Glial Glutamate Transporters

Glial Glutamate Transporters 793

Glial Glutamate Transporters A Nieoullon, CNRS – Universite´ de la Me´diterrane´e, Marseille, France

could be their real contribution to neurodegenerative diseases?

ã 2009 Elsevier Ltd. All rights reserved.

The EAA Transporter Family as Key Membrane Proteins Introduction At glutamatergic synapses, excitatory amino acid (EAA) transporters are involved in the removal of the neurotransmitter glutamate (Glu) from the synaptic cleft to stop intercellular signaling following the release of the neurotransmitter and action on receptors (Figure 1). EAAs are major brain neurotransmitters. They are primarily involved in the so-called excitatory ‘fast signaling,’ which supports rapid transfer of excitatory information in neuronal networks. Glu is the main EAA. It is generally accepted to be present in approximately 30–40% of total brain synapses and probably much more in selected cortical and hippocampal areas involved in cognitive processes. One of the most interesting properties of the EAA synapse, however, is its contribution to neuronal plasticity through cellular mechanisms known as long-term potentiation and long-term depression processes, which are generally accepted to represent basic mechanisms for learning and memory. Indeed, numerous reports have shown specific impairments of learning and memory when synaptic transmission involving the NMDA receptor subtypes is altered in vivo. In addition to the key contribution to the transfer of excitatory information in the brain, EAAs and especially Glu exhibit the unique property of representing a very potent endogenous neurotoxin. Pharmacological stimulation of EAA receptors using various Glu agonists has been shown both in vitro and in vivo to induce neuronal death, suggesting that an overconcentration of EAAs in the synaptic space is deleterious for the cells due to an excess of excitation. This process is called excitotoxicity. However, excitotoxicity is not the only mechanism that induces neuronal death; the depolarizing action of Glu is concomitant with a massive increase in Naþ in the cell, which can lead to cell death through swelling of the cell in the case of overstimulation of the EAA receptors. Therefore, although such a consideration of neuropathology can be viewed as highly reductionistic, dysfunctions of brain neurotransmission involving EAAs have been reported in select brain diseases with neuronal death and their putative contribution to many others is often proposed. However, what is the basic nature of the mechanisms involved in the clearance of EAAs at the synapse? What is the role of the transporters? What

Five members of the Glu transporter family have been characterized. The human subtypes are designed EAAT1–EAAT5. EAAT1, -2, and -3 are the human homologs, respectively, of GLAST, GLT-1, and EAAC1, the three main Glu transporters in the rat brain cloned in 1992. GLT-1 was initially purified as a 73 kDa glycoprotein and GLAST as a 66 kDa protein. When comparing the different Glu transporters, the identity of amino acid sequence is in the range of 44–55%. Drosophila EAAT homologs dEAAT1 and dEAAT2 have also been identified in the central nervous system. However, only dEAAT1 can transport Glu since dEAAT2 was characterized as an aspartate/ taurine transporter. EAAT1/GLAST and EAAT2/GLT-1 are primarily astrocytic, although some authors have suggested a limited neuronal expression of EAAT2/GLT-1. Conversely, EAAT3/EAAC1 and EAAT4 and -5 are considered to be neuronal transporters. However, EAAT3/EAAC1 expression was proposed to occur in some astrocytes and even oligodendrocytes, but such glial expression is considered as exceptional and limited. Genetic approaches have been used following biochemical characterization of the transporters. Five different genes related to the solute carrier family 1 (SLC1) genes have been identified in humans. For example, the human EAAT2 gene is localized on chromosome 11p13–12 and consists of approximately 100 kb of DNA. The gene contains 11 exons. At least three different splice variants of the EAAT2/ GLT-1 protein are issued from the reading of the sequence as the result of alternative splicing. In this respect, it has been shown that in an animal model of amyotrophic lateral sclerosis (ALS) exhibiting a mutation in the superoxide dismutase 1 (SOD1) gene associated with some forms of the human disease, the expression of five different splice variants of EAAT2/ GLT-1 is affected. Regarding the neuronal transporters, very little is known about EAAT4 and -5. The contribution of EAAT3/EAAC1 to glutamatergic transmission is still under investigation. Briefly, its postsynaptic localization and early expression in the fetus suggest a contribution to brain development. Because of its apparent capacity to also contribute to neuronal uptake of

794 Glial Glutamate Transporters

Glutamatergic terminal Glucose Glucose Glycolysis a-ketoglutarate GLUTAMINE

Glutaminase ATP

H+

H +

ASTROCYTE

GLUTAMINE

GLUTAMATE

Glutamine Synthetase

?

Glutathione

GLUTAMATE (1 µM)

mGlu R

iGlu R

EAAT3/ EAAC1

Cystine 3Glu Na+

EAAT2/ GLT-1

Cystine

XcK+

GLUTAMATE

EAAT1/GLAST

Na+

TCA cycle

GLU

iGluR ATP

mGluR

Na+, K+ ATPase ADP

Postsynaptic neuron K+

Figure 1 Main features of a brain synapse using excitatory amino acids as neurotransmitter. The neurotransmitter glutamate (Glu) is released from nerve terminals by an active process. In the synaptic space, Glu acts on receptors of both ionotropic (iGluR) and metabotropic (mGluR) subtypes. The termination of Glu action is assumed by quick removal from synaptic space either by the neuronal EAAT3/EAAC1 transporter, primarily located on the postsynaptic membrane, or by astrocytic EAAT1/GLAST and EAAT2/GLT-1 transporters. The question of the presence of a presynaptic Glu transporter is still a matter of debate. The diagram also shows the ion dependence of Glu transport into cells through transporter systems and the destination of Glu in the astrocytes. Glu is first subjected to transformation into glutamine, which acts as a Glu precursor when transported into the neuron (referred as the Glu–glutamine cycle). Glu is also transformed into glutathione, which constitutes one of the main antioxidative defenses of astrocytes. In fact, glutathione is even more dependent on Glu since Glu is also involved in the uptake of cystine through the transporter Xc-, which also acts as a precursor for glutathione. Newly synthesized Glu from glucose also contributes to neurotransmitter biosynthesis. The electrochemical gradient is maintained by Naþ/Kþ ATPase.

cysteine, which acts as a key substrate for the synthesis of glutathione, such a transporter may contribute to neuroprotective mechanisms against oxidative stress. Interestingly, numerous regulatory mechanisms involving both extracellular signals and intracellular signaling cascades have been evidenced, thus suggesting that the fine tuning of EAA neuronal uptake is a highly regulated process, which influences glutamatergic excitatory transmission.

