CEREBRAL OXIDATIVE METABOLISM IN HYPOXIC NEWBORNS

描述（由申请人提供）：我们研究计划的长期目标是阐明新生儿缺氧诱导脑损伤的细胞和分子机制。目前的研究将集中于在缺氧过程中，启动程序性细胞死亡的关键步骤，procaspase-9激活的机制。我们推测，在缺氧期间，caspase-9的活化是由于增加的表达和凋亡蛋白的翻译后修饰导致细胞溶质室中促凋亡/抗凋亡蛋白（Bax/Bcl-2）的比例增加。此外，我们推测过量的Bax蛋白通过凋亡蛋白酶激活因子-1（Apaf-1）激活半胱氨酸天冬氨酸蛋白酶原-9。我们建议，缺氧诱导的增加，导致增加的促凋亡蛋白，Bax和坏的表达的核内Cainflux，介导的Ca++依赖性激酶IV（CaM激酶IV）级联的激活增加，并导致磷酸化的环AMP反应元件结合（CREB）蛋白在Ser 133。此外，我们提出，缺氧诱导的Apaf-1结合结构域的ATP和细胞色素c的亲和力的修改，有利于Apaf-1介导的半胱氨酸蛋白酶原-9的激活。使用已建立的技术和新生仔猪模型，我们将证明在缺氧期间a）促凋亡蛋白Bax的表达增加介导了线粒体复合物的激活（蛋白酶原-9-Apaf-1-Bcl-2/Bax）导致蛋白酶原-9的活化，B）促凋亡蛋白Bax和Bad的表达增加是由于钙激活的增加，c）缺氧诱导的Apaf-1分子对ATP和细胞色素c和Bcl-2/Bax的调节位点的修饰导致半胱氨酸天冬氨酸蛋白酶原-9的活化，和d）通过抑制核Cainflux，通过抑制促凋亡蛋白的合成和抑制caspase-9的活性，我们将防止缺氧诱导的新生猪脑神经元死亡。拟议的研究将建立缺氧诱导的促凋亡蛋白表达增加和Apaf-1结合结构域的修饰之间的联系，并激活半胱氨酸天冬氨酸蛋白酶原-9，这是启动新生儿大脑中程序性神经元死亡级联反应的关键步骤。

DESCRIPTION (provided by applicant): The long-term objective of our research program is to elucidate cellular and molecular mechanisms of brain injury induced by hypoxia in the newborn. The present study will focus on the mechanisms of procaspase-9 activation, a key step for initiating programmed cell death, during hypoxia. We hypothesize that during hypoxia, activation of procaspase-9 is due to increased expression and to post-translational modification of apoptotic proteins leading to an increased ratio of proapoptotic/antiapoptotic proteins (Bax/Bcl-2) in the cytosolic compartment. In addition, we hypothesize that excess Bax protein activates procaspase-9 through apoptotic protease activating factor-1 (Apaf-1). We propose that the hypoxia-induced increase in intranuclear Cainflux leading to increased expression of the proapoptotic proteins, Bax and Bad, is mediated through the increased activation of the Ca++-dependent kinase IV (CaM kinase IV) cascade, and results in phosphorylation of cyclic AMP-responsive element binding (CREB) protein at ser 133. In addition, we propose that hypoxia induces a modification of the affinity of Apaf-1 binding domains for ATP and cytochrome c favoring Apaf-1-mediated activation of procaspase-9. Using established techniques and the newborn piglet model, we will demonstrate that during hypoxia a) the increased expression of the proapoptotic protein Bax mediates activation of the apoptosome complex (procaspase-9-Apaf-1-Bcl-2/Bax) resulting in the activation of procaspase-9, b) the increased expression of proapoptotic proteins Bax and Bad is due to increased activation of the calcium-dependent CaM kinase IV cascade and increased phosphorylation of CREB in neuronal nuclei of newborn piglets, c) the hypoxia-induced modification of modulatory sites of the Apaf-1 molecule for ATP and cytochrome c and Bcl-2/Bax results in activation of procaspase-9 and d) by inhibiting nuclear Cainflux, inhibiting synthesis of proapoptotic proteins and inhibiting caspase-9 activity, we will prevent hypoxia-induced neuronal death in the newborn piglet brain. The proposed studies will establish a link between the hypoxia-induced increased expression of proapoptotic proteins and the modification of Apaf-1 binding domains, with activation of procaspase-9, a critical step that initiates the cascade of programmed neuronal death in the newborn brain.