which share many surface molecules with microglia.

Adenosine Triphosphate↗
PubMed2004

Microglia, a potential source of neurons, astrocytes, and oligodendrocytes.

Microglia are considered the only cell population of mesodermal origin in the brain, although their role is not fully understood. The present study demonstrated that rat primary microglial cells expressed nestin, A2B5, and O4 antigens, which are markers for oligodendrocyte precursor cells. Based on these findings, we investigated whether microglial cells generated neurons or macroglial cells. Purified microglial cells were cultured in the presence of 10% fetal bovine serum for 3 days, followed by culture in the presence of 70% serum for 2 days. During the two-step culture, microglial cells became highly proliferative and strongly expressed inhibitor of DNA binding (Id) genes, indicative of dedifferentiation of the cells. The dedifferentiated cells also expressed transcription factors that promote differentiation into neurons or macroglial cells. When the dedifferentiated cells were transferred into serum-free medium on poly-L-lysine-coated substrate, a substantial number of the cells rapidly turned into long process-bearing cells, which expressed microtubule-associated protein 2, synapsin I, neurofilament proteins, glial fibrillary acidic protein, or galactocerebroside. When microglial cells were fluorescently labeled through acetylated low-density lipoprotein (LDL) receptors or by a phagocytosis-dependent mechanism, fluorescence-bearing neurons, astrocytes, or oligodendrocytes were observed. Neurospheres, aggregates of neural stem cells, expressed Musashi 1 and epidermal growth factor receptor, but the microglia-derived cells did not. These results suggest a novel role of microglia as multipotential stem cells to give rise to neurons, astrocytes, or oligodendrocytes.

Animals↗
PubMed2004

Activation of p38 mitogen-activated protein kinase in spinal hyperactive microglia contributes to pain hypersensitivity following peripheral nerve injury.

Neuropathic pain is an expression of pathological operation of the nervous system, which commonly results from nerve injury and is characterized by pain hypersensitivity to innocuous stimuli, a phenomenon known as tactile allodynia. The mechanisms by which nerve injury creates tactile allodynia have remained largely unknown. We report that the development of tactile allodynia following nerve injury requires activation of p38 mitogen-activated protein kinase (p38MAPK), a member of the MAPK family, in spinal microglia. We found that immunofluorescence and protein levels of the dually phosphorylated active form of p38MAPK (phospho-p38MAPK) were increased in the dorsal horn ipsilateral to spinal nerve injury. Interestingly, the phospho-p38MAPK immunofluorescence in the dorsal horn was found exclusively in microglia, but not in neurons or astrocytes. The level of phospho-p38MAPK immunofluorescence in individual microglial cells was much higher in the hyperactive phenotype in the ipsilateral dorsal horn than the resting one in the contralateral side. Intrathecal administration of the p38MAPK inhibitor, 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580), suppresses development of the nerve injury-induced tactile allodynia. Taken together, our results demonstrate that nerve injury-induced pain hypersensitivity depends on activation of the p38MAPK signaling pathway in hyperactive microglia in the dorsal horn following peripheral nerve injury.

Animals↗
PubMed2004

Heterogeneity and potentiation of AMPA type of glutamate receptors in rat cultured microglia.

