Deficient brain GABA metabolism leads to widespread impairments of astrocyte and oligodendrocyte function.

The neurometabolic disorder succinic semialdehyde dehydrogenase (SSADH) deficiency leads to great neurochemical imbalances and severe neurological manifestations. The cause of the disease is loss of function of the enzyme SSADH, leading to impaired metabolism of the principal inhibitory neurotransmitter GABA. Despite the known identity of the enzymatic deficit, the underlying pathology of SSADH deficiency remains unclear. To uncover new mechanisms of the disease, we performed an untargeted integrative analysis of cerebral protein expression, functional metabolism, and lipid composition in a genetic mouse model of SSADH deficiency (ALDH5A1 knockout mice). Our proteomic analysis revealed a clear regional vulnerability, as protein alterations primarily manifested in the hippocampus and cerebral cortex of the ALDH5A1 knockout mice. These regions displayed aberrant expression of proteins linked to amino acid homeostasis, mitochondria, glial function, and myelination. Stable isotope tracing in acutely isolated brain slices demonstrated an overall maintained oxidative metabolism of glucose, but a selective decrease in astrocyte metabolic activity in the cerebral cortex of ALDH5A1 knockout mice. In contrast, an elevated capacity of oxidative glutamine metabolism was observed in the ALDH5A1 knockout brain, which may serve as a neuronal compensation of impaired astrocyte glutamine provision. In addition to reduced expression of critical oligodendrocyte proteins, a severe depletion of myelin-enriched sphingolipids was found in the brains of ALDH5A1 knockout mice, suggesting degeneration of myelin. Altogether, our study highlights that impaired astrocyte and oligodendrocyte function is intimately linked to SSADH deficiency pathology, suggesting that selective targeting of glial cells may hold therapeutic potential in this disease.

Preface: Special issue: 14th International Conference on Brain Energy Metabolism: Energy substrates and microbiome govern brain bioenergetics and cognitive function with aging.

This Preface introduces the Special Issue entitled, "Energy Substrates and Microbiome Govern Brain Bioenergetics and Cognitive Function with Aging", which is comprised of manuscripts contributed by invited speakers and program/organizing committee members who participated in the 14th International Conference on Brain Energy Metabolism (ICBEM) held on October 24-27, 2022 in Santa Fe, New Mexico, USA. The conference covered the latest developments in research related to neuronal energetics, emerging roles for glycogen in higher brain functions, the impact of dietary intervention on aging, memory, and Alzheimer's disease, roles of the microbiome in gut-brain signaling, astrocyte-neuron interactions related to cognition and memory, novel roles for mitochondria and their metabolites, and metabolic neuroimaging in aging and neurodegeneration. The special issue contains 25 manuscripts on these topics plus three tributes to outstanding scientists who have made important contributions to brain energy metabolism and participated in numerous ICBEM conferences. In addition, two of the manuscripts describe important directions and the rationale for future research in many thematic areas covered by the conference.

Milestone Review: Metabolic dynamics of glutamate and GABA mediated neurotransmission - The essential roles of astrocytes.

Since it was first generally accepted that the two amino acids glutamate and GABA act as principal neurotransmitters, several landmark discoveries relating to this function have been uncovered. Synaptic homeostasis of these two transmitters involves several cell types working in close collaboration and is facilitated by specialized cellular processes. Notably, glutamate and GABA are extensively recycled between neurons and astrocytes in a process known as the glutamate/GABA-glutamine cycle, which is essential to maintain synaptic transmission. The glutamate/GABA-glutamine cycle is intimately coupled to cellular energy metabolism and relies on the metabolic function of both neurons and astrocytes. Importantly, astrocytes display unique metabolic features allowing extensive metabolite release, hereby providing metabolic support for neurons. Furthermore, astrocytes undergo complex metabolic adaptations in response to injury and pathology, which may greatly affect the glutamate/GABA-glutamine cycle and synaptic transmission during disease. In this Milestone Review we outline major discoveries in relation to synaptic balancing of glutamate and GABA signaling, including cellular uptake, metabolism, and recycling. We provide a special focus on how astrocyte function and metabolism contribute to sustain neuronal transmission through metabolite transfer. Recent advances on cellular glutamate and GABA homeostasis are reviewed in the context of brain pathology, including glutamate toxicity and neurodegeneration. Finally, we consider how pathological astrocyte metabolism may serve as a potential target of metabolic intervention. Integrating the multitude of fine-tuned cellular processes supporting neurotransmitter recycling, will aid the next generation of major discoveries on brain glutamate and GABA homeostasis.

