Glutamate/aspartate Transporter mRNA Research Articles

Naringin provides neuroprotection in CCL2-induced cognition impairment by attenuating neuronal apoptosis in the hippocampus

BackgroundChemokine C–C motif ligand 2 (CCL2) is one of the most widely recognised proinflammatory chemokines in cognitive disorders. Currently, CCL2-targeting drugs are extremely limited. Thus, this study aimed to explore the neuroprotection afforded by naringin in CCL2-induced cognitive impairment in rats.MethodsBefore the CCL2 intra-hippocampal injection, rats were treated with naringin for 3 consecutive days via intraperitoneal injection. Two days post-surgery, the Morris water maze (MWM) and novel object recognition (NORT) tests were performed to detect spatial learning and memory and object cognition, respectively. Nissl staining and dUTP nick-end labelling (TUNEL) staining were performed to assess histopathological changes in the hippocampus. Commercial kits were used to measure the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and the content of malondialdehyde (MDA). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to examine the relative mRNA expression of interleukin 1β, (IL-1β), interleukin 6 (IL-6), glutamate/aspartate transporter (GLAST), glutamate transporter-1 (GLT-1), phosphate-activated glutaminase (PAG), cysteine aspartic acid-specific protease 8 (caspase-8), cysteine aspartic acid-specific protease 3 (caspase-3), cell lymphoma/leukaemia-2 (Bcl-2), and Bcl-2 associated X protein (Bax).ResultsIn the MWM, the average escape latency and average swimming distance were significantly reduced and the crossing times were increased in the naringin-treated groups, compared with the CCL2 group. The NORT results revealed that, compared with the CCL2 rats, the discrimination index in the naringin-treated rats increased significantly. Nissl and TUNEL staining revealed that naringin protected the structure and survival of the neurons in the CA1 zone of the hippocampus. In the naringin-treated groups, the SOD and GSH-Px activities were increased, whereas the MDA levels were decreased. Furthermore, in the naringin-treated groups, the relative mRNA expression of IL-1β and IL-6 was significantly decreased; GLAST and GLT-1 mRNA expression levels were increased, whereas PAG was decreased. In the naringin-treated groups, the relative mRNA expression levels of caspase-8, caspase-3, and Bax were decreased, whereas that of Bcl-2 was increased.ConclusionCollectively, these data indicated that naringin alleviated the CCL2-induced cognitive impairment. The underlying mechanisms could be associated with the inhibition of neuroinflammation, oxidative stress, apoptosis, and the regulation of glutamate metabolism.

Cyanin Chloride Inhibits Hyperbaric Pressure-Induced Decrease of Intracellular Glutamate-Aspartate Transporter in Rat Retinal Müller Cells.

Purpose Glaucoma is the leading cause of irreversible blindness throughout the world. The pathogenesis of glaucoma is complex, and neuroprotection is a crucial aspect of therapy. High concentrations of extracellular glutamate are toxic to the optic nerve. The glutamate-aspartate transporter (GLAST) in retinal Müller cells is involved in the development of glaucoma. Anthocyanin has been reported to protect retinal neurons. We hypothesize that cyanin chloride, a type of anthocyanin, can inhibit hyperbaric pressure-induced GLAST decreases in cultured rat retinal Müller cells and may serve as a potential neuroprotective agent in glaucoma treatment. Materials and Methods Sprague Dawley rat Müller cells were cultured in a hyperbaric pressure device at 60 mmHg additional pressure and treated with cyanin chloride (10 μmol/L, 30 μmol/L, or 50 μmol/L) or vehicle for 2 hours. Cell survival rates (SRs) were evaluated by an MTT assay. GLAST mRNA and protein expression were determined by western blot and RT-PCR analyses, respectively. Results Cell SR was significantly decreased in the 60 mmHg additional hyperbaric pressure group compared to the control group (P < 0.01). Cyanin chloride treatment significantly improved SR under 60 mmHg additional pressure (P < 0.01). GLAST mRNA and protein expression levels in Müller cells were significantly reduced in the 60 mmHg hyperbaric pressure group compared to the control group (P < 0.01), but cyanin chloride significantly inhibited hyperbaric pressure-induced decreases in GLAST expression (P < 0.01). Conclusion Our results support our hypothesis and demonstrate that cyanin chloride can protect rat retinal Müller cells from hyperbaric pressure-induced decreases of GLAST.

