CEREBRAL OXIDATIVE METABOLISM IN HYPOXIC NEWBORNS

描述(申请人提供)：我们研究计划的长期目标是阐明新生儿缺氧所致脑损伤的细胞和分子机制。本研究将集中在低氧时原天冬氨酸酶-9激活的机制，这是启动细胞程序性死亡的关键步骤。我们推测，在缺氧过程中，原天冬氨酸氨基转移酶-9的激活是由于表达增加和翻译后对凋亡蛋白的修饰，导致胞浆中促凋亡/抗凋亡蛋白(Bax/Bcl2)的比率增加。此外，我们还假设过量的Bax蛋白通过凋亡蛋白激活因子-1(APAF-1)激活原天冬氨酸蛋白酶-9。我们认为，低氧诱导的核内钙离子通量增加导致促凋亡蛋白Bax和Bad的表达增加，是通过钙依赖的蛋白IV(CaM Kinase IV)级联通路的激活，并导致SER133处的环状AMP反应元件结合(CREB)蛋白的磷酸化。此外，我们认为，低氧诱导APAF-1结合区与ATP和细胞色素c的亲和力发生改变，有利于APAF-1介导的原天冬氨酸酶-9的激活。利用已建立的技术和新生仔猪模型，我们将证明：a)在缺氧期间，促凋亡蛋白Bax的表达增加介导了凋亡体复合体(proaspase-9-APAF-1-Bcl2/Bax)的激活，导致了proaspase-9的激活，b)促凋亡蛋白Bax和Bad的表达增加是由于新生仔猪神经元核中钙依赖的CaM激酶IV级联激活和CREB磷酸化增加所致，C)低氧诱导APAF-1分子对ATP和细胞色素c的调节位点的修饰，以及Bcl2/Bax的修饰导致原aspase-9的激活；d)通过抑制核流、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仔猪脑内神经元死亡。拟议的研究将建立低氧诱导的促凋亡蛋白表达增加与APAF-1结合域的修饰之间的联系，并激活Proaspase-9，这是启动新生儿大脑中神经元程序性死亡的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.