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.

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.

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.

Using stable isotope tracing to unravel the metabolic components of neurodegeneration: Focus on neuron-glia metabolic interactions.

Disrupted brain metabolism is a critical component of several neurodegenerative diseases. Energy metabolism of both neurons and astrocytes is closely connected to neurotransmitter recycling via the glutamate/GABA-glutamine cycle. Neurons and astrocytes hereby work in close metabolic collaboration which is essential to sustain neurotransmission. Elucidating the mechanistic involvement of altered brain metabolism in disease progression has been aided by the advance of techniques to monitor cellular metabolism, in particular by mapping metabolism of substrates containing stable isotopes, a technique known as isotope tracing. Here we review key aspects of isotope tracing including advantages, drawbacks and applications to different cerebral preparations. In addition, we narrate how isotope tracing has facilitated the discovery of central metabolic features in neurodegeneration with a focus on the metabolic cooperation between neurons and astrocytes.

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.

ß-Hydroxybutyrate and Medium-Chain Fatty Acids are Metabolized by Different Cell Types in Mouse Cerebral Cortex Slices.

Ketogenic diets and medium-chain triglycerides are gaining attention as treatment of neurological disorders. Their major metabolites, ß-hydroxybutyrate (ßHB) and the medium-chain fatty acids (MCFAs) octanoic acid (C8) and decanoic acid (C10), are auxiliary brain fuels. To which extent these fuels compete for metabolism in different brain cell types is unknown. Here, we used acutely isolated mouse cerebral cortical slices to (1) compare metabolism of 200 µM [U-13C]C8, [U-13C]C10 and [U-13C]ßHB and (2) assess potential competition between metabolism of ßHB and MCFAs by quantifying metabolite 13C enrichment using gas chromatography-mass spectrometry (GC-MS) analysis. The 13C enrichment in most metabolites was similar with [U-13C]C8 and [U-13C]C10 as substrates, but several fold lower with [U-13C]ßHB. The 13C enrichment in glutamate was in a similar range for all three substrates, whereas the 13C enrichments in citrate and glutamine were markedly higher with both [U-13C]C8 and [U-13C]C10 compared with [U-13C]ßHB. As citrate and glutamine are indicators of astrocytic metabolism, the results indicate active MCFA metabolism in astrocytes, while ßHB is metabolized in a different cellular compartment. In competition experiments, 12C-ßHB altered 13C incorporation from [U-13C]C8 and [U-13C]C10 in only a few instances, while 12C-C8 and 12C-C10 only further decreased the low [U-13C]ßHB-derived 13C incorporation into citrate and glutamine, signifying little competition for oxidative metabolism between ßHB and the MCFAs. Overall, the data demonstrate that ßHB and MCFAs are supplementary fuels in different cellular compartments in the brain without notable competition. Thus, the use of medium-chain triglycerides in ketogenic diets is likely to be beneficial in conditions with carbon and energy shortages in both astrocytes and neurons, such as GLUT1 deficiency.

Pharmacological inhibition of mitochondrial soluble adenylyl cyclase in astrocytes causes activation of AMP-activated protein kinase and induces breakdown of glycogen.

Mobilization of astrocyte glycogen is key for processes such as synaptic plasticity and memory formation but the link between neuronal activity and glycogen breakdown is not fully known. Activation of cytosolic soluble adenylyl cyclase (sAC) in astrocytes has been suggested to link neuronal depolarization and glycogen breakdown partly based on experiments employing pharmacological inhibition of sAC. However, several studies have revealed that sAC located within mitochondria is a central regulator of respiration and oxidative phosphorylation. Thus, pharmacological sAC inhibition is likely to affect both cytosolic and mitochondrial sAC and if bioenergetic readouts are studied, the observed effects are likely to stem from inhibition of mitochondrial rather than cytosolic sAC. Here, we report that a pharmacologically induced inhibition of sAC activity lowers mitochondrial respiration, induces phosphorylation of the metabolic master switch AMP-activated protein kinase (AMPK), and decreases glycogen stores in cultured primary murine astrocytes. From these data and our discussion of the literature, mitochondrial sAC emerges as a key regulator of astrocyte bioenergetics. Lastly, we discuss the challenges of investigating the functional and metabolic roles of cytosolic versus mitochondrial sAC in astrocytes employing the currently available pharmacological tool compounds.

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.

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.

