Knockout of GAD65 has major impact on synaptic GABA synthesized from astrocyte-derived glutamine.
γ-Aminobutyric acid (GABA) synthesis from glutamate is catalyzed by glutamate decarboxylase (GAD) of which two isoforms, GAD65 and GAD67, have been identified. The GAD65 has repeatedly been shown to be important during intensified synaptic activity. To specifically elucidate the significance of GAD65 for maintenance of the highly compartmentalized intracellular and intercellular GABA homeostasis, GAD65 knockout and corresponding wild-type mice were injected with [1-(13)C]glucose and the astrocyte-specific substrate [1,2-(13)C]acetate. Synthesis of GABA from glutamine in the GABAergic synapses was further investigated in GAD65 knockout and wild-type mice using [1,2-(13)C]acetate and in some cases γ-vinylGABA (GVG, Vigabatrin), an inhibitor of GABA degradation. A detailed metabolic mapping was obtained by nuclear magnetic resonance (NMR) spectroscopic analysis of tissue extracts of cerebral cortex and hippocampus. The GABA content in both brain regions was reduced by ∼20%. Moreover, it was revealed that GAD65 is crucial for maintenance of biosynthesis of synaptic GABA particularly by direct synthesis from astrocytic glutamine via glutamate. The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism. Furthermore, a severe neuronal hypometabolism, involving glycolysis and tricarboxylic acid (TCA) cycle activity, was observed in cerebral cortex of GAD65 knockout mice.
Walls
Anne B
AB
Department of Neuroscience, Faculty of Medicine, Norwegian University of Science and Technology (NTNU), Trondheim, Norway.
Eyjolfsson
Elvar M
EM
Smeland
Olav B
OB
Nilsen
Linn Hege
LH
Schousboe
Inger
I
Schousboe
Arne
A
Sonnewald
Ursula
U
Waagepetersen
Helle S
HS
D000085:Acetates / Q000378:metabolism; D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D015153:Blotting, Western; D002540:Cerebral Cortex / Q000378:metabolism; D002952:Citric Acid Cycle / Q000502:physiology; D018682:GABA Agents / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D005947:Glucose / Q000378:metabolism; D005968:Glutamate Decarboxylase / Q000235:genetics* / Q000502:physiology*; D018698:Glutamic Acid / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism*; D006624:Hippocampus / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D013569:Synapses / Q000378:metabolism*; D020888:Vigabatrin / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000096:biosynthesis* / Q000235:genetics*
D000085:Acetates; D018682:GABA Agents; D005973:Glutamine; D018698:Glutamic Acid; D005680:gamma-Aminobutyric Acid; D005968:Glutamate Decarboxylase; C401141:glutamate decarboxylase 2; D020888:Vigabatrin; D005947:Glucose