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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.  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
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.  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
6.  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
7.  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
8.  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
9.  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
10.  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
11.  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
12.  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
13.  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
14.  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
15.  Physical and Functional Interaction of NCX1 and EAAC1 Transporters Leading to Glutamate-Enhanced ATP Production in Brain Mitochondria 
PLoS ONE  2012;7(3):e34015.
Glutamate is emerging as a major factor stimulating energy production in CNS. Brain mitochondria can utilize this neurotransmitter as respiratory substrate and specific transporters are required to mediate the glutamate entry into the mitochondrial matrix. Glutamate transporters of the Excitatory Amino Acid Transporters (EAATs) family have been previously well characterized on the cell surface of neuronal and glial cells, representing the primary players for glutamate uptake in mammalian brain. Here, by using western blot, confocal microscopy and immunoelectron microscopy, we report for the first time that the Excitatory Amino Acid Carrier 1 (EAAC1), an EAATs member, is expressed in neuronal and glial mitochondria where it participates in glutamate-stimulated ATP production, evaluated by a luciferase-luciferin system. Mitochondrial metabolic response is counteracted when different EAATs pharmacological blockers or selective EAAC1 antisense oligonucleotides were used. Since EAATs are Na+-dependent proteins, this raised the possibility that other transporters regulating ion gradients across mitochondrial membrane were required for glutamate response. We describe colocalization, mutual activity dependency, physical interaction between EAAC1 and the sodium/calcium exchanger 1 (NCX1) both in neuronal and glial mitochondria, and that NCX1 is an essential modulator of this glutamate transporter. Only NCX1 activity is crucial for such glutamate-stimulated ATP synthesis, as demonstrated by pharmacological blockade and selective knock-down with antisense oligonucleotides. The EAAC1/NCX1-dependent mitochondrial response to glutamate may be a general and alternative mechanism whereby this neurotransmitter sustains ATP production, since we have documented such metabolic response also in mitochondria isolated from heart. The data reported here disclose a new physiological role for mitochondrial NCX1 as the key player in glutamate-induced energy production.
doi:10.1371/journal.pone.0034015
PMCID: PMC3316532  PMID: 22479505
16.  Increased expression of cholesterol 24S-hydroxylase results in disruption of glial glutamate transporter EAAT2 association with lipid rafts: a potential role in Alzheimer’s disease 
Journal of neurochemistry  2010;113(4):978-989.
The glial glutamate transporter EAAT2 is the major mediator of glutamate clearance that terminates glutamate-mediated neurotransmission. Loss of EAAT2 and associated glutamate uptake function has been reported in the brains of patients with Alzheimer’s disease (AD). We previously reported that EAAT2 is associated with lipid raft microdomains of the plasma membrane. In the present study, we demonstrated that association of EAAT2 with lipid rafts is disrupted in AD brains. This abnormality is not a consequence of neuron degeneration, oxidative stress, or amyloid beta toxicity. In AD brains, cholesterol 24S-hydroxylase (CYP46), a key enzyme in maintenance of cholesterol homeostasis in the brain, is markedly increased in astrocytes but decreased in neurons. We demonstrated that increased expression of CYP46 in primary astrocytes results in a reduction of membrane cholesterol levels and leads to the dissociation of EAAT2 from lipid rafts and the loss of EAAT2 and associated glutamate uptake function. These results suggest that a disturbance of cholesterol metabolism may contribute to loss of EAAT2 in AD.
doi:10.1111/j.1471-4159.2010.06661.x
PMCID: PMC3010752  PMID: 20193040
glutamate transporter EAAT2; lipid raft microdomain; Alzheimer’s disease; cholesterol 24S-hydroxylase; excitotoxicity
17.  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
18.  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
19.  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
20.  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
21.  Loss of Astrocytic Glutamate Transporters in Wernicke Encephalopathy 
Glia  2010;58(2):148-156.
