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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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Adenosine A1 receptor mediates neuro-protection against MeHg via suppressing excitatory neurotransmission.(A) Newly synthesized proteins participate in the IL-6-mediated neuro-protection by recombinant IL-6 (100 pg/ml) was suppressed by CHX (1 µM). **P<0.01 vs. MeHg/IL-6. (B) Upregulation of adenosine A1 receptor mRNA by recombinant IL-6 (200 pg/ml, 2 hr) in the cortical neurons. *P<0.05 vs. no treatment. (C) Changes in [Ca2+]i in control and IL-6-treated cortical neurons, showing effect of A1 receptors. (i) The basal [Ca2+]i level in IL-6-treated (100 pg/ml, 24 hr) neurons was significantly lower than that in control neurons. This decrease was restored by DPCPX (1 µM). *P<0.05 vs. IL-6 alone. (ii) Representative traces of the glutamate-evoked increases in [Ca2+]i in non-treated control (left), IL-6-treated (100 pg/ml, 24 hr) (right) and IL-6-treated neurons in the presence of 1 µM DPCPX. Glutamate (10 µM) was added to the neurons for 10 s. Bold line in each panel showed averaged changes in [Ca2+]i in neurons, which was summarized in (iii). The glutamate-evoked increase in [Ca2+]i in IL-6-treated neurons was significantly lower than that in control neurons, which was restored by DPCPX. **P<0.01 vs. Glu/IL-6. (D) A1 receptor-mediated neuro-protection by IL-6. The protective effect of IL-6 (100 pg/ml) was suppressed by DPCPX (1 µM). **P<0.01 vs. MeHg/IL-6. (E) ATP-induced neuro-protection is mediated by A1 receptor. Exogenously applied ATP (100 µM) restored the MeHg (1 µM, 48 hr)-reduced neuronal viability, and this effect was blocked by DPCPX (1 µM). *P<0.05, **P<0.01 vs. MeHg/ATP. (F) The MeHg-evoked increase in activity of ecto-ATPases in astrocytes. Activity of ecto-ATPases was analyzed by an enzyme histochemical assay. When stimulated with MeHg (3 µM), the activity (shown as brown signals) was increased, which peaked at around 6 to 12 hr. Scale bar, 200 µm.
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pone-0057898-g006: Adenosine A1 receptor mediates neuro-protection against MeHg via suppressing excitatory neurotransmission.(A) Newly synthesized proteins participate in the IL-6-mediated neuro-protection by recombinant IL-6 (100 pg/ml) was suppressed by CHX (1 µM). **P<0.01 vs. MeHg/IL-6. (B) Upregulation of adenosine A1 receptor mRNA by recombinant IL-6 (200 pg/ml, 2 hr) in the cortical neurons. *P<0.05 vs. no treatment. (C) Changes in [Ca2+]i in control and IL-6-treated cortical neurons, showing effect of A1 receptors. (i) The basal [Ca2+]i level in IL-6-treated (100 pg/ml, 24 hr) neurons was significantly lower than that in control neurons. This decrease was restored by DPCPX (1 µM). *P<0.05 vs. IL-6 alone. (ii) Representative traces of the glutamate-evoked increases in [Ca2+]i in non-treated control (left), IL-6-treated (100 pg/ml, 24 hr) (right) and IL-6-treated neurons in the presence of 1 µM DPCPX. Glutamate (10 µM) was added to the neurons for 10 s. Bold line in each panel showed averaged changes in [Ca2+]i in neurons, which was summarized in (iii). The glutamate-evoked increase in [Ca2+]i in IL-6-treated neurons was significantly lower than that in control neurons, which was restored by DPCPX. **P<0.01 vs. Glu/IL-6. (D) A1 receptor-mediated neuro-protection by IL-6. The protective effect of IL-6 (100 pg/ml) was suppressed by DPCPX (1 µM). **P<0.01 vs. MeHg/IL-6. (E) ATP-induced neuro-protection is mediated by A1 receptor. Exogenously applied ATP (100 µM) restored the MeHg (1 µM, 48 hr)-reduced neuronal viability, and this effect was blocked by DPCPX (1 µM). *P<0.05, **P<0.01 vs. MeHg/ATP. (F) The MeHg-evoked increase in activity of ecto-ATPases in astrocytes. Activity of ecto-ATPases was analyzed by an enzyme histochemical assay. When stimulated with MeHg (3 µM), the activity (shown as brown signals) was increased, which peaked at around 6 to 12 hr. Scale bar, 200 µm.

