Regulation of the pro-apoptotic protein Bax during neuronal apoptosis

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，发生在急性功能衰竭（即坏死）和不适当的基因死亡程序激活（即细胞凋亡）。后一个过程被解决，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通过促进线粒体细胞色素c的释放在细胞凋亡中起着至关重要的作用，其结果是细胞的蛋白水解破坏。这一提议验证了线粒体外膜Bax孔只有在两个关键事件发生后才会形成的假设——细胞质Bax转化的诱导，即6A7构象变化，以及Bax线粒体结合位点的揭露。本研究的长期目标是了解触发神经元凋亡的调控机制。以新生大鼠小脑颗粒神经元为模型，研究bax依赖性细胞凋亡。剥夺这些神经元的血清生长因子并使细胞外K+去极化（即，在没有血清的情况下用3.5 mM K+孵育，称为K+/血清剥夺）启动死亡程序。第一个具体目标是建立由K+和/或血清剥夺引起的Bax的线粒体和细胞质分布，量化Bax的结合量，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到线粒体之前或之后。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上与线粒体结合。己糖激酶和肌酸激酶是这些位点的调控成分，可能阻止或竞争Bax的结合。如果这是正确的，则预测一种或两种激酶的线粒体含量在凋亡过程中减少。第三个具体目的是研究己糖激酶的细胞质/线粒体分布，作为评估Bax可用的VDAC位点数量变化的一种方法，并确定凋亡过程中接触位点的蛋白质组成。从这些目标中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。解决这些问题的实验方法包括定量免疫荧光、免疫沉淀和免疫印迹。

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.