Astrocytes aged in vitro show a decreased neuroprotective capacity

Alterations in astrocyte function that may affect neuronal viability occur with brain aging. In this study, we evaluate the neuroprotective capacity of astrocytes in an experimental model of in vitro aging. Changes in oxidative stress, glutamate uptake and protein expression were evaluated in rat cortical astrocytes cultured for 10 and 90 days in vitro (DIV). Levels of glial fibrillary acidic protein and S100beta increased at 90 days when cells were positive for the senescence beta-galactosidase marker. In long-term astrocyte cultures, the generation of reactive oxygen species was enhanced and mitochondrial activity decreased. Simultaneously, there was an increase in proteins that stained positively for nitrotyrosine. The expression of Cu/Zn-superoxide dismutase (SOD-1) and haeme oxygenase-1 (HO-1) proteins and inducible nitric oxide synthase (iNOS) increased in aged astrocytes. Glutamate uptake in 90-DIV astrocytes was higher than in 10 DIV ones, and was more vulnerable to inhibition by H2O2 exposure. Enhanced glutamate uptake was probably because of up-regulation of the glutamate/aspartate transporter protein. Aged astrocytes had a reduced ability to maintain neuronal survival. These findings indicate that astrocytes may partially loose their neuroprotective ability during aging. The results also suggest that aged astrocytes may contribute to exacerbating neuronal injury in age-related neurodegenerative processes.     

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.     

Cytotoxicity of the Hypoxanthine-Xanthine Oxidase System on V79 Cells: Comparison of the Effects of Sod and Cudips

The hypoxanthine — xanthine oxidase system generates an extracellular flux of superoxide anion radical (O₂^?) and hydrogen peroxide (H₂O₂). Catalase but not superoxide dismutase (SOD) protects V79 cells exposed to the hypoxanthine — xanthine oxidase system, showing that H₂O₂ is the major reactive oxygen species involved in the cytotoxicity of such a system. In contrast to SOD, the lipophilic SOD like compound CuII (diisopropylsalicylate)₂ (CuDIPS) exhibits some protection at non cytotoxic concentration. It is also found that methanol partially protects cells exposed to the hypoxanthine-xanthine oxidase system. It appears that in our experimental conditions (temperature, ionic strength and pH) the protective effect afforded by methanol and CuDIPS is due to the inhibition of the xanthine oxidase activity.     

Morin hydrate protects cultured rat glomerular mesangial cells against oxyradical damage

Cultured rat glomerular mesangial cells were damaged when exposed to oxyradicals generated either from xanthine oxidase plus hypoxanthine, or by superoxide radicals formed from menadione. Morin hydrate is an antioxidant extracted from yellow Brazil wood. When morin hydrate was added to cultured rat glomerular mesangial cells which were attacked by oxyradicals generated by xanthine oxidase plus hypoxanthine, the survival time of the cells was doubled. However, this protective effect of morin hydrate was less marked when the cells were attacked by menadione. Note that the protective effects of Trolox which is a polar analogue of vitamin E were miniscule relative to those of morin hydrate with both oxidants.     

Metabolic activation of 1-naphthol and phenol by a simple superoxide-generating system and human leukocytes

Phenol and 1-naphthol, products of benzene and naphthalene biotransformation, are metabolized during O2- generation by xanthine oxidase/hypoxanthine and phorbol myristate acetate (PMA)-stimulated human neutrophils. The addition of 1-naphthol to xanthine oxidase/hypoxanthine incubations resulted in the formation of 1,4-naphthoquinone (1,4-NQ) whereas phenol addition yielded only small quantities of hydroquinone, catechol and a unidentified reducible product but not 1,4-benzoquinone. This formation of 1,4-NQ was dependent upon hypoxanthine, xanthine oxidase, and 1-naphthol and was inhibited by the addition of superoxide dismutase (SOD) demonstrating that the conversion was O2-mediated. During O2- generation by PMA-stimulated neutrophils, the addition of phenol interfered with luminol-dependent chemiluminescence and resulted in covalent binding of phenol to protein. Protein binding was 80% inhibited by the addition of azide or catalase to the incubations indicating that bioactivation was peroxidase-mediated. In contrast, the addition of 1-naphthol to PMA-stimulated neutrophils interfered with superoxide-dependent cytochrome c reduction as well as luminol-dependent chemiluminescence and also resulted in protein binding. Protein binding was only partially inhibited by azide or catalase. The addition of SOD in combination with catalase resulted in a significantly greater inhibition of binding when compared to that of catalase alone. The results of these experiments indicate that phenol and 1-naphthol are converted to reactive metabolites during superoxide generating conditions but by different mechanisms. The formation of reactive metabolites from phenol was almost exclusively peroxidase-mediated whereas the bioactivation of 1-naphthol could occur by two different mechanisms, a peroxidase-dependent and a direct superoxide-dependent mechanism.     

