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Susan G Amara - One of the best experts on this subject based on the ideXlab platform.
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cysteine transport through Excitatory amino acid Transporter 3 eaat3
PLOS ONE, 2014Co-Authors: Spencer D Watts, Christopher B Divito, Delany Torressalazar, Susan G AmaraAbstract: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.
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amphetamine modulates Excitatory neurotransmission through endocytosis of the glutamate Transporter eaat3 in dopamine neurons
Neuron, 2014Co-Authors: Suzanne M Underhill, Susan L Ingram, Susan G Amara, David S Wheeler, Spencer D WattsAbstract:Summary Amphetamines modify the brain and alter behavior through mechanisms generally attributed to their ability to regulate extracellular dopamine concentrations. However, the actions of amphetamine are also linked to adaptations in glutamatergic signaling. We report here that when amphetamine enters dopamine neurons through the dopamine Transporter, it stimulates endocytosis of an Excitatory amino acid Transporter, EAAT3, in dopamine neurons. Consistent with this decrease in surface EAAT3, amphetamine potentiates Excitatory synaptic responses in dopamine neurons. We also show that the process of internalization is dynamin- and Rho-mediated and requires a unique sequence in the cytosolic C terminus of EAAT3. Introduction of a peptide based on this motif into dopamine neurons blocks the effects of amphetamine on EAAT3 internalization and its action on Excitatory responses. These data indicate that the internalization of EAAT3 triggered by amphetamine increases glutamatergic signaling and thus contributes to the effects of amphetamine on neurotransmission.
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large collective motions regulate the functional properties of glutamate Transporter trimers
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Jie Jiang, Spencer D Watts, Indira H Shrivastava, Ivet Bahar, Susan G AmaraAbstract:Glutamate Transporters clear synaptically released glutamate to maintain precise communication between neurons and limit glutamate neurotoxicity. Although much progress has been made on the topology, structure, and function of these carriers, few studies have addressed large-scale structural motions collectively associated with substrate transport. Here we show that a series of single cysteine substitutions in the helical hairpin HP2 of Excitatory amino acid Transporter 1 form intersubunit disulfide cross-links within the trimer. After cross-linking, substrate uptake, but not substrate-activated anion conductance, is completely inhibited in these mutants. These disulfide bridges link residue pairs > 40 A apart in the outward-facing crystal structure, and can be explained by concerted subunit movements predicted by the anisotropic network model (ANM). The existence of these global motions is further supported by the observation that single cysteine substitutions at the extracellular part of the transmembrane domain 8 can also be cross-linked by copper phenanthroline as predicted by the ANM. Interestingly, the transport domain in the un-cross-linked subunit of the trimer assumes an inward-facing orientation, suggesting that individual subunits potentially undergo separate transitions between outward- and inward-facing forms, rather than an all-or-none transition of the three subunits, a mechanism also supported by ANM-predicted intrinsic dynamics. These results shed light on how large collective motions contribute to the functional dynamics of glutamate Transporters.
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Excitatory amino acid Transporter 5 a retinal glutamate Transporter coupled to a chloride conductance
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Jeffrey L Arriza, Michael P Kavanaugh, Scott Eliasof, Susan G AmaraAbstract:Although a glutamate-gated chloride conductance with the properties of a sodium-dependent glutamate Transporter has been described in vertebrate retinal photoreceptors and bipolar cells, the molecular species underlying this conductance has not yet been identified. We now report the cloning and functional characterization of a human Excitatory amino acid Transporter, EAAT5, expressed primarily in retina. Although EAAT5 shares the structural homologies of the EAAT gene family, one novel feature of the EAAT5 sequence is a carboxy-terminal motif identified previously in N-methyl-d-aspartate receptors and potassium channels and shown to confer interactions with a family of synaptic proteins that promote ion channel clustering. Functional properties of EAAT5 were examined in the Xenopus oocyte expression system by measuring radiolabeled glutamate flux and two-electrode voltage clamp recording. EAAT5-mediated l-glutamate uptake is sodium- and voltage-dependent and chloride-independent. Transporter currents elicited by glutamate are also sodium- and voltage-dependent, but ion substitution experiments suggest that this current is largely carried by chloride ions. These properties of EAAT5 are similar to the glutamate-elicited chloride conductances previously described in retinal neurons, suggesting that the EAAT5-associated chloride conductance may participate in visual processing.
