Dopamine and glutamate neurotoxicity in cultured chick telencephali cells: effects of NMDA antagonists, antioxidants and MAO inhibitors

In a recent study, it was found that the intrastriatal administration to rats of the organophosphorous compound soman and kainic acid produced a rapid release not only of glutamate but also of dopamine in this brain region. Dopamine is a potent source of free radicals and is known to produce cytotoxic effects, per se. This raises the possibility that the released glutamate and dopamine act synergistically to produce the neurotoxicity found after soman administration. In order to investigate the feasibility of this hypothesis in an in vitro system, the effects of dopamine and glutamate upon cell survival were investigated using chick neurons (7 DIV) in serum-free primary culture. The neurons were treated with dopamine and/or glutamate for up to 24 h and cell toxicity was then assessed either by determination of cell densities, by the release of cytoplasmic LDH or by the MTT cytotoxicity assay. L-Glutamate produced a concentration-dependent cytotoxicity that was seen as early as after 30 min of exposure, and was accompanied by an increased level of lipid peroxidation. The L-glutamate toxicity could to a large extent by prevented by NMDA receptor antagonists and to a lesser extent by catalase, superoxide dismutase or glutathione ethyl ester added 30 min before the glutamate. Dopamine was also cytotoxic, and the cytotoxicity was reduced by the combination of catalase and glutathione ethyl ester but not by the MAO inhibitors clorgyline or L-deprenyl, or by the selective dopamine uptake inhibitor GBR 12783. The cytotoxic effects of dopamine and L-glutamate were additive rather than synergistic, regardless of the incubation time used. It is concluded that chick neurons in serum-free culture are a useful in vitro model system for the study of cell toxicity produced by oxidative stress and by glutamate. The cytotoxic effects of dopamine in this model are not due to the monoamine oxidase-mediated production of hydrogen peroxide but appear at least in part to be related to oxidative stress.     

Cooperative action of glutamate transporters and cystine/glutamate antiporter system Xc- protects from oxidative glutamate toxicity

Oxidative glutamate toxicity in the neuronal cell line HT22 is a model for cell death by oxidative stress. In this paradigm, an excess of extracellular glutamate blocks the glutamate/cystine-antiporter system Xc-, depleting the cell of cysteine, a building block of the antioxidant glutathione. Loss of glutathione leads to the accumulation of reactive oxygen species and eventually cell death. We selected cells resistant to oxidative stress, which exhibit reduced glutamate-induced glutathione depletion mediated by an increase in the antiporter subunit xCT and system Xc- activity. Cystine uptake was less sensitive to inhibition by glutamate and we hypothesized that glutamate import via excitatory amino acid transporters and immediate re-export via system Xc- underlies this phenomenon. Inhibition of glutamate transporters by l-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) and DL-threo-beta-benzyloxyaspartic acid (TBOA) exacerbated glutamate-induced cell death. PDC decreased intracellular glutamate accumulation and exacerbated glutathione depletion in the presence of glutamate. Transient overexpression of xCT and the glutamate transporter EAAT3 cooperatively protected against glutamate. We conclude that EAATs support system Xc- to prevent glutathione depletion caused by high extracellular glutamate. This knowledge could be of use for the development of novel therapeutics aimed at diseases associated with depletion of glutathione like Parkinson's disease.     

Hyperthermia assists survival of astrocytes from oxidative-mediated necrotic cell death.

In response to many stresses and pathologic states, including different models of nervous system injury, cells synthesize a variety of proteins, most notably the inducible 72 kDa heat shock protein 70 (Hsp70), which plays important roles in maintaining cellular integrity and viability. We report here that cultured astrocytes from rat diencephalon express high levels of Hsp70 upon exposure to elevated temperatures, and are less vulnerable to a subsequent oxidative stress. Complex oxidative stress was induced by exposure of astrocytes to an aqueous extract of tobacco smoke. This resulted in both glutathione and ATP depletion, along with cell death that proceeded through a necrotic pathway. Pretreatment of cultures with the glutathione replenishing agent, N-acetyl-L-cysteine, prevented glutathione and ATP loss as well as necrotic cell death. Thermal stress also protected astrocytes from necrotic cell death but without affecting glutathione or ATP levels. We propose that heat shock protects astrocytes from necrosis induced by oxidative stress, probably as a result of Hsp70 synthesis, through an antioxidant-ATP independent mechanism. As Hsp70 may transfer from glial to neuronal cells, its synthesis by astrocytes may represent an important survival mechanism by which astrocytes protect neurons against oxidative-mediated cell death.     