Peroxynitrite formation from the simultaneous reduction of nitrite and oxygen by xanthine oxidase

One electron reductions of oxygen and nitrite by xanthine oxidase form peroxynitrite. The nitrite and oxygen reducing activities of xanthine oxidase are regulated by oxygen with K(oxygen) 26 and 100 microM and K(nitrite) 1.0 and 1.1 mM with xanthine and NADH as donor substrates. Optimal peroxynitrite formation occurs at 70 microM oxygen with purine substrates. Kinetic parameters: V(max) approximately 50 nmol/min/mg and K(m) of 22, 36 and 70 microM for hypoxanthine, pterin and nitrite respectively. Peroxynitrite generation is inhibited by allopurinol, superoxide dismutase and diphenylene iodonium. A role for this enzyme activity can be found in the antibacterial activity of milk and circulating xanthine oxidase activity.     

Allopurinol-insensitive oxygen radical formation by milk xanthine oxidase systems.

Oxygen radical generation in the xanthine- and NADH-oxygen reductase reactions by xanthine oxidase, was demonstrated using the ESR spin trap 5,5'-dimethyl-1- pyrroline-N-oxide. No xanthine-dependent oxygen radical formation was observed when allopurinol-treated xanthine oxidase was used. The significant superoxide generation in the NADH-oxygen reductase reaction by the enzyme was increased by the addition of menadione and adriamycin. The NADH-menadione and -adriamycin reductase activities of xanthine oxidase were assessed in terms of NADH oxidation. From Lineweaver-Burk plots, the Km and Vmax of xanthine oxidase were estimated to be respectively 51 microM and 5.5 s-1 for menadione and 12 microM and 0.4 s-1 for adriamycin. Allopurinol-inactivated xanthine oxidase generates superoxide and OH.radicals in the presence of NADH and menadione or adriamycin to the same extent as the native enzyme. Adriamycin radicals were observed when the reactions were carried out under an atmosphere of argon. The effects of superoxide dismutase and catalase revealed that OH.radicals were mainly generated through the direct reaction of H2O2 with semiquinoid forms of menadione and adriamycin.     

Role of superoxide in hemorrhagic shock-induced P-selectin expression

Superoxide has been implicated in the regulation of endothelial cell adhesion molecule expression and the subsequent initiation of leukocyte-endothelial cell adhesion in different experimental models of inflammation. The objective of this study was to assess the contribution of oxygen radicals to P-selectin expression in a murine model of whole body ischemia-reperfusion, i.e., hemorrhage-resuscitation (H/R), with the use of different strategies that interfere with either the production (allopurinol, CD11/CD18-deficient or p47(phox)-/- mice) or accumulation [intravenous superoxide dismutase (SOD), mutant mice that overexpress SOD] of oxygen radicals. P-selectin expression was quantified in different regional vascular beds by use of the dual-radiolabeled monoclonal antibody technique. H/R elicited a significant increase in P-selectin expression in all vascular beds. This response was blunted in SOD transgenic mice and in wild-type mice receiving either intravenous SOD or the xanthine oxidase inhibitor allopurinol. Mice genetically deficient in either a subunit of NADPH oxidase or the leukocyte adhesion molecule CD11/CD18 also exhibited a reduced P-selectin expression. These results implicate superoxide, derived from both xanthine oxidase and NADPH oxidase, as mediators of the increased P-selectin expression observed in different regional vascular beds exposed to hemorrhage and retransfusion.     

Identification of Hypoxanthine Transport and Xanthine Oxidase Activity in Brain Capillaries

Microvessel segments were isolated from rat brain and used for studies of hypoxanthine transport and metabolism. Compared to an homogenate of cerebral cortex, the isolated microvessels were 3.7-fold enriched in xanthine oxidase. Incubation of the isolated microvessels with labeled hypoxanthine resulted in its rapid uptake followed by the slower accumulation of hypoxanthine metabolites including xanthine and uric acid. The intracellular accumulation of these metabolites was inhibited by the xanthine oxidase inhibitor allopurinol. Hypoxanthine transport into isolated capillaries was inhibited by adenine but not by representative pyrimidines or nucleosides. Similar results were obtained when blood to brain transport of hypoxanthine in vivo was measured using the intracarotid bolus injection technique. Thus, hypoxanthine is transported into brain capillaries by a transport system shared with adenine. Once inside the cell, hypoxanthine can be metabolized to xanthine and uric acid by xanthine oxidase. Since this reaction leads to the release of oxygen radicals, it is suggested that brain capillaries may be susceptible to free radical mediated damage. This would be most likely to occur in conditions where the brain hypoxanthine concentration is increased as following ischemia.     

