Methylglyoxal,glyoxalases and the development of diabetic complications

Summary The formation of the reactive,-dicarbonyl metabolite, methylglyoxal, is increased during hyperglycaemia associated with diabetes mellitus. Methylglyoxal is metabolised to S-D-lactoylglutathione and D-lactate by the glyoxalase system and to hydroxyacetone (95%) and D-lactaldehyde by aldose reductase. Methylglyoxal and hydroxyacetone bind and modify protein, producing fluorescent products. Red blood cell activities of glyoxalase enzymes are risk factors for the development of clinical complications of diabetes. Aldose reductase inhibitors decrease the concentration of methylglyoxal in experimental diabetic rats to normal levels, aminoguanidine and L-arginine scavenge methylglyoxal; these effects may be involved in their prospective preventive therapy of diabetic complications. Biochemical and clinical evidence suggests that the metabolism of methylglyoxal in diabetes mellitus is linked to the development of diabetic complications. A causal relationship may involve modification of protein by methylglyoxal and hydroxyacetone.     

生长分化因子11对甲醛诱导的海马神经细胞凋亡的影响

目的探讨生长分化因子11（GDF11）对甲醛诱导的海马神经（HT22）细胞毒性的影响。 方法把HT22细胞分为对照组（细胞未做任何处理）、甲醛组（50、100、200 μmol/ L甲醛处理细胞）和GDF11+甲醛组（GDF11转染细胞后用100 μmol/L甲醛处理）。细胞计数试剂盒（CCK8）法检测HT22细胞的活力;蛋白免疫印迹法检测HT22细胞凋亡相关蛋白Bax以及Bcl-2的变化;caspase-3活性检测试剂盒检测HT22细胞内caspase-3活性;DCFDA染色流式细胞仪检测HT22细胞中活性氧（ROS）水平。三组间比较采用单因素方差分析,组间两两比较采用LSD-t检验。 结果与对照组比较,甲醛组HT22细胞活力（92.23±0.20比56.12±0.61）和Bcl-2蛋白表达（220.32±2.21比150.25±0.31）水平均降低,差异具有统计学意义（P均< 0.05）;而caspase-3活性（95.36±1.74比190.17±2.14）、Bax蛋白表达（132.19±1.21比150.17±1.06）和ROS水平（1099.32±75.47比2802.17±126.49）均升高,差异具有统计学意义（P均< 0.05）。GDF11转染HT22细胞后,与甲醛组比较,GDF11+甲醛组HT22细胞活力升高（56.12±0.61比83.11±1.64）,Bax蛋白表达（270.03±0.17比150.17±1.06）降低,Bcl-2蛋白表达（150.25±0.31比187.34±1.52）升高,caspase-3活性降低（190.17±2.14比105.31±4.12）和ROS水平降低（2802.17±126.49比1305.36±68.45）,差异具有统计学意义（P均< 0.05）。 结论GDF11能够逆转甲醛对HT22细胞凋亡的诱导作用以及降低甲醛对HT22细胞ROS水平的增加作用,此机制对防治甲醛的神经毒性具有重要意义。     

Identification of thermostable glyoxalase I in the fission yeast <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

Glyoxalase I is a ubiquitous enzyme that detoxifies methylglyoxal, which is derived from glycolysis but inhibits the growth of cells from microorganisms to mammals. Here, the structural gene for glyoxalase I (glo1⁺) from the fission yeast Schizosaccharomyces pombe was identified. Disruption of glo1⁺ enhanced susceptibility to methylglyoxal, while expression of glo1⁺ in a glo1 mutant of Saccharomyces cerevisiae restored tolerance to this aldehyde. The glo1⁺ gene product was purified. The glyoxalase I of S. pombe was a monomeric enzyme with a molecular weight of 34,000 and the k_cat/K_m value for methylglyoxal was 4.3×10⁷ M^–1 min^–1. Treatment of purified enzyme with EDTA in imidazole buffer completely abolished enzyme activity, whereas the EDTA-treated enzyme was reactivated by several divalent metal ions, such as Zn²⁺, Co²⁺, Ni²⁺ and Mn²⁺. The glyoxalase I of S. pombe exhibited fairly high thermal stability, and almost 100% activity was retained after incubating the enzyme at 60°C for 4 h.     

