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1.  Epigenetic Regulation of Neuron-Dependent Induction of Astroglial Synaptic Protein GLT1 
Glia  2010;58(3):277-286.
Astroglial glutamate transporter EAAT2/GLT1 prevents glutamate-induced excitotoxicity in the central nervous system. Expression of EAAT2/GLT1 is dynamically regulated by neurons. The pathogenesis of amyotrophic lateral sclerosis (ALS) involves astroglial dysfunction, including dramatic loss of EAAT2/GLT1. DNA methylation of gene promoters represents one of the most important epigenetic mechanisms in regulating gene expression. The involvement of DNA methylation in the regulation of astroglial EAAT2/GLT1 expression in different conditions, especially in ALS has not been explored. In this study, we established a procedure to selectively isolate a pure astrocyte population in vitro and in vivo from BAC GLT1 eGFP mice using an eGFP-based fluorescence-activated cell sorting approach. Astrocytes isolated from this procedure are GFAP+ and GLT1+ and respond to neuronal stimulation, enabling direct methylation analysis of GLT1 promoter in these astrocytes. To investigate the role of DNA methylation in physiological and pathological EAAT2/GLT1 expression, methylation status of the EAAT2/GLT1 promoter was analyzed in astrocytes from in vitro and in vivo paradigms or postmortem ALS motor cortex by bisulfite sequencing method. DNA demethylation on selective CpG sites of the GLT1 promoter was highly correlated to increased GLT1 mRNA levels in astrocytes in response to neuronal stimulation; however, low level of methylation was found on CpG sites of EAAT2 promoter from postmortem motor cortex of human amyotrophic lateral sclerosis patients. In summary, hypermethylation on selective CpG sites of the GLT1 promoter is involved in repression of GLT1 promoter activation, but this regulation does not play a role in astroglial dysfunction of EAAT2 expression in patients with ALS.
doi:10.1002/glia.20922
PMCID: PMC2794958  PMID: 19672971
epigenetic; astrocyte; GLT1
2.  Harmine, A Natural Beta-Carboline Alkaloid, Upregulates Astroglial Glutamate Transporter Expression 
Neuropharmacology  2010;60(7-8):1168-1175.
Glutamate is the predominant excitatory amino acid neurotransmitter in the mammalian central nervous system (CNS). Glutamate transporter EAAT2 /GLT-1 is the physiologically dominant astroglial protein that inactivates synaptic glutamate. Previous studies have shown that EAAT2 dysfunction leads to excessive extracellular glutamate and may contribute to various neurological disorders including amyotrophic lateral sclerosis (ALS). The recent discovery of the neuroprotective properties of ceftriaxone, a beta lactam antibiotic, suggested that increasing EAAT2 /GLT-1 gene expression might be beneficial in ALS and other neurological/psychiatric disorders by augmenting astrocytic glutamate uptake. Here we report our efforts to develop a new screening assay for identifying compounds that activate EAAT2 gene expression. We generated fetal derived-human immortalized astroglial cells that are stably expressing a firefly luciferase reporter under the control of the human EAAT2 promoter. When screening a library of 1040 FDA approved compounds and natural products, we identified harmine, a naturally occurring beta-carboline alkaloid, as one of the top hits for activating the EAAT2 promoter. We further tested harmine in our in vitro cell culture systems and confirmed its ability to increase EAAT2/GLT1 gene expression and functional glutamate uptake activity. We next tested its efficacy in both wild type animals and in an ALS animal model of disease and demonstrated that harmine effectively increased GLT-1 protein and glutamate transporter activity in vivo. Our studies provide potential novel neurotherapeutics by modulating the activity of glutamate transporters via gene activation.
doi:10.1016/j.neuropharm.2010.10.016
PMCID: PMC3220934  PMID: 21034752
harmine; GLT-1; EAAT2; glutamate transporter; astroglia; ALS
3.  Role of Excitatory Amino Acid Transporter-2 (EAAT2) and Glutamate in Neurodegeneration: Opportunities for Developing Novel Therapeutics 
Journal of Cellular Physiology  2011;226(10):2484-2493.
Glutamate is an essential excitatory neurotransmitter regulating brain functions. Excitatory amino acid transporter (EAAT)-2 is one of the major glutamate transporters expressed predominantly in astroglial cells and is responsible for 90% of total glutamate uptake. Glutamate transporters tightly regulate glutamate concentration in the synaptic cleft. Dysfunction of EAAT2 and accumulation of excessive extracellular glutamate has been implicated in the development of several neurodegenerative diseases including Alzheimer’s disease, Huntington’s disease, and amyotrophic lateral sclerosis. Analysis of the 2.5-kb human EAAT2 promoter showed that NF-κB is an important regulator of EAAT2 expression in astrocytes. Screening of approximately 1,040 FDA-approved compounds and nutritionals led to the discovery that many β-lactam antibiotics are transcriptional activators of EAAT2 resulting in increased EAAT2 protein levels. Treatment of animals with ceftriaxone (CEF), a β-lactam antibiotic, led to an increase of EAAT2 expression and glutamate transport activity in the brain. CEF has neuroprotective effects in both in vitro and in vivo models based on its ability to inhibit neuronal cell death by preventing glutamate excitotoxicity. CEF increases EAAT2 transcription in primary human fetal astrocytes (PHFA) through the NF-κB signaling pathway. The NF-κB binding site at −272 position was critical in CEF-mediated EAAT2 protein induction. These studies emphasize the importance of transcriptional regulation in controlling glutamate levels in the brain. They also emphasize the potential utility of the EAAT2 promoter for developing both low and high throughput screening assays to identify novel small molecule regulators of glutamate transport with potential to ameliorate pathological changes occurring during and causing neurodegeneration.
