Brain Energy Metabolism

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Cell Metabolism 14, December 7, 2011 ©2011 Elsevier Inc. 724

Brain Energy Metabolism:Focus on Astrocyte-Neuron Metabolic CooperationMireille Belanger,1'3 Igor Ailaman,13 and Pierre J. Magistretti1,2 * 1Laboratory of Neuroenergetics and Cellular Dynamics, Brain Mind Institute, Ecole Polytechnique Federale de Lausanne,Lausanne CH-1015, Switzerland2Centre de Neurosciences Psychiatriques, CHUV, Departement de Psychiatrie, Site de Cery, Prilly/Lausanne CH-1008, Switzerland 3These authors contributed equally to this work 'Correspondence: [email protected] DOI 10.1016/j.cmet.2011.08.016

The energy requirements of the brain are very high, and tight regulatory mechanisms operate to ensure adequate spatial and temporal delivery of energy substrates in register with neuronal activity. Astrocytes—a type of glial cell—have emerged as active players in brain energy delivery, production, utilization, and storage. Our understanding of neuroenergetics is rapidly evolving from a “neurocentric” view to a more integrated picture involving an intense cooperativity between astrocytes and neurons. This review focuses on the cellular aspects of brain energy metabolism, with a particular emphasis on the metabolic interactions between neurons and astrocytes.

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725 Cell Metabolism 14, December 7, 2011 ©2011 Elsevier Inc.

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ReviewIntroductionGlucose is the obligatory energy substrate of the adult brain. Nevertheless, under particular circumstances the brain has the capacity to use other blood-derived energy substrates, such as ketone bodies during development and starvation (Nehlig, 2004; Magistretti, 2008) or lactate during periods of intense physical activity (van Hall et al., 2009). Glucose enters cells trough specific glucose transporters (GLUTs) and is phosphorylated by hexokinase (HK) to produce glucose-6-phosphate. As in other organs, glucose 6-phosphate can be processed via different metabolic pathways (Figure 1A), the main ones being(1) glycolysis (leading to lactate production or mitochondrial metabolism), (2) the pentose phosphate pathway (PPP), and (3) glycogenesis (in astrocytes only, see below). Overall, glucose is almost entirely oxidized to CO2 and water in the brain (Clarke and Sokoloff, 1999). Nevertheless, as evidenced by the different metabolic routes that glucose can follow, each individual brain cell does not necessarily metabolize glucose to CO2 and water. Indeed, a wide range of metabolic intermediates formed from glucose in the brain can subsequently be oxidized for energy production (e.g., lactate, pyruvate, glutamate, or acetate) (Zielke et al., 2009).

The brain has high energy requirements. About 20% of the oxygen and 25% of the glucose consumed by the human body are dedicated to cerebral functions, yet the brain represents only 2% of the total body mass. Maintenance and restoration of ion gradients dissipated by signaling processes such as postsynaptic and action potentials, as well as uptake and recycling of neurotransmitters, are the main processes contributing to the high brain energy needs (Attwell and Laughlin, 2001; Alle et al.,2009) . Among them, synaptic potentials, rather than action potentials, appear to represent by far the main energetic cost related to maintenance of excitability (Alle et al., 2009). Excitatory synapses largely dominate in the gray matter—glutamatergic ones alone represent at least 80% of cortical synapses—suggesting that excitatory neurotransmission accounts for most of the energy requirements at the cortical level. Correspondingly, it has been estimated that glutamate-mediated neurotransmission is responsible for most (~80%) of the energy expended in the gray matter (Sibson et al., 1998; Hyder et al., 2006; Shulman et al., 2004; Attwell and Laughlin, 2001), highlighting the close relationship between brain activity, glutamatergic neurotransmission, energy requirements, and glucose utilization.

A striking feature of brain energy metabolism is the tight coupling that exists between energy demand and supply (reflected by glucose and oxygen delivery from the vasculature). Indeed, task- dependent increases in cerebral activity are invariably accompanied by changes in local blood flow and glucose utilization—these processes being referred to as neurovascular and neurometabolic coupling, respectively (Figures 1B, 2B, and 2C). These close rela-tionships constitute the basis for functional brain imaging techniques which have enabled “functional mapping’’ studies in the brain. Among them, positron emission tomography (PET) allows determination of the cerebral metabolic rate of glucose consumption (CMRglc), the cerebral metabolic rate of oxygen consumption (CMRO2), or the cerebral blood flow (CBF), while functional magnetic resonance imaging (fMRI) mostly measures brain oxygenation and blood volume (Magistretti and Pellerin, 1996; Raichleand Mintun, 2006; Figleyand Stroman, 2011).

Since neurons account for most of the energy consumption

during brain activation, it was first rationally assumed that CMRglc measurements from 18F-fluoro-2-deoxyglucose (FDG)- PET signals directly reflected the neuronal use of glucose (Sokol off et al., 1977 ). In addition, neurometabolism was postulated to be a strictly oxidative (i.e., oxygen-depending) process—an assumption based on the higher efficiency to produce ATP from glucose oxidation in the mitochondria (oxidative process) compared to glycolysis (nonoxidative process; i.e., lactate producing) (Figure 1A).

In the mid-1980s, an important series of PET studies conducted by Fox and Raichle challenged this assumption, and led to a major breakthrough in our understanding of the mechanisms underlying task-induced increases in glucose metabolism. In awake adult humans, they observed that the activity-dependent


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(ii)Blood flow Oxygen utilization

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Figure 1. Brain Glucose Utilization(A) Schematic representation ofglucose metabolism. Glucoseenters cellstrough glucosetransporters (GLUTs) and is phosphorylated by HKto produce glucose- 6-phosphate (glucose-6P). Glucose-6Pcan be processed intothree main metabolic pathways. First, it can be metabolized through glycolysis(i), giving riseto two molecules of pyruvate and producing ATP and NADH. Pyruvate can then enter mitochondria, where it is metabolized through the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (Ox Phos), producing ATP and CO2 while consuming oxygen. Pyruvate can otherwise be reduced to lactate by lactate dehydrogenase (LDH). This lactate can be released in the extracellular space through monocarboxylate transporters (MCTs). The complete oxidation of glucose produces larger amounts of energy in the form of ATP in the mitochondria (30-34 ATP) compared to glycolysis (2 ATP). Alternatively, glucose-6P can be pro cessed through the pentose phosphate pathway (PPP) (ii), leading to the production of reducing equivalent in the form of NADPH. Note that the PPP and glycolysis are linked at the level of glyceraldehyde-3-phosphate (GA3P) and fructose-6-phosphate (fructose-6P). Finally, in astrocytes, glucose-6P can also be used to store glucosyl units as glycogen (iii). Abbreviations are as follows: GPI, glucose-6-phosphate isomerase; PFK, phosphofructokinase-1; Fructose-1,6-P2, fructose-1,6-bisphosphate; DHAP, dihydroxyacetone phosphate; TPI, triose phosphate isomerase; G6PDH, glucose-6-phosphate dehydrogenase; 6-PGL, 6-phosphoglucono-5-lactone; 6-PG, 6-phosphogluconate; 6 PGDH, 6-phosphogluconate dehydrogenase; ribulose-5P, ribulose-5-phosphate; ribose-5P, ribose-5-phosphate; xylulose-5P, xylulose-5-phosphate; TK, transketolase; sedoheptulose-7P, sedoheptulose-7-phosphate; TA, transaldolase; and erythrose- 4P, erythrose-4-phosphate.(B) Differential regulation ofCBF, CMRO2, and CMRglc during brain activation in human. Stimulation ofthe human visual cortexwas performed experimentally by presenting a visual stimulus in theform of a reversing annular chequerboard (i), and determination ofCBF, CMRO2, and CMRglc was obtained by PET (ii). When compared with viewing a blank screen, visual stimulation produces marked changes in activity in visual areas of the brain, as shown in PET images using pseudocolors (from small increase, blue, to major increase, red). Increases in both blood flow and glucose use in the visual cortex could be observed which are unaccompanied by similar increases in oxygen use, suggesting thatthe metabolic needsassociated with brain activation are in part met by anaerobic glycolysis. As a result, the local oxygen availability increases because the increased supply of oxygen by flowing blood exceeds the increased local demand for oxygen. Such effect accounts for the blood-oxygen-level-dependent (BOLD) signal of fMRI. Reproduced with permission from Gusnard and Raichle (2001).