Deletion of Neuronal GLT-1 in Mice Reveals Its Role in Synaptic Glutamate Homeostasis and Mitochondrial Function.

The glutamate transporter GLT-1 is highly expressed in astrocytes but also in neurons, primarily in axon terminals. We generated a conditional neuronal GLT-1 KO using synapsin 1-Cre (synGLT-1 KO) to elucidate the metabolic functions of GLT-1 expressed in neurons, here focusing on the cerebral cortex. Both synaptosomal uptake studies and electron microscopic immunocytochemistry demonstrated knockdown of GLT-1 in the cerebral cortex in the synGLT-1 KO mice. Aspartate content was significantly reduced in cerebral cortical extracts as well as synaptosomes from cerebral cortex of synGLT-1 KO compared with control littermates. 13C-Labeling of tricarboxylic acid cycle intermediates originating from metabolism of [U-13C]-glutamate was significantly reduced in synGLT-1 KO synaptosomes. The decreased aspartate content was due to diminished entry of glutamate into the tricarboxylic acid cycle. Pyruvate recycling, a pathway necessary for full glutamate oxidation, was also decreased. ATP production was significantly increased, despite unaltered oxygen consumption, in isolated mitochondria from the synGLT-1 KO. The density of mitochondria in axon terminals and perisynaptic astrocytes was increased in the synGLT-1 KO. Intramitochondrial cristae density of synGLT-1 KO mice was increased, suggesting increased mitochondrial efficiency, perhaps in compensation for reduced access to glutamate. SynGLT-1 KO synaptosomes exhibited an elevated oxygen consumption rate when stimulated with veratridine, despite a lower baseline oxygen consumption rate in the presence of glucose. GLT-1 expressed in neurons appears to be required to provide glutamate to synaptic mitochondria and is linked to neuronal energy metabolism and mitochondrial function.SIGNIFICANCE STATEMENT All synaptic transmitters need to be cleared from the extracellular space after release, and transporters are used to clear glutamate released from excitatory synapses. GLT-1 is the major glutamate transporter, and most GLT-1 is expressed in astrocytes. Only 5%-10% is expressed in neurons, primarily in axon terminals. The function of GLT-1 in axon terminals remains unknown. Here, we used a conditional KO approach to investigate the significance of the expression of GLT-1 in neurons. We found multiple abnormalities of mitochondrial function, suggesting impairment of glutamate utilization by synaptic mitochondria in the neuronal GLT-1 KO. These data suggest that GLT-1 expressed in axon terminals may be important in maintaining energy metabolism and biosynthetic activities mediated by presynaptic mitochondria.

AMP-activated protein kinase (AMPK) regulates astrocyte oxidative metabolism by balancing TCA cycle dynamics.

AMP-activated protein kinase (AMPK) is an important energy sensor located in cells throughout the human body. From the periphery, AMPK is known to be a metabolic master switch controlling the use of energy fuels. The energy sensor is activated when the energy status of the cell is low, initiating energy-producing pathways and deactivating energy-consuming pathways. All brain cells are crucially dependent on energy production for survival, and the availability of energy substrates must be closely regulated. Intriguingly, the role of AMPK in the regulation of brain cell metabolism has been sparsely investigated, particularly in astrocytes. By investigating metabolism of 13 C-labeled energy substrates in acutely isolated hippocampal slices and cultured astrocytes, with subsequent mass spectrometry analysis, we here show that activation of AMPK increases glycolysis as well as the capacity of the TCA cycle, that is, anaplerosis, through the activity of pyruvate carboxylase (PC) in astrocytes. In addition, we demonstrate that AMPK activation leads to augmented astrocytic glutamate oxidation via pyruvate recycling (i.e., cataplerosis). This regulatory mechanism induced by AMPK activation is mediated via glutamate dehydrogenase (GDH) shown in a CNS-specific GDH knockout mouse. Collectively, these findings demonstrate that AMPK regulates TCA cycle dynamics in astrocytes via PC and GDH activity. AMPK functionality has been shown to be hampered in Alzheimer's and Parkinson's disease and our findings may therefore add to the toolbox for discovery of new metabolic drug targets.

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.

Conditional Knockout of GLT-1 in Neurons Leads to Alterations in Aspartate Homeostasis and Synaptic Mitochondrial Metabolism in Striatum and Hippocampus.

