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Microglia trigger astrocyte-mediated neuroprotection via purinergic gliotransmission

Abstract

Microglia are highly sensitive to even small changes in the brain environment, such as invasion of non-hazardous toxicants or the presymptomatic state of diseases. However, the physiological or pathophysiological consequences of their responses remain unknown. Here, we report that cultured microglia sense low concentrations of the neurotoxicant methylmercury (MeHglow) and provide neuroprotection against MeHg, for which astrocytes are also required. When exposed to MeHglow, microglia exocytosed ATP via p38 MAPK- and vesicular nucleotide transporter (VNUT)-dependent mechanisms. Astrocytes responded to the microglia-derived ATP via P2Y1 receptors and released interleukin-6 (IL-6), thereby protecting neurons against MeHglow. These neuroprotective actions were also observed in organotypic hippocampal slices from wild-type mice, but not in slices prepared from VNUT knockout or P2Y1 receptor knockout mice. These findings suggest that microglia sense and respond to even non-hazardous toxicants such as MeHglow and change their phenotype into a neuroprotective one, for which astrocytic support is required.

Introduction

Microglia, the resident immune cells in the brain, continuously monitor the brain microenvironment1,2 and immediately respond to CNS disorders. Microglia are characterized by a very low threshold of activation and their activation occurs within a few tens of minutes3. Microglial activation is a common feature of the early stage of CNS disorders4,5, during which they migrate toward the lesion, phagocytose debris and mediate inflammatory reactions6. The P2 receptor, a receptor for extracellular nucleotides, is highly expressed in microglia and participates in these reactions7,8. Extracellular ATP and other nucleotides are released or leak from injured cells under pathological conditions. For example, ATP released from apoptotic neurons contributes a ‘find-me’ signal9 that activates microglial P2Y12 receptors and induces process extension8. In contrast, UDP functions as an ‘eat-me’ signal and triggers microglial phagocytosis via P2Y6 receptors7. In addition to the nucleotide-activated microglial function, microglia can release ATP10,11,12. Furthermore, microglia-derived ATP has recently been shown to activate the astrocytic P2Y1 receptor and trigger ATP release, which in turn regulates synaptic transmission13. P2Y1 receptor activation in astrocytes has been reported to induce neuroprotective effects against oxidative stress14, photothrombosis15, trauma16 and neurotoxicants17. Although the physiological consequences of purinergic signal-mediated gliotransmission between microglia and astrocytes have begun to be identified, their neuroprotective roles are mostly unknown.

Methylmercury (MeHg), a well-known environmental pollutant, readily crosses the blood–brain barrier18,19 inducing serious neuronal damage20. Our previous report demonstrated that MeHg evoked ATP release from astrocytes, thereby activating their P2Y1 receptors, which was followed by interleukin-6 (IL-6) release17. Additionally, the ATP release from astrocytes required a relatively high concentration of MeHg (≥1 μM), whereas neuronal damage was observed at lower concentrations of MeHg (<1 μM), to which astrocytes showed no responses. Thus, other mechanisms appear to be required for neuroprotection against low concentrations of MeHg (MeHglow). It has been reported that microglia respond to lower concentrations of MeHg than do astrocytes in vitro21. In addition, long-term exposure of adult female Macaca fascicularis to a subclinical dose of MeHg (50 μg/kg body weight/day) induced microglial activation but no changes in other brain cell types (i.e., neurons, astrocytes, oligodendrocytes, endothelia or pericytes)22. Furthermore, it has been demonstrated that human microglia are activated in the absence of significant neuronal loss in the presence of a low concentration of MeHg (i.e., <300 ppb)19, indicating potential neuroprotective roles of microglia. In fact, recent reports have shown microglia-mediated neuroprotective actions including neurotrophic support during brain development23, neuroprotection against NMDA-induced neurotoxicity24 and global brain ischemia25. Here we report that microglia can detect MeHglow, but their neuroprotective action against MeHglow requires astrocytes. MeHglow triggered VNUT-mediated microglial ATP release, which activated P2Y1 receptors in astrocytes thereby inducing IL-6 production and neuroprotection.

Results

Astrocytes upregulate IL-6 expression in response to MeHghigh but not MeHglow

Because we previously demonstrated that interleukin-6 (IL-6) production from astrocytes was essential for neuroprotection against MeHg17, we first evaluated the concentration-dependency of MeHg-induced IL-6 expression in a rat primary astrocyte monoculture (Rt astrocytes). Although a high concentration of MeHg (MeHghigh, ≥1 μM) induced a significant increase in the amount of IL-6 production from the astrocyte monoculture, a low concentration of MeHg (MeHglow) (i.e., 0.01 or 0.1 μM for 24 h) did not increase the amount of IL-6 production from Rt astrocytes (Fig. 1a) in accordance with previous reports17,31. In contrast to astrocytes, neurons were more sensitive to MeHg and their viabilities decreased in the presence of even MeHglow (0.1 μM), at which concentration astrocytes do not induce IL-6 production17,31. Because it has been reported that microglia respond to MeHg at concentrations too low to induce neuronal damage in vitro21, in macaques22 and in humans19, we next examined IL-6 production in a microglia monoculture. The results of ELISA analysis also showed no IL-6 release from rat (Rt) microglia monocultures in the presence of any concentration of MeHg (Fig. 1b). Serum starvation for 24 h before MeHg treatment for IL-6 measurement did not affect microglial viability (Fig. S2a), so it is unlikely that the lack of IL-6 production from microglia is a result of cell damage owing to serum starvation. In contrast to these Rt microglial monocultures, mixed glial cultures of Rt primary astrocytes and microglia (at a ratio of 1 : 5) released IL-6 in response to MeHglow (Fig. 1c). In accordance with IL-6 production from the glial mixed culture in response to MeHglow, conditioned media from the Rt glial mixed culture (Microglia/Astrocytes-Conditioned Medium, MACM) exhibited neuroprotective effects against MeHglow (Fig. 1d). MACM itself did not affect neuronal viability.

