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The initial microglial responses that occur after brain injury and in various neurological diseases are characterized by microglial accumulation in the affected sites of brain that results from the migration and proliferation of these cells. The early-phase signal responsible for this accumulation is likely to be transduced by rapidly diffusible factors. In this study, the possibility of ATP released from injured neurons and nerve terminals affecting cell motility was determined in rat primary cultured microglia. Extracellular ATP and ADP induced membrane ruffling and markedly enhanced chemokinesis in Boyden chamber assay. Further analyses using the Dunn chemotaxis chamber assay, which allows direct observation of cell movement, revealed that both ATP and ADP induced chemotaxis of microglia. The elimination of extracellular calcium or treatment with pyridoxalphosphate-6-azophenyl-2Ј,4Ј-disulphonic acid, suramin, or adenosine-3Ј-phosphate-5Ј-phosphosulfate did not inhibit ATP-or ADP-induced membrane ruffling, whereas AR-C69931MX or pertussis toxin treatments clearly did so. As an intracellular signaling molecule underlying these phenomena, the small G-protein Rac was activated by ATP and ADP stimulation, and its activation was also inhibited by pretreatment with pertussis toxin. These results strongly suggest that membrane ruffling and chemotaxis of microglia induced by ATP or ADP are mediated by G i/o -coupled P2Y receptors. Key words: microglia; ATP; ADP; membrane ruffling; chemotaxis; G i/o -coupled P2Y receptorsAccumulated evidence suggests that extracellular ATP functions in various tissues and cells (Dubyak and El-Moatassim, 1993). The roles of extracellular ATP as a neurotransmitter and neuromodulator in the CNS have been well documented. For example, ATP induces excitation and increases in calcium in various neurons in the brain (Edwards et al., 1992;Shen and North, 1993;Chen et al., 1994;Inoue et al., 1995;Nabekura et al., 1995). In addition to the role played by ATP in neurons, effects of ATP on glial cells have also been demonstrated. In astrocytes, for example, DNA synthesis, process formation, and the increase in the expression of glial fibrillary acidic protein (Neary et al., 1994), arachidonic acid release (Chen and Chen, 1998), Erk activation (Neary et al., 1999), and calcium wave propagation (Scemes et al., 2000) were reported to be stimulated by ATP. Ca 2ϩ release from internal stores by ATP stimulation was also reported in oligodendrocytes (Kirischuk et al., 1995). This evidence suggests diverse roles of extracellular ATP in the CNS.Reports have shown that ATP stimulates microglia, another kind of glial cell in the CNS, to release various biologically active substances, such as interleukin-1␤ (Ferrari et al., 1996(Ferrari et al., , 1997, plasminogen (Inoue et al., 1998), and tumor necrosis factor-␣ (Hide et al., 2000). Microglial cell death was also demonstrated after stimulation with high-dose ATP (Ferrari et al., 1999). After neuronal damage, microglia migrate to the affected sites, where the...

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.

Microglia are reactively activated by various environmental stimulations caused by brain injury or disease. Activated microglia exhibit morphological transformation, proliferation, migration, phagocytosis, and the production of bioactive molecules. Various molecules are reported and suggested to activate microglia. Among them, macrophage-colony-stimulating factor (M-CSF) is considered one of the most convincing candidates responsible for maintaining activation properties of microglia. Therefore, the focus of the present study is on intracellular molecular events that arise downstream of M-CSF stimulation. M-CSF activates its receptor, Fms tyrosine kinase, and Fms sequentially activates a number of signaling molecules, including PI3K or phospholipase C␥ (PLC␥). Stimulation of continuing signaling cascades results in the activation of a small GTPase, Rac, the key molecule in microglia activation. Rac is known to be activated downstream of receptor tyrosine kinases and to regulate reorganization of the actin cytoskeleton, which profoundly underlies the above-mentioned properties of activated microglia. Iba1, a macrophage/microglia-specific calcium-binding protein, was identified by our group and was shown to be involved in the Rac signaling pathway. Further, we introduce a novel signaling pathway in which Rac is activated, dependent on PLC␥ and Iba1. However, to understand the molecular details of microglia activation, future work is required. GLIA 40: 164 -174, 2002.