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1.  Epigenetic Regulation of Neuron-Dependent Induction of Astroglial Synaptic Protein GLT1 
Glia  2010;58(3):277-286.
Astroglial glutamate transporter EAAT2/GLT1 prevents glutamate-induced excitotoxicity in the central nervous system. Expression of EAAT2/GLT1 is dynamically regulated by neurons. The pathogenesis of amyotrophic lateral sclerosis (ALS) involves astroglial dysfunction, including dramatic loss of EAAT2/GLT1. DNA methylation of gene promoters represents one of the most important epigenetic mechanisms in regulating gene expression. The involvement of DNA methylation in the regulation of astroglial EAAT2/GLT1 expression in different conditions, especially in ALS has not been explored. In this study, we established a procedure to selectively isolate a pure astrocyte population in vitro and in vivo from BAC GLT1 eGFP mice using an eGFP-based fluorescence-activated cell sorting approach. Astrocytes isolated from this procedure are GFAP+ and GLT1+ and respond to neuronal stimulation, enabling direct methylation analysis of GLT1 promoter in these astrocytes. To investigate the role of DNA methylation in physiological and pathological EAAT2/GLT1 expression, methylation status of the EAAT2/GLT1 promoter was analyzed in astrocytes from in vitro and in vivo paradigms or postmortem ALS motor cortex by bisulfite sequencing method. DNA demethylation on selective CpG sites of the GLT1 promoter was highly correlated to increased GLT1 mRNA levels in astrocytes in response to neuronal stimulation; however, low level of methylation was found on CpG sites of EAAT2 promoter from postmortem motor cortex of human amyotrophic lateral sclerosis patients. In summary, hypermethylation on selective CpG sites of the GLT1 promoter is involved in repression of GLT1 promoter activation, but this regulation does not play a role in astroglial dysfunction of EAAT2 expression in patients with ALS.
doi:10.1002/glia.20922
PMCID: PMC2794958  PMID: 19672971
epigenetic; astrocyte; GLT1
2.  GLT-1 Promoter Activity in Astrocytes and Neurons of Mouse Hippocampus and Somatic Sensory Cortex 
GLT-1 eGFP BAC reporter transgenic adult mice were used to detect GLT-1 gene expression in individual cells of CA1, CA3 and SI, and eGFP fluorescence was measured to analyze quantitatively GLT-1 promoter activity in different cells of neocortex and hippocampus. Virtually all GFAP+ astrocytes were eGFP+; we also found that about 80% of neurons in CA3 pyramidal layer, 10–70% of neurons in I-VI layers of SI and rare neurons in all strata of CA1 and in strata oriens and radiatum of CA3 were eGFP+. Analysis of eGFP intensity showed that astrocytes had a higher GLT-1 promoter activity in SI than in CA1 and CA3, and that neurons had the highest levels of GLT-1 promoter activity in CA3 stratum pyramidale and in layer VI of SI. Finally, we observed that the intensity of GLT-1 promoter activity in neurons is 1–20% of that measured in astrocytes. These results showed that in the hippocampus and neocortex GLT-1 promoter activity is observed in astrocytes and neurons, detailed the distribution of GLT-1 expressing neurons, and indicated that GLT-1 promoter activity in both astrocytes and neurons varies in different brain regions.
doi:10.3389/neuro.05.031.2009
PMCID: PMC2813724  PMID: 20161698
glutamate transporters; GLT-1/EAAT2; neurons; astrocytes; hippocampus; neocortex
3.  Molecular Comparison of GLT1+ and ALDH1L1+ Astrocytes In Vivo In Astroglial Reporter Mice 
Glia  2011;59(2):200-207.
Astrocyte heterogeneity remains largely unknown in the CNS due to lack of specific astroglial markers. In this study, molecular identity of in vivo astrocytes was characterized in BAC ALDH1L1 and BAC GLT1 eGFP promoter reporter transgenic mice. ALDH1L1 promoter is selectively activated in adult cortical and spinal cord astrocytes, indicated by the overlap of eGFP expression with ALDH1L1 and GFAP, but not with NeuN, APC, Olig2, IbaI, PDGFRα immunoreactivity in BAC ALDH1L1 eGFP reporter mice. Interestingly, ALDH1L1 expression levels (protein, mRNA, and promoter activity) in spinal cord were selectively decreased during postnatal maturation. In contrast, its expression was up-regulated in reactive astrocytes in both acute neural injury and chronic neurodegenerative (G93A mutant SOD1) conditions, similar to GFAP, but opposite of GLT1. ALDH1L1+ and GLT1+ cells isolated through fluorescence activated cell sorting (FACS) from BAC ALDH1L1 and BAC GLT1 eGFP mice share a highly similar gene expression profile, suggesting ALDH1L1 and GLT1 are co-expressed in the same population of astrocytes. This observation was further supported by overlap of the eGFP driven by the ALDH1L1 genomic promoter and the tdTomato driven by a 8.3kb EAAT2 promoter fragment in astrocytes of BAC ALDH1L1 eGFP X EAAT2-tdTomato mice. These studies support ALDH1L1 as a general CNS astroglial marker and investigated astrocyte heterogeneity in the CNS by comparing the molecular identity of the ALDH1L1+ and GLT1+ astrocytes from astroglial reporter mice. These astroglial reporter mice provide useful in vivo tools for the molecular analysis of astrocytes in physiological and pathological conditions.
doi:10.1002/glia.21089
PMCID: PMC3199134  PMID: 21046559
astroglia; BAC; ALDH1L1; GLT1; GFAP; oligodendroglia; ALS
4.  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
5.  Neuronal influences are necessary to produce mitochondrial co-localization with glutamate transporters in astrocytes 
Journal of Neurochemistry  2014;130(5):668-677.
