Regional Deafferentiation Down-Regulates Subtypes of Glutamate Transporter Proteins

Abstract: Low extracellular glutamate content is maintained primarily by high-affinity sodium-dependent glutamate transport. Three glutamate transporter proteins have been cloned: GLT-1 and GLAST are astroglial, whereas EAAC1 is neuronal. The effects of axotomy on glutamate transporter expression was evaluated in adult rats following unilateral fimbria-fornix and corticostriatal lesions. The hippocampus and striatum were collected at 3, 7, 14, and 30 days postlesion. Homogenates were immunoblotted using antibodies directed against GLT-1, GLAST, EAAC1, and glial fibrillary acidic protein and assayed for glutamate transport by d -[³H]aspartate binding. GLT-1 immunoreactivity was decreased within the ipsilateral hippocampus and striatum at 14 days postlesion. GLAST immunoreactivity was decreased within the ipsilateral hippocampus and striatum at 7 and 14 days postlesion. No alterations in EAAC1 immunoreactivity were observed. d -[³H]Aspartate binding was decreased at 14 days postlesion within the ipsilateral hippocampus and at 7 and 14 days postlesion within the ipsilateral striatum. By 30 days postlesion, glutamate transporters and d -[³H]aspartate binding returned to control levels. This study demonstrates the down-regulation of primarily glial, and not neuronal, glutamate transporters following regional disconnection.     

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.     

Changes in expression of neuronal and glial glutamate transporters in lead-exposed adult rat brain

Excitatory amino acid transporters (EAATs) are membrane-bound proteins localized in glial and neuronal cells which transport glutamate (Glu) in a process essential for terminating its action and protecting neurons from excitotoxic damage. Since Pb-induced neurotoxicity has a glutamatergic component and astrocytes serve as a cellular Pb deposition site, it was of interest to investigate the response of main glutamate transporters to short-term lead exposure in the adult rat brain (25mg/kg b.w. of lead acetate, i.p. for 3 days). We examined the expression of mRNA and protein of GLAST, GLT-1 and EAAC1 in homogenates obtained from cerebellum, hippocampus and forebrain. Molecular evidence is provided which indicates that, of the two glial transporters, GLT-1 is more susceptible than GLAST to the neurotoxic effect arising from Pb. RT-PCR analysis revealed highly decreased expression of GLT-1 mRNA in forebrain and hippocampus. In contrast, GLAST was overexpressed in forebrain and in cerebellum. In the case of EAAC1, the enhanced expression of mRNA and protein of transporter was observed only in forebrain. The results demonstrate regional differences in the expression of glutamate transporters after short-term exposure to Pb. In forebrain, downregulation of GLT-1 is compensated by enhanced expression of GLAST, while in hippocampus, the expression of both is lowered. This observation suggests that under conditions of Pb toxicity in adult rat brain, the hippocampus is most vulnerable to the excitotoxic cell damage arising from impaired clearance of the released glutamate.     

Selective induction of glial glutamate transporter GLT-1 by hypertonic stress in C6 glioma cells.

Glial glutamate transporter GLT-1 mRNA was selectively induced in C6 glioma cells exposed to hypertonic stress (HS), while the expression of two other subtypes, GLAST and EAAC1, was suppressed. HS increased phosphorylation of the MAPK family, ERK, p38 MAPK, and JNK. Treatment with a PKC inhibitor showed that phosphorylation of both p38 MAPK and JNK is PKC-dependent but ERK phosphorylation is independent. Inhibition of either ERK or p38 MAPK did not abolish GLT-1 mRNA induction. Inhibition of PKC also had no effect. These findings indicate that the induction of GLT-1 mRNA by HS is independent of the MAPK pathways. This is the first report that the expression of glial glutamate transporters is osmotically regulated.     

