Search

Hyponatremia is the most common clinical electrolyte disorder. Chronic hyponatremia has been recently reported to be associated with falls, fracture, osteoporosis, neurocognitive impairment, and mental manifestations. In the treatment of chronic hyponatremia, overly rapid correction of hyponatremia can cause osmotic demyelination syndrome (ODS), a central demyelinating disease that is also associated with neurological morbidity and mortality. Using a rat model, we have previously shown that microglia play a critical role in the pathogenesis of ODS. However, the direct effect of rapid correction of hyponatremia on microglia is unknown. Furthermore, the effect of chronic hyponatremia on microglia remains elusive. Using microglial cell lines BV-2 and 6-3, we show here that low extracellular sodium concentrations (36 mmol/L decrease; LS) suppress Nos2 mRNA expression and nitric oxide (NO) production of microglia. On rapid correction of low sodium concentrations, NO production was significantly increased in both cells, suggesting that acute correction of hyponatremia partly directly contributes to increased Nos2 mRNA expression and NO release in ODS pathophysiology. LS also suppressed expression and nuclear translocation of nuclear factor of activated T cells-5 (NFAT5), a transcription factor that regulates the expression of genes involved in osmotic stress. Furthermore, overexpression of NFAT5 significantly increased Nos2 mRNA expression and NO production in BV-2 cells. Expressions of Nos2 and Nfat5 mRNA were also modulated in microglia isolated from cerebral cortex in chronic hyponatremia model mice. These data indicate that LS modulates microglial NO production dependent on NFAT5 and suggest that microglia contribute to hyponatremia-induced neuronal dysfunctions., Competing Interests: Declaration of competing interest The authors have nothing to disclose., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

A substantial and constitutive expression of translocator protein (TSPO) in cerebral blood vessels hampers the sensitive detection of neuroinflammation characterized by greatly induced TSPO expression in activated glia. Here, we conducted in vivo positron emission tomography (PET) and in vitro autoradiographic imaging of normal and TSPO-deficient mouse brains to compare the binding properties of 18 F-FEBMP, a relatively novel TSPO radioligand developed for human studies based on its insensitivity to a common polymorphism, with 11 C-PK11195, as well as other commonly used TSPO radioligands including 11 C-PBR28, 11 C-Ac5216 and 18 F-FEDAA1106. TSPO in cerebral vessels of normal mice was found to provide a major binding site for 11 C-PK11195, 11 C-PBR28 and 18 F-FEDAA1106, in contrast to no overt specific binding of 18 F-FEBMP and 11 C-Ac5216 to this vascular component. In addition, 18 F-FEBMP yielded PET images of microglial TSPO with a higher contrast than 11 C-PK11195 in a tau transgenic mouse modeling Alzheimer's disease (AD) and allied neurodegenerative tauopathies. Moreover, TSPO expression examined by immunoblotting was significantly increased in AD brains compared with healthy controls, and was well correlated with the autoradiographic binding of 18 F-FEBMP but not 11 C-PK11195. Our findings support the potential advantage of comparatively glial TSPO-selective radioligands such as 18 F-FEBMP for PET imaging of inflammatory glial cells.

To differentiate subtypes of microglia (MG), we developed a novel monoclonal antibody, 9F5, against one subtype (type 1) of rat primary MG. The 9F5 showed high selectivity for this cell type in Western blot and immunocytochemical analyses and no cross-reaction with rat peritoneal macrophages (Mφ). We identified the antigen molecule for 9F5: the 50- to 70-kDa fragments of rat glycoprotein nonmetastatic melanoma protein B (GPNMB)/osteoactivin, which started at Lys(170) . In addition, 9F5 immunoreactivity with GPNMB depended on the activity of furin-like protease(s). More important, rat type 1 MG expressed the GPNMB fragments, but type 2 MG and Mφ did not, although all these cells expressed mRNA and the full-length protein for GPNMB. These results suggest that 9F5 reactivity with MG depends greatly on cleavage of GPNMB and that type 1 MG, in contrast to type 2 MG and Mφ, may have furin-like protease(s) for GPNMB cleavage. In neonatal rat brain, amoeboid 9F5+ MG were observed in specific brain areas including forebrain subventricular zone, corpus callosum, and retina. Double-immunοstaining with 9F5 antibody and anti-Iba1 antibody, which reacts with MG throughout the CNS, revealed that 9F5+ MG were a portion of Iba1+ MG, suggesting that MG subtype(s) exist in vivo. We propose that 9F5 is a useful tool to discriminate between rat type 1 MG and other subtypes of MG/Mφ and to reveal the role of the GPNMB fragments during developing brain. GLIA 2016;64:1938-1961., (© 2016 The Authors. Glia Published by Wiley Periodicals, Inc.)

As acidic glycocalyx on primary mouse microglial cells and a mouse microglial cell line Ra2, expression of polysialic acid (polySia/PSA), a polymer of the sialic acid Neu5Ac (N-acetylneuraminic acid), was demonstrated. PolySia is known to modulate cell adhesion, migration, and localization of neurotrophins mainly on neural cells. PolySia on Ra2 cells disappeared very rapidly after an inflammatory stimulus. Results of knockdown and inhibitor studies indicated that rapid surface clearance of polySia was achieved by secretion of endogenous sialidase Neu1 as an exovesicular component. Neu1-mediated polySia turnover was accompanied by the release of brain-derived neurotrophic factor normally retained by polySia molecules. Introduction of a single oxygen atom change into polySia by exogenous feeding of the non-neural sialic acid Neu5Gc (N-glycolylneuraminic acid) caused resistance to Neu1-induced polySia turnover and also inhibited the associated release of brain-derived neurotrophic factor. These results indicate the importance of rapid turnover of the polySia glycocalyx by exovesicular sialidases in neurotrophin regulation., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Lipid resuscitation has become a standard treatment for local anesthetic (LA) systemic toxicity, but its mechanisms remain to be fully elucidated. Although the partitioning effect is one of the proposed mechanisms, it is difficult to evaluate its impact independently from several other mechanisms or to examine the intracellular concentration of a LA, which is primarily responsible for LA systemic toxicity. We recently reported that LAs as weak bases reduced voltage-gated proton currents by increasing intracellular pH, which could be estimated from the reversal potentials of the channels (Vrev). Using this characteristic, we examined the partitioning effect in detail and showed its impact on lipid resuscitation., Methods: A whole-cell voltage clamp technique was used to record proton channel currents in a rat microglial cell line (GMI-R1). We used Intralipid® 20% as lipid emulsion. The effects of lipid emulsion on the intracellular concentrations of LAs were evaluated by measuring the current amplitude and the Vrev. The intracellular concentrations of LAs were calculated by the Henderson-Hasselbalch equation, using estimated intracellular pH. To confirm the importance of partitioning, we separated lipid by centrifugation. Data are means ± SD unless otherwise stated., Results: Bupivacaine (1 mM) decreased proton currents to 43% ± 10% of the control and shifted the Vrev to positive voltages (from -88.0 ± 4.1 to -76.0 ± 5.5 mV, n = 5 each, P = 0.02). An addition of the lipid emulsion recovered the currents to 79% ± 2% of the control and returned the Vrev toward the control value (to -86.0 ± 7.1 mV, n = 5, P = 0.03). Both recoveries of the current and Vrev in the centrifuged aqueous extract were almost the same as in the 4% lipid solution (-85.6 ± 4.9 mV, n = 5, P = 0.9, 95% confidence interval for difference = -9.3 to 8.6). When 1 mM bupivacaine was applied extracellularly, the intracellular concentration of the charged form of bupivacaine was estimated to reach about 18.1 ± 3.9 mM but decreased to 5.4 ± 1.8 mM by the 4% lipid solution., Conclusions: Here we quantitatively evaluated for the first time the partitioning effect of lipid emulsion therapy on the intracellular concentration of bupivacaine in real-time settings by analyzing behaviors of voltage-gated proton channels. Our results suggested that lipid emulsion markedly reduced the intracellular concentration of bupivacaine, which was mostly due to the partitioning effect. This could contribute to our understanding of the mechanisms underlying lipid resuscitation, especially the importance of the partitioning effect.