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(visual or somatosensory) increases in blood flow and glucose utilization were only marginally matched by parallel increases in oxygen consumption (Fox and Raichle, 1986; Fox et al., 1988) (Figure 1B). Such uncoupling between CBF and CMRO2 created the rationale for developing blood oxygenation level- dependent (BOLD) fMRI contrast (Ogawaet al., 1992; Magistretti and Pellerin, 1996; Raichle and Mintun, 2006). These fundamental observations also brought support to the notion that the metabolic needs of active neural tissue are met, at least partially, by nonoxidative glucose metabolism (i.e., glycolysis). This hypothesis was then further supported by various 1H nuclear magnetic resonance studies showing activity-dependent increases in lactate levels in different brain areas (Figley and Stroman, 2011, and references therein), giving empirical demonstration that glycolytic metabolism increases with corresponding elevations in brain activity. It is now generally accepted that following transient changes in neural activity, (1) blood delivery increases with metabolic demand, (2) CBF and CMRglc

increase more than oxygen utilization, and (3) both oxidative and nonoxidative processes are involved to meet the increased metabolic requirements (Figley and Stroman, 2011).

These observations raised fundamental questions as to the molecular and cellular mechanisms that could reconcile the coexistence of increases in oxidative and nonoxidative glucose metabolism during synaptic activity. Specifically, the open questions were as follows: are the changes of glucose metabolism (oxidative versus glycolytic) taking place in different cell compartments; and to which precise cellular processes are they linked? The evidence pointing at a major contribution of astrocytes to neuroenergetics (Magistretti et al., 1981; Pellerin and Magistretti, 1994) has provided a key to the understanding of these questions.

Cytoarchitectural Organization of the BrainAstrocytes outnumber neurons in the human brain (Nedergaard et al., 2003). They play a key role in numerous functions of the


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ReviewFigure 2. Role ofAstrocytes inNeurovascular Coupling(A) Morphological features of astrocytes. (i)Astrocytic endfeet are in close contact with bloodvessels and almost entirely cover their surface(glial fibrillary acidic protein [GFAP] immunostain-ing [white] in the rat brain). Scale bar, 50 mm. Re-produced with permission from Kacem et al.(1998). (ii) Single astrocytes occupy virtuallydistinct territories and enwrap different parts ofthedendritic arborization of a single neuron. Recon-struction showing a biocytin-filled cortical neuron(red) and EGFP-expressing cortical astrocytes(green) in a mouse brain slice. Reproduced withpermission from Halassa et al. (2007). (iii) As-trocytic processes (in blue, marked by an asterisk)closely ensheathing a synapse as revealed byelectronic microscopy of mouse brain tissue (S,spine; and B, bouton). Scale bar, 0.5 mm. Repro-duced with permission from Genoud et al. (2006).(iv) 3D reconstruction ofasingle astroglial process(blue) interdigitating among many dendrites (fourare shown in gold, yellow, red, and purple) in therat hippocampus. Reproduced with permissionfrom Witcher et al. (2007).(B) Photolysis of caged Ca2+ in astrocytic endfeettriggers vasodilation. Time series images of thecross-section of a penetrating artery in mousebrain in vivo. Astrocytes were loaded with the Ca2+indicator dye rhod-2 AM (red) and with DMNP-EDTA AM, whereas the vasculature was stainedwith FITC-dextran (green). Ca2+ uncaging (DMNP-EDTA photolysis) triggered a rapid increase ofCa2+ in the astrocytic endfoot associated witha transient arterial vasodilation (basal arterialdiameter isdelineated byawhite dashed line). Thearrow indicates the position of photostimulation.Scale bar, 10 mm. Reproduced with permissionfrom Takano et al. (2006).(C) Schematic representation of the main path-ways by which astrocytes regulate CBF. Neuronalactivity-dependent glutamate release raises[Ca2+]i in astrocytes by activating metabotropicglutamate receptors (mGluR). Ca2+ transients inastrocytes activate cytosolic phospholipase A2(PLA2), thus producing arachidonic acid (AA). AAcan generate different types of vasodilatingmetabolites in astrocytes such as prostaglandins,in particular PGE2 (via cyclooxygenases) and ep-oxyeicosatrienoic acids (EETs) (by P450 epox-ygenases). In parallel, AA can diffuse to arteriolarsmooth muscle cells, where it is converted to 20-hydroxyeicosatetraenoic acid (20-HETE) by u-hydroxylase, which has a vasoconstricting effect.Low [O2], as observed during brain activation,promotes vasodilation through the increase of

___ ___ extracellular lactate and adenosine concentra-tions. Indeed, lactate favors PGE2 accumulation inthe extracellular space and thus vasodilation bydecreasing the efficacy of the prostaglandin

transporter(PGT) in astrocytes, whereas adenosine has an inhibitory effect on 20-HETE-mediated arteriolar constriction. These specific mechanisms aswell ascomplementary ones involving astrocytes and/or other cell types are reviewed in detail elsewhere (ladecola and Nedergaard, 2007; Carmignoto and Gomez-Gonzalo, 2010; Attwell et al., 2010).

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central nervous system, including glutamate, ion and water homeostasis, defense against oxidative stress, energy storage in the form of glycogen, scar formation, tissue repair, modulation of synaptic activity via the release of gliotransmitters, and synapse formation and remodeling (Belanger and Magistretti, 2009; Volterra and Meldolesi, 2005). Astrocytes possess unique cytoarchitectural and phenotypic features that ideally position them to sense their surroundings and dynamically respond to changes in their microenvironment. They extend two different

types of processes: fine perisynaptic processes that ensheath most synapses, and vascular processes (endfeet) that are larger in diameter and closely apposed onto intraparenchymal blood vessels (Figure 2A). Perisynaptic processes express a wide range of receptors for neurotransmitters, cytokines, and growth factors, as well as various transporters and ion channels. Among these, receptors and transporters for glutamate are of particular interest, since they may act as ‘‘sensors’’ of neuronal glutamatergic neurotransmission (as discussed below). Astrocytic


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endfeet almost entirely enwrap the intracerebral blood vessels (ladecola and Nedergaard, 2007; Kacem et al., 1998; Oberheim et al., 2009). Endfeet express several proteins at their luminal surface, including glucose transporters and aquaporin 4 (ladecola and Nedergaard, 2007). Interestingly, human astrocytes have been shown to present more complex morphological and functional features than their rodent counterparts (Oberheim et al., 2009), which suggests a phylogenetic evolution of astrocytic complexity. For example, human neocortical astrocytes are 2.6 times larger and extend 10 times more processes than mouse astrocytes (Oberheim et al., 2009).

One striking aspect of glial biology is that astrocytes are territorial cells: fine astrocytic processes ‘‘cover’’ a defined territory with little overlap between neighboring astrocytes (Oberheim et al., 2009; Halassa et al., 2007) (Figure 2A), thereby forming highly organized anatomical domains which are interconnected into functional networks via gap junctions (Giaume et al.,2010) . It has been demonstrated that a single mature rodent astrocyte in the gray matter associates with 300-600 neuronal processes (dendrites) (Halassa et al., 2007) and contacts an esti-mated 20,000-120,000 synapses (up to 2,000,000 in humans) (Oberheim et al., 2006, 2009).

Role of Astrocyte-Neuron Interactions in the Regulation of Cerebral Blood FlowThe morphological and phenotypical characteristics of astrocytes are thus tailored to ideally position them (among other important astrocytic functions) to sense neuronal activity at the synapse and respond with the appropriate metabolic supply via their astrocytic endfeet, which enwrap the intracerebral blood vessels. In line with this, an increasing body of evidence suggests that astrocytes play a key role in neurovascular coupling, or in other words, the spatiotemporal coupling between neuronal activity and increased CBF (also known as functional hyperemia).