Recent evidence suggests that the predominant astrocyte glutamate transporter, GLT-1/ Excitatory Amino Acid Transporter 2 (EAAT2) is associated with mitochondria. We used primary cultures of mouse astrocytes to assess co-localization of GLT-1 with mitochondria, and tested whether the interaction was dependent on neurons, actin polymerization or the kinesin adaptor, TRAK2. Mouse primary astrocytes were transfected with constructs expressing V5-tagged GLT-1, pDsRed1-Mito with and without dominant negative TRAK2. Astrocytes were visualized using confocal microscopy and co-localization was quantified using Volocity software. Image analysis of confocal z-stacks revealed no co-localization between mitochondria and GLT-1 in pure astrocyte cultures. Co-culture of astrocytes with primary mouse cortical neurons revealed more mitochondria in processes and a positive correlation between mitochondria and GLT-1. This co-localization was not further enhanced after neuronal depolarization induced by 1 h treatment with 15 mM K+. In pure astrocytes, a rho kinase inhibitor, Y27632 caused the distribution of mitochondria to astrocyte processes without enhancing GLT-1/mitochondrial co-localization, however, in co-cultures, Y27632 abolished mitochondrial:GLT-1 co-localization. Disrupting potential mitochondrial: kinesin interactions using dominant negative TRAK2 did not alter GLT-1 distribution or GLT-1: mitochondrial co-localization. We conclude that the association between GLT-1 and mitochondria is modest, is driven by synaptic activity and dependent on polymerized actin filaments.Mitochondria have limited co-localization with the glutamate transporter GLT-1 in primary astrocytes in culture. Few mitochondria are in the fine processes where GLT-1 is abundant. It is necessary to culture astrocytes with neurones to drive a significant level of co-localization, but co-localization is not further altered by depolarization, manipulating sodium ion gradients or Na/K ATPase activity.
doi:10.1111/jnc.12759
PMCID: PMC4283053  PMID: 24814819
co-culture; EAAT2; Glial cell; glutamate transporter; rho kinase; TRAK2
6.  The Glutamate Transporter GLT1a Is Expressed in Excitatory Axon Terminals of Mature Hippocampal Neurons 
GLT1 is the major glutamate transporter of the brain and has been thought to be expressed exclusively in astrocytes. Although excitatory axon terminals take up glutamate, the transporter responsible has not been identified. GLT1 is expressed in at least two forms varying in the C termini, GLT1a and GLT1b. GLT1 mRNA has been demonstrated in neurons, without associated protein. Recently, evidence has been presented, using specific C terminus-directed antibodies, that GLT1b protein is expressed in neurons in vivo. These data suggested that the GLT1 mRNA detected in neurons encodes GLT1b and also that GLT1b might be the elusive presynaptic transporter. To test these hypotheses, we used variant-specific probes directed to the 3′-untranslated regions for GLT1a and GLT1b to perform in situ hybridization in the hippocampus. Contrary to expectation, GLT1a mRNA was the more abundant form. To investigate further the expression of GLT1 in neurons in the hippocampus, antibodies raised against the C terminus of GLT1a and against the N terminus of GLT1, found to be specific by testing in GLT1 knock-out mice, were used for light microscopic and EM-ICC. GLT1a protein was detected in neurons, in 14–29% of axons in the hippocampus, depending on the region. Many of the labeled axons formed axo-spinous, asymmetric, and, thus, excitatory synapses. Labeling also occurred in some spines and dendrites. The antibody against the N terminus of GLT1 also produced labeling of neuronal processes. Thus, the originally cloned form of GLT1, GLT1a, is expressed as protein in neurons in the mature hippocampus and may contribute significantly to glutamate uptake into excitatory terminals.
doi:10.1523/JNEUROSCI.1586-03.2004
PMCID: PMC2849838  PMID: 14762132
uptake; trafficking; alternative splicing; excitotoxicity; PDZ domain; synapse
7.  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
8.  Transforming growth factor-α mediates estrogen-induced upregulation of glutamate transporter GLT-1 in rat primary astrocytes 
Glia  2012;60(7):1024-1036.