doi:10.1002/jcp.22609
PMCID: PMC3130100  PMID: 21792905
4.  Evidence for change in current flux coupling of GLT1 at high glutamate concentrations in primary forebrain neurons and GLT1a expressing COS7 cells 
Glutamate is the major excitatory neurotransmitter of the central nervous system and is toxic to neurons even at low concentrations. GLT1, the rodent analog of human EAAT2, is primarily responsible for glutamate clearance in the cerebrum. GLT1 was thought to be expressed exclusively in astrocytes in the mature brain. Recently, however, GLT1a was demonstrated in excitatory axon terminals where synaptic glutamate concentration rises above 1 mM during excitatory transmission. However, GLT1 function in neurons with accurate control of both intracellular and extracellular solutions mimicking synaptic concentration gradients has never been studied. Here we characterized the kinetics of coupled glutamate transporter current in whole-cell configuration and [3H]-L-glutamate uptake in cultured rat cerebral neurons across the entire range of synaptic glutamate concentrations. In both neurons and GLT1a transfected COS-7 cells, the kinetics were similar and revealed two specific components: a high affinity component with glutamate kD value around 15 μM and low affinity component with kD value around 0.2 mM. The specific low affinity component was discovered due to significant deviation of the transporter current from Michaelis-Menten kinetics in the 100 – 300 μM concentration range. Activation of the specific low affinity component led to a twofold decrease in the current/flux ratio implying a change in the transport coupling. Our data indicate that GLT1 endogenously expressed in cultured rat forebrain neurons displays high and low glutamate affinity uptake components that are different in current/flux coupling ratios. This property is intrinsic to the protein because it was also observed in GLT1a transfected COS-7 cells.
doi:10.1111/j.1460-9568.2009.06809.x
PMCID: PMC3690583  PMID: 19614985
GLT1; patch-clamp; whole cell; current/flux coupling; excitotoxicity; presynaptic; synapse
5.  Molecular Dynamics Simulations of the Mammalian Glutamate Transporter EAAT3 
PLoS ONE  2014;9(3):e92089.
Excitatory amino acid transporters (EAATs) are membrane proteins that enable sodium-coupled uptake of glutamate and other amino acids into neurons. Crystal structures of the archaeal homolog GltPh have been recently determined both in the inward- and outward-facing conformations. Here we construct homology models for the mammalian glutamate transporter EAAT3 in both conformations and perform molecular dynamics simulations to investigate its similarities and differences from GltPh. In particular, we study the coordination of the different ligands, the gating mechanism and the location of the proton and potassium binding sites in EAAT3. We show that the protonation of the E374 residue is essential for binding of glutamate to EAAT3, otherwise glutamate becomes unstable in the binding site. The gating mechanism in the inward-facing state of EAAT3 is found to be different from that of GltPh, which is traced to the relocation of an arginine residue from the HP1 segment in GltPh to the TM8 segment in EAAT3. Finally, we perform free energy calculations to locate the potassium binding site in EAAT3, and find a high-affinity site that overlaps with the Na1 and Na3 sites in GltPh.
doi:10.1371/journal.pone.0092089
PMCID: PMC3958442  PMID: 24643009
6.  Nuclear Factor-κB Contributes to Neuron-Dependent Induction of GLT-1 Expression in Astrocytes 
The GLT-1 (EAAT2) subtype of glutamate transporter ensures crisp excitatory signaling and limits excitotoxicity in the CNS. Astrocytic expression of GLT-1 is regulated during development, by neuronal activity, and in neurodegenerative diseases. Although neurons activate astrocytic expression of GLT-1, the mechanisms involved have not been identified. In the present study, astrocytes from transgenic mice that express enhanced green fluorescent protein (eGFP) under the control of a bacterial artificial chromosome (BAC) containing a very large region of DNA surrounding the GLT-1 gene (BAC GLT-1 eGFP mice) were used to assess the role of nuclear factor-κB (NF-κB) in neuron-dependent activation of the GLT-1 promoter. We provide evidence that neurons activate NF-κB signaling in astrocytes. Transduction of astrocytes from the BAC GLT-1 eGFP mice with dominant-negative inhibitors of NF-κB signaling completely blocked neuron-dependent activation of a NF-κB reporter construct and attenuated induction of eGFP. Exogenous expression of p65 and/or p50 NF-κB subunits induced expression of eGFP or GLT-1 and increased GLT-1-mediated transport activity. Using wild type and mutant GLT-1 promoter reporter constructs, we found that NF-κB sites at −583 or −251 relative to the transcription start site eliminated neuron-dependent reporter activation. Electrophoretic mobility shift and supershift assays reveal that p65 and p50 interact with these same sites ex vivo. Finally, chromatin immunoprecipitation (ChIP) showed that p65 and p50 interact with these sites in adult cortex, but not in kidney (a tissue that expresses no detectable GLT-1). Together, these studies strongly suggest that NF-κB contributes to neuron-dependent regulation of astrocytic GLT-1 transcription.
