Orla M. Larsson

Trafficking between glia and neurons of TCA cycle intermediates and related metabolites

Net synthesis of the neurotransmitter amino acids glutamate and GABA can take place either from glutamine or from α‐ketoglutarate or another tricarboxylic acid (TCA) cycle intermediate plus an amino acid as donor of the amino group. Since neurons lack the enzymes glutamine synthetase and pyruvate carboxylase that are expressed only in astrocytes, trafficking of these metabolites must take place between neurons and astrocytes. Moreover, it is likely that astrocytes play an important role in maintaining the energy status in neurons supplying energy substrates, e.g., in the form of lactate. The role of trafficking of glutamine, TCA cycle constituents as well as the role of lactate as an energy source in neurons is discussed. Using [U‐13C]lactate and NMR spectroscopy, it is shown that lactate that can be produced in astrocytes can be taken up into neurons and metabolized through the TCA‐cycle leading to labeling of TCA cycle intermediates plus amino acids derived from these. The labeling pattern of glutamate and GABA indicates that C atoms from lactate remain in the cycle for several turns and that GABA formation may involve more than one glutamate pool, i.e., that compartmentation may exist. Additionally, a possible role of citrate as a chelator of Zn++ with regard to neuronal excitation is discussed. Astrocytes produce large quantities of citrate which by chelation of Zn++ alters the excitable state of neurons via regulation of N‐methyl‐D‐aspartate receptor activity. Thus, astrocytes may regulate neuronal activity at a number of different levels. GLIA 21:99–105, 1997.

Characterization of uptake and release processes ford- andl-aspartate in primary cultures of astrocytes and cerebellar granule cells

The uptake ofl-andd-aspartate was studied in astrocytes cultured from prefrontal cortex and in granule cells cultured from cerebellum. A high affinity uptake system forl- andd-aspartate was found in both cell types, and the two stereoisomers exhibited essentially the sameKm- andVmax-values in bouth astrocytes (l-aspartate:Km 77 μM;Vmax 11.8 nmol×min−1×mg−1;d-aspartate:Km 83 μM;Vmax 14.0 nmol×min−1×mg−1) and granule cells (l-aspartate:Km 32 μM;Vmax 2.8 nmol ×min−1×mg−1;d-aspartate:Km 26 μM;Vmax 3.0 nmol×min−1×mg−1). To investigate whetherl-glutamate,l-aspartate andd-aspartate use the same uptake system a detailed kenetic analysis was performed. The uptake kinetics of each one of the three amino acids was studied in the presence of the two other amino acids, and no essential differences between the uptake characteristics of the amino acids were found. In addition to the uptake studies the release ofD-aspartate from cerebellar granule cells was investigated and compared withl-glutamate release. A Ca2+-dependent, K+-induced release was found for both amino acids.

A possible role of alanine for ammonia transfer between astrocytes and glutamatergic neurons

The metabolism of [U‐13C]lactate (1 mM) in the presence of unlabeled glucose (2.5 mM) was investigated in glutamatergic cerebellar granule cells, cerebellar astrocytes, and corresponding co‐cultures. It was evident that lactate is primarily a neuronal substrate and that lactate produced glycolytically from glucose in astrocytes serves as a substrate in neurons. Alanine was highly enriched with 13C in the neurons, whereas this was not the case in the astrocytes. Moreover, the cellular content and the amount of alanine released into the medium were higher in neurons than astrocytes. On incubation of the different cell types in medium containing alanine (1 mM), the astrocytes exhibited the highest level of accumulation. Altogether, these results indicate a preferential synthesis and release of alanine in glutamatergic neurons and uptake in cerebellar astrocytes. A new functional role of alanine may be suggested as a carrier of nitrogen from glutamatergic neurons to astrocytes, a transport that may operate to provide ammonia for glutamine synthesis in astrocytes and dispose of ammonia generated by the glutaminase reaction in glutamatergic neurons. Hence, a model of a glutamate‐glutamine/lactate‐alanine shuttle is presented. To elucidate if this hypothesis is compatible with the pattern of alanine metabolism observed in the astrocytes and neurons from cerebellum, the cells were incubated in a medium containing [15N]alanine (1 mM) and [5‐15N]glutamine (0.5 mM), respectively. Additionally, neurons were incubated with [U‐13C]glutamine to estimate the magnitude of glutamine conversion to glutamate. Alanine was labeled from [5‐15N]glutamine to 3.3% and [U‐13C]glutamate generated from [U‐13C]glutamine was labeled to 16%. In spite of the modest labeling in alanine, it is clear that nitrogen from ammonia is transferred to alanine via transamination with glutamate formed by reductive amination of α‐ketoglutarate. With regard to the astrocytic part of the shuttle, glutamine was labeled to 22% in one nitrogen atom whereas 3.2% was labeled in two when astrocytes were incubated in [15N]alanine. Moreover, in co‐cultures, [U‐13C]alanine labeled glutamate and glutamine equally, whereas [U‐13C]lactate preferentially labeled glutamate. Altogether, these results support the role proposed above of alanine as a possible ammonia nitrogen carrier between glutamatergic neurons and surrounding astrocytes and they show that lactate is preferentially metabolized in neurons and alanine in astrocytes.