Dual cell protective mechanisms activated by differing levels of oxidative stress in HT22 murine hippocampal cells

Oxidative stress is recognized as one of the pathogenic mechanisms involved in neurodegenerative disease. However, recent evidence has suggested that regulation of cellular fate in response to oxidative stress appears to be dependent on the stress levels. In this study, using HT22 cells, we attempted to understand how an alteration in the oxidative stress levels would influence neuronal cell fate. HT22 cell viability was reduced with exposure to high levels of oxidative stress, whereas, low levels of oxidative stress promoted cell survival. Erk1/2 activation induced by a low level of oxidative stress played a role in this cell protective effect. Intriguingly, subtoxic level of H₂O₂ induced expression of a growth factor, progranulin (PGRN), and exogenous PGRN pretreatment attenuated HT22 cell death induced by high concentrations of H₂O₂ in Erk1/2-dependent manner. Together, our study indicates that two different cell protection mechanisms are activated by differing levels of oxidative stress in HT22 cells.     

Methylglyoxal levels in plants under salinity stress are dependent on glyoxalase I and glutathione

Methylglyoxal (MG), a cytotoxic by-product produced mainly from triose phosphates, is used as a substrate by glyoxalase I. In this paper, we report on the estimation of MG level in plants which has not been reported earlier. We show that MG concentration varies in the range of 30-75 microM in various plant species and it increases 2- to 6-fold in response to salinity, drought, and cold stress conditions. Transgenic tobacco underexpressing glyoxalase I showed enhanced accumulation of MG which resulted in the inhibition of seed germination. In the glyoxalase I overexpressing transgenic tobacco, MG levels did not increase in response to stress compared to the untransformed plants, however, with the addition of exogenous GSH there was a decrease in MG levels in both untransformed and transgenic plants. The exogenous application of GSH reduced MG levels in WT to 50% whereas in the transgenic plants a 5-fold decrease was observed. These studies demonstrate an important role of glyoxalase I along with GSH concentration in maintaining MG levels in plants under normal and abiotic stress conditions.     

Indirubin derivatives protect against endoplasmic reticulum stress-induced cytotoxicity and down-regulate CHOP levels in HT22 cells

Indirubin and its derivatives have been reported to exhibit anti-cancer and anti-inflammatory activities. Recently, some of its derived analogs have been shown to have neuroprotective potential. Endoplasmic reticulum (ER) stress has been demonstrated to contribute to the pathogenesis of various neurodegenerative diseases, whereas the effects of indirubin derivatives on ER stress-induced cell death have not been addressed. In the present study, a series of 44 derivatives of indirubin was prepared to search for a novel class of neuroprotective agents against ER stress-induced neuronal death. The MTT reduction assay indicated that tunicamycin (TM), an inducer of ER stress, significantly decreased the viability of hippocampal neuronal HT22 cells. Among the compounds tested, eight showed significant inhibitory activity against TM-induced cell death. Western blot analysis showed that application of these analogs to the cells simultaneously with TM reduced the TM-induced expression of CHOP, an established mediator of ER stress. Our results suggest that the preventive effect of these indirubin derivatives against ER stress-induced neuronal death may be due, at least in part, to attenuation of the CHOP-dependent signaling system.     

Proteomic analysis of glutamate-induced toxicity in HT22 cells

In the present study, we have investigated the proteome changes associated with glutamate-induced HT22 cell death, a model system to study oxidative stress-mediated toxicity. Among a number of HT22 proteins exhibiting altered expression, several molecular chaperones demonstrated substantial changes. For example, the levels of Hsp90 and Hsp70 decreased as cell death progressed whereas that of Hsp60 increased dramatically. Interestingly, cytosolic Hsp60 increased more prominently than mitochondrial Hsp60. Concomitantly, the accumulation of poly-ubiquitylated proteins and differential regulation of the peptidase activities and the subunits of 26S proteasomes were observed in glutamate-treated HT22 cells. Our findings that the molecular chaperones and the ubiquitin-proteasome system undergo changes during glutamate-induced HT22 cell death may suggest the importance of a protein quality control system in oxidative damage-mediated toxicity.     

