Deletion of Neuronal GLT-1 in Mice Reveals Its Role in Synaptic Glutamate Homeostasis and Mitochondrial Function

Laura F. McNair, Jens V. Andersen, Blanca I. Aldana, Michaela C. Hohnholt, Jakob D. Nissen, Yan Sun, Kathryn D. Fischer, Ursula Sonnewald, Nils Nyberg, Sophie C. Webster, Kush Kapur, Theresa S. Rimmele, Ilaria Barone, Hannah Hawks-Mayer, Jonathan O. Lipton, Nathaniel W. Hodgson, Takao K. Hensch, Chiye Aoki, Paul A. Rosenberg, Helle S. Waagepetersen

Research output: Contribution to journalArticle

Abstract

The glutamate transporter GLT-1 is highly expressed in astrocytes but also in neurons, primarily in axon terminals. We generated a conditional neuronal GLT-1 KO using synapsin 1-Cre (synGLT-1 KO) to elucidate the metabolic functions of GLT-1 expressed in neurons, here focusing on the cerebral cortex. Both synaptosomal uptake studies and electron microscopic immunocytochemistry demonstrated knockdown of GLT-1 in the cerebral cortex in the synGLT-1 KO mice. Aspartate content was significantly reduced in cerebral cortical extracts as well as synaptosomes from cerebral cortex of synGLT-1 KO compared with control littermates. 13C-Labeling of tricarboxylic acid cycle intermediates originating from metabolism of [U-13C]-glutamate was significantly reduced in synGLT-1 KO synaptosomes. The decreased aspartate content was due to diminished entry of glutamate into the tricarboxylic acid cycle. Pyruvate recycling, a pathway necessary for full glutamate oxidation, was also decreased. ATP production was significantly increased, despite unaltered oxygen consumption, in isolated mitochondria from the synGLT-1 KO. The density of mitochondria in axon terminals and perisynaptic astrocytes was increased in the synGLT-1 KO. Intramitochondrial cristae density of synGLT-1 KO mice was increased, suggesting increased mitochondrial efficiency, perhaps in compensation for reduced access to glutamate. SynGLT-1 KO synaptosomes exhibited an elevated oxygen consumption rate when stimulated with veratridine, despite a lower baseline oxygen consumption rate in the presence of glucose. GLT-1 expressed in neurons appears to be required to provide glutamate to synaptic mitochondria and is linked to neuronal energy metabolism and mitochondrial function.SIGNIFICANCE STATEMENT All synaptic transmitters need to be cleared from the extracellular space after release, and transporters are used to clear glutamate released from excitatory synapses. GLT-1 is the major glutamate transporter, and most GLT-1 is expressed in astrocytes. Only 5%-10% is expressed in neurons, primarily in axon terminals. The function of GLT-1 in axon terminals remains unknown. Here, we used a conditional KO approach to investigate the significance of the expression of GLT-1 in neurons. We found multiple abnormalities of mitochondrial function, suggesting impairment of glutamate utilization by synaptic mitochondria in the neuronal GLT-1 KO. These data suggest that GLT-1 expressed in axon terminals may be important in maintaining energy metabolism and biosynthetic activities mediated by presynaptic mitochondria.

Original languageEnglish (US)
Pages (from-to)4847-4863
Number of pages17
JournalThe Journal of neuroscience : the official journal of the Society for Neuroscience
Volume39
Issue number25
DOIs
StatePublished - Jun 19 2019

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Glutamic Acid
Homeostasis
Presynaptic Terminals
Mitochondria
Synaptosomes
Neurons
Oxygen Consumption
Astrocytes
Cerebral Cortex
Amino Acid Transport System X-AG
Citric Acid Cycle
Aspartic Acid
Energy Metabolism
Synapsins
Veratridine
Multiple Abnormalities
Extracellular Space
Recycling
Pyruvic Acid
Synapses

Keywords

  • anaplerosis
  • aspartate
  • bioenergetics
  • Huntington's
  • neurodegeneration
  • TCA cycle

ASJC Scopus subject areas

  • Neuroscience(all)

Cite this

Deletion of Neuronal GLT-1 in Mice Reveals Its Role in Synaptic Glutamate Homeostasis and Mitochondrial Function. / McNair, Laura F.; Andersen, Jens V.; Aldana, Blanca I.; Hohnholt, Michaela C.; Nissen, Jakob D.; Sun, Yan; Fischer, Kathryn D.; Sonnewald, Ursula; Nyberg, Nils; Webster, Sophie C.; Kapur, Kush; Rimmele, Theresa S.; Barone, Ilaria; Hawks-Mayer, Hannah; Lipton, Jonathan O.; Hodgson, Nathaniel W.; Hensch, Takao K.; Aoki, Chiye; Rosenberg, Paul A.; Waagepetersen, Helle S.

