Biomedical subjects

Ursula Sonnewald

Publications and source records attributed to Ursula Sonnewald.

At least 19 recordsLinked to original sources

PubMed2007

Glutamine in the central nervous system: function and dysfunction.

Glutamine (Gln) abounds in the central nervous system (CNS), and its interstitial and cerebrospinal fluid (CSF) concentrations are at least one order of magnitude higher than of any other amino acid. Gln transport from blood to the brain is insufficient to meet the demand of the brain tissues for this amino acid. This demand is met by intracerebral Gln synthesis from glutamate (Glu), a reaction carried out by glutamine synthetase (GS), an enzyme residing in astrocytes. A major proportion of astroglia-derived Gln is shuttled to neurons where it is degraded by phosphate-activated glutaminase (PAG) giving rise to the excitatory neurotransmitter amino acid Glu, which is also a precursor of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). Glu released from neurons is taken up by astrocytes, and reconverted to Gln, closing the so called "glutamate-glutamine" cycle. A portion of Gln serves as an energy metabolite, and part of it leaves the brain to blood. Gln efflux from astrocytes, its neuronal uptake and egress to the blood via the cerebral capillary endothelial cells is mediated by different amino acid carriers showing i) considerable preference for Gln, ii) distribution between astrocytes and neurons that favors astrocyte-to-neuron fluxes of the amino acid. The Gln-specific carriers also largely contribute to Gln efflux from the brain to the vascular bed. Excessive accumulation of Gln in brain cells may be deleterious to brain function. In hyperammonemia associated with acute liver failure, excess Gln leads to cerebral edema, which largely results from its interference with mitochondrial function and partly from its osmotic action. Future analyses of the roles of Gln in both normal and abnormal cerebral metabolism and function will have to account for its newly recognized direct involvement in the regulation of gene transcription and/or translation.

Animals↗

PubMed2006

Long-term kainic acid exposure reveals compartmentation of glutamate and glutamine metabolism in cultured cerebellar neurons.

Glutamate neurotoxicity is implicated in most neurodegenerative diseases, and in the present study the long-term effects of the glutamate agonist kainic acid (KA) on cerebellar neurons are investigated. Primary cell cultures, mainly consisting of glutamatergic granule neurons, were cultured in medium containing 0.05 or 0.50mM KA for 7 days and subsequently incubated in medium containing [U-13C]glutamate or [U-13C]glutamine. The amount of protein and number of cells were greatly reduced in cultures exposed to 0.50 mM KA compared to those exposed to 0.05 mM KA. Glutamine consumption was not affected by KA concentration, whereas that of glutamate was decreased by high KA, confirming reduction in glutamate transport reported earlier. Neurons cultured with 0.50 mM KA and incubated with glutamate contained decreased amounts of glutamate, aspartate and GABA compared to those cultured with 0.05 mM KA. Incubation of cells exposed to 0.50 mM KA with glutamine led to an increased amount of glutamate compared to cells exposed to 0.05 mM KA, whereas the intracellular amounts of aspartate and GABA remained unaffected by KA concentration. Furthermore, mitochondrial metabolism of alpha-[U-13C]ketoglutarate derived from [U-13C]glutamate and [U-13C]glutamine was significantly reduced by 0.50 mM KA. The results presented illustrate differential vulnerability to KA and can only be understood in terms of inter- and intracellular compartmentation.

Animals↗

PubMed2006

How do glial-neuronal interactions fit into current neurotransmitter hypotheses of schizophrenia?

