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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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Neuro-protective effect of recombinant IL-6.(A) The experimental schedule. Recombinant IL-6 (100 pg/ml) was pretreated to the cell culture 24 hr before MeHg exposure (48 hr). Cells were then fixed for immunostaining or used for WST-1 assay. (B) MeHg-induced morphological changes in neurons. MAP2 signals in the processes were disrupted by MeHg (3 µM), whereas the GFAP signals exhibited no differences with or without MeHg. Scale bar: 50 µm. (C) MeHg-decreased the neuronal viability. Neuronal viability was significantly reduced by MeHg in a concentration-dependent fashion at a concentration range of from 0.01 to 3 µM (gray columns). MeHg did not affect the astrocytic viability (white columns). *P<0.05, **P<0.01 vs. control. (D) Recombinant IL-6 restored the MeHg-induced morphological changes in neurons. The collapsed MAP2 signals in neuronal processes were recovered in the presence of recombinant IL-6 (100 ng/ml). IL-6 did not change the neuronal morphology in controls. Scale bar: 50 µm. (E) IL-6 protects neurons against MeHg. Recombinant IL-6 (100 ng/ml) significantly restored the MeHg (1 or 3 µM)-reduced neuronal viability. **P<0.01 vs. MeHg.
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pone-0057898-g004: Neuro-protective effect of recombinant IL-6.(A) The experimental schedule. Recombinant IL-6 (100 pg/ml) was pretreated to the cell culture 24 hr before MeHg exposure (48 hr). Cells were then fixed for immunostaining or used for WST-1 assay. (B) MeHg-induced morphological changes in neurons. MAP2 signals in the processes were disrupted by MeHg (3 µM), whereas the GFAP signals exhibited no differences with or without MeHg. Scale bar: 50 µm. (C) MeHg-decreased the neuronal viability. Neuronal viability was significantly reduced by MeHg in a concentration-dependent fashion at a concentration range of from 0.01 to 3 µM (gray columns). MeHg did not affect the astrocytic viability (white columns). *P<0.05, **P<0.01 vs. control. (D) Recombinant IL-6 restored the MeHg-induced morphological changes in neurons. The collapsed MAP2 signals in neuronal processes were recovered in the presence of recombinant IL-6 (100 ng/ml). IL-6 did not change the neuronal morphology in controls. Scale bar: 50 µm. (E) IL-6 protects neurons against MeHg. Recombinant IL-6 (100 ng/ml) significantly restored the MeHg (1 or 3 µM)-reduced neuronal viability. **P<0.01 vs. MeHg.

Mentions: We [18] and others [19], [46], [47], [48], [49], [50], [51], [52] have already reported that IL-6 had neuro-protective effects against several types of insults. Using immunocytochemical analysis and WST-1 assay, we evaluated whether IL-6 showed neuro-protection against MeHg. As shown in Fig. 4B, healthy cortical neurons exhibited clear cell bodies and extended dendrites when stained with anti-MAP2 antibody (Fig. 4B, MAP2-control). After MeHg treatment (3 µM, 48 hr), the anti-MAP2 signals were dramatically changed into signals with only cell body-like structures and fragmented or bead-like process structures (Fig. 4B, MAP2-MeHg). In contrast to neurons, anti-GFAP signals exhibited no significant changes with or without MeHg (Fig. 4B, GFAP). WST-1 assay revealed that MeHg (48 hr) decreased neuronal viability in a concentration-dependent manner over a range of from 0.01 to 3 µM (control, 100±5.0%; 0.01 µM, 84.9±4.3%; 0.1 µM, 75.2±4.3%; 1 µM, 50.0±3.2%; 3 µM, 19.9±3.2%) (Fig. 4C, gray columns), whereas astrocytes showed no decrease in cell viability (control, 100±2.4%; 0.01 µM, 96.6±2.9%; 0.1 µM, 96.7±1.8%; 1 µM, 100.6±4.1%; 3 µM, 103.9±4.6%) (Fig. 4C, white columns). Recombinant IL-6 protein (100 pg/ml, 24 hr pretreatment) suppressed the MeHg-induced morphological changes in neurons (Fig. 4D, MeHg+IL-6). IL-6 itself had no significant morphological effect on neurons (Fig. 4D, IL-6). Recombinant IL-6 protein also restored the MeHg (1 or 3 µM, 48 hr)-reduced neuronal viability (MeHg 1 µM, 35.5±3.9%; MeHg 3 µM, 16.4±2.4%; MeHg 1 µM/IL-6, 49.7±4.2%; MeHg 3 µM/IL-6, 46.7±4.0%, n = 20) (Fig. 4E). IL-6 itself had no significant effect on neuronal cell viability (93.2±3.3%, n = 14). When IL-6 was added to the neuronal culture 12 hr after MeHg treatment, it did not show neuro-protection (data not shown). As it is known that neuronal culture contains small portion of astrocytes, we estimated glial contamination in the culture of cortical neurons by immunocytochemical analysis. In our culture condition, almost all cells were positive to MAP2 (98.8±1.2%, n = 780), and remained 1.2±0.2% of cells were positive to GFAP, suggesting that glial contamination could be negligible.