doi:10.1523/JNEUROSCI.0302-11.2011
PMCID: PMC3138498  PMID: 21697367
glutamate transport; NF-κB; astrocytes; p65; p50; EAAT2; GLT-1; IκBα
7.  The Glutamate Transporter GLT1a Is Expressed in Excitatory Axon Terminals of Mature Hippocampal Neurons 
GLT1 is the major glutamate transporter of the brain and has been thought to be expressed exclusively in astrocytes. Although excitatory axon terminals take up glutamate, the transporter responsible has not been identified. GLT1 is expressed in at least two forms varying in the C termini, GLT1a and GLT1b. GLT1 mRNA has been demonstrated in neurons, without associated protein. Recently, evidence has been presented, using specific C terminus-directed antibodies, that GLT1b protein is expressed in neurons in vivo. These data suggested that the GLT1 mRNA detected in neurons encodes GLT1b and also that GLT1b might be the elusive presynaptic transporter. To test these hypotheses, we used variant-specific probes directed to the 3′-untranslated regions for GLT1a and GLT1b to perform in situ hybridization in the hippocampus. Contrary to expectation, GLT1a mRNA was the more abundant form. To investigate further the expression of GLT1 in neurons in the hippocampus, antibodies raised against the C terminus of GLT1a and against the N terminus of GLT1, found to be specific by testing in GLT1 knock-out mice, were used for light microscopic and EM-ICC. GLT1a protein was detected in neurons, in 14–29% of axons in the hippocampus, depending on the region. Many of the labeled axons formed axo-spinous, asymmetric, and, thus, excitatory synapses. Labeling also occurred in some spines and dendrites. The antibody against the N terminus of GLT1 also produced labeling of neuronal processes. Thus, the originally cloned form of GLT1, GLT1a, is expressed as protein in neurons in the mature hippocampus and may contribute significantly to glutamate uptake into excitatory terminals.
doi:10.1523/JNEUROSCI.1586-03.2004
PMCID: PMC2849838  PMID: 14762132
uptake; trafficking; alternative splicing; excitotoxicity; PDZ domain; synapse
8.  Impaired glutamate recycling and GluN2B-mediated neuronal calcium overload in mice lacking TGF-β1 in the CNS 
Glia  2013;61(6):985-1002.
Transforming growth factor β1 (TGF-β1) is a pleiotropic cytokine expressed throughout the CNS. Previous studies demonstrated that TGF-β1 contributes to maintain neuronal survival, but mechanistically this effect is not well understood. We generated a CNS-specific TGF-β1-deficient mouse model to investigate the functional consequences of TGF-β1-deficiency in the adult mouse brain. We found that depletion of TGF-β1 in the CNS resulted in a loss of the astrocyte glutamate transporter (GluTs) proteins GLT-1 (EAAT2) and GLAST (EAAT1) and decreased glutamate uptake in the mouse hippocampus. Treatment with TGF-β1 induced the expression of GLAST and GLT-1 in cultured astrocytes and enhanced astroglial glutamate uptake. Similar to GLT-1-deficient mice, CNS-TGF-β1-deficient mice had reduced brain weight and neuronal loss in the CA1 hippocampal region. CNS-TGF-β1-deficient mice showed GluN2B-dependent aberrant synaptic plasticity in the CA1 area of the hippocampus similar to the glutamate transport inhibitor DL-TBOA and these mice were highly sensitive to excitotoxic injury. In addition, hippocampal neurons from TGF-β1-deficient mice had elevated GluN2B-mediated calcium signals in response to extrasynaptic glutamate receptor stimulation, whereas cells treated with TGF-β1 exhibited reduced GluN2B-mediated calcium signals. In summary, our study demonstrates a previously unrecognized function of TGF-β1 in the CNS to control extracellular glutamate homeostasis and GluN2B-mediated calcium responses in the mouse hippocampus.
doi:10.1002/glia.22490
PMCID: PMC3981075  PMID: 23536313
TGF-β1; glutamate uptake; hippocampus; neuronal calcium; extrasynaptic; astrocytes
9.  Regional Distribution of Sodium-Dependent Excitatory Amino Acid Transporters in Rat Spinal Cord 
Background/Objective:
The excitatory amino acid transporters (EAATs), or sodium-dependent glutamate transporters, provide the primary mechanism for glutamate removal from the synaptic cleft. EAAT distribution has been determined in the rat brain, but it is only partially characterized in the spinal cord.
Methods:
The regional anatomic distribution of EAATs in spinal cord was assessed by radioligand autoradiography throughout cervical, thoracic, and lumbar cord levels in female Sprague-Dawley rats. EAAT subtype regional distribution was evaluated by inclusion of pharmacologic transport inhibitors in the autoradiography assays and by immunohistochemistry using subtype-specific polyclonal antibodies to rat GLT1 (EAAT2), GLAST (EAAT1), and EAAC1 (EAAT3) rat transporter subtypes.
Results:
[3H]-D-Aspartate binding was distributed throughout gray matter at the 3 spinal cord levels, with negligible binding in white matter. Inclusion of pharmacologic transport inhibitors indicates that the EAAT2/GLT1 subtype represents 21% to 40% of binding. Both EAAT1/GLAST and EAAT3/EAAC1 contributed the remainder of binding. Immunoreactivity to subtype-specific antibodies varied, depending on cord level, and was present in both gray and white matter. All 3 subtypes displayed prominent immunoreactivity in the dorsal horn. EAAT3/EAAC1 and to a lesser extent EAAT1/GLAST immunoreactivity also occurred in a punctate pattern in the ventral horn.
Conclusions:
The results indicate heterogeneity of EAAT distribution among spinal cord levels and regions. The presence of these transporters throughout rat spinal cord suggests the importance of their contributions to spinal cord function.
PMCID: PMC2031954  PMID: 17684893
Spinal cord; Glutamate plasma membrane proteins; Autoradiography; Immunohistochemistry; Amino acid transporters, Excitatory
10.  Glutamate transporter EAAT2: a new target for the treatment of neurodegenerative diseases 
Future medicinal chemistry  2012;4(13):1689-1700.
