活化ARE通路对运动神经元保护作用的研究
|
摘要
肌萎缩侧索硬化(Amyotrophic Lateral Sclerosis ALS)是一个致命的神经变性疾病。它的主要病理改变是选择性地损伤运动神经元(包括大脑皮层运动神经元、脑干运动神经核和脊髓前角运动神经元)。临床表现为缓慢进展的四肢无力,累及呼吸肌,多于发病3-5年内死亡。ALS可分为家族型肌萎缩侧索硬化(Familial Amyotrophic Lateral Sclerosis,FALS)和散发型肌萎缩侧索硬化(Sporadic Amyotrophic Lateral Sclerosis,SALS),90%以上为散发型不足10%为家族型。
其中FALS主要是由于编码Cu/Zn超氧化物歧化酶(SOD1)的基因突变引起,其突变位点在21号染色体长臂Cu/Zn SOD基因内,即21q22.1-22.2。与ALS有关的SOD1基因的突变大约有100余种。在这些突变中SOD酶的活性基本正常或升高,SOD基因敲除的小鼠则没有发病。这些发现提示SOD1相关的ALS不是因为功能缺失而引起,而是可能由一种获得性毒性引起。
在运动神经元变性过程中牵涉到几个机制:兴奋毒性、免疫反应、线粒体功能障碍及自由基损伤、蛋白质异常聚积、蛋白酶体功能改变和凋亡。虽然这些通路对运动神经元损伤的启动和进展都起作用,但是这些通路之间的相互反应以及哪个起主要作用还不明了。
这些机制不是互相排斥的而是可以被一个共同的反应所激活,这一共同的反应的协调者正是氧化应激。尽管确切机制尚不明了,但在导致运动神经元损伤的过程中,增加的氧化应激是出现较早且持久的事件。氧化应激不仅在ALS的发病中起了突出的作用,并且它通过活化其它各个通路,引起更新一轮的氧化应激,促进了疾病的进展。所以许多研究的焦点集中在氧化应激和自由基损伤。
哺乳动物细胞内还原型谷胱甘肽(glutathione GSH)是细胞内含量最丰富、最有效的反应氧族(reactive oxygen species ROS)的直接清除剂。通过GSH与氧化型谷胱甘肽(GSSG)的转换来调节ROS水平,GSH减少则ROS产生增加并促进氧化损伤。
另外GSH也是许多抗氧化酶的关键底物,这些酶解毒过氧化氢和脂质过氧化的产物。正如CHI.L和KE.Y等应用离体和在体的实验证实那样,GSH的缺失加强了运动神经元的变性。
GSH的合成是一个ATP依赖的两步酶促反应。第一步由γ-谷氨酸半胱氨酸连接酶(γ- Glutamate Cysteine ligase GCL)催化完成,第二步由GSH合成酶(GSH synthetase)催化完成。前者是重要的限速酶。分别有GCLC和GCLM两个亚单位组成。随着年龄的老化,在大鼠中枢神经系统组织GCLM基因表达的下调,伴随着GCL酶活力的减低和GSH水平的降低。说明在与年龄相关的神经变性疾病中GCL及GSH发挥重要作用。
由谷胱甘肽S一转移酶(Glutathione S-transferase GST)家族是一个分布广泛,包括近百种同工酶在内的多基因大家族。GSTs属Ⅱ相酶防御系统。具有多种生物学功能如催化包括亲电子试剂(electrophiles)、致癌物(carcinogens)和有基因毒性、细胞毒性的异生化合物(xenobiotics)与还原型谷胱甘肽(GSH)连接,使其转变为亲水的易排泄物质。GST在神经细胞氧化防御方面起重要作用。所以诱导内源性Ⅱ相酶,如GST已经被提议作为治疗PD的方法和癌症的化学预防。
本课题根据谷氨酸的慢性兴奋毒性机制,应用谷氨酸转运体抑制剂苏-羟天冬氨酸(THA),抑制星形胶质细胞对细胞外谷氨酸的转运,最终导致运动神经元死亡。制备成慢性选择性运动神经元损伤的脊髓器官型培养模型
总之,慢性选择性运动神经元损伤的脊髓器官型培养模型为神经保护剂在人类运动神经元疾病的临床试验和临床前试验间提供了密切的联系。该模型已经成功地预言了力如唑(riluzole)和加巴喷丁(neurontin)有效的运动神经元保护作用,这两种药已经用于人类运动神经元病。故本课题利用该模型,以诱导内源性抗氧化剂为切入点寻求理想的治疗靶点。
在许多神经变性疾病的病因涉及到神经细胞内ROS的堆积,细胞中和这些反应中间产物的能力很大程度上依赖一个顺式作用元件的活化,这个顺式作用元件被命名为抗氧化反应元件(antioxidant response element ARE)。它存在于抗氧化蛋白和解毒酶基因的5’端区域。在人类、大鼠、小鼠的多种组织中及原代培养的星形胶质细胞、神经元中都已证实,ARE驱动的靶基因包括GCLC、GLCM和GSTs等。
所以,本课题应用ARE活化剂,观察其在该模型中对运动神经元的作用,并进一步探讨作用机制。论文共分四部分:第一部分证实ARE活化剂对运动神经元确实存在保护作用。第二部分探讨这种保护作用伴随着该模型中GSH水平和Ca~(2+)水平的变化,而与P75NTR水平无关。第三部分进一步证实GSH合成的限速酶GCL在该模型中存在异常,随着ARE的保护作用,GCL的基因水平发生改变,说明GSH水平的改变是其上游合成酶变化引起。第四部分探讨了GSH相关酶:GST在该模型中及应用ARE活化剂后基因和蛋白水平的变化。总之,本课题证实,在慢性选择性运动神经元损伤的脊髓器官型培养模型中存在着GSH及其相关酶的异常,通过ARE活化剂能够改变它们的表达,这种改变伴随着对运动神经元的保护作用。
第一部分在脊髓器官型培养模型中ARE活化剂对慢性选择性运动神经元损伤的保护作用
目的:在慢性选择性运动神经元损伤的脊髓器官型培养模型中,应用不同类型的ARE活化剂,不同剂量、不同方式进行干预,观察它是否能保护运动神经元免受慢性谷氨酸兴奋毒性损伤。并摸索出恰当的干预方法。为保护运动神经元寻找一个新的靶点。
方法:取出生后8天乳鼠的腰段脊髓组织切片做脊髓器官型培养,培养7天后,在培养液中分别加入不同浓度CPDT、D3T和tBHQ(5μmol/L、15μmol/L、30μmol/L),并且提前干预组(提前48小时)和与THA同时干预组,培养4周后,用神经元的特异性SMI-32免疫组化染色,对不同时点脊髓腹角α运动神经元进行记数,用透射电镜观察神经元超微结构变化,与对照组做比较。
结果:正常对照组脊髓片形态完好,α运动神经元数目恒定,模型组α运动神经元数目减少。在提前干预的CPDT、D3T和tBHQ组15μmol/L、30μmol/L浓度,培养4周后α运动神经元数目较模型组运动神经元数目增多,与正常对照组相似。电镜下观察显示超微结构损伤也不明显,细胞器保持相对完好。而5μmol/L的CPDT、D3T和THA同时干预的各组,脊髓片损伤与模型组相似,α运动神经元明显减少。
结论:利用ARE活化剂CPDT、D3T和tBHQ能够明显地保护运动神经元免受慢性谷氨酸兴奋毒损伤,为进一步研究ALS的发病机制及探讨神经保护治疗提供了有效的方法。
第二部分ARE活化剂对慢性运动神经元损伤模型GSH、Ca~(2+)及P75NTR的影响
目的:在慢性选择性运动神经元损伤的脊髓器官型培养模型中,观察GSH、Ca~(2+)及P75NTR的变化。进一步应用ARE活化剂观察,在其对运动神经元的保护作用的同时对这些指标的影响。
方法:取出生后8天乳鼠的腰段脊髓组织切片做脊髓器官型培养,培养7天后,同第一部分,分别加入不同浓度CPDT、D3T。培养4周时取出。借助细胞内硫醇的分子探针(mCB)应用多功能酶标仪荧光法,检测脊髓组织细胞内还原型谷胱甘肽(GSH)浓度。借助Flow 3-AM(膜通透的细胞内Ca~(2+)指示剂)应用流式细胞仪检测脊髓组织细胞内Ca~(2+)相对浓度。提取组织匀浆蛋白,用免疫印迹检测脊髓组织P75NTR的蛋白水平。
结果:GSH在48h组30μmol/L的CPDT明显较MS组增高,而15μmol/L的CPDT组略高。30CPDT96h组和15CPDT96h组的GSH水平均高于MS组,说明CPDT引起的GSH的升高是时间和剂量依赖的。在THA组存在GSH水平的降低,应用两个浓度的CPDT提前干预后可以阻止这种降低,并使GSH水平升高3-4倍。
THA引起细胞内Ca~(2+)水平升高,提前48小时应用CPDT和D3T后,纠正了细胞内Ca~(2+)水平的升高。但THA组和CPDT提前组均无P75NTR蛋白的表达。
结论: ARE活化剂CPDT、D3T对运动神经元的保护作用,伴随着提高细胞内GSH水平和降低细胞内Ca~(2+)。该模型中不存在对P75NTR依赖的凋亡,也未发现CPDT对该途径的影响。
第三部分慢性选择性运动神经元损伤模型中GCL的基因表达及ARE活化剂对其影响
目的:验证在体外培养的脊髓薄片中,能否如在其它组织和器官中一样, GCLC、GCLM基因作为ARE的靶基因被上调。进一步验证慢性选择性运动神经元损伤模型中是否有该目的基因的异常,以及ARE活化剂能否阻止这种异常的改变。
方法:取出生后8天乳鼠的腰段脊髓组织切片做脊髓器官型培养,培养7天后,同第一部分,分别加入不同浓度CPDT。培养4周时取出。采用TRIZOL一步法从培养的脊髓组织中提取总RNA。应用RT-PCR法检测两个目的基因GCLC和GCLM以β-actin基因作为对照。
结果:30μmol/L CPDT提前48h干预组GCLCmRNA表达明显高于正常对照组(MS组),15μmol/L CPDT提前48h干预组与MS组比较略高于MS组,但无统计学意义。但在干预48小时各组GCLMmRNA的表达与MS在比较无统计学意义。
在培养4周的模型中,两个浓度的CPDT提前干预48小时,然后再同时加入THA。GCLCmRNA的表达在两个浓度的CPDT提前组均明显高于MS组和THA组。MS组和THA组之间无差异。而GCLMmRNA的表达,仅THA组明显低于MS组,而15、30μmol/L CPDT提前干预对其无影响。
结论: ARE活化剂CPDT可以升高体外培养的脊髓器官型模型中GCLC的基因水平,而对GCLM基因无影响。在THA造成的模型中存在GCLM基因表达的降低。与其下游的GSH的含量的改变相一致。
第四部分应用ARE活化剂对慢性选择性运动神经元损伤模型中GSTM的影响
目的:检测与GSH相关的酶, ARE活化剂的靶基因之一GSTM的蛋白和基因水平在该模型中的的表达。进一步验证,ARE活化剂在对运动神经元保护作用的同时是否伴随GSTM的变化。
方法:取出生后8天乳鼠的腰段脊髓组织切片做脊髓器官型培养,培养7天后,同第一部分,分别加入不同浓度的ARE活化剂:CPDT、tBHQ。继续培养48小时或继续培养3周后取出。提取组织总蛋白和总RNA。应用RT-PCR和Western blot分别检测各组组织中GSTM的基因和蛋白水平。
结果:在ARE活化剂CPDT和tBHQ干预48小时可以刺激体外培养的脊髓片模型中GSTM蛋白水平的升高。在培养4周的模型中,CPDT干预后GSTM的蛋白和基因水平均比正常对照组增高。THA组也较正常对照组增高。
结论:作为ARE的靶基因之一,GSTM可以在ARE活化剂的刺激下,在体外培养的脊髓片模型中表达升高。伴随着运动神经元的保护作用,这种升高可以持续4周。
Amyotrophic lateral sclerosis (ALS) is a common progressive neurodegenerative disease in central nervous system. It is characterized by selective degeneration of motor neurons in brain ,brain stem and spinal cord, which clinical features are delayed onset, chronic progression, weakness of limbs’muscles. Most patients died of respiratory failure 3-5 years later. ALS can be divided into familial amyotrophic lateral sclerosis (FALS) and sporadic amyotrophic lateral sclerosis (SALS). FALS is less than 10% of the total ALS patients, while SALS is more than 90%.
FALS is mainly caused by the mutation of copper/zinc superoxide dismutase (Cu/Zn SOD) gene, whose site is at 21q22.1～22.2. More than 100 different mutations of the SOD1 gene have been linked to familial ALS. For many of these mutations, SOD1 enzyme activity is actually normal or elevated, and SOD1 knockout mice have no disease phenotype. These findings indicate that SOD1-associated ALS is not caused by a loss of function, but rather a toxic gain of function.
Several mechanisms are implicated in the pathogenesis of motor neuron degeneration, including excitotoxicity, immune activation, mitochondrial dysfunction and oxidative stress, protein aggregation, altered proteosomal function and apoptosis. Although disturbances in each of these pathways may contribute to amplification or even initiation of motor neuron injury, the temporal relation of these pathways and their primacy in dictating disease onset and progression are unclear. These mechanisms are not mutually exclusive but are activated as a communal response that may be coordinated by oxidative stress. Increased oxidative stress appears to be an early and sustained event in association with motor neuron death in ALS, although the specific mechanism leading to oxidative damage on motor neurons remains to be defined. Oxidative stress has a prominent role in the initiation of ALS and is capable of activating pathways that elicit additional oxidative stress and propagate disease.
GSH is the most abundant and effective scavenger against ROS directly in mammalian cells. In addition, GSH is also a key substrate for antioxidant enzymes that detoxify hydrogen peroxide and lipid peroxide products. CHI.L和KE.Y show that depletion of reduced glutathione enhances motor neuron degeneration in vitro and vivo.
The synthesis of GSH involves the actions of two ATP-dependent enzymes,γ-glutamylcysteine ligase (GCL) and GSH synthetase. GCL, the rate-controlling enzyme in the overall pathway, is a heterodimer composed of a catalytic (GCLC) and a modulatory (GCLM).There is a down-regulation of GCLM gene expression in rat CNS tissue during aging, accompany by reduced activity of GCL and GSH level. This means, GCL and GSH play an important role in age-related neuron degeneration disease.
Glutathione S-transferase (GST) is a multigene family of more than 100 isoenzymes that catalyze the conjugation of GSH to a variety of electrophilic compounds。Glutathione transferases (GSTs; EC, 2.5.1.18) are phase II enzymes of defense that catalyze the conjugation of reduced glutathione to a wide range of electrophiles, carcinogens and other xenobiotics with genotoxic and cytotoxic activities。
GSTs play an important role in protecting neurons against oxidative stress damage. Many study suggest induction of endogenous phase II enzymes,e g,GST may be a strategy for PD and AD.
