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.
glutamate transport; NF-κB; astrocytes; p65; p50; EAAT2; GLT-1; IκBα
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 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
Glutamate is an essential excitatory neurotransmitter regulating brain functions. Excitatory amino acid transporter (EAAT)-2 is one of the major glutamate transporters expressed predominantly in astroglial cells and is responsible for 90% of total glutamate uptake. Glutamate transporters tightly regulate glutamate concentration in the synaptic cleft. Dysfunction of EAAT2 and accumulation of excessive extracellular glutamate has been implicated in the development of several neurodegenerative diseases including Alzheimer’s disease, Huntington’s disease, and amyotrophic lateral sclerosis. Analysis of the 2.5-kb human EAAT2 promoter showed that NF-κB is an important regulator of EAAT2 expression in astrocytes. Screening of approximately 1,040 FDA-approved compounds and nutritionals led to the discovery that many β-lactam antibiotics are transcriptional activators of EAAT2 resulting in increased EAAT2 protein levels. Treatment of animals with ceftriaxone (CEF), a β-lactam antibiotic, led to an increase of EAAT2 expression and glutamate transport activity in the brain. CEF has neuroprotective effects in both in vitro and in vivo models based on its ability to inhibit neuronal cell death by preventing glutamate excitotoxicity. CEF increases EAAT2 transcription in primary human fetal astrocytes (PHFA) through the NF-κB signaling pathway. The NF-κB binding site at −272 position was critical in CEF-mediated EAAT2 protein induction. These studies emphasize the importance of transcriptional regulation in controlling glutamate levels in the brain. They also emphasize the potential utility of the EAAT2 promoter for developing both low and high throughput screening assays to identify novel small molecule regulators of glutamate transport with potential to ameliorate pathological changes occurring during and causing neurodegeneration.
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.
GLT1; patch-clamp; whole cell; current/flux coupling; excitotoxicity; presynaptic; synapse
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.
EAAT2; neuroprotection; citicholine; Parkinson’s Disease; glutamate uptake; glutamate transporters
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.
astrocyte; GLT-1; EAAT2; biolistics; time-lapse; activity
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.
glutamate transporters; interleukin-1β; viral encephalomyelitis; motor neuron; excitotoxicity
The astrocyte glutamate transporter, GLT1, is responsible for the vast majority of glutamate uptake in the adult central nervous system (CNS), thereby regulating extracellular glutamate homeostasis and preventing excitotoxicity. Glutamate dysregulation plays a central role in outcome following traumatic spinal cord injury (SCI). To determine the role of GLT1 in secondary cell loss following SCI, mice heterozygous for the GLT1 astrocyte glutamate transporter (GLT1+/−) and wild-type mice received thoracic crush SCI. Compared to wild-type controls, GLT1+/− mice had an attenuated recovery in hindlimb motor function, increased lesion size, and decreased tissue sparing. GLT1+/− mice showed a decrease in intraspinal GLT1 protein and functional glutamate uptake compared to wild-type mice, accompanied by increased apoptosis and neuronal loss following crush injury. These results suggest that astrocyte GLT1 plays a role in limiting secondary cell death following SCI, and also show that compromise of key astrocyte functions has significant effects on outcome following traumatic CNS injury. These findings also suggest that increasing intraspinal GLT1 expression may represent a therapeutically relevant target for SCI treatment.
secondary injury; GLT1+/− mice; crush injury; glutamate uptake; excitotoxicity
In the central nervous system, excitatory amino acid transporters (EAATs) localized to neurons and glia terminate the actions of synaptically released glutamate. Whereas glial transporters are primarily responsible for maintaining low ambient levels of extracellular glutamate, neuronal transporters have additional roles in shaping excitatory synaptic transmission. Here we test the hypothesis that the expression level of the Purkinje cell (PC)-specific transporter, EAAT4, near parallel fiber (PF) release sites controls the extrasynaptic glutamate concentration transient following synaptic stimulation. Expression of EAAT4 follows a parasagittal banding pattern that allows us to compare regions of high and low EAAT4-expressing PCs. Using EAAT4 promoter driven eGFP reporter mice together with pharmacology and genetic deletion, we show that the level of neuronal transporter expression influences extrasynaptic transmission from PFs to adjacent Bergmann glia (BG). Surprisingly, a twofold difference in functional EAAT4 levels is sufficient to alter signaling to BG although EAAT4 may only be responsible for removing a fraction of released glutamate. These results demonstrate that physiological regulation of neuronal transporter expression can alter extrasynaptic neuro-glial signaling.
