Mercuric chloride inhibits the in vitro uptake of glutamate in GLAST- and GLT-1-transfected mutant CHO-K1 cells

Glutamate is removed mainly by astrocytes from the extracellular fluid via high-affinity astroglial Na⁺-dependent excitatory amino acid transporters, glutamate/aspartate transporter (GLAST), and glutamate transporter-1 (GLT-1). Mercuric chloride (HgCl₂) is a highly toxic compound that inhibits glutamate uptake in astrocytes, resulting in excessive extracellular glutamate accumulation, leading to excitotoxicity and neuronal cell death. The mechanisms associated with the inhibitory effects of HgCl₂ on glutamate uptake are unknown. This study examines the effects of HgCl₂ on the transport of ³H-d-aspartate, a nonmetabolizable glutamate analog, using Chinese hamster ovary cells (CHO) transfected with two glutamate transporter subtypes, GLAST (EAAT1) and GLT-1 (EAAT2), as a model system. Additionally, studies were undertaken to determine the effects of HgCl₂ on mRNA and protein levels of these transporters. The results indicate that (1) HgCl₂ leads to significant (p<0.001) inhibition of glutamate uptake via both transporters, but is a more potent inhibitor of glutamate transport via GLAST and (2) the effect of HgCl₂ on inhibition of glutamate uptake in transfected CHO cells is not associated with changes in transporter protein levels despite a significant decrease in mRNA expression; thus, (3) HgCl₂ inhibition is most likely related to its direct binding to the functional thiol groups of the transporters and interference with their uptake function.     

Neuronal Soluble Factors Differentially Regulate the Expression of the GLT1 and GLAST Glutamate Transporters in Cultured Astroglia

Abstract: The glutamate transporters in the plasma membranes of neural cells secure termination of the glutamatergic synaptic transmission and keep the glutamate levels below toxic concentrations. Astrocytes express two types of glutamate transporters, GLAST (EAAT1) and GLT1 (EAAT2). GLT1 predominates quantitatively and is responsible for most of the glutamate uptake activity in the juvenile and adult brain. However, GLT1 is severely down-regulated in amyotrophic lateral sclerosis, a progressive neurodegenerative disease. Furthermore, selective loss of this transporter occurs in cultured astroglia. Expression of GLAST, but not of GLT1, seems to be regulated via the glutamate receptor signalling. The present study was undertaken to examine whether neuronal factors, other than glutamate, influence the expression of astroglial glutamate transporters. The expression of GLT1 and GLAST was examined in primary cultures of cerebellar granule neurons, cortical neurons, and astrocytes under different experimental conditions, including those that mimic neuron-astrocyte interactions. Pure astroglial cultures expressed only GLAST, whereas astrocytes grown in the presence of neurons expressed both GLAST (at increased levels) and GLT1. The induction of GLT1 protein and its mRNA was reproduced in pure cortical astroglial cultures supplemented with conditioned media from cortical neuronal cultures or from mixed neuron-glia cultures. This treatment did not change the levels of GLAST. These results suggest that soluble neuronal factors differentially regulate the expression of GLT1 and GLAST in cultured astroglia. Further elucidation of the molecular nature of the secreted neuronal factors and corresponding signalling pathways regulating the expression of the astroglial glutamate transporters in vitro may reveal mechanisms important for the understanding and treatment of neurological diseases.     

