siRNA knock down of glutamate dehydrogenase in astrocytes affects glutamate metabolism leading to extensive accumulation of the neuroactive amino acids glutamate and aspartate

Dorte M. Skytt; Anna M. Klawonn; Malin Helena Strid; Kamilla Pajecka; Yasar Patruss; Ruben Quintana-Cabrera; Juan P. Bolanos; Arne Schousboe; Helle S. Waagepetersen

doi:10.1016/j.neuint.2012.04.014

siRNA knock down of glutamate dehydrogenase in astrocytes affects glutamate metabolism leading to extensive accumulation of the neuroactive amino acids glutamate and aspartate

Dorte M. Skytt, Anna M. Klawonn, Malin Helena Strid, Kamilla Pajecka, Yasar Patruss, Ruben Quintana-Cabrera, Juan P. Bolanos, Arne Schousboe, Helle S. Waagepetersen

24 Citations (Scopus)

Abstract

Glutamate is the most abundant excitatory neurotransmitter in the brain and astrocytes are key players in sustaining glutamate homeostasis. Astrocytes take up the predominant part of glutamate after neurotransmission and metabolism of glutamate is necessary for a continuous efficient removal of glutamate from the synaptic area. Glutamate may either be amidated by glutamine synthetase or oxidatively metabolized in the mitochondria, the latter being at least to some extent initiated by oxidative deamination by glutamate dehydrogenase (GDH). To explore the particular importance of GDH for astrocyte metabolism we have knocked down GDH in cultured cortical astrocytes employing small interfering RNA (siRNA) achieving a reduction of the enzyme activity by approximately 44%. The astrocytes were incubated for 2 h in medium containing either 1.0 mM [ ¹⁵NH₄⁺] or 100 μM [¹⁵N]glutamate. For those exposed to [¹⁵N]glutamate an additional 100 μM was added after 1 h. Metabolic mapping was performed from isotope incorporation measured by mass spectrometry into relevant amino acids of cell extracts and media. The contents of the amino acids were measured by HPLC. The ¹⁵N incorporation from [¹⁵NH₄⁺] into glutamate, aspartate and alanine was decreased in astrocytes exhibiting reduced GDH activity. However, the reduced GDH activity had no effect on the cellular contents of these amino acids. This supports existing in vivo and in vitro studies that GDH is predominantly working in the direction of oxidative deamination and not reductive amination. In contrast, when exposing the astrocytes to [¹⁵N]glutamate, the reduced GDH activity led to an increased ¹⁵N incorporation into glutamate, aspartate and alanine and a large increase in the content of glutamate and aspartate. Surprisingly, this accumulation of glutamate and net-synthesis of aspartate were not reflected in any alterations in either the glutamine content or labeling, but a slight increase in mono labeling of glutamine in the medium. We suggest that this extensive net-synthesis of aspartate due to lack of GDH activity is occurring via the concerted action of AAT and the part of TCA cycle operating from α-ketoglutarate to oxaloacetate, i.e. the truncated TCA cycle.

Original language	English
Journal	Neurochemistry International
Volume	61
Issue number	4
Pages (from-to)	490-497
ISSN	0197-0186
DOIs	https://doi.org/10.1016/j.neuint.2012.04.014
Publication status	Published - Sept 2012

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10.1016/j.neuint.2012.04.014

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Skytt, D. M., Klawonn, A. M., Strid, M. H., Pajecka, K., Patruss, Y., Quintana-Cabrera, R., Bolanos, J. P., Schousboe, A., & Waagepetersen, H. S. (2012). siRNA knock down of glutamate dehydrogenase in astrocytes affects glutamate metabolism leading to extensive accumulation of the neuroactive amino acids glutamate and aspartate. Neurochemistry International, 61(4), 490-497. https://doi.org/10.1016/j.neuint.2012.04.014

siRNA knock down of glutamate dehydrogenase in astrocytes affects glutamate metabolism leading to extensive accumulation of the neuroactive amino acids glutamate and aspartate. / Skytt, Dorte M.; Klawonn, Anna M.; Strid, Malin Helena et al.

