| 1998 |
GLT-1/EAAT2 functions as a Na+-dependent high-affinity glutamate transporter responsible for the bulk of forebrain glutamate uptake, and biophysical studies of cloned transporters revealed that some subtypes also function as ligand-gated ion channels. |
Expression systems, biophysical/electrophysiological recordings, subtype-specific antibodies |
Neurochemistry international |
High |
10098717
|
| 1998 |
GLT-1/EAAT2 protein is expressed in neurons (hippocampal microcultures), primarily in dendrites of excitatory neurons, and neuronal GLT-1 can participate in clearance of synaptically released glutamate as shown by dihydrokainate-sensitive transport currents and prolonged autaptic currents after glial inactivation. |
Immunocytochemistry with N- and C-terminal antibodies, whole-cell electrophysiology, pharmacological blockade with dihydrokainate |
The Journal of neuroscience |
High |
9614226
|
| 1998 |
GLT-1 (EAAT2) mRNA and protein are expressed in cultured hippocampal neurons (not restricted to glia), demonstrated by single-cell mRNA amplification and immunocytochemistry. |
Single-cell mRNA amplification, immunocytochemistry with subtype-specific antibodies |
Neurochemistry international |
Medium |
9761452
|
| 1998 |
A splice variant of GLT1/EAAT2 (GLT1v) generated by alternative splicing at the 3'-end is preferentially expressed in neurons (CNS and PNS) rather than astrocytes, and immunolabeling shows a cytoplasmic/granular localization suggesting vesicle membrane association. |
cDNA cloning, RT-PCR, Northern blot, in situ hybridization, immunocytochemistry |
Neuroscience |
High |
11784699
|
| 1998 |
Alternative splicing of human EAAT2 produces two novel transcripts (EAAT2-C1, lacking exon 8; EAAT2-C2, with internal splice sites in exons 5 and 6) resulting in deletions of 45 and 107 amino acids in the C-terminal and central protein regions, respectively. |
RT-PCR cloning, sequence analysis |
Neuroscience letters |
Medium |
9502218
|
| 2003 |
EAAT2 (GLT-1) is the predominant nerve terminal glutamate transporter in adult rodent CNS; Western blotting detected EAAT2 in synaptosomes, and pharmacological inhibition with dihydrokainate failed to unmask any non-EAAT2 uptake sites in nerve terminals. |
Western blotting of synaptosomes and glial plasmalemmal vesicles, [3H]D-aspartate and [3H]L-glutamate uptake assays, pharmacological inhibition |
Journal of neurochemistry |
High |
12558972
|
| 2005 |
NF-κB positively regulates EAAT2 transcription (EGF-dependent activation), while TNFα mediates repression of EAAT2 through a distinct NF-κB pathway requiring recruitment of N-myc to N-myc binding sites in the EAAT2 promoter; EGF activates NF-κB independently of IκB signaling. |
Reporter gene assays, ChIP, promoter mutagenesis, IKKβ/p65 overexpression, N-myc overexpression |
The EMBO journal |
High |
15660126
|
| 2007 |
DNA methylation of the EAAT2 promoter CpG island silences EAAT2 expression in glial cells; bisulfite analysis showed dense methylation in EAAT2-negative glioma lines vs. hypomethylation in EAAT2-positive brain tissue; in vitro methylation reduced promoter activity and altered nuclear factor binding. |
Bisulfite sequencing, EMSA, reporter gene assay, DNA methyltransferase inhibition |
Glia |
High |
17311293
|
| 2007 |
PKC activation causes clathrin-dependent endocytosis and subsequent lysosomal degradation of GLT-1; dominant-negative dynamin1, clathrin heavy chain, and Rab7 constructs blocked these effects, while cholesterol depletion, caveolin-1 DN, Eps15 DN, and Arf6 DN had no effect. |
Surface biotinylation, dominant-negative constructs (dynamin, clathrin, Rab7, caveolin, Eps15, Arf6), lysosomal inhibitors (chloroquine, ammonium chloride) |
Neurochemistry international |
High |
17919781
|
| 2008 |
PKC activation induces ubiquitination of GLT-1 at redundant lysine residues in the carboxyl terminus (specifically C7K-R region), which is required for PKC-dependent internalization and degradation; mutation of all 11 cytoplasmic lysines abolished ubiquitination. |