Based on the chronic excitotoxicity pathogenesis, incubation of organotypic spinal cord cultures in presence of threo-hydroxyaspartate (THA), the inhibitor of astrocyte glutamate transporter, causes death of motor neurons. We established the model of chronic motor neuron degeneration.
Isummary, the organotypic spinal cord model of chronic motor neuron degeneration promises preclinical feasibility testing of potential neuroprotectants with enhanced relevance for clinical trials in human motor neuron disease. This model has already successfully predicted the efficacy of motor neuron protection by riluzole and neurontin, which are being used for human motor neuron disease. In this model, we want to study neuroprotective effects by inducing endogenous antioxidant agents.
The intraneuronal accumulation of reactive oxygen species has been implicated in the pathogenesis of many neurodegenerative diseases .The ability of a cell to neutralize reactive intermediates is, in part, dependent on the activation of a cis-acting regulatory element termed the antioxidant response element (ARE). The ARE is located in the 5'-flanking region of many genes essential for both detoxification and antioxidant proteins. In many tissues of human, rat, mice and primary culture neurons and astrocytes, there are many ARE-drived gene including DCLC, GCLM and GSTs.
The organotypic spinal cord model of chronic motor neuron degeneration were used to study the effect of ARE activator on motor neuron cell death and mechanism. The first part: we show that ARE activator completely inhibit glutamate-induced motor neuron death in these explants. The second part: we show that ARE activator inhibits glutamate-induced intracellular Ca~(2+) rise and decrease of tissue glutathione, but don’t affect P75NTR pathway.
The third part: the rate-controlling enzyme of GSH synthesis, GCL is abnormal in the model, but ARE activator correct the abnormal, which is corresponding to GSH alteration. The forth part: we investigate GSH related enzyme-GSTM protein and mRNA level in the model, ARE activator can change its level, which is accompanied by motor neuron protection.
PartⅠARE activator protects motor neuron against glutamate excitotoxity-induced motor neuron death in organotypic spinal cord model of chronic motor neuron degeneration ,
Objective: In organotypic spinal cord model of chronic motor neuron degeneration , to investigate if ARE activator could protect motor neurons against glutamate excitotoxicity.We use three ARE activator :CPDT ,D3T and tBHQ at different concentration,at different time points. To explore new neuroprotective treatment.
Methods: Organotypic spinal cord cultures were prepared using lumbar spinal cord slices from 8-day-old rat. Various concentrations of CPDT ,D3T and tBHQ(5μmol/L、15μmol/L、30μmol/L) were continuously added into the culture medium after cultured seven days. Spinal cord slices were treated with THA, a combination of THA and ARE activator , or treatment with the ARE activator without THA for 48 hours .Then we use monoclonal antibody SMI-32, a nonphosphorylated neurofilament marker, immunohistochemistry staining compared with different group.
Results: The results showed that the spinal cord explants in control group could maintain excellent organotypic cellular organization and a stable population of ventralα-motor neurons. THA could produce a slow loss ofα-motor neurons. The complete neuron protection was achieved when the explants were pretreated by ARE activator: CPDT, D3T, tBHQ for 48 hour prior to initiating the combination treatment. Whereas ARE activator was only able to offer very limited neuron protection against THA-induced motor neuron death when the two agents were always added together to the culture medium,and same as 5μmol/L ARE activator.
Conclusions: It is possibility that ARE activator may block glutamate toxicity to protects motor neuron against glutamate excitotoxity-induced motor neuron death. This subject could provide an effective studying pathogenesis and neuroprotection of ALS.
PartⅡTissue glutathione (GSH) contents, intracellular Ca2+ levels and P75NTR protein in organotypic spinal cord model of chronic motor neuron degeneration and alternation by ARE activator
Objective: To study if CPDT stimulates GSH content in rat spinal cord explants Proctention motor neuron survival in CPDT-treated spinal cord explants is accompanied by GSH, intracellular Ca2+ levels and P75NTR protein alteration.
Methods: Organotypic spinal cord cultures were prepared using lumbar spinal cord slices from 8-day-old rat. Various concentrations of CPDT, D3T were continuously added into the culture medium after cultured seven days. GSH in tissue homogenates was derivatized by monochlorobimane, then the derivatives (GSH-monochlorobimane) were measured using a fluorescence plate reader. Measurement of intracellular Ca~(2+) level with 8μM Flou3-AM, then them were immediately analyzed by flow cytometry to determine fluorescence intensity.Tissue P75NTR protein was measured by Western blot.
Results: After on week recover, the spinal cord tissues were treated with 15μM , 30μMCPDT for 48hours, then collected , measured ,GSH level increase only in 30CPDT group, but continue culture for 96 hours,GSH level was increase eather in 15CPDT group or in 30CPDT group.
In the present experiment, the spinal cord tissues were first treated with CPDT at 15 and 30μM for 48 h before combined treatment of CPDT and THA for 3 weeks. We show that THA treatment might deplete tissue GSH. Indeed, 3-week THA treatment markedly reduced GSH level. Significantly, CPDT not only prevented THA-induced GSH depletion but actually elevated tissue GSH level 3-4 fold over the control. We found that CPDT fully prevented both intracellular Ca~(2+) rise.
In the model, we could not find any specific P75NTR protein expression , but we could see it on one day old rat, which is control. After treatment with CPDT, there is no any specific protein expression at 75KD marker can be seen.
Conclusions: CPDT could increase GSH level in spinal cord tissues ,which is time-depented and dose-depented.THA treatment reduced GSH level and CPDT not only prevented that but all so elevated GSH level 3-4 fold .CPDT,D3T fully prevented intracellular Ca~(2+) rise. CPDT have no effection on P75NTR pathway.
PartⅢARE target gene-GCL expression in organotypic spinal cord model of chronic motor neuron degeneration and alternation by ARE activator
Objective: to investigate one of ARE target gene GCL (include two subunit GCLC and GCLM) expression in organotypic spinal cord model of chronic motor neuron degeneration. If their alteration are accompany by motor neuron protection and ARE activitor can prevented THA-induced alteration.
Methods: Various concentrations of CPDT were continuously added into the culture medium after cultured seven days. In the present study, lumber spinal cord explants prepared from 7-day old rats, after one week of culture, were treated with CPDT for 48 h and then harvested for measurement of GCLC, GCLMmRNA.
Thus, explants after one week of culture were exposed to either solvent, THA or THA plus CPDT. In the THA plus CPDT group, the explants were first treated with CPDT for 48 h before the combination treatment, since this treatment schedule allowed CPDT to fully protect motor neurons.
RT-PCR was used to measure the expression of two genes, including GCLCand GCLMalong withβ-actin gene as a control. The Trizol method was used to extract total RNA from the rat spinal cord explants.
Results: At treat for 48hours group, only the rat spinal cord explants treated with 30μmol/LCPDT can increase GCLCmRNA expression, 15μmol/LCPDT could not affect it. On the other hand, two concentration CPDT treatment spinal cord explants show no alteration in GCLMmRNA expression at all.
In the THA plus CPDT group,. In THA group, GCLMmRNA expression is lower than others. However, THA seems to have no effect on GCLC expression. Just as above, the combination treatment of THA with CPDT could not cause increase in GCLMmRNA expression. But this way caused a more significant increase in GCLCmRNA expression.
Conclusions: Hight concentration CPDT (30μM) could cause increase expression of GCLC, but could not affect GCLM. However THA decrease GCLMmRNA expression, but can not affect GCLC.CPDT could increase GCLCmRNA last for 4 weeks, accompany by protection motor neuron.
PartⅣGSTM protein and gene expression in organotypic spinal cord model of chronic motor neuron degeneration and alternation by ARE activator
Objective: to investigate one of ARE target gene GSTM protein and mRNA expression in organotypic spinal cord model of chronic motor neuron degeneration, If their alteration are accompany by motor neuron protection and ARE activator can prevented THA-induced alteration.
Methods: Organotypic spinal cord cultures were prepared using lumbar spinal cord slices from 8-day-old rat. Various concentrations of CPDT were continuously added into the culture medium after cultured seven days. In the present study, lumber spinal cord explants prepared from 7-day old rats, after one week of culture, were treated with CPDT for 48 h and then harvested for measurement of GSTM protein.
Thus, explants after one week of culture were exposed to either solvent, THA or THA plus CPDT. In the THA plus CPDT group, the explants were first treated with CPDT for 48 h before the combination treatment, since this treatment schedule allowed CPDT to fully protect motor neurons.
Prepare whole tissue extracts for Western Blot analysis. The Trizol method was used to extract total RNA from the rat spinal cord explants.
Results: In lumber spinal cord explants treated with CPDT for 48 h shows CPDT and tBHQ CPDT at 15 and 30μM caused significant increase in expression of GSTM protein.
In cultured for 4 weeks group, exposure of the explants to CPDT in the presence of THA lead to increased expression of GSTM in mRNA and protein .Interestingly, THA itself also seems to positively modulate some of the genes, GSTM.
Conclusions: CPDT and tBHQ could stimulates ARE target genes in rat spinal cord explants. Increased motor neuron survival in CPDT-treated spinal cord explants is accompanied by activation of ARE targen gene GSTM expression and protein level.
其中FALS主要是由于编码Cu/Zn超氧化物歧化酶(SOD1)的基因突变引起,其突变位点在21号染色体长臂Cu/Zn SOD基因内,即21q22.1-22.2。与ALS有关的SOD1基因的突变大约有100余种。在这些突变中SOD酶的活性基本正常或升高,SOD基因敲除的小鼠则没有发病。这些发现提示SOD1相关的ALS不是因为功能缺失而引起,而是可能由一种获得性毒性引起。
在运动神经元变性过程中牵涉到几个机制:兴奋毒性、免疫反应、线粒体功能障碍及自由基损伤、蛋白质异常聚积、蛋白酶体功能改变和凋亡。虽然这些通路对运动神经元损伤的启动和进展都起作用,但是这些通路之间的相互反应以及哪个起主要作用还不明了。
这些机制不是互相排斥的而是可以被一个共同的反应所激活,这一共同的反应的协调者正是氧化应激。尽管确切机制尚不明了,但在导致运动神经元损伤的过程中,增加的氧化应激是出现较早且持久的事件。氧化应激不仅在ALS的发病中起了突出的作用,并且它通过活化其它各个通路,引起更新一轮的氧化应激,促进了疾病的进展。所以许多研究的焦点集中在氧化应激和自由基损伤。
哺乳动物细胞内还原型谷胱甘肽(glutathione GSH)是细胞内含量最丰富、最有效的反应氧族(reactive oxygen species ROS)的直接清除剂。通过GSH与氧化型谷胱甘肽(GSSG)的转换来调节ROS水平,GSH减少则ROS产生增加并促进氧化损伤。
另外GSH也是许多抗氧化酶的关键底物,这些酶解毒过氧化氢和脂质过氧化的产物。正如CHI.L和KE.Y等应用离体和在体的实验证实那样,GSH的缺失加强了运动神经元的变性。
GSH的合成是一个ATP依赖的两步酶促反应。第一步由γ-谷氨酸半胱氨酸连接酶(γ- Glutamate Cysteine ligase GCL)催化完成,第二步由GSH合成酶(GSH synthetase)催化完成。前者是重要的限速酶。分别有GCLC和GCLM两个亚单位组成。随着年龄的老化,在大鼠中枢神经系统组织GCLM基因表达的下调,伴随着GCL酶活力的减低和GSH水平的降低。说明在与年龄相关的神经变性疾病中GCL及GSH发挥重要作用。
由谷胱甘肽S一转移酶(Glutathione S-transferase GST)家族是一个分布广泛,包括近百种同工酶在内的多基因大家族。GSTs属Ⅱ相酶防御系统。具有多种生物学功能如催化包括亲电子试剂(electrophiles)、致癌物(carcinogens)和有基因毒性、细胞毒性的异生化合物(xenobiotics)与还原型谷胱甘肽(GSH)连接,使其转变为亲水的易排泄物质。GST在神经细胞氧化防御方面起重要作用。所以诱导内源性Ⅱ相酶,如GST已经被提议作为治疗PD的方法和癌症的化学预防。
本课题根据谷氨酸的慢性兴奋毒性机制,应用谷氨酸转运体抑制剂苏-羟天冬氨酸(THA),抑制星形胶质细胞对细胞外谷氨酸的转运,最终导致运动神经元死亡。制备成慢性选择性运动神经元损伤的脊髓器官型培养模型
总之,慢性选择性运动神经元损伤的脊髓器官型培养模型为神经保护剂在人类运动神经元疾病的临床试验和临床前试验间提供了密切的联系。该模型已经成功地预言了力如唑(riluzole)和加巴喷丁(neurontin)有效的运动神经元保护作用,这两种药已经用于人类运动神经元病。故本课题利用该模型,以诱导内源性抗氧化剂为切入点寻求理想的治疗靶点。
在许多神经变性疾病的病因涉及到神经细胞内ROS的堆积,细胞中和这些反应中间产物的能力很大程度上依赖一个顺式作用元件的活化,这个顺式作用元件被命名为抗氧化反应元件(antioxidant response element ARE)。它存在于抗氧化蛋白和解毒酶基因的5’端区域。在人类、大鼠、小鼠的多种组织中及原代培养的星形胶质细胞、神经元中都已证实,ARE驱动的靶基因包括GCLC、GLCM和GSTs等。
所以,本课题应用ARE活化剂,观察其在该模型中对运动神经元的作用,并进一步探讨作用机制。论文共分四部分:第一部分证实ARE活化剂对运动神经元确实存在保护作用。第二部分探讨这种保护作用伴随着该模型中GSH水平和Ca~(2+)水平的变化,而与P75NTR水平无关。第三部分进一步证实GSH合成的限速酶GCL在该模型中存在异常,随着ARE的保护作用,GCL的基因水平发生改变,说明GSH水平的改变是其上游合成酶变化引起。第四部分探讨了GSH相关酶:GST在该模型中及应用ARE活化剂后基因和蛋白水平的变化。总之,本课题证实,在慢性选择性运动神经元损伤的脊髓器官型培养模型中存在着GSH及其相关酶的异常,通过ARE活化剂能够改变它们的表达,这种改变伴随着对运动神经元的保护作用。
第一部分在脊髓器官型培养模型中ARE活化剂对慢性选择性运动神经元损伤的保护作用
目的:在慢性选择性运动神经元损伤的脊髓器官型培养模型中,应用不同类型的ARE活化剂,不同剂量、不同方式进行干预,观察它是否能保护运动神经元免受慢性谷氨酸兴奋毒性损伤。并摸索出恰当的干预方法。为保护运动神经元寻找一个新的靶点。