Glutamate is the primary excitatory amino acid neurotransmitter in the CNS. The concentration of glutamate in the synaptic cleft is tightly controlled by interplay between glutamate release and glutamate clearance. Abnormal glutamate release and/or dysfunction of glutamate clearance can cause overstimulation of glutamate receptors and result in neuronal injury known as excitotoxicity. The glial glutamate transporter EAAT2 plays a major role in glutamate clearance. Dysfunction or reduced expression of EAAT2 has been documented in many neurodegenerative diseases. In addition, many studies in animal models of disease indicate that increased EAAT2 expression provides neuronal protection. Here, we summarize these studies and suggest that EAAT2 is a potential target for the prevention of excitotoxicity. EAAT2 can be upregulated by transcriptional or translational activation. We discuss current progress in the search for EAAT2 activators, which is a promising direction for the treatment of neurodegenerative diseases.
doi:10.4155/fmc.12.122
PMCID: PMC3580837  PMID: 22924507
11.  Riluzole elevates GLT-1 activity and levels in striatal astrocytes 
Neurochemistry international  2011;60(1):31-38.
Drugs which upregulate astrocyte glutamate transport may be useful neuroprotective compounds by preventing excitotoxicity. We set up a new system to identify potential neuroprotective drugs which act through GLT-1. Primary mouse striatal astrocytes grown in the presence of the growth-factor supplement G5 express high levels of the functional glutamate transporter, GLT-1 (also known as EAAT2) as assessed by Western blotting and 3H-glutamate uptake assay, and levels decline following growth factor withdrawal. The GLT-1 transcriptional enhancer dexamethasone (0.1 or 1 μM) was able to prevent loss of GLT-1 levels and activity following growth factor withdrawal. In contrast, ceftriaxone, a compound previously reported to enhance GLT-1 expression, failed to regulate GLT-1 in this system. The neuroprotective compound riluzole (100 μM) upregulated GLT-1 levels and activity, through a mechanism that was not dependent on blockade of voltage-sensitive ion channels, since zonasimide (1 mM) did not regulate GLT-1. Finally, CDP-choline (10 μM – 1 mM), a compound which promotes association of GLT-1/EAAT2 with lipid rafts was unable to prevent GLT-1 loss under these conditions. This observation extends the known pharmacological actions of riluzole, and suggests that this compound may exert its neuroprotective effects through an astrocyte-dependent mechanism.
doi:10.1016/j.neuint.2011.10.017
PMCID: PMC3430367  PMID: 22080156
EAAT2; neuroprotection; citicholine; Parkinson’s Disease; glutamate uptake; glutamate transporters
12.  Motor neuron impairment mediated by a sumoylated fragment of the glial glutamate transporter EAAT2 
Glia  2011;59(11):1719-1731.
Dysregulation of glutamate handling ensuing downregulation of expression and activity levels of the astroglial glutamate transporter EAAT2 is implicated in excitotoxic degeneration of motor neurons in amyotrophic lateral sclerosis (ALS). We previously reported that EAAT2 (a.k.a. GLT-1) is cleaved by caspase-3 at its cytosolic carboxy-terminus domain. This cleavage results in impaired glutamate transport activity and generates a proteolytic fragment (CTE) that we found to be post-translationally conjugated by SUMO1. We show here that this sumoylated CTE fragment accumulates in the nucleus of spinal cord astrocytes of the SOD1-G93A mouse model of ALS at symptomatic stages of disease. Astrocytic expression of CTE, artificially tagged with SUMO1 (CTE-SUMO1) to mimic the native sumoylated fragment, recapitulates the nuclear accumulation pattern of the endogenous EAAT2-derived proteolytic fragment. Moreover, in a co-culture binary system, expression of CTE-SUMO1 in spinal cord astrocytes initiates extrinsic toxicity by inducing caspase-3 activation in motor neuron-derived NSC-34 cells or axonal growth impairment in primary motor neurons. Interestingly, prolonged nuclear accumulation of CTE-SUMO1 is intrinsically toxic to spinal cord astrocytes, although this gliotoxic effect of CTE-SUMO1 occurs later than the indirect, non-cell autonomous toxic effect on motor neurons. As more evidence on the implication of SUMO substrates in neurodegenerative diseases emerges, our observations strongly suggest that the nuclear accumulation in spinal cord astrocytes of a sumoylated proteolytic fragment of the astroglial glutamate transporter EAAT2 could participate to the pathogenesis of ALS and suggest a novel, unconventional role for EAAT2 in motor neuron degeneration.
doi:10.1002/glia.21218
PMCID: PMC3896305  PMID: 21769946
Amyotrophic lateral sclerosis; post-translational modification; SUMO; excitotoxicity
13.  The Inflammatory Cytokine, Interleukin-1 Beta, Mediates Loss of Astroglial Glutamate Transport and Drives Excitotoxic Motor Neuron Injury in the Spinal Cord During Acute Viral Encephalomyelitis 
Journal of neurochemistry  2008;105(4):1276-1286.
Astrocytes remove glutamate from the synaptic cleft via specific transporters, and impaired glutamate reuptake may promote excitotoxic neuronal injury. In a model of viral encephalomyelitis caused by neuroadapted Sindbis virus (NSV), mice develop acute paralysis and spinal motor neuron degeneration inhibited by the AMPA receptor antagonist, NBQX. To investigate disrupted glutamate homeostasis in the spinal cord, expression of the main astroglial glutamate transporter, GLT-1, was examined. GLT-1 levels declined in the spinal cord during acute infection while GFAP expression was preserved. There was simultaneous production of inflammatory cytokines at this site, and susceptible animals treated with drugs that blocked IL-1β release also limited paralysis and prevented the loss of GLT-1 expression. Conversely, infection of resistant mice that develop mild paralysis following NSV challenge showed higher baseline GLT-1 levels as well as lower production of IL-1β and relatively preserved GLT-1 expression in the spinal cord compared to susceptible hosts. Finally, spinal cord GLT-1 expression was largely maintained following infection of IL-1β-deficient animals. Together, these data show that IL-1β inhibits astrocyte glutamate transport in the spinal cord during viral encephalomyelitis. They provide one of the strongest in vivo links between innate immune responses and the development of excitotoxicity demonstrated to date.
doi:10.1111/j.1471-4159.2008.05230.x
PMCID: PMC2579753  PMID: 18194440
glutamate transporters; interleukin-1β; viral encephalomyelitis; motor neuron; excitotoxicity
14.  Glutamate spillover augments GABA synthesis and release from axodendritic synapses in rat hippocampus 
Hippocampus  2010;20(1):134-144.
