Glutamate-induced Oxidative Toxicity Research Articles

Novel Probucol Analogue, 4,4'-Diselanediylbis (2,6-Di-tert-Butylphenol), Prevents Oxidative Glutamate Neurotoxicity In Vitro and Confers Neuroprotection in a Rodent Model of Ischemic Stroke.

Oxidative glutamate toxicity is regarded as one of the injurious mechanisms associated with ischemic stroke, which represents a major health problem and requires improved pharmacological treatments. We designed and synthesized two new probucol analogues [2,6-di-tert-butyl-4-selenocyanatophenol (C1) and 4,4'-diselanediylbis (2,6-di-tert-butylphenol) (C2)] and investigated their effects against glutamate-induced neuronal oxidative toxicity in vitro in cultured HT22 cells, compared with their parental compound (probucol). In addition, C2, which exhibited the lowest toxicity, was investigated in an in vivo rodent model of ischemic stroke. Glutamate caused concentration- and time-dependent cytotoxicity in HT22 neuronal cells, which was preceded by increased levels of oxidants and depletion of the antioxidant glutathione. The analogues (C1 and C2), but not probucol, significantly decreased the levels of oxidants (including mitochondrial superoxide anion and lipid reactive oxygen species (ROS)) and protected against glutamate-induced cytotoxicity. In the in vivo model of ischemic stroke, which was based on central injections of the vasoconstrictor agent endothelin-1 (800 pmol/site), C2 (20 or 50 mg/kg/day, intraperitoneally, for 4 consecutive days after stroke) displayed significant beneficial effects against ischemic injury in vivo, improving rats' motor-related behavioral skills and decreasing stroke-related striatal gliosis. This is the first study to design, synthesize, and present a probucol analogue (C2) with in vivo beneficial effects against ischemic stroke. This novel compound, which was able to mitigate glutamate-induced oxidative toxicity in vitro, represents a promising neuroprotective drug.

Metabolically Activated Proteostasis Regulators Protect against Glutamate Toxicity by Activating NRF2.

The extracellular accumulation of glutamate is a pathologic hallmark of numerous neurodegenerative diseases including ischemic stroke and Alzheimer’s disease. At high extracellular concentrations, glutamate causes neuronal damage by promoting oxidative stress, which can lead to cellular death. This has led to significant interest in developing pharmacologic approaches to mitigate the oxidative toxicity caused by high levels of glutamate. Here, we show that the small molecule proteostasis regulator AA147 protects against glutamate-induced cell death in a neuronal-derived cell culture model. While originally developed as an activator of the activating transcription factor 6 (ATF6) arm of the unfolded protein response, this AA147-dependent protection against glutamate toxicity is primarily mediated through activation of the NRF2-regulated oxidative stress response. We demonstrate that AA147 activates NRF2 selectively in neuronal-derived cells through a mechanism involving metabolic activation to a reactive electrophile and covalent modification of KEAP1—a mechanism analogous to that involved in the AA147-dependent activation of ATF6. These results define the potential for AA147 to protect against glutamate-induced oxidative toxicity and highlight the potential for metabolically activated proteostasis regulators like AA147 to activate both protective ATF6 and NRF2 stress-responsive signaling pathways to mitigate oxidative damage associated with diverse neurologic diseases.

Thunbergia laurifolia Leaf Extract Inhibits Glutamate-Induced Neurotoxicity and Cell Death through Mitophagy Signaling.

Oxidative stress plays a crucial role in neurodegeneration. Therefore, reducing oxidative stress in the brain is an important strategy to prevent neurodegenerative disorders. Thunbergia laurifolia (Rang-jued) is well known as an herbal tea in Thailand. Here, we aimed to determine the protective effects of T. laurifolia leaf extract (TLE) on glutamate-induced oxidative stress toxicity and mitophagy-mediated cell death in mouse hippocampal cells (HT-22). Our results reveal that TLE possesses a high level of bioactive antioxidants by LC–MS technique. We found that the pre-treatment of cells with TLE prevented glutamate-induced neuronal death in a concentration-dependent manner. TLE reduced the intracellular ROS and maintained the mitochondrial membrane potential caused by glutamate. Moreover, TLE upregulated the gene expression of antioxidant enzymes (SOD1, SOD2, CAT, and GPx). Interestingly, glutamate also induced the activation of the mitophagy process. However, TLE could reverse this activity by inhibiting autophagic protein (LC3B-II/LC3B-I) activation and increasing a specific mitochondrial protein (TOM20). Our results suggest that excessive glutamate can cause neuronal death through mitophagy-mediated cell death signaling in HT-22 cells. Our findings indicate that TLE protects cells from neuronal death by stimulating the endogenous antioxidant enzymes and inhibiting glutamate-induced oxidative toxicity via the mitophagy–autophagy pathway. TLE might have potential as an alternative or therapeutic approach in neurodegenerative diseases.