The in vitro uptake of glutamate in GLAST and GLT-1 transfected mutant CHO-K1 cells is inhibited by manganese

In the central nervous system (CNS), extracellular concentrations of amino acids (e.g., aspartate, glutamate) and divalent metals (e.g., zinc, copper, manganese) are primarily regulated by astrocytes. Adequate glutamate homeostasis and control over extracellular concentrations of these excitotoxic amino acids are essential for the normal functioning of the brain. Not only is glutamate of central importance for nitrogen metabolism but, along with aspartate, it is the primary mediator of excitatory pathways in the brain. Similarly, the maintenance of proper Mn levels is important for normal brain function. Brain glutamate is removed from the extracellular fluid mainly by astrocytes via high affinity astroglial Na+-dependent excitatory amino acid transporters, glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1). The effects of Mn on specific glutamate transporters have yet to be determined. As a first step in this process, we examined the effects of Mn on the transport of [D-2, 3-3H]D-aspartate, a non-metabolizable glutamate analog, in Chinese hamster ovary cells (CHO) transfected with two glutamate transporter subtypes, GLAST (EAAT1) or GLT-1 (EAAT2). Mn-mediated inhibition of glutamate transport in the CHO-K1 cell line DdB7 was pronounced in both the GLT-1 and GLAST transfected cells. This resulted in a statistically significant inhibition (p<0.05) of glutamate uptake compared with transfected control in the absence of Mn treatment. These studies suggest that Mn accumulation in the CNS might contribute to dysregulation of glutamate homeostasis.     

Differential Regulation of Glutamate Transporter Subtypes by Pro-Inflammatory Cytokine TNF-α in Cortical Astrocytes from a Rat Model of Amyotrophic Lateral Sclerosis

Dysregulation of the astroglial glutamate transporters GLAST and GLT-1 has been implicated in several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) where a loss of GLT-1 protein expression and activity is reported. Furthermore, the two principal C-terminal splice variants of GLT-1 (namely GLT-1a and GLT-1b) show altered expression ratio in animal models of this disease. Considering the putative link between inflammation and excitotoxicity, we have here characterized the influence of TNF-α on glutamate transporters in cerebral cortical astrocyte cultures from wild-type rats and from a rat model of ALS (hSOD1^G93A). Contrasting with the down-regulation of GLAST, a 72 h treatment with TNF-α substantially increased the expression of GLT-1a and GLT-1b in both astrocyte cultures. However, as the basal level of GLT-1a appeared considerably lower in hSOD1^G93A astrocytes, its up-regulation by TNF-α was insufficient to recapitulate the expression observed in wild-type astrocytes. Also the glutamate uptake activity after TNF-α treatment was lower for hSOD1^G93A astrocytes as compared to wild-type astrocytes. In the presence of the protein synthesis inhibitor cycloheximide, TNF-α did not influence GLT-1 isoform expression, suggesting an active role of dynamically regulated protein partners in the adaptation of astrocytes to the inflammatory environment. Confirming the influence of inflammation on the control of glutamate transmission by astrocytes, these results shed light on the regulation of glutamate transporter isoforms in neurodegenerative disorders.     

Effects of estrogen treatment on glutamate uptake in cultured human astrocytes derived from cortex of Alzheimer's disease patients

Estrogen is thought to play a protective role against neurodegeneration through a variety of mechanisms including the activation of growth factors, the control of synaptic plasticity, and the reduction of response to various insults, such as iron and glutamate. Increasing evidence indicates an increased level of extracellular glutamate and a down-regulation of glutamate transporters in Alzheimer's disease (AD). In this study, we show that glutamate uptake in astrocytes derived from Alzheimer's patients is significantly lower than that from non-demented controls. Estrogen treatment increases glutamate uptake in a dose-dependent pattern. Two glutamate transporters, GLT-1 and GLAST, are expressed in the astrocytes. Up-regulation of the glutamate transporters is induced by estrogen treatment in AD astrocytes only. Our data suggest that the action of estrogen on glutamate uptake by astrocytes might contribute to its potential neuroprotective role in AD.     