Figure 1
figure 1

IL-6 release from astrocytes, microglia and mixed glial cultures.

(a) ELISA analysis of MeHg-induced IL-6 release from astrocytes. MeHghigh (1 or 3 μM, 24 h) increased in the amount of IL-6 release from Rt astrocytes but not by MeHglow (0.01 or 0.1 μM, 24 h) (n = 26, **p < 0.01, one-way ANOVA followed by Fisher's LSD test). (b) Microglia released no IL-6 in response to MeHg. At no concentration did Rt microglia monocultures show significant IL-6 release (n = 12, p > 0.05, one-way ANOVA followed by Fisher's LSD test). (c) Rt microglia/astrocyte mixed cultures showed IL-6 release in response to both MeHglow and MeHghigh (0.01–3 μM) (n = 8, *p < 0.05, **p < 0.01 vs. control, one-way ANOVA followed by Fisher's LSD test). For evaluating IL-6 production from glial cell cultures, the cells were serum starved for 24 h before MeHg stimulation. (d) Conditioned media from mixed glial cultures showed a neuroprotective effect on primary cortical neurons against MeHg. Reduced neuronal viability evoked by MeHglow (0.1 μM, 48 h) was restored by MACM (n = 4–8, **p < 0.01 vs. no treatment group; ##p < 0.01 vs. MeHglow, one-way ANOVA followed by Fisher's LSD test). Without MeHg, MACM did not affect basal neuronal viability. Values are the means ± SEM for all groups.

Microglia release ATP in response to MeHglow

Because we previously showed that extracellular ATP is essential for IL-6 production from astrocytes17, we then measured ATP release from individual monocultures or mixed glial cultures. Although our previous data showed that MeHghigh evoked ATP release from Rt astrocytes17, MeHglow (i.e., 0.01 or 0.1 μM, 3 h) induced no ATP release from the astrocyte monoculture (Fig. 2a). In contrast, Rt microglia released ATP in response to MeHglow but not to MeHghigh (1 or 3 μM, 3 h)(Fig. 2b). At higher concentrations (i.e., ≥1 μM), microglia in monoculture exhibited necrotic cell death or decreased cell viability (Fig. S2b and S2c). The Rt mixed glial culture showed ATP release in response to both MeHglow and MeHghigh (i.e., 0.1–3 μM, 3 h) (Fig. 2c).

Figure 2
figure 2

ATP is released from astrocytes, microglia and mixed glial cultures.

(a) Rt astrocyte monocultures released ATP in response to MeHghigh (1 or 3 μM for 3 h) but not to MeHglow (i.e., 0.01 or 0.1 μM for 3 h) (n = 15–24, **p < 0.01 vs. control, one-way ANOVA followed by Fisher's LSD test). (b) Microglia released ATP in response to a limited concentration window. MeHghigh did not induce ATP release from Rt microglia monocultures. At 0.1 μM, MeHg significantly increased the extracellular ATP level (n = 24–28, **p < 0.01 vs. control, one-way ANOVA followed by Fisher's LSD test). (c) Mixed glial cultures released ATP in response to both MeHglow and MeHghigh. Microglia/astrocyte mixed cultures released ATP in response to 0.1–3 μM of MeHg (for 3 h) (n = 10–16, *p < 0.05, **p < 0.01 vs. control, one-way ANOVA followed by Fisher's LSD test). Glial cells were serum starved for 24 h before MeHg treatment. Values are the means ± SEM for all groups.

MeHglow triggers ATP-mediated Ca2+ oscillations in astrocytes

Because astrocytes have been reported to show ATP-mediated intracellular Ca2+ oscillations (i.e., repetitive [Ca2+]i transients in a single astrocyte) in response to MeHghigh17, we examined whether microglia-derived ATP triggers astrocytic Ca2+ responses. We found that MeHglow (0.1 μM, 15-min-treatment) increased the frequency of Ca2+ oscillations in the Rt astrocytes in the mixed glial culture (Fig. 3a and 3b). The increased frequency of astrocytic Ca2+ oscillations in the mixed glial culture was maintained for at least 3 h. In contrast to the mixed glial culture, in the Rt astrocyte monoculture, MeHglow induced no changes in the frequencies of astrocytic Ca2+ oscillations (Fig. 3b). The increase in MeHglow-induced Ca2+ oscillations in the Rt astrocytes in the mixed glial culture was significantly suppressed by the broad P2 receptor antagonist suramin (100 μM, 30 min pretreatment) (Fig. 3b). Based on these data, we surmised that ATP released from microglia in response to MeHglow evoked Ca2+ oscillations in astrocytes via P2 receptor activation.

Figure 3
figure 3

MeHglow increases astrocytic Ca2+ oscillations in mixed glial cultures via P2 receptor activation.

(a) Typical Ca2+ responses in microglia/astrocyte mixed cultures in response to MeHglow (0.1 μM, 15 min). Ca2+ images before (control) and after (MeHg) MeHglow are shown. Scale bar: 50 μm. (b) The MeHglow(0.1 μM, 15 min treatment)-evoked increase in the frequencies of Ca2+ oscillations seen in astrocytes in the mixed glial cultures, which was suppressed by the P2 receptor antagonist suramin (100 μM, 30 min pretreatment) (n = 110–240, **p < 0.01 vs. astrocytes/microglia without MeHg, ##p < 0.01 vs. astrocytes/microglia with MeHglow, one-way ANOVA followed by Fisher's LSD test). Astrocytes in the absence of microglia showed no Ca2+ responses to MeHglow (0.1 μM, 15 min). Values are the means ± SEM for (b).