Methylglyoxal induces apoptosis through activation of p38 MAPK in rat Schwann cells

The formation of glucose-derived methylglyoxal (MG), a highly reactive dicarbonyl compound, is accelerated under diabetic conditions. We examined whether MG was capable of inducing apoptosis in Schwann cells (SCs), since recent studies have suggested a potential involvement of apoptotic cell death in the development of diabetic neuropathy. MG induced apoptosis in SCs in a dose-dependent manner, accompanied by a reduction of intracellular glutathione content and activation of the p38 MAPK. Inhibiting the p38 MAPK activation by SB203580 successfully suppressed the MG-induced apoptosis in SCs. Aminoguanidine and N-acetyl-l-cysteine also inhibited the MG-induced p38 MAPK activation and apoptosis along with restoration of the intracellular glutathione content. These results suggest a potential role for MG in SC injury through oxidative stress-mediated p38 MAPK activation under diabetic conditions, and it may serve as a novel insight into therapeutic strategies for diabetic neuropathy.     

Protective effect of casuarinin against glutamate-induced apoptosis in HT22 cells through inhibition of oxidative stress-mediated MAPK phosphorylation

Glutamate is the major excitatory neurotransmitter in the central nervous system and is involved in oxidative stress during neurodegeneration. In the present study, casuarinin prevented glutamate-induced HT22 murine hippocampal neuronal cell death by inhibiting intracellular reactive oxygen species (ROS) production. Moreover, casuarinin reduced chromatin condensation and annexin-V-positive cell production induced by glutamate. We also confirmed the underlying protective mechanism of casuarinin against glutamate-induced neurotoxicity. Glutamate markedly increased the phosphorylation of extracellular signal regulated kinase (ERK)-1/2 and p38, which are crucial in oxidative stress-mediated neuronal cell death. Conversely, treatment with casuarinin diminished the phosphorylation of ERK1/2 and P38. In conclusion, the results of this study suggest that casuarinin, obtained from natural products, acts as potent neuroprotective agent by suppressing glutamate-mediated apoptosis through the inhibition of ROS production and activation of the mitogen activated protein kinase (MAPK) pathway. Thus, casuarinin can be a potential therapeutic agent in the treatment of neurodegenerative diseases.     

Expression,Purification and Molecular Structure Modeling of Thioredoxin (Trx) and Thioredoxin Reductase (TrxR) from <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis>

The thioredoxin system consists of thioredoxin (Trx), thioredoxin reductase (TrxR) and NADPH, which plays several key roles in maintaining the redox environment of the cell. In Acidithiobacillus ferrooxidans, thioredoxin system may play important functions in the activity regulation of periplasmic proteins and energy metabolism. Here, we cloned thioredoxin (trx) and thioredoxin reductase (trxR) genes from Acidithiobacillus ferrooxidans, and expressed the genes in Escherichia coli. His-Trx and His-TrxR were purified to homogeneity with one-step Ni-NTA affinity column chromatography. Site-directed mutagenesis results confirmed that Cys33, Cys36 of thioredoxin, and Cys142, Cys145 of thioredoxin reductase were active-site residues.     

Glutamate levels control HT22 murine hippocampal cell death by regulating biphasic patterns of Erk1/2 activation: role of metabolic glutamate receptor 5

Extracellular glutamate concentration is a critical determinant of neuronal cell fate. We recently demonstrated that HT22 murine hippocampal cell viability was reduced by exposure to high concentrations of glutamate, whereas low concentrations promoted cell survival. Extracellular signal-regulated kinase (Erk)1/2 activation by glutamate is important for both glutamate-induced cell death and survival. In this study, we investigated the role of glutamate-induced or hydrogen peroxide (H₂O₂)-induced Erk1/2 activation in HT22 cell fate determination. Glutamate and H₂O₂ treatment similarly induced early (<1 h) Erk1/2 phosphorylation regardless of concentration. On the other hand, persistent Erk1/2 phosphorylation (16–24 h) was observed only in the presence of excess glutamate. Only the latter contributed to glutamate-induced cell death, which involved metabolic glutamate receptor 5. Our findings suggest that glutamate concentration modulates two distinct phases of Erk1/2 activation, which can explain the glutamate concentration-dependent determination of HT22 cell fate.     

Redox atlas of the mouse : Immunohistochemical detection of glutaredoxin-, peroxiredoxin-, and thioredoxin-family proteins in various tissues of the laboratory mouse

Background

Oxidoreductases of the thioredoxin family of proteins have been thoroughly studied in numerous cellular and animal models mimicking human diseases. Despite of their well documented role in various disease conditions, no systematic information on the presence of these proteins is available.