EAA Transport Shows Selective Ion Dependence One of the main characteristics of the transport involving members of the SLC1 gene family is the

dependence of the transport of extracellular sodium concentration. The electrogenicity of the transport of Glu was first demonstrated approximately 30 years ago using brain synaptosomes, although in such a preparation the contribution of each selective ion was not easy to establish. Data from studies using cell expression of the cloned transporters and patch clamp analysis have identified the precise stoichiometry of the transport. For each Glu molecule transported into the cell, the transport involves two or three sodium ions (Naþ) and one hydrogen (Hþ) ion entering the cell together with the molecule of Glu, in exchange for one potassium (Kþ) ion. A sodium-activated chloride (Cl) conductance is also mediated through EAATs, but such a conductance is

Glial Glutamate Transporters 795

not thermodynamically linked to Glu translocation into the cell. Site-directed mutagenesis has been used to identify amino acid residues related to ion transports associated with EAA translocation, verifying that Cl movement is not necessary for EAA transport. Consequently, it appears that the transport of EAAs into the cells is driven by the electrochemical gradient existing through the membrane. Such a transport is highly efficient to concentrate the neurotransmitter into the cell. The transport is electrogenic, generating a net depolarization of the membrane, which can be measured by using electrophysiological recordings. Detailed analysis of currents produced by the transport has led to the proposal of hypothetic kinetic models which describe the different steps of Glu transport assuming that EAATs behave like sequential exchangers. Differences may be related to variations in the cycle and efficacy in the transport regarding the heterogeneity of the transporters. In this respect, although there are some differences, Glu transport appears to be rather slow since one complete transport cycle is believed to range from 60 to 80 ms.

Molecular Structure and Presumed Topology of EAATs After purification of the first EAATs was achieved, sodium dodecyl sulfate–polyacrylamide gel electrophoresis demonstrated that these proteins have a tendency to form oligomers. In contrast to the pentameric structure proposed for EAAT3/EAAC1, the glial transporter contributing to the maturation of the pore was proposed to involve a trimeric structure. Such a proposal was verified by the crystallization of bacterial homologs of the SLC1 Glu transporters. The Glu transporters in eukaryotic cells are in fact members of a more general membrane transport protein family also involving prokaryotic transporters. Both types of proteins share a high degree of amino acid homology, similar substrate transport ionic specificity, and close transmembrane topology. The crystallized transporter from Pyrococcus horikoshii (GltPh) shows a 37% sequence identity with the human EAAT2/GLT-1. In agreement with the previously proposed multimeric structure, which emphasized that trimers and dimers predominate, in the presence of Glu the GltPh transporter appears to consist of three functional units, also in agreement with previous results obtained from the purification of Glu transporters from Bacillus caldotenax and Bacillus stearothermophilus. The high-resolution topology of each subunit, however, remains to be clarified. Although two main models have been proposed based on studies using site-directed-mutagenesis, the controversy persists. The latest developments suggest that the protein,

which exhibits intracellular localization of N- and C-terminals, consists of six transmembrane (TM) domains thought to be organized as a-helices, followed by two reentrant loops corresponding to hairpin loops in the proximity of the C-terminal part of the protein (Figure 2). Regarding GLT-1 topology, several amino acid residues have been shown to be critical for Glu transport, for example, at the levels of the two hairpin loops. The protein can be glycosylated in the large extracellular hydrophilic loops between TM3 and TM4 (two sites) and shows putative consensus phosphorylation sites in its intracellular part, suggesting the possibility of posttraductional regulation of activity. Furthermore, because of interactions between the transporters and proteins located in the immediate vicinity, there is a strong possibility that such partners contribute to intracellular trafficking to and from the plasma membrane. Regarding EAAT2/GLT-1, the transporter was shown, for example, to interact at its N-terminal ending with a protein of the LIM family Ajuba, which may be involved in regulation of intracellular signaling cascades or acting on the cytoskeleton. Similarly, the C-terminal part of the EAAT1/ GLAST protein may be preferentially linked to the protein septine 2, a member of the GTPase family, and such an interaction may be involved in the intracellular trafficking of the transporter and, specifically, its presence at the membrane. Finally, because depletion of membrane cholesterol by methylb-cyclodextrin induced primarily marked reduction of glial Glu uptake in primary cortical cultures, it has been proposed that the Glu transporters, including EAAT3/ EAAC1 but especially EAAT2/GLT-1, are associated with cholesterol-enriched lipid raft microdomains, and that such a localization is important for function.

Glial EAATs Are a Target for Drugs: Toward a New Molecular Pharmacology? Because of high structural homology, it is difficult to selectively act on the different transporter subtypes. The initial generation of compounds that inhibit EAA transport of Glu focused on competitive substrates such as pyrrolidine decarboxylate (PDC) derivatives, which induce an efflux of endogenous Glu due to heteroexchange. Further development of nontransportable compounds, such as DL-TBOA or dihydrokainic acid (DHK), contributed to a more precise identification of transporter properties. DHK is one of the most interesting compounds because of its rather selective binding to EAAT2/GLT-1 but not to EAAT1/ GLAST or EAAT3/EAAC1. However, it has the major drawback of also binding to the kainic acid EAA receptor subtype. Conversely, the serine derivative serine-O-sulfate was proposed to inhibit preferentially

796 Glial Glutamate Transporters

Hairpin loop 2 Out

Glutamate 1

2

3

4

5

6

Hairpin loop 2 Hairpin loop 1

a

C

Hairpin loop 1

N

b

Hairpin loop 2

c

d

Hairpin loop 1

þ

Figure 2 High-resolution structure of the Na -coupled glutamate transporter homolog (SLC1 family) from Pyrococcu horikoshii (GltPh). (a) Schematic representation of membrane topology. 1–6, the transmembrane segments of the protein. (b) Predicted form of the glutamate binding site bordering the two hairpin loops. The hairpin loops could form the gate at both extracellular and intracellular levels to control access to the substrate binding site. (c) View parallel to the membrane. (d) View from the extracellular side. Reproduced from Gether U, Andersen PH, Larsson OM, and Schousboe A (2006) Neurotransmitter transporters: Molecular function of important drug targets. Trends in Pharmacological Sciences 27: 375–383, with permission from Elsevier.

