Transformation of Astrocytes to a Neuroprotective Phenotype by Microglia via P2Y1 Receptor Downregulation

Article

Transformation of Astrocytes to a Neuroprotective Phenotype by Microglia via P2Y1 Receptor Downregulation Graphical Abstract

Authors Youichi Shinozaki, Keisuke Shibata, Keitaro Yoshida, ..., Kazuhiro Ikenaka, Kenji F. Tanaka, Schuichi Koizumi

Correspondence [email protected]

In Brief Shinozaki et al. show that microglia transform astrocytes into a neuroprotective phenotype via downregulation of astrocytic P2Y1 receptors. This is critical for neuroprotection against traumatic brain injury.

Highlights d

Microglia transform astrocytes into a neuroprotective phenotype after brain trauma

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Microglia downregulate astrocytic P2Y1 receptors

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Downregulation of P2Y1 receptors in astrocytes is critical for their transformation

Shinozaki et al., 2017, Cell Reports 19, 1151–1164 May 9, 2017 ª 2017 The Authors. http://dx.doi.org/10.1016/j.celrep.2017.04.047

Cell Reports

Article Transformation of Astrocytes to a Neuroprotective Phenotype by Microglia via P2Y1 Receptor Downregulation Youichi Shinozaki,1 Keisuke Shibata,1 Keitaro Yoshida,2 Eiji Shigetomi,1 Christian Gachet,3 Kazuhiro Ikenaka,4 Kenji F. Tanaka,2 and Schuichi Koizumi1,5,* 1Department of Neuropharmacology, Interdisciplinary Graduate School of Medicine, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi 409-3898, Japan 2Department of Neuropsychiatry, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo 160-8582, Japan 3Institut National de la Sante ´ et de la Recherche Me´dicale (INSERM), U.311, Etablissement de Transfusion Sanguine, 10, rue Spielmann, B.P. 36, 67065 Strasbourg, France 4Division of Neurobiology and Bioinformatics, National Institute for Physiological Sciences, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi 444-8787, Japan 5Lead Contact *Correspondence: [email protected] http://dx.doi.org/10.1016/j.celrep.2017.04.047

SUMMARY

Microglia and astrocytes become reactive following traumatic brain injury (TBI). However, the coordination of this reactivity and its relation to pathophysiology are unclear. Here, we show that microglia transform astrocytes into a neuroprotective phenotype via downregulation of the P2Y1 purinergic receptor. TBI initially caused microglial activation in the injury core, followed by reactive astrogliosis in the peri-injured region and formation of a neuroprotective astrocyte scar. Equivalent changes to astrocytes were observed in vitro after injury. This change in astrocyte phenotype resulted from P2Y1 receptor downregulation, mediated by microglia-derived cytokines. In mice, astrocyte-specific P2Y1 receptor overexpression (Astro-P2Y1OE) counteracted scar formation, while astrocyte-specific P2Y1 receptor knockdown (Astro-P2Y1KD) facilitated scar formation, suggesting critical roles of P2Y1 receptors in the transformation. Astro-P2Y1OE and AstroP2Y1KD mice showed increased and reduced neuronal damage, respectively. Altogether, our findings indicate that microglia-astrocyte interaction, involving a purinergic signal, is essential for the formation of neuroprotective astrocytes. INTRODUCTION Under pathological conditions in the CNS, two types of glial cells, microglia and astrocytes, become reactive. Although the association of pathology with reactivity is widely recognized, it is unclear how these glial cells cooperate with each other and what the consequences of this cooperation are for pathophysi-

ology. Microglia are resident immune cells in the brain that continuously monitor the brain microenvironment (Nimmerjahn et al., 2005). They have a very low threshold of activation and rapidly respond within 20–40 min of injury (Davalos et al., 2005). Astrocytes, another major type of glial cell, are known to participate in CNS homeostasis (Allaman et al., 2011), synapse formation (Clarke and Barres, 2013), and synapse function (Halassa and Haydon, 2010). Under pathological conditions, astrocytes also change their phenotype from a resting to a reactive form, characterized by hypertrophic cell bodies and processes. These cells are referred to as reactive astrocytes (Sofroniew, 2009). In severe cases, the reactive astrocytes form a characteristic structure called an astrocyte scar, which prevents infiltration of peripheral inflammatory cells or molecules, thereby protecting normal tissue (Bush et al., 1999; Herrmann et al., 2008; Okada et al., 2006; Wanner et al., 2013). Although many reports have shown that reactive astrogliosis is a hallmark of the neuroprotective reaction of astrocytes against CNS disorders such as traumatic brain injury, the detailed mechanisms underlying reactive astrogliosis and induction of the neuroprotective effect have not been fully determined. Regarding the temporal pattern of glial changes, microglia are the first cell type to respond following CNS disruption, and this is followed by reactive astrogliosis. Microglia are activated before astrocytes in cases of neuropathic pain (Shibata et al., 2011), amyotrophic lateral sclerosis (Alexianu et al., 2001), ischemia or reperfusion (Lambertsen et al., 2005; Petito et al., 1990), Huntington’s disease (Faideau et al., 2010; Palazuelos et al., 2009; Sapp et al., 2001; Tai et al., 2007; Tong et al., 2014), and traumatic brain injury (Batchelor et al., 1999; d’Avila et al., 2012). Although the initial activation of microglia is well characterized, it is unclear whether or how this activation affects reactive astrogliosis. Extracellular ATP and P2 receptors are essential for the microglial activation mechanism (Inoue, 2008). Under pathological conditions, nucleotides such as ATP are released or leaked from injured cells and function as ‘‘find me’’ (Elliott et al., 2009)

Cell Reports 19, 1151–1164, May 9, 2017 ª 2017 The Authors. 1151 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Figure 1. Minocycline Alters the Response of Astrocytes to Brain Injury (A) Temporal analysis of expression changes in GFAP and IBA1. The level of IBA1, but not of GFAP, was increased at 1 dpi. Bar graph shows relative intensities for GFAP and IBA1 bands (*p < 0.05, **p < 0.01 for GFAP, ##p < 0.01 for IBA1 versus sham control, one-way ANOVA followed by Fisher’s LSD test). (B) Spatiotemporal patterns for reactive astrocytes and activated microglia. No GFAP signals were observed in naive or 1 dpi brains. GFAP+ astrocytes surrounded the injured core area at 3 dpi. No GFAP+ cells were observed in the core (asterisk). Microglia already showed amoeboid morphology in the injury core at 1 dpi and showed accumulation at 3 dpi. (C) Fluoro-Jade (FJ) signals were observed only in the ipsilateral side at 3 dpi (asterisk). (D) Reactive astrocytes surround damaged neurons at 3 dpi. (E) Microglial localization. At 3 dpi, microglia adjacent to FJ+ cells showed activated forms, including amoeboid (area 1), phagocytosing (areas 2 and 3), and process extended (area 4, arrows).