Methods

Here, we have systematically analyzed the presence of some of the major constituents of the glutaredoxin (Grx)-, peroxiredoxin (Prx)-, and thioredoxin (Trx)-systems, i.e. Grx1, Grx2, Grx3 (TXNL-2/PICOT), Grx5, nucleoredoxin (Nrx), Prx1, Prx2, Prx3, Prx4, Prx5, Prx6, Trx1, thioredoxin reductase 1 (TrxR1), Trx2, TrxR2, and γ-glutamyl cysteine synthetase (γ-GCS) in various tissues of the mouse using immunohistochemistry.

Results

The identification of the Trx family proteins in the central nervous system, sensory organs, digestive system, lymphatic system, reproductive system, urinary system, respiratory system, endocrine system, skin, heart, and muscle revealed a number of significant differences between these proteins with respect to their distribution in these tissues.

Conclusion

Our results imply more specific functions and interactions between the proteins of this family than previously assumed.

General significance

Crucial functions of Trx family proteins have been demonstrated in various disease conditions. A detailed overview on their distribution in various tissues will be helpful to fully comprehend their potential role and the interactions of these proteins in the most thoroughly studied model for human diseases—the laboratory mouse.This article is part of a Special Issue entitled Human and Murine Redox Protein Atlases.     

白藜芦醇减轻神经元氧化应激损伤的作用机制研究

目的:观察白藜芦醇(RSV)对过氧化氢(H2O2)所致的海马神经元HT22细胞损伤的保护作用,并探讨超氧化物歧化酶2(Mn-SOD)在其中的作用。方法:采用体外培养HT22小鼠海马神经元细胞系,H2O2作为损伤因素模拟氧化应激损伤。将细胞分为5组,分别为正常培养组(Control)、150μM H2O2损伤组(H2O2)、25μM白藜芦醇保护组(RSV+H2O2)、SOD2-si RNA干扰组(SOD2-si RNA+RSV+H2O2)和乱序RNA组(SC-si RNA+RSV+H2O2),药物暴露24 h后,应用MTT法检测HT22细胞活力、比色法检测乳酸脱氢酶(Lactate Dehydrogenase,LDH)释放量、相差显微镜观测细胞形态。结果:与对照组相比,H2O2组的活力显著下降(P0.05),LDH释放量明显增加(P0.05),细胞形态明显破坏;25μM的RSV显著恢复了HT22细胞的活力、减少了LDH释放、改善了细胞形态,而SOD2-si RNA显著逆转了RSV引起的上述保护作用,乱序RNA(SC-si RNA)未对上述保护作用产生明显影响。结论:白藜芦醇可能通过上调SOD2减轻H2O2对HT22细胞的氧化应激损伤。     

Biochemical and thermodynamic comparison of the selenocysteine containing and non-containing thioredoxin glutathione reductase of Fasciola gigantica

The thiol-disulfide redox metabolism in platyhelminth parasites depends entirely on a single selenocysteine (Sec) containing flavoenzyme, thioredoxin glutathione reductase (TGR) that links the classical thioredoxin (Trx) and glutathione (GSH) systems. In the present study, we investigated the catalytic and structural properties of different variants of Fasciola gigantica TGR to understand the role of Sec. The recombinant full-length Sec containing TGR (FgTGRsec), TGR without Sec (FgTGR) and TGRsec without the N-terminal glutaredoxin (Grx) domain (?NTD-FgTGRsec) were purified to homogeneity. Biochemical studies revealed that Sec597 is responsible for higher thioredoxin reductase (TrxR) and glutathione reductase (GR) activity of FgTGRsec. The N-terminal Grx domain was found to positively regulate the DTNB-based TrxR activity of FgTGRsec. The FgTGRsec was highly sensitive to inhibition by auranofin (AF). The structure of FgTGR was modeled, and the inhibitor AF was docked, and binding sites were identified. Unfolding studies suggest that all three proteins are highly cooperative molecules since during GdnHCl-induced denaturation, a monophasic unfolding of the proteins without stabilization of any intermediate is observed. The Cm for GdnHCl induced unfolding of FgTGR was higher than FgTGRsec and ?NTD-FgTGRsec suggesting that FgTGR without Sec was more stable in solution than the other protein variants. The free energy of stabilization for the proteins was also determined. To our knowledge, this is also the first report on unfolding and stability analysis of any TGR.     