Microglial ATP release is mediated by p38 activation

We then examined which intracellular signaling pathways mediate ATP release from Rt microglia. Because mitogen-activated protein kinases (MAPKs) are important for microglial activation32, we examined MAPK phosphorylation in a MeHg-treated Rt microglia monoculture. As shown in Figure 4b, neither ERK1/2 nor JNK showed enhanced phosphorylation (Fig. 4b), whereas p38 MAPK phosphorylation was increased in the presence of either 0.1 or 0.5 μM MeHg (2 h treatment) (Fig. 4a). Consistent with a previous report33, the Rt microglia exhibited necrotic cell death (Fig. S2b) or decreased cell viability (Fig. S2c) in the presence of higher MeHg concentrations (i.e., ≥0.5 μM), resulting in reduced levels of β-actin (Fig. 4b) and total p38 MAPK (Fig. 4a). To confirm p38 MAPK phosphorylation in Rt microglia, we next performed immunocytochemical analysis. Phosphorylated p38 MAPK signal was detected in MeHglow (0.1 μM, 2 h)-treated microglia in both the Rt microglial monoculture (Fig. 4c) and the Rt mixed glial culture (Fig. 4d), but was not observed in the absence of MeHg (Fig. 4c and 4d). No change in signal intensity for either phosphorylated ERK1/2 or JNK was observed in the presence of MeHg in either the monoculture or the mixed culture (Fig. 4c and 4d). We then examined whether activated p38 contributes to the MeHg-evoked ATP release from Rt microglia. The ATP release induced by MeHglow (0.1 μM, 3 h) from the Rt microglia monoculture was significantly suppressed in the presence of the p38 inhibitor SB203580 (10 μM, 15 min pretreatment) (Fig. 4e), indicating that p38 is essential for microglial ATP release. The MeHglow (0.1 μM, 15 min)-induced increase in Ca2+ oscillations in astrocytes in the Rt mixed glial culture was also suppressed by SB203580 (Fig. 4f). Furthermore, MeHglow (0.1 μM, 24 h)-induced IL-6 production from the Rt mixed glial culture was also blocked by SB203580 (Fig. 4g). These data show that p38-mediated ATP release from microglia induces P2 receptor-mediated Ca2+ oscillations in and IL-6 production by astrocytes.

Figure 4
figure 4

Phosphorylation of p38 MAPK contributes to ATP release from microglia.

(a) MeHg (2 h treatment) at 0.1 or 0.5 μM evoked phosphorylation of p38 MAPK (P-p38 MAPK) in Rt microglia monocultures. (b) JNK MAPK was not phosphorylated by MeHg. P-ERK1/2 MAPK was rather decreased by MeHg at concentrations over 1.0 μM. The β-actin level was also decreased at concentrations of MeHg over 0.5 μM. Western blot data were cropped and the selected areas were shown here and full-length data are presented in Supplementary Figure 5. The gels have been run under the same experimental conditions. (c) Immunocytochemistry of phosphorylated MAPKs in a Rt microglia monoculture. P-p38 MAPK signal was increased (arrows) after MeHglow treatment (0.1 μM, 2 h). P-ERK1/2 or P-JNK MAPKs did not show enhanced signals in the presence of MeHglow. Scale bar: 10 μm. (d) Phosphorylation of MAPKs in a Rt mixed glial culture. The intensity of P-p38 MAPK signal was increased in microglia (arrows) by MeHglow. P-ERK1/2 MAPK signals were also enhanced but were localized in CD11b-negative cells. The P-JNK MAPK signal intensity was not changed. Scale bar: 20 μm. (e) ATP release is regulated by p38 MAPK. The MeHglow (0.1 μM, 3 h)-induced Rt microglial ATP release was significantly suppressed by a p38 MAPK inhibitor SB203580 (10 μM, 15 min pretreatment) (n = 8, **p < 0.01, Mann–Whitney U-test). (f) The MeHglow-evoked astrocytic Ca2+ oscillation in the Rt glial mixed cultures is regulated by p38 MAPK. The increase in astrocytic Ca2+ oscillations induced by MeHglow (0.1 μM, 15 min) was reduced in the presence of SB203580 (10 μM, 15 min pretreatment) (n = 50, **p < 0.01, Mann–Whitney U-test). (g) IL-6 production from glial mixed cultures is regulated by p38 MAPK. IL-6 production in MeHglow (0.1 μM, 24 h)-treated Rt mixed glial cultures was suppressed by SB203580 (10 μM, 15 min pretreatment) (n = 8, **p < 0.01, Mann–Whitney U-test). Values are the means ± SEM for (e–g). Microglia monocultures and glial mixed cultures were serum starved for 24 h before each experiment.

MeHglow stimulates the exocytosis of ATP from microglia via a VNUT-mediated pathway(s)

MeHg has been reported to both facilitate and suppress exocytosis17,34,35. In addition, we recently reported that microglia release ATP by exocytosis via a vesicular nucleotide transporter (VNUT)-mediated pathway(s)11; thus, we next investigated VNUT-mediated ATP release evoked by MeHg. Botulinum toxin A (BoNT) (5 unit/ml, 24 h pretreatment), a toxin that cleaves SNAPs36,37 and thereby prevents exocytosis, significantly reduced MeHglow (0.1 μM, 3 h)-evoked ATP release from the Rt microglia monoculture (Fig. 5a). We then compared MeHglow-evoked ATP release from mouse (Ms) microglia monocultures prepared from WT and VNUT knockout mice (WT and VNUTKO microglia, respectively). MeHglow evoked significant ATP release from WT microglia but not from a VNUTKO microglia monoculture (Fig. 5b). VNUTKO microglia exhibited similar morphology to WT microglia (Fig. S3a) and normal adhesion to culture dishes (Fig. S3b). MeHglow (0.1 μM, 24 h) did not affect the number of adherent cells (Fig. S3c) or the viability of VNUTKO microglia (Fig. S3d), so it is unlikely that the lack of ATP release from VNUTKO microglia is due to damage caused by MeHglow. In the Ms mixed glial culture, the MeHglow (0.1 μM, 3 h)-evoked ATP release was totally dependent on microglial VNUT (Fig. 5c). As shown in Fig. 3, MeHglow evoked microglial ATP release, leading to subsequent astrocytic Ca2+ oscillations. MeHglow (0.1 μM, 15 min incubate) significantly increased Ca2+ oscillations in astrocytes in mixed cultures of WT microglia/WT astrocytes but not in mixed cultures of VNUTKO microglia/WT astrocytes (Fig. 5d). In accordance with ATP release and Ca2+ oscillations, the MeHglow-evoked IL-6 production from the mixed culture was also dependent on microglial VNUT (Fig. 5e). In the mixed culture, phase contrast images of VNUTKO microglia on WT astrocytes revealed them to be as normal as WT microglia and the numbers of microglia on astrocytes were not different, regardless of VNUT expression in microglia (Fig. S3e). Similarly, the morphology and numbers of Iba1 (a microglial marker)-positive cells in the mixed culture did not change in response to MeHglow (0.1 μM, 24 h) (Fig. S3f). Taken together, these findings suggest that the lack of ATP release, Ca2+ oscillations and IL-6 release in VNUTKO microglia/WT astrocytes is unlikely to be due to MeHg-induced cell death of VNUTKO microglia.

