Glutamate-aspartate Transporter Protein Research Articles

Effects of GLAST gene knockout on phenotype and hearing in mice

To investigate the effects of glutamate aspartate transporter (GLAST)deletion on the normal auditory function of mice. We hybridized GLAST+/- mice with C57BL/6J background and identified the genotypes of their offspring by agarose gel electrophoresis. 9-10-week-old mice were selected to detect the expression of GLAST protein in the cochlea by immunofluorescence staining and to verify the knockout results(n=3). The changes in weight from 7 days to 30 days after birth and the 30-day body length of male and female mice were compared(n=8). The auditory brainstem response(ABR) was used to detect the auditory threshold and the amplitude of wave I in 9-10-week-old male and female mice(n=5). Male GLAST-/- mice had shown significantly lower weight and body length compared to male GLAST+/+ and GLAST+/- mice(P＜0.01), and male GLAST-/- mice showed significant differences compared to GLAST+/+ from P7 to P30 statistical time. Male GLAST-/- mice exhibited a significant reduction in weight after P15 compared to male GLAST+/- mice. In contrast, no significant differences in weight and body length were observed in female GLAST-/- mice compared with female GLAST+/+ and GLAST+/- mice. There was no difference in the hearing threshold detected by ABR between the three genotypes in both male and female mice, but the amplitude of wave I in GLAST-/- mice was significantly lower than that in male GLAST+/+ mice(P＜0.01). In contrast, the amplitude of wave I in females was reduced throughout the stimulus intensity but was most significant only at high-intensity stimulation (e.g.80 dB, 90 dB) (P＜0.05). GLAST knockout affects the normal growth and development of male mice, and decreases the amplitude of wave I, but do not change the threshold, suggesting that GLAST knockout may lead to synaptic pathological changes, and there are gender differences in this effect.

Disruption of Glutamate Release and Uptake-Related Protein Expression After Noise-Induced Synaptopathy in the Cochlea.

High-intensity noise can cause permanent hearing loss; however, short-duration medium-intensity noise only induces a temporary threshold shift (TTS) and damages synapses formed by inner hair cells (IHCs) and spiral ganglion nerves. Synaptopathy is generally thought to be caused by glutamate excitotoxicity. In this study, we investigated the expression levels of vesicle transporter protein 3 (Vglut3), responsible for the release of glutamate; glutamate/aspartate transporter protein (GLAST), responsible for the uptake of glutamate; and Na+/K+-ATPase α1 coupled with GLAST, in the process of synaptopathy in the cochlea. The results of the auditory brainstem response (ABR) and CtBP2 immunofluorescence revealed that synaptopathy was induced on day 30 after 100 dB SPL noise exposure in C57BL/6J mice. We found that GLAST and Na+/K+-ATPase α1 were co-localized in the cochlea, mainly in the stria vascularis, spiral ligament, and spiral ganglion cells. Furthermore, Vglut3, GLAST, and Na+/K+-ATPase α1 expression were disrupted after noise exposure. These results indicate that disruption of glutamate release and uptake-related protein expression may exacerbate the occurrence of synaptopathy.

Calcineurin Controls Expression of EAAT1/GLAST in Mouse and Human Cultured Astrocytes through Dynamic Regulation of Protein Synthesis and Degradation.

Alterations in the expression of glutamate/aspartate transporter (GLAST) have been associated with several neuropathological conditions including Alzheimer’s disease and epilepsy. However, the mechanisms by which GLAST expression is altered are poorly understood. Here we used a combination of pharmacological and genetic approaches coupled with quantitative PCR and Western blot to investigate the mechanism of the regulation of GLAST expression by a Ca2+/calmodulin-activated phosphatase calcineurin (CaN). We show that treatment of cultured hippocampal mouse and fetal human astrocytes with a CaN inhibitor FK506 resulted in a dynamic modulation of GLAST protein expression, being downregulated after 24–48 h, but upregulated after 7 days of continuous FK506 (200 nM) treatment. Protein synthesis, as assessed by puromycin incorporation in neo-synthesized polypeptides, was inhibited already after 1 h of FK506 treatment, while the use of a proteasome inhibitor MG132 (1 μM) shows that GLAST protein degradation was only suppressed after 7 days of FK506 treatment. In astrocytes with constitutive genetic ablation of CaN both protein synthesis and degradation were significantly inhibited. Taken together, our data suggest that, in cultured astrocytes, CaN controls GLAST expression at a posttranscriptional level through regulation of GLAST protein synthesis and degradation.

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.

Effect of subarachnoid nerve block anesthesia on glutamate transporter GLAST and GLT-1 expressions in rabbits.