Expression of the glutamate transporter GLT-1 in neurons has been shown to be important for synaptic mitochondrial function in the cerebral cortex. Here we determined whether neuronal GLT-1 plays a similar role in the hippocampus and striatum, using conditional GLT-1 knockout mice in which GLT-1 was inactivated in neurons by expression of synapsin-Cre (synGLT-1 KO). Ex vivo 13C-labelling using [1,2-13C]acetate, representing astrocytic metabolism, yielded increased [4,5-13C]glutamate levels, suggesting increased astrocyte-neuron glutamine transfer, in the striatum but not in the hippocampus of the synGLT-1 KO. Moreover, aspartate concentrations were reduced - 38% compared to controls in the hippocampus and the striatum of the synGLT-1 KO. Mitochondria isolated from the hippocampus of synGLT-1 KO mice exhibited a lower oxygen consumption rate in the presence of oligomycin A, indicative of a decreased proton leak across the mitochondrial membrane, whereas the ATP production rate was unchanged. Electron microscopy revealed reduced mitochondrial inter-cristae distance within excitatory synaptic terminals in the hippocampus and striatum of the synGLT-1 KO. Finally, dilution of 13C-labelling originating from [U-13C]glucose, caused by metabolism of unlabelled glutamate, was reduced in hippocampal synGLT-1 KO synaptosomes, suggesting that neuronal GLT-1 provides glutamate for synaptic tricarboxylic acid cycle metabolism. Collectively, these data demonstrate an important role of neuronal expression of GLT-1 in synaptic mitochondrial metabolism in the forebrain.

Enhanced cerebral branched-chain amino acid metabolism in R6/2 mouse model of Huntington's disease.

Huntington's disease (HD) is a hereditary and fatal disease causing profound neurodegeneration. Deficits in cerebral energy and neurotransmitter metabolism have been suggested to play a central role in the neuronal dysfunction and death associated with HD. The branched-chain amino acids (BCAAs), leucine, isoleucine and valine, are important for cerebral nitrogen homeostasis, neurotransmitter recycling and can be utilized as energy substrates in the tricarboxylic acid (TCA) cycle. Reduced levels of BCAAs in HD have been validated by several reports. However, it is still unknown how cerebral BCAA metabolism is regulated in HD. Here we investigate the metabolism of leucine and isoleucine in the R6/2 mouse model of HD. Acutely isolated cerebral cortical and striatal slices of control and R6/2 mice were incubated in media containing 15N- or 13C-labeled leucine or isoleucine and slice extracts were analyzed by gas chromatography-mass spectrometry (GC-MS) to determine isotopic enrichment of derived metabolites. Elevated BCAA transamination was found from incubations with [15N]leucine and [15N]isoleucine, in both cerebral cortical and striatal slices of R6/2 mice compared to controls. Metabolism of [U-13C]leucine and [U-13C]isoleucine, entering oxidative metabolism as acetyl CoA, was maintained in R6/2 mice. However, metabolism of [U-13C]isoleucine, entering the TCA cycle as succinyl CoA, was elevated in both cerebral cortical and striatal slices of R6/2 mice, suggesting enhanced metabolic flux via this anaplerotic pathway. To support the metabolic studies, expression of enzymes in the BCAA metabolic pathway was assessed from a proteomic resource. Several enzymes related to BCAA metabolism were found to exhibit augmented expression in the R6/2 brain, particularly related to isoleucine metabolism, suggesting an increase in the BCAA metabolic machinery. Our results show that the capacity for cerebral BCAA metabolism, predominantly of isoleucine, is amplified in the R6/2 brain and indicates that perturbations in cerebral BCAA homeostasis could have functional consequences for HD pathology.

The energetic brain - A review from students to students.

The past 20 years have resulted in unprecedented progress in understanding brain energy metabolism and its role in health and disease. In this review, which was initiated at the 14th International Society for Neurochemistry Advanced School, we address the basic concepts of brain energy metabolism and approach the question of why the brain has high energy expenditure. Our review illustrates that the vertebrate brain has a high need for energy because of the high number of neurons and the need to maintain a delicate interplay between energy metabolism, neurotransmission, and plasticity. Disturbances to the energetic balance, to mitochondria quality control or to glia-neuron metabolic interaction may lead to brain circuit malfunction or even severe disorders of the CNS. We cover neuronal energy consumption in neural transmission and basic ('housekeeping') cellular processes. Additionally, we describe the most common (glucose) and alternative sources of energy namely glutamate, lactate, ketone bodies, and medium chain fatty acids. We discuss the multifaceted role of non-neuronal cells in the transport of energy substrates from circulation (pericytes and astrocytes) and in the supply (astrocytes and microglia) and usage of different energy fuels. Finally, we address pathological consequences of disrupted energy homeostasis in the CNS.