Importance of astrocytic inactivation of synaptically released glutamate for cell survival in the central nervous system--are astrocytes vulnerable to low intracellular glutamate concentrations?

Multiple levels of neuron-astrocyte interactions do exist at glutamatergic synapses, glial glutamate transporters being involved in most of them. Inactivation of synaptically released glutamate is not only important for the phasic aspect of glutamatergic transmission but also for astrocyte metabolism, which supply neurons with different metabolic precursors, and for cell survival in the central nervous system. Alteration of glutamate transport, which leads to abnormally high extracellular glutamate levels, has been involved in numerous neurodegenerative diseases. There are different ways by which elevated extracellular levels of glutamate can be toxic. Excitotoxic mechanisms, involving overstimulation of glutamate receptors, have been shown to induce the death of neurons and oligodendrocytes, but not of astrocytes. Oxidative glutamate toxicity, which can affect every cell type of the central nervous system, is currently viewed as the consequence of altered cystine transport, leading in turn to reduced glutathione synthesis and oxidative stress. This review summarizes the functional implications of astroglial glutamate transport and the consequences of its alteration. Emphasis is laid on our recent finding that alteration of glutamate transport, by depleting intracellular stores of glutamate, can induce oxidative toxicity in astrocytes. The consequences for the other cell types of the central nervous system are discussed in terms of neuron dependency on astrocytes for glutathione synthesis and therefore oxidative stress protection.     

L-glutamate suppresses amyloid beta-protein-induced stellation of cultured rat cortical astrocytes

Alzheimer's amyloid beta-protein (Abeta) has been reported to potentiate glutamate toxicity in neurons, but very little is known about interaction between Abeta and glutamate in astrocytes. Therefore, in the present study, we investigated the effects of Abeta and glutamate on morphology of astrocytes. Cultured rat cortical astrocytes exhibited polygonal morphology in the absence of stimulation and differentiated into process-bearing stellate cells following exposure to Abeta (20 microM). L-Glutamate (30-1,000 microM) had no effect on astrocyte morphology in the absence of stimulation but strongly suppressed Abeta-induced stellation. The suppressive effect of L-glutamate on Abeta-induced stellation was not mimicked by glutamate receptor agonists and not blocked by glutamate receptor antagonists. In contrast, the suppressive effect of L-glutamate was mimicked by D- and L-aspartate and transportable glutamate uptake inhibitors. These results suggest that Abeta-induced astrocyte stellation is suppressed by a mechanism related to glutamate transporters.     

Activation of stimulatory heterotrimeric G proteins increases glutathione and protects neuronal cells against oxidative stress

Oxidative glutamate toxicity in the neuronal cell line HT22 is a model for cell death by oxidative stress, where an excess of extracellular glutamate inhibits import of cystine, a building block of the antioxidant glutathione. The subsequent decrease in glutathione then leads to the accumulation of reactive oxygen species (ROS) and programmed cell death. We used pharmacological compounds known to interact with heterotrimeric G-protein signalling and studied their effects on cell survival, morphology, and intracellular events that ultimately lead to cell death. Cholera toxin and phorbol esters were most effective and prevented cell death through independent pathways. Treating HT22 cells with cholera toxin attenuated the glutamate-induced accumulation of ROS and calcium influx. This was, at least in part, caused by an increase in glutathione due to improved uptake of cystine mediated by the induction of the glutamate/cystine-antiporter subunit xCT or, additionally, by the up-regulation of the antiapoptotic protein Bcl-2. Gs activation also protected HT22 cells from hydrogen peroxide or inhibition of glutathione synthesis by buthionine sulfoximine, and immature cortical neurones from oxidative glutamate toxicity. Thus, this pathway might be more generally implicated in protection from neuronal death by oxidative stress.     