Voltage-gated proton channels (H(+) channels) are highly proton-selective transmembrane pathways. Although the primary determinants for activation are the pH and voltage gradients across the membrane, the current amplitudes fluctuate often when these gradients are constant. The aim of this study was to investigate the role of the intracellular pH (pHi) in regulating the availability of H(+) channels in osteoclasts and microglia. In whole-cell clamp recordings, the pHi was elevated after exposure to NH4Cl and returned to the control level after washout. However, the H(+) channel conductance did not recover fully when the exposure was prolonged (>5 min). Similar results were observed in osteoclasts and microglia, but not in COS7 cells expressing a murine H(+) channel gene (mVSOP). As other electrophysiological properties, like the gating kinetics and voltage dependence for activation, were unchanged, the decreases in the H(+) channel conductance were probably due to the decreases in H(+) channels available at the plasma membrane. The decreases in the H(+) channel conductances were accompanied by reductions in the cell capacitance. Exposure to NH4Cl increased the uptake of the endocytosis marker FM1-43, substantiating the idea that pHi increases facilitated endocytosis. In osteoclasts, whose plasma membrane expresses V-ATPases and H(+) channels, pHi increases by these H(+)-transferring molecules in part facilitated endocytosis. The endocytosis and decreases in the H(+) channel conductance were reduced by dynasore, a dynamin blocker. These results suggest that pHi increases in osteoclasts and microglia decrease the numbers of H(+) channels available at the plasma membrane through facilitation of dynamin-dependent endocytosis.

Microglia, macrophage-like cells in the CNS, are multi-functional cells; they play an important role in removal of dead cells or their remnants by phagocytosis in the CNS degeneration as well as are one of important cells in the CNS cytokine network. They are thought to be originated from mesoderm, and to be similar cells to other tissue-resident macrophages. As macrophages, activated microglia have been shown to remove potentially deleterious debris and promote tissue repair by secreting neurotrophic factors at the neuronal injury sites, however, they can release potentially cytotoxic substances in vitro, and at least so-called fully activated form of microglia which are observed at the injury site in AIDS dementia is neurotoxic. These suggest that some factor(s) may contribute to change microglial phenotype from protective to toxic, but the detail is not clear. Recently we generated channelrhodopsin-mutant protein expressing microglia, Ra2_GR and 6-3_GR. Channelrhodopsin is an ion channel activated by light irradiation. Intracellular sodium ion increased by light irradiation in both Ra2_GR and 6-3_GR accompanied by increase of mRNA expression such as pro-inflammatory cytokines, chemokines and iNOS. This technique can control microglial activation, therefore, it may provide a new strategy for repair/regeneration of neural and oligodendrocytic damages.

The mammalian tachykinins, substance P (SP) and hemokinin-1 (HK-1), are widely distributed throughout the nervous system and/or peripheral organs, and function as neurotransmitters or chemical modulators by activating their cognate receptor NK(1). The TAC1 gene encoding SP is highly expressed in the nervous system, while the TAC4 gene encoding HK-1 is uniformly expressed throughout the body, including a variety of peripheral immune cells. Since TAC4 mRNA is also expressed in microglia, the resident immune cells in the central nervous system, HK-1 may be involved in the inflammatory processes mediated by these cells. In the present study, we found that TAC4, rather than TAC1, was the predominant tachykinin gene expressed in primary cultured microglia. TAC4 mRNA expression was upregulated in the microglia upon their activation by lipopolysaccharide, a well-characterized Toll-like receptor 4 agonist, while TAC1 mRNA expression was downregulated. Furthermore, both nuclear factor-κB and p38 mitogen-activated protein kinase intracellular signaling pathways were required for the upregulation of TAC4 mRNA expression, but not for the downregulation of TAC1 mRNA expression. These findings suggest that HK-1, rather than SP, plays dominant roles in the pathological conditions associated with microglial activation, such as neurodegenerative and neuroinflammatory disorders.

Purpose: To evaluate the utility of new Translocator protein 18 kDa (TSPO)-targeted fluorescent probes for in vivo molecular imaging of activated microglia., Methods: Compounds 2-4 were synthesized; their stability and affinity for TSPO were determined. Compounds 2-4 were incubated both with Ra2 cells in the presence of LPS, a potent activator of microglia, and with tissue sections of normal and chemically injured brains. Compounds 2-4 were injected into carotid artery or directly in striatum of mice. Cells and tissue sections from these in vitro and in vivo studies were observed by fluorescence microscopy after histochemical treatments., Results: Compounds 2-4 are stable in both buffer and physiological medium and showed high affinity for TSPO and were found to stain live Ra2 microglial cells effectively. Double staining with Mito Tracker Red suggested that binding sites of compounds 2 and 3 may exist on mitochondria. In vivo studies showed that compounds 2-4 may penetrate in part into brain; moreover, cells in mouse striatum were stained with compounds 2-4 and microglial marker CD11b., Conclusion: Compounds 2-4 can fluorescently label activated microglia in vitro and in vivo.

We first verified that a single chain Fv fragment against prion protein (anti-PrP scFv) was secreted by HEK293T cells and prevented prion replication in infected cells. We then stably expressed anti-PrP scFv in brain-engraftable murine microglial cells and intracerebrally injected these cells into mice before or after infection with prions. Interestingly, the injection before or at an early time point after infection attenuated the infection marginally but significantly prolonged survival times of the mice. These suggest that the ex vivo gene transfer of anti-PrP scFvs using brain-engraftable cells could be a possible immunotherapeutic approach against prion diseases.

Several studies have suggested the involvement of neuroinflammation in the pathomechanism of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). We recently demonstrated increased levels of protein-bound 4-hydroxy-2-nonenal (HNE) as a highly reactive lipid peroxidation product and cytosolic phospholipase A(2) (cPLA(2)) as a proinflammatory enzyme in glial cells as well as motor neurons in the spinal cord of sporadic ALS patients. However, a link between HNE and cPLA(2) in ALS remains to be determined. To address this issue, we investigated effects of HNE stimulation on the state of cPLA(2) expression in cultured microglial cell line (Ra2). Exposure of Ra2 cells to HNE significantly increased expression levels of cPLA(2) and its activated form phosphorylated at amino acid residue S(505) (p-cPLA(2)) on immunoblots. Pretreatment of Ra2 cells with the antioxidant N-acetylcysteine, the extracellular signal-regulated kinase (ERK) inhibitor PD98059 or the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 prevented the HNE-induced increased expression of cPLA(2) and p-cPLA(2). Immunocytochemical analysis revealed that staining for p-cPLA(2) in Ra2 cells was localized in the cytoplasm and more intense in the HNE-stimulated group than in the vehicle group. The present results provide in vitro evidence that HNE upregulates and phosphorylates cPLA(2) in microglia via the ERK and p38 MAPK pathways., (© 2010 Japanese Society of Neuropathology.)