A tribute to Leif Hertz: The historical context of his pioneering studies of the roles of astrocytes in brain energy metabolism, neurotransmission, cognitive functions, and pharmacology identifies important, unresolved topics for future studies.

Leif Hertz, M.D., D.Sc. (honoris causa) (1930-2018), was one of the original and noteworthy participants in the International Conference on Brain Energy Metabolism (ICBEM) series since its inception in 1993. The biennial ICBEM conferences are organized by neuroscientists interested in energetics and metabolism underlying neural functions; they have had a high impact on conceptual and experimental advances in these fields and on promoting collaborative interactions among neuroscientists. Leif made major contributions to ICBEM discussions and understanding of metabolic and signaling characteristics of astrocytes and their roles in brain function. His studies ranged from uptake of K+ from extracellular fluid and its stimulation of astrocytic respiration, identification, and regulation of enzymes specifically or preferentially expressed in astrocytes in the glutamate-glutamine cycle of excitatory neurotransmission, a requirement for astrocytic glycogenolysis for fueling K+ uptake, involvement of glycogen in memory consolidation in the chick, and pharmacology of astrocytes. This tribute to Leif Hertz highlights his major discoveries, the high impact of his work on astrocyte-neuron interactions, and his unparalleled influence on understanding the cellular basis of brain energy metabolism. His work over six decades has helped integrate the roles of astrocytes into neurotransmission where oxidative and glycogenolytic metabolism during neurotransmitter glutamate turnover are key aspects of astrocytic energetics. Leif recognized that brain astrocytic metabolism is greatly underestimated unless the volume fraction of astrocytes is taken into account. Adjustment for pathway rates expressed per gram tissue for volume fraction indicates that astrocytes have much higher oxidative rates than neurons and astrocytic glycogen concentrations and glycogenolytic rates during sensory stimulation in vivo are similar to those in resting and exercising muscle, respectively. These novel insights are typical of Leif's astute contributions to the energy metabolism field, and his publications have identified unresolved topics that provide the neuroscience community with challenges and opportunities for future research.

Deletion of CaMKIIα disrupts glucose metabolism, glutamate uptake, and synaptic energetics in the cerebral cortex.

Ca2+ /calmodulin-dependent protein kinase II alpha (CaMKIIα) is a key regulator of neuronal signaling and synaptic plasticity. Synaptic activity and neurotransmitter homeostasis are closely coupled to the energy metabolism of both neurons and astrocytes. However, whether CaMKIIα function is implicated in brain energy and neurotransmitter metabolism remains unclear. Here, we explored the metabolic consequences of CaMKIIα deletion in the cerebral cortex using a genetic CaMKIIα knockout (KO) mouse. Energy and neurotransmitter metabolism was functionally investigated in acutely isolated cerebral cortical slices using stable 13 C isotope tracing, whereas the metabolic function of synaptosomes was assessed by the rates of glycolytic activity and mitochondrial respiration. The oxidative metabolism of [U-13 C]glucose was extensively reduced in cerebral cortical slices of the CaMKIIα KO mice. In contrast, metabolism of [1,2-13 C]acetate, primarily reflecting astrocyte metabolism, was unaffected. Cellular uptake, and subsequent metabolism, of [U-13 C]glutamate was decreased in cerebral cortical slices of CaMKIIα KO mice, whereas uptake and metabolism of [U-13 C]GABA were unaffected, suggesting selective metabolic impairments of the excitatory system. Synaptic metabolic function was maintained during resting conditions in isolated synaptosomes from CaMKIIα KO mice, but both the glycolytic and mitochondrial capacities became insufficient when the synaptosomes were metabolically challenged. Collectively, this study shows that global deletion of CaMKIIα significantly impairs cellular energy and neurotransmitter metabolism, particularly of neurons, suggesting a metabolic role of CaMKIIα signaling in the brain.