Figure 5
figure 5

VNUT-mediated ATP release from microglia is essential for Ca2+ signaling and IL-6 production from astrocytes in the mixed glial cultures.

(a) Dependency of of the MeHglow-evoked ATP release on exocytosis. BoNT (5 units/ml, 24 h pretreatment) significantly suppressed the MeHglow-induced ATP release from microglia (n = 5, **p < 0.01, Mann–Whitney U-test). (b) Dependency of the MeHglow-evoked ATP release on VNUT. In contrast to ATP release from WT microglia, VNUTKO microglia showed no ATP release in response to MeHglow (0.1 μM, 3 h) (n = 6–11, **p < 0.01 vs. WT microglia without MeHg, ##p < 0.01 vs. WT microglia with MeHglow, one-way ANOVA followed by Fisher's LSD test). (c) Microglial VNUT is essential for the MeHglow-evoked ATP release in Ms mixed glial cultures. MeHglow(0.1 μM, 3 h) evoked ATP release in WT microglia/WT astrocyte mixed cultures but not in VNUTKO microglia/WT astrocyte cultures (n = 8, **p < 0.01 vs. WT microglia/WT astrocytes without MeHg, ##p < 0.01 vs. WT microglia/WT astrocytes with MeHglow, one-way ANOVA followed by Fisher's LSD test). (d) Microglial VNUT is indispensable for MeHglow-triggered Ca2+ oscillations in astrocytes in the Ms mixed glial cultures. MeHglow (0.1 μM, 15 min) evoked a dramatic increase in the frequency of Ca2+ oscillations in astrocytes in WT microglia/WT astrocyte mixed cultures, but a small increase in VNUTKO microglia/WT astrocytes (n = 98, **p < 0.01 vs. WT microglia/WT astrocytes without MeHg, ##p < 0.01 vs. WT microglia/WT astrocytes with MeHglow, one-way ANOVA followed by Fisher's LSD test). (e) Microglial VNUT-dependent IL-6 production in Ms mixed glial cultures. MeHglow (0.1 μM, 24 h) evoked IL-6 production in WT microglia/WT astrocyte mixed cultures, but not in VNUTKO microglia/WT astrocytes (n = 3, **p < 0.01 vs. WT microglia/WT astrocytes without MeHg, ##p < 0.01 vs. WT microglia/WT astrocytes with MeHglow, one-way ANOVA followed by Fisher's LSD test). Values are the means ± SEM for all groups.

Activation of P2Y1 receptors in astrocytes is essential for IL-6 production from the mixed glial cultures

Microglial ATP acted on P2 receptors and then caused Ca2+ oscillations in astrocytes (Fig. 3). In our previous study, MeHg-evoked Ca2+ signaling was mediated by P2Y1 receptors in astrocytes17 and the P2Y1 receptor-mediated Ca2+ oscillation was essential for IL-6 production. Similarly, the MeHglow-stimulated Ca2+ oscillations in astrocytes in the mixed glial culture were dependent on astrocytic P2Y1 receptors because MeHglow significantly increased Ca2+ oscillations in astrocytes in the WT microglia/WT astrocyte culture, but not in astrocytes in the WT microglia/P2Y1KO astrocyte culture (Fig. 6a). The MeHglow-evoked IL-6 production in the glial mixed culture was also dependent on P2Y1 receptors in astrocytes (Fig. 6b).

Figure 6
figure 6

Astrocytic P2Y1 receptors are essential for IL-6 production from mixed glial cultures.

(a) The enhancement of Ca2+ oscillations in astrocytes in the Ms mixed glial culture is mediated by astrocytic P2Y1 receptors. MeHglow (0.1 μM, 15 min treatment) dramatically increased the frequency of astrocytic Ca2+ oscillations in WT microglia/WT astrocyte cultures but not in co-cultures of WT microglia and P2Y1KO astrocytes (n = 98, **p < 0.01 vs. WT microglia/WT astrocytes without MeHg, ##p < 0.01 vs. WT microglia/WT astrocytes with MeHglow, one-way ANOVA followed by Fisher's LSD test). (b) Activation of P2Y1 receptors in astrocytes is essential for IL-6 production in Ms mixed glial cultures. MeHglow (0.1 μM, 24 h) increased IL-6 production in WT microglia/WT astrocytes but not in WT microglia/P2Y1KO astrocytes (n = 3, **p < 0.01 vs. WT microglia/WT astrocytes without MeHg, ##p < 0.01 vs. WT microglia/WT astrocytes with MeHglow, one-way ANOVA followed by Fisher's LSD test). Cells were serum starved for 24 h before MeHg treatment. Values are the means ± SEM for all groups.

