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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：
Mika Jekabsons
金额：
$ 21.18万
依托单位：
UNIVERSITY OF MISSISSIPPI
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

来源：
https://reporter.nih.gov/project-details/7515007
关键词：
Acetoacetates Acute Address Adenine Nucleotide Translocase Alzheimer&apos s Disease Amyotrophic Lateral Sclerosis Antioxidants Apoptosis Attention Binding Binding Sites Biochemical Brain Injuries Carbon Cell Death Cells Cessation of life Complex Condition Creatine Kinase Cytoplasmic Granules Data Development Event Failure Free Radicals Genetic Glucose Glycolysis Goals Growth Factor Hydroxybutyrates Immunofluorescence Immunologic Immunoprecipitation Incidence Inner mitochondrial membrane Lipid Binding Mitochondria Modeling Molecular Conformation Nature Necrosis Nerve Degeneration Nervous system structure Neurodegenerative Disorders Neurons Newborn Infant Nitric Oxide Numbers Outer Mitochondrial Membrane Pathology Phosphotransferases Play Process Production Proteins Public Health Pyruvate Pyruvates Rate Rattus Reactive Oxygen Species Regulation Research Resistance Role Serum Severities Signal Transduction Site Staging Stroke Survivors Testing Voltage-Dependent Anion Channel Western Blotting coping cytochrome c deprivation drug development extracellular hexokinase improved neuron apoptosis pro-apoptotic protein programs research study

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)：由于中风或神经退行性疾病导致的神经元死亡，既有急性功能衰竭(即坏死)，也有基因死亡程序的不适当激活(即细胞凋亡)。后一个过程被讨论，重点是了解促进Bax依赖的细胞死亡的调控事件。促凋亡蛋白Bax通过促进线粒体释放细胞色素c而在细胞凋亡中发挥重要作用，最终导致细胞的蛋白分解破坏。这一提议检验了这样的假设，即线粒体外膜Bax孔只有在两个关键事件发生后才会形成--诱导细胞质Bax转换，即6A7构象变化，以及揭示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控机制。从新生大鼠分离的小脑颗粒神经元将作为研究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去极化细胞外K+(即在没有血清的情况下与3.5 mM K+孵育，称为K+/血清剥夺)启动死亡程序。第一个特定的目的是建立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的量，并进一步确定与细胞色素c释放相关的构象变化是在移位到线粒体之前还是之后发生的。在神经细胞凋亡过程中，常可观察到稳态自由基/活性氧物种的增加，但其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的转换，特别是关注易位步骤。线粒体靶向抗氧化剂将被用来测试这种可能性。Bax可能通过线粒体外膜电压依赖性阴离子通道(VDAC)在特定的连接复合体(称为接触部位)与线粒体结合。己糖激酶和肌酸激酶是这些位点的受调控成分，它们可能排除或竞争Bax结合。如果是真的，则可以预测，在细胞凋亡过程中，一种或两种激酶的线粒体含量都会减少。第三个具体目的是研究己糖激酶的细胞质/线粒体分布，以此来评估BAX可能可用的VDAC位点数量的变化，并另外确定细胞凋亡过程中接触部位的蛋白质组成。从这些目标获得的数据将提高我们对调控Bax在细胞凋亡早期阶段的调控事件的理解。解决这些目的的实验方法包括定量免疫荧光、免疫沉淀和Western blotting。公共卫生相关性：不适当的神经元凋亡会导致中风和神经退行性疾病(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。这些情况的发生率很高：大约400万中风幸存者必须应对神经元死亡的衰弱影响，目前约有450万人患有阿尔茨海默病，这一数字自1980年以来翻了一番。从这一提议中获得的数据将有助于理解神经元凋亡的机制，从而有助于药物开发以限制这些病理的严重性。