alpha-amino-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptor in rat cultured microglia were analyzed precisely using flop- and flip-preferring allosteric modulators of AMPA receptors, 4-[2-(phenylsulfonylamino)ethylthio]-2,6-difluoro-phenoxyacetamide (PEPA) and cyclothiazide (CTZ), respectively. Glutamate (Glu)- or kainite (KA)-induced currents were completely inhibited by a specific blocker of AMPA receptor, LY300164, indicating that functional Glu-receptors in cultured microglia are mostly AMPA receptor but not KA receptor in many cells. Glu- and KA-induced currents were potentiated by PEPA and CTZ in a concentration-dependent manner. The ratio of the potentiation by PEPA to the potentiation by cyclothiazide varied with cells between 0.1 and 0.9, suggesting cell-to-cell heterogeneity of AMPA receptor subunits expressed in microglia. Quantitative RT-PCR revealed that GluR1-3 mainly occurred in the flip forms, which agreed with the stronger potentiation of receptor currents by CTZ vs. PEPA. Finally, the potentiation of microglial AMPA receptors by PEPA and CTZ inhibited the Glu-induced release of tumor necrosis factor-alpha (TNF-alpha) unpredictably. The increase in TNF-alpha release by Glu or KA required extracellular Na+ and Ca2+ ions but not mitogen-activated protein kinase (MAPK), suggesting the effects of PEPA and CTZ were not due to the inhibition of MAPK. These results suggest that potentiation of microglial AMPA receptors serves as a negative feedback mechanism for the regulation of TNF-alpha release and may contribute to the ameliorating effects of allosteric modulators of AMPA receptors.

Allosteric Regulation↗
PubMed2005

c-Jun N-terminal kinases (JNKs) mediate pro-inflammatory actions of microglia.

The activation and function of c-Jun N-terminal kinases (JNKs) were investigated in primary microglia cultures from neonatal rat brain, which express all three JNK isoforms. Lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-alpha), and thrombin preparations induced a rapid and lasting activation of JNKs in the cytoplasm. In the nucleus, the activation patterns were rather complex. In untreated microglia, the small pool of nuclear JNKs was strongly activated, while the high-affinity JNK substrate c-Jun was only weakly phosphorylated. Stimulation with LPS increased the total amount of nuclear JNKs and the phosphorylation of the transcription factor c-Jun. Levels of activated JNKs in the nucleus, however, rapidly decreased. Analysis of the nuclear JNK isoforms revealed that the amount of JNK1 declined, while JNK2 increased, and the weakly expressed JNK3 did not vary. This observation suggests that JNK2 is mainly responsible for the activation of c-Jun in this context. Upstream of JNKs, LPS induced a lasting activation of the constitutively present JNK kinase MKK4. The function of JNKs in LPS-triggered cellular reactions was investigated using SP600125 (0.5-5 microM), a direct inhibitor of JNKs. Inhibition of JNKs reduced the LPS-induced metabolic activity and induction of the AP-1 target genes cyclooxygenase-2 (Cox-2), TNF-alpha, monocyte chemoattractant protein-1 (MCP-1), and interleukin-6 (IL-6) in response to LPS, while ERK1/2 and p38 alpha had a more pronounced effect on LPS-induced cellular enlargement than JNKs. In summary, JNKs are essential mediators of relevant pro-inflammatory functions in microglia with different contributions of the JNK isoforms.

Animals↗
PubMed2005

Proteomic fingerprints distinguish microglia, bone marrow, and spleen macrophage populations.

Mononuclear phagocytes (MP; dendritic cells, monocytes, tissue macrophages, and microglia) maintain tissue homeostasis and provide a first line of defense against invading pathogens. In specific circumstances, MPs also induce inflammatory responses and as such affect disease onset and progression. Despite intensive research into MP biology, little is known of the functional and molecular properties of individual MP subtypes. Using a novel proteomics platform, unique protein patterns and protein identities were observed among populations of spleen and bone marrow macrophages and microglia. Cells were obtained from C57BL/6 mice and were cultivated in macrophage colony-stimulating factor. MP subtypes were indistinguishable by morphological or antigenic criteria. Protein profiling by Surface Enhanced Laser Desorption Ionization-Time of Flight (SELDI-TOF) ProteinChip assays with weak cationic exchange chips showed unique MP spectral profiles. Corresponding protein fractions were recovered by high performance liquid chromatography and identified by liquid chromatography tandem mass spectrometry. The results provide a unique means to distinguish microglia from other MP subtypes.

Animals↗
PubMed2006

Lipopolysaccharide sensitizes microglia toward Ca(2+)-induced cell death: mode of cell death shifts from apoptosis to necrosis.