Evidence is accumulating that the exclusive dopamine hypothesis of schizophrenia has to be abandoned. Instead, a more integrative approach combines different neurotransmitter systems, in which glutamatergic, GABAergic and dopaminergic pathways interact. This paradigm shift coincides with the recognition that, while typical and modern atypical antipsychotic drugs have efficiently controlled the dramatic psychotic symptoms of schizophrenia, their impact on negative and cognitive symptoms is negligible. Indeed, cognitive decline is now believed to represent the core of schizophrenic morbidity and in this context, impairment of glutamate and more specifically NMDA function is of major importance. Given that astrocytes are important in controlling glutamate homeostasis, it is necessary to assign a significant role to glial-neuronal interactions in the pathophysiology of schizophrenia. Indeed, recent data from several animal and human studies corroborate this notion. This review outlines current neurotransmitter hypotheses and evidence for glial impairment in schizophrenia. Furthermore, findings from recent studies of (13)C nuclear magnetic resonance spectroscopy in experimental models of schizophrenia and NMDA hypofunction are presented and their implications for future research on glial-neuronal interactions discussed.

Animals↗

PubMed2006

Glutamate is preferred over glutamine for intermediary metabolism in cultured cerebellar neurons.

The glutamate-glutamine cycle is thought to be of paramount importance in the mature brain; however, its significance is likely to vary with regional differences in distance between astrocyte and synapse. The present study is aimed at evaluating the role of this cycle in cultures of cerebellar neurons, mainly consisting of glutamatergic granule cells. Cells were incubated in medium containing [U-13C]glutamate or [U-13C]glutamine in the presence and absence of unlabeled glutamine and glutamate, respectively. Cell extracts and media were analyzed using high-performance liquid chromatography (HPLC) and gas chromatography combined with mass spectrometry (GC/MS). Both [U-13C]glutamate and [U-13C]glutamine were shown to be excellent precursors for synthesis of neuroactive amino acids and tricarboxylic acid (TCA) cycle intermediates. Labeling from [U-13C]glutamate was higher than that from [U-13C]glutamine in all metabolites measured. The presence of [U-13C]glutamate plus unlabeled glutamine in the experimental medium led to labeling very similar to that from [U-13C]glutamate alone. However, incubation in medium containing [U-13C]glutamine in the presence of unlabeled glutamate almost abolished labeling of metabolites. Thus, it could be shown that glutamate is the preferred substrate for intermediary metabolism in cerebellar neurons. Label distribution indicating TCA cycle activity showed more prominent cycling from [U-13C]glutamine than from [U-13C]glutamate. Labeling of succinate was lower than that of the other TCA cycle intermediates, indicating an active role of the gamma-amino butyric acid shunt in these cultures. It can be concluded that the cerebellar neurons rely more on reuptake of glutamate than supply of glutamine from astrocytes for glutamate homeostasis.

Amino Acids↗

PubMed2006

Complex glutamate labeling from [U-13C]glucose or [U-13C]lactate in co-cultures of cerebellar neurons and astrocytes.

Glutamate metabolism was studied in co-cultures of mouse cerebellar neurons (predominantly glutamatergic) and astrocytes. One set of cultures was superfused (90 min) in the presence of either [U-(13)C]glucose (2.5 mM) and lactate (1 mM) or [U-(13)C]lactate (1 mM) and glucose (2.5 mM). Other sets of cultures were incubated in medium containing [U-(13)C]lactate (1 mM) and glucose (2.5 mM) for 4 h. Regardless of the experimental conditions cell extracts were analyzed using mass spectrometry and nuclear magnetic resonance spectroscopy. (13)C labeling of glutamate was much higher than that of glutamine under all experimental conditions indicating that acetyl-CoA from both lactate and glucose was preferentially metabolized in the neurons. Aspartate labeling was similar to that of glutamate, especially when [U-(13)C]glucose was the substrate. Labeling of glutamate, aspartate and glutamine was lower in the cells incubated with [U-(13)C]lactate. The first part of the pyruvate recycling pathway, pyruvate formation, was detected in singlet and doublet labeling of alanine under all experimental conditions. However, full recycling, detectable in singlet labeling of glutamate in the C-4 position was only quantifiable in the superfused cells both from [U-(13)C]glucose and [U-(13)C]lactate. Lactate and alanine were mostly uniformly labeled and labeling of alanine was the same regardless of the labeled substrate present and higher than that of lactate when superfused in the presence of [U-(13)C]glucose. These results show that metabolism of pyruvate, the precursor for lactate, alanine and acetyl-CoA is highly compartmentalized.