Fimbria-Fornix Transections Selectively Down-Regulate Subtypes of Glutamate Transporter and Glutamate Receptor Proteins in Septum and Hippocampus

Abstract: The effects of CNS axotomy on glutamate transporter and glutamate receptor expression were evaluated in adult rats following unilateral fimbria-fornix transections. The septum and hippocampus were collected at 3, 7, 14, and 30 days postlesion. Homogenates were immunoblotted by using antibodies directed against glutamate transporters (GLT-1, GLAST, and EAAC1) and glutamate receptors (GluR1, GluR2/3, GluR6/7, and NMDAR1), and they were assayed for glutamate transport by d -[³H]aspartate binding. GLT-1 was decreased at 7 and 14 days postlesion within the ipsilateral septum and at 7 days postlesion in the hippocampus. GLAST was decreased within the ipsilateral septum and hippocampus at 7 and 14 days postlesion. No postlesion alterations in EAAC1 immunoreactivity were observed. d -[³H]Aspartate binding was decreased at 7, 14, and 30 days postlesion within the ipsilateral septum and 14 days postlesion in the hippocampus. GluR2/3 expression was down-regulated at 30 days postlesion within the ipsilateral septum, whereas GluR1, GluR6/7, and NMDAR1 immunoreactivity was unchanged. In addition, no alterations in glutamate receptor expression were detected within hippocampal homogenates. This study demonstrates a selective down-regulation of primarily glial, and not neuronal, glutamate transporters and a delayed, subtype-specific down-regulation of septal GluR2/3 receptor expression after regional deafferentation within the CNS.     

Glial glutamate transporter GLT-1 down-regulation precedes delayed neuronal death in gerbil hippocampus following transient global cerebral ischemia

Glial (GLT-1 and GLAST) and neuronal (EAAC1) high-affinity transporters mediate the sodium dependent glutamate reuptake in mammalian brain. Their dysfunction leads to neuronal damage by allowing glutamate to remain in the synaptic cleft for a longer duration. The purpose of the present study is to understand their contribution to the ischemic delayed neuronal death seen in gerbil hippocampus following transient global cerebral ischemia. The protein levels of these three transporters were studied by immunoblotting as a function of reperfusion time (6 h to 7 days) following a 10 min occlusion of bilateral common carotid arteries in gerbils. In the vulnerable hippocampus, there was a significant decrease in the protein levels of GLT-1 (by 36-46%, P < 0.05; between 1 and 3 days of reperfusion) and EAAC1 (by 42-68%, P < 0.05; between 1 and 7 days of reperfusion). Histopathological evaluation showed no neuronal loss up to 2 days of reperfusion but an extensive neuronal loss (by approximately 84%, P < 0.01) at 7 days of reperfusion in the hippocampal CA1 region. The time frame of GLT-1 dysfunction (1-3 days of reperfusion) precedes the initiation of delayed neuronal death (2-3 days of reperfusion). This suggests GLT-1 dysfunction as a contributing factor for the hippocampal neuronal death following transient global cerebral ischemia. Furthermore, decreased EAAC1 levels may contribute to GABAergic dysfunction and excitatory/inhibitory imbalance following transient global ischemia.     

The Rates of Postmortem Proteolysis of Glutamate Transporters Differ Dramatically between Cells and between Transporter Subtypes

Glutamate transporters (GLT-1, GLAST, EAAC1) limit the actions of excitatory amino acids. Because a disturbed transporter operation can cause or aggravate neurological diseases, transporters are of considerable neuropathological interest. Human samples, however, are seldom obtained fresh. Here, we used mice brains to study how fast glutamate transporters are degraded after death. Immunoblots showed that terminal GLT-1 epitopes (within residues 1–26 and 518–573) had mostly disappeared after 24 hr. GLAST termini (1–25 and 522–543) degraded slightly slower. In contrast, epitopes within central parts of GLT-1 (493–508) and the EAAC1 C-terminus (510–523) were readily detectable after 72 hr. The decline in immunoreactivity of the GLT-1 and GLAST termini was also seen in tissue sections, but proteolysis did not happen synchronously in all cells. At 24 hr, scattered cells remained strongly immunopositive, while the majority of cells were completely immunonegative. GLAST and GLT-1 co-localized in neocortical tissue, but at 12 hr, many GLAST-positive cells had lost the GLT-1 termini. The uneven disappearance of labeling was not observed with the antibodies to GLT-1 residues 493–508. The immunoreactivity to this epitope correlated better with the reported glutamate uptake activity. Thus, postmortem delay may affect epitopes differently, possibly causing erroneous conclusions about relative expression levels.     

