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1.  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
2.  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
3.  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
4.  Pre-synaptic regulation of astroglial excitatory neurotransmitter transporter GLT1 
Neuron  2009;61(6):880-894.
SUMMARY
The neuron-astrocyte synaptic complex is a fundamental operational unit of the nervous system. Astroglia play a central role in the regulation of synaptic glutamate, via neurotransmitter transport by GLT1/EAAT2. The astroglial mechanisms underlying this essential neuron-glial communication are not known. Here we show that presynaptic terminals are sufficient and necessary for GLT1/EAAT2 transcriptional activation and have identified the molecular pathway that regulates astroglial responses to presynaptic input. Presynaptic terminals regulate astroglial GLT1/EAAT2 via kappa B-motif binding phosphoprotein (KBBP), the mouse homologue of human heterogeneous nuclear ribonucleoprotein K (hnRNP K), which binds to an essential element of GLT1/EAAT2 promoter. This neuron-stimulated factor is required for GLT1/EATT2 transcriptional activation and is responsible for astroglial alterations in neural injury. Denervation of neuron-astrocyte signaling in vivo, by acute corticospinal tract transection, ricin-induced motor neuron death, or chronic neurodegeneration in amyotrophic lateral sclerosis (ALS) all result in reduced astroglial KBBP expression and transcriptional dysfunction of astroglial transporter expression. Our studies indicate that presynaptic elements dynamically coordinate normal astroglial function and also provide a fundamental signaling mechanism by which altered neuronal function and injury leads to dysregulated astroglia in CNS disease.
doi:10.1016/j.neuron.2009.02.010
PMCID: PMC2743171  PMID: 19323997
5.  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
6.  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
7.  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
8.  Klotho Sensitivity of the Neuronal Excitatory Amino Acid Transporters EAAT3 and EAAT4 
PLoS ONE  2013;8(7):e70988.
Klotho, a transmembrane protein, which can be cleaved off as β-glucuronidase and hormone, is released in both, kidney and choroid plexus and encountered in blood and cerebrospinal fluid. Klotho deficiency leads to early appearance of age-related disorders and premature death. Klotho may modify transport by inhibiting 1,25(OH)2D3 formation or by directly affecting channel and carrier proteins. The present study explored whether Klotho influences the activity of the Na+-coupled excitatory amino acid transporters EAAT3 and EAAT4, which are expressed in kidney (EAAT3), intestine (EAAT3) and brain (EAAT3 and EAAT4). To this end, cRNA encoding EAAT3 or EAAT4 was injected into Xenopus oocytes with and without additional injection of cRNA encoding Klotho. EAAT expressing Xenopus oocytes were further treated with recombinant human β-Klotho protein with or without β-glucuronidase inhibitor D-saccharic acid 1,4-lactone monohydrate (DSAL). Electrogenic excitatory amino acid transport was determined as L-glutamate-induced current (Iglu) in two electrode voltage clamp experiments. EAAT3 and EAAT4 protein abundance in the Xenopus oocyte cell membrane was visualized by confocal microscopy and quantified utilizing chemiluminescence. As a result, coexpression of Klotho cRNA significantly increased Iglu in both, EAAT3 or EAAT4-expressing Xenopus oocytes. Klotho cRNA coexpression significantly increased the maximal current and cell membrane protein abundance of both EAAT3 and EAAT4. The effect of Klotho coexpression on EAAT3 and EAAT4 activity was mimicked by treating EAAT3 or EAAT4-expressing Xenopus oocytes with recombinant human β-Klotho protein. The effects of Klotho coexpression and of treatment with recombinant human β-Klotho protein were both abrogated in the presence of DSAL (10 µM). In conclusion, Klotho is a novel, powerful regulator of the excitatory amino acid transporters EAAT3 and EAAT4.
doi:10.1371/journal.pone.0070988
PMCID: PMC3726597  PMID: 23923038
9.  ENT1 Regulates Ethanol-Sensitive EAAT2 Expression and Function in Astrocytes 
Background
Equilibrative nucleoside transporter 1 (ENT1) and excitatory amino acid transporter 2 (EAAT2) are predominantly expressed in astrocytes where they are thought to regulate synaptic adenosine and glutamate levels. Because mice lacking ENT1 display increased glutamate levels in the ventral striatum, we investigated whether ENT1 regulates the expression and function of EAAT2 in astrocytes, which could contribute to altered glutamate levels in the striatum.