In: Neurochemistry International, Vol. 61, No. 4, 09.2012, p. 490-497.

Research output: Contribution to journal › Journal article › Research › peer-review

Skytt, DM, Klawonn, AM, Strid, MH, Pajecka, K, Patruss, Y, Quintana-Cabrera, R, Bolanos, JP, Schousboe, A & Waagepetersen, HS 2012, 'siRNA knock down of glutamate dehydrogenase in astrocytes affects glutamate metabolism leading to extensive accumulation of the neuroactive amino acids glutamate and aspartate', Neurochemistry International, vol. 61, no. 4, pp. 490-497. https://doi.org/10.1016/j.neuint.2012.04.014

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title = "siRNA knock down of glutamate dehydrogenase in astrocytes affects glutamate metabolism leading to extensive accumulation of the neuroactive amino acids glutamate and aspartate",

abstract = "Glutamate is the most abundant excitatory neurotransmitter in the brain and astrocytes are key players in sustaining glutamate homeostasis. Astrocytes take up the predominant part of glutamate after neurotransmission and metabolism of glutamate is necessary for a continuous efficient removal of glutamate from the synaptic area. Glutamate may either be amidated by glutamine synthetase or oxidatively metabolized in the mitochondria, the latter being at least to some extent initiated by oxidative deamination by glutamate dehydrogenase (GDH). To explore the particular importance of GDH for astrocyte metabolism we have knocked down GDH in cultured cortical astrocytes employing small interfering RNA (siRNA) achieving a reduction of the enzyme activity by approximately 44%. The astrocytes were incubated for 2 h in medium containing either 1.0 mM [ 15NH4+] or 100 μM [15N]glutamate. For those exposed to [15N]glutamate an additional 100 μM was added after 1 h. Metabolic mapping was performed from isotope incorporation measured by mass spectrometry into relevant amino acids of cell extracts and media. The contents of the amino acids were measured by HPLC. The 15N incorporation from [15NH4+] into glutamate, aspartate and alanine was decreased in astrocytes exhibiting reduced GDH activity. However, the reduced GDH activity had no effect on the cellular contents of these amino acids. This supports existing in vivo and in vitro studies that GDH is predominantly working in the direction of oxidative deamination and not reductive amination. In contrast, when exposing the astrocytes to [15N]glutamate, the reduced GDH activity led to an increased 15N incorporation into glutamate, aspartate and alanine and a large increase in the content of glutamate and aspartate. Surprisingly, this accumulation of glutamate and net-synthesis of aspartate were not reflected in any alterations in either the glutamine content or labeling, but a slight increase in mono labeling of glutamine in the medium. We suggest that this extensive net-synthesis of aspartate due to lack of GDH activity is occurring via the concerted action of AAT and the part of TCA cycle operating from α-ketoglutarate to oxaloacetate, i.e. the truncated TCA cycle.",

author = "Skytt, {Dorte M.} and Klawonn, {Anna M.} and Strid, {Malin Helena} and Kamilla Pajecka and Yasar Patruss and Ruben Quintana-Cabrera and Bolanos, {Juan P.} and Arne Schousboe and Waagepetersen, {Helle S.}",

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T1 - siRNA knock down of glutamate dehydrogenase in astrocytes affects glutamate metabolism leading to extensive accumulation of the neuroactive amino acids glutamate and aspartate

AU - Skytt, Dorte M.

AU - Klawonn, Anna M.

AU - Strid, Malin Helena

AU - Pajecka, Kamilla

AU - Patruss, Yasar

AU - Quintana-Cabrera, Ruben

AU - Bolanos, Juan P.

AU - Schousboe, Arne

AU - Waagepetersen, Helle S.

