RESEARCH ARTICLE

Different Danger Signals Differently Impact on Microglial Proliferation Through Alterations of ATP Release and Extracellular Metabolism Jimmy George,1 Francisco Q. Gonc¸alves,1 Gonc¸alo Crist ov~ ao,1 Lisa Rodrigues,1,2 Jos e Roberto Meyer Fernandes,3 Teresa Gonc¸alves,1,2 Rodrigo A. Cunha,1,2 and Catarina A. Gomes1,2 Microglia rely on their ability to proliferate in the brain parenchyma to sustain brain innate immunity and participate in the reaction to brain damage. We now studied the influence of different danger signals activating microglia, both internal (typified by glutamate, associated with brain damage) and external (using a bacterial lipopolysaccharide, LPS), on the proliferation of microglia cells. We found that LPS (100 ng/mL) increased, whereas glutamate (0.5 mM) decreased proliferation. Notably, LPS decreased whereas glutamate increased the extracellular levels of ATP. In contrast, LPS increased whereas glutamate decreased the extracellular catabolism of ATP into adenosine through ecto-nucleotidases and ecto-5’-nucleotidase. Finally, apyrase (degrades extracellular ATP) abrogated glutamate-induced inhibition of microglia proliferation; conversely, inhibitors of ecto-nucleotidases (ARL67156 or a,b-methylene ADP) and adenosine deaminase (degrades extracellular adenosine) abrogated the LPS-induced increase of microglia proliferation, which was blocked by a selective A2A receptor antagonist, SCH58261 (50 nM). Overall, these results highlight the importance of the extracellular purinergic metabolism to format microglia proliferation and influence the spatio-temporal profile of neuroinflammation in different conditions of brain damage. GLIA 2015;00:000–000

Key words: microglia, ATP, adenosine, A2A receptor

Introduction

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icroglia cells function as sensors, continuously monitoring the brain parenchyma: they play a major role in mounting neuroinflammatory responses upon brain injury (Kettenmann et al., 2011; Saijo and Glass, 2011), but even subtle changes in the neuronal workload are detected and rectified by microglia (Davalos et al., 2005; Kondo et al., 2011; Li et al., 2012; Nimmerjahn et al., 2005; Wake et al., 2013). Thus, microglia play a key role in responding to extrinsic non-neuronal stimuli, such as bacterial antigens, but they also respond to neuronal danger signals (e.g. glutamate, ATP) released upon neurodegenerative conditions (reviewed in Kettenmann et al., 2011; Saijo and Glass, 2011). One critical

aspect of microglia responses is their pleiotropism and plasticity (Benarroch, 2013; Gomez-Nicola and Perry, 2015): this involves a control of their proliferation, migration and the release of pro- and anti-inflammatory mediators, which is expected to be different according to the triggering stimuli that engage microglial responses. The signalling to control each of these microglial functions needs to be orchestrated in a precise spatio-temporal manner: for instance, microglia proliferation occurs in the brain parenchyma after an insult, but microglia cannot proliferate in the close vicinity of a brain lesion (Wang et al., 2004). In accordance with the chemotactic recruitment of microglia to cope with brain damage, microglia must be recruited from remote areas of the brain,

View this article online at wileyonlinelibrary.com. DOI: 10.1002/glia.22833 Published online Month 00, 2015 in Wiley Online Library (wileyonlinelibrary.com). Received June 24, 2014, Accepted for publication Mar 17, 2015. Address correspondence to Catarina A. Gomes, Center for Neuroscience and Cell Biology, University of Coimbra, Rua Larga, Coimbra 3004-504, Portugal. E-mail: [email protected] From the 1CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Portugal; 2FMUC-Faculty of Medicine, University of Coimbra, Portugal; 3Instituto de Bioquımica M edica Leopoldo De Meis, Universidade Federal Do Rio De Janeiro, Rio De Janeiro, Brazil

