FRET probes for analysis of serpin-mediated apoptosis in focal brain ischemia

描述（申请人提供）：FRET探针用于分析局灶性脑缺血中蛇形蛋白介导的细胞凋亡。鉴于细胞凋亡研究的深远意义，有必要找到能够区分不同凋亡途径的探针。丝氨酸蛋白酶依赖途径是最近发现的凋亡程序之一，目前仍缺乏特异性的工具来原位可视化。Serpin 1B是该通路的关键成员，是神经组织凋亡死亡的重要效应因子。在代谢应激下，它转化为活性内切酶。这种独特的转变发生在缺氧条件下，同时暴露了一个活跃的dna酶位点和一个核定位信号。新形成的DNA降解酶迅速进入细胞核，导致细胞死亡。尽管这种非caspase凋亡机制非常重要，但目前还没有方法在组织切片和活细胞培养中选择性地标记它。在本项目中，我们将开发这些方法并将其应用于局灶性脑缺血中蛇形蛋白介导的细胞凋亡的研究。具体目标：1；建立新的基于fret的方法来选择性标记蛇形蛋白介导的组织切片细胞凋亡。该方法将同时共同检测丝氨酸蛋白酶1b衍生的核酸内切酶蛋白及其产生的特征DNA断裂。2. 开发新的基于fret的方法来选择性标记蛇形蛋白介导的活细胞凋亡。FRET探针只有在检测到蛇形蛋白介导途径的特定标记后才会荧光。3. 应用新开发的分子工具研究局灶性脑缺血。探讨蛇蛋白介导的细胞凋亡在大鼠局灶性脑缺血模型中的作用；研究其产生机制、动态和分布模式。该项目将为细胞凋亡的研究，特别是缺血的研究引入使能技术。它们在局灶性脑缺血中的首次系统应用将为未来脑卒中的临床和研究调查以及有效治疗干预的发展提供有用的信息。

DESCRIPTION (provided by applicant): FRET probes for analysis of serpin-mediated apoptosis in focal brain ischemia. In light of the profound significance of apoptosis research, it s essential to have probes which can discriminate between different apoptotic pathways. The serine protease- dependent pathway is one of the most recently identified apoptotic programs, which still lacks specific tools for its visualization in situ. Serpin 1B is a key member of this pathway and is an important effector of apoptotic death in neural tissue. In metabolic stress it transforms into an active endonuclease. This unique transition occurs in hypoxia and simultaneously exposes an active DNase site and a nuclear localization signal. The newly formed DNA degrading enzyme promptly moves into the nucleus causing cell death. In spite of the high importance of this non-caspase apoptosis mechanism, there are currently no methods to selectively label it in tissue sections and in live cell cultures. In this project we will develp such methods and will apply them to study serpin-mediated apoptosis in focal brain ischemia. Specific aims: 1. To develop the new FRET-based approach for selective labeling of serpin-mediated apoptosis in tissue sections. The approach will simultaneously co-detect serpin 1B-derived endonuclease protein and characteristic DNA breaks which it produces. 2. To develop the new FRET-based approach for selective labeling of serpin-mediated apoptosis in live cell cultures. The FRET probes will become fluorescent only after they detect a specific marker of serpin-mediated pathway. 3. To apply the newly developed molecular tools to study focal cerebral ischemia. To evaluate the role of serpin-mediated apoptosis in focal brain ischemia in the rat model; to investigate its initiation mechanisms, dynamics, and patterns of distribution. The project will introduce enabling technologies for apoptosis research in general, and for studies in ischemia in particular. Their first systematic application to focal brain ischemia will provide information useful for the future clinical and research investigations of stroke, and for the development of effective therapeutic interventions.