Microglioma in a child - a further case in support of the microglioma entity and distinction from histiocytic sarcoma.

Microglia are not generally known to cause brain tumors but one bona fide case of adult microglioma has been published [9]. This tumor was highly malignant. We now report on a second, juvenile case, which showed a less aggressive course. Microglioma is a primary central nervous system (CNS) neoplasm distinct from glioma and other known brain tumor entities, based on its strong immunoreactivity for the macrophage marker CD163, the microglia marker Iba1, and the complete absence of neural as well as lymphocyte antigens. Furthermore, we have analyzed the literature and identified a number of cases that qualify as primary parenchymal histiocytic sarcomas of the CNS, which lack microglial morphology. Considering the non-hematopoietic developmental origin of the vast majority of microglia and the distinct morphological as well as immunophenotypic similarity of their neoplastic counterparts, we suggest using the term microglioma. More cases will be required along with appropriately-collected tissue to establish the molecular genetic profile of this extremely rare entity.

Purinergic receptors in microglia: functional modal shifts of microglia mediated by P2 and P1 receptors.

Microglia are sensitive to environmental changes and are immediately transformed into several phenotypes. For such dynamic "modal shifts", purinergic receptors have central roles. When microglia sense ATP/ADP leaked from injured cells by P2Y(12) receptors, they are transformed into a moving phenotype, showing process extension and migration toward the injured sites. Microglia upregulate adenosine A(2A) receptors, by which they retract the processes showing an amoeboid-shaped, activated phenotype. Microglia also upregulate P2Y(6) receptors, and if they meet UDP leaked from dead cells, microglia start to engulf and eat the dead cells as a phagocytic phenotype. The P2Y(12) receptor-mediated responses are modulated by other P2 or P1 receptors. In contrast, the P2Y(6) receptor-mediated responses were not influenced by P2Y(12) receptors and vice versa. Microglia appear to use purinergic signals either cooperatively or distinctively to cause their modal shifts.

UDP acting at P2Y6 receptors is a mediator of microglial phagocytosis.

Microglia, brain immune cells, engage in the clearance of dead cells or dangerous debris, which is crucial to the maintenance of brain functions. When a neighbouring cell is injured, microglia move rapidly towards it or extend a process to engulf the injured cell. Because cells release or leak ATP when they are stimulated or injured, extracellular nucleotides are thought to be involved in these events. In fact, ATP triggers a dynamic change in the motility of microglia in vitro and in vivo, a previously unrecognized mechanism underlying microglial chemotaxis; in contrast, microglial phagocytosis has received only limited attention. Here we show that microglia express the metabotropic P2Y6 receptor whose activation by endogenous agonist UDP triggers microglial phagocytosis. UDP facilitated the uptake of microspheres in a P2Y6-receptor-dependent manner, which was mimicked by the leakage of endogenous UDP when hippocampal neurons were damaged by kainic acid in vivo and in vitro. In addition, systemic administration of kainic acid in rats resulted in neuronal cell death in the hippocampal CA1 and CA3 regions, where increases in messenger RNA encoding P2Y6 receptors that colocalized with activated microglia were observed. Thus, the P2Y6 receptor is upregulated when neurons are damaged, and could function as a sensor for phagocytosis by sensing diffusible UDP signals, which is a previously unknown pathophysiological function of P2 receptors in microglia.

Involvement of activated microglia in increased vulnerability of motoneurons after facial nerve avulsion in presymptomatic amyotrophic lateral sclerosis model rats.