Comparison of Lactate and Glucose Metabolism in Cultured Neocortical Neurons and Astrocytes Using 13C-NMR Spectroscopy

In cerebral cortical neurons, synthesis of the tricarboxylic acid (TCA) cycle-derived amino acids, glutamate and aspartate as well as the neurotransmitter of these neurons, γ-aminobutyrate (GABA), was studied incubating the cells in media containing 0.5 mM [U-13C]glucose in the absence or presence of glutamine (0.5 mM). Lyophilized cell extracts were analyzed by 13C nuclear magnetic resonance (NMR) spectroscopy and HPLC. The present findings were compared to results previously obtained using 1.0 mM [U-13C]lactate as the labeled substrate for the neurons. Regardless of the amino acids studied, incubation periods of 1 and 4 h resulted in identical amounts of 13C incorporated. Furthermore, the metabolism of lactate was studied under analogous conditions in cultured cerebral cortical astrocytes. The incorporation of 13C from lactate into glutamate was much lower in the astrocytes than in the neurons. In cerebral cortical neurons the total amount of 13C in GABA, glutamate and aspartate was independent of the labeled substrate. The enrichment in glutamate and aspartate was, however, higher in neurons incubated with lactate. Thus, lactate appears to be equivalent to glucose with regard to its access to the TCA cycle and subsequent labeling of glutamate, aspartate and GABA. It should be noted, however, that incubation with lactate in place of glucose led to lower cellular contents of glutamate and aspartate. The presence of glutamine affected the metabolism of glucose and lactate differently, suggesting that the metabolism of these substrates may be compartmentalized.

Role of astrocytic transport processes in glutamatergic and GABAergic neurotransmission.

The fine tuning of both glutamatergic and GABAergic neurotransmission is to a large extent dependent upon optimal function of astrocytic transport processes. Thus, glutamate transport in astrocytes is mandatory to maintain extrasynaptic glutamate levels sufficiently low to prevent excitotoxic neuronal damage. In GABA synapses hyperactivity of astroglial GABA uptake may lead to diminished GABAergic inhibitory activity resulting in seizures. As a consequence of this the expression and functional activity of astrocytic glutamate and GABA transport is regulated in a number of ways at transcriptional, translational and post-translational levels. This opens for a number of therapeutic strategies by which the efficacy of excitatory and inhibitory neurotransmission may be manipulated.

Acetoacetate and Glucose as Lipid Precursors and Energy Substrates in Primary Cultures of Astrocytes and Neurons from Mouse Cerebral Cortex

Abstract: Primary cultures of astrocytes and neurons derived from neonatal and embryonic mouse cerebral cortex, respectively, were incubated with [3‐14C]acetoacetate or [2‐14C]glucose. The utilization of glucose and acetoacetate, the production of lactate, d‐3‐hydroxybu‐tyrate, and 14CO2 and the incorporation of 14C and of 3H from 3H2O into lipids and lipid fractions were measured. Both cell types used acetoacetate as an energy substrate and as a lipid precursor; lactate was the major product of glucose metabolism. About 60% of the acetoacetate that was utilized by neurons was oxidized to CO2, whereas this was only ∼20% in the case of cultured astrocytes. This indicates that the rate at which 14C‐labeled Krebs cycle intermediates exchange with pools of unlabeled intermediates is much higher in astrocytes than in neurons. Acetoacetate is a better precursor for the synthesis of fatty acids and cholesterol than glucose, presumably because it can be used directly in the cytosol for these processes; preferential incorporation into cholesterol was not observed in these in vitro systems. We conclude that ketone bodies can be metabolized both by the glial cells and by the neuronal cells of developing mouse brain.