Transient focal cerebral ischemia down-regulates glutamate transporters GLT-1 and EAAC1 expression in rat brain

Transient focal cerebral ischemia leads to extensive excitotoxic neuronal damage in rat cerebral cortex. Efficient reuptake of the released glutamate is essential for preventing glutamate receptor over-stimulation and neuronal death. Present study evaluated the expression of the glial (GLT-1 and GLAST) and neuronal (EAAC1) subtypes of glutamate transporters after transient middle cerebral artery occlusion (MCAO) induced focal cerebral ischemia in rats. Between 24h to 72h of reperfusion after transient MCAO, GLT-1 and EAAC1 protein levels decreased significantly (by 36% to 56%, p < 0.05) in the ipsilateral cortex compared with the contralateral cortex or sham control. GLT-1 and EAAC1 mRNA expression also decreased in the ipsilateral cortex of ischemic rats at both 24h and 72h of reperfusion, compared with the contralateral cortex or sham control. Glutamate transporter down-regulation may disrupt the normal clearance of the synaptically-released glutamate and may contribute to the ischemic neuronal death.     

A carboxyl-terminal determinant of the neuronal glutamate transporter, EAAC1, is required for platelet-derived growth factor-dependent trafficking

The neuronal glutamate transporter, EAAC1 (excitatory amino acid carrier 1), undergoes rapid regulation after treatment with platelet-derived growth factor (PDGF) or phorbol ester in C6 glioma cells and neurons. A large intracellular pool of EAAC1 exists, from which transporters are redistributed to the cell surface in response to these signals. Here we show that PDGF had no effect on subcellular localization of the glial glutamate transporter, GLT-1, after transfection into C6 glioma cells. Chimeras consisting of domains from EAAC1 or GLT-1 were used to investigate structural motifs involved in PDGF-dependent redistribution of EAAC1. PDGF did not induce trafficking of an EAAC1 chimera containing the carboxyl-terminal domain of GLT-1; however, it did induce trafficking of a GLT-1 chimera containing the carboxyl-terminal domain of EAAC1. A truncated mutant of EAAC1 lacking 10 carboxyl-terminal amino acids was responsive to PDGF, whereas a mutant lacking 20 residues was not. Alanine substitution mutagenesis in this region revealed a short motif, (502)YVN(504), necessary for regulated trafficking. This motif was also involved in protein kinase C-dependent trafficking, as mutant transporters exhibited an attenuated response to phorbol ester. Interestingly, the presence of YVN in the homologous region of a nonresponsive chimera was not sufficient to confer regulated trafficking; however, the presence of a 12-amino acid motif starting at this Tyr residue was sufficient to confer responsiveness to PDGF. These studies identify a novel motif within the carboxyl terminus of EAAC1 which is required for regulated trafficking. The possibility that this motif targets EAAC1 to an intracellular, "regulated pool" is discussed.     

Neurotoxic and neuroprotective effects of the glutamate transporter inhibitor DL-threo-beta-benzyloxyaspartate (DL-TBOA) during physiological and ischemia-like conditions