Osmotic demyelination syndrome (ODS) is a serious demyelinating disease in the central nervous system usually caused by rapid correction of hyponatremia. In an animal model of ODS, we previously reported microglial accumulation expressing proinflammatory cytokines. Microglia and astrocytes secreting proinflammatory cytokines and neurotrophic factors are reported to be involved in the pathogenesis of demyelinative diseases. Therefore, to clarify the role of microglial and astrocytic function in ODS, we examined the time-dependent changes in distribution, morphology, proliferation, and mRNA/protein expression of proinflammatory cytokines, neurotrophic factors, and matrix metalloproteinase (MMP) in microglia and astrocytes 2 days (early phase) and 5 days (late phase) after the rapid correction of hyponatremia in ODS rats. The number of microglia time dependently increased at demyelinative lesion sites, proliferated, and expressed tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, inducible nitric oxide synthase, and MMP2, 9, and 12 at the early phase. Microglia also expressed leukemia inhibitory factor (a neurotrophic factor) and phagocytosed myelin debris at the late phase. The number of astrocytes time dependently increased around demyelinative lesions, extended processes to lesions, proliferated, and expressed nerve growth factor and glial cell line-derived neurotrophic factor at the late phase. Moreover, treatment with infliximab, a monoclonal antibody against TNF-α, significantly attenuated neurological impairments. Our results suggest that the role of microglia in ODS is time dependently shifted from detrimental to protective and that astrocytes play a protective role at the late phase. Modulation of excessive proinflammatory responses in microglia during the early phase after rapid correction may represent a therapeutic target for ODS., (Copyright © 2010 Wiley-Liss, Inc.)

Rapid correction of chronic hyponatremia can lead to osmotic demyelination syndrome (ODS), a severe demyelination disease. The microglia that accumulate in the demyelinative lesions may play a detrimental role in the pathogenesis of ODS by producing proinflammatory cytokines, suggesting that they may be a target for therapeutic intervention. Here, we investigated whether minocycline, a selective and potent inhibitor of microglial activation, could protect against ODS in rats. We induced hyponatremia by liquid diet feeding and dDAVP infusion. Rapid correction of the hyponatremia 7 days later resulted in neurologic impairment with severe demyelinative lesions. Activated microglia accumulated at the site of demyelination. Treatment with minocycline within 24 hours of rapid correction, however, was protective: rats exhibited minimal neurologic impairment, and survival improved. Histologic analysis showed that minocycline inhibited demyelination and suppressed the accumulation of microglia at the site of demyelination. Real-time RT-PCR and immunohistochemical analyses showed that minocycline inhibited the activity of microglia and the expression of inflammatory cytokines (e.g. IL-1β, inducible nitric-oxide synthase, and TNF-α), monocyte chemoattractant protein-1, and matrix metalloproteinase-12 in microglia. These results demonstrate that minocycline can protect against ODS by inhibiting the activation and accumulation of microglia at the site of demyelinative lesions, suggesting its possible use in clinical practice.

Most CNS diseases begin with inflammation with subsequent neural damage eventually occurring; however, the process leading from the onset of inflammation to neural damage remains obscure. We used an artificial brain injury mouse model and examined how neural damage occurred in the brain parenchyma. The damaged area in each mouse was clearly observed by magnetic resonance imaging (MRI), and the progression of damage was observed to occur in a biphasic manner (acute damage, within 1 week; delayed damage, after 2 weeks). We found that the delayed neural damage was absent in iNOS-deficient mice (iNOS-KO mice). Then, we analyzed brain tissues and determined that delayed neural damage was accompanied by an increase in the levels of NO end products and iNOS expression, with accumulation of iNOS-expressing microglia around the injured area. In addition, the expression of IL-1beta mRNA was increased in areas affected by acute damage, but not in those affected by delayed damage. These findings suggest that delayed neural damage might arise from NO production by iNOS-expressing activated microglia and that such activated microglia might become a therapeutic target for many CNS diseases.

Objective: The transition of microglia from the normal resting state to the activated state is associated with an increased expression of peripheral benzodiazepine receptors (PBR). The extent of PBR expression is dependent on the level of microglial activation. A PBR ligand, [(11)C]PK11195, has been used for imaging of the activation of microglia in vivo. We evaluated whether [(11)C]PK11195 PET can indicate differences of microglial activation between no treatment and lipopolysaccharide (LPS) treatment in a rat artificial injury model of brain inflammation., Methods: On day 1, a small aliquot of absolute ethanol was injected into the rat right striatum (ST) to produce artificial brain injury. On day 3, MRI scans were performed to evaluate and select rats showing a similar degree of brain injury. Then LPS or vehicle was administered intraperitoneally. On day 4, PET scans were performed after a bolus injection of [(11)C]PK11195. Eleven rats (7 LPS administered rats, 4 LPS non-administered rats) were evaluated. We used uptake ratios of the integral of right and left striatum from 0 to 60 min as an estimate of PBR distribution volume (V (60)). The number of activated microglia and mRNA expression of inflammatory cytokines (TNFalpha, IL-1beta) were assessed by isolectin-B4 staining and RT-PCR, respectively., Results: Right/left ST V (60) ratios of LPS group were significantly higher than those of non-LPS group (P < 0.03). Although there were no significant differences in the number of activated microglia between the two groups, LPS group showed higher expression of inflammatory cytokines (TNFalpha, IL-1beta) than the non-treated group indicating that further activation was induced by LPS treatment., Conclusion: The results suggest that intensity of PBR signals in [(11)C]PK11195 PET may be related to the level of microglial activation rather than the number in activated microglia at least in an artificial brain injury model.

Activated microglia play important roles in the inflammatory process and progression in Parkinson's disease. These cells produce various cytokines, nitric oxide, and neurotrophins, which are pleiotropic in their action, i.e., neuroprotective or neurotoxic. In an in vivo study on a mouse model with nigrostriatal lesions produced by the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), microglia activated by systemic lipopolysaccharide (LPS) were neurotoxic toward dopamine neurons in aged mice, but unexpectedly, neuroprotective in neonatal mice. In contrast to microglia in the MPTP model, LPS-activated microglia in neonatal mice in a model made by the stereotaxic injection of ethanol into the striatum were neurotoxic, and systemic LPS administration in the ethanol-injury model caused marked increases both in the volume of necrotic lesions and in the number of degenerative neurons in the striatum. Thus, activated microglia in the neonatal mouse brain play either a neuroprotective or neurotoxic role depending on the type of brain injury., (Copyright 2010 S. Karger AG, Basel.)

Voltage-gated proton channels are found in many different types of cells, where they facilitate proton movement through the membrane. The mechanism of proton permeation through the channel is an issue of long-term interest, but it remains an open question. To address this issue, we examined the temperature dependence of proton permeation. Under whole cell recordings, rapid temperature changes within a few milliseconds were imposed. This method allowed for the measurement of current amplitudes immediately before and after a temperature jump, from which the ratios of these currents (Iratio) were determined. The use of Iratio for evaluating the temperature dependence minimized the contributions of factors other than permeation. Temperature jumps of various degrees (DeltaT, -15 to 15 degrees C) were applied over a wide temperature range (4-49 degrees C), and the Q10s for the proton currents were evaluated from the Iratios. Q10 exhibited a high temperature dependence, varying from 2.2 at 10 degrees C to 1.3 at 40 degrees C. This implies that processes with different temperature dependencies underlie the observed Q10. A novel resistivity pulse method revealed that the access resistance with its low temperature dependence predominated in high temperature ranges. The measured temperature dependence of Q10 was decomposed into Q10 of the channel and of the access resistances. Finally, the Q10 for proton permeation through the voltage-gated proton channel itself was calculated and found to vary from 2.8 at 5 degrees C to 2.2 at 45 degrees C, as expected for an activation enthalpy of 64 kJ/mol. The thermodynamic features for proton permeation through proton-selective channels were discussed for the underlying mechanism.