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but this migratory wave to sites of lesion is preceded by a proliferative period (Wang et al., 2004). This indicates that the potential for microglia self-replenishing is strictly dependent on specific and regional environmental signals. Several microglial functions are modulated by purines, namely ATP and one of the end-products of ATP catabolism, adenosine (Inoue, 2008; Kettenmann et al., 2011; Koizumi et al., 2013). In fact, the activation of microglia (Haynes et al., 2006; Koizumi et al., 2007; Ohsawa et al., 2007) and, in particular, their migration and chemotaxis to focal lesions in the brain, is governed by ATP released by damaged neurons and astrocytes (Davalos et al., 2005; F€arber et al., 2008; Honda et al., 2001; Nimmerjahn et al., 2005). Importantly, ATP diffusion in the parenchyma is limited by rapid ATP degradation by ecto-phosphatases (Cunha et al., 1998; Zhang et al., 2003; Zimmermann 1996), raising the hypothesis that neuronal or astrocytic-derived ATP would evoke further ATP release from neighbour microglial cells, in order to establish a long-range gradient required for remote microglial chemotaxis (Dou et al., 2012). An additional consequence of the efficient extracellular catabolism of ATP is the formation of its metabolite, adenosine, which can also modulate different microglial functions (Dai et al., 2010; Rebola et al., 2011; Saura et al., 2005; van der Putten et al., 2009), namely motility (Gyoneva et al., 2014; Orr et al., 2009) and proliferation (Gomes et al., 2013). However, the autocrine role of purines (locally released ATP and its catabolism into adenosine) in microglia has not yet been clarified. As a first step to explore if the purinergic system might contribute to orchestrate the spatio-temporal constraints of microglia proliferation, we now investigated if different danger signals (bacterial components as exogenous danger signals and glutamate as an internally-generated danger signal) could trigger a different release and extracellular metabolism of ATP into adenosine and how this might impact on microglia proliferation.

Materials and Methods Microglial Cell Culture A murine microglia cell line, N9 (kindly provided by Claudia Verderio, CNR Institute of Neuroscience, Cellular and Molecular Pharmacology, Milan, Italy), was grown in an RPMI 1640 (Sigma) medium supplemented with 30 mM glucose, 5% heat-inactivated foetal bovine serum, 100 lg/mL streptomycin and 1 U/mL penicillin (all from GIBCO, Invitrogen). Primary microglia cultures were prepared as previously described (Gomes et al., 2013). Briefly, primary cultures of glial cells were obtained from postnatal (P1-P5) C57BL6 mouse and maintained for 15 days in DMEM-F12 medium with glutamax (InvitroAbbreviations

ANOVA LPS

2

analysis of variance lipopolysaccharide

gen) containing 10% fetal bovine serum (Invitrogen), 0.25% gentamycin (Invitrogen) and 0.25 ng/mL M-CSF (murine-colony stimulating factor, Peprotech). Microglia were then separated from the mixed primary culture by shaking (200 rpm for 2 hours), and plated in DMEM-F12 medium with glutamax containing 0.25% gentamycin (Invitrogen). N9 cells and primary microglia were kept at 37  C under a humidified atmosphere with 95% O2 and 5% CO2. Viable cells (excluding trypan blue-stained cellular elements) were plated at a density of 5 3 105 cells/cm2 in 6-well trays for ATP quantification and enzymatic activity, and at 105 cells/cm2 in 12-well trays for proliferation assays.

Pharmacological Treatment To test the ability of different microglial inducers (bacterial versus neuronal) to trigger ATP release from microglia, cell cultures were challenged with 100 ng/mL LPS (from Escherichia coli, serotype 055:B5; Sigma) or 0.5 mM glutamate (Sigma) for 6 hours. These particular concentrations and time point were selected based on their reported ability to trigger microglia proliferation (Gomes et al., 2013) and pro-inflammatory microglia responses (Dai et al., 2010). To test the role of A2AR, microglia cells were pre-incubated (20 min before LPS or glutamate) with a supra-maximal concentration (50 nM) of the selective A2AR antagonist, SCH 58261 (Tocris) (Lopes et al., 2004), which was present throughout the incubation time, and was previously shown to control both LPS- and glutamate-induced responses in microglia (Dai et al., 2010; Gomes et al., 2013). To test the role of ATP extracellular catabolism into adenosine, the following drugs were added to the cultures, 20 min before LPS or glutamate, in supra-maximal and selective concentrations, based on our previous experience in different preparations: 1 and 5 nM adenosine-50 -(b,g-imido)-triphosphate (b,g-imATP, a non-hydrolysable ATP analogue; Sigma) (Cunha et al., 1998); 20 U/ mL apyrase (enzyme degrading ATP; Sigma) (Rodrigues et al., 2005); 2 U/mL adenosine deaminase (converts endogenous adenosine into inosine; Sigma) (Gomes et al., 2013); 100 lM ARL 67156 (6-N, N-diethyl-D-b,g-dibromomethylene ATP, a non-selective inhibitor of several ecto-nucleoside triphosphate diphosphohydrolases catabolizing ATP; Tocris) (Rebola et al., 2008); 50 lM AOPCP (a,b-methylene adenosine 50 -diphosphate, an inhibitor of ecto-50 nucleotidase, which converts extracellular AMP into adenosine; Sigma) (Cunha et al., 2000); 50 mM D-AP5 (D-2-amino-5-phosphonopentanoate, a N-methyl-D-aspartate (NMDA) glutamate receptor antagonist) (Rebola et al., 2011); 20 mM NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f ]quinoxaline-2,3-dione, an alphaamino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor antagonist) (Rebola et al., 2008); 50 mM MTEP (3[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine, a metabotropic glutamate receptor (mGluR5) antagonist) (Lea and Faden, 2006).

