ARTICLES The P2Y12 receptor regulates microglial activation by extracellular nucleotides Sharon E Haynes1, Gunther Hollopeter1,4, Guang Yang2, Dana Kurpius3, Michael E Dailey3, Wen-Biao Gan2 & David Julius1 Microglia are primary immune sentinels of the CNS. Following injury, these cells migrate or extend processes toward sites of tissue damage. CNS injury is accompanied by release of nucleotides, serving as signals for microglial activation or chemotaxis. Microglia express several purinoceptors, including a Gi-coupled subtype that has been implicated in ATP- and ADP-mediated migration in vitro. Here we show that microglia from mice lacking Gi-coupled P2Y12 receptors exhibit normal baseline motility but are unable to polarize, migrate or extend processes toward nucleotides in vitro or in vivo. Microglia in P2ry12–/– mice show significantly diminished directional branch extension toward sites of cortical damage in the living mouse. Moreover, P2Y12 http://www.nature.com/natureneuroscience expression is robust in the ‘resting’ state, but dramatically reduced after microglial activation. These results imply that P2Y12 is a primary site at which nucleotides act to induce microglial chemotaxis at early stages of the response to local CNS injury. In the spinal cord and brain, microglia migrate or project cellular candidate for mediating morphological responses of microglia to processes toward sites of mechanical injury or tissue damage1–3,where extracellular nucleotides. 4,5 they clear debris and release neurotrophic or neurotoxic agents .As The P2Y12 receptor was initially identified on platelets, where it such, microglial activation, or lack thereof, has been proposed to regulates their conversion from the inactive to active state during the influence degenerative and regenerative processes in the brain and clotting process12,14,15. Thus, growth of a nascent clot is dependent on 16 spinal cord. Some aspects of microglial response to injury can be the release of ADP (and other factors such as thromboxane A2) from mimicked by injection of ATP or attenuated by broad-spectrum activated platelets or damaged red blood cells, thereby promoting a Nature Publishing Group Group Nature Publishing 1 6 inhibitors of purinergic signaling , indicating that nucleotides may feed-forward mechanism to recruit platelets into the active, clotting- function as regulators of microglial behavior in vivo. competent state. Indeed, peripherally active P2Y12 antagonists such as 200 Microglia express an array of ionotropic (P2X4 and P2X7)and clopidogrel (Plavix) block platelet activation and reduce the risk of © 17 metabotropic (P2Y1,P2Y2 and P2Y12) purinergic receptors that have recurrent heart attack or stroke . Moreover, mutations in the P2Y12 been proposed to have important roles in activation, movement or receptor (in humans or mice) result in prolonged bleeding times due to 6,7 paracrine signaling by these cells . For example, activation of the the reduced ability of P2Y12-deficient platelets to change shape and 14 P2X4 receptor has been implicated in the process whereby nerve undergo coagulation in response to ADP released after vascular injury . damage produces pain hypersensitivity (neuropathic pain), a pathway In light of the importance of this receptor in platelet activation, we that may involve nucleotide-evoked release of neurotrophins (BDNF) asked whether P2Y12 might have a similar role in microglia by from microglia8. contributing to their ability to respond to injury through their capacity One hallmark of myeloid cells is their ability to undergo to detect nucleotide release. The paucity of subtype-selective agonists morphological changes in response to signaling molecules released or antagonists has hindered efforts to rigorously examine physiological after injury or disease9. In this regard, ATP- or ADP-evoked roles for specific P2 receptor subtypes in vivo, and we have therefore membrane ruffling and chemotaxis of cultured microglia have taken a genetic approach to address this question. By examining been shown to occur through a pertussis toxin–sensitive signaling microglial behavior in a variety of in vitro and in vivo systems, we pathway, thus implicating Gi-coupled receptor(s)–mediated inhibition now show that P2Y12 is indeed an essential component of the signaling of cyclic AMP synthesis10. Whereas the vast majority of metabo- pathway through which extracellular purines promote directed micro- tropic P2Y receptors transduce their signals through Gq-phospholipase glial movement following CNS injury. Moreover, our findings imply 11 Cpathways , the P2Y12 subtype is somewhat unique in that it that, as in platelets, signaling in microglia through P2Y12 is most 12,13 couples to Gi-adenylyl cyclase pathways , making it an attractive relevant during early stages of the activation process. 