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.     

Postsynaptic scaffold protein Homer 1a protects against traumatic brain injury via regulating group I metabotropic glutamate receptors

Traumatic brain injury (TBI) produces excessive glutamate, leading to excitotoxicity via the activation of glutamate receptors. Postsynaptic density scaffold proteins have crucial roles in mediating signal transduction from glutamate receptors to their downstream mediators. Therefore, studies on the mechanisms underlying regulation of excitotoxicity by scaffold proteins can uncover new treatments for TBI. Here, we demonstrated that the postsynaptic scaffold protein Homer 1a was neuroprotective against TBI in vitro and in vivo, and this neuroprotection was associated with its effects on group I metabotropic glutamate receptors (mGluRs). Upon further study, we found that Homer 1a mainly affected neuronal injury induced by mGluR1 activation after TBI and also influenced mGluR5 function when its activity was restored. The ability of Homer 1a to disrupt mGluR-ERK signaling contributed to its ability to regulate the functions of mGluR1 and mGluR5 after traumatic injury. Intracellular Ca²⁺ and PKC were two important factors involved in the mediation of mGluR-ERK signaling by Homer 1a. These results define Homer 1a as a novel endogenous neuroprotective agent against TBI.     

Metabotropic glutamate receptor 3 protects neurons from glucose-induced oxidative injury by increasing intracellular glutathione concentration

High glucose concentrations cause oxidative injury and programmed cell death in neurons, and can lead to diabetic neuropathy. Activating the type 3 metabotropic glutamate receptor (mGluR3) prevents glucose-induced oxidative injury in dorsal root ganglion neurons co-cultured with Schwann cells. To determine the mechanisms of protection, studies were performed in rat dorsal root ganglion neuron-Schwann cell co-cultures. The mGluR3 agonist 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate prevented glucose-induced inner mitochondrial membrane depolarization, reactive oxygen species accumulation, and programmed cell death, and increased glutathione (GSH) concentration in co-cultured neurons and Schwann cells, but not in neurons cultured without Schwann cells. Protection was diminished in neurons treated with the GSH synthesis inhibitor l-buthionine-sulfoximine, suggesting that mGluR-mediated protection requires GSH synthesis. GSH precursors and the GSH precursor GSH-ethyl ester also protected neurons from glucose-induced injury, indicating that GSH synthesis in Schwann cells, and transport of reaction precursors to neurons, may underlie mGluR-mediated neuroprotection. These results support the conclusions that activating glial mGluR3 protects neurons from glucose-induced oxidative injury by increasing free radical scavenging and stabilizing mitochondrial function, through increased GSH antioxidant defense.     

Schisandrin B protects against tert-butylhydroperoxide induced cerebral toxicity by enhancing glutathione antioxidant status in mouse brain

Schisandrin B (Sch B), a dibenzocyclooctadiene derivative isolated from Fructus Schisandrae, has been shown to produce antioxidant effect on rodent liver and heart. A mouse model of tert-butylhydroperoxide (t-BHP) induced cerebral toxicity was adopted for examining the antioxidant potential of Sch B in the brain. Intracerebroventricular injection of t-BHP caused a time-dependent increase in mortality rate in mice. The t-BHP toxicity was associated with an increase in the extent of cerebral lipid peroxidation and an impairment in cerebral glutathione antioxidant status, as evidenced by the abrupt decrease in reduced glutathione (GSH) level and the inhibition of Se-glutathione peroxidase activity at 5 min following t-BHP challenge. Sch B pretreatment (1 or 2 mmol/kg/day × 3) produced a dose-dependent protection against t-BHP induced mortality. The protection was associated with a decrease in the extent of lipid peroxidation and an enhancement in glutathione antioxidant status in brain tissue detectable at 5 min post t-BHP challenge, with the assessed biochemical parameters being returned to normal values at 60 min in Sch B pretreated mice at a dose of 2 mmol/kg. The ensemble of results suggests the antioxidant potential of Sch B pretreatment in protecting against cerebral oxidative stress.     

