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是由于功能的急性衰竭（即坏死）和遗传死亡程序的不适当激活（即，凋亡）。后一个过程是解决的，重点是了解的监管事件，促进细胞凋亡依赖性细胞死亡。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起关键作用，其结果是细胞的蛋白水解破坏。该提议测试了线粒体外膜Bax孔仅在两个关键事件发生后形成的假设--细胞质Bax转化（称为6A 7构象变化）的诱导，以及Bax线粒体结合位点的暴露。本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被用作研究细胞凋亡的模型。剥夺这些神经元的血清生长因子和去极化细胞外K+（即，在没有血清的情况下与3.5mM K+孵育，称为K+/血清剥夺）启动死亡程序。第一个具体的目的是建立线粒体和细胞质分布的Bax引起的K+和/或血清剥夺，定量的Bax结合的量，并进一步确定是否发生构象变化与细胞色素c的释放之前或之后易位到线粒体。在神经元凋亡过程中通常观察到稳态自由基/活性氧增加，但其在凋亡中的作用尚不清楚。第二个具体的目的是确定在何种程度上线粒体内膜自由基调节Bax的过渡，特别注意被集中在易位步骤。线粒体靶向抗氧化剂将用于测试这种可能性。Bax可以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组分，其可排除Bax结合或与Bax结合竞争。如果为真，则预测在细胞凋亡期间一种或两种激酶的线粒体含量降低。第三个具体的目的是调查的细胞质/线粒体分布的己糖激酶作为一种方式来评估的VDAC网站，可能是Bax的数量的变化，并另外确定在细胞凋亡过程中的接触点的蛋白质组成。从这些目标获得的数据将提高我们的理解，在细胞凋亡的早期阶段控制Bax的调节事件。解决这些目标的实验方法包括定量免疫荧光，免疫沉淀和Western印迹。 公共卫生相关性：不适当的神经元凋亡导致中风和神经退行性疾病如阿尔茨海默病和肌萎缩性侧索硬化症导致的脑损伤的严重性。这些病症的发生率很高：大约4，000，000名中风幸存者必须科普神经元死亡的衰弱影响，目前大约4，500，000人患有阿尔茨海默病，自1980年以来，这一数字翻了一番。从该提案中获得的数据将有助于理解负责神经元凋亡的机制，从而有助于药物开发，以限制这些病理的严重程度。

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.