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an Excitatory amino acid Transporter with properties of a ligand gated chloride channel
Nature, 1995Co-Authors: Wendy A Fairman, Michael P Kavanaugh, Robert J Vandenberg, Jeffrey L Arriza, Susan G AmaraAbstract:Excitatory Amino-Acid Transporters (EAATs) in the central nervous system maintain extracellular glutamate concentrations below excitotoxic levels and may limit the activation of glutamate receptors. Here we report the cloning of a novel human aspartate/ glutamate Transporter, EAAT4, which is expressed predominantly in the cerebellum. The transport activity encoded by EAAT4 has high apparent affinity for L-aspartate and L -glutamate, and has a pharmacological profile consistent with previously described cere-bellar transport activities. In Xenopus oocytes expressing EAAT4, L -aspartate and i -glutamate elicited a current predominantly carried by chloride ions. This chloride conductance was not blocked by components that block endogenous oocyte chloride channels. Thus EAAT4 combines the re-uptake of neurotransmitter with a mechanism for increasing chloride permeability, both of which could regulate Excitatory neurotransmission.
Jeffrey D. Rothstein - One of the best experts on this subject based on the ideXlab platform.
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motor neuron derived micrornas cause astrocyte dysfunction in amyotrophic lateral sclerosis
Brain, 2018Co-Authors: Mariah L Hoye, Jeffrey D. Rothstein, Svetlana Vidensky, Melissa R Regan, Leah Jensen, Allison M Lake, Linga V Reddy, Jean Philippe Richard, Nicholas J Maragakis, Joseph D DoughertyAbstract:: We recently demonstrated that microRNA-218 (miR-218) is greatly enriched in motor neurons and is released extracellularly in amyotrophic lateral sclerosis model rats. To determine if the released, motor neuron-derived miR-218 may have a functional role in amyotrophic lateral sclerosis, we examined the effect of miR-218 on neighbouring astrocytes. Surprisingly, we found that extracellular, motor neuron-derived miR-218 can be taken up by astrocytes and is sufficient to downregulate an important glutamate Transporter in astrocytes [Excitatory amino acid Transporter 2 (EAAT2)]. The effect of miR-218 on astrocytes extends beyond EAAT2 since miR-218 binding sites are enriched in mRNAs translationally downregulated in amyotrophic lateral sclerosis astrocytes. Inhibiting miR-218 with antisense oligonucleotides in amyotrophic lateral sclerosis model mice mitigates the loss of EAAT2 and other miR-218-mediated changes, providing an important in vivo demonstration of the relevance of microRNA-mediated communication between neurons and astrocytes. These data define a novel mechanism in neurodegeneration whereby microRNAs derived from dying neurons can directly modify the glial phenotype and cause astrocyte dysfunction.
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neuronal exosomal mirna dependent translational regulation of astroglial glutamate Transporter glt1
Journal of Biological Chemistry, 2013Co-Authors: Lydie Morel, Jeffrey D. Rothstein, Svetlana Vidensky, Melissa R Regan, Haruki Higashimori, Seng Kah Ng, Christine Esau, Yongjie YangAbstract:Perisynaptic astrocytes express important glutamate Transporters, especially Excitatory amino acid Transporter 2 (EAAT2, rodent analog GLT1) to regulate extracellular glutamate levels and modulate synaptic activation. In this study, we investigated an exciting new pathway, the exosome-mediated transfer of microRNA (in particular, miR-124a), in neuron-to-astrocyte signaling. Exosomes isolated from neuron-conditioned medium contain abundant microRNAs and small RNAs. These exosomes can be directly internalized into astrocytes and increase astrocyte miR-124a and GLT1 protein levels. Direct miR-124a transfection also significantly and selectively increases protein (but not mRNA) expression levels of GLT1 in cultured astrocytes. Consistent with our in vitro findings, intrastriatal injection of specific antisense against miR-124a into adult mice dramatically reduces GLT1 protein expression and glutamate uptake levels in striatum without reducing GLT1 mRNA levels. MiR-124a-mediated regulation of GLT1 expression appears to be indirect and is not mediated by its suppression of the putative GLT1 inhibitory ligand ephrinA3. Moreover, miR-124a is selectively reduced in the spinal cord tissue of end-stage SOD1 G93A mice, the mouse model of ALS. Subsequent exogenous delivery of miR-124a in vivo through stereotaxic injection significantly prevents further pathological loss of GLT1 proteins, as determined by GLT1 immunoreactivity in SOD1 G93A mice. Together, our study characterized a new neuron-to-astrocyte communication pathway and identified miRNAs that modulate GLT1 protein expression in astrocytes in vitro and in vivo.