Wernicke encephalopathy (WE), a neurological disorder caused by thiamine deficiency (TD), is characterized by structural damage in brain regions that include the thalamus and cerebral cortex. The basis for these lesions is unclear, but may involve a disturbance of glutamatergic neurotransmission. We have therefore investigated levels of the astrocytic glutamate transporters EAAT1 and EAAT2 in order to evaluate their role in the pathophysiology of this disorder. Histological assessment of the frontal cortex revealed a significant loss of neurons in neuropathologically confirmed cases of WE compared with age-matched controls, concomitant with decreases in α-internexin and synaptophysin protein content of 67 and 52% by immunoblotting. EAAT2 levels were diminished by 71% in WE, with levels of EAAT1 also reduced by 62%. Loss of both transporter sites was confirmed by immunohistochemical methods. Development of TD in rats caused a profound loss of EAAT1 and EAAT2 in the thalamus accompanied by decreases in other astrocyte-specific proteins. Treatment of TD rats with N-acetylcysteine prevented the downregulation of EAAT2 in the medial thalamus, and ameliorated the loss of several other astrocyte proteins, concomitant with increased neuronal survival. Our results suggest that (1) loss of EAAT1 and EAAT2 glutamate transporters is associated with structural damage to the frontal cortex in patients with WE, (2) oxidative stress plays an important role in this process, and (3) TD has a profound effect on the functional integrity of astrocytes. Based on these findings, we recommend that early treatment using a combination of thiamine AND antioxidant approaches should be an important consideration in cases of WE.
doi:10.1002/glia.20908
PMCID: PMC3388119  PMID: 19565658
thiamine deficiency; glutamate; astrocyte; vitamin B1; EAAT; excitotoxicity; oxidative stress
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.  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
24.  Selective Over Expression Of EAAT2 In Astrocytes Enhances Neuroprotection From Moderate But Not Severe Hypoxia-Ischemia 
Neuroscience  2008;155(4):1204-1211.
Attempts have been made to elevate EAAT2 expression in effort to compensate for loss of function and expression associated with disease or pathology. Increased EAAT2 expression has been noted following treatment with β-lactam antibiotics, and during ischemic preconditioning (IPC). However, both of these conditions induce multiple changes in addition to alterations in EAAT2 expression that could potentially contribute to neuroprotection. Therefore, the aim of this study was to selectively overexpress EAAT2 in astrocytes and characterize the cell type specific contribution of this transporter to neuroprotection. To accomplish this we used a recombinant Adeno-associated virus vector, AAV1-GFAP-EAAT2, designed to selectively drive the overexpression of EAAT2 within astrocytes. Both viral mediated gene delivery and β-lactam antibiotic (penicillin-G) treatment of rat hippocampal slice cultures resulted in a significant increase in both the expression of EAAT2, and dihydrokainate (DHK) sensitive glutamate uptake. Penicillin-G provided significant neuroprotection in rat hippocampal slice cultures under conditions of both moderate and severe oxygen glucose deprivation (OGD). In contrast, the overexpression of EAAT2 in astrocytes provided enhanced neuroprotection only following a moderate OGD insult. These results indicate that functional EAAT2 can be selectively overexpressed in astrocytes, leading to enhanced neuroprotection. However, this cell type specific-increase in EAAT2 expression offers only limited protection compared to treatment with penicillin-G.
doi:10.1016/j.neuroscience.2008.05.059
PMCID: PMC2729515  PMID: 18620031
25.  Potential therapeutic role of glutamate transporter 1 for the treatment of alcohol dependence 
OA alcohol  2013;1(1):6-.
Introduction
Evidence has demonstrated that deficits in glutamate transmission impair neurocircuits involved in drug abuse or drug-seeking behaviour and affect many aspects of neuroplasticity associated with alcohol and drug addiction. Alcohol-seeking behaviour is promoted by increased glutamate transmission in key regions of the mesocorticolimbic reward circuit, including the nucleus accumbens and prefrontal cortex. Glutamate transmission or glutamate uptake is regulated by a number of glutamate transporters in the brain regions. Among these glutamate transporters, glutamate transporter 1 (GLT1; its human homolog is the excitatory amino acid transporter 2, EAAT2) regulates the removal of majority of the extracellular glutamate. The role of GLT1 has been tested in alcohol and other drugs of abuse models with dysfunction in glutamate transmission. We recently reported that treatment of alcohol-preferring rats with compounds ceftriaxone and GPI-1046, known to upregulate GLT1 levels, showed reduction in alcohol intake and attenuation of relapse-like ethanol-drinking behaviour. Furthermore, we demonstrated that upregulation of GLT1 was associated with attenuation of cue-induced cocaine relapse. Together, we suggest that GLT1 is considered as a potential therapeutic target for the treatment of drug dependence, including alcohol. The aim of this critical review was to discuss the potential therapeutic role of GLT1 for the treatment of alcohol dependence.
Conclusion
Dysfunction of glutamate transmission has been suggested to impair neurocircuits involved in alcohol dependence, which affect neuroplasticity that is associated with ethanol intake.
PMCID: PMC3883353  PMID: 24409344

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