Journal of Neuroscience Research 95:2140–2151 (2017) Review Astroglial Glutamate Transporters in the Brain: Regulating Neurotransmitter Homeostasis and Synaptic Transmission Ciaran Murphy-Royal,1,2 Julien Dupuis,2,3 Laurent Groc,2,3 and Stephane H. R. Oliet 1,2 1Neurocentre Magendie, Inserm U1215, Bordeaux, France 2Universite de Bordeaux, Bordeaux, France 3Interdisciplinary Institute for Neuroscience, CNRS UMR 5297, Bordeaux, France Astrocytes, the major glial cell type in the central nervous compartments. As an example, the extracellular glutamate system (CNS), are critical for brain function and have concentration is maintained at low values lying most like- been implicated in various disorders of the central ner- ly below 100 nM (Hamberger and Nystrom,€ 1984; vous system. These cells are involved in a wide range of Cavelier and Attwell, 2005; Herman and Jahr, 2007), cerebral processes including brain metabolism, control of while it is much more concentrated inside cells. As there central blood flow, ionic homeostasis, fine-tuning synap- are no extracellular enzymes degrading glutamate, this tic transmission, and neurotransmitter clearance. Such low extracellular concentration can be fully accounted for varied roles can be efficiently carried out due to the inti- by specific transporters, which are widely expressed mate interactions astrocytes maintain with neurons, the throughout the brain (Furuta et al., 1997). Glutamate vasculature, as well as with other glial cells. Arguably, one transporters working under physiological conditions have of the most important functions of astrocytes in the brain the capacity to establish concentration gradients of 106 is their control of neurotransmitter clearance. This is across the plasma membrane, theoretically resulting in a particularly true for glutamate whose timecourse in the glutamate concentration of 10 nM extracellularly and 10 synaptic cleft needs to be controlled tightly under physio- mM intracellularly (Zerangue and Kavanaugh, 1996a). logical conditions to maintain point-to-point excitatory They also allow the maintenance of gradients with subcel- transmission, thereby limiting spillover and activation lular compartments, as in synaptic vesicles where gluta- of more receptors. Most importantly, accumulation of mate has been estimated to be present at concentrations glutamate in the extracellular space can trigger excessive up to 60 mM due to highly efficient vesicular glutamate activation of glutamatergic receptors and lead to transporters (Burger et al., 1989; Shupliakov et al., 1992; excitotoxicity, a trademark of many neurodegenerative diseases. It is thus of utmost importance for both physio- for review see El Mestikawy et al., 2011). In this review, logical and pathophysiological reasons to understand the processes that control glutamate time course within the SIGNIFICANCE synaptic cleft and regulate its concentrations in the extra- This review is aimed at summarizing our present knowledge on astro- cellular space. VC 2017 Wiley Periodicals, Inc. cytes and their role in glutamate clearance. Astrocytes are non-neuronal Key words: GLT-1; Astrocytes; surface trafficking; gluta- cell present in our brain where they ensure several key fonctions. One mate uptake of the most important role played by these glial cells is to clear the neu- rotransmitter glutamate from the extracellular space. This is essential to maintain an appropriate cerebral communication. Such clearance is made possible by highly-selective transporters that are expressed on INTRODUCTION astrocytes and which are endowed with specific properties and expres- Since the initial discovery of its excitatory action on sion profile. They could represent a target of choice for therapuetic neurons (Hayashi, 1954; Curtis and Watkins, 1960), glu- strategies in the context of neurodegenerative diseases. tamate has been found to be highly concentrated in the central nervous system of mammals, with estimates in the range of 5-15 mmol/kg (Danbolt, 2001). Yet this neuro- Received 13 October 2016; Revised 20 December 2016; Accepted 2 transmitter is not uniformly distributed. Indeed, vast dif- January 2017 ferences in glutamate concentrations of have been Published online 2 February 2017 in Wiley Online Library reported in extracellular, intracellular and subcellular (wileyonlinelibrary.com). DOI: 10.1002/jnr.24029 VC 2017 Wiley Periodicals, Inc. Astroglial Glutamate Uptake 2141 we discuss how these unique properties allow glutamate be differences between subtypes (Haugeto et al., 1996; transporters to fulfil critical functions such as ensuring the Yernool et al., 2003, 2004). All five EAATs transport kinetics and specificity of synaptic transmissions or pre- L-glutamate (and DL-aspartate) with high affinities venting