Distinct differences in rates of oxygen consumption and ATP synthesis of regionally isolated non-synaptic mouse brain mitochondria.

Brain mitochondrial dysfunction has been implicated in several neurodegenerative diseases. The distribution and efficiency of mitochondria display large heterogeneity throughout the regions of the brain. This may imply that the selective regional susceptibility of neurodegenerative diseases could be mediated through inherent differences in regional mitochondrial function. To investigate regional cerebral mitochondrial energetics, the rates of oxygen consumption and adenosine-5'-triphosphate (ATP) synthesis were assessed in isolated non-synaptic mitochondria of the cerebral cortex, hippocampus, and striatum of the male mouse brain. Oxygen consumption rates were assessed using a Seahorse XFe96 analyzer and ATP synthesis rates were determined by an online luciferin-luciferase coupled luminescence assay. Complex I- and complex II-driven respiration and ATP synthesis, were investigated by applying pyruvate in combination with malate, or succinate, as respiratory substrates, respectively. Hippocampal mitochondria exhibited the lowest basal and adenosine-5'-diphosphate (ADP)-stimulated rate of oxygen consumption when provided pyruvate and malate. However, hippocampal mitochondria also exhibited an increased proton leak and an elevated relative rate of oxygen consumption in response to the uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), showing a large capacity for uncoupled respiration in the presence of pyruvate. When the complex II-linked substrate succinate was provided, striatal mitochondria exhibited the highest respiration and ATP synthesis rate, whereas hippocampal mitochondria had the lowest. However, the mitochondrial efficiency, determined as ATP produced/O2 consumed, was similar between the three regions. This study reveals inherent differences in regional mitochondrial energetics and may serve as a tool for further investigations of regional mitochondrial function in relation to neurodegenerative diseases.

Alterations in Cerebral Cortical Glucose and Glutamine Metabolism Precedes Amyloid Plaques in the APPswe/PSEN1dE9 Mouse Model of Alzheimer's Disease.

Alterations in brain energy metabolism have been suggested to be of fundamental importance for the development of Alzheimer's disease (AD). However, specific changes in brain energetics in the early stages of AD are poorly known. The aim of this study was to investigate cerebral energy metabolism in the APPswe/PSEN1dE9 mouse prior to amyloid plaque formation. Acutely isolated cerebral cortical and hippocampal slices of 3-month-old APPswe/PSEN1dE9 and wild-type control mice were incubated in media containing [U-13C]glucose, [1,2-13C]acetate or [U-13C]glutamine, and tissue extracts were analyzed by mass spectrometry. The ATP synthesis rate of isolated whole-brain mitochondria was assessed by an on-line luciferin-luciferase assay. Significantly increased 13C labeling of intracellular lactate and alanine and decreased tricarboxylic acid (TCA) cycle activity were observed from cerebral cortical slices of APPswe/PSEN1dE9 mice incubated in media containing [U-13C]glucose. No changes in glial [1,2-13C]acetate metabolism were observed. Cerebral cortical slices from APPswe/PSEN1dE9 mice exhibited a reduced capacity for uptake and oxidative metabolism of glutamine. Furthermore, the ATP synthesis rate tended to be decreased in isolated whole-brain mitochondria of APPswe/PSEN1dE9 mice. Thus, several cerebral metabolic changes are evident in the APPswe/PSEN1dE9 mouse prior to amyloid plaque deposition, including altered glucose metabolism, hampered glutamine processing and mitochondrial dysfunctions.

Metabolic Characterization of Acutely Isolated Hippocampal and Cerebral Cortical Slices Using [U-13C]Glucose and [1,2-13C]Acetate as Substrates.