EAAT1/ GLAST and EAAT3/EAAC1, and L-b-benzylaspartate was shown to act as a preferential inhibitor of EAAT3/EAAC1 compared to glial transporters.

The Brain Regional Distribution of EAATs Is Uneven and Their Expression Is Developmentally Regulated In addition to brain expression, mRNA encoding EAAT1/GLAST and, more frequently, EAAT2/GLT-1, has been detected in the heart, kidney, pancreas, bone cells, and mammary glands. However, the functional role of such tissue expression is not completely understood. In the brain, since the demonstration that most Glu uptake activity is attributable to the glial transporters, the key role of astrocytes in EAA transport has been emphasized because EAAT1/GLAST and EAAT2/GLT-1 proteins are selectively expressed by these glial cell subtypes in the adult. Some ependymal cells and neurons in the retina, however, have also been shown to express these transporters. Thus, the debate is still open regarding especially EAAT2/ GLT-1 since mRNA cellular localization has been described to occur not only in astrocytes but also in

neurons from the adult brain in hippocampus and cortical areas. Moreover, transient EAAT2/GLT-1 expression has been reported in Purkinje cells or in neurons in the spinal cord during development. This is apparently not the case in rat cortical primary neuronal cultures: Astrocytes selectively express EAAT1/GLAST and EAAT2/GLT-1, whereas EAAT3/ EAAC1 is expressed in the neurons (Figure 3). Finally, a truncated variant of the EAAT2/GLT-1 protein cloned from mouse liver (GLT-1v) is possibly expressed in astrocytes and certain neurons. However, the level of such neuronal expression of EAAT2/GLT-1 is low, and there is a consensus that EAAT2/GLT-1 expression is generally restricted to astrocytes in the adult brain. The regional brain distribution of EAAT glial transporters is uneven, in contrast to that for EAAT3/ EAAC1. In the rat brain, EAAT1/GLAST is primarily expressed in the cerebellum. The olfactory bulb, hippocampus, and striatal and cortical areas also express the transporter. Moreover, the distribution of the transporter is not restricted to brain areas; the EAAT1/GLAST is also present in the inner ear, for example. EAAT1/GLAST expression is restricted to

Glial Glutamate Transporters 797

Figure 3 Cell selectivity and developmental profile of the expression of different EAATs in rat primary cortical cultures. Confocal doublelabeling immunocytochemistry was used to study the localization of the three main EAA transporter proteins to neurons identified by neuronal neurofilament (NF) labeling or to astrocytes labeled by the glial fibrillary acidic protein (GFAP). (a–c) Data from cortical cultures on day in vitro (DIV) 7 treated for double labeling using GFAP or NF antibodies, which are combined on the same preparation with antibodies EAAT3/EAAC1 (a), EAAT1/GLAST (b), and EAAT2/GLT-1 (c), respectively. Data from similar experiments performed on DIV 14 (d–f) confirmed the selective neuronal expression of EAAT3/EAAC1 and astrocytic expression of EAAT1/GLAST and EAAT2/GLT-1. Interestingly, the expression of EAAT2/GLT-1 was only detected at DIV 14, in agreement with data from in vivo experiments showing delayed expression of the transporter. Reproduced from Guillet B, Lortet S, Masmejean F, Samuel D, Nieoullon A, and Pisano P (2002) Developmental expression and activity of high affinity glutamate transporters in rat cortical primary cultures. Neurochemistry International 40: 661–671, with permission from Elsevier.

798 Glial Glutamate Transporters

astrocytes and Muller cells in the retina. Developmental analysis in vivo shows a progressive increase in EAAT1/GLAST expression from birth to adulthood. Conversely, in the newborn rodent EAAT2/GLT-1 brain expression is undetectable, whereas EAAT1/ GLAST is present. Thus, the expression of both transporters increases dramatically with synaptogenesis during the first month after birth, which supports the proposal for the essential contribution of EAA to brain maturation. Interestingly, the expression of EAAT1/GLAST in cortical primary cultures occurs in the early stages of the cultures in vitro (day in vitro (DIV) 3), whereas EAAT2/GLT-1 expression in similar conditions occurs later (DIV 10). In such an experimental model, EAAT3/EAAC1 expression occurs as early as DIV 1. EAAT2/GLT-1 is actually the major EAAT expressed in the forebrain, especially in cortical areas, hippocampus, and striatal areas. However, the transporter is expressed at significant levels in the entire brain. EAAT2/GLT-1 is also present in the retina, where it is expressed in photoreceptor cells and in some bipolar ganglionic neurons. As mentioned previously, brain EAAT2/GLT-1 is expressed at very low levels at birth, and it progressively increases during the second week of life and reaches the adult level after approximately 1 month, in agreement with what is observed in primary cultures. The same astrocytes were shown to be able to simultaneously express EAAT1/GLAST and EAAT2/GLT-1. Finally, regarding the subcellular localization of the glial transporters in astrocytes, basal expression is preferentially membranar (70%). The density of the proteins is also thought to be higher in the vicinity of glutamatergic synapses. However, this changes with stimulation or pharmacological manipulation, thus showing dynamic regulation of trafficking of the transporters.

