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Physical and functional interaction of NCX1 and EAAC1 transporters leading to glutamate-enhanced ATP production in brain mitochondria.

Magi S, Lariccia V, Castaldo P, Arcangeli S, Nasti AA, Giordano A, Amoroso S - PLoS ONE (2012)

Bottom Line: Only NCX1 activity is crucial for such glutamate-stimulated ATP synthesis, as demonstrated by pharmacological blockade and selective knock-down with antisense oligonucleotides.The EAAC1/NCX1-dependent mitochondrial response to glutamate may be a general and alternative mechanism whereby this neurotransmitter sustains ATP production, since we have documented such metabolic response also in mitochondria isolated from heart.The data reported here disclose a new physiological role for mitochondrial NCX1 as the key player in glutamate-induced energy production.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences and Public Health, University Politecnica delle Marche, Ancona, Italy.

ABSTRACT
Glutamate is emerging as a major factor stimulating energy production in CNS. Brain mitochondria can utilize this neurotransmitter as respiratory substrate and specific transporters are required to mediate the glutamate entry into the mitochondrial matrix. Glutamate transporters of the Excitatory Amino Acid Transporters (EAATs) family have been previously well characterized on the cell surface of neuronal and glial cells, representing the primary players for glutamate uptake in mammalian brain. Here, by using western blot, confocal microscopy and immunoelectron microscopy, we report for the first time that the Excitatory Amino Acid Carrier 1 (EAAC1), an EAATs member, is expressed in neuronal and glial mitochondria where it participates in glutamate-stimulated ATP production, evaluated by a luciferase-luciferin system. Mitochondrial metabolic response is counteracted when different EAATs pharmacological blockers or selective EAAC1 antisense oligonucleotides were used. Since EAATs are Na(+)-dependent proteins, this raised the possibility that other transporters regulating ion gradients across mitochondrial membrane were required for glutamate response. We describe colocalization, mutual activity dependency, physical interaction between EAAC1 and the sodium/calcium exchanger 1 (NCX1) both in neuronal and glial mitochondria, and that NCX1 is an essential modulator of this glutamate transporter. Only NCX1 activity is crucial for such glutamate-stimulated ATP synthesis, as demonstrated by pharmacological blockade and selective knock-down with antisense oligonucleotides. The EAAC1/NCX1-dependent mitochondrial response to glutamate may be a general and alternative mechanism whereby this neurotransmitter sustains ATP production, since we have documented such metabolic response also in mitochondria isolated from heart. The data reported here disclose a new physiological role for mitochondrial NCX1 as the key player in glutamate-induced energy production.

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Glutamate-stimulated ATP synthesis in isolated mitochondria from cells.(A) Effects of DL-TBOA on glutamate-stimulated ATP synthesis in mitochondria from SH-SY5Y neuroblastoma and C6 glioma cells. (B) EAAC1 expression in SH-SY5Y neuroblastoma and C6 glioma cell mitochondria. Samples enriched with anti-EAAC1 antibody by selective immunoprecipitation (IP) were also loaded onto the gel. Protein extracts from rat brain and from BHK cells stably expressing NCX1 were used as controls. In each IP lane, the lower band represents the immunoglobulin. (C) Effect of anti-EAAC1 (As-EAAC1) and anti-Citrin/AGC22 (As-AGC2) antisense (D) ODNs, on glutamate-stimulated ATP synthesis in SH-SY5Y neuroblastoma and C6 glioma cells. Data from cells treated with Lipofectamine (Ctl) and sense ODNs (S-EAAC1 and S-AGC2) are also reported. Each bar in panels A, C and D represents the mean ± SEM of 14 different determinations. * p<0.05 vs control; ** p<0.01 vs control; *** p<0.001 vs control; ## p<0.01 vs 0.5 or 1 mM glutamate; ### p<0.001 vs 0.5 or 1 mM glutamate; n.s.: not significant vs control. In ODNs experiments, *** p<0.001 vs S+glutamate.
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pone-0034015-g003: Glutamate-stimulated ATP synthesis in isolated mitochondria from cells.(A) Effects of DL-TBOA on glutamate-stimulated ATP synthesis in mitochondria from SH-SY5Y neuroblastoma and C6 glioma cells. (B) EAAC1 expression in SH-SY5Y neuroblastoma and C6 glioma cell mitochondria. Samples enriched with anti-EAAC1 antibody by selective immunoprecipitation (IP) were also loaded onto the gel. Protein extracts from rat brain and from BHK cells stably expressing NCX1 were used as controls. In each IP lane, the lower band represents the immunoglobulin. (C) Effect of anti-EAAC1 (As-EAAC1) and anti-Citrin/AGC22 (As-AGC2) antisense (D) ODNs, on glutamate-stimulated ATP synthesis in SH-SY5Y neuroblastoma and C6 glioma cells. Data from cells treated with Lipofectamine (Ctl) and sense ODNs (S-EAAC1 and S-AGC2) are also reported. Each bar in panels A, C and D represents the mean ± SEM of 14 different determinations. * p<0.05 vs control; ** p<0.01 vs control; *** p<0.001 vs control; ## p<0.01 vs 0.5 or 1 mM glutamate; ### p<0.001 vs 0.5 or 1 mM glutamate; n.s.: not significant vs control. In ODNs experiments, *** p<0.001 vs S+glutamate.