The history of Danish neuroscience.

The history of Danish neuroscience starts with an account of impressive contributions made at the 17th century. Thomas Bartholin was the first Danish neuroscientist, and his disciple Nicolaus Steno became internationally one of the most prominent neuroscientists in this period. From the start, Danish neuroscience was linked to clinical disciplines. This continued in the 19th and first half of the 20th centuries with new initiatives linking basic neuroscience to clinical neurology and psychiatry in the same scientific environment. Subsequently, from the middle of the 20th century, basic neuroscience was developing rapidly within the preclinical university sector. Clinical neuroscience continued and was even reinforced during this period with important translational research and a close co-operation between basic and clinical neuroscience. To distinguish 'history' from 'present time' is not easy, as many historical events continue in present time. Therefore, we decided to consider 'History' as new major scientific developments in Denmark, which were launched before the end of the 20th century. With this aim, scientists mentioned will have been born, with a few exceptions, no later than the early 1960s. However, we often refer to more recent publications in documenting the developments of initiatives launched before the end of the last century. In addition, several scientists have moved to Denmark after the beginning of the present century, and they certainly are contributing to the present status of Danish neuroscience-but, again, this is not the History of Danish neuroscience.

Glial Glutamine Homeostasis in Health and Disease.

Glutamine is an essential cerebral metabolite. Several critical brain processes are directly linked to glutamine, including ammonia homeostasis, energy metabolism and neurotransmitter recycling. Astrocytes synthesize and release large quantities of glutamine, which is taken up by neurons to replenish the glutamate and GABA neurotransmitter pools. Astrocyte glutamine hereby sustains the glutamate/GABA-glutamine cycle, synaptic transmission and general brain function. Cerebral glutamine homeostasis is linked to the metabolic coupling of neurons and astrocytes, and relies on multiple cellular processes, including TCA cycle function, synaptic transmission and neurotransmitter uptake. Dysregulations of processes related to glutamine homeostasis are associated with several neurological diseases and may mediate excitotoxicity and neurodegeneration. In particular, diminished astrocyte glutamine synthesis is a common neuropathological component, depriving neurons of an essential metabolic substrate and precursor for neurotransmitter synthesis, hereby leading to synaptic dysfunction. While astrocyte glutamine synthesis is quantitatively dominant in the brain, oligodendrocyte-derived glutamine may serve important functions in white matter structures. In this review, the crucial roles of glial glutamine homeostasis in the healthy and diseased brain are discussed. First, we provide an overview of cellular recycling, transport, synthesis and metabolism of glutamine in the brain. These cellular aspects are subsequently discussed in relation to pathological glutamine homeostasis of hepatic encephalopathy, epilepsy, Alzheimer's disease, Huntington's disease and amyotrophic lateral sclerosis. Further studies on the multifaceted roles of cerebral glutamine will not only increase our understanding of the metabolic collaboration between brain cells, but may also aid to reveal much needed therapeutic targets of several neurological pathologies.

Low cerebral energy metabolism in hepatic encephalopathy reflects low neuronal energy demand. Role of ammonia-induced increased GABAergic tone.

Hepatic encephalopathy (HE) is a frequent and devastating but generally reversible neuropsychiatric complication secondary to chronic and acute liver failure. During HE, brain energy metabolism is markedly reduced and it remains unclear whether this is due to external or internal energy supply limitations, or secondary to depressed neuronal cellular functions - and if so, which mechanisms that are in play. The extent of deteriorated cerebral function correlates to blood ammonia levels but the metabolic link to ammonia is not clear. Early studies suggested that high levels of ammonia inhibited key tricarboxylic acid (TCA) cycle enzymes thus limiting mitochondrial energy production and oxygen consumption; however, later studies by us and others showed that this is not the case in vivo. Here, based on a series of translational studies from our group, we advocate the view that the low cerebral energy metabolism of HE is likely to be caused by neuronal metabolic depression due to an elevated GABAergic tone rather than by restricted energy availability. The increased GABAergic tone seems to be secondary to synthesis of large amounts of glutamine in astrocytes for detoxification of ammonia with the glutamine acting as a precursor for elevated neuronal synthesis of vesicular GABA.

