Impairment of glutamate transport and increased vulnerability to oxidative stress in neuroblastoma SH-SY5Y cells expressing a Cu,Zn superoxide dismutase typical of familial amyotrophic lateral sclerosis

Human neuroblastoma SH-SY5Y cells transfected with either familial amyotrophic lateral sclerosis-typical G93A mutant or wild-type copper/zinc superoxide dismutase were compared to untransfected cells in term of glutamate transport. Vmax of glutamate uptake was reduced in mutant cells, with no change in Km. No difference in EAAT1, EAAT2 and EAAT3 glutamate transporter mRNAs and immunoreactive proteins was found, suggesting that one or more transporters are functionally inactivated, possibly due to increased oxidative stress induced by the G93A mutation. Mutant cells showed a marked sensitivity to oxidants, resulting in a more pronounced reduction of glutamate uptake. Short-term antioxidant treatment did not reverse the impairment of glutamate uptake in G93A cells. Interestlingly, N-acetylcysteine was partially effective in preventing glutamate uptake reduction due to exogenous oxidative insults. Since the inhibition of the EAAT2 transporter subtype had no effect on glutamate re-uptake in this model, our study suggests an impaired function of the EAAT1/3 transporter subtypes, possibly due to oxidative inactivation, in the presence of mutant copper/zinc superoxide dismutase. Therefore, this model might prove to be a valuable tool to study the effects of mutant copper/zinc superoxide dismutase associated with amyotrophic lateral sclerosis on glutamate transport in neuronal cells, without the specific contribution of glial cells. These findings might lead to the identification of new therapeutic strategies aimed at preventing the damage associated with ALS.     

PI3K/Akt/mTOR signaling regulates glutamate transporter 1 in astrocytes

Reduction in or dysfunction of glutamate transporter 1 (GLT1) is linked to several neuronal disorders such as stroke, Alzheimer’s disease, and amyotrophic lateral sclerosis. However, the detailed mechanism underlying GLT1 regulation has not been fully elucidated. In the present study, we first demonstrated the effects of mammalian target of rapamycin (mTOR) signaling on GLT1 regulation. We prepared astrocytes cultured in astrocyte-defined medium (ADM), which contains several growth factors including epidermal growth factor (EGF) and insulin. The levels of phosphorylated Akt (Ser473) and mTOR (Ser2448) increased, and GLT1 levels were increased in ADM-cultured astrocytes. Treatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor or an Akt inhibitor suppressed the phosphorylation of Akt (Ser473) and mTOR (Ser2448) as well as decreased ADM-induced GLT1 upregulation. Treatment with the mTOR inhibitor rapamycin decreased GLT1 protein and mRNA levels. In contrast, rapamycin did not affect Akt (Ser473) phosphorylation. Our results suggest that mTOR is a downstream target of the PI3K/Akt pathway regulating GLT1 expression.     

Human adipose-derived stem cells enhance the glutamate uptake function of GLT1 in SOD1-bearing astrocytes

Impaired glutamate uptake function of astrocytes associated with accumulation of extracellular glutamate is a well-documented feature of amyotrophic lateral sclerosis (ALS). Enhancing the uptake function of astrocytic glutamate transport 1 (GLT1) may be a potential treatment for this disease. Human adipose-derived stem cells (hADSCs) are capable of secreting a large number of cytokines which exhibit diverse pharmacological effects. Therefore, we investigate the influence of the soluble factors released by hADSCs on the GLT1 in primary astrocytes cultured from SOD1^G93A mice, a widely studied mutant human SOD1 transgenic model of ALS. Our data indicate that soluble factors from hADSCs significantly upregulate the expression of GLT1 in SOD1^G93A-bearing astrocytes, which result in enhanced glutamate uptake function. The upregulation of GLT1 is accompanied by the inhibition of caspase-3 activation in mutant astrocytes. In addition, we find that hADSCs cocultured with SOD1^G93A-bearing astrocytes produce more VEGF, HGF and IGF-1, which are reported to have neuroprotective effects. Our results suggest that hADSCs may be a potential candidate in cellular therapy for ALS.     