There may be two subtypes of microglia (MG) at least in the CNS. We separated the two types from rat mixed glial culture. mRNAs and proteins for inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNFalpha) were more induced in CD40(+) MG than CD40(-) MG after LPS stimulation. Although the expression level of LPS receptors showed a little difference between the subtypes, LPS-induced degradation of phosphorylated IkappaBalpha was marked in CD40(+) MG. These results strongly suggest that CD40(+) MG produce larger amount of NO and TNFalpha to exhibit neurotoxic action under certain pathological conditions in brains.

After injury to the adult central nervous system, levels of extracellular matrix molecules increase at the injury site and may inhibit the repair of injured axons. Among these molecules, the importance of proteoglycans, particularly their chondroitin sulfate chains, has been highlighted. We have recently reported that keratan sulfate-deficient mice show better axonal regeneration after injury. Here, we investigated the regulation of keratan sulfate and chondroitin sulfate biosynthesis after neuronal injuries. Several key enzymes required for glycosaminoglycan biosynthesis (beta3GlcNAcT-7 and GlcNAc6ST-1 for keratan sulfate; CS synthase-1 and C6ST-1 for chondroitin sulfate) were expressed at significantly higher levels in the lesion 7 days after a knife-cut injury was made to the cerebral cortex in adult mice. These increases were accompanied by increased expression of TGF-beta(1) and bFGF. Since microglias at the injury sites expressed both keratan sulfate and chondroitin sulfate, the effects of these cytokines were examined in microglias. TGF-beta(1) induced the expression of the above-named enzymes in microglias, and consequently induced keratan sulfate and chondroitin sulfate biosynthesis as well as the expression of the chondroitin/keratan sulfate proteoglycan aggrecan in these cells. TGF-beta(1) also induced bFGF expression in microglias. bFGF in turn induced TGF-beta(1) expression in astrocytes. Astrocyte-conditioned medium following bFGF stimulation indeed induced keratan sulfate and chondroitin sulfate production in microglias. This production was blocked by TGF-beta(1)-neutralizing antibody. Taken together, our data indicate that the biosyntheses of keratan sulfate and chondroitin sulfate are upregulated in common by TGF-beta(1) in microglias after neuronal injuries.

Microglia are macrophage-like cells in the CNS. As macrophages, activated microglia remove potentially deleterious debris and promote tissue repair. However, they can release potentially cytotoxic substances in vitro. So-called fully activated microglia, observed at the injury site in many neurodegenerative conditions, are neurotoxic. This suggests that some factor(s) may contribute to change microglial phenotype from protective to toxic, but details are not clear. Recently, we generated HIV-derived Nef protein-transduced microglia. They increase the potential to produce O(-)(2) and MPO-like peroxidase activity, resulting in neurotoxicity. Therefore, the target protein(s) of Nef might be involved in the control of microglial neurotoxicity.

We demonstrate the significance of peripheral benzodiazepine receptor (PBR) imaging in living mouse models of Alzheimer's disease (AD) as biomarkers and functional signatures of glial activation. By radiochemically and immunohistochemically analyzing murine models of the two pathological hallmarks of AD, we found that AD-like Abeta deposition is concurrent with astrocyte-dominant PBR expression, in striking contrast with nonastroglial PBR upregulation in accumulations of AD-like phosphorylated tau. Because tau-induced massive neuronal loss was distinct from the marginal neurodegeneration associated with Abeta plaques in these models, cellular localization of PBR reflected deleterious and beneficial glial reactions to tau versus Abeta pathologies, respectively. This notion was subsequently examined in models of various non-AD neuropathologies, revealing the following reactive glial dynamics underlying differential PBR upregulation: (1) PBR(-) astrogliosis uncoupled with microgliosis or coupled with PBR(+) microgliosis associated with irreversible neuronal insults; and (2) PBR(+) astrogliosis coupled with PBR(- or +/-) microgliosis associated with minimal or reversible neuronal toxicity. Intracranial transplantation of microglia also indicated that nontoxic microglia drives astroglial PBR expression. Moreover, levels of glial cell line-derived neurotrophic factor (GDNF) in astrocytes were correlated with astroglial PBR, except for increased GDNF in PBR(-) astrocytes in the model of AD-like tau pathology, thereby suggesting that PBR upregulation in astrocytes is an indicator of neurotrophic support. Together, PBR expressions in astrocytes and microglia reflect beneficial and deleterious glial reactions, respectively, in diverse neurodegenerative disorders including AD, pointing to new applications of PBR imaging for monitoring the impact of gliosis on the pathogenesis and treatment of AD.

A hallmark of immunopathology associated with Alzheimer's disease is the presence of activated microglia (MG) surrounding senile plaque deposition of beta-amyloid (Abeta) peptides. Abeta peptides are believed to be potent activators of MG, which leads to Alzheimer's disease pathology, but the role of MG subtypes in Abeta clearance still remains unclear. In this study, we found that IL-4 treatment of rat primary-type 2 MG enhanced uptake and degradation of oligomeric Abeta(1-42) (o-Abeta(1-42)). IL-4 treatment induced significant expression of the scavenger receptor CD36 and the Abeta-degrading enzymes neprilysin (NEP) and insulin-degrading enzyme (IDE) but reduced expression of certain other scavenger receptors. Of cytokines and stimulants tested, the anti-inflammatory cytokines IL-4 and IL-13 effectively enhanced CD36, NEP, and IDE. We demonstrated the CD36 contribution to IL-4-induced Abeta clearance: Chinese hamster ovary cells overexpressing CD36 exhibited marked, dose-dependent degradation of (125)I-labeled o-Abeta(1-42) compared with controls, the degradation being blocked by anti-CD36 Ab. Also, we found IL-4-induced clearance of o-Abeta(1-42) in type 2 MG from CD36-expressing WKY/NCrj rats but not in cells from SHR/NCrj rats with dysfunctional CD36 expression. NEP and IDE also contributed to IL-4-induced degradation of Abeta(1-42), because their inhibitors, thiorphan and insulin, respectively, significantly suppressed this activity. IL-4-stimulated uptake and degradation of o-Abeta(1-42) were selectively enhanced in type 2, but not type 1 MG that express CD40, which suggests that the two MG types may play different neuroimmunomodulating roles in the Abeta-overproducing brain. Thus, selective o-Abeta(1-42) clearance, which is induced by IL-4, may provide an additional focus for developing strategies to prevent and treat Alzheimer's disease.

Glutamate released by activated microglia induces excito-neurotoxicity and may contribute to neurodegeneration in numerous neurological diseases including ischemia, inflammation, epilepsy, and neurodegenerative diseases. We observed that the gap junction blocker carbenoxolone (CBX) or the glutaminase inhibitor 6-diazo-5-oxo-L-norleucine (DON) decreased glutamate release from activated microglia and rescued neuronal death in a dose-dependent manner in vitro. In gerbils, treatment with CBX or DON also prevented the delayed death of hippocampal neurons following transient global ischemia. Thus, blockade of microglial glutamate release may be an effective therapeutic strategy against neurodegeneration after ischemic injury.