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or ‘‘eat me’’ (Koizumi et al., 2007) signals to evoke process extension, chemotaxis, and phagocytosis by microglia. P2 receptor activation also induces cytokine production from microglia, including interleukin-1b (IL-1b), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-a) (Ferrari et al., 1997; Friedle et al., 2011; Suzuki et al., 2004). Such pro-inflammatory mediators have been shown to dynamically change G proteincoupled receptor (GPCR) expression and function in astrocytes (Hamby et al., 2012). Because inositol triphosphate (IP3)dependent Ca2+ signaling, a downstream target of GTP-binding protein subunit alpha q (Gq)-coupled GPCRs, is essential for reactive astrogliosis (Kanemaru et al., 2013), it is conceivable that microglial activation can affect astrocyte responses after brain injury. Here, we demonstrate that the initial microglia response to traumatic brain injury (TBI) dynamically changes the astrocyte response to TBI. During this response, microglia-derived proinflammatory cytokines play a central role. Astrocytes become reactive, elongate their processes, and acquire a neuroprotective capacity, mechanisms for which downregulation of the P2Y1 receptor is critical. RESULTS Microglial Activation Occurs before Reactive Astrogliosis but after TBI We first investigated spatiotemporal patterns of microglial and astrocytic changes after TBI (Figure S1A). Western blot analysis showed that glial fibrillary acidic protein (GFAP), a reactive astrocyte marker, was not changed at 1 day post-injury (dpi) but was upregulated at 3 dpi in the ipsilateral side of the cortex (Figure 1A). Ionized calcium-binding adaptor molecule 1 (IBA1), a microglia marker, was already upregulated at 1 dpi. Immunohistochemistry showed that cortical GFAP+ cells were not observed in naive brains or at 1 dpi but were observed at 3 dpi (Figure 1B, GFAP) and formed an astrocyte scar surrounding the injury core (Figure 1B, GFAP, 3 dpi, asterisk). GFAP signals were co-localized with other astrocyte markers such as S100b, Vimentin, and ALDH1L1 (Figure S1B) (Cahoy et al., 2008), indicating that these GFAP+ cells surrounding the lesion were reactive astrocytes. Microglia in the injury core at 1 dpi were already activated with retracted processes and hypertrophic cell bodies (Figure 1B, IBA1, 1 dpi). At 3 dpi, they were accumulated at the injury core (Figure 1B, IBA1, 3 dpi, asterisk). No reactive astrocytes or microglia were observed in the contralateral side of the cortex (Figures S1C and S1D). Staining with Fluoro-Jade (FJ), which selectively labels dying neurons (Schmued and Hopkins, 2000), revealed an increased number of damaged neurons in the ipsilat-

eral side at 3 dpi (Figure 1C, asterisk; Figure S1E). The FluoroJade-positive (FJ+) cells were co-localized with a neuronal marker, NeuN, and with the apoptosis markers single-stranded DNA (ssDNA) and active caspase-3 (cas3) (Figure S1F). The number of FJ+ cells was increased after TBI and peaked around 3–5 dpi (Figures S1G and S1H). At 3 dpi, reactive astrocytes surrounded FJ+ neurons (Figures 1D and 1F) and the activated microglia were in close apposition to FJ+ neurons (Figures 1E and 1F). The distances of astrocytes from the core and the number of FJ+ cells were positively correlated (Figure S1I). To confirm that microglia respond earlier than astrocytes, we employed an in vitro traumatic injury model (Figure S2A). After injury, astrocytes elongated their processes toward the scratched area (Figure S2B, in vitro), resembling those in vivo (Figure S2B, in vivo). In contrast, microglia migrated to the injured area in vitro (Figure S2C). The speed of microglial migration within 1 hr after injury was faster than that of process elongation by astrocytes. Astrocytes started process elongation 6 hr after injury (Figure S2D). Microglial migration was clearly blocked by apyrase (ATP-degrading enzyme, 10 U/mL), clopidogrel (P2Y12 receptor antagonist, 1 mM), and minocycline (a microglial inhibitor, 100 mM) (Figures S2E and S2F) (Yrja¨nheikki et al., 1998), indicating involvement of P2Y12 receptors (Haynes et al., 2006). The extracellular level of ATP in the culture transiently increased during microglial migration (<3 hr) (Figure S3A), and in vivo microdialysis showed a transient increase in extracellular ATP levels when only microglia were activated (i.e., 6 hr
1 dpi) after brain injury (Figure S3B). In accordance with the reduced level of extracellular ATP, the level of Ntpdase1 mRNA was upregulated at 3 dpi, whereas Ntpdase2 mRNA levels did not change (Figure S3C). Enzyme histochemistry also showed increased ATPase activity in the injury core (Figure S3D). Small and strong ATPase signals were co-localized with a marker for activated microglia, CD11b (Figure S3E). Microglia Enhance the Astrocyte Reaction In Vitro and In Vivo To explore the microglia-regulated astrocyte reaction in vivo, we employed minocycline (50 mg/kg intraperitoneally [i.p.]). Minocycline increased the size of the injury core area and the distances of surrounding reactive astrocytes from the injury core (Figures 1G–1I) and suppressed microglial activation (Figures 1L and 1M). Inhibition of microglial function by clopidogrel (25 mg/kg i.p.), a selective P2Y12 receptor antagonist, also increased the core size (Figures 1G and 1J). Furthermore, depletion of microglia by PLX5622 (1,200 ppm in chow diet from 7 days before to 3 days after TBI) (Valdearcos et al., 2014), antagonist for colony-stimulating factor 1 receptor, also increased the injury

(F) Quantitative data for distances of FJ+-damaged neurons, IBA1+ activated microglia, and GFAP+ reactive astrocytes from the center of the lesion (n = 306–464, **p < 0.01, one-way ANOVA followed by Fisher’s LSD test). (G) Representative images for astrocyte scars after TBI treated without (control), with minocycline (mino), clopidogrel (clo), or PLX5622 (PLX). (H and I) Mino significantly increased the distance of bordering astrocytes to the injury core (H) and the injury core size (I). (J and K) Clo-treated (J) or PLX-treated (K) mice also showed an increase in the injury core size (n = 5, *p < 0.05, **p < 0.01, Mann-Whitney U test). (L) Minocycline (50 mg/kg i.p.) administration transformed the amoeboid shape of microglia in the injury core at 3 dpi into cells that have a small cell body with long branched processes (areas 1 and 3). The microglia far from the injury site showed ramified morphology regardless of minocycline administration. (M) Minocycline (mino) administration decreased the number of activated microglia (n = 5, **p < 0.01, unpaired t test). Values are the mean ± SEM for (A), (F), (H)–(K), and (M). Scale bars, 20 mm for (B), (E), and (L); 50 mm for (D) and (E); 100 mm for (B), (G), and (L); and 500 mm for (C).

