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Astrocytes protect neurons against methylmercury via ATP/P2Y(1) receptor-mediated pathways in astrocytes.

Noguchi Y, Shinozaki Y, Fujishita K, Shibata K, Imura Y, Morizawa Y, Gachet C, Koizumi S - PLoS ONE (2013)

Bottom Line: MeHg-treated astrocyte-conditioned medium (ACM) showed neuro-protective effects against MeHg, which was blocked by anti-IL-6 antibody and was mimicked by the application of recombinant IL-6.As for the mechanism of neuro-protection by IL-6, an adenosine A1 receptor-mediated pathway in neurons seems to be involved.Taken together, when astrocytes sense MeHg, they release ATP that autostimulates P2Y1 receptors to upregulate IL-6, thereby leading to A1 receptor-mediated neuro-protection against MeHg.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuropharmacology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Yamanashi, Japan.

ABSTRACT
Methylmercury (MeHg) is a well known environmental pollutant that induces serious neuronal damage. Although MeHg readily crosses the blood-brain barrier, and should affect both neurons and glial cells, how it affects glia or neuron-to-glia interactions has received only limited attention. Here, we report that MeHg triggers ATP/P2Y1 receptor signals in astrocytes, thereby protecting neurons against MeHg via interleukin-6 (IL-6)-mediated pathways. MeHg increased several mRNAs in astrocytes, among which IL-6 was the highest. For this, ATP/P2Y1 receptor-mediated mechanisms were required because the IL-6 production was (i) inhibited by a P2Y1 receptor antagonist, MRS2179, (ii) abolished in astrocytes obtained from P2Y1 receptor-knockout mice, and (iii) mimicked by exogenously applied ATP. In addition, (iv) MeHg released ATP by exocytosis from astrocytes. As for the intracellular mechanisms responsible for IL-6 production, p38 MAP kinase was involved. MeHg-treated astrocyte-conditioned medium (ACM) showed neuro-protective effects against MeHg, which was blocked by anti-IL-6 antibody and was mimicked by the application of recombinant IL-6. As for the mechanism of neuro-protection by IL-6, an adenosine A1 receptor-mediated pathway in neurons seems to be involved. Taken together, when astrocytes sense MeHg, they release ATP that autostimulates P2Y1 receptors to upregulate IL-6, thereby leading to A1 receptor-mediated neuro-protection against MeHg.

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IL-6 upregulation by MeHg is mediated by P2Y1 receptors followed by p38 activation.(A) P2Y1 receptor blockade suppresses IL-6 mRNA expression induced by MeHg. MeHg (3 µM, 2 hr)-increased IL-6 mRNA expression was inhibited by either suramin (sur, 100 µM) or MRS2179 (MRS, 10 µM). *P<0.05 vs. MeHg. (B) P2Y1 receptor mediates MeHg-induced IL-6 mRNA expression. P2Y1R KO astrocytes exhibited no increase in IL-6 mRNA with MeHg (3 µM, 2 hr). **P<0.01 vs. WT. (C) Contribution of p38 in MeHg-induced IL-6 mRNA expression. The IL-6 mRNA expression evoked by MeHg (3 µM, 2 hr) was inhibited by SB203580 (SB, 10 µM) but not by PD98059 (PD, 10 µM) or SP600125 (SP, 10 µM). *P<0.05 vs. MeHg. (D) Downstream signaling molecule of P2Y1 receptor is p38. MeHg (3 µM, 30 min)-induced p38 phosphorylation was inhibited by suramin (sur, 100 µM). ATP (100 µM) also induced p38 phosphorylation. *P<0.05, **P<0.01 vs. control, #P<0.05 vs. MeHg.
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pone-0057898-g002: IL-6 upregulation by MeHg is mediated by P2Y1 receptors followed by p38 activation.(A) P2Y1 receptor blockade suppresses IL-6 mRNA expression induced by MeHg. MeHg (3 µM, 2 hr)-increased IL-6 mRNA expression was inhibited by either suramin (sur, 100 µM) or MRS2179 (MRS, 10 µM). *P<0.05 vs. MeHg. (B) P2Y1 receptor mediates MeHg-induced IL-6 mRNA expression. P2Y1R KO astrocytes exhibited no increase in IL-6 mRNA with MeHg (3 µM, 2 hr). **P<0.01 vs. WT. (C) Contribution of p38 in MeHg-induced IL-6 mRNA expression. The IL-6 mRNA expression evoked by MeHg (3 µM, 2 hr) was inhibited by SB203580 (SB, 10 µM) but not by PD98059 (PD, 10 µM) or SP600125 (SP, 10 µM). *P<0.05 vs. MeHg. (D) Downstream signaling molecule of P2Y1 receptor is p38. MeHg (3 µM, 30 min)-induced p38 phosphorylation was inhibited by suramin (sur, 100 µM). ATP (100 µM) also induced p38 phosphorylation. *P<0.05, **P<0.01 vs. control, #P<0.05 vs. MeHg.