Abnormal exocytotic release of glutamate in a mouse model of amyotrophic lateral sclerosis

Glutamate-mediated excitotoxicity plays a major role in the degeneration of motor neurons in amyotrophic lateral sclerosis and reduced astrocytary glutamate transport, which in turn increases the synaptic availability of the amino acid neurotransmitter, was suggested as a cause. Alternatively, here we report our studies on the exocytotic release of glutamate as a possible source of excessive glutamate transmission. The basal glutamate efflux from spinal cord nerve terminals of mice-expressing human soluble superoxide dismutase (SOD1) with the G93A mutation [SOD1/G93A(+)], a transgenic model of amyotrophic lateral sclerosis, was elevated when compared with transgenic mice expressing the wild-type human SOD1 or to non-transgenic controls. Exposure to 15 mM KCl or 0.3 μM ionomycin provoked Ca(2+)-dependent glutamate release that was dramatically increased in late symptomatic and in pre-symptomatic SOD1/G93A(+) mice. Increased Ca(2+) levels were detected in SOD1/G93A(+) mouse spinal cord nerve terminals, accompanied by increased activation of Ca(2+)/calmodulin-dependent kinase II and increased phosphorylation of synapsin I. In line with these findings, release experiments suggested that the glutamate release augmentation involves the readily releasable pool of vesicles and a greater capability of these vesicles to fuse upon stimulation in SOD1/G93A(+) mice.     

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.     

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.     

Loss of metabotropic glutamate receptor-mediated regulation of glutamate transport in chemically activated astrocytes in a rat model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a selective loss of motor neurones accompanied by intense gliosis in lesioned areas of the brain and spinal cord. Glutamate-mediated excitotoxicity resulting from impaired astroglial uptake constitutes one of the current pathophysiological hypotheses explaining the progression of the disease. In this study, we examined the regulation of glutamate transporters by type 5 metabotropic glutamate receptor (mGluR5) in activated astrocytes derived from transgenic rats carrying an ALS-related mutated human superoxide dismutase 1 (hSOD1(G93A)) transgene. Cells from transgenic animals and wild-type littermates showed similar expression of glutamate-aspartate transporter and glutamate transporter 1 (GLT-1) after in vitro activation, whereas cells carrying the hSOD1 mutation showed a three-fold higher expression of functional mGluR5, as observed in the spinal cord of end-stage animals. In cells from wild-type animals, (S)-3,5-dihydroxyphenylglycine (DHPG) caused an immediate protein kinase C (PKC)-dependent up-regulation of aspartate uptake that reflected the activation of GLT-1. Although this effect was mimicked in both cultures by direct activation of PKC using phorbol myristate acetate, DHPG failed to up-regulate aspartate uptake in cells derived from the transgenic rats. The failure of activated mGluR5 to increase glutamate uptake in astrocytes derived from this animal model of ALS supports the theory of glutamate excitotoxicity in the pathogenesis of the disease.     

Functional Modulation of the Glutamate Transporter Variant GLT1b by the PDZ Domain Protein PICK1

The dominant glutamate transporter isoform in the mammalian brain, GLT1, exists as at least three splice variants, GLT1a, GLT1b, and GLT1c. GLT1b interacts with the scaffold protein PICK1 (protein interacting with kinase C1), which is implicated in glutamatergic neurotransmission via its regulatory effect on trafficking of AMPA-type glutamate receptors. The 11 extreme C-terminal residues specific for the GLT1b variant are essential for its specific interaction with the PICK1 PDZ domain, but a functional consequence of this interaction has remained unresolved. To identify a functional effect of PICK1 on GLT1a or GLT1b separately, we employed the Xenopus laevis expression system. GLT1a and GLT1b displayed similar electrophysiological properties and EC₅₀ for glutamate. Co-expressed PICK1 localized efficiently to the plasma membrane and resulted in a 5-fold enhancement of the leak current in GLT1b-expressing oocytes with only a minor effect on [³H]glutamate uptake. Three different GLT1 substrates all caused a slow TBOA-sensitive decay in the membrane current upon prolonged application, which provides support for the leak current being mediated by GLT1b itself. Leak and glutamate-evoked currents in GLT1a-expressing oocytes were unaffected by PICK1 co-expression. PKC activation down-regulated GLT1a and GLT1b activity to a similar extent, which was not affected by co-expression of PICK1. In conclusion, PICK1 may not only affect glutamatergic neurotransmission by its regulatory effect on glutamate receptors but may also affect neuronal excitability via an increased GLT1b-mediated leak current. This may be particularly relevant in pathological conditions such as amyotrophic lateral sclerosis and cerebral hypoxia, which are associated with neuronal GLT1b up-regulation.     