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Figure 2. Microglia-Derived Factors Accelerate Astrocyte Reaction In Vitro (A) Process extension of astrocytes in the absence or presence of microglia after traumatic injury in vitro. Microglia dramatically enhanced astrocyte process extension speed toward the scratched area. Scale bar, 20 mm. (B) Enhancement of astrocyte process extension by microglia was suppressed by minocycline (mino, 100 mM) (n = 63–108, **p < 0.01 versus astrocytes, ##p < 0.01 versus astrocytes plus microglia, one-way ANOVA followed by Fisher’s LSD test). Without microglia, minocycline did not affect the speed of astrocyte process extension. (C) Co-culture of microglia and astrocytes without direct contact accelerates astrocyte process extension. In the presence of microglia in a culture insert, astrocyte process elongation was significantly promoted (n = 107–606, **p < 0.01, unpaired t test). (D and E) Conditioned media from astrocyte and microglia mixed culture (D) or microglial monoculture (E) significantly enhanced astrocyte process extension (n = 350–606 for mixed culture and 110–130 for microglial culture, **p < 0.01, unpaired t test). Values are the mean ± SEM for (B)–(E).

core size at 3 dpi (Figures 1G and 1K). Under this condition, more than 90% of microglia were ablated (data not shown). These data show that suppression of microglia can change astrocyte responses. We further tested microglia-regulated reactive astrogliosis using an in vitro traumatic injury model. In the presence of microglia, the speed of astrocyte process elongation was accelerated (Figure 2A; Movies S1 and S2); however, the accelerated elongation was blocked by minocycline (100 mM) (Figure 2B). The effect of astrocyte co-culture with microglia was reproduced by co-culture on a culture insert or culturing astrocytes with conditioned media from a mixed glial culture or a microglial monoculture (Figures 2C–2E; Figure S4A). Our data show that early microglial activation promotes astrocyte responses after brain injury. We then asked what molecule or molecules mediate the regulation of astrocytes by microglia. Because it is known that cytokines regulate reactive astrogliosis (Robel et al., 2011) and that extracellular ATP induces cytokine release from microglia (Ferrari et al., 1997; Friedle et al., 2011; Suzuki et al., 2004), we next investigated cytokine expression at 1 dpi in vivo, at which time only microglia are activated. We found significant increases in mRNA levels for Tnfa, Il6, and Il1b, but not for Ifng and Tgfb1, in the ipsilateral cortex (Figure S4B). Immunohistochemistry showed that most TNF-a, IL-1b, and IL-6 signals overlapped with those of IBA1 in close apposition to the injury core (TNF-a, 72.9% ± 4.8%; IL-1b, 78.3% ± 6.8%; IL-6, 82.5% ± 3.6%) (Figure S4C). The enhancement of astrocyte process elongation by microglia was suppressed by a blocking antibody mixture against TNF-a, IL-6, and IL-1b (Figures S4A and S4D). Conversely, a recombinant cytokine mixture (IL-6, IL-1b, and TNF-a) promoted process extension (Figures S4A and S4E). These data show that inflammatory cytokines are essential for microgliaenhanced reactive astrogliosis.

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Blockade of P2Y1 Receptors Promotes Astrocyte Responses after Traumatic Injury In Vitro We then asked what molecule or molecules modify astrocyte responses after traumatic injury. It has been reported that inflammatory cytokines change GPCR expression levels and function in astrocytes (Hamby et al., 2012), and that downstream signaling of GPCRs is essential for astrocyte responses after TBI (Kanemaru et al., 2013). We, therefore, examined the roles of gliotransmitter-activated GPCRs on astrocytes in vitro. PPADS (100 mM) and RB2 (10 mM), P2 receptor antagonists, enhanced the astrocyte response; however, the astrocyte response was not enhanced by XAC (P1 receptor antagonist, 10 mM) (Figure 3A). Among selective P2 receptor antagonists, only the P2Y1 receptor antagonist, MRS2179 (1 mM), promoted astrocyte responses; responses were not observed following treatment with MRS2578 (P2Y6 receptor, 3 mM), NF340 (P2Y11 receptor, 10 mM), brilliant blue G (BBG) (P2X7 receptor, 1 mM), or pertussis toxin (PTX) (to block GTP-binding protein subunit alpha i [Gi]-coupled P2Y12 and P2Y13 receptors, 100 ng/mL) (Figure 3B). In addition, astrocytes prepared from P2Y1 receptor knockout (P2Y1KO) mice exhibited faster process extension (Figure 3C). No further enhancement of process extension was observed in P2Y1KO astrocytes mixed with wild-type (WT) microglia (Figure 3D). Therefore, we considered downregulation of the P2Y1 receptor to be essential for the enhanced responses of astrocytes against traumatic injury. Microglia Control P2Y1 Receptor Levels of Astrocytes In Vitro and Mediate the TBI-Induced Reduction of P2Y1 Receptor Levels of Astrocytes In Vivo To address the possible interaction between inflammatory cytokines and P2Y1 receptors, we examined the role of inflammatory cytokines on the microglia-mediated P2Y1 receptor