Despite intense research, the exact physiological mechanisms underlying functional hyperemia are still incompletely understood. Over the past several decades, a variety of vasoactive agents, including H+, K+, neurotransmitters, adenosine, arachidonic acid metabolites, and nitric oxide (NO), released from different cellular compartments (i.e., neurons, astrocytes, pericytes, and smooth muscle cells), have been implicated in the activity-dependent increase in CBF (ladecola and Nedergaard, 2007; Gordon et al., 2008; Carmignoto and Gomez-Gonzalo, 2010; Attwell et al., 2010). Nevertheless, there is now strong in vitro and in vivo evidence indicating that astrocytes (mainly through neuron-to-astrocyte and glutamate signaling) are essential contributors to vasomotor responses, participating in both vasoconstriction and vasodilation (Carmignoto and Gomez- Gonzalo, 2010; Attwell et al., 2010; ladecola and Nedergaard, 2007; Takano et al., 2006). Interestingly, these opposite effects on the vascular tone were shown to involve different but parallel signaling cascades in astrocytes. Indeed, Ca2+

transients in astrocytes resulting from mGluR activation activate cytosolic phospholipase A2, triggering the formation of arachidonic acid which is converted into vasodilating agents such as epoxyeico- satrienoic acids (EETs) and prostaglandin E2 (PGE2) (Zonta et al., 2003; Takano et al., 2006; Attwell et al., 2010) (Figures 2B and 2C). Alternatively, arachidonic acid can diffuse in smooth muscle cells and cause vasoconstriction after its conversion in 20-hydroxyeicosatetraenoic acid (20-HETE) (Mulligan and MacVicar, 2004; Metea and Newman, 2006) (Figure 2C). Interestingly, Gordon

and collaborators demonstrated in rat brain slices that the balance between vasoconstriction and vasodilation may be dictated by brain metabolism. They observed that neuronal activation leads to arteriolar dilatation under low- oxygen conditions, whereas in high-oxygen conditions the same stimulus induces the constriction of arterioles (Gordon et al., 2008). The authors also demonstrated that increased astrocytic activity-reflected by increased astrocytic Ca2+

concentrations—led to the activation of glycolysis and an elevation of extracellular lactate and adenosine concentrations, particularly during lower-oxygen conditions. They further elucidated the underlying mechanisms by showing that high external lactate hinders PGE2 clearance (by attenuating the efficacy of the prostaglandin transporter), thus increasing extracellular PGE2, which induces arteriolar dilatation (Figure 2C). In addition, adenosine released under low oxygen inhibits astrocyte-mediated vasoconstrictions at the level of smooth muscle cells by blocking the effect of arachidonic acid (Figure 2C).

Although they were obtained in specific conditions (i.e., ex vivo preparations with oxygen tensions that are high compared to the ones measured in vivo) and remain to be confirmed in more physiological conditions, these observations suggest that under resting conditions when oxygen is not being rapidly consumed, astrocytic Ca2+ signals would induce a constricting tone, keeping CBF at an appropriate lower level. On the opposite, during activation, the drop in pO2 due to oxygen consumption (Ances et al., 2001; Offenhauser et al., 2005) and the rise in extracellular lactate and adenosine would promote astrocyte-mediated vasodilation. In support of this, it was recently observed that increased blood flow to focally active brain regions is highly correlated with elevations in lactate concentrations but negatively correlated with CMRO2, further suggesting that increased CBF is driven by glycolytic, as opposed to oxidative, metabolism under normal physiological conditions (Lin et al., 2010). Overall, astrocytes now appear as important players in the neurovascular unit, acting as intermediaries in neuronal signaling to blood vessels. This in turn firmly supports the notion that astrocytes are important actors of functional neuroimaging signals in relation to synaptic activity (Magistretti and Pellerin, 1996; Figley and Stroman, 2011).

Metabolic Specialization of Neurons and GliaWhile the brain is a high energy-consuming organ, it contains little energy reserves and is therefore highly dependent upon the uninterrupted supply of energy substrates from the circulation. Impairment in this process results in perturbation of neurological functions, loss of consciousness, and coma within minutes. As already mentioned, brain cells can efficiently utilize various energy substrates in addition to glucose, including lactate, pyruvate, glutamate, and glutamine (Zielke et al., 2009). Most of these metabolites are formed endogenously using glucose as the source of carbon, and—because cerebral metabolism is a compartmentalized process-complex intercellular trafficking of these metabolites occurs within the brain.

Among these substrates, lactate has been the center of much attention in recent years. Lactate is present in the extracellular space in concentrations similar to those of glucose (between 0.5 and 1.5 mM), and while it has long been considered a metabolic dead end, this view has drastically changed in light of the growing evidence indicating that it represents an important energy source for the brain (Schurr et al., 1999; Gallagher et al., 2009; Smith et al., 2003; Boumezbeur et al., 2010b). Interestingly, emerging evidence


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Importantly, both astrocytes and neurons have the capacity to fully oxidize glucose and/or lactate (Zielke et al., 2009), which is in accordance with the observation that both cell type processes contain equivalent numbers of mitochondria (Lovatt et al., 2007). However, as will be described in the following sections, neurons and astrocytes preferentially use different metabolic pathways in physiological conditions, which is in part due to cell type-specific expression patterns of key genes regulating energy metabolism (Lovatt et al., 2007; Herrero-Mendez et al., 2009; Vilchez et al., 2007). As a result, neurons and astrocytes present different (but complementary) metabolic profiles, paving the way for extensive metabolic cooperativity.

Metabolic Profile of NeuronsNeurons Rely on Oxidative Metabolism to Meet Their High Energy NeedsConsistent with their higher energy requirements, neurons sustain a high rate of oxidative metabolism compared to glial cells (Lebon et al., 2002; Itoh et al., 2003; Bouzier-Sore et al., 2006; Boumezbeur et al., 2010a). Interestingly, a large body of evidence shows that neurons can efficiently use lactate as an energy substrate (Schurr et al., 1997; Bouzier et al., 2000; Qu et al., 2000; Serres et al., 2005; Boumezbeur et al., 2010b) and even show a preference for lactate over glucose when both substrates are present (Itoh et al., 2003; Bouzier-Sore et al., 2006).

Recent evidence provides insights into the mechanisms underlying these neuronal features. Indeed, it has been reported that the enzyme 6-phosphofructose-2-kinase/fructose-2,6- bisphosphatase-3 (Pfkfb3) is virtually absent in neurons, due to its constant proteasomal degradation, which is in stark contrast with the high expression levels observed in astrocytes (Almeida et al., 2004; Herrero-Mendez et al., 2009). This enzyme is responsible for the generation of fructose-2,6-bisphosphate (fructose-2,6-P2), a potent activator of the glycolytic enzyme phosphofructokinase-1 (PFK). As a result of a low production of fructose-2,6-P2, neurons display a slower glycolytic rate and, unlike astrocytes, are unable to upregulate this pathway in response to NO-induced cellular stress (Almeida et al., 2004; Herrero-Mendez et al., 2009). Remarkably, the activation of neuronal glycolysis via Pfkfb3 overexpression leads to oxidative stress and apoptosis (Herrero-Mendez et al., 2009), suggesting that neurons cannot afford to sustain a high glycolytic rate. The authors showed that the increase in glucose flux via the glycolytic pathway occurs at the expense of metabolism through the PPP—which is essential for the production of NADPH and therefore for the maintenance of the cellular antioxidant potential (see below). While the exact contribution of the pentose phosphate and glycolytic pathways to neuronal glucose consumption remains to be directly established in normal conditions both in vitro and in vivo, it appears that a fine balance between the glycolytic pathway and the PPP has to be maintained in neurons in order to meet their energy needs while maintaining their antioxidant potential—both aspects being essential for their survival. Consistent with this, the use of lactate as an oxidative substrate may provide a convenient means for neurons to produce high amounts of ATP while circumventing the glycolytic pathway, thereby sparing glucose for the PPP (Bolanos et al.,

2010).