To observe the effect of subarachnoid nerve block anesthesia on glutamate transporter glutamate-aspartate transporter (GLAST) and GLT-1 expressions in rabbits, and to investigate the effect of peripheral nerve anesthesia on the morphology and function of the spinal cord. Twenty healthy New Zealand white rabbits were randomly divided into two groups: the experimental group and control group; with 10 rabbits in each group. For spinal nerve anesthesia, 5g/L of bupivacaine was used in the experimental group, and sterile saline was used in the control group. After 30min of cardiac perfusion, GLAST and GLT-1 protein expression in spinal neurons were detected by immunohistochemistry and immunofluorescence staining. GLAST and GLT-1 protein-positive cells increased in neurons in the experimental group, compared with the control group (P<0.05). After subarachnoid nerve block anesthesia, rabbit glutamate transporter GLAST and GLT-1 expression is increased; and spinal cord nerve cell function is inhibited.

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.

Laminar and subcellular heterogeneity of GLAST and GLT‐1 immunoreactivity in the developing postnatal mouse hippocampus

Astrocytes express two sodium-coupled transporters, glutamate-aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1), which are essential for the maintenance of low extracellular glutamate levels. We performed a comparative analysis of the laminar and subcellular expression profile of GLAST and GLT-1 in the developing postnatal mouse hippocampus by using immunohistochemistry and western blotting and employing high-resolution fluorescence microscopy. Astrocytes were identified by costaining with glial fibrillary acidic protein (GFAP) or S100β. In CA1, the density of GFAP-positive cells and GFAP expression rose during the first 2 weeks after birth, paralleled by a steady increase in GLAST immunoreactivity and protein content. Upregulation of GLT-1 was completed only at postnatal days (P) P20-25 and was thus delayed by about 10 days. GLAST staining was highest along the stratum pyramidale and was especially prominent in astrocytes at P3-5. GLAST immunoreactivity indicated no preferential localization to a specific cellular compartment. GLT-1 exhibited a laminar expression pattern from P10-15 on, with the highest immunoreactivity in the stratum lacunosum-moleculare. At the cellular level, GLT-1 immunoreactivity did not entirely cover astrocyte somata and exhibited clusters at processes. In neonatal and juvenile animals, discrete clusters of GLT-1 were also detected at perivascular endfeet. From these results, we conclude there is a remarkable subcellular heterogeneity of GLAST and GLT-1 expression in the developing hippocampus. The clustering of GLT-1 at astrocyte endfeet indicates that it might serve a specialized functional role at the blood-brain barrier during formation of the hippocampal network.

Upregulation of Glutamate–Aspartate Transporter by Glial Cell Line–Derived Neurotrophic Factor Ameliorates Cell Apoptosis in Neural Retina in Streptozotocin‐Induced Diabetic Rats

Dysfunction of glutamate uptake, largely mediated by the glutamate-aspartate transporter (GLAST), may lead to retinal cell apoptosis in diabetic retinopathy. The aim of this study is to examine how cell apoptosis and the expression level of GLAST in neural retina of a diabetic rat model are changed and whether the neuroretinal apoptosis could be ameliorated by the administration of glial cell line-derived neurotrophic factor (GDNF). Diabetes was induced by intraperitoneal injection of streptozotocin (STZ) in Sprague-Dawley rats. GLAST protein expression levels were determined by Western blotting, whereas apoptosis of retinal neurons was evaluated by TUNEL staining. To assess the role of GDNF in ameliorating the STZ-induced retinal changes, GDNF/GDNF with siRNA directed against GLAST was injected into the vitreous after STZ injection. In rat retinas 4weeks after the onset of STZ-induced diabetes, TUNEL-positive cells were significantly increased, whereas GLAST levels were significantly reduced. Intraocular administration of GDNF at the early stage of diabetes remarkably increased the GLAST levels and decreased TUNEL-positive signals in the retinas. These effects of GDNF were largely abolished by coadministration of GLAST siRNA. GDNF, administrated at the early stage of diabetes, could rescue retinal cells from neurodegeneration by upregulating the expression of GLAST.

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.

Plasticity in expression of the glutamate transporters GLT-1 and GLAST in spinal dorsal horn glial cells following partial sciatic nerve ligation.