In: The Journal of neuroscience : the official journal of the Society for Neuroscience, Vol. 39, No. 25, 19.06.2019, p. 4847-4863.

Research output: Contribution to journalArticle

McNair, LF, Andersen, JV, Aldana, BI, Hohnholt, MC, Nissen, JD, Sun, Y, Fischer, KD, Sonnewald, U, Nyberg, N, Webster, SC, Kapur, K, Rimmele, TS, Barone, I, Hawks-Mayer, H, Lipton, JO, Hodgson, NW, Hensch, TK, Aoki, C, Rosenberg, PA & Waagepetersen, HS 2019, 'Deletion of Neuronal GLT-1 in Mice Reveals Its Role in Synaptic Glutamate Homeostasis and Mitochondrial Function', The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 39, no. 25, pp. 4847-4863. https://doi.org/10.1523/JNEUROSCI.0894-18.2019
McNair, Laura F. ; Andersen, Jens V. ; Aldana, Blanca I. ; Hohnholt, Michaela C. ; Nissen, Jakob D. ; Sun, Yan ; Fischer, Kathryn D. ; Sonnewald, Ursula ; Nyberg, Nils ; Webster, Sophie C. ; Kapur, Kush ; Rimmele, Theresa S. ; Barone, Ilaria ; Hawks-Mayer, Hannah ; Lipton, Jonathan O. ; Hodgson, Nathaniel W. ; Hensch, Takao K. ; Aoki, Chiye ; Rosenberg, Paul A. ; Waagepetersen, Helle S. / Deletion of Neuronal GLT-1 in Mice Reveals Its Role in Synaptic Glutamate Homeostasis and Mitochondrial Function. In: The Journal of neuroscience : the official journal of the Society for Neuroscience. 2019 ; Vol. 39, No. 25. pp. 4847-4863.
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T1 - Deletion of Neuronal GLT-1 in Mice Reveals Its Role in Synaptic Glutamate Homeostasis and Mitochondrial Function

AU - McNair, Laura F.

AU - Andersen, Jens V.

AU - Aldana, Blanca I.

AU - Hohnholt, Michaela C.

AU - Nissen, Jakob D.

AU - Sun, Yan

AU - Fischer, Kathryn D.

AU - Sonnewald, Ursula

AU - Nyberg, Nils

AU - Webster, Sophie C.

AU - Kapur, Kush

AU - Rimmele, Theresa S.

AU - Barone, Ilaria

AU - Hawks-Mayer, Hannah

AU - Lipton, Jonathan O.

AU - Hodgson, Nathaniel W.

AU - Hensch, Takao K.

AU - Aoki, Chiye

AU - Rosenberg, Paul A.

AU - Waagepetersen, Helle S.

