Loss or Mislocalization of Aquaporin-4 Affects Diffusion Properties and Intermediary Metabolism in Gray Matter of Mice.

The first aim of this study was to determine how complete or perivascular loss of aquaporin-4 (AQP4) water channels affects membrane permeability for water in the mouse brain grey matter in the steady state. Time-dependent diffusion magnetic resonance imaging was performed on global Aqp4 knock out (KO) and α-syntrophin (α-syn) KO mice, in the latter perivascular AQP4 are mislocalized, but still functioning. Control animals were corresponding wild type (WT) mice. By combining in vivo diffusion measurements with the effective medium theory and previously measured extra-cellular volume fractions, the effects of membrane permeability and extracellular volume fraction were uncoupled for Aqp4 and α-syn KO. The second aim was to assess the effect of α-syn KO on cortical intermediary metabolism combining in vivo [1-13C]glucose and [1,2-13C]acetate injection with ex vivo 13C MR spectroscopy. Aqp4 KO increased the effective diffusion coefficient at long diffusion times by 5%, and a 14% decrease in membrane water permeability was estimated for Aqp4 KO compared with WT mice. α-syn KO did not affect the measured diffusion parameters. In the metabolic analyses, significantly lower amounts of [4-13C]glutamate and [4-13C]glutamine, and percent enrichment in [4-13C]glutamate were detected in the α-syn KO mice. [1,2-13C]acetate metabolism was unaffected in α-syn KO, but the contribution of astrocyte derived metabolites to GABA synthesis was significantly increased. Taken together, α-syn KO mice appeared to have decreased neuronal glucose metabolism, partly compensated for by utilization of astrocyte derived metabolites.

Early differences in dorsal hippocampal metabolite levels in males but not females in a transgenic rat model of Alzheimer's disease.

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.

A possible role of the non-GAT1 GABA transporters in transfer of GABA from GABAergic to glutamatergic neurons in mouse cerebellar neuronal cultures.

Cultures of dissociated cerebellum from 7-day-old mice were used to investigate the mechanism involved in synthesis and cellular redistribution of GABA in these cultures consisting primarily of glutamatergic granule neurons and a smaller population of GABAergic Golgi and stellate neurons. The distribution of GAD, GABA and the vesicular glutamate transporter VGlut-1 was assessed using specific antibodies combined with immunofluorescence microscopy. Additionally, tiagabine, SKF 89976-A, betaine, beta-alanine, nipecotic acid and guvacine were used to inhibit the GAT1, betaine/GABA (BGT1), GAT2 and GAT3 transporters. Only a small population of cells were immuno-stained for GAD while many cells exhibited VGlut-1 like immuno-reactivity which, however, never co-localized with GAD positive neurons. This likely reflects the small number of GABAergic neurons compared to the glutamatergic granule neurons constituting the majority of the cells. GABA uptake exhibited the kinetics of high affinity transport and could be partly (20%) inhibited by betaine (IC(50) 142 microM), beta-alanine (30%) and almost fully (90%) inhibited by SKF 89976-A (IC(50) 0.8 microM) or nipecotic acid and guvacine at 1 mM concentrations (95%). Essentially all neurons showed GABA like immunostaining albeit with differences in intensity. The results indicate that GABA which is synthesized in a small population of GAD-positive neurons is redistributed to essentially all neurons including the glutamatergic granule cells. GAT1 is not likely involved in this redistribution since addition of 15 microM tiagabine (GAT1 inhibitor) to the culture medium had no effect on the overall GABA content of the cells. Likewise the BGT1 transporter cannot alone account for the redistribution since inclusion of 3 mM betaine in the culture medium had no effect on the overall GABA content. The inhibitory action of beta-alanine and high concentrations of nipecotic acid and guvacine on GABA transport strongly suggests that also GAT2 or GAT3 (HUGO nomenclature) could play a role.

Gray matter metabolism in acute and chronic hydrocephalus.