ATP Quantification The extracellular levels of ATP were assessed by the high sensitivity luciferin-luciferase bioluminescence assay, as previously described (Cunha et al., 1996). Briefly, after an incubation period of 6 hours with different drugs, cell supernatants were collected and kept at -80

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George et al.: ATP Metabolism Tunes Microglia Proliferation 

C. Cell supernatants (80 lL) were added to 40 lL of ATP assay mix (Sigma) in white 96-well plates kept inside a VICTOR multilabel plate reader (Perkin ElmerTM) for 1 min at room temperature before starting luminescence recording (5 sec of acquisition). ATP levels were quantified by extrapolation of a standard curve (2 3 10212 – 8 3 1025 M) and released ATP was normalized by the total protein content by the bicinchonic acid (Thermo Scientific) method.

by 5 mM p-nitrophenyl phosphate as substrate. To determine the concentration of p-nitrophenol formed by ecto-phosphatase activity, the reaction was stopped by the addition of 2 mL of NaOH (1 M), followed by spectrophotometric analysis at 425 nm to interpolate a p-nitrophenol standard curve (Fernandes et al., 1997). In both enzymatic assays, inter-assay variations in the number of cells were normalized by the total protein content measured by the bicinchonic acid (Thermo Scientific) method.

Proliferation Assay

Data Analysis

Microglia proliferation was evaluated as previously described (Gomes et al., 2013) by measuring the incorporation of 5-bromo-20 deoxyuridine (BrdU), a synthetic nucleoside that is incorporated into newly synthesized DNA, replacing thymidine, during cell replication. Cells were incubated with BrdU (10 lM; Sigma) for the last 2 hours of pharmacological treatments, fixed in 4% paraformaldehyde, washed in Tris-buffered saline with 0.3% Triton X-100 and maintained in 1 M HCl at 37  C for 30 min. Non-specific binding was determined by incubation in Tris-buffered saline with 3% bovine serum albumin and 1% Triton X-100 for 1 hour. Cells were incubated overnight at 4  C with a rat primary anti-BrdU antibody (1:100, Serotec) in Tris-buffered saline with 0.1% Triton X-100 and 0.3% bovine serum albumin, washed and incubated for 2 hours at room temperature with a donkey anti-rat secondary antibody labelled with Alexa Fluor 594 (1:200, Molecular Probes). Nuclear staining was achieved with incubation with DAPI (Invitrogen) for 5 min at room temperature. Preparations were mounted in Dakocytomation fluorescent medium (Dakocytomation Inc.) and fluorescent images were acquired using an Axioskop 2 Plus fluorescence microscope (Zeiss; PG-Hitec). The number of proliferating cells (BrdUpositive) was counted and expressed as a percentage of the total cells stained with DAPI.

Values are presented as mean6standard error of the mean (SEM) of n experiments performed in duplicates. Either a Student0 s t test for independent means or a one-way analysis of variance (ANOVA) followed by a Newman-Keuls post hoc test, were carried out to define statistical differences between absolute values, which were considered significant at P < 0.05, unless otherwise specified. Note that, although the impact of several drugs and modulators are presented as percentage values for the sake of clarity, the statistical comparisons were always carried out using absolute values.

