ElF4g Cleavage and Persistent Protein Synthesis Inhibition Following Ischemia

描述（由申请人提供）： 脑缺血事件（即中风）后的神经系统并发症会导致严重的临床疾病。因此，我们的长期目标是阐明缺血后产生临床疾病的机制，以潜在地确定新的治疗策略。缺血和再灌注引起所有受缺血影响的神经元中蛋白质合成的立即和全面停滞。最初的蛋白质合成抑制（PSI）之后是蛋白质合成的几乎完全恢复，或者PSI持续存在。正是这种持续的PSI与神经变性和最终的神经元死亡高度相关。因此，我们的工作假设是，持续PSI介导脑缺血后的细胞死亡。我们的具体假设是，蛋白酶如钙蛋白酶对真核起始因子4G（eIF4G）的蛋白水解裂解可能介导持续性PSI，并且防止eIF4G裂解可能阻断持续性PSI，从而提供针对缺血性损伤的神经保护。这一假设基于以下数据：1）eIF4G裂解在体外和体内其他蛋白质合成起始因子减少之前开始，2）裂解的时间过程与体外持续PSI的时间过程相关，3）体外缺血导致产生新的大小特异性裂解产物，和4）通过其内源性抑制剂钙蛋白酶抑制素的过表达抑制钙蛋白酶活性部分恢复体外缺血后的蛋白质合成。作为这些数据的结果，我们的具体重点是确定钙蛋白酶在eIF4G裂解中的作用，并确定eIF4G裂解在持续PSI和细胞死亡中的作用。我们的具体目的是：1）检验体外缺血后eIF4G切割和持续PSI由钙蛋白酶激活介导的假设。我们将i）确定在体外缺血后，（i）通过过表达内源性钙蛋白酶抑制剂钙蛋白酶抑制素和（ii）使用源自钙蛋白酶敲除小鼠的神经元是否阻止eIF4G裂解。我们还将（iii）确定通过钙蛋白酶抑制来防止eIF4G裂解是否可以在体外缺血后挽救蛋白质合成。2)为了检验以下假设：防止eIF4G裂解增强持续PSI并赋予针对体外缺血的神经保护。我们将（i）过表达eIF4G以试图恢复基线eIF4G水平，（ii）确定eIF4G的过表达是否将在体外缺血后赋予神经保护，和（iii）使用重组蛋白在体外鉴定钙蛋白酶对eIF4G的切割位点。 公共卫生相关性：最终，持续性PSI的机制的描绘可能导致脑缺血的新的治疗策略。

DESCRIPTION (provided by applicant): The neurological complications following cerebral ischemic events (i.e. stroke) produce severe clinical disease. Our long term goal is thus to elucidate mechanisms following ischemia that produce clinical disease to potentially identify novel therapeutic strategies. Ischemia and reperfusion cause immediate and global arrest of protein synthesis in all ischemia-affected neurons. Initial protein synthesis inhibition (PSI) is either followed by near complete restoration of protein synthesis or there is a persistence of PSI. It is this persistent PSI that is highly correlated with neurodegeneration and eventual neuronal death. Thus our working hypothesis is that persistent PSI mediates cell death following cerebral ischemia. Our specific hypothesis is that proteolytic cleavage of eukaryotic initiation factor 4G (elF4G) by proteases such as calpain may mediate persistent PSI, and prevention of elF4G cleavage may block persistent PSI and thus provide neuroprotection against ischemic injury. This hypothesis is based on the following data: 1) elF4G cleavage begins prior to decreases in other protein synthesis initiation factors in vitro and in vivo, 2) the time course of cleavage is correlated with that of persistent PSI in vitro, 3) in vitro ischemia results in the production of a novel size-specific cleavage product, and 4) inhibition of calpain activity by overexpression of its endogenous inhibitor calpastatin partially restores protein synthesis following in vitro ischemia. As a result of these data, our specific focus is to determine the role of calpain in elF4G cleavage and to identify the role of elF4G cleavage in persistent PSI and cell death. Our specific aims are: 1) to test the hypothesis that elF4G cleavage and persistent PSI after in vitro ischemia is mediated by calpain activation. We will i) determine if elF4G cleavage is prevented following in vitro ischemia (i) by overexpression the endogenous calpain inhibitor calpastatin and (ii) using neurons derived from calpain knock out mice. We will also (iii) determine if prevention of elF4G cleavage via calpain inhibition can rescue protein synthesis following in vitro ischemia. 2) To test the hypothesis that prevention of elF4G cleavage alleviates persistent PSI and confers neuroprotection against in vitro ischemia. We will (i) overexpress elF4G to attempt to restore baseline elF4G levels, (ii) determine if overexpression of elF4G will confer neuroprotection following in vitro ischemia and (iii) identify the cleavage sites of elF4G by calpain using recombinant proteins in vitro. PUBLIC HEALTH RELEVANCE: Ultimately, the delineation of the mechanism governing persistent PSI may lead to a novel therapeutic strategy for cerebral ischemia.