Stress Granules Cause Translation Arrest In Ischemic Vulunerable Neurons

描述(申请人提供)：脑缺血和再灌注(I/R)损伤发生在心脏骤停和中风复苏后，导致高发病率和死亡率。由于对导致细胞死亡的细胞损伤级联反应的不完全了解，目前尚无临床有效的治疗方法。我的实验室的长期目标是研究脑I/R引起的神经元死亡的机制，以便开发有效的治疗方法。在短暂性全脑I/R后，蛋白质合成抑制与海马CA1区锥体神经元的选择性死亡密切相关。这种不可逆的翻译抑制及其与细胞死亡的关系尚不清楚。应激颗粒是细胞质颗粒，在细胞应激过程中隔离不活跃的翻译机械。我们提出了令人信服的证据，即应激颗粒的改变是脑缺血脆弱的海马区CA1神经元持续翻译停滞的核心。我们的具体目标是：1.探讨不可逆翻译停滞的机制。我们将利用互补的显微镜和生化方法来分析应激颗粒的功能组成。我们将比较缺血抵抗的CA3和缺血的脆弱的CA1从再灌流早期到脆弱神经元细胞死亡的应激颗粒。2.探讨缺血预适应(IPC)对再灌流神经元应激颗粒组成和行为的影响。IPC可防止脆弱的CA1神经元中的细胞死亡和持续翻译停滞。我们将评估IPC对CA1神经元应激颗粒行为和成分、蛋白质合成率和细胞死亡的影响。3.为了证明持续性翻译停滞与脑I/R后神经元死亡的因果关系，我们将使用抗生素蛋白质合成抑制剂来预见性地改变再灌流海马神经元中的应激颗粒，并观察其对再灌流海马神经元中蛋白质合成率、应激颗粒组成和行为以及细胞死亡的影响。通过对持续翻译停滞和I/R诱导的细胞死亡之间的关系进行综合研究，我们的具体目标是解决阻碍该领域进展的一个问题：再灌流神经元中不可逆转的蛋白质合成抑制如何导致细胞死亡。公共卫生相关性：每年，数以百万计的人因心脏骤停或中风导致的脑损伤而受伤或死亡。目前还没有预防这种脑损伤的治疗方法，因为医生和科学家不知道血液停止在大脑中流动一段时间后，细胞是如何死亡的，然后又恢复了。这项提案中的工作旨在加深我们对大脑中神经元在低血流量或无血流量(缺血)一段时间后恢复正常血流(再灌注)后死亡的方式的理解。

DESCRIPTION (provided by applicant): Ischemia and reperfusion (I/R) injury of the brain occurs following resuscitation from cardiac arrest and stroke, and results in high morbidity and mortality. There is no clinically effective treatment because of an incomplete understanding of the cellular injury cascades leading to cell death. The long- term goal of my laboratory is to investigate the mechanisms of neuronal death caused by brain I/R to allow for the development of effective treatments. There is a striking correlation between protein synthesis inhibition and the selective death of hippocampal CA1 pyramidal neurons following transient global brain I/R. The mechanism of this irreversible translation arrest and its relationship to cell death is unknown. Stress granules are cytoplasmic particles that sequester inactive translational machinery during cellular stress. We present compelling evidence that stress granule alterations are central to persistent translation arrest in ischemic-vulnerable hippocampal CA1 neurons. Our Specific Aims are: 1. To investigate the mechanism of irreversible translation arrest. We will analyze the functional composition of stress granules utilizing complementary microscopic and biochemical approaches. We will compare stress granules in ischemic resistant CA3 and ischemic vulnerable CA1 from early reperfusion to the point of cell death of vulnerable neurons. 2. To identify the effect of ischemic preconditioning (IPC) on stress granule composition and behavior in reperfused neurons. IPC prevents both cell death and persistent translation arrest in vulnerable CA1 neurons. We will assess the effect of IPC on stress granule behavior and composition, protein synthesis rates, and cell death in CA1 neurons. 3. To show that persistent translation arrest is causally related to neuronal death following brain I/R. Antibiotic protein synthesis inhibitors will be used to predictably alter stress granules in reperfused hippocampal neurons, and we will examine the effect on protein synthesis rates, stress granule composition and behavior and cell death in reperfused hippocampal neurons. By providing an integrated examination of the relationship between persistent translation arrest and I/R-induced cell death, our Specific Aims address a problem that has been a barrier to progress in the field: how irreversible inhibition of protein synthesis in reperfused neurons causes cell death. PUBLIC HEATH RELEVANCE: Every year, millions of people are injured or die from brain damage caused by cardiac arrest or stroke. There are no treatments to prevent this brain damage because physicians and scientists do not understand how the cells die following a period when blood has stopped flowing in the brain and subsequently resumed. The work in this proposal seeks to further our understanding of the way in which neurons in the brain die following a period of low or no blood flow (ischemia) followed by resumption of normal blood flow (reperfusion).