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.

Microglia are immune cells in the brain; their activation, migration, and proliferation have pivotal roles in brain injuries and diseases. Microglia are known to attach firmly to fibronectin, the upregulation of which is associated with several pathological conditions in the CNS, through beta1 integrin and become activated. Extracellular nucleotides can serve as potent signaling molecules. Recently, ATP and ADP were revealed to possess chemoattractive properties to microglia via Gi-coupled P2Y receptors. In the present study, we report that the ADP-induced chemotaxis of microglia is mediated by P2Y12/13 receptors and is beta1 integrin-dependent in the presence of fibronectin. Signals from P2Y12/13 receptors also cause beta1 integrin translocation to the membrane ruffle regions, but this redistribution was lost when the intracellular cyclic AMP (cAMP) was increased by forskolin or dibutyryl cAMP. This inhibitory effect of cAMP-elevating agents did not appear when microglia were co-incubated with a protein kinase A (PKA) inhibitor, KT-5720, suggesting that PKA is a negative regulator of the beta1 integrin translocation. We also show that the engagement of beta1 integrin enhanced microglial proliferation. Signals from P2Y12/13 receptors attenuated the proliferation, whereas ADP itself had no effect on microglial growth. Furthermore, beta1 integrin-induced proliferation is positively regulated by the cAMP-dependent PKA. Together, these results indicate the involvement of beta1 integrin in microglial proliferation and chemotaxis, both of which have clinical importance. The data also suggest that PKA is inversely involved in these two cellular functions.

We have recently demonstrated that the P2X4 receptor, an ATP-gated cation channel, in spinal microglia is a key molecule that mediates the mechanical allodynia induced by peripheral nerve injury. Although microglial P2X4 receptor expression is increased after peripheral nerve injury, the molecular mechanism(s) underlying its upregulation remains largely unknown. Fibronectin is a member of the extracellular matrix molecules and is actively produced in response to injury and diseases in the CNS. Here, we describe the influence of fibronectin on P2X4 receptor expression in microglia and the upregulation of fibronectin after peripheral nerve injury. Microglia that were cultured on fibronectin-coated dishes showed a marked increase in P2X4 receptor expression, both at the mRNA and protein levels, as compared to those cultured on control dishes. Fibronectin also enhanced the microglial Ca2+ responses mediated by P2X4 receptors. Moreover, Western blot examination of the spinal cord from a rat with spinal nerve injury indicated that fibronectin was upregulated on the ipsilateral side. Interestingly, intrathecal injection of ATP-stimulated microglia to the rat lumber spinal cord revealed that microglia cultured on fibronectin-coated dishes was more effective in the induction of allodynia than microglia cultured on control dishes. Taken together, our results suggest that spinal fibronectin is elevated after the peripheral nerve injury and it may be involved in the upregulation of the P2X4 receptor in microglia, which leads to the induction of neuropathic pain.

We have previously shown that activation of the ATP-gated ion channel subtype P2X(4) receptors (P2X(4)Rs) in the spinal cord, the expression of which is upregulated in microglia after nerve injury, is necessary for producing neuropathic pain. The upregulation of P2X(4)Rs in microglia is, therefore, a key process in neuropathic pain, but the mechanism remains unknown. Here, we find a fibronectin/integrin-dependent mechanism in the upregulation of P2X(4)Rs. Microglia cultured on dishes coated with fibronectin, an extracellular matrix molecule, expressed a higher level of P2X(4)R protein when compared with those cultured on control dishes. The increase was suppressed by echistatin, a peptide that selectively blocks beta(1) and beta(3)-containing integrins, and with a function-blocking antibody of beta(1) integrin. In in vivo studies, the upregulation of P2X(4)Rs in the spinal cord after spinal nerve injury was significantly suppressed by intrathecal administration of echistatin. Tactile allodynia in response to nerve injury and intrathecal administration of ATP- and fibronectin-stimulated microglia was inhibited by echistatin. Furthermore, intrathecal administration of fibronectin in normal rats increased the level of P2X(4)R protein in the spinal cord and produced tactile allodynia. Moreover, the fibronectin-induced allodynia was not observed in mice lacking P2X(4)R. Taken together with the results of our previous study showing an increase in the spinal fibronectin level after nerve injury, the present results suggest that the fibronectin/integrin system participates in the upregulation of P2X(4)R expression after nerve injury and subsequent neuropathic pain.

Glial cells have various important roles in regulation of brain functions. For such events, extracellular nucleotides/P2 receptors have central roles. Although there have been huge amount of literature about activation of P2 receptors and glial functions, little is known about what happens in glia or the brain if glial P2 receptor is inhibited. Here we show that the inhibition of P2 receptors in astrocytes, the most abundant glial cells and cause a constitutive release of nucleotides, resulted in secretion of metalloproteinase-9 (MMP-9), a metal-dependent endopeptidase that degrades extracellular matrix molecules and is important in regulation of brain remodeling. When cultured astrocytes were treated with apyrase (ecto-nucleotidase), reactive blue 2 (P2 receptor antagonist), and pertussis toxin, they secreted MMP-9, suggesting that Gi-coupled P2Y receptor-mediated signals constitutively suppress the production of MMP-9. Among Gi-coupled P2Y receptors, we found that an inhibition of P2Y(14) receptor, a receptor for nucleotide-sugars such as UDP-glucose, is responsible for the production of MMP-9 by pharmacological and molecular biochemical analysis. As for the mechanisms, the inhibition of P2Y(14) receptors resulted in the release of tumor necrosis factor (TNF)-α which then acted on astrocytes to induce MMP-9. Taken together, our results suggest that the constitutive releases of nucleotide-sugars in astrocytes should play an important role in maintaining the normal status of the cell, through Gi-coupled P2Y(14) receptors, and when the signal is removed, the cells start to release TNF-α, which then acts on astrocytes in a feedback fashion to boost MMP-9 synthesis and secretion.