Figure 3. Downregulation of P2Y1 Receptors Promotes Astrocyte Process Extension (A) P2 receptor antagonists accelerate the astrocytic reaction. RB2 and PPADS (10 and 100 mM), but not XAC (10 mM), promoted process extension after injury in vitro (n = 206–606, **p < 0.01 versus control, one-way ANOVA followed by Fisher’s LSD test). (B) P2Y1 receptor regulates the astrocyte reaction. MRS2179 (1 mM) promoted astrocyte process extension, but MRS2578 (3 mM), NF340 (10 mM), pertussis toxin (PTX, 100 ng/mL), and brilliant blue G (BBG, 1 mM) did not (n = 140–606, **p < 0.01 versus control, one-way ANOVA followed by Fisher’s LSD test). All antagonists were applied 30 min before scratching. (C) P2Y1KO accelerated the extension of processes (n = 140–606, **p < 0.01, unpaired t test). (D) No additional enhancement of process extension in P2Y1KO astrocytes was observed with the addition of WT microglia (n = 102–129, p = 0.11, unpaired t test). (E and F) P2Y1 receptor expression in astrocytes is regulated by microglia-derived cytokines. P2Y1 receptor levels (estimated at 50 kDa) were decreased in astrocytes following addition of (E) microglia or (F) conditioned medium from microglia and astrocyte culture. The effect of microglia was blocked by a mixture of antibodies against cytokines (Cyto Abs) (IL-6, IL-1b, and TNF-a, 1 ng/mL) (n = 5, **p < 0.01, **p < 0.01 versus control, one-way ANOVA followed by Fisher’s LSD test). (G) A mixture of recombinant cytokines (Cyto mix) (IL-6, IL-1b, and TNF-a, 1 ng/mL) also reduced levels of P2Y1 receptors in astrocytes (n = 3, *p < 0.05, MannWhitney U test). (H–K) P2Y1 receptor-mediated calcium transients in astrocytes are dynamically regulated by microglia. MRS2365 (10 nM, 10 s)-evoked Ca2+ transients in astrocytes were significantly reduced in the presence of microglia. The inhibition by microglia was diminished by (H) minocycline (100 mM) or (I) a mixture of antibodies against TNF-a, IL-6, and IL-1b (Cyto Abs, 1 ng/mL) (n = 80–95, **p < 0.01 versus control, ##p < 0.01 versus +microglia, one-way ANOVA followed by Fisher’s LSD test). Minocycline did not affect P2Y1 receptor-mediated Ca2+ responses in astrocyte monoculture. (J) The inhibitory effect on P2Y1 receptors by microglia was reproduced by conditioned medium from a glial mixed culture, which was also restored by Cyto Abs treatment (n = 157–233, **p < 0.01 versus control, ##p < 0.01 versus +microglia, one-way ANOVA followed by Fisher’s LSD test). (K) Suppression of P2Y1 receptor-mediated Ca2+ transients was reproduced by a mixture of recombinant cytokines (i.e., TNF-a, IL-6, and IL-1b, 1 ng/mL) (n = 230, **p < 0.01 versus control, unpaired t test). Values are the mean ± SEM for all groups.

downregulation in astrocytes. Treatment with mixed glial cultureconditioned medium or co-culture with microglia reduced P2Y1 receptor expression in astrocytes; however, expression was restored by the addition of a cytokine blocking antibody mixture (Figures 3E and 3F). Furthermore, a recombinant cytokine mixture significantly decreased P2Y1 receptor expression in as-

trocytes (Figure 3G). P2Y1 receptor-mediated Ca2+ transients in astrocytes were also suppressed by microglial co-culture or cytokine administration. MRS2365 (10 nM)-evoked Ca2+ transients in astrocytes were suppressed by microglial co-culture or by treatment with conditioned medium (Figures 3H–3J). These suppressions were annulled by treatment with minocycline or

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Figure 4. Microglia-Mediated Downregulation of P2Y1 Receptors In Vivo (A and B) The P2Y1 receptor is downregulated after brain injury. At 3 dpi, both (A) mRNA and (B) protein levels (estimated at 100 kDa) of the P2Y1 receptor were decreased in the ipsilateral cortex (n = 4, *p < 0.05, **p < 0.01 versus sham, Mann-Whitney U test). (C and D) Minocycline (C) and clopidogrel (D) blocked TBI-induced downregulation of P2Y1 receptors (n = 5, Mann-Whitney U test). (E) Astrocyte-specific downregulation of P2Y1 receptors after TBI in vivo. Signals for P2Y1 receptors were significantly and specifically decreased in S100b+ astrocytes after TBI (3 dpi) (n = 5–9, *p < 0.05, unpaired t test). (F) P2ry1 mRNA level in astrocytes was reduced after TBI (n = 5, *p < 0.05, unpaired t test). Values are the mean ± SEM for all groups. Scale bar, 10 mm in (E).

a cytokine-blocking antibody mixture. A mixture of recombinant cytokines also suppressed P2Y1 receptor-mediated Ca2+ transients in astrocytes (Figure 3K). In addition to the in vitro data, both mRNA and protein levels of the P2Y1 receptor were downregulated in the ipsilateral brain at 3 dpi (Figures 4A and 4B). These responses were blocked by minocycline or clopidogrel treatment (Figures 4C and 4D). Although it was unclear whether P2Y1 receptors were selectively downregulated in astrocytes estimated by co-labeling of P2ry1 mRNA and S100b (Figure S5), immunohistochemical analysis and purification of astrocytes using magnetic-activated cell sorting (MACS) associated with qPCR revealed that P2Y1 receptor levels in S100b-positive (S100b+) astrocytes were clearly reduced in the scar area at 3 dpi (Figures 4E and 4F). These data show that microglia downregulate astrocytic P2Y1 receptor expression and function via cytokine signaling. P2Y1 Receptor Downregulation Is Essential for Acceleration of Reactive Astrogliosis To investigate the role of P2Y1 receptor downregulation, we used P2Y1KO mice. P2Y1KO mice showed earlier reactive astrogliosis compared to WT mice, and the level of GFAP was upregulated at 1 dpi (Figure 5A). In addition, phosphorylation of signal transducer and activator of transcription 3 (P-STAT3), a critical regulator of reactive astrogliosis (Herrmann et al., 2008; Okada et al., 2006), was increased at 1 dpi. Immunohistochemistry showed GFAP signals as dot-like structures in WT mice at 1 dpi; however, cell skeleton-like structures were observed in P2Y1KO mice (Figure 5B), indicating accelerated reactive astro-