Gliopreventive effects of guanosine against glucose deprivation in vitro

Guanosine, a guanine-based purine, is recognized as an extracellular signaling molecule that is released from astrocytes and confers neuroprotective effects in several in vivo and in vitro studies. Astrocytes regulate glucose metabolism, glutamate transport, and defense mechanism against oxidative stress. C6 astroglial cells are widely used as an astrocyte-like cell line to study the astrocytic function and signaling pathways. Our previous studies showed that guanosine modulates the glutamate uptake activity, thus avoiding glutamatergic excitotoxicity and protecting neural cells. The goal of this study was to determine the gliopreventive effects of guanosine against glucose deprivation in vitro in cultured C6 cells. Glucose deprivation induced cytotoxicity, an increase in reactive oxygen and nitrogen species (ROS/RNS) levels and lipid peroxidation as well as affected the metabolism of glutamate, which may impair important astrocytic functions. Guanosine prevented glucose deprivation-induced toxicity in C6 cells by modulating oxidative and nitrosative stress and glial responses, such as the glutamate uptake, the glutamine synthetase activity, and the glutathione levels. Glucose deprivation decreased the level of EAAC1, the main glutamate transporter present in C6 cells. Guanosine also prevented this effect, most likely through PKC, PI3K, p38 MAPK, and ERK signaling pathways. Taken together, these results show that guanosine may represent an important mechanism for protection of glial cells against glucose deprivation. Additionally, this study contributes to a more thorough understanding of the glial- and redox-related protective properties of guanosine in astroglial cells.     

Apolipoprotein E protects against oxidative stress in mixed neuronal-glial cell cultures by reducing glutamate toxicity

Apolipoprotein E (ApoE) deficiency has been shown to adversely affect outcome after transient cerebral ischemia and head trauma. Since oxidative stress contributes to these injuries, the ability of ApoE to reduce irreversible oxidative damage was studied in primary mixed neuronal-glial cell cultures. Cells (13-16 days in vitro) were exposed to 50 microM hydrogen peroxide (H2O2) for 30 min, and toxicity was determined by the release of lactate dehydrogenase (LDH) 24 h after exposure. The presence of recombinant human ApoE2 (100, 300, or 1000 nM) in the culture media partially protected against oxidative injury. This protection was not reversed by pre-treatment with receptor associated protein. The NMDA receptor antagonist, MK-801, also provided partial protection against H2O2 toxicity. The degree of protection was similar to that conferred by ApoE treatment. The protective effects of ApoE and MK-801 were not additive; no ApoE protection was observed in cultures treated with MK-801 prior to H2O2 exposure. ApoE treatment had no effect on H2O2 stimulated glutamate release, but did increase the rate of glutamate uptake via the high affinity glutamate transporter in H2O2 treated cultures. Pre-treatment with ApoE also conferred partial protection against glutamate-induced LDH release. Taken together, these findings suggest that ApoE protects mixed neuronal-glial cell cultures against irreversible oxidative injury from H2O2 by reducing secondary glutamate excitotoxicity.     

Neuroprotective immunity: T cell-derived glutamate endows astrocytes with a neuroprotective phenotype

A well-controlled T cell response to CNS injury may result in increased neuronal survival. However, the precise mechanism of T cell-induced neuroprotection is unknown. In this study, we report the unexpected finding that during culture of T cells, high levels of glutamate accumulate, which are efficiently cleared if T cells are cocultured with astrocytes. The T cell-derived glutamate elicits in turn, the release of neuroprotective thiols (cysteine, glutathione, and cysteinyl-glycine) and lactate from astrocytes. Media obtained from astrocytes conditioned in the presence of T cells reduce neuronal apoptosis induced by oxidative stress in primary neuronal cultures from 48 +/- 14 to 9 +/- 4% (p < 0.001). Inhibition of glutamate-dependent signaling during astrocyte-T cell cocultivation by a glutamate uptake inhibitor, l-aspartic acid beta-hydroxamate, abolishes this neuroprotective effect. The ability of astrocytes to clear extracellular glutamate is impaired under conditions of oxidative stress. We demonstrate that T cells, via secreted cytokines, restore glutamate clearance capacity of astrocytes under oxidative conditions. Furthermore, under normoxic conditions, glutamate-buffering capacity of astrocytes is increased upon cocultivation with T cells. It is known that, following CNS injury, astrocytes can respond with beneficial or destructive effects on neurons. However, the context and signaling mechanisms for this dual astrocytic response are unknown. Our results implicate T cells as potential determinants of the context that elicits a protective role for astrocytes in the damaged CNS.     