neurotoxicity associated with excessive activation (30 lM for GLT-1; Arriza et al., 1994, 1997; Fairman of glutamate receptors (Tzingounis and Wadiche, 2007). et al., 1995), using energy stored in the concentration gra- dients of sodium and potassium. During one individual Distribution and Functional Properties of transport cycle, each transporter binds one extracellular Glutamate Transporters molecule of glutamate as well as three sodium ions and The family of brain glutamate transporters is com- one proton. The transporter then undergoes a conforma- posed of five members – namely excitatory amino acid tional change towards an inward facing conformation transporters 1 to 5 (EAAT1-5)—which have been exten- where these substrates are released into the cytoplasm. sively reviewed elsewhere (Danbolt, 2001). EAAT1 This step is followed by the binding of one internal potas- (GLAST) and EAAT2 (GLT-1) are ubiquitously sium ion and a switch back to an outward facing state, expressed in the brain, with GLT-1 being particularly completing the transport cycle (Zerangue and Kavanaugh, abundant in the hippocampus and the cortex whereas 1996a; Reyes et al., 2009). To note, EAAT3 also trans- GLAST is highly concentrated in the cerebellum (Roth- ports cysteine (Zerangue and Kavanaugh, 1996b). More- stein et al., 1994; Lehre et al., 1995; Chaudhry et al., over, uncoupled fluxes can occur and EAATs can 1995; Schmitt et al., 1997; Furuta et al., 1997). EAAT3 function as chloride channels, particularly EAAT4 and (EAAC1) is also ubiquitous in the central nervous system EAAT5 which display considerable chloride conductance and is particularly concentrated in the hippocampus and may thus be alternatively envisioned as inhibitory glu- where it is targeted to somatic and dendritic locations on tamate receptors (Dehnes et al., 1998; Veruki et al., 2006). neurons (Rothstein et al., 1994; Shashidharan et al., 1997; Holmseth et al., 2012). To note, EAAT3 is also expressed Importance of Glutamate Transporters: Lessons on GABAergic nerve terminals where it provides gluta- From Knock-Out Models and Human Mutations mate as a precursor for GABA synthesis (Sepkuty et al., It was not until the 1990’s that the different gluta- 2002; Mathews and Diamond, 2003; Stafford et al., mate transporters were identified and cloned (Storck 2010). EAAT4 expression is essentially found in the cere- et al., 1992; Kanai and Hediger, 1992; Pines et al., 1992; bellum on the dendrites of Purkinje cells (Fairman et al., Arriza et al., 1994, 1997; Fairman et al., 1995), which 1995; Dehnes et al., 1998). Finally, EAAT5 is very weak- eventually led to the generation of subtype-specific gluta- ly expressed in the central nervous system and is preferen- mate transporter knockout mice. Overwhelming evi- tially found in the retina (Arriza et al., 1997; Barnett and dence from glutamate transporter knockout models Pow, 2000; Lee et al., 2012). demonstrated that glial glutamate transporters are critical The EAAT family can be roughly divided based on for normal brain function (see Table I). Indeed, impaired cell-specific expression patterns, with EAAT1 (GLAST) GLT-1 activity causes a massive increase in extracellular and EAAT2 (GLT-1) mainly located on astrocytes while glutamate levels in GLT-1 knockouts (Tanaka et al., EAAT3-5 are exclusively neuronal. However, it is impor- 1997; Mitani and Tanaka, 2003; Takasaki et al., 2008). tant to note that there is evidence supporting GLT-1 While mice appear to be normal at birth, they show low- expression on cortical and hippocampal neurons, either er body weight and start to suffer from hyperactivity as from in situ hybridization or immunodetection well as severe epileptic seizures after two to three weeks, approaches ex vivo and in cultured neurons (Schmitt a time-window when synaptic clearing of neurotransmit- et al., 1996; Wang et al., 1998; Mennerick et al., 1998; ters shifts from passive diffusion to uptake by transporters Melone et al., 2009). In the hippocampal CA1 area for (Ullensvang et al., 1997; Furuta et al., 1997; Thomas example, glutamatergic axon terminals from CA3 pyrami- et al., 2011). About half of GLT-1-deficient animals die dal cells express functional GLT-1 with full glutamate within the first month of life (Tanaka et al., 1997; Matsu- transport ability which are believed to represent 5-10% of gami et al., 2006). Interestingly, cell-specific knock-outs total hippocampal GLT-1 (Chen et al., 2004; Berger models recently allowed to distinguish the relative impor- et al., 2005; Furness et al., 2008). While neuronal GLT-1 tance of astrocytic versus neuronal GLT-1 (Petr et al., allow noticeable glutamate uptake within hippocampal 2015) and confirmed that deletion of astrocytic GLT-1 nerve terminals, evidence is still lacking on whether