Protective Effect of Osmundacetone against Neurological Cell Death Caused by Oxidative Glutamate Toxicity.

Oxidative stress is one of the main causes of brain cell death in neurological disorders. The use of natural antioxidants to maintain redox homeostasis contributes to alleviating neurodegeneration. Glutamate is an excitatory neurotransmitter that plays a critical role in many brain functions. However, excessive glutamate release induces excitotoxicity and oxidative stress, leading to programmed cell death. Our study aimed to evaluate the effect of osmundacetone (OAC), isolated from Elsholtzia ciliata (Thunb.) Hylander, against glutamate-induced oxidative toxicity in HT22 hippocampal cells. The effect of OAC treatment on excess reactive oxygen species (ROS), intracellular calcium levels, chromatin condensation, apoptosis, and the expression level of oxidative stress-related proteins was evaluated. OAC showed a neuroprotective effect against glutamate toxicity at a concentration of 2 μM. By diminishing the accumulation of ROS, as well as stimulating the expression of heat shock protein 70 (HSP70) and heme oxygenase-1 (HO-1), OAC triggered the self-defense mechanism in neuronal cells. The anti-apoptotic effect of OAC was demonstrated through its inhibition of chromatin condensation, calcium accumulation, and reduction of apoptotic cells. OAC significantly suppressed the phosphorylation of mitogen-activated protein kinases (MAPKs), including c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 kinases. Thus, OAC could be a potential agent for supportive treatment of neurodegenerative diseases.

Lipidomics-based study on the neuroprotective effect of geissoschizine methyl ether against oxidative stress-induced cytotoxicity

Ethnopharmacological relevanceLipid homoeostasis is important for neurodevelopment, cell signaling and neurotransmission. Alteration of lipid metabolism has been demonstrated in many neurological disorders and neurodegenerative diseases. Geissoschizine methyl ether (GM) is an active alkaloid ingredient in the traditional Chinese medicine Uncaria hook. It has been shown that GM has strong potency in neuroprotective activity and GM reduces the production of reactive oxygen species by regulating glucose metabolism, which protects neurons against oxidative stress-induced cell death. However, it is unknown whether GM could regulate neuronal lipid metabolism during oxidative challenge. Aim of the studyThe current study aimed to explore whether GM regulates lipid metabolism in oxidative damaged neurons and to determine the underlying mechanism involved in this neuro-protection. Materials and methodsUsing a glutamate-induced oxidative toxicity model in mouse hippocampal neuronal cell line (HT-22 cells), we investigated the effect of GM on glutamate-induced lipid peroxidation, lipotoxicity and mitochondrial dysfunction. In order to clarify the mechanism underlying the neuroprotection by GM, lipid metabolomics was performed to investigate whether GM prevent oxidative stress-induced lipid metabolism disruption. Furthermore, the expression of lipid metabolism-related genes was measured. ResultsThe results show the protective effect of GM against oxidative stress through blocking glutamate-induced lipid peroxidation and lipotoxicity. Overall, lipidomics analysis revealed that glutamate treatment resulted in different extents of changes in a wide range of lipid classes such as fatty acids (FA), triacylglycerol (TG), sphingomyelin (SM), cardiolipin (CL), lysophosphatidylcholines (LPC). However, GM treatment can significantly reverse glutamate-induced lipids disorder to the homeostasis level. GM prevented the disruption of lipid metabolism by regulating the expression of lipid homeostasis related genes, which contributes to preserve mitochondrial function under oxidative damage. ConclusionThese findings clearly demonstrated a novel protective mechanism of GM against glutamate-induced oxidative toxicity in neurons via regulating lipid metabolism. GM may provide an effective approach for the prevention and treatment of oxidative damaged neurons.