Deficient astrocyte metabolism impairs glutamine synthesis and neurotransmitter homeostasis in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) leads to cerebral accumulation of insoluble amyloid-ß plaques causing synaptic dysfunction and neuronal death. Neurons rely on astrocyte-derived glutamine for replenishment of the amino acid neurotransmitter pools. Perturbations of astrocyte glutamine synthesis have been described in AD, but whether this functionally affects neuronal neurotransmitter synthesis is not known. Since the synthesis and recycling of neurotransmitter glutamate and GABA are intimately coupled to cellular metabolism, the aim of this study was to provide a functional investigation of neuronal and astrocytic energy and neurotransmitter metabolism in AD. To achieve this, we incubated acutely isolated cerebral cortical and hippocampal slices from 8-month-old female 5xFAD mice, in the presence of 13C isotopically enriched substrates, with subsequent gas chromatography-mass spectrometry (GC-MS) analysis. A prominent neuronal hypometabolism of [U-13C]glucose was observed in the hippocampal slices of the 5xFAD mice. Investigating astrocyte metabolism, using [1,2-13C]acetate, revealed a marked reduction in glutamine synthesis, which directly hampered neuronal synthesis of GABA. This was supported by an increased metabolism of exogenously supplied [U-13C]glutamine, suggesting a neuronal metabolic compensation of the reduced astrocytic glutamine supply. In contrast, astrocytic metabolism of [U-13C]GABA was reduced, whereas [U-13C]glutamate metabolism was unaffected. Finally, astrocyte de novo synthesis of glutamate and glutamine was hampered, whereas the enzymatic capacity of glutamine synthetase for ammonia fixation was maintained. Collectively, we demonstrate that deficient astrocyte metabolism leads to reduced glutamine synthesis, directly impairing neuronal GABA synthesis in the 5xFAD brain. These findings suggest that astrocyte metabolic dysfunction may be fundamental for the imbalances of synaptic excitation and inhibition in the AD brain.

Two Metabolic Fuels, Glucose and Lactate, Differentially Modulate Exocytotic Glutamate Release from Cultured Astrocytes.

Astrocytes have a prominent role in metabolic homeostasis of the brain and can signal to adjacent neurons by releasing glutamate via a process of regulated exocytosis. Astrocytes synthesize glutamate de novo owing to the pyruvate entry to the citric/tricarboxylic acid cycle via pyruvate carboxylase, an astrocyte specific enzyme. Pyruvate can be sourced from two metabolic fuels, glucose and lactate. Thus, we investigated the role of these energy/carbon sources in exocytotic glutamate release from astrocytes. Purified astrocyte cultures were acutely incubated (1 h) in glucose and/or lactate-containing media. Astrocytes were mechanically stimulated, a procedure known to increase intracellular Ca2+ levels and cause exocytotic glutamate release, the dynamics of which were monitored using single cell fluorescence microscopy. Our data indicate that glucose, either taken-up from the extracellular space or mobilized from the intracellular glycogen storage, sustained glutamate release, while the availability of lactate significantly reduced the release of glutamate from astrocytes. Based on further pharmacological manipulation during imaging along with tandem mass spectrometry (proteomics) analysis, lactate alone, but not in the hybrid fuel, caused metabolic changes consistent with an increased synthesis of fatty acids. Proteomics analysis further unveiled complex changes in protein profiles, which were condition-dependent and generally included changes in levels of cytoskeletal proteins, proteins of secretory organelle/vesicle traffic and recycling at the plasma membrane in aglycemic, lactate or hybrid-fueled astrocytes. These findings support the notion that the availability of energy sources and metabolic milieu play a significant role in gliotransmission.

Downregulation of GABA Transporter 3 (GAT3) is Associated with Deficient Oxidative GABA Metabolism in Human Induced Pluripotent Stem Cell-Derived Astrocytes in Alzheimer's Disease.