Glial EAATs Are Essential Actors of the Excitatory Synapse: Evidence for Multiple Functional Implications Importance of Glu Transport in Maintaining Synaptic Glu Concentration within Physiological Limits

As mentioned previously, glial EAATs are major contributors to the removal of EAAs and especially Glu from the synaptic space. The relative contribution of the different transporters to Glu uptake was examined in vivo using specific antisense oligonucleotides administered in the rat brain to transiently block the expression of the transporter subtypes. Data showed that the extracellular concentration of Glu

is highly increased in selected brain structures (hippocampus and striatum). Comparative studies found that EAAT1/GLAST and especially EAAT2/GLT-1 are the major actors of Glu uptake, contributing approximately 80% of EAA uptake in the striatum and 60% in the hippocampus, although the resulting increase in extracellular Glu could be due to cytotoxic effects related to elevated Glu and subsequent excitotoxicity. Glu transport is believed to be primarily involved in regulating excitatory transmission. Because of the desensitization properties of EAA receptors involved in fast signaling, the rapid removal of the neurotransmitter from the synapse is an essential step of neurotransmission to ensure the functionality of intercellular communication. Interestingly, Glu uptake is thought to also play a key role in limiting the spillover of the excitatory transmitter to EAA receptors located at a distance from the releasing site. Two hypotheses have been proposed. First, limiting the spillover of Glu could contribute to limit activation of receptors of the NMDA subtype present at neighboring synapses and thus to focus excitatory transmission, which may only involve active synapses and not neighboring, so-called ‘silent synapses.’ Second, because extrasynaptic NMDA activity would presumably contribute to cytotoxicity and neuronal death in contrast to synaptic NMDA receptors, limiting Glu spillover through primary EAA glial uptake could be neuroprotective. In this respect, in vivo experiments in the rat using antisense oligonucleotides administered in the brain with miniosmotic pumps showed that the loss of the glial transporters EAAT1/GLAST and EAAT2/GLT-1 produced neurodegeneration in the striatum or hippocampus similar to that observed in excitotoxicity. The progressive motor deficit observed in the treated animals was considered to be the consequence of neuronal cytotoxicity. However, it has to be taken into account that the lack of glial Glu uptake could have differential effects in the brain regarding regional brain differences in neuronal–astroglial relationships. For example, in the cerebellar cortex, the excitatory synapses on Purkinje cells are closely surrounded by Bergmann glia. Thus, glial processes are in a position to efficiently limit Glu extracellular synaptic diffusion and receptor influence. In contrast, this is not the case at the cortical level and in the hippocampus since very few synapses are entirely surrounded by astrocytes. Consequently, limiting Glu uptake will influence EAA transmission in these brain structures less than in the cerebellar cortex, further suggesting that following synaptic release Glu can rapidly diffuse out of the synaptic cleft and act at a distance.

Glial Glutamate Transporters 799 Glial EAATs Provide Neurons with Glu

Glu uptake into astrocytes provides a major contribution to the so-called ‘Glu–glutamine cycle’ (Figure 1). Numerous data indicate that Glu released in the synaptic space is taken up by surrounding astrocytes and subsequently transformed in glutamine by glutamine synthetase. The astrocytes release glutamine into the extracellular space, and glutamine is secondarily taken up by excitatory nerve terminals. In the nerve terminals, glutamine is transformed in Glu by the enzyme glutaminase phosphate, which contributes to increase the cytosolic and vesicular pool of the neurotransmitter. Approximately 30% of total Glu entering astrocytes is subjected to oxidative processes and 70% contributes to the Glu–glutamine cycle, which indicates that biosynthesis of Glu continuously occurs in the neurons, supplying nerve terminals with sufficient neurotransmitter. Interestingly, glutamine is not selectively taken up by excitatory terminals: Inhibitory GABA-containing neurons can also take up glutamine. In the inhibitory nerve terminals, glutamine is metabolized into Glu, which represents a metabolic precursor of GABA synthesized from Glu by glutamate decarboxylase. Thus, through the contribution of glial EAATs, the Glu–glutamine cycle contributes to biosynthesis of both the excitatory neurotransmitter Glu and the inhibitory transmitter GABA. EAA Glial Transporters as Active Contributors to Energetic and Metabolic Processes in Astrocytes and Neurons

It is worth discussing another possible major destination of Glu taken up into astrocytes through glial EAATs. Glu transformed into a-cetoglutarate can be considered as an energetic substrate for the cell. Multiple transamination and oxidative deamination processes contribute to the production of this energetic substrate from Glu. Thus, when necessary, pyruvate produced in the astrocytes can be transferred as lactate to neurons through monocarboxylate transporters and used as pyruvate again in the tricarboxylic acid cycle as an energy source for the cell. The increase in glial Glu uptake occurs concurrently with glucose uptake and consumption and an increase in lactate release, which supports the concept of a net increase in Glu biosynthesis from glucose in astrocytes and increased metabolism when Glu uptake is activated. Lactate provides the energy to the cells. Glu uptake activates the Naþ/Kþ-ATPase a2 subunit, which is specifically expressed in the astrocytes. This mechanism has been suggested to contribute to coupling excitatory neuronal transmission to an increase in astrocytic glucose metabolism. Such a mechanism emphasizes the

contribution of glial EAATs to results obtained with functional brain imaging methods (functional magnetic resonance imaging or positron emission tomography). The metabolic response to sensory stimuli is reduced in transgenic mice in which the expression of EAAT1/ GLAST or EAAT2/GLT-1 is suppressed or following reduced expression of EAAT1/GLAST using antisense oligonucleotides. The Glial EAATs as Key Actors for Antioxidative Defenses in Astrocytes