Mentions: The neuro-protection by recombinant IL-6 was significantly inhibited by 1 µM cycloheximide (CHX), an inhibitor of protein synthesis (MeHg, 65.7±7.6%; MeHg/IL-6, 108.3±4.5%; MeHg/IL-6/CHX, 86.1±4.2%, n = 5) (Fig. 6A). CHX (1 µM, added to the culture 24 hr after IL-6 treatment) alone had no effect on neuronal cell viability (107.2±2.2%, n = 5). Thus, it appears that IL-6 would newly synthesize neuro-protective molecules that would account for the neuro-protection against MeHg. IL-6 has been reported to increase A1 receptor expression thereby inducing neuro-protection against cytotoxicity [53], [54]. We found that recombinant IL-6 (200 pg/ml, 2 hr) significantly increased adenosine A1 receptor mRNA in cortical neurons (control, 100±6.7%; IL-6, 173.1±23.9%, n = 7) (Fig. 6B). We further tested whether the IL-6-mediated A1 receptor upregulation contributes to excitability of cortical neurons. Firstly, we investigated whether pre-treatment with IL-6 might affect basal level of [Ca2+]i in the cortical neurons, because this can reflect a baseline activity of synaptic transmission. Thus, when excitatory synaptic transmission is inhibited by either TTX, antagonists of glutamate receptors, or agonists of presynaptic auto-receptors such as A1 receptors, the basal [Ca2+]i is decreased [20], [21]. As shown in Fig. 6C (i), the basal [Ca2+]i in IL-6-treated neurons (100 pg/ml, 24 hr) was lower than that in control neurons, which was restored by an A1 receptor antagonist DPCPX (1 µM, 5 min) (F340/F380: no treatment, 0.72±0.06; IL-6, 0.56±0.01; IL-6/DPCPX, 0.74±0.05, n = 99). Secondly, we tested whether the glutamate-evoked responses were affected by the IL-6-treatment. The glutamate (10 µM)-evoked increase in [Ca2+]i in IL-6-treated neurons was smaller than that in control neurons(F340/F380: no treatment, 3.2±0.2; IL-6, 2.2±0.2, n = 99), which was also restored by 1 µM DPCPX (IL-6/DPCPX, 3.0±0.2, n = 99) (Fig. 6C (ii) (iii)). DPCPX alone (1 µM) never affected the neuronal viability (control, 100.0±2.9%; DPCPX, 95.6±6.0%, n = 5). All these findings suggest that IL-6 could regulate excitability of cortical neurons by increasing both expression and function of adenosine A1 receptors, which might contribute to neuro-protection against MeHg.


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)

Adenosine A1 receptor mediates neuro-protection against MeHg via suppressing excitatory neurotransmission.(A) Newly synthesized proteins participate in the IL-6-mediated neuro-protection by recombinant IL-6 (100 pg/ml) was suppressed by CHX (1 µM). **P<0.01 vs. MeHg/IL-6. (B) Upregulation of adenosine A1 receptor mRNA by recombinant IL-6 (200 pg/ml, 2 hr) in the cortical neurons. *P<0.05 vs. no treatment. (C) Changes in [Ca2+]i in control and IL-6-treated cortical neurons, showing effect of A1 receptors. (i) The basal [Ca2+]i level in IL-6-treated (100 pg/ml, 24 hr) neurons was significantly lower than that in control neurons. This decrease was restored by DPCPX (1 µM). *P<0.05 vs. IL-6 alone. (ii) Representative traces of the glutamate-evoked increases in [Ca2+]i in non-treated control (left), IL-6-treated (100 pg/ml, 24 hr) (right) and IL-6-treated neurons in the presence of 1 µM DPCPX. Glutamate (10 µM) was added to the neurons for 10 s. Bold line in each panel showed averaged changes in [Ca2+]i in neurons, which was summarized in (iii). The glutamate-evoked increase in [Ca2+]i in IL-6-treated neurons was significantly lower than that in control neurons, which was restored by DPCPX. **P<0.01 vs. Glu/IL-6. (D) A1 receptor-mediated neuro-protection by IL-6. The protective effect of IL-6 (100 pg/ml) was suppressed by DPCPX (1 µM). **P<0.01 vs. MeHg/IL-6. (E) ATP-induced neuro-protection is mediated by A1 receptor. Exogenously applied ATP (100 µM) restored the MeHg (1 µM, 48 hr)-reduced neuronal viability, and this effect was blocked by DPCPX (1 µM). *P<0.05, **P<0.01 vs. MeHg/ATP. (F) The MeHg-evoked increase in activity of ecto-ATPases in astrocytes. Activity of ecto-ATPases was analyzed by an enzyme histochemical assay. When stimulated with MeHg (3 µM), the activity (shown as brown signals) was increased, which peaked at around 6 to 12 hr. Scale bar, 200 µm.
