Keiko Ohsawa

Microglia-specific localisation of a novel calcium binding protein, Iba1

Recently it has been shown that mRNA of Iba1 (ionized calcium binding adaptor molecule 1), which was a novel calcium binding protein cDNA-cloned by our group, is specifically expressed in microglia in cultures of rat brain cells [Imai et al. Biophys. Biochem. Res. Commun., 224 (1996) 855-862]. In the present study, immunocytochemical and immunohistochemical examinations demonstrated that Iba1 protein is expressed in microglia alone both in cultured brain cells and in the brain, respectively. In a mixed cell culture of embryonic rat brain, immunocytochemically positive for Iba1 protein were the microglia but it was not detectable in neurons, astroglia, or oligodendroglia. Immunohistochemical staining of adult rat brain sections showed Iba1 protein to be specifically localised in ramified microglia. In addition, immunohistochemical staining and immunoblot analysis of activated microglia in the facial nucleus after facial nerve axotomy shows that expression of Iba1 protein was upregulated and peaked at 7 days. These results indicated that localisation of Iba1 protein is restricted to microglia both in vitro and in vivo, and that Iba1 protein plays a role in regulating the function of microglia, especially in the activated microglia.

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 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 P2Y12 receptor (P2Y12R). Here we report discovering that the P2X4 receptor (P2X4R) is also involved in ATP‐induced microglial chemotaxis. To understand the intracellular signaling pathway downstream of P2Y12R 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 P2Y12R antagonist. These results indicate that P2Y12R‐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 P2X4R antagonists on the chemotaxis, and the results showed that pharmacological blockade of P2X4R significantly inhibited it. Knockdown of the P2X4 receptor in microglia by RNA interference through the lentivirus vector system also suppressed the microglial chemotaxis. These results indicate that P2X4R as well as P2Y12R is involved in ATP‐induced microglial chemotaxis.

Mechanisms underlying extracellular ATP-evoked interleukin-6 release in mouse microglial cell line, MG-5

Microglia play various important roles in the CNS via the synthesis of cytokines. The ATP‐evoked production of interleukin‐6 (IL‐6) and its intracellular signals were examined using a mouse microglial cell line, MG‐5. ATP, but not its metabolites, produced IL‐6 in a concentration‐dependent manner. Although ATP activated two mitogen‐activated protein kinases, i.e. p38 and extracellular signal‐regulated protein kinase, only p38 was involved in the IL‐6 induction. However, the activation of p38 was not sufficient for the IL‐6 induction because 2′‐ and 3′‐O‐(4‐benzoylbenzoyl) ATP, an agonist to P2X7 receptors, failed to produce IL‐6 despite the fact that it activated p38. Unlike in other cytokines in microglial cells, P2Y rather than P2X7 receptors seem to have a major role in the IL‐6 production by the cells. The ATP‐evoked IL‐6 production was attenuated by Gö6976, an inhibitor of Ca2+‐dependent protein kinase C (PKC). The P2Y receptor responsible for these responses was insensitive to pertussis toxin (PTX) and was linked to phospholipase C. Taken together, ATP acting on PTX‐insensitive P2Y receptors activates p38 and Ca2+‐dependent PKC, thereby resulting in the mRNA expression and release of IL‐6 in MG‐5. This is a novel pathway for the induction of cytokines in microglia.

Visualization of microglia in living tissues using Iba1-EGFP transgenic mice

Microglia are thought to play important roles not only in repairing injured tissue but in regulating neuronal activity, and visualizing the cells is very useful as a means of further investigating the function of microglia in vivo. We previously cloned the ionized calcium‐binding adaptor molecule 1 (Iba1) gene, which is expressed selectively in microglia/microphages. To generate new transgenic mice to visualize microglia with enhanced green fluorescent protein (EGFP), we here constructed a plasmid carrying EGFP cDNA under control of the Iba1 promoter. This construct was injected into C57B/6 mouse zygotes, and the Iba1‐EGFP transgenic line was developed. Fluorescent in‐situ hybridization analysis revealed that the Iba1‐EGFP transgene was located on chromosome 11D. No obvious defects were observed during development or in adulthood, and the EGFP fluorescence remained invariant over the course of at least four generations. Judging from the immunoreactivity with anti‐Iba1 antibody, all EGFP‐positive cells in the adult brain were ramified microglia. In the developing transgenic embryos, EGFP signals were detected as early as embryonic Day 10.5. The most prominent EGFP signals were found in forebrain, spinal cord, eye, foreleg, yolk sac, liver, and vessel walls. At postnatal Day 6, clear EGFP signals were observed in the supraventricular corpus callosum, known as “fountain of microglia,” where ameboid microglia migrate into the brain parenchyma and mature into ramified microglia. Iba1‐EGFP transgenic mice thus permit observation of living microglia under a fluorescence microscope and provide a useful tool for studying the function of microglia in vivo.

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.

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 P2Y12 receptors, they are transformed into a moving phenotype, showing process extension and migration toward the injured sites. Microglia upregulate adenosine A2A receptors, by which they retract the processes showing an amoeboid‐shaped, activated phenotype. Microglia also upregulate P2Y6 receptors, and if they meet UDP leaked from dead cells, microglia start to engulf and eat the dead cells as a phagocytic phenotype. The P2Y12 receptor‐mediated responses are modulated by other P2 or P1 receptors. In contrast, the P2Y6 receptor‐mediated responses were not influenced by P2Y12 receptors and vice versa. Microglia appear to use purinergic signals either cooperatively or distinctively to cause their modal shifts.

P2Y12 receptor‐mediated integrin‐β1 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 P2Y12 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‐β1. An immunoprecipitation analysis with an antibody against the active form of integrin‐β1 showed that P2Y12 mediated the integrin‐β1 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‐β1 accumulated in the tips of the microglial processes in rat hippocampal slices stimulated with ADP. These findings indicate that ATP induces the integrin‐β1 activation in microglia through P2Y12 and suggest that the integrin‐β1 activation is involved in the directional process extension by microglia in brain tissue.

Generation and characterization of a microglial cell line, MG5, derived from a p53-deficient mouse

We have established a cell line cloned from primary‐cultured microglia obtained from p53‐deficient mice. The cell line, MG5, could be grown in astrocyte‐conditioned medium and has been maintained for more than a year. MG5 cells are immunocytochemically positive for Mac‐1 and F4/80 antibody and express the major histocompatibility complex (MHC) class I antigen, leukocyte function‐associated antigen‐1, leukocyte common antigen, and intercellular adhesion molecular‐1 mRNA. Interferon‐γ enhanced the expression of MHC class II antigen mRNA in MG5 cells. We previously identified a novel calcium‐binding protein, Iba1 (ionized calcium‐binding adapter molecule 1), which is highly and specifically expressed in cultured microglia. Iba1 protein was also immunocytochemically demonstrated in MG5 cells. The cells retained non‐specific esterase activity, 5′‐nucleotidase activity, acid phosphatase activity, and phagocytic ability. Like primary cultured microglia from wild‐type mice, MG5 cells released nitric oxide in response to lipopolysaccharide, and actively proliferated in the presence of mitogenic factors such as macrophage‐colony stimulating factor (M‐CSF), granulocyte/macrophage‐CSF (GM‐CSF), and interleukin‐3 (IL‐3). Tyrosine‐phosphorylation of M‐CSF receptor in MG5 cells was induced by the addition of M‐CSF or astrocyte‐conditioned medium. These findings indicate that MG5 cells preserve the morphological, biochemical, and physiological properties of primary‐cultured microglia well. The MG5 cell line will be a useful tool for studying microglial function. GLIA 21:285–298, 1997.

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.