Chebulinic acid attenuates glutamate-induced HT22 cell death by inhibiting oxidative stress,calcium influx and MAPKs phosphorylation

Glutamate-induced excitotoxicity and oxidative stress is a major causative factor in neuronal cell death in acute brain injuries and chronic neurodegenerative diseases. The prevention of oxidative stress is a potential therapeutic strategy. Therefore, in the present study, we aimed to examine a potential therapeutic agent and its protective mechanism against glutamate-mediated cell death. We first found that chebulinic acid isolated from extracts of the fruit of Terminalia chebula prevented glutamate-induced HT22 cell death. Chebulinic acid significantly reduced intracellular reactive oxygen species (ROS) production and Ca²⁺ influx induced by glutamate. We further demonstrated that chebulinic acid significantly decreased the phosphorylation of mitogen-activated protein kinases (MAPKs), including ERK1/2, JNK, and p38, as well as inhibiting pro-apoptotic Bax and increasing anti-apoptotic Bcl-2 protein expression. Moreover, we demonstrated that chebulinic acid significantly reduced the apoptosis induced by glutamate in HT22 cells. In conclusion, our results in this study suggest that chebulinic acid is a potent protectant against glutamate-induced neuronal cell death via inhibiting ROS production, Ca²⁺ influx, and phosphorylation of MAPKs, as well as reducing the ratio of Bax to Bcl-2, which contribute to oxidative stress-mediated neuronal cell death.     

Methylglyoxal induces endoplasmic reticulum stress and DNA demethylation in the Keap1 promoter of human lens epithelial cells and age-related cataracts

Age-related cataracts are a leading cause of blindness. Previously, we have demonstrated the association of the unfolded protein response with various cataractogenic stressors. However, DNA methylation alterations leading to suppression of lenticular antioxidant protection remains unclear. Here, we report the methylglyoxal-mediated sequential events responsible for Keap1 promoter DNA demethylation in human lens epithelial cells, because Keap1 is a negative regulatory protein that regulates the Nrf2 antioxidant protein. Methylglyoxal induces endoplasmic reticulum stress and activates the unfolded protein response leading to overproduction of reactive oxygen species before human lens epithelial cell death. Methylglyoxal also suppresses Nrf2 and DNA methyltransferases but activates the DNA demethylation pathway enzyme TET1. Bisulfite genomic DNA sequencing confirms the methylglyoxal-mediated Keap1 promoter DNA demethylation leading to overexpression of Keap1 mRNA and protein. Similarly, bisulfite genomic DNA sequencing shows that human clear lenses (n = 15) slowly lose 5-methylcytosine in the Keap1 promoter throughout life, at a rate of 1% per year. By contrast, diabetic cataractous lenses (n = 21) lose an average of 90% of the 5-methylcytosine regardless of age. Overexpressed Keap1 protein is responsible for decreasing Nrf2 by proteasomal degradation, thereby suppressing Nrf2-dependent stress protection. This study demonstrates for the first time the associations of unfolded protein response activation, Nrf2-dependent antioxidant system failure, and loss of Keap1 promoter methylation because of altered active and passive DNA demethylation pathway enzymes in human lens epithelial cells by methylglyoxal. As an outcome, the cellular redox balance is altered toward lens oxidation and cataract formation.     

Methylglyoxal accumulation de-regulates HoxA5 expression,thereby impairing angiogenesis in glyoxalase 1 knock-down mouse aortic endothelial cells