Tight coupling between GABA synthesis and vesicle filling suggests that the presynaptic supply of precursor glutamate could dynamically regulate inhibitory synapses. Although the neuronal glutamate transporter Excitatory Amino Acid Transporter 3 (EAAT3) has been proposed to mediate such a metabolic role, highly efficient astrocytic uptake of synaptically released glutamate normally maintains low extracellular glutamate levels. We examined whether axodendritic inhibitory synapses in stratum radiatum of hippocampal area CA1, which are closely positioned among excitatory glutamatergic synapses, are regulated by synaptic glutamate release via presynaptic uptake. Under conditions of spatially and temporally coordinated release of glutamate and GABA within pyramidal cell dendrites, blocking glial glutamate uptake enhanced quantal release of GABA in a transporter-dependent manner. These physiological findings correlated with immunohistochemical studies revealing expression of EAAT3 by interneurons and uptake of D-asparate into putative axodendritic inhibitory terminals only when glial uptake was blocked. These results indicate that spillover of glutamate between adjacent excitatory and inhibitory synapses can occur under conditions when glial uptake incompletely clears synaptically released glutamate. Our anatomical studies also suggest that perisomatic inhibitory synapses, unlike synapses within dendritic layers of hippocampus, are not capable of glutamate uptake and therefore transporter-mediated dynamic regulation of inhibition is a unique feature of axodendritic synapses that may play a role in maintaining a homeostatic balance of inhibition and excitation.
doi:10.1002/hipo.20600
PMCID: PMC2810457  PMID: 19338018
15.  Co-Compartmentalization of the Astroglial Glutamate Transporter, GLT-1, with Glycolytic Enzymes and Mitochondria 
The Journal of Neuroscience  2011;31(50):18275-18288.
Efficient excitatory transmission depends on a family of transporters that utilize the Na+-electrochemical gradient to maintain low synaptic concentrations of glutamate. These transporters consume substantial energy in the spatially restricted space of fine astrocytic processes. GLT-1 (EAAT2) mediates the bulk of this activity in forebrain. To date, relatively few proteins have been identified that associate with GLT-1. In the present study, GLT-1 immunoaffinity isolates were prepared from rat cortex using three strategies and analyzed by LC coupled tandem mass spectrometry. In addition to known interacting proteins, the analysis identified glycolytic enzymes and outer mitochondrial proteins. Using double label immunofluorescence, GLT-1 was shown to co-localize with the mitochondrial matrix protein, ubiquinol-cytochrome c reductase core protein II (UQCRC2) or the inner mitochondrial membrane protein, ADP/ATP translocase (ANT), in rat cortex. In biolistically transduced hippocampal slices, fluorescently tagged GLT-1 puncta overlapped with fluorescently tagged mitochondria along fine astrocytic processes. In a Monte Carlo-type computer simulation, this overlap was significantly more frequent than would occur by chance. Furthermore, fluorescently tagged hexokinase-1 overlapped with mitochondria or GLT-1, strongly suggesting that GLT-1, mitochondria, and the first step in glycolysis are co-compartmentalized in astrocytic processes. Acute inhibition of glycolysis or oxidative phosphorylation had no effect on glutamate uptake in hippocampal slices, but simultaneous inhibition of both processes significantly reduced transport. Together with previous results, these studies show that GLT-1 co-compartmentalizes with Na+/K+ ATPase, glycolytic enzymes, and mitochondria, providing a mechanism to spatially match energy and buffering capacity to the demands imposed by transport.
doi:10.1523/JNEUROSCI.3305-11.2011
PMCID: PMC3259858  PMID: 22171032
glutamate transport; GLT-1; mitochondria; glycolysis
16.  Na+–H+ Exchanger Regulatory Factor 1 is a PDZ Scaffold for the Astroglial Glutamate Transporter GLAST 
Glia  2007;55(2):119-129.
Glutamate is a key neurotransmitter and its levels in the synaptic cleft are tightly regulated by reuptake mechanisms that primarily involve transporters in astrocytes. This requires that the glutamate transporters be spatially constrained to effect maximum glutamate transport. GLAST (EAAT1) is the predominant astroglial transporter and contains a class I PDZ-binding consensus (ETKM) in its C-terminus. The epithelial Na+/H+ exchanger regulatory factors NHERF1 and NHERF2 are PDZ proteins that contain two tandem PDZ domains and a C-terminal domain that binds members of the ERM (ezrin–radixin–moesin) family of membrane-cytoskeletal adaptors. NHERF proteins have been extensively characterized in renal epithelia and their expression in brain has recently been reported; however, their function in the brain remains unknown. The aims of the current study were to (1) determine the distribution of NHERF1/2 in the rodent brain and (2) investigate whether GLAST was a physiological ligand for NHERF1/2. Immunohistochemistry revealed that NHERF1 expression was widespread in rat brain (abundant in cerebellum, cerebral cortex, hippocampus, and thalamus) and primarily restricted to astrocytes whereas NHERF2 expression was primarily restricted to endothelial cells of blood vessels and capillaries. Importantly, NHERF1 distribution closely matched that of GLAST and confocal imaging demonstrated co-localization of the two proteins. Co-immunoprecipitation demonstrated that GLAST, NHERF1, and ezrin associate in vivo. In vitro binding assays showed that GLAST bound directly to the PDZ1 domain of NHERF1 via the C-terminal ETKM motif of GLAST. These findings implicate the GLAST–NHERF1 complex in the regulation of glutamate homeostasis in astrocytes.