方法:取出生后8天乳鼠的腰段脊髓组织切片做脊髓器官型培养,培养7天后,在培养液中分别加入不同浓度CPDT、D3T和tBHQ(5μmol/L、15μmol/L、30μmol/L),并且提前干预组(提前48小时)和与THA同时干预组,培养4周后,用神经元的特异性SMI-32免疫组化染色,对不同时点脊髓腹角α运动神经元进行记数,用透射电镜观察神经元超微结构变化,与对照组做比较。
结果:正常对照组脊髓片形态完好,α运动神经元数目恒定,模型组α运动神经元数目减少。在提前干预的CPDT、D3T和tBHQ组15μmol/L、30μmol/L浓度,培养4周后α运动神经元数目较模型组运动神经元数目增多,与正常对照组相似。电镜下观察显示超微结构损伤也不明显,细胞器保持相对完好。而5μmol/L的CPDT、D3T和THA同时干预的各组,脊髓片损伤与模型组相似,α运动神经元明显减少。
结论:利用ARE活化剂CPDT、D3T和tBHQ能够明显地保护运动神经元免受慢性谷氨酸兴奋毒损伤,为进一步研究ALS的发病机制及探讨神经保护治疗提供了有效的方法。
第二部分ARE活化剂对慢性运动神经元损伤模型GSH、Ca~(2+)及P75NTR的影响
目的:在慢性选择性运动神经元损伤的脊髓器官型培养模型中,观察GSH、Ca~(2+)及P75NTR的变化。进一步应用ARE活化剂观察,在其对运动神经元的保护作用的同时对这些指标的影响。
方法:取出生后8天乳鼠的腰段脊髓组织切片做脊髓器官型培养,培养7天后,同第一部分,分别加入不同浓度CPDT、D3T。培养4周时取出。借助细胞内硫醇的分子探针(mCB)应用多功能酶标仪荧光法,检测脊髓组织细胞内还原型谷胱甘肽(GSH)浓度。借助Flow 3-AM(膜通透的细胞内Ca~(2+)指示剂)应用流式细胞仪检测脊髓组织细胞内Ca~(2+)相对浓度。提取组织匀浆蛋白,用免疫印迹检测脊髓组织P75NTR的蛋白水平。
结果:GSH在48h组30μmol/L的CPDT明显较MS组增高,而15μmol/L的CPDT组略高。30CPDT96h组和15CPDT96h组的GSH水平均高于MS组,说明CPDT引起的GSH的升高是时间和剂量依赖的。在THA组存在GSH水平的降低,应用两个浓度的CPDT提前干预后可以阻止这种降低,并使GSH水平升高3-4倍。
THA引起细胞内Ca~(2+)水平升高,提前48小时应用CPDT和D3T后,纠正了细胞内Ca~(2+)水平的升高。但THA组和CPDT提前组均无P75NTR蛋白的表达。
结论: ARE活化剂CPDT、D3T对运动神经元的保护作用,伴随着提高细胞内GSH水平和降低细胞内Ca~(2+)。该模型中不存在对P75NTR依赖的凋亡,也未发现CPDT对该途径的影响。
第三部分慢性选择性运动神经元损伤模型中GCL的基因表达及ARE活化剂对其影响
目的:验证在体外培养的脊髓薄片中,能否如在其它组织和器官中一样, GCLC、GCLM基因作为ARE的靶基因被上调。进一步验证慢性选择性运动神经元损伤模型中是否有该目的基因的异常,以及ARE活化剂能否阻止这种异常的改变。
方法:取出生后8天乳鼠的腰段脊髓组织切片做脊髓器官型培养,培养7天后,同第一部分,分别加入不同浓度CPDT。培养4周时取出。采用TRIZOL一步法从培养的脊髓组织中提取总RNA。应用RT-PCR法检测两个目的基因GCLC和GCLM以β-actin基因作为对照。
结果:30μmol/L CPDT提前48h干预组GCLCmRNA表达明显高于正常对照组(MS组),15μmol/L CPDT提前48h干预组与MS组比较略高于MS组,但无统计学意义。但在干预48小时各组GCLMmRNA的表达与MS在比较无统计学意义。
在培养4周的模型中,两个浓度的CPDT提前干预48小时,然后再同时加入THA。GCLCmRNA的表达在两个浓度的CPDT提前组均明显高于MS组和THA组。MS组和THA组之间无差异。而GCLMmRNA的表达,仅THA组明显低于MS组,而15、30μmol/L CPDT提前干预对其无影响。
结论: ARE活化剂CPDT可以升高体外培养的脊髓器官型模型中GCLC的基因水平,而对GCLM基因无影响。在THA造成的模型中存在GCLM基因表达的降低。与其下游的GSH的含量的改变相一致。
第四部分应用ARE活化剂对慢性选择性运动神经元损伤模型中GSTM的影响
目的:检测与GSH相关的酶, ARE活化剂的靶基因之一GSTM的蛋白和基因水平在该模型中的的表达。进一步验证,ARE活化剂在对运动神经元保护作用的同时是否伴随GSTM的变化。
方法:取出生后8天乳鼠的腰段脊髓组织切片做脊髓器官型培养,培养7天后,同第一部分,分别加入不同浓度的ARE活化剂:CPDT、tBHQ。继续培养48小时或继续培养3周后取出。提取组织总蛋白和总RNA。应用RT-PCR和Western blot分别检测各组组织中GSTM的基因和蛋白水平。
结果:在ARE活化剂CPDT和tBHQ干预48小时可以刺激体外培养的脊髓片模型中GSTM蛋白水平的升高。在培养4周的模型中,CPDT干预后GSTM的蛋白和基因水平均比正常对照组增高。THA组也较正常对照组增高。
结论:作为ARE的靶基因之一,GSTM可以在ARE活化剂的刺激下,在体外培养的脊髓片模型中表达升高。伴随着运动神经元的保护作用,这种升高可以持续4周。
Amyotrophic lateral sclerosis (ALS) is a common progressive neurodegenerative disease in central nervous system. It is characterized by selective degeneration of motor neurons in brain ,brain stem and spinal cord, which clinical features are delayed onset, chronic progression, weakness of limbs’muscles. Most patients died of respiratory failure 3-5 years later. ALS can be divided into familial amyotrophic lateral sclerosis (FALS) and sporadic amyotrophic lateral sclerosis (SALS). FALS is less than 10% of the total ALS patients, while SALS is more than 90%.
FALS is mainly caused by the mutation of copper/zinc superoxide dismutase (Cu/Zn SOD) gene, whose site is at 21q22.1～22.2. More than 100 different mutations of the SOD1 gene have been linked to familial ALS. For many of these mutations, SOD1 enzyme activity is actually normal or elevated, and SOD1 knockout mice have no disease phenotype. These findings indicate that SOD1-associated ALS is not caused by a loss of function, but rather a toxic gain of function.
Several mechanisms are implicated in the pathogenesis of motor neuron degeneration, including excitotoxicity, immune activation, mitochondrial dysfunction and oxidative stress, protein aggregation, altered proteosomal function and apoptosis. Although disturbances in each of these pathways may contribute to amplification or even initiation of motor neuron injury, the temporal relation of these pathways and their primacy in dictating disease onset and progression are unclear. These mechanisms are not mutually exclusive but are activated as a communal response that may be coordinated by oxidative stress. Increased oxidative stress appears to be an early and sustained event in association with motor neuron death in ALS, although the specific mechanism leading to oxidative damage on motor neurons remains to be defined. Oxidative stress has a prominent role in the initiation of ALS and is capable of activating pathways that elicit additional oxidative stress and propagate disease.
GSH is the most abundant and effective scavenger against ROS directly in mammalian cells. In addition, GSH is also a key substrate for antioxidant enzymes that detoxify hydrogen peroxide and lipid peroxide products. CHI.L和KE.Y show that depletion of reduced glutathione enhances motor neuron degeneration in vitro and vivo.
The synthesis of GSH involves the actions of two ATP-dependent enzymes,γ-glutamylcysteine ligase (GCL) and GSH synthetase. GCL, the rate-controlling enzyme in the overall pathway, is a heterodimer composed of a catalytic (GCLC) and a modulatory (GCLM).There is a down-regulation of GCLM gene expression in rat CNS tissue during aging, accompany by reduced activity of GCL and GSH level. This means, GCL and GSH play an important role in age-related neuron degeneration disease.
Glutathione S-transferase (GST) is a multigene family of more than 100 isoenzymes that catalyze the conjugation of GSH to a variety of electrophilic compounds。Glutathione transferases (GSTs; EC, 2.5.1.18) are phase II enzymes of defense that catalyze the conjugation of reduced glutathione to a wide range of electrophiles, carcinogens and other xenobiotics with genotoxic and cytotoxic activities。
GSTs play an important role in protecting neurons against oxidative stress damage. Many study suggest induction of endogenous phase II enzymes,e g,GST may be a strategy for PD and AD.
Based on the chronic excitotoxicity pathogenesis, incubation of organotypic spinal cord cultures in presence of threo-hydroxyaspartate (THA), the inhibitor of astrocyte glutamate transporter, causes death of motor neurons. We established the model of chronic motor neuron degeneration.
Isummary, the organotypic spinal cord model of chronic motor neuron degeneration promises preclinical feasibility testing of potential neuroprotectants with enhanced relevance for clinical trials in human motor neuron disease. This model has already successfully predicted the efficacy of motor neuron protection by riluzole and neurontin, which are being used for human motor neuron disease. In this model, we want to study neuroprotective effects by inducing endogenous antioxidant agents.
The intraneuronal accumulation of reactive oxygen species has been implicated in the pathogenesis of many neurodegenerative diseases .The ability of a cell to neutralize reactive intermediates is, in part, dependent on the activation of a cis-acting regulatory element termed the antioxidant response element (ARE). The ARE is located in the 5'-flanking region of many genes essential for both detoxification and antioxidant proteins. In many tissues of human, rat, mice and primary culture neurons and astrocytes, there are many ARE-drived gene including DCLC, GCLM and GSTs.
The organotypic spinal cord model of chronic motor neuron degeneration were used to study the effect of ARE activator on motor neuron cell death and mechanism. The first part: we show that ARE activator completely inhibit glutamate-induced motor neuron death in these explants. The second part: we show that ARE activator inhibits glutamate-induced intracellular Ca~(2+) rise and decrease of tissue glutathione, but don’t affect P75NTR pathway.
The third part: the rate-controlling enzyme of GSH synthesis, GCL is abnormal in the model, but ARE activator correct the abnormal, which is corresponding to GSH alteration. The forth part: we investigate GSH related enzyme-GSTM protein and mRNA level in the model, ARE activator can change its level, which is accompanied by motor neuron protection.
PartⅠARE activator protects motor neuron against glutamate excitotoxity-induced motor neuron death in organotypic spinal cord model of chronic motor neuron degeneration ,
Objective: In organotypic spinal cord model of chronic motor neuron degeneration , to investigate if ARE activator could protect motor neurons against glutamate excitotoxicity.We use three ARE activator :CPDT ,D3T and tBHQ at different concentration,at different time points. To explore new neuroprotective treatment.
Methods: Organotypic spinal cord cultures were prepared using lumbar spinal cord slices from 8-day-old rat. Various concentrations of CPDT ,D3T and tBHQ(5μmol/L、15μmol/L、30μmol/L) were continuously added into the culture medium after cultured seven days. Spinal cord slices were treated with THA, a combination of THA and ARE activator , or treatment with the ARE activator without THA for 48 hours .Then we use monoclonal antibody SMI-32, a nonphosphorylated neurofilament marker, immunohistochemistry staining compared with different group.
Results: The results showed that the spinal cord explants in control group could maintain excellent organotypic cellular organization and a stable population of ventralα-motor neurons. THA could produce a slow loss ofα-motor neurons. The complete neuron protection was achieved when the explants were pretreated by ARE activator: CPDT, D3T, tBHQ for 48 hour prior to initiating the combination treatment. Whereas ARE activator was only able to offer very limited neuron protection against THA-induced motor neuron death when the two agents were always added together to the culture medium,and same as 5μmol/L ARE activator.
Conclusions: It is possibility that ARE activator may block glutamate toxicity to protects motor neuron against glutamate excitotoxity-induced motor neuron death. This subject could provide an effective studying pathogenesis and neuroprotection of ALS.
PartⅡTissue glutathione (GSH) contents, intracellular Ca2+ levels and P75NTR protein in organotypic spinal cord model of chronic motor neuron degeneration and alternation by ARE activator
Objective: To study if CPDT stimulates GSH content in rat spinal cord explants Proctention motor neuron survival in CPDT-treated spinal cord explants is accompanied by GSH, intracellular Ca2+ levels and P75NTR protein alteration.
Methods: Organotypic spinal cord cultures were prepared using lumbar spinal cord slices from 8-day-old rat. Various concentrations of CPDT, D3T were continuously added into the culture medium after cultured seven days. GSH in tissue homogenates was derivatized by monochlorobimane, then the derivatives (GSH-monochlorobimane) were measured using a fluorescence plate reader. Measurement of intracellular Ca~(2+) level with 8μM Flou3-AM, then them were immediately analyzed by flow cytometry to determine fluorescence intensity.Tissue P75NTR protein was measured by Western blot.
Results: After on week recover, the spinal cord tissues were treated with 15μM , 30μMCPDT for 48hours, then collected , measured ,GSH level increase only in 30CPDT group, but continue culture for 96 hours,GSH level was increase eather in 15CPDT group or in 30CPDT group.
In the present experiment, the spinal cord tissues were first treated with CPDT at 15 and 30μM for 48 h before combined treatment of CPDT and THA for 3 weeks. We show that THA treatment might deplete tissue GSH. Indeed, 3-week THA treatment markedly reduced GSH level. Significantly, CPDT not only prevented THA-induced GSH depletion but actually elevated tissue GSH level 3-4 fold over the control. We found that CPDT fully prevented both intracellular Ca~(2+) rise.
In the model, we could not find any specific P75NTR protein expression , but we could see it on one day old rat, which is control. After treatment with CPDT, there is no any specific protein expression at 75KD marker can be seen.
Conclusions: CPDT could increase GSH level in spinal cord tissues ,which is time-depented and dose-depented.THA treatment reduced GSH level and CPDT not only prevented that but all so elevated GSH level 3-4 fold .CPDT,D3T fully prevented intracellular Ca~(2+) rise. CPDT have no effection on P75NTR pathway.
PartⅢARE target gene-GCL expression in organotypic spinal cord model of chronic motor neuron degeneration and alternation by ARE activator
Objective: to investigate one of ARE target gene GCL (include two subunit GCLC and GCLM) expression in organotypic spinal cord model of chronic motor neuron degeneration. If their alteration are accompany by motor neuron protection and ARE activitor can prevented THA-induced alteration.
