Glutamate is the predominant excitatory amino acid neurotransmitter in the mammalian central nervous system (CNS). Glutamate transporter EAAT2 /GLT-1 is the physiologically dominant astroglial protein that inactivates synaptic glutamate. Previous studies have shown that EAAT2 dysfunction leads to excessive extracellular glutamate and may contribute to various neurological disorders including amyotrophic lateral sclerosis (ALS). The recent discovery of the neuroprotective properties of ceftriaxone, a beta lactam antibiotic, suggested that increasing EAAT2 /GLT-1 gene expression might be beneficial in ALS and other neurological/psychiatric disorders by augmenting astrocytic glutamate uptake. Here we report our efforts to develop a new screening assay for identifying compounds that activate EAAT2 gene expression. We generated fetal derived-human immortalized astroglial cells that are stably expressing a firefly luciferase reporter under the control of the human EAAT2 promoter. When screening a library of 1040 FDA approved compounds and natural products, we identified harmine, a naturally occurring beta-carboline alkaloid, as one of the top hits for activating the EAAT2 promoter. We further tested harmine in our in vitro cell culture systems and confirmed its ability to increase EAAT2/GLT1 gene expression and functional glutamate uptake activity. We next tested its efficacy in both wild type animals and in an ALS animal model of disease and demonstrated that harmine effectively increased GLT-1 protein and glutamate transporter activity in vivo. Our studies provide potential novel neurotherapeutics by modulating the activity of glutamate transporters via gene activation.
harmine; GLT-1; EAAT2; glutamate transporter; astroglia; ALS
Dysregulation of the glutamate transporters EAAT1 and EAAT2 and their isoforms have been implicated in schizophrenia. EAAT1 and EAAT2 expression has been studied in different brain regions but the prevalence of astrocytic glutamate transporter expression masks the more subtle changes in excitatory amino acid transporters (EAATs) isoforms in neurons in the cortex. Using laser capture microdissection, pyramidal neurons were cut from the anterior cingulate cortex of postmortem schizophrenia (n=20) and control (n=20) subjects. The messenger RNA (mRNA) levels of EAAT1, EAAT2 and the splice variants EAAT1 exon9skipping, EAAT2 exon9skipping and EAAT2b were analyzed by real time PCR (RT-PCR) in an enriched population of neurons. Region-level expression of these transcripts was measured in postmortem schizophrenia (n=25) and controls (n=25). The relationship between selected EAAT polymorphisms and EAAT splice variant expression was also explored. Anterior cingulate cortex pyramidal cell expression of EAAT2b mRNA was increased (P<0.001; 67%) in schizophrenia subjects compared with controls. There was no significant change in other EAAT variants. EAAT2 exon9skipping mRNA was increased (P<0.05; 38%) at region level in the anterior cingulate cortex with no significant change in other EAAT variants at region level. EAAT2 single-nucleotide polymorphisms were significantly associated with changes in EAAT2 isoform expression. Haloperidol decanoate-treated animals, acting as controls for possible antipsychotic effects, did not have significantly altered neuronal EAAT2b mRNA levels. The novel finding that EAAT2b levels are increased in populations of anterior cingulate cortex pyramidal cells further demonstrates a role for neuronal glutamate transporter splice variant expression in schizophrenia.
EAAT2 is a predominantly astroglial glutamate transporter responsible for the majority of synaptic glutamate clearance in the mammalian CNS. Its dysfunction has been linked to many neurological disorders, including amyotrophic lateral sclerosis (ALS). Decreases in EAAT2 expression and function have been implicated in causing motor neuron excitotoxic death in ALS. Nevertheless, increasing EAAT2 expression does not significantly improve ALS phenotype in mouse models or in clinical trials. In the SOD1-G93A mouse model of inherited ALS, the cytosolic carboxy-terminal domain is cleaved from EAAT2, conjugated to SUMO1, and accumulated in astrocytes where it triggers astrocyte-mediated neurotoxic effects as disease progresses. However, it is not known whether this fragment is sumoylated after cleavage or if full-length EAAT2 is already sumoylated prior to cleavage as part of physiological regulation. In this study, we show that a fraction of full-length EAAT2 is constitutively sumoylated in primary cultures of astrocytes in vitro and in the CNS in vivo. Furthermore, the extent of sumoylation of EAAT2 does not change during the course of ALS in the SOD1-G93A mouse and is not affected by the expression of ALS-causative mutant SOD1 proteins in astrocytes in vitro, indicating that EAAT2 sumoylation is not driven by pathogenic mechanisms. Most interestingly, sumoylated EAAT2 localizes to intracellular compartments, while non-sumoylated EAAT2 resides on the plasma membrane. In agreement, promoting desumoylation in primary astrocytes causes increased EAAT2–mediated glutamate uptake. These findings could have implications for optimizing therapeutic approaches aimed at increasing EAAT2 activity in the dysfunctional or diseased CNS.