Alanine↗

PubMed2006

Neuronal-glial interactions in rats fed a ketogenic diet.

Glucose is the preferred energy substrate for the adult brain. However, during periods of fasting and consumption of a high fat, low carbohydrate (ketogenic) diet, ketone bodies become major brain fuels. The present study was conducted to investigate how the ketogenic diet influences neuronal-glial interactions in amino acid neurotransmitter metabolism. Rats were kept on a standard or ketogenic diet. After 21 days all animals received an injection of [1-(13)C]glucose plus [1,2-(13)C]acetate, the preferential substrates of neurons and astrocytes, respectively. Extracts from cerebral cortex and plasma were analyzed by (13)C and (1)H nuclear magnetic resonance spectroscopy and HPLC. Increased amounts of valine, leucine and isoleucine and a decreased amount of glutamate were found in the brains of rats receiving the ketogenic diet. Glycolysis was decreased in ketotic rats compared with controls, evidenced by the reduced amounts of [3-(13)C]alanine and [3-(13)C]lactate. Additionally, neuronal oxidative metabolism of [1-(13)C]glucose was decreased in ketotic rats compared with controls, since amounts of [4-(13)C]glutamate and [4-(13)C]glutamine were lower than those of controls. Although the amount of glutamate from [1-(13)C]glucose was decreased, this was not the case for GABA, indicating that relatively more [4-(13)C]glutamate is converted to GABA. Astrocytic metabolism was increased in response to ketosis, shown by increased amounts of [4,5-(13)C]glutamine, [4,5-(13)C]glutamate, [1,2-(13)C]GABA and [3,4-(13)C]-/[1,2-(13)C]aspartate derived from [1,2-(13)C]acetate. The pyruvate carboxylation over dehydrogenation ratio for glutamine was increased in the ketotic animals compared to controls, giving further indication of increased astrocytic metabolism. Interestingly, pyruvate recycling was higher in glutamine than in glutamate in both groups of animals. An increase in this pathway was detected in glutamate in response to ketosis. The decreased glycolysis and oxidative metabolism of glucose as well as the increased astrocytic metabolism, may reflect adaptation of the brain to ketone bodies as major source of fuel.

Acetic Acid↗

PubMed2006

Cortical glutamate metabolism is enhanced in a genetic model of absence epilepsy.

Disturbances in GABAergic and glutamatergic neurotransmission in the thalamocortical loop are involved in absence seizures. Here, we examined potential disturbances in metabolism and interactions between neurons and glia in 5-month-old genetic absence epilepsy rats from Strasbourg (GAERS) and nonepileptic rats (NER). Animals received [1-(13)C]glucose and [1,2-(13)C]acetate, the preferential substrates of neurons and astrocytes, respectively. Extracts from cerebral cortex, thalamus, and hippocampus were analyzed by (13)C nuclear magnetic resonance spectroscopy. Most changes were detected in the cortex. Pyruvate metabolism was enhanced as evidenced by increases of lactate, and labeled and unlabeled alanine. Neuronal mitochondrial metabolism was also enhanced as detected by elevated amounts of N-acetylaspartate and nicotinamide adenine dinucleotide as well as increased incorporation of label from [2-(13)C]acetyl CoA into glutamate, glutamine, and aspartate. Likewise, mitochondrial metabolism in astrocytes was increased. Changes in thalamus were restricted to increased concentration and labeling of glutamine. Changes in the hippocampus were similar to those in the cortex. This increase in glutamate-glutamine metabolism in cortical neurons and astrocytes accompanied by a decreased gamma aminobyturic acid level may lead to impaired thalamic filter function. Hence, reduced sensory input to cortex could allow the occurrence of spike-and-wave discharges in the thalamocortical loop. Increased glutamatergic output from the cortex to hippocampus may be the underlying cause of improved learning in GAERS.