Ferrostatin-1 protects HT-22 cells from oxidative toxicity.

Ferroptosis is a type of programmed cell death dependent on iron. It is different from other forms of cell death such as apoptosis, classic necrosis and autophagy. Ferroptosis is involved in many neurodegenerative diseases. The role of ferroptosis in glutamate-induced neuronal toxicity is not fully understood. To test its toxicity, glutamate (1.25-20 mM) was applied to HT-22 cells for 12 to 48 hours. The optimal experimental conditions occurred at 12 hours after incubation with 5 mM glutamate. Cells were cultured with 3-12 μM ferrostatin-1, an inhibitor of ferroptosis, for 12 hours before exposure to glutamate. The cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Autophagy was determined by monodansylcadaverine staining and apoptosis by caspase 3 activity. Damage to cell structures was observed under light and by transmission electron microscopy. The release of lactate dehydrogenase was detected by the commercial kit. Reactive oxygen species were measured by flow cytometry. Glutathione peroxidase activity, superoxide dismutase activity and malondialdehyde level were detected by the appropriate commercial kit. Prostaglandin peroxidase synthase 2 and glutathione peroxidase 4 gene expression was detected by real-time quantitative polymerase chain reaction. Glutathione peroxidase 4 and nuclear factor erythroid-derived-like 2 protein expression was detected by western blot analysis. Results showed that ferrostatin-1 can significantly counter the effects of glutamate on HT-22 cells, improving the survival rate, reducing the release of lactate dehydrogenase and reducing the damage to mitochondrial ultrastructure. However, it did not affect the caspase-3 expression and monodansylcadaverine-positive staining in glutamate-injured HT-22 cells. Ferrostatin-1 reduced the levels of reactive oxygen species and malondialdehyde and enhanced superoxide dismutase activity. It decreased gene expression of prostaglandin peroxidase synthase 2 and increased gene expression of glutathione peroxidase 4 and protein expressions of glutathione peroxidase 4 and nuclear factor (erythroid-derived)-like 2 in glutamate-injured HT-22 cells. Treatment of cultured cells with the apoptosis inhibitor Z-Val-Ala-Asp (OMe)-fluoromethyl ketone (2-8 μM), autophagy inhibitor 3-methyladenine (100-400 μM) or necrosis inhibitor necrostatin-1 (10-40 μM) had no effect on glutamate induced cell damage. However, the iron chelator deferoxamine mesylate salt inhibited glutamate induced cell death. Thus, the results suggested that ferroptosis is caused by glutamate-induced toxicity and that ferrostatin-1 protects HT-22 cells from glutamate-induced oxidative toxicity by inhibiting the oxidative stress.

Acanthus ebracteatus leaf extract provides neuronal cell protection against oxidative stress injury induced by glutamate

BackgroundAcanthus ebracteatus (AE), an herb native to Asia, has been recognized in traditional folk medicine not only for its antioxidant properties and various pharmacological activities but also as an ingredient of longevity formulas. However, its anti-neurodegenerative potential is not yet clearly known. This work aimed to evaluate the protective effect of AE leaf extract against glutamate-induced oxidative damage in mouse hippocampal HT22 cells, a neurodegenerative model system due to a reduction in glutathione levels and an increase in reactive oxygen species (ROS).MethodsCell viability, apoptosis, and ROS assays were performed to assess the protective effect of AE leaf extract against glutamate-induced oxidative toxicity in HT22 cells. The antioxidant capacity of AE was evaluated using in vitro radical scavenging assays. The subcellular localization of apoptosis-inducing factor (AIF) and the mRNA and protein levels of genes associated with the nuclear factor erythroid 2–related factor 2 (Nrf2) antioxidant system were determined to elucidate the mechanisms underlying the neuroprotective effect of AE leaf extract.ResultsWe demonstrated that AE leaf extract is capable of attenuating the intracellular ROS generation and HT22 cell death induced by glutamate in a concentration-dependent manner. Co-treatment of glutamate with the extract significantly reduced apoptotic cell death via inhibition of AIF nuclear translocation. The increases in Nrf2 levels in the nucleus and gene expression levels of antioxidant-related downstream genes under Nrf2 control were found to be significant in cells treated with the extract.ConclusionsThe results suggested that AE leaf extract possesses neuroprotective activity against glutamate-induced oxidative injury and may have therapeutic potential for the treatment of neurodegenerative diseases associated with oxidative stress.