Metabolic Profile of Astrocytes Astrocytes Are Highly GlycolyticAlthough astrocytes display lower rates of oxidative metabolism compared to neurons, they avidly take up glucose and charac-teristically present a high glycolytic rate (Itoh et al., 2003; Her rero- Mendez et al., 2009; Bittner et al., 2010). A large portion of the glucose entering the glycolytic pathway in astrocytes is released as lactate in the extracellular space (Itoh et al., 2003; Bouzier-Sore et al., 2006; Lovatt et al., 2007; Pellerin and Magistretti, 1994; Serres et al., 2005). The glycolytic nature of astrocytes and their preference for the production and release of lactate over the entry of pyruvate in the tricarboxylic acid (TCA) cycle are the result of a specific gene expression profile which involves several enzymes and transporters acting in concert to produce this phenotype.

For instance, the substantial expression level of Pfkfb3 in astrocytes favors a high glycolytic rate via the allosteric activation of PFK by fructose-2,6-P2. Another feature of astrocytes, at the mitochondrial level, is the low expression levels of the aspartate/glutamate carrier (AGC), a component of the malate-aspartate shuttle which operates the transfer of reducing equivalents (i.e., NADH) from the cytosol to the mitochondria (Berkich et al., 2007; Ramos et al., 2003). In this context, the conversion of glycolytically derived pyruvate to lactate in the cytosol provides a means by which to ensure the maintenance of a high NAD+/ NADH ratio, which is essential to sustain a high glycolytic rate (Figure 1A). Finally, the conversion of pyruvate to lactate in astrocytes may also be favored by low expression levels (Laughton et al., 2007) and/or phosphorylation-mediated inactivation (Itoh et al., 2003; Halim et al., 2010) of pyruvate dehydrogenase, a key enzyme regulating the entry of pyruvate in the TCA cycle. Astrocytes Play a Key Role in Neurotransmitter Recycling and AnaplerosisOne of the best-characterized functions of astrocytes is the rapid removal of neurotransmitters released into the synaptic cleft, an essential process for the termination of synaptic transmission and maintenance of neuronal excitability. This is especially critical for glutamate, the primary excitatory neurotransmitter in the brain, as overstimulation of glutamate receptors is highly toxic to neurons (a phenomenon referred to as excitotoxicity). Glutamate uptake is primarily achieved by the astrocyte-specific sodium-dependent high-affinity glutamate transporters glutamate transporter 1 (GLT-1) and glutamate aspartate transporter (GLAST) (corresponding to human EAAT2 and EAAT1, respectively) (Bak et al., 2006). Following glutamate uptake via these transporters, astrocytes also play an important role in transferring this neurotransmitter back to neurons, which is important to maintain the neuronal neurotransmitter pool of glutamate. This transfer is achieved by a process called the

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A Neurons Astrocyte Capillary

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Figure 3. Astrocyte-Neuron Metabolic Interactions(A) Schematic representation of the astrocyte-neuron lactate shuttle (ANLS).

Glutamate (Glu) released at the synapse activates

glutamatergic receptors (GluR) and is associated with important energy expenditures in neuronal compartments. A large proportion of the glutamate released at the synapse is taken up by astrocytes via excitatory amino acid transporters (EAATs, more specifically GLT-1 and GLAST) together with 3 Na+ ions. This Na+ is extruded by the action of the Na+/K+ ATPase, consuming ATP. This triggers nonoxidative glucose utilization in astrocytes and glucose uptake from the circulation through the glucose transporter GLUT1 expressed by both capil lary endothelial cells and astrocytes. Glycolytically derived pyruvate is converted to lactate by lactate dehydrogenase 5 (LDH5; mainly expressed in astrocytes) and shuttled to neurons through monocarboxylate transporters (mainly MCT1 and MCT4 in astrocytes and MCT2 in neurons). In neurons, this lactate can be used as an energy substrate following its conversion to pyruvate (Pyr) by LDH1 (mainly expressed in neurons). Neurons can also take up glucose via the neuronal glucose transporter 3 (GLUT3). Concomitantly, astrocytes participate in the recycling of synaptic glutamate via the glutamate-glutamine cycle. Following its uptake by astrocytes, glutamate is converted to glutamine (gln) by the action of glutamine synthetase (GS) and shuttled to neurons, where it is converted back to glutamate by gluta- minases (GLS).(B) Glial glutamate transporters play a central role in neurometabolic coupling. Glucose utilization (as assessed by the 2-deoxyglucose [2-DG] uptake technique) was measured in the somatosensory cortex of P10 GLAST mutant mice following unilateral whisker stimulation. Representative pseudocolored digitized autoradiograms of anteroposterior coronal sections at the level of the somatosensory barrel field are shown. Whisker stimulation in GLAST+/+ mice produced a local increase in glucose utilization in the activated somatosensory cortex (i, white square). Such an increase in glucose utilization was not observed in coronal sections from GLAST'~ mice (ii, white square). Similar

(C) glutamate-glutamine cycle (Bak et al., 2006; McKenna, 2007) (Figure 3A). In short, glutamate is converted to glutamine by the astrocyte-specific enzyme glutamine synthetase. Glutamine is then transferred to neurons and converted back to glutamate via deamination by phosphate-activated glutaminase. It is important to note that glutamate can also be metabolized in a number of different pathways in astrocytes and neurons, including oxidation in the TCA cycle (McKenna, 2007). Astrocytes are responsible for the replenishment of brain glutamate, as they are the only neural cell type expressing pyruvate carboxylase, a key enzyme in the main

anaplerotic pathway in the brain, effectively allowing them to synthesize glutamate from glucose (Magistretti, 2008).

The marked differences in the metabolic profile of astrocytes and neurons strongly support the notion that brain energy metabolism must be, at least to a certain extent, a compartmentalized process. Importantly, the metabolic phenotypes of these two cell types appear to be largely complementary.

The Astrocyte-Neuron Lactate ShuttleAstrocytes are generally considered to account for only 5%-15% of the brain’s energy expenditure (Attwell and Laugh- lin, 2001; Magistretti, 2008). Recent evidence suggesting that the energetic cost of action potentials is lower that previously proposed (Alle et al., 2009) leaves open the possibility that the contribution of astrocytes to the overall brain energetic costs may have been underestimated. Nevertheless, experimental evidence demonstrates that the amount of glucose that astrocytes actually take up is disproportionally high in comparison to their energy requirements. For example, in acute cerebellar slices, the uptake of fluorescent glucose analogs is severalfold higher in Bergmann glia than in Purkinje cells (Barros et al., 2009). In the resting rat brain, other studies have shown that astrocytes are responsible for approximately half of glucose uptake (Nehlig et al., 2004; Chuquet et al., 2010), and that this proportion increases even further upon functional activation (Chuquet et al., 2010).

How can these data be reconciled with the fact that neurons— not astrocytes—have the highest energy needs? The transfer of energy substrates from astrocytes to neurons could provide a simple explanation for this apparent paradox. This is a central point of the astrocyte-neuron lactate shuttle (ANLS) model proposed over a decade ago by Pellerin and Magistretti (Fig ure 3 A) (Pellerin and Magistretti, 1994). The essence of this model

results were obtained in GLT-1 mutant mice. Reproduced with permission from

Voutsinos-Porche et al. (2003).(D) Astrocytes play an important role in glutathione metabolism in the brain. The tripeptide glutathione (GSH) is synthetized by the successive actions of glutamate cysteine ligase (GCL) and GSH synthetase. Astrocytes can efficiently use oxidized cysteine (cystine) for GSH synthesis. Astrocytes release a large amount of GSH in the extracellular space, where it is cleaved by the astrocytic ectoenzyme g-glutamyl transpeptidase (gGT). The resulting dipeptide CysGly is cleaved bythe neuronal ectopeptidase aminopeptidase N (ApN), forming cysteine (Cys) and glycine (Gly), which serve as precursors for neuronal GSH synthesis. GSH is an electron donor in many reactions in both neurons and astrocytes, including the detoxification of ROS (e.g., the reduction of peroxides [ROOH] by glutathione peroxidase [GPx]). The oxidized glutathione (GSSG) formed as a result is recycled back to glutathione by

- Cystine

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the action of glutathione reductase (GR) using NADPH as an electron donor. X represents an acceptor for the g-glutamyl moiety (gGlu) in the reaction catalyzed by gGT.