The glial EAATs may contribute to antioxidative defenses in astrocytes. Glu acts as a metabolic precursor of glutathione (GSH), the major neuroprotective agent against the deleterious action of reactive oxygen species (ROS). Glu is metabolized into g-glutamylcysteine and then into GSH by selective enzymes. Interestingly, the contribution of Glu to GSH biosynthesis is also indirect since Glu is involved in the incorporation of cystine into the astrocytes from outside through the Xc- transport system. The incorporation of cystine provides astrocytes with cysteine used to synthesize GSH. In such a transport system, incorporation of cystine is associated with an efflux of Glu. Consequently, a decreased concentration of Glu in astrocytes may significantly effect the antioxidative defenses of the cell. This occurs when the transport of EAA is pharmacologically blocked: In cell cultures, we measured a progressive and massive death of astrocytes in the presence of PDC, which was correlated with increased production of ROS and further depletion in GSH. We verified that the death of astrocytes in the presence of PDC is neither due to excitotoxicity nor due to oxidative properties of Glu because increasing extracellular Glu concentrations, for example, is protective. Moreover, N-acetylcysteine was also shown to exert a protective action against PDC, which further supports the hypothesis that the key contribution of Glu transport into astrocytes is to antioxidative defenses through GSH production. Further Evidence for an Essential Contribution of Glial EAATs to Brain Homeostasis

Evidences for a functional role of glial EAATs has been obtained from mutations inducing loss of function of the transporters. Mice lacking expression of the Glu transporter EAAT2/GLT-1 showed marked neurodegeneration in the hippocampus and spontaneous seizures. These mice generally died within 6 weeks after birth, further suggesting that such a glial transporter has a key role in controlling excitatory synaptic activity and EAA-induced cytotoxicity. The phenotype resulting from mutation of EAAT1/GLASTshowed impaired

800 Glial Glutamate Transporters

motor coordination and increased susceptibility to cerebellar injury, illustrating the importance of the transporter in cerebellar physiology, at least in mice. These experiments emphasize the major contribution to brain functioning of EAAT2/GLT-1 compared to EAAT1/GLAST or EAAT3/EAAC1. Furthermore, in the SOD1 mutant mouse developed as an animal model of some forms of ALS, in which different splice variants of EAAT2/GLT-1 are affected, histological analysis showed alterations in astrocytes concomitant with progressive hind limb paralysis and animal death. Does Inversion of Glu Uptake Represent a Physiological Process Contributing to Regulation of Excitatory Transmission?

Since not only neurons but also astrocytes exhibit vesicles that contain neurotransmitter and can apparently contribute to exocytosis, the previous observations raise the question of the possible contribution to physiological processes of Glu released by the glial cells. The actual contribution of astrocytes to exocytosis is still under investigation. However, there is another way to obtain an increase in extracellular Glu from these cells. Depending on the electrochemical gradient across the membrane, the transport of Glu into astrocytes can be reversed. Although such a process was primarily assumed to be involved in pathological conditions, there is also evidence that this occurs in physiological conditions in intact cells. The transmembrane gradient of amino acids may be the driving force of the transport, and the reversion of the transport likely results from weakening of such a driving force. The reversed transport in this situation is independent of both ATP and Ca2þ ions, and it contributes to increasing extracellular EAA levels, further reinforcing the neurotransmitter action on synaptic receptors and excitatory synaptic signaling when necessary. However, the reversion of Glu transport may be more frequently relevant in pathological conditions.

EAA Transport in Glial Cells Is a Highly Regulated Cellular Process Fine-Tuning of Glu Uptake Rate at the Excitatory EAA Synapse

In the early 1980s, our laboratory developed the concept of a possible short-term regulation of Glu uptake rate with regard to changes in neuronal activity. Such a concept of activity-dependent regulation of neurotransmitter uptake rate was further extended since modulations of Glu uptake have been shown in the rat, for example, in relation to stress or even estrous cycle. Numerous studies have supported the proposal

for a fine-tuning of EAA transport, especially in astrocytes. Regarding EAAT1/GLAST and EAAT2/GLT-1, numerous data have been obtained suggesting regulatory processes which involve both transcriptional and posttraductional mechanisms. This latter hypothesis was also supported by experiments from our group showing that Glu uptake may be modulated in response to activation of neurotransmitter receptors. Furthermore, numerous trophic factors have been shown to increase Glu uptake. Arachidonic acid, NO (indirect action through cytokines production), Zn2þ ions, as well as pharmacological activation or inhibition of various protein kinases were conversely shown to decrease Glu uptake rate, supporting the concept of posttraductional regulation of the activity of transporter molecules. Such a concept was further supported by the demonstration that EAATs exhibit putative consensus phosphorylation sites in their molecular structure. Finally, it is worth mentioning the special role of oxidative conditions in altering Glu transport. Oxidative conditions severely decrease transporter activity, possibly through oxidization of the cysteine residues of EAAT1/GLAST and EAAT2/ GLT-1. This occurs in the presence of ROS in the cells, further emphasizing the deleterious role of oxidative processes on the functionality of the transporter molecules in addition to the action of cell integrity. Regulation of Cell Expression of Glial EAATs

In addition to evidence for the regulation of Glu transport rate, numerous experiments have also shown that EAA transport can be influenced at the transcriptional level. Astrocytes in culture provided a useful model to assess such regulatory influence on EAAT expression. Data have shown, for example, that EAAT2/GLT-1 expression is induced by various trophic factors, such as epidermal growth factor, transforming growth factor-a, fibroblast growth factor-2, platelet-derived growth factor, or even brain-derived neurotrophic factor, suggesting a contribution of tyrosine kinase pathways. Interestingly, insulin-like growth factor-2 was evidenced to maintain cellular EAAT1/GLAST and EAAT2/GLT-1 expression. Transcriptional effects on EAAT genes thus primarily involve protein kinase (PK) activation. For example, in the HEK293 cell line, increasing PKC activity induced a decreased expression of EAAT1/GLAST. Conversely, activation of PKC in HeLa cells induced an increase in EAAT2/GLT-1 expression; in this case, the authors originally demonstrated that the transcriptional effect was due to direct phosphorylation of the transporter. Co-culturing astrocytes together with neurons also increased the expression of the two glial transporters. Different mechanisms have been proposed to explain such