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gliosis. P2Y1KO mice had smaller injury cores (Figures 5C–5E) and a smaller number of FJ+ cells (Figures 5F and 5G). The P2Y1 receptor is also expressed in neurons; however, the neuronal P2Y1 receptor displayed no protective role in in vitro experiments (data not shown). These data show that downregulation of the P2Y1 receptor, probably on astrocytes, can be essential for neuroprotection. Knockdown of P2Y1 Receptors in Astrocytes Is Critical for Neuroprotection To further clarify the role of astrocytic P2Y1 receptors in TBI, we specifically manipulated P2ry1 gene expression in astrocytes. We used the Flexible Accelerated STOP Tetracycline OperatorKnockin (FAST) system (Tanaka et al., 2010). For astrocytespecific gene overexpression, we generated a double-transgenic mouse with astrocyte-specific tetracycline transactivator (tTA) and P2ry1 tetracycline operator (tetO) knockin (Mlc1tTA::P2ry1tetO/tetO) (Figure S6A; see also Supplemental Experimental Procedures). Levels of cortical P2Y1 receptor protein were significantly increased (>7-fold) (Figure S6C). Enhanced immunoreactivity for P2Y1 receptor (P2Y1R) proteins and signals for P2ry1 mRNA were observed in S100b+ astrocytes by immunohistochemistry and in situ hybridization (ISH), respectively (Figures S6E–S6H and S5A). Using MACS, purified astrocytes from adult Mlc1-tTA::P2ry1tetO/tetO brains also showed a dramatic increase in P2ry1 mRNA levels (>25-fold) (Figure S6K). No significant changes in P2ry1 mRNA levels were observed in other cell type-enriched fractions (Figure S6K). Hereafter, we refer to Mlc1-tTA::P2ry1tetO/tetO mice as Astro-P2Y1OE mice. Astrocytes prepared from Astro-P2Y1OE mice showed slower process extension in vitro compared with WT littermates (Figure 6A). Astro-P2Y1OE mice showed greatly increased injury core size, distances of peri-lesion astrocytes from the core (Figure 6B), and numbers of FJ+-damaged neurons (Figure 6C).

Figure 5. Downregulation of P2Y1 Receptors Accelerates Reactive Astrogliosis and the Neuroprotective Phenotype (A) Accelerated reactive astrogliosis in P2Y1KO mice. In WT mice, GFAP and P-STAT3 levels were increased at 3 dpi but only slightly increased at 1 dpi. In P2Y1KO mice, both GFAP and P-STAT3 levels were already upregulated at 1 dpi. (B) Immunohistochemical analysis. At 1 dpi, only dot-like GFAP signals were observed in WT mice, but cytoskeleton-like signals were observed in P2Y1KO mice. (C–E) Knockout of P2Y1 receptor diminishes the size of the astrocyte-formed scar-like structure (C). The distances of reactive astrocytes from the injury locus was small in P2Y1KO mice at 3 dpi (n = 535–995, **p < 0.01 versus WT, Mann-Whitney U test) (D). The P2Y1KO mice also showed a smaller injury core area (n = 5, *p < 0.05, MannWhitney U test) (E). (F and G) P2Y1 receptor modulates neuronal damage after brain injury. The number of FluoroJade-positive (FJ+) cells at 3 dpi was significantly smaller in P2Y1KO mice compared with WT mice (n = 7–12, *p < 0.05, unpaired t test). Scale bars, 100 mm for (B), (C), and (F). Values are the mean ± SEM for (D), (E), and (G).

These data demonstrate that overexpression of P2Y1 receptors in astrocytes prevents scar-forming astrogliosis and exacerbate TBI-evoked neuronal injury. For astrocyte-specific gene knockdown, we generated a double-transgenic mouse from an astrocyte-specific tTS (tetracycline trans-silencer) line and a P2ry1 tetO knockin line (Mlc1-tTS::P2ry1tetO/tetO) (Figure S6B). In Mlc1-tTS:: P2ry1tetO/tetO mice, the tTS protein binds to a tetO site near the endogenous P2ry1 promoter and blocks P2ry1 mRNA transcription. tTS-mediated suppression of P2ry1 transcription halved the cortical P2Y1 receptor protein level (Figure S6D). Mlc1tTS::P2ry1tetO/tetO mice had a 90% reduction in P2Y1R immunoreactivity within S100b+ astrocytes but no changes in S100b area (Figures S6E–S6G; see also Supplemental Experimental Procedures). Astrocytes, purified from the adult brain of Mlc1tTS::P2ry1tetO/tetO mice using MACS, exhibited significantly reduced P2ry1 mRNA levels (
90%), whereas no reduction was detected in other cell types (Figure S6L). Hereafter, we refer to Mlc1-tTS::P2ry1tetO/tetO mice as Astro-P2Y1KD mice. Astro-P2Y1KD mice showed smaller injury core size and shorter distances from peri-lesion astrocytes to the core (Figure 6D), which is consistent with the responses of astrocytes in P2Y1KO mice (Figure 5). Associated with the accelerated astrocyte scar formation, the number of FJ+-damaged neurons at 3 dpi was significantly decreased in Astro-P2Y1KD mice (Figure 6E). These data show that knockdown of P2Y1 receptors in astrocytes promotes reactive astrogliosis and astrocyte scar formation, thereby enhancing the neuroprotective effects of astrocytes after TBI. Because trauma-evoked infiltration of peripheral leukocytes into the brain aggravates inflammation and neuronal death, processes that are limited by reactive astrocytes (Bush et al., 1999), we further investigated the role of the astrocytic P2Y1 receptor

on barrier functions. Astro-P2Y1OE mice showed an increased number of CD45+ cells (Figure S7A), whereas decreased infiltration was observed in Astro-P2Y1KD mice (Figure S7B). In addition, blood-brain barrier (BBB) disruption after brain trauma is a cause of neuronal death (Obermeier et al., 2013), and astrocytes are essential for BBB regulation (Alvarez et al., 2013). AstroP2Y1OE mice showed enhanced extravasation of Evans blue (EB) into the parenchyma (Figure S7C), but no significant leakage was observed in Astro-P2Y1KD mice (Figure S7D). These data demonstrate that astrocyte-specific P2Y1 receptor knockdown is critical for neuroprotection. DISCUSSION We found that microglia are activated very early following brain injury and that these activated microglia are responsible for signaling the changes that confer astrocytes with neuroprotective capacity. In this process, astrocyte-specific downregulation of P2Y1 receptors was found to be crucial. In many brain disorders, microglial activation occurs before reactive astrogliosis (Alexianu et al., 2001; Batchelor et al., 1999; d’Avila et al., 2012; Faideau et al., 2010; Lambertsen et al., 2005; Palazuelos et al., 2009; Petito et al., 1990; Shibata et al., 2011). However, before this study, it was not known how these cells cooperate and how these interactions contribute to pathophysiology. The crucial insight gleaned from our data is that activated microglia induce a neuroprotective phenotype in astrocytes. Inhibition of microglial activity (by minocycline), migration (by clopidogrel), or ablation (by PLX5622) significantly disrupted the engagement of peripheral astrocytes from the injury core in vivo, suggesting a microglia-to-astrocyte control after brain injury. Our in vitro data showed that microglia-derived soluble factor or factors are important because the effect on