Methods: Various concentrations of CPDT were continuously added into the culture medium after cultured seven days. In the present study, lumber spinal cord explants prepared from 7-day old rats, after one week of culture, were treated with CPDT for 48 h and then harvested for measurement of GCLC, GCLMmRNA.
Thus, explants after one week of culture were exposed to either solvent, THA or THA plus CPDT. In the THA plus CPDT group, the explants were first treated with CPDT for 48 h before the combination treatment, since this treatment schedule allowed CPDT to fully protect motor neurons.
RT-PCR was used to measure the expression of two genes, including GCLCand GCLMalong withβ-actin gene as a control. The Trizol method was used to extract total RNA from the rat spinal cord explants.
Results: At treat for 48hours group, only the rat spinal cord explants treated with 30μmol/LCPDT can increase GCLCmRNA expression, 15μmol/LCPDT could not affect it. On the other hand, two concentration CPDT treatment spinal cord explants show no alteration in GCLMmRNA expression at all.
In the THA plus CPDT group,. In THA group, GCLMmRNA expression is lower than others. However, THA seems to have no effect on GCLC expression. Just as above, the combination treatment of THA with CPDT could not cause increase in GCLMmRNA expression. But this way caused a more significant increase in GCLCmRNA expression.
Conclusions: Hight concentration CPDT (30μM) could cause increase expression of GCLC, but could not affect GCLM. However THA decrease GCLMmRNA expression, but can not affect GCLC.CPDT could increase GCLCmRNA last for 4 weeks, accompany by protection motor neuron.
PartⅣGSTM protein and gene expression in organotypic spinal cord model of chronic motor neuron degeneration and alternation by ARE activator
Objective: to investigate one of ARE target gene GSTM protein and mRNA expression in organotypic spinal cord model of chronic motor neuron degeneration, If their alteration are accompany by motor neuron protection and ARE activator can prevented THA-induced alteration.
Methods: Organotypic spinal cord cultures were prepared using lumbar spinal cord slices from 8-day-old rat. Various concentrations of CPDT were continuously added into the culture medium after cultured seven days. In the present study, lumber spinal cord explants prepared from 7-day old rats, after one week of culture, were treated with CPDT for 48 h and then harvested for measurement of GSTM protein.
Thus, explants after one week of culture were exposed to either solvent, THA or THA plus CPDT. In the THA plus CPDT group, the explants were first treated with CPDT for 48 h before the combination treatment, since this treatment schedule allowed CPDT to fully protect motor neurons.
Prepare whole tissue extracts for Western Blot analysis. The Trizol method was used to extract total RNA from the rat spinal cord explants.
Results: In lumber spinal cord explants treated with CPDT for 48 h shows CPDT and tBHQ CPDT at 15 and 30μM caused significant increase in expression of GSTM protein.
In cultured for 4 weeks group, exposure of the explants to CPDT in the presence of THA lead to increased expression of GSTM in mRNA and protein .Interestingly, THA itself also seems to positively modulate some of the genes, GSTM.
Conclusions: CPDT and tBHQ could stimulates ARE target genes in rat spinal cord explants. Increased motor neuron survival in CPDT-treated spinal cord explants is accompanied by activation of ARE targen gene GSTM expression and protein level.
引文
1. CHI L, KE Y, LUO C. Depletion of reduced glutathione enhances motor neuron degeneration in vitro and vivo. Neuroscience, 2007, 144 :991~1003.
2. Rothstein JD, Jin L, Dykes-Hoberg M, et al. Chronic inhibition of gluta- mine uptake produces a model of slow neurotoxicity. Proc Natl Acad Sci USA, 1993, 90:6591~6595.
3. Westerlaak W M, Joosten EAJ, Gribnau A M, et al. Chronic mito- chondrial inhibition induces glutamate-mediated cortico motoneuron death in an organotypic culture model. Exp Neurol , 2001, 167:393~400.
4. Yew DT, Luo CB, Heizmann CW, et al. Differential expression of calretinin, calbindin D28K and parvalbumin in the developing human cerebellum. Brain Res Dev Brain Res, 1997, 103:37~45.
5. Abe K, Pan L-H, Watanabe M, Kato T, et al , Induction of nitrotyrosine-like immunoreactivity in the lower motor neuron of amyotrophic lateral sclerosis. Neurosci Lett , 1995,199 :152~154.
6. Beal MF, Ferrante RJ, Browne SE, et al. Increased 3-nitrotyrosine in both sporadic and familial amyotrophic lateral sclerosis, Ann Neurol , 1997,42 :644~654.
7. Bowling AC, Schulz JB, Brown RH, et al. Superoxide dismutase activity, oxidative damage and mitochondrial energy metabolism in familial and sporadic amyotrophic lateralsclerosis. J Neurochem, 1993, 64:2366~2369.
8. Ferrante RJ, Browne SE, Shinobu LA, et al Evidence of increased oxidative damage in both sporadic and familial amyotrophic lateral sclerosis. J. Neurochem, 1997, 9 :2064~2074.
9. Couratier P, Hugon J, Sindou P, et al. Cell culture evidence for neuronal degeneration in anyotrofic lateral sclerosis being linked to glutamate AMPA/kainite receptors. Lancet, 1993, 341:265~268.
1. CHI L, KE Y, LUO C. Depletion of reduced glutathione enhances motor neuron degeneration in vitro and vivo. Neuroscience, 2007, 144 :991~1003.
2. Rothstein JD, Jin L, Dykes-Hoberg M, et al. Chronic inhibition of gluta- mine uptake produces a model of slow neurotoxicity. Proc Natl Acad Sci USA, 1993, 90:6591~6595.
3. Westerlaak W M, Joosten EAJ, Gribnau A M, et al. Chronic mito- chondrial inhibition induces glutamate-mediated cortico motoneuron death in an organotypic culture model. Exp Neurol , 2001, 167:393~400.
4. Yew DT, Luo CB, Heizmann CW, et al. Differential expression of calretinin, calbindin D28K and parvalbumin in the developing human cerebellum. Brain Res Dev Brain Res, 1997, 103:37~45.
5. Abe K, Pan L-H, Watanabe M, Kato T, et al , Induction of nitrotyrosine-like immunoreactivity in the lower motor neuron of amyotrophic lateral sclerosis. Neurosci Lett , 1995,199 :152~154.
6. Beal MF, Ferrante RJ, Browne SE, et al. Increased 3-nitrotyrosine in both sporadic and familial amyotrophic lateral sclerosis, Ann Neurol , 1997,42 :644~654.
7. Bowling AC, Schulz JB, Brown RH, et al. Superoxide dismutase activity, oxidative damage and mitochondrial energy metabolism in familial and sporadic amyotrophic lateralsclerosis. J Neurochem, 1993, 64:2366~2369.
8. Ferrante RJ, Browne SE, Shinobu LA, et al Evidence of increased oxidative damage in both sporadic and familial amyotrophic lateral sclerosis. J. Neurochem, 1997, 9 :2064~2074.
9. Couratier P, Hugon J, Sindou P, et al. Cell culture evidence for neuronal degeneration in anyotrofic lateral sclerosis being linked to glutamate AMPA/kainite receptors. Lancet, 1993, 341:265~268.element. Activation by oxidative stress and identification of the DNA consensus sequence required for functional acticity. J Biol Chem, 1991, 266:11632~11639.
22. Moi P, Chan K, Asunis I, et al. Isolation of NF-E2-related factor 2(Nrf2), a NF-E2-like basic leucine zipper transcripyional activator that binfs to the tandem NF-E2/AP1 repeat of theβ-globin locus control region . Proc Natl Acad ci USA, 1994, 91:9926~9930.
23. Andy YS, Delinda AJ, Gloria W, et al. Coordinate regulation of glutathione biosynthesis and release by Nrf2-expression Glia potently protects Neurones from Oxidative Stress. J. Neurosci, 2003, 23:3394~3406.
24. Egner PA, Kensler TW. Regulation of phrase 2 enzymeinduction by oltipraz and other dithiolethione. Carcinogenesis, 1994, 15:177~181.
25. Munday R, Zhang Y, Munday CM, et al. Structure-activity relationships in the induction of Phase II enzymes by derivatives of 3H-1,2-dithiole-3-thione in rats. Chem Biol Interact, 2006, 160:115~122.
26. Zhuo XC, Seema H, Harry Z, et al. Induction of endogenous glutathione by the chemoprotective agent, 3H-1,2-dithiole-3-thione, in human neuroblastoma SH-SY5Y cells protection against peroxynitrite- induced cytotoxicity. Biochem and Biophys Res Commun, 2004, 316:1043~1049.
27. Ommen B, Koster A, Verhagen H, et al. The glutathione conjugates of tert-butylhydroquinone as potent redox cycling agents and possible reactive agents underlying the toxicity of butylated hydroxyanisole, Biochem Biophys Res Commun, 1992, 189: 309~314.
1. Kamenicic H, Lyon A, Paterson PG, et al. Monochlorobimane fluorometric method to measure tissue glutathione. Anal Biochem, 2000, 286:35~37.
2. Rothstein JD, Jin L, Dykes-Hoberg M, et al. Chronic inhibition of gluta- mine uptake produces a model of slow neurotoxicity. Proc Natl Acad Sci USA, 1993, 90:6591~6595.
3. Hissin PJ, Hilf R A. Fuorometric method for determination of oxidized and reduced glutathione in tissues. Anal Biochem, 1976, 74:214~26.
4. Floreani M, Petrone M, Debetto P, et al. A comparison between different methods for the determination of reduced and oxidized glutathione in mammalian tissues. Free Radic Res, 1997 , 26:449~55.
5. Zhang L, Li XR, Xu H , et al. Detection of intracelluar calcium ion of
6. lymphocytes of type Ⅱdiabetes patients and the effect of medicament on the concentration of intracellular calcium ion. J Chin Pharm Univ, 1999, 30:451 ~455.
7. Fuller CL , Braciale VL. Selective induction of CD8 + cytotoxic T
8. lymphocyte effector function by Staphylococcus Enterotoxin B. J Immunol, 1998, 161:5179 ~ 5186.
9. Merritt JE, McCarthy SA, Davies MP, et al. Use of fluo-3 to measure cytosolic Ca2+ in platelets and neutrophils. Loading cells with the dye,calibration of traces, measurements in the presence of plasma, and buffering of cytosolic Ca2+. Biochem J, 1990, 269:513~519.
10. Lanius RA, Krieger C, Wagey, R, et al. Increased glutathione binding sites in spinal cords from patients with sporadic amyotrophic lateral sclerosis. Neurosci. Lett. 1993,163: 89~92.
11. Moumen R, Nouvelot A, Duval D, et al. Plasma superoxide dismutase and glutathione peroxidase activity in sporadic amyotrophic lateral sclerosis. J. Neurol. Sci., 1997, 151:35~39.
12. Lee M, Hyun D, Jenner P, et al. Effect of overexpression of wild-type and mutant Cu/Zn-superoxide dismutases on oxidative damage and antioxidant defences: relevance to Down’s syndrome and familial amyotrophic lateral sclerosis. J Neurochem, 2001,76: 957~965.
13. Bains JS, Shaw CA, Neurodegenerative disorders in humans: The role of glutathione in oxidative stress-mediated neuronal death. Brain Res Brain Res Rev,1997,25:335~358.
14. Grima G, Benz B, Parpura V, et al. Dopamineinduced oxidative stress in neurons with glutathione deficit: implication for schizophrenia. Schizophr Res, 2003,62:213~224.
15. Janaky R, Varga V, Saransaari P. Glutathione modulates the -methyl-D-aspartate receptor-activated calcium influx into cultured rat cerebellar granule cells. Neurosci Lett, 1993, 156: 153~157.
16. Chi L, Ke AY, Luo BC, et al. Deleption of reduced glutathione enhanced motor neuron degeneration in vitro and vivo. Neuroscience, 2007, 144: 991~1003.
17. Carriedo SG, Yin HZ, Weiss JH, et al. Motor neurons are selectively vulnerable to AMPA/Kainate receptor~mediated injury in vitro.J Neurosci, 1996, 16: 4069~4079.
18. Mattson MP, Mark RJ. Excitotoxicity and excitoprotection in vitro. Adv Neurol, 1996, 71:1~37.
19. Saroff D, Delfs J, Kuznetsov D, et al. Selective vulnerability of spinal cord motor neurons to non-NMDA toxicity. Neuroreport, 2000, 11:1117~1121.
20. Shaw PJ, Williams TL, Slade JY, et al. Low expression of GluR2 AMPA receptor subunit protein by human motor neurons. Neuroreport, 1999, 10:261~265.
21. Lee J M, Shih A Y, Murphy TH, et al. NF-E2-related Factor-2 Mediates Neuroprotection against Mitochondrial Complex I Inhibitors and Increased Concentrations of Intracellular Calcium in Primary Cortical Neurons. J Bio chem, 2003:37948~37956.
22. Guegan C, Przedborski S. Programmed cell death in amyotrophic lateral sclerosis. J Clin Invest, 2003, 111:153~161.
23. Seeburger J L, Tarras S. Spinal cord motoneurons express p75NGFR and p145 trk B mRNA in amyotropjic lateral sclerosis. Brain Res, 1993, 621: 111~115.
24. Lowry K S, Murray S S, Mclean C A, et al. A pontential role for the p75 low-affinity neurotrophin receptor in spinal motor neuron degeneration in murine and human amyotrophic lateral sclerosis. Amyotroph Lateral Scler Other Motor Neuron Disord, 2005, 2:127~134.
25. Marcelo RV, Mariana P, Patricia C, et al. Increased glutathione biosynthesis by Nrf2 activation in astrocytes prevents p75NTR-dependent motor neuron apoptosis. J Neurochem, 2006, 97:687~693.
1. Bains JS, Shaw CA , Neurodegenerative disorders in humans: the role of glutathione in oxidative stress-mediated neuronal death,Brain Res Brain Res Rev, 1997,25:335~358.
2. Grima G, Benz B, Parpura V, et al. Dopamineinduced oxidative stress in neurons with glutathione deficit: implication for schizophrenia. Schizophr Res,2003,62:213~224.
3. Rothstein JD, Jin L, Dykes-Hoberg M, et al. Chronic inhibition of gluta-mine uptake produces a model of slow neurotoxicity. Proc Natl Acad Sci USA, 1993, 90:6591~6595.
4. Liu RM, Hu H, Robison TW ,Dfferential enhancement of gamma-glutamyl traspeptidase and gamma-glutamylcysteine synthetase by tert-butylhydroquinone in rat lung epithelial L2 cells. Am .J Respir Cell Mol Biol. 1996, 14:186~191.
5. Zipper LM, Mulcahy RT, Erk Activation Is Required for Nrf2 Nuclear Localization during Pyrrolidine Dithiocarbamate Induction of Glutamate Cysteine Ligase Modulatory Gene Expression in HepG2 Cells. Toxicological Sciences,2003, 73:124~134.