MCP-1 (CCL2) protects human neurons and astrocytes from NMDA or HIV-tat-induced apoptosis

Acquired immunodeficiency syndrome (AIDS)-associated dementia is often characterized by chronic inflammation, with infected macrophage infiltration of the CNS resulting in the production of human immunodeficiency virus type 1 (HIV-1) products, including tat, and neurotoxins that contribute to neuronal loss. In addition to their established role in leukocyte recruitment and activation, we identified an additional role for chemokines in the CNS. Monocyte chemoattractant protein-1 (MCP-1 or CCL2) and regulated upon activation normal T cell expressed and secreted (RANTES) were found to protect mixed cultures of human neurons and astrocytes from tat or NMDA-induced apoptosis. Neuronal and astrocytic apoptosis in these cultures was significantly inhibited by co-treatment with MCP-1 or RANTES but not IP-10. The protective effect of RANTES was blocked by antibodies to MCP-1, indicating that RANTES protection is mediated by the induction of MCP-1. The NMDA blocker, MK801, also abolished the toxic effects of both tat and NMDA. Tat or NMDA treatment of mixed cultures for 24 h resulted in increased extracellular glutamate ([Glu]e) and NMDA receptor 1 (NMDAR1) expression, potential contributors to apoptosis. Co-treatment with MCP-1 inhibited tat and NMDA-induced increases in [Glu]e and NMDAR1, and also reduced the levels and number of neurons containing intracellular tat. These data indicate that MCP-1 may play a novel role as a protective agent against the toxic effects of glutamate and tat.     

Protection against pulmonary oxygen toxicity by interleukin-1 and tumor necrosis factor: Role of antioxidant enzymes and effect of cyclooxygenase inhibitors

Rats injected with interleukin-1 (10 g) and tumor necrosis factor (10 g) and then exposed continuously to hyperoxia (> 99% O₂, 1 atm) survived longer, had increased lung reduced/oxidized glutathione ratios, smaller pleural effusions, less pulmonary hypertension and improv+++ed arterial blood gases. The percentage of animals surviving for 72 hours in hyperoxia increased from 8% to 94%. Although relatively small increases in glutathione redox cycle enzymes occurred four and sixteen hours following cytokine injection, dramatic increases in all major antioxidant enzymes including superoxide dismutase, glucose-6-phosphate dehydrogenase, glutathione reductase, glutathione peroxidase, and catalase had occurred following 72 hours of exposure to hyperoxia. The protective effect of IL-1 + TNF against lethal pulmonary O₂ toxicity could be partially inhibited by pre-injection of lysine acetylsalicylate or, less effectively, of ibuprofen.Recent studies have suggested that both IL-1 and TNF can induce manganese (mitochondrial) superoxide dismutase mRNA and protein synthesis in a variety of cell types. Preliminary studies suggest that IL-1 alone, in ample dosage, can provide protection against lethal pulmonary O₂ toxicity. Future studies should be directed toward identification of acute phase changes in lung antioxidant enzymes, surfactant proteins and/or lipid components, enzymes needed for synthesis of surfactant phospholipids, and/or other protective proteins. Additional work also needs to be done in identifying the lung cell types in which early enzyme induction occurs. These studies should provide a better understanding of mechanisms whereby protection against pulmonary O₂ toxicity can occur. An understanding of the molecular mechanisms inducing protective proteins should lead to more precise pharmacologic control of these processes.     

Group II metabotropic glutamate receptor activation protects striatal dopaminergic nerve terminals against MPP+-induced neurotoxicity along with brain-derived neurotrophic factor induction

We have studied the in vivo effect of the selective agonist for group II metabotropic glutamate receptors (2S, 2'R, 3'R)-2-(2'3'-dicarboxycyclopropyl)glycine (DCG-IV) against MPP+-induced toxicity on rat striatal dopaminergic nerve terminals by using both microdialysis and immunohistochemical techniques. Perfusion of 1 mM DCG-IV during 1 h protected dopaminergic nerve terminals against the degeneration induced by a 15-minute perfusion of 1 mM MPP+. In addition, the microglial cell population was markedly activated 24 h after DCG-IV perfusion. The astroglial cell population was only markedly activated around the microdialysis probe. This protective effect seems to be dependent on protein synthesis since 1 mM cycloheximide, an inhibitor of protein synthesis, abolished the neuroprotective effect of 1 mM DCG-IV against MPP+ toxicity. Perfusion of DCG-IV induced an upregulation of striatal brain-derived neurotrophic factor (BDNF) mRNA expressing cells which were confined precisely around the microdialysis probe. Taken together, our results suggest that the induction and release of brain-derived neurotrophic factor (BDNF) by activated glial cells induced by DCG-IV perfusion may account for its protective action against MPP+-induced dopaminergic terminal degeneration.     

Insulin increases glutamate transporter GLT1 in cultured astrocytes

The astroglial cell-specific glutamate transporter subtype 2 (excitatory amino acid transporter 2, GLT1) plays an important role in excitotoxicity that develops after damage to the central nervous system (CNS) is incurred. Both the protein kinase C signaling pathway and the epidermal growth factor (EGF) pathway have been suggested to participate in the modulation of GLT1, but the modulatory mechanisms of GLT1 expression are not fully understood. In the present study, we aimed to evaluate the effects of insulin on GLT1 expression. We found that short-term stimulation of insulin led to the upregulation of both total and surface expressions of GLT1. Akt phosphorylation increased after insulin treatment, and triciribine, the inhibitor of Akt phosphorylation, significantly inhibited the effects of insulin. We also found that the upregulation of GLT1 expression correlated with increased kappa B motif-binding phosphoprotein (KBBP) and GLT1 mRNA levels. Our results suggest that insulin may modulate the expression of astrocytic GLT1, which might play a role in reactive astrocytes after CNS injuries.     

