SUPPRESSION OF PROTEIN SYNTHESIS IN THE REPERFUSED BRAIN

再灌注大脑中蛋白质合成的抑制

• 批准号: 2393122
• 负责人: GARY S KRAUSE
• 金额: $ 19.36万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2393122
• 关键词: brain injury; brain metabolism; calcium flux; cardiopulmonary resuscitation; cerebral ischemia /hypoxia; disease /disorder model; genetic translation; hippocampus; immunocytochemistry; insulin; laboratory rat; neuroprotectants; neurotoxicology; oxidative stress; phosphorylation; protein biosynthesis; proteolysis; reperfusion; tissue /cell culture; translation factor

More than 90% of the 70,000 patients resuscitated annually from cardiac arrest suffer permanent, often severe, brain injury as a sequela to the ischemia associated with the cardiac arrest. The broad, long-term objectives of our work are to understand the damage mechanisms operative in brain ischemia and reperfusion and thereby identify clinically effective therapeutic interventions to forestall the frequent occurrence of severe post-resuscitation brain injury. In those brain regions most vulnerable to damage by ischemia and reperfusion, neurons exhibit a substantial inhibition of post-ischemic protein synthesis. We propose that inhibition of protean synthesis (1) occurs at the level of formation of the initiation complex, (2) as a consequence of proteolysis and oxygen radical reaction products, (3) which lead to decreased levels or altered phosphorylation of specific translation initiation factors. We further propose that (4) translation competence during reperfusion can be protected or restored by either (a) providing the active initiation factor(s) involved (in vitro) or (b) by growth factor-mediated reversal of the reperfusion-induced altered phosphorylation of initiation factors. The specific aims of this project are to (l) examine the effect of global brain ischemia and reperfusion on the rate of initiation of protein translation. (2) examine key eukaryotic translation initiation factors (eIFs) for ischemia or reperfusion-induced alterations in levels, phosphorylation, and activity, and tissue-map observed eIF alterations, (3) examine the effects of calcium overload or radical damage on cultured neurons with respect to the above parameters (4) examine the effects of adding purified eIFs on in vitro protein translation in homogenates obtained from normal and reperfused brains and from radical-damaged or calcium overloaded cultured neurons. and (5) studs the effects of insulin as an exogenous growth factor on translation competence and phosphorylation of eIFs in the cell culture model and in the reperfused brain. Our preliminary data indicates that after 10-min. ischemia and 90- min reperfusion (1) translation initiation is inhibited. (2) eIF-4gamma is partially fragmented, and (3) eIF-2alpha is serine phosphorylated, and during more prolonged ischemia (4) eIF-4E undergoes degradation that in preliminary immunohistochemical studies is pronounced in vulnerable hippocampal neurons. The experimental design utilizes a rat model of cardiac arrest and resuscitation to characterize the effects of ischemia and reperfusion on the eIFs that control the initiation of protein synthesis (eIF-2, eIF-2B, eIF-4E, and eIF-4gamma), and utilizes a cultured rat neuroblastoma cell line (NB104 in which neuronal differentiation can be induced) to characterize the effects of controlled radical or calcium overload insults on translation and eIFs. Insulin is a clinically relevant growth factor that has been shown to improve post-ischemic neurologic outcome and to enhance translation by increased activity of eIF-4E and eIF-2alpha, and we will study its effects on translation and eIFs in both model systems.

每年从心脏复苏的70,000名患者中，超过90％ 逮捕遭受永久性的，通常严重的脑损伤，作为后遗症 与心脏骤停有关的缺血。宽阔，长期 我们工作的目标是了解损害机制的操作 在脑缺血和再灌注中，从而在临床上识别 有效的治疗干预措施以阻止频繁发生 严重的响应后脑损伤。 在那些最容易受到缺血和损害的大脑区域中 再灌注，神经元表现出极大的抑制后缺血性 蛋白质合成。我们建议抑制蛋白质合成（1） 发生在起始复合物的形成水平，（2）作为一个 蛋白水解和氧自由基反应产物的结果（3） 导致特定水平降低或磷酸化的变化 翻译起始因素。我们进一步建议（4）翻译 再灌注过程中的能力可以受到（a）的保护或恢复 通过（体外）提供主动启动因子或（b） 生长因子介导的再灌注诱导的逆转变化 起始因子的磷酸化。 该项目的具体目的是（l）检查全球的影响 脑缺血和对蛋白质起始率的再灌注 翻译。 （2）检查关键真核翻译起始因子 （EIF）用于缺血或再灌注诱导的水平改变， 磷酸化，活性以及组织图观察到的EIF改变， （3）检查钙超负荷或根治损伤对培养的影响 关于上述参数的神经元（4）检查 在匀浆中的体外蛋白质翻译上添加纯化的EIF 从普通和重新注射的大脑以及自由基损坏或 钙超载培养的神经元。 （5）螺母胰岛素的作用 作为翻译能力的外源增长因素和 EIF在细胞培养模型中的磷酸化和重复的磷酸化 脑。我们的初步数据表明10分钟后。缺血和90- 最小再灌注（1）翻译起始被抑制。 （2）EIF-4GAMMA是 部分碎片，（3）EIF-2Alpha是丝氨酸磷酸化的，并且 在较长的缺血期间（4）EIF-4E经历降解 初步免疫组织化学研究在脆弱 海马神经元。 实验设计利用心脏骤停的大鼠模型和 复苏以表征缺血和再灌注对 控制蛋白质合成启动的EIF（EIF-2，EIF-2B，， EIF-4E和EIF-4GAMMA），并使用培养的大鼠神经母细胞瘤细胞 线（可以诱导神经元分化的NB104） 表征受控自由基或钙超负荷侮辱的影响 关于翻译和EIF。胰岛素是临床相关的生长因子 已显示可改善缺血后神经系统结局，并 通过增加EIF-4E和EIF-2Alpha的活性来增强翻译，我们 将研究其对两个模型系统中翻译和EIF的影响。