Alterations in neurotransmitter homeostasis, primarily of glutamate and GABA, is strongly implicated in the pathophysiology of Alzheimer's disease (AD). Homeostasis at the synapse is maintained by neurotransmitter recycling between neurons and astrocytes. Astrocytes support neuronal transmission through glutamine synthesis, which can be derived from oxidative metabolism of GABA. However, the precise implications of astrocytic GABA metabolism in AD remains elusive. The aim of this study was to investigate astrocytic GABA metabolism in AD pathology implementing human induced pluripotent stem cells derived astrocytes. Metabolic mapping of GABA was performed with [U-13C]GABA stable isotopic labeling using gas chromatography coupled to mass spectrometry (GC-MS). Neurotransmitter and amino acid content was quantified via high performance liquid chromatography (HPLC) and protein expression was investigated by Western blot assay. Cell lines carrying mutations in either amyloid precursor protein (APP) or presenilin1 (PSEN-1) were used as AD models and were compared to a control cell line of the same genetic background. AD astrocytes displayed a reduced oxidative GABA metabolism mediated by a decreased uptake capacity of GABA, as GABA transporter 3 (GAT3) was downregulated in AD astrocytes compared to the controls. Interestingly, the carbon backbone of GABA in AD astrocytes was utilized to a larger extent to support glutamine synthesis compared to control astrocytes. The results strongly indicate alterations in GABA uptake and metabolism in AD astrocytes linked to reduced GABA transporter expression, hereby contributing further to neurotransmitter disturbances.

Extensive astrocyte metabolism of γ-aminobutyric acid (GABA) sustains glutamine synthesis in the mammalian cerebral cortex.

Synaptic transmission is closely linked to brain energy and neurotransmitter metabolism. However, the extent of brain metabolism of the inhibitory neurotransmitter γ-aminobutyric acid (GABA), and the relative metabolic contributions of neurons and astrocytes, are yet unknown. The present study was designed to investigate the functional significance of brain GABA metabolism using isolated mouse cerebral cortical slices and slices of neurosurgically resected neocortical human tissue of the temporal lobe. By using dynamic isotope labeling, with [15 N]GABA and [U-13 C]GABA as metabolic substrates, we show that both mouse and human brain slices exhibit a large capacity for GABA metabolism. Both the nitrogen and the carbon backbone of GABA strongly support glutamine synthesis, particularly in the human cerebral cortex, indicative of active astrocytic GABA metabolism. This was further substantiated by pharmacological inhibition of the primary astrocytic GABA transporter subtype 3 (GAT3), by (S)-SNAP-5114 or 1-benzyl-5-chloro-2,3-dihydro-1H-indole-2,3-dione (compound 34), leading to significant reductions in oxidative GABA carbon metabolism. Interestingly, this was not the case when tiagabine was used to specifically inhibit GAT1, which is predominantly found on neurons. Finally, we show that acute GABA exposure does not directly stimulate glycolytic activity nor oxidative metabolism in cultured astrocytes, but can be used as an additional substrate to enhance uncoupled respiration. These results clearly show that GABA is actively metabolized in astrocytes, particularly for the synthesis of glutamine, and challenge the current view that synaptic GABA homeostasis is maintained primarily by presynaptic recycling.

Astrocytic Metabolism Focusing on Glutamate Homeostasis: A Short Review Dedicated to Vittorio Gallo.

A large number of studies have during the last several decades shown that astrocytes play a significant role in brain energy metabolism accounting for a considerable part of the oxygen uptake and the corresponding oxidative metabolism of glucose and lactate. Interestingly, it has become clear that in addition to these two major energy substrates, glutamate may be considered as an important alternative energy substrate and this is tightly coupled to its role as an excitatory neurotransmitter. Hence, this short review will link these events and provide an account of the role that Vittorio Gallo came to play as he coauthored a publication which demonstrated the usefulness of cultured cerebellar granule cells for studies of glutamate neurotransmission. Just by chance this study was published the same year that my own group published a similar study of glutamate uptake and release in a corresponding preparation of cultured neurons and astrocytes from cerebellum and cerebral cortex. Thus, it is a pleasure to dedicate this account of the role of astrocytes in glutamate neurotransmission to Vittorio Gallo whom I have had the pleasure of knowing for more than three decades.

Pharmacological Characterization of a Betaine/GABA Transporter 1 (BGT1) Inhibitor Displaying an Unusual Biphasic Inhibition Profile and Anti-seizure Effects.