6. Liu RM, Down-regulation of gamma-glutamylcysteine synthetase regulatory subunit gene expression in rat brain tissue during aging. JNeurosci Res, 2002, 68 : 344~351.
7. Shi MM, Iwamoto T, Forman HJ. Gamma-Glutamylcysteine synthetase and GSH increase in quinone-induced oxidative stress in BPAEC. Am J Physiol, 1994 ,267: 414~21.
8. Borroz KI, Buetler TM, Eaton DL,Modulation of gamma-glutamylcysteine synthetase large subunit mRNA expression by butylated hydroxyanisole. , 1994 126:150~155.
9. Woods JS, Davis HA, Baer RP, Enhancement of gamma-glutamylcysteine synthetase mRNA in rat kidney by methyl mercury.Arch Biochem Bio,1992 ,296:350~3.
10. Chinta SJ, Andersen JK. Reversible inhibition of mitochondrial complex I activity following chronic dopaminergic glutathione depletion in vitro: implications for Parkinson's disease. Free Radic Biol Med. 2006, 41: 1442~1448.
11. Okouchi M, Okayama N, Alexander JS, et al. NRF2-dependent glutamate-L-cysteine ligase catalytic subunit expression mediates insulin protection against hyperglycemia- induced brain endothelial cell apoptosis. Curr Neurovasc Res ,2006 3:249~61.
12. Erickson A M, Nevarea Z, Gipp JJ, Identification of a Variant Antioxidant Response Element in the Promoter of the Human Glutamate-Cysteine Ligase Modifier Subunit Gene. Biol. Chem., 2002, 34:30730~30737.
13. Liu R M, Gao L, Choi J.γ-Glutamylcysteine synthetase: mRNA stabilization and independent subunit transcription by4-hydroxy-2-nonenal. Am J Physiol Lung Cell Mol Physiol,1998,275:861~869.
14. Suh J H, Shenvi S V, Dixon B M. Decline in transcriptional activity of Nrf2 causes age-related loss of glutathione synthesis, which is reversible with lipoic acid,Natl. Acad. Sci ,2004, 101: 3381~3386.
1. Mulcahy RT and Gipp JJ. Indetification of putative antioxidant response element in 5’-flanking region of the human gamma-glutamylcystine synthetase heavy subunit gene ,Biochem Biophy Res Commun ,1995, 209:227~233.
2. Jaiswal AK, Antioxidant response element. Bio Phama,1994.48:439~444.
3. Rushmore T.H. and Pickett C.B. Transcripional regulation of rat glutathione S-transferase Ya subuint gene .Characterization of a xenobiotic-responsive element controlling inducible expression by phenolic antioxidant ,J .Bio Chem ,1990, 265:14648~14653.
4. Rothstein JD, Jin L, Dykes-Hoberg M, et al. Chronic inhibition of gluta- mine uptake produces a model of slow neurotoxicity. Proc Natl Acad Sci USA. 1993, 90:6591~6595.
5. Mannervik B, Awasthi YC, board PG, et al. Nomenclature for human glutathione transferases. Biochem. J. 1992, 282: 305~306.
6. Tsuchida S, Sato K. Glutathione transferases and cancer. Crit Rev. Biochem. Mol. Biol. 1992, 27: 337~384.
7. Scarpato R, Hirvonen A, Migliore L, et al. Influence of GSTM 1 and GSTTI polymorphisms on the frequency of chromosome aberrations in lymphocytes of smokers and pesticide exposed greenhouse workers,[J]. Mutat Res. 1997, 389: 27~235.
8. Sofia B, Juan S AR et al Gutathione transferase catalyze t he detxication of oxidized metabolities of catechlamines and may serve as an antioxidant system preventiong degenerative cellular processes. Bioch. J. 1997, 324:25~28.
9. 9Wikinson J and Clapper M L, Detoxification enzymes and chemoprevention. Proc. Soc. Exp. Biol Med. 1997, 216:192~200.
10. Drukarch B and Muiswinkel FL, Drug treatment of Parkinson’s disease. Time for phrase Ⅱ .Bioch Pharmarol,2000, 59:1023~1031.
11. Usarek E, Gajewska B, Kazmierczak B, et alA study of glutathione S-transferase pi expression in central nervous system of subjects with amyotrophic lateral sclerosis using RNA extraction from formalin-fixed, paraffin-embedded material, Neurochem Res. 2005, 30(8):1003~1007.
12. Allen S, Heath PR, Kirby J,et al, Analysis of the cytosolic proteome in a cell culture model of familial amyotrophic lateral sclerosis reveals alterations to the proteasome, antioxidant defenses, and nitric oxide synthetic pathways. J Biol Chem. 2003, 278(8):6371~6383.
13. Kuzma M, Jamrozik Z, Baranczyk-Kuzma A, Activity and expression of glutathione S-transferase pi in patients with amyotrophic lateral sclerosis. Clin Chim Acta. 2006, 364:217~220.
14. Murphy T.H., J.Yu, Preferential expression of antioxidant responsive element mediate gene expression in astrocytes. J Neurochemistry, 2001,76: 1670~1678.
15. Jong-Min L, Andy Y. Sand Jeffrey A. J, NF-E2-related Factor-2 Mediates Neuroprotection against Mitochondrial Complex I Inhibitors and Increased Concentrations of Intracellular Calcium in Primary Cortical Neurons, J BIO Chem. 2003, 278: 37948~37956.
16. Dinkova-Kostova AT, Holtzcaw WD, Kensler TW,The role of Keap1 in cellular protective response.,Chem Res Toxicol. 2005, 18:1779~1791.
1 Dringen R, Gutterer JM,Hirrlinger J. Glutathione metabolism in brain. Eur. J. Biochem., 2000, 267, 4912~4916.
2 Gerlach M, Ben-Shachar D, Riederer P, et al. Altered brain metabolism of iron as a cause of neurodegenerative diseases? J. Neurochem., 1994, 63: 793~807.
3 Cooper, A.J.L. Glutathione in the brain: disorders of glutathione metabolism. In The Molecular and Genetic Basis of Neurological Disease (Rosenberg, RN, Prusiner, SB, DiMauro, S, Barchi, RL & Kunk, LM, eds),1997, pp. 1195~1230.
4 Chinta SJ,Rajagopalaw S,Butterfield DA,etal In vitro and in vivo neuroprotection by gamma-glutamylcysteine ethyl ester against MPTP: relevance to the role of glutathione in Parkinson's disease.Neurosci Lett. 2006 , 402:137~41.
5 Bartov O, Sultana R, Butterfield DA, et al, Low molecular weight thiol amides attenuate MAPK activity and protect primary neurons from Abeta(1-42) toxicity,Brain Res. 2006 ,19,:198~206.
6 Bharath S, Andersen JK,Glutathione depletion in a midbrain-derived immortalized dopaminergic cell line results in limited tyrosine nitration of mitochondrial complex I subunits: implications for Parkinson's disease,Antioxid Redox Signal,2005 ,7:900~910.
7 Ericka PS, Albert AY, Stanley HA. Oxidative Stress: a common denominator in the pathogenesis of amyotrophic lateral sclerosis. Curr Opin Rheumatol, 2003, 15:730~736.
8 Oteiza PI, Uchitel OD, Carrasquedo F, et al. Evaluation of antioxidants, protein, and lipid oxidation products in blood from sporadic amyotrophic lateral sclerosis patients. Neurochem Res., 1997, 22:535~539.
9 Beal MF, Ferrante RJ, Browne SE, et al. Increased 3-nitrotyrosine in both sporadic and familial amyotrophic lateral sclerosis. Ann. Neurol., 1997, 42: 644~654.
10 Bruijn LI, Beal MF, Becher MW, et al. Elevated free nitrotyrosine levels, but not protein-bound nitrotyrosine or hydroxyl radicals, throughoutamyotrophic lateral sclerosis (ALS)-like disease implicate tyrosinenitration as an aberrant in vivo property of one familial ALS-linkedsuperoxide dismutase 1 mutant. Proc. Natl Acad. Sci. USA., 1997,94: 7606~7611.
11 Tohgi H, Abe T, Yamazaki K, et al. Remarkable increase in cerebrospinal fluid 3-nitrotyrosine in patients with sporadic amyotrophic lateral sclerosis. Ann. Neurol., 1999, 46:129~131.
12 Pedersen WA, Fu W, Keller JN, et al. Protein modification by the lipidperoxidation product 4-hydroxynonenal in the spinal cords of amyotrophic lateral sclerosis patients. Ann. Neurol., 1998, 44: 819~824.
13 Smith RG, Henry YK, Mattson MP, et al. Presence of 4-hydroxynonenal in cerebrospinal fluid of patients with sporadic amyotrophic lateral sclerosis. Ann. Neurol., 1998, 44:696~699.
14 Heath PR, Shaw PJ. Update on the glutamatergic neurotransmitter system and the role of excitotoxicity in amyotrophic lateral sclerosis. Muscle Nerve, 2002, 26:438~458.
15 Kawahara Y, Kwak S, Sun H, et al. Human spinal motoneurons express low relative abundance of GluR2 mRNA: an implication for excitotoxicity in ALS. J Neurochem, 2003, 85:680～689.
16 Takuma H, Kwak S, Yoshizawa T, et al. Reduction of GluR2 RNA editing, a molecular change that increases calcium influx through AMPA receptors, selective in the spinal ventral gray of patients with amyotrophic lateral sclerosis. Ann Neurol., 1999, 46:806~815.
17 Murphy T H,Miyamoto M,Sastre A,et al,Glutamate toxicity in a neuronal cell line involves inhibition of cystine transport leading to oxidative stress,Neuron,1989,2:1547~1558.
18 Wyatt I,Gyte A,Simpson M Get al,The role of glutathione in L-2-chloropropionic acid-induced cerebellar granule cell necrosis in the rat,Arch.Toxicol,1996,70:724~735.
19 Spina M B, and Cohen G, Dopamine turnover and glutathione oxidation: Implications for Parkinson’disease.Proc.Natl.Acad.Sci.U S A,1989,86:1398~1400.
20 Winuta Y,Kikuchi H,Ishikawa M,et al Lipid peroxidation in focial cerebral ischemia,J.Neurosug,1989,71:421~429.
21 Makar T K,Nedergaard M,Preuss A S,Perumal A S,et al Vitamin E,ascorbate,glutathione,glutathione disulfide, and enzymes of glutathione metabolism in cultures of chick astrocytes:Evidence that astrocytes play an important role in antioxidative processes in the brain,J.Neurochem,1994,62:45~53.
22 Lowndes H E,Philbert M A,Beiswanger C M,et al Xenobiotic metabolism in the brain as mechanistic bases for neurotoxicity,In handbook of Neurotoxicology(L W Chang and R S Dyer,Eds),pp 1~27.
23 Philber M A,Beiswanger C M,Water D K,et al,Cellular and regional distribution of reduced glutathione in the nervous system of tha rat:Histochemical localization by mercury orange and 0-phthaldialdehyde-inducedhistofluorescence,Toxicol.Appl.Pharm ,1991,107:215~227.
24 Abramovitz M,Homma H,Ishigaki S,et alCharacterization and localization of glutathione –S-transferases in rat brain and binding of hormones,neurotransmitter,and drugs,J.Neurochem,1988,50:50~57.
25 Cammer W,Tansey F,Abramovitz M, et al,Differential localization of glutathione S-transferases Yp and Yb subunits in oligodendrocytes and astrocytes of brain. J. .Neurochem, 1989, 53:876~883.
26 Johnson J A,Barbary A,Kornguth S E, et al,Glutathione S-transferase isoenzymes in rat brain neurons and glia,J Neurosic,1993,13:2013~2023.
27 PhilbertM A,BeiswangerCM,Manson M M,et al,Glutathione S-transferases and γ-glutamyl transpeptidase in rat nervous system:A basis for differential susceptibility to neurotoxicants, Neurotoxicology, 1995,16:349~362.
28 Dringen R, Hamprecht B,Glutathione content as an indicator for the presence of metabolic pathways of amino acid in astroglial cultures,1996,J.Neurochem,67:1375~1382.
29 Leslie S W,Brown L M,Trent,R D,et al,Stimulation of N-methyl-D-aspartate receptor-mediated calcium entry into dissociated neurons by reduced and oxidized glutathione,Mol.Pharmacol,1992,41:308~314.
30 Sagara J,Miura K,and Bannai S,maintenance of neuronal glutathione by glial cells,1993,J.Neurochem,61:1672~1676.
31 Ogita K,Ogaw Y,and Yoneda Y ,Apparent binding activity of [3H]-glutathione in rat central and peripheraltissues.Neurochem,Int,1988.13;493~497.
32 Liu Y F,and Quirion R,Modulatory role of glutathione on μ-opioid,subatant P/neurokinin-1,and kainic acid receptor binding sites,J. Neurochem,1992,59:1024~1032.
33 Bains JS, Shaw CA. Neurodegenerative disorders in humans: the role of glutathione in oxidative stress-mediated neuronal death. Brain Res Rev, 1997,25:335~358.
34 Grima G, Benz B, Parpura V, et al. Dopamineinduced oxidative stress in neurons with glutathione deficit: implication for schizophrenia. Schizophr Res., 2003, 62:213～224.
35 Janaky R, Varga V, Saransaari P, et al. Glutathione modulates the N-methyl-D-aspartate receptor-activated calcium influx into cultured rat cerebellar granule cells. Neurosci Lett., 1993, 156:153~157.
36 Dringen, R., Pfeiffer, B. & Hamprecht, B.,Synthesis of the antioxidant glutathione in neurons: supply by astrocytes of CysGly as precursor for neuronal glutathione. J. Neurosci,1999,19: 562~569.
37 Holopainen, I. & Kontro, P. Uptake and release of glycine in cerebellar granule cells and astrocytes in primary culture: potassiumstimulated release from granule cells is calcium-dependent. J. Neurosci.Res. 1989,24:374~383.
38 Sagara, J., Miura, K. & Bannai, S. Cystine uptake and glutathione level in fetal brain cells in primary culture and in suspension. J. Neurochem. 1993 ,61:1667~1671.
39 Monks MJ, Chersi-Egea JF, Philbert M, et al. Symposium overview: the role of glutathione in neuroprotection and neurotoxicity. Toxicol sci, 1999, 51:161~177.
40Desagher S, Glowinski, and PremontJ, Astrocytes protect neurons from hydrogen peroxide toxicity. J. Neurosci. 1996,16, 2553~2562.
41Drukarch, B., Schepens, E., Stoof, J.C,et al, Astrocyte-enhanced neuronal survival is mediated by scavenging of extracellular reactive oxygen species.Free Rad. Biol. Med. 1998,25: 217~220.
42Drukarch, B., Schepens, E., Jongenelen, C.A.M., et al, Astrocyte-mediated enhancement of neuronal survival is abolished by glutathione deficiency. Brain Res. 1997, 770:123~130.