Maintenance of low extracellular glutamate ([Glu](O)) preventing excitotoxic cell death requires fast removal of glutamate from the synaptic cleft. This clearance is mainly provided by high affinity sodium-dependent glutamate transporters. These transporters can, however, also be reversed and release glutamate to the extracellular space in situations with energy failure. In this study the cellular localisation of the glutamate transporters GLAST and GLT-1 in organotypic hippocampal slice cultures was studied by immunofluorescence confocal microscopy, under normal culture conditions, and after a simulated ischemic insult, achieved by oxygen and glucose deprivation (OGD). In accordance with in vivo findings, GLAST and GLT-1 were primarily expressed by astrocytes under normal culture conditions, but after OGD some damaged neurons also expressed GLAST and GLT-1. The potential damaging effect of inhibition of the glutamate transporters by DL-threo-beta-benzyloxyaspartate (DL-TBOA) was studied using cellular uptake of propidium iodide (PI) as a quantitative marker for the cell death. Addition of DL-TBOA for 48 h was found to induce significant cell death in all hippocampal regions, with EC(50) values ranging from 38 to 48 microM for the different hippocampal subregions. The cell death was prevented by addition of the glutamate receptor antagonists NBQX and MK-801, together with an otherwise saturating concentration of DL-TBOA (100 microM). Finally, the effect of inhibition of glutamate release, via reverse operating transporters during OGD, was investigated. Addition of a sub-toxic (10 microM) dose of DL-TBOA during OGD, but not during the subsequent 48 h recovery period, significantly reduced the OGD-induced PI uptake. It is concluded: (1) that the cellular expression of the glutamate transporters GLAST and GLT-1 in hippocampal slice cultures in general corresponds to the expression in vivo, (2) that inhibition of the glutamate transporters induces cell death in the slice cultures, and (3) that partial inhibition during simulation of ischemia by OGD protects against the induced PI uptake, most likely by blocking the reverse operating transporters otherwise triggered by the energy failure.     

Protein kinase C activation decreases cell surface expression of the GLT-1 subtype of glutamate transporter. Requirement of a carboxyl-terminal domain and partial dependence on serine 486

Na(+)-dependent glutamate transporters are required for the clearance of extracellular glutamate and influence both physiological and pathological effects of this excitatory amino acid. In the present study, the effects of a protein kinase C (PKC) activator on the cell surface expression and activity of the GLT-1 subtype of glutamate transporter were examined in two model systems, primary co-cultures of neurons and astrocytes that endogenously express GLT-1 and C6 glioma cells transfected with GLT-1. In both systems, activation of PKC with phorbol ester caused a decrease in GLT-1 cell surface expression. This effect is opposite to the one observed for the EAAC1 subtype of glutamate transporter (Davis, K. E., Straff, D. J., Weinstein, E. A., Bannerman, P. G., Correale, D. M., Rothstein, J. D., and Robinson, M. B. (1998) J. Neurosci. 18, 2475-2485). Several recombinant chimeric proteins between GLT-1 and EAAC1 transporter subtypes were generated to identify domains required for the subtype-specific redistribution of GLT-1. We identified a carboxyl-terminal domain consisting of 43 amino acids (amino acids 475-517) that is required for PKC-induced GLT-1 redistribution. Mutation of a non-conserved serine residue at position 486 partially attenuated but did not completely abolish the PKC-dependent redistribution of GLT-1. Although we observed a phorbol ester-dependent incorporation of (32)P into immunoprecipitable GLT-1, mutation of serine 486 did not reduce this signal. We also found that chimeras containing the first 446 amino acids of GLT-1 were not functional unless amino acids 475-517 of GLT-1 were also present. These non-functional transporters were not as efficiently expressed on the cell surface and migrated to a smaller molecular weight, suggesting that a subtype-specific interaction is required for the formation of functional transporters. These studies demonstrate a novel effect of PKC on GLT-1 activity and define a unique carboxyl-terminal domain as an important determinant in cellular localization and regulation of GLT-1.     

高亲和力谷氨酸转运体结构和功能的研究进展

真核生物高亲和力谷氨酸转运体(excitatory amino acid transporters,EAATs)分为GLAST(EAAT1)、GLT-1(EAAT2)、EAAC1(EAAT3)、EAAT4和EAAT5等5个亚型.高亲和力谷氨酸转运体结构学的研究,揭示了谷氨酸转运体的跨膜拓扑结构、真核和原核生物EAATs结构的差异,以及在底物转运过程中的一些底物和协同转运离子的结合位点.其功能学的研究发现,EAATs在参与突触的传递,避免兴奋性氨基酸的毒性效应中发挥重要作用,同时也参与了对学习、记忆以及运动行为的调控.结合我们既往的工作,就近几年EAATs的结构和功能研究做一综述.     

