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.