Role of astrocytes in trimethyltin neurotoxicity

Although the neurotoxicity of trimethyltin (TMT) is well known, mechanisms are still not clear. Glia have been proposed to mediate the toxic action of TMT on nerve cells. Accordingly, the effects of TMT were tested in primary neuronal cultures from rat cerebellum and compared to effects in astrocytes and mixed cultures. Neuronal damage observed following TMT exposure was less in the presence of astrocytes and astrocytes alone were resistant to TMT. Thus, astrocytes have a protective effect against TMT-induced neurotoxicity. TMT caused an oxidative stress in granule cell cultures involving a variety of oxidative species (O2)*-, H2O2, NO), but astrocytes were less sensitive to TMT-induced oxidative species generation. Antioxidants, glutathione and 7-nitroindazole attenuated neuronal cell death induced by TMT. It appears that oxidative stress mediates a large part of the destructive action of TMT in neuronal cultures. The presence of astrocytes appears to modulate TMT-induced oxidative stress so that TMT causes only a small increase in lipid peroxidation in mouse brain after systemic administration. Thus, TMT induces a pronounced oxidative stress in cultured neurons, but when astrocytes are present, oxidative species play a lesser role in the neurotoxic action of TMT.     

The glio-toxic mechanism of α-aminoadipic acid on cultured astrocytes

The mechanism of action of the glutamate analogue α-aminoadipic (A A A) acid was investigated in terms of its toxicity to cultured astrocytes. A A A was more toxic to type 1 astrocytes than type 2 astrocytes. Also the higher toxicity of the L-isomer as compared to the D-isomer was seen on type 1 astrocytes but not type 2. The toxicity of A A A can be reduced by co-culture of type 1 astrocytes with microglia. This inhibition may be due to glutamate release by microglia. No such effect is seen for type 2 astrocytes. The major uptake route for A A A by type 1 astrocytes is through the sodium dependent glutamate port. Both isomers of A A A are toxic to dividing astrocytes. The D-isomer appears to be toxic only for mitotic cells. The mechanism of this toxicity is protein synthesis dependent. It is suggested that A A A is toxic to mitotic astrocytes by interference with protein synthesis needed for cell division. D-A A A as opposed to L-A A A may prove a valuable tool for investigation of astrocyte proliferation in development and disease.     

Upregulation of cerebral cortical glutathione synthesis by ammonia in vivo and in cultured glial cells: the role of cystine uptake

Glutathione (GSH) is a major antioxidant in the brain and ammonia neurotoxicity is associated with oxidative stress. In this study, we show that intracerebral administration of ammonium chloride (“ammonia”, final concentration 5 mM) via a microdialysis probe, increases by 80% the glutathione content in cerebral cortical microdialysates, and tends to increase its content in striatal microdialysates. Treatment with ammonia in vitro dose-dependently increased the glutathione content in cultured cerebral cortical astrocytes and a C6 glioma cell line. Significant effects have been observed after 1 h (astrocytes) or 3 h (C6 cells) of exposure and were sustained up to 72 h of incubation. A gradual decrease of the GSH/GSSG ratio noted during 3 h (astrocytes) or 24 h (C6 cells) of exposure, was followed by an partial recovery after 24 h of incubation, the latter phase possibly reflecting increased availability of de novo synthesized glutathione. In our hands, cystine, the precursor for astrocytic glutathione synthesis, was transported to astrocytes almost exclusively by system X_AG⁻, while in C6 cells the transport engaged both system x_c⁻ (60% of uptake) and X_AG⁻ (40% of uptake). Ammonia in either cell type stimulated cystine uptake without changing the relative contribution of the uptake systems. The results are consistent with the concept of increased astrocytic glutathione synthesis as an adaptive response of the brain to ammonia challenge, and emphasize upregulation of cystine uptake as a factor contributing to this response.     

Enhanced NAD(P)H:Quinone Reductase Activity Prevents Glutamate Toxicity Produced by Oxidative Stress

Glutamate toxicity in the N18-RE-105 neuronal cell line results from the inhibition of high-affinity cystine uptake, which leads to a depletion of glutathione and the accumulation of oxidants. Production of superoxides by one-electron oxidation/reduction of quinones is decreased by NAD(P)H:quinone reductase, an enzyme with DT-diaphorase activity. Using glutamate toxicity in N18-RE-105 cells as a model of neuronal oxidative stress, we report that the degree of glutamate toxicity observed is inversely proportional to quinone reductase activity. Induction of quinone reductase activity by treatment with t-butylhydroquinone reduced glutamate toxicity by up to 80%. In contrast, treatment with the quinone reductase inhibitor dicumarol potentiated the toxic effect of glutamate. Measurement of cellular glutathione indicates that increases in its levels are not responsible for the protective effect of t-butylhydroquinone treatment. Because many types of cell death may involve the formation of oxidants, induction of quinone reductase may be a new strategy to combat neurodegenerative disease.     