PY - 2019/6/19

Y1 - 2019/6/19

N2 - The glutamate transporter GLT-1 is highly expressed in astrocytes but also in neurons, primarily in axon terminals. We generated a conditional neuronal GLT-1 KO using synapsin 1-Cre (synGLT-1 KO) to elucidate the metabolic functions of GLT-1 expressed in neurons, here focusing on the cerebral cortex. Both synaptosomal uptake studies and electron microscopic immunocytochemistry demonstrated knockdown of GLT-1 in the cerebral cortex in the synGLT-1 KO mice. Aspartate content was significantly reduced in cerebral cortical extracts as well as synaptosomes from cerebral cortex of synGLT-1 KO compared with control littermates. 13C-Labeling of tricarboxylic acid cycle intermediates originating from metabolism of [U-13C]-glutamate was significantly reduced in synGLT-1 KO synaptosomes. The decreased aspartate content was due to diminished entry of glutamate into the tricarboxylic acid cycle. Pyruvate recycling, a pathway necessary for full glutamate oxidation, was also decreased. ATP production was significantly increased, despite unaltered oxygen consumption, in isolated mitochondria from the synGLT-1 KO. The density of mitochondria in axon terminals and perisynaptic astrocytes was increased in the synGLT-1 KO. Intramitochondrial cristae density of synGLT-1 KO mice was increased, suggesting increased mitochondrial efficiency, perhaps in compensation for reduced access to glutamate. SynGLT-1 KO synaptosomes exhibited an elevated oxygen consumption rate when stimulated with veratridine, despite a lower baseline oxygen consumption rate in the presence of glucose. GLT-1 expressed in neurons appears to be required to provide glutamate to synaptic mitochondria and is linked to neuronal energy metabolism and mitochondrial function.SIGNIFICANCE STATEMENT All synaptic transmitters need to be cleared from the extracellular space after release, and transporters are used to clear glutamate released from excitatory synapses. GLT-1 is the major glutamate transporter, and most GLT-1 is expressed in astrocytes. Only 5%-10% is expressed in neurons, primarily in axon terminals. The function of GLT-1 in axon terminals remains unknown. Here, we used a conditional KO approach to investigate the significance of the expression of GLT-1 in neurons. We found multiple abnormalities of mitochondrial function, suggesting impairment of glutamate utilization by synaptic mitochondria in the neuronal GLT-1 KO. These data suggest that GLT-1 expressed in axon terminals may be important in maintaining energy metabolism and biosynthetic activities mediated by presynaptic mitochondria.

AB - The glutamate transporter GLT-1 is highly expressed in astrocytes but also in neurons, primarily in axon terminals. We generated a conditional neuronal GLT-1 KO using synapsin 1-Cre (synGLT-1 KO) to elucidate the metabolic functions of GLT-1 expressed in neurons, here focusing on the cerebral cortex. Both synaptosomal uptake studies and electron microscopic immunocytochemistry demonstrated knockdown of GLT-1 in the cerebral cortex in the synGLT-1 KO mice. Aspartate content was significantly reduced in cerebral cortical extracts as well as synaptosomes from cerebral cortex of synGLT-1 KO compared with control littermates. 13C-Labeling of tricarboxylic acid cycle intermediates originating from metabolism of [U-13C]-glutamate was significantly reduced in synGLT-1 KO synaptosomes. The decreased aspartate content was due to diminished entry of glutamate into the tricarboxylic acid cycle. Pyruvate recycling, a pathway necessary for full glutamate oxidation, was also decreased. ATP production was significantly increased, despite unaltered oxygen consumption, in isolated mitochondria from the synGLT-1 KO. The density of mitochondria in axon terminals and perisynaptic astrocytes was increased in the synGLT-1 KO. Intramitochondrial cristae density of synGLT-1 KO mice was increased, suggesting increased mitochondrial efficiency, perhaps in compensation for reduced access to glutamate. SynGLT-1 KO synaptosomes exhibited an elevated oxygen consumption rate when stimulated with veratridine, despite a lower baseline oxygen consumption rate in the presence of glucose. GLT-1 expressed in neurons appears to be required to provide glutamate to synaptic mitochondria and is linked to neuronal energy metabolism and mitochondrial function.SIGNIFICANCE STATEMENT All synaptic transmitters need to be cleared from the extracellular space after release, and transporters are used to clear glutamate released from excitatory synapses. GLT-1 is the major glutamate transporter, and most GLT-1 is expressed in astrocytes. Only 5%-10% is expressed in neurons, primarily in axon terminals. The function of GLT-1 in axon terminals remains unknown. Here, we used a conditional KO approach to investigate the significance of the expression of GLT-1 in neurons. We found multiple abnormalities of mitochondrial function, suggesting impairment of glutamate utilization by synaptic mitochondria in the neuronal GLT-1 KO. These data suggest that GLT-1 expressed in axon terminals may be important in maintaining energy metabolism and biosynthetic activities mediated by presynaptic mitochondria.

KW - anaplerosis

KW - aspartate

KW - bioenergetics

KW - Huntington's

KW - neurodegeneration

KW - TCA cycle

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U2 - 10.1523/JNEUROSCI.0894-18.2019

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