Although hydrocephalus is usually considered a disorder of periventricular white matter, disturbance of gray matter is probably also involved. However, so far gray matter metabolism has not been studied in experimental hydrocephalus using high resolution in vivo magnetic resonance spectroscopy (MRS). Therefore 15 rats were made hydrocephalic by injection of 0.1 ml kaolin into the cisterna magna, whereas 10 sham-operated rats served as controls. (1)H MRS and magnetic resonance imaging were performed longitudinally in acute hydrocephalus 2 and 4 weeks after kaolin treatment and in chronic hydrocephalus after 6 weeks. Volumes of interest included the gray matter regions cortex, thalamus and hippocampus. In hydrocephalic animals, (1)H MRS revealed decreased glutamate levels in all examined areas at all time points. Moreover, in acute hydrocephalus disturbances were noted in the hippocampus with decreased concentrations of N-acetyl aspartate, creatine, inositol and taurine, and in the cortex with decreased taurine levels. A clear lactate peak was detected in CSF spectra from hydrocephalic rats. In addition, T2-weighted images showed increase of free water in the hippocampus. It can be concluded that glutamate metabolism is deranged in gray matter in acute and chronic hydrocephalus in rats. If confirmed in humans, early detection of glutamatergic disturbances and lactate accumulation using in vivo(1)H MRS might serve as an indication for surgical treatment of hydrocephalus before irreversible neuronal damage develops.

Expression of a Patatin-like Protein in the Anthers of Potato and Sweet Pepper Flowers.

Patatin, the major glycoprotein in potato tubers, is encoded by a multigene family. RNA and protein analyses reveal that a homologous mRNA and an immunologically cross-reacting protein can be found in potato flowers, which is similar to patatin in that it displays a lipid acyl hydrolase activity. The patatin-like protein found in flowers has a higher molecular weight than the authentic tuber patatin. Deglycosylation experiments show that this is not due to differences in the glycosylation pattern. Immunocytochemical analysis shows the patatin-like protein to be present only in the epidermal cell layer of the anther, the exothecium, and in petals of potato flowers. Furthermore, the fact that a patatin-like protein can be detected in a similar tissue in sweet pepper, another solanaceous plant, could give a clue concerning the evolutionary origin of patatin.

Systemic Acquired Resistance Mediated by the Ectopic Expression of Invertase: Possible Hexose Sensing in the Secretory Pathway.

Systemic acquired resistance (SAR) has been reported to be associated with lesion-mimic mutants. Tobacco plants expressing vacuolar and apoplastic yeast-derived invertase (vaclnv and cwlnv, respectively) develop spontaneous necrotic lesions similar to hypersensitive responses caused by avirulent pathogens. Therefore, SAR and metabolic alterations leading to the activation of defense-related responses were studied in these plants. Defense-related gene transcripts, callose content, peroxidase activities, and levels of salicylic acid were found to be elevated. The defense reactions were accompanied by increased resistance toward potato virus Y and were measured as decreased viral spreading and reduced multiplication in systemic leaves of the transgenic plants. Interestingly, the accumulation of pathogenesis-related (PR) protein transcripts (PR-Q) and repression of photosynthetic gene transcripts (chlorophyll a/b binding protein) were inversely correlated and required the same threshold level of hexoses for induction and repression. Expression of a cytosolic yeast-derived invertase in transgenic tobacco plants with equally increased levels of sugars neither displayed SAR responses nor showed decreased levels of photosynthetic genes. It is suggested that hexose sensing in the secretory pathway is essential for mediating the activation of defense-related genes as well as repression of photosynthetic genes in vaclnv and cwlnv plants.

Effect of the pyrrolopyrimidine lipid peroxidation inhibitor U-101033E on neuronal and astrocytic metabolism and infarct volume in rats with transient middle cerebral artery occlusion.