synaptic transmission; Purkinje cell; parallel fiber; EAAT4
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
GLT-1 eGFP BAC reporter transgenic adult mice were used to detect GLT-1 gene expression in individual cells of CA1, CA3 and SI, and eGFP fluorescence was measured to analyze quantitatively GLT-1 promoter activity in different cells of neocortex and hippocampus. Virtually all GFAP+ astrocytes were eGFP+; we also found that about 80% of neurons in CA3 pyramidal layer, 10–70% of neurons in I-VI layers of SI and rare neurons in all strata of CA1 and in strata oriens and radiatum of CA3 were eGFP+. Analysis of eGFP intensity showed that astrocytes had a higher GLT-1 promoter activity in SI than in CA1 and CA3, and that neurons had the highest levels of GLT-1 promoter activity in CA3 stratum pyramidale and in layer VI of SI. Finally, we observed that the intensity of GLT-1 promoter activity in neurons is 1–20% of that measured in astrocytes. These results showed that in the hippocampus and neocortex GLT-1 promoter activity is observed in astrocytes and neurons, detailed the distribution of GLT-1 expressing neurons, and indicated that GLT-1 promoter activity in both astrocytes and neurons varies in different brain regions.
glutamate transporters; GLT-1/EAAT2; neurons; astrocytes; hippocampus; neocortex
OBJECTIVES—To investigate if sequence alterations
of the excitatory amino acid transporter gene EAAT2 (GLT-1) may be a
contributory factor to the pathogenesis of motor system degeneration.
EAAT2 serves as a candidate gene as its reduced expression was reported
in patients with amyotrophic lateral sclerosis (ALS). Furthermore, neurolathyrism, a motor neuron disease clinically related to hereditary spastic paraplegia (HSP), has been associated with an exogenous excitotoxin.
METHODS—Sequence alterations were screened for in
the coding region of EAAT2 in 55 patients with ALS and one family with
autosomal dominant HSP (AD-HSP).
RESULTS—In ALS, no sequence alteration in the
EAAT2 gene have been found. Interestingly, a heterozygous A79G mutation
of the EAAT2 gene was detected in two of seven affected patients with
AD-HSP in the same kindred. The absence of cosegregation with the
familial disease showed that the detected variant was not the cause of disease. The A79G sequence variant was not found in 55 patients with
ALS or in 50 non-neurological controls.
CONCLUSION—The allelic variant of the EAAT2 gene
in conjunction with the primary gene defect may be a modifying factor
for the highly variable AD-HSP phenotype.
Glutamate is a regulated molecule in the mammalian testis. Extracellular regulation of glutamate in the body is determined largely by the expression of plasmalemmal glutamate transporters. We have examined by PCR, western blotting and immunocytochemistry the expression of a panel of sodium-dependent plasmalemmal glutamate transporters in the rat testis. Proteins examined included: glutamate aspartate transporter (GLAST), glutamate transporter 1 (GLT1), excitatory amino acid carrier 1 (EAAC1), excitatory amino acid transporter 4 (EAAT4) and EAAT5. We demonstrate that many of the glutamate transporters in the testis are alternately spliced. GLAST is present as exon-3- and exon-9-skipping forms. GLT1 was similarly present as the alternately spliced forms GLT1b and GLT1c, whereas the abundant brain form (GLT1a) was detectable only at the mRNA level. EAAT5 was also strongly expressed, whereas EAAC1 and EAAT4 were absent. These patterns of expression were compared with the patterns of endogenous glutamate localization and with patterns of 𝒹-aspartate accumulation, as assessed by immunocytochemistry. The presence of multiple glutamate transporters in the testis, including unusually spliced forms, suggests that glutamate homeostasis may be critical in this organ. The apparent presence of many of these transporters in the testis and sperm may indicate a need for glutamate transport by such cells.