Short-term alterations in hippocampal glutamate transport system caused by one-single neonatal seizure episode: implications on behavioral performance in adulthood

Impairment in the activity and expression of glutamate transporters has been found in experimental models of epilepsy in adult animals. However, there are few studies investigating alterations on glutamate transporters caused by epilepsy in newborn animals, especially in the early periods after seizures. In this study, alterations in the hippocampal glutamate transporters activity and immunocontent were investigated in neonatal rats (7 days old) submitted to kainate-induced seizures model. Glutamate uptake, glutamate transporters (GLT-1, GLAST, EAAC1) and glutamine synthetase (GS) were assessed in hippocampal slices obtained 12 h, 24 h, 48 h, 72 h and 60 days after seizures. Immunoreactivity for hippocampal GFAP, NeuN and DAPI were assessed 24 h after seizure. Behavioral analysis (elevated-plus maze and inhibitory avoidance task) was also investigated in the adult animals (60 days old). The decrease on glutamate uptake was observed in hippocampal slices obtained 24 h after seizures. The immunocontent of GLT-1 increased at 12 h and decreased at 24 h (+62% and −20%, respectively), while GLAST increased up to 48 h after seizures. No alterations were observed for EAAC1 and GS. It should be mentioned that there were no long-term changes in tested glutamate transporters at 60 days after kainate treatment. GFAP immunoreactivity increased in all hippocampal subfields (CA1, CA3 and dentate gyrus) with no alterations in NeuN and DAPI staining. In the adulthood, kainate-treated rats showed anxiety-related behavior and lower performance in the inhibitory avoidance task. Our findings indicate that acute modifications on hippocampal glutamate transporters triggered by a single convulsive event in early life may play a role in the behavioral alterations observed in adulthood.     

Transport of L-[14C]cystine and L-[14C]cysteine by subtypes of high affinity glutamate transporters over-expressed in HEK cells

Transport of L-cystine across the cell membrane is essential for synthesis of the major cellular antioxidant, glutathione (gamma-glutamylcysteinylglycine). In this study, uptake of L-[14C]cystine by three of the high affinity sodium-dependent mammalian glutamate transporters (GLT1, GLAST and EAAC1) individually expressed in HEK cells has been determined. All three transporters display saturable uptake of L-[14C]cystine with Michaelis affinity (K(m)) constants in the range of 20-110 microM. L-glutamate and L-homocysteate are potent inhibitors of sodium-dependent L-[14C]cystine uptake in HEK(GLAST), HEK(GLT1) and HEK(EAAC1) cells. Reduction of L-[14C]cystine to L-[14C]cysteine in the presence of 1mM cysteinylglycine increases the uptake rate in HEK(GLT1), HEK(GLAST) and HEK(EAAC1) cells, but only a small proportion (<10%) of L-[14C]cysteine uptake in HEK(GLT1) and HEK(GLAST) cells occurs by the high affinity glutamate transporters. The majority (>90%) of L-[14C]cysteine transport in these cells is mediated by the ASC transport system. In HEK(EAAC1) cells, on the other hand, L-[14C]cysteine is transported equally by the ASC and EAAC1 transporters. L-homocysteine inhibits L-[14C]cysteine transport in both HEK(GLAST) and HEK(GLT1) cells, but not in HEK(EAAC1) cells. It is concluded that the quantity of L-[14C]cyst(e)ine taken up by individual high affinity sodium-dependent glutamate transporters is determined both by the extracellular concentration of amino acids, such as glutamate and homocysteine, and by the extracellular redox potential, which will control the oxidation state of L-cystine.     