Measurement of the Activities of Ecto-50 -Nucleotidase and Ecto-Nucleotidases After incubation with LPS or glutamate for 6 hours, the drugs were washed and microglia cells were incubated for 1 hour at 37 C and 5% CO2 in 1 mL of reaction mixture containing 116 mM NaCl, 5.4 mM glucose, 50 mM HEPES-MES-Tris buffer (pH 7.2) and 5 mM 50 -AMP (Sigma) as substrate for ecto-50 -nucleotidase. Cell supernatants were collected to quantify inorganic phosphate (Pi) (Guilherme et al., 1991). Briefly, the enzymatic reaction was stopped by the addition of 2 mL of 25% charcoal in 0.1 M HCl to the supernatants, followed by a centrifugation at 1500 g for 15 min at 4  C. Supernatants (100 lL) were added to 100 lL of FiskeSubbarow mixture (Fiske and Subbarow, 1925) and the absorbance was measured at 650 nm. Ecto-50 -nucleotidase activity was calculated by subtracting the non-specific 50 -AMP hydrolysis measured in the absence of cells from the total Pi released, both determined by extrapolation of a concentration curve of standard Pi. Differences in total ecto-phosphatases activity (which includes the classical NTPDase ecto-nucleotidases, ecto-nucleotide pyrophosphatase/phosphodiesterases, and alkaline phosphatases) were determined as previously described (Fernandes et al., 1997), using the same conditions as for ecto-50 -nucleotidase, but replacing 5’-AMP

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Results LPS and Glutamate Differentially Modulated the Extracellular Levels of ATP Released by Microglia Figure 1 shows the quantitative analysis of extracellular ATP levels in N9 cells (A) and in primary cultures of microglia (B). LPS (100 ng/mL) decreased the extracellular ATP levels (58 6 6%, n 5 6, P < 0.05, as compared with non-treated N9 cells, Fig. 1A), whereas glutamate (0.5 mM) increased the extracellular ATP levels (195 6 22%, n 5 6, P < 0.05, as compared with non-treated N9 cells, Fig. 1A). Although the levels of ATP measured in the supernatants of primary microglia (in the range of 1027 M) are higher than those obtained from N9 cells (in the range of 1029M) in basal conditions (lack of added drugs), the effects of LPS and glutamate were still observed: LPS decreased (80 6 3%, n 5 4, P < 0.05, as compared with non-treated cells, Fig. 1B), whereas glutamate (0.5 mM) increased extracellular ATP levels (184 6 32%, n 5 5, P < 0.05, as compared with non-treated cells, Fig. 1B). Microglia Proliferation is Enhanced by LPS and Decreased by Glutamate We previously reported (Gomes et al., 2013) that the magnitude of proliferation in the absence of a proliferative stimulus (e.g. LPS) is higher in the microglial cell line than in the microglia primary culture. However, the effect of drugs (namely LPS and adenosine receptor ligands) upon proliferation is similar between the cell line and the primary microglial cultures (Gomes et al., 2013). In line with these observations, N9 cells are considered a suitable model to assess microglial proliferation. LPS increased microglia proliferation (183 6 25%, n 5 4, P < 0.05, as compared with non-treated cells, Fig. 2A,B; nontreated cells exhibit a ratio BrdU-positive nuclei/DAPI of 3

FIGURE 1: LPS and glutamate oppositely regulate the extracellular levels of ATP released by N9 cells (A) and primary cultured microglia cells (B). Microglia cells were exposed to LPS (100 ng/mL) or glutamate (0.5 mM) for 6 hours and ATP levels were measured in the supernatants using the luciferin-luciferase bioluminescence assay. LPS decreased and glutamate increased ATP levels. Results are mean6SEM of six independent experiments performed in duplicate (*P < 0.05, Student0 s t test).

0.360.02, n 5 4 and this value was taken as the reference to which the ratio obtained in the presence of LPS or glutamate was compared, in order to calculate % of effects). On the other hand, microglia cells exposed to glutamate (0.5 mM) displayed a decrease of proliferation (7369%, n 5 4, P < 0.05 as compared with non-treated cells, Fig. 2A,B). In order to evaluate which type of glutamate receptors was involved in the control of microglia proliferation, N9 cells were co-incubated with glutamate and selective antagonists of NMDA (D-APV, 50 lM), AMPA (NBQX, 20 mM) or metabotropic group 5 glutamate receptors (MTEP, 50 mM). Interestingly, all these glutamate receptors seem to contribute to the effect of glutamate, since all of the tested antagonists prevented the proliferation arrest observed in the presence of glutamate (in the presence of glutamate, D-APV:121 6 22%, n 5 5; NBQX:131 6 14%, n 5 4; MTEP:104 6 8%, n 5 5, P 0.05, as compared with non-treated cells, Fig. 3A). However, A2AR blockade prevented the LPS- and glutamate-induced changes in extracellular ATP levels (Fig. 3A), suggesting that A2AR may function as a normalizer of altered extracellular ATP levels in the vicinity of microglial cells. Figure 3B shows the ability of A2AR blockade to modulate the effects of LPS or glutamate on the extracellular ATP levels in primary microglia cultures: as observed with N9 cells, SCH58261 (50 nM) prevented both the LPS- (103 6 12%, n 5 4, P < 0.05, as compared with LPS-treated cells) and glutamateinduced changes (77 6 16%, n 5 4, P < 0.05, as compared with glutamate-treated cells) of the extracellular ATP levels. SCH58261 prevented the LPS-induced increase in proliferation levels (66618%, n 5 4, P < 0.001, compared with LPS-treated cells, Fig. 3C), as previously reported (Gomes et al., 2013). By contrast, A2AR blockade did not modify the glutamate-induced decrease of microglia proliferation (n 5 4, P > 0.05, compared with the absence of SCH58261, Fig. 3C). Arrest of Proliferation in the Presence of High Concentrations of ATP is not a Direct Effect of ATP The observations that A2AR blockade normalized both the LPS- and glutamate-induced changes of extracellular ATP levels, but only prevented the LPS-induced increase of microglia proliferation, without affecting glutamate-induced proliferation “arrest”, suggest that changes of microglia proliferation may not be directly regulated by extracellular ATP levels. Since the extracellular ATP levels result from a balance of its release and extracellular metabolism, and that LPS-induced increase of proliferation depends on A2AR activation by adenosine (Gomes et al., 2013 and present results, Fig. 3), we hypothesized that the increased microglia proliferation may Volume 00, No. 00