Animals↗

PubMed2006

Repeated injection of MK801: an animal model of schizophrenia?

Glutamate-induced neurotoxicity plays an important role in neurological and psychiatric diseases. Thus, much attention has been given to the potential neuroprotective role of glutamate receptor antagonists, especially to those acting on the N-methyl-d-aspartate (NMDA) subtype. However, in addition to their neuroprotective potential, these compounds have also neurotoxic and psychotogenic properties. In the present study we used repeated injections of MK801 to examine if this non-competitive NMDA receptor antagonist could be used to produce schizophrenia-like alterations in behavior and brain metabolism in animals. Rats were given injections of MK801 (0.1 mg/kg) on six consecutive days, the last dose together with [1-(13)C]glucose and [1,2-(13)C]acetate, to probe neuronal and astrocytic metabolism, respectively. Analyses of extracts from parts of the frontal cortex plus cingulate and retrosplenial cortices and temporal lobes were performed using (13)C and (1)H magnetic resonance spectroscopy. Changes in glutamate and glutamine were restricted to the temporal lobe, in which amounts and labeling from [1-(13)C]glucose and [1,2-(13)C]acetate were increased compared to control. Locomotor activity was slightly higher in rats treated with MK801 compared to untreated animals. Metabolic changes did not resemble the alterations occurring in schizophrenia and those after repeated high dose (0.5 mg/kg) [Kondziella, D., Brenner, E., Eyjolfsson, E.M., Markinhuhta, K.R., Carlsson, M., Sonnewald, U., 2005. Glial-neuronal interactions are impaired in the schizophrenia model of repeated MK801 exposure. Neuropsychopharmacology, Epub ahead of print] but rather those caused by MK801 seen after a single high dose (0.5 mg/kg) [Brenner, E., Kondziella, D., Haberg, A., Sonnewald, U., 2005. Impaired glutamine metabolism in NMDA receptor hypofunction induced by MK801. J. Neurochem. 94, 1594-1603.].

Acetic Acid↗

PubMed2006

Demonstration of extensive GABA synthesis in the small population of GAD positive neurons in cerebellar cultures by the use of pharmacological tools.

Cultures of dissociated cerebella from 7-day-old mice were maintained in vitro for 1-13 days. GABA biosynthesis and degradation were studied during development in culture and pharmacological agents were used to identify the enzymes involved. The amount of GABA increased, whereas that of glutamate was unchanged during the first 5 days and both decreased thereafter. The presence of aminooxyacetic acid (AOAA, 10 microM) which inhibits transaminases and other pyridoxal phosphate dependent enzymes including GABA-transaminase (GABA-T), in the culture medium caused an increase in the intracellular amount of GABA and a decrease in glutamate. The GABA content was also increased following exposure to the specific GABA-T inhibitor gamma-vinyl GABA. From day 6 in culture (day 4 when cultured in the presence of AOAA) GABA levels in the medium were increased compared to that in medium from 1-day-old cultures. Synthesis of GABA during the first 3 days was demonstrated by the finding that incubation with either [1-(13)C]glucose or [U-(13)C]glutamine led to formation of labeled GABA. Synthesis of GABA after 1 week in culture, when the enzymatic machinery is considered to be at a more differentiated level, was shown by labeling from [U-(13)C]glutamine added on day 7. Altogether the findings show continuous GABA synthesis and degradation throughout the culture period in the cerebellar neurons. At 10 microM AOAA, GABA synthesis from [U-(13)C]glutamine was not affected, indicating that transaminases are not involved in GABA synthesis and thus excluding the putrescine pathway. At a concentration of 5 mM AOAA GABA labeling was, however, abolished, showing that glutamate decarboxylase, which is inhibited at this level of AOAA, is responsible for GABA synthesis in the cerebellar cultures. In conclusion, the present study shows that GABA synthesis is taking place via GAD in a subpopulation of the cerebellar neurons, throughout the culture period.