Antagonists of protein kinase C inhibit rat retinal glutamate transport activity in situ

Neuronal and glial high‐affinity transporters regulate extracellular glutamate concentration, thereby terminating synaptic transmission and preventing neuronal excitotoxicity. Glutamate transporter activity has been shown to be modulated by protein kinase C (PKC) in cell culture. This is the first study to demonstrate such modulation in situ, by following the fate of the non‐metabolisable glutamate transporter substrate, d ‐aspartate. In the rat retina, pan‐isoform PKC inhibition with chelerythrine suppressed glutamate uptake by GLAST (glutamate/aspartate transporter), the dominant excitatory amino acid transporter localized to the glial Müller cells. This effect was mimicked by rottlerin but not by Gö6976, suggesting the involvement of the PKCδ isoform, but not PKCα, β or γ. Western blotting and immunohistochemical labeling revealed that the suppression of glutamate transport was not due to a change in transporter expression. Inhibition of PKCδ selectively suppressed GLAST but not neuronal glutamate transporter activity. These data suggest that the targeting of specific glutamate transporters with isoform‐specific modulators of PKC activity may have significant implications for the understanding of neurodegenerative conditions arising from compromised glutamate homeostasis, e.g. glaucoma and amyotrophic lateral sclerosis.     

Expression of glutamate transporters in rat cardiomyocytes and their localization in the T-tubular system.

Glutamate and aspartate play important roles in the intermediary metabolism of the myocardium and have been shown to improve cardiac recovery after hypoxia or ischemia. Limited data are available about the expression of glutamate transporters that are involved in the uptake of glutamate and aspartate in cardiomyocytes. In this study, non-radioactive in situ hybridization (ISH) using complementary RNA probes was applied to detect the glutamate transporters GLT1 variant (GLT1v) and EAAC1 mRNA in rat cardiomyocytes. The transporter proteins were demonstrated by Western blotting and immunocytochemistry using affinity-purified antibodies against transporter peptides. ISH and immunocytochemistry showed that both glutamate transporters are coexpressed in cardiomyocytes. The ISH labeling indicates the distribution of transporter mRNA throughout the cytoplasm of cardiomyocytes. GLT1v and EAAC1 proteins, which showed in Western blots a molecular mass of approximately 60 kD, are strongly enriched and colocalized in the transverse (T)-tubular system of cardiomyocytes. These results may indicate that glutamate/aspartate uptake into cardiomyocytes could be mediated by the high-affinity transporters GLT1v and EAAC1. A high efficiency of glutamate/aspartate transport into cardiomyocytes could be achieved by their localization in the T-tubular system, which consists of tubular invaginations of the sarcolemma extending deep into the cell.     

Splice variants of the glutamate transporter GLT1 form hetero-oligomers that interact with PSD-95 and NMDA receptors

The glutamate transporter GLT1 is expressed in at least two isoforms, GLT1a and GLT1b, which differ in their C termini. As GLT1 is an oligomeric protein, we have investigated whether GLT1a and GLT1b might associate as hetero-oligomers. Differential tagging (HA-GLT1a and YFP-GLT1b) revealed that these isoforms form complexes that could be immunoprecipitated when co-expressed in heterologous systems. The association of GLT1a and GLT1b was also observed in mixed primary cultures of rat brain and in the adult rat brain, where specific antibodies for GLT1a immunoprecipitated GLT1b and vice versa. Dual immunofluorescence in mixed cultures demonstrated the partial co-localization of both isoforms in neurons and in glial cells. Because GLT1b interacts with an organizer of post-synaptic densities, PSD-95, we examined the capacity of GLT1a to associate with this protein. GLT1a was immunoprecipitated from the rat brain in protein complexes that contained not only GLT1b but also PSD-95 and NMDAR. The interaction between GLT1a with PSD-95 and NMDAR was reproduced in transfected COS7 cells and it appears to be indirect as it requires the presence of GLT1b. These results indicate that the major isoform of the glutamate transporter, GLT1a, can acquire the capacity to interact with PDZ proteins through its inclusion in hetero-oligomers containing GLT1b.     