1Departments of Physiology & Cellular and Molecular Pharmacology, University of California, San Francisco (UCSF), 600 16th Street, San Francisco, California 94158- 2517, USA. 2Department of Physiology and Neuroscience, Skirball Institute Program in Molecular Neurobiology, New York University School of Medicine, 540 FirstAvenue, New York, New York 10016, USA. 3Department of Biological Sciences, University of Iowa, 369 Biology Building, Iowa City, Iowa 52242-1324, USA. 4Present address: Department of Biology, University of Utah, 257 South 1400 East, Salt Lake City, Utah 84112-0840, USA. Correspondence should be addressed to D.J. ([email protected]). Received 31 August; accepted 26 October; published online 19 November 2006; doi:10.1038/nn1805 1512 VOLUME 9 [ NUMBER 12 [ DECEMBER 2006 NATURE NEUROSCIENCE ARTICLES RESULTS Expression of various cell surface markers and cellular phenotypes are 20 P2Y12 is expressed on microglia but not macrophages known to change following CNS injury , and we therefore asked We, and others, have previously shown that P2Y12 transcripts are whether P2Y12 expression varies with the extent of microglial activa- expressed by glial cells of the spinal cord and brain, specifically tion. Toaddress this question, we first used an ex vivo tissue preparation 7,12 microglia . To determine whether receptor protein is located on to examine the time-course of P2Y12 expression following neural the microglial cell surface, we generated an antibody that recognizes the injury. To induce neural injury and facilitate microscopic examination C terminus of mouse P2Y12 and used this polyclonal antiserum to stain over an extended period, thick (400 mm) tissue slices were cut from sections from spinal cord and brain. Indeed, robust signals were clearly hippocampi of Cx3cr1+/GFP-expressing neonatal (postnatal days (P) observed in a population of glial cells evenly distributed throughout 4–7) mice. Over a 24-h time-course, we observed trauma-induced both white and gray matter. Identification of these cells as microglia was activation of microglia as typically characterized by their transforma- 3 confirmed by colocalization with two independent microglial markers, tion from a highly ramified to amoeboid morphology . Notably, P2Y12 including enhanced green fluorescent protein (GFP) expressed from expression showed a dramatic and continuous decrease, such that by 18 the CX3CR1 fractalkine receptor promoter and the integrin CD11b 24 h, expression was barely observable (Fig. 2a). This correlated with a (Fig. 1a,b). Moreover, P2Y12 immunoreactivity was not observed in decrement in the number of primary projections emanating from the GFAP-expressing astrocytes (Fig. 1a). Higher magnification images cell body as microglia progressed toward a more amoeboid state showed that P2Y12 protein was localized predominantly to the cell (Fig. 2b). Indeed, when P2Y12 expression was quantified on a single- surface, including the elaborate ramified processes emanating from the cell level, a linear correlation was revealed between expression level and cell body (Fig. 1c). Thus P2Y12 is poised to enable microglia to detect the morphological state of the cell (Fig. 2c–h). changes in extracellular nucleotide concentration as they survey their We next asked whether P2Y12 expression changes as a function of local environment. Notably, P2Y12-deficient mice were devoid of microglial activation in vivo. To accomplish this, we examined P2Y12 receptor immunoreactivity (Fig. 1a), but showed normal prevalence, immunoreactivity following a bolus injection of lipopolysaccharide distribution and morphology of ‘resting’ microglia (Supplementary (LPS) into the striatum, a treatment that activates microglia within the Fig. 1 online), indicating that the receptor may not be required for injected area21 and which has been shown to decrease 2-methylthio- proper development or CNS localization of these cells. ADP (2MeSADP)-evoked calcium increases in cultured mouse micro- http://www.nature.com/natureneuroscience Few markers exist that can differentiate between microglia of the glia22. Four days after LPS injection, virtually all microglia in the central nervous system and macrophages that reside in peripheral proximity of the injection site exhibited an amoeboid morphology, and tissues or infiltrate the CNS after injury. Previous in situ hybridization P2Y12 immunoreactivity was virtually undetectable in these cells studies have shown that P2Y12 transcripts are observed in microglia but (Fig. 3a). Reduced P2Y12 expression was confirmed at the transcrip- not splenic macrophages7. Here, we confirm this observation at the tional level by in situ hybridization histochemistry and northern protein level by showing that