Focal epileptic seizures can in some patients be managed by inhibiting γ-aminobutyric acid (GABA) uptake via the GABA transporter 1 (GAT1) using tiagabine (Gabitril®). Synergistic anti-seizure effects achieved by inhibition of both GAT1 and the betaine/GABA transporter (BGT1) by tiagabine and EF1502, compared to tiagabine alone, suggest BGT1 as a target in epilepsy. Yet, selective BGT1 inhibitors are needed for validation of this hypothesis. In that search, a series of BGT1 inhibitors typified by (1R,2S)-2-((4,4-bis(3-methylthiophen-2-yl)but-3-en-yl)(methyl)amino)cyclohexanecarboxylic acid (SBV2-114) was developed. A thorough pharmacological characterization of SBV2-114 using a cell-based [3H]GABA uptake assay at heterologously expressed BGT1, revealed an elusive biphasic inhibition profile with two IC50 values (4.7 and 556 µM). The biphasic profile was common for this structural class of compounds, including EF1502, and was confirmed in the MDCK II cell line endogenously expressing BGT1. The possibility of two binding sites for SBV2-114 at BGT1 was assessed by computational docking studies and examined by mutational studies. These investigations confirmed that the conserved residue Q299 in BGT1 is involved in, but not solely responsible for the biphasic inhibition profile of SBV2-114. Animal studies revealed anti-seizure effects of SBV2-114 in two mouse models, supporting a function of BGT1 in epilepsy. However, as SBV2-114 is apparent to be rather non-selective for BGT1, the translational relevance of this observation is unknown. Nevertheless, SBV2-114 constitutes a valuable tool compound to study the molecular mechanism of an emerging biphasic profile of BGT1-mediated GABA transport and the putative involvement of two binding sites for this class of compounds.

Astrocytic glycogen metabolism in the healthy and diseased brain.

The brain contains a fairly low amount of glycogen, mostly located in astrocytes, a fact that has prompted the suggestion that glycogen does not have a significant physiological role in the brain. However, glycogen metabolism in astrocytes is essential for several key physiological processes and is adversely affected in disease. For instance, diminished ability to break down glycogen impinges on learning, and epilepsy, Alzheimer's disease, and type 2 diabetes are all associated with abnormal astrocyte glycogen metabolism. Glycogen metabolism supports astrocytic K+ and neurotransmitter glutamate uptake and subsequent glutamine synthesis-three fundamental steps in excitatory signaling at most brain synapses. Thus, there is abundant evidence for a key role of glycogen in brain function. Here, we summarize the physiological brain functions that depend on glycogen, discuss glycogen metabolism in disease, and investigate how glycogen breakdown is regulated at the cellular and molecular levels.

Astrocytic pyruvate carboxylation: Status after 35 years.

The first two publications dealing with the question of the cellular localization of the enzyme pyruvate carboxylase (PC) which in the brain represents the most important metabolic pathway to allow anaplerosis of TCA cycle constituents were published in 1983 and 1985. Hence, 2018 marks the 35th anniversary of the notion based on the results of the publications provided above that PC-catalyzed anaplerosis in the brain is an astrocytic process. This review will provide the background for investigating this enzymatic pathway as well as a discussion of cataplerosis, the degradation of products from anaplerosis, and the current status of the functional significance of pyruvate carboxylation in brain metabolism.

Correction to: Delineation of the Role of Astroglial GABA Transporters in Seizure Control.

The original version of this article unfortunately contained a mistake. In Fig. 1 two chemical structures are incorrect, namely exo-THPO and N-methyl-exo-THPO. The hydroxyl group (-OH) in the isoxazole ring is missing. The corrected Fig. 1 is given below.

Expression of the human isoform of glutamate dehydrogenase, hGDH2, augments TCA cycle capacity and oxidative metabolism of glutamate during glucose deprivation in astrocytes.

