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

作者：Skytt Dorte M; Klawonn Anna M; Stridh Malin H; Pajecka Kamilla; Patruss Yasar; Quintana Cabrera Ruben; Bolanos Juan P; Schou**oe Arne; Waagepetersen Helle S^*

来源：Neurochemistry International, 2012, 61(4): 490-497.

DOI：10.1016/j.neuint.2012.04.014

• Glutamate
• Aspartate
• Homeostasis
• Metabolism
• Mass spectrometry
• N-15
• Glutamine
• siRNA
• Astrocyte
• Alanine
• TCA cycle

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 neuro-transmission 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 [(NH4+)-N-15] or 100 mu M [N-15]glutamate. For those exposed to [N-15]glutamate an additional 100 mu M 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 N-15 incorporation from [(NH4+)-N-15] 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 [N-15]glutamate, the reduced GDH activity led to an increased N-15 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 alpha-ketoglutarate to oxaloacetate, i.e. the truncated TCA cycle.

• 出版日期2012-9