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is that (1) neuronal activity increases extracellular glutamate (via glutamatergic neurotransmission), which is avidly taken up via a Na+-dependent mechanism by specific glial glutamate transporters; (2) the resulting increase in [Na+]i activates the Na+/K+ ATPase (in particular by mobilizing its alpha2 subunit), thereby increasing ATP consumption (Magistretti and Chatton, 2005), glucose uptake, and glycolysis in astrocytes; (3) this in turn leads to a large increase in the production of lactate which is released in the extracellular space; and (4) lactate can be used as an energy substrate for neurons for oxidative-derived ATP production (for review, see Pellerin et al., 2007; Magistretti, 2009).

The concept of a lactate shuttle—which was first introduced by Brooks, who described the transfer of lactate between glycolytic and oxidative muscle fibers as early as the mid-1980s—has now been extended to several other biological systems and includes intracellular lactate shuttles, cell-cell lactate shuttles, and interorgan lactate shuttles (see Brooks, 2009 for review). In brain cells, early experimental evidence suggesting a role of astrocytes in coupling glutamatergic transmission and energy metabolism via a lactate shuttle was obtained in vitro by Pellerin and Magistretti (1994), who have shown that glutamate uptake into astrocytes increases glucose utilization and lactate release from these cells. The stimulation of astrocytic glucose utilization by glutamate was subsequently confirmed by several independent groups (reviewed in Pellerin et al., 2007; Barros and Deitmer, 2010). Evidence that such a mechanism also occurs in vivo during functional brain activation was provided by the demonstration that reduced expression of astrocyte-specific glutamate transporters strongly impairs the activity-dependent increase in glucose utilization (Cholet et al., 2001; Voutsinos- Porche et al., 2003; Herard et al., 2005) (Figure 3B). More recently, a real-time two-photon microscopy study has shown that the fluorescent glucose analog 6-[N-(7-nitrobenz-2-oxa- 1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (6-NBDG) is preferentially taken up in astrocytes rather than in neurons in the rat barrel cortex following whisker stimulation (Chuquet et al., 2010).

Interestingly, following exposure to glutamate in mixed cultured cells, the stimulation of glucose utilization in astrocytes is concomitant with a rapid inhibition of glucose transport in neurons, which is more pronounced in the presence of lactate (Porras et al., 2004). In other words, following glutamatergic activity, there is a shift of glucose utilization away from neurons and toward astrocytes, suggesting that an energy substrate other than glucose must be used by neurons to meet the increased energy requirements associated with activation. According to the redox switch hypothesis (Cerdan et al., 2006), the inhibition of glucose transport (and consequently of glycolysis) induced by glutamate in neurons would create thermodynamically favorable conditions for the use of lactate as an energy substrate in this cell type, since these two processes compete for intracellular NAD+ (Figure 1A).

Using hippocampal slice preparations, it has been possible to obtain some evidence for a spatial and temporal compartmentation of energy metabolism, by monitoring changes in NADH levels by means of two-photon microscopy. In these preparations, activation of the intrahippocampal Schaffer collateral pathway induced a biphasic NADH fluorescence response which consisted of a rapid and transient decrease of NADH levels in neuronal dendrites, reaching a minimum 10 s after stimulation, followed by a sustained overshoot of NADH levels in astrocytic processes, peaking at 60 s (Kasischke et al., 2004). These observations suggest that, following

stimulation, the rapid activation of oxidative metabolism in neurons causes a decrease in NADH levels, which rapidly recovers following the activation of the TCA cycle; in contrast, astrocytes display a delayed but sustained increase of their glycolytic rate (Kasischke et al., 2004). Although their study did not identify the substrates used by neurons to produce NADH, it is interesting to note that the sequence of events observed by Kasischke and collaborators closely parallels the temporal changes in extracellular lactate levels observed in vivo, i.e., a transient decrease in extracellular lactate levels after stimulation both in the human and rodent brain (Hu and Wilson, 1997; Mangia et al., 2003) followed by a sustained increase in extracellular lactate (Prichard et al., 1991; Hu and Wilson, 1997; Lin et al., 2010). Together, these studies suggest that as a result of neuronal activation, extracellular lactate is rapidly oxidized by neurons to meet their energy needs, a process closely followed by a sustained activation of the glycolytic pathway in astrocytes, allowing for the replenishment of the extracellular lactate pool. It should be mentioned, however, that a recent report using two-photon imaging in vivo was unable to demonstrate similar cell-specific changes of NADH intensity in the cortex of mice following focal activation (Kasischke et al.,2011) . Further studies are warranted to clarify this issue.

Additional support for the shuttling of lactate from astrocytes to neurons and its use by the latter as an energy substrate was recently provided by an elegant study by Rouach and collaborators (2008). Results of this study demonstrated that during glucose deprivation—which resulted in a 50% depression of synaptic transmission in hippocampal slices—glucose delivery into a single astrocyte and its subsequent diffusion through the astrocytic syncitium could rescue neuronal activity. This effect was mimicked by lactate but was abolished in the presence of the monocarboxylate transporter (MCT) inhibitor a-cyano-4- hydroxycinnamic acid, suggesting that glucose present in the astrocytic network is metabolized to lactate, transported out of astrocytes, and used by neurons to sustain their synaptic activity (Rouach et al., 2008).

Interestingly, it has also been demonstrated in primary cultured astrocytes that electrical or mechanical stimulation triggers ‘‘metabolic waves,’’ in addition to the well-described Ca2+ waves that travel through the astrocytic network (Bernardinelli et al., 2004). These metabolic waves are mediated by the Ca2+-dependent release of glutamate, which in turn triggers a Na+ wave in the astrocytic network resulting from glutamate uptake via Na+-dependent glutamate transporters. Remarkably, the propagating Na+ signal is spatially and temporally correlated with an increase in glucose uptake, which is also dependent on the activity of astrocytic glutamate transporters. Such metabolic waves could mediate the propagation of the metabolic response within the astrocytic network away from the site of activation, allowing for a concerted neurometabolic coupling within the activated area (Bernardinelli et al., 2004).

By coupling glucose utilization (via astrocyte glycolysis) to neuronal activity and oxidative phosphorylation, the ANLS is consistent with previous studies that have implicated a nonoxidative metabolic component during focal brain activation (see the Introduction and Fox et al., 1988). Moreover, these data are consistent with the view that glucose PET signals actually mainly reflect glucose consumption in astrocytes rather than in neurons (Magistretti and Pellerin, 1996; Chuquetetal., 2010; Fig- ley and Stroman, 2011). This in turn raises important questions about the

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interpretation of PET data showing modifications of glucose consumption, as observed in Alzheimer’s disease patients, for example (e.g., should it be attributed to a neuronal or an astrocytic component, or both) (Mosconi et al., 2008).

It is important to note, however, that the ANLS does not exclude the possibility that some of the energy needs of the brain are met by the astrocytic TCA cycle and by the glycolytic pathway in neurons. In this regard, it should be mentioned that there are still divergent point of views concerning some aspects of the ANLS (Simpson et al., 2007; Mangia et al., 2009; DiNuzzo et al., 2010; Chih and Roberts, 2003). In particular, based mainly on modeling studies, some reports suggest a higher glucose transport capacity in neurons compared to astrocytes (DiNuzzo et al., 2010; Mangia et al., 2009; Simpson et al., 2007). From this prediction, neurons were considered to be mainly (if not exclusively) dependent on glucose uptake, as opposed to astrocytic lactate delivery, to fuel their energy needs in both resting and activating states. This theoretical conclusion based on modeling studies is, however, inconsistent with the observation that mice presenting a haploinsufficiency of the neuron-specific glucose transporter GLUT3 do not present any neurological or brain energy metabolism abnormalities. Furthermore, their brain glucose utilization is not different from wild-type animals (Schmidt et al., 2008; Stuart et al., 2011). In contrast, haploinsufficiency for GLUT1, which is absent in neurons, results in a severe neurological phenotype (Wang et al., 2006). A further critical discussion about the ANLS has been presented in Jolivet et al. (2010).