doi:10.1002/glia.20439
PMCID: PMC2773615  PMID: 17048262
NHERF1; NHERF2; GLAST; astrocytes; glutamate transport
17.  Expression of multiple glutamate transporter splice variants in the rodent testis 
Asian Journal of Andrology  2010;13(2):254-265.
Glutamate is a regulated molecule in the mammalian testis. Extracellular regulation of glutamate in the body is determined largely by the expression of plasmalemmal glutamate transporters. We have examined by PCR, western blotting and immunocytochemistry the expression of a panel of sodium-dependent plasmalemmal glutamate transporters in the rat testis. Proteins examined included: glutamate aspartate transporter (GLAST), glutamate transporter 1 (GLT1), excitatory amino acid carrier 1 (EAAC1), excitatory amino acid transporter 4 (EAAT4) and EAAT5. We demonstrate that many of the glutamate transporters in the testis are alternately spliced. GLAST is present as exon-3- and exon-9-skipping forms. GLT1 was similarly present as the alternately spliced forms GLT1b and GLT1c, whereas the abundant brain form (GLT1a) was detectable only at the mRNA level. EAAT5 was also strongly expressed, whereas EAAC1 and EAAT4 were absent. These patterns of expression were compared with the patterns of endogenous glutamate localization and with patterns of 𝒹-aspartate accumulation, as assessed by immunocytochemistry. The presence of multiple glutamate transporters in the testis, including unusually spliced forms, suggests that glutamate homeostasis may be critical in this organ. The apparent presence of many of these transporters in the testis and sperm may indicate a need for glutamate transport by such cells.
doi:10.1038/aja.2010.99
PMCID: PMC3739219  PMID: 21170079
excitatory amino acid transporter; glutamate aspartate transporter; glutamate transporter 1; sperm; splice variant; testis; transporter
18.  Neuronal glutamate transporters regulate glial excitatory transmission 
In the central nervous system, excitatory amino acid transporters (EAATs) localized to neurons and glia terminate the actions of synaptically released glutamate. Whereas glial transporters are primarily responsible for maintaining low ambient levels of extracellular glutamate, neuronal transporters have additional roles in shaping excitatory synaptic transmission. Here we test the hypothesis that the expression level of the Purkinje cell (PC)-specific transporter, EAAT4, near parallel fiber (PF) release sites controls the extrasynaptic glutamate concentration transient following synaptic stimulation. Expression of EAAT4 follows a parasagittal banding pattern that allows us to compare regions of high and low EAAT4-expressing PCs. Using EAAT4 promoter driven eGFP reporter mice together with pharmacology and genetic deletion, we show that the level of neuronal transporter expression influences extrasynaptic transmission from PFs to adjacent Bergmann glia (BG). Surprisingly, a twofold difference in functional EAAT4 levels is sufficient to alter signaling to BG although EAAT4 may only be responsible for removing a fraction of released glutamate. These results demonstrate that physiological regulation of neuronal transporter expression can alter extrasynaptic neuro-glial signaling.
doi:10.1523/JNEUROSCI.5232-11.2012
PMCID: PMC3532047  PMID: 22302796
synaptic transmission; Purkinje cell; parallel fiber; EAAT4
19.  Abnormal Expression of Glutamate Transporter and Transporter Interacting Molecules in Prefrontal Cortex in Elderly Patients with Schizophrenia 
Schizophrenia research  2008;104(1-3):108-120.
Glutamate cycling is critically important for neurotransmission, and may be altered in schizophrenia. The excitatory amino acid transporters (EAATs) facilitate the reuptake of glutamate from the synaptic cleft and have a key role in glutamate cycling. We hypothesized that expression of the EAATs and the EAAT regulating proteins ARHGEF11, JWA, G protein suppressor pathway 1 (GPS1), and KIAA0302 are altered in the brain in schizophrenia. To test this, we measured expression of EAAT1, EAAT2, EAAT3, and EAAT interacting proteins in postmortem tissue from the dorsolateral prefrontal and anterior cingulate cortex of patients with schizophrenia and a comparison group using in situ hybridization and Western blot analysis. We found increased EAAT1 transcripts and decreased protein expression, increased EAAT3 transcripts and protein, and elevated protein expression of both GPS1 and KIAA0302 protein. We did not find any changes in expression of EAAT2. These data indicate that proteins involved in glutamate reuptake and cycling are altered in the cortex in schizophrenia, and may provide potential targets for future treatment strategies.
doi:10.1016/j.schres.2008.06.012
PMCID: PMC2656372  PMID: 18678470
GPS1; anterior cingulate cortex; dorsolateral prefrontal cortex; postmortem; Western blot; in situ hybridization
20.  Time-resolved Mechanism of Extracellular Gate Opening and Substrate Binding in a Glutamate Transporter*S⃞ 
The Journal of Biological Chemistry  2008;283(42):28680-28690.