43 Bains, J.S. & Shaw, C.A, Neurodegenerative disorders in humans: the role of glutathione in oxidative stress-mediated neuronal death. Brain Res. Rev. 1997, 25: 335~358.
44 Lanius, R.A., Krieger, C., Wagey, R. et al. Increased [35S] glutathione binding sites in spinal cords from patients with sporadic amyotrophic lateral sclerosis. Neurosci. Lett, 1993,163: 89~92.
45Moumen, R, Nouvelot, A , Duval, D, et al, Plasma superoxide dismutase and glutathione peroxidase activity in sporadic amyotrophic lateral sclerosis. J. Neurol. Sci. 1997, 151:35~39.
46Apostolski, S., Marinkovic, Z., Nikolic, A., et al, Glutathione peroxidase in amyotrophic lateral sclerosis: the effects of selenium supplementation.J. Environ. Pathol. Toxicol. Oncol. 1998, 17:325~329.
47 Macho A, Hirsch T, Marzo I, et al. Glutathione depletion is an early and calcium elevation is a late event of thymocyte apoptosis. J. Immunol., 1997, 158:4612~4619.
48Beaver JP, Waring PA. Decrease in intracellular glutathione concentration precedes the onset of apoptosis in murine thymocytes. Eur. J. Cell Biol., 1995, 68:47~54.
49 Chi L, Ke AY, Luo BC, et al. Depletion of Reduced Glutahione Enhances Motor Neuron Degeneration in Vitro and in Vivo. Neuroscience, 2007, 144: 991~1003.
50Li Y, Maher P, Schubert D, A role for 12-lipoxygenase in nerve cell death caused by glutathione depletion. Neuron, 1997, 19:453~463.
51 Yamamoto, M., Sakamoto, N., Iwai, A., et al, Protective actions of YM737, a new glutathione analog, against cerebral ischemia in rats. Res. Commun. Chem. Pathol. Pharmacol. 1993,81: 221~232.
52Henderson, J.T., Javaheri, M., Kopko, S. et al,.Reduction of lower motor neuron degeneration in wobbler mice by N-acetyl-l-cysteine. J. Neurosci.1996, 16:7574~7582.
53Jain.A,Madsen.D.C,Auld.P.A.,etal,l-2-oxothiazolidine-4-carboxylate,a cysteine precursor, stimulates growth and normalizes tissue glutathione concentrations in rats fed a sulfur amino acid-deficient diet. J. Nutr. 1995 ,125: 851~856.
54Cudkowicz, M.E., Sexton, P.M., Ellis, T., et al,The pharmacokinetics and pharmaco-dynamics of procysteine in amyotrophic lateral sclerosis.Neurology 1999,52: 1492~1494.
55Kanai Y, Segawa H, Miyamoto K,et al, Expression cloning and characterization of a transporter for large neutral amino acids activated by the heavy chain of 4F2 antigen, J Biol Chem,1998 ,273:23629~23632 .
56Sato H, Tamba M, Ishii T, et al, Cloning and expression of a plasma membrane cystine/glutamate exchange transporter composed of two distinct proteins. J Biol Chem, 1999,274: 11455~11458.
57Coyle JT, Bird SJ, Evans RH, et al, Excitatory amino acid neurotoxins: selectivity, specificity, and mechanisms of action. Neurosci Res Program Bull, 1981,19:331~427.
58Sagara Y, Schubert D The activation of metabotropic glutamate receptors protects nerve cells from oxidative stress. J Neurosci , 1998,18:6662~6671.
59Murphy TH, Miyamoto M, Sastre A, et al, Glutamate toxicity in a neuronal cell line involves inhibition of cystine transport leading to oxidative stress. Neuron, 1989,2: 1547~1558.
60Trotti D, Danbolt NC, Volterra A, Glutamate transporters are oxidant-vulnerable: a molecular link between oxidative and excitotoxic neurodegeneration? Trends Pharmacol Sci, 1998,19:328~334.
61Mannervi I , Mannervik B, Awasthi YC, et al.Nomenclature for human glutathione transferases. J Biochem, 1992, 282: 305~306.
62 Tsuchida S, Sato K,Glutathione transferases and cancer. Crit. Rev. Biochem. Mol. Biol., 1992, 27: 337～384.
63Scarpato R, Hirvonen A, Migliore L, et al. Influence of GSTM 1 and GSTTI polymorphisms on the frequency of chromosome aberrations inlymphocytes of smokers and pesticide exposed greenhouse workers. Mutat Res,1997, 389:227~235.
64 Sofia BAEZ, Juan, SEGURA-AGUILAR et al. Glutathione transferase catalyse the detxication of oxidized metabolities of catechlamines and may serve as an antioxidant system preventiong degenerative cellular processes. Bioch. J, 1997, 324:25~28.
65Usarek E, Gajewska B, Kazmierczak B, et al. A study of glutathione S-transferase pi expression in central nervous system of subjects with amyotrophic lateral sclerosis using RNA extraction from formalin-fixed, paraffin-embedded material. Neurochem Res., 2005, 30:1003~1007.
66Kuzma M,Jamrozik Z, Baranczyk-Kuzma A. Activity and expression of glutathione S-transferase pi in patients with amyotrophic lateral sclerosis. Clin Chim Acta, 2006, 364:217~221.
67Allen S, Heath PR, Kirby J, et al. Analysis of the cytosolic proteome in a cell culture model of familial amyotrophic lateral sclerosis reveals alterations to the proteasome, antioxidant defenses, and nitric oxide synthetic pathways. J Biol Chem., 2003, 278:6371~6383.
68Venugopal R,Jasiwal AK, Positirely response element—mediated expression of NADH ( P ) : quinone oxidoreductasedgene ,Proc .Natl .Acad.Sci.USA,1996,93:14960~14965.
69 Huang CS, Chang LS, Anderson ME, Meister A.Catalytic and regulatory properties of the heavy subunit of rat kidney gamma-glutamylcysteine synthetase, J Biol Chem.,1993, 268(26):19675~19680.
70Yao K, Godwin AK, Ozols RF, Hamilton TC, O'Dwyer PJVariable baseline gamma-glutamylcysteine synthetase messenger RNA expression in peripheral mononuclear cells of cancer patients, and its induction by buthionine sulfoximine treatment.Cancer Res. 1993 Aug 15;53(16):3662~3666.
71Liu RM, Gao L, Choi J, Forman HJ gamma-glutamylcysteine synthetase: mRNA stabilization and independent subunit transcription by 4-hydroxy-2-nonenal.Am J Physiol. 1998 Nov;275:861~869.72Dickinson DA, Iles KE, Watanabe N, Iwamoto T, Zhang H, Krzywanski DM, Forman HJ4-hydroxynonenal induces glutamate cysteine ligase through JNK in HBE1 cells.Free Radic Biol Med. 2002 Oct 1;33(7):974.
73Shi MM, Kugelman A, Iwamoto T et al,Quinone-induced oxidative stress elevates glutathione and induces gamma-glutamylcysteine synthetase activity in rat lung epithelial L2 cells.J Biol Chem. 1994 Oct 21;269(42):26512~26517.
74Day RM, Suzuki YJ, Lum JM,Bleomycin upregulates expression of gamma-glutamylcysteine synthetase in pulmonary artery endothelial cells.Am J Physiol Lung Cell Mol Physiol. 2002, 282: 1349~1357.
75Morimitsu Y, Nakagawa Y, Hayashi K,A, sulforaphane analogue that potently activates the Nrf2-dependent detoxification pathway, 2002 ,277:3456~63.
76David M. K, Dale A.D, Karen E. I, Variable regulation of glutamate cysteine ligase subunitproteins affects glutathione biosynthesis in response to oxidative stress ,Arch Biochemand Bioph, 2004,423: 116~125.
77Solis WA ,Dalton TP, Dieter MZ, Glutamate-cysteine ligase modifier subunit: mouse Gclm gene structure and regulation by agents that cause oxidative stress,Biochem Pharmacol.,2002, 63(9):1739~54.
78Dinkova-Kostova AT, Holtzclaw WD, Kensler TW, et al,The role of Keap1 in cellular protective responses. Chem Res Toxicol, 2005,18: 1779~1791.
79Allen S, Heath PR, Kirby J, et al. Analysis of the cytosolic proteome in a cell culture model of familial amyotrophic lateral sclerosis reveals alterations to the proteasome, antioxidant defenses, and nitric oxide synthetic pathways. J Biol Chem., 2003, 278:6371~6383.
80Murphy TH,Yu J,Preferential expression of antioxidant responsive element mediate gene expression in astrocytes. J Neurochem, 2001, 76: 1670~1678.
81Lee JM, Andy Y, Shih, et al. NF-E2-related Factor-2 Mediates Neuroprotection against Mitochondrial Complex I Inhibitors and Increased Concentrations of Intracellular Calcium in Primary Cortical Neurons. The Journal of Biological Chemistry, 2003, 278: 37948~37956.
82Dinkova-Kostova AT, Holtzclaw WD, Kensler TW, et al,The role of Keap1 in cellular protective responses. Chem Res Toxicol, 2005,18 :1779~1791.
2. Rothstein JD, Jin L, Dykes-Hoberg M, et al. Chronic inhibition of gluta- mine uptake produces a model of slow neurotoxicity. Proc Natl Acad Sci USA, 1993, 90:6591~6595.
3. Westerlaak W M, Joosten EAJ, Gribnau A M, et al. Chronic mito- chondrial inhibition induces glutamate-mediated cortico motoneuron death in an organotypic culture model. Exp Neurol , 2001, 167:393~400.
4. Yew DT, Luo CB, Heizmann CW, et al. Differential expression of calretinin, calbindin D28K and parvalbumin in the developing human cerebellum. Brain Res Dev Brain Res, 1997, 103:37~45.
5. Abe K, Pan L-H, Watanabe M, Kato T, et al , Induction of nitrotyrosine-like immunoreactivity in the lower motor neuron of amyotrophic lateral sclerosis. Neurosci Lett , 1995,199 :152~154.
6. Beal MF, Ferrante RJ, Browne SE, et al. Increased 3-nitrotyrosine in both sporadic and familial amyotrophic lateral sclerosis, Ann Neurol , 1997,42 :644~654.
7. Bowling AC, Schulz JB, Brown RH, et al. Superoxide dismutase activity, oxidative damage and mitochondrial energy metabolism in familial and sporadic amyotrophic lateralsclerosis. J Neurochem, 1993, 64:2366~2369.
8. Ferrante RJ, Browne SE, Shinobu LA, et al Evidence of increased oxidative damage in both sporadic and familial amyotrophic lateral sclerosis. J. Neurochem, 1997, 9 :2064~2074.
9. Couratier P, Hugon J, Sindou P, et al. Cell culture evidence for neuronal degeneration in anyotrofic lateral sclerosis being linked to glutamate AMPA/kainite receptors. Lancet, 1993, 341:265~268.
1. CHI L, KE Y, LUO C. Depletion of reduced glutathione enhances motor neuron degeneration in vitro and vivo. Neuroscience, 2007, 144 :991~1003.
2. Rothstein JD, Jin L, Dykes-Hoberg M, et al. Chronic inhibition of gluta- mine uptake produces a model of slow neurotoxicity. Proc Natl Acad Sci USA, 1993, 90:6591~6595.
3. Westerlaak W M, Joosten EAJ, Gribnau A M, et al. Chronic mito- chondrial inhibition induces glutamate-mediated cortico motoneuron death in an organotypic culture model. Exp Neurol , 2001, 167:393~400.
4. Yew DT, Luo CB, Heizmann CW, et al. Differential expression of calretinin, calbindin D28K and parvalbumin in the developing human cerebellum. Brain Res Dev Brain Res, 1997, 103:37~45.
5. Abe K, Pan L-H, Watanabe M, Kato T, et al , Induction of nitrotyrosine-like immunoreactivity in the lower motor neuron of amyotrophic lateral sclerosis. Neurosci Lett , 1995,199 :152~154.
6. Beal MF, Ferrante RJ, Browne SE, et al. Increased 3-nitrotyrosine in both sporadic and familial amyotrophic lateral sclerosis, Ann Neurol , 1997,42 :644~654.
7. Bowling AC, Schulz JB, Brown RH, et al. Superoxide dismutase activity, oxidative damage and mitochondrial energy metabolism in familial and sporadic amyotrophic lateralsclerosis. J Neurochem, 1993, 64:2366~2369.
8. Ferrante RJ, Browne SE, Shinobu LA, et al Evidence of increased oxidative damage in both sporadic and familial amyotrophic lateral sclerosis. J. Neurochem, 1997, 9 :2064~2074.
9. Couratier P, Hugon J, Sindou P, et al. Cell culture evidence for neuronal degeneration in anyotrofic lateral sclerosis being linked to glutamate AMPA/kainite receptors. Lancet, 1993, 341:265~268.element. Activation by oxidative stress and identification of the DNA consensus sequence required for functional acticity. J Biol Chem, 1991, 266:11632~11639.
22. Moi P, Chan K, Asunis I, et al. Isolation of NF-E2-related factor 2(Nrf2), a NF-E2-like basic leucine zipper transcripyional activator that binfs to the tandem NF-E2/AP1 repeat of theβ-globin locus control region . Proc Natl Acad ci USA, 1994, 91:9926~9930.
23. Andy YS, Delinda AJ, Gloria W, et al. Coordinate regulation of glutathione biosynthesis and release by Nrf2-expression Glia potently protects Neurones from Oxidative Stress. J. Neurosci, 2003, 23:3394~3406.
24. Egner PA, Kensler TW. Regulation of phrase 2 enzymeinduction by oltipraz and other dithiolethione. Carcinogenesis, 1994, 15:177~181.
25. Munday R, Zhang Y, Munday CM, et al. Structure-activity relationships in the induction of Phase II enzymes by derivatives of 3H-1,2-dithiole-3-thione in rats. Chem Biol Interact, 2006, 160:115~122.
26. Zhuo XC, Seema H, Harry Z, et al. Induction of endogenous glutathione by the chemoprotective agent, 3H-1,2-dithiole-3-thione, in human neuroblastoma SH-SY5Y cells protection against peroxynitrite- induced cytotoxicity. Biochem and Biophys Res Commun, 2004, 316:1043~1049.
27. Ommen B, Koster A, Verhagen H, et al. The glutathione conjugates of tert-butylhydroquinone as potent redox cycling agents and possible reactive agents underlying the toxicity of butylated hydroxyanisole, Biochem Biophys Res Commun, 1992, 189: 309~314.
1. Kamenicic H, Lyon A, Paterson PG, et al. Monochlorobimane fluorometric method to measure tissue glutathione. Anal Biochem, 2000, 286:35~37.
2. Rothstein JD, Jin L, Dykes-Hoberg M, et al. Chronic inhibition of gluta- mine uptake produces a model of slow neurotoxicity. Proc Natl Acad Sci USA, 1993, 90:6591~6595.