George et al.: ATP Metabolism Tunes Microglia Proliferation

FIGURE 2: LPS triggers, whereas glutamate decreases microglia proliferation. Microglia N9 cells were exposed to LPS (100 ng/mL) or glutamate (0.5 mM) for 6 hours in the presence of 10 lM 5-bromo-2’-deoxyuridine (BrdU) in the last 2 hours. (A) Representative images illustrating the ability of LPS to enhance and of glutamate to decrease cell proliferation, which was quantified as the number of BrdUlabelled nuclei (pink) and expressed as a percentage of the total number of DAPI-labelled nuclei (blue). (B) Average quantitative analysis showing that LPS increases, while glutamate decreases cell proliferation. Results are mean6SEM of 4 independent experiments performed in duplicate (*P < 0.05, compared with control conditions; Student0 s t test). (C) Average quantitative analysis showing that glutamate receptor antagonists (metabotropic and ionotropic) prevent glutamate-induced decrease of proliferation. Results are mean6SEM of 4-5 independent experiments performed in duplicate (*P < 0.05, compared with glutamate-treated cells; Student’s t test).

result from an increased extracellular catabolism of ATP. To address this question, microglia N9 cells were incubated with b,g-ImATP, a non-hydrolysable ATP analogue, used in concentrations of 1 nM and 5 nM, similar to the extracellular ATP concentrations measured in the presence of LPS and glutamate, respectively (Fig. 4A). Notably, both concentrations of b,g-ImATP decreased microglia proliferation (at 1 nM: 21 6 4%, n 5 3, P < 0.05 compared with non-treated cells; at 5 nM: 38 6 7%, n 5 3, P < 0.05 compared with non-treated cells, Fig. 4B). Further supporting our hypothesis, apyrase (20 U/mL), which degrades extracellular ATP, prevented the glutamate-induced decrease of microglia proliferation (223 6 20%, n 5 3, P < 0.05 compared with glutamatetreated cells, Fig. 4C). Microglia Proliferation Induced by LPS Depends on ATP Catabolism into Adenosine To clarify if the metabolic conversion of ATP into adenosine is required for LPS-induced microglia proliferation, we used two different approaches: decrease of endogenous extracellular adenosine and inhibition of the extracellular catabolism of ATP. The pharmacological blockade of the extracellular catabolism of ATP into ADP, AMP and ultimately, adenosine with Month 2015

100 lM ARL67156, prevented LPS-induced proliferation (64 6 16%, n 5 3, P < 0.05 compared with LPS-treated cells, Fig. 5). Likewise, the selective blockade with 50 lM AOPCP of the last step of extracellular ATP catabolism into adenosine, also prevented LPS-mediated increase in proliferation (40 6 8%, n 5 3, P < 0.05 compared with LPS-treated cells, Fig. 5). Finally, removing endogenous extracellular adenosine with adenosine deaminase (ADA 2 U/mL) prevented the LPS-induced proliferation (6666%, n 5 3, P < 0.05 compared with LPS-treated cells, Fig. 5). LPS and Glutamate Differentially Modulate ATP Metabolism The results described above prompted us to hypothesize that LPS may increase the extracellular catabolism of ATP into adenosine, whereas glutamate may instead depress adenosine formation from extracellular ATP. Thus, we directly quantified the activity of the ecto-enzymes dephosphorylating extracellular ATP and forming extracellular adenosine. The activity of ecto-nucleotidases or ecto-phosphatases, a group of enzymes able to degrade extracellular ATP and its metabolites, in microglia N9 cells is 2662 nmol Pi/h (n 5 3). As shown in Figure 6B, LPS increased (47 6 5 nmol 5