Glial Glutamate Transporters 801

a neuronal influence on astrocytes, possibly involving cAMP, CREB transcription factor, MAP/Erk kinase signaling pathway, or pituitary adenylate cyclaseactivating peptide. Additional Regulatory Processes of Glial EAATs Acting on Intracellular Trafficking of the Transporter Protein and Membrane Stabilization

A recent view of the processes involved in the regulation of EAA transport is related to changes in the trafficking and stabilization of the transporter proteins at the membrane from intracellular stores. Such a mechanism is well documented for the neuronal transporter EAAT3/EAAC1, which is subjected to numerous influences. For example, PKC activation facilitates the association of the transporter with the aPKC subunit, which contributes to stabilization of the transporter at the membrane. Biotinylation and immunoblotting experiments show similar processes for the glial transporters. Interestingly, EAAT1/ GLAST and EAAT2/GLT-1 are differentially regulated but possibly expressed in the same astrocyte. For example, inhibition of PKA activity using the compound H89 or stimulation of PKC using PMA both resulted in decreased EAAT1/GLAST at the astrocytic membrane, whereas wortmannin acting as a specific inhibitor of PI3 kinase produced the opposite response. Conversely, H89 induced an increase in EAAT2/GLT-1 at the membrane, and PMA and wortmannin produced the opposite effect. These results illustrate differential posttraductional regulation of the two transporters. Interestingly, assuming that increasing the presence of the transporter at the membrane correlates with increased EAA uptake activity, in our experimental model in vitro (primary cultures from rat cerebral cortex) PI3K inhibition using wortmannin is only active to reduce the uptake process at 14 DIV, when EAAT2/GLT-1 is fully active. At 7 DIV, wortmannin is inactive but EAAT2/GLT-1 is not yet expressed by the astrocytes. The molecular mechanisms by which the different PKs contribute to transporter trafficking, membrane stabilization, and cytosolic sequestration remain obscure. As in the case of EAAT3/EAAC1, regulatory cytosolic proteins may be involved. Since the protein septin 2 acting as a GTPase has been shown to interact with the C-terminal part of the EAAT1/GLAST protein, septin 2 may represent one of the cytosolic regulatory factors for stabilization of the transporter at the membrane and activation of astroglial Glu uptake rate. Similarly, the protein LIM associated with the N-terminal part of EAAT2/GLT1 may contribute to changes in its intracellular trafficking. In summary, the concept of fine tuning the uptake of EAAs to further regulate extracellular

concentrations of the neurotransmitter with regard to nerve activity is well documented. Regulatory processes involve both transcriptional and posttraductional mechanisms and have been demonstrated to influence Glu uptake rate. Interestingly, the activity of the excitatory neurons may be a critical factor for regulatory processes. Such a proposal is further reinforced by the fact that treatment of cell cultures with high concentrations of NMDA to activate Glu transmission downregulates EAAT2/GLT-1 expression but upregulates EAAT1/GLAST expression, further illustrating differential regulatory mechanisms for both transporters. Conversely, low extracellular concentration of Glu increases EAAT2/GLT-1 expression in astrocytic cultures. Possible EAAT2/GLT-1 expression in neurons has also been reported, but because the uptake process is not sensitive to DHK, it is likely that the transporter is inactive or represents an inactive variant of EAAT2/GLT-1.

Glial EAATs Are Likely Involved in Neurodegenerative Diseases Numerous observations primarily from animal models have implicated alterations of Glu transport in various brain diseases (Table 1). EAAs have been associated with acute brain lesions related to stroke (ischemic, anoxic, and traumatic events) and to the so-called functional diseases involving an excess of excitatory transmission, such as different states of epilepsy or, possibly, some forms of schizophrenia. EAAs have also been proposed to be involved in numerous neurodegenerative diseases, such as Huntington’s disease, Parkinson’s disease, or even Alzheimer’s disease, although direct evidence for the contribution of EAAs is generally lacking, except for ALS. The involvement of EAAs in brain pathological events has also been proposed in HIV-associated dementia, malignant glioma, or hepatic encephalopathy due to an increased level of ammonia in the brain since decreased expression of EAAT2/GLT-1 has been reported. Putative Contribution of EAAT2/GLT-1 to ALS

Approximately 10 years ago, a selective loss of EAAT2/GLT-1 isoforms in motor and sensory cortical areas and concomitant decreased Glu transport activity in patients suffering sporadic ALS was shown. Aberrant splicing of the EAAT2/GLT-1 transcript, and also vulnerability of the protein to oxidative stress, was suggested to be the cause of decreased EAAT2/GLT-1 expression/activity. Furthermore, a mutation which substitutes an asparagine for a serine at position 206 in the EAAT2/GLT-1 gene (mutation N206S) was reported in sporadic ALS. Because this

802 Glial Glutamate Transporters Table 1 Putative implication of excitatory amino acid transporters and brain pathologies highlighted from experimental approaches Disease

Experimental model

Transporter subtype

Resulting phenotype

ALS

SOD1 mutants

EAAT2/GLT-1 reduced

EAAT2/GLT-1 antisense strategy in rat

EAAT2/GLT-1 reduced

Mutant APP over expression in mice

EAAT2/GLT-1 knockout mice

EAAT1/GLAST and EAAT2/GLT-1 protein reduced (mRNA levels normals) Peptide b-amyloid reversal of Glu uptake L-DOPA induced increase in striatal EAAT2/GLT-1 protein EAAT1/GLAST and EAAT2/GLT-1 striatal reduction Reduction of EAAT2/GLT-1 mRNA and striatal protein EAAT2/GLT-1 protein reduced

Progressive hind limb paralysis and death Progressive motor neuron loss and hind limb paralysis Amyloid plaque formation; behavioral changes

EAAT1/GLAST knockout mice

EAAT1/GLAST protein reduced

EAAT3/EAAC1 antisense strategy Seven-day-old-rat hypoxia–ischemia (cortex; hippocampus) Hippocampal slices from EAAT2/GLT-1 knockout mice Rat MCA occlusion

Reduced EAAT3/EAAC1 and GABA All the three main transporters decreased at 12 h Reduced Glu uptake but no reversal related to EAAT2/GLT-1 Decreased EAAT2/GLT-1 and EAAT3/ EEAC1

