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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.
PMCID: PMC2794958  PMID: 19672971
epigenetic; astrocyte; GLT1
2.  Nuclear Factor-κB Contributes to Neuron-Dependent Induction of GLT-1 Expression in Astrocytes 
The GLT-1 (EAAT2) subtype of glutamate transporter ensures crisp excitatory signaling and limits excitotoxicity in the CNS. Astrocytic expression of GLT-1 is regulated during development, by neuronal activity, and in neurodegenerative diseases. Although neurons activate astrocytic expression of GLT-1, the mechanisms involved have not been identified. In the present study, astrocytes from transgenic mice that express enhanced green fluorescent protein (eGFP) under the control of a bacterial artificial chromosome (BAC) containing a very large region of DNA surrounding the GLT-1 gene (BAC GLT-1 eGFP mice) were used to assess the role of nuclear factor-κB (NF-κB) in neuron-dependent activation of the GLT-1 promoter. We provide evidence that neurons activate NF-κB signaling in astrocytes. Transduction of astrocytes from the BAC GLT-1 eGFP mice with dominant-negative inhibitors of NF-κB signaling completely blocked neuron-dependent activation of a NF-κB reporter construct and attenuated induction of eGFP. Exogenous expression of p65 and/or p50 NF-κB subunits induced expression of eGFP or GLT-1 and increased GLT-1-mediated transport activity. Using wild type and mutant GLT-1 promoter reporter constructs, we found that NF-κB sites at −583 or −251 relative to the transcription start site eliminated neuron-dependent reporter activation. Electrophoretic mobility shift and supershift assays reveal that p65 and p50 interact with these same sites ex vivo. Finally, chromatin immunoprecipitation (ChIP) showed that p65 and p50 interact with these sites in adult cortex, but not in kidney (a tissue that expresses no detectable GLT-1). Together, these studies strongly suggest that NF-κB contributes to neuron-dependent regulation of astrocytic GLT-1 transcription.
PMCID: PMC3138498  PMID: 21697367
glutamate transport; NF-κB; astrocytes; p65; p50; EAAT2; GLT-1; IκBα
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.
PMCID: PMC3199134  PMID: 21046559
astroglia; BAC; ALDH1L1; GLT1; GFAP; oligodendroglia; ALS
4.  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.
PMCID: PMC3676901  PMID: 16175560
uptake; presynaptic; alternative splicing; excitotoxicity; excitatory; synapse
5.  Plasticity in expression of the glutamate transporters GLT-1 and GLAST in spinal dorsal horn glial cells following partial sciatic nerve ligation 
Molecular Pain  2009;5:15.
Clearance of synaptically released glutamate, and hence termination of glutamatergic neurotransmission, is carried out by glutamate transporters, most especially glutamate transporter-1 (GLT-1) and the glutamate-aspartate transporter (GLAST) that are located in astrocytes. It is becoming increasingly well appreciated that changes in the function and expression of GLT-1 and GLAST occur under different physiological and pathological conditions. Here we investigated the plasticity in expression of GLT-1 and GLAST in the spinal dorsal horn using immunohistochemistry following partial sciatic nerve ligation (PSNL) in rats.
Animals were confirmed to develop hypersensitivity to mechanical stimulation by 7 days following PSNL. Baseline expression of GLT-1 and GLAST in naive animals was only observed in astrocytes and not in either microglia or neurons. Microglia and astrocytes showed evidence of reactivity to the nerve injury when assessed at 7 and 14 days following PSNL evidenced by increased expression of OX-42 and GFAP, respectively. In contrast, the total level of GLT-1 and GLAST protein decreased at both 7 and 14 days after PSNL. Importantly, the cellular location of GLT-1 and GLAST was also altered in response to nerve injury. Whereas activated astrocytes showed a marked decrease in expression of GLT-1 and GLAST, activated microglia showed de novo expression of GLT-1 and GLAST at 7 days after PSNL and this was maintained through day 14. Neurons showed no expression of GLT-1 or GLAST at any time point.
These results indicate that the expression of glutamate transporters in astrocytes and microglia are differentially regulated following nerve injury.
PMCID: PMC2676254  PMID: 19323820
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.
PMCID: PMC2849838  PMID: 14762132
uptake; trafficking; alternative splicing; excitotoxicity; PDZ domain; synapse
7.  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
8.  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.
PMCID: PMC3430367  PMID: 22080156
EAAT2; neuroprotection; citicholine; Parkinson’s Disease; glutamate uptake; glutamate transporters
9.  Characterization of nestin expression in astrocytes in the rat hippocampal CA1 region following transient forebrain ischemia 
Anatomy & Cell Biology  2013;46(2):131-140.