FIGURE 3: Adenosine A2A receptor blockade normalizes the modification of microglia ATP release caused by LPS and glutamate, and prevents LPS, but not glutamate-mediated effects upon microglial proliferation. Microglia N9 cells (A) or primary microglia (B) were exposed to LPS (100 ng/mL) or glutamate (0.5 mM) for 6 hours in the absence or in the presence of the selective A2AR antagonist, SCH 58261 (50 nM), with 10 lM 5-bromo-2’-deoxyuridine (BrdU) present in the last 2 hours. (A,B) ATP levels, measured in the supernatants using the luciferin-luciferase bioluminescence assay, were decreased by LPS and increased by glutamate, and previous incubation with the A2AR antagonist SCH58261 (50 nM, added 15 minutes before the stimuli and present until the end of the experiment) prevented both LPS and glutamate-mediated changes upon ATP levels. (C) Average quantitative analysis of N9 proliferation (quantified as the number of BrdU-labelled nuclei expressed as a percentage of the total number of cells) showing the ability of SCH 58261 (50 nM) to prevent LPS-induced proliferation, but not glutamate-induced decrease of proliferation. Results are mean 6 SEM of 4-6 independent experiments performed in duplicate (*P < 0.05, one way ANOVA followed by Newman-Keuls multiple comparison test).

Pi/h, n 5 3, P < 0.05, compared with non-treated cells) and glutamate decreased (18 6 1 nmol Pi/h, n 5 3, P < 0.05, compared with non-treated cells) the activity of ectophosphatases. Further confirming our hypothesis, glutamate also decreased the activity of ecto-50 -nucleotidase, the last enzyme of the sequential conversion of extracellular ATP into adenosine (102 6 11 nmol Pi/h, n 5 4, P < 0.05, compared with basal conditions, where ecto-5’-nucleotidase activity was 180 6 22 nmol Pi/h, n 5 4). Ecto-50 -nucleotidase activity was not altered in the presence of LPS (172 6 24 nmol Pi/h, n 5 4, P > 0.05, compared with non-treated cells) (Fig. 6A).

Discussion This study shows that different danger signals in the brain may cause an opposite impact on microglia proliferation through an opposite alteration of the release of ATP and of its extracellular metabolism into adenosine. Thus, it was shown that: (1) glutamate decreases, whereas LPS increases 6

the activity of enzymes able to metabolize ATP into adenosine; (2) glutamate decreases, whereas LPS increases microglia proliferation; (3) microglia proliferation requires ATP conversion into adenosine and subsequent activation of A2AR. Thus, this places the ecto-enzymes converting extracellular ATP into adenosine at the heart of the mechanism underlying the control of microglia proliferation by environmental factors. In the brain, extracellular levels of ATP result from a regulated balance between ATP released by different cells, and its extracellular catabolism (Kukulski et al., 2011). Neurons and astrocytes are considered the main cellular source of extracellular ATP (Burnstock et al., 2011), whereas microglia was mostly identified as an important responsive cellular element to ATP (Anderson et al., 2004; Davalos et al., 2005; Honda et al., 2001). Although it is now accepted that microglia can release ATP (Dou et al., 2012; Fujita et al., 2008; Higashi et al., 2011; Kim et al., 2007; Liu et al., 2006), the role of ATP released from microglia has not been clarified. Volume 00, No. 00

George et al.: ATP Metabolism Tunes Microglia Proliferation

FIGURE 4: A non-hydrolysable ATP analogue decreases microglia proliferation and bolstering ATP degradation reverts glutamateinduced decrease of proliferation. (A) Absolute values of extracellular ATP measured in the supernatants of N9 cells exposed to LPS (100 ng/mL) or glutamate (0.5 nM) using the luciferinluciferase bioluminescence assay. (B) Microglia N9 cells were exposed for 6 hours to a non-hydrolysable ATP analogue, b,c-imidoATP (b,c-ImATP) at two concentrations: 1 nM (similar to ATP levels in the presence of LPS) or 5 nM (similar to ATP levels in the presence of glutamate), both causing a decrease of proliferation, evaluated as the number of 5-bromo-2’-deoxyuridine (BrdU)-labelled nuclei expressed as a percentage of the total number of cells. Data are mean6SEM of three experiments; *P < 0.05, as compared with control conditions, Student0 s t test. (C) The exposure of microglia N9 cells to glutamate (0.5 mM) in the absence or in the presence of apyrase (20 U/mL) showed that bolstering ATP degradation and catabolism converted the inhibitory impact of glutamate on microglia proliferation into a facilitatory effect. Data are mean 6 SEM of three independent experiments performed in duplicate; *P < 0.05, one way ANOVA followed by Newman-Keuls multiple comparison test.