Rat MCA occlusion Rat MCA occlusion with antisense strategy to EAAT1/GLAST and EAAT2/GLT-1

EAAT1/GLAST increased The two transporter proteins are reduced

Hypoxic neonatal pig

EAAT2/GLT-1 and EAAT3/EAAC1 reduced at 24 h (striatum)

Alzheimer’s disease

Cultured rat astrocytes Parkinson’s disease

Nigro-striatal dopamine denervation in rat Striatal dopamine denervation

Huntington’s disease Epilepsy

Stroke

Expression of mutant huntingtin

Seizures and death at 6 weeks Increased sensitivity to convulsive agents EEG episodes with seizures

Contralateral hemiparesis Contralateral hemiparesis

45% increase in stroke volume and increased mortality

Adapted from Maragakis NJ and Rothstein JD (2004) Glutamate transporters: Animal models to neurological diseases. Neurobiology of Diseases 15: 461–473.

mutation was demonstrated to alter Glu transport, it appeared that the excess of Glu in the extracellular space along with clearance deficiency of the neurotransmitter contributed to excitotoxicity and induced the death of motor neurons. The supranormal levels of Glu measured in the blood and possibly in cerebrospinal fluid from patients may be indicative of changes in EAA metabolism in ALS. The putative contribution of EAAs to neuronal death in ALS was further emphasized by the positive effect of the antiglutamatergic compound riluzole, which was shown to slow the rate of the disease. The case of ALS pointed to the possible contribution of the EAAT system, and endogenous Glu, to the neurodegenerative processes. Because of the main glial expression of such transporters, results from ALS patients also emphasize the possible contribution of glial cells and especially astrocytes to motor neuron degeneration. Finally, the modest but real clinical

effects of riluzole in ALS also point to the glutamatergic synapse as a possible target for development of further neuroprotective strategies. In the SOD1 mutant mice in which the function of EAAT2/GLT-1 is highly reduced, damaged astrocytes are detected in the spinal cord. Such astrocytic alteration is concomitant with motor neuron degeneration, which supports impaired locomotion. In this respect, as mentioned previously, increasing extracellular Glu levels using chronically implanted osmotic minipumps for local injections of the EAAT inhibitor PDC in vivo resulted in a loss of striatal neurons and astrocytes in the rat. The critical role of EAATs in neurodegeneration was also evidenced in the pioneer experiments of Rothstein and group, who elegantly showed that decreasing glial EAAT expression using antisense oligonucleotides in the rat resulted in elevated extracellular Glu levels, neurodegeneration, and progressive paralysis. As also mentioned previously, contribution of glial EAATs to

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brain pathology was obtained from transgenic mice lacking expression of the transporters showing impaired motor coordination, cerebellar alterations, and spontaneous seizures depending on the transporter expressed. Interestingly, the decreased antioxidative defenses in astrocytes subjected to Glu depletion would explain the death of astrocytes. Therefore, such a hypothesis may be proposed for ALS in humans and for neurodegenerative processes in the SOD1 mutant mice. If such a hypothesis is correct, the death of motor neurons may therefore be secondary to lack of Glu transport in astrocytes and subsequent excitotoxicity. Do the Glial EAATs Contribute to Alzheimer’s Disease?

With the exception of ALS, the contribution of EAATs in other neurodegenerative diseases is not so evident. In Alzheimer’s disease, it is generally accepted that the level of EAATs is normal, although specific reductions of EAAT2/GLT-1 have been reported in frontal cortex. However, abnormal neuronal expression of the two glial transporters has also been reported, notably in neurons concerned with tau protein accumulation. Interestingly, in vitro experiments have shown an inhibitory effect of the b-amyloid peptide on Glu transport, further indicating that in Alzheimer’s disease the pathological process can contribute to neuronal death through impaired EAA transport. Consequently, the negative influence of the b-amyloid peptide on Glu transport may be viewed as initiating a ‘cytotoxic cascade’ which may be amplified by direct overstimulation of EAA receptors mediating neuronal death. The associated Glu astrocytic depletion, which may therefore decrease oxidative defenses of the cell, is then thought to induce astrocyte death and secondarily contribute to increase extracellular Glu concentrations since astrocytes cannot proceed to EAA uptake. However, such a hypothesis remains to be documented, and the contribution of EAAs to pathological processes in Alzheimer’s disease has not been proved. Brain Ischemia and Anoxia: Does Reversal of EAATs Represent One of the Key Steps of Degenerative Processes?

The involvement of EAATs in brain pathology may be related to a decreased uptake rate, decreased transporter tissue expression and/or altered trafficking of transporter proteins, and their putative implication in the transport of cytotoxic substances and also in the reversal of the transport direction, which likely contributes to increase extracellular Glu levels and decrease Glu astrocytic contents. EAA neuronal toxicity in severe ischemia is generally accepted to result from

reversal of EAATs, especially the neuronal transporter EAAT3/EAAC1. However, the implication of the glial transporters is also evident since the driving force of transport is related primarily to the electrochemical gradient of ions associated with the uptake process in general. For example, the increase in extracellular Glu in experimental anoxic conditions is highly sensitive to DHK, suggesting a major contribution from EAAT2/GLT-1. Transient or more permanent disruptions of such a gradient in the case of deficient energy supply, as observed in ischemia or anoxia processes, will decrease EAA uptake and induce reversed Glu transport. Indeed, ATP depletion will result in rapid Naþ/Kþ ATPase loss of activity and reversal of the Naþ/Kþ gradient. The contribution of neurons to the increased extracellular Glu is related to their high content in the neurotransmitter compared to astrocytes, but depleting intracellular Glu stores in astrocytes is likely to induce a rapid decrease in GSH levels, thus reducing the antioxidative defenses of the cells. In this respect, in the ischemic area, cell death involves both neurons and glial cells as shown in many reports. Consequently, pharmacologically blocking the Glu transport to prevent reversal of the transport of Glu without altering uptake capacities may be of therapeutic interest to maintain low extracellular Glu in stroke. Considerable delayed alterations of the EAATs have been observed in experimental models of ischemia following reestablishment of the blood supply. Although there is great variability due to major differences in animal models and experimental conditions in vivo and in vitro, the general feature is a decreased expression of most of the transporters, both glial and neuronal, until 72 h following the ischemic procedure. Interestingly, during the postischemic period abnormal neuronal expression of the glial transporters EAAT1/GLAST and EAAT2/GLT-1 as well as overexpression of EAAT1/GLAST have been reported in the region surrounding the ischemic areas, further supporting the idea that EAATs are involved either in long-lasting neurodegenerative processes following brain ischemia or in compensatory protective mechanisms that limit cellular damage.