Valproate and sodium butyrate attenuate manganese-decreased locomotor activity and astrocytic glutamate transporters expression in mice

Manganese (Mn) is an essential trace element, but chronic overexposure to this metal, either environmentally or occupationally may cause manganism, a disease analogous to Parkinson’s disease. Inhibitors of histone deacetylases, such as valproic acid (VPA) and sodium butyrate (NaB) exert neuroprotective effects in various animal models of neurological disorders. Thus, the present study investigated whether VPA or NaB prevent Mn-induced neurotoxicity by assessing locomotor activities and expression of astrocytic glutamate transporters, glutamate transporter 1 (GLT-1) and glutamate aspartate transporter (GLAST), in C57BL/6 mice. C57BL/6 mice were pretreated with VPA (200mg/kg, i.p.) or NaB (1200mg/kg, i.p.) prior to intranasal instillation of Mn (30mg/kg) continually for 21days, followed by open-field and rota-rod behavioral tests and analyses of astrocytic glutamate transporters GLT-1 and GLAST protein/mRNA levels. The results showed that Mn significantly decreased locomotor activity as determined by total distance travelled, stereotypic and ambulatory counts. Mn also significantly decreased rota-rod activity reflecting altered motor coordination. Pretreatment with VPA and NaB with Mn reversed the effects of Mn on the locomotor activity and motor coordination. VPA and NaB also attenuated the Mn-induced decrease in GLT-1 and GLAST mRNA and protein levels in the cerebral cortical and cerebellar regions of mice. These results suggest that VPA and NaB exert protective effects against Mn toxicity seem in vitro are also shown in vivo. VPA and NaB pretreatment in mice enhancing astrocytic glutamate transporter GLT-1 expression as well as locomotor activities. Future research endeavors are warranted to determine if the therapeutic potential of VPA and NaB is via common molecular mechanism, namely, inhibition of histone deacetylases.

Effects of erythropoietin preconditioning on rat cerebral ischemia-reperfusion injury and the GLT-1/GLAST pathway.

The aim of the present study was to investigate whether erythropoietin (EPO) preconditioning affects the expression of glutamate transporter 1 (GLT-1) and glutamate aspartate transporter (GLAST) and protects against rat cerebral ischemia-reperfusion injury. A total of 140 Sprague Dawley rats were randomly assigned to one of the following four groups: Sham, EPO-sham, middle cerebral artery occlusion (MCAO) and EPO-MCAO. Neurological function scores were obtained 24, 36 and 72 h after reperfusion. Seventy-two hours after the induction of cerebral ischemia-reperfusion, the number of apoptotic neural cells and the cerebral infarct volume of each group were measured. The mRNA levels of GLT-1 and GLAST were determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis, while the GLT-1 and GLAST protein levels were assessed using western blotting. The cerebral infarct volume was significantly increased in the MCAO group compared with that in the sham group (P<0.01); however, the infarct volume of the EPO-MCAO group was significantly lower than that of the MCAO group (P<0.01). In addition, the number of apoptotic cells found in the MCAO group was higher than that in the sham group (P<0.01), but the number of apoptotic cells in the EPO-MCAO group was significantly lower than that in the MCAO group (P<0.01). The GLT-1 and GLAST mRNA and protein levels were significantly decreased 72 h after the cerebral ischemia-reperfusion (P<0.01) compared with those in the sham group, whereas the same levels were increased significantly in the EPO-MCAO group relative to those in the MCAO group (P<0.01). In conclusion, EPO preconditioning protected against cerebral ischemia-reperfusion injury and upregulated the GLT-1 and GLAST expression.

Stimulation of α7 nicotinic acetylcholine receptor regulates glutamate transporter GLAST via basic fibroblast growth factor production in cultured cortical microglia

The α7 nicotinic acetylcholine (nACh) receptor expressed in microglia has a crucial role in neuroprotection. Simulation of α7 nACh receptor leads to increased expression of glutamate/aspartate transporter (GLAST), which in turn decreases synaptic glutamate levels. However, the upregulation of GLAST in cultured rat cortical microglia appears long after (over 18h) stimulation of the α7 nACh receptor with nicotine. Thus, the current study elucidated the pathway responsible for the induction of GLAST expression in cultured cortical microglia. Nicotine-induced GLAST mRNA expression was significantly inhibited by cycloheximide pretreatment, indicating that a protein intermediary, such as a growth factor, is required for GLAST expression. The expression of fibroblast growth factor-2 (FGF-2) mRNA in cortical microglia was significantly increased 6 and 12h after treatment with nicotine, and this increase was potently inhibited by pretreatment with methyllycaconitine, a selective α7 nACh receptor antagonist. The treatment with nicotine also significantly increased FGF-2 protein expression. Furthermore, treatment with recombinant FGF-2 increased GLAST mRNA, protein expression and 14C-glutamate uptake, a functional measurement of GLAST activity. Conversely, pretreatment with PD173074, an inhibitor of FGF receptor (FGFR) tyrosine kinase, significantly prevented the nicotine-induced expression of GLAST mRNA, its protein and 14C-glutamate uptake. Reverse transcription polymerase chain reaction confirmed FGFR1 mRNA expression was confined to cultured cortical microglia. Together, the current findings demonstrate that the neuroprotective effect of activation of microglial α7 nACh receptors could be due to the expression of FGF-2, which in turn increases GLAST expression, thereby clearing glutamate from synapse and decreasing glutamate neurotransmission.