Amyotrophic lateral sclerosis; excitotoxicity; protein trafficking; GLT-1
Excitotoxicity has been implicated as the mechanism of neuronal damage resulting from acute insults such as stroke, epilepsy, and trauma, as well as during the progression of adult-onset neurodegenerative disorders such as Alzheimer’s disease and amyotrophic lateral sclerosis (ALS). Excitotoxicity is defined as excessive exposure to the neurotransmitter glutamate or overstimulation of its membrane receptors, leading to neuronal injury or death. One potential approach to protect against excitotoxic neuronal damage is enhanced glutamate reuptake. The glial glutamate transporter EAAT2 is the quantitatively dominant glutamate transporter and plays a major role in clearance of glutamate. Expression of EAAT2 protein is highly regulated at the translational level. In an effort to identify compounds that can induce translation of EAAT2 transcripts, a cell-based enzyme-linked immunosorbent assay was developed using a primary astrocyte line stably transfected with a vector designed to identify modulators of EAAT2 translation. This assay was optimized for high-throughput screening, and a library of approximately 140,000 compounds was tested. In the initial screen, 293 compounds were identified as hits. These 293 hits were retested at 3 concentrations, and a total of 61 compounds showed a dose-dependent increase in EAAT2 protein levels. Selected compounds were tested in full 12-point dose-response experiments in the screening assay to assess potency as well as confirmed by Western blot, immunohistochemistry, and glutamate uptake assays to evaluate the localization and function of the elevated EAAT2 protein. These hits provide excellent starting points for developing therapeutic agents to prevent excitotoxicity.
excitotoxicity; glutamate transporter; EAAT2; high-throughput screen; neurodegeneration
In multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE), impairment of glial “Excitatory Amino Acid Transporters” (EAATs) together with an excess glutamate-release by invading immune cells causes excitotoxic damage of the central nervous system (CNS). In order to identify pathways to dampen excitotoxic inflammatory CNS damage, we assessed the effects of a β-lactam antibiotic, ceftriaxone, reported to enhance expression of glial EAAT2, in “Myelin Oligodendrocyte Glycoprotein” (MOG)-induced EAE. Ceftriaxone profoundly ameliorated the clinical course of murine MOG-induced EAE both under preventive and therapeutic regimens. However, ceftriaxone had impact neither on EAAT2 protein expression levels in several brain areas, nor on the radioactive glutamate uptake capacity in a mixed primary glial cell-culture and the glutamate-induced uptake currents in a mammalian cell line mediated by EAAT2. Moreover, the clinical effect of ceftriaxone was preserved in the presence of the EAAT2-specific transport inhibitor, dihydrokainate, while dihydrokainate alone caused an aggravated EAE course. This demonstrates the need for sufficient glial glutamate uptake upon an excitotoxic autoimmune inflammatory challenge of the CNS and a molecular target of ceftriaxone other than the glutamate transporter. Ceftriaxone treatment indirectly hampered T cell proliferation and proinflammatory INFγ and IL17 secretion through modulation of myelin-antigen presentation by antigen-presenting cells (APCs) e.g. dendritic cells (DCs) and reduced T cell migration into the CNS in vivo. Taken together, we demonstrate, that a β-lactam antibiotic attenuates disease course and severity in a model of autoimmune CNS inflammation. The mechanisms are reduction of T cell activation by modulation of cellular antigen-presentation and impairment of antigen-specific T cell migration into the CNS rather than or modulation of central glutamate homeostasis.
Background and Purpose
Excitatory amino acid transporters (EAATs) in the CNS contribute to the clearance of glutamate released during neurotransmission. The aim of this study was to explore the role of EAATs in the regulation of locus coeruleus (LC) neurons by glutamate.
We measured the effect of different EAAT subtype inhibitors/enhancers on glutamate- and KCl-induced activation of LC neurons in rat slices. EAAT2–3 expression in the LC was also characterized by immunohistochemistry.