4-Aminobutyrate Transaminase↗

PubMed2006

Inhibitors of the alpha-ketoglutarate dehydrogenase complex alter [1-13C]glucose and [U-13C]glutamate metabolism in cerebellar granule neurons.

Diminished activity of the alpha-ketoglutarate dehydrogenase complex (KGDHC), an important component of the tricarboxylic acid (TCA) cycle, occurs in several neurological diseases. The effect of specific KGDHC inhibitors [phosphonoethyl ester of succinyl phosphonate (PESP) and the carboxy ethyl ester of succinyl phosphonate (CESP)] on [1-13C]glucose and [U-13C]glutamate metabolism in intact cerebellar granule neurons was investigated. Both inhibitors decreased formation of [4-13C]glutamate from [1-13C]glucose, a reduction in label in glutamate derived from [1-13C]glucose/[U-13C]glutamate through a second turn of the TCA cycle and a decline in the amounts of gamma-aminobutyric acid (GABA), aspartate, and alanine. PESP decreased formation of [U-13C]aspartate and total glutathione, whereas CESP decreased concentrations of valine and leucine. The findings are consistent with decreased KGDHC activity; increased alpha-ketoglutarate formation; increased transamination of alpha-ketoglutarate with valine, leucine, and GABA; and new equilibrium position of the aspartate aminotransferase reaction. Overall, the findings also suggest that some carbon derived from alpha-ketoglutarate may bypass the block in the TCA cycle at KGDHC by means of the GABA shunt and/or conversion of valine to succinate. The results suggest the potential of succinyl phosphonate esters for modeling the biochemical and pathophysiological consequences of reduced KGDHC activity in brain diseases.

Animals↗

PubMed2006

Glucose is necessary to maintain neurotransmitter homeostasis during synaptic activity in cultured glutamatergic neurons.

Glucose is the primary energy substrate for the adult mammalian brain. However, lactate produced within the brain might be able to serve this purpose in neurons. In the present study, the relative significance of glucose and lactate as substrates to maintain neurotransmitter homeostasis was investigated. Cultured cerebellar (primarily glutamatergic) neurons were superfused in medium containing [U-13C]glucose (2.5 mmol/L) and lactate (1 or 5 mmol/L) or glucose (2.5 mmol/L) and [U-13C]lactate (1 mmol/L), and exposed to pulses of N-methyl-D-aspartate (300 micromol/L), leading to synaptic activity including vesicular release. The incorporation of 13C label into intracellular lactate, alanine, succinate, glutamate, and aspartate was determined by mass spectrometry. The metabolism of [U-13C]lactate under non-depolarizing conditions was high compared with that of [U-13C]glucose; however, it decreased significantly during induced depolarization. In contrast, at both concentrations of extracellular lactate, the metabolism of [U-13C]glucose was increased during neuronal depolarization. The role of glucose and lactate as energy substrates during vesicular release as well as transporter-mediated influx and efflux of glutamate was examined using preloaded D-[3H]aspartate as a glutamate tracer and DL-threo-beta-benzyloxyaspartate to inhibit glutamate transporters. The results suggest that glucose is essential to prevent depolarization-induced reversal of the transporter (efflux), whereas vesicular release was unaffected by the choice of substrate. In conclusion, the present study shows that glucose is a necessary substrate to maintain neurotransmitter homeostasis during synaptic activity and that synaptic activity does not induce an upregulation of lactate metabolism in glutamatergic neurons.

Animals↗

PubMed2006

Mercuric chloride inhibits the in vitro uptake of glutamate in GLAST- and GLT-1-transfected mutant CHO-K1 cells.