Effects of sinomenine on expression of glutamate transporter-1 mRNA and glutamate aspartate transporter mRNA in spinal cord of rats with neuropathic pain

Objective To evaluate the effectsof sinomenine on the expression of glutamate transporter-1 (GLT-1) mRNA and glutamate aspartate transporter (GLAST) mRNA in the spinal cord of rats with neuropathic pain (NP).Methods Seventy-two male Wistar rats,weighing 180-220 g,aged 6-8 weeks,were divided into 3 groups (n =24 each) using a random number table:sham operation group (group S); NP group; sinomenine group (group SIN).The animals were anesthetized with intraperitoneal 10％ chloral hydrate 350 mg/kg.NP was induced by chronic constrictive injury.The right sciatic nerve was exposed and 4 loose ligatures were placed on the sciatic nerve at 1 mm intervals with 4-0 silk thread.In group S,the right sciatic nerves were only exposed,but not ligated.In group SIN,sinomenine injection 40 mg/kg was injected intraperitoneally once a day starting from the end of operation until 1 day before the animals were sacrificed,while the equal volume of normal saline was given instead in S and NP groups.Mechanical paw withdrawal threshold to von Frey stimuli (MWT) and paw withdrawal latency to thermal nociceptive stimulus (TWL) were measured at 1 day before operation and 1,3,7 and 14 days after operation (T1-4).The rats were sacrificed after measurement of pain threshold,and their lumbar segments (L4-6) of the spinal cord were immediately removed for determination of GLT-1 mRNA and GLAST mRNA expression in the spinal dorsal horn (by real-time fluorescent quantitative PCR).Results Compared with group S,MWT was significantly decreased and TWL was shortened at T1-4 in NP and SIN groups,the expression of GLT-1 mRNA and GLAST mRNA was up-regulated at T1.2 in group NP,and the expression of GLT-1 mRNA and GLAST mRNA was up-regulated at T1-3 in group SIN.Compared with group NP,MWT was significantly increased,TWL was prolonged,and the expression of GLT-1 mRNA and GLAST mRNA was up-regulated at T1.3 in group SIN.Conclusion Sinomenine can mitigate NP in rats and up-regulation of GLT-1 mRNA and GLAST mRNA expression is involved in the mechanism. Key words: Sinomenium; Neuralgia; Glutamate plasma membrane transport proteins; Spinal cord

Primary cultures of rat cortical microglia treated with nicotine increases in the expression of excitatory amino acid transporter 1 (GLAST) via the activation of the α7 nicotinic acetylcholine receptor

Although the clearance of glutamate from the synapse under physiological conditions is performed by astrocytic glutamate transporters, their expression might be diminished under pathological conditions. Microglia glutamate transporters, however, might serve as a back-up system when astrocytic glutamate uptake is impaired, and could have a prominent neuroprotective function under pathological conditions. In the current study, the effect of nicotine, well known as a neuroprotective molecule, on the function of glutamate transporters in cultured rat cortical microglia was examined. Reverse transcription polymerase chain reaction and pharmacological approaches demonstrated that, glutamate/aspartate transporter (GLAST), not glutamate transporter 1 (GLT-1), is the major functional glutamate transporter in cultured cortical microglia. Furthermore, the α7 subunit was demonstrated to be the key subunit comprising nicotinic acetylcholine (nACh) receptors in these cells. Treatment of cortical microglia with nicotine led to a significant increase of GLAST mRNA expression and 14C-glutamate uptake in a concentration- and time-dependent manner, which were markedly inhibited by pretreatment with methyllycaconitine, a selective α7 nACh receptor antagonist. The nicotine-induced expression of GLAST mRNA and protein is mediated through an inositol trisphosphate (IP3) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) depend intracellular pathway, since pretreatment with either xestospongin C, an IP3 receptor antagonist, or KN-93, a CaMKII inhibitor, blocked GLAST expression. Together, these findings indicate that activation of nACh receptors, specifically those expressing the α7 subunit, on cortical microglia could be a key mechanism of the neuroprotective effect of nACh receptor ligands such as nicotine.