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Figure 6. Knockout of the P2Y1 Receptor in Astrocytes Is Essential for Neuroprotection (A) P2Y1OE astrocytes showed slower process extension after traumatic injury in vitro compared with WT littermates (n = 198–264, **p < 0.01, Mann-Whitney U test). (B) Astro-P2Y1OE mice showed an increase in the distances of reactive astrocytes from the injury core at 3 dpi (n = 262–562, **p < 0.01, Mann-Whitney U test) and in the injured core size compared with WT littermates (n = 6–8, **p < 0.01, Mann-Whitney U test). (C) In Astro-P2Y1OE mice, the FJ+ cell number at 3 dpi was significantly larger than that of littermate control mice (n = 4, *p < 0.05, Mann-Whitney U test). (D) Astro-P2Y1KD mice showed smaller distances of reactive astrocytes from the injury core (n = 93–127, *p < 0.05 versus WT, Mann-Whitney U test) and smaller injured core size compared with WT littermates (n = 7, **p < 0.01 versus WT, Mann-Whitney U test). (E) The Astro-P2Y1KD mice showed a smaller number of FJ+ cells at 3 dpi (n = 5, **p < 0.01 versus WT, MannWhitney U test). Values are the mean ± SEM. Scale bars, 50 mm for (A) and 100 mm for (B)–(E).

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astrocytes occurred without direct contact with microglia. To investigate these molecules in vivo, we chose the time point of 1 dpi, because only microglia are activated at this time, and we found that TNF-a, IL-1b, and IL-6 were preferentially upregulated in microglia at 1 dpi in vivo. In vitro, TNF-a, IL-1b, and IL-6 also dramatically changed the astrocyte phenotype into a more reactive one, and blocking antibodies for these cytokines clearly inhibited microglia-enhanced responses. Previous reports also show that microglia modulate astrocyte activities via other molecules (Bezzi et al., 2001; Pascual et al., 2012; Shinozaki et al., 2014). Recently, it has been reported that microglia can induce either neurotoxic or neuroprotective phenotype of astrocytes depending on the context of microglial activation (i.e., lipopolysaccharide or middle cerebral artery occlusion) (Liddelow et al., 2017). Thus, it is conceivable that microglia regulate reactive astrogliosis. Our data demonstrate that microglia-derived pro-inflammatory cytokines result in neuroprotection; however, many reports have shown that they trigger neurotoxic effects. One explanation for these conflicting results is that these cytokines act as a double-edged sword. For example, IL-6 signals through two distinct pathways: classical and trans-signaling pathways (Rothaug et al., 2016). The classical pathway contributes to neuroprotective and regenerative roles (Chucair-Elliott et al., 2014; Hirota et al., 1996; Yang et al., 2012), whereas the trans-signaling pathway induces neurodegeneration (Campbell et al., 1993, 2014). IL-1, including IL-1b, also elicits both neurodegenerative (Betz et al., 1995; Relton and Rothwell, 1992) and neuroprotective effects (Ohtsuki et al., 1996). IL-1 especially contributes to preconditioning effects. TNF-a binds to two types of receptor: TNFR1 and TNFR2. TNFR1 transduces its signal to the adaptor protein TNF receptor-associated death domain (FADD). This causes caspase-8 activation and apoptosis (Weiss et al., 1998). TNFR2 recruits TNF receptor-associated factor 2 (TRAF2) and cellular inhibitor of apoptosis protein 1 and 2 (cIAP-1 and cIAP-2), resulting in an anti-apoptotic effect (Mukhopadhyay et al., 2001). In our experimental conditions, these molecules may trigger neuroprotective mechanisms rather than neurotoxic ones. Extracellular ATP acting through P2 receptors is thought to be a crucial component of the mechanism for triggering microglial activation (Ferrari et al., 1997; Friedle et al., 2011; Suzuki et al., 2004). A report showed that after TBI, acute purinergic signaling by microglia and inflammatory responses contribute to neuroprotection (Roth et al., 2014). Our data showed that traumatic injury increased extracellular ATP levels during the limited window when only microglia are activated (i.e.,
1 dpi in vivo and
60 min in vitro). Traumatic injury-evoked microglial migration in vitro was mediated by P2Y12 receptors, and administration of clopidogrel to the TBI-treated mouse increased the distance of reactive astrocytes from the injury core, indicating that the initially released or leaked ATP triggers microglial activation, migration, and cytokine production, thereby changing the astrocyte phenotype. Clopidogrel is known to inhibit platelet activation; therefore, we tested the effect of heparin, a P2Y12 receptor-independent platelet inhibitor, on astrocyte scar formation. Heparin (200 U/kg, intravenously [i.v.]) did not affect astrocyte scar size (data not shown), indicating that the effect of

