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UDP acting at P2Y6 receptors is a mediator of microglial phagocytosis

Abstract

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 stimulated1,2 or injured3,4, extracellular nucleotides are thought to be involved in these events. In fact, ATP triggers a dynamic change in the motility of microglia in vitro5,6 and in vivo3,4, a previously unrecognized mechanism underlying microglial chemotaxis5,6; 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.

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Figure 1: Expression of P2Y6 receptor and UDP-evoked increase in [Ca2+]i in cultured microglia.
Figure 2: Changes in cell motilities of microglia.
Figure 3: Increase in P2Y 6 receptors in the hippocampus after kainic acid (KA)-treatment.
Figure 4: KA-evoked increases in extracellular uridine nucleotides and P2Y6-receptor-mediated microglial phagocytosis in vitro and in vivo. a, Schematic diagram of the experiments in vitro.

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Acknowledgements

We thank T. Shimizu and Dr. S. Ishii for providing CysLT1 receptor-expressed Chinese hamster ovary cells; K. Sakemi for technical assistance; Y. Sasaki for helpful suggestions; K. Suzuki and R. Adachi for allowing us to use the Pascal confocal microscope system; and T. Nishimaki-Mogami, Y. Ohno and T. Nagao for continuous encouragement. This study was supported in part by a grant from The National Institute of Biomedical Innovation, a grant from Uehara Memorial Foundation, a Grant-in-Aid for Scientific Research on Priority Areas, for Creative Scientific Research, Scientific Research (A and B), and for Young Scientists (A) from the Ministry of Education, Science, Sports and Culture of Japan.

Author Contributions S.K. designed most experiments, performed Ca2+ imaging and in vivo experiments and wrote the paper. Y.S.M. conducted major parts of the experiments. K.N.T. and Y.S. carried out the FACS assay and the HPLC analysis, respectively. K.O. and S.K. performed the chemotaxis analysis. B.V.J. and K.A.J. made the P2Y6 receptor antagonist MRS2578. M.T. analysed the data. K.I. analysed the data and coordinated the project. K.I. also designed the project. All authors discussed the results and commented on the manuscript.

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Correspondence to Kazuhide Inoue.

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Supplementary information

Supplementary Information

This file contains Supplementary Methods and Supplementary Figures 1-6 with Legends. (PDF 895 kb)

Supplementary Movie

This file contains Supplementary Movie showing a time-lapse image of the effect of UDP on the microglial morphogenic changes and uptake of fluorescent zymozan particles (green). This Supplementary Movie corresponds to Fig. 2c in the manuscript. (MOV 776 kb)

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Koizumi, S., Shigemoto-Mogami, Y., Nasu-Tada, K. et al. UDP acting at P2Y6 receptors is a mediator of microglial phagocytosis. Nature 446, 1091–1095 (2007). https://doi.org/10.1038/nature05704

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