Mentions: The mechanisms underlying the MeHg-evoked IL-6 production were investigated. Since astrocytes release or leak ATP that functions as a gliotransmitter or inflammatory mediator in response to various environmental changes, we firstly focused on ATP/P2 receptor-mediated signals. The MeHg (3 µM, 2 hr)-evoked increase in IL-6 mRNA was significantly suppressed by the broad P2 receptor antagonist suramin (100 µM) (MeHg/suramin, 58.7±8.1% of MeHg, n = 8) (Fig. 2A). The selective P2Y1 receptor antagonist MRS2179 (10 µM) also inhibited the IL-6 mRNA upregulation to a similar extent (MeHg/MRS2179, 64.0±4.0% of MeHg, n = 9), suggesting the predominant involvement of P2Y1 receptors in the MeHg-evoked IL-6 production. In addition, MeHg (3 µM, 2 hr) failed to increase IL-6 mRNA in P2Y1R KO astrocytes (WT, 5.8±0.7; P2Y1R KO, 1.0±0.7 fold increase vs. control, n = 3) (Fig. 2B), confirming that P2Y1 receptors are necessary in this process. As for intracellular signaling mechanisms, it is known that ATP activates mitogen-activated protein kinases (MAPKs) including ERK1/2, JNK, and p38 in glial cells via several types of P2 receptors [24], [25], [26], [27]. PD98059 (10 µM), an inhibitor of mitogen-activated protein kinase kinase, the upstream activator of ERK [28] and SP600125 (10 µM), an inhibitor of SAPK/JNK [29], exhibited no effect on the IL-6 expression (MeHg/PD98059, 78.3±37.5% of MeHg, n = 4; MeHg/SP600125, 127.5±19.5% of MeHg, n = 6) (Fig. 2C). In contrast, a p38 inhibitor, SB203580 (10 µM) [30], significantly suppressed the MeHg-mediated increase in IL-6 mRNA (MeHg/SB203580, 54.7±13.9% of MeHg, n = 6). Western blotting analysis revealed that MeHg (3 µM, 30 min) induced p38 phosphorylation, which was strongly blocked by suramin (control, 100.0±8.5; MeHg, 210.0±36.3; MeHg/suramin, 114.0±19.7% of control, n = 4) (Fig. 2D). The phosphorylation of p38 in astrocytes was mimicked by ATP (100 µM, 30 min) (246.3±58.0% of control, n = 4). Overall, the data suggest that the activation of P2Y1 receptors and subsequent phosphorylation of p38 MAPK pathway should be involved in the MeHg-evoked IL-6 production.


Astrocytes protect neurons against methylmercury via ATP/P2Y(1) receptor-mediated pathways in astrocytes.

Noguchi Y, Shinozaki Y, Fujishita K, Shibata K, Imura Y, Morizawa Y, Gachet C, Koizumi S - PLoS ONE (2013)