Objective: To investigate whether [(11)C]PK-11195, a specific peripheral benzodiazepine receptors (PBRs) ligand for positron emission tomography (PET), can show activated microglia in a rat brain injury model., Methods: On day 1, ethanol was injected into the rat's right striatum (ST) using a stereotaxic operative procedure. On day 3, head magnetic resonance imaging (MRI) scans for surgically treated rats were performed to evaluate ethanol injury morphologically. On day 4, dynamic PET scans (17 injured rats and 7 non-injured controls) were performed for 60 min with an animal PET scanner under chloral hydrate anesthesia following a bolus injection of [(11)C]PK-11195 through tail vein. Because PBRs are present throughout the brain, there is no suitable receptor-free reference region. The reference tissue model may not be applicable because of low target to background ratio for low affinity of [(11)C]PK-11195 to PBRs. We evaluated the PBRs binding with regions of interest (ROIs)-based approach to estimate total distribution volume (V). We used an integral from 0 min to 60 min (V (60)) as an estimate of V. On the coronal PET image, ROIs were placed on bilateral ST. Differences in right/left ST V (60) ratios between lesioned and unlesioned control rats were compared using unpaired t tests. Immunohistochemical staining was performed for confirming the presence of activated microglia following decapitation on the PET experiment day., Results: The right/left ST V (60) ratios in lesioned rats (1.07 +/- 0.08) were significantly higher than those in unlesioned control rats (1.00 +/- 0.06, P < 0.05). On immunohistochemical staining, activated microglia were exclusively observed in the injured right ST but not in the noninjured left ST of the injury rats and the bilateral ST of the non-injured control rats., Conclusions: These results suggest that [(11)C]PK-11195 PET imaging would be a useful tool for evaluating microglial activation in a rat brain injury model.

Voltage-gated proton (H+) channels play a pivotal role in compensating charge and pH imbalances during respiratory bursts in phagocytes. Lactic acidosis is a clinically important metabolic condition accompanying various tissue disorders in which the extracellular pH and the intracellular pH often change in parallel. In this study, we investigated the responses of the H+ channel in microglia to lactate-induced pH disturbances using the perforated-patch recordings. Na-lactate (pH 6.8) acidified the cells and activated the H+ channel within 5 min. This early activation was correlated with increases in the pH gradient across the plasma membrane (DeltapH) and was dose-dependent over a concentration range of 10-150 mM. At 10 mM, the change in DeltapH was only slight, but the H+ currents continued to increase over an hour after the cell acidosis was stabilized. Prolonged exposure to lactate (10-20 mM, >1 h) increased the amplitude by two to threefold. The late activation was not explained by increased DeltapH but by changes in the property of the channel per se. Pretreatment with staurosporine and chelerythrine, inhibitors for protein kinase C, prevented the late activation. These results suggest that the H+ channel could be activated greatly during long-lasting lactic acidosis through both DeltapH-dependent and -independent mechanisms.

The inflammatory process in the brain, which is accompanied by changes in the levels of proinflammatory cytokines and neurotrophins, along with the presence of activated microglia, has recently gained much attention in neurodegenerative diseases. Activated microglia produce either neuroprotective or neurotoxic factors. Many reports indicate that activated microglia promote degeneration of dopamine (DA) neurons in Parkinson's disease (PD). On the other hand, there are several lines of evidence that microglia also have a neuroprotective function. Microglia activated with lipopolysaccharide in the nigrostriatum of neonatal mice protect DA neurons against the PD-producing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), whereas activated microglia in aged mice promote DA cell death by MPTP. These results suggest that the function of activated microglia may change in vivo from neuroprotective to neurotoxic during aging as neurodegeneration progresses in the PD brain., (2008 S. Karger AG, Basel)

The aim of this work was to develop new fluorescent probes for the localization and function of the peripheral benzodiazepine receptor (PBR). This receptor is primarily expressed on the mitochondria, and it is overexpressed in a variety of different states including glioma, breast cancer, Alzeheimer's disease, and activated microglia. For the mentioned purpose, imidazopyridine and imidazopyrimidine compounds 5-20 were synthesized, and their affinity for PBR was determined. Although some intrinsically fluorescent imidazopyrimidine compounds 12-20 possess good binding affinity, they cannot be used for visualizing PBR due to their unfavorable fluorescence characteristics. Among the imidazopyridine-7-nitrofurazan conjugates 5-11, compound 10 was the most active, and it was found to stain live Ra2 microglial cells effectively. An in vivo biodistribution study carried out on compound 10 showed that this imidazopyridine derivative, injected in the carotid artery, is able to penetrate to liver parenchyma, whereas fluorescein isothiocyanate labeled dextran (FITC-dextran), used as a control dye, hardly penetrated from blood vessels to tissues. On the other hand, as for the distribution to brain, the patterns of staining with 10 and FITC-dextran are similar, indicating that both of them hardly penetrate into the brain because of the existence of the blood-brain barrier. The obtained results indicate that compound 10 represents a new useful fluorescent probe for visualization of activated microglia and PBR.

Circumstantial evidence has suggested that activated microglia may be associated with the pathogenesis of depression. Pro-inflammatory cytokines may also be involved. Therefore, we examined the effects of various types of antidepressants, as well as the mood-stabilizer lithium chloride, on interferon-gamma (IFN-gamma)-induced microglial production of the pro-inflammatory mediators interleukin-6 (IL-6) and nitric oxide (NO). Treatment of the murine microglial 6-3 cells with 100 U/ml of IFN-gamma resulted in an eightfold increase in IL-6 and a tenfold increase in NO into the culture medium. Pretreatment with the selective serotonin reuptake inhibitor fluvoxamine, the relatively selective noradrenaline reuptake inhibitor reboxetine, or the non-selective monoaminergic reuptake inhibitor imipramine, significantly inhibited IL-6 and NO production in a dose-dependent manner. These inhibitions were reversed significantly by SQ 22536, a cyclic adenosine monophosphate (cAMP) inhibitor, and, except for reboxetine, by the protein kinase A (PKA) inhibitor Rp-adenosine3',5'-cyclic monophosphorothioate triethylammonium salt (Rp-3',5'-cAMPS). Lithium chloride, which is believed to act by inhibiting the calcium-dependent release of noradrenaline, had a different spectrum of action on microglial 6-3 cells. It enhanced IFN-gamma-stimulated IL-6 production and inhibited NO production. The inhibitory effect of lithium chloride was not reversed by either SQ 22536 or Rp-3',5'-cAMPS. These results suggest that antidepressants have inhibitory effects on IFN-gamma-activated microglia and these effects are, at least partially, mediated by the cAMP-dependent PKA pathway. On the other hand, the mood stabilizer and anti-manic agent lithium chloride has mixed effects on IFN-gamma-induced microglial activation.

Microglia play an important role in the inflammatory process that occurs in Parkinson's disease (PD). Activated microglia produce cytokines and neurotrophins and may have neurotoxic or neurotrophic effects. Because microglia are most proliferative and easily activated during the neonatal period, we examined the effects of neonatal microglia activated with lipopolysaccharide (LPS) on the nigro-striatal dopamine neurons in mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), in comparison with activated microglia from the aged mice. By MPTP administration to neonatal mice, the number of dopamine neurons in the substantia nigra (SN) was decreased significantly, whereas that in the mice treated with LPS and MPTP was recovered to normal, along with significant microglial activation. Tyrosine hydroxylase (TH) activity, the levels of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC), and the levels of pro-inflammatory cytokines IL-1beta and IL-6 in the midbrain were elevated in the neonates treated with LPS and MPTP. On the contrary, although the number of dopamine neurons in the 60-week-old mice treated with MPTP was also decreased significantly, the microglial activation by LPS treatment caused a further decrease in their number. These results suggest that the activated microglia in neonatal mice are different from those in aged mice, with the former having neurotrophic potential toward the dopamine neurons in the SN, in contrast to the neurotoxic effect of the latter., (Copyright (c) 2007 Wiley-Liss, Inc.)