Recent studies have suggested that nestin facilitates cellular structural remodeling in vasculature-associated cells in response to ischemic injury. The current study was designed to investigate the potential role of post-ischemic nestin expression in parenchymal astrocytes. With this aim, we characterized ischemia-induced nestin expression in the CA1 hippocampal region, an area that undergoes a delayed neuronal death, followed by a lack of neuronal generation after transient forebrain ischemia. Virtually all of the nestin-positive cells in the ischemic CA1 hippocampus were reactive astrocytes. However, induction of nestin expression did not correlate simply with astrogliosis, but rather showed characteristic time- and strata-dependent expression patterns. Nestin induction in astrocytes of the pyramidal cell layer was rapid and transient, while a long-lasting induction of nestin was observed in astrocytes located in the CA1 dendritic subfields, such as the stratum oriens and radiatum, until at least day 28 after ischemia. There was no detectable expression in the stratum lacunosum moleculare despite the evident astroglial reaction. Almost all of the nestin-positive cells also expressed a transcription factor for neural/glial progenitors, i.e., Sox-2 or Sox-9, and some cells were also positive for Ki-67. However, all of the nestin-positive astrocytes expressed the calcium-binding protein S100β, which is known to be expressed in a distinct, post-mitotic astrocyte population. Thus, our data indicate that in the ischemic CA1 hippocampus, nestin expression was induced in astroglia that were becoming reactive, but not in a progenitor/stem cell population, suggesting that nestin may allow for the structural remodeling of these cells in response to ischemic injury.
PMCID: PMC3713277  PMID: 23869260
Nestin; Transient forebrain ischemia; Reactive astrocytes; Hippocampus
10.  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.
PMCID: PMC2423012  PMID: 18358622
Spinal glia; Neuropathic pain; Neuroimmune; Peripheral nerve injury; Mice
11.  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.
PMCID: PMC4283053  PMID: 24814819
co-culture; EAAT2; Glial cell; glutamate transporter; rho kinase; TRAK2
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.
PMCID: PMC3232333  PMID: 22052455
astrocyte; GLT-1; EAAT2; biolistics; time-lapse; activity
13.  Selective dysfunction of hippocampal CA1 astrocytes contributes to delayed neuronal damage after transient forebrain ischemia 
Transient global ischemia, as with cardiac arrest, causes loss of CA1 hippocampal neurons 2–4 days later, while nearby dentate gyrus (DG) neurons are relatively resistant. Whether differential astrocyte vulnerability to ischemic injury contributes to CA1 neuronal death is uncertain. Here we find that CA1 astrocytes are more sensitive to ischemia than DG astrocytes. In rats subjected to transient forebrain ischemia, CA1 astrocytes lose glutamate transport activity and immunoreactivity for GFAP, S100β and glutamate transporter GLT-1, within a few hours of reperfusion, but without astrocyte cell death. Oxidative stress may contribute to the observed selective CA1 changes as CA1 astrocytes show early increases in mitochondrial free radicals and reduced mitochondrial membrane potential. Similar changes were not observed in DG astrocytes. Upregulation of GLT-1 expression in astrocytes with ceftriaxone protected CA1 neurons from forebrain ischemia. We suggest that greater oxidative stress and loss of GLT-1 function selectively in CA1 astrocytes is central to the well known delayed death of CA1 neurons.
PMCID: PMC3140959  PMID: 17442809
astrocyte; global ischemia; glutamate transporter; hippocampus; mitochondria; oxidative stress
14.  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.
PMCID: PMC3633372  PMID: 23396537
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.
PMCID: PMC3775433  PMID: 24078881
16.  Riluzole neuroprotection in a parkinson's disease model involves suppression of reactive astrocytosis but not GLT-1 regulation 
BMC Neuroscience  2012;13:38.
Riluzole is a neuroprotective drug used in the treatment of motor neurone disease. Recent evidence suggests that riluzole can up-regulate the expression and activity of the astrocyte glutamate transporter, GLT-1. Given that regulation of glutamate transport is predicted to be neuroprotective in Parkinson's disease, we tested the effect of riluzole in parkinsonian rats which had received a unilateral 6-hydroxydopamine injection into the median forebrain bundle.