Methods
We examined the effect of ENT1 inhibition and overexpression on the expression of EAAT2 using quantitative real-time PCR and measured glutamate uptake activity in cultured astrocytes. We also examined the effect of 0 to 200 mM ethanol doses for 0 to 24 hours of ethanol exposure on EAAT2 expression and glutamate uptake activity. We further examined the effect of ENT1 knockdown by a specific siRNA on ethanol-induced EAAT2 expression.
Results
An ENT1-specific antagonist and siRNA treatments significantly reduced both EAAT2 expression and glutamate uptake activity while ENT1 overexpression up-regulated EAAT2 mRNA expression. Interestingly, 100 or 200 mM ethanol exposure increased EAAT2 mRNA expression as well as glutamate uptake activity. Moreover, we found that ENT1 knockdown inhibited the ethanol-induced EAAT2 up-regulation.
Conclusions
Our results suggest that ENT1 regulates glutamate uptake activity by altering EAAT2 expression and function, which might be implicated in ethanol intoxication and preference.
doi:10.1111/j.1530-0277.2010.01187.x
PMCID: PMC2913860  PMID: 20374202
Excitatory Amino Acid Transporter 2 (EAAT2); Equilibrative Nucleoside Transporter 1 (ENT1); Glutamate Uptake; Adenosine Uptake
10.  Small-molecule activator of glutamate transporter EAAT2 translation provides neuroprotection 
The Journal of Clinical Investigation  2014;124(3):1255-1267.
Glial glutamate transporter EAAT2 plays a major role in glutamate clearance in synaptic clefts. Several lines of evidence indicate that strategies designed to increase EAAT2 expression have potential for preventing excitotoxicity, which contributes to neuronal injury and death in neurodegenerative diseases. We previously discovered several classes of compounds that can increase EAAT2 expression through translational activation. Here, we present efficacy studies of the compound LDN/OSU-0212320, which is a pyridazine derivative from one of our lead series. In a murine model, LDN/OSU-0212320 had good potency, adequate pharmacokinetic properties, no observed toxicity at the doses examined, and low side effect/toxicity potential. Additionally, LDN/OSU-0212320 protected cultured neurons from glutamate-mediated excitotoxic injury and death via EAAT2 activation. Importantly, LDN/OSU-0212320 markedly delayed motor function decline and extended lifespan in an animal model of amyotrophic lateral sclerosis (ALS). We also found that LDN/OSU-0212320 substantially reduced mortality, neuronal death, and spontaneous recurrent seizures in a pilocarpine-induced temporal lobe epilepsy model. Moreover, our study demonstrated that LDN/OSU-0212320 treatment results in activation of PKC and subsequent Y-box–binding protein 1 (YB-1) activation, which regulates activation of EAAT2 translation. Our data indicate that the use of small molecules to enhance EAAT2 translation may be a therapeutic strategy for the treatment of neurodegenerative diseases.
doi:10.1172/JCI66163
PMCID: PMC3938250  PMID: 24569372
11.  Volatile anesthetics attenuate oxidative stress-reduced activity of glutamate transporter type 3 
Anesthesia and analgesia  2009;109(5):1506-1510.
Background
Volatile anesthetics enhance the activity of glutamate transporter type 3 (also called excitatory amino acid transporter type 3, EAAT3), the major neuronal EAAT. In addition to glutamate, EAAT3 can also uptake L-cysteine, the rate-limiting substrate for the synthesis of glutathione. Our previous study showed that oxidative stress inhibited glutamate-induced EAAT3 activity. We determined whether oxidative stress would reduce L-cysteine-induced EAAT3 activity and whether this reduction would be attenuated by volatile anesthetics.
Methods
Rat EAAT3 was expressed in Xenopus oocytes. L-glutamate- and L-cysteine-induced membrane currents were recorded using the two-electrode voltage clamp technique. The peak current was quantified to reflect the amount of transported substrates because transport of substrates via EAATs is electrogenic.