Immunoprecipitation, site-directed mutagenesis of lysine residues, ubiquitin incorporation assay in C6 glioma and primary cortical cultures |
Neurochemistry international |
High |
18805448
|
| 2009 |
Presynaptic terminals regulate astroglial GLT-1/EAAT2 expression via kappa B-motif binding phosphoprotein (KBBP/hnRNP K), which binds the GLT-1 promoter and is required for transcriptional activation; denervation reduces KBBP expression and causes astroglial transporter dysfunction. |
Promoter binding assays, neuron-astrocyte co-culture, in vivo denervation models (corticospinal tract transection, ricin-induced motor neuron death), ALS mouse model |
Neuron |
High |
19323997
|
| 2009 |
PIKfyve (phosphatidylinositol-3-phosphate-5-kinase) enhances EAAT2-mediated glutamate transport current and increases EAAT2 protein abundance at the cell membrane; this effect depends on SGK1 phosphorylation of PIKfyve at S318. |
Xenopus oocyte expression, electrophysiology (glutamate-induced inward currents), confocal microscopy, mutagenesis (S318A PIKfyve), kinase-dead SGK1 construct |
Cellular physiology and biochemistry |
High |
19910676
|
| 2010 |
DNA demethylation of selective CpG sites in the GLT-1 promoter correlates with increased GLT-1 mRNA in astrocytes in response to neuronal stimulation; hypermethylation at selective CpG sites represses GLT-1 promoter activation, but this mechanism does not account for EAAT2 dysregulation in ALS. |
Bisulfite sequencing of FACS-isolated astrocytes from BAC GLT-1 eGFP mice, in vitro and in vivo neuronal stimulation paradigms, postmortem ALS motor cortex analysis |
Glia |
High |
19672971
|
| 2011 |
GLT-1 co-compartmentalizes with mitochondria and glycolytic enzymes (including hexokinase-1) in fine astrocytic processes; immunoaffinity isolation identified these interacting proteins by LC-MS/MS, and simultaneous inhibition of both glycolysis and oxidative phosphorylation (but not either alone) significantly reduces glutamate transport. |
GLT-1 immunoaffinity isolation from rat cortex, LC-MS/MS proteomics, double-label immunofluorescence, biolistic transfection in hippocampal slices, Monte Carlo simulation, acute metabolic inhibition assays |
The Journal of neuroscience |
High |
22171032
|
| 2012 |
Constitutive GLT-1 internalization occurs via clathrin-dependent endocytosis into EEA1/Rab4-positive recycling endosomes (not Rab11 or Rab7 compartments); ubiquitination (at lysines 517 and 526) drives internalization, and deubiquitination by UCH-L1 promotes recycling to the plasma membrane. |
Clathrin inhibitors, dominant-negative Rab constructs, E1 ubiquitin enzyme inhibitor, site-directed mutagenesis (K517, K526), UCH-L1 inhibitor (LDN-57444), endosomal marker co-localization in heterologous system and primary astrocytes |
Glia |
High |
22593014
|
| 2012 |
The transcription factor Pax6 is expressed in astrocytes and contributes to neuron-induced GLT-1 expression by binding to a conserved distal enhancer element ~8 kb upstream of the GLT-1 translation start site. |
Lentiviral Pax6 overexpression in BAC GLT-1 eGFP reporter astrocytes, shRNA knockdown, EMSA, ChIP, GLT-1 protein and uptake assays |
Journal of neurochemistry |
High |
26485579
|
| 2013 |
Astroglial FMRP positively regulates mGluR5 protein translation in astrocytes (FMRP associates with mGluR5 mRNA); loss of astroglial FMRP reduces mGluR5 protein and Ca2+ responses, which in turn attenuates neuron-dependent GLT-1 expression and glutamate uptake in fmr1-/- mice. |
Mismatched astrocyte-neuron co-cultures, FMRP immunoprecipitation + qRT-PCR, Western blot, Ca2+ imaging, dihydrokainate-sensitive GLT-1 inhibition in cortical slices |
Human molecular genetics |
High |
23396537
|
| 2014 |
EAAT2 undergoes constitutive sumoylation (SUMO1 conjugation) in astrocytes in vitro and in vivo; sumoylated EAAT2 localizes to intracellular compartments while non-sumoylated EAAT2 resides on the plasma membrane; promoting desumoylation increases EAAT2-mediated glutamate uptake. |