Both VNUT and P2Y1 receptors are essential for an endogenous neuroprotective mechanism against MeHglowin situ

To examine glial communication-mediated neuroprotection in situ, we employed an organotypic hippocampal culture. Neuronal cell death was assessed using propidium iodide (PI) (5 μg/ml, 1 h at 37°C) incorporation, a widely used indicator of neuronal death in hippocampal slices38. In WT slices, MeHglow (0.1 μM, 48 h) induced no increase in PI incorporation (Fig. 7a). MeHghigh (3 μM, 48 h)-induced PI-positive cells were co-localized with the neuronal marker NeuN, but not with the astrocyte marker glial fibrillary acidic protein (GFAP) or Iba1 (Fig. S4a–4c). Some NeuN signals in MeHghigh-treated slices were co-localized with single-stranded DNA (ssDNA), a marker of apoptosis (Fig. S4d). When microglia or astrocytes were suppressed by minocycline or fluorocitrate (10 and 1 μM, respectively, 24 h pretreatment), MeHglow-induced PI incorporation was dramatically increased (Fig. 7b), indicating that both microglial and astrocytic activities were required for the endogenous neuroprotective mechanism against MeHglow. We then examined the role of P2Y1 receptor activity in this endogenous neuroprotection. The P2Y1 receptor antagonist MRS2179 (10 μM, 24 h pretreatment) also increased PI incorporation. In addition, when extracellular IL-6 was blocked by applying an IL-6 antibody (200 pg/ml, simultaneous treatment), the PI signal was increased in the presence of MeHglow. In fact, MeHglow evoked the release of ATP and the production of IL-6 protein from WT slices (3 h and 24 h treatment for ATP and IL-6, respectively) (Fig. 7c and 7d). These data indicate that glial activity, P2Y1 receptor activation and IL-6 production are essential for the endogenous neuroprotective mechanism against MeHg in WT slices. None out of minocycline, fluorocitrate, MRS2179 and the IL-6 blocking antibody affected basal PI incorporation in the absence of MeHg (data not shown). PI signals showed a characteristic spatial pattern that corresponded well to the distribution of pyramidal neurons from the CA1 region to the CA3 region (Fig. 7a and Fig. S4e).

Figure 7
figure 7

ATP-triggered trilateral communication pathways among microglia, astrocytes and neurons in organotypic hippocampal slice cultures.

(a), (b) The endogenous neuroprotection is mediated by glial cells, P2Y1 receptors and IL-6. MeHglow (0.1 μM, 48 h) did not cause an increase in PI incorporation in WT slices. The PI incorporation was increased by minocycline (mino, 10 μM, 24 h pretreatment) or fluorocitrate (FC, 1 μM, 24 h pretreatment), MRS2179 (MRS, 10 μM, 24 h pretreatment) and an IL-6 blocking antibody (IL6Ab, 200 pg/ml, simultaneous treatment) (n = 8–11, **p < 0.01 vs. control). (c) MeHglow (0.1 μM, 24 h) increased IL-6 production from WT slices (n = 4, **p < 0.01 vs. control). (d) MeHglow (0.1 μM, 3 h) increased ATP release from WT slices (n = 5, **p < 0.01 vs. control). (e), (f) MeHglow (0.1 μM, 48 h) increased PI incorporation in VNUTKO slices, which was prevented by ATP (100 μM, 30 min pretreatment) or recombinant IL-6 protein (100 pg/ml, simultaneous treatment) (n = 17–22, **p < 0.01 vs. control, ##p < 0.01 vs. MeHglow). (g) VNUTKO slices did not release ATP in response to MeHglow (0.1 μM, 3 h) (n = 6, p = 0.41). (h) MeHglow (0.1 μM, 24 h) did not induce any increase in IL-6 production from VNUTKO slices (n = 4, p = 0.87). (i), (j) Similar to the findings in VNUTKO slices, MeHglow (0.1 μM, 48 h) increased PI incorporation in P2Y1KO slices; however, unlike the results in VNUTKO slices, exogenously applied ATP (100 μM, 30 min pretreatment) had no effect on MeHglow-increased PI incorporation. Recombinant IL-6 protein (100 pg/ml, simultaneous treatment) significantly reduced the PI signals (n = 8–11, **p < 0.01 vs. control, ##p < 0.01 vs. MeHglow). (k) MeHglow (0.1 μM, 24 h) did not induced IL-6 production from P2Y1KO slices (n = 4, p = 0.19). Values are the mean ± SEM for (b–d), (f–h), (j) and (k). Student's t-test and one-way ANOVA followed by Fisher's LSD test were used for comparisons of two and multiple groups, respectively.

To confirm the impact of VNUT-mediated ATP release on the neuroprotection observed in the in vitro study, we used hippocampal slices obtained from VNUT-KO mice (VNUTKO slices). We did not find any significant morphological abnormality or PI incorporation in the VNUTKO slices in the absence of MeHg. However, upon stimulation with MeHglow (0.1 μM, 48 h), VNUTKO slices showed increased PI incorporation (Fig. 7e and 7f), which was restored by the exogenous application of ATP (100 μM, 30 min pretreatment) or recombinant IL-6 protein (100 pg/ml, simultaneous treatment). In addition, MeHglow failed to stimulate the release of ATP or production of IL-6 in the VNUTKO slices (Fig. 7g and 7h). Similar to the VNUTKO slices, the organotypic hippocampal slices from P2Y1R-KO mice (P2Y1KO slices) also showed pronounced increases in PI signals in response to MeHglow (0.1 μM, 48 h) (Fig. 7i and 7j). Without MeHg, PI signals were hardly detected in the P2Y1KO slices. Unlike the situation in VNUTKO slices, ATP did not rescue the MeHglow-induced neuronal damage in the P2Y1KO slices, but recombinant IL-6 protein (100 pg/ml) did (Fig. 7i and 7j), indicating that the release of ATP or IL-6 production is essential for the neuroprotection as an signal upstream or downstream of P2Y1 receptors, respectively, in situ. The finding that MeHglow did not increase IL-6 production in P2Y1KO slices also supports this idea (Fig. 7k).

Discussion

The main findings in the present study are as follows. (i) Microglia detected MeHglow and showed activation of p38 MAPK followed by ATP release via a VNUT-dependent pathway. (ii) Although astrocytes did not sense MeHglow, they responded to the released ATP via P2Y1 receptors and produced IL-6, by which they protected neurons against MeHg. (iii) These microglia-originated trilateral talks among microglia, astrocytes and neurons seen in the mixed cultures were mimicked in situ and were essential for neuroprotection against MeHglow.