clopidogrel is mediated not by platelet inhibition but by P2Y12 receptor inhibition. The P2Y1 receptor was found to be responsible for the accelerated astrocyte reaction by microglia and cytokines. Downregulation of P2Y1 receptors was essential for the phenotypic changes observed in astrocytes. In addition to the receptor downregulation, we observed a decreased level of extracellular ATP and increased nucleoside triphosphate diphosphohydrolases (NTPDase) 1 levels and ATPase activity when astrocytes are reactive (i.e., 3 dpi). NTPDase 1 hydrolyzes ATP to AMP, whereas NTPDase 2 hydrolyzes ATP to ADP (Heine et al., 2001; Zimmermann, 2001); therefore, the increased levels of NTPDase 1 effectively degrade extracellular ATP, thus bypassing ADP, an endogenous ligand for the P2Y1 receptor. The ATPase signals co-localized with the microglial marker CD11b. Our results were consistent with those of previous reports showing selective NTPDase 1 expression in microglia (Braun et al., 2000) and upregulation after TBI (Nedeljkovic et al., 2006). These observations suggest that microglia suppress astrocytic P2Y1 receptor signaling by downregulating P2Y1 receptor levels and by increasing NTPDase 1 expression and ATPase activity. A report has shown that downregulation of P2Y1 receptors in astrocytes also occurred in reactive astrocytes after spinal cord injury (Anderson et al., 2016) (see also their website at https://astrocyte.rnaseq.sofroniewlab.neurobio.ucla.edu). P2Y1 receptor downregulation for reactive astrogliosis may not be limited in TBI but can be broaden into other pathological situations. In the present study, P2ry1 mRNA levels were reduced by inflammatory cytokines, so transcriptional suppression may be one mechanism for this effect. TNF-a has been reported to reduce mRNA expression of target genes via inhibition of CCAAT-enhancer-binding proteins (C/EBPs) (Foka et al., 2009; Stephens and Pekala, 1992; Vig et al., 2012). IL-1b and IL-6 have also been reported to suppress C/EBPs, thereby reducing target gene expression (Schmid et al., 2001). It has been shown that C/EBPs control expression levels of several subtypes of P2 receptor (Bilodeau et al., 2015; Degagne´ et al., 2012). Therefore, pro-inflammatory cytokines may reduce P2ry1 mRNA expression via a C/EBP-mediated mechanism. Our results show that P2Y1 receptor downregulation changes the astrocyte phenotype. In the P2Y1KO mouse, accelerated upregulation of GFAP and P-STAT3 levels at 1 dpi were observed. Previous reports have shown that upregulated P-STAT3 levels at the early phase (12 hr
3 dpi) induced efficient glial scar formation at a late phase (i.e., >3 dpi) (Okada et al., 2006). This observation indicates that accelerated activation during the early phase after injury is important for inducing the neuroprotective phenotype of astrocytes. In addition, a report has shown that signal transducer and activator of transcription 3 (STAT3) signaling is essential for formation of astrocyte scar (Anderson et al., 2016) and that the P2ry1 mRNA level is specifically downregulated by 4-fold in scar-forming astrocytes after spinal cord injury (see also https://astrocyte.rnaseq.sofroniewlab.neurobio. ucla.edu). Downregulation in P2y1 levels of astrocytes may contribute to astrocyte scar formation not only in TBI but also in models including stroke and spinal cord injury. One cause of P2Y1 receptor-induced neuronal damage might be reactive oxygen species (ROS) (Roth et al., 2014). The P2Y1

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Figure 7. Diagram Depicting the Findings of the Present Study (A) In the naive and contralateral brain, microglia show ramified morphology with small cell bodies and highly branched processes. Astrocytes in the undamaged tissue show no or faint GFAP expression. (B) After TBI, ATP is released or leaked from the damaged tissue and microglia are activated. (C and D) The activated microglia release inflammatory cytokines (i.e., IL-6, TNF-a and IL-1b) (C), which is followed by reactive astrogliosis and astrocyte scar formation (D). The astrocyte scar separates healthy tissue from damaged tissue, thereby endowing neuroprotective effects. (E) Microglia block P2Y1 receptor signaling in astrocytes via dual pathways. (1) Microglia-derived cytokines downregulate P2Y1 receptor expression in astrocytes. (2) At the same time, microglia upregulate levels of NTPDase 1 and ATPase activity, thereby decreasing extracellular ATP levels and bypassing ADP, an endogenous ligand for the P2Y1 receptor. P2Y1 receptor downregulation changes the phenotype of astrocytes: enhanced process extension (in vitro), accelerated reactive astrogliosis (i.e., increased GFAP and P-STAT3 expression at 1 dpi), enhanced astrocyte scar formation, restored BBB functions, suppression of leukocyte infiltration, and induction of neuroprotective effects.

receptor mediates ROS production in astrocytes (Safiulina et al., 2006). Therefore, downregulation of P2Y1 receptors may counteract the neurotoxic action of astrocytes. P2Y1 receptors in astrocytes also affect leukocyte infiltration and BBB function. Preventing infiltration of peripheral inflammatory cells is important for protecting normal tissue (Sofroniew, 2009), and STAT3-mediated reactive astrogliosis is indispensable for blocking infiltration of inflammatory cells both in vivo and in vitro (Wanner et al., 2013). BBB disruption after brain trauma is also a key issue for neuronal death (Obermeier et al., 2013), and astrocytes are essential for BBB regulation (Alvarez

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et al., 2013). Our data show that the P2Y1 receptor in astrocytes is a key regulator responsible for blocking leukocyte infiltration and restoring BBB function after brain injury. As summarized in Figure 7, our findings characterize one component of the complex mechanism of microglia-to-astrocyte regulation after brain injury. In this process, microglia-derived inflammatory cytokines were found to be crucial. P2Y1 receptor downregulation was essential for enhanced formation of the astrocyte scar, suppression of leukocyte infiltration, BBB repair, and induction of neuroprotection. Because both microglia and astrocytes are known to become reactive under pathological

conditions, understanding the complex connection between these cells will reveal mechanisms of various neurological diseases and support the establishment of new therapeutic approaches. EXPERIMENTAL PROCEDURES Full methods are provided in the Supplemental Information. Animals All animals used in this study were obtained, housed, cared for, and used in accordance with the Guiding Principles in the Care and Use of Animals in the Field of Physiological Sciences published by the Physiological Society of Japan and with the approval of the Animal Care Committee of Yamanashi University. Male C57BL/6J mice were obtained from Japan SLC. P2Y1KO mice (male, C57BL/6J background) were generated as previously reported (Le´on et al., 1999). Astrocyte-specific P2ry1 overexpression (Astro-P2Y1OE) and knockdown (Astro-P2Y1KD) transgenic mice were generated as described in Supplemental Information. Primary Neuronal Culture Neuronal cultures were prepared as previously described (Noguchi et al., 2013; Shinozaki et al., 2014). Cerebral cortices were dissected from embryonic day (E) 17 mice and digested in 50 U/mL of papain. Neurons were dispersed in neuronal culture medium and maintained under an atmosphere of 10% CO2 at 37 C. The culture medium was changed twice a week, and neurons were used 14 days after plating. Primary Glial Cell Culture Glial cultures were prepared as previously reported (Shinozaki et al., 2014). Cortices from newborn mice were digested in 0.1% trypsin-EDTA, and the cells were cultured in flasks. To obtain microglia, the flask was shaken at 100 rpm for 1 min, and floating microglia were subcultured on cultured astrocytes. For astrocyte monoculture, the flask was subjected to 24 hr of continuous shaking. Ca2+ Imaging Changes in intracellular Ca2+ concentration were measured by the fura 2 method (Noguchi et al., 2013; Shinozaki et al., 2014). In brief, cells were loaded with fura 2-acetoxymethyl ester (fura 2-AM, 10 mM) at room temperature for 45 min. A microscope equipped with a 75-W xenon lamp and band-pass filters of 340- and 380-nm wavelengths was used for measurement of Ca2+-dependent signals (F340 and F380 nm). Image data were recorded by a chargecoupled device (CCD) camera. The F340/F380 ratio was used to evaluate [Ca2+]i. TBI In Vivo Mice were anesthetized, and the head was immobilized in a stereotactic frame. The skull bone was thinned with a 1.0-mm bit on a high-speed drill. TBI was performed by compression injury (Roth et al., 2014). Traumatic Injury In Vitro Primary glial cell cultures were physically damaged by scratching cells with a plastic pipette. For pharmacological analysis, reagents were added to the cell culture 30 min or 24 hr before injury. Astrocyte process extension was imaged every 5 min for 24 hr. For microglial migration, images were obtained every 1 min during the first 60 min after scratching. Process extension or cell migration speeds were estimated using ImageJ software with a Manual Tracking program from Ibidi. Western Blotting Cell or tissue lysates were electrophoresed on 10% or 15% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. The membranes were blocked and incubated with primary antibodies. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies with agitation for 1 hr at room temperature. Protein bands were visualized by

