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* Transgenic Animal Research Center, National Institute of Agrobiological Sciences, Tsukuba, Japan;
Laboratory for Chemistry and Metabolism, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan;
Research Center for Prion Diseases, National Institute of Animal Health, Tsukuba, Japan;
Department of Physiology, Nippon Medical School, Tokyo, Japan; and
¶ Faculty of Pharmaceutical Sciences, Tokyo University of Science, Noda-shi, Japan
Recently, autophagy has been associated with the TLR signaling pathway to eliminate intracellular pathogens in the innate immune system. However, it is unknown if other pathways regulate autophagy during the immunologic response. Given the critical role of the purinergic P2X7 receptor (P2X7R) pathway during various immunologic functions (i.e., caspase activation and IL-1β secretion), the principal objective here was to determine whether the P2X7R pathway may regulate autophagy in immune cells. We observed in both MG6 mouse microglial cells and primary microglia that activation of P2X7R by ATP increases the expression of microtubule-associated protein 1 light chain 3 (LC3)-II, the autophagosomal membrane-associated form of LC3, in an extracellular Ca2+-dependent manner. Consistent with this, immunohistochemistry showed extensive formation of LC3-immunopositive dots, and electron microscopy demonstrated accumulation of autophagosomes and autophagolysosomes in ATP-treated cells. Importantly, the up-regulation of LC3-II by P2X7R activation was not affected by autophagy inhibitors, such as 3-methyladenine and PI3K inhibitors. Furthermore, while lysosomal functions were impaired by ATP treatment, autophagolysosomal components were released into the extracellular space. Similarly, a phagocytosis assay using Escherichia coli BioParticles showed that phagosome maturation was impaired in ATP-treated cells and a robust release of LC3-immunopositive phagolysosomes was induced along with a radial extension of microtubule bundles. Taken together, the data suggest a novel mechanism whereby the P2X7R signaling pathway may negatively regulate autophagic flux through the impairment of lysosomal functions, leading to stimulation of a release of autophagolysosomes/phagolysosomes into the extracellular space.
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1 This work was supported by a research grant and a Grant-in-Aid from the BSE Control Project of the Ministry of Agriculture, Forestry, and Fisheries in Japan; by Grants-in-Aid for Young Scientists (category B) and Exploratory Research from the Ministry of Education, Science, Sports and Technology of Japan; and by research grants from The Naito Foundation and The Takeda Foundation.
2 Address correspondence and reprint requests to Dr. Makoto Hashimoto, Laboratory for Chemistry and Metabolism, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo 183-8526, Japan. E-mail address: hashimoto-mk{at}igakuken.or.jp
3 Abbreviations used in this paper: P2X7R, P2X7 receptor; BzATP, 3'-O-(4-benzoyl)benzoyl-ATP; BBG, brilliant blue G; oATP, oxidized ATP; LC3B, microtubule-associated protein 1 light chain 3B; 3-MA, 3-methyladenine; 2,3-DPG, 2,3-diphosphoglycerate; AMPK, AMP-activated protein kinase; S6K, S6 kinase; mTOR, mammalian targent of rapamycin; TfR, transferrin receptor; HBSS, HEPES-buffered salt solution; PLD, phospholipase D; [Ca2+]i, intracellular Ca2+ concentration.
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