We have recently shown that astrocytes protect neurons against MeHghigh by producing IL-617. In the present study, we focused on the effects induced by a much lower concentration of MeHg, to which astrocytes cannot respond and investigated the role of microglia, which are highly sensitive cells, in the brain microenvironment. There are many lines of evidence suggesting that microglia are activated even in a pre-symptomatic state or in the early stages of various diseases and after insults39,40. However, the physiological or pathophysiological consequences of such microglial activations in the presymptomatic state have remained largely unknown. With regard to MeHg, it is reported that although a low MeHg concentration does not cause any sign of brain damage in intoxicated human individuals, microglia are already activated19, suggesting a potential role of microglia in the neuroprotection against MeHg. In fact, recent reports have described the neuroprotective roles of microglia24,41,42. Here, we demonstrated that microglia function as a highly sensitive sensor of MeHg and reveal neuroprotective effects for which astrocytes are also required.

MeHglow evoked ATP release but did not induce production of IL-6 in a microglia monoculture (Figs. 1b and 2b); by contrast, it increased neither ATP release nor IL-6 production in an astrocyte monoculture (Figs. 1a and 2a), although MeHghigh could evoke both responses17 (Figs 1a and 2a). Interestingly, MeHg, even at a low concentration, could evoke both ATP release and IL-6 production in the microglia/astrocyte mixed cultures (Figs. 1c and 2c). The simplest interpretation of these findings is that there is some form of communication between microglia and astrocytes. Eskes et al. previously showed that microglia evoke IL-6 production from astrocytes31; however, the detailed molecular mechanisms underlying the communication between these cell types remain to be clarified. Our findings that pharmacological and genetic inhibition of both microglial ATP release and astrocytic P2Y1 receptors abolished these responses seen in the mixed glial cultures, suggest that microglia can sense MeHglow, which evoked release of ATP that functions as a paracrine signal and stimulates P2Y1 receptors in astrocytes to induce IL-6 upregulation. Although a previous report has shown that ATP induces production of IL-6 in microglia via P2X7 receptors43, MeHglow did not induce IL-6 production from microglia (Fig. 1b). This discrepancy may be explained by the fact that the amount of released ATP in the present study was too low to activate P2X7 receptors because their activation requires a relatively high concentration of ATP (i.e., >100 μM)43.

The findings that (1) MeHglow-evoked ATP release was significantly decreased by BoNT or in VNUTKO microglia (Figs. 5a and 5b) and (2) microglial ATP-induced Ca2+ oscillations in astrocytes were abolished in the mixed cultures of VNUTKO microglia/WT astrocytes (Fig. 5d), indicated that the microglial ATP release in response to MeHglow should be mediated by exocytosis. Because a high concentration of ATP would be stored in exocytotic vesicles (e.g., ~100 mM in synaptic vesicles44), a locally released high concentration of ATP may be able to activate P2X7 receptors and induce IL-6 production, but in our condition this was not the case. We recently demonstrated that microglia exocytosed ATP via VNUT-dependent mechanisms and this exocytosis was dramatically increased in inflammatory conditions11. Thus, if microglial ATP exocytosis is enhanced in some pathological conditions such as inflammation, the released ATP might function as an autocrine signal to activate P2X7 receptors. However, the P2X7 receptor antagonist brilliant blue G (1 μM) did not inhibit MeHglow-induced IL-6 production (data not shown). Therefore in the present experimental condition, instead of acting as an autocrine signal, microglial ATP seemed to function as a paracrine signal to activate P2Y1 receptors in astrocytes.

We also found that p38 MAPK is involved in microglial ATP release (Fig. 4e). Many reports have demonstrated that p38 MAPK is a critical regulator of microglial activation45,46. Although p38 MAPK activation seems to be a common pathway for microglial activation, by which various microglial responses are regulated, to date its involvement in ATP exocytosis remains unknown. However, the activation of p38 MAPK is essential for the exocytosis of matrix metalloproteinase-9 and myeloperoxidase in neutrophils47 and for the nucleotide-evoked exocytosis of elastase in neutrophils48. Thus, p38 MAPK may somehow affect the machinery of exocytosis. An initial mechanism of microglial activation by MeHglow may be mediated by oxidative stress, because MeHg has been shown to induce oxidative stress49,50,51,52,53 and reactive oxygen species are well known to contribute to activation of both p38 MAPK54,55,56 and microglia57,58. Therefore, MeHglow may change the redox state of microglia, thereby causing their activation. Although the molecular mechanisms underlying the microglial sensing of MeHglow and the subsequent activation of p38 MAPK and ATP exocytosis have not been fully clarified, we clearly showed that microglia sense MeHglow and exocytose ATP via p38 MAPK- and VNUT-dependent mechanisms, which is especially important because these microglial events initiate the subsequent trilateral communication among microglia, astrocytes and neurons.

Finally, we demonstrated that the MeHglow-evoked microglia-to-astrocyte communication by ATP seen in the mixed glial cultures was mimicked in situ using organotypic hippocampal slice cultures (Fig. 7). In the slice culture experiments, we rather focused on the relationship between this form of communication and neuronal damage. Again, (1) MeHglow evoked microglial ATP exocytosis; and (2) this microglial ATP activated P2Y1 receptors leading to the upregulation of IL-6 in the astrocytes of mixed cultures. Similarly, (1) MeHglow evoked ATP release in WT slices, which was abolished by genetic inhibition of VNUT; and (2) MeHglow evoked IL-6 production, which was abolished by genetic inhibition of either VNUT or P2Y1 receptors (Fig. 7). More importantly, these genetic and pharmacological inhibitions resulted in neuronal damage induced by MeHglow, which was prevented by the addition of recombinant IL-6. Regarding the mechanism of IL-6-mediated neuroprotection, we and other groups have shown that both upregulation of adenosine A1 receptors and suppression of glutamate-mediated excitotoxicity are involved17,59,60. These results suggest that an endogenous neuroprotective mechanism(s) against brain insults is formed by the communication network among microglia, astrocytes and neurons. Microglia are especially important in this mechanism because their sensing and responses can be a trigger of a subsequent chain of responses.