Glutamate transporter-1 (GLT-1) plays a central role in preventing excitotoxicity by removing excess glutamate from the synaptic clefts. 17β-estradiol (E2) and tamoxifen (TX), a selective estrogen receptor modulator (SERM), afford neuroprotection in a range of experimental models. However, the mechanisms that mediate E2 and TX neuroprotection have yet to be elucidated. We tested the hypothesis that E2 and TX enhance GLT-1 function by increasing transforming growth factor (TGF)-α expression and thus, attenuate manganese (Mn)-induced impairment in astrocytic GLT-1 expression and glutamate uptake in rat neonatal primary astrocytes. The results showed that E2 (10 nM) and TX (1 μM) increased GLT-1 expression and reversed the Mn-induced reduction in GLT-1, both at the mRNA and protein levels. E2/TX also concomitantly reversed the Mn-induced inhibition of astrocytic glutamate uptake. E2/TX activated the GLT-1 promoter and attenuated the Mn-induced repression of the GLT-1 promoter in astrocytes. TGF-α knock-down (siRNA) abolished the E2/TX effect on GLT-1 expression, and inhibition of epidermal growth factor receptor (TGF-α receptor) suppressed the effect of E2/TX on GLT-1 expression and GLT-1 promoter activity. E2/TX also increased TGF-α mRNA and protein levels with a concomitant increase in astrocytic glutamate uptake. All estrogen receptors (ERs: ER-α ER-β and GPR30) were involved in mediating E2 effects on the regulation of TGF-α, GLT-1, and glutamate uptake. These results indicate that E2/TX increase GLT-1 expression in astrocytes via TGF-α signaling, thus offering an important putative target for the development of novel therapeutics for neurological disorders.
doi:10.1002/glia.22329
PMCID: PMC3353324  PMID: 22488924
GLT-1; TGF-α; glutamate uptake; glutamate transporter; manganese; tamoxifen; 17β-estradiol; astrocytes
9.  NEURONAL ACTIVITY REGULATES GLUTAMATE TRANSPORTER DYNAMICS IN DEVELOPING ASTROCYTES 
Glia  2011;60(2):175-188.
Glutamate transporters maintain a low ambient level of glutamate in the CNS and shape the activation of glutamate receptors at synapses. Nevertheless, the mechanisms that regulate the trafficking and localization of transporters near sites of glutamate release are poorly understood. Here we examined the subcellular distribution and dynamic remodeling of the predominant glutamate transporter GLT-1 (EAAT2) in developing hippocampal astrocytes. Immunolabeling revealed that endogenous GLT-1 is concentrated into discrete clusters along branches of developing astrocytes that were apposed preferentially to synapsin-1 positive synapses. GFP-GLT-1 fusion proteins expressed in astrocytes also formed distinct clusters that lined the edges of astrocyte processes, as well as the tips of filopodia and spine-like structures. Time-lapse 3D confocal imaging in tissue slices revealed that GFP-GLT-1 clusters were dynamically remodeled on a timescale of minutes. Some transporter clusters moved within developing astrocyte branches as filopodia extended and retracted, while others maintained stable positions at the tips of spine-like structures. Blockade of neuronal activity with tetrodotoxin reduced both the density and perisynaptic localization of GLT-1 clusters. Conversely, enhancement of neuronal activity increased the size of GLT-1 clusters and their proximity to synapses. Together, these findings indicate that neuronal activity influences both the organization of glutamate transporters in developing astrocyte membranes and their position relative to synapses.
doi:10.1002/glia.21249
PMCID: PMC3232333  PMID: 22052455
astrocyte; GLT-1; EAAT2; biolistics; time-lapse; activity
10.  Astroglial FMRP-dependent translational down-regulation of mGluR5 underlies glutamate transporter GLT1 dysregulation in the fragile X mouse 
Human Molecular Genetics  2013;22(10):2041-2054.
Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by the loss-of-function of fragile X mental retardation protein (FMRP). The loss of FMRP function in neurons abolishes its suppression on mGluR1/5-dependent dendritic protein translation, enhancing mGluR1/5-dependent synaptic plasticity and other disease phenotypes in FXS. In this study, we describe a new activation function of FMRP in regulating protein expression in astroglial cells. We found that astroglial glutamate transporter subtype glutamate transporter 1 (GLT1) and glutamate uptake is significantly reduced in the cortex of fmr1−/− mice. Correspondingly, neuronal excitability is also enhanced in acute fmr1−/− (but not in fmr1+/+ control) cortical slices treated with low doses (10 μm) of the GLT1-specific inhibitor dihydrokainate (DHK). Using mismatched astrocyte and neuron co-cultures, we demonstrate that the loss of astroglial (but not neuronal) FMRP particularly reduces neuron-dependent GLT1 expression and glutamate uptake in co-cultures. Interestingly, protein (but not mRNA) expression and the (S)-3,5-dihydroxyphenylglycine-dependent Ca2+ responses of astroglial mGluR5 receptor are also selectively reduced in fmr1−/− astrocytes and brain slices, attenuating neuron-dependent GLT1 expression. Subsequent FMRP immunoprecipitation and QRT–PCR analysis showed that astroglial mGluR5 (but not GLT1) mRNA is associated with FMRP. In summary, our results provide evidence that FMRP positively regulates translational expression of mGluR5 in astroglial cells, and FMRP-dependent down-regulation of mGluR5 underlies GLT1 dysregulation in fmr1−/− astrocytes. The dysregulation of GLT1 and reduced glutamate uptake may potentially contribute to enhanced neuronal excitability observed in the mouse model of FXS.