Glutamate Neurotoxicity in Rat Cerebellar Granule Cells: A Major Role for Xanthine Oxidase in Oxygen Radical Formation

Abstract: To gain insight into the mechanism through which the neurotransmitter glutamate causally participates in several neurological diseases, in vitro cultured cerebellar granule cells were exposed to glutamate and oxygen radical production was investigated. To this aim, a novel procedure was developed to detect oxygen radicals; the fluorescent dye 2',7'-dichlorofluorescein was used to detect production of peroxides, and a specific search for the possible conversion of the enzyme xanthine dehydrogenase into xanthine oxidase after the excitotoxic glutamate pulse was undertaken. A 100 µ M glutamate pulse administered to 7-day-old cerebellar granule cells is accompanied by the onset of neuronal death, the appearance of xanthine oxidase, and production of oxygen radicals. Xanthine oxidase activation and superoxide (O₂^•−) production are completely inhibited by concomitant incubation of glutamate with MK-801, a specific NMDA receptor antagonist, or by chelation of external calcium with EGTA. Partial inhibition of both cell death and parallel production of reactive oxygen species is achieved with allopurinol, a xanthine oxidase inhibitor, leupeptin, a protease inhibitor, reducing agents such as glutathione or dithiothreitol, antioxidants such as vitamin E and vitamin C, and externally added superoxide dismutase. It is concluded that glutamate-triggered, NMDA-mediated, massive Ca²⁺ influx induces rapid conversion of xanthine dehydrogenase into xanthine oxidase with subsequent production of reactive oxygen species that most probably have a causal involvement in the initial steps of the series of intracellular events leading to neuronal degeneration and death.     

Lysosomal enzyme leakage during the hypoxanthine/xanthine oxidase reaction

Impairment of lysosomal stability due to reactive oxygen species generated during the oxidation of hypoxanthine by xanthine oxidase was studied in rat liver lysosomes isolated in a discontinuous Nycodenz gradient. Production of O2.- and H2O2 during the hypoxanthine/xanthine oxidase reaction occurred for at least 5 min, while lysosomal damage, indicated by the release of N-acetyl-beta-glucosaminidase, occurred within 30 s, there being no further damage to these organelles thereafter. The extent of lysosomal enzyme release increased with increasing xanthine oxidase concentration. Superoxide dismutase and catalase did not prevent lysosomal damage during the hypoxanthine/xanthine oxidase reaction. Lysosomes reduced xanthine oxidase activity, as assessed in terms of O2 consumption, only slightly but substantially inhibited in a competitive manner the O2.- -mediated reduction of cytochrome c. This inhibition was almost completely reversed by potassium cyanide, thus pointing to the presence of a cyanide-sensitive superoxide dismutase in the lysosomal fraction. However, potassium cyanide did not affect the hypoxanthine/xanthine oxidase-mediated lysosomal damage, thus suggesting an inability of the lysosomal superoxide dismutase to protect the organelles. Negligible malondialdehyde formation was observed in the lysosomes either during the hypoxanthine/xanthine oxidase reaction or with different selective experimental approaches known to produce lipid peroxidation in other organelles such as microsomes and mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of Gap Junction Elevates Glutamate Uptake in Cultured Astrocytes

Glutamate uptake is a main function of astrocytes to keep extracellular glutamate levels low and protect neurons against glutamate-induced excitotoxicity. On the other hand, astrocyte networks formed by gap junctions, which are consisted with connexins and connecting neighboring cells, are reported to play a critical role in maintaining the homeostasis in the brain. In the present study, we examined the effects of gap junction inhibitors on the glutamate uptake activity in cultured rat cortical astrocytes. At first, we confirmed the effects of gap junction inhibitors, 1-octanol and carbenoxolone, on cell–cell communication by the scrape-loading assay using a fluorescent dye Lucifer yellow. Both of 1-octanol and carbenoxolone treatments for 20 min in cultured astrocytes significantly suppressed the cell–cell communication assessed as the distance of dye-spreading. 1-octanol and carbenoxolone increased the glutamate uptake by astrocytes and glutamate aspartate transporter (GLAST) expression on the cell membrane. These results suggest that gap junction inhibitors increase the glutamate uptake activity through the increase of GLAST proteins located on the cell membrane. The regulation of gap junction in astrocytes might protect neurons against glutamate-induced excitotoxicity.     