Conclusion Although direct evidence is lacking, numerous data from experimental models point to the contribution of EAAs to pathophysiological processes in several neurological and psychiatric disorders. However, the concept of the possible involvement of EAATs in such pathological processes is quite recent; for years, the involvement of EAAs was focused on excitotoxicity and the EAA receptors. Of course, excitotoxicity can

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represent one of the final steps of neuronal death, but astrocytes are generally reported to be insensitive or less sensitive than neurons to excitotoxicity. Thus, the contribution to EAATs to degenerative processes in general is not restricted to simple impaired uptake into either neurons or glial cells. The dependence of astroglial cell survival on maintenance of sufficient intracellular Glu levels illustrates the key contribution of EAATs. Defective transport results in GSH depletion and increased susceptibility to oxidative stress, likely resulting in astrocytic death. Stimulating Glu uptake may therefore be a way to protect the neurons. Finally, because of the dependence of EAA transport on the electrochemical gradient, impaired energy results in reversal of the transport, which consequently can increase both excitotoxicity and astrocyte depletion in Glu. Therefore, the conservation of functional glial EAATs could contribute to neuroprotection. In this respect, developing new compounds aimed at keeping such EAATs active may be a useful strategy in the case of neurodegenerative diseases. Experiments in progress will contribute to developing this concept. See also: Excitotoxicity in Neurodegenerative Disease; Glial Glutamate Transporters: Electrophysiology; Glial Ion Homeostasis: A Fluorescence Microscopy Approach; Glial Glutamate and GABA Metabolism; Glutamate; Ionic Channels in Glia.

Further Reading Bergles DE, Diamond JS, and Jarh CE (1999) Clearance of glutamate inside the synapse and beyond. Current Opinion in Neurobiology 9: 293–298. Bonvento G, Sibson N, and Pellerin L (2002) Does glutamate image your thoughts? Trends in Neurosciences 25: 359–364. Bridges RJ and Esslinger CS (2005) The excitatory amino acid transporters: Pharmacological insights on substrate and inhibitor specificity of the EAAT subtypes. Pharmacological Therapeutics 107: 271–285. Danbolt NC (2001) Glutamate uptake. Progress in Neurobiology 65: 1–105. Dringen R, Gutterer JM, and Hirrlinger J (2000) Glutathione metabolism in brain metabolic interaction between astrocytes

and neurons in the defense against reactive oxygen species. European Journal of Biochemistry 267: 4912–4916. Gadea A and Lopez-Colome AM (2001) Glial transporters for glutamate, glycine and GABA: I. Glutamate transporters. Journal of Neuroscience Research 15: 453–460. Gether U, Andersen PH, Larsson OM, and Schousboe A (2006) Neurotransmitter transporters: Molecular function of important drug targets. Trends in Pharmacological Sciences 27: 375–383. Guillet B, Lortet S, Masmejean F, Samuel D, Nieoullon A, and Pisano P (2002) Developmental expression and activity of high affinity glutamate transporters in rat cortical primary cultures. Neurochemistry International 40: 661–671. Hertz L and Zielke R (2004) Astrocytic control of glutamatergic activity: Astrocytes as stars of the show. Trends in Neurosciences 27: 735–743. Hinoi E, Takarada T, Tsuchihashi Y, and Yoneda Y (2005) Glutamate transporters as drug targets. Current Drug Targets – CNS Neurological Disorders 4: 211–220. Kanai Y and Hediger MA (2003) The glutamate and neutral amino acid transporter family: Physiological and pharmacological implications. European Journal of Pharmacology 479: 237–247. Maragakis NJ and Rothstein JD (2004) Glutamate transporters: Animal models to neurological diseases. Neurobiology of Diseases 15: 461–473. Nieoullon A, Canolle B, Masmejean F, Guillet B, Pisano P, and Lortet S (2006) The neuronal excitatory amino acid transporter EAAC1/EAAT3: Does it represent a major actor at the brain excitatory synapse? Journal of Neurochemistry 98: 1007–1018. Pellerin L and Magistretti P (2004) Neuroenergetics: Calling upon astrocytes to satisfy hungry neurons. Neuroscientist 10: 53–62. Re´ DB, Boucraut J, Samuel D, Birman S, Kerkerian-LeGoff L, and Had-Aissouni L (2003) Glutamate transport alteration triggers differentiation-state selective oxidative death of cultured astrocytes: A mechanism different from excitotoxicity depending on intracellular GSH contents. Journal of Neurochemistry 85: 1159–1170. Re´ DB, Nafia I, Melon C, Shimamoto K, Kerkerian-LeGoff L, and Had-Aissouni L (2006) Glutamate leakage from a compartmentalized intracellular metabolic pool and activation of the lipoxygenase pathway mediate oxidative astrocyte death by reversed glutamate transport. Glia 54: 47–57. Rothstein JD, Dykes-Hoberg M, Pardo CA, et al. (1996) Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate. Neuron 16: 675–686. Seifert G, Schilling K, and Steinha¨user C (2006) Astrocyte dysfunction in neurological disorders: A molecular perspective. Nature Reviews Neuroscience 7: 194–206. Shigeri Y, Seal RP, and Shimamoto K (2004) Molecular pharmacology of glutamate transporters, EAATs and VGLUTs. Brain Research Reviews 45: 250–265.