Overall, a considerable body of evidence supports the notion of a net transfer of energy from astrocytes to neurons in the form of lactate as a result of glutamatergic transmission, as proposed by the ANLS.

Astrocytic Glycogen MetabolismDespite its relatively low level in the CNS compared to peripheral tissues, glycogen is the largest energy reserve of the brain. It represents an advantageous form of glucose storage, as it can be rapidly metabolized without requirements for ATP, and unlike fatty acids it can yield ATP under anaerobic conditions. Interestingly, at the cellular level, glycogen has been found to be almost exclusively localized in astrocytes in the adult brain (Magistretti et al., 1993; Brown, 2004). Although neurons express the enzymatic machinery for synthesizing glycogen (i.e., glycogen synthase) both in vitro and in vivo (Brown, 2004; Vilchez et al., 2007), recent evidence shows that this machinery is kept inactive in neurons through proteasomal-dependent mechanisms (Vilchez et al., 2007). More surprisingly, manipulations that drive glycogen deposition in cultured neurons cause apoptosis (Vilchez et al., 2007), further demonstrating that glycogen constitutes a specific feature of astrocytic glucose metabolism. The restricted cellular localization of glycogen raises an obvious question: why is the main energy reserve of the brain found in astrocytes instead of neurons, which are the most energy-requiring neural cell type? This suggests that glycogen metabolism may involve metabolic interactions between astrocytes and neurons. A first set of evidences was presented in the early 1980s by the demonstration that a restricted set of neurotransmitters, i.e., neuronal-derived molecules, could promote glycogenolysis in cortical slices and primary astrocyte cultures (Magistretti et al., 1981,1983). Furthermore, studies showing that increasing astrocytic glycogen stores preserves neuronal function and viability in conditions of limited energy availability, such as hypoglycemia,

provided additional evidence for neuron-glia metabolic coupling involving glycogen (Brown and Ransom, 2007; Brown, 2004; and references therein). While glycogen mobilization may also fulfill the astrocytes’ own metabolic needs (Sickmann et al., 2009; Walls et al., 2009), glycogen breakdown typically results in lactate production and release in the extracellular space (Walls et al., 2009; Dringen et al., 1993). Considering the role of astrocytic glucose-derived lactate in fuelling neuronal energy needs (described above in the ANLS section), this positions lactate as a likely candidate to account for the protective effects of glycogen. In support of this, axonal function was demonstrated to be preserved during aglycemia through the transfer of glycogen-derived lactate from astrocytes to axons in an optic nerve preparation (Tekkok et al., 2005; Brown et al., 2005).

In addition to its role as an emergency energy reserve, there is compelling evidence indicating other important roles for glycogen in normal brain functions. A physiological role of brain glycogen was first suggested on the basis of the following observations: (1) glycogen content is under the dynamic control of neurotransmitters (e.g., noradrenaline, serotonin, and vasoactive intestinal peptide [VIP]), neurohumoral agents (e.g., insulin), and local energy status (e.g., glucose levels) (Magistretti et al., 1981, 1993; Brown, 2004); (2) decreased neuronal activity, as observed during sleep and anesthesia, correlates with increased levels of brain glycogen, consistent with the notion that the awake brain utilizes glycogen (Magistretti etal., 1993; Brown, 2004); and (3) increased neuronal activity induced by sensory stimulation is associated with a decrease in glycogen levels in the activated areas, demonstrating a tight coupling between neuronal activity and glycogen mobilization (Swanson et al., 1992; Dienel et al., 2007). It was also demonstrated that astrocytic glycogen mobilization (likely associated with the transfer of lactate from astrocytes to neurons) is required to sustain neuronal activity during intense stimulation in a mouse optic nerve preparation (Brown et al., 2005; Tekkok et al., 2005) and to maintain glutamatergic synaptic transmission (i.e., neurotransmitter release) in coculture models (Sickmann et al., 2009; Mozrzymas et al., 2011).

A demonstration of the implication of glycogen metabolism in higher brain functions was first obtained by Gibbs and collaborators, who demonstrated that pharmacological inhibition of glycogenolysis in astrocytes (using 1,4-dideoxy-1,4-imino-D- arabinitol [DAB], a potent inhibitor of glycogen phosphorylase) interrupts memory consolidation in young chickens in a bead discrimination learning task (Gibbs et al., 2006). Very recently, the involvement of astrocytic glycogen-derived lactate in longterm memory formation, and for the in vivo maintenance of long-term potentiation (LTP) of synaptic strength in the mammalian brain, was demonstrated by Suzuki and colleagues (Suzuki et al., 2011). In this study, it was observed that intrahippocampal injection of DAB specifically impairs long-term memory formation (while leaving unaffected short-term memory) in an inhibitory avoidance test in rats (Figure 4A). Among other evidence, it was shown that this effect can be rescued by the addition of

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741 Cell Metabolism 14, December7, 2011 ©2011 Elsevier Inc.


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B

DABSTM

T Impaired ImpairedLTM LTM

DAB + * Intact IntactL-Lactate r\ LTM LTM

MCT4 1 Impaired Impairedantisense N LTM LTM

MCT4 Intact Intactantisense LTM LTM

MCT2 N Impaired Impairedantisense G LTM LTM

MCT2 L-Lactate Impaired Impairedantisense LTM LTM

EXPERMENTS

Figure 4. Mobilization of the Astrocytic Glycogen Stores Is Necessary for Long-Term Memory Formation in Rats(A) Summary ofthe main findings reported bySuzuki et al. (2011). Rows represent individual experiments, and columns indicatetreatments and results obtained. Prior to inhibitory avoidance training, rats received intrahippocampal injections of 1,4-dideoxy-1,4-imino-D-arabinitol (DAB, a potent inhibitor of glycogen phosphorylase), lactate, or antisense oligodeoxynucleotides, as indicated. Short-term memory (STM) was assessed 1 hr later (experiment 1), while long-term memory (LTM) formation was assessed after 1 and 7 days (experiments 2-7). Results and conclusion are given for each individual experiment. Note that monocarboxylate transporter (MCT)-1 knockdown produced similar results to those obtained with MCT4 knockdown (experiments 4 and 5).(B) Schematic representation of the cellular mechanisms involving glycogen mobilization in long-term memory formation. Synaptic activity associated with a learningtask(1)triggersastrocyticglycogen breakdown (2) andthe release oflactatevia MCT1 and MCT4 (3) in rat hippocampus. Lactate istaken up by neurons via MCT2 (4) and triggersan increase in the expression ofthe activity-dependent geneArc as well asthe phosphorylation ofCREB (a nucleartranscription factor) and cofilin (an actin-binding protein) (5), which are all known to contribute to memory consolidation processes. These events are likely involved in the structural synaptic changes necessary for the establishment of long-term memory (6). The mechanisms by which lactate causes these effects are yet to be determined (indicated by question marks).

exogenous lactate in normal conditions, but not when the expression of the main neuronal lactate transporter (MCT2) was disrupted using antisense oligodeoxynucleotides, demon

CONCLUSIONS

Glycogen mobilization is not necessary for shortterm memoryGlycogen mobilization is necessary for long-term memoryLactate can rescue long-term memory when glycogen mobilization is inhibited Monocarboxylate transport through MCT4 (expressed almost exclusively in astrocytes)plays an essential role in long-term memory formation Lactate can rescue long-term memory when monocarboxylate transport through MCT4 isimpaired __________________________Monocarboxylate transport through MCT2 (expressed almost exclusively in neurons) plays an essential role in long-term memory formation Extracellular lactate cannot rescue long-term memory formation when monocarboxylate transport through MCT2 is impaired, suggesting that the import of lactate in neurons is essential for this process_________________________________

NEURON

L-Lactate

MCT2

CREB ^Transcriptioi

Structural synaptic changes

LTM formation

Synaptic activity (learning) (7}

strating the importance of glycogen-derived lactate transport into neuronsfor memory consolidation (Figure 4A). Interestingly, glycogenolysis and astrocytic lactate transporters were also


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shown to be critical for the induction of molecular changes required for memory formation, including the induction of phospho-cAMP response element-binding (CREB), activity-regulated cytoskeletal-associated protein (Arc), and phospho-cofilin (Su zuki et al., 2011), suggesting that lactate may also act as a signaling molecule in this context (Figure 4B).