The Protective Effects of Riluzole on Manganese-Induced Disruption of Glutamate Transporters and Glutamine Synthetase in the Cultured Astrocytes

Chronic exposure to excessive manganese (Mn) can lead to manganism, a type of neurotoxicity accomplished with extracellular glutamate (Glu) accumulation. To investigate this accumulation, this study focused on the role of astrocyte glutamate transporters (GluTs) and glutamine synthetase (GS), which have roles in Glu transport and metabolism, respectively. And the possible protective effects of riluzole (a glutamatergic modulator) were studied in relation to Mn exposure. At first, the astrocytes were exposed to 0, 125, 250, and 500 μM MnCl(2) for 24 h, and 100 μM riluzole was pretreated to astrocytes for 6 h before 500 μM MnCl(2) exposure. Then, [(3)H]-glutamate uptake was measured by liquid scintillation counting; Na(+)-K(+) ATPase and GS activities were determined by a colorimetric method; glutamate/aspartate transporter (GLAST), glutamate transporter-1 (GLT-1), and GS mRNA expression were determined by RT-PCR and protein levels were measured by western blotting. The results showed that Mn inhibited Glu uptake, Na(+)-K(+) ATPase and GS activities, GLAST, GLT-1, and GS mRNA, and protein in a concentration-dependent manner. And they were significantly higher for astrocytes pretreated with 100 μM riluzole than the group exposed to 500 μM MnCl(2). The results suggested that Mn disrupted Glu transport and metabolism by inhibiting GluTs and GS. Riluzole activated protective effects on enhancing GluTs and GS to reverse Glu accumulation. In conclusion, Mn exposure results in the disruption of GLAST, GLT-1, and GS expression and function. Furthermore, riluzole attenuates this Mn toxicity.

Riluzole-triggered GSH synthesis via activation of glutamate transporters to antagonize methylmercury-induced oxidative stress in rat cerebral cortex.

Objective. This study was to evaluate the effect of riluzole on methylmercury- (MeHg-) induced oxidative stress, through promotion of glutathione (GSH) synthesis by activating of glutamate transporters (GluTs) in rat cerebral cortex. Methods. Eighty rats were randomly assigned to four groups, control group, riluzole alone group, MeHg alone group, and riluzole + MeHg group. The neurotoxicity of MeHg was observed by measuring mercury (Hg) absorption, pathological changes, and cell apoptosis of cortex. Oxidative stress was evaluated via determining reactive oxygen species (ROS), 8-hydroxy-2-deoxyguanosine (8-OHdG), malondialdehyde (MDAs), carbonyl, sulfydryl, and GSH in cortex. Glutamate (Glu) transport was studied by measuring Glu, glutamine (Gln), mRNA, and protein of glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1). Result. (1) MeHg induced Hg accumulation, pathological injury, and apoptosis of cortex; (2) MeHg increased ROS, 8-OHdG, MDA, and carbonyl, and inhibited sulfydryl and GSH; (3) MeHg elevated Glu, decreased Gln, and downregulated GLAST and GLT-1 mRNA expression and protein levels; (4) riluzole antagonized MeHg-induced downregulation of GLAST and GLT-1 function and expression, GSH depletion, oxidative stress, pathological injury, and apoptosis obviously. Conclusion. Data indicate that MeHg administration induced oxidative stress in cortex and that riluzole could antagonize this situation through elevation of GSH synthesis by activating of GluTs.

Ca2+‐dependent reduction of glutamate aspartate transporter GLAST expression in astrocytes by P2X7 receptor‐mediated phosphoinositide 3‐kinase signaling