Effects of lipopolysaccharide on glial phenotype and activity of glutamate transporters: Evidence for delayed up-regulation and redistribution of GLT-1

Excitatory amino acid transporters (EAATs) are responsible for homeostasis of extracellular L-glutamate, and the glial transporters are functionally dominant. EAAT expression or function is altered in acute and chronic neurological conditions, but little is known about the regulation of EAATs in reactive astroglia found in such neuropathologies. These studies examined the effects of the bacterial endotoxin lipopolysaccharide (LPS) on glial EAATs in vitro. The effects of LPS (1 microg/ml, 24-72 h) on EAAT activity and expression were examined in primary cultures of mouse astrocytes. [(3)H]D-aspartate uptake increased to 129% of control by 72 h treatment with LPS. Saturation analysis revealed that apparent K(m) was unchanged whilst V(max) was significantly increased to 172% of control by 72 h LPS treatment. Biotinylation and Western blotting indicated that cell-surface expression of GLT-1 was significantly elevated (146% control) by LPS treatment whereas GLAST expression was unchanged. Confocal analyses revealed that LPS treatment resulted in cytoskeletal changes and stellation of astrocytes, with rearrangement of F-actin (as shown by phalloidin labelling). Immunocytochemistry revealed clustering of GLAST, and increased expression and redistribution of GLT-1 to the cell-surface following treatment with LPS. Similar experiments were conducted in microglia, where LPS (50 ng/ml) was found to up-regulate expression of GLT-1 at 24 and 72 h in concert with cytoskeletal changes accompanying activation. These findings suggest an association of cytoskeletal changes in glia with EAAT activity, with the predominant adaptation involving up-regulation and redistribution of GLT-1.     

In vitro uptake of glutamate in GLAST-and GLT-1-transfected mutant CHO-K1 cells is inhibited by the ethylmercury-containing preservative thimerosal

Thimerosal, also known as thimersal, Merthrolate, or sodiumethyl-mercurithiosalicylate, is an organic mercurial compound that is used in a variety of commercial as well as biomedical applications. As a preservative, it is used in a number of vaccines and pharmaceutical products. Its active ingredient is ethylmercury. Both inorganic and organic mercurials are known to interfere with glutamate homeostasis. Brain glutamate is removed mainly by astrocytes from the extracellular fluid via high-affinity astroglial Na⁺-dependent excitatory amino acid transporters, glutamate/ aspartats transporter (GLAST) and glutamate transporter-1 (GLT-1). The effects of thimerosal on glutamate homeostasis have yet to be determined. As a first step in this process, we examined the effects of thimerosal on the transport of [³H]-D-aspartate, a nonmetabolizable glutamate analog, in Chinese hamster ovary (CHO) cells transfected with two glutamate transporter subtypes, GLAST (EAAT1) and GLT-1 (EAAT2). Additionally, studies were undertaken to determine the effects of thimerosal on mRNA and protein levels of these transporters. The results indicate that thimerosal treatment caused significant but selective changes in both glutamate transporter mRNA and protein expression in CHO cells. Thimerosal-mediated inhibition of glutamate transport in the CHO-K1 cell line DdB7 was more pronounced in the GLT-1-transfected cells compared with the GLAST-transfected cells. These studies suggest that thimerosal accumulation in the central nervous system might contribute to dysregulation of glutamate homeostasis.     

The GLT-1 and GLAST glutamate transporters are expressed on morphologically distinct astrocytes and regulated by neuronal activity in primary hippocampal cocultures

The GLT-1 and GLAST astroglial transporters are the glutamate transporters mainly involved in maintaining physiological extracellular glutamate concentrations. Defects in neurotransmitter glutamate transport may represent an important component of glutamate-induced neurodegenerative disorders (such as amyotrophic lateral sclerosis) and CNS insults (ischemia and epilepsy). We characterized the protein expression of GLT-1 and GLAST in primary astrocyte-neuron cocultures derived from rat hippocampal tissues during neuron differentiation/maturation. GLT-1 and GLAST are expressed by morphologically distinct glial fibrillary acidic protein-positive astrocytes, and their expression correlates with the status of neuron differentiation/maturation and activity. Up-regulation of the transporters paralleled the content of the synaptophysin synaptic vesicle marker p38, and down-regulation was a consequence of glutamate-induced neuronal death or the reduction of synaptic activity. Finally, soluble factors in neuronal-conditioned media prevented the down-regulation of the GLT-1 and GLAST proteins. Although other mechanisms may participate in regulating GLT-1 and GLAST in the CNS, our data indicate that soluble factors dependent on neuronal activity play a major regulating role in hippocampal cocultures.     