Results
Exposure of oocytes to 5 mM tert-butyl hydroperoxide, an organic oxidant, for 10 min reduced the Vmax, but did not affect the Km, of EAAT3 for L-cysteine. The volatile anesthetics isoflurane, sevoflurane and desflurane at concentrations from 1 to 3% attenuated the tert-butyl hydroperoxide-reduced EAAT3 activity for L-glutamate and L-cysteine.
Conclusions
Our results suggest that volatile anesthetics preserve EAAT3 function to transport L-glutamate and L-cysteine under oxidative stress, which may be a mechanism for the neuroprotective effects of volatile anesthetics.
doi:10.1213/ANE.0b013e3181b6709a
PMCID: PMC2773695  PMID: 19843789
12.  Structure-activity-relationship study of N-acyl-N-phenylpiperazines as potential inhibitors of the Excitatory Amino Acid Transporters (EAATs): improving the potency of a micromolar screening Hit is not truism 
SpringerPlus  2013;2:112.
The excitatory amino acid transporters (EAATs) are transmembrane proteins responsible for the uptake of (S)-glutamate from the synaptic cleft. To date, five subtypes EAAT1-5 have been identified for which selective inhibitors have been discovered for EAAT1 and EAAT2. By screening of a commercially available compound library consisting of 4,000 compounds, N-acyl-N-phenylpiperazine analog (±)-exo-1 was identified to be a non-selective inhibitor at EAAT1-3 displaying IC50 values in the mid-micromolar range (10 μM, 40 μM and 30 μM at EAAT1, 2 and 3, respectively). Subsequently, we designed and synthesized a series of analogs to explore the structure-activity-relationship of this scaffold in the search for analogs characterized by increased inhibitory potency and/or EAAT subtype selectivity. Despite extensive efforts, all analogs of (±)-exo-1 proved to be either inactive or to have least 3-fold lower inhibitory potency than the lead, and furthermore none of the active analogs displayed selectivity for a particular subtype amongst the EAAT1-3. On the basis of our findings, we speculate that (±)-exo-1 binds to a recess (deepening) on the EAAT proteins than a well-defined pocket.
doi:10.1186/2193-1801-2-112
PMCID: PMC4225009  PMID: 25530930
Excitatory amino acid transporters; EAATs; Rational ligand design; Medicinal chemistry
13.  Cysteine Transport through Excitatory Amino Acid Transporter 3 (EAAT3) 
PLoS ONE  2014;9(10):e109245.
Excitatory amino acid transporters (EAATs) limit glutamatergic signaling and maintain extracellular glutamate concentrations below neurotoxic levels. Of the five known EAAT isoforms (EAATs 1–5), only the neuronal isoform, EAAT3 (EAAC1), can efficiently transport the uncharged amino acid L-cysteine. EAAT3-mediated cysteine transport has been proposed to be a primary mechanism used by neurons to obtain cysteine for the synthesis of glutathione, a key molecule in preventing oxidative stress and neuronal toxicity. The molecular mechanisms underlying the selective transport of cysteine by EAAT3 have not been elucidated. Here we propose that the transport of cysteine through EAAT3 requires formation of the thiolate form of cysteine in the binding site. Using Xenopus oocytes and HEK293 cells expressing EAAT2 and EAAT3, we assessed the transport kinetics of different substrates and measured transporter-associated currents electrophysiologically. Our results show that L-selenocysteine, a cysteine analog that forms a negatively-charged selenolate ion at physiological pH, is efficiently transported by EAATs 1–3 and has a much higher apparent affinity for transport when compared to cysteine. Using a membrane tethered GFP variant to monitor intracellular pH changes associated with transport activity, we observed that transport of either L-glutamate or L-selenocysteine by EAAT3 decreased intracellular pH, whereas transport of cysteine resulted in cytoplasmic alkalinization. No change in pH was observed when cysteine was applied to cells expressing EAAT2, which displays negligible transport of cysteine. Under conditions that favor release of intracellular substrates through EAAT3 we observed release of labeled intracellular glutamate but did not detect cysteine release. Our results support a model whereby cysteine transport through EAAT3 is facilitated through cysteine de-protonation and that once inside, the thiolate is rapidly re-protonated. Moreover, these findings suggest that cysteine transport is predominantly unidirectional and that reverse transport does not contribute to depletion of intracellular cysteine pools.