The EAAT2–5 inhibitor DL-threo-β-benzyloxaspartic acid (100 μM), but not the EAAT2, 4, 5 inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (100 μM) or the EAAT2 inhibitor dihydrokainic acid (DHK; 100 μM), enhanced the glutamate- and KCl-induced activation of the firing rate of LC neurons. These effects were blocked by ionotropic, but not metabotrobic, glutamate receptor antagonists. DHK (100 μM) was the only EAAT inhibitor that increased the spontaneous firing rate of LC cells, an effect that was due to inhibition of EAAT2 and subsequent AMPA receptor activation. Chronic treatment with ceftriaxone (200 mg·kg−1 i.p., once daily, 7 days), an EAAT2 expression enhancer, increased the actions of glutamate and DHK, suggesting a functional impact of EAAT2 up-regulation on the glutamatergic system. Immuhistochemical data revealed the presence of EAAT2 and EAAT3 surrounding noradrenergic neurons and EAAT2 on glial cells in the LC.
Conclusions and Implications
These results remark the importance of EAAT2 and EAAT3 in the regulation of rat LC by glutamate. Neuronal EAAT3 would be responsible for terminating the action of synaptically released glutamate, whereas glial EAAT2 would regulate tonic glutamate concentrations in this nucleus.
locus coeruleus; EAAT2; EAAT3; KCl; glutamate; firing
Klotho, a transmembrane protein, which can be cleaved off as β-glucuronidase and hormone, is released in both, kidney and choroid plexus and encountered in blood and cerebrospinal fluid. Klotho deficiency leads to early appearance of age-related disorders and premature death. Klotho may modify transport by inhibiting 1,25(OH)2D3 formation or by directly affecting channel and carrier proteins. The present study explored whether Klotho influences the activity of the Na+-coupled excitatory amino acid transporters EAAT3 and EAAT4, which are expressed in kidney (EAAT3), intestine (EAAT3) and brain (EAAT3 and EAAT4). To this end, cRNA encoding EAAT3 or EAAT4 was injected into Xenopus oocytes with and without additional injection of cRNA encoding Klotho. EAAT expressing Xenopus oocytes were further treated with recombinant human β-Klotho protein with or without β-glucuronidase inhibitor D-saccharic acid 1,4-lactone monohydrate (DSAL). Electrogenic excitatory amino acid transport was determined as L-glutamate-induced current (Iglu) in two electrode voltage clamp experiments. EAAT3 and EAAT4 protein abundance in the Xenopus oocyte cell membrane was visualized by confocal microscopy and quantified utilizing chemiluminescence. As a result, coexpression of Klotho cRNA significantly increased Iglu in both, EAAT3 or EAAT4-expressing Xenopus oocytes. Klotho cRNA coexpression significantly increased the maximal current and cell membrane protein abundance of both EAAT3 and EAAT4. The effect of Klotho coexpression on EAAT3 and EAAT4 activity was mimicked by treating EAAT3 or EAAT4-expressing Xenopus oocytes with recombinant human β-Klotho protein. The effects of Klotho coexpression and of treatment with recombinant human β-Klotho protein were both abrogated in the presence of DSAL (10 µM). In conclusion, Klotho is a novel, powerful regulator of the excitatory amino acid transporters EAAT3 and EAAT4.
Excitatory amino acid transporters (EAAT) uptake extracellular glutamate, the major excitatory neurotransmitter in the brain. EAAT type 3 (EAAT3), the main neuronal EAAT, is expressed widely in the central nervous system. We have shown that the volatile anesthetic isoflurane increases EAAT3 activity and trafficking to the plasma membrane. Thus, we hypothesize that EAAT3 mediates isoflurane-induced anesthesia. To test this hypothesis, the potency of isoflurane to induce immobility and hypnosis, two major components of general anesthesia, was compared in the CD-1 wild-type mice and EAAT knockout mice that had a CD-1 strain gene background. Hypnosis was assessed by loss of righting reflex in this study. The expression of EAAT1 and EAAT2, two widely expressed EAATs in the central nervous system, in the cerebral cortex and spinal cord was not different between the EAAT3 knockout mice and wild-type mice. The concentration required for isoflurane to cause immobility to painful stimuli, a response involving primarily reflex loops in the spinal cord, was not changed by EAAT3 knockout. However, the EAAT3 knockout mice were more sensitive to isoflurane-induced hypnotic effects, which may be mediated by hypothalamic sleep neural circuits. Interestingly, the EAAT3 knockout mice did not have an altered sensitivity to the hypnotic effects caused by ketamine, an intravenous anesthetic that is a glutamate receptor antagonist and does not affect EAAT3 activity. These results suggest that EAAT3 modulates the sensitivity of neural circuits to isoflurane. These results, along with our previous findings that isoflurane increases EAAT3 activity, indicate that EAAT3 may regulate isoflurane-induced behavioral changes, including anesthesia.