Glutamate transporters, also referred to as excitatory amino acid transporters (EAATs), are membrane proteins that regulate glutamatergic signal transmission by clearing excess glutamate after its release at synapses. A structure-based understanding of their molecular mechanisms of function has been elusive until the recent determination of the x-ray structure of an archaeal transporter, GltPh. GltPh exists as a trimer, with each subunit containing a core region that mediates substrate translocation. In the present study a series of molecular dynamics simulations have been conducted and analyzed in light of new experimental data on substrate binding properties of EAATs. The simulations provide for the first time a full atomic description of the time-resolved events that drive the recognition and binding of substrate. The core region of each subunit exhibits an intrinsic tendency to open the helical hairpin HP2 loop, the extracellular gate, within tens of nanoseconds exposing conserved polar residues that serve as attractors for substrate binding. The NMDGT motif on the partially unwound part of the transmembrane helix TM7 and the residues Asp-390 and Asp-394 on TM8 are also distinguished by their important role in substrate binding and close interaction with mediating water molecules and/or sodium ions. The simulations reveal a Na+ binding site comprised in part of Leu-303 on TM7 and Asp-405 on TM8 and support a role for sodium ions in stabilizing substrate-bound conformers. The functional importance of Leu-303 or its counterpart Leu-391 in human EAAT1 (hEAAT1) is confirmed by site-directed mutagenesis and Na+ dependence assays conducted with hEAAT1 mutants L391C and L391A.
doi:10.1074/jbc.M800889200
PMCID: PMC2568915  PMID: 18678877
21.  Inhibitors of Glutamate Dehydrogenase Block Sodium-Dependent Glutamate Uptake in Rat Brain Membranes 
We recently found evidence for anatomic and physical linkages between the astroglial Na+-dependent glutamate transporters (GLT-1/EAAT2 and GLAST/EAAT1) and mitochondria. In these same studies, we found that the glutamate dehydrogenase (GDH) inhibitor, epigallocatechin-monogallate (EGCG), inhibits both glutamate oxidation and Na+-dependent glutamate uptake in astrocytes. In the present study, we extend this finding by exploring the effects of EGCG on Na+-dependent l-[3H]-glutamate (Glu) uptake in crude membranes (P2) prepared from rat brain cortex. In this preparation, uptake is almost exclusively mediated by GLT-1. EGCG inhibited l-[3H]-Glu uptake in cortical membranes with an IC50 value of 230 μM. We also studied the effects of two additional inhibitors of GDH, hexachlorophene (HCP) and bithionol (BTH). Both of these compounds also caused concentration-dependent inhibition of glutamate uptake in cortical membranes. Pre-incubating with HCP for up to 15 min had no greater effect than that observed with no pre-incubation, showing that the effects occur rapidly. HCP decreased the Vmax for glutamate uptake without changing the Km, consistent with a non-competitive mechanism of action. EGCG, HCP, and BTH also inhibited Na+-dependent transport of d-[3H]-aspartate (Asp), a non-metabolizable transporter substrate, and [3H]-γ-aminobutyric acid (GABA). In contrast to the forebrain, glutamate uptake in crude cerebellar membranes (P2) is likely mediated by GLAST (EAAT1). Therefore, the effects of these compounds were examined in cerebellar membranes. In this region, none of these compounds had any effect on uptake of either l-[3H]-Glu or d-[3H]-Asp, but they all inhibited [3H]-GABA uptake. Together these studies suggest that GDH is preferentially required for glutamate uptake in forebrain as compared to cerebellum, and GDH may be required for GABA uptake as well. They also provide further evidence for a functional linkage between glutamate transport and mitochondria.
doi:10.3389/fendo.2013.00123
PMCID: PMC3775299  PMID: 24062726
glutamate; GLT-1; EAAT2; GLAST; GABA; glutamate dehydrogenase; sodium-dependent uptake; epigallocatechin-monogallate
22.  Vesicular glutamate transporter and cognition in stroke 
Neurology  2010;75(20):1803-1809.
Objectives:
Vascular dementia (VaD) accounts for approximately 15%–20% of all dementias, but the relationship of progressive cognitive impairment to neurochemical changes is poorly understood. We have therefore investigated glutamatergic synaptic markers in VaD.
Methods:
We used homogenates prepared from gray matter from 2 neocortical regions (Brodmann area [BA] 9 and BA 20) and Western blotting to determine the concentrations of key components of the glutamatergic neurotransmitter system, vesicular glutamate transporter 1 (VGLUT1) and excitatory amino acid transporter EAAT2 (GLT-1), and the ubiquitous synaptic protein, synaptophysin, in 73 individuals—48 patients with cerebrovascular disease with and without dementia, 10 patients with AD, and 15 controls—in a case-control design.
Results:
VGLUT1 concentrations in BA 20 and BA 9 were correlated with CAMCOG total (Rs 0.525, p = 0.018, n = 20; Rs 0.560, p = 0.002, n = 27) and CAMCOG memory scores (Rs 0.616, p = 0.004, n = 20; Rs 0.675, p = 0.000, n = 27). VGLUT1 concentration in BA 9 differed between the different dementia groups and the stroke no dementia group (1-way analysis of variance F = 6.69, p = 0.001 and Bonferroni p < 0.01 in each case), with subjects with stroke who did not develop dementia exhibiting the highest mean value for VGLUT1.
Conclusions:
These data suggest that loss of glutamatergic synapses is a feature of VaD and Alzheimer disease but the preservation of synapses, in particular glutamatergic synapses, in the frontal cortex against the temporal cortex plays a role in sustaining cognition and protecting against dementia following a stroke.