Transport and toxic mechanism for aluminum citrate in human neuroblastoma SH-SY5Y cells

Aluminum (Al) is thought to be a risk factor for neurodegenerative disorders, but the molecular mechanism has been not clarified yet. In this study, we examined how a transport system handled transport of Al citrate, the major Al species in brain, and effect of Al citrate treatment on expression of the transporter and on susceptibility to oxidative stress in human neuroblastoma SH-SY5Y cells. Uptake of Al citrate by the cells was temperature- and concentration-dependent, and inwardly-directed Na(+)-gradient-independent. Simultaneous application and preloading of L-cystine or L-glutamate inhibited and stimulated, respectively, the Al citrate uptake by SH-SY5Y cells, demonstrating kinetically that Na(+)-independent L-cystine/L-glutamate exchanger, system Xc(-), is involved in its uptake. When the cells were treated with Al citrate, but not citrate, for 2 weeks, but not a day, the expression of the transporter was decreased. Although the cell viability and glutathione content of the cells were not altered by the treatment with Al citrate alone, the number of dead cells among the Al citrate-treated cells increased on exposure to oxidative stress caused by a glucose deprivation/reperfusion treatment. These findings demonstrate that Al citrate is a substrate for system Xc(-), and that chronic treatment with Al citrate causes downregulation of the transporter and increases the vulnerability of the cells to oxidative stress without a direct effect on the viability or GSH content.     

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.     

Characterization of cystine uptake in cultured astrocytes

Glutathione is involved in the maintenance of the structural and functional integrity of membrane proteins, in protection against free radicals and oxidative stress, and in the detoxification of xenobiotics. The cellular uptake of cystine is the rate limiting step in the biosynthesis of glutathione. The precise mechanism for such uptake is not clear as some reports indicate that the uptake occurs through a glutamate-cystine antiporter (system X(c)(-)), whereas, others suggest that it is taken up by the glutamate transporter (system X(AG)). Our studies in cultured astrocytes derived from neonatal rats showed that glutamate, D- and L-aspartate inhibited cystine uptake; that factors that increased intracellular glutamate levels, which would have enhanced the activity of the antiporter, did not stimulate cystine uptake; that the uptake was sodium dependent and partially chloride dependent; that the b(o,+) and ASC systems, which have been shown to carry cystine in some cells, did not mediate cystine uptake in astrocytes; that glutamate uptake blockers such as L-aspartate-beta-hydroxamate (AbetaH) and L-trans-pyrrolidine-2,4-dicarboxylate (PDC), as well as cystine uptake inhibitor L-alpha-aminoadipate (AAA) potently reduced cystine uptake. Additionally, deferoxamine (100 microM) as well as ammonium chloride (5 mM), both of which inhibit glutamate uptake, also inhibited cystine uptake. Taken together, our findings indicate that astrocytes take up cystine through a similar, if not identical, system used to take up glutamate. Interference of cystine uptake by astrocytes through the glutamate transport system may have profound effects on the redox state and the structural and functional integrity of the CNS.     

Role of beta-carotene in ameliorating the cadmium-induced oxidative stress in rat brain and testis

The role of oxidative stress in chronic cadmium (Cd) toxicity and its prevention by cotreatment with beta-carotene was investigated. Adult male rats were intragastrically administered 2 mg CdCl2/kg body weight three times a week intragastrically for 3 and 6 weeks. Brain and testicular thiobarbituric acid reactive substances (TBARS) was elevated after 3 and 6 weeks of Cd administration, indicating increased lipid peroxidation (LPO) and oxidative stress. Cellular damage was indicated by inhibition of adenosine triphosphatase (ATPase) activity and increased lactate dehydrogenase (LDH) activity in brain and testicular tissues. Chronic Cd administration resulted in a decline in glutathione (GSH) content and a decrease of superoxide dismutase (SOD) and glutathione S-transferase (GST) activity in both organs. Administration of beta-carotene (250 IU/kg i.g.) concurrent with Cd ameliorated Cd-induced LPO. The brain and testicular antioxidants, SOD, GST, and GSH, decreased by Cd alone, were restored by beta-carotene cotreatment. Concurrent treatment with beta-carotene also ameliorated the decrease in ATPase activity and the increase in LDH activity in brain and testis of Cd-treated rats, indicating a prophylactic action of beta-carotene on Cd toxicity. Therefore, the results indicate that the nutritional antioxidant beta-carotene ameliorated oxidative stress and the loss of cellular antioxidants and suggest that beta-carotene may control Cd-induced brain and testicular toxicity.     