Astrocytes are responsible for clearance of extracellular glutamate, primarily through glial-specific glutamate transporter-1 and the Na(+)-dependent glutamate/aspartate transporter (GLAST). After traumatic injury to the CNS, such as spinal cord injury, persistent release of ATP from damaged neurons and activated glial cells occurs, inducing detrimental and/or beneficial effects via activation of ionotropic (P2XR) and metabotropic purinergic receptors. In this study, we show a decrease in GLAST mRNA in the lesion center and caudal portions at 24 h post-spinal cord injury. In an in vitro system, the ability of astrocytes to take up glutamate and astrocytic GLAST mRNA levels were significantly decreased after exposure to ATP and its P2X(7)R agonist, 2'-3'-O-(4-benzoylbenzoyl)-ATP. ATP- or 2'-3'-O-(4-benzoylbenzoyl)-ATP-induced inhibitory effect on GLAST mRNA expression was blocked by the irreversible P2X(7)R blocker, oxidized ATP, or when P2X(7)R mRNA expression was reduced by the lentivirus-short hairpin RNA knockdown approach. Furthermore, deletion of the GLAST promoter and RNA decay assays showed that P2X(7)R signaling triggered post-transcriptional regulation of GLAST expression via the phosphoinositide 3-kinase cascade. The signaling pathway participating in the P2X(7)R effect on GLAST mRNA expression was identified as a Ca(2+)-dependent phosphoinositide 3-kinase-phospholipase Cgamma involving the inositol 1,4,5-trisphosphate receptor, calcium/calmodulin-dependent kinase II, and protein kinase C. We conclude that P2X(7)R activation by sustained release of ATP in the injured CNS may decrease GLAST mRNA stability via Ca(2+)-dependent signaling, suggesting that inhibition of P2X(7)R may allow for recovery of astrocytic GLAST function and protect neurons from glutamate-induced excitotoxicity.

Development of a β-Lactamase Reporter Gene Assay for Metabotropic Glutamate Receptor 1 by Using Coexpression of Glutamate Transporter

mGluR1 antagonists have been postulated to be novel CNS drugs, including antipsychotics. Toward this end, the authors developed a beta-lactamase reporter assay to identify mGluR1 antagonists. beta-Lactamase has several interesting features for high-throughput screening, including very high sensitivity and less well-to-well variation than other reporter enzymes. mGluR1-expressing Chinese hamster ovary (CHO) cells with the beta-lactamase gene under control of the nuclear factor of activated T cells (NFAT) promoter (CHO-NFAT-bla-hmGluR1b) exhibited very high basal activity, resulting in an inadequate signal-to-basal (S/B) ratio. Coexpression of glutamate/aspartate transporter (GLAST) with mGluR1 in the cell line (CHO-NFAT-bla-hmGluR1b-GLAST) dramatically decreased basal activity and improved the S/B ratio (from 2- to 20-fold). The contribution of GLAST to lowering basal activity and increasing the S/B ratio was validated by the expression level of GLAST mRNA and by a GLAST inhibitor. Antagonistic activities of known mGluR1 antagonists in the beta-lactamase reporter assay were comparable with those in the conventional Ca(2+) mobilization assay. The Z' factor of the beta-lactamase reporter assay was 0.89 under optimized conditions. Taken together, the beta-lactamase reporter assay with CHO-NFAT-bla-hmGluR1b-GLAST could be a novel high-throughput assay for mGluR1 antagonist screening. This is the first description of a successful beta-lactamase reporter assay among all mGluR subtypes.

Estrogen and tamoxifen reverse manganese‐induced glutamate transporter impairment in astrocytes

Chronic exposure to manganese (Mn) can cause manganism, a neurodegenerative disorder similar to Parkinson's disease. The toxicity of Mn includes impairment of astrocytic glutamate transporters. 17beta-Estradiol (E2) has been shown to be neuroprotective in various neurodegenerative diseases including Parkinson's disease and Alzheimer's disease, and some selective estrogen receptor modulators, including tamoxifen (TX), also possess neuroprotective properties. We have tested our hypothesis that E2 and TX reverse Mn-induced glutamate transporter impairment in astrocytes. The results established that E2 and TX increased glutamate transporter function and reversed Mn-induced glutamate uptake inhibition, primarily via the up-regulation of glutamate/aspartate transporter (GLAST). E2 and TX also increased astrocytic GLAST mRNA levels and attenuated the Mn-induced inhibition of GLAST mRNA expression. In addition, E2 and TX effectively increased the expression of transforming growth factor beta1, a potential modulator of the stimulatory effects of E2/TX on glutamate transporter function. This effect was mediated by the activation of MAPK/extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)/Akt signaling pathways. These novel findings suggest, for the first time, that E2 and TX enhance astrocytic glutamate transporter expression via increased transforming growth factor beta1 expression. Furthermore, the present study is the first to show that both E2 and TX effectively reverse Mn-induced glutamate transport inhibition by restoring its expression and activity, thus offering a potential therapeutic modality in neurodegenerative disorders characterized by altered glutamate homeostasis.