Alzheimer's disease is characterized by the presence of senile plaques in the brain composed primarily of amyloid-beta peptide. Microglia have been reported to surround these Abeta plaques, which have opposite roles, provoking a microglia-mediated inflammatory response that contributes to neuronal cell loss or the removal of Abeta and damaged neurons. We herein analyzed the process of expression of Matrix metalloproteinases induced by Abeta stimulation. We found that Abeta1-42 induces a high level of MMP3, MMP12 and MMP13 in the microglia. The signal transduction pathway for the expression of these MMPs mRNA induced by Abeta1-42 depends on PI3K/Akt.

Background: Microglial cells play important roles in coordinating the inflammatory brain responses to hypoxia and trauma. Ionotropic P2X receptors and metabotropic P2Y receptors (P2YRs) expressed in microglia can be activated by extracellular adenosine triphosphate (ATP) derived from damaged cells or astrocytes, and participate in the signaling pathways evoked in brain insult. Although several inhaled and IV anesthetics produce neuroprotective effects through neuronal mechanisms, little is known about how general anesthetics modulate microglial responses in the pathological state. We examined the effects of various general anesthetics on purinergic responses in a rat microglial cell line., Methods: Currents were consistently activated by applications of ATP via a U-tube system under the whole-cell configuration. ATP-induced nondesensitizing currents observed after several applications of ATP exhibited characteristics of P2X7 receptors. The P2YRs-mediated mobilization of intracellular Ca2+ was measured using a Ca2+-sensitive fluorescent dye (fura-2)., Results: Inhaled anesthetics (sevoflurane, isoflurane, and halothane) at doses three times as high as minimum alveolar concentrations had no effect on the P2X7Rs-mediated currents. IV anesthetics (ketamine, propofol, and thiopental) enhanced the P2X7Rs-mediated currents reversibly. The potencies for activation of P2X7Rs were not correlated with the octanol/buffer partition coefficients. Thiopental, at low concentrations, slightly inhibited the P2X7Rs-mediated currents, suggesting its dual actions on P2X7Rs. The P2YRs-mediated mobilization of intracellular Ca2+ was not affected by any of the general anesthetics tested., Conclusions: Our results suggest that IV anesthetics, particularly thiopental and propofol, may modulate microglial functions through P2X7Rs in pathological conditions.

There is increasing evidence that microglial activation is one of the major pathogenic factors for Alzheimer's disease (AD) and the inhibition of the inflammatory activation of the microglia thus appears to be neuroprotective and a potentially useful treatment for AD. Phospholipids such as phosphatidylserine (PS) and phosphatidylcholine (PC) have been reported to modulate the immune function of phagocytes. In addition, PS has been reported to be a nootropics that can be used as nonprescription memory or cognitive enhancers. We therefore evaluated the effects of liposomes, which comprise both PS and PC (PS/PC liposomes), on the microglial production of tumor necrosis factor-alpha (TNF-alpha), nitric oxide (NO), and superoxide (*O(2)-) induced by amyloid beta (Abeta) and interferon-gamma (IFN-gamma). Pretreatment of microglia with PS/PC liposomes considerably inhibited the TNF-alpha, NO and *O(2)- production induced by Abeta/IFN-gamma. These results suggest that PS/PC liposomes have both neuroprotective and antioxidative properties through the inhibition of microglial activation, thus supporting the nootropic and antidementia effect of PS.

Exogenous microglia pass through the blood-brain barrier and migrate to ischemic hippocampal lesions when injected into the circulation. We investigated the effect of exogenous microglia on ischemic CA1 pyramidal neurons. Microglia were isolated from neonatal mixed brain cultures, labeled with the fluorescent dye PKH26, and injected into the subclavian artery of Mongolian gerbils subjected to ischemia reperfusion neuronal injury. PKH26-labeled microglia migrated to the ischemic hippocampal lesion, resulting in increased numbers of surviving neurons compared with control animals, even when injected 24 h after ischemia. Interferon-gamma stimulation of isolated microglia enhanced the neuroprotective effect. Administration of exogenous microglia resulted in normal performance in a passive avoidance-learning task. Additionally, administration of exogenous microglia increased the expression of brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor in the ischemic hippocampus, and thus might have induced neurotrophin-dependent protective activity in damaged neurons. Peripherally injected microglia exhibited a specific affinity for ischemic brain lesions, and protected against ischemic neuronal injury in vivo. It is possible that administration of exogenous microglia can be developed as a potential candidate therapy for central nervous system repair after transitory global ischemia.

Microglial activation and inflammatory processes have been implicated in the pathogenesis of a number of neurodegenerative disorders. Recently, peroxynitrite (ONOO(-)), the reaction product of superoxide (O(2)(-)) and nitric oxide (NO) both of which can be generated by activated microglia, has been demonstrated to act as a major mediator in the neurotoxicity induced by activated microglia. On the other hand, phospholipids such as phosphatidylserine (PS) and phosphatidylcholine (PC) have been reported to modulate the immune function of phagocytes. We therefore evaluated the effects of liposomes which comprise both PS and PC (PS/PC liposomes) or PC only (PC liposomes) regarding the production of both O(2)(-) and NO by lipopolysaccharide (LPS)/phorbol 12-myristate-13-acetate (PMA)-activated microglia using electron spin resonance (ESR) spin trap technique with a DEPMPO and Griess reaction, respectively. Pretreatment with PS/PC liposomes or PC liposomes considerably inhibited the signal intensity of O(2)(-) adduct associated with LPS/PMA-activated microglia in a dose-dependent manner. In addition, pretreatment with PS/PC liposomes also significantly reduced LPS/PMA-induced microglial NO production. In contrast, pretreatment with PC liposomes had no effect on the NO production. These results indicate that PS/PC liposomes can inhibit the microglial production of both NO and O(2)(-), and thus presumably prevent a subsequent formation of ONOO(-). Therefore, PS/PC liposomes appear to have both neuroprotective and anti-oxidative properties through the inhibition of microglial activation.

Alzheimer's disease (AD) is characterized by the presence of senile plaques composed primarily of amyloid-beta peptide (Abeta) in the brain. Microglia have been reported to surround these Abeta plaques, which have opposite roles, provoking a microglia-mediated inflammatory response that contributes to neuronal cell loss or the removal of Abeta and damaged neurons. To perform these tasks microglia migrate to the sites of Abeta secretion. We herein analyzed the process of chemokine expression induced by Abeta stimulation in primary murine microglia and Ra2 microglial cell line. We found that Abeta1-42 induced the expressions of CCL7, CCL2, CCL3, CCL4 and CXCL2 in the microglia. The signal transduction pathway for the expression of CCL2 and CCL7 mRNA induced by Abeta1-42 was found to depend on phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK), whereas the pathway for CCL4 depended only on PI3K/Akt. These inflammatory chemokine expressions by Abeta stimulation emphasize the contribution of neuroinflammatory mechanisms to the pathogenesis of AD.