The present observation that both bacterial endotoxin (LPS) and an internally generated danger signal (glutamate) trigger opposite effects on ATP release from microglia provides the first evidence that environmental factors control ATP release. Furthermore, the additional demonstration that bolstering ATP catabolism, inhibiting ecto-nucleotidases and removing extracellular adenosine critically affect the different proliferative response of microglia to LPS and glutamate provides the first evidence of an autocrine role for ATP in microglia, with a particular novel emphasis on the balanced conversion of extracellular ATP into adenosine (see also F€arber et al., 2008). We have previously reported that LPS, in the same experimental conditions used in the present work, increased Month 2015

microglia proliferation, an effect dependent on the activation of adenosine A2A receptors (Gomes et al., 2013). This observation, and the known ability of A2AR to modulate several microglia functions, led us to test if A2AR were able to control the extracellular levels of ATP and their eventual impact upon microglial proliferation in the presence of LPS and glutamate. A2AR blockade per se did not affect extracellular ATP levels, but A2AR blockade prevented the changes of extracellular ATP levels induced by LPS and glutamate (Fig. 3). This normalization of the extracellular ATP levels by A2AR blockade allows anticipating that A2AR blockade should also normalize microglia proliferation in both conditions, which would be in agreement with the general ability of A2AR blockade to restore brain function upon different brain disorders (reviewed in Cunha, 2005; Gomes et al., 2011). Interestingly, although A2AR blockade prevented the LPS-induced increase of microglia proliferation, it did not interfere with the microglia proliferation “arrest” caused by exposure to glutamate. This indicates that the changes in extracellular ATP levels, although temporally correlated (inverse correlation) with microglia proliferation, might not directly control microglia proliferation, suggesting that this role may instead depend on

FIGURE 5: Blockade of the extracellular catabolism of ATP and removal of endogenous extracellular adenosine prevent LPSinduced increase in microglia proliferation. Microglia N9 cells were exposed for 6 hours to LPS (100 ng/mL), in the absence or in the presence of inhibitors of ecto-nucleotidases responsible for the extracellular ATP catabolism, ARL 67156 (100 lM) or AOPCP (50 lM), or of adenosine deaminase (ADA, 2 U/mL, which removes endogenous adenosine). The average quantitative analysis of microglia proliferation evaluated as the number of 5-bromo-2’-deoxyuridine (BrdU)-labelled nuclei expressed as a percentage of the total number of cells, showed that the blockade of ATP extracellular catabolism or the removal of extracellular adenosine prevented LPS-induced microglia proliferation. Results are mean 6 SEM of three independent experiments performed in duplicate (*P < 0.05 one way ANOVA followed by Newman-Keuls multiple comparison test).

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FIGURE 6: LPS increases, whereas glutamate decreases the activity of ecto-enzymes involved in ATP extracellular metabolism. Microglia N9 cells were exposed to LPS (100 ng/mL) or glutamate (0.5 mM) for 6 hours and the activities of ecto-50 -nucleotidase or of ectophosphatases were measured in the presence of either 50 -AMP or of p-nitrophenyl phosphate, as respective substrates. (A) LPS increases, whereas glutamate decreases the enzymatic activity of ecto-phosphatases. (B) Glutamate, but not LPS, decreases the activity of ecto-50 -nucleotidase. Results are mean 6SEM of 3 or 4 independent experiments performed in duplicate (*P < 0.05; Student’s t test).