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the endoplasmic reticulum exit of glutamate Transporter is regulated by the inducible mammalian yip6b gtrap3 18 protein
Journal of Biological Chemistry, 2008Co-Authors: Alicia M Ruggiero, Yiting Liu, Svetlana Vidensky, Susanne Maier, Elizabeth Jung, Hesso Farhan, Michael B Robinson, Harald H Sitte, Jeffrey D. RothsteinAbstract:GTRAP3-18 interacts with and reduces the activity of the neuronal specific Na+/K+ glutamate Transporter, EAAC1 both in vitro and in vivo. GTRAP3-18 and the related isoform, JM4, are distant relatives of the Rab GTPase-interacting factor PRA1, and share a topology of four transmembrane domains and cytosolic termini. GTRAP3-18 and JM4 are resident endoplasmic reticulum (ER) proteins. The physiological role of GTRAP3-18 is poorly understood. We demonstrate for the first time that GTRAP3-18 is a regulator of ER protein trafficking. Expression of GTRAP3-18 delays the ER exit of EAAC1, as well as other members of the Excitatory amino acid Transporter family. GTRAP3-18 uses hydrophobic domain interactions in the ER membrane to self-associate and cytoplasmic interactions at the C terminus to regulate trafficking. The features of GTRAP3-18 activity are consistent with recent phylogenic sequence analyses suggesting GTRAP3-18 and JM4 be reclassified as mammalian isoforms of the yeast protein family Yip, Yip6b, and Yip6a, respectively.
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the glutamate aspartate Transporter glast mediates glutamate uptake at inner hair cell afferent synapses in the mammalian cochlea
The Journal of Neuroscience, 2006Co-Authors: Elisabeth Glowatzki, Kohichi Tanaka, Jeffrey D. Rothstein, Ning Cheng, Hakim Hiel, Graham C R Ellisdavies, Dwight E BerglesAbstract:Ribbon synapses formed between inner hair cells (IHCs) and afferent dendrites in the mammalian cochlea can sustain high rates of release, placing strong demands on glutamate clearance mechanisms. To investigate the role of Transporters in glutamate removal at these synapses, we made whole-cell recordings from IHCs, afferent dendrites, and glial cells adjacent to IHCs [inner phalangeal cells (IPCs)] in whole-mount preparations of rat organ of Corti. Focal application of the Transporter substrate d-aspartate elicited inward currents in IPCs, which were larger in the presence of anions that permeate the Transporter-associated anion channel and blocked by the Transporter antagonist d,l-threo-β-benzyloxyaspartate. These currents were produced by glutamate–aspartate Transporters (GLAST) (Excitatory amino acid Transporter 1) because they were weakly inhibited by dihydrokainate, an antagonist of glutamate Transporter-1 (Excitatory amino acid Transporter 2) and were absent from IPCs in GLAST−/− cochleas. Furthermore, d-aspartate-induced currents in outside-out patches from IPCs exhibited larger steady-state currents than responses elicited by l-glutamate, a prominent feature of GLAST, and examination of cochlea from GLAST–Discosoma red (DsRed) promoter reporter mice revealed that DsRed expression was restricted to IPCs and other supporting cells surrounding IHCs. Saturation of Transporters by photolysis of caged d-aspartate failed to elicit Transporter currents in IHCs, as did local application of d-aspartate to afferent terminals, indicating that neither presynaptic nor postsynaptic membranes are major sites for glutamate removal. These data indicate that GLAST in supporting cells is responsible for transmitter uptake at IHC afferent synapses.