P2Y1 receptor-mediated glutamate release from cultured dorsal spinal cord astrocytes

P2 receptors have been implicated in the release of neurotransmitter and proinflammatory cytokines by the response to neuroexcitatory substances in astrocytes. In the present study, we examined the mechanisms of ADP and adenosine 5'-O-2-thiodiphosphate (ADPbetaS, ADP analogue) on glutamate release from cultured dorsal spinal cord astrocytes by using confocal laser scanning microscopy and HPLC. Immunofluorescence activity showed that P2Y₁ receptor protein is expressed in cultured astrocytes. ADP and ADPbetaS-induced [Ca²⁺]_i increase and glutamate release are mediated by P2Y₁ receptor. Ca²⁺ release from IP₃-sensitive calcium stores and protein kinase C (PKC) activation is important for glutamate release from astrocytes. Furthermore, P2Y₁ receptor-evoked glutamate release is regulated by volume-sensitive Cl⁻ channels and anion co-transporter, which open up the possibility that P2Y₁ receptor activation causes the increase of cell volume. Release of glutamate by ADPbetaS was abolished by 5-nitro-2 (3-phenyl propy lamino)–benzoate plus furosemide but was unaffected by botulinum toxin A. These observations indicate that P2Y₁ receptor-evoked glutamate may be mediated via volume-sensitive Cl⁻ channel but not via exocytosis of glutamate containing vesicles. We speculate that P2Y₁ receptors-evoked glutamate efflux, occurring under pathological condition, may modulate the activity of synapses in spinal cord.     

Hypoxia-inducible factor 1alpha regulates lung adenocarcinoma cell invasion

We studied the role of hypoxia-inducible factor-1alpha (HIF-1alpha) in human lung adenocarcinoma cell invasion using a metastatic cell model composed of low invasive CL1 and highly invasive CL1-5 cells. We showed that HIF-1alpha was expressed in CL1-5 but not in CL1 cells under normoxic condition, and that inhibition of HIF-1alpha expression by a small interfering RNA decreased invasiveness of CL1-5 cells. Complementary, overexpression of HIF-1alpha increased the invasiveness of CL1 and gastric cancer SC-M1 cells. Subsequently, we showed that urokinase-type plasminogen activator receptor (uPAR), and matrix metalloproteinases (MMPs) 1 and 2 were critical in HIF-1alpha-induced invasion. Mechanistic studies revealed that HIF-1alpha overexpression could increase the expression of uPAR and MMP1, but not MMP2. However, ELISA assays on the conditioned media generated from control CL1 and CL1 cells overexpressing HIF-1alpha showed that overexpression of HIF-1alpha increased the levels of endogenous free active MMP2 and total free MMP2, and the former was blocked by inhibition of MMP1 expression. We conclude that (i) HIF-1alpha overexpression enhances lung cancer cell invasion at least through up-regulating the expression and activities of uPAR, MMP1, and MMP2; and (ii) induction of MMP1 participates in cell invasion and also plays an important role in HIF-1alpha-induced activation of MMP2.     

Hypoxia-inducible factor 1-alpha and vascular endothelial growth factor in cartilage tumors

Neoangiogenesis has been demonstrated in chondrosarcoma. Anti-angiogenic therapies are being tested in clinical trials for chondrosarcomas. Studies of the underlying mechanisms have been performed almost exclusively in cell lines. We immunostained 20 samples of chondrosarcoma and 20 samples of enchondromas with antibodies against hypoxia-inducible factor 1-alpha (HIF-1-alpha) and vascular endothelial growth factor (VEGF). The immunohistochemical staining of HIF-1-alpha and VEGF were highly correlated. Enchondromas were HIF-1-alpha and VEGF negative, whereas all chondrosarcoma exhibited HIF-1-alpha and VEGF immunostaining. HIF-1-alpha/VEGF double positive cases were almost exclusively chondrosarcomas with a high tumor grade. We suggest that HIF-1-alpha is a marker of malignancy in chondrosarcomas that correlates with tumor neo-angiogenesis. Our findings also suggest that a HIF-1-alpha/VEGF angiogenic pathway may exist in chondrosarcoma in vivo as in other malignant tumors. The inclusion of novel inhibitors to HIF-1-alpha and other factors may optimize anti-angiogenic interventions in chondrosarcoma.     