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pone-0057898-g006: Adenosine A1 receptor mediates neuro-protection against MeHg via suppressing excitatory neurotransmission.(A) Newly synthesized proteins participate in the IL-6-mediated neuro-protection by recombinant IL-6 (100 pg/ml) was suppressed by CHX (1 µM). **P<0.01 vs. MeHg/IL-6. (B) Upregulation of adenosine A1 receptor mRNA by recombinant IL-6 (200 pg/ml, 2 hr) in the cortical neurons. *P<0.05 vs. no treatment. (C) Changes in [Ca2+]i in control and IL-6-treated cortical neurons, showing effect of A1 receptors. (i) The basal [Ca2+]i level in IL-6-treated (100 pg/ml, 24 hr) neurons was significantly lower than that in control neurons. This decrease was restored by DPCPX (1 µM). *P<0.05 vs. IL-6 alone. (ii) Representative traces of the glutamate-evoked increases in [Ca2+]i in non-treated control (left), IL-6-treated (100 pg/ml, 24 hr) (right) and IL-6-treated neurons in the presence of 1 µM DPCPX. Glutamate (10 µM) was added to the neurons for 10 s. Bold line in each panel showed averaged changes in [Ca2+]i in neurons, which was summarized in (iii). The glutamate-evoked increase in [Ca2+]i in IL-6-treated neurons was significantly lower than that in control neurons, which was restored by DPCPX. **P<0.01 vs. Glu/IL-6. (D) A1 receptor-mediated neuro-protection by IL-6. The protective effect of IL-6 (100 pg/ml) was suppressed by DPCPX (1 µM). **P<0.01 vs. MeHg/IL-6. (E) ATP-induced neuro-protection is mediated by A1 receptor. Exogenously applied ATP (100 µM) restored the MeHg (1 µM, 48 hr)-reduced neuronal viability, and this effect was blocked by DPCPX (1 µM). *P<0.05, **P<0.01 vs. MeHg/ATP. (F) The MeHg-evoked increase in activity of ecto-ATPases in astrocytes. Activity of ecto-ATPases was analyzed by an enzyme histochemical assay. When stimulated with MeHg (3 µM), the activity (shown as brown signals) was increased, which peaked at around 6 to 12 hr. Scale bar, 200 µm.
Mentions: The neuro-protection by recombinant IL-6 was significantly inhibited by 1 µM cycloheximide (CHX), an inhibitor of protein synthesis (MeHg, 65.7±7.6%; MeHg/IL-6, 108.3±4.5%; MeHg/IL-6/CHX, 86.1±4.2%, n = 5) (Fig. 6A). CHX (1 µM, added to the culture 24 hr after IL-6 treatment) alone had no effect on neuronal cell viability (107.2±2.2%, n = 5). Thus, it appears that IL-6 would newly synthesize neuro-protective molecules that would account for the neuro-protection against MeHg. IL-6 has been reported to increase A1 receptor expression thereby inducing neuro-protection against cytotoxicity [53], [54]. We found that recombinant IL-6 (200 pg/ml, 2 hr) significantly increased adenosine A1 receptor mRNA in cortical neurons (control, 100±6.7%; IL-6, 173.1±23.9%, n = 7) (Fig. 6B). We further tested whether the IL-6-mediated A1 receptor upregulation contributes to excitability of cortical neurons. Firstly, we investigated whether pre-treatment with IL-6 might affect basal level of [Ca2+]i in the cortical neurons, because this can reflect a baseline activity of synaptic transmission. Thus, when excitatory synaptic transmission is inhibited by either TTX, antagonists of glutamate receptors, or agonists of presynaptic auto-receptors such as A1 receptors, the basal [Ca2+]i is decreased [20], [21]. As shown in Fig. 6C (i), the basal [Ca2+]i in IL-6-treated neurons (100 pg/ml, 24 hr) was lower than that in control neurons, which was restored by an A1 receptor antagonist DPCPX (1 µM, 5 min) (F340/F380: no treatment, 0.72±0.06; IL-6, 0.56±0.01; IL-6/DPCPX, 0.74±0.05, n = 99). Secondly, we tested whether the glutamate-evoked responses were affected by the IL-6-treatment. The glutamate (10 µM)-evoked increase in [Ca2+]i in IL-6-treated neurons was smaller than that in control neurons(F340/F380: no treatment, 3.2±0.2; IL-6, 2.2±0.2, n = 99), which was also restored by 1 µM DPCPX (IL-6/DPCPX, 3.0±0.2, n = 99) (Fig. 6C (ii) (iii)). DPCPX alone (1 µM) never affected the neuronal viability (control, 100.0±2.9%; DPCPX, 95.6±6.0%, n = 5). All these findings suggest that IL-6 could regulate excitability of cortical neurons by increasing both expression and function of adenosine A1 receptors, which might contribute to neuro-protection against MeHg.

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