Microglia are reported to have alpha-amino-hydroxy-5-methyl-isoxazole-4-propionate/kainate (KA) types. However, only small population of primary cultured rat microglia (approximately 20%) responded to KA. In the present study, we have attempted to elucidate the regulatory mechanism of responsiveness to KA in GMIR1 rat microglial cell line. When the GMIR1 cells were plated at a low density in the presence of granulocyte macrophage colony-stimulating factor, the proliferation rate increased and reached the peak after 2 days in culture and then gradually decreased because of density-dependent inhibition. At cell proliferation stage, approximately 80% of the GMIR1 cells exhibited glutamate (Glu)- and KA-induced inward currents at cell proliferation stage, whereas only 22.5% of the cells showed responsiveness to Glu and KA at cell quiescent stage. Furthermore, the mean amplitudes of inward currents induced by Glu and KA at cell proliferation stage (13.8+/-3.0 and 8.4+/-0.6 pA) were significantly larger than those obtained at cell quiescent stage (4.7+/-0.8 and 6.2+/-1.2 pA). In the GMIR1 cells, KA-induced inward currents were markedly inhibited by (RS)-3-(2-carboxybenzyl) willardiine (UBP296), a selective antagonist for KA receptors. The KA-responsive cells also responded to (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA), a selective agonist for GluR5, in both GMIR1 cells and primary cultured rat microglia. Furthermore, mRNA levels of the KA receptor subunits, GluR5 and GluR6, at the cell proliferation stage were significantly higher than those at the cell quiescent stage. Furthermore, the immunoreactivity for GluR6/7 was found to increase in activated microglia in the post-ischemic hippocampus. These results strongly suggest that microglia have functional KA receptors mainly consisting of GluR5 and GluR6, and the expression levels of these subunits are closely regulated by the cell cycle mechanism.

During neurotropic virus infection, microglia act as a source of chemokines, thereby regulating the recruitment of peripheral leukocytes and the multicellular immune response within the CNS. Herein, we present a comprehensive study on the chemokine production by microglia in response to double-stranded RNA (dsRNA), a conserved molecular pattern of virus infection. Transcriptional analyses of chemokine genes revealed that dsRNA strongly induces the expression of CXC chemokine ligand 10 (CXCL10) and CC chemokine ligand 5 (CCL5) in microglia. We also observed that the dsRNA stimulation triggered the activation of signaling pathways mediated by nuclear factor kappaB (NF-kappaB) and mitogen-activated protein kinases (MAPK), including extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38, and c-Jun N-terminal kinase (JNK). The microglial CXCL10 response to dsRNA was induced via NF-kappaB, p38, and JNK pathways, whereas the dsRNA-induced CCL5 production was dependent on JNK, but not on the other signal-transducing molecules tested. In addition, the acidic environment of intracellular vesicles was required for the activation of cellular signaling in response to dsRNA. Taken together, these results suggest that the recognition of dsRNA structure selectively induces the CXCL10 and CCL5 responses in microglia through vacuolar pH-dependent activation of NF-kappaB and MAPK signaling pathways.

Following virus infection of the central nervous system, microglia, the ontogenetic and functional equivalents of macrophages in somatic tissues, act as sources of chemokines, thereby recruiting peripheral leukocytes into the brain parenchyma. In the present study, we have systemically examined the growth characteristics of rabies virus (RV) in microglia and the activation of cellular signaling pathways leading to chemokine expression upon RV infection. In RV-inoculated microglia, the synthesis of the viral genome and the production of virus progenies were significantly impaired, while the expression of viral proteins was observed. Transcriptional analyses of the expression profiles of chemokine genes revealed that RV infection, but not exposure to inactivated virions, strongly induces the expression of CXC chemokine ligand 10 (CXCL10) and CC chemokine ligand 5 (CCL5) in microglia. RV infection triggered the activation of signaling pathways mediated by mitogen-activated protein kinases, including p38, extracellular signal-regulated kinases 1 and 2 (ERK1/2), and c-Jun N-terminal kinase, and nuclear factor kappaB (NF-kappaB). RV-induced expression of CXCL10 and CCL5 was achieved by the activation of p38 and NF-kappaB pathways. In contrast, the activation of ERK1/2 was found to down-regulate CCL5 expression in RV-infected microglia, despite the fact that it was involved in partial induction of CXCL10 expression. Furthermore, NF-kappaB signaling upon RV infection was augmented via a p38-mediated mechanism. Taken together, these results indicate that the strong induction of CXCL10 and CCL5 expression in microglia is precisely regulated by the activation of multiple signaling pathways through the recognition of RV infection.

Alzheimer's disease is characterized by numerous amyloid-beta peptide (Abeta) plaques surrounded by microglia. Here we report that Abeta induces the proliferation of the mouse microglial cell line Ra2 by increasing the expression of macrophage colony-stimulating factor (M-CSF). We examined signal cascades for Abeta-induced M-CSF mRNA expression. The induction of M-CSF was blocked by a phosphatidylinositol 3 kinase (PI3-kinase) inhibitor (LY294002), a Src family tyrosine kinase inhibitor (PP1) and an Akt inhibitor. Electrophoretic mobility shift assays showed that Abeta enhanced NF-kappaB binding activity to the NF-kappaB site of the mouse M-CSF promoter, which was blocked by LY294002. These results indicate that Abeta induces M-CSF mRNA expression via the PI3-kinase/Akt/NF-kappaB pathway.

Dementia is a frequent complication of Parkinson's disease (PD) and usually occurs late in the protracted course of the illness. We have already reported numerous MHC class II-positive microglia in the hippocampus in PD patients, and that this phenomenon may be responsible for functional changes in the neurons and the cognitive decline in PD patients. In this study, we have investigated the distribution of activated microglia and the immunohistochemical and the mRNA expression of several cytokines and neurotrophic factors of the hippocampus in PD and dementia with Lewy bodies (DLB). The brains from five cases of PD and five cases of DLB that were clinically and neuropathologically diagnosed, and those from four normal controls (NC) were evaluated by immunohistochemistry using anti-HLA-DP, -DQ, -DR (CR3/43), anti-alpha-synuclein, anti-brain-derived neurotrophic factor (BDNF), and anti-glial fibrillary acidic protein antibodies. In addition, the mRNA expressions of cytokines (IL-1alpha, IL-1beta, TNF-alpha, IL-6, TGF-beta) and neurotrophic factors (BDNF, GDNF, NGF, NT-3) of these brains were evaluated by the reverse transcription-PCR method. MHC class II-positive microglia were distributed diffusely in the hippocampus of PD and DLB brains. Although the cytoplasm of pyramidal and granular cells of the hippocampus in NC brains was strongly stained by anti-BDNF antibodies, it was only weakly stained in PD and DLB brains. The mRNA expression of IL-6 was significantly increased in the hippocampus of PD and DLB brains, and that of BDNF was significantly decreased in the hippocampus of DLB brains. The increased number of activated microglia and the production of neurotrophic cytokines such as IL-6, together with the decreased expression of the neurotrophic factors of neurons in the hippocampus of PD and DLB brains, may be related to functional cellular changes associated with dementia.