Activated microglia are observed in various neurodegenerative diseases and are thought to be involved in the processes of neuronal cell death. Motoneuron damage in the facial nuclei after facial nerve avulsion is accelerated in presymptomatic transgenic rats expressing human mutant Cu(2+) /Zn(2+) superoxide dismutase 1 (SOD1), compared with that in wild-type rats. To reveal the functional role of microglia in motoneuronal death, we investigated the microglial response after facial nerve avulsion in presymptomatic mutant SOD1(H46R) (mSOD1(H46R) ) rats. At 3 days after avulsion, microglial clusters were observed in the facial nuclei of both wild-type and mSOD1(H46R) rats. The numbers of microglial clusters, proliferating microglia, and microglial attachments to motoneurons were significantly higher in mSOD1(H46R) rats, compared with those in wild-type rats. Immunopositive signals for the phagocytic marker ED1 were significantly stronger in mSOD1(H46R) rats, compared with that in wild-type rats, at 2 weeks after avulsion. Furthermore, primary microglia prepared from mSOD1(H46R) rats showed enhanced phagocytic activity, compared with that in wild-type rats. The expression of P2Y(12) mRNA was higher in the facial nuclei of mSOD1(H46R) rats, compared with that in wild-type rats. A laser microdissection system revealed that the expression of ATF3 mRNA was higher in the motoneurons of mSOD1(H46R) rats, compared with that in wild-type rats, at 2 days after avulsion. These results indicate that microglial activation in response to early neuronal damage increased in mSOD1(H46R) rats and suggest that the enhanced activation of microglia may lead to an increase in the vulnerability of motoneurons after avulsion in mSOD1(H46R) rats.

Adenosine A3 receptor is involved in ADP-induced microglial process extension and migration.

The extension of microglial processes toward injured sites in the brain is triggered by the stimulation of the purinergic receptor P2Y(12) by extracellular ATP. We recently showed that P2Y(12) stimulation by ATP induces microglial process extension in collagen gels. In the present study, we found that a P2Y(12) agonist, 2-methylthio-ADP (2MeSADP), failed to induce the process extension of microglia in collagen gels and that co-stimulation with adenosine, a phosphohydrolytic derivative of ATP, and 2MeSADP restored the chemotactic process extension. An adenosine A3 receptor (A3R)-selective agonist restored the chemotactic process extension, but other receptor subtype agonists did not. The removal of adenosine by adenosine deaminase and the blocking of A3R by an A3R-selective antagonist inhibited ADP-induced process extension. The A3R antagonist inhibited ADP-induced microglial migration, and an A3R agonist promoted 2MeSADP-stimulated migration. ADP and the A3R agonist activated Jun N-terminal kinase in microglia, and a Jun N-terminal kinase inhibitor inhibited the ADP-induced process extension. An RT-PCR analysis showed that A1R and A3R were expressed by microglia sorted from adult rat brains and that the A2AR expression level was very low. These results suggested that A3R signaling may be involved in the ADP-induced process extension and migration of microglia.

Dynamic motility of microglia: purinergic modulation of microglial movement in the normal and pathological brain.

Microglia have highly branched and motile cell processes and constantly screen the brain parenchyma under physiological conditions. In response to pathological stimuli, microglia exhibit morphological changes and migrate toward the lesioned site, where they play important roles in inflammatory reactions and neuronal damage. Within minutes of brain damage, microglial processes rapidly extend toward the injured site. The chemoattractive response is triggered by ATP released at the site of injury and the consequent activation of the purinergic receptor P2Y12R on microglia. In addition to the purinergic signals, various neuronal signaling molecules actively and negatively control microglial motility, which is important for regulating the functional activation of microglia in response to pathology. In this review, we focus on the dynamic motion of microglia and describe several key molecules regulating microglial motility in normal and pathological brain tissues.

P2Y12 receptor-mediated integrin-beta1 activation regulates microglial process extension induced by ATP.

Microglia are the primary immune surveillance cells in the brain, and when activated they play critical roles in inflammatory reactions and tissue repair in the damaged brain. Microglia rapidly extend their processes toward the damaged areas in response to stimulation of the metabotropic ATP receptor P2Y(12) by ATP released from damaged tissue. This chemotactic response is a highly important step that enables microglia to function properly at normal and pathological sites in the brain. To investigate the molecular pathways that underlie microglial process extension, we developed a novel method of modeling microglial process extension that uses transwell chambers in which the insert membrane is coated with collagen gel. In this study, we showed that ATP increased microglial adhesion to collagen gel, and that the ATP-induced process extension and increase in microglial adhesion were inhibited by integrin blocking peptides, RGD, and a functional blocking antibody against integrin-beta1. An immunoprecipitation analysis with an antibody against the active form of integrin-beta1 showed that P2Y(12) mediated the integrin-beta1 activation by ATP. In addition, time-lapse imaging of EGFP-labeled microglia in mice hippocampal slices showed that RGD inhibited the directional process extension toward the nucleotide source, and immunohistochemical staining showed that integrin-beta1 accumulated in the tips of the microglial processes in rat hippocampal slices stimulated with ADP. These findings indicate that ATP induces the integrin-beta1 activation in microglia through P2Y(12) and suggest that the integrin-beta1 activation is involved in the directional process extension by microglia in brain tissue.