Glutamate is removed mainly by astrocytes from the extracellular fluid via high-affinity astroglial Na+ -dependent excitatory amino acid transporters, glutamate/aspartate transporter (GLAST), and glutamate transporter-1 (GLT-1). Mercuric chloride (HgCl2) is a highly toxic compound that inhibits glutamate uptake in astrocytes, resulting in excessive extracellular glutamate accumulation, leading to excitotoxicity and neuronal cell death. The mechanisms associated with the inhibitory effects of HgCl2 on glutamate uptake are unknown. This study examines the effects of HgCl2 on the transport of 3H-D-aspartate, a nonmetabolizable glutamate analog, using Chinese hamster ovary cells (CHO) transfected with two glutamate transporter subtypes, GLAST (EAAT1) and GLT-1 (EAAT2), as a model system. Additionally, studies were undertaken to determine the effects of HgCl2 on mRNA and protein levels of these transporters. The results indicate that (1) HgCl2 leads to significant (p < 0.001) inhibition of glutamate uptake via both transporters, but is a more potent inhibitor of glutamate transport via GLAST and (2) the effect of HgCl2 on inhibition of glutamate uptake in transfected CHO cells is not associated with changes in transporter protein levels despite a significant decrease in mRNA expression; thus, (3) HgCl2 inhibition is most likely related to its direct binding to the functional thiol groups of the transporters and interference with their uptake function.

Animals↗

PubMed2005

Neuronal and astrocytic shuttle mechanisms for cytosolic-mitochondrial transfer of reducing equivalents: current evidence and pharmacological tools.

The malate-aspartate shuttle and the glycerol phosphate shuttle act to transfer reducing equivalents from NADH in the cytosol to the mitochondria since the inner mitochondrial membrane is impermeable to NADH and NAD+. This transfer of reducing equivalents is essential for maintaining a favorable NAD+/NADH ratio required for the oxidative metabolism of glucose and synthesis of neurotransmitters in brain. There is evidence that both the malate-aspartate shuttle and glycerol phosphate shuttle function in brain; however, there is controversy about the relative importance and cellular localization of these shuttles. The malate-aspartate shuttle is considered the most important shuttle in brain. It is particularly important in neurons and may be extremely low, or even non-existent in brain astrocytes. Several studies provide evidence of glycerol phosphate shuttle activity in brain cells; however, the activity of this shuttle in brain has been questioned. A number of pharmacological tools, including aminooxyacetic acid, beta-methyleneaspartate, phenylsuccinate, and 3-nitropropionic acid, have been used to inhibit the four enzymes and two carrier proteins that participate in the malate-aspartate shuttle. Although no drugs completely inhibit the glycerol phosphate shuttle, evidence for the existence of this shuttle is provided by studies using drugs to inhibit the malate-aspartate shuttle. This report evaluates the evidence for each shuttle in brain cells and the drugs that can be used as pharmacological tools to study these shuttles.

Animals↗

PubMed2005

Glial-neuronal interactions are impaired in the schizophrenia model of repeated MK801 exposure.

Schizophrenia-mimicking compounds such as phencyclidine (PCP) and MK801 are antagonists at the N-methyl-D-aspartate (NMDA) receptor and produce the whole spectrum of positive, negative, and cognitive symptoms. This is one of the most important pillars of the hypoglutamatergic hypothesis of schizophrenia. Since the synthesis of glutamate and GABA in neurons is closely connected to astrocyte metabolism, the study of astrocytic function is essential in this context. Dizocilpine-maleate (MK801) (0.5 mg/kg) was injected into rats every day for 6 days. The last dose was given together with [1-(13)C]glucose and [1,2-(13)C]acetate. Extracts from frontal, retrosplenial, and cingulate cortices (CRFC) and temporal lobes were examined by (13)C nuclear magnetic resonance spectroscopy, high pressure liquid chromatography, and light microscopy. In CRFC, significant increases in the levels of glutamate, glutathione, and taurine were seen, whereas amounts and turnover of noradrenaline, dopamine, and serotonin were unchanged. Glutamate and glutamine, derived from [1,2-(13)C]acetate and thus astrocytes, were significantly decreased in CRFC as compared to controls. Labeling from [1-(13)C]glucose and thus mostly neuronal metabolism was affected in the same brain region with decreased labeling of glutamate and GABA. The present model mimics the increased glutamate/glutamine activity found in drug-naive patients with first episode schizophrenia. Moreover, the decreased labeling indicates the transition to lower glutamatergic function seen in chronic schizophrenia patients. The disturbance in astrocytic function and the glutamine-glutamate-GABA cycle are of significant importance and might add to the malfunction of the cortico-striato-thalamo-cortical loop caused by NDMA receptor blockade.