Hypoxia regulates glutamate metabolism and membrane transport in rat PC12 cells

We investigated the effect of hypoxia on glutamate metabolism and uptake in rat pheochromocytoma (PC12) cells. Various key enzymes relevant to glutamate production, metabolism and transport were coordinately regulated by hypoxia. PC12 cells express two glutamate-metabolizing enzymes, glutamine synthetase (GS) and glutamate decarboxylase (GAD), as well as the glutamate-producing enzyme, phosphate-activated glutaminase (PAG). Exposure to hypoxia (1% O(2)) for 6 h or longer increased expression of GS mRNA and protein and enhanced GS enzymatic activity. In contrast, hypoxia caused a significant decrease in expression of PAG mRNA and protein, and also decreased PAG activity. In addition, hypoxia led to an increase in GAD65 and GAD67 protein levels and GAD enzymatic activity. PC12 cells express three Na(+)-dependent glutamate transporters; EAAC1, GLT-1 and GLAST. Hypoxia increased EAAC1 and GLT-1 protein levels, but had no effect on GLAST. Chronic hypoxia significantly enhanced the Na(+)-dependent component of glutamate transport. Furthermore, chronic hypoxia decreased cellular content of glutamate, but increased that of glutamine. Taken together, the hypoxia-induced changes in enzymes related to glutamate metabolism and transport are consistent with a decrease in the extracellular concentration of glutamate. This may have a role in protecting PC12 cells from the cytotoxic effects of glutamate during chronic hypoxia.     

Corticosterone decreases the activity of rat glutamate transporter type 3 expressed in Xenopus oocytes

Glucocorticoids can increase the extracellular concentrations of glutamate, the major excitatory neurotransmitter. We investigated the effects of corticosterone on the activity of a glutamate transporter, excitatory amino acid carrier 1 (EAAC1; also called excitatory amino acid transporter type 3 [EAAT3]), and the roles of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) in regulating these effects. Rat EAAC1 was expressed in Xenopus oocytes by injecting mRNA. l-Glutamate (30 μM)-induced membrane currents were measured using the two-electrode voltage clamp technique. Exposure of these oocytes to corticosterone (0.01–1 μM) for 72 h decreased EAAC1 activity in a dose-dependent fashion, and this inhibition was incubation time-dependent. Corticosterone (0.01 μM for 72 h) significantly decreased the V_max, but not the K_m, of EAAC1 for glutamate. Furthermore, pretreatment of oocytes with staurosporine, a PKC inhibitor, significantly decreased EAAC1 activity (1.00 ± 0.06 to 0.70 ± 0.05 μC; P < 0.05). However, no statistical differences were observed between oocytes treated with staurosporine, corticosterone, or corticosterone plus staurosporine. Similar patterns of responses were achieved by chelerythrine or calphostin C, other PKC inhibitors. Phorbol-12-myristate-13-acetate (PMA), a PKC activator, inhibited corticosterone-induced reduction in EAAC1 activity. Pretreating oocytes with wortmannin or LY294002, PI3K inhibitors, also significantly reduced EAAC1 activity, but no difference was observed between oocytes treated with wortmannin, corticosterone, or wortmannin plus corticosterone. The above results suggest that corticosterone exposure reduces EAAC1 activity and this effect is PKC- and PI3K-dependent.     

Platelet-derived growth factor rapidly increases activity and cell surface expression of the EAAC1 subtype of glutamate transporter through activation of phosphatidylinositol 3-kinase

Na(+)-dependent glutamate transporters are the primary mechanism for removal of excitatory amino acids (EAAs) from the extracellular space of the central nervous system and influence both physiologic and pathologic effects of these compounds. Recent evidence suggests that the activity and cell surface expression of a neuronal subtype of glutamate transporter, EAAC1, are rapidly increased by direct activation of protein kinase C and are decreased by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-K). We hypothesized that this regulation could be analogous to insulin-induced stimulation of the GLUT4 subtype of glucose transporter, which is dependent upon activation of PI3-K. Using C6 glioma, a cell line that endogenously and selectively expresses EAAC1, we report that platelet-derived growth factor (PDGF) increased Na(+)-dependent L-[(3)H]-glutamate transport activity within 30 min. This effect of PDGF was not due to a change in total cellular EAAC1 immunoreactivity but was instead correlated with an increase cell surface expression of EAAC1, as measured using a membrane impermeant biotinylation reagent combined with Western blotting. A decrease in nonbiotinylated intracellular EAAC1 was also observed. These studies suggest that PDGF causes a redistribution of EAAC1 from an intracellular compartment to the cell surface. These effects of PDGF were accompanied by a 35-fold increase in PI3-K activity and were blocked by the PI3-K inhibitors, wortmannin and LY 294002, but not by an inhibitor of protein kinase C. Other growth factors, including insulin, nerve growth factor, and epidermal growth factor had no effect on glutamate transport nor did they increase PI3-K activity. These studies suggest that, as is observed for insulin-mediated translocation of GLUT4, EAAC1 cell surface expression can be rapidly increased by PDGF through activation of PI3-K. It is possible that this PDGF-mediated increase in EAAC1 activity may contribute to the previously demonstrated neuroprotective effects of PDGF.     