Appearance of phagocytic microglia adjacent to motoneurons in spinal cord tissue from a presymptomatic transgenic rat model of amyotrophic lateral sclerosis.

Microglial activation occurs early during the pathogenesis of amyotrophic lateral sclerosis (ALS). Recent evidence indicates that the expression of mutant Cu(2+)/Zn(2+) superoxide dismutase 1 (SOD1) in microglia contributes to the late disease progression of ALS. However, the mechanism by which microglia influence the neurodegenerative process and disease progression in ALS remains unclear. In this study, we revealed that activated microglia aggregated in the lumbar spinal cord of presymptomatic mutant SOD1(H46R) transgenic rats, an animal model of familial ALS. The aggregated microglia expressed a marker of proliferating cell, Ki67, and phagocytic marker proteins ED1 and major histocompatibility complex (MHC) class II. The motoneurons near the microglial aggregates showed weak choline acetyltransferase (ChAT) immunoreactivity and contained reduced granular endoplasmic reticulum and altered nucleus electron microscopically. Furthermore, immunopositive signals for tumor necrosis factor-alpha (TNFalpha) and monocyte chemoattractant protein-1 (MCP-1) were localized in the aggregated microglia. These results suggest that the activated and aggregated microglia represent phagocytic features in response to early changes in motoneurons and possibly play an important role in ALS disease progression during the presymptomatic stage.

Akt activation is involved in P2Y12 receptor-mediated chemotaxis of microglia.

Microglia play a variety of significant roles in the central nervous system (CNS), and in one of those roles they undergo morphological change in response to neural injury and migrate to the injured region. We previously reported that ATP/ADP promotes microglial chemotaxis via the Gi/o-coupled P2Y12 receptor; however, the intracellular signaling underlying P2Y12-receptor-mediated microglial chemotaxis is not fully understood. In this study, we examined the role of phospholipase C (PLC) and calcium signaling in ADP-induced microglial chemotaxis. A PLC inhibitor, U73122, significantly suppressed the chemotaxis and completely blocked the ADP-evoked intracellular calcium response, and a calcium chelator, BAPTA-AM, inhibited the chemotaxis. These results indicate that ADP-induced microglial chemotaxis is regulated by a PLC-mediated calcium pathway. ADP stimulation induced Akt phosphorylation in microglia, and the phosphorylation was inhibited by a P2Y12 receptor antagonist, AR-C69931MX. The Akt phosphorylation was blocked by U73122 and BAPTA-AM as well as by a phosphatidylinositol 3-kinase (PI3K) inhibitor, wortmannin, and inhibition of the Akt activation resulted in failure of chemotaxis. These results indicate that Akt activation is dependent on the PI3K pathway and a PLC-mediated increase in intracellular calcium and suggest that Akt activation is involved in ADP-induced microglial chemotaxis.

X-ray structures of the microglia/macrophage-specific protein Iba1 from human and mouse demonstrate novel molecular conformation change induced by calcium binding.