360 POSTER Ror2 in renal cell carcinoma: evaluating its role in RCC tumorigenesis

The α7 nicotinic acetylcholine (nACh) receptor expressed in microglia has a crucial role in neuroprotection. Simulation of α7 nACh receptor leads to increased expression of glutamate/aspartate transporter (GLAST), which in turn decreases synaptic glutamate levels. However, the upregulation of GLAST in cultured rat cortical microglia appears long after (over 18 h) stimulation of the α7 nACh receptor with nicotine. Thus, the current study elucidated the pathway responsible for the induction of GLAST expression in cultured cortical microglia. Nicotine-induced GLAST mRNA expression was significantly inhibited by cycloheximide pretreatment, indicating that a protein intermediary, such as a growth factor, is required for GLAST expression. The expression of fibroblast growth factor-2 (FGF-2) mRNA in cortical microglia was significantly increased 6 and 12 h after treatment with nicotine, and this increase was potently inhibited by pretreatment with methyllycaconitine, a selective α7 nACh receptor antagonist. The treatment with nicotine also significantly increased FGF-2 protein expression. Furthermore, treatment with recombinant FGF-2 increased GLAST mRNA, protein expression and 14C-glutamate uptake, a functional measurement of GLAST activity. Conversely, pretreatment with PD173074, an inhibitor of FGF receptor (FGFR) tyrosine kinase, significantly prevented the nicotine-induced expression of GLAST mRNA, its protein and 14C-glutamate uptake. Reverse transcription polymerase chain reaction confirmed FGFR1 mRNA expression was confined to cultured cortical microglia. Together, the current findings demonstrate that the neuroprotective effect of activation of microglial α7 nACh receptors could be due to the expression of FGF-2, which in turn increases GLAST expression, thereby clearing glutamate from synapse and decreasing glutamate neurotransmission.

Expression of glutamate transporters GLT-1 and GLAST in different regions of rat brain during the course of experimental autoimmune encephalomyelitis

Demyelination and oligodendroglial cell death accompanied by axonal injury are dominating features of multiple sclerosis (MS) a chronic demyelinating disease of the CNS. Accumulation of extracellular glutamate, observed during MS, is implicated in excitotoxic injury of nerve and oligodendroglial cells as a result of over-activation of glutamate receptors. The appropriate concentration of extracellular glutamate is maintained by glutamate transporters, the most predominant of which is glial transporter GLT-1 (excitatory amino acid transporter (EAAT) 2). The aim of this study is to determine the time-course of GLT-1 and glutamate-aspartate transporter (GLAST) expression in forebrain and cerebellum of rats subjected to experimental autoimmune encephalomyelitis (EAE). Our findings revealed that: (1) GLT-1 mRNA and to a lower extent GLAST mRNA are overexpressed in forebrain and cerebellum of EAE rats (2) expression of GLT-1 transporter mRNA shows a similar temporal pattern throughout the course of EAE in both structures examined, and is closely correlated with the appearance of neurological symptoms; and (3) the expression of GLT-1 and GLAST protein does not mirror mRNA changes during EAE and exhibits a differential spatial pattern. The protein levels of GLT-1 in cerebellum and GLAST in both structures are significantly reduced just before the acute phase and later during the recovery. The results imply that transcriptional up-regulation of the GLT-1 gene occurs early in both the forebrain and the cerebellum of the EAE rat model. This up-regulation is associated with the severity of symptoms but tends to precede the onset of maximal neurological deficits. The observations confirm the involvement of glutamate in the pathogenesis of EAE and provide an indication of the protective role of this glutamate transporter. However, changes in protein expression of both transporters suggest the existence of post-translational disturbances or the influence of regulating factors connecting with EAE conditions that may lead to the insufficient protection against glutamate excitotoxicity.

Inhibitory regulation of glutamate aspartate transporter (GLAST) expression in astrocytes by cadmium‐induced calcium influx

After injury to the CNS, the accumulation of extracellular glutamate induces neuronal excitotoxicity, leading to secondary tissue damage. Astrocytes can reduce excess extracellular glutamate primarily through the astrocytic glutamate transporter-1 and the Na(+)-dependent glutamate/aspartate transporter (GLAST). In this study, we used an in vitro model of cadmium-induced cellular stress and found that glutamate uptake activity of astrocytes was suppressed because of cadmium-induced inhibition of GLAST expression. The blockage of cadmium-triggered Ca(2+) influx by Ca(2+) chelators elevated GLAST transcription and glutamate uptake activity in astrocytes, suggesting that the suppression of GLAST expression in cadmium-treated astrocytes was Ca(2+)-dependent. This was supported by the findings showing the reduction of GLAST mRNA in astrocytes after treatment with Ca(2+)-ionophore A23187. Cadmium reduced human GLAST promoter activity; however, it increased the binding of Ca(2+)-sensitive activator protein-1 (AP-1) and cAMP response element binding protein (CREB) to their specific elements derived from the human GLAST promoter. These results demonstrate that AP-1 and CREB may be coupled with Ca(2+)-dependent pathway triggered by cadmium to mediate the inhibition of GLAST transcription. Our results suggest that Ca(2+) influx into astrocytes after CNS injury could cause the down-regulation of GLAST expression, thus reducing the astrocytic glutamate uptake function, which in turn may exacerbate secondary damage after CNS injury.