Neuroprotective Effects of Kinetin Against Glutamate-Induced Oxidative Cytotoxicity in HT22 Cells: Involvement of Nrf2 and Heme Oxygenase-1.

Oxidative stress is considered as one of key factors related to Alzheimer's disease (AD), while kinetin (KT) has been reported to exert anti-oxidative activities as well as neuroprotective effects both in vivo and in vitro. Thus, in this study, the neuroprotective effects of KT against glutamate-induced oxidative toxicity in HT22 cells were investigated. To evaluate the anti-oxidative capabilities of KT itself, several anti-oxidative assays in vitro were conducted. To evaluate the neuroprotective effects of KT, the levels of intracellular reactive oxygen species (ROS) and calcium influx, mitochondrial membrane potential (MMP), and cell death were measured by flow cytometry. Nuclear translocation of apoptosis inducing factor (AIF) and content of intracellular ATP were also determined. In addition, the phosphorylation levels of apoptosis signal-regulating kinase 1 (ASK-1), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinases (p38) were evaluated as well. Besides, nuclear translocation of nuclear factor-E2-related factor 2 (Nrf2) and the expression of heme oxygenase-1 (HO-1) were also examined to reveal underlying mechanisms. Results showed that KT rescued cell death, and suppressed the accumulation of intracellular ROS and the increase of intracellular calcium influx. In addition, KT maintained normal function of mitochondria and inhibited the phosphorylation of ASK-1, JNK, and p38. KT also promoted nuclear translocation of Nrf2 and enhanced the expression of HO-1 both at protein and mRNA level. Importantly, blockage of Nrf2 almost completely abolished the neuroprotective effects of KT, while blockage of HO-1 expression partly neutralized its neuroprotective effects. Our results indicated that KT can protect HT22 cells from glutamate-induced cell death by activating Nrf2 pathway and inducing expression of HO-1, suggesting KT might be a drug candidate for treatment of AD and other neurodegenerative disorders related to oxidative stress.

Neuroprotective compounds from Reynoutria sachalinensis.

Glutamate is a neurotransmitter in central nervous system. Overexpression of glutamate leads to oxidative stress, resulting in several neurodegenerative disorders that include Alzheimer's disease. The n-hexane fraction of stems and ethyl acetate (EtOAc) fraction of flowers of Reynoutria sachalinensis provide neuroprotection against glutamate-induced oxidative toxicity in HT22 cells. In this study, 1-decanol (1), β-amyrin (2), dammaran-3β-ol (3), campesterol (4), daucosterol (5), ergosterol peroxide (6), emodin 8-O-β-D-glucopyranoside (7), quercetin (8) and isoquercitrin (9) were isolated from n-hexane fractions of stems and EtOAc fractions of flowers of R. sachalinensis. Their neuroprotective activity was evaluated by MTT assay. 1-Decanol, campesterol, ergosterol peroxide, quercetin and isoquercitrin exhibited neuroprotective activity. These compounds decreased reactive oxygen species level, showed anti-oxidant activity with DPPH radical and in a H2O2 scavenging assay. Therefore, the neuroprotective activity of 1-decanol, campesterol, ergosterol peroxide, quercetin and isoquercitrin are associated with antioxidant activity.

Metabolic Alterations and the Protective Effect of Punicalagin Against Glutamate-Induced Oxidative Toxicity in HT22 Cells.

