c-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 c-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 B20%. 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. Blood Flow & Metabolism (2011) 31, 494-503; doi:10.1038/jcbfm.2010; published online 28 July 2010 Journal of Cerebral Keywords:13 C isotopes; g-vinylGABA (GVG, vigabatrin); glutamate decarboxylase; hypometabolism; neuronal-glial trafficking; nuclear magnetic resonance IntroductionThe most abundant neurotransmitters in the brain are glutamate and g-aminobutyric acid (GABA) for excitatory and inhibitory transmission, respectively.A tight regulation of the synthesis and degradation of these two compounds is therefore crucial. Disturbances in this regulation are likely involved in GABA-glutamate imbalances characteristic for a number of neurodegenerative and psychiatric disorders (Sonnewald and Kondziella, 2003). The GABA synthesis from glutamate is catalyzed by the enzyme glutamate decarboxylase (GAD, EC 4.1.1.15) of which two isoforms, GAD65 and GAD67, have been identified. These isoforms are encoded for by separate genes and differ with regard to regulation and intracellular localization (Erlander et al, 1991;Kaufman et al, 1991;Esclapez et al, 1994). The GAD65 appears to exist predominantly as a dormant apoenzyme, being rapidly activated on binding of pyridoxal phosphate. In contrast, GAD67 is present as the active holoenzyme having the coenzyme bound . The GAD67 is localized throughout the cytosol of GABAergic neurons, Correspondence: Dr HS Waagepetersen, Department of Pharmacology and Pharmacotherapy, Faculty of Pharmaceutical Sciences, University of Copenhagen, 2 Universitetsparken, DK-2100 Copenhagen, Denmark. E-mail: hsw@farma.ku.dk 5 The major part of the wo...

We investigated metabolite levels during the progression of pathology in McGill-R-Thy1-APP rats, a transgenic animal model of Alzheimer's disease, and in healthy age-matched controls. Rats were subjected to in vivo 1 H magnetic resonance spectroscopy (MRS) of the dorsal hippocampus at age 3, 9 and 12 months and of frontal cortex at 9 and 12 months. At 3 months, a stage in which only Ab oligomers are present, lower glutamate, myo-inositol and total choline content were apparent in McGill-R-Thy1-APP rats. At age 9 months, lower levels of glutamate, GABA, N-acetylaspartate and total choline and elevated myo-inositol and taurine were found in dorsal hippocampus, whereas lower levels of glutamate, GABA, glutamine and N-acetylaspartate were found in frontal cortex.At age 12 months, only the taurine level was significantly different in dorsal hippocampus, whereas taurine, myo-inositol, N-acetylaspartate and total creatine levels were significantly higher in frontal cortex. McGill-R-Thy1-APP rats did not show the same changes in metabolite levels with age as displayed in the controls, and overall, prominent and complex metabolite differences were evident in this transgenic rat model of Alzheimer's disease. The findings also demonstrate that in vivo 1 H MRS is a powerful tool to investigate diseaserelated metabolite changes in the brain. Keywords: biomarkers, GABA, glutamate, metabolism, N-acetylaspartate, transgenic rats. Alzheimer's disease (AD) is a progressive neurodegenerative disease and the most common cause of dementia in the elderly. It is characterized by accumulation of extracellular plaques containing aggregated amyloid b (Ab) peptides and intracellular neurofibrillary tangles composed of hyperphosphorylated tau proteins. In addition, regional loss of neurons and synapses, progressive cognitive decline and regional hypometabolism occurs (Mosconi 2005; SerranoPozo et al. 2011). Emerging evidence also suggest that, intraneuronal Ab oligomers may contribute substantially to AD disease progression (Haass and Selkoe 2007).There is no definite biomarker for the diagnosis of AD, which motivates the search for neuroimaging markers that may facilitate early detection of the disease. Using 1 H magnetic resonance spectroscopy (MRS), the regional concentration of low-molecular-weight metabolites can be measured non-invasively and provides insight into neurochemical processes of normal and pathological conditions in vivo. Performing 1 H MRS of patients with AD has revealed a consistent pattern of decreased levels of Nacetylaspartate (NAA) or NAA/total creatine (tCr) and increased myo-inositol (mIns) or mIns/tCr (Kantarci et al. 2003;Shiino et al. 2012). NAA is synthesised in neurons (Wiame et al. 2010) Abbreviations used: AD, Alzheimer's disease; APP, amyloid precursor protein; Ab, amyloid beta; CMR glc , cerebral metabolic rate of glucose; CRLB, Cramer-Rao lower bounds; DH, dorsal hippocampus; FCX, frontal cortex; GS, glutamine synthetase; mIns, myo-inositol; MRI, magnetic resonance imaging; MRS, magnetic resona...