Methylmercury Alters the In Vitro Uptake of Glutamate in GLAST- and GLT-1-Transfected Mutant CHO-K1 Cells

In order to maintain normal functioning of the brain, glutamate homeostasis and extracellular levels of excitotoxic amino acids (EAA) must be tightly controlled. This is accomplished, in large measure, by the astroglial high-affinity Na+-dependent EAA transporters glutamate/ aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1). Methylmercury (MeHg) is a potent neurotoxicant. Astrocytes are known targets for MeHg toxicity, representing a site for mercury localization. MeHg is known to cause astrocytic swelling, EAA release, and uptake inhibition in astrocytes, leading to increased extracellular glutamate levels and ensuing neuronal excitotoxicity and degeneration. However, the mechanisms and contribution of specific glutamate transporters to MeHg-induced glutamate dyshomeostasis remain unknown. Accordingly, the present study was carried out to investigate the effects of MeHg on the transport of [d-2, 3-3H]-d-aspartate, a nonmetabolizable glutamate analog in Chinese hamster ovary cells (CHO) transfected with the glutamate transporter subtypes GLAST or GLT-1. Additional studies examined the effects of MeHg on mRNA and protein levels of these transporters. Our results indicate the following (1) MeHg selectively affects glutamate transporter mRNA expression. MeHg treatment (6 h) led to no discernible changes in GLAST mRNA expression; however, GLT-1 mRNA expression significantly (p < 0.001) increased following treatments with 5 or 10 microM MeHg. (2) Selective changes in the expression of glutamate transporter protein levels were also noted. GLAST transporter protein levels significantly (p < 0.001, both at 5 and 10 microM MeHg) increased and GLT-1 transporter protein levels significantly (p < 0.001) decreased following MeHg exposure (5 microM). (3) MeHg exposure led to significant inhibition (p < 0.05) of glutamate uptake by GLAST (both 5 and 10 microM MeHg), whereas GLT-1 transporter activity was significantly (p < 0.01) increased following exposure to 5 and 10 microM MeHg. These studies suggest that MeHg contributes to the dysregulation of glutamate homeostasis and that its effects are distinct for GLAST and GLT-1.

The acute effects of acrylamide on astrocyte functions.

We assessed biochemical endpoints indicative of acute toxicity in neonatal rat primary astrocyte cultures exposed to acrylamide. Metallothionein (MT), glutamine synthetase (GS), glutamate/aspartate transporter (GLAST), and taurine transporter (tau-T) mRNA expression levels as well as cell volume were determined in astrocytes acutely treated with 0.1 and 1.0 mM acrylamide. Statistically significant changes in acrylamide treated astrocytes were noted for GS (0.1 mM) and GLAST (1.0 mM) mRNA expression levels. All other measurements were insignificant in comparison with controls, suggesting that astrocytic function is minimally compromised even at exceedingly high levels of acute acrylamide exposure.

Glutamate/Aspartate Transporter (GLAST), Taurine Transporter and Metallothionein mRNA Levels are Differentially Altered in Astrocytes Exposed to Manganese Chloride, Manganese Phosphate or Manganese Sulfate