As summarized in Fig. 8, we demonstrated the following findings. Microglia, a highly sensitive microenvironmental sensor, senses MeHglow and responds via exoytosis of ATP, for which p38 MAPK and VNUT are essential. Although astrocytes do not respond to MeHglow, they receive microglial ATP through P2Y1 receptors, produce IL-6 and thereby provide neuroprotection against MeHglow. Microglia respond to non-hazardous insults or change their phenotypes even in the presymptomatic state of diseases. Such early responses in microglia might be key events in protecting neurons and should be studied further toward clarifying the causes of brain diseases.

Figure 8
figure 8

Schematic diagram illustrating the communication pathways among microglia, astrocytes and neurons responsible for neuroprotection.

When exposed to MeHglow, microglia sense MeHglow and activation of p38 MAPK occurs. This is followed by VNUT-dependent ATP exocytosis. The microglia-derived ATP in turn activates P2Y1 receptors and induces Ca2+ oscillations in astrocytes. The activation of P2Y1 receptors induces IL-6 release from astrocytes, thereby leading to neuroprotection against MeHg.

Methods

Detailed methods are provided in the Supplementary 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 Physiologic Sciences” published by the Physiologic Society of Japan and with the previous approval of the Animal Care Committee of Yamanashi University (Chuo, Yamanashi, Japan). Wistar rats and C57BL/6 mice (17-day-old fetal) were obtained from Japan SLC (Shizuoka, Japan). P2Y1 receptor knockout mice (C57BL/6 background) were developed as previously reported26. Vesicular nucleotide transporter knockout (VNUTKO) mice were generated by the standard gene disruption procedure. For genotyping polymerase chain reaction (PCR), the primers used were as follows: WT forward, 5′-CTA TGT GTA GCC CTG GAT GG-3′; WT reverse, 5′-GTG TAC CCT TCG GGG AAA GT-3′; VNUTKO forward (corresponding to a partial sequence inside PGK promoter that drives the Neo gene), 5′-CTT GTG TTT GGG ATC CTG GT-3′; and VNUTKO reverse, 5′-GGG AGG ATT GGG AAG ACA AT-3′. They were backcrossed onto a C57BL/6 background and used.

Cell culture

Glial monocultures were prepared as previously reported7,27. Briefly, cerebral cortices dissected from newborn Wistar rats or C57BL/6 mice were digested in 0.1% trypsin-EDTA and the cells were cultured in a flask. For microglia monoculture, cells were collected by gentle shaking of the flask. For astrocyte monoculture, the flask was subjected to 24 h of continuous shaking. For the mixed glial culture, microglia were added to a confluently cultured astrocyte culture. Glial cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 10 units/ml penicillin and 10 μg/ml streptomycin under conditions of 10% CO2 at 37°C.

Neuronal culture was prepared as previously described28. Cerebral cortices dissected from 17-day-old fetal mice were digested in 50 units/ml papain. Neurons were maintained in neuronal culture medium (DMEM supplemented with 5% horse serum, 5% FBS, 10 units/ml penicillin and 10 μg/ml streptomycin) under conditions of 10% CO2 at 37°C and were treated with cytosine β-D-arabinofuranoside (ara-C, 10 μM) two days after plating. The culture medium was changed twice a week and neurons were used 14 days after plating.

Assay of cell survival

Cell survival was estimated by WST-1 assay using a cell counting kit (Dojindo, Kumamoto, Japan), which determines cell viability based on the extent of cleavage of a soluble tetrazolium salt WST-1 by mitochondrial dehydrogenases. Briefly, cells were incubated for 1 h with the WST-1 reagent at a 1:10 dilution at 37°C. The absorbance of media was then measured using a microplate reader at 450 nm with 650 nm as a reference wavelength.

Organotypic slice cultures

Organotypic hippocampal slice cultures were made as previously reported29,30. Briefly, hippocampi from 5- to 7-day-old mice were dissected, placed in ice-cold HBSS containing 6 mg/ml of D-(+)-glucose for 15 min and transverse slices (300 μm) were obtained using a McIlwain tissue chopper (Mickle Laboratory Engineering Co. Ltd., Gomshall, UK). Six slices were placed on a Millicell-CM culture plate insert (PICM0RG50, Millipore, Billerica, MA, USA) and maintained in slice culture medium (50% MEM, 25% Hanks BSS, 25% heat-inactivated horse serum, 2 mM L-glutamine, 6.5 g/l glucose, 100 units/ml penicillin and 100 μg/ml streptomycin) under conditions of 5% CO2 at 37°C and used for experiments 7 days after incubation with medium change every few days. Neuronal cell death was evaluated by propidium iodide (PI) incorporation and PI fluorescence images were obtained using a Keyence BV-8000 fluorescence microscope (Tokyo, Japan) at an exposure time of 0.5 s. The PI intensity was estimated using Image J software (http://rsbweb.nih.gov/ij/).

Quantitative RT-PCR

Total RNA was isolated and purified from cultured cells using RNeasy kits (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Before RNA isolation, cells were serum-starved for 24 h. Reverse transcription (RT)-PCR was performed using a one step PrimeScript® RT-PCR Kit (Takara Bio Inc., Shiga, Japan) according to the manufacturer's protocol. The reaction mix contained 40 ng of total RNA, 200 nM primers, 100 nM TaqMan probe, Takara EX Taq® HS and PrimeScript™ RT enzyme Mix. RT-PCR amplification and real-time detection were performed using an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Reverse transcription was performed at 42°C for 5 min followed by inactivation at 95°C for 10 s. The temperature profile consisted of 40 cycles of denaturation at 95°C for 5 s and annealing/extension at 60°C for 34 s. The sequences of the primers and probe for rat IL-6 were as follows: TaqMan probe, 5′-CAGAATTGCCATTGCACAACTCTTTTCTCA-3′; forward primer, 5′-CAGTGCATCATCGCTGTTCA-3′; and reverse primer, 5′-CATATGTTCTCAGGGAGATCTTGGA-3′. The primers and TaqMan probe for GAPDH were obtained from Rodent GAPDH Control Reagents (Applied Biosystems). Mouse IL-6 expression was estimated using the probe set (Mm0046190-m1) from Applied Biosystems.