doi:10.1371/journal.pone.0109245
PMCID: PMC4183567  PMID: 25275463
14.  Modulation of Glutamate Transport and Receptor Binding by Glutamate Receptor Antagonists in EAE Rat Brain 
PLoS ONE  2014;9(11):e113954.
The etiology of multiple sclerosis (MS) is currently unknown. However, one potential mechanism involved in the disease may be excitotoxicity. The elevation of glutamate in cerebrospinal fluid, as well as changes in the expression of glutamate receptors (iGluRs and mGluRs) and excitatory amino acid transporters (EAATs), have been observed in the brains of MS patients and animals subjected to experimental autoimmune encephalomyelitis (EAE), which is the predominant animal model used to investigate the pathophysiology of MS. In the present paper, the effects of glutamatergic receptor antagonists, including amantadine, memantine, LY 367583, and MPEP, on glutamate transport, the expression of mRNA of glutamate transporters (EAATs), the kinetic parameters of ligand binding to N-methyl-D-aspartate (NMDA) receptors, and the morphology of nerve endings in EAE rat brains were investigated. The extracellular level of glutamate in the brain is primarily regulated by astrocytic glutamate transporter 1 (GLT-1) and glutamate-aspartate transporter (GLAST). Excess glutamate is taken up from the synaptic space and metabolized by astrocytes. Thus, the extracellular level of glutamate decreases, which protects neurons from excitotoxicity. Our investigations showed changes in the expression of EAAT mRNA, glutamate transport (uptake and release) by synaptosomal and glial plasmalemmal vesicle fractions, and ligand binding to NMDA receptors; these effects were partially reversed after the treatment of EAE rats with the NMDA antagonists amantadine and memantine. The antagonists of group I metabotropic glutamate receptors (mGluRs), including LY 367385 and MPEP, did not exert any effect on the examined parameters. These results suggest that disturbances in these mechanisms may play a role in the processes associated with glutamate excitotoxicity and the progressive brain damage in EAE.
doi:10.1371/journal.pone.0113954
PMCID: PMC4245246  PMID: 25426719
15.  Striatal Adenosine Signaling Regulates EAAT2 and Astrocytic AQP4 Expression and Alcohol Drinking in Mice 
Neuropsychopharmacology  2012;38(3):437-445.
Adenosine signaling is implicated in several neuropsychiatric disorders, including alcoholism. Among its diverse functions in the brain, adenosine regulates glutamate release and has an essential role in ethanol sensitivity and preference. However, the molecular mechanisms underlying adenosine-mediated glutamate signaling in neuroglial interaction remain elusive. We have previously shown that mice lacking the ethanol-sensitive adenosine transporter, type 1 equilibrative nucleoside transporter (ENT1), drink more ethanol compared with wild-type mice and have elevated striatal glutamate levels. In addition, ENT1 inhibition or knockdown reduces glutamate transporter expression in cultured astrocytes. Here, we examined how adenosine signaling in astrocytes contributes to ethanol drinking. Inhibition or deletion of ENT1 reduced the expression of type 2 excitatory amino-acid transporter (EAAT2) and the astrocyte-specific water channel, aquaporin 4 (AQP4). EAAT2 and AQP4 colocalization was also reduced in the striatum of ENT1 null mice. Ceftriaxone, an antibiotic compound known to increase EAAT2 expression and function, elevated not only EAAT2 but also AQP4 expression in the striatum. Furthermore, ceftriaxone reduced ethanol drinking, suggesting that ENT1-mediated downregulation of EAAT2 and AQP4 expression contributes to excessive ethanol consumption in our mouse model. Overall, our findings indicate that adenosine signaling regulates EAAT2 and astrocytic AQP4 expressions, which control ethanol drinking in mice.
doi:10.1038/npp.2012.198
PMCID: PMC3547194  PMID: 23032072
ENT1; astrocytes; aquaporin 4; EAAT2; Alcoholism; ceftriaxone; ENT1; Astrocytes; Aquaporin 4; EAAT2; Alcoholism; Ceftriaxone
16.  A β-Lactam Antibiotic Dampens Excitotoxic Inflammatory CNS Damage in a Mouse Model of Multiple Sclerosis 
PLoS ONE  2008;3(9):e3149.
In multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE), impairment of glial “Excitatory Amino Acid Transporters” (EAATs) together with an excess glutamate-release by invading immune cells causes excitotoxic damage of the central nervous system (CNS). In order to identify pathways to dampen excitotoxic inflammatory CNS damage, we assessed the effects of a β-lactam antibiotic, ceftriaxone, reported to enhance expression of glial EAAT2, in “Myelin Oligodendrocyte Glycoprotein” (MOG)-induced EAE. Ceftriaxone profoundly ameliorated the clinical course of murine MOG-induced EAE both under preventive and therapeutic regimens. However, ceftriaxone had impact neither on EAAT2 protein expression levels in several brain areas, nor on the radioactive glutamate uptake capacity in a mixed primary glial cell-culture and the glutamate-induced uptake currents in a mammalian cell line mediated by EAAT2. Moreover, the clinical effect of ceftriaxone was preserved in the presence of the EAAT2-specific transport inhibitor, dihydrokainate, while dihydrokainate alone caused an aggravated EAE course. This demonstrates the need for sufficient glial glutamate uptake upon an excitotoxic autoimmune inflammatory challenge of the CNS and a molecular target of ceftriaxone other than the glutamate transporter. Ceftriaxone treatment indirectly hampered T cell proliferation and proinflammatory INFγ and IL17 secretion through modulation of myelin-antigen presentation by antigen-presenting cells (APCs) e.g. dendritic cells (DCs) and reduced T cell migration into the CNS in vivo. Taken together, we demonstrate, that a β-lactam antibiotic attenuates disease course and severity in a model of autoimmune CNS inflammation. The mechanisms are reduction of T cell activation by modulation of cellular antigen-presentation and impairment of antigen-specific T cell migration into the CNS rather than or modulation of central glutamate homeostasis.
doi:10.1371/journal.pone.0003149
PMCID: PMC2522272  PMID: 18773080
17.  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
18.  Yin Yang 1 Is a Repressor of Glutamate Transporter EAAT2, and It Mediates Manganese-Induced Decrease of EAAT2 Expression in Astrocytes 
Molecular and Cellular Biology  2014;34(7):1280-1289.
Impairment of astrocytic glutamate transporter (GLT-1; EAAT2) function is associated with multiple neurodegenerative diseases, including Parkinson's disease (PD) and manganism, the latter being induced by chronic exposure to high levels of manganese (Mn). Mn decreases EAAT2 promoter activity and mRNA and protein levels, but the molecular mechanism of Mn-induced EAAT2 repression at the transcriptional level has yet to be elucidated. We reveal that transcription factor Yin Yang 1 (YY1) is critical in repressing EAAT2 and mediates the effects of negative regulators, such as Mn and tumor necrosis factor alpha (TNF-α), on EAAT2. YY1 overexpression in astrocytes reduced EAAT2 promoter activity, while YY1 knockdown or mutation of the YY1 consensus site of the EAAT2 promoter increased its promoter activity and attenuated the Mn-induced repression of EAAT2. Mn increased YY1 promoter activity and mRNA and protein levels via NF-κB activation. This led to increased YY1 binding to the EAAT2 promoter region. Epigenetically, histone deacetylase (HDAC) classes I and II served as corepressors of YY1, and, accordingly, HDAC inhibitors increased EAAT2 promoter activity and reversed the Mn-induced repression of EAAT2 promoter activity. Taken together, our findings suggest that YY1, with HDACs as corepressors, is a critical negative transcriptional regulator of EAAT2 and mediates Mn-induced EAAT2 repression.
doi:10.1128/MCB.01176-13
PMCID: PMC3993574  PMID: 24469401
19.  The Glutamate Transporter Subtypes EAAT4 and EAATs 1-3 Transport Glutamate with Dramatically Different Kinetics and Voltage Dependence but Share a Common Uptake Mechanism 
The Journal of General Physiology  2005;126(6):571-589.