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cellular and synaptic localization of the neuronal glutamate Transporters Excitatory amino acid Transporter 3 and 4
Neuroscience, 1997Co-Authors: Akiko Furuta, Lee J Martin, C L G Lin, M Dykeshoberg, Jeffrey D. RothsteinAbstract:Glutamate transport is a primary mechanism for the synaptic inactivation of glutamate. Excitatory amino acid Transporter 4 (EAAT4) is a novel glutamate Transporter with properties of a ligand-gated chloride channel that was recently cloned from human brain. The present study was an investigation of the protein expression and cellular localization of EAAT4 in human and rat brain, and comparison with another neuronal glutamate Transporter, EAAT3 (rabbit Excitatory amino acid carrier 1; EAAC1). Regional immunoblot analysis of EAAT4, using a monospecific oligopeptide (carboxy-terminal) affinity-purified polyclonal antibody, revealed that the protein was restricted to the central nervous system. The EAAT4 protein was largely expressed in cerebellum, with a much lower expression in hippocampus, neocortex, striatum, brain stem and thalamus. Immunohistochemical studies showed intense EAAT4 immunoreactivity in the human and rat cerebellar Purkinje cells with a somatodendritic localization. Other brain regions including neocortex, hippocampus, striatum showed faint neuropil staining of EAAT4. Immunogold localization identified EAAT4 protein at plasma membranes of Purkinje cell dendrites and spines. In the hippocampus and neocortex, EAAT4 immunoreactivity was found mainly at small calibre dendrites. Rarely, EAAT4 immunoreactivity was found in astrocytic cell processes of forebrain. In the cerebellum, EAAT4 localization partly overlapped with the neuronal localization of EAAT3 (EAAC1). Immunoreactivity for EAAT3 was enriched in the somatodendritic compartment of the Purkinje cells like EAAT4, but EAAT3 was also found in Purkinje cell axons and in boutons in deep cerebellar nuclei, as well as in granular cells and stellate cells. Our results indicate that EAAT4 protein is largely localized to cerebellar cortex and lower levels of EAAT4 protein are present in forebrain by immunoblot and immunohistochemistry. Both neuronal glutamate Transporter EAAT3 (EAAC1) and EAAT4 are located at somatodendritic compartment of Purkinje cells, and probably contribute to glutamate re-uptake mechanisms at Purkinje cell synapses.
Wolfgang Schwarz - One of the best experts on this subject based on the ideXlab platform.
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different functional roles of arginine residues 39 and 61 and tyrosine residue 98 in transport and channel mode of the glutamate Transporter eaac1
Biochimica et Biophysica Acta, 2004Co-Authors: Larisa A Vasilets, Wolfgang SchwarzAbstract:The Excitatory amino acid Transporter EAAC1 is an electrogenic Na+- and K+-gradient-driven Transporter. In addition, the Transporter mediates in the presence of Na+ and glutamate an anion conductance uncoupled from the transport of the glutamate. The first two N-terminal domains, important for forming the conductance mode, are extracellularly bordered by positively charged arginine residues, R39 and R61, being completely conserved throughout the Transporter family. Also the conserved tyrosine residue Y98 could be important for Cl- conductance. We have investigated, by measurements of glutamate uptake and glutamate-induced currents, the effects of mutation of the arginines and the tyrosine to alanine. The mutation R39A hardly affects transport and channel mode. The mutation R61A, on the other hand, reduces the activity of transport but stimulates the channel conductance. In addition, the apparent K, values for glutamate uptake and for the glutamate-activated current are reduced. Glutamate stimulation of current seems to be associated with a voltage-dependent step, and the apparent valence of charge moved during binding is reduced in the R61A mutant. The mutation Y98A leads to reduced function with reduced apparent K-m value for glutamate, and with strong reduction of the selectivity ration between NO3- and Cl-_ of the conductance mode. (C) 2004 Elsevier B.V. All rights reserved.