Impaired angiogenesis leads to long-term complications and is a major contributor of the high morbidity in patients with Diabetes Mellitus (DM). Methylglyoxal (MGO) is a glycolysis byproduct that accumulates in DM and is detoxified by the Glyoxalase 1 (Glo1). Several studies suggest that MGO contributes to vascular complications through mechanisms that remain to be elucidated. In this study we have clarified for the first time the molecular mechanism involved in the impairment of angiogenesis induced by MGO accumulation.Angiogenesis was evaluated in mouse aortic endothelial cells isolated from Glo1-knockdown mice (Glo1KD MAECs) and their wild-type littermates (WT MAECs). Reduction in Glo1 expression led to an accumulation of MGO and MGO-modified proteins and impaired angiogenesis of Glo1KD MAECs. Both mRNA and protein levels of the anti-angiogenic HoxA5 gene were increased in Glo1KD MAECs and its silencing improved both their migration and invasion. Nuclear NF-?B-p65 was increased 2.5-fold in the Glo1KD as compared to WT MAECs. Interestingly, NF-?B-p65 binding to HoxA5 promoter was also 2-fold higher in Glo1KD MAECs and positively regulated HoxA5 expression in MAECs. Consistent with these data, both the exposure to a chemical inhibitor of Glo1 “SpBrBzGSHCp2” (GI) and to exogenous MGO led to the impairment of migration and the increase of HoxA5 mRNA and NF-?B-p65 protein levels in microvascular mouse coronary endothelial cells (MCECs).This study demonstrates, for the first time, that MGO accumulation increases the antiangiogenic factor HoxA5 via NF-?B-p65, thereby impairing the angiogenic ability of endothelial cells.     

Functionalized acridin-9-yl phenylamines protected neuronal HT22 cells from glutamate-induced cell death by reducing intracellular levels of free radical species

The in vitro neuronal cell death model based on the HT22 mouse hippocampal cell model is a convenient means of identifying compounds that protect against oxidative glutamate toxicity which plays a role in the development of certain neurodegenerative diseases. Functionalized acridin-9-yl-phenylamines were found to protect HT22 cells from glutamate challenge at submicromolar concentrations. The Aryl¹-NH-Aryl² scaffold that is embedded in these compounds was the minimal pharmacophore for activity. Mechanistically, protection against the endogenous oxidative stress generated by glutamate did not involve up-regulation of glutathione levels but attenuation of the late stage increases in mitochondrial ROS and intracellular calcium levels. The NH residue in the pharmacophore played a crucial role in this regard as seen from the loss of neuroprotection when it was structurally modified or replaced. That the same NH was essential for radical scavenging in cell-free and cell-based systems pointed to an antioxidant basis for the neuroprotective activities of these compounds.     

Glutamate-induced cell death in neuronal HT22 cells is attenuated by extracts from St. John''s wort (Hypericum perforatum L.)

Glutamate-induced cell death of hippocampal HT22 cells is a model system for neuronal disorders due to depletion of glutathione levels and increase of intracellular reactive oxygen species. Standardized extracts of Hypericum perforatum (HPE) contain flavonoids known for antioxidative properties. In the above model, cytoprotective effects at a concentration of 0.05% HPE by attenuation of calcium fluxes and cellular energy statuses are reported.     

Protective effects of the thioredoxin and glutaredoxin systems in dopamine-induced cell death

Although the etiology of sporadic Parkinson disease (PD) is unknown, it is well established that oxidative stress plays an important role in the pathogenic mechanism. The thioredoxin (Trx) and glutaredoxin (Grx) systems are two central systems upholding the sulfhydryl homeostasis by reducing disulfides and mixed disulfides within the cell and thereby protecting against oxidative stress. By examining the expression of redox proteins in human postmortem PD brains, we found the levels of Trx1 and thioredoxin reductase 1 (TrxR1) to be significantly decreased. The human neuroblastoma cell line SH-SY5Y and the nematode Caenorhabditis elegans were used as model systems to explore the potential protective effects of the redox proteins against 6-hydroxydopamine (6-OHDA)-induced cytotoxicity. 6-OHDA is highly prone to oxidation, resulting in the formation of the quinone of 6-OHDA, a highly reactive species and powerful neurotoxin. Treatment of human cells with 6-OHDA resulted in an increased expression of Trx1, TrxR1, Grx1, and Grx2, and small interfering RNA for these genes significantly increased the cytotoxic effects exerted by the 6-OHDA neurotoxin. Evaluation of the dopaminergic neurons in C. elegans revealed that nematodes lacking trxr-1 were significantly more sensitive to 6-OHDA, with significantly increased neuronal degradation. Importantly, both the Trx and the Grx systems were also found to directly mediate reduction of the 6-OHDA-quinone in vitro and thus render its cytotoxic effects. In conclusion, our results suggest that the two redox systems are important for neuronal survival in dopamine-induced cell death.