Decreasing glutamate buffering capacity triggers oxidative stress and neuropil degeneration in the Drosophila brain

L-glutamate is both the major brain excitatory neurotransmitter and a potent neurotoxin in mammals. Glutamate excitotoxicity is partly responsible for cerebral traumas evoked by ischemia and has been implicated in several neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). In contrast, very little is known about the function or potential toxicity of glutamate in the insect brain. Here, we show that decreasing glutamate buffering capacity is neurotoxic in Drosophila. We found that the only Drosophila high-affinity glutamate transporter, dEAAT1, is selectively addressed to glial extensions that project ubiquitously through the neuropil close to synaptic areas. Inactivation of dEAAT1 by RNA interference led to characteristic behavior deficits that were significantly rescued by expression of the human glutamate transporter hEAAT2 or the administration in food of riluzole, an anti-excitotoxic agent used in the clinic for human ALS patients. Signs of oxidative stress included hypersensitivity to the free radical generator paraquat and rescue by the antioxidant melatonin. Inactivation of dEAAT1 also resulted in shortened lifespan and marked brain neuropil degeneration characterized by widespread microvacuolization and swollen mitochondria. This suggests that the dEAAT1-deficient fly provides a powerful genetic model system for molecular analysis of glutamate-mediated neurodegeneration.     

The glutamate transporter GLT1b interacts with the scaffold protein PSD-95

The glutamate transporter (GLT1) regulates glutamate concentrations in glutamatergic synapses and it is expressed in at least two isoforms, GLT1a and GLT1b. In this work, we show that the C-terminus of GLT1b is able to interact with the PDZ domains of a number of proteins. Notably, one of them might be the scaffold protein post-synaptic density (PSD-95). GLT1b formed co-immunoprecipitable complexes with PSD-95 in solubilizated rat brain extracts, complexes that also contained NMDA receptors. Co-transfection of GLT1b, PSD-95, and NMDA receptor subunits in heterologous expression systems recapitulated in vitro the interactions among these proteins that had been observed in the rat brain extracts and revealed the importance of the GLT1b C-terminal PDZ binding motif in tethering this transporter to PSD-95. Significantly, co-expression of GLT1b and PSD-95 increased the V _max of the transporter by decreasing the rate of GLT1b endocytosis. Moreover, GLT1b transfected into primary cultured neurons or glia formed protein clusters that co-localized with co-transfected PSD-95, clusters that in these neurons accumulated preferentially in dendritic spines. We hypothesize that the GLT1b/PSD-95 interaction, characterized here in vitro , might anchor this transporter close to the post-synaptic glutamate receptors, thereby permitting the fine regulation of glutamate concentrations in this microenvironment. This tight association might also facilitate the regulation of GLT1b through the signaling pathways initiated by the activation of glutamate receptors.     

Cooperation of the conserved aspartate 439 and bound amino acid substrate is important for high-affinity Na+ binding to the glutamate transporter EAAC1

The neuronal glutamate transporter EAAC1 contains several conserved acidic amino acids in its transmembrane domain, which are possibly important in catalyzing transport and/or binding of co/countertransported cations. Here, we have studied the effects of neutralization by site-directed mutagenesis of three of these amino acid side chains, glutamate 373, aspartate 439, and aspartate 454, on the functional properties of the transporter. Transport was analyzed by whole-cell current recording from EAAC1-expressing mammalian cells after applying jumps in voltage, substrate, or cation concentration. Neutralization mutations in positions 373 and 454, although eliminating steady-state glutamate transport, have little effect on the kinetics and thermodynamics of Na(+) and glutamate binding, suggesting that these two positions do not constitute the sites of Na(+) and glutamate association with EAAC1. In contrast, the D439N mutation resulted in an approximately 10-fold decrease of apparent affinity of the glutamate-bound transporter form for Na(+), and an approximately 2,000-fold reduction in the rate of Na(+) binding, whereas the kinetics and thermodynamics of Na(+) binding to the glutamate-free transporter were almost unchanged compared to EAAC1(WT). Furthermore, the D439N mutation converted l-glutamate, THA, and PDC, which are activating substrates for the wild-type anion conductance, but not l-aspartate, into transient inhibitors of the EAAC1(D439) anion conductance. Activation of the anion conductance by l-glutamate was biphasic, allowing us to directly analyze binding of two of the three cotransported Na(+) ions as a function of time and [Na(+)]. The data can be explained with a model in which the D439N mutation results in a dramatic slowing of Na(+) binding and a reduced affinity of the substrate-bound EAAC1 for Na(+). We propose that the bound substrate controls the rate and the extent of Na(+) interaction with the transporter, depending on the amino acid side chain in position 439.     