doi:10.1093/hmg/ddt055
PMCID: PMC3633372  PMID: 23396537
11.  Expression of a Variant Form of the Glutamate Transporter GLT1 in Neuronal Cultures and in Neurons and Astrocytes in the Rat Brain 
To identify glutamate transporters expressed in forebrain neurons, we prepared a cDNA library from rat forebrain neuronal cultures, previously shown to transport glutamate with high affinity and capacity. Using this library, we cloned two forms, varying in the C terminus, of the glutamate transporter GLT1. This transporter was previously found to be localized exclusively in astrocytes in the normal mature brain. Specific antibodies against the C-terminal peptides were used to show that forebrain neurons in culture express both GLT1a and GLT1b proteins. The pharmacological properties of glutamate transport mediated by GLT1a and GLT1b expressed in COS-7 cells and in neuronal cultures were indistinguishable. Both GLT1a and GLT1b were upregulated in astrocyte cultures by exposure to dibutyryl cAMP. We next investigated the expression of GLT1b in vivo. Northern blot analysis of forebrain RNA revealed two transcripts of ~3 and 11 kb that became more plentiful with developmental age. Immunoblot analysis showed high levels of expression in the cortex, hippocampus, striatum, thalamus, and midbrain. Pre-embedding electron microscopic immunocytochemistry with silver-enhanced immunogold detection was used to localize GLT1b in vivo. In the rat somatosensory cortex, GLT1b was clearly expressed in neurons in presynaptic terminals and dendritic shafts, as well as in astrocytes. The presence of GLT1b in neurons may offer a partial explanation for the observed uptake of glutamate by presynaptic terminals, for the preservation of input specificity at excitatory synapses, and may play a role in the pathophysiology of excitotoxicity.
PMCID: PMC2849837  PMID: 11896154
glutamate; transport; dihydrokainate; presynaptic; astrocytes; synapse; excitotoxicity
12.  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
13.  Cellular and Subcellular mRNA Localization of Glutamate Transporter Isoforms GLT1a and GLT1b in Rat Brain by In Situ Hybridization 
GLT1, the predominant glutamate transporter of the forebrain, exists in two splice variant isoforms, i.e. GLT1a and GLT1b. Although GLT1 was originally only detected in astrocytes, we have recently demonstrated that GLT1a protein is expressed by neurons in the hippocampus as well. In the present study, the mRNA distribution patterns for the two isoforms were examined throughout the rat brain using non-isotopic in situ hybridization and variant specific RNA probes. Both isoforms were expressed in neuronal subgroups outside the hippocampus such as in the cerebral cortex layer VI, or the neurons in the olfactory tubercle. Similar to the hippocampus, GLT1a was the predominant transcript in neurons. Both GLT1 isoforms were widely expressed in astrocytes throughout the brain. GLT1a mRNA expression in astrocytes showed noticeable variation in labeling intensity in subregions of the hippocampus and other areas, whereas GLT1b expression in astrocytes was relatively homogeneous. On the subcellular level, GLT1a mRNA was primarily expressed in astrocyte processes, whereas GLT1b mRNA was more restricted to the astrocyte cell body. The two isoforms showed similar distributions in the subfornical organ and in tanycytes of the third ventricle. However, GLT1 expression in the pineal gland and the retina was primarily due to GLT1b, whereas GLT1a was more strongly expressed in Bergman glia in the cerebellum. These findings suggest that the expression of the two GLT1 isoforms is regulated by different mechanisms. Moreover, the function of the two isoforms may be subject to different regulatory processes.
doi:10.1002/cne.20737
PMCID: PMC3676901  PMID: 16175560
uptake; presynaptic; alternative splicing; excitotoxicity; excitatory; synapse
14.  The Inflammatory Cytokine, Interleukin-1 Beta, Mediates Loss of Astroglial Glutamate Transport and Drives Excitotoxic Motor Neuron Injury in the Spinal Cord During Acute Viral Encephalomyelitis 
Journal of neurochemistry  2008;105(4):1276-1286.
Astrocytes remove glutamate from the synaptic cleft via specific transporters, and impaired glutamate reuptake may promote excitotoxic neuronal injury. In a model of viral encephalomyelitis caused by neuroadapted Sindbis virus (NSV), mice develop acute paralysis and spinal motor neuron degeneration inhibited by the AMPA receptor antagonist, NBQX. To investigate disrupted glutamate homeostasis in the spinal cord, expression of the main astroglial glutamate transporter, GLT-1, was examined. GLT-1 levels declined in the spinal cord during acute infection while GFAP expression was preserved. There was simultaneous production of inflammatory cytokines at this site, and susceptible animals treated with drugs that blocked IL-1β release also limited paralysis and prevented the loss of GLT-1 expression. Conversely, infection of resistant mice that develop mild paralysis following NSV challenge showed higher baseline GLT-1 levels as well as lower production of IL-1β and relatively preserved GLT-1 expression in the spinal cord compared to susceptible hosts. Finally, spinal cord GLT-1 expression was largely maintained following infection of IL-1β-deficient animals. Together, these data show that IL-1β inhibits astrocyte glutamate transport in the spinal cord during viral encephalomyelitis. They provide one of the strongest in vivo links between innate immune responses and the development of excitotoxicity demonstrated to date.
doi:10.1111/j.1471-4159.2008.05230.x
PMCID: PMC2579753  PMID: 18194440
glutamate transporters; interleukin-1β; viral encephalomyelitis; motor neuron; excitotoxicity
15.  Lesion-Induced Alterations in Astrocyte Glutamate Transporter Expression and Function in the Hippocampus 
ISRN Neurology  2013;2013:893605.