Regional hypometabolism of glucose in the brain is a hallmark of Alzheimer's disease (AD). However, little is known about the specific alterations of neuronal and astrocytic metabolism involved in homeostasis of glutamate and GABA in AD. Here, we investigated the effects of amyloid β (Aβ) pathology on neuronal and astrocytic metabolism and glial-neuronal interactions in amino acid neurotransmitter homeostasis in the transgenic McGill-R-Thy1-APP rat model of AD compared with healthy controls at age 15 months. Rats were injected with [1-(13)C]glucose and [1,2-(13)C]acetate, and extracts of the hippocampal formation as well as several cortical regions were analyzed using (1)H- and (13)C nuclear magnetic resonance spectroscopy and high-performance liquid chromatography. Reduced tricarboxylic acid cycle turnover was evident for glutamatergic and GABAergic neurons in hippocampal formation and frontal cortex, and for astrocytes in frontal cortex. Pyruvate carboxylation, which is necessary for de novo synthesis of amino acids, was decreased and affected the level of glutamine in hippocampal formation and those of glutamate, glutamine, GABA, and aspartate in the retrosplenial/cingulate cortex. Metabolic alterations were also detected in the entorhinal cortex. Overall, perturbations in energy- and neurotransmitter homeostasis, mitochondrial astrocytic and neuronal metabolism, and aspects of the glutamate-glutamine cycle were found in McGill-R-Thy1-APP rats.

In neurodegenerative diseases including Alzheimer's disease and frontotemporal dementia, the protein tau is hyperphosphorylated and eventually aggregates to develop neurofibrillary tangles. Here, the consequences of tau hyperphosphorylation on both neuronal and astrocytic metabolism and amino-acid neurotransmitter homeostasis were assessed in transgenic mice expressing the pathogenic mutation P301L in the human tau gene (pR5 mice) compared with nontransgenic littermate controls. Mice were injected with the neuronal and astrocytic substrate [1-13 C]glucose and the astrocytic substrate [1,2-13 C]acetate. Hippocampus and cerebral cortex extracts were analyzed using 1 H and 13 C nuclear magnetic resonance spectroscopy, gas chromatography-mass spectrometry and high-performance liquid chromatography. The glutamate level was reduced in the hippocampus of pR5 mice, accompanied by reduced incorporation of 13 C label derived from [1-13 C]glucose in glutamate. In the cerebral cortex, glucose utilization as well as turnover of glutamate, glutamine, and GABA, were increased. This was accompanied by a relative increase in production of glutamate via the pyruvate carboxylation pathway in cortex. Overall, we revealed that astrocytes as well as glutamatergic and GABAergic neurons in the cortex of pR5 mice were in a hypermetabolic state, whereas in the hippocampus, where expression levels of mutant human tau are the highest, glutamate homeostasis was impaired.

J. Neurochem. (2010) 115, 1398–1408. Abstract GABA is synthesized from glutamate by glutamate decarboxylase (GAD), which exists in two isoforms, that is, GAD65 and GAD67. In line with GAD65 being located in the GABAergic synapse, several studies have demonstrated that this isoform is important during sustained synaptic transmission. In contrast, the functional significance of GAD65 in the maintenance of GABA destined for extrasynaptic tonic inhibition is less well studied. Using GAD65−/− and wild type GAD65+/+ mice, this was examined employing the cortical wedge preparation, a model suitable for investigating extrasynaptic GABAA receptor activity. An impaired tonic inhibition in GAD65−/− mice was revealed demonstrating a significant role of GAD65 in the synthesis of GABA acting extrasynaptically. The correlation between an altered tonic inhibition and metabolic events as well as the functional and metabolic role of GABA synthesized by GAD65 was further investigated in vivo. Tonic inhibition and the demand for biosynthesis of GABA were augmented by injection of kainate into GAD65−/− and GAD65+/+ mice. Moreover, [1‐13C]glucose and [1,2‐13C]acetate were administered to study neuronal and astrocytic metabolism concomitantly. Subsequently, cortical and hippocampal extracts were analyzed by NMR spectroscopy and mass spectrometry, respectively. Although seizure activity was induced by kainate, neuronal hypometabolism was observed in GAD65+/+ mice. In contrast, kainate evoked hypermetabolism in GAD65−/− mice exhibiting deficiencies in tonic inhibition. These findings underline the importance of GAD65 for synthesis of GABA destined for extrasynaptic tonic inhibition, regulating epileptiform activity.