ATP metabolites, namely adenosine. Indeed, different pharmacological approaches confirmed that it was the extracellular conversion of ATP into adenosine, rather than ATP itself that drives the modifications of microglia proliferation observed in the presence of LPS and glutamate, namely: (1) LPS increased microglia proliferation (Fig. 2) and simultaneously increased the activity of ecto-phosphatases (Fig. 6); (2) LPS-induced increase of microglia proliferation was prevented by the removal of endogenous extracellular adenosine, by the blockade of several steps of the extracellular metabolism of ATP into adenosine (Fig. 5) and by A2AR blockade (Fig. 3); (3) a stable ATP analogue, not degradable into metabolites (Cunha et al., 1998), induced a concentration-independent “arrest” of microglia proliferation (Fig. 4A); 4) glutamate decreased microglia proliferation (Fig. 2), and simultaneously decreased the activities of ecto-phosphatases (Fig. 6A) and of ecto-50 -nucleotidase (Fig. 6B); 5) bolstering ATP catabolism (with exogenously added apyrase) in the presence of glutamate triggered an increase in proliferation (Fig. 4B). The pathophysiological relevance of the present observations resides in the key role of purines in the control of microglia proliferation to sustain neuroinflammatory responses (Gomez-Nicola and Perry, 2015; Kettenmann et al., 2011). In fact, in the region surrounding focal brain lesions where ATP levels are high, microglia cells are unable to proliferate (Wang et al., 2004). Thus, the recruitment of microglia from areas far from the lesion is critical to cope with damage over time. Accordingly, it has been shown that a proliferative event precedes the migratory wave of microglia (Wang et al., 2004). Therefore, the dual purinergic control of proliferation and migration is of critical importance to mount a spatio-temporally organized microglia response (Koizumi

8

et al., 2013). In this respect, it is tempting to note the key role of A2AR, which blockade re-establishes microglia motility towards an injury site that is impaired in inflammatory conditions (Gyoneva et al., 2014a,b), while simultaneously refraining microglia proliferation (Gomes et al., 2013). This heralds a working hypothesis where the currently proposed ATPderived adenosine, acting through A2AR, would play a key switching role between proliferation and migration of microglia. Future ex vivo and in vivo studies are required to clarify if the proliferative peak preceding migration towards damaged areas also requires A2AR activation by ATP-derived adenosine, as suggested by our in vitro study. A final relevant finding is the functional association between ATP-derived adenosine and the activation of A2AR. This is in notable agreement with a recent study showing that ecto-50 -nucleotidase-mediated formation of extracellular adenosine is responsible for the selective activation of striatal A2AR (Augusto et al., 2013), prompting the suggestion that the manipulation of ecto-50 -nucleotidase activity would be a novel strategy to regulate A2AR activity in physiological and pathological conditions of the brain (Augusto et al., 2013). Our current findings linking the extracellular catabolism of ATP with A2AR function in microglia lends further support to this proposal and prompt the possibility that a similar rationale may be used to manipulate the purinergic control of microglia proliferation. In summary, the present study shows that the nature of the “danger” signal (in the present study, glutamate and LPS) differently modifies purinergic metabolism (in particular ATP conversion into adenosine), subsequently formatting microglia proliferation. Thus, the purinergic modulation system is ideally positioned to control the context-dependent shifts in

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George et al.: ATP Metabolism Tunes Microglia Proliferation

microglial-mediated inflammatory responses. Indeed a similar stimulus-specific regulation of purinergic metabolism was also observed in astrocytes (Brisevac et al., 2012), bone marrow stromal cells (Costa et al., 2011), macrophages (Zanin et al., 2012) and nerve terminals (reviewed in Cunha, 2001). This prompts the new concept that the extracellular purinergic metabolism, by balancing ATP and adenosine signalling, may be designed to format the spatio-temporal response of cells with potential pleiotropic responses to different stimuli or conditions.

Acknowledgment Grant sponsor: DARPA; Grant number: 09-68-ESR-FP-010; Grant sponsor: FCT; Grant number: PTDC/SAU-TOX/ 122005/2010; Grant sponsor: QREN; Grant numbers: 0968-ESR-FP-010, W911NF-10-1-0059; Grant sponsors: European Neuroscience Campus (Marie-Curie-Cycle 2-2011-PT), Gabinete de Apoio a Investigac¸~ao (FMUC), CNPq (Ci^encia sem Fronteiras). CNC.IBILI - UID/NEU/04539/2013 - covered the publication costs (color figures expenses). The authors thank Claudia Verderio (National Research Council, Institute of Neuroscience and Department of Medical Pharmacology, Milan, Italy) for generously providing the murine N9 microglia cell line, which was kept at the facilities of the Department of Microbiology of the Faculty of Medicine, University of Coimbra, Portugal.

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Volume 00, No. 00

Different danger signals differently impact on microglial proliferation through alterations of ATP release and extracellular metabolism.

Microglia rely on their ability to proliferate in the brain parenchyma to sustain brain innate immunity and participate in the reaction to brain damag...
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