Microglial self-defence mediated through GLT-1 and glutathione

Glutamate is stored in synaptic vesicles in presynaptic neurons. It is released into the synaptic cleft to provide signalling to postsynaptic neurons. Normally, the astroglial glutamate transporters GLT-1 and GLAST take up glutamate to mediate a high signal-to-noise ratio in the synaptic signalling, and also to prevent excitotoxic effects by glutamate. In astrocytes, glutamate is transformed into glutamine, which is safely transported back to neurons. However, in pathological conditions, such as an ischemia or virus infection, astroglial transporters are down-regulated which could lead to excitotoxicity. Lately, it was shown that even microglia can express glutamate transporters during pathological events. Microglia have two systems for glutamate transport: GLT-1 for transport into the cells and the x_c⁻ system for transport out of the cells. We here review results from our work and others, which demonstrate that microglia in culture express GLT-1, but not GLAST, and transport glutamate from the extracellular space. We also show that TNF-α can induce increased microglial GLT-1 expression, possibly associating the expression with inflammatory systems. Furthermore, glutamate taken up through GLT-1 may be used for direct incorporation into glutathione and to fuel the intracellular glutamate pool to allow cystine uptake through the x_c⁻ system. This can lead to a defence against oxidative stress and have an antiviral function.     

Molecular and functional characterisation of glutamate transporters in rat cortical astrocytes exposed to a defined combination of growth factors during in vitro differentiation

In vitro culture of astroglial progenitors can be obtained from early post-natal brain tissues and several methods have been reported for promoting their maturation into differentiated astrocytes. Hence, a combination of several nutriments/growth factors -- the G5 supplement (insulin, transferrin, selenite, biotin, hydrocortisone, fibroblast growth factor and epidermal growth factor) -- is widely used as a culture additive favouring the growth, differentiation and maturation of primary cultured astrocytes. Considering the key role played by glial cells in the clearance of glutamate in the synapses, cultured astrocytes are frequently used as a model for the study of glutamate transporters. Indeed, it has been shown that when tested separately, growth factors influence the expression and activity of the GLAST and GLT-1. The present study aimed at characterising the functional expression of these transporters during the time course of differentiation of cultured cortical astrocytes exposed to the supplement G5. After a few days, the vast majority of cells exposed to this supplement adopted a typical stellate morphology (fibrous or type II astrocytes) and showed intense expression of the glial fibrillary acidic protein. Both RT-PCR and immunoblotting studies revealed that the expression of both GLAST and GLT-1 rapidly increased in these cells. While this was correlated with a significant increase in specific uptake of radiolabelled aspartate, fluorescence monitoring of the Na+ influx associated with glutamate transporters activity revealed that the exposure to the G5 supplement considerably increased the percentage of cells participating in the uptake. Biochemical and pharmacological studies revealed that this activity did not involve GLT-1 but most likely reflected an increase in GLAST-mediated uptake. Together, these data indicate that the addition of this classical combination of growth factors and nutriments drives the rapid differentiation toward a homogenous culture of fibrous astrocytes expressing functional glutamate transporters.     

In vivo evidence that truncated trkB.T1 participates in nociception

Background

Clearance 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.

Results

Animals 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.

Conclusion

These results indicate that the expression of glutamate transporters in astrocytes and microglia are differentially regulated following nerve injury.     