Oxidative stress is involved in many neurological diseases, including Alzheimer's disease. Punicalagin (PC) is a hydrolysable polyphenol derived from Punica granatum and a potent antioxidant. In this study, the neuroprotective effect of PC on glutamate-induced oxidative stress was evaluated in the mouse hippocampal cell line, HT22. PC treatment protected HT22 cells from glutamate-induced cell death in a concentration-dependent manner, potentially attenuated glutamate-induced intracellular reactive oxygen species (ROS) and restored the mitochondrial membrane depolarization. Metabolic alterations after glutamate-induced oxidative stress and the protective effect of PC were evaluated with HPLC and GC-MS profiling methods with multivariate statistical analyses. Alterations in ten metabolites were identified, including amino acids, aspartic acid, asparagine, threonine, anserine, cysteine, tryptophan, lysine, as well as fatty acids palmitic acid, stearic acid, and palmitoleic acid. Metabolic pathway analysis revealed the involvement of multiple affected pathways, such as cysteine and methionine metabolism, tryptophan metabolism, alanine, aspartate, and glutamate and fatty acid oxidation. These results clearly demonstrate that PC is a promising therapeutic agent for oxidative stress-associated diseases.

Tanshinone IIA Inhibits Glutamate-Induced Oxidative Toxicity through Prevention of Mitochondrial Dysfunction and Suppression of MAPK Activation in SH-SY5Y Human Neuroblastoma Cells.

Glutamate excitotoxicity is associated with many neurological diseases, including cerebral ischemia and neurodegenerative diseases. Tanshinone IIA, a diterpenoid naphthoquinone from Salvia miltiorrhiza, has been shown to suppress presynaptic glutamate release, but its protective mechanism against glutamate-induced neurotoxicity is lacking. Using SH-SY5Y human neuroblastoma cells, we show here that excessive glutamate exposure decreases cell viability and proliferation and increases LDH release. Pretreatment with tanshinone IIA, however, prevents the decrease in cell viability and proliferation and the increase in LDH release induced by glutamate. Tanshinone IIA also attenuates glutamate-induced oxidative stress by reducing reactive oxygen species level and malondialdehyde and protein carbonyl contents and by enhancing activities and protein levels of superoxide dismutase and catalase. We then show that tanshinone IIA prevents glutamate-induced mitochondrial dysfunction by increasing mitochondrial membrane potential and ATP content and by reducing mitochondrial protein carbonyl content. Moreover, tanshinone IIA can inhibit glutamate-induced apoptosis through regulation of apoptosis-related protein expression and MAPK activation, including elevation of Bcl-2 protein level, decrease in Bax and cleaved caspase-3 levels, and suppression of JNK and p38 MAPK activation. Collectively, our findings demonstrate that tanshinone IIA protects SH-SY5Y cells against glutamate toxicity by reducing oxidative stress and regulating apoptosis and MAPK pathways.

Neuroprotective effects of 2,3,5,4′-tetrahydoxystilbene-2-O-β-D-glucoside from Polygonum multiflorum against glutamate-induced oxidative toxicity in HT22 cells

Ethnopharmacological relevanceSince ancient times, Polygonum multiflorum Thunb. has been used to treat premature grey hair, dizziness, and blurred vision in East Asia. A major bioactive constituent of this medicinal herb, 2,3,5,4'-tetrahydoxystilbene-2-O-β-D-glucoside (THSG), has antioxidant activity and exerts beneficial effects on cognition and memory. Aim of the studyThe purpose of the current study was to determine if THSG affects hippocampal neuronal cell death and mitochondrial function following exposure to oxidative stress. Materials and methodsHT22 hippocampal cells with or without THSG pretreatment were exposed to glutamate, and the effects on cell viability and expression of molecules related to apoptotic cell death were examined using biochemical techniques, flow cytometry, western immunoblotting, and real-time polymerase chain reaction. ResultsPretreatment with THSG significantly attenuated glutamate-induced loss of cell viability and release of lactate dehydrogenase as well as apoptotic cell death. THSG inhibited generation of reactive oxygen species (ROS), expression of heme oxygenase-1, and activation of caspase-3 and calpain-1 proteases, all of which were increased by glutamate. THSG inhibited glutamate-induced disruption of mitochondrial membrane potential (MMP) and voltage-dependent anion channel-1. It also regulated the ratio of Bax to Bcl-2. ConclusionsThese results indicate that THSG has a marked neuroprotective effect against glutamate-induced hippocampal damage by decreasing ROS production and stabilizing MMP. These findings suggest the potential of THSG as a new therapeutic agent for the treatment of cognitive disorders.

