Leif Hertz

Energy Metabolism in Astrocytes: High Rate of Oxidative Metabolism and Spatiotemporal Dependence on Glycolysis/Glycogenolysis

Astrocytic energy demand is stimulated by K+ and glutamate uptake, signaling processes, responses to neurotransmitters, Ca2+ fluxes, and filopodial motility. Astrocytes derive energy from glycolytic and oxidative pathways, but respiration, with its high-energy yield, provides most adenosine 5 triphosphate (ATP). The proportion of cortical oxidative metabolism attributed to astrocytes (~30%) in in vivo nuclear magnetic resonance (NMR) spectroscopic and autoradiographic studies corresponds to their volume fraction, indicating similar oxidation rates in astrocytes and neurons. Astrocyte-selective expression of pyruvate carboxylase (PC) enables synthesis of glutamate from glucose, accounting for two-thirds of astrocytic glucose degradation via combined pyruvate carboxylation and dehydrogenation. Together, glutamate synthesis and oxidation, including neurotransmitter turnover, generate almost as much energy as direct glucose oxidation. Glycolysis and glycogenolysis are essential for astrocytic responses to increasing energy demand because astrocytic filopodial and lamellipodial extensions, which account for 80% of their surface area, are too narrow to accommodate mitochondria; these processes depend on glycolysis, glycogenolysis, and probably diffusion of ATP and phosphocreatine formed via mitochondrial metabolism to satisfy their energy demands. High glycogen turnover in astrocytic processes may stimulate glucose demand and lactate production because less ATP is generated when glucose is metabolized via glycogen, thereby contributing to the decreased oxygen to glucose utilization ratio during brain activation. Generated lactate can spread from activated astrocytes via low-affinity monocarboxylate transporters and gap junctions, but its subsequent fate is unknown. Astrocytic metabolic compartmentation arises from their complex ultrastructure; astrocytes have high oxidative rates plus dependence on glycolysis and glycogenolysis, and their energetics is underestimated if based solely on glutamate cycling.

Astrocytic control of glutamatergic activity: astrocytes as stars of the show

It is a major recent finding that astrocytes can influence synaptic activity by release of glutamate, but many other glutamate-mediated activities are also controlled by astrocytes. Even the most obvious neuronal function of glutamate - its release as a transmitter - is regulated by astrocytes; these cells are needed for formation of precursors for glutamate synthesis, for reuptake of released transmitter, and for disposal of excess glutamate. Without astrocytic involvement, normal function of glutamatergic neurons is not possible, as exemplified by almost instantaneous abrogation of normal vision and learning upon inhibition of astrocyte-specific metabolic pathways. In addition, astrocytes are essential for production of the neuroprotectant glutathione, yet they can also contribute to neuronal death during ischemia by maintaining glutamine synthesis, enabling neuronal formation of neurotoxic glutamate.

Inhibition of glycogenolysis in astrocytes interrupts memory consolidation in young chickens

Glycolysis and glycogenolysis are involved in memory processing in day‐old chickens and, aside from the provision of energy for neuronal and astrocytic energy metabolism these pathways enable astrocytes to supply neurones with precursor for transmitter glutamate by glucose‐based de novo synthesis. We have previously shown that memory processing for bead discrimination learning is dependent on glycolysis; however, the metabolic inhibitor used, iodoacetate, inhibits pyruvate formation from both glucose and glycogen. At specific time points after training transient reductions in brain glycogen content occur, mirrored by increases in glutamate/glutamine content. In the present study, we used intracerebral injection of a glycogen phosphorylase inhibitor, 1,4‐dideoxy‐1,4‐imino‐D‐arabinitol (DAB), which does not affect glucose breakdown, to evaluate the role of glycogen metabolism in memory consolidation. Dose‐dependent inhibition of learning occurred when DAB was administered at specific time periods in relation to training: (i) 5 min before training, (ii) around 30 min posttraining, and (iii) 55 min posttraining. After injection at either of the two earlier periods, memory disappeared after consolidation 30 min postlearning, and after injection 55 min after learning memory was absent at 70 min. The memory loss caused by early administration could be prevented after training by central injection of the glutamate precursor glutamine or the astrocyte‐specific substrate acetate together with aspartate, substituting for pyruvate carboxylation. Thus, glycogenolysis is essential for learning in this paradigm and, aside from energy supply considerations, we suggest that an important role for glycogenolysis is to provide neurones with glutamine as the precursor for neuronal glutamate and GABA.