Immunoprecipitation for SUMO1-EAAT2, immunofluorescence, subcellular fractionation, desumoylation promotion assay with functional glutamate uptake measurement in primary astrocytes and SOD1-G93A mouse |
Glia |
High |
24753081
|
| 2014 |
EAAT2 heteroexchange and net uptake have comparable rates; net uptake is sensitive to membrane potential and stimulated by external permeable anions (uncoupled anion conductance); a sodium leak is also present in EAAT2; the voltage sensitivity of exchange is caused by voltage-dependent third Na+ binding. |
Reconstituted liposome system with rat and mouse EAAT2 protein, electrophysiological voltage manipulation, anion substitution, computational modeling |
The Journal of neuroscience |
High |
25274824
|
| 2014 |
Small-molecule LDN/OSU-0212320 activates EAAT2 translation through PKC activation and subsequent Y-box-binding protein 1 (YB-1) phosphorylation, which drives EAAT2 translational activation. |
Cell-based translational activation assays, PKC pathway inhibitors, YB-1 phosphorylation analysis, in vivo pharmacokinetics and ALS/epilepsy mouse models |
The Journal of clinical investigation |
High |
24569372
|
| 2017 |
Novel positive allosteric modulators of EAAT2 interact with residues at the interface between the trimerization domain and the substrate-binding transport domain; mutagenesis of these residues abolished activator effects; activators enhance glutamate translocation rate without affecting substrate binding, confirming an allosteric mechanism. |
Virtual screening, in vitro transport assay in heterologous expression system, site-directed mutagenesis at trimerization/transport domain interface, selectivity assays |
ACS chemical neuroscience |
High |
29140675
|
| 2017 |
Brain endothelial cells induce astrocytic GLT-1 expression through a contact-dependent, Notch-dependent mechanism; γ-secretase inhibition blocks endothelia-induced Notch intracellular domain nuclear accumulation and GLT-1 upregulation; shRNA against RBPJκ (Notch effector) reduces endothelial induction of GLT-1. |
Astrocyte-endothelial co-culture with transwells (contact dependence), γ-secretase inhibitor (DAPT), shRNA against RBPJκ, BAC GLT-1 eGFP reporter mice, GLT-1 protein and glutamate uptake assays |
Journal of neurochemistry |
High |
28771710
|
| 2018 |
Neuronal EAAT2 in axon terminals is present throughout multiple forebrain regions (not just hippocampus); conditional neuronal deletion (synapsin1-Cre) disproportionately reduces glutamate accumulation in homogenates relative to protein loss, and increases 13C-labeling of glutamine and GABA suggesting neuronal EAAT2 partially short-circuits the glutamate-glutamine cycle. |
Conditional KO (synapsin1-Cre × EAAT2-flox), Western blot, glutamate accumulation in tissue homogenates, U-13C-glucose isotope tracing, Ai9 Cre-reporter |
Neurochemistry international |
High |
29530756
|
| 2019 |
Neuronal GLT-1 (synGLT-1 KO) is required to supply glutamate to synaptic mitochondria for TCA cycle metabolism; its loss reduces aspartate content, diminishes [U-13C]-glutamate-derived TCA labeling, decreases pyruvate recycling, increases mitochondrial ATP production efficiency, and increases mitochondrial cristae density in axon terminals. |
Conditional KO (synapsin1-Cre), synaptosomal uptake, electron microscopy immunocytochemistry, 13C-isotope tracing, isolated mitochondria ATP/oxygen consumption assays |
The Journal of neuroscience |
High |
30926746
|
| 2019 |
Astrocytic conditional EAAT2 deletion causes early deficits in short-term and spatial long-term memory with transcriptomic signatures overlapping human AD and aging (inflammatory/synaptic pathways); neuronal EAAT2 deletion causes late-onset spatial memory deficit with kynurenine pathway dysregulation—demonstrating cell-type-specific roles. |
Conditional KO mice (astrocytic vs. neuronal EAAT2 deletion), behavioral testing (Morris water maze, novel object recognition), transcriptomics |
Proceedings of the National Academy of Sciences |
High |
31591195
|
| 2021 |
