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Glutamate transporter-dependent mTOR phosphorylation in Müller glia cells.

María López-Colomé A, Martínez-Lozada Z, Guillem AM, López E, Ortega A - ASN Neuro (2012)

Bottom Line: The results showed that D-Asp transport induces the time- and dose-dependent phosphorylation of mTOR, mimicked by the transportable GLAST inhibitor THA (threo-β-hydroxyaspartate).Signalling leading to mTOR phosphorylation includes Ca2+ influx, the activation of p60src, phosphatidylinositol 3-kinase, protein kinase B, mTOR and p70S6K.Interestingly, GLAST activity promoted AP-1 (activator protein-1) binding to DNA, supporting a function for transporter signalling in retinal long-term responses.

View Article: PubMed Central - PubMed

Affiliation: *División de Neurociencias, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, México D.F., México.

ABSTRACT
Glu (glutamate), the excitatory transmitter at the main signalling pathway in the retina, is critically involved in changes in the protein repertoire through the activation of signalling cascades, which regulate protein synthesis at transcriptional and translational levels. Activity-dependent differential gene expression by Glu is related to the activation of ionotropic and metabotropic Glu receptors; however, recent findings suggest the involvement of Na+-dependent Glu transporters in this process. Within the retina, Glu uptake is aimed at the replenishment of the releasable pool, and for the prevention of excitotoxicity and is carried mainly by the GLAST/EAAT-1 (Na+-dependent glutamate/aspartate transporter/excitatory amino acids transporter-1) located in Müller radial glia. Based on the previous work showing the alteration of GLAST expression induced by Glu, the present work investigates the involvement of GLAST signalling in the regulation of protein synthesis in Müller cells. To this end, we explored the effect of D-Asp (D-aspartate) on Ser-2448 mTOR (mammalian target of rapamycin) phosphorylation in primary cultures of chick Müller glia. The results showed that D-Asp transport induces the time- and dose-dependent phosphorylation of mTOR, mimicked by the transportable GLAST inhibitor THA (threo-β-hydroxyaspartate). Signalling leading to mTOR phosphorylation includes Ca2+ influx, the activation of p60src, phosphatidylinositol 3-kinase, protein kinase B, mTOR and p70S6K. Interestingly, GLAST activity promoted AP-1 (activator protein-1) binding to DNA, supporting a function for transporter signalling in retinal long-term responses. These results add a novel receptor-independent pathway for Glu signalling in Müller glia, and further strengthen the critical involvement of these cells in the regulation of glutamatergic transmission in the retina.

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D-Asp-induced mTOR phosphorylation is a PI3K-PKB/Akt pathway-dependent eventMGC were pre-incubated for 30 min with 10 nM of the PI3K inhibitor wortmannin (Wort) (A), with 100 nM of the PKB inhibitor (PKBI-IV) (B), or the p60src inhibitor PP2 (10 nM) in the absence or presence of the RTK inhibitor genistein at 25 μM (C); and then treated with D-Asp (1 mM) or THA (100 μM). Levels of mTOR phosphorylation were detected as for Figure 1. (D) Levels of Tyr-527 phosphorylated p60src were detected after treatment with 1 mM D-Asp in the presence/absence of W7 (25 μM) or genistein (25 μM). Phospho-p60src levels were analysed by Western blot, Ponceau S was used as a loading control. The results are the mean ±S.E. of at least three independent experiments. In each panel, a representative Western blot is shown. Statistical analysis was performed comparing against non-stimulated cells using a non-parametric one-way ANOVA (Kruskal-Wallis test) and Dunn's post-hoc test (*P<0.05, ***P<0.001).
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Figure 6: D-Asp-induced mTOR phosphorylation is a PI3K-PKB/Akt pathway-dependent eventMGC were pre-incubated for 30 min with 10 nM of the PI3K inhibitor wortmannin (Wort) (A), with 100 nM of the PKB inhibitor (PKBI-IV) (B), or the p60src inhibitor PP2 (10 nM) in the absence or presence of the RTK inhibitor genistein at 25 μM (C); and then treated with D-Asp (1 mM) or THA (100 μM). Levels of mTOR phosphorylation were detected as for Figure 1. (D) Levels of Tyr-527 phosphorylated p60src were detected after treatment with 1 mM D-Asp in the presence/absence of W7 (25 μM) or genistein (25 μM). Phospho-p60src levels were analysed by Western blot, Ponceau S was used as a loading control. The results are the mean ±S.E. of at least three independent experiments. In each panel, a representative Western blot is shown. Statistical analysis was performed comparing against non-stimulated cells using a non-parametric one-way ANOVA (Kruskal-Wallis test) and Dunn's post-hoc test (*P<0.05, ***P<0.001).

Mentions: To establish the molecular signalling cascade involved in GLAST-mediated mTOR phosphorylation, we first explored the participation of PI3K, known to act upstream of mTOR in several systems. Pretreatment of MGC for 15 min with 10 nM of the PI3K inhibitor wortmannin, completely prevented the increase in mTOR phosphorylation induced by either D-Asp or THA (Figure 6A).