Rats were treated with intraperitoneal riluzole (4 mg/kg or 8 mg/kg), 1 hour before the lesion then once daily for seven days. Riluzole produced a modest but significant attenuation of dopamine neurone degeneration, assessed by suppression of amphetamine-induced rotations, preservation of tyrosine hydroxylase positive neuronal cell bodies in the substantia nigra pars compacta and attenuation of striatal tyrosine hydroxylase protein loss. Seven days after 6-hydroxydopamine lesion, reactive astrocytosis was observed in the striatum, as determined by increases in expression of glial fibrillary acidic protein, however the glutamate transporter, GLT-1, which is also expressed in astrocytes was not regulated by the lesion.
The results confirm that riluzole is a neuroprotective agent in a rodent model of parkinson's disease. Riluzole administration did not regulate GLT-1 levels but significantly reduced GFAP levels, in the lesioned striatum. Riluzole suppression of reactive astrocytosis is an intriguing finding which might contribute to the neuroprotective effects of this drug.
PMCID: PMC3349538  PMID: 22480308
EAAT2; GLT-1; Neuroprotection; Parkinson's Disease; GFAP; Glial cell; 6-hydroxydopamine
17.  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.
PMCID: PMC3353324  PMID: 22488924
GLT-1; TGF-α; glutamate uptake; glutamate transporter; manganese; tamoxifen; 17β-estradiol; astrocytes
18.  The chemokine, macrophage inflammatory protein-2γ, reduces the expression of glutamate transporter-1 on astrocytes and increases neuronal sensitivity to glutamate excitotoxicity 
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.
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.
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.
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.
PMCID: PMC3545864  PMID: 23234294
Glutamate transporter; Chemokines; MIP-2γ; Astrocyte; Lipid rafts
19.  Exocytosis of ATP From Astrocytes Modulates Phasic and Tonic Inhibition in the Neocortex 
PLoS Biology  2014;12(1):e1001747.
Astrocytes secrete ATP by exocytosis from synaptic-like vesicles, activating neuronal P2X receptors, which contribute to postsynaptic GABA receptor down-regulation, ultimately mediating the communication between astrocytes and neurons required for brain function.
Communication between neuronal and glial cells is important for many brain functions. Astrocytes can modulate synaptic strength via Ca2+-stimulated release of various gliotransmitters, including glutamate and ATP. A physiological role of ATP release from astrocytes was suggested by its contribution to glial Ca2+-waves and purinergic modulation of neuronal activity and sleep homeostasis. The mechanisms underlying release of gliotransmitters remain uncertain, and exocytosis is the most intriguing and debated pathway. We investigated release of ATP from acutely dissociated cortical astrocytes using “sniff-cell” approach and demonstrated that release is vesicular in nature and can be triggered by elevation of intracellular Ca2+ via metabotropic and ionotropic receptors or direct UV-uncaging. The exocytosis of ATP from neocortical astrocytes occurred in the millisecond time scale contrasting with much slower nonvesicular release of gliotransmitters via Best1 and TREK-1 channels, reported recently in hippocampus. Furthermore, we discovered that elevation of cytosolic Ca2+ in cortical astrocytes triggered the release of ATP that directly activated quantal purinergic currents in the pyramidal neurons. The glia-driven burst of purinergic currents in neurons was followed by significant attenuation of both synaptic and tonic inhibition. The Ca2+-entry through the neuronal P2X purinoreceptors led to phosphorylation-dependent down-regulation of GABAA receptors. The negative purinergic modulation of postsynaptic GABA receptors was accompanied by small presynaptic enhancement of GABA release. Glia-driven purinergic modulation of inhibitory transmission was not observed in neurons when astrocytes expressed dn-SNARE to impair exocytosis. The astrocyte-driven purinergic currents and glia-driven modulation of GABA receptors were significantly reduced in the P2X4 KO mice. Our data provide a key evidence to support the physiological importance of exocytosis of ATP from astrocytes in the neocortex.
Author Summary
Brain function depends on the interaction between two major types of cells: neurons transmitting electrical signals and glial cells, which control cerebral circulation and neuronal homeostasis. There is a growing evidence of the participation of astrocytes in regulating neuronal excitability and synaptic plasticity via the release of “gliotransmitters,” which include glutamate and ATP. The importance of ATP release from astrocytes was suggested by studies that demonstrated its contribution to neuronal activity and sleep homeostasis via modulation of known “purinergic” receptors. But the mechanisms underlying gliotransmitter release and the physiological significance of direct glia-to-neuron communication remain unknown and intensively debated. Here, we investigate the release of ATP from astrocytes of brain neocortex and demonstrate that astrocytes can release ATP by Ca2+-dependent exocytosis, most likely from synaptic-like microvesicles. We also find that vesicular release of ATP from astrocytes can directly activate excitatory signaling in the neighboring neurons, operating through purinergic P2X receptors. We saw that activation of these P2X receptors by astrocyte-driven ATP down-regulated the inhibitory synaptic signaling in the neocortical neurons. Our results imply that exocytosis of gliotransmitters is important for the communication between astrocytes and neurons in the neocortex.