BackgroundClearance of synaptically released glutamate, and hence termination of glutamatergic neurotransmission, is carried out by glutamate transporters, most especially glutamate transporter-1 (GLT-1) and the glutamate-aspartate transporter (GLAST) that are located in astrocytes. It is becoming increasingly well appreciated that changes in the function and expression of GLT-1 and GLAST occur under different physiological and pathological conditions. Here we investigated the plasticity in expression of GLT-1 and GLAST in the spinal dorsal horn using immunohistochemistry following partial sciatic nerve ligation (PSNL) in rats.ResultsAnimals were confirmed to develop hypersensitivity to mechanical stimulation by 7 days following PSNL. Baseline expression of GLT-1 and GLAST in naive animals was only observed in astrocytes and not in either microglia or neurons. Microglia and astrocytes showed evidence of reactivity to the nerve injury when assessed at 7 and 14 days following PSNL evidenced by increased expression of OX-42 and GFAP, respectively. In contrast, the total level of GLT-1 and GLAST protein decreased at both 7 and 14 days after PSNL. Importantly, the cellular location of GLT-1 and GLAST was also altered in response to nerve injury. Whereas activated astrocytes showed a marked decrease in expression of GLT-1 and GLAST, activated microglia showed de novo expression of GLT-1 and GLAST at 7 days after PSNL and this was maintained through day 14. Neurons showed no expression of GLT-1 or GLAST at any time point.ConclusionThese results indicate that the expression of glutamate transporters in astrocytes and microglia are differentially regulated following nerve injury.

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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.

Increase in excitatory amino acid concentration and transporters expression in osteoarthritic knees of anterior cruciate ligament transected rabbits

The present study aimed to determine the role of excitatory amino acids (EAAs) and EAA transporters (EAATs) in an osteoarthritis (OA) model of rabbit knees. OA was induced in New Zealand white male rabbits by anterior cruciate ligament transection (ACLT) in one knee of one hind limb; the other knee left unoperated. Rabbits that received ACLT of knee were assigned to the ACLT group (n=6), while a sham-operated group (n=6) underwent arthrotomy with no ACLT. Six naïve rabbits that received no surgery were used as normal control. The width of the knee joint was measured to determine the severity of joint inflammation. Before operation and at 10, 20, and 30 weeks after operation, knee joint dialysates were collected by microdialysis and assayed for EAAs by high-performance liquid chromatography. Gross morphology and histopathology and EAATs glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) expression in the articular cartilage of the knees were evaluated by immunohistochemistry and western blot analysis. In the ACLT knees, a significant increase in the joint width was observed (5.3+/-0.9 mm, P<0.05) at 30 weeks after operation, while the sham-operated and naïve knees showed no difference as compared with the basal values. The concentrations (microM) of aspartate and glutamate in knee dialysates at 30 weeks after ACLT in naïve, sham, and ACLT were 0.36+/-0.07 and 4.5+/-1.10; 0.38+/-0.09 and 4.61+/-1.11; 0.67+/-0.18 and 9.71+/-2.89, respectively. Levels of glutamate and aspartate in the dialysates obtained from the ACLT knees increased by 213.3+/-29.6% and 187.5+/-33.8% (P<0.05) when compared to those in the sham-operated knees. Both naïve and ACLT chondrocytes were positively stained by antibodies against GLAST and GLT-1. GLAST and GLT-1 protein expressions were significantly increased in the ACLT knees (P<0.05). Our findings indicate an involvement of EAAs and EAATs in the pathogenesis of OA in ACLT rabbits.

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.

Variations in Promoter Activity Reveal a Differential Expression and Physiology of Glutamate Transporters by Glia in the Developing and Mature CNS

Glutamate transporters regulate excitatory neurotransmission and prevent glutamate-mediated excitotoxicity in the CNS. To better study the cellular and temporal dynamics of the expression of these transporters, we generated bacterial artificial chromosome promoter Discosoma red [glutamate-aspartate transporter (GLAST)] and green fluorescent protein [glutamate transporter-1 (GLT-1)] reporter transgenic mice. Analysis of these mice revealed a differential activation of the transporter promoters not previously appreciated. GLT-1 promoter activity in the adult CNS is almost completely restricted to astrocytes, often and unexpectedly in a nonoverlapping pattern with GLAST. Spinal cord GLT-1 promoter reporter, protein density, and physiology were 10-fold lower than in brain, suggesting a possible mechanism for regional sensitivity seen in disease. The GLAST promoter is active in both radial glia and many astrocytes in the developing CNS but is downregulated in most astrocytes as the mice mature. In the adult CNS, the highest GLAST promoter activity was observed in radial glia, such as those located in the subgranular layer of the dentate gyrus. The continued expression of GLAST by these neural progenitors raises the possibility that GLAST may have an unanticipated role in regulating their behavior. In addition, GLAST promoter activation was observed in oligodendrocytes in white matter throughout many (e.g., spinal cord and corpus callosum), but not all (e.g., cerebellum), CNS fiber tracts. Overall, these studies of GLT-1 and GLAST promoter activity, protein expression, and glutamate uptake revealed a close correlation between transgenic reporter signals and uptake capacity, indicating that these mice provide the means to monitor the expression and regulation of glutamate transporters in situ.