SuperSignal West Pico Chemiluminescent Substrate, and images were obtained using an LAS-4000. Immunohistochemistry Mice were anesthetized and transcardially perfused with 4% paraformaldehyde (PFA) in 0.1 M PBS. Brains were dissected and immersed in 30% sucrose for a few days at 4 C. Brains were coronally sliced, embedded in optimal cutting temperature (OCT) compound, and frozen on dry ice. Brain sections were prepared and blocked with 3% goat serum for 1 hr at room temperature. Sections were incubated with primary antibodies overnight at 4 C. The sections were then washed and incubated with secondary antibodies for 1 hr at room temperature. Secondary antibodies included Alexa-conjugated goat anti-rabbit, anti-mouse, or anti-rat immunoglobulin G (IgG) antibodies. Co-labeling for P2ry1 mRNA and an Astrocyte Marker Cryosections from 4% paraformaldehyde-perfused brains were used. Detailed methods for in situ hybridization are described elsewhere (Tanaka et al., 2010). Cryosections were treated with proteinase K for 30 min. After they were washed and acetylated, sections were incubated with a digoxigenin-labeled mouse P2ry1 cRNA probe. After the sections were washed in buffers with serial differences in stringency, they were incubated with an alkaline phosphatase-conjugated anti-digoxigenin (anti-DIG) antibody. The cRNA probes were visualized with freshly prepared colorimetric substrate (nitro blue tetrazolium/5-bromo-4-chloro-3-indolylphosphate [NBT/BCIP]). After in situ hybridization, S100b, an astrocyte marker, was labeled with an antiS100b antibody overnight. Following incubation with a biotin-tagged secondary antibody for 90 min, sections were treated with an avidin-biotin complex for 30 min and 3,30 -diaminobenzidine. FJ Staining Brain slices were treated sequentially with 100% EtOH, 70% EtOH, and distilled water followed by immersion in 0.06% KMnO4 for 15 min at room temperature. Slices were then stained in 0.0001% FJ B solution containing 0.09% acetic acid for 20 min at room temperature. In Vivo Microdialysis We performed in vivo microdialysis as previously reported (Koizumi et al., 2007). Briefly, a microdialysis probe (A-I type probe; Eicom) was inserted into the injured site and used to perfuse artificial cerebrospinal fluid at a flow rate of 4.0 mL/mL (collected for 50 min). The collected samples were immediately heated to 95 C for 5 min. Measurement of Extracellular ATP The ATP concentration was determined with an ATP bioluminescence assay kit CLS II. Samples were heated to 95 C for 5 min immediately after collection. All standards and samples were measured with a Lumat LB9501 tube luminometer. Assay of Neuronal Viability In Vitro Neuronal viability in vitro was estimated by water-soluble tetrazolium salt-1 (WST-1) assays using a cell counting kit. Cells were incubated for 1 hr with the WST-1 reagent at 1:10 dilution at 37 C. The absorbance of media was then measured by a microplate reader at 450 nm, with 650 nm as the reference wavelength. Real-Time RT-PCR Total RNA was isolated and purified from astrocytes and neurons using an RNeasy kit according to the manufacturer’s instructions. RT-PCR was performed using a One-Step Primescript RT-PCR Kit according to the manufacturer’s protocol. Estimation of BBB Function TBI-induced dysfunction of the BBB was estimated by EB extravasation as previously reported (Uyama et al., 1988). Cortices were homogenized and centrifuged (10,000 3 g for 5 min), and the fluorescence intensity of EB (excitation, 540 nm; emission, 680 nm) (Hed et al., 1983) in the supernatants was measured using a Spectra MAX Gemini EM.

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Statistics Data are expressed as means ± SEM. Unpaired or paired t tests or the MannWhitney U test was used for comparison of two groups. One-way ANOVA followed by Fisher’s least significant difference (LSD) test was applied for multiple comparisons. The differences were considered significant when the p value was less than 5%. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, seven figures, and two movies and can be found with this article online at http://dx.doi.org/10.1016/j.celrep.2017.04.047. AUTHOR CONTRIBUTIONS Y.S. designed the project. Y.S., K.S., K.Y., and K.F.T. performed the experiments. Y.S., K.F.T., and S.K. wrote the paper. K.F.T., C.G., E.S., and K.I. contributed new reagents or analytical tools. S.K. coordinated and directed the project. ACKNOWLEDGMENTS We thank Drs. Morizawa, Hirayama, and Imura and Mr. Hayashi and Mr. Komatsu (University of Yamanashi) for fruitful discussions. PLX5622 was provided under Materials Transfer Agreement (MTA) by Plexxikon Inc. This study was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI grants 23700442 to Y.S. and 16H04669, 25670622, and 25117003 to S.K., the Uehara Foundation to Y.S., grants-in-aid from the Japan Agency for Medical Research and Development (AMED) to S.K. Received: May 7, 2016 Revised: March 7, 2017 Accepted: April 14, 2017 Published: May 9, 2017

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