Polysaccharides purified from Cordyceps cicadae protects PC12 cells against glutamate-induced oxidative damage

Two polysaccharides CPA-1 and CPB-2 were isolated purified from Cordyceps cicadae by hot water extraction, ethanol precipitation and purification using anion exchange and gel filtration chromatography. Preliminary structural characterization of CPA-1 and CPB-2 were performed. The protective effect of CPA-1 and CPB-2 against glutamate-induced oxidative toxicity in PC12 cells was analyzed. The results indicated that pretreatment of PC12 cells with CPA-1 and CPB-2 significantly increased cell survival, Ca2+ overload and ROS generation. CPA-1 and CPB-2 also markedly up-regulated the antioxidant status of pretreated PC12 cells. Our results suggested that Cordyceps cicadae polysaccharides can protect PC12 cells against glutamate excitotoxicity and might serve as therapeutic agents for neuronal disorders.

Emodin from Polygonum multiflorum ameliorates oxidative toxicity in HT22 cells and deficits in photothrombotic ischemia

Ethnopharmacological relevancePolygonum multiflorum Thunb. has been used widely in East Asia in treatment of diseases associated with aging. Emodin, an active component from Polygonum multiflorum Thunb., provides benefits for brain disturbances induced by severe cerebral injury. Aim of the studyWe investigated the neuroprotective effect of emodin from Polygonum multiflorum Thunb. against glutamate-induced oxidative toxicity and cerebral ischemia. Materials and methodsFor examination of neuroprotective effects of emodin, cell viability, cytotoxicity, flow cytometry, and Western blot were performed in HT22 cells and infarct volume, behavioral tests and Western blot in a mouse model of photothrombotic ischemic stroke. ResultsPretreatment with emodin resulted in significantly reduced glutamate-induced apoptotic cell death in HT22 cells. However, blocking of phosphatidylinositol-3 kinase (PI3K) activity with LY294002 resulted in significantly inhibited cell survival by emodin. Exposure of glutamate-treated cells to emodin induced an increase in the level of Bcl-2 expression, whereas the expression of Bax and active caspase-3 proteins was significantly reduced. In addition, treatment with emodin resulted in increased phosphorylation of Akt and cAMP response element binding protein (CREB), and expression of mature brain-derived neurotrophic factor (BDNF). This expression by emodin was also significantly inhibited by blocking of PI3K activity. In a photothrombotic ischemic stroke model, treatment with emodin resulted in significantly reduced infarct volume and improved motor function. We confirmed the critical role of the expression levels of Bcl-2/Bax, active caspase-3, phosphorylated (p)Akt, p-CREB, and mature BDNF for potent neuroprotective effects of emodin in cerebral ischemia. ConclusionsThese results suggest that emodin may afford a significant neuroprotective effect against glutamate-induced apoptosis through activation of the PI3K/Akt signaling pathway, and subsequently enhance behavioral function in cerebral ischemia.

Curcumin-Protected PC12 Cells Against Glutamate-Induced Oxidative Toxicity.

Glutamate is a major excitatory neurotransmitter present in the central nervous system. The glutamate/cystine antiporter system x c- connects the antioxidant defense with neurotransmission and behaviour. Overactivation of ionotropic glutamate receptors induces neuronal death, a pathway called excitotoxicity. Glutamate-induced oxidative stress is a major contributor to neurodegenerative diseases including cerebral ischemia, Alzheimer's and Huntington's disease. Curcuma has a wide spectrum of biological activities regarding neuroprotection and neurocognition. By reducing the oxidative damage, curcumin attenuates a spinal cord ischemia-reperfusion injury, seizures and hippocampal neuronal loss. The rat pheochromocytoma (PC12) cell line exhibits many characteristics useful for the study of the neuroprotection and neurocognition. This investigation was carried out to determine whether the neuroprotective effects of curcumin can be observed via the glutamate-PC12 cell model. Results indicate that glutamate (20 mM) upregulated glutathione peroxidase 1, glutathione disulphide, Ca2+ influx, nitric oxide production, cytochrome c release, Bax/Bcl-2 ratio, caspase-3 activity, lactate dehydrogenase release, reactive oxygen species, H 2 O 2 , and malondialdehyde; and downregulated glutathione, glutathione reductase, superoxide dismutase and catalase, resulting in enhanced cell apoptosis. Curcumin alleviates all these adverse effects. Conclusively, curcumin can effectively protect PC12 cells against the glutamate-induced oxidative toxicity. Its mode of action involves two pathways: the glutathione-dependent nitric oxide-reactive oxygen species pathway and the mitochondria-dependent nitric oxide-reactive oxygen species pathway.