Localization of the gene encoding the human L-glutamate transporter (GLT-1) to 11p11.2–p13 by fluorescence in situ hybridization

High affinity glutamate transporters regulate levels of extracellular glutamate in the central nervous system. Impaired glutamate transport has been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). The glutamate transporter subtypes GluT-1 and EAAC1 have previously been mapped to human chromosomes 5p13 and 9p24, respectively. In the present study, the GLT-1 subtype was mapped to human chromosome 11p11.2–p13 by fluorescence in situ hybridization. The possible clinical implications of this finding are discussed.     

Beta-lactam antibiotics are multipotent agents to combat neurological diseases

Recently, Rothstein et al. reported that beta-lactam antibiotics, including penicillin and ceftriaxone, are potential therapeutic drugs to treat some neurological disorders, e.g., amyotrophic lateral sclerosis (ALS), by modulating the expression of glutamate transporter GLT1 via gene activation. However, considering the facts that: (i) many neurological diseases (including ALS) are associated with transition metal ions and redox stress, and ALS can be efficiently prevented by metal chelators, e.g., diethyl-dithiocarbamate (DDC); (ii) beta-lactam antibiotics have long been known as metal chelators, we argue that the beneficial effect of beta-lactam antibiotics on ALS likely involves Cu(II)-attenuating ability. This is partially supported by our theoretical calculations.     

Expression of SOD1 G93A or wild-type SOD1 in primary cultures of astrocytes down-regulates the glutamate transporter GLT-1: lack of involvement of oxidative stress

Glutamate excitotoxicity is implicated in the aetiology of amyotrophic lateral sclerosis (ALS) with impairment of glutamate transport into astrocytes a possible cause of glutamate-induced injury to motor neurons. It is possible that mutations of Cu/Zn superoxide dismutase (SOD1), responsible for about 20% of familial ALS, down-regulates glutamate transporters via oxidative stress. We transfected primary mouse astrocytes to investigate the effect of the FALS-linked mutant hSOD1(G93A) and wild-type SOD1 (hSOD1wt) on the glutamate uptake system. Using western blotting, immunocytochemistry and RT-PCR it was shown that expression of either hSOD1(G93A) or hSOD1wt in astrocytes produced down-regulation of the levels of a glutamate transporter GLT-1, without alterations in its mRNA level. hSOD1(G93A) or hSOD1wt expression caused a decrease of the monomeric form of GLT-1 without increasing oxidative multimers of GLT-1. The effects were selective to GLT-1, since another glutamate transporter GLAST protein and mRNA levels were not altered. Reflecting the decrease in GLT-1 protein, [3H]d-aspartate uptake was reduced in cultures expressing hSOD1(G93A) or hSOD1wt. The hSOD1-induced decline in GLT-1 protein and [3H]d-aspartate uptake was not blocked by the antioxidant Trolox nor potentiated by antioxidant depletion using catalase and glutathione peroxidase inhibitors. Measurement of 2',7'-dichlorofluorescein (DCF)-induced fluorescence revealed that expression of hSOD1(G93A) or hSOD1wt in astrocytes does not lead to detectable increase of intracellular reactive oxygen species. This study suggests that levels of GLT-1 protein in astrocytes are reduced rapidly by overexpression of hSOD1, and is due to a property shared between the wild-type and G93A mutant form, but does not involve the production of intracellular oxidative stress.