Using a modified MK-801 (dizocilpine) N-methyl-D-aspartic acid (NMDA) receptor hypofunction model for schizophrenia, we analyzed glycolysis, as well as glutamatergic, GABAergic, and monoaminergic neurotransmitter synthesis and degradation. Rats received an injection of MK-801 daily for 6 days and on day 6, they also received an injection of [1-(13)C]glucose. Extracts of frontal cortex (FCX), parietal and temporal cortex (PTCX), thalamus, striatum, nucleus accumbens (NAc), and hippocampus were analyzed using (13)C nuclear magnetic resonance spectroscopy, high-performance liquid chromatography, and gas chromatography-mass spectrometry. A pronounced reduction in glycolysis was found only in PTCX, in which (13)C labeling of glucose, lactate, and alanine was decreased. (13)C enrichment in lactate, however, was reduced in all areas investigated. The largest reductions in glutamate labeling were detected in FCX and PTCX, whereas in hippocampus, striatum, and Nac, (13)C labeling of glutamate was only slightly but significantly reduced. The thalamus was the only region with unaffected glutamate labeling. γ-Aminobutyric acid (GABA) labeling was reduced in all areas, but most significantly in FCX. Glutamine and aspartate labeling was unchanged. Mitochondrial metabolites were also affected. Fumarate labeling was reduced in FCX and thalamus, whereas malate labeling was reduced in FCX, PTCX, striatum, and NAc. Dopamine turnover was decreased in FCX and thalamus, whereas that of serotonin was unchanged in all regions. In conclusion, neurotransmitter metabolism in the cortico-striato-thalamo-cortical loop is severely impaired in the MK-801 (dizocilpine) NMDA receptor hypofunction animal model for schizophrenia.

McGill-R-Thy1-APP rats express the human amyloid precursor protein carrying the Swedish and Indiana mutations. We examined the neurochemical content of the dorsal hippocampus in three-months-old male and female transgenic rats and healthy age- and gender-matched controls using in vivo (1)H MRS in order to assess early metabolite alterations and whether these were similar for both genders. Whereas male and female controls had similar levels of all metabolites, differences were evident between male and female McGill-R-Thy1-APP rats. Compared with McGill-R-Thy1-APP females, McGill-R-Thy1-APP males had lower levels of myo-inositol and N-acetylaspartate (NAA). No differences in metabolite levels were evident when female control and McGill-R-Thy1-APP rats were compared, whereas McGill-R-Thy1-APP males had lower levels of glutamate, NAA and total choline compared with male controls. In addition to metabolite concentrations, metabolite ratios are reported as these are widely used. The results from this preliminary study demonstrate early metabolite alterations in the dorsal hippocampus of males in this rat model of Alzheimer's disease, and imply that very early possible neurochemical markers of the disease are different for males and females.

The activity of the α-ketoglutarate dehydrogenase complex (KGDHC), a mitochondrial enzyme complex which mediates the oxidative decarboxylation of α-ketoglutarate in the TCA cycle, is reduced in Alzheimer’s Disease. We investigated the metabolic effects of a partial KGDHC activity reduction on brain glucose metabolism using mice with disrupted expression of dihydrolipoyl succinyltransferase (DLST; gene encoding the E2k subunit of KGDHC). Brain tissue extracts from cortex and cerebellum of 6-week –old heterozygote DLST knockout mice (DLST+/−) and corresponding wild type mice injected with [U-13C]glucose and decapitated 15 minutes later were analyzed. An increase in the concentration of glucose in cortex suggested a decrease in the cortical utilization of glucose in DLST +/− mice. Furthermore, the concentration and 13C labelling of aspartate in cortex were reduced in DLST+/− mice. This decline was likely caused by a decrease in the pool of oxaloacetate. In contrast to results from cell culture studies, no indications of altered glycolysis or GABA shunt activity were found. Glucose metabolism in the cerebellum was unaffected by the decrease in KGDHC activity. Among metabolites not related to glucose metabolism, the concentration of taurine was decreased in the cortex, and that of tyrosine was increased in the cerebellum. These results imply that diminished KGDHC activity has the potential to induce the reduction in glucose utilization that is seen in several neurodegenerative diseases.