anesthesia; glutamate transporter; gene expression; hypnosis; isoflurane
Adenosine signaling is implicated in several neuropsychiatric disorders, including alcoholism. Among its diverse functions in the brain, adenosine regulates glutamate release and has an essential role in ethanol sensitivity and preference. However, the molecular mechanisms underlying adenosine-mediated glutamate signaling in neuroglial interaction remain elusive. We have previously shown that mice lacking the ethanol-sensitive adenosine transporter, type 1 equilibrative nucleoside transporter (ENT1), drink more ethanol compared with wild-type mice and have elevated striatal glutamate levels. In addition, ENT1 inhibition or knockdown reduces glutamate transporter expression in cultured astrocytes. Here, we examined how adenosine signaling in astrocytes contributes to ethanol drinking. Inhibition or deletion of ENT1 reduced the expression of type 2 excitatory amino-acid transporter (EAAT2) and the astrocyte-specific water channel, aquaporin 4 (AQP4). EAAT2 and AQP4 colocalization was also reduced in the striatum of ENT1 null mice. Ceftriaxone, an antibiotic compound known to increase EAAT2 expression and function, elevated not only EAAT2 but also AQP4 expression in the striatum. Furthermore, ceftriaxone reduced ethanol drinking, suggesting that ENT1-mediated downregulation of EAAT2 and AQP4 expression contributes to excessive ethanol consumption in our mouse model. Overall, our findings indicate that adenosine signaling regulates EAAT2 and astrocytic AQP4 expressions, which control ethanol drinking in mice.
ENT1; astrocytes; aquaporin 4; EAAT2; Alcoholism; ceftriaxone; ENT1; Astrocytes; Aquaporin 4; EAAT2; Alcoholism; Ceftriaxone
Impairment of astrocytic glutamate transporter (GLT-1; EAAT2) function is associated with multiple neurodegenerative diseases, including Parkinson's disease (PD) and manganism, the latter being induced by chronic exposure to high levels of manganese (Mn). Mn decreases EAAT2 promoter activity and mRNA and protein levels, but the molecular mechanism of Mn-induced EAAT2 repression at the transcriptional level has yet to be elucidated. We reveal that transcription factor Yin Yang 1 (YY1) is critical in repressing EAAT2 and mediates the effects of negative regulators, such as Mn and tumor necrosis factor alpha (TNF-α), on EAAT2. YY1 overexpression in astrocytes reduced EAAT2 promoter activity, while YY1 knockdown or mutation of the YY1 consensus site of the EAAT2 promoter increased its promoter activity and attenuated the Mn-induced repression of EAAT2. Mn increased YY1 promoter activity and mRNA and protein levels via NF-κB activation. This led to increased YY1 binding to the EAAT2 promoter region. Epigenetically, histone deacetylase (HDAC) classes I and II served as corepressors of YY1, and, accordingly, HDAC inhibitors increased EAAT2 promoter activity and reversed the Mn-induced repression of EAAT2 promoter activity. Taken together, our findings suggest that YY1, with HDACs as corepressors, is a critical negative transcriptional regulator of EAAT2 and mediates Mn-induced EAAT2 repression.
Glutamate transport through astrocytic excitatory amino-acid transporters (EAAT)-1 and EAAT-2 is paramount for neural homeostasis. EAAT-1 has been reported in secreted extracellular microvesicles (eMV, such as exosomes) and because the protein kinase C (PKC) family controls the sub-cellular distribution of EAATs, we have explored whether PKCs drive EAATs into eMV. Using rat primary astrocytes, confocal immunofluorescence and ultracentrifugation on sucrose gradient we here report that PKC activation by phorbol myristate acetate (PMA) reorganizes EAAT-1 distribution and reduces functional [3H]-aspartate reuptake. Western-blots show that EAAT-1 is present in eMV from astrocyte conditioned medium, together with NaK ATPase and glutamine synthetase all being further increased after PMA treatment. However, nanoparticle tracking analysis reveals that PKC activation did not change particle concentration. Functional analysis indicates that eMV have the capacity to reuptake [3H]-aspartate. In vivo, we demonstrate that spinal astrocytic reaction induced by peripheral nerve lesion (spared nerve injury, SNI) is associated with a phosphorylation of PKC δ together with a shift of EAAT distribution ipsilaterally. Ex vivo, spinal explants from SNI rats release eMV with an increased content of NaK ATPase, EAAT-1 and EAAT-2. These data indicate PKC and cell activation as important regulators of EAAT-1 incorporation in eMV, and raise the possibility that microvesicular EAAT-1 may exert extracellular functions. Beyond a putative role in neuropathic pain, this phenomenon may be important for understanding neural homeostasis and a wide range of neurological diseases associated with astrocytic reaction as well as non-neurological diseases linked to eMV release.
