Knockout of GAD65 has major impact on synaptic GABA synthesized from astrocyte-derived glutamine

Abstract

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.

Figure 1

Western blot analysis of expression of GAD65 in extracts of cerebral cortex from wild-type (WT) and GAD65 knockout (KO) mice. Cortex was excised from decapitated mice (WT and KO) and homogenized in phosphate-buffered saline (PBS) by sonication (see Materials and methods). Cytoplasmic GAD65 was saved as the supernatant after centrifugation of 10% homogenates (fraction 1). Membrane-bound GAD65 was extracted from the precipitate (fraction 2) by resolubilization in PBS containing 1% Triton X-100 followed by centrifugation. The extracts were diluted 10-fold in PBS containing 0.1% Triton X-100 and analyzed for content of GAD65 by Western blotting (see Materials and methods). GAD, glutamate decarboxylase.

Figure 2

The amount of metabolites (μmol/g tissue) in extracts from cerebral cortex of wild-type (WT; open bars) and GAD65 knockout (GAD65 KO; filled bars) mice was determined by ¹H-nuclear magnetic resonance spectroscopy. The inset shows the amount of γ-aminobutyric acid (GABA; μmol/g tissue) after treatment with γ-vinylGABA (GVG). Results are averages±s.e.m. (n=5), and statistically significant differences (P<0.05) between GAD65 knockout and wild-type mice are indicated with an asterisk. GAD, glutamate decarboxylase.

Figure 3

Percent enrichment of [4,5-¹³C]glutamate, [4,5-¹³C]glutamine, and [1,2-¹³C]GABA labeled from [1,2-¹³C]acetate in wild-type (WT; open bars) and GAD65 knockout (GAD65 KO; filled bars) mice. The animals were injected with [1-¹³C]glucose and [1,2-¹³C]acetate and killed 15 minutes later as detailed in Materials and methods. The enrichment was determined from ¹³C-nuclear magnetic resonance spectroscopic analysis of brain extracts as detailed in Materials and methods. The inset shows the amount of [1,2-¹³C]GABA (nmol/g tissue). Results are averages±s.e.m. (n=5), and the asterisk indicates a statistically significant difference between GAD65 knockout and wild-type mice (P<0.05). GABA, γ-aminobutyric acid; GAD, glutamate decarboxylase.

Figure 4

Percent γ-aminobutyric acid (GABA) formed via direct synthesis from [4,5-¹³C]glutamine (A) and percent of glutamate and glutamine synthesized in the first turn of the tricarboxylic acid (TCA) cycle from [1,2-¹³C]acetate (B) in wild-type (WT; open bars) and GAD65 knockout (GAD65 KO; filled bars) mice. The animals were injected with [1,2-¹³C]acetate and killed 15 minutes later as detailed in Materials and methods. The amount of ¹³C incorporation was determined from ¹³C-nuclear magnetic resonance spectroscopic analysis of brain extracts as detailed in Materials and methods. The percent direct synthesis of GABA from glutamine was calculated by dividing the amount of [1,2-¹³C]GABA by the sum of [1,2-¹³C]GABA, [3-¹³C]GABA, and [4-¹³C]GABA. The percent synthesis of glutamate was calculated by dividing the amount of [4,5-¹³C]glutamate by the sum of [4,5-¹³C]glutamate, [2-¹³C]glutamate, [3-¹³C]glutamate, and [1,2-¹³C]glutamate. The corresponding calculation was performed for glutamine. Results are averages±s.e.m. (n=5), and the asterisk indicates a statistically significant difference between GAD65 knockout and wild-type mice (P=0.03). GAD, glutamate decarboxylase.

Figure 5

Percent enrichment of [4-¹³C]glutamate, [4-¹³C]glutamine, [2-¹³C]GABA, [3-¹³C]aspartate, [3-¹³C]alanine, and [3-¹³C]lactate labeled from [1-¹³C]glucose in wild-type (WT; open bars) and GAD65 knockout (GAD65 KO; filled bars) mice. The animals were injected with [1-¹³C]glucose and [1,2-¹³C]acetate and killed 15 minutes later as detailed in Materials and methods. The enrichment was determined from ¹³C-nuclear magnetic resonance spectroscopic analysis of brain extracts as detailed in Materials and methods. Results are averages±s.e.m. (n=5) and statistically significant differences between GAD65 knockout and wild-type mice are indicated with asterisks (^*P<0.05; ^**P<0.01). GABA, γ-aminobutyric acid; GAD, glutamate decarboxylase.

Figure 6

Schematic illustration of synthesis of γ-aminobutyric acid (GABA) from glutamine directly and via tricarboxylic acid (TCA) cycle metabolism. Glutamine is converted to glutamate by phosphate-activated glutaminase (PAG) and pathway 1 illustrates direct synthesis of GABA from glutamate in the cytosol. GABA synthesis via this pathway is as indicated governed by both GAD65 and GAD67. Pathway 2 shows the deamination of glutamate and metabolism of α-ketoglutarate in the TCA cycle before GABA synthesis catalyzed by GAD67. GAD, glutamate decarboxylase.