Loss of neuronal GLT-1 in presynaptic terminals causes excitotoxic failure of synaptic transmission in CA1; NMDA receptor blockade (MK801) or glutamate scavenging prevents fEPSP failure, indicating that neuronal GLT-1 protects against NMDA receptor-mediated excitotoxicity; metabolic perturbations (reduced glutamate utilization by synaptic mitochondria) contribute to vulnerability. |
Conditional neuronal GLT-1 KO, field potential recordings in hippocampal slices, extracellular FRET-based glutamate sensor, MK801 pharmacology, electron microscopy for mitochondrial cristae density |
Frontiers in cellular neuroscience |
High |
35035352
|
| 2011 |
CD44-SLC1A2 gene fusion in gastric cancer (caused by paracentric chromosomal inversion) places SLC1A2 under CD44 regulatory elements, driving its overexpression; silencing CD44-SLC1A2 reduces intracellular glutamate, proliferation, invasion, and anchorage-independent growth, while overexpression promotes these pro-oncogenic traits. |
Genomic breakpoint analysis, RT-PCR for fusion transcript, siRNA knockdown, overexpression in gastric cell lines, proliferation/invasion/anchorage-independent growth assays, intracellular glutamate measurement |
Science translational medicine |
High |
21471434
|
| 2006 |
PPARγ is a transcriptional regulator of GLT-1/EAAT2; rosiglitazone (PPARγ agonist) increases GLT-1 mRNA and protein and [3H]glutamate uptake; six PPAR response elements (PPREs) were identified in the GLT-1 promoter; rosiglitazone increased GLT-1 promoter activity 4-fold; PPARγ antagonist blocks ischemic preconditioning-induced GLT-1 upregulation. |
Reporter gene assay, [3H]glutamate uptake, Western blot, RT-PCR, PPARγ antagonist/agonist pharmacology, in vitro oxygen-glucose deprivation model |
Journal of cerebral blood flow and metabolism |
High |
17213861
|
| 2012 |
GPR30 activation (by G1 agonist) increases GLT-1 protein and mRNA in astrocytes through MAPK and PI3K signaling, TGF-α receptor transactivation, PKA, and NF-κB (both p50 and p65 subunits bind the GLT-1 promoter); GPR30 silencing reduces GLT-1 and TGF-α expression. |
GPR30 siRNA knockdown, G1 agonist treatment, MAPK/PI3K inhibitors, PKA inhibitor, NF-κB inhibitor, CREB and NF-κB ChIP on GLT-1 promoter, glutamate uptake assay in rat primary astrocytes |
The Journal of biological chemistry |
High |
22645130
|
| 2011 |
Equilibrative nucleoside transporter 1 (ENT1) regulates EAAT2 expression and function in astrocytes; ENT1 antagonist or siRNA reduces EAAT2 mRNA and glutamate uptake; ENT1 overexpression upregulates EAAT2; ENT1 knockdown inhibits ethanol-induced EAAT2 upregulation. |
ENT1-specific antagonist, siRNA knockdown, overexpression, qRT-PCR, glutamate uptake assay in cultured astrocytes |
Alcoholism, clinical and experimental research |
Medium |
20374202
|
| 2006 |
Glucocorticoid receptor activation by corticosterone inhibits microglial GLT-1 expression and glutamate uptake, likely through suppression of TNF-α release; mifepristone (glucocorticoid receptor antagonist) blocks this effect; exogenous TNF-α counteracts corticosterone's inhibitory effect on GLT-1. |
Primary microglial cultures, LPS stimulation, corticosterone dose-response, mifepristone antagonism, TNF-α measurement, GLT-1 expression (Western blot/immunostaining), glutamate uptake assay |
Neuroscience |
Medium |
16473474
|
| 2020 |
Glial GLT-1 determines susceptibility to cortical spreading depression (CSD); conditional GLT-1 KO mice show enhanced CSD frequency and velocity, more rapid extracellular glutamate accumulation during early CSD phase (measured by enzyme-based biosensor), while EAAC1 and GLAST germline KOs show no such effect. |
Conditional GLT-1 KO mice, electrophysiological CSD recording, hemodynamic imaging, enzyme-based extracellular glutamate biosensor |
Glia |
High |
32585762
|
| 2021 |
Astrocytic REST transcription factor positively regulates EAAT2 expression by recruiting the epigenetic co-activator CBP/p300 to REST binding sites in the EAAT2 promoter; REST overexpression in astrocytes attenuates Mn-induced reduction of EAAT2 and prevents excitotoxic dopaminergic neuronal death in co-culture. |