glutamate transporters; extracellular microvesicles; astrocyte; glia; protein kinase C; nerve injury; spinal cord
The major regulators of synaptic glutamate in the cerebral cortex are the excitatory amino acid transporters 1–3 (EAAT1–3). In this study, we determined the cellular and temporal expression of EAAT1–3 in the developing human cerebral cortex. We applied single- and double-label immunocytochemistry to normative frontal or parietal (associative) cortex samples from 14 cases ranging in age from 23 gestational weeks to 2.5 postnatal years. The most striking finding was the transient expression of EAAT2 in layer V pyramidal neuronal cell bodies up until 8 postnatal months prior to its expression in protoplasmic astrocytes at 41 postconceptional weeks onward. EAAT2 was also expressed in neurons in layer I (presumed Cajal-Retzius cells), and white matter (interstitial) neurons. This expression in neurons in the developing human cortex contrasts with findings by others of transient expression exclusively in axon tracts in the developing sheep and rodent brain. With western blotting, we found that EAAT2 was expressed as a single band until two postnatal months after which it was expressed as two bands. The expression of EAAT2 in pyramidal neurons during human brain development may contribute to cortical vulnerability to excitotoxicity during the critical period for perinatal hypoxic-ischemic encephalopathy. In addition, by studying the expression of EAAT1 and EAAT2 glutamate transporters it was possible to document the development of protoplasmic astrocytes.
Cajal-Retzius cell; ischemia; periventricular leukomalacia; prematurity; pyramidal; subplate; cerebral palsy
L-Glutamate (Glu) is the major excitatory neurotransmitter in the mammalian CNS and five types of high-affinity Glu transporters (EAAT1–5) have been identified. The transporters EAAT1 and EAAT2 in glial cells are responsible for the majority of Glu uptake while neuronal EAATs appear to have specialized roles at particular types of synapses. Dysfunction of EAATs is specifically implicated in the pathology of neurodegenerative conditions such as amyotrophic lateral sclerosis, epilepsy, Huntington's disease, Alzheimer's disease and ischemic stroke injury, and thus treatments that can modulate EAAT function may prove beneficial in these conditions. Recent advances have been made in our understanding of the regulation of EAATs, including their trafficking, splicing and post-translational modification. This article summarises some recent developments that improve our understanding of the roles and regulation of EAATs.
L-Glutamate; transport; EAAT; trafficking; neurodegeneration; molecular pharmacology; splicing; glia; neurons
Glutamate cycling is critically important for neurotransmission, and may be altered in schizophrenia. The excitatory amino acid transporters (EAATs) facilitate the reuptake of glutamate from the synaptic cleft and have a key role in glutamate cycling. We hypothesized that expression of the EAATs and the EAAT regulating proteins ARHGEF11, JWA, G protein suppressor pathway 1 (GPS1), and KIAA0302 are altered in the brain in schizophrenia. To test this, we measured expression of EAAT1, EAAT2, EAAT3, and EAAT interacting proteins in postmortem tissue from the dorsolateral prefrontal and anterior cingulate cortex of patients with schizophrenia and a comparison group using in situ hybridization and Western blot analysis. We found increased EAAT1 transcripts and decreased protein expression, increased EAAT3 transcripts and protein, and elevated protein expression of both GPS1 and KIAA0302 protein. We did not find any changes in expression of EAAT2. These data indicate that proteins involved in glutamate reuptake and cycling are altered in the cortex in schizophrenia, and may provide potential targets for future treatment strategies.