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different functional roles of arginine residues 39 and 61 and tyrosine residue 98 in transport and channel mode of the glutamate Transporter eaac1
Biochimica et Biophysica Acta, 2004Co-Authors: Yani Zhu, Larisa A Vasilets, Jian Fei, Lihe Guo, Wolfgang SchwarzAbstract:The Excitatory amino acid Transporter EAAC1 is an electrogenic Na+ - and K+ -gradient-driven Transporter. In addition, the Transporter mediates in the presence of Na+ and glutamate an anion conductance uncoupled from the transport of the glutamate. The first two N-terminal domains, important for forming the conductance mode, are extracellularly bordered by positively charged arginine residues, R39 and R61, being completely conserved throughout the Transporter family. Also the conserved tyrosine residue Y98 could be important for Cl- conductance. We have investigated, by measurements of glutamate uptake and glutamate-induced currents, the effects of mutation of the arginines and the tyrosine to alanine. The mutation R39A hardly affects transport and channel mode. The mutation R61A, on the other hand, reduces the activity of transport but stimulates the channel conductance. In addition, the apparent Km values for glutamate uptake and for the glutamate-activated current are reduced. Glutamate stimulation of current seems to be associated with a voltage-dependent step, and the apparent valence of charge moved during binding is reduced in the R61A mutant. The mutation Y98A leads to reduced function with reduced apparent Km value for glutamate, and with strong reduction of the selectivity ration between NO3- and Cl- of the conductance mode.
Chienliang Glenn Lin - One of the best experts on this subject based on the ideXlab platform.
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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, 2010Co-Authors: Guilian Tian, Qiongman Kong, Liching Lai, Abhik Raychaudhury, Chienliang Glenn LinAbstract:J. Neurochem. (2010) 113, 978–989. Abstract The glial glutamate Transporter EAAT2 (Excitatory amino acid Transporter 2) 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.
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association of Excitatory amino acid Transporters especially eaat2 with cholesterol rich lipid raft microdomains importance for Excitatory amino acid Transporter localization and function
Journal of Biological Chemistry, 2004Co-Authors: Matthew E R Butchbach, Guilian Tian, Hong Guo, Chienliang Glenn LinAbstract:Abstract In the present study, we investigated the role of membrane cholesterol in the function of glutamate Transporters. Depletion of membrane cholesterol by methyl-β-cyclodextrin resulted in reduced Na+-dependent glutamate uptake in primary cortical cultures. Glial glutamate Transporter EAAT2-mediated uptake was more sensitive to this effect. Cell surface biotinylation and immunostaining experiments revealed that the loss of cholesterol significantly altered the trafficking of EAAT2 to the plasma membrane as well as their membrane distribution. These effects were also observed in neuronal glutamate Transporter EAAT3 but to a lesser extent. Furthermore, the treatment of mouse brain plasma membrane vesicles with methyl-β-cyclodextrin resulted in a significant reduction in glutamate uptake, suggesting that cholesterol depletion has a direct effect on the function of the glutamate Transporters. Plasma membrane cholesterol is localized within discreet microdomains known as lipid rafts. Analyses of purified lipid raft microdomains revealed that a large portion of total EAAT2 and a minor portion of total EAAT1, EAAT3, and EAAT4 were associated with lipid rafts. Artificial aggregation of lipid rafts in vivo resulted in the formation of larger EAAT2-immunoreactive clusters on the cell surface. The purified lipid raft-associated fractions were capable of Na+-dependent glutamate uptake. Our data suggest that the glutamate Transporters, especially EAAT2, are associated with cholesterol-rich lipid raft microdomains of the plasma membrane and that the association with these cholesterol-rich microdomains is important for Excitatory amino acid Transporter localization and function.
David J Volsky - One of the best experts on this subject based on the ideXlab platform.