excitatory amino acid transporter; glutamate aspartate transporter; glutamate transporter 1; sperm; splice variant; testis; transporter
Efficient excitatory transmission depends on a family of transporters that utilize the Na+-electrochemical gradient to maintain low synaptic concentrations of glutamate. These transporters consume substantial energy in the spatially restricted space of fine astrocytic processes. GLT-1 (EAAT2) mediates the bulk of this activity in forebrain. To date, relatively few proteins have been identified that associate with GLT-1. In the present study, GLT-1 immunoaffinity isolates were prepared from rat cortex using three strategies and analyzed by LC coupled tandem mass spectrometry. In addition to known interacting proteins, the analysis identified glycolytic enzymes and outer mitochondrial proteins. Using double label immunofluorescence, GLT-1 was shown to co-localize with the mitochondrial matrix protein, ubiquinol-cytochrome c reductase core protein II (UQCRC2) or the inner mitochondrial membrane protein, ADP/ATP translocase (ANT), in rat cortex. In biolistically transduced hippocampal slices, fluorescently tagged GLT-1 puncta overlapped with fluorescently tagged mitochondria along fine astrocytic processes. In a Monte Carlo-type computer simulation, this overlap was significantly more frequent than would occur by chance. Furthermore, fluorescently tagged hexokinase-1 overlapped with mitochondria or GLT-1, strongly suggesting that GLT-1, mitochondria, and the first step in glycolysis are co-compartmentalized in astrocytic processes. Acute inhibition of glycolysis or oxidative phosphorylation had no effect on glutamate uptake in hippocampal slices, but simultaneous inhibition of both processes significantly reduced transport. Together with previous results, these studies show that GLT-1 co-compartmentalizes with Na+/K+ ATPase, glycolytic enzymes, and mitochondria, providing a mechanism to spatially match energy and buffering capacity to the demands imposed by transport.
glutamate transport; GLT-1; mitochondria; glycolysis
Currently, the cellular and molecular mechanisms that underlie radiation-induced damage in the CNS are unclear. The present study began investigations of the underlying mechanism(s) for radiation-induced neurotoxicity by characterizing glutamate transport expression and function in neurons and astrocytes after exposure to γ rays. NTera2-derived neurons and astrocytes, isolated as pure cultures, were exposed to doses of 10 cGy, 50 cGy and 2 Gy γ rays, and transporter expression and function were assessed 3 h, 2 days and 7 days after exposure. In neurons, at 7 days after exposure, a significant increase was detected in EAAT3 after 50 cGy (P < 0.05) and a dose-dependent increase in GLT-1 expression was seen between doses of 10 and 50 cGy (P < 0.05). Functional assays of glutamate uptake revealed that neurons and astrocytes respond in a reciprocal manner after irradiation. Neurons responded to radiation exposure by increased glutamate uptake, an effect still evident at our last time (7 days) after exposure (P < 0.05). The astrocyte response to γ radiation was an initial decrease in uptake followed by recovery to baseline levels at 2 days after exposure (P < 0.05). The observations made in this study demonstrate that neurons and astrocytes, while part of the same multifunctional unit, have distinct functional and reciprocal responses. The response in neurons appears to indicate a protracted response with potential long-term effects after irradiation.
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.
glutamate; transport; dihydrokainate; presynaptic; astrocytes; synapse; excitotoxicity
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.
astroglia; BAC; ALDH1L1; GLT1; GFAP; oligodendroglia; ALS
We recently found evidence for anatomic and physical linkages between the astroglial Na+-dependent glutamate transporters (GLT-1/EAAT2 and GLAST/EAAT1) and mitochondria. In these same studies, we found that the glutamate dehydrogenase (GDH) inhibitor, epigallocatechin-monogallate (EGCG), inhibits both glutamate oxidation and Na+-dependent glutamate uptake in astrocytes. In the present study, we extend this finding by exploring the effects of EGCG on Na+-dependent l-[3H]-glutamate (Glu) uptake in crude membranes (P2) prepared from rat brain cortex. In this preparation, uptake is almost exclusively mediated by GLT-1. EGCG inhibited l-[3H]-Glu uptake in cortical membranes with an IC50 value of 230 μM. We also studied the effects of two additional inhibitors of GDH, hexachlorophene (HCP) and bithionol (BTH). Both of these compounds also caused concentration-dependent inhibition of glutamate uptake in cortical membranes. Pre-incubating with HCP for up to 15 min had no greater effect than that observed with no pre-incubation, showing that the effects occur rapidly. HCP decreased the Vmax for glutamate uptake without changing the Km, consistent with a non-competitive mechanism of action. EGCG, HCP, and BTH also inhibited Na+-dependent transport of d-[3H]-aspartate (Asp), a non-metabolizable transporter substrate, and [3H]-γ-aminobutyric acid (GABA). In contrast to the forebrain, glutamate uptake in crude cerebellar membranes (P2) is likely mediated by GLAST (EAAT1). Therefore, the effects of these compounds were examined in cerebellar membranes. In this region, none of these compounds had any effect on uptake of either l-[3H]-Glu or d-[3H]-Asp, but they all inhibited [3H]-GABA uptake. Together these studies suggest that GDH is preferentially required for glutamate uptake in forebrain as compared to cerebellum, and GDH may be required for GABA uptake as well. They also provide further evidence for a functional linkage between glutamate transport and mitochondria.