GPS1; anterior cingulate cortex; dorsolateral prefrontal cortex; postmortem; Western blot; in situ hybridization
The etiology of multiple sclerosis (MS) is currently unknown. However, one potential mechanism involved in the disease may be excitotoxicity. The elevation of glutamate in cerebrospinal fluid, as well as changes in the expression of glutamate receptors (iGluRs and mGluRs) and excitatory amino acid transporters (EAATs), have been observed in the brains of MS patients and animals subjected to experimental autoimmune encephalomyelitis (EAE), which is the predominant animal model used to investigate the pathophysiology of MS. In the present paper, the effects of glutamatergic receptor antagonists, including amantadine, memantine, LY 367583, and MPEP, on glutamate transport, the expression of mRNA of glutamate transporters (EAATs), the kinetic parameters of ligand binding to N-methyl-D-aspartate (NMDA) receptors, and the morphology of nerve endings in EAE rat brains were investigated. The extracellular level of glutamate in the brain is primarily regulated by astrocytic glutamate transporter 1 (GLT-1) and glutamate-aspartate transporter (GLAST). Excess glutamate is taken up from the synaptic space and metabolized by astrocytes. Thus, the extracellular level of glutamate decreases, which protects neurons from excitotoxicity. Our investigations showed changes in the expression of EAAT mRNA, glutamate transport (uptake and release) by synaptosomal and glial plasmalemmal vesicle fractions, and ligand binding to NMDA receptors; these effects were partially reversed after the treatment of EAE rats with the NMDA antagonists amantadine and memantine. The antagonists of group I metabotropic glutamate receptors (mGluRs), including LY 367385 and MPEP, did not exert any effect on the examined parameters. These results suggest that disturbances in these mechanisms may play a role in the processes associated with glutamate excitotoxicity and the progressive brain damage in EAE.
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.
epigenetic; astrocyte; GLT1
Equilibrative nucleoside transporter 1 (ENT1) and excitatory amino acid transporter 2 (EAAT2) are predominantly expressed in astrocytes where they are thought to regulate synaptic adenosine and glutamate levels. Because mice lacking ENT1 display increased glutamate levels in the ventral striatum, we investigated whether ENT1 regulates the expression and function of EAAT2 in astrocytes, which could contribute to altered glutamate levels in the striatum.
We examined the effect of ENT1 inhibition and overexpression on the expression of EAAT2 using quantitative real-time PCR and measured glutamate uptake activity in cultured astrocytes. We also examined the effect of 0 to 200 mM ethanol doses for 0 to 24 hours of ethanol exposure on EAAT2 expression and glutamate uptake activity. We further examined the effect of ENT1 knockdown by a specific siRNA on ethanol-induced EAAT2 expression.
An ENT1-specific antagonist and siRNA treatments significantly reduced both EAAT2 expression and glutamate uptake activity while ENT1 overexpression up-regulated EAAT2 mRNA expression. Interestingly, 100 or 200 mM ethanol exposure increased EAAT2 mRNA expression as well as glutamate uptake activity. Moreover, we found that ENT1 knockdown inhibited the ethanol-induced EAAT2 up-regulation.
Our results suggest that ENT1 regulates glutamate uptake activity by altering EAAT2 expression and function, which might be implicated in ethanol intoxication and preference.
Excitatory Amino Acid Transporter 2 (EAAT2); Equilibrative Nucleoside Transporter 1 (ENT1); Glutamate Uptake; Adenosine Uptake
BACKGROUND AND PURPOSE
Glutamate transporters play a major role in maintaining brain homeostasis and the astrocytic transporters, EAAT1 and EAAT2, are functionally dominant. Astrocytic excitatory amino acid transporters (EAATs) play important roles in various neuropathologies wherein astrocytes undergo cytoskeletal changes. Astrocytic plasticity is well documented, but the interface between EAAT function, actin and the astrocytic cytoskeleton is poorly understood. Because Rho kinase (ROCK) is a key determinant of actin polymerization, we investigated the effects of ROCK inhibitors on EAAT activity and astrocytic morphology.
The functional activity of glutamate transport was determined in murine cultured astrocytes after exposure to the ROCK inhibitors Fasudil (HA-1077) and Y27632 using biochemical, molecular and morphological approaches. Cytochemical analyses assessed changes in astrocytic morphology, F-/G-actin, and localizations of EAAT1/2.
Fasudil and Y27632 increased [3H]-d-aspartate (d-Asp) uptake into astrocytes, and the action of Fasudil was time-dependent and concentration-related. The rapid stellation of astrocytes (glial fibrillary acidic protein immunocytochemistry) induced by Fasudil was accompanied by reduced phalloidin staining of F-actin and increased Vmax for [3H]-d-Asp uptake. Immunoblotting after biotinylation demonstrated that Fasudil increased the expression of EAAT1 and EAAT2 on the cell surface. Immunocytochemistry indicated that Fasudil induced prominent labelling of astrocytic processes by EAAT1/2.