As a whole, these observations demonstrate that astrocytic glycogen is more than a simple emergency reserve, and plays an important and active role in complex brain physiological functions, in particular through an astrocyte-to-neuron transfer of energy metabolites in the form of lactate. It thus appears that both glucose- and glycogen-derived lactate are important to sustain neuronal function, as postulated by the ANLS.

The Role of Astrocyte-Neuron Interactions for the Defense against Oxidative StressThe brain is particularly susceptible to oxidative damage. This is illustrated by the fact that oxidative injury is a key feature of several neuropatholological conditions such as stroke, traumatic brain injury, and neurodegenerative diseases (including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis) (Wilson, 1997; Dringen, 2000). Several factors contribute to increased brain vulnerability to oxidative stress, including its high rate of oxidative energy metabolism (a process inevitably generating reactive oxygen species, ROS, as a byproduct), its high unsaturated fatty acids content (which are prone to lipid peroxidation), and its relatively low intrinsic antioxidant capacity (Dringen, 2000). Paradoxically, despite the fact that the greater part of the brain’s oxidative metabolism occurs in neurons, this cell type displays limited defense mechanisms against oxidative stress compared to astrocytes. Indeed, astrocytes have significantly higher levels of various antioxidant molecules and ROS-detoxifying enzymes, including glutathione, heme-oxygenase 1, glutatione peroxidase, glutathione S-transferase, catalase, and thioredoxin reductase (Shih et al., 2003; Dringen, 2000; Belanger and Magistretti, 2009; Wilson, 1997). The greater antioxidant potential of astrocytes is further evidenced by the fact that they are much more resistant than neurons to cellular damage induced by pro-oxidant compounds such asH2O2, NO, peroxinitrites,and6-hydroxydopamine (Wilson, 1997; Dringen, 2000; Almeida et al., 2001). Importantly, in coculture systems, astrocytes also protect neighboring neurons from toxic doses of NO, H2O2, superoxide anion combined with NO, iron, or 6-hydroxydopamine (Wilson, 1997; Vargas and Johnson, 2009; Dringen, 2000; Belanger and Magistretti, 2009), suggesting that neurons are dependent upon the high antioxidant potential of astrocytes for their own defense against oxidative stress.

The shuttling of glutathione (GSH) precursors from astrocytes to neurons appears to be instrumental in the neuroprotective effects of astrocytes (Figure 3C). GSH—the most abundant antioxidant molecule in the brain—acts either directly as a ROS scavenger or can be used as a substrate for glutathione S-transferase or glutathione peroxidase (Dringen, 2000). Both neurons and astrocytes can synthesize the GSH tripeptide (g-L-glutamyl- L-cysteinylglycine); however, neurons are highly dependent on astrocytes for the supply of the precursor amino acids necessary for their own GSH synthesis. This is especially crucial for cysteine, the rate-limiting substrate for GSH synthesis, since neurons, unlike astrocytes, have a poor capacity to use extracellular cystine (the oxidized form of cysteine) as a precursor (Dringen, 2000). Cultured

astrocytes release large amounts of GSH into the extracellular space—up to 10% of their total content within 1 hr (Dringen, 2000). GSH is then cleaved by the astrocytic ectoenzyme g-glutamyl transpeptidase (gGT) to produce CysGly, which in turn is cleaved by the extracellular neuronal aminopeptidase N, producing glycine and cysteine readily available to neurons for their own GSH synthesis (Dringen, 2000). As a result of this shuttling process, the GSH content of neurons is strongly increased when they are cultured in the presence of astrocytes (Dringen, 2000). In contrast, GSH- depleted astrocytes display a reduced ability to protect neurons against oxidative injury, highlighting the importance of this coop-erative mechanism for neuronal defense against oxidative stress (Chen et al., 2001; Shih et al., 2003). The differential antioxidant response of neurons and astrocytes is in part explained by the preferential astrocytic expression and activation of the nuclear factor erythroid-2-related factor 2 (Nrf2), a redox-sensitive transcription factor playing a key role in orchestrating the cellular antioxidant response (Vargas and Johnson, 2009).

Relationship between Antioxidant Defense Mechanisms and Energy MetabolismThe reduction of peroxides catalyzed by glutathione peroxidase generates glutathione disulfide (GSSG, the oxidized form of GSH). GSH can subsequently be regenerated from GSSG by the action of the enzyme glutathione reductase, using NADPH as an electron donor (Dringen, 2000). This process is essential for the maintenance of GSH in its reduced form, and thus for its availability for the detoxification of ROS. As a consequence, constant NADPH supply is essential for the maintenance of the cellular redox state. NADPH production is mainly achieved by the metabolism of glucose via the PPP, thereby coupling glutathione recycling to glucose utilization. Interestingly, it has been demonstrated that NADPH is more abundant in astrocytes than in neurons, and that astrocytes have a higher basal PPP activity rate and a better capacity to stimulate this pathway in response to oxidative stress (Garcia-Nogales et al., 2003; Ben Yoseph et al., 1996). Consistent with this, transcriptome analysis in astrocytes and neurons freshly isolated from the mouse brain also reveal higher expression levels of glucose-6-phosphate dehydrogenase, the rate-limiting enzyme of the PPP, in astrocytes compared to neurons (Lovatt et al., 2007; Cahoy et al., 2008). These features also contribute to the higher antioxidant potential of astrocytes.

An ascorbic acid metabolic switch has been proposed as another level of interaction between antioxidant mechanisms and neuroenergetic pathways (Castro et al., 2009). According to this model, astrocytes release the antioxidant molecule ascorbic acid in response to glutamatergic activity. This ascorbic acid is taken up by neurons and modifies local energy metabolism by inhibiting glucose consumption and increasing lactate uptake (Castro et al., 2009). Thus, in addition to contributing to the antioxidant capacity of neurons, ascorbic acid shuttling may provide another mechanism by which neuronal lactate utilization is favored (over glucose) in order to meet the energy needs associated with glutamatergic activation (as proposed by the ANLS).Metabolic Plasticity in Astrocytes as a Protective MechanismA large body of experimental evidence suggests that astrocytes have a greater metabolic plasticity than neurons, i.e., they can better adapt their energy metabolism to face various cellular chal-lenges. A striking example is the differential response of astrocytes and neurons following the inhibition of mitochondrial respiration


CellCell Metabolism

Reviewinduced by NO. Astrocytes respond to NO with an increase in glucose metabolism through the glycolytic pathway, thereby limiting the fall in ATP levels and preventing apoptosis. In neurons, however, this response does not seem to be present, and a similar NO challenge causes a massive ATP depletion, leading to apoptosis (Almeida et al., 2001). Another strong indication of the limited metabolic plasticityof neurons is provided by recent studies which have shown that driving a higher glycolytic rate (Herrero- Mendez et al., 2009) or glycogen synthesis (Vilchez et al., 2007) in neurons causes apoptotic cell death, even though these pathways are generally innocuous (or even beneficial) in astrocytes.