Enzyme-linked immunosorbent assay of IL-6

MeHg-induced IL-6 production from astrocytes, microglia, astrocytes/microglia mixed culture or organotypic hippocampal slice cultures was measured using a Quantikine® rat or mouse IL-6 immunoassay kit (R&D Systems, MN, USA). The assay was performed according to the manufacturer's instructions. All standards and samples were measured using a microplate reader at a wavelength of 450 nm.

Ca2+-imaging

Changes in intracellular Ca2+ were measured using the fura 2 method with minor modifications28. In brief, the culture medium was replaced with balanced salt solution (BSS) of the following composition (in mM): NaCl 150, KCl 5.0, CaCl2 1.8, MgCl2 1.2, HEPES 25 and D-glucose 10 (pH 7.4). Cells were loaded with fura 2 by incubation with 10 μM fura 2-acetoxymethyl ester (fura 2-AM) at room temperature in BSS for 45 min. After loading, the samples were mounted on a microscope (ECLIPSE TE2000-U, Nikon, Tokyo, Japan) equipped with a 75-W xenon lamp and band-pass filters of 340 and 380 nm wavelengths for measurement of the Ca2+-dependent signals (F340 and F380 nm). Image data were recorded using a CCD camera (ORCA-ER, Hamamatsu Photonics, Shizuoka, Japan). For evaluation, we used the F340/F380 ratio. To estimate the frequency of Ca2+ oscillations in astrocytes, we compared the numbers of Ca2+ oscillations in a single astrocyte within 5 minutes.

Immunocytochemistry and immunohistochemistry

Cells cultured in μ-dish (Ibidi GmbH, Munich, Germany) or slices were serum starved for 24 h followed by MeHglow treatment (2 h) and fixed with 4% PFA for 30 min at room temperature. After permeabilization/blocking in PBS with 0.3% Triton X-100 (PBST) containing 3% goat serum for 1 h at room temperature, samples were incubated with the primary antibodies (CD11b, GFAP, Iba1, NeuN, phospho-p38, phospho-ERK1/2, phospho-JNK and ssDNA at 1:1000) in Can Get Signal A (TOYOBO, Osaka, Japan) for 24 h at 4°C. Then, the cells were further incubated with Alexa dye-conjugated secondary antibodies (at 1:1000) for 1 h at room temperature. Fluorescence images were obtained using a laser scanning confocal microscope FV-1000 (Olympus, Tokyo, Japan). For nuclear staining, 4′,6-Diamidino-2-phenylindole, dihydrochloride (DAPI) (1 μg/ml) was used.

Measurement of extracellular ATP

The extracellular ATP concentrations of MeHg-treated cells or slices were determined using an ATP bioluminescence assay kit CLS II (Roche Applied Science, Mannheim, Germany). Cells or slices were serum starved for 24 h followed by incubation for 3 h with MeHg (0.01, 0.1, 1 or 3 μM) in serum-free medium and the supernatants were collected and immediately boiled at 95°C for 5 min. Equal volumes of luciferin/luciferase reagents and samples (100 μl each) were mixed a few times by gentle pipetting. All standards and samples were measured using a Lumat LB9501 tube luminometer (Berthold, Wildbad, Germany). The ATP concentrations were calculated from the intensities of a series of standard ATP. For glial cells, samples were collected from 24-well plates (i.e., 1 × 105 cells in astrocyte monocultures, 6 × 104 cells in microglia monocultures, or mixed cultures of 1 × 105 astrocytes and 2 × 104 microglia). For hippocampal slices, samples were collected from 12-well plates (i.e., one slice/well).

Western blotting

Cells for western blotting were serum starved for 24 h before MeHg treatment (2 h). Cell lysates were electrophoresed by 10% SDS-PAGE gels and transferred to PVDF membranes. The membranes were blocked for 1 h in Tris-buffered saline containing 0.1% Tween-20 and 5% BSA at room temperature and were incubated with primary antibodies (1:1000) overnight at 4°C. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:20,000) for 1 h at room temperature. Protein bands were visualized by rinsing the membrane with SuperSignal West Pico Chemiluminescence Substrate (Thermo Scientific, PA, USA). Images were obtained using a LAS-4000 imaging system (Fujifilm, Tokyo, Japan). Full-length data of each blot are shown in Figure S5.

Statistics

Data are expressed as means ± SEM. The Student's t-test or Mann–Whitney U-test was used for comparisons of two groups. For multiple comparisons, one way parametric ANOVA followed by Fisher's least significant difference (LSD) test was used. Differences were considered to be significant when the P value was less than 0.05.

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Acknowledgements

We thank Dr. Shigetomi, Dr. Shibata, Dr. Imura, Ms. Hirayama, Mr. Morizawa, Mr. Komatsu, Ms. Kobayashi and Mr. Taguchi for fruitful discussion. This work was supported by a grant for the Study (Group) of the Health Effects of Heavy Metals Organized by the Ministry of the Environment, Japan (to S.K.) and KAKENHI on Innovative Areas (25116512 & 25117003)(to S.K.) and on Challenging Exploratory Research (25670622)(to S.K.).

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S.K. designed the experiments and supervised the studies. Y.S. performed the experiments. Y.S. and S.K. prepared the manuscript. M.N., K.I. and Y.M. made the VNUTKO mice. C.G. made P2Y1KO mice. All authors reviewed the manuscript.

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Shinozaki, Y., Nomura, M., Iwatsuki, K. et al. Microglia trigger astrocyte-mediated neuroprotection via purinergic gliotransmission. Sci Rep 4, 4329 (2014). https://doi.org/10.1038/srep04329

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