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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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EAAC1 Pre-embedding Immunoelectron Microscopy.(A–E) Specific staining (granular electron-dense reaction product) is present on the membrane of some mitochondria (arrows) contained into the dendrites (a,b), neuronal somata (b,d) and astrocyte soma (c) and perivascular processes (e) of both rat cerebral cortex (a–c) and hippocampus (d,e). Labeling is also scattered in the cytoplasm and present into cisterns of the rough endoplasmic reticulum and Golgi apparatus (arrowheads, inset of b). Insets are the enlargement of the corresponding framed area. Den, dendrite; Nuc, nucleus; G, Golgi apparatus; RER, rough endoplasmic reticulum; E, endothelium; F, filaments; Cap, capillary. Bar: a, 3 µm; b, 4 µm; c, 6 µm; d, 1.5 µm; e 2.5 µm; inset of a, 1 µm; inset of b, 2 µm; inset of c, 2.5 µm.
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pone-0034015-g005: EAAC1 Pre-embedding Immunoelectron Microscopy.(A–E) Specific staining (granular electron-dense reaction product) is present on the membrane of some mitochondria (arrows) contained into the dendrites (a,b), neuronal somata (b,d) and astrocyte soma (c) and perivascular processes (e) of both rat cerebral cortex (a–c) and hippocampus (d,e). Labeling is also scattered in the cytoplasm and present into cisterns of the rough endoplasmic reticulum and Golgi apparatus (arrowheads, inset of b). Insets are the enlargement of the corresponding framed area. Den, dendrite; Nuc, nucleus; G, Golgi apparatus; RER, rough endoplasmic reticulum; E, endothelium; F, filaments; Cap, capillary. Bar: a, 3 µm; b, 4 µm; c, 6 µm; d, 1.5 µm; e 2.5 µm; inset of a, 1 µm; inset of b, 2 µm; inset of c, 2.5 µm.

Mentions: Altogether, these findings lend support to the hypothesis that glutamate triggers metabolic activation by enhancing ATP synthesis in glial and neuronal mitochondria, in a process involving a specific glutamate transporter subtype, EAAC1. Additional support for the mitochondrial localization of EAAC1 came from immunoelectron microscopy, showing the presence of specific staining in neuronal and glial mitochondria in rat cerebral cortex and hippocampus (Figures 5A–E). Notably, the specificity of EAAC1 antibody was verified by looking for reactivity in different rat tissues by western blot. As previously described EAAC1 was not detected in rat testis [40] (Figure 1E). Moreover, the lack of immunoreactivity demonstrated no cross-reaction with GLAST and GLT-1, known to be expressed in the same tissue [40].


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)

EAAC1 Pre-embedding Immunoelectron Microscopy.(A–E) Specific staining (granular electron-dense reaction product) is present on the membrane of some mitochondria (arrows) contained into the dendrites (a,b), neuronal somata (b,d) and astrocyte soma (c) and perivascular processes (e) of both rat cerebral cortex (a–c) and hippocampus (d,e). Labeling is also scattered in the cytoplasm and present into cisterns of the rough endoplasmic reticulum and Golgi apparatus (arrowheads, inset of b). Insets are the enlargement of the corresponding framed area. Den, dendrite; Nuc, nucleus; G, Golgi apparatus; RER, rough endoplasmic reticulum; E, endothelium; F, filaments; Cap, capillary. Bar: a, 3 µm; b, 4 µm; c, 6 µm; d, 1.5 µm; e 2.5 µm; inset of a, 1 µm; inset of b, 2 µm; inset of c, 2.5 µm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3316532&req=5

pone-0034015-g005: EAAC1 Pre-embedding Immunoelectron Microscopy.(A–E) Specific staining (granular electron-dense reaction product) is present on the membrane of some mitochondria (arrows) contained into the dendrites (a,b), neuronal somata (b,d) and astrocyte soma (c) and perivascular processes (e) of both rat cerebral cortex (a–c) and hippocampus (d,e). Labeling is also scattered in the cytoplasm and present into cisterns of the rough endoplasmic reticulum and Golgi apparatus (arrowheads, inset of b). Insets are the enlargement of the corresponding framed area. Den, dendrite; Nuc, nucleus; G, Golgi apparatus; RER, rough endoplasmic reticulum; E, endothelium; F, filaments; Cap, capillary. Bar: a, 3 µm; b, 4 µm; c, 6 µm; d, 1.5 µm; e 2.5 µm; inset of a, 1 µm; inset of b, 2 µm; inset of c, 2.5 µm.
Mentions: Altogether, these findings lend support to the hypothesis that glutamate triggers metabolic activation by enhancing ATP synthesis in glial and neuronal mitochondria, in a process involving a specific glutamate transporter subtype, EAAC1. Additional support for the mitochondrial localization of EAAC1 came from immunoelectron microscopy, showing the presence of specific staining in neuronal and glial mitochondria in rat cerebral cortex and hippocampus (Figures 5A–E). Notably, the specificity of EAAC1 antibody was verified by looking for reactivity in different rat tissues by western blot. As previously described EAAC1 was not detected in rat testis [40] (Figure 1E). Moreover, the lack of immunoreactivity demonstrated no cross-reaction with GLAST and GLT-1, known to be expressed in the same tissue [40].

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