GLOSSARY
= Alzheimer disease;
= analysis of variance;
= Brodmann area;
= cerebral amyloid angiopathy;
= Cambridge Assessment of Mental Health for the Elderly, Section B;
= Consortium to Establish a Registry for Alzheimer's Disease;
= Diagnostic and Statistical Manual of Mental Disorders, 4th edition;
= glial fibrillary acidic protein;
= hematoxylin & eosin;
= Luxol fast blue;
= stroke no dementia;
= vascular dementia;
= vesicular glutamate transporter 1.
doi:10.1212/WNL.0b013e3181fd6328
PMCID: PMC2995382  PMID: 21079182
23.  Identification of Translational Activators of Glial Glutamate Transporter EAAT2 through Cell-Based High-Throughput Screening: An Approach to Prevent Excitotoxicity 
Journal of biomolecular screening  2010;15(6):653-662.
Excitotoxicity has been implicated as the mechanism of neuronal damage resulting from acute insults such as stroke, epilepsy, and trauma, as well as during the progression of adult-onset neurodegenerative disorders such as Alzheimer’s disease and amyotrophic lateral sclerosis (ALS). Excitotoxicity is defined as excessive exposure to the neurotransmitter glutamate or overstimulation of its membrane receptors, leading to neuronal injury or death. One potential approach to protect against excitotoxic neuronal damage is enhanced glutamate reuptake. The glial glutamate transporter EAAT2 is the quantitatively dominant glutamate transporter and plays a major role in clearance of glutamate. Expression of EAAT2 protein is highly regulated at the translational level. In an effort to identify compounds that can induce translation of EAAT2 transcripts, a cell-based enzyme-linked immunosorbent assay was developed using a primary astrocyte line stably transfected with a vector designed to identify modulators of EAAT2 translation. This assay was optimized for high-throughput screening, and a library of approximately 140,000 compounds was tested. In the initial screen, 293 compounds were identified as hits. These 293 hits were retested at 3 concentrations, and a total of 61 compounds showed a dose-dependent increase in EAAT2 protein levels. Selected compounds were tested in full 12-point dose-response experiments in the screening assay to assess potency as well as confirmed by Western blot, immunohistochemistry, and glutamate uptake assays to evaluate the localization and function of the elevated EAAT2 protein. These hits provide excellent starting points for developing therapeutic agents to prevent excitotoxicity.
doi:10.1177/1087057110370998
PMCID: PMC3016154  PMID: 20508255
excitotoxicity; glutamate transporter; EAAT2; high-throughput screen; neurodegeneration
24.  Glutamate transporter type 3 knockout mice have a decreased isoflurane requirement to induce loss of righting reflex 
Neuroscience  2010;171(3):788-793.
Excitatory amino acid transporters (EAAT) uptake extracellular glutamate, the major excitatory neurotransmitter in the brain. EAAT type 3 (EAAT3), the main neuronal EAAT, is expressed widely in the central nervous system. We have shown that the volatile anesthetic isoflurane increases EAAT3 activity and trafficking to the plasma membrane. Thus, we hypothesize that EAAT3 mediates isoflurane-induced anesthesia. To test this hypothesis, the potency of isoflurane to induce immobility and hypnosis, two major components of general anesthesia, was compared in the CD-1 wild-type mice and EAAT knockout mice that had a CD-1 strain gene background. Hypnosis was assessed by loss of righting reflex in this study. The expression of EAAT1 and EAAT2, two widely expressed EAATs in the central nervous system, in the cerebral cortex and spinal cord was not different between the EAAT3 knockout mice and wild-type mice. The concentration required for isoflurane to cause immobility to painful stimuli, a response involving primarily reflex loops in the spinal cord, was not changed by EAAT3 knockout. However, the EAAT3 knockout mice were more sensitive to isoflurane-induced hypnotic effects, which may be mediated by hypothalamic sleep neural circuits. Interestingly, the EAAT3 knockout mice did not have an altered sensitivity to the hypnotic effects caused by ketamine, an intravenous anesthetic that is a glutamate receptor antagonist and does not affect EAAT3 activity. These results suggest that EAAT3 modulates the sensitivity of neural circuits to isoflurane. These results, along with our previous findings that isoflurane increases EAAT3 activity, indicate that EAAT3 may regulate isoflurane-induced behavioral changes, including anesthesia.
doi:10.1016/j.neuroscience.2010.09.044
PMCID: PMC3401886  PMID: 20875840
anesthesia; glutamate transporter; gene expression; hypnosis; isoflurane
25.  Abnormal Glycosylation of EAAT1 and EAAT2 in Prefrontal Cortex of Elderly Patients with Schizophrenia 
Schizophrenia research  2009;117(1):92.
The excitatory amino acid transporters (EAATs) are a family of molecules that are essential for regulation of synaptic glutamate levels. The EAATs may also be regulated by N-glycosylation, a posttranslational modification that is critical for many cellular functions including localization in the plasma membrane. We hypothesized that glycosylation of the EAATs is abnormal in schizophrenia. To test this hypothesis, we treated postmortem tissue from the dorsolateral prefrontal and anterior cingulate cortices of patients with schizophrenia and comparison subjects with deglycosylating enzymes. We then measured the resulting shifts in molecular weight of the EAATs using Western blot analysis to determine the mass of glycans cleaved from the transporter. We found evidence for less glycosylation of both EAAT1 and EAAT2 in schizophrenia. We did not detect N-linked glycosylation of EAAT3 in either schizophrenia or the comparison subjects in these regions. Our data suggest an abnormality of posttranslational modification of glutamate transporters in schizophrenia that suggests a decreased capacity for glutamate reuptake.
doi:10.1016/j.schres.2009.07.025
PMCID: PMC2822023  PMID: 19716271
GLAST; GLT-1; EAAC1; deglycosylation; anterior cingulate cortex; dorsolateral prefrontal cortex

Results 1-25 (390126)