Gene Repair in signal Transduction after CNS Injury

描述(申请人提供)：由心脏骤停和中风引起的缺血/再灌注型脑损伤是美国死亡和残疾的主要原因，每分钟攻击一人，每年造成50多万人死亡。脑缺血导致脂质、蛋白质和核酸的氧化损伤，减少能量来源，从而导致功能恶化，导致细胞死亡。修复过程通常会修复基因，几乎不会出错。然而，尽管有这些修复机制，缺血仍会导致氧化DNA损伤(ODL)增加。这些事件与Fos的表达和信号转导途径中其他晚期基因的关键激活同时发生。到目前为止，ODLS对大脑基因功能的影响还不完全清楚。我们将使用雄性C57Black/6小鼠的前脑缺血(30-90分钟)/再灌注(FblR)模型来研究基因损伤是否会影响c-fos基因的功能。这种模型导致海马区神经元死亡，类似于心脏骤停后观察到的情况和一些神经疾病。我们的假设是，修复信号转导中的基因有利于氧化应激后神经元的恢复。1.确定Fos活性是否诱导FblR后的基因修复功能，我们将使用3-溴-7-硝基吲唑来检测Fos蛋白、修复活性和神经元死亡。3-溴-7-硝基吲唑抑制FblR后脑内一氧化氮，但促进c-FOS mRNA的表达。(B)使用特异性取消FblR后Fos表达的反义技术，研究海马区Fos活性、修复活性和神经元死亡之间的相关性。2.确定基因损伤修复是否对海马区具有保护作用，我们将在FblR后确定c-fos转录本中的ODL和ORL(碱基修饰)。(D)FblR后修复活性、Fos活性与迟效基因表达的相关性。我们已经通过开发一系列技术来检测大脑中Fos的表达和DNA修复过程，从而证明了研究的可行性。我们已经开发出有效的方法来传递反义cDNA来消除大脑中Fos的活性。几个终点(FblR诱导的Fos/AP-1活性、神经生长因子mRNA、基因修复活性和神经元死亡)将被测量。未来的发展方向包括在用FblR治疗的动物中检测额外的基因激活剂，以及在这些基因激活剂上应用额外的反义cDNA来阐明它们在脑修复过程中的作用。

DESCRIPTION (provided by applicant): Brain damage of the ischemia/reperfusion type resulting from cardiac arrest and stroke is a major cause of mortality and disability in the United States, attacking one person every minute, and creating more than 500,000 victims every year. Cerebral ischemia causes oxidative damage to lipids, proteins and nucleic acids, reduces energy source with consequent functional deterioration leading to cell death. Restoration processes normally repair genes with few errors. However, ischemia causes elevated oxidative DNA lesions (ODLs) despite these repair mechanisms. These episodes occur concurrently with Fos expression and critical activation of other late genes in the signal transduction pathway. To date, the effect of ODLs on gene function of the brain is not totally understood. We will investigate whether gene damage could affect the function of the c-fos gene using a forebrain ischemia (30-90 min)/reperfusion (FblR) model in male C57black/6 mice. This model induces neuronal death in the hippocampus, similar to that observed after cardiac arrest and in some neurological disorders. Our hypothesis is that repair of genes in the signal transduction benefits neuronal recovery after oxidative stress. The specific aims are to: 1. Establish whether Fos activity induces gene repair function after FblR, we will determine (a) Fos protein, repair activity and neuronal death in the hippocampus using 3-bromo-7nitroindazole that inhibits brain nitric oxide but enhances the expression of c-fos mRNA after FblR. (b) Correlation in Fos activity, repair activity and neuronal death in the hippocampus using antisense technology that specifically abolishes the expression of Fos after FblR. 2. Establish whether repair of gene damage protects the hippocampus, we will determine (c) ODLs and ORLs (base modifications) in the c-fos transcript after FblR. (d) Correlation between repair activity, Fos activity and the expression of late effector genes after FblR. We have shown the feasibility of the studies by developing an array of technology to detect Fos expression and DNA repair processes in the brain. We have developed effective means to deliver antisense cDNA that abolishes Fos activity to the brain. Several end-points (FblR-induced Fos/AP-1 activity, nerve growth factor mRNA, gene repair activity and neuronal death) will be measured. Future directions include detections of additional gene activators in animals treated with FblR, and application of additional antisense cDNA to these gene activators to elucidate their roles in repair process of the brain.