CONCLUSION AND IMPLICATIONS
These data show for the first time that ROCK plays a major role in determining the cell surface expression of EAAT1/2, providing new evidence for an association between transporter function and astrocytic phenotype. ROCK inhibitors, via the actin cytoskeleton, effect a consequent elevation of glutamate transporter function – this activity profile may contribute to their beneficial actions in neuropathologies.
glutamate transporters; astrocytic phenotype; Rho kinase; Fasudil; glial scarring; actin; cell surface expression
Genetic polymorphisms in Solute carrier family 1 (glial high affinity glutamate transporter), member 2 (SLC1A2) have been linked with essential tremor. SLC1A2 encodes excitatory amino acid transporter type 2 (EAAT2), which clears glutamate from the synaptic cleft. One postulated mechanism for essential tremor is the over-excitation of glutamatergic olivo-cerebellar climbing fibers, leading to excitotoxic death of Purkinje cells. Other glutamatergic excitatory signals are transmitted to Purkinje cells via parallel fibers of cerebellar granule neurons. Therefore, the expression level of glutamate transporters could be important in essential tremor pathogenesis. Using Western blotting, we compared the expression levels of the two main glutamate transporters in the cerebellar cortex, EAAT1 and EAAT2, in postmortem tissue from 16 essential tremor cases and 13 age-matched controls. We also studied the localization of EAAT1 and EAAT2 using immunohistochemistry in 10 essential tremor cases and 12 controls. EAAT1 protein levels were similar in cases and controls (1.12 ± 0.83 vs. 1.01 ± 0.69, p =0.71) whereas EAAT2 protein levels in essential tremor cases were only 1/3 of that in controls (0.35 ± 0.23 vs. 1.00 ± 0.62, p < 0.01). Interestingly, EAAT2, but not EAAT1, was expressed in astrocytic processes surrounding the Purkinje cell axon initial segment, a region of previously observed pathological changes in essential tremor. Our main finding, a significant reduction in cerebellar cortical EAAT2 protein levels in essential tremor, suggests that Purkinje cells in essential tremor might be more vulnerable to excitotoxic damage than those of controls.
Electronic supplementary material
The online version of this article (doi:10.1186/s40478-014-0157-z) contains supplementary material, which is available to authorized users.
Essential tremor; EAAT1; EAAT2; Excitotoxicitiy; Purkinje cell; Neurodegenerative
A loss of the glutamate transporter EAAT2 has been reported in
the neoplastic transformation of astrocytic cells and astrocytoma. The
RNA expression of EAAT2 and five 5'-regulatory splice variants was
investigated to identify alterations of the post-transcriptional EAAT2
gene regulation in human astrocytic tumours.
Three known (EAAT2, HBGTII, and HBGTIIC) and two novel (EAAT2/3
and EAAT2/31) EAAT2 transcripts originating from alternative splicing
of 5'-regulatory sequences were subject to an RNA expression analysis
using reverse transcription and competitive PCR. Specimens of
astrocytoma World Health Organisation (WHO) grade I-IV in 14patients
and control brain tissue obtained from three normal persons were studied.
The main EAAT2 RNA was found to be equally expressed in normal
human brain and astrocytic tumour samples. By contrast, the expression
pattern of four 5'-variants of the transporter transcript was altered
in the investigated series of astrocytoma compared with normal brain.
HBGTII, HBGTIIC, and EAAT2/3 were amplified from seven and four tumours
and one sample, respectively. EAAT2/31 was expressed in none of the
tumour specimens studied.
in astrocytic tumours of different
histopathological grades there was a substantial reduction of RNA
splicing events in EAAT2. The impairment of EAAT2 splicing indicates an altered expression which is not primarily involved in the tumorigenesis but may contribute to some biological properties of astrocytoma such as
oedema, necrosis, and tumour related seizures.
The mammalian genome contains four genes encoding GABA transporters (GAT1, slc6a1; GAT2, slc6a13; GAT3, slc6a11; BGT1, slc6a12) and five glutamate transporter genes (EAAT1, slc1a3; EAAT2, slc1a2; EAAT3, slc1a1; EAAT4, slc1a6; EAAT5, slc1a7). These transporters keep the extracellular levels of GABA and excitatory amino acids low and provide amino acids for metabolic purposes. The various transporters have different properties both with respect to their transport functions and with respect to their ability to act as ion channels. Further, they are differentially regulated. To understand the physiological roles of the individual transporter subtypes, it is necessary to obtain information on their distributions and expression levels. Quantitative data are important as the functional capacity is limited by the number of transporter molecules. The most important and most abundant transporters for removal of transmitter glutamate in the brain are EAAT2 (GLT-1) and EAAT1 (GLAST), while GAT1 and GAT3 are the major GABA transporters in the brain. EAAT3 (EAAC1) does not appear to play a role in signal transduction, but plays other roles. Due to their high uncoupled anion conductance, EAAT4 and EAAT5 seem to be acting more like inhibitory glutamate receptors than as glutamate transporters. GAT2 and BGT1 are primarily expressed in the liver and kidney, but are also found in the leptomeninges, while the levels in brain tissue proper are too low to have any impact on GABA removal, at least in normal young adult mice. The present review will provide summary of what is currently known and will also discuss some methodological pitfalls.