Little is known about the effect of microglial activation on cell death. This study examines the effects of lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma), triggers of microglial activation, on cell death induced by several agents in cultured rat microglia. For comparison, the effect of LPS on cell death is also examined in cultured astrocytes. LPS or IFN-gamma enhanced cell death induced by thapsigargin or ionomycin, an agent that increases intracellular Ca2+ concentration, although LPS or IFN-gamma alone did not affect cell viability. Thapsigargin or ionomycin induced apoptosis in LPS-untreated microglia, while they induced necrosis in LPS-treated microglia, which were partially reversed by O,O'-bis(2-aminophenyl)ethyleneglycol-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM, an intracellular Ca2+ chelator). In contrast, LPS treatment did not affect tunicamycin- or staurosporine-induced apoptosis, while it inhibited S-nitroso-N-acetylpenicillamine-induced apoptosis. The effect of LPS on thapsigargin or ionomycin-induced apoptosis was not observed in astrocytes. These results indicate that microglial activation sensitizes the cells toward cell death induced by the change in intracellular Ca2+ concentration and shifts the mode of cell death from apoptosis to necrosis.

Animals↗
PubMed2006

Mouse model for ablation of proliferating microglia in acute CNS injuries.

Activation of microglia, the primary immune effectors of the CNS and proinflammatory signaling, is a hallmark of brain damage. However, it remains controversial whether microglial cells have beneficial or detrimental functions in various neuropathological conditions. We report the generation of transgenic mice that express a mutant form of herpes simplex virus type 1 thymidine kinase (HSV-1 TK(mt-30)) driven by the myeloid-specific CD11b promoter. Using two paradigms of nervous system damage, hypoglossal nerve axotomy, and cortical stab injury, we show that specific ablation of proliferating microglia in CD11b-TK(mt-30) mice can be achieved by administration of ganciclovir. For example, after hypoglossal nerve injury, a 75% reduction in proliferating microglial cells was observed at the site of injury. The CD11b-TK(mt-30) transgenic mouse should provide a valuable tool for studying the role of microglia in CNS damage and repair.

Animals↗
PubMed2006

Minocycline affects microglia activation, Abeta deposition, and behavior in APP-tg mice.

Activated microglia and reactive astrocytes invade and surround cerebral beta amyloid (Abeta) plaques in Alzheimer's disease (AD), but the role of microglia in plaque development is still unclear. In this study, minocycline was administered for 3 months, prior to and early in Abeta plaque formation in amyloid precursor protein transgenic mice (APP-tg). When minocycline was given to younger mice, there was a small but significant increase in Abeta deposition in the hippocampus, concurrent with improved cognitive performance relative to vehicle treated mice. If APP-tg mice received minocycline after Abeta deposition had begun, microglial activation was suppressed but this did not affect Abeta deposition or improve cognitive performance. In vitro studies demonstrated that minocycline suppressed microglial production of IL-1beta, IL-6, TNF, and NGF. Thus, minocycline has different effects on Abeta plaque deposition and microglia activation depending on the age of administration. Our data suggest that this may be due to the effects of minocycline on microglial function. Therefore, anti-inflammatory therapies to suppress microglial activation or function may reduce cytokine production but enhance Abeta plaque formation early in AD.

Age Factors↗
PubMed1994

Sodium channel in isolated human brain macrophages (microglia).

Human brain macrophages (microglia) have been isolated from mixed brain cell cultures initiated from explants of neurosurgical adult human tissue in one step according to a method developed for rat microglia. Cells were characterized enzyme-histochemically (NDPase) in mixed and immunocytochemically (anti-CD 14) in mixed and isolated cultures. Purified cells were used to investigate in more detail membrane currents by the patch clamp technique. In 14 cells microdialyzed with a standard, K(+)-containing intracellular solution there was no indication for a hyperpolarization-induced K(+)-inward current characteristic for newborn rat microglia. However, in 12 cells depolarizing pulses initiated a rapidly inactivating inward current which was followed by an outward current (in 4 cells). The outward current appeared to be carried by K+, since it was absent in another 18 cells, recorded by micropipettes containing Cs+ instead of K+ as the main intracellular cation. The depolarization-induced inward current persisted under these conditions. This current was inhibited by tetrodotoxin (5 microM) and by substitution of Na+ by choline in the bath solution. It is suggested that this Na(+)-current is specifically expressed in macrophages derived from adult brain.