Astrocytes express the sodium-dependent glutamate transporters GLAST and GLT-1, which are critical to maintain low extracellular glutamate concentrations. Here, we analyzed changes in their expression and function following a mechanical lesion in the CA1 area of organotypic hippocampal slices. 6-7 days after lesion, a glial scar had formed along the injury site, containing strongly activated astrocytes with increased GFAP and S100β immunoreactivity, enlarged somata, and reduced capability for uptake of SR101. Astrocytes in the scar's periphery were swollen as well, but showed only moderate upregulation of GFAP and S100β and efficiently took up SR101. In the scar, clusters of GLT-1 and GLAST immunoreactivity colocalized with GFAP-positive fibers. Apart from these, GLT-1 immunoreactivity declined with increasing distance from the scar, whereas GLAST expression appeared largely uniform. Sodium imaging in reactive astrocytes indicated that glutamate uptake was strongly reduced in the scar but maintained in the periphery. Our results thus show that moderately reactive astrocytes in the lesion periphery maintain overall glutamate transporter expression and function. Strongly reactive astrocytes in the scar, however, display clusters of GLAST and GLT-1 immunoreactivity together with reduced glutamate transport activity. This reduction might contribute to increased extracellular glutamate concentrations and promote excitotoxic cell damage at the lesion site.
doi:10.1155/2013/893605
PMCID: PMC3775433  PMID: 24078881
16.  Overexpression of the Astrocyte Glutamate Transporter GLT1 Exacerbates Phrenic Motor Neuron Degeneration, Diaphragm Compromise, and Forelimb Motor Dysfunction following Cervical Contusion Spinal Cord Injury 
The Journal of Neuroscience  2014;34(22):7622-7638.
A major portion of spinal cord injury (SCI) cases affect midcervical levels, the location of the phrenic motor neuron (PhMN) pool that innervates the diaphragm. While initial trauma is uncontrollable, a valuable opportunity exists in the hours to days following SCI for preventing PhMN loss and consequent respiratory dysfunction that occurs during secondary degeneration. One of the primary causes of secondary injury is excitotoxic cell death due to dysregulation of extracellular glutamate homeostasis. GLT1, mainly expressed by astrocytes, is responsible for the vast majority of functional uptake of extracellular glutamate in the CNS, particularly in spinal cord. We found that, in bacterial artificial chromosome-GLT1-enhanced green fluorescent protein reporter mice following unilateral midcervical (C4) contusion SCI, numbers of GLT1-expressing astrocytes in ventral horn and total intraspinal GLT1 protein expression were reduced soon after injury and the decrease persisted for ≥6 weeks. We used intraspinal delivery of adeno-associated virus type 8 (AAV8)-Gfa2 vector to rat cervical spinal cord ventral horn for targeting focal astrocyte GLT1 overexpression in areas of PhMN loss. Intraspinal delivery of AAV8-Gfa2-GLT1 resulted in transduction primarily of GFAP+ astrocytes that persisted for ≥6 weeks postinjury, as well as increased intraspinal GLT1 protein expression. Surprisingly, we found that astrocyte-targeted GLT1 overexpression increased lesion size, PhMN loss, phrenic nerve axonal degeneration, and diaphragm neuromuscular junction denervation, and resulted in reduced functional diaphragm innervation as assessed by phrenic nerve-diaphragm compound muscle action potential recordings. These results demonstrate that GLT1 overexpression via intraspinal AAV-Gfa2-GLT1 delivery exacerbates neuronal damage and increases respiratory impairment following cervical SCI.
doi:10.1523/JNEUROSCI.4690-13.2014
PMCID: PMC4035523  PMID: 24872566
adeno-associated virus; gene therapy; glutamate transporter; phrenic motor neuron; respiratory; spinal cord injury
17.  Regulation of Glutamate Transport in Developing Rat Oligodendrocytes 
Glutamate released from synaptic vesicles mediates excitatory neurotransmission by stimulating glutamate receptors. Glutamate transporters maintain low synaptic glutamate levels critical for this process, a role primarily attributed to astrocytes. Recently, vesicular release of glutamate from unmyelinated axons in the rat corpus callosum has been shown to elicit AMPA receptor-mediated currents in glial progenitor cells. Glutamate transporters are the only mechanism of glutamate clearance, yet very little is known about the role of glutamate transporters in normal development of oligodendrocytes (OLs) or in excitotoxic injury to OLs. We found that OLs in culture are capable of sodium-dependent glutamate uptake with a Km of 10 ± 2 μm and a Vmax of 2.6, 5.0, and 3.8 nmol · min−1 · mg−1 for preoligodendrocytes, immature, and mature OLs, respectively. Surprisingly, EAAC1, thought to be exclusively a neuronal transporter, contributes more to [3H]l-glutamate uptake in OLs than GLT1 or GLAST. These data suggest that glutamate transporters on oligodendrocytes may serve a critical role in maintaining glutamate homeostasis at a time when unmyelinated callosal axons are engaging in glutamatergic signaling with glial progenitors. Furthermore, GLT1 was significantly increased in cultured mature OLs contrary to in vivo data in which we have shown that, although GLT1 is present on developing OLs when unmyelinated axons are prevalent in the developing rat corpus callosum, after myelination, GLT1 is not expressed on mature OLs. The absence of GLT1 in mature OLs in the rat corpus callosum and its presence in mature rat cultured OLs may indicate that a signaling process in vivo is not activated in vitro.