3. Hissin PJ, Hilf R A. Fuorometric method for determination of oxidized and reduced glutathione in tissues. Anal Biochem, 1976, 74:214~26.
4. Floreani M, Petrone M, Debetto P, et al. A comparison between different methods for the determination of reduced and oxidized glutathione in mammalian tissues. Free Radic Res, 1997 , 26:449~55.
5. Zhang L, Li XR, Xu H , et al. Detection of intracelluar calcium ion of
6. lymphocytes of type Ⅱdiabetes patients and the effect of medicament on the concentration of intracellular calcium ion. J Chin Pharm Univ, 1999, 30:451 ~455.
7. Fuller CL , Braciale VL. Selective induction of CD8 + cytotoxic T
8. lymphocyte effector function by Staphylococcus Enterotoxin B. J Immunol, 1998, 161:5179 ~ 5186.
9. Merritt JE, McCarthy SA, Davies MP, et al. Use of fluo-3 to measure cytosolic Ca2+ in platelets and neutrophils. Loading cells with the dye,calibration of traces, measurements in the presence of plasma, and buffering of cytosolic Ca2+. Biochem J, 1990, 269:513~519.
10. Lanius RA, Krieger C, Wagey, R, et al. Increased glutathione binding sites in spinal cords from patients with sporadic amyotrophic lateral sclerosis. Neurosci. Lett. 1993,163: 89~92.
11. Moumen R, Nouvelot A, Duval D, et al. Plasma superoxide dismutase and glutathione peroxidase activity in sporadic amyotrophic lateral sclerosis. J. Neurol. Sci., 1997, 151:35~39.
12. Lee M, Hyun D, Jenner P, et al. Effect of overexpression of wild-type and mutant Cu/Zn-superoxide dismutases on oxidative damage and antioxidant defences: relevance to Down’s syndrome and familial amyotrophic lateral sclerosis. J Neurochem, 2001,76: 957~965.
13. Bains JS, Shaw CA, Neurodegenerative disorders in humans: The role of glutathione in oxidative stress-mediated neuronal death. Brain Res Brain Res Rev,1997,25:335~358.
14. Grima G, Benz B, Parpura V, et al. Dopamineinduced oxidative stress in neurons with glutathione deficit: implication for schizophrenia. Schizophr Res, 2003,62:213~224.
15. Janaky R, Varga V, Saransaari P. Glutathione modulates the -methyl-D-aspartate receptor-activated calcium influx into cultured rat cerebellar granule cells. Neurosci Lett, 1993, 156: 153~157.
16. Chi L, Ke AY, Luo BC, et al. Deleption of reduced glutathione enhanced motor neuron degeneration in vitro and vivo. Neuroscience, 2007, 144: 991~1003.
17. Carriedo SG, Yin HZ, Weiss JH, et al. Motor neurons are selectively vulnerable to AMPA/Kainate receptor~mediated injury in vitro.J Neurosci, 1996, 16: 4069~4079.
18. Mattson MP, Mark RJ. Excitotoxicity and excitoprotection in vitro. Adv Neurol, 1996, 71:1~37.
19. Saroff D, Delfs J, Kuznetsov D, et al. Selective vulnerability of spinal cord motor neurons to non-NMDA toxicity. Neuroreport, 2000, 11:1117~1121.
20. Shaw PJ, Williams TL, Slade JY, et al. Low expression of GluR2 AMPA receptor subunit protein by human motor neurons. Neuroreport, 1999, 10:261~265.
21. Lee J M, Shih A Y, Murphy TH, et al. NF-E2-related Factor-2 Mediates Neuroprotection against Mitochondrial Complex I Inhibitors and Increased Concentrations of Intracellular Calcium in Primary Cortical Neurons. J Bio chem, 2003:37948~37956.
22. Guegan C, Przedborski S. Programmed cell death in amyotrophic lateral sclerosis. J Clin Invest, 2003, 111:153~161.
23. Seeburger J L, Tarras S. Spinal cord motoneurons express p75NGFR and p145 trk B mRNA in amyotropjic lateral sclerosis. Brain Res, 1993, 621: 111~115.
24. Lowry K S, Murray S S, Mclean C A, et al. A pontential role for the p75 low-affinity neurotrophin receptor in spinal motor neuron degeneration in murine and human amyotrophic lateral sclerosis. Amyotroph Lateral Scler Other Motor Neuron Disord, 2005, 2:127~134.
25. Marcelo RV, Mariana P, Patricia C, et al. Increased glutathione biosynthesis by Nrf2 activation in astrocytes prevents p75NTR-dependent motor neuron apoptosis. J Neurochem, 2006, 97:687~693.
1. Bains JS, Shaw CA , Neurodegenerative disorders in humans: the role of glutathione in oxidative stress-mediated neuronal death,Brain Res Brain Res Rev, 1997,25:335~358.
2. Grima G, Benz B, Parpura V, et al. Dopamineinduced oxidative stress in neurons with glutathione deficit: implication for schizophrenia. Schizophr Res,2003,62:213~224.
3. Rothstein JD, Jin L, Dykes-Hoberg M, et al. Chronic inhibition of gluta-mine uptake produces a model of slow neurotoxicity. Proc Natl Acad Sci USA, 1993, 90:6591~6595.
4. Liu RM, Hu H, Robison TW ,Dfferential enhancement of gamma-glutamyl traspeptidase and gamma-glutamylcysteine synthetase by tert-butylhydroquinone in rat lung epithelial L2 cells. Am .J Respir Cell Mol Biol. 1996, 14:186~191.
5. Zipper LM, Mulcahy RT, Erk Activation Is Required for Nrf2 Nuclear Localization during Pyrrolidine Dithiocarbamate Induction of Glutamate Cysteine Ligase Modulatory Gene Expression in HepG2 Cells. Toxicological Sciences,2003, 73:124~134.
6. Liu RM, Down-regulation of gamma-glutamylcysteine synthetase regulatory subunit gene expression in rat brain tissue during aging. JNeurosci Res, 2002, 68 : 344~351.
7. Shi MM, Iwamoto T, Forman HJ. Gamma-Glutamylcysteine synthetase and GSH increase in quinone-induced oxidative stress in BPAEC. Am J Physiol, 1994 ,267: 414~21.
8. Borroz KI, Buetler TM, Eaton DL,Modulation of gamma-glutamylcysteine synthetase large subunit mRNA expression by butylated hydroxyanisole. , 1994 126:150~155.
9. Woods JS, Davis HA, Baer RP, Enhancement of gamma-glutamylcysteine synthetase mRNA in rat kidney by methyl mercury.Arch Biochem Bio,1992 ,296:350~3.
10. Chinta SJ, Andersen JK. Reversible inhibition of mitochondrial complex I activity following chronic dopaminergic glutathione depletion in vitro: implications for Parkinson's disease. Free Radic Biol Med. 2006, 41: 1442~1448.
11. Okouchi M, Okayama N, Alexander JS, et al. NRF2-dependent glutamate-L-cysteine ligase catalytic subunit expression mediates insulin protection against hyperglycemia- induced brain endothelial cell apoptosis. Curr Neurovasc Res ,2006 3:249~61.
12. Erickson A M, Nevarea Z, Gipp JJ, Identification of a Variant Antioxidant Response Element in the Promoter of the Human Glutamate-Cysteine Ligase Modifier Subunit Gene. Biol. Chem., 2002, 34:30730~30737.
13. Liu R M, Gao L, Choi J.γ-Glutamylcysteine synthetase: mRNA stabilization and independent subunit transcription by4-hydroxy-2-nonenal. Am J Physiol Lung Cell Mol Physiol,1998,275:861~869.
14. Suh J H, Shenvi S V, Dixon B M. Decline in transcriptional activity of Nrf2 causes age-related loss of glutathione synthesis, which is reversible with lipoic acid,Natl. Acad. Sci ,2004, 101: 3381~3386.
1. Mulcahy RT and Gipp JJ. Indetification of putative antioxidant response element in 5’-flanking region of the human gamma-glutamylcystine synthetase heavy subunit gene ,Biochem Biophy Res Commun ,1995, 209:227~233.
2. Jaiswal AK, Antioxidant response element. Bio Phama,1994.48:439~444.
3. Rushmore T.H. and Pickett C.B. Transcripional regulation of rat glutathione S-transferase Ya subuint gene .Characterization of a xenobiotic-responsive element controlling inducible expression by phenolic antioxidant ,J .Bio Chem ,1990, 265:14648~14653.
4. Rothstein JD, Jin L, Dykes-Hoberg M, et al. Chronic inhibition of gluta- mine uptake produces a model of slow neurotoxicity. Proc Natl Acad Sci USA. 1993, 90:6591~6595.
5. Mannervik B, Awasthi YC, board PG, et al. Nomenclature for human glutathione transferases. Biochem. J. 1992, 282: 305~306.
6. Tsuchida S, Sato K. Glutathione transferases and cancer. Crit Rev. Biochem. Mol. Biol. 1992, 27: 337~384.
7. Scarpato R, Hirvonen A, Migliore L, et al. Influence of GSTM 1 and GSTTI polymorphisms on the frequency of chromosome aberrations in lymphocytes of smokers and pesticide exposed greenhouse workers,[J]. Mutat Res. 1997, 389: 27~235.
8. Sofia B, Juan S AR et al Gutathione transferase catalyze t he detxication of oxidized metabolities of catechlamines and may serve as an antioxidant system preventiong degenerative cellular processes. Bioch. J. 1997, 324:25~28.
9. 9Wikinson J and Clapper M L, Detoxification enzymes and chemoprevention. Proc. Soc. Exp. Biol Med. 1997, 216:192~200.
10. Drukarch B and Muiswinkel FL, Drug treatment of Parkinson’s disease. Time for phrase Ⅱ .Bioch Pharmarol,2000, 59:1023~1031.
11. Usarek E, Gajewska B, Kazmierczak B, et alA study of glutathione S-transferase pi expression in central nervous system of subjects with amyotrophic lateral sclerosis using RNA extraction from formalin-fixed, paraffin-embedded material, Neurochem Res. 2005, 30(8):1003~1007.
12. Allen S, Heath PR, Kirby J,et al, Analysis of the cytosolic proteome in a cell culture model of familial amyotrophic lateral sclerosis reveals alterations to the proteasome, antioxidant defenses, and nitric oxide synthetic pathways. J Biol Chem. 2003, 278(8):6371~6383.
13. Kuzma M, Jamrozik Z, Baranczyk-Kuzma A, Activity and expression of glutathione S-transferase pi in patients with amyotrophic lateral sclerosis. Clin Chim Acta. 2006, 364:217~220.
14. Murphy T.H., J.Yu, Preferential expression of antioxidant responsive element mediate gene expression in astrocytes. J Neurochemistry, 2001,76: 1670~1678.
15. Jong-Min L, Andy Y. Sand Jeffrey A. J, NF-E2-related Factor-2 Mediates Neuroprotection against Mitochondrial Complex I Inhibitors and Increased Concentrations of Intracellular Calcium in Primary Cortical Neurons, J BIO Chem. 2003, 278: 37948~37956.
16. Dinkova-Kostova AT, Holtzcaw WD, Kensler TW,The role of Keap1 in cellular protective response.,Chem Res Toxicol. 2005, 18:1779~1791.
1 Dringen R, Gutterer JM,Hirrlinger J. Glutathione metabolism in brain. Eur. J. Biochem., 2000, 267, 4912~4916.
2 Gerlach M, Ben-Shachar D, Riederer P, et al. Altered brain metabolism of iron as a cause of neurodegenerative diseases? J. Neurochem., 1994, 63: 793~807.
3 Cooper, A.J.L. Glutathione in the brain: disorders of glutathione metabolism. In The Molecular and Genetic Basis of Neurological Disease (Rosenberg, RN, Prusiner, SB, DiMauro, S, Barchi, RL & Kunk, LM, eds),1997, pp. 1195~1230.
4 Chinta SJ,Rajagopalaw S,Butterfield DA,etal In vitro and in vivo neuroprotection by gamma-glutamylcysteine ethyl ester against MPTP: relevance to the role of glutathione in Parkinson's disease.Neurosci Lett. 2006 , 402:137~41.
5 Bartov O, Sultana R, Butterfield DA, et al, Low molecular weight thiol amides attenuate MAPK activity and protect primary neurons from Abeta(1-42) toxicity,Brain Res. 2006 ,19,:198~206.
6 Bharath S, Andersen JK,Glutathione depletion in a midbrain-derived immortalized dopaminergic cell line results in limited tyrosine nitration of mitochondrial complex I subunits: implications for Parkinson's disease,Antioxid Redox Signal,2005 ,7:900~910.
7 Ericka PS, Albert AY, Stanley HA. Oxidative Stress: a common denominator in the pathogenesis of amyotrophic lateral sclerosis. Curr Opin Rheumatol, 2003, 15:730~736.
8 Oteiza PI, Uchitel OD, Carrasquedo F, et al. Evaluation of antioxidants, protein, and lipid oxidation products in blood from sporadic amyotrophic lateral sclerosis patients. Neurochem Res., 1997, 22:535~539.
9 Beal MF, Ferrante RJ, Browne SE, et al. Increased 3-nitrotyrosine in both sporadic and familial amyotrophic lateral sclerosis. Ann. Neurol., 1997, 42: 644~654.
10 Bruijn LI, Beal MF, Becher MW, et al. Elevated free nitrotyrosine levels, but not protein-bound nitrotyrosine or hydroxyl radicals, throughoutamyotrophic lateral sclerosis (ALS)-like disease implicate tyrosinenitration as an aberrant in vivo property of one familial ALS-linkedsuperoxide dismutase 1 mutant. Proc. Natl Acad. Sci. USA., 1997,94: 7606~7611.
11 Tohgi H, Abe T, Yamazaki K, et al. Remarkable increase in cerebrospinal fluid 3-nitrotyrosine in patients with sporadic amyotrophic lateral sclerosis. Ann. Neurol., 1999, 46:129~131.
12 Pedersen WA, Fu W, Keller JN, et al. Protein modification by the lipidperoxidation product 4-hydroxynonenal in the spinal cords of amyotrophic lateral sclerosis patients. Ann. Neurol., 1998, 44: 819~824.
13 Smith RG, Henry YK, Mattson MP, et al. Presence of 4-hydroxynonenal in cerebrospinal fluid of patients with sporadic amyotrophic lateral sclerosis. Ann. Neurol., 1998, 44:696~699.
14 Heath PR, Shaw PJ. Update on the glutamatergic neurotransmitter system and the role of excitotoxicity in amyotrophic lateral sclerosis. Muscle Nerve, 2002, 26:438~458.
15 Kawahara Y, Kwak S, Sun H, et al. Human spinal motoneurons express low relative abundance of GluR2 mRNA: an implication for excitotoxicity in ALS. J Neurochem, 2003, 85:680～689.