Acid Anhydride Hydrolases↗
PubMed1994

Transitory disappearance of microglia during the regeneration of the lizard medial cortex.

In normal lizards, microglial cells populate the medial cortex (a zone homologous to the hippocampal fascia dentata), with a preferential distribution along the border between the granular cell layer and the plexiform layers. Intraperitoneal injection of the neurotoxin 3-acetylpyridine (3AP) induces a selective lesion in the medial cortex with a rapid degeneration of the granular layer and its zinc-enriched axonal projection. Within 6-8 weeks, the granular layer is, however, repopulated by a new set of neurons generated in the subjacent ependyma and the cell debris is removed. The aim of this study was to determine to what extent microglia were involved in the scavenging processes during the regeneration process. To this end we studied the brains of regenerating lizards at different times after 3AP lesion, visualising microglial cells by the nucleoside diphosphatase (NDPase) histochemical reaction. Surprisingly, we found that stained microglial cells disappeared 6-8 hours after 3AP injection and remained absent until 10-15 days after injection. One month postlesion an increased population of microglial cells was found scattered throughout all plexiform layers of the cortex. Thorough examination of semithin and ultrathin sections confirmed the absence of microglia in the medial cortex of recent lesioned animals but the presence of an exuberant population after 1 month postlesion. In the tissue, phagocytotic scavenging was carried out by radial ependymocytes, not by microglia.

Acid Anhydride Hydrolases↗
PubMed1995

Change of phosphotyrosine immunoreactivity on microglia in the rat substantia nigra following striatal ischemic injury.

Using immunohistochemistry, we investigated changes in phosphotyrosine (P-Tyr) immunoreactivity on the microglia of the rat substantia nigra (SN) following striatal ischemic injury produced by transient middle cerebral artery (MCA) occlusion. Anterograde axonal degeneration in the SN due to striatal ischemic injury was detected by depletion of calcineurin immunoreactivity in that region from 1 day after operation. From 3 days to 1 month (the longest period examined in this study) after MCA occlusion, there was a significant increase in P-Tyr immunoreactivity in the SN ipsilateral to the MCA occlusion. Also, light microscopic observation showed that the microglia exhibited an increased immunoreactivity for P-Tyr and characteristic morphological changes in the ipsilateral SN. The present results indicate that a signal transducing cascade(s) associated with tyrosine phosphorylation may be involved in the activation of the microglia in the SN responding to anterograde degeneration of the striatonigral pathway.

Animals↗
PubMed2003

Downregulation of inducible nitric oxide synthetase by neurotrophin-3 in microglia.

Microglia activated after many neurological degeneration of the central nervous system (CNS) act as important regulators for neuropathogenesis in the injured CNS via producing proinflammatory mediators, such as nitric oxide (NO), TNF-alpha, and IL-1beta. Neurotrophin-3 (NT-3) is a well-known trophic factor for neural survival, development, and plasticity. Activated microglia are NT-3-producing cells in the injured CNS, and express its receptor-TrkC. However, little is known about the effect of NT-3 on activated microglia. In this study, pre-treatment of a mouse microglial cell line, BV2, with NT-3 for 24 h indicated that NT-3 reduced the inducible form of NO synthase (iNOS), NO, and TNF-alpha in BV2 stimulated with lipopolysaccharide (LPS). NT-3 exerted less effect on the reduction of these proinflammatory mediators when it was added to BV2 cultures either simultaneously with LPS or post LPS treatment. These findings indicate that NT-3 may serve as an anti-inflammatory factor to suppress microglial activation.

Animals↗