Ontogenetic development of glutamate and gaba metabolizing enzymes in cultured cerebral cortex interneurons and in cerebral cortex in vivo

The development of the enzymes phosphate activated glutaminase (PAG), glutamate dehydrogenase (GLDH), glutamic‐oxaloacetic‐transaminase (GOT), glutamine synthetase (GS), GABA‐transaminase (GABA‐T) and ornithine‐δ‐aminotransferase (Orn‐T) was followed in mouse cerebral cortex in vivo and in cultured mouse cerebral cortex interneurons. It was found that GLDH, GOT and Orn‐T exhibited an enhanced developmental pattern in the cultured neurons compared to cerebral cortex. The activities of PAG and GABA‐T developed in parallel in vivo and in culture but the activity of GS remained low in the cultured neurons compared to the increasing activity of this enzyme found in vivo. Compared to cerebral cortex the cultured neurons exhibited higher activities of PAG, GLDH and Orn‐T, whereas the activities of GABA‐T and GOT were lower in the cultured cells. The activity of GS in the cultured neurons was only 5–10% of the activity in cerebral cortex in vivo. It is concluded that neurons from cerebral cortex represent a reliable model system by which the metabolism and function of GABAergic neurons can be conveniently studied in a physiologically meaningful way.

Utilization of Glutamine and of TCA Cycle Constituents as Precursors for Transmitter Glutamate and GABA

In the present review evidence is presented that (1) glutamine synthesis in astrocytes is essential for synthesis of GABA in neurons; (2) alpha-ketoglutarate in the presence of alanine (as an amino group donor) can replace glutamine as a precursor for synthesis of transmitter glutamate, but maybe not as a precursor for transmitter GABA; (3) differences exist in the intraneuronal metabolic pathways for utilization of alpha-ketoglutarate plus alanine and of glutamine, and (4) alanine also functions as a substrate for oxidative metabolism in glutamatergic neurons. It should be emphasized that the supply of precursors for transmitter glutamate and GABA in glutamatergic and GABAergic neurons depends on metabolic processes in astrocytes regardless whether glutamine or alpha-ketoglutarate plus L-alanine function as the transmitter precursors. The key reason that an interaction with astrocytes is essential is that both pyruvate carboxylase, the major enzyme in the brain for net synthesis of tricarboxylic acid cycle intermediates, and glutamine synthetase, the enzyme forming glutamine from glutamate, are specifically located in astrocytes, but not in neurons.

Ontogenetic development of glutamate metabolizing enzymes in cultured cerebellar granule cells and in cerebellumin vivo

The ontogenetic development of the enzymes phosphate activated glutaminase (PAG), glutamate dehydrogenase (GLDH), glutamic-oxaloacetic-transaminase (GOT), glutamine synthetase (GS), and ornithine-δ-aminotransferase (Orn-T) was followed in cerebellum in vivo and in cultured cerebellar granule cells. It was found that PAG, GLDH, and GOT exhibited similar developmental patterns in the cultured neurons compared to cerebellum. PAG showed, however, a more pronounced phosphate activation in the cultured granule cells compared to in vivo. The activity of GS remained low in the cultured neurons compared to the increasing activity of this enzyme found in vivo. On the other hand Orn-T exhibited an increase in its specific activity in the cultured cells as a function of time in culture in contrast to the non-changing activity of this enzyme in vivo. Compared to cerebellum the cultured neurons exhibited higher activities of GLDH, GOT, and Orn-T whereas the activity of PAG was only slightly higher in the cultured cells. The activity of GS in the cultured neurons was only 5–10% of the activity in cerebellum in vivo. It is concluded that cultured cerebellar granule cells represent a reliable model system by which the metabolism and function of glutamatergic neurons can be conveniently studied in a physiologically meaningful way.

GABA transporters and GABA-transaminase as drug targets.

The fine-tuning and homeostatic balance of the GABAergic inhibitory tone in the central nervous system (CNS) is a prerequisite for controlling the excitatory neurotransmission. This principal mechanism for controlling excitation is inhibition which has been the topic of intensive research covering all known functional entities of the GABAergic synapse. The therapeutical scope for targeting the GABA system covers a large number of neurological and psychiatric disorders. This review focuses on the major inactivation systems for GABAergic neurotransmission, the GABA transporters (GATs) and the GABA catabolic enzyme GABA -transaminase (GABA-T) as drug targets. Tiagabin and Vigabatrin, two anti-epileptic drugs on the market today, specifically inhibit GABA transport and metabolism, respectively. However, previous and recent evidence has clearly demonstrated the importance and differential functional roles of glial and neuronal GABA uptake and the metabolic fate of the sequestered neurotransmitter GABA in these cells. Moreover, the diverse expression patterns of the GABA transporters, in combination with development of GAT inhibitors with novel pharmacological profiles may initiate a renaissance for these inactivation systems as drugs targets. In particular, further research to elucidate the specialized physiological function of the GATs combined with their differential spatial expression could be of fundamental importance for the understanding of concerted action with regard to the fine-tuning of the GABAergic inhibitory tone. As such, selective targeting and modulation of GABA transporter subtypes and cell-specific GABA uptake and metabolism is of therapeutical interest in GABA-related CNS disorders, including epilepsy.