Brain slice preparations from rats, mice and guinea pigs have served as important tools for studies of neurotransmission and metabolism. While hippocampal slices routinely have been used for electrophysiology studies, metabolic processes have mostly been studied in cerebral cortical slices. Few comparative characterization studies exist for acute hippocampal and cerebral cortical slices, hence, the aim of the current study was to characterize and compare glucose and acetate metabolism in these slice preparations in a newly established incubation design. Cerebral cortical and hippocampal slices prepared from 16 to 18-week-old mice were incubated for 15-90 min with unlabeled glucose in combination with [U-13C]glucose or [1,2-13C]acetate. Our newly developed incubation apparatus allows accurate control of temperature and is designed to avoid evaporation of the incubation medium. Subsequent to incubation, slices were extracted and extracts analyzed for 13C-labeling (%) and total amino acid contents (µmol/mg protein) using gas chromatography-mass spectrometry and high performance liquid chromatography, respectively. Release of lactate from the slices was quantified by analysis of the incubation media. Based on the measured 13C-labeling (%), total amino acid contents and relative activity of metabolic enzymes/pathways, we conclude that the slice preparations in the current incubation apparatus exhibited a high degree of metabolic integrity. Comparison of 13C-labeling observed with [U-13C]glucose in slices from cerebral cortex and hippocampus revealed no significant regional differences regarding glycolytic or total TCA cycle activities. On the contrary, results from the incubations with [1,2-13C]acetate suggest a higher capacity of the astrocytic TCA cycle in hippocampus compared to cerebral cortex. Finally, we propose a new approach for assessing compartmentation of metabolite pools between astrocytes and neurons using 13C-labeling (%) data obtained from mass spectrometry. Based on this approach we suggest that cellular metabolic compartmentation in hippocampus and cerebral cortex is very similar.

Astrocytes regulate inhibitory neurotransmission through GABA uptake, metabolism, and recycling.

Synaptic regulation of the primary inhibitory neurotransmitter γ-aminobutyric acid (GABA) is essential for brain function. Cerebral GABA homeostasis is tightly regulated through multiple mechanisms and is directly coupled to the metabolic collaboration between neurons and astrocytes. In this essay, we outline and discuss the fundamental roles of astrocytes in regulating synaptic GABA signaling. A major fraction of synaptic GABA is removed from the synapse by astrocytic uptake. Astrocytes utilize GABA as a metabolic substrate to support glutamine synthesis. The astrocyte-derived glutamine is subsequently transferred to neurons where it serves as the primary precursor of neuronal GABA synthesis. The flow of GABA and glutamine between neurons and astrocytes is collectively termed the GABA-glutamine cycle and is essential to sustain GABA synthesis and inhibitory signaling. In certain brain areas, astrocytes are even capable of synthesizing and releasing GABA to modulate inhibitory transmission. The majority of oxidative GABA metabolism in the brain takes place in astrocytes, which also leads to synthesis of the GABA-related metabolite γ-hydroxybutyric acid (GHB). The physiological roles of endogenous GHB remain unclear, but may be related to regulation of tonic inhibition and synaptic plasticity. Disrupted inhibitory signaling and dysfunctional astrocyte GABA handling are implicated in several diseases including epilepsy and Alzheimer's disease. Synaptic GABA homeostasis is under astrocytic control and astrocyte GABA uptake, metabolism, and recycling may therefore serve as relevant targets to ameliorate pathological inhibitory signaling.

Astrocyte energy and neurotransmitter metabolism in Alzheimer's disease: Integration of the glutamate/GABA-glutamine cycle.

Astrocytes contribute to the complex cellular pathology of Alzheimer's disease (AD). Neurons and astrocytes function in close collaboration through neurotransmitter recycling, collectively known as the glutamate/GABA-glutamine cycle, which is essential to sustain neurotransmission. Neurotransmitter recycling is intimately linked to astrocyte energy metabolism. In the course of AD, astrocytes undergo extensive metabolic remodeling, which may profoundly affect the glutamate/GABA-glutamine cycle. The consequences of altered astrocyte function and metabolism in relation to neurotransmitter recycling are yet to be comprehended. Metabolic alterations of astrocytes in AD deprive neurons of metabolic support, thereby contributing to synaptic dysfunction and neurodegeneration. In addition, several astrocyte-specific components of the glutamate/GABA-glutamine cycle, including glutamine synthesis and synaptic neurotransmitter uptake, are perturbed in AD. Integration of the complex astrocyte biology within the context of AD is essential for understanding the fundamental mechanisms of the disease, while restoring astrocyte metabolism may serve as an approach to arrest or even revert clinical progression of AD.