GDNF pre-treatment aggravates neuronal cell loss in oxygen-glucose deprived hippocampal slice cultures: a possible effect of glutamate transporter up-regulation

Besides its neurotrophic and neuroprotective effects on dopaminergic neurons and spinal motoneurons, glial cell line-derived neurotrophic factor (GDNF) has potent neuroprotective effects in cerebral ischemia. The protective effect has so far been related to reduced activation of N-methyl-D-aspartate receptors (NMDAr). This study tested the effects of GDNF on glutamate transporter expression, with the hypothesis that modulation of glutamate transporter activity would affect the outcome of cerebral ischemia. Organotypic hippocampal slice cultures, derived from 1-week-old rats, were treated with 100 ng/ml GDNF for either 2 or 5 days, followed by Western blot analysis of NMDAr subunit 1 (NR1) and two glutamate transporter subtypes, GLAST and GLT-1. After 5-day exposure to GDNF, expression of GLAST and GLT-1 was up-regulated to 169 and 181% of control values, respectively, whereas NR1 was down-regulated to 64% of control. However, despite these changes that potentially would support neuronal resistance to excitotoxicity, the long-term treatment with GDNF was found to aggravate the neuronal damage induced by oxygen-glucose deprivation (OGD). The increased cell death, assessed by propidium iodide (PI) uptake, occurred not only among the most susceptible CA1 pyramidal cells, but also in CA3 and fascia dentata. Given that glutamate transporters are able to release glutamate by reversed action during energy failure, it is suggested that the observed increase in OGD-induced cell death in the GDNF-pretreated cultures was caused by the build-up of excitotoxic concentrations of extracellular glutamate released through the glutamate transporters, which were up-regulated by GDNF. Although the extent and consequences of glutamate release via reversal of GLAST and GLT-1 transporters seem to vary in different energy failure models, the present findings should be taken into account in clinical trials of GDNF.     

Elevated cortical extracellular fluid glutamate in transgenic mice expressing human mutant (G93A) Cu/Zn superoxide dismutase

Transgenic mice expressing a mutated (G93A) human Cu/Zn superoxide dismutase (SOD1) develop motor neuron pathology and clinical symptoms similar to those seen in patients with amyotrophic lateral sclerosis. Loss of motor neurons is most prominent in lumbar, followed by cervical cord and then brainstem. No significant cell death has been reported in motor cortex. The integrity of the cortical glutamate reuptake systems was evaluated using intracerebral microdialysis and western immunoblot assays for the glutamate transporters GLT-1, GLAST, and EAAC1. The basal extracellular fluid levels of aspartate, glutamate, glutamine, 3,4-dihydroxyphenylacetic acid, and 5-hydroxyindole-3-acetic acid were evaluated by HPLC. The extraction fraction of L-3H]glutamate, corrected with [14C]mannitol, was also evaluated. GLT-1, EAAC1, and GLAST protein levels were determined by semiquantitative chemiluminescence immunoblot of proteins from membrane-enriched fractions. The relative optical density of film was translated into relative protein level by comparison with a standard control mouse. The SOD1 mutant mice demonstrated a significant (p < 0.05) increase in basal levels of extracellular aspartate and glutamate. In addition, when the glutamate extraction fraction was challenged with exogenous unlabeled glutamate (500 microM) by reversed microdialysis, the glutamate extraction fraction in the mutant SOD1 mice was decreased significantly from control levels. The SOD1 mutant mice demonstrated no difference in the cortical protein levels of the glutamate transporter subtypes. This study demonstrates that in areas of no visible pathology and no loss of glutamate transporter proteins, SOD1 mutant mice have elevated extracellular fluid aspartate and glutamate levels and a decreased capacity to clear glutamate from the extracellular space.