Glutamate transporter-dependent mTOR phosphorylation in Müller glia cells.

María López-Colomé A, Martínez-Lozada Z, Guillem AM, López E, Ortega A - ASN Neuro (2012)

D-Asp-induced mTOR phosphorylation is a PI3K-PKB/Akt pathway-dependent eventMGC were pre-incubated for 30 min with 10 nM of the PI3K inhibitor wortmannin (Wort) (A), with 100 nM of the PKB inhibitor (PKBI-IV) (B), or the p60src inhibitor PP2 (10 nM) in the absence or presence of the RTK inhibitor genistein at 25 μM (C); and then treated with D-Asp (1 mM) or THA (100 μM). Levels of mTOR phosphorylation were detected as for Figure 1. (D) Levels of Tyr-527 phosphorylated p60src were detected after treatment with 1 mM D-Asp in the presence/absence of W7 (25 μM) or genistein (25 μM). Phospho-p60src levels were analysed by Western blot, Ponceau S was used as a loading control. The results are the mean ±S.E. of at least three independent experiments. In each panel, a representative Western blot is shown. Statistical analysis was performed comparing against non-stimulated cells using a non-parametric one-way ANOVA (Kruskal-Wallis test) and Dunn's post-hoc test (*P<0.05, ***P<0.001).
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Figure 6: D-Asp-induced mTOR phosphorylation is a PI3K-PKB/Akt pathway-dependent eventMGC were pre-incubated for 30 min with 10 nM of the PI3K inhibitor wortmannin (Wort) (A), with 100 nM of the PKB inhibitor (PKBI-IV) (B), or the p60src inhibitor PP2 (10 nM) in the absence or presence of the RTK inhibitor genistein at 25 μM (C); and then treated with D-Asp (1 mM) or THA (100 μM). Levels of mTOR phosphorylation were detected as for Figure 1. (D) Levels of Tyr-527 phosphorylated p60src were detected after treatment with 1 mM D-Asp in the presence/absence of W7 (25 μM) or genistein (25 μM). Phospho-p60src levels were analysed by Western blot, Ponceau S was used as a loading control. The results are the mean ±S.E. of at least three independent experiments. In each panel, a representative Western blot is shown. Statistical analysis was performed comparing against non-stimulated cells using a non-parametric one-way ANOVA (Kruskal-Wallis test) and Dunn's post-hoc test (*P<0.05, ***P<0.001).
Mentions: To establish the molecular signalling cascade involved in GLAST-mediated mTOR phosphorylation, we first explored the participation of PI3K, known to act upstream of mTOR in several systems. Pretreatment of MGC for 15 min with 10 nM of the PI3K inhibitor wortmannin, completely prevented the increase in mTOR phosphorylation induced by either D-Asp or THA (Figure 6A).

Bottom Line: The results showed that D-Asp transport induces the time- and dose-dependent phosphorylation of mTOR, mimicked by the transportable GLAST inhibitor THA (threo-β-hydroxyaspartate).Signalling leading to mTOR phosphorylation includes Ca2+ influx, the activation of p60src, phosphatidylinositol 3-kinase, protein kinase B, mTOR and p70S6K.Interestingly, GLAST activity promoted AP-1 (activator protein-1) binding to DNA, supporting a function for transporter signalling in retinal long-term responses.

View Article: PubMed Central - PubMed

Affiliation: *División de Neurociencias, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, México D.F., México.

ABSTRACT
Glu (glutamate), the excitatory transmitter at the main signalling pathway in the retina, is critically involved in changes in the protein repertoire through the activation of signalling cascades, which regulate protein synthesis at transcriptional and translational levels. Activity-dependent differential gene expression by Glu is related to the activation of ionotropic and metabotropic Glu receptors; however, recent findings suggest the involvement of Na+-dependent Glu transporters in this process. Within the retina, Glu uptake is aimed at the replenishment of the releasable pool, and for the prevention of excitotoxicity and is carried mainly by the GLAST/EAAT-1 (Na+-dependent glutamate/aspartate transporter/excitatory amino acids transporter-1) located in Müller radial glia. Based on the previous work showing the alteration of GLAST expression induced by Glu, the present work investigates the involvement of GLAST signalling in the regulation of protein synthesis in Müller cells. To this end, we explored the effect of D-Asp (D-aspartate) on Ser-2448 mTOR (mammalian target of rapamycin) phosphorylation in primary cultures of chick Müller glia. The results showed that D-Asp transport induces the time- and dose-dependent phosphorylation of mTOR, mimicked by the transportable GLAST inhibitor THA (threo-β-hydroxyaspartate). Signalling leading to mTOR phosphorylation includes Ca2+ influx, the activation of p60src, phosphatidylinositol 3-kinase, protein kinase B, mTOR and p70S6K. Interestingly, GLAST activity promoted AP-1 (activator protein-1) binding to DNA, supporting a function for transporter signalling in retinal long-term responses. These results add a novel receptor-independent pathway for Glu signalling in Müller glia, and further strengthen the critical involvement of these cells in the regulation of glutamatergic transmission in the retina.

Show MeSH
Related in: MedlinePlus