The aim of the present study was to assess the effect of post ictal administration of the pyrrolopyrimidine lipid peroxidation inhibitor, U-101033E, on infarct volume and neuronal and astrocytic metabolism in rats with transient middle cerebral artery occlusion (MCAO). Rats were subjected to 120 min of MCAO followed by 140 min of reperfusion and randomly assigned to control (n=17) or U-101033E treatment (n=16). Drug infusion started 5 min after MCAO and lasted 220 min with a 15 min interruption during the reperfusion procedure. Sixteen rats underwent diffusion weighted imaging 260 min after ictus, from which the apparent diffusion coefficient (ADC) was determined. Seventeen rats received an iv bolus injection of [1-13C]glucose and [1,2-13C]acetate 245 min after ictus. Tissue extracts from two brain regions representing penumbra and ischemic core were analyzed with 13C NMRS and HPLC. U-101033E did not affect the volume of ischemic tissue estimated from the ADC maps. In the penumbra, U-101033E specifically decreased mitochondrial pyruvate metabolism via both pyruvate dehydrogenase and pyruvate carboxylase pathways. Thus, U-101033E impaired both neuronal and astrocytic mitochondrial pyruvate metabolism. At the same time anaerobic glucose usage was increased, leading to increased lactate labeling and content. Also alanine labeling was increased. The data do not support lactate as an important substrate for neuronal mitochondria in ischemia-reperfusion. A similar pattern of reduced mitochondrial pyruvate metabolism and increased cytosolic pyruvate metabolism was found in the irreversibly damaged ischemic core. The present study highlights the importance of other outcome measures than ischemic tissue volume for evaluation of drug efficacy in animal models, which in turn could increase the likelihood of success in clinical trials.

Ectopic expression of a tobacco invertase inhibitor homolog prevents cold-induced sweetening of potato tubers.

We have transformed potato with Nt-inhh cDNA, encoding a putative vacuolar homolog of a tobacco cell wall invertase inhibitor, under the control of the CaMV 35S promoter. In transgenic tubers, cold-induced hexose accumulation was reduced by up to 75%, without any effect on potato tuber yield. Processing quality of tubers was greatly improved without changing starch quantity or quality, an important prerequisite for the biotechnological use of Nt-inhh for potato transformation.

Increased potato tuber size resulting from apoplastic expression of a yeast invertase.

The role of sucrose cleavage in determining sink strength in potato was investigated by generating transgenic potato plants that expressed a yeast invertase in either the cytosol or apoplast of tubers. Cytosolic localization gave rise to a reduction in tuber size and an increase in tuber number per plant whereas apoplastic targeting led to an increase in tuber size and a decrease in tuber number per plant. Sink organ size can be manipulated through modification of sucrose metabolism.

An ethanol inducible gene switch for plants used to manipulate carbon metabolism.

Many transgenic plant studies use constitutive promoters to express transgenes. For certain genes, deleterious effects arise from constant expression in all tissues throughout development. We describe a chemically inducible plant gene expression system, with negligible background activity, that obviates this problem. We demonstrate its potential by showing inducible manipulation of carbon metabolism in transgenic plants. Upon rapid induction of yeast cytosolic invertase, a marked phenotype appears in developing leaves that is absent from leaves that developed before induction or after it has ceased.

Molecular determinants of sink strength.

The manipulation of sink to source relations has been subject to extensive plant breeding programs aiming to improve harvest index and thereby crop yield. The introduction of molecular and biochemical tools has enabled scientists to investigate the underlying principles. This has opened up the fascinating possibility of identifying molecular determinants of sink strength and to further increase yield on a rational basis. In the past, transgenic plants with alterations in the activity of only one putative molecular determinant have been created and this strategy has not resulted in substantial and reliable increases in yield. Yet, careful molecular and biochemical investigations have provided valuable insight about carbon flux into different metabolic pathways at different stages of sink development and it has become apparent that this metabolic channelling needs to be exploited by using stage- and cell-specific promoters in attempts to increase sink strength.

Glutamate and GABA metabolism in transient and permanent middle cerebral artery occlusion in rat: importance of astrocytes for neuronal survival.