Regulation of astrocytic glutamate transporter expression by Akt: evidence for a selective transcriptional effect on the GLT‐1/EAAT2 subtype

In the nervous system, astrocytes express different ratios of the two glial glutamate transporters, glutamate transporter subtype 1 (GLT-1) and glutamate/aspartate transporter (GLAST), but little is known about the signaling pathways that independently regulate their expression. Treatment with dibutyryl-cAMP, epidermal growth factor (EGF) or other growth factors both induces expression of GLT-1 and increases expression of GLAST in astrocyte cultures. The induction of GLT-1 is correlated with morphological and biochemical changes that are consistent with astrocyte maturation. Pharmacological studies suggest that phosphatidylinositol 3-kinase (PI-3K) and the nuclear transcription factor-kappaB (NF-kappaB) may be involved in the induction of GLT-1 expression. In several signaling systems Akt, also known as protein kinase B (PKB), functions downstream of PI-3K. In these present studies we used lentiviral vectors engineered to express dominant-negative (DN), constitutively active (CA), or null variants of Akt to study the possible involvement of Akt in the regulation of GLT-1. Expression of DN-Akt attenuated the EGF-dependent induction of GLT-1. Expression of CA-Akt caused a dose- and time-dependent increase in GLT-1 protein, increased GLT-1 mRNA levels, increased dihydrokainate-sensitive (presumably GLT-1 mediated) transport activity, and caused a change in astrocyte morphology to a more stellate shape, but had no effect on GLAST protein levels. Finally, the expression of CA-Akt increased the expression of a reporter construct containing a putative promoter fragment from the human homolog of GLT-1, called EAAT2. From these studies, we conclude that Akt induces the expression of GLT-1 through increased transcription and that Akt can regulate GLT-1 expression without increasing GLAST expression in astrocytes.

Differential regulation of GLAST immunoreactivity and activity by protein kinase C: evidence for modification of amino and carboxyl termini

Many neurotransmitter transporters, including the GLT-1 and EAAC1 subtypes of the glutamate transporter, are regulated by protein kinase C (PKC) and these effects are associated with changes in cell surface expression. In the present study, the effects of PKC activation on the glutamate aspartate transporter (GLAST) subtype of glutamate transporter were examined in primary astrocyte cultures. Acute (30 min) exposure to the phorbol 12-myristate 13-acetate (PMA) increased (approximately 20%) transport activity but had the opposite effect on both total and cell surface immunoreactivity. Chronic treatment (6 or 24 h) with PMA had no effect on transport activity but caused an even larger decrease in total and cell surface immunoreactivity. This loss of immunoreactivity was observed using antibodies directed against three different cytoplasmic epitopes, and was blocked by the PKC antagonist, bisindolylmaleimide II. We provide biochemical and pharmacological evidence that the activity observed after treatment with PMA is mediated by GLAST. Two different flag-tagged variants of the human homolog of GLAST were introduced into astrocytes using lentiviral vectors. Although treatment with PMA caused a loss of transporter immunoreactivity, flag immunoreactivity did not change in amount or size. Together, these studies suggest that activation of PKC acutely up-regulates GLAST activity, but also results in modification of several different intracellular epitopes so that they are no longer recognized by anti-GLAST antibodies. We found that exposure of primary cultures of neurons/astrocytes to transient hypoxia/glucose deprivation also caused a loss of GLAST immunoreactivity that was attenuated by the PKC antagonist, bisindolylmaleimide II, suggesting that some acute insults previously thought to cause a loss of GLAST protein may mimic the phenomenon observed in the present study.

Müller cell response to laser-induced increase in intraocular pressure in rats.

The goal of this study was to investigate the reaction of the Müller cells to elevated intraocular pressure (IOP). Elevated IOP is one of the risk factors in glaucomatous retinal ganglion cell (RGC) degeneration. Müller cells play an important role in retinal homeostasis. The reaction of Müller cells was examined by evaluating temporal changes in glutamate aspartate transporter (GLAST), glutamine synthase (GS), glial fibrillary acidic protein (GFAP), and the B-cell lymphoma (Bcl-2) using immunoblotting and immunohistochemical techniques. After IOP was elevated for 4-60 days, there was a time-related decrease in RGC ranging from 6% to 44%. There was also a time-related increase in GLAST protein reaching maximum after 3 weeks of elevated IOP. On the other hand, there was very little change in the expression of GS during the first 2 weeks followed by some increase between 21 and 60 days. An increase in Bcl-2 was biphasic with maximum increase after 4 days followed by decline after 15 and 21 days. GFAP, which is usually not expressed in normal Müller cells, was present at all time points. In all cases, the increase was most intense in the vicinity of the ganglion cells where the astrocytes and endfeet of the Müller cells are located. These results indicate that Müller cells react to the insult of elevated IOP by expressing GFAP and Bcl-2, proteins that are expressed in reactive gliosis and other pathological conditions. The increase in GLAST along with minimum change in GS indicates a disturbance in glutamate homeostasis.