While the metabolic plasticity of astrocytes is an important feature for their homeostatic and neuroprotective functions in physiological conditions, it may become a double-edged sword in pathological conditions. Indeed, considering the extensive functional cooperativity taking place between neurons and astrocytes, any significant alteration of astrocytic pathways caused by pathological stimuli could potentially contribute to neuronal dysfunction. In support of this, our group has reported that the internalization by astrocytes of amyloid-beta (Ap), a peptide associated with Alzheimer’s disease, induces profound changes in their metabolic phenotype, in terms of both energy metabolism and oxidative status. Exposure of naive neurons to these “Ap-filled” astrocytes resulted in neuronal cell death (Allaman et al., 2010). The contribution of astrocytic dysfunction to neurodegenerative processes is currently under intense investigation, and a rapidly growing body of evidence indicates that enhancing or reestablishing astrocytic functions may represent promising therapeutic avenues for neurodegenerative disease (see Allaman et al., 2011, for review).

Astrocyte-Neuron Energetic Interactions Influence Neuronal ExcitabilityAstrocytes are known to modulate synaptic transmission by the Ca2+-dependent release of neuroactive molecules (called gliotransmitters) such as glutamate, D-serine, ATP, or adenosine. Description of these mechanisms is beyond the scope of this article, but they have been reviewed by several authors (see, e.g., Volterra and Meldolesi, 2005). In addition to this, there are now strong evidences demonstrating that astrocyte-neuron interactions at the energy metabolism level (in particular through lactate release by astrocytes) also influence higher brain functions (Ainscow et al., 2002; Lam et al., 2005; Shimizu et al., 2007; Erlichman et al., 2008; Parsons and Hirasawa, 2010; Suzuki et al., 2011). We have already described the role of astrocytic glycogen breakdown and associated lactate release for long-term memory formation (Suzuki et al., 2011). Here we will provide additional examples illustrating the role of astrocyte-derived lactate in controlling neuronal activity.

(1) Glucose-sensing neurons within satiety and feeding centers of the hypothalamus increase their firing rate in response to elevation of central glucose levels, ultimately resulting in decreased blood glucose and insulin levels and the suppression of hepatic gluconeogenesis (Lam et al., 2005, 2009). This central regulation of glucose homeostasis can be recapitulated by intra- cerebroventricular lactate infusions, showing that these neurons also function as ‘‘lactate sensors’’ (Lam et al., 2005, 2009). More importantly, the regulation of hypothalamic ‘‘glucose-sensing’’ neuron activity has been shown to implicate lactate trafficking between astrocytes and neurons (Lam et al., 2005, 2009).

(2) Orexin neurons also function as lactate sensors (Parsons and Hirasawa, 2010). In hypothalamic slice preparations, the firing

rate of orexin neurons is specifically regulated by astrocyte-derived lactate. Even in the presence of glucose, lactate disinhibits and sensitizes orexin neurons, priming them for subsequent excitation (Parsons and Hirasawa, 2010). These observations highlight lactate as an important regulator of the orexin system, which is known to participate in the control of arousal and feeding behavior (Tsujino and Sakurai, 2009).

(3) The firing rate activity of g-aminobutyric acid (GABA)-ergic neurons of the subfornical organ (SFO), which control salt-intake behavior, is also influenced by extracellular astrocyte-derived lactate (Shimizu et al., 2007) (Figure 5A). This process involves Na+ sensing by astrocytes and ependymal cells via specific Na+ channels, which in turn activate the Na+/K+ ATPase pump. This results in increased glycolysis and concomitant lactate release into the extracellular space and activation of inhibitory GABAergic neurons (Shimizu et al., 2007).

Interestingly, the underlying mechanisms for the effects of lactate on neuronal excitability in the three examples described above (glucose-sensing, orexin, and SFO GABAergic neurons) appear to critically involve ATP-sensitive K+ channels (KATP) (Ainscow et al., 2002; Lam et al., 2005; Shimizu et al., 2007; Parsons and Hirasawa, 2010). These channels close with increased ATP levels, a process that suppresses the efflux of K+ ions, leading to depolarization of the plasma membrane, and the firing of action potentials (Tarasov et al., 2004). Thus, increased free cytosolic ATP concentration, derived from lactate oxidation, triggers the closure of KATP channels leading to neuronal activation, providing a mechanism that links lactate release by astrocytes, lactate utilization by neurons, and modulation of neuronal excitability (Figure 5B). Other regulatory processes of neuronal excitability by lactate shuttling between astrocytes and neurons may also operate. For example, a pH- dependent effect of lactate has been reported to modulate the activity of the chemosensory (pH-dependent) neurons of the retrotrapezoid nucleus in the ventral medulla which control the breathing rate (Erlichman et al., 2008).

ConclusionsOverall the observations presented in this review demonstrate that brain energy metabolism involves more complex cellular and molecular mechanisms than previously thought. The field of neuroenergetics has evolved from a neurocentric view into a more integrated one in which complementarities and coopera- tivities between astrocytes and neurons play a central role. A striking demonstration is the coordination of both the CBF response (neurovascular coupling) and the stimulation of metabolism (neurometabolic coupling), which are necessary to meet the increased energy requirements of active neurons and astrocytes during brain activation. In addition to this, metabolic


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A

Figure 5. Glial-Released Lactate Can Control Neuronal Activity(A) Control by lactate of salt-intake behavior by the subfornical organ (SFO). In the SFO, specific Na+ channels (Nax) populate perineural processes of astrocytes and ependymal cells (facing the third ventricle). When animals are dehydrated, Na+ concentration in plasmaand thecerebrospinal fluid increases abovethe usual level (1). This leadsto Naxchannelsopening in ependymal and glial cells (2), resulting in the increase in intracellular Na+ concentration and the activation of Na+/K+ ATPase (3). To fuel Na+/K+ ATPase with ATP, glucose uptake and glycolysis are enhanced (4) consistently with the ANLS model. Lactate is released and supplied to neurons, including GABAergic neurons (5). Lactate stimulates the activity of the GABAergic neurons through the production of ATP (see below), which presumably regulates neurons involved in the control of salt-intake behavior (6). Ependymal cells are depicted in this figure for simplicity; however, the same scheme is applicable for intraparenchymal Nax-positive astrocytes. Reproduced with permission from Shimizu et al. (2007).(B) Proposed mechanism by which lactate can contribute to the control of neuronal activity. Stimulation of glucose utilization in astrocytes results in the increase of extracellular lactate levels. Lactate is taken up by neurons, where it is oxidized in the mitochondria, thus increasing neuronal ATP levels. This leads to an increased [ATP]/[ADP] ratio and the closure of the KATP channels, thus promoting membrane depolarization and neuronal electrical activity.

interactions—in particular in the form of lactate shuttling from astrocytes to neurons—appear to play an important role in the control of neuronal activity and excitability.

Although these observations open novel perspectives for the understanding of neuroenergetics, some critical questions remain at the moment unresolved. Are these metabolic interactions relevant for all brain structures, and do they generally apply to all conditions?

Are other neural cell types, such as oligoden

B glucose


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intriguing question concernsthefundamental functional benefits afforded by the metabolic compartmentation between astrocytes and neurons as opposed to an ‘‘every cell for itself’’ use of energy substrate(s). In line with this, there is now evidence that these neuroenergetic interactions are not only dedicated to fuelling energy-demanding cells but also act as signaling events participating in the control of important brain functions such as whole-body homeostasis or memory consolidation.

The importance of neuron-astrocyte metabolic interactions is illustrated by a large body of evidence indicating that disturbances in these processes contribute to neurodegenerative disorders. Accordingly, new therapeutic avenues aimed at preserving or enhancing astrocytic function in order to harness their intrinsic neuroprotective properties are beginning to be envisioned and will certainly attract further attention from the pharma and biotech world.

ACKNOWLEDGMENTS

We apologize to those authors whose original work could not be referenced due to space constraints. This work was supported by a grant from the Swiss National Science Foundation (FNRS) to P.J.M. (number 3100AO-108336/1). P.J.M. is the recipient of the Asterion Foundation Chair.

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Cell Metabolism

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Cell Metabolism

ReviewCell

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