High-Glucose and S100B Stimulate Glutamate Uptake in C6 Glioma Cells

Diabetes mellitus is a disease associated with several changes in the central nervous system, including oxidative stress and abnormal glutamatergic neurotransmission, and the astrocytes play an essential role in these alterations. In vitro studies of astroglial function have been performed using cultures of primary astrocytes or C6 glioma cells. Herein, we investigated glutamate uptake, glutamine synthetase and S100B secretion in C6 glioma cells cultured in a high-glucose environment, as well as some parameters of oxidative stress and damage. C6 glioma cells, cultured in 12 mM glucose medium, exhibited signals of oxidative and nitrosative stress similar to those found in diabetes mellitus and other models of diabetic disease (decrease in glutathione, elevated NO, DNA damage). Interestingly, we found an increase in glutamate uptake and S100B secretion, and a decrease in glutamine synthetase, which might be linked to the altered glutamatergic communication in diabetes mellitus. Moreover, glutamate uptake in C6 glioma cells, like primary astrocytes, was stimulated by extracellular S100B. Aminoguanidine partially prevented the glial alterations induced by the 12 mM glucose medium. Together, these data emphasize the relevance of astroglia in diabetes mellitus, as well as the importance of glial parameters in the evaluation of diabetic disease progression and treatment.     

Glutamate transport alteration triggers differentiation-state selective oxidative death of cultured astrocytes: a mechanism different from excitotoxicity depending on intracellular GSH contents

Recent evidence has been provided for astrocyte degeneration in experimental models of neurodegenerative insults associated with glutamate transport alteration. To determine whether astrocyte death can directly result from altered glutamate transport, we here investigated the effects of L-trans-pyrrolidine-2,4-dicarboxylate (PDC) on undifferentiated or differentiated cultured rat striatal astrocytes. PDC induced death of differentiated astrocytes without affecting undifferentiated astrocyte viability. Death of differentiated astrocytes was also triggered by another substrate inhibitor but not by blockers of glutamate transporters. The PDC-induced death was delayed and apoptotic, and death rate was dose and treatment duration-dependent. Although preceded by extracellular glutamate increase, this death was not mediated through glutamate receptor stimulation, as antagonists did not provide protection. It involves oxidative stress, as a decrease in glutathione contents and a dramatic raise in reactive oxygen species preceded cell loss, and as protection was provided by antioxidants. PDC induced a similar percentage of GSH depletion in the undifferentiated astrocytes, but only a slight increase in reactive oxygen species. Interestingly, undifferentiated astrocytes exhibited twofold higher basal GSH content compared with the differentiated ones, and depleting their GSH content was found to render them susceptible to PDC. Altogether, these data demonstrate that basal GSH content is a critical factor of astrocyte vulnerability to glutamate transport alteration with possible insights onto concurrent death of astrocytes and gliosis in neurodegenerative insults.     

Prion protein regulates glutathione metabolism and neural glutamate and cysteine uptake via excitatory amino acid transporter 3

Prion protein (PrP) plays crucial roles in regulating antioxidant systems to improve cell defenses against cellular stress. Here, we show that the interactions of PrP with the excitatory amino acid transporter 3 (EAAT3), γ‐glutamyl transpeptidase (γ‐GT), and multi‐drug resistance protein 1 (MRP1) in astrocytes and the interaction between PrP and EAAT3 in neurons regulate the astroglial and neuronal metabolism of the antioxidant glutathione. Ablation of PrP in astrocytes and cerebellar neurons leads to dysregulation of EAAT3‐mediated uptake of glutamate and cysteine, which are precursors for the synthesis of glutathione. In PrP‐deficient astrocytes, levels of intracellular glutathione are increased, and under oxidative stress, levels of extracellular glutathione are increased, due to (i) increased glutathione release via MRP1 and (ii) reduced activity of the glutathione‐degrading enzyme γ‐GT. In PrP‐deficient cerebellar neurons, cell death is enhanced under oxidative stress and glutamate excitotoxicity, when compared to wild‐type cerebellar neurons. These results indicate a functional interplay of PrP with EAAT3, MRP1 and γ‐GT in astrocytes and of PrP and EAAT3 in neurons, suggesting that these interactions play an important role in the metabolic cross‐talk between astrocytes and neurons and in protection of neurons by astrocytes from oxidative and glutamate‐induced cytotoxicity.