Mentions: Since mitochondrial preparations from hippocampal and cortical regions contained mitochondria from both neurons and glia, we wondered whether the effect of glutamate was to be ascribed solely to neuronal mitochondria, or whether glial mitochondria were also involved. To dissect this effect we measured the ATP synthesis stimulated by exogenous glutamate in mitochondria isolated from two different continuous cell lines, one purely neuronal, human SH-SY5Y neuroblastoma cells, and one purely glial, rat C6 glioma cells, and found a significant increase in ATP production in both systems (Figure 3A). These preparations were not contaminated by cytosol, since glucose was unable to stimulate ATP production whereas it was effective in intact cells (Figure S1). In addition LDH activity was virtually absent (data not shown). Glutamate therefore appears to stimulate mitochondrial metabolism both in neurons and in glia, suggesting that an increase in oxidative metabolism may be a general response mechanism to the activation of glutamatergic neurotransmission.


Physical and functional interaction of NCX1 and EAAC1 transporters leading to glutamate-enhanced ATP production in brain mitochondria.

Magi S, Lariccia V, Castaldo P, Arcangeli S, Nasti AA, Giordano A, Amoroso S - PLoS ONE (2012)

Glutamate-stimulated ATP synthesis in isolated mitochondria from cells.(A) Effects of DL-TBOA on glutamate-stimulated ATP synthesis in mitochondria from SH-SY5Y neuroblastoma and C6 glioma cells. (B) EAAC1 expression in SH-SY5Y neuroblastoma and C6 glioma cell mitochondria. Samples enriched with anti-EAAC1 antibody by selective immunoprecipitation (IP) were also loaded onto the gel. Protein extracts from rat brain and from BHK cells stably expressing NCX1 were used as controls. In each IP lane, the lower band represents the immunoglobulin. (C) Effect of anti-EAAC1 (As-EAAC1) and anti-Citrin/AGC22 (As-AGC2) antisense (D) ODNs, on glutamate-stimulated ATP synthesis in SH-SY5Y neuroblastoma and C6 glioma cells. Data from cells treated with Lipofectamine (Ctl) and sense ODNs (S-EAAC1 and S-AGC2) are also reported. Each bar in panels A, C and D represents the mean ± SEM of 14 different determinations. * p<0.05 vs control; ** p<0.01 vs control; *** p<0.001 vs control; ## p<0.01 vs 0.5 or 1 mM glutamate; ### p<0.001 vs 0.5 or 1 mM glutamate; n.s.: not significant vs control. In ODNs experiments, *** p<0.001 vs S+glutamate.
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pone-0034015-g003: Glutamate-stimulated ATP synthesis in isolated mitochondria from cells.(A) Effects of DL-TBOA on glutamate-stimulated ATP synthesis in mitochondria from SH-SY5Y neuroblastoma and C6 glioma cells. (B) EAAC1 expression in SH-SY5Y neuroblastoma and C6 glioma cell mitochondria. Samples enriched with anti-EAAC1 antibody by selective immunoprecipitation (IP) were also loaded onto the gel. Protein extracts from rat brain and from BHK cells stably expressing NCX1 were used as controls. In each IP lane, the lower band represents the immunoglobulin. (C) Effect of anti-EAAC1 (As-EAAC1) and anti-Citrin/AGC22 (As-AGC2) antisense (D) ODNs, on glutamate-stimulated ATP synthesis in SH-SY5Y neuroblastoma and C6 glioma cells. Data from cells treated with Lipofectamine (Ctl) and sense ODNs (S-EAAC1 and S-AGC2) are also reported. Each bar in panels A, C and D represents the mean ± SEM of 14 different determinations. * p<0.05 vs control; ** p<0.01 vs control; *** p<0.001 vs control; ## p<0.01 vs 0.5 or 1 mM glutamate; ### p<0.001 vs 0.5 or 1 mM glutamate; n.s.: not significant vs control. In ODNs experiments, *** p<0.001 vs S+glutamate.