The ionized calcium-binding adaptor molecule 1 (Iba1) with 147 amino acid residues has been identified as a calcium-binding protein, expressed specifically in microglia/macrophages, and is expected to be a key factor in membrane ruffling, which is a typical feature of activated microglia. We have determined the crystal structure of human Iba1 in a Ca(2+)-free form and mouse Iba1 in a Ca(2+)-bound form, to a resolution of 1.9 A and 2.1 A, respectively. X-ray structures of Iba1 revealed a compact, single-domain protein with two EF-hand motifs, showing similarity in overall topology to partial structures of the classical EF-hand proteins troponin C and calmodulin. In mouse Iba1, the second EF-hand contains a bound Ca(2+), but the first EF-hand does not, which is often the case in S100 proteins, suggesting that Iba1 has S100 protein-like EF-hands. The molecular conformational change induced by Ca(2+)-binding of Iba1 is different from that found in the classical EF-hand proteins and/or S100 proteins, which demonstrates that Iba1 has an unique molecular switching mechanism dependent on Ca(2+)-binding, to interact with target molecules.

Cell-permeable p38 MAP kinase promotes migration of adult neural stem/progenitor cells.

Endogenous neural stem/progenitor cells (NPCs) can migrate toward sites of injury, but the migration activity of NPCs is insufficient to regenerate damaged brain tissue. In this study, we showed that p38 MAP kinase (p38) is expressed in doublecortin-positive adult NPCs. Experiments using the p38 inhibitor SB203580 revealed that endogenous p38 participates in NPC migration. To enhance NPC migration, we generated a cell-permeable wild-type p38 protein (PTD-p38WT) in which the HIV protein transduction domain (PTD) was fused to the N-terminus of p38. Treatment with PTD-p38WT significantly promoted the random migration of adult NPCs without affecting cell survival or differentiation; this effect depended on the cell permeability and kinase activity of the fusion protein. These findings indicate that PTD-p38WT is a novel and useful tool for unraveling the roles of p38, and that this protein provides a reasonable approach for regenerating the injured brain by enhancing NPC migration.

Arachidonic acid release and prostaglandin F2alpha formation induced by phenylarsine oxide in PC12 cells: possible involvement of secretory phospholipase A2 activity.

Activation of phospholipase A2 (PLA2) causing arachidonic acid (AA) release is involved in neuronal cell functions. Previously, we reported AA release and prostaglandin F(2alpha) (PGF(2alpha)) formation via activation of cytosolic PLA2 by orthovanadate (Na3VO4), an inhibitor of tyrosine phosphatases, in rat pheochromocytoma PC12 cells. We investigated the effects of phenylarsine oxide (PAO), which reacts with sulfhydryl groups of proteins and thus acts as an inhibitor of tyrosine phosphatases, on AA release and PGF(2alpha) formation in PC12 cells. PAO stimulated [3H]AA release from the prelabeled cells and PGF(2alpha) formation. The PAO responses were dependent upon the concentrations used (10 microM to 0.5mM) and on extracellular CaCl2. [3H]AA release induced by PAO was decreased significantly by inhibition of secretory, but not cytosolic, PLA2. [3H]AA release by PAO was not reversed by washing the cells, but the addition of dithiol compounds such as 2,3-dimercapto-1-propanol decreased the release from the PAO-treated cells. The existence of mRNA of types IB and IIC secretory PLA2 in PC12 cells was detected by reverse transcriptase-polymerase chain reaction using specific primers. Addition of secretory PLA2 from bee venom to the assay mixture stimulated [3H]AA release, and PAO enhanced the response synergistically. The addition of 0.1mM PAO directly enhanced the activity of secretory PLA2 from bee venom. These findings suggest that PAO stimulates AA release and PGF(2alpha) formation probably via activation of secretory PLA2 in PC12 cells.

Modification of a fiber protein in an adenovirus vector improves in vitro gene transfer efficiency to the mouse microglial cell line.

In microglia, it is difficult to introduce exogenous genes of interest even by recombinant adenovirus vectors (Ad) which can infect with high efficiency only to the cells expressing coxackievirus and adenovirus receptors (CAR). We found a lack of CAR expression in primary cultured murine microglia (PCMG) and its immortalized cell line MG5 by reverse transcription-polymerase chain reaction. In order to improve the efficiency of gene transfer, we generated a novel Ad (Ad-RGD) by an incorporation of the Arg-Gly-Asp motif (RGD) containing peptide in the HI loop of the viral fiber knob domain, which enables the virus to contact target cells through alpha V integrins which are known to be ubiquitously expressed on the surface of mammalian cells. Ad-RGD showed a remarkable improvement (13-18-fold) in the delivery of Escherichia coli LacZ gene in MG5 cells and a moderate increase in PCMG cells under the treatment with granulocyte-macrophage colony stimulating factor. These results suggest that Ad-RGD may be a potent tool for the delivery of genes to microglia activated by optimum stimulation, and thus analyzing the function of microglia with utilization of MG5 and PCMG cells.