The neuroprotective effects of cordycepin inhibit glutamate-induced oxidative and ER stress-associated apoptosis in hippocampal HT22 cells

Glutamate toxicity increases the formation of reactive oxygen species (ROS) and intracellular calcium levels, resulting in neuronal dysfunction, neurodegenerative disorders, and death. Cordycepin is a derivative of the nucleoside adenosine, and is believed to exert neuroprotective effects against glutamate-induced oxidative toxicity in HT22 neuronal cells. Excessive glutamate induces oxidative and endoplasmic reticulum (ER) stress, gradually increasing ER-related pro-apoptotic transcription factor C/EBP homologous protein (CHOP) expression, and eventually up-regulating expression of the pro-apoptotic factor Bax. Cordycepin inhibits CHOP and Bax expressions, as well as p-ERK, p-JNK, and p-p38, all of which are involved in oxidative or ER stress-induced apoptosis. In addition, the increased production of ROS from excessive glutamate leads to elevation of mitochondrial membrane potential (MMP), a hallmark of mitochondrial dysfunction. Cordycepin retains MMP and reduces the elevated levels of ROS and Ca2+ induced by glutamate. Caspases are crucial mediators involved in mitochondrial apoptosis, and while glutamate disrupts mitochondrial function, it does not change expression levels of caspase 3 and caspase 9. Similarly, cordycepin has no effect on caspase 3 and caspase 9 expressions; however, it decreases the expression of ER stress-specific caspase 12, which plays a key role in the initiation of ER stress-induced apoptosis. Finally, we found that the anti-apoptotic effects of cordycepin are partially dependent on activation of the adenosine A1 receptor, whereas an antagonist selectively attenuated the neuroprotective effects of cordycepin. Collectively, these results suggest that cordycepin could be a potential future therapeutic agent for neuronal disorders.

N-Stearoyltyrosine Protects Against Glutamate-Induced Oxidative Toxicity by an Apoptosis-Inducing Factor (AIF)-Mediated Caspase-Independent Cell Death Pathway

N-Stearoyltyrosine (NsTyr), a synthesized anandamide (AEA) analogue, could exert potent neuroprotective effects on cerebral ischemia models both in vivo and in vitro via intervening in multiple injuries. Glutamate, a major excitatory neurotransmitter, plays a critical role during stroke/cerebral ischemia. In this study, we explored the protective effects of NsTyr on glutamate neurotoxicity in PC12 cells and investigated its underlying mechanisms. NsTyr treatment attenuated glutamate-induced oxidative toxicity in a dose-dependent manner and the best performance was observed at 10 μΜ. NsTyr treatment suppressed glutamate-induced upregulation of lipoxygenase 12/15 (LOX 12/15) activity and reactive oxygen species (ROS) elevation, attenuated the increase of BH3-interacting domain death agonist (Bid) in the mitochondria, prevented the loss of mitochondria membrane potential and consequently inhibited apoptosis-inducing factor (AIF) translocation into the nucleus. The results demonstrated that NsTyr could protect cells against AIF-mediated caspase-independent cell death induced by glutamate, which may be due to the blockage of Bid-mediated mitochondrial damage via reducing LOX 12/15 activity and ROS accumulation.

Neuroprotective effects of Polygonum multiflorum extract against glutamate-induced oxidative toxicity in HT22 hippocampal cells