Protein kinase C (PKC) plays a prominent role in immune signaling. To elucidate the signal transduction in a respiratory burst and isoform-specific function of PKC during FcgammaR-mediated phagocytosis, we used live, digital fluorescence imaging of mouse microglial cells expressing GFP-tagged molecules. betaI PKC, epsilonPKC, and diacylglycerol kinase (DGK) beta dynamically and transiently accumulated around IgG-opsonized beads (BIgG). Moreover, the accumulation of p47(phox), an essential cytosolic component of NADPH oxidase and a substrate for betaI PKC, at the phagosomal cup/phagosome was apparent during BIgG ingestion. Superoxide (O(2)(-)) production was profoundly inhibited by Gö6976, a cPKC inhibitor, and dramatically increased by the DGK inhibitor, R59949. Ultrastructural analysis revealed that BIgG induced O(2)(-) production at the phagosome but not at the intracellular granules. We conclude that activation/accumulation of betaI PKC is involved in O(2)(-) production, and that O(2)(-) production is primarily initiated at the phagosomal cup/phagosome. This study also suggests that DGKbeta plays a prominent role in regulation of O(2)(-) production during FcgammaR-mediated phagocytosis.

Hyperplasia of microglia is one characteristic of reactive gliosis, which is observed in various pathologic conditions in the central nervous system (CNS). To investigate the regulation mechanisms of microglial proliferation, the effect of granulocyte macrophage colony-stimulating factor (GM-CSF) on the expression of cell cycle-associated proteins was examined in the microglial cell line GMI-M6-3. After GM-CSF administration, cyclins D1, E, and A and cyclin-dependent kinase inhibitor p21Cip1 were increased, and another cyclin-dependent kinase inhibitor, p27Kip1, was decreased with morphologic transformation into ameboid form. By contrast, downregulation of these cyclins and p21Cip1, and strong upregulation of p27Kip1 accompanied by ramification were observed with GM-CSF deprivation. We also found that GMI-M6-3 exhibited homotypic contact inhibition of proliferation without any morphologic transformation. The increase of p27Kip1 and the decrease of cyclin A were suggested to play an important role in microglial contact inhibition. In addition, the direct effect of p27Kip1 to inhibit microglial proliferation was demonstrated both in vitro and in vivo by overexpression of p27Kip1., (Copyright 2003 Wiley-Liss, Inc.)

There are numerous observations confirming that microglia expressing major histocompatibility complex (MHC) class II molecules are associated with the central nervous system (CNS) in aging and pathological conditions. In this study, we investigated the distribution of MHC class II-positive microglia in Parkinson's disease (PD) brains. The number of MHC class II-positive microglia in the substantia nigra (SN) and putamen increased as the neuronal degeneration of the SN proceeded. These cells were also ICAM-1 (CD54) and LFA-1 (CD11a) positive. The number of activated microglia not only in the SN and putamen but also in the hippocampus, transentorhinal cortex, cingulate cortex and temporal cortex in PD was significantly higher than that in the normal control. Most activated microglia persisted regardless of the presence or absence of Lewy bodies. They were frequently associated not only with alpha-synuclein-positive Lewy neurites, but also with TH-16-positive dopaminergic and WH-3-positive serotonergic neurites, as well as MAP-2- and SMI-32-positive neurites. These activated microglia were also positive for TNF-alpha and interleukin-6, which are known to have a neuroprotective function. We conclude that MHC class II-positive microglia are a sensitive index of neuropathological change and are actively associated with damaged neurons and neurites.

The extrusion of protons is considered a very general parameter of the activation of many kinds of membrane or intracellular molecules, such as receptors, ion channels, and enzymes. We found that pepstatin A caused a reproducible, concentration-related increase in the extracellular acidification rate in two microglial cell lines, Ra2 and 6-3. Washing abolished pepstatin A-induced acidification immediately. However, pepstatin A did not cause the extracellular acidification in other cell types, such as CHO, C6 glioma, and NIH3T3 cells. These observations strongly suggest that pepstatin A interacts with certain membrane proteins specific to both Ra2 and 6-3 cells from outside. N-methylmaleimide and N,N'-dicyclohexylcarbodiimide, inhibitors of H(+)-ATPase, were found to reduce pepstatin A-induced response strongly, while bafilomycin A1, a vacuolar H(+)-ATPase inhibitor, vanadate, a P-type H(+)-ATPase inhibitor, and NaN3, an F1 ATPase inhibitor, virtually did not. 5-(N-ethyl-N-isopropyl) amiloride, an inhibitor of Na(+)/H(+) exchanger isoform 1, greatly enhanced pepstatin-induced response, while amiloride did not. Zn(2+), a voltage-dependent proton channel blocker, did not affect pepstatin-induced response neither. Staurosporine, a nonspecific inhibitor of protein kinase C, inhibited pepstatin A-induced response, while chelerythrine, more selective inhibitor of protein kinase C, greatly enhanced it. H-7 and H-8 did not affected the response. These findings suggest that pepstatin A induces extracellular acidification in microglia cell lines, Ra2 and 6-3, through an N-methylmaleimide- and N,N'-dicyclohexylcarbodiimide-sensitive, but bafilomycin A1-insensitive, ATPase, which seems to be distinct from protein kinase C-dependent process., (Copyright 2003 Wiley-Liss, Inc.)

We examined the expression and function of beta-adrenergic receptor (beta-AR) subtypes in both isolated primary rat microglia and a rat microglial cell line. RT-PCR analyses revealed that microglia expressed beta(1)- and beta(2)-ARs but not beta(3)-ARs, whereas rat primary peritoneal macrophages expressed only beta(2)-ARs. Stimulation of beta-ARs on microglia by norepinephrine (NE) resulted in an increase in the level of intracellular cAMP and the subsequent expression of interleukin-1beta mRNA. These effects were prevented by propranolol. Similar results were obtained with other selective beta(1)-AR agonists and antagonists. beta(2)-ARs on microglia were also functional. It is possible that noradrenergic innervations participate in the control of microglial functions via beta(1)-ARs on microglia in the brain, because NE has high affinity for beta(1)- and beta(3)-ARs but little or no affinity for beta(2)-ARs. It seems physiologically significant that microglia can be controlled by NE, which predominates over epinephrine in the brain, whereas macrophages in peripheral tissues can be controlled by epinephrine, which is at higher levels in peripheral tissues., (Copyright 2002 Wiley-Liss, Inc.)

DA strain of Theiler's murine encephalomyelitis virus (TMEV) persists in the mouse central nervous system (CNS) and induces demyelination while GDVII strain fails to persist or demyelinate. L* protein, which is synthesized only in DA but not GDVII, is believed important in virus persistence and demyelination. Because a major reservoir for DA persistence is infiltrated macrophages or microglia, a resident macrophage of the CNS, we investigated TMEV infection of Ra2 cells, a murine microglial cell line. We found that DA strain grew well in Ra2 cells, but not GDVII strain or DAL*-1 virus (which fails to synthesize L* protein), suggesting that L* protein plays an important role in virus growth in microglia. Interestingly, in contrast to virus growth, most Ra2 cells infected with DA strain survived with no evidence of virus-induced apoptosis. These results may be important in clarifying the pathogenesis of DA-induced demyelinating disease.

Microglial activation and proliferation occur in nearly all forms of brain injury. The aim of this study was to investigate the influence of glial cell-line derived neurotrophic factor (GDNF) on proliferation and/or survival in a GMIR1 rat microglial cell line, which proliferates in response to granulocyte-macrophage-colony stimulating factor (GM-CSF). Endogenous GDNF and its receptor, GFRalpha-1, were detected in GMIR1 cells by ELISA and immunohistochemistry/Western blot, respectively. Recombinant GDNF strongly enhanced GMIR1 cell numbers and BrdU-incorporation, an effect inhibited by GDNF blocking antibodies. Inhibition of cAMP/cGMP dependent protein kinase enhanced the GDNF-induced GMIR1 cell number. The results suggest that GDNF has synergistic survival promoting effects on microglia potentially via autocrine mechanisms.