Accumulating microglia phagocytose injured neurons in hippocampal slice cultures: involvement of p38 MAP kinase.

In this study, microglial migration and phagocytosis were examined in mouse organotypic hippocampal slice cultures, which were treated with N-methyl-D-aspartate (NMDA) to selectively injure neuronal cells. Microglial cells were visualized by the expression of enhanced green fluorescent protein. Daily observation revealed microglial accumulation in the pyramidal cell layer, which peaked 5 to 6 days after NMDA treatment. Time-lapse imaging showed that microglia migrated to the pyramidal cell layer from adjacent and/or remote areas. There was no difference in the number of proliferating microglia between control and NMDA-treated slices in both the pyramidal cell layer and stratum radiatum, suggesting that microglial accumulation in the injured areas is mainly due to microglial migration, not to proliferation. Time-lapse imaging also showed that the injured neurons, which were visualized by propidium iodide (PI), disappeared just after being surrounded by microglia. Daily observation revealed that the intensity of PI fluorescence gradually attenuated, and this attenuation was suppressed by pretreatment with clodronate, a microglia toxin. These findings suggest that accumulating microglia phagocytosed injured neurons, and that PI fluorescence could be a useful indicator for microglial phagocytosis. Using this advantage to examine microglial phagocytosis in living slice cultures, we investigated the involvements of mitogen-activated protein (MAP) kinases in microglial accumulation and phagocytosis. p38 MAP kinase inhibitor SB203580, but not MAP kinase/extracellular signal-regulated kinase inhibitor PD98059 or c-Jun N-terminal kinase inhibitor SP600125, suppressed the attenuation of PI fluorescence. On the other hand, microglial accumulation in the injured areas was not inhibited by any of these inhibitors. These data suggest that p38 MAP kinase plays an important role in microglial phagocytosis of injured neurons.

Involvement of P2X4 and P2Y12 receptors in ATP-induced microglial chemotaxis.

We previously reported that extracellular ATP induces membrane ruffling and chemotaxis of microglia and suggested that their induction is mediated by the Gi/o-protein coupled P2Y(12) receptor (P2Y(12)R). Here we report discovering that the P2X(4) receptor (P2X(4)R) is also involved in ATP-induced microglial chemotaxis. To understand the intracellular signaling pathway downstream of P2Y(12)R that underlies microglial chemotaxis, we examined the effect of two phosphatidylinositol 3'-kinase (PI3K) inhibitors, wortmannin, and LY294002, on chemotaxis in a Dunn chemotaxis chamber. The PI3K inhibitors significantly suppressed chemotaxis without affecting ATP-induced membrane ruffling. ATP stimulation increased Akt phosphorylation in the microglia, and the increase was reduced by the PI3K inhibitors and a P2Y(12)R antagonist. These results indicate that P2Y(12)R-mediated activation of the PI3K pathway is required for microglial chemotaxis in response to ATP. We also found that the Akt phosphorylation was reduced when extracellular calcium was chelated, suggesting that ionotropic P2X receptors are involved in microglial chemotaxis by affecting the PI3K pathway. We therefore tested the effect of various P2X(4)R antagonists on the chemotaxis, and the results showed that pharmacological blockade of P2X(4)R significantly inhibited it. Knockdown of the P2X(4) receptor in microglia by RNA interference through the lentivirus vector system also suppressed the microglial chemotaxis. These results indicate that P2X(4)R as well as P2Y(12)R is involved in ATP-induced microglial chemotaxis.

Reversible activation of secretory phospholipase A2 by sulfhydryl reagents.

