Yui Koga

Activation of P2X7 receptors induces CCL3 production in microglial cells through transcription factor NFAT

Microglia are implicated as a source of diverse proinflammatory factors in the CNS. Extracellular nucleotides are well known to be potent activators of glial cells and trigger the release of cytokines from microglia through purinergic receptors. However, little is known about the role of purinoceptors in microglial chemokine release. In this study, we found that high concentrations of ATP evoked release of CC‐chemokine ligand 3 (CCL3)/macrophage inflammatory protein‐1α from MG‐5 cells, a mouse microglial cell line, and rapid up‐regulation of CCL3 mRNA was elicited within 30 min of ATP stimulation. The release of CCL3 was also stimulated by 2′‐ and 3′‐O‐(4‐benzoylbenzoyl) ATP, an agonist of P2X7 receptors. Brilliant Blue G, an antagonist of P2X7 receptors, strongly inhibited this ATP‐induced CCL3 release. Similar pharmacological profile was observed in primary microglia. In MG‐5 cells, ATP caused de‐phosphorylation and nuclear translocation of the transcription factor nuclear factor of activated T cells (NFAT). ATP‐induced NFAT de‐phosphorylation was also dependent on P2X7 receptor activation. Furthermore, ATP‐induced CCL3 release and production were prevented by a selective inhibitor of NFAT. Taken together, the results of this study demonstrate an involvement of NFAT in the mechanism underlying P2X7 receptor‐mediated CCL3 release.

Involvement of protein kinase D in uridine diphosphate-induced microglial macropinocytosis and phagocytosis.

The clearance of tissue debris by microglia is a crucial component of maintaining brain homeostasis. Microglia continuously survey the brain parenchyma and utilize extracellular nucleotides to trigger the initiation of their dynamic responses. Extracellular uridine diphosphate (UDP), which leaks or is released from damaged neurons, has been reported to stimulate the phagocytotic activity of microglia through P2Y6 receptor activation. However, the intracellular mechanisms underlying microglial P2Y6 receptor signals have not been identified. In this study, we demonstrated that UDP stimulation induced immediate and long‐lasting dynamic movements in the cell membrane. After 60 min of UDP stimulation, there was an upregulation in the number of large vacuoles formed in the cell that incorporate extracellular fluorescent‐labeled dextran, which indicates microglial macropinocytosis. In addition, UDP‐induced vacuole formation and continuous membrane motility were suppressed by the protein kinase D (PKD) inhibitors, Gö6976 and CID755673, unlike Gö6983, which is far less sensitive to PKD. The inhibition of PKD also reduced UDP‐induced incorporation of fluorescent‐labeled dextran and soluble β‐amyloid and phagocytosis of microspheres. UDP induced rapid phosphorylation and membrane translocation of PKD, which was abrogated by the inhibition of protein kinase C (PKC) with Gö6983. However, Gö6983 failed to suppress UDP‐induced incorporation of microspheres. Finally, we found that inhibition of PKD by CID755673 significantly suppressed UDP‐induced engulfment of IgG‐opsonized microspheres. These data suggest that a PKC‐independent function of PKD regulates UDP‐induced membrane movement and contributes to the increased uptake of extracellular fluid and microspheres in microglia.

Age-specific onset and distribution of the natural anticoagulant deficiency in pediatric thromboembolism

Background:The early diagnosis of inherited thrombophilia in children is challenging because of the rarity and hemostatic maturation.Methods:We explored protein C (PC), protein S (PS), and antithrombin (AT) deficiencies in 306 thromboembolic patients aged ≤20 y using the screening of plasma activity and genetic analysis.Results:Reduced activities were determined in 122 patients (40%). Low PC patients were most frequently found in the lowest age group (0–2 y, 45%), while low PS or low AT patients were found in the highest age group (16–20 y; PS: 30% and AT: 20%). Genetic study was completed in 62 patients having no other causes of thromboembolism. Mutations were determined in 18 patients (8 PC, 8 PS, and 2 AT genes). Six of eight patients with PC gene mutation were found in age 0–2 y (75%), while six of eight patients with PS gene mutation were in 7–20 y. Two AT gene–mutated patients were older than 4 y. Four PC-deficient and two PS-deficient patients carried compound heterozygous mutations. All but one PC gene–mutated patient suffered from intracranial thromboembolism, while PS/AT gene–mutated patients mostly developed extracranial venous thromboembolism.Conclusion:Stroke in low PC infants and deep vein thrombosis in low PS/AT school age children could be targeted for genetic screening of pediatric thrombophilias.

Involvement of vasodilator-stimulated phosphoprotein in UDP-induced microglial actin aggregation via PKC- and Rho-dependent pathways

Microglia are major immunocompetent cells in the central nervous system and retain highly dynamic motility. The processes which allow these cells to move, such as chemotaxis and phagocytosis, are considered part of their functions and are closely related to purinergic signaling. Previously, we reported that the activation of the P2Y6 receptor by UDP stimulation in microglia evoked dynamic cell motility which enhanced their phagocytic capacity, as reported by Koizumi et al. (Nature 446(7139):1091–1095, 2007). These responses require actin cytoskeletal rearrangement, which is seen after UDP stimulation. However, the intracellular signaling pathway has not been defined. In this study, we found that UDP in rat primary microglia rapidly induced the transient phosphorylation at Ser157 of vasodilator-stimulated phosphoprotein (VASP). VASP, one of actin binding protein, accumulated at the plasma membrane where filamentous (F)-actin aggregated in a time-dependent manner. The phosphorylation of VASP was suppressed by inhibition of PKC. UDP-induced local actin aggregations were also abrogated by PKC inhibitors. The Rho inhibitor CT04 and the expression of p115-RGS, which suppresses G12/13 signaling, attenuated UDP-induced phosphorylation of VASP and actin aggregation. These results indicate that PKC- and Rho-dependent phosphorylation of VASP is involved in UDP-induced actin aggregation of microglia.

The clinical presentation and genotype of protein C deficiency with double mutations of the protein C gene

Severe protein C (PC) deficiency is a rare heritable thrombophilia leading to thromboembolic events during the neonatal period. It remains unclear how individuals with complete PC gene (PROC) defects develop or escape neonatal stroke or purpura fulminans (PF).