Glial soluble factors regulate the activity and expression of the neuronal glutamate transporter EAAC1: implication of cholesterol

A co-ordinated regulation between neurons and astrocytes is essential for the control of extracellular glutamate concentration. Here, we have investigated the influence of astrocytes and glia-derived cholesterol on the regulation of glutamate transport in primary neuronal cultures from rat embryonic cortices. Glutamate uptake rate and expression of the neuronal glutamate transporter EAAC1 were low when neurons were grown without astrocytes and neurons were unable to clear extracellular glutamate. Treatment of the neuronal cultures with glial conditioned medium (GCM) increased glutamate uptake Vmax, EAAC1 expression and restored the capacity of neurons to eliminate extracellular glutamate. Thus, astrocytes up-regulate the activity and expression of EAAC1 in neurons. We further showed that cholesterol, present in GCM, increased glutamate uptake activity when added directly to neurons and had no effect on glutamate transporter expression. Furthermore, part of the GCM-induced effect on glutamate transport activity was lost when cholesterol was removed from GCM (low cholesterol-GCM) and was restored when cholesterol was added to low cholesterol-GCM. This demonstrates that glia-derived cholesterol regulates glutamate transport activity. With these experiments, we provide new evidences for neuronal glutamate transport regulation by astrocytes and identified cholesterol as one of the factors implicated in this regulation.     

Dysfunction of astrocytes in senescence-accelerated mice SAMP8 reduces their neuroprotective capacity

Early onset increases in oxidative stress and tau pathology are present in the brain of senescence-accelerated mice prone (SAMP8). Astrocytes play an essential role, both in determining the brain's susceptibility to oxidative damage and in protecting neurons. In this study, we examine changes in tau phosphorylation, oxidative stress and glutamate uptake in primary cultures of cortical astrocytes from neonatal SAMP8 mice and senescence-accelerated-resistant mice (SAMR1). We demonstrated an enhancement of abnormally phosphorylated tau in Ser(199) and Ser(396) in SAMP8 astrocytes compared with that of SAMR1 control mice. Gsk3beta and Cdk5 kinase activity, which regulate tau phosphorylation, was also increased in SAMP8 astrocytes. Inhibition of Gsk3beta by lithium or Cdk5 by roscovitine reduced tau phosphorylation at Ser(396). Moreover, we detected an increase in radical superoxide generation, which may be responsible for the corresponding increase in lipoperoxidation and protein oxidation. We also observed a reduced mitochondrial membrane potential in SAMP8 mouse astrocytes. Glutamate uptake in astrocytes is a critical neuroprotective mechanism. SAMP8 astrocytes showed a decreased glutamate uptake compared with those of SAMR1 controls. Interestingly, survival of SAMP8 or SAMR1 neurons cocultured with SAMP8 astrocytes was significantly reduced. Our results indicate that alterations in astrocyte cultures from SAMP8 mice are similar to those detected in whole brains of SAMP8 mice at 1-5 months. Moreover, our findings suggest that this in vitro preparation is suitable for studying the molecular and cellular processes underlying early aging in this murine model. In addition, our study supports the contention that astrocytes play a key role in neurodegeneration during the aging process.     

Extracellular Superoxide Dismutase in Cultured Astrocytes: Decrease in Cell-Surface Activity and Increase in Medium Activity by Lipopolysaccharide-Stimulation

Under pathological conditions such as ischemia/reperfusion, a large amount of superoxide anion (O(2) (-)) is produced and released in brain. Among three isozymes of superoxide dismutase (SOD), extracellular (EC)-SOD, known to be excreted outside cells and bound to extracellular matrix, should play a role to detoxify O(2) (-) in extracellular space; however, a little is known about EC-SOD in brain. In order to evaluate the SOD activity in extracellular space of CNS as direct as possible, we attempted to measure the cell-surface SOD activity on primary cultured rat brain cells by the inhibition of color development of a water-soluble tetrazolium due to O(2) (-) generation by xanthine oxidase/hypoxanthine added into extracellular medium of intact cells. The cell-surface SOD activity on cultured neuron and microglia was below the detection limit; however, that on cultured astrocyte was high enough to measure. By means of RT-PCR, all mRNA of three isozymes of SOD could be detected in the three types of the cells examined; however, the semi-quantitative analysis revealed that the level of EC-SOD mRNA in astrocytes was significantly higher than that in neurons and microglia. When astrocytes were stimulated with lipopolysaccharide (LPS) for 12-24?h, the cell-surface SOD activity decreased to a half, whereas the activity recovered after 36-48?h. The decrease in the activity was dependent on the LPS concentration. On the other hand, the SOD activity in the medium increased by the LPS-stimulation in a dose dependent manner; suggesting that the SOD protein localized on cell-surface, probably EC-SOD, was released into the medium. These results suggest that EC-SOD of astrocyte play a role for detoxification of extracellular O(2) (-) and the regulation of EC-SOD in astrocytes may contribute to the defensive mechanism against oxidative stress in brain.     