Secretory phospholipase A(2)s (sPLA(2)s) have been implicated in physiological and pathological events, but the regulatory mechanism(s) of their activities in cells remains to be solved. Previously, we reported that phenylarsine oxide (PAO), a sulfhydryl reagent, stimulated arachidonic acid (AA) release in rat pheochromocytoma PC12 cells. In this study, we examined the effects of thimerosal, another sulfhydryl reagent, to clarify the sulfhydryl modification and activation of sPLA(2) molecules in cells. Like PAO, thimerosal-stimulated AA release in an irreversible manner and the responses were not additive. Dithiol compounds such as dithiothreitol inhibited AA release from both the thimerosal- and the PAO-treated cells, and monothiol compounds (l-Cys and glutathione) decreased the thimerosal response. Both sulfhydryl reagents stimulated AA release from the HEK293T cells expressing human sPLA(2)X, and stimulated the sPLA(2) activities of bee venom sPLA(2) and the soluble fraction of sPLA(2)X-expressing cells. Our results suggest that the sPLA(2)s in cells are inactive and modification of disulfide bonds in the molecules can be a trigger of sPLA(2) activation in cells. Sulfhydryl reagents are useful tools for studying the regulatory mechanism(s) of sPLA(2) activity in cells.

Microglia/macrophage-specific protein Iba1 binds to fimbrin and enhances its actin-bundling activity.

Ionized calcium binding adaptor molecule 1 (Iba1) is a microglia/macrophage-specific calcium-binding protein. Iba1 has the actin-bundling activity and participates in membrane ruffling and phagocytosis in activated microglia. In order to understand the Iba1-related intracellular signalling pathway in greater detail, we employed a yeast two-hybrid screen to isolate an Iba1-interacting molecule and identified another actin-bundling protein, L-fimbrin. In response to stimulation, L-fimbrin accumulated and co-localized with Iba1 in membrane ruffles induced by M-CSF-stimulation and phagocytic cups formed by IgG-opsonized beads in microglial cell line MG5. L-fimbrin was shown to associate with Iba1 in cell lysate of COS-7 expressing L-fimbrin and Iba1. By using purified proteins, direct binding of Iba1 to L-fimbrin was demonstrated by immunoprecipitation, glutathione S-transferase pull-down assays and ligand overlay assays. The binding of Iba1 was also found to increase the actin-bundling activity of L-fimbrin. These results indicate that Iba1 forms complexes with L-fimbrin in membrane ruffles and phagocytic cups, and suggest that Iba1 co-operates with L-fimbrin in modulating actin reorganization to facilitate cell migration and phagocytosis by microglia.

Macrophage/microglia-specific protein Iba1 enhances membrane ruffling and Rac activation via phospholipase C-gamma -dependent pathway.

Iba1 is a macrophage/microglia-specific calcium-binding protein that is involved in RacGTPase-dependent membrane ruffling and phagocytosis. In this study, we introduced Iba1 into Swiss 3T3 fibroblasts and demonstrated the enhancement of platelet-derived growth factor (PDGF)-induced membrane ruffling and chemotaxis. Wortmannin treatment did not completely suppressed this enhanced membrane ruffling in Iba1-expressing cells, whereas it did in Iba1-nonexpressing cells, suggesting that the enhancement is mediated through a phosphatidylinositol 3-kinase (PI3K)-independent signaling pathway. Porcine aorta endothelial cells transfected with expression constructs of Iba1 and PDGF receptor add-back mutants were used to analyze the signaling pathway responsible for the Iba1-induced enhancement of membrane ruffling. In the absence of Iba1 expression, PDGF did not induced membrane ruffling in cells expressing the Tyr-1021 receptor mutant, which is capable of activating phospholipase C-gamma (PLC-gamma) but not PI3K. In contrast, in the presence of Iba1 expression, membrane ruffling was formed in cells expressing the Tyr-1021 mutant. In addition, Rac was shown to be activated during membrane ruffling in cells expressing Iba1 and the Tyr-1021 mutant. Furthermore, dominant negative forms of PLC-gamma completely suppressed PDGF-induced Iba1-dependent membrane ruffling and Rac activation. These results indicate the existence of a novel signaling pathway where PLC-gamma activates Rac in a manner dependent on Iba1.