GABA uptake; glutamate uptake; BGT1; GAT1; GAT3; GAT2; EAAT2; EAAT1
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.
Glutamate is the primary excitatory amino acid neurotransmitter in the CNS. The concentration of glutamate in the synaptic cleft is tightly controlled by interplay between glutamate release and glutamate clearance. Abnormal glutamate release and/or dysfunction of glutamate clearance can cause overstimulation of glutamate receptors and result in neuronal injury known as excitotoxicity. The glial glutamate transporter EAAT2 plays a major role in glutamate clearance. Dysfunction or reduced expression of EAAT2 has been documented in many neurodegenerative diseases. In addition, many studies in animal models of disease indicate that increased EAAT2 expression provides neuronal protection. Here, we summarize these studies and suggest that EAAT2 is a potential target for the prevention of excitotoxicity. EAAT2 can be upregulated by transcriptional or translational activation. We discuss current progress in the search for EAAT2 activators, which is a promising direction for the treatment of neurodegenerative diseases.
Aggressive tumor growth, diffuse tissue invasion and neurodegeneration are hallmarks of malignant glioma. Although glutamate excitotoxicity is considered to play a key role in glioma-induced neurodegeneration, the mechanism(s) controlling this process is poorly understood. AEG-1 is an oncogene overexpressed in multiple types of human cancers including >90% of brain tumors. AEG-1 also promotes gliomagenesis particularly in the context of tumor growth and invasion, two primary characteristics of glioma. In the present study, we investigated the contribution of AEG-1 to glioma-induced neurodegeneration. Pearson correlation coefficient analysis in normal brain tissues and glioma patient samples indicated a strong negative correlation between expression of AEG-1 and a primary glutamate transporter of astrocytes EAAT2. Gain and loss of function studies in normal primary human fetal astrocytes and T98G glioblastoma multiforme cells revealed that AEG-1 repressed EAAT2 expression at a transcriptional level by inducing YY1 activity to inhibit CBP function as a coactivator on the EAAT2 promoter. In addition, AEG-1-mediated EAAT2 repression caused a reduction of glutamate uptake by glial cells, resulting in induction of neuronal cell death. These findings were also confirmed in glioma patient samples demonstrating that AEG-1 expression negatively correlated with NeuN expression. Taken together, our findings suggest that AEG-1 contributes to glioma-induced neurodegeneration, a hallmark of this fatal tumor, through regulation of EAAT2 expression.
AEG-1; glioma; EAAT2; glutamate; glioma-induced neurodegeneration
In the central nervous system (CNS), glutamate rapidly upregulates the activities of different excitatory amino-acid transporter subtypes (EAATs) in order to help protect neurons from excitotoxicity. Since human platelets display a specific sodium-dependent glutamate uptake activity, and express the three major glutamate transporters, which may be affected in neurological disorders, we investigated whether platelets are subject to substrate-induced modulation as described for CNS.A time- and dose-dependent upregulation of [3H]-glutamate uptake (up to two-fold) was observed in platelets preincubated with glutamate. There was an increase in maximal velocity rate without affinity changes. Glutamate receptor agonists and antagonists did not modulate this upregulation and preincubation with glutamate analogues failed to mimic the glutamate effect. Only aspartate preincubation increased the uptake, albeit ∼35% less with respect to glutamate.The effect of glutamate preincubation on the expression of the three major transporters was studied by Western blotting, showing an increase of ∼70% in EAAT1 immunoreactivity that was completely blocked by cycloheximide (CEM). However, L-serine-O-sulphate, at a concentration (200 μM) known to block EAAT1/3 selectively, did not completely inhibit the effect of glutamate stimulation, indicating the possible involvement of EAAT2.In fact, glutamate stimulation was completely abolished only when, following CEM pre-incubation, the experiment was run in the presence of the selective EAAT2 inhibitor dihydrokainic acid. Since surface biotinylation experiments failed to show evidence of EAAT2 translocation, our results suggest the existence of a different way of regulating EAAT2 activity.These findings indicate that human platelets display a substrate-dependent modulation of glutamate uptake mediated by different molecular mechanisms and confirm that ex vivo platelets are a reliable model to investigate the dysfunction of glutamate uptake regulation in patients affected by neurological disorders.
Glutamate uptake; human platelets; substrate modulation; EAAT1