doi:10.1523/JNEUROSCI.6129-08.2009
PMCID: PMC2926807  PMID: 19535601
18.  Gene transfer of GLT-1, a glial glutamate transporter, into the spinal cord by recombinant adenovirus attenuates inflammatory and neuropathic pain in rats 
Molecular Pain  2008;4:65.
Background
The glial glutamate transporter GLT-1 is abundantly expressed in astrocytes and is crucial for glutamate removal from the synaptic cleft. Decreases in glutamate uptake activity and expression of spinal glutamate transporters are reported in animal models of pathological pain. However, the lack of available specific inhibitors and/or activators for GLT-1 makes it difficult to determine the roles of spinal GLT-1 in inflammatory and neuropathic pain. In this study, we examined the effect of gene transfer of GLT-1 into the spinal cord with recombinant adenoviruses on the inflammatory and neuropathic pain in rats.
Results
Intraspinal infusion of adenoviral vectors expressing the GLT-1 gene increased GLT-1 expression in the spinal cord 2–21 days after the infusion. Transgene expression was primarily localized to astrocytes. The spinal GLT-1 gene transfer had no effect on acute mechanical and thermal nociceptive responses in naive rats, whereas it significantly reduced the inflammatory mechanical hyperalgesia induced by hindlimb intraplantar injection of carrageenan/kaolin. Spinal GLT-1 gene transfer 7 days before partial sciatic nerve ligation recovered the extent of the spinal GLT-1 expression in the membrane fraction that was decreased following the nerve ligation, and prevented the induction of tactile allodynia. However, the partial sciatic nerve ligation-induced allodynia was not reversed when the adenoviruses were infused 7 or 14 days after the nerve ligation.
Conclusion
These results suggest that overexpression of GLT-1 on astrocytes in the spinal cord by recombinant adenoviruses attenuates the induction, but not maintenance, of inflammatory and neuropathic pain, probably by preventing the induction of central sensitization, without affecting acute pain sensation. Upregulation or functional enhancement of spinal GLT-1 could be a novel strategy for the prevention of pathological pain.
doi:10.1186/1744-8069-4-65
PMCID: PMC2628654  PMID: 19108711
19.  Role of astrocytes in delayed neuronal death: GLT-1 and its novel regulation by microRNAs 
Advances in neurobiology  2014;11:171-188.
Astrocytes have been shown to protect neurons from delayed neuronal death and increase their survival in cerebral ischemia. One of the main mechanisms of astrocyte protection is rapid removal of excess glutamate from synaptic sites by astrocytic plasma membrane glutamate transporters such as GLT-1/EAAT-2, reducing excitotoxicity. Astrocytic mitochondrial function is essential for normal GLT-1 function. Manipulating astrocytic mitochondrial and GLT-1 function is thus an important strategy to enhance neuronal survival and improve outcome following cerebral ischemia. Increasing evidence supports the involvement of microRNAs (miRNA), some of them being astrocyte-enriched, in the regulation of cerebral ischemia. This chapter will first update the information about astrocytes, GLT-1, astrocytic mitochondria, and delayed neuronal death. Then we will focus on two recently reported astrocyte-enriched miRNAs (miR-181 and miR-29 families), their effects on astrocytic mitochondria and GLT-1 as well as on outcome after cerebral ischemia.
doi:10.1007/978-3-319-08894-5_9
PMCID: PMC4283583  PMID: 25236729
Astrocyte; glutamate transporter; GLT-1; microRNA; miR-181; miR-29; cerebral ischemia; delayed neuronal death
20.  Differential Regulation of Glutamate Transporter Subtypes by Pro-Inflammatory Cytokine TNF-α in Cortical Astrocytes from a Rat Model of Amyotrophic Lateral Sclerosis 
PLoS ONE  2014;9(5):e97649.
Dysregulation of the astroglial glutamate transporters GLAST and GLT-1 has been implicated in several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) where a loss of GLT-1 protein expression and activity is reported. Furthermore, the two principal C-terminal splice variants of GLT-1 (namely GLT-1a and GLT-1b) show altered expression ratio in animal models of this disease. Considering the putative link between inflammation and excitotoxicity, we have here characterized the influence of TNF-α on glutamate transporters in cerebral cortical astrocyte cultures from wild-type rats and from a rat model of ALS (hSOD1G93A). Contrasting with the down-regulation of GLAST, a 72 h treatment with TNF-α substantially increased the expression of GLT-1a and GLT-1b in both astrocyte cultures. However, as the basal level of GLT-1a appeared considerably lower in hSOD1G93A astrocytes, its up-regulation by TNF-α was insufficient to recapitulate the expression observed in wild-type astrocytes. Also the glutamate uptake activity after TNF-α treatment was lower for hSOD1G93A astrocytes as compared to wild-type astrocytes. In the presence of the protein synthesis inhibitor cycloheximide, TNF-α did not influence GLT-1 isoform expression, suggesting an active role of dynamically regulated protein partners in the adaptation of astrocytes to the inflammatory environment. Confirming the influence of inflammation on the control of glutamate transmission by astrocytes, these results shed light on the regulation of glutamate transporter isoforms in neurodegenerative disorders.