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)

Neuro-protective effect of recombinant IL-6.(A) The experimental schedule. Recombinant IL-6 (100 pg/ml) was pretreated to the cell culture 24 hr before MeHg exposure (48 hr). Cells were then fixed for immunostaining or used for WST-1 assay. (B) MeHg-induced morphological changes in neurons. MAP2 signals in the processes were disrupted by MeHg (3 µM), whereas the GFAP signals exhibited no differences with or without MeHg. Scale bar: 50 µm. (C) MeHg-decreased the neuronal viability. Neuronal viability was significantly reduced by MeHg in a concentration-dependent fashion at a concentration range of from 0.01 to 3 µM (gray columns). MeHg did not affect the astrocytic viability (white columns). *P<0.05, **P<0.01 vs. control. (D) Recombinant IL-6 restored the MeHg-induced morphological changes in neurons. The collapsed MAP2 signals in neuronal processes were recovered in the presence of recombinant IL-6 (100 ng/ml). IL-6 did not change the neuronal morphology in controls. Scale bar: 50 µm. (E) IL-6 protects neurons against MeHg. Recombinant IL-6 (100 ng/ml) significantly restored the MeHg (1 or 3 µM)-reduced neuronal viability. **P<0.01 vs. MeHg.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585279&req=5

pone-0057898-g004: Neuro-protective effect of recombinant IL-6.(A) The experimental schedule. Recombinant IL-6 (100 pg/ml) was pretreated to the cell culture 24 hr before MeHg exposure (48 hr). Cells were then fixed for immunostaining or used for WST-1 assay. (B) MeHg-induced morphological changes in neurons. MAP2 signals in the processes were disrupted by MeHg (3 µM), whereas the GFAP signals exhibited no differences with or without MeHg. Scale bar: 50 µm. (C) MeHg-decreased the neuronal viability. Neuronal viability was significantly reduced by MeHg in a concentration-dependent fashion at a concentration range of from 0.01 to 3 µM (gray columns). MeHg did not affect the astrocytic viability (white columns). *P<0.05, **P<0.01 vs. control. (D) Recombinant IL-6 restored the MeHg-induced morphological changes in neurons. The collapsed MAP2 signals in neuronal processes were recovered in the presence of recombinant IL-6 (100 ng/ml). IL-6 did not change the neuronal morphology in controls. Scale bar: 50 µm. (E) IL-6 protects neurons against MeHg. Recombinant IL-6 (100 ng/ml) significantly restored the MeHg (1 or 3 µM)-reduced neuronal viability. **P<0.01 vs. MeHg.
Mentions: We [18] and others [19], [46], [47], [48], [49], [50], [51], [52] have already reported that IL-6 had neuro-protective effects against several types of insults. Using immunocytochemical analysis and WST-1 assay, we evaluated whether IL-6 showed neuro-protection against MeHg. As shown in Fig. 4B, healthy cortical neurons exhibited clear cell bodies and extended dendrites when stained with anti-MAP2 antibody (Fig. 4B, MAP2-control). After MeHg treatment (3 µM, 48 hr), the anti-MAP2 signals were dramatically changed into signals with only cell body-like structures and fragmented or bead-like process structures (Fig. 4B, MAP2-MeHg). In contrast to neurons, anti-GFAP signals exhibited no significant changes with or without MeHg (Fig. 4B, GFAP). WST-1 assay revealed that MeHg (48 hr) decreased neuronal viability in a concentration-dependent manner over a range of from 0.01 to 3 µM (control, 100±5.0%; 0.01 µM, 84.9±4.3%; 0.1 µM, 75.2±4.3%; 1 µM, 50.0±3.2%; 3 µM, 19.9±3.2%) (Fig. 4C, gray columns), whereas astrocytes showed no decrease in cell viability (control, 100±2.4%; 0.01 µM, 96.6±2.9%; 0.1 µM, 96.7±1.8%; 1 µM, 100.6±4.1%; 3 µM, 103.9±4.6%) (Fig. 4C, white columns). Recombinant IL-6 protein (100 pg/ml, 24 hr pretreatment) suppressed the MeHg-induced morphological changes in neurons (Fig. 4D, MeHg+IL-6). IL-6 itself had no significant morphological effect on neurons (Fig. 4D, IL-6). Recombinant IL-6 protein also restored the MeHg (1 or 3 µM, 48 hr)-reduced neuronal viability (MeHg 1 µM, 35.5±3.9%; MeHg 3 µM, 16.4±2.4%; MeHg 1 µM/IL-6, 49.7±4.2%; MeHg 3 µM/IL-6, 46.7±4.0%, n = 20) (Fig. 4E). IL-6 itself had no significant effect on neuronal cell viability (93.2±3.3%, n = 14). When IL-6 was added to the neuronal culture 12 hr after MeHg treatment, it did not show neuro-protection (data not shown). As it is known that neuronal culture contains small portion of astrocytes, we estimated glial contamination in the culture of cortical neurons by immunocytochemical analysis. In our culture condition, almost all cells were positive to MAP2 (98.8±1.2%, n = 780), and remained 1.2±0.2% of cells were positive to GFAP, suggesting that glial contamination could be negligible.

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