The aim of the present study was to identify the distinguishing metabolic characteristics of brain tissue salvaged by reperfusion following focal cerebral ischemia. Rats were subjected to 120 min of middle cerebral artery occlusion followed by 120 min of reperfusion. The rats received an intravenous bolus injection of [1-(13)C]glucose plus [1,2-(13)C]acetate. Subsequently two brain regions considered to represent penumbra and ischemic core, i.e. the frontoparietal cortex and the lateral caudoputamen plus lower parietal cortex, respectively, were analyzed with (13)C NMRS and HPLC. The results demonstrated four metabolic events that distinguished the reperfused penumbra from the ischemic core. (1) Improved astrocytic metabolism demonstrated by increased amounts of [4,5-(13)C]glutamine and improved acetate oxidation. (2) Neuronal mitochondrial activity was better preserved although the flux of glucose via pyruvate dehydrogenase into the tricarboxylic acid (TCA) cycle in glutamatergic and GABAergic neurons was halved. However, NAA content was at control level. (3) Glutamatergic and GABAergic neurons used relatively more astrocytic metabolites derived from the pyruvate carboxylase pathway. (4) Lactate synthesis was not increased despite decreased glucose metabolism in the TCA cycle via pyruvate dehydrogenase. In the ischemic core both neuronal and astrocytic TCA cycle activity declined significantly despite reperfusion. The utilization of astrocytic precursors originating from the pyruvate carboxylase pathway was markedly reduced compared the pyruvate dehydrogenase pathway in glutamate, and completely stopped in GABA. The NAA level fell significantly and lactate accumulated. The results demonstrate that preservation of astrocytic metabolism is essential for neuronal survival and a predictor for recovery.

Decreased expression of sucrose phosphate synthase strongly inhibits the water stress-induced synthesis of sucrose in growing potato tubers.

Water stress stimulates sucrose synthesis and inhibits starch synthesis in wild-type tubers. Antisense and co-suppression potato transformants with decreased expression of sucrose-phosphate synthase (SPS) have been used to analyse the importance of SPS for the regulation of this water-stress induced change in partitioning. (i) In the absence of water stress, a 70-80% decrease in SPS activity led to a 30-50% inhibition of sucrose synthesis and a slight (10-20%) increase of starch synthesis in tuber discs in short-term labelling experiments with low concentrations of labelled glucose. Similar changes were seen in short-term labelling experiments with intact tubers attached to well-watered plants. Provided plants were grown with ample light and water, transformant tubers had a slightly lower water and sucrose content and a similar or even marginally higher starch content than wild-type tubers. (ii) When wild-type tuber slices were incubated with labelled glucose in the presence of mannitol to generate a moderate water deficit (between -0.12 and -0.72 MPa), there was a marked stimulation of sucrose synthesis and inhibition of starch synthesis. A similar stimulation was seen in labelling experiments with wild-type tubers that were attached to water-stressed wild-type plants. These changes were almost completely suppressed in transformants with a 70-80% reduction of SPS activity. (iii) Decreased irrigation led to an increase in the fraction of the dry-matter allocated to tubers in wild-type plants. This shift in allocation was prevented in transformants with reduced expression of SPS. (iv) The results show that operation of SPS and the sucrose cycle in growing potato tubers may lead to a marginal decrease in starch accumulation in non-stressed plants. However, SPS becomes a crucial factor in water-stressed plants because it is required for adaptive changes in tuber metabolism and whole plant allocation.

Production of new/modified proteins in transgenic plants.

In recent years, plant biotechnology has almost reached maturity. Transgenic plants engineered to be herbicide- or insect-resistant are outcompeting conventional crop plants and pest managing strategies leading to a major rethinking of the chemical industry. Due to worldwide efforts to study genome function, almost any gene of interest is, or will soon be available. Thus, identification of gene function will be the major challenge of the next few years. In combination with established gene-delivery systems and desired promoter and targetting sequences, gene discovery will open a fascinating and new field of crop plant design. Transgenic plants engineered to produce superior polypeptides have already been created and the first examples are entering clinical and industrial trials.

Manipulating metabolic partitioning in transgenic plants.