16 Takuma H, Kwak S, Yoshizawa T, et al. Reduction of GluR2 RNA editing, a molecular change that increases calcium influx through AMPA receptors, selective in the spinal ventral gray of patients with amyotrophic lateral sclerosis. Ann Neurol., 1999, 46:806~815.
17 Murphy T H,Miyamoto M,Sastre A,et al,Glutamate toxicity in a neuronal cell line involves inhibition of cystine transport leading to oxidative stress,Neuron,1989,2:1547~1558.
18 Wyatt I,Gyte A,Simpson M Get al,The role of glutathione in L-2-chloropropionic acid-induced cerebellar granule cell necrosis in the rat,Arch.Toxicol,1996,70:724~735.
19 Spina M B, and Cohen G, Dopamine turnover and glutathione oxidation: Implications for Parkinson’disease.Proc.Natl.Acad.Sci.U S A,1989,86:1398~1400.
20 Winuta Y,Kikuchi H,Ishikawa M,et al Lipid peroxidation in focial cerebral ischemia,J.Neurosug,1989,71:421~429.
21 Makar T K,Nedergaard M,Preuss A S,Perumal A S,et al Vitamin E,ascorbate,glutathione,glutathione disulfide, and enzymes of glutathione metabolism in cultures of chick astrocytes:Evidence that astrocytes play an important role in antioxidative processes in the brain,J.Neurochem,1994,62:45~53.
22 Lowndes H E,Philbert M A,Beiswanger C M,et al Xenobiotic metabolism in the brain as mechanistic bases for neurotoxicity,In handbook of Neurotoxicology(L W Chang and R S Dyer,Eds),pp 1~27.
23 Philber M A,Beiswanger C M,Water D K,et al,Cellular and regional distribution of reduced glutathione in the nervous system of tha rat:Histochemical localization by mercury orange and 0-phthaldialdehyde-inducedhistofluorescence,Toxicol.Appl.Pharm ,1991,107:215~227.
24 Abramovitz M,Homma H,Ishigaki S,et alCharacterization and localization of glutathione –S-transferases in rat brain and binding of hormones,neurotransmitter,and drugs,J.Neurochem,1988,50:50~57.
25 Cammer W,Tansey F,Abramovitz M, et al,Differential localization of glutathione S-transferases Yp and Yb subunits in oligodendrocytes and astrocytes of brain. J. .Neurochem, 1989, 53:876~883.
26 Johnson J A,Barbary A,Kornguth S E, et al,Glutathione S-transferase isoenzymes in rat brain neurons and glia,J Neurosic,1993,13:2013~2023.
27 PhilbertM A,BeiswangerCM,Manson M M,et al,Glutathione S-transferases and γ-glutamyl transpeptidase in rat nervous system:A basis for differential susceptibility to neurotoxicants, Neurotoxicology, 1995,16:349~362.
28 Dringen R, Hamprecht B,Glutathione content as an indicator for the presence of metabolic pathways of amino acid in astroglial cultures,1996,J.Neurochem,67:1375~1382.
29 Leslie S W,Brown L M,Trent,R D,et al,Stimulation of N-methyl-D-aspartate receptor-mediated calcium entry into dissociated neurons by reduced and oxidized glutathione,Mol.Pharmacol,1992,41:308~314.
30 Sagara J,Miura K,and Bannai S,maintenance of neuronal glutathione by glial cells,1993,J.Neurochem,61:1672~1676.
31 Ogita K,Ogaw Y,and Yoneda Y ,Apparent binding activity of [3H]-glutathione in rat central and peripheraltissues.Neurochem,Int,1988.13;493~497.
32 Liu Y F,and Quirion R,Modulatory role of glutathione on μ-opioid,subatant P/neurokinin-1,and kainic acid receptor binding sites,J. Neurochem,1992,59:1024~1032.
33 Bains JS, Shaw CA. Neurodegenerative disorders in humans: the role of glutathione in oxidative stress-mediated neuronal death. Brain Res Rev, 1997,25:335~358.
34 Grima G, Benz B, Parpura V, et al. Dopamineinduced oxidative stress in neurons with glutathione deficit: implication for schizophrenia. Schizophr Res., 2003, 62:213～224.
35 Janaky R, Varga V, Saransaari P, et al. Glutathione modulates the N-methyl-D-aspartate receptor-activated calcium influx into cultured rat cerebellar granule cells. Neurosci Lett., 1993, 156:153~157.
36 Dringen, R., Pfeiffer, B. & Hamprecht, B.,Synthesis of the antioxidant glutathione in neurons: supply by astrocytes of CysGly as precursor for neuronal glutathione. J. Neurosci,1999,19: 562~569.
37 Holopainen, I. & Kontro, P. Uptake and release of glycine in cerebellar granule cells and astrocytes in primary culture: potassiumstimulated release from granule cells is calcium-dependent. J. Neurosci.Res. 1989,24:374~383.
38 Sagara, J., Miura, K. & Bannai, S. Cystine uptake and glutathione level in fetal brain cells in primary culture and in suspension. J. Neurochem. 1993 ,61:1667~1671.
39 Monks MJ, Chersi-Egea JF, Philbert M, et al. Symposium overview: the role of glutathione in neuroprotection and neurotoxicity. Toxicol sci, 1999, 51:161~177.
40Desagher S, Glowinski, and PremontJ, Astrocytes protect neurons from hydrogen peroxide toxicity. J. Neurosci. 1996,16, 2553~2562.
41Drukarch, B., Schepens, E., Stoof, J.C,et al, Astrocyte-enhanced neuronal survival is mediated by scavenging of extracellular reactive oxygen species.Free Rad. Biol. Med. 1998,25: 217~220.
42Drukarch, B., Schepens, E., Jongenelen, C.A.M., et al, Astrocyte-mediated enhancement of neuronal survival is abolished by glutathione deficiency. Brain Res. 1997, 770:123~130.
43 Bains, J.S. & Shaw, C.A, Neurodegenerative disorders in humans: the role of glutathione in oxidative stress-mediated neuronal death. Brain Res. Rev. 1997, 25: 335~358.
44 Lanius, R.A., Krieger, C., Wagey, R. et al. Increased [35S] glutathione binding sites in spinal cords from patients with sporadic amyotrophic lateral sclerosis. Neurosci. Lett, 1993,163: 89~92.
45Moumen, R, Nouvelot, A , Duval, D, et al, Plasma superoxide dismutase and glutathione peroxidase activity in sporadic amyotrophic lateral sclerosis. J. Neurol. Sci. 1997, 151:35~39.
46Apostolski, S., Marinkovic, Z., Nikolic, A., et al, Glutathione peroxidase in amyotrophic lateral sclerosis: the effects of selenium supplementation.J. Environ. Pathol. Toxicol. Oncol. 1998, 17:325~329.
47 Macho A, Hirsch T, Marzo I, et al. Glutathione depletion is an early and calcium elevation is a late event of thymocyte apoptosis. J. Immunol., 1997, 158:4612~4619.
48Beaver JP, Waring PA. Decrease in intracellular glutathione concentration precedes the onset of apoptosis in murine thymocytes. Eur. J. Cell Biol., 1995, 68:47~54.
49 Chi L, Ke AY, Luo BC, et al. Depletion of Reduced Glutahione Enhances Motor Neuron Degeneration in Vitro and in Vivo. Neuroscience, 2007, 144: 991~1003.
50Li Y, Maher P, Schubert D, A role for 12-lipoxygenase in nerve cell death caused by glutathione depletion. Neuron, 1997, 19:453~463.
51 Yamamoto, M., Sakamoto, N., Iwai, A., et al, Protective actions of YM737, a new glutathione analog, against cerebral ischemia in rats. Res. Commun. Chem. Pathol. Pharmacol. 1993,81: 221~232.
52Henderson, J.T., Javaheri, M., Kopko, S. et al,.Reduction of lower motor neuron degeneration in wobbler mice by N-acetyl-l-cysteine. J. Neurosci.1996, 16:7574~7582.
53Jain.A,Madsen.D.C,Auld.P.A.,etal,l-2-oxothiazolidine-4-carboxylate,a cysteine precursor, stimulates growth and normalizes tissue glutathione concentrations in rats fed a sulfur amino acid-deficient diet. J. Nutr. 1995 ,125: 851~856.
54Cudkowicz, M.E., Sexton, P.M., Ellis, T., et al,The pharmacokinetics and pharmaco-dynamics of procysteine in amyotrophic lateral sclerosis.Neurology 1999,52: 1492~1494.
55Kanai Y, Segawa H, Miyamoto K,et al, Expression cloning and characterization of a transporter for large neutral amino acids activated by the heavy chain of 4F2 antigen, J Biol Chem,1998 ,273:23629~23632 .
56Sato H, Tamba M, Ishii T, et al, Cloning and expression of a plasma membrane cystine/glutamate exchange transporter composed of two distinct proteins. J Biol Chem, 1999,274: 11455~11458.
57Coyle JT, Bird SJ, Evans RH, et al, Excitatory amino acid neurotoxins: selectivity, specificity, and mechanisms of action. Neurosci Res Program Bull, 1981,19:331~427.
58Sagara Y, Schubert D The activation of metabotropic glutamate receptors protects nerve cells from oxidative stress. J Neurosci , 1998,18:6662~6671.
59Murphy TH, Miyamoto M, Sastre A, et al, Glutamate toxicity in a neuronal cell line involves inhibition of cystine transport leading to oxidative stress. Neuron, 1989,2: 1547~1558.
60Trotti D, Danbolt NC, Volterra A, Glutamate transporters are oxidant-vulnerable: a molecular link between oxidative and excitotoxic neurodegeneration? Trends Pharmacol Sci, 1998,19:328~334.
61Mannervi I , Mannervik B, Awasthi YC, et al.Nomenclature for human glutathione transferases. J Biochem, 1992, 282: 305~306.
62 Tsuchida S, Sato K,Glutathione transferases and cancer. Crit. Rev. Biochem. Mol. Biol., 1992, 27: 337～384.
63Scarpato R, Hirvonen A, Migliore L, et al. Influence of GSTM 1 and GSTTI polymorphisms on the frequency of chromosome aberrations inlymphocytes of smokers and pesticide exposed greenhouse workers. Mutat Res,1997, 389:227~235.
64 Sofia BAEZ, Juan, SEGURA-AGUILAR et al. Glutathione transferase catalyse the detxication of oxidized metabolities of catechlamines and may serve as an antioxidant system preventiong degenerative cellular processes. Bioch. J, 1997, 324:25~28.
65Usarek E, Gajewska B, Kazmierczak B, et al. A study of glutathione S-transferase pi expression in central nervous system of subjects with amyotrophic lateral sclerosis using RNA extraction from formalin-fixed, paraffin-embedded material. Neurochem Res., 2005, 30:1003~1007.
66Kuzma M,Jamrozik Z, Baranczyk-Kuzma A. Activity and expression of glutathione S-transferase pi in patients with amyotrophic lateral sclerosis. Clin Chim Acta, 2006, 364:217~221.
67Allen S, Heath PR, Kirby J, et al. Analysis of the cytosolic proteome in a cell culture model of familial amyotrophic lateral sclerosis reveals alterations to the proteasome, antioxidant defenses, and nitric oxide synthetic pathways. J Biol Chem., 2003, 278:6371~6383.
68Venugopal R,Jasiwal AK, Positirely response element—mediated expression of NADH ( P ) : quinone oxidoreductasedgene ,Proc .Natl .Acad.Sci.USA,1996,93:14960~14965.
69 Huang CS, Chang LS, Anderson ME, Meister A.Catalytic and regulatory properties of the heavy subunit of rat kidney gamma-glutamylcysteine synthetase, J Biol Chem.,1993, 268(26):19675~19680.
70Yao K, Godwin AK, Ozols RF, Hamilton TC, O'Dwyer PJVariable baseline gamma-glutamylcysteine synthetase messenger RNA expression in peripheral mononuclear cells of cancer patients, and its induction by buthionine sulfoximine treatment.Cancer Res. 1993 Aug 15;53(16):3662~3666.
71Liu RM, Gao L, Choi J, Forman HJ gamma-glutamylcysteine synthetase: mRNA stabilization and independent subunit transcription by 4-hydroxy-2-nonenal.Am J Physiol. 1998 Nov;275:861~869.72Dickinson DA, Iles KE, Watanabe N, Iwamoto T, Zhang H, Krzywanski DM, Forman HJ4-hydroxynonenal induces glutamate cysteine ligase through JNK in HBE1 cells.Free Radic Biol Med. 2002 Oct 1;33(7):974.
73Shi MM, Kugelman A, Iwamoto T et al,Quinone-induced oxidative stress elevates glutathione and induces gamma-glutamylcysteine synthetase activity in rat lung epithelial L2 cells.J Biol Chem. 1994 Oct 21;269(42):26512~26517.
74Day RM, Suzuki YJ, Lum JM,Bleomycin upregulates expression of gamma-glutamylcysteine synthetase in pulmonary artery endothelial cells.Am J Physiol Lung Cell Mol Physiol. 2002, 282: 1349~1357.
75Morimitsu Y, Nakagawa Y, Hayashi K,A, sulforaphane analogue that potently activates the Nrf2-dependent detoxification pathway, 2002 ,277:3456~63.
76David M. K, Dale A.D, Karen E. I, Variable regulation of glutamate cysteine ligase subunitproteins affects glutathione biosynthesis in response to oxidative stress ,Arch Biochemand Bioph, 2004,423: 116~125.
77Solis WA ,Dalton TP, Dieter MZ, Glutamate-cysteine ligase modifier subunit: mouse Gclm gene structure and regulation by agents that cause oxidative stress,Biochem Pharmacol.,2002, 63(9):1739~54.
78Dinkova-Kostova AT, Holtzclaw WD, Kensler TW, et al,The role of Keap1 in cellular protective responses. Chem Res Toxicol, 2005,18: 1779~1791.
79Allen S, Heath PR, Kirby J, et al. Analysis of the cytosolic proteome in a cell culture model of familial amyotrophic lateral sclerosis reveals alterations to the proteasome, antioxidant defenses, and nitric oxide synthetic pathways. J Biol Chem., 2003, 278:6371~6383.
80Murphy TH,Yu J,Preferential expression of antioxidant responsive element mediate gene expression in astrocytes. J Neurochem, 2001, 76: 1670~1678.
81Lee JM, Andy Y, Shih, et al. NF-E2-related Factor-2 Mediates Neuroprotection against Mitochondrial Complex I Inhibitors and Increased Concentrations of Intracellular Calcium in Primary Cortical Neurons. The Journal of Biological Chemistry, 2003, 278: 37948~37956.
82Dinkova-Kostova AT, Holtzclaw WD, Kensler TW, et al,The role of Keap1 in cellular protective responses. Chem Res Toxicol, 2005,18 :1779~1791.