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.     

Amyloid β Peptide (25–35) Inhibits Na+-Dependent Glutamate Uptake in Rat Hippocampal Astrocyte Cultures

Abstract: Large numbers of neuritic plaques surrounded by reactive astrocytes are characteristic of Alzheimer's disease (AD). There is a large body of research supporting a causal role for the amyloid β peptide (Aβ), a main constituent of these plaques, in the neuropathology of AD. Several hypotheses have been proposed to explain the toxicity of Aβ including free radical injury and excitotoxicity. It has been reported that treatment of neuronal/astrocytic cultures with Aβ increases the vulnerability of neurons to glutamate-induced cell death. One mechanism that may explain this finding is inhibition of the astrocyte glutamate transporter by Aβ. The aim of the current study was to determine if Aβs inhibit astrocyte glutamate uptake and if this inhibition involves free radical damage to the transporter/astrocytes. We have previously reported that Aβ can generate free radicals, and this radical production was correlated with the oxidation of neurons in culture and inhibition of astrocyte glutamate uptake. In the present study, Aβ (25–35) significantly inhibited l -glutamate uptake in rat hippocampal astrocyte cultures and this inhibition was prevented by the antioxidant Trolox. Decreases in astrocyte function, in particular l -glutamate uptake, may contribute to neuronal degeneration such as that seen in AD. These results lead to a revised excitotoxicity/free radical hypothesis of Aβ toxicity involving astrocytes.     

Comparison of the effects of superoxide dismutase and cytochrome c on luminol chemiluminescence produced by xanthine oxidase-catalyzed reactions

Superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1) (SOD) and ferricytochrome c are used to check the effects on luminol chemiluminescence induced by a xanthine or hypoxanthine/xanthine oxidase/oxygen system. Luminol chemiluminescence has been attributed to superoxide anion radical (O2.-) in this system. From kinetic studies on the light intensity vs. time curves it is demonstrated that addition of SOD into the system does not affect the mechanism of O2.- generation, whilst ferricytochrome c dramatically alters the time-course of the reaction. This is interpreted as the effect of cytochrome c redox cycling by reaction with H2O2, modifying oxy-radical generation in the reaction medium. Also, an alternative mechanism for luminol chemiexcitation is proposed under certain experimental conditions.     

Glutamate/glutamine metabolism coupling between astrocytes and glioma cells: Neuroprotection and inhibition of glioma growth

Glioma glutamate release has been shown to promote the growth of glioma cells and induce neuronal injuries from epilepsy to neuronal death. However, potential counteractions from normal astrocytes against glioma glutamate release have not been fully evaluated. In this study, we investigated the glutamate/glutamine cycling between glioma cells and astrocytes and their impact on neuronal function. Co-cultures of glioma cells with astrocytes (CGA) in direct contact were established under different mix ratio of astrocyte/glioma. Culture medium conditioned in these CGAs were sampled for HPLC measurement, for neuronal ratiometric calcium imaging, and for neuronal survival assay. We found: (1) High levels of glutaminase expression in glioma cells, but not in astrocytes, glutaminase enables glioma cells to release large amount of glutamate in the presence of glutamine. (2) Glutamate levels in CGAs were directly determined by the astrocyte/glioma ratios, indicating a balance between glioma glutamate release and astrocyte glutamate uptake. (3) Culture media from CGAs of higher glioma/astrocyte ratios induced stronger neuronal Ca²⁺ response and more severe neuronal death. (4) Co-culturing with astrocytes significantly reduced the growth rate of glioma cells. These results indicate that normal astrocytes in the brain play pivotal roles in glioma growth inhibition and in reducing neuronal injuries from glioma glutamate release. However, as tumor growth, the protective role of astrocytes gradually succumb to glioma cells.     

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.