A key enzyme in brain glutamate homeostasis is glutamate dehydrogenase (GDH) which links carbohydrate and amino acid metabolism mediating glutamate degradation to CO2 and expanding tricarboxylic acid (TCA) cycle capacity with intermediates, i.e. anaplerosis. Humans express two GDH isoforms, GDH1 and 2, whereas most other mammals express only GDH1. hGDH1 is widely expressed in human brain while hGDH2 is confined to astrocytes. The two isoforms display different enzymatic properties and the nature of these supports that hGDH2 expression in astrocytes potentially increases glutamate oxidation and supports the TCA cycle during energy-demanding processes such as high intensity glutamatergic signaling. However, little is known about how expression of hGDH2 affects the handling of glutamate and TCA cycle metabolism in astrocytes. Therefore, we cultured astrocytes from cerebral cortical tissue of hGDH2-expressing transgenic mice. We measured glutamate uptake and metabolism using [3 H]glutamate, while the effect on metabolic pathways of glutamate and glucose was evaluated by use of 13 C and 14 C substrates and analysis by mass spectrometry and determination of radioactively labeled metabolites including CO2 , respectively. We conclude that hGDH2 expression increases capacity for uptake and oxidative metabolism of glutamate, particularly during increased workload and aglycemia. Additionally, hGDH2 expression increased utilization of branched-chain amino acids (BCAA) during aglycemia and caused a general decrease in oxidative glucose metabolism. We speculate, that expression of hGDH2 allows astrocytes to spare glucose and utilize BCAAs during substrate shortages. These findings support the proposed role of hGDH2 in astrocytes as an important fail-safe during situations of intense glutamatergic activity. GLIA 2017;65:474-488.

Misconceptions regarding basic thermodynamics and enzyme kinetics have led to erroneous conclusions regarding the metabolic importance of lactate dehydrogenase isoenzyme expression.

Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate involving the coenzyme NAD+ . Part of the foundation for the proposed shuttling of lactate from astrocytes to neurons during brain activation is the differential distribution of LDH isoenzymes between the two cell types. In this short review, we outline the basic kinetic properties of the LDH isoenzymes expressed in neurons and astrocytes, and argue that the distribution of LDH isoenzymes does not in any way govern directional flow of lactate between the two cellular compartments. The two main points are as follows. First, in line with the general concept of chemical catalysis, enzymes do not influence the thermodynamic equilibrium of a chemical reaction but merely the speed at which equilibrium is obtained. Thus, differential distribution of LDH isoenzymes with different kinetic parameters does not predict which cells are producing and which are consuming lactate. Second, the thermodynamic equilibrium of the reaction is toward the reduced substrate (i.e., lactate), which is reflected in the concentrations measured in brain tissue, suggesting that the reaction is at near-equilibrium at steady state. To conclude, the cellular distribution of LDH isoenzymes is of little if any consequence in determining any directional flow of lactate between neurons and astrocytes. © 2017 Wiley Periodicals, Inc.

Specificity of exogenous acetate and glutamate as astrocyte substrates examined in acute brain slices from female mice using methionine sulfoximine (MSO) to inhibit glutamine synthesis.

Removal of endogenously released glutamate is mediated primarily by astrocytes and exogenous 13 C-labeled glutamate has been applied to study glutamate metabolism in astrocytes. Likewise, studies have clearly established the relevance of 13 C-labeled acetate as an astrocyte specific metabolic substrate. Recent studies have, however, challenged the arguments used to anchor this astrocyte specificity of acetate and glutamate. The aim of the current study was to evaluate the specificity of acetate and glutamate as astrocyte substrates in brain slices. Acutely isolated hippocampal and cerebral cortical slices from female NMRI mice were incubated in media containing [1,2-13 C]acetate or [U-13 C]glutamate, with or without methionine sulfoximine (MSO) to inhibit glutamine synthetase (GS). Tissue extracts were analyzed by gas chromatography-mass spectrometry. Blocking GS abolished the majority of glutamine 13 C-labeling from [1,2-13 C]acetate as intended. However, 13 C-labeling of GABA was only 40-50% reduced by MSO, suggesting considerable neuronal uptake of acetate. Moreover, labeling of glutamate from [1,2-13 C]acetate in the presence of MSO exceeded the level probable from exclusive labeling of the astrocytic pool, which likewise suggests neuronal acetate metabolism. Approximately 50% of glutamate was uniformly labeled in slices incubated with [U-13 C]glutamate in the presence of MSO, suggesting that neurons exhibit substantial uptake of exogenously provided glutamate. © 2017 Wiley Periodicals, Inc.