IL-6 upregulation by MeHg is mediated by P2Y1 receptors followed by p38 activation.(A) P2Y1 receptor blockade suppresses IL-6 mRNA expression induced by MeHg. MeHg (3 µM, 2 hr)-increased IL-6 mRNA expression was inhibited by either suramin (sur, 100 µM) or MRS2179 (MRS, 10 µM). *P<0.05 vs. MeHg. (B) P2Y1 receptor mediates MeHg-induced IL-6 mRNA expression. P2Y1R KO astrocytes exhibited no increase in IL-6 mRNA with MeHg (3 µM, 2 hr). **P<0.01 vs. WT. (C) Contribution of p38 in MeHg-induced IL-6 mRNA expression. The IL-6 mRNA expression evoked by MeHg (3 µM, 2 hr) was inhibited by SB203580 (SB, 10 µM) but not by PD98059 (PD, 10 µM) or SP600125 (SP, 10 µM). *P<0.05 vs. MeHg. (D) Downstream signaling molecule of P2Y1 receptor is p38. MeHg (3 µM, 30 min)-induced p38 phosphorylation was inhibited by suramin (sur, 100 µM). ATP (100 µM) also induced p38 phosphorylation. *P<0.05, **P<0.01 vs. control, #P<0.05 vs. MeHg.
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pone-0057898-g002: IL-6 upregulation by MeHg is mediated by P2Y1 receptors followed by p38 activation.(A) P2Y1 receptor blockade suppresses IL-6 mRNA expression induced by MeHg. MeHg (3 µM, 2 hr)-increased IL-6 mRNA expression was inhibited by either suramin (sur, 100 µM) or MRS2179 (MRS, 10 µM). *P<0.05 vs. MeHg. (B) P2Y1 receptor mediates MeHg-induced IL-6 mRNA expression. P2Y1R KO astrocytes exhibited no increase in IL-6 mRNA with MeHg (3 µM, 2 hr). **P<0.01 vs. WT. (C) Contribution of p38 in MeHg-induced IL-6 mRNA expression. The IL-6 mRNA expression evoked by MeHg (3 µM, 2 hr) was inhibited by SB203580 (SB, 10 µM) but not by PD98059 (PD, 10 µM) or SP600125 (SP, 10 µM). *P<0.05 vs. MeHg. (D) Downstream signaling molecule of P2Y1 receptor is p38. MeHg (3 µM, 30 min)-induced p38 phosphorylation was inhibited by suramin (sur, 100 µM). ATP (100 µM) also induced p38 phosphorylation. *P<0.05, **P<0.01 vs. control, #P<0.05 vs. MeHg.
Mentions: The mechanisms underlying the MeHg-evoked IL-6 production were investigated. Since astrocytes release or leak ATP that functions as a gliotransmitter or inflammatory mediator in response to various environmental changes, we firstly focused on ATP/P2 receptor-mediated signals. The MeHg (3 µM, 2 hr)-evoked increase in IL-6 mRNA was significantly suppressed by the broad P2 receptor antagonist suramin (100 µM) (MeHg/suramin, 58.7±8.1% of MeHg, n = 8) (Fig. 2A). The selective P2Y1 receptor antagonist MRS2179 (10 µM) also inhibited the IL-6 mRNA upregulation to a similar extent (MeHg/MRS2179, 64.0±4.0% of MeHg, n = 9), suggesting the predominant involvement of P2Y1 receptors in the MeHg-evoked IL-6 production. In addition, MeHg (3 µM, 2 hr) failed to increase IL-6 mRNA in P2Y1R KO astrocytes (WT, 5.8±0.7; P2Y1R KO, 1.0±0.7 fold increase vs. control, n = 3) (Fig. 2B), confirming that P2Y1 receptors are necessary in this process. As for intracellular signaling mechanisms, it is known that ATP activates mitogen-activated protein kinases (MAPKs) including ERK1/2, JNK, and p38 in glial cells via several types of P2 receptors [24], [25], [26], [27]. PD98059 (10 µM), an inhibitor of mitogen-activated protein kinase kinase, the upstream activator of ERK [28] and SP600125 (10 µM), an inhibitor of SAPK/JNK [29], exhibited no effect on the IL-6 expression (MeHg/PD98059, 78.3±37.5% of MeHg, n = 4; MeHg/SP600125, 127.5±19.5% of MeHg, n = 6) (Fig. 2C). In contrast, a p38 inhibitor, SB203580 (10 µM) [30], significantly suppressed the MeHg-mediated increase in IL-6 mRNA (MeHg/SB203580, 54.7±13.9% of MeHg, n = 6). Western blotting analysis revealed that MeHg (3 µM, 30 min) induced p38 phosphorylation, which was strongly blocked by suramin (control, 100.0±8.5; MeHg, 210.0±36.3; MeHg/suramin, 114.0±19.7% of control, n = 4) (Fig. 2D). The phosphorylation of p38 in astrocytes was mimicked by ATP (100 µM, 30 min) (246.3±58.0% of control, n = 4). Overall, the data suggest that the activation of P2Y1 receptors and subsequent phosphorylation of p38 MAPK pathway should be involved in the MeHg-evoked IL-6 production.

Bottom Line: MeHg-treated astrocyte-conditioned medium (ACM) showed neuro-protective effects against MeHg, which was blocked by anti-IL-6 antibody and was mimicked by the application of recombinant IL-6.As for the mechanism of neuro-protection by IL-6, an adenosine A1 receptor-mediated pathway in neurons seems to be involved.Taken together, when astrocytes sense MeHg, they release ATP that autostimulates P2Y1 receptors to upregulate IL-6, thereby leading to A1 receptor-mediated neuro-protection against MeHg.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuropharmacology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Yamanashi, Japan.

ABSTRACT
Methylmercury (MeHg) is a well known environmental pollutant that induces serious neuronal damage. Although MeHg readily crosses the blood-brain barrier, and should affect both neurons and glial cells, how it affects glia or neuron-to-glia interactions has received only limited attention. Here, we report that MeHg triggers ATP/P2Y1 receptor signals in astrocytes, thereby protecting neurons against MeHg via interleukin-6 (IL-6)-mediated pathways. MeHg increased several mRNAs in astrocytes, among which IL-6 was the highest. For this, ATP/P2Y1 receptor-mediated mechanisms were required because the IL-6 production was (i) inhibited by a P2Y1 receptor antagonist, MRS2179, (ii) abolished in astrocytes obtained from P2Y1 receptor-knockout mice, and (iii) mimicked by exogenously applied ATP. In addition, (iv) MeHg released ATP by exocytosis from astrocytes. As for the intracellular mechanisms responsible for IL-6 production, p38 MAP kinase was involved. MeHg-treated astrocyte-conditioned medium (ACM) showed neuro-protective effects against MeHg, which was blocked by anti-IL-6 antibody and was mimicked by the application of recombinant IL-6. As for the mechanism of neuro-protection by IL-6, an adenosine A1 receptor-mediated pathway in neurons seems to be involved. Taken together, when astrocytes sense MeHg, they release ATP that autostimulates P2Y1 receptors to upregulate IL-6, thereby leading to A1 receptor-mediated neuro-protection against MeHg.

Show MeSH
Related in: MedlinePlus