Neuronal-induced and glutamate-dependent activation of glial glutamate transporter function

The activity of high-affinity glutamate transporters is essential for the normal function of the mammalian central nervous system. Using a combined pharmacological, confocal immunocytochemical, enzyme-based microsensor and fluorescence imaging approach, we examined glutamate uptake and transporter protein localization in single astrocytes of neuron-containing and neuron-free microislands prior to pre-synaptic transmitter secretion and during functional neuronal activity. Here, we report that the presence or absence of neurons strikingly affects the uptake capacity of the astroglial glutamate transporters GLT1 and GLAST1. Induction of transporter function is activated by neurons and this effect is mimicked by pre-incubation of astrocytes with micromolar concentrations of glutamate. Moreover, increased glutamate transporter activation is reproduced by endogenous release of glutamate via activation of neuronal nicotinic receptors. The increase in transport activity is dependent on neuronal release of glutamate, is associated with the local redistribution (clustering) of GLT1 and GLAST1 but is independent of transporter synthesis and of glutamate receptor activation. Together, these results suggest an activity-dependent neuronal feedback system for rapid astroglial glutamate transporter regulation where neuron-derived glutamate is the physiological signal that triggers transporter function.     

The L-glutamate transporters GLAST (EAAT1) and GLT-1 (EAAT2): expression and regulation in rat lactating mammary gland

The Na(+)-dependent L-glutamate transporters GLAST (EAAT1) and GLT-1 (EAAT2), were expressed in rat lactating mammary gland, but EAAC1 (EAAT3) was not. GLT-1 expression in rat lactating mammary gland was constant in all the physiological situations studied; however, the GLAST expression is under tight regulation. Fasting for 24 h decreased the GLAST expression which returned to control values after refeeding. Weaning for 24 h produced a decrease in GLAST expression through a mechanism independent of prolactin deficiency. Resuckling for 6 h returned the expression of this transporter to control values. There is a correlation between the levels of GLAST (mRNA and protein) and the in vivo uptake of L-glutamate by the lactating mammary gland during the starvation/refeeding cycle and milk accumulation process.     

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 μM 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 μM MeHg) increased and GLT-1 transporter protein levels significantly (p<0.001) decreased followign MeHg exposure (5 μM). (3) MeHg exposure led to significant inhibition (p<0.05) of glutamate uptake by GLAST (both 5 and 10 μM MeHg), whereas GLT-1 transporter activity was significantly (p<0.01) increased following exposure to 5 and 10 μM MeHg. These studies suggest that MeHg contributes to the dysregulation of glutamate homeostasis and that its effects are distinct for GLAST and GLT-1.     

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.     

Effect of topiramate and dBcAMP on expression of the glutamate transporters GLAST and GLT-1 in astrocytes cultured separately, or together with neurons

The mechanism of the antiepileptic drug topiramate is not fully understood, but interaction with the excitatory neurotransmission, e.g. glutamate receptors, is believed to be part of its anticonvulsant effect. The glutamate transporters GLAST and GLT-1 are responsible for the inactivation of glutamate as a neurotransmitter and it was therefore investigated if topiramate might affect the expression of GLAST and GLT-1 in astrocytes cultured separately or together with neurons. Since expression and membrane trafficking of glutamate transporters are affected by the protein kinase C system as well as by dBcAMP it was also investigated if these signalling pathways might play a role. In astrocyte cultures expressing mainly GLAST treatment with dBcAMP (0.25 mM) led to an increased expression of the total amount of GLAST as well as of its membrane association. The enhanced expression in the membrane was particularly pronounced for the oligomeric form of GLAST. No detectable effect on the expression of GLAST in astrocytes treated with topiramate in the presence and absence of protein kinase C activators or inhibitors was observed. Astrocytes co-cultured with neurons expressed both GLAST and GLT-1. In these cultures prolonged exposure to 30 muM topiramate (10 days) led to a statistically significant increase (P<0.025) in the membrane expression of GLAST. In case of GLT-1, culture in the presence of 30 microM topiramate for 1 and 10 days led to alterations in the total, cytoplamic and membrane expression of the oligomeric form of the transporter.     

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.