Mentions: Since mitochondrial preparations from hippocampal and cortical regions contained mitochondria from both neurons and glia, we wondered whether the effect of glutamate was to be ascribed solely to neuronal mitochondria, or whether glial mitochondria were also involved. To dissect this effect we measured the ATP synthesis stimulated by exogenous glutamate in mitochondria isolated from two different continuous cell lines, one purely neuronal, human SH-SY5Y neuroblastoma cells, and one purely glial, rat C6 glioma cells, and found a significant increase in ATP production in both systems (Figure 3A). These preparations were not contaminated by cytosol, since glucose was unable to stimulate ATP production whereas it was effective in intact cells (Figure S1). In addition LDH activity was virtually absent (data not shown). Glutamate therefore appears to stimulate mitochondrial metabolism both in neurons and in glia, suggesting that an increase in oxidative metabolism may be a general response mechanism to the activation of glutamatergic neurotransmission.

Bottom Line: Only NCX1 activity is crucial for such glutamate-stimulated ATP synthesis, as demonstrated by pharmacological blockade and selective knock-down with antisense oligonucleotides.The EAAC1/NCX1-dependent mitochondrial response to glutamate may be a general and alternative mechanism whereby this neurotransmitter sustains ATP production, since we have documented such metabolic response also in mitochondria isolated from heart.The data reported here disclose a new physiological role for mitochondrial NCX1 as the key player in glutamate-induced energy production.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences and Public Health, University Politecnica delle Marche, Ancona, Italy.

ABSTRACT
Glutamate is emerging as a major factor stimulating energy production in CNS. Brain mitochondria can utilize this neurotransmitter as respiratory substrate and specific transporters are required to mediate the glutamate entry into the mitochondrial matrix. Glutamate transporters of the Excitatory Amino Acid Transporters (EAATs) family have been previously well characterized on the cell surface of neuronal and glial cells, representing the primary players for glutamate uptake in mammalian brain. Here, by using western blot, confocal microscopy and immunoelectron microscopy, we report for the first time that the Excitatory Amino Acid Carrier 1 (EAAC1), an EAATs member, is expressed in neuronal and glial mitochondria where it participates in glutamate-stimulated ATP production, evaluated by a luciferase-luciferin system. Mitochondrial metabolic response is counteracted when different EAATs pharmacological blockers or selective EAAC1 antisense oligonucleotides were used. Since EAATs are Na(+)-dependent proteins, this raised the possibility that other transporters regulating ion gradients across mitochondrial membrane were required for glutamate response. We describe colocalization, mutual activity dependency, physical interaction between EAAC1 and the sodium/calcium exchanger 1 (NCX1) both in neuronal and glial mitochondria, and that NCX1 is an essential modulator of this glutamate transporter. Only NCX1 activity is crucial for such glutamate-stimulated ATP synthesis, as demonstrated by pharmacological blockade and selective knock-down with antisense oligonucleotides. The EAAC1/NCX1-dependent mitochondrial response to glutamate may be a general and alternative mechanism whereby this neurotransmitter sustains ATP production, since we have documented such metabolic response also in mitochondria isolated from heart. The data reported here disclose a new physiological role for mitochondrial NCX1 as the key player in glutamate-induced energy production.

Show MeSH
Related in: MedlinePlus