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mechanism of ceftriaxone induction of Excitatory amino acid Transporter 2 expression and glutamate uptake in primary human astrocytes
Journal of Biological Chemistry, 2008Co-Authors: Seokgeun Lee, Devanand Sarkar, David J Volsky, Luni Emdad, Pankaj Gupta, Alejandra BorjabadAbstract:Glutamate is an essential neurotransmitter regulating brain functions. Excitatory amino acid Transporter (EAAT)-2 is one of the major glutamate Transporters primarily expressed in astroglial cells. Dysfunction of EAAT2 is implicated in acute and chronic neurological disorders, including stroke/ischemia, temporal lobe epilepsy, amyotrophic lateral sclerosis, Alzheimer disease, human immunodeficiency virus 1-associated dementia, and growth of malignant gliomas. Ceftriaxone, one of the β-lactam antibiotics, is a stimulator of EAAT2 expression with neuroprotective effects in both in vitro and in vivo models based in part on its ability to inhibit neuronal cell death by glutamate excitotoxicity. Based on this consideration and its lack of toxicity, ceftriaxone has potential to manipulate glutamate transmission and ameliorate neurotoxicity. We investigated the mechanism by which ceftriaxone enhances EAAT2 expression in primary human fetal astrocytes (PHFA). Ceftriaxone elevated EAAT2 transcription in PHFA through the nuclear factor-κB (NF-κB) signaling pathway. The antibiotic promoted nuclear translocation of p65 and activation of NF-κB. The specific NF-κB binding site at the -272 position of the EAAT2 promoter was responsible for ceftriaxone-mediated EAAT2 induction. In addition, ceftriaxone increased glutamate uptake, a primary function of EAAT2, and EAAT2 small interference RNA completely inhibited ceftriaxone-induced glutamate uptake activity in PHFA. Taken together, our data indicate that ceftriaxone is a potent modulator of glutamate transport in PHFA through NF-κB-mediated EAAT2 promoter activation. These findings suggest a mechanism for ceftriaxone modulation of glutamate transport and for its potential effects on ameliorating specific neurodegenerative diseases through modulation of extracellular glutamate.
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cloning and characterization of hiv 1 inducible astrocyte elevated gene 1 aeg 1
Gene, 2005Co-Authors: Dongchul Kang, Devanand Sarkar, David J Volsky, Luni Emdad, Zaozhong Su, Paul B FisherAbstract:Abstract We presently describe the full-length cloning and functional characterization of an HIV-1-inducible gene, astrocyte elevated gene (AEG)-1. Additionally, a novel method is outlined for producing tag-free recombinant protein in a baculovirus system and its use in producing AEG-1 protein. AEG-1 mRNA is expressed ubiquitously with higher expression in tissues containing muscular actin and its expression is increased in astrocytes infected with HIV-1 or treated with gp120 or tumor necrosis factor (TNF)-α. The mRNA encodes a single pass transmembrane protein of predicted molecular mass of 64-kDa and p I 9.3 that predominantly localizes in the endoplasmic reticulum and perinuclear region. Ectopic expression of AEG-1 inhibits Excitatory amino acid Transporter 2 (EAAT2) promoter activity with the potential to promote glutamate excitotoxicity and consequently HIV-1-associated dementia (HAD). AEG-1 expression is elevated in subsets of breast carcinomas, malignant gliomas and melanomas and it synergizes with oncogenic Ha- ras to enhance soft agar colony forming ability of non-tumorigenic immortalized melanocytes, documenting its tumor promoting activity. AEG-1 may affect tumor progression in multiple cell lineages by augmenting expression of the transformed phenotype and/or by inducing glutamate excitotoxicity in malignant glioma. In these contexts, an HIV-1-inducible gene, AEG-1, may contribute to multiple brain abnormalities, including HAD and tumor formation, by both common and distinct mechanisms.
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insights into glutamate transport regulation in human astrocytes cloning of the promoter for Excitatory amino acid Transporter 2 eaat2
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Magdalena Leszczyniecka, Dongchul Kang, Devanand Sarkar, Wei Chao, David J Volsky, Paul B FisherAbstract:Glutamate transport is central to neurotransmitter functions in the brain. Impaired glutamate transport induces neurotoxicity associated with numerous pathological processes, including stroke/ischemia, temporal lobe epilepsy, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, HIV-1-associated dementia, and growth of malignant gliomas. Excitatory amino acid Transporter-2 (EAAT2) is a major glutamate Transporter in the brain expressed primarily in astrocytes. We presently describe the cloning and characterization of the human EAAT2 promoter, demonstrating elevated expression in astrocytes. Regulators of EAAT2 transport, both positive and negative, alter EAAT2 transcription, promoter activity, mRNA, and protein. These findings imply that transcriptional processes can regulate EAAT2 expression. Moreover, they raise the intriguing possibility that the EAAT2 promoter may be useful for targeting gene expression in the brain and for identifying molecules capable of modulating glutamate transport that could potentially inhibit, ameliorate, or prevent various neurodegenerative diseases.