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

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

Stromal cell-derived factor 1 (SDF-1) and its receptor CXCR4 improves diabetic retinopathy

Diabetes induced a serious of complications including diabetic retinopathy. Our study aimed to investigate the role of Stromal cell-derived factor 1 (SDF-1) and its receptor CXCR4 in diabetic retinopathy. A mice model of diabetic retinopathy was established, and expression of SDF-1 and CXCR4 in retina was examined by Real-time quantitative PCR (qRT-PCR). Cells of human retinal pigment epithelial cell line ARPE-19 were treated with CXCR4 siRNAs and expression vector, and cell viability was detected by MTT assay. We found that expression of SDF-1 and CXCR4 in retina was significantly downregulated in mice with diabetic retinopathy than in normal healthy mice. High glucose treatment downregulated the expression of SDF-1 and CXCR4 in ARPE-19 cells at both mRNA and protein levels. Transfection with CXCR4 siRNAs decreased, while transfection with CXCR4 expression vector increased cell viability under high glucose treatment. We concluded that SDF-1/CXCR4 pathway improved diabetic retinopathy possibly by increasing cell viability.

Abbreviations: SDF-1: Stromal cell-derived factor 1; CXCL12: C-X-C motif chemokine 12; qRT-PCR: Real-time quantitative PCR     

Necrostatin-1 protects against glutamate-induced glutathione depletion and caspase-independent cell death in HT-22 cells

Glutamate, a major excitatory neurotransmitter in the CNS, plays a critical role in neurological disorders such as stroke and Parkinson's disease. Recent studies have suggested that glutamate excess can result in a form of cell death called glutamate-induced oxytosis. In this study, we explore the protective effects of necrostatin-1 (Nec-1), an inhibitor of necroptosis, on glutamate-induced oxytosis. We show that Nec-1 inhibits glutamate-induced oxytosis in HT-22 cells through a mechanism that involves an increase in cellular glutathione (GSH) levels as well as a reduction in reactive oxygen species production. However, Nec-1 had no protective effect on free radical-induced cell death caused by hydrogen peroxide or menadione, which suggests that Nec-1 has no antioxidant effects. Interestingly, the protective effect of Nec-1 was still observed when cellular GSH was depleted by buthionine sulfoximine, a specific and irreversible inhibitor of glutamylcysteine synthetase. Our study further demonstrates that Nec-1 significantly blocks the nuclear translocation of apoptosis-inducing factor (a marker of caspase-independent programmed cell death ) and inhibits the integration of Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (a pro-death member of the Bcl-2 family) into the mitochondrial membrane. Taken together, these results demonstrate for the first time that Nec-1 prevents glutamate-induced oxytosis in HT-22 cells through GSH related as well as apoptosis-inducing factor and Bcl-2/adenovirus E1B 19 kDa-interacting protein 3-related pathways.     

Epidermal growth factor and insulin-like growth factor-1 preserve cell viability in the absence of protein synthesis

Summary Prolonged exposure of cells to the potent protein synthesis inhibitor cycloheximide (CHX) terminates in cell death. In the present study we investigated the effect of epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and insulin on cell death induced by CHX in the human cancerous cell lines MDA-231 and MCF-7 (breast), KB (oral epidermoid), HEP-2 (larynx epidermoid), and SW-480 (colon), and correlated this effect to the inhibition rate of protein synthesis. Cell death was evaluated by measuring either dead cells by trypan blue dye exclusion test or by the release of lactic dehydrogenase into the culture medium. CHX was shown to induce cell death in a concentration (1 to 60 μg/ml) and time (24 to 72 h)-dependent manner in each of the five cell lines. EGF at physiologic concentrations (2 to 40 ng/ml) reduced cell death close to control level (without CHX) in the cell lines HEP-2, KB, MDA-231, and SW-480, but had almost no effect on cell death in the MCF-7 cells. IGF-1 at physiologic concentrations (2 to 40 ng/ml) reduced cell death nearly to control level in the MCF-7 cells, but had only a partial effect in the other four cell lines. Insulin at supraphysiologic concentration (10 000 ng/ml) mimicked the effect of IGF-1 in each of the cell lines. CHX at concentrations that induced about 60% cell death, inhibited about 90% of protein synthesis as measured by [³H]leucine incorporation. Protein synthesis remained inhibited although cell viability was preserved by EGF or IGF-1. These results indicated that the mechanism by which EGF or IGF-1 preserve cell viability does not require new protein synthesis and may be mediated via a posttranslational modification effect.