PY - 2012/9

Y1 - 2012/9

N2 - Glutamate is the most abundant excitatory neurotransmitter in the brain and astrocytes are key players in sustaining glutamate homeostasis. Astrocytes take up the predominant part of glutamate after neurotransmission and metabolism of glutamate is necessary for a continuous efficient removal of glutamate from the synaptic area. Glutamate may either be amidated by glutamine synthetase or oxidatively metabolized in the mitochondria, the latter being at least to some extent initiated by oxidative deamination by glutamate dehydrogenase (GDH). To explore the particular importance of GDH for astrocyte metabolism we have knocked down GDH in cultured cortical astrocytes employing small interfering RNA (siRNA) achieving a reduction of the enzyme activity by approximately 44%. The astrocytes were incubated for 2 h in medium containing either 1.0 mM [ 15NH4+] or 100 μM [15N]glutamate. For those exposed to [15N]glutamate an additional 100 μM was added after 1 h. Metabolic mapping was performed from isotope incorporation measured by mass spectrometry into relevant amino acids of cell extracts and media. The contents of the amino acids were measured by HPLC. The 15N incorporation from [15NH4+] into glutamate, aspartate and alanine was decreased in astrocytes exhibiting reduced GDH activity. However, the reduced GDH activity had no effect on the cellular contents of these amino acids. This supports existing in vivo and in vitro studies that GDH is predominantly working in the direction of oxidative deamination and not reductive amination. In contrast, when exposing the astrocytes to [15N]glutamate, the reduced GDH activity led to an increased 15N incorporation into glutamate, aspartate and alanine and a large increase in the content of glutamate and aspartate. Surprisingly, this accumulation of glutamate and net-synthesis of aspartate were not reflected in any alterations in either the glutamine content or labeling, but a slight increase in mono labeling of glutamine in the medium. We suggest that this extensive net-synthesis of aspartate due to lack of GDH activity is occurring via the concerted action of AAT and the part of TCA cycle operating from α-ketoglutarate to oxaloacetate, i.e. the truncated TCA cycle.

AB - Glutamate is the most abundant excitatory neurotransmitter in the brain and astrocytes are key players in sustaining glutamate homeostasis. Astrocytes take up the predominant part of glutamate after neurotransmission and metabolism of glutamate is necessary for a continuous efficient removal of glutamate from the synaptic area. Glutamate may either be amidated by glutamine synthetase or oxidatively metabolized in the mitochondria, the latter being at least to some extent initiated by oxidative deamination by glutamate dehydrogenase (GDH). To explore the particular importance of GDH for astrocyte metabolism we have knocked down GDH in cultured cortical astrocytes employing small interfering RNA (siRNA) achieving a reduction of the enzyme activity by approximately 44%. The astrocytes were incubated for 2 h in medium containing either 1.0 mM [ 15NH4+] or 100 μM [15N]glutamate. For those exposed to [15N]glutamate an additional 100 μM was added after 1 h. Metabolic mapping was performed from isotope incorporation measured by mass spectrometry into relevant amino acids of cell extracts and media. The contents of the amino acids were measured by HPLC. The 15N incorporation from [15NH4+] into glutamate, aspartate and alanine was decreased in astrocytes exhibiting reduced GDH activity. However, the reduced GDH activity had no effect on the cellular contents of these amino acids. This supports existing in vivo and in vitro studies that GDH is predominantly working in the direction of oxidative deamination and not reductive amination. In contrast, when exposing the astrocytes to [15N]glutamate, the reduced GDH activity led to an increased 15N incorporation into glutamate, aspartate and alanine and a large increase in the content of glutamate and aspartate. Surprisingly, this accumulation of glutamate and net-synthesis of aspartate were not reflected in any alterations in either the glutamine content or labeling, but a slight increase in mono labeling of glutamine in the medium. We suggest that this extensive net-synthesis of aspartate due to lack of GDH activity is occurring via the concerted action of AAT and the part of TCA cycle operating from α-ketoglutarate to oxaloacetate, i.e. the truncated TCA cycle.

U2 - 10.1016/j.neuint.2012.04.014

DO - 10.1016/j.neuint.2012.04.014

M3 - Journal article

C2 - 22542772

SN - 0197-0186

VL - 61

SP - 490

EP - 497

JO - Neurochemistry International

JF - Neurochemistry International

IS - 4

ER -

siRNA knock down of glutamate dehydrogenase in astrocytes affects glutamate metabolism leading to extensive accumulation of the neuroactive amino acids glutamate and aspartate

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