PMCID: PMC3883644  PMID: 24409095
20.  Evidence for change in current flux coupling of GLT1 at high glutamate concentrations in primary forebrain neurons and GLT1a expressing COS7 cells 
Glutamate is the major excitatory neurotransmitter of the central nervous system and is toxic to neurons even at low concentrations. GLT1, the rodent analog of human EAAT2, is primarily responsible for glutamate clearance in the cerebrum. GLT1 was thought to be expressed exclusively in astrocytes in the mature brain. Recently, however, GLT1a was demonstrated in excitatory axon terminals where synaptic glutamate concentration rises above 1 mM during excitatory transmission. However, GLT1 function in neurons with accurate control of both intracellular and extracellular solutions mimicking synaptic concentration gradients has never been studied. Here we characterized the kinetics of coupled glutamate transporter current in whole-cell configuration and [3H]-L-glutamate uptake in cultured rat cerebral neurons across the entire range of synaptic glutamate concentrations. In both neurons and GLT1a transfected COS-7 cells, the kinetics were similar and revealed two specific components: a high affinity component with glutamate kD value around 15 μM and low affinity component with kD value around 0.2 mM. The specific low affinity component was discovered due to significant deviation of the transporter current from Michaelis-Menten kinetics in the 100 – 300 μM concentration range. Activation of the specific low affinity component led to a twofold decrease in the current/flux ratio implying a change in the transport coupling. Our data indicate that GLT1 endogenously expressed in cultured rat forebrain neurons displays high and low glutamate affinity uptake components that are different in current/flux coupling ratios. This property is intrinsic to the protein because it was also observed in GLT1a transfected COS-7 cells.
PMCID: PMC3690583  PMID: 19614985
GLT1; patch-clamp; whole cell; current/flux coupling; excitotoxicity; presynaptic; synapse
21.  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.
PMCID: PMC4035523  PMID: 24872566
adeno-associated virus; gene therapy; glutamate transporter; phrenic motor neuron; respiratory; spinal cord injury
22.  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.
PMCID: PMC2579753  PMID: 18194440
glutamate transporters; interleukin-1β; viral encephalomyelitis; motor neuron; excitotoxicity
23.  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.
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.
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.
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.
PMCID: PMC2628654  PMID: 19108711
24.  Physiological properties of enkephalin-containing neurons in the spinal dorsal horn visualized by expression of green fluorescent protein in BAC transgenic mice 
BMC Neuroscience  2011;12:36.
Enkephalins are endogenous opiates that are assumed to modulate nociceptive information by mediating synaptic transmission in the central nervous system, including the spinal dorsal horn.
To develop a new tool for the identification of in vitro enkephalinergic neurons and to analyze enkephalin promoter activity, we generated transgenic mice for a bacterial artificial chromosome (BAC). Enkephalinergic neurons from these mice expressed enhanced green fluorescent protein (eGFP) under the control of the preproenkephalin (PPE) gene (penk1) promoter. eGFP-positive neurons were distributed throughout the gray matter of the spinal cord, and were primarily observed in laminae I-II and V-VII, in a pattern similar to the distribution pattern of enkephalin-containing neurons. Double immunostaining analysis using anti-enkephalin and anti-eGFP antibodies showed that all eGFP-expressing neurons contained enkephalin. Incubation in the presence of forskolin, an activator of adenylate cyclase, increased the number of eGFP-positive neurons. These results indicate that eGFP expression is controlled by the penk1 promoter, which contains cyclic AMP-responsive elements. Sections obtained from sciatic nerve-ligated mice exhibited increased eGFP-positive neurons on the ipsilateral (nerve-ligated side) compared with the contralateral (non-ligated side). These data indicate that PPE expression is affected by peripheral nerve injury. Additionally, single-neuron RT-PCR analysis showed that several eGFP positive-neurons in laminae I-II expressed glutamate decarboxylase 67 mRNA and that some expressed serotonin type 3 receptors.
These results suggest that eGFP-positive neurons in laminae I-II coexpress enkephalin and γ-aminobutyric acid (GABA), and are activated by forskolin and in conditions of nerve injury. The penk1-eGFP BAC transgenic mouse contributes to the further characterization of enkephalinergic neurons in the transmission and modulation of nociceptive information.
PMCID: PMC3115906  PMID: 21548966
25.  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.
PMCID: PMC4023965  PMID: 24836816

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