Here, we report the application of glutamate concentration jumps and voltage jumps to determine the kinetics of rapid reaction steps of excitatory amino acid transporter subtype 4 (EAAT4) with a 100-μs time resolution. EAAT4 was expressed in HEK293 cells, and the electrogenic transport and anion currents were measured using the patch-clamp method. At steady state, EAAT4 was activated by glutamate and Na+ with high affinities of 0.6 μM and 8.4 mM, respectively, and showed kinetics consistent with sequential binding of Na+-glutamate-Na+. The steady-state cycle time of EAAT4 was estimated to be >300 ms (at −90 mV). Applying step changes to the transmembrane potential, Vm, of EAAT4-expressing cells resulted in the generation of transient anion currents (decaying with a τ of ∼15 ms), indicating inhibition of steady-state EAAT4 activity at negative voltages (<−40 mV) and activation at positive Vm (>0 mV). A similar inhibitory effect at Vm < 0 mV was seen when the electrogenic glutamate transport current was monitored, resulting in a bell-shaped I-Vm curve. Jumping the glutamate concentration to 100 μM generated biphasic, saturable transient transport and anion currents (Km ∼ 5 μM) that decayed within 100 ms, indicating the existence of two separate electrogenic reaction steps. The fast electrogenic reaction was assigned to Na+ binding to EAAT4, whereas the second reaction is most likely associated with glutamate translocation. Together, these results suggest that glutamate uptake of EAAT4 is based on the same molecular mechanism as transport by the subtypes EAATs 1–3, but that its kinetics and voltage dependence are dramatically different from the other subtypes. EAAT4 kinetics appear to be optimized for high affinity binding of glutamate, but not rapid turnover. Therefore, we propose that EAAT4 is a high-affinity/low-capacity transport system, supplementing low-affinity/high-capacity synaptic glutamate uptake by the other subtypes.
doi:10.1085/jgp.200509365
PMCID: PMC2266596  PMID: 16316976
20.  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
21.  GABA and Glutamate Transporters in Brain 
The mammalian genome contains four genes encoding GABA transporters (GAT1, slc6a1; GAT2, slc6a13; GAT3, slc6a11; BGT1, slc6a12) and five glutamate transporter genes (EAAT1, slc1a3; EAAT2, slc1a2; EAAT3, slc1a1; EAAT4, slc1a6; EAAT5, slc1a7). These transporters keep the extracellular levels of GABA and excitatory amino acids low and provide amino acids for metabolic purposes. The various transporters have different properties both with respect to their transport functions and with respect to their ability to act as ion channels. Further, they are differentially regulated. To understand the physiological roles of the individual transporter subtypes, it is necessary to obtain information on their distributions and expression levels. Quantitative data are important as the functional capacity is limited by the number of transporter molecules. The most important and most abundant transporters for removal of transmitter glutamate in the brain are EAAT2 (GLT-1) and EAAT1 (GLAST), while GAT1 and GAT3 are the major GABA transporters in the brain. EAAT3 (EAAC1) does not appear to play a role in signal transduction, but plays other roles. Due to their high uncoupled anion conductance, EAAT4 and EAAT5 seem to be acting more like inhibitory glutamate receptors than as glutamate transporters. GAT2 and BGT1 are primarily expressed in the liver and kidney, but are also found in the leptomeninges, while the levels in brain tissue proper are too low to have any impact on GABA removal, at least in normal young adult mice. The present review will provide summary of what is currently known and will also discuss some methodological pitfalls.