REST overexpression/knockdown in astrocytes, ChIP for CBP/p300 at EAAT2 promoter, glutamate uptake assay, astrocyte-neuron co-culture dopaminergic death assay |
The Journal of biological chemistry |
High |
34756885
|
| 2012 |
The CpG island shore of the GLT-1 gene acts as a methylation-sensitive enhancer; in vitro methylation of shore CpG sites abolishes dexamethasone-stimulated transcriptional activity; the shore region shows higher methylation and repressive histone marks (H3K27me3) in cerebellum (low GLT-1) vs. cortex (high GLT-1) astrocytes. |
Reporter gene assay, targeted in vitro CpG methylation, ChIP for H3K27me3 and H4ac, bisulfite sequencing, HDAC inhibitor treatment in region-specific astrocytes |
Glia |
High |
22593010
|
| 2016 |
The laforin/malin complex (mutated in Lafora disease) slows endocytic recycling of GLT-1; overexpression of laforin and malin causes accumulation of GLT-1 at the plasma membrane and reduces GLT-1 ubiquitination; primary astrocytes from Lafora disease mice have reduced GLT-1 at plasma membrane and reduced glutamate transport capacity. |
Laforin/malin overexpression in cellular models, surface GLT-1 quantification, ubiquitination assays, glutamate transport assay in primary Lafora disease mouse astrocytes |
Biochimica et biophysica acta |
Medium |
26976331
|
| 2009 |
Up-regulation of GLT-1 by ceftriaxone severely impairs mGluR-dependent LTD at mossy fiber-CA3 synapses and reduces LTP at the same synapses; postembedding immunogold shows increased GLT-1a density at mossy fiber terminals near and within active zones, revealing that presynaptic GLT-1 prevents activation of presynaptic receptors needed for plasticity. |
Ceftriaxone treatment, field potential recordings, dihydrokainate rescue, gamma-DGG assay for synaptic glutamate transient, postembedding immunogold electron microscopy, GLT-1 KO mice as specificity controls |
The Journal of physiology |
High |
19651762
|
| 2006 |
GLAST/GLT-1 double knockout mice show multiple brain developmental defects (cortical, hippocampal, olfactory bulb disorganization), impaired neural stem cell proliferation, radial migration, neuronal differentiation, and survival of subplate neurons, demonstrating that GLAST and GLT-1 are essential for brain development through regulation of extracellular glutamate. |
GLAST/GLT-1 double knockout mouse, histology, immunohistochemistry, analysis of cortical development |
Proceedings of the National Academy of Sciences |
High |
16880397
|
| 2013 |
Proteome analysis of pancreatic islets detected that EAAT2 levels are too low to support any proposed glutamate transport functions in islets; conditional pancreatic EAAT2 deletion did not affect survival, growth, glucose tolerance, or β-cell number, ruling out a role for EAAT2 in insulin secretion. |
Conditional pancreatic EAAT2 KO (RIP-Cre, IPF1-Cre), LC-MS/MS proteomics of islet proteins (>7000 proteins detected), TaqMan RT-PCR, immunoblotting, immunocytochemistry, glucose tolerance testing |
The Journal of biological chemistry |
High |
24280215
|
| 2014 |
Restoring GLT-1 expression (but not xCT/cystine-glutamate exchange) in nucleus accumbens is the key mechanism by which chronic N-acetylcysteine (NAC) inhibits cue-induced cocaine reinstatement; suppressing NAC-induced GLT-1 restoration with vivo-morpholino antisense augmented reinstatement via increased extracellular glutamate activating mGluR5. |
Rat self-administration/extinction/reinstatement model, vivo-morpholino antisense oligomers targeting GLT-1 or xCT, mGluR5 blockade, intra-accumbal microinjection |
Addiction biology |
High |
24612076
|
| 2004 |
Adenoviral overexpression of GLT-1 specifically in the locus coeruleus inhibits naloxone-precipitated morphine withdrawal signs, demonstrating that GLT-1 in the locus coeruleus plays an inhibitory role in morphine physical dependence. |
Recombinant adenovirus-mediated GLT-1 gene transfer into bilateral locus coeruleus, morphine pellet implantation, naloxone-precipitated withdrawal scoring in rats |
The European journal of neuroscience |
Medium |
14750980
|