Regulation of the pro-apoptotic protein Bax during neuronal apoptosis

神经元凋亡过程中促凋亡蛋白 Bax 的调节

基本信息

  • 批准号:
    7515007
  • 负责人:
  • 金额:
    $ 21.18万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2008
  • 资助国家:
    美国
  • 起止时间:
    2008-07-01 至 2012-06-30
  • 项目状态:
    已结题

项目摘要

DESCRIPTION (provided by applicant): Neuronal death as a consequence of stroke or neurodegenerative disorders occurs by both acute failure in function (i.e, necrosis) and inappropriate activation of a genetic death program (i.e., apoptosis). The latter process is addressed, with the focus being to understand the regulatory events that promote Bax-dependent cell death. The pro-apoptotic protein Bax plays a crucial role in apoptosis through facilitating cytochrome c release from mitochondria, the result of which is proteolytic destruction of the cell. This proposal tests the hypothesis that mitochondrial outer membrane Bax pores form only after two crucial events occur- induction of a cytoplasmic Bax transformation known as the 6A7 conformational change, and unmasking of a Bax mitochondrial binding site. The long term goal of this research is to understand the regulatory mechanisms that trigger neuronal apoptosis. Cerebellar granule neurons isolated from newborn rats will be used as a model to study Bax-dependent apoptosis. Depriving these neurons of serum growth factors and depolarizing extracellular K+ (i.e., incubation with 3.5 mM K+ without serum, referred to as K+/serum deprivation) initiates the death program. The first specific aim is to establish the mitochondrial and cytoplasmic distributions of Bax elicited by K+ and/or serum deprivation, to quantify the amount of Bax that binds, and to further determine if a conformational change associated with cytochrome c release occurs before or after translocation to the mitochondria. Increased steady- state free radicals/reactive oxygen species are commonly observed during neuronal apoptosis, but their role in apoptosis is unclear. The second specific aim is to determine to what extent mitochondrial inner membrane free radicals regulate the Bax transition, with particular attention being focused on the translocation step. Mitochondrial-targeted antioxidants will be used to test this possibility. Bax may associate with mitochondria through the mitochondrial outer membrane voltage-dependent anion channel, or VDAC, at specific junctional complexes known as contact sites. Hexokinase and creatine kinase are regulated components of these sites that may preclude or compete with Bax binding. If true, it is predicted that the mitochondrial content of one or both kinases decreases during apoptosis. The third specific aim is to investigate the cytoplasmic/mitochondrial distribution of hexokinase as a way to assess changes in the number of VDAC sites that may be available for Bax, and to additionally determine the protein composition of contact sites during apoptosis. Data obtained from these aims will improve our understanding of the regulatory events controlling Bax in the early stages of apoptosis. The experimental approaches to address these aims include quantitative immunofluorescence, immunoprecipitation, and Western blotting. PUBLIC HEALTH RELEVANCE: Inappropriate neuronal apoptosis contributes to the severity of brain damage resulting from stroke and neurodegenerative disorders such as Alzheimer's disease and Amyotrophic Lateral Sclerosis. The incidence of these conditions is high: approximately 4,000,000 stroke survivors must cope with the debilitating effects of neuronal death and approximately 4,500,000 people currently suffer from Alzheimer's disease, a number that has doubled since 1980. Data obtained from this proposal will contribute to understanding the mechanisms responsible for neuronal apoptosis, thus aiding drug development to limit the severity of these pathologies.
描述(由申请人提供):中风或神经退行性疾病导致的神经元死亡是由急性功能衰竭(即坏死)和遗传死亡程序的不适当激活(即细胞凋亡)引起的。后一个过程得到解决,重点是了解促进 Bax 依赖性细胞死亡的调控事件。促凋亡蛋白 Bax 通过促进细胞色素 c 从线粒体释放而在细胞凋亡中发挥关键作用,其结果是细胞的蛋白水解破坏。该提案测试了这样的假设:线粒体外膜 Bax 孔仅在两个关键事件发生后才形成 - 诱导细胞质 Bax 转化(称为 6A7 构象变化),以及揭开 Bax 线粒体结合位点。这项研究的长期目标是了解触发神经元凋亡的调节机制。从新生大鼠中分离出的小脑颗粒神经元将用作研究 Bax 依赖性细胞凋亡的模型。剥夺这些神经元的血清生长因子并使细胞外 K+ 去极化(即与 3.5 mM K+ 无血清一起孵育,称为 K+/血清剥夺)启动死亡程序。第一个具体目标是确定 K+ 和/或血清剥夺引起的 Bax 的线粒体和细胞质分布,量化结合的 Bax 量,并进一步确定与细胞色素 c 释放相关的构象变化是否发生在易位之前或之后到线粒体。在神经元凋亡过程中通常观察到稳态自由基/活性氧的增加,但它们在细胞凋亡中的作用尚不清楚。第二个具体目标是确定线粒体内膜自由基在多大程度上调节 Bax 转变,特别关注易位步骤。线粒体靶向抗氧化剂将用于测试这种可能性。 Bax 可能通过线粒体外膜电压​​依赖性阴离子通道 (VDAC) 在称为接触位点的特定连接复合物处与线粒体结合。己糖激酶和肌酸激酶是这些位点的受调节成分,可能会阻止或竞争 Bax 结合。如果属实,则预测一种或两种激酶的线粒体含量在细胞凋亡过程中减少。第三个具体目标是研究己糖激酶的细胞质/线粒体分布,以此评估 Bax 可用的 VDAC 位点数量的变化,并另外确定细胞凋亡期间接触位点的蛋白质组成。从这些目标获得的数据将提高我们对细胞凋亡早期控制 Bax 的调控事件的理解。实现这些目标的实验方法包括定量免疫荧光、免疫沉淀和蛋白质印迹。 公共健康相关性:不适当的神经细胞凋亡会导致中风和神经退行性疾病(例如阿尔茨海默病和肌萎缩侧索硬化症)造成的脑损伤的严重程度。这些疾病的发生率很高:大约 4,000,000 名中风幸存者必须应对神经元死亡带来的衰弱影响,目前大约有 4,500,000 人患有阿尔茨海默病,这一数字自 1980 年以来增加了一倍。从该提案中获得的数据将有助于理解其机制负责神经元凋亡,从而帮助药物开发以限制这些病理的严重程度。

项目成果

期刊论文数量(2)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
A systems biology approach to investigating apoptotic stimuli as effectors of cell metabolism: practical application of top-down control analysis to attached neurons.
研究细胞凋亡刺激作为细胞代谢效应物的系统生物学方法:自上而下控制分析对附着神经元的实际应用。
  • DOI:
  • 发表时间:
    2009-02
  • 期刊:
  • 影响因子:
    5.6
  • 作者:
    Jekabsons; Mika B
  • 通讯作者:
    Mika B
(13)C metabolic flux analysis in neurons utilizing a model that accounts for hexose phosphate recycling within the pentose phosphate pathway.
(13)C 神经元代谢通量分析,利用模型解释磷酸戊糖途径中的磷酸己糖循环。
  • DOI:
  • 发表时间:
    2016-02
  • 期刊:
  • 影响因子:
    4.2
  • 作者:
    Gebril HM;Avula B;Wang YH;Khan IA;Jekabsons MB
  • 通讯作者:
    Jekabsons MB
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Mika Jekabsons其他文献

Mika Jekabsons的其他文献

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