glutamate; GLT-1; EAAT2; GLAST; GABA; glutamate dehydrogenase; sodium-dependent uptake; epigallocatechin-monogallate
Glutamate transporters, also called excitatory amino acid transporters (EAATs), uptake extracellular glutamate and regulate neurotransmission. Activation of protein kinase C (PKC) increases the activity of EAAT type 3 (EAAT3), the major neuronal EAAT. We designed this study to determine which amino acid residue(s) in EAAT3 may be involved in this PKC effect. Selective potential PKC phosphorylation sites were mutated. These EAAT3 mutants were expressed in the Xenopus oocytes. Phorbol 12-myristate 13-acetate, a PKC activator, significantly increased wild-type EAAT3 activity. Mutation of serine 465 to alanine or to aspartic acid, but not the mutation of threonine 5 to alanine, abolished PKC-increased EAAT3 activity. Our results suggest a critical role of serine 465 in the increased EAAT3 activity by PKC activation.
Glutamate; glutamate transporter; protein kinase C; site-directed mutagenesis
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.
Saccharomyces cerevisiae glutamate synthase (GOGAT) is an oligomeric enzyme composed of three 199-kDa identical subunits encoded by GLT1. In this work, we analyzed GLT1 transcriptional regulation. GLT1-lacZ fusions were prepared and GLT1 expression was determined in a GDH1 wild-type strain and in a gdh1 mutant derivative grown in the presence of various nitrogen sources. Null mutants impaired in GCN4, GLN3, GAT1/NIL1, or UGA43/DAL80 were transformed with a GLT1-lacZ fusion to determine whether the above-mentioned transcriptional factors had a role in GLT1 expression. A collection of increasingly larger 5′ deletion derivatives of the GLT1 promoter was constructed to identify DNA sequences that could be involved in GLT1 transcriptional regulation. The effect of the lack of GCN4, GLN3, or GAT1/NIL1 was also tested in the pertinent 5′ deletion derivatives. Our results indicate that (i) GLT1 expression is negatively modulated by glutamate-mediated repression and positively regulated by Gln3p- and Gcn4p-dependent transcriptional activation; (ii) two cis-acting elements, a CGGN15CCG palindrome and an imperfect poly(dA-dT), are present and could play a role in GLT1 transcriptional activation; and (iii) GLT1 expression is moderately regulated by GCN4 under amino acid deprivation. Our results suggest that in a wild-type strain grown on ammonium, GOGAT constitutes an ancillary pathway for glutamate biosynthesis.
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.
uptake; trafficking; alternative splicing; excitotoxicity; PDZ domain; synapse
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.
GLT-1; TGF-α; glutamate uptake; glutamate transporter; manganese; tamoxifen; 17β-estradiol; astrocytes
Glutamate is a key neurotransmitter and its levels in the synaptic cleft are tightly regulated by reuptake mechanisms that primarily involve transporters in astrocytes. This requires that the glutamate transporters be spatially constrained to effect maximum glutamate transport. GLAST (EAAT1) is the predominant astroglial transporter and contains a class I PDZ-binding consensus (ETKM) in its C-terminus. The epithelial Na+/H+ exchanger regulatory factors NHERF1 and NHERF2 are PDZ proteins that contain two tandem PDZ domains and a C-terminal domain that binds members of the ERM (ezrin–radixin–moesin) family of membrane-cytoskeletal adaptors. NHERF proteins have been extensively characterized in renal epithelia and their expression in brain has recently been reported; however, their function in the brain remains unknown. The aims of the current study were to (1) determine the distribution of NHERF1/2 in the rodent brain and (2) investigate whether GLAST was a physiological ligand for NHERF1/2. Immunohistochemistry revealed that NHERF1 expression was widespread in rat brain (abundant in cerebellum, cerebral cortex, hippocampus, and thalamus) and primarily restricted to astrocytes whereas NHERF2 expression was primarily restricted to endothelial cells of blood vessels and capillaries. Importantly, NHERF1 distribution closely matched that of GLAST and confocal imaging demonstrated co-localization of the two proteins. Co-immunoprecipitation demonstrated that GLAST, NHERF1, and ezrin associate in vivo. In vitro binding assays showed that GLAST bound directly to the PDZ1 domain of NHERF1 via the C-terminal ETKM motif of GLAST. These findings implicate the GLAST–NHERF1 complex in the regulation of glutamate homeostasis in astrocytes.
NHERF1; NHERF2; GLAST; astrocytes; glutamate transport