Manganese (Mn)-induced neurotoxicity can occur due to environmental exposure (air pollution, soil, water) and/or metabolic aberrations (decreased biliary excretion). High brain manganese levels lead to oxidative stress, as well as alterations in neurotransmitter metabolism with concurrent neurobehavioral deficits. Based on the few existing studies that have examined brain regional Mn concentration, it is likely that in pathological conditions, Mn concentration can reach between 100 and 500 μM. Environmental Mn exposure as a result of methylcyclopentadienyl manganese tricarbonyl (MMT) combustion is in the form of phosphate or sulfate (MnPO 4, MnSO 4, respectively). Pharmacokinetic studies have shown that the Mn salt will determine the rate of transport into the brain: MnCl 2> MnSO 4> MnPO 4. The salt-specific neurotoxicity of these species is unknown. The primary goal of this study was to examine gene expression of glutamate/aspartate transporter (GLAST), taurine transporter (tau-T), and metallothionein-I (MT-I) in astrocytes exposed to manganese chloride (MnCl 2), manganese sulfate (MnSO 4), and manganese phosphate (MnPO 4). We hypothesized that the effects of MnPO 4 and MnSO 4 exposure on GLAST expression in astrocytes would be similar to those induced by MnCl 2, since irrespective of salt species exposure, once internalized by astrocytes, the Mn ion would be identically complexed. At the same time, we hypothesized that the magnitude of the effect would be salt-dependent, since the chemical speciation would determine the rate of intracellular uptake of Mn. MnCl 2 caused a significant overall decrease ( P<0.0001) in astrocytic GLAST mRNA levels with MnSO 4 causing a moderate decrease. MnPO 4 exposure did not alter GLAST mRNA in astrocytes. We also sought to examine astrocytic metallothionein and taurine transporter gene expression as markers of manganese exposure. Our findings suggest that manganese chloride significantly decreased ( P<0.0001) astrocytic metallothionein mRNA compared to both the sulfate and phosphate species. However, astrocytic taurine transporter mRNA was not affected by Mn exposure, irrespective of the salt species. These data are consistent with the hypothesis that astrocytic neurotoxicity due to Mn exposure is dependent upon its species, with solubility, and by inference, intracellular concentration, representing a major determinant of its neurotoxicity.

Degradation of glial glutamate transporter mRNAs is selectively blocked by inhibition of cellular transcription.

Recent studies have demonstrated that the expression of the glial glutamate transporters GLT-1 (glutamate transporter 1) and GLAST (glutamate aspartate transporter) is regulated both in vivo and in vitro. For example, co-culturing with neurons, treatment with N:(6), 2'-O:-dibutyryladenosine 3':5'-cyclic monophosphate (dbcAMP), and treatment with epidermal growth factor all increase the steady-state levels of GLT-1 and GLAST protein in astrocyte cultures. These changes in protein expression are correlated with increased mRNA levels. In the present study, the degradation of GLT-1 and GLAST mRNAs was examined in control and dbcAMP-treated astrocyte cultures after inhibiting transcription with actinomycin D. Although one would predict that inhibition of transcription would cause a decrease in GLT-1 and GLAST mRNAs and that this decrease would depend on the rate of mRNA degradation, the levels of GLT-1 and GLAST mRNAs did not decrease even after 24 h of treatment with actinomycin D. Withdrawal of dbcAMP caused the levels of GLT-1 and GLAST mRNAs to fall to basal levels within 24 h, but this degradation was blocked if actinomycin D was added at the time of dbcAMP withdrawal. Importantly, actinomycin D did not block the degradation of c-fos mRNA also induced by dbcAMP in these cultures. Inhibition of translation with cycloheximide did not stabilize GLT-1 but partially attenuated the degradation of GLAST mRNA. Although the mechanism of this effect remains to be defined, these studies suggest that GLT-1 and GLAST mRNAs belong to a select class of inducible mRNAs stabilized by inhibitors of transcription. The possible relevance of these data to astrocyte differentiation is briefly discussed.

Inherited Defects of Sodium-dependent Glutamate Transport Mediated by Glutamate/Aspartate Transporter in Canine Red Cells Due to a Decreased Level of Transporter Protein Expression

Canine red cells have a high affinity Na(+)/K(+)-dependent glutamate transporter. We herein demonstrate that this transport is mediated by the canine homologue of glutamate/aspartate transporter (GLAST), one of the glutamate transporter subtypes abundant in the central nervous system. We also demonstrate that GLAST is the most ubiquitous glutamate transporter among the transporter subtypes that have been cloned to date. The GLAST protein content was extremely reduced in variant red cells, low glutamate transport (LGlut) red cells characterized by an inherited remarkable decrease in glutamate transport activity. All LGluT dogs carried a missense mutation of Gly(492) to Ser (G492S) in either the heterozygous or homozygous state. The GLAST protein with G492S mutation was fully functional in glutamate transport in Xenopus oocytes. However, G492S GLAST exhibited a marked decrease in activity after the addition of cycloheximide, while the wild type showed no significant change, indicating that G492S GLAST was unstable compared with the wild-type transporter. Moreover, LGluT dogs, but not normal dogs, heterozygous for the G492S mutation showed a selective decrease in the accumulation of GLAST mRNA from the normal allele. Based on these findings, we conclude that a complicated heterologous combination of G492S mutation and some transcriptional defect contributes to the pathogenesis of the LGluT red cell phenotype.