Bioenergetics of cerebral ischemia: a cellular perspective.

In cerebral ischemia survival of neurons, astrocytes, oligodendrocytes and endothelial cells is threatened during energy deprivation and/or following re-supply of oxygen and glucose. After a brief summary of characteristics of different cells types, emphasizing the dependence of all on oxidative metabolism, the bioenergetics of focal and global ischemia is discussed, distinguishing between events during energy deprivation and subsequent recovery attempt after re-circulation. Gray and white matter ischemia are described separately, and distinctions are made between mature and immature brains. Next comes a description of bioenergetics in individual cell types in culture during oxygen/glucose deprivation or exposure to metabolic inhibitors and following re-establishment of normal aerated conditions. Due to their expression of NMDA and non-NMDA receptors neurons and oligodendrocytes are exquisitely sensitive to excitotoxicity by glutamate, which reaches high extracellular concentrations in ischemic brain for several reasons, including failing astrocytic uptake. Excitotoxicity kills brain cells by energetic exhaustion (due to Na(+) extrusion after channel-mediated entry) combined with mitochondrial Ca(2+)-mediated injury and formation of reactive oxygen species. Many (but not all) astrocytes survive energy deprivation for extended periods, but after return to aerated conditions they are vulnerable to mitochondrial damage by cytoplasmic/mitochondrial Ca(2+) overload and to NAD(+) deficiency. Ca(2+) overload is established by reversal of Na(+)/Ca(2+) exchangers following Na(+) accumulation during Na(+)-K(+)-Cl(-) cotransporter stimulation or pH regulation, compensating for excessive acid production. NAD(+) deficiency inhibits glycolysis and eventually oxidative metabolism, secondary to poly(ADP-ribose)polymerase (PARP) activity following DNA damage. Hyperglycemia can be beneficial for neurons but increases astrocytic death due to enhanced acidosis.

Astrocytic contributions to bioenergetics of cerebral ischemia.

Astrocytes are multifunctional cells that interact with neurons and other astrocytes in signaling and metabolic functions, and their resistance to pathophysiological conditions can help restrict loss of tissue after an ischemic event provided adequate nutrients are supplied to support their requirements. Astrocytes have substantial oxidative capacity and mechanisms to upregulate glycolytic capability when respiration is impaired. An astrocytic enzyme that synthesizes a powerful activator of glycolysis is not present in neurons, endowing astrocytes with the ability to sustain ATP production under restrictive conditions. The monocarboxylic acid transporter (MCT) isoforms predominating in astrocytes are optimized to facilitate very large increases in lactate flux as lactate concentration increases within (1–3 mM) and above (>3 mM) the normal range. In sharp contrast, the major neuronal MCT serves as a barrier to increased transmembrane transport as lactate rises above 1 mM, restricting both entry and efflux. Lactate can serve as fuel during recovery from ischemia but direct evidence that lactate is oxidized by neurons (vs. astrocytes) to maintain synaptic function is lacking. Astrocytes have critical roles in regulation of ionic homeostasis and control of extracellular glutamate levels, and spreading depression associated with ischemia places high demands on energy supplies in astrocytes and contributes to metabolic exhaustion and demise. Disruption of Ca2+ homeostasis, generation of oxygen free radicals and nitric oxide, and mitochondrial depolarization contribute to astrocyte death during and after a metabolic insult. Novel pharmaceutical agents targeted to astrocytes and hyperoxic therapy that restores penumbral oxygen level during energy failure might improve postischemic outcome.

Lactate transport and transporters: General principles and functional roles in brain cells