Acetates↗

PubMed2005

Impaired glutamine metabolism in NMDA receptor hypofunction induced by MK801.

Paradoxically, glutamate receptor antagonists have neurotoxic and psychotogenic properties in addition to their neuroprotective potential during excessive glutamate release. In the present study the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist MK801 was used to examine glial-neuronal interactions in NMDA receptor hypofunction. Rats were given a subanesthetic dose of MK801 together with [1-13C]glucose and [1,2-13C]acetate, and brains were removed 20 min later. Analyses of extracts from cingulate, retrosplenial plus middle frontal cortices (CRFC) and temporal lobe were performed using HPLC and 13C and 1H nuclear magnetic resonance spectroscopy. Hypofunction of the NMDA receptor induced similar changes in both brain areas investigated; however, the changes were most pronounced in the temporal lobe. Generally, only labeling from [1-13C]glucose was affected by MK801. In CRFC and temporal lobe amounts of both labeled and unlabeled glutamine were increased, whereas those of aspartate were decreased. In the CRFC the decrease in labeling of aspartate was greater than the decrease in concentration, leading to decreased 13C enrichment. In temporal lobe, not in CRFC, increased concentrations of glutamate, GABA, succinate, glutathione and inositol were detected together with increased labeling of GABA and succinate from [1-13C]glucose. 13C Enrichment was decreased in glutamate and increased in succinate. The results point towards a disturbance in glutamate-glutamine cycling and thus interaction between neurons and glia, since labeling of glutamate and glutamine from glucose was affected differently.

Animals↗

PubMed2005

Effects of methylmercury on primary brain cells in mono- and co-culture.

We report on the uptake of MeHg in astrocytes and neurons, as well as specific indicators of neurotoxicity. Cerebellar granule neurons and astrocytes separately and in co-culture were cultured in the presence of MeHg and changes in 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyltetrazolium bromide (MTT)-reduction, lactate dehydrogenase (LDH) leakage, and cellular content of glutathione and amino acids were used as indicators of MeHg toxicity. Mitochondria in cortical astrocytes were slightly more sensitive than those in cerebellar astrocytes to the toxic effects of MeHg; furthermore, cellular integrity was better preserved in cerebellar astrocytes. When neurons and astrocytes from cerebellum were incubated in separable co-cultures using inserts, the astrocytes showed cellular damage at lower exposure to MeHg while neurons showed less changes compared to respective cell types in mono-cultures. Mercury uptake studies at 25 microM MeHg (10% serum present) showed that for neurons in co-culture the uptake was 1/3 compared to mono-cultures. In contrast, for astrocytes in co-culture, uptake was increased by 75%. A MeHg concentration-dependent increase of glutamate content in mono-cultures was noted. When MeHg concentration was increased to 10, 25, or 50 microM, neurons in co-cultures decreased their glutamate content, whereas astrocytes showed an increase. Other amino acids, such as glutamine, serine, valine, isoleucine, taurine, and phenylalanine were unaffected by MeHg. Glutathione content showed MeHg concentration-dependent changes in astrocytes and was increased in neurons in co-culture incubated with 5 microM MeHg. In conclusion, astrocytes appear to increase neuronal resistance, indicating a possible protective role for astrocytes in MeHg neurotoxicity.