doi:10.3389/fendo.2013.00165
PMCID: PMC3822327  PMID: 24273530
GABA uptake; glutamate uptake; BGT1; GAT1; GAT3; GAT2; EAAT2; EAAT1
22.  Critical role of S465 in protein kinase C-increased rat glutamate transporter type 3 activity 
Glutamate transporters, also called excitatory amino acid transporters (EAATs), uptake extracellular glutamate and regulate neurotransmission. Activation of protein kinase C (PKC) increases the activity of EAAT type 3 (EAAT3), the major neuronal EAAT. We designed this study to determine which amino acid residue(s) in EAAT3 may be involved in this PKC effect. Selective potential PKC phosphorylation sites were mutated. These EAAT3 mutants were expressed in the Xenopus oocytes. Phorbol 12-myristate 13-acetate, a PKC activator, significantly increased wild-type EAAT3 activity. Mutation of serine 465 to alanine or to aspartic acid, but not the mutation of threonine 5 to alanine, abolished PKC-increased EAAT3 activity. Our results suggest a critical role of serine 465 in the increased EAAT3 activity by PKC activation.
PMCID: PMC2780444  PMID: 19922365
Glutamate; glutamate transporter; protein kinase C; site-directed mutagenesis
23.  The EAAT2 (GLT-1) gene in motor neuron disease: absence of mutations in amyotrophic lateral sclerosis and a point mutation in patients with hereditary spastic paraplegia 
OBJECTIVES—To investigate if sequence alterations of the excitatory amino acid transporter gene EAAT2 (GLT-1) may be a contributory factor to the pathogenesis of motor system degeneration. EAAT2 serves as a candidate gene as its reduced expression was reported in patients with amyotrophic lateral sclerosis (ALS). Furthermore, neurolathyrism, a motor neuron disease clinically related to hereditary spastic paraplegia (HSP), has been associated with an exogenous excitotoxin.
METHODS—Sequence alterations were screened for in the coding region of EAAT2 in 55 patients with ALS and one family with autosomal dominant HSP (AD-HSP).
RESULTS—In ALS, no sequence alteration in the EAAT2 gene have been found. Interestingly, a heterozygous A79G mutation of the EAAT2 gene was detected in two of seven affected patients with AD-HSP in the same kindred. The absence of cosegregation with the familial disease showed that the detected variant was not the cause of disease. The A79G sequence variant was not found in 55 patients with ALS or in 50 non-neurological controls.
CONCLUSION—The allelic variant of the EAAT2 gene in conjunction with the primary gene defect may be a modifying factor for the highly variable AD-HSP phenotype.


PMCID: PMC2170294  PMID: 9771796
24.  Differential Regulation of Glutamate Transporter Subtypes by Pro-Inflammatory Cytokine TNF-α in Cortical Astrocytes from a Rat Model of Amyotrophic Lateral Sclerosis 
PLoS ONE  2014;9(5):e97649.
Dysregulation of the astroglial glutamate transporters GLAST and GLT-1 has been implicated in several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) where a loss of GLT-1 protein expression and activity is reported. Furthermore, the two principal C-terminal splice variants of GLT-1 (namely GLT-1a and GLT-1b) show altered expression ratio in animal models of this disease. Considering the putative link between inflammation and excitotoxicity, we have here characterized the influence of TNF-α on glutamate transporters in cerebral cortical astrocyte cultures from wild-type rats and from a rat model of ALS (hSOD1G93A). Contrasting with the down-regulation of GLAST, a 72 h treatment with TNF-α substantially increased the expression of GLT-1a and GLT-1b in both astrocyte cultures. However, as the basal level of GLT-1a appeared considerably lower in hSOD1G93A astrocytes, its up-regulation by TNF-α was insufficient to recapitulate the expression observed in wild-type astrocytes. Also the glutamate uptake activity after TNF-α treatment was lower for hSOD1G93A astrocytes as compared to wild-type astrocytes. In the presence of the protein synthesis inhibitor cycloheximide, TNF-α did not influence GLT-1 isoform expression, suggesting an active role of dynamically regulated protein partners in the adaptation of astrocytes to the inflammatory environment. Confirming the influence of inflammation on the control of glutamate transmission by astrocytes, these results shed light on the regulation of glutamate transporter isoforms in neurodegenerative disorders.
doi:10.1371/journal.pone.0097649
PMCID: PMC4023965  PMID: 24836816
25.  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

Results 1-25 (1148553)