doi:10.1371/journal.pone.0097649
PMCID: PMC4023965  PMID: 24836816
21.  Babesia bovis: A bipartite signal directs the glutamyl-tRNA synthetase to the apicoplast 
Experimental Parasitology  2012;131(2):261-266.
Graphical abstract
Highlights
► Protein traffic into the apicoplastic lumen requires a bipartite signal. ► Glutamyl tRNA synthetase (GltX) is a putative apicoplast targeted protein. ► GltX contains a bipartite signal sequence, a pre-requisite for apicoplast traffic. ► GltX bipartite sequence successfully directed GFP into the apicoplast. ► Native GltX may utilize its bipartite sequence for apicoplast entry.
Babesia bovis contains a prokaryotic derived organelle known as the apicoplast. Many participants of the metabolic pathways within the apicoplast are encoded in the nuclear genome and post-translationally imported with the help of a bipartite signal. Recently, an all encompassing algorithm was derived to predict apicoplast targeted proteins for many non-Plasmodium apicomplexans in which it reported the presence of 260 apicoplast targeted proteins in Babesia. One of these proteins is glutamyl tRNA synthetase (GltX). This study investigates if the putative bipartite signal of GltX alone is sufficient to direct proteins into the apicoplast. Using a transient transfection system consisting of a green fluorescent protein as the reporter, we tested the signal and transit portions of the bipartite signal in apicoplastic transport. We first identified the transcript of gltX to be expressed during the asexual blood stages and subsequently confirmed that the complete bipartite signal is responsible for directing the reporter protein into a compartment distinct from the nucleus and the mitochondrion. As GltX bipartite signal successfully guided the reporter protein into the apicoplast, our finding implies that it also directs native GltX into the same organelle.
doi:10.1016/j.exppara.2012.04.013
PMCID: PMC3377962  PMID: 22561041
Babesia bovis; Apicoplast; Traffic; Glutamyl tRNA synthetase; Transfection
22.  The chemokine, macrophage inflammatory protein-2γ, reduces the expression of glutamate transporter-1 on astrocytes and increases neuronal sensitivity to glutamate excitotoxicity 
Background
Changes in glutamatergic neurotransmission via decreased glutamate transporter (GLT) activity or expression contributes to multiple neurological disorders. Chemokines and their receptors are involved in neurological diseases but the role of chemokines in the expression of glutamate transporters is unclear.
Methods
Primary astrocytes were prepared from neonatal (<24 hours old) SJL/J mouse brains and incubated with 5 μg/ml lipopolysaccharide (LPS) or 50 ng/ml tumor necrosis factor α (TNF-α) for 24 hours. Soluble macrophage inflammatory protein-2γ (MIP-2γ) in culture supernatants was determined using a sandwich ELISA. The MIP-2γ effect on the expression of GLT-1 was measured by quantitative RT-PCR, flow cytometric analysis or western blot assay. Detergent-resistant membranes from astrocytes were isolated on the basis of their ability to float in density gradients. Raft-containing fractions were tracked by the enrichment of caveolin-1 and the dendritic lipid raft marker, flotillin-1. Cell viability was determined by measuring either the leakage of lactate dehydrogenase or the reduction of 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide by viable cells and confirmed by visual inspection.
Results
The production of the chemokine MIP-2γ by mouse cortical astrocytes increased significantly after stimulation with LPS or TNF-α in vitro. Astrocytes over-expressing MIP-2γ down-regulated the expression of GLT-1 at the mRNA and protein level and caused redistribution of GLT-1 out of the lipid rafts that mediate glutamate uptake. We used pharmacological inhibitors to identify the downstream signaling pathways underlying MIP-2γ activity. We also found complementary results by knocking down MIP-2γ activity in astrocytes with MIP-2γ small interfering RNA (siRNA). MIP-2γ overexpression in astrocytes enhanced the neuronal toxicity of glutamate by decreasing GLT-1 activity, but MIP-2γ itself was not toxic to neurons.
Conclusions
These results suggest that MIP-2γ mediates the pathogenesis of central nervous system disorders associated with neutrophil infiltration in the brain and decreased GLT-1 activity.
doi:10.1186/1742-2094-9-267
PMCID: PMC3545864  PMID: 23234294
Glutamate transporter; Chemokines; MIP-2γ; Astrocyte; Lipid rafts
23.  Reduction in expression of the astrocyte glutamate transporter, GLT1, worsens functional and histological outcomes following traumatic spinal cord injury 
Glia  2011;59(12):1996-2005.