Lactate is transported across cell membranes by diffusional, saturable cotransport with protons, mediated by monocarboxylate transporters (MCTs). This transport is bidirectional and in the absence of a transcellular H+ gradient, it can increase the intracellular concentration of lactate up to but not beyond the extracellular level (or vice versa). If extra‐ and intracellular pH differ, however, the equilibrium level is determined by the gradients of both lactate anions and protons. Rates of lactate uptake are determined most often by measuring uptake of labeled lactate, e.g., [U‐14C]lactate. In the case of lactate and other compounds that are metabolized, errors are introduced easily because continuing inwardly directed diffusional net transport of label can be achieved by intracellular metabolism, reducing the intracellular level of the nonmetabolized lactate and thus maintaining a concentration gradient between extra‐ and intracellular concentrations of the nonmetabolized compound (metabolism‐driven uptake). For measurement of facilitated diffusion kinetics, it is essential that the period during which the uptake is measured is short enough that little or no metabolism‐driven uptake contributes to the measured uptake (or that first‐order regression analysis is carried out to obtain initial uptake rates from nonlinear traces). To achieve initial uptake rates, incubation periods well below 1 min are generally required. Lactate uptake is fast in astrocytes, which express powerful, low‐affinity MCTs, i.e., MCT1 and MCT4. Due to the low affinity of these transporters, they respond to increased lactate gradients with enhanced transporter activity. The predominant MCT in neurons is the high‐affinity MCT2, which can only increase its activity to a limited extent in the face of an increased lactate gradient. This is reflected by a high‐affinity lactate uptake, although most investigators also have demonstrated a component of lactate uptake with lower affinity. In both neurons and astrocytes, however, facilitated diffusion is fast enough that under most conditions lactate fluxes will be determined mainly by the rate of metabolism‐driven uptake, and MCT‐mediated transport only will be rate‐limiting after establishment of large transmembrane gradients.

Astrocytic involvement in learning and memory consolidation

Astrocytes play fundamental roles in brain function, interacting with neurons and other astrocytes, yet their role in learning is not widely recognized. This review focuses on astrocytic involvement in memory consolidation following bead discrimination learning in day-old chick and draws parallels to mammalian learning, providing strong empirical support for the conclusion that the described neuronal-astrocytic interactions are universally valid. It identifies specific mechanisms whereby astrocytes support memory consolidation. Uptake of glucose, stimulated in astrocytes by beta(3)-noradrenergic receptor activation, provides energy by glycolytic/oxidative metabolism. Unlike neurons, astrocytes carry out net synthesis of tricarboxylic acid cycle intermediates needed for synthesis of transmitter glutamate formed by rapid degradation of glucose-derived glycogen and stimulated by beta(2)-noradrenergic receptor activation. This makes learning dependent on glycogenolysis and its stimulation by noradrenaline. Astrocytes take up most synaptically released glutamate, terminating transmitter activity and returning glutamate to neurons in a glutamate-glutamine cycle, interference with which abolishes learning. The various astrocytic activities follow a rigidly controlled time schedule, easily determined after bead discrimination learning but also detectable in other paradigms.

Adrenoceptors in brain: Cellular gene expression and effects on astrocytic metabolism and [Ca2+]i

Recent in vivo studies have established astrocytes as a major target for locus coeruleus activation (Bekar et al., 2008), renewing interest in cell culture studies on noradrenergic effects on astrocytes in primary cultures and calling for additional information about the expression of adrenoceptor subtypes on different types of brain cells. In the present communication, mRNA expression of alpha(1)-, alpha(2)- and beta-adrenergic receptors and their subtypes was determined in freshly isolated, cell marker-defined populations of astrocytes, NG2-positive cells, microglia, endothelial cells, and Thy1-positive neurons (mainly glutamatergic projection neurons) in murine cerebral cortex. Immediately after dissection of frontal, parietal and occipital cortex of 10-12-week-old transgenic mice, which combined each cell-type marker with a specific fluorescent signal, the tissue was digested, triturated and centrifuged, yielding a solution of dissociated cells of all types, which were separated by fluorescence-activated cell sorting (FACS). mRNA expression in each cell fraction was determined by microarray analysis. alpha(1A)-Receptors were unequivocally expressed in astrocytes and NG2-positive cells, but absent in other cell types, and alpha(1B)-receptors were not expressed in any cell population. Among alpha(2)-receptors only alpha(2A)-receptors were expressed, unequivocally in astrocytes and NG-positive cells, tentatively in microglia and questionably in Thy1-positive neurons and endothelial cells. beta(1)-Receptors were unequivocally expressed in astrocytes, tentatively in microglia, and questionably in neurons and endothelial cells, whereas beta(2)-adrenergic receptors showed tentative expression in neurons and astrocytes and unequivocal expression in other cell types. This distribution was supported by immunochemical data and its relevance established by previous studies in well-differentiated primary cultures of mouse astrocytes, showing that stimulation of alpha(2)-adrenoceptors increases glycogen formation and oxidative metabolism, the latter by a mechanism depending on intramitochondrial Ca(2+), whereas alpha(1)-adrenoceptor stimulation enhances glutamate uptake, and beta-adrenoceptor activation causes glycogenolysis and increased Na(+), K(+)-ATPase activity. The Ca(2+)- and cAMP-mediated association between energy-consuming and energy-yielding processes is emphasized.

