Gene Repair in signal Transduction after CNS Injury

描述（由申请人提供）：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，每年造成超过500,000名受害者。脑缺血引起脂质、蛋白质和核酸的氧化损伤，减少能量来源，导致功能恶化，导致细胞死亡。修复过程通常修复基因的错误很少。然而，尽管存在这些修复机制，缺血仍会导致氧化性DNA损伤（odl）升高。这些事件与Fos表达和信号转导通路中其他晚期基因的关键激活同时发生。迄今为止，odl对大脑基因功能的影响尚未完全了解。我们将利用雄性C57black/6小鼠的前脑缺血(30-90 min)/再灌注（FblR）模型研究基因损伤是否会影响c-fos基因的功能。该模型诱导海马神经元死亡，类似于在心脏骤停后和一些神经系统疾病中观察到的情况。我们的假设是信号转导基因的修复有利于氧化应激后神经元的恢复。具体目标是：1。为了确定FblR后Fos活性是否诱导基因修复功能，我们将使用抑制脑一氧化氮但增强FblR后c-fos mRNA表达的3-溴-7硝基茚唑测定(a) Fos蛋白、修复活性和海马神经元死亡。(b)利用特异性消除FblR后Fos表达的反义技术研究海马Fos活性、修复活性和神经元死亡之间的相关性。2. 为了确定基因损伤修复是否能保护海马，我们将测定FblR后c-fos转录物中的ODLs和ORLs（碱基修饰）。(d) FblR后修复活性、Fos活性与晚期效应基因表达的相关性。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，证明了这些研究的可行性。我们已经开发出一种有效的方法来将反义cDNA传递到大脑中，从而消除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。未来的研究方向包括在FblR治疗的动物中检测额外的基因激活因子，以及在这些基因激活因子上应用额外的反义cDNA来阐明它们在大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.