The astrocyte glutamate transporter, GLT1, is responsible for the vast majority of glutamate uptake in the adult central nervous system (CNS), thereby regulating extracellular glutamate homeostasis and preventing excitotoxicity. Glutamate dysregulation plays a central role in outcome following traumatic spinal cord injury (SCI). To determine the role of GLT1 in secondary cell loss following SCI, mice heterozygous for the GLT1 astrocyte glutamate transporter (GLT1+/−) and wild-type mice received thoracic crush SCI. Compared to wild-type controls, GLT1+/− mice had an attenuated recovery in hindlimb motor function, increased lesion size, and decreased tissue sparing. GLT1+/− mice showed a decrease in intraspinal GLT1 protein and functional glutamate uptake compared to wild-type mice, accompanied by increased apoptosis and neuronal loss following crush injury. These results suggest that astrocyte GLT1 plays a role in limiting secondary cell death following SCI, and also show that compromise of key astrocyte functions has significant effects on outcome following traumatic CNS injury. These findings also suggest that increasing intraspinal GLT1 expression may represent a therapeutically relevant target for SCI treatment.
doi:10.1002/glia.21241
PMCID: PMC3269541  PMID: 21882244
secondary injury; GLT1+/− mice; crush injury; glutamate uptake; excitotoxicity
24.  Propentofylline-Induced Astrocyte Modulation Leads to Alterations in Glial Glutamate Promoter Activation Following Spinal Nerve Transection 
Neuroscience  2008;152(4):1086-1092.
We have previously shown that the atypical methylxanthine, propentofylline, reduces mechanical allodynia after peripheral nerve transection in a rodent model of neuropathy. In the present study, we sought to determine whether propentofylline-induced glial modulation alters spinal glutamate transporters, GLT-1 and GLAST in vivo, which may contribute to reduced behavioral hypersensitivity after nerve injury. In order to specifically examine the expression of the spinal glutamate transporters, a novel line of double transgenic GLT-1-eGFP/GLAST-DsRed promoter mice was used. Adult mice received propentofylline (10 mg/kg) or saline via intraperitoneal injection starting 1-hour prior to L5-spinal nerve transection and then daily for 12 days. Mice receiving saline exhibited punctate expression of both eGFP (GLT-1 promoter activation) and DsRed (GLAST promoter activation) in the dorsal horn of the spinal cord, which was decreased ipsilateral to nerve injury on day 12. Propentofylline administration reinstated promoter activation on the injured side as evidenced by an equal number of eGFP (GLT-1) and DsRed (GLAST) puncta in both dorsal horns. As demonstrated in previous studies, propentofylline induced a concomitant reversal of L5 spinal nerve transection-induced expression of Glial Fibrillary Acidic Protein (GFAP). The ability of propentofylline to alter glial glutamate transporters highlights the importance of controlling aberrant glial activation in neuropathic pain and suggests one possible mechanism for the anti-allodynic action of this drug.
doi:10.1016/j.neuroscience.2008.01.065
PMCID: PMC2423012  PMID: 18358622
Spinal glia; Neuropathic pain; Neuroimmune; Peripheral nerve injury; Mice
25.  Integration of regulatory signals through involvement of multiple global regulators: control of the Escherichia coli gltBDF operon by Lrp, IHF, Crp, and ArgR 
BMC Microbiology  2007;7:2.
Background
The glutamate synthase operon (gltBDF) contributes to one of the two main pathways of ammonia assimilation in Escherichia coli. Of the seven most-global regulators, together affecting expression of about half of all E. coli genes, two were previously shown to exert direct, positive control on gltBDF transcription: Lrp and IHF. The involvement of Lrp is unusual in two respects: first, it is insensitive to the usual coregulator leucine, and second, Lrp binds more than 150 bp upstream of the transcription starting point. There was indirect evidence for involvement of a third global regulator, Crp. Given the physiological importance of gltBDF, and the potential opportunity to learn about integration of global regulatory signals, a combination of in vivo and in vitro approaches was used to investigate the involvement of additional regulatory proteins, and to determine their relative binding positions and potential interactions with one another and with RNA polymerase (RNAP).
Results
Crp and a more local regulator, ArgR, directly control gltBDF transcription, both acting negatively. Crp-cAMP binds a sequence centered at -65.5 relative to the transcript start. Mutation of conserved nucleotides in the Crp binding site abolishes the Crp-dependent repression. ArgR also binds to the gltBDF promoter region, upstream of the Lrp binding sites, and decreases transcription. RNAP only yields a defined DNAse I footprint under two tested conditions: in the presence of both Lrp and IHF, or in the presence of Crp-cAMP. The DNAse I footprint of RNAP in the presence of Lrp and IHF is altered by ArgR.
Conclusion
The involvement of nearly half of E. coli's most-global regulatory proteins in the control of gltBDF transcription is striking, but seems consistent with the central metabolic role of this operon. Determining the mechanisms of activation and repression for gltBDF was beyond the scope of this study. However the results are consistent with a model in which IHF bends the DNA to allow stabilizing contacts between Lrp and RNAP, ArgR interferes with such contacts, and Crp introduces an interfering bend in the DNA and/or stabilizes RNAP in a poised but inactive state.
doi:10.1186/1471-2180-7-2
PMCID: PMC1784095  PMID: 17233899

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