Various strategies can be used to influence the partitioning of metabolites, both between competing pathways and within a given biochemical pathway. Changes in metabolic partitioning in transgenic plants can be brought about by either expressing heterologous genes or suppressing endogenous ones. Strategies for altering metabolic partitioning can include accelerating, circumventing or inhibiting enzymatic, regulatory or transport steps. In addition, metabolic pathways can be modified, through the use of collections of novel enzymes, to synthesize novel products.

Mitochondrial heterogeneity in the brain at the cellular level.

Classically, compartmentation of glutamate metabolism in the brain is associated with the fact that neurons and glia exhibit distinct differences with regard to metabolism of this amino acid. The recent use of 13C-labeled compounds to study this metabolism in conjunction with the availability of cell type-specific tissue culture modes has led to the notion that such compartmentation may even be present in individual cell types, neurons as well as glia. To better understand and explain this, it is proposed that mitochondrial heterogeneity may exist resulting in tricarboxylic acid cycles with different properties regarding cycling rates and ratio as well as coupling to amino acid biosynthesis, primarily involving glutamate and aspartate. These hypotheses are evaluated in the light of current knowledge about mitochondrial structure and function.

Trafficking of amino acids between neurons and glia in vivo. Effects of inhibition of glial metabolism by fluoroacetate.

Glial-neuronal interchange of amino acids was studied by 13C nuclear magnetic resonance spectroscopy of brain extracts from fluoroacetate-treated mice that received [1,2-(13)C]acetate and [1-(13)C]glucose simultaneously. [13C]Acetate was found to be a specific marker for glial metabolism even with the large doses necessary for nuclear magnetic resonance spectroscopy. Fluoroacetate, 100 mg/kg, blocked the glial, but not the neuronal tricarboxylic acid cycles as seen from the 13C labeling of glutamine, glutamate, and gamma-aminobutyric acid. Glutamine, but not citrate, was the only glial metabolite that could account for the transfer of 13C from glia to neurons. Massive glial uptake of transmitter glutamate was indicated by the labeling of glutamine from [1-(13)C]glucose in fluoroacetate-treated mice. The C-3/C-4 enrichment ratio, which indicates the degree of cycling of label, was higher in glutamine than in glutamate in the presence of fluoroacetate, suggesting that transmitter glutamate (which was converted to glutamine after release) is associated with a tricarboxylic acid cycle that turns more rapidly than the overall cerebral tricarboxylic acid cycle.

In vivo injection of [1-13C]glucose and [1,2-13C]acetate combined with ex vivo 13C nuclear magnetic resonance spectroscopy: a novel approach to the study of middle cerebral artery occlusion in the rat.

Astrocytes play a pivotal role in cerebral glutamate homeostasis. After 90 minutes of middle cerebral artery occlusion in the rat, the changes induced in neuronal and astrocytic metabolism and in the neuronal-astrocytic interactions were studied by combining in vivo injection of [1-13C]glucose and [1,2-13C]acetate with ex vivo 13C nuclear magnetic resonance spectroscopy and HPLC analysis of amino acids of the lateral caudoputamen and lower parietal cortex, representing the putative ischemic core, and the upper frontoparietal cortex, corresponding to the putative penumbra. In the putative ischemic core, evidence of compromised de novo glutamate synthesis located specifically in the glutamatergic neurons was detected, and a larger proportion of glutamate was derived from astrocytic glutamine. In the same region, pyruvate carboxylase activity, representing the anaplerotic pathway in the brain and exclusively located in astrocytes, was abolished. However, astrocytic glutamate uptake and conversion to glutamine took place, and cycling of intermediates in the astrocytic tricarboxylic acid cycle was elevated. In the putative penumbra, glutamate synthesis was improved compared with the ischemic core, the difference appeared to be brought on by better neuronal de novo glutamate synthesis, combined with normal levels of glutamate formed from astrocytic glutamine. In both ischemic regions, gamma-aminobutyric acid synthesis directly from glucose was reduced to about half, indicating impaired pyruvate dehydrogenase activity; still, gamma-aminobutyric acid reuptake and cycling was increased. The results obtained in the current study demonstrate that by combining in vivo injection of [1-13C]glucose and [1,2-13C]acetate with ex vivo 13C nuclear magnetic resonance spectroscopy, specific metabolic alterations in small regions within the rat brain suffering a focal ischemic lesion can be studied.