Glycogen is a preferred glutamate precursor during learning in 1-day-old chick: Biochemical and behavioral evidence

Bead discrimination training in chicks sets in motion a tightly timed series of biochemical events, including glutamate release, increase in forebrain level of glutamate and utilization of glycogen and glucose. Inhibition of glycogen breakdown by the glycogen phosphorylase inhibitor 1,4‐dideoxy‐1,4‐imino‐D‐arabinitol (DAB) around the time of training abolishes the increase in glutamate 5 min posttraining in the left hemisphere, in spite of uninhibited glucose metabolism. It also reduces the contents of glutamate, glutamine, and aspartate in the right hemisphere. Behavioral evidence supports the conclusion that glucose breakdown serves to provide energy, whereas glycogen acts as a substrate for glutamate, glutamine, and aspartate formation, requiring both pyruvate dehydrogenation to acetyl coenzyme A and pyruvate carboxylation in astrocytes. Inhibition of memory consolidation caused by DAB or 2‐deoxyglucose (2‐DG), an inhibitor of glucose phosphorylation without effect on glycogen metabolism, was challenged by intracerebral administration of acetate, aspartate, glutamine, lactate or glucose. DAB‐mediated memory inhibition was successfully challenged by administration at 0 or 20 min posttraining of acetate (an astrocyte‐specific acetyl CoA precursor) together with aspartate, substituting for pyruvate carboxylation, or of glutamine at 0–2.5 or 30 min posttraining. 2‐DG‐mediated memory impairment was not challenged by acetate with or without aspartate at 0 time but was challenged by acetate without aspartate at 20 min. Lactate, a substrate for both dehydrogenation and pyruvate carboxylation challenged both DAB and 2‐DG. Doses of DAB and 2‐DG which, on their own were subeffective, were not additive, further supporting the existence of one pathway using glucose and another using glycogen.

Fluoxetine-mediated 5-HT2B receptor stimulation in astrocytes causes EGF receptor transactivation and ERK phosphorylation.

RationaleFluoxetine has relatively high affinity for Gq/11 protein-coupled 5-HT2 receptors. Part of these receptors in brain are on astrocytes, where fluoxetine causes an increase in free cytosolic calcium concentration ([Ca2+]i) and phosphorylation of extracellular regulated kinase 1 and 2 (ERK1/2).ObjectiveThe objectives of the study are to identify subtype of the 5-HT2 receptor involved, to establish whether ERK1/2 phosphorylation is a result of 5-HT2-mediated transactivation of epidermal growth factor (EGF) receptors (EGFRs), and to determine signaling pathways up- and downstream of ERK1/2.Materials and methodsPrimary cultures of mouse astrocytes, which express all three subtypes of the 5-HT2 receptor but no 5-HT2 transporter, were used. ERK1/2 phosphorylation and c-Fos and FosB protein expression were determined with Western blotting, and c-fos and fosB mRNA expression with reverse transcription polymerase chain reaction. Receptor subtype was investigated with subtype-specific 5-HT antagonists and 5-HT2B receptor depletion and signaling pathways by EGFR phosphorylation, using immunoprecipitation and Western blotting, inhibition of protein kinase C (PKC), and [Ca2+]i chelation by BAPTA/AM.ResultsERK1/2 phosphorylation was abolished by SB204741, a universal 5-HT2 receptor antagonist, and in 5-HT2B receptor-depleted cells, but unaffected by 5-HT2A or 5-HT2C receptor antagonists (M100907 and SB242084). Phosphorylation of ERK1/2 and EGFRs was abolished by AG 1478, an inhibitor of EGFR tyrosine kinases, and GM 6001, an inhibitor of Zn-dependent metalloproteinases, suggesting growth factor “shedding” and transactivation of EGFRs. Chelation of [Ca2+]i or PKC inhibition with GF 109203X abrogated ERK1/2 phosphorylation. Up-regulated mRNA and protein expression of c-fos and fosB was abolished by SB204741, AG1478, and by U0126, an inhibitor of ERK phosphorylation by MAP kinase/ERK kinase.