Ethnopharmacological relevanceDried roots of Polygonum multiflorum have traditionally been used in the retarding of aging process in East Asian countries and its extracts exhibit anti-oxidative activities. Materials and methodsNeuroprotective effects of ethyl acetate extract from Polygonum multiflorum (EEPM) were investigated against glutamate-induced oxidative cell death in HT22 hippocampal cells. Cell viability, cytotoxicity, morphological, flow cytometry, and Western blot assays were performed in order to observe alterations of neuronal cell survival or death related pathways. ResultsPretreatment with EEPM resulted in significantly decreased glutamate-induced neurotoxicity and also resulted in drastically inhibited glutamate-induced apoptotic and necrotic neuronal death. To elucidate possible pathways of neuroprotection by EEPM, we explored the activation of mitogen activated protein kinases (MAPKs), phosphatidylinositol-3-kinase, and cAMP responsive element binding protein (CREB). Treatment with glutamate alone led to activation of extracellular regulated kinase (ERK), Jun N-terminal kinase, and p38 during the late phase after glutamate exposure, but pretreatment with EEPM resulted in significantly attenuated activation of these proteins. Pretreatment with EEPM resulted in increased activation of CREB. The specific inhibitors of ERK and p38, PD98059 and SB203580, abrogated the neuroprotective effects of EEPM. When we evaluated calpain I and striatal-enriched protein tyrosine phosphatase (STEP), active form of calpain I was significantly increased after glutamate exposure, and, along with this, active form of STEP showed a decrease. Pretreatment with EEPM resulted in significant recovery of pro-calpain I and active form of STEP caused by glutamate. Co-treatment with calpain inhibitor ALLN and EEPM had a synergistic effect on neuronal death and contributed to blockade of activation of both ERK and p38 with increased activation of CREB. ConclusionsThese results suggest that Polygonum multiflorum extract may have neuroprotective effects through both alleviation of ERK and p38 activation with increased activation of CREB under oxidative stress and has potential as a therapeutic intervention for treatment of oxidative neuronal death.

Crambescidin 800, a pentacyclic guanidine alkaloid, protects a mouse hippocampal cell line against glutamate-induced oxidative stress

Oxidative stress is implicated in the pathogenesis of neuronal degenerative diseases. In our search for protective substances against glutamate-induced oxidative stress using mouse hippocampal cell line HT22, we have isolated crambescidin 800, a pentacyclic guanidine alkaloid, from the Indonesian marine sponge, Monanchora ungiculata. Crambescidin 800 (1) protected HT22 cells against glutamate-induced oxidative toxicity at 0.06 μM (EC50) concentration. Crambescidin 800 (1) also protected HT22 and neuroblastoma cells from the oxidative stress induced by a hypoxic condition or nitric oxide (NO). Crambescidin 800 (1) showed no effect on the depletion of intracellular glutathione (GSH) of HT22 cells induced by glutamate, and production of reactive oxygen species (ROS) and activation of mitogen-activated protein kinase (MAPK) were inhibited only by the high concentration (1 μM) of 1.

Opposing Roles for ERK1/2 in Neuronal Oxidative Toxicity: DISTINCT MECHANISMS OF ERK1/2 ACTION AT EARLY VERSUS LATE PHASES OF OXIDATIVE STRESS

Glutamate-induced oxidative toxicity is mediated by glutathione depletion in the HT22 mouse hippocampal cell line. Previous results with pharmacological agents implicated the extracellular signal-regulated kinases-1/2 (ERK1/2) in glutamate toxicity in HT22 cells and immature embryonic rat cortical neurons. In this report, we definitively establish a role for ERK1/2 in oxidative toxicity using dominant negative MEK1 expression in transiently transfected HT22 cells to block glutamate-induced cell death. In contrast, chronic activation of ERK (i.e. brought about by transfection of constitutively active ERK2 chimera) is not sufficient to trigger HT22 cell death demonstrating that ERK1/2 activation is not sufficient for toxicity. Activation of ERK1/2 in HT22 cells has a distinct kinetic profile with an initial peak occurring between 30 min and 1 h of glutamate treatment and a second peak typically emerging after 6 h. We demonstrate here that the initial phase of ERK1/2 induction is because of activation of metabotropic glutamate receptor type I (mGluRI). ERK1/2 activation by mGluRI contributes to an HT22 cell adaptive response to oxidative stress as glutamate-induced toxicity is enhanced upon pharmacological inhibition of mGluRI. The protective effect of ERK1/2 activation at early times after glutamate treatment is mediated by a restoration of glutathione (GSH) levels that are reduced because of depletion of intracellular cysteine pools. Thus, ERK1/2 appears to play dual roles in HT22 cells acting as part of a cellular adaptive response during the initial phases of glutamate-induced oxidative stress and contributing to toxicity during later stages of stress.