Metabolism of lactate in cultured GABAergic neurons studied by 13C nuclear magnetic resonance spectroscopy.

Primary cultures of mouse cerebral cortical neurons (GABAergic) were incubated for 4 hours in media without glucose containing 1.0 mmol/L [U-13C]lactate in the absence or presence of 0.5 mmol/L glutamine. Redissolved, lyophilized cell extracts were analyzed by 13C nuclear magnetic resonance spectroscopy to investigate neuronal metabolism of lactate and by HPLC for determination of the total amounts of glutamate (Glu), gamma-aminobutyric acid (GABA), and aspartate (Asp). The 13C nuclear magnetic resonance spectra of cell extracts exhibited multiplets for Glu, GABA, and Asp, indicating pronounced recycling of labeled tricarboxylic acid cycle constituents. There was extensive incorporation of 13C label into amino acids in neurons incubated without glutamine, with the percent enrichments being approximately 60% for Glu and Asp, and 27% for GABA. When 0.5 mmol/L glutamine was added to the incubation medium, the enrichments for Asp, Glu, and GABA were 25%, 35%, and 25%, respectively. This strongly suggests that glutamine is readily converted to Glu and Asp but that conversion to GABA may be complex. The observation that enrichment in GABA was identical in the absence and presence of glutamine whereas cycling was decreased in the presence of glutamine indicates that only C-2 units derived from glutamine are used for GABA synthesis, that is, that metabolism through the tricarboxylic acid cycle is a prerequisite for GABA synthesis from glutamine. The current study gives further support to the hypothesis that cellular metabolism is compartmentalized and that lactate is an important fuel for neurons in terms of energy metabolism and extensively labels amino acids synthesized from tricarboxylic acid cycle intermediates (Asp and Glu) as well as the neurotransmitter in these neurons (GABA).

Differences in neurotransmitter synthesis and intermediary metabolism between glutamatergic and GABAergic neurons during 4 hours of middle cerebral artery occlusion in the rat: the role of astrocytes in neuronal survival.

Astrocytes are intimately involved in both glutamate and gamma-aminobutyric acid (GABA) synthesis, and ischemia-induced disruption of normal neuroastrocytic interactions may have important implications for neuronal survival. The effects of middle cerebral artery occlusion (MCAO) on neuronal and astrocytic intermediary metabolism were studied in rats 30, 60, 120, and 240 minutes after MCAO using in vivo injection of [1-13C]glucose and [1,2- 13C]acetate combined with ex vivo 13C magnetic resonance spectroscopy and high-performance liquid chromatography analysis of the ischemic core (lateral caudoputamen and lower parietal cortex) and penumbra (upper frontoparietal cortex). In the ischemic core, both neuronal and astrocytic metabolism were impaired from 30 minutes MCAO. There was a continuous loss of glutamate from glutamatergic neurons that was not replaced as neuronal glucose metabolism and use of astrocytic precursors gradually declined. In GABAergic neurons astrocytic precursors were not used in GABA synthesis at any time after MCAO, and neuronal glucose metabolism and GABA-shunt activity declined with time. No flux through the tricarboxylic acid cycle was found in GABAergic neurons at 240 minutes MCAO, indicating neuronal death. In the penumbra, the neurotransmitter pool of glutamate coming from astrocytic glutamine was preserved while neuronal metabolism progressively declined, implying that glutamine contributed significantly to glutamate excitotoxicity. In GABAergic neurons, astrocytic precursors were used to a limited extent during the initial 120 minutes, and tricarboxylic acid cycle activity was continued for 240 minutes. The present study showed the paradoxical role that astrocytes play in neuronal survival in ischemia, and changes in the use of astrocytic precursors appeared to contribute significantly to neuronal death, albeit through different mechanisms in glutamatergic and GABAergic neurons.