Animals↗

PubMed2005

Role of glutamine and neuronal glutamate uptake in glutamate homeostasis and synthesis during vesicular release in cultured glutamatergic neurons.

Glutamate exists in a vesicular as well as a cytoplasmic pool and is metabolically closely related to the tricarboxylic acid (TCA) cycle. Glutamate released during neuronal activity is most likely to a large extent accumulated by astrocytes surrounding the synapse. A compensatory flux from astrocytes to neurons of suitable precursors is obligatory as neurons are incapable of performing a net synthesis of glutamate from glucose. Glutamine appears to play a major role in this context. Employing cultured cerebellar granule cells, as a model system for glutamatergic neurons, details of the biosynthetic machinery have been investigated during depolarizing conditions inducing vesicular release. [U-13C]Glucose and [U-13C]glutamine were used as labeled precursors for monitoring metabolic pathways by nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) technologies. To characterize release mechanisms and influence of glutamate transporters on maintenance of homeostasis in the glutamatergic synapse, a quantification was performed by HPLC analysis of the amounts of glutamate and aspartate released in response to depolarization by potassium (55 mM) in the absence and presence of DL-threo-beta-benzyloxyaspartate (TBOA) and in response to L-trans-pyrrolidine-2,4-dicarboxylate (t-2,4-PDC), a substrate for the glutamate transporter. Based on labeling patterns of glutamate the biosynthesis of the intracellular pool of glutamate from glutamine was found to involve the TCA cycle to a considerable extent (approximately 50%). Due to the mitochondrial localization of PAG this is unlikely only to reflect amino acid exchange via the cytosolic aspartate aminotransferase reaction. The involvement of the TCA cycle was significantly lower in the synthesis of the released vesicular pool of glutamate. However, in the presence of TBOA, inhibiting glutamate uptake, the difference between the intracellular and the vesicular pool with regard to the extent of involvement of the TCA cycle in glutamate synthesis from glutamine was eliminated. Surprisingly, the intracellular pool of glutamate was decreased after repetitive release from the vesicular pool in the presence of TBOA indicating that neuronal reuptake of released glutamate is involved in the maintenance of the neurotransmitter pool and that 0.5 mM glutamine exogenously supplied is inadequate to sustain this pool.

Amino Acid Transport System X-AG↗

PubMed2005

Metabolism is normal in astrocytes in chronically epileptic rats: a (13)C NMR study of neuronal-glial interactions in a model of temporal lobe epilepsy.

The aim of the present work was to study potential disturbances in metabolism and interactions between neurons and glia in the lithium-pilocarpine model of temporal lobe epilepsy. Rats chronically epileptic for 1 month received [1-(13)C]glucose, a substrate for neurons and astrocytes, and [1,2-(13)C]acetate, a substrate for astrocytes only. Analyses of extracts from cerebral cortex, cerebellum, and hippocampal formation (hippocampus, amygdala, entorhinal, and piriform cortices) were performed using (13)C and (1)H nuclear magnetic resonance spectroscopy and HPLC. In the hippocampal formation of epileptic rats, levels of glutamate, aspartate, N-acetyl aspartate, adenosine triphosphate plus adenosine diphosphate and glutathione were decreased. In all regions studied, labeling from [1,2-(13)C]acetate was similar in control and epileptic rats, indicating normal astrocytic metabolism. However, labeling of glutamate, GABA, aspartate, and alanine from [1-(13)C]glucose was decreased in all areas possibly reflecting neuronal loss. The labeling of glutamine from [1-(13)C]glucose was decreased in cerebral cortex and cerebellum and unchanged in hippocampal formation. In conclusion, no changes were detected in glial-neuronal interactions in the hippocampal formation while in cortex and cerebellum the flow of glutamate to astrocytes was decreased, indicating a disturbed glutamate-glutamine cycle. This is, to our knowledge, the first study showing that metabolic disturbances are confined to neurons inside the epileptic circuit.

Acetates↗