FRET assay for in situ assessment of nucleolytic lysosomal cell death in Alzheimer's neurodegeneration

原位FRET法检测溶酶体核溶解细胞死亡 阿尔茨海默氏神经变性 摘要 溶酶体进行性衰竭是阿尔茨海默病(AD)的一个显著特征。它链接到 神经变性作为神经细胞死亡的主要诱因，既有凋亡的，也有非凋亡的 机械装置。通过移除凋亡效应器来防止细胞死亡的失败暴露了底层的非 细胞凋亡的溶酶体死亡途径。其中，溶酶体的核溶解途径仍然是最直接的。 同时，也是研究最少的人。其关键事件是DNase II从溶酶体中逃逸并进入 进入细胞核，导致核DNA断裂。这种快速的细胞死亡机制是平行激活的 可有坏死性、凋亡性或混合性形态。因此，它特别困难 在组织切片上进行区分。因此，尽管这种细胞死亡机制具有很高的重要性，但它仍然 缺乏选择性原位标记的特定工具。 在这个项目中，我们将通过开发第一个选择性标签技术来缩小这一技术差距 组织切片核溶酶体细胞死亡(LCD)。这项技术将采用一种新的标签方法 使用交错寡核苷酸探针检测仅由溶酶体核酸酶产生的特征性DNA损伤。这 标记将与溶酶体DNase II的FRET共定位相结合。DNase II只有在 离它的断裂处很近。这些方法将共同标记逃逸的溶酶体核酸酶 以及它所产生的特定的DNA损伤。这项技术将使检测和评估主要 组织切片中的细胞死亡机制。然后我们将通过共同标记另一种来扩展新的检测方法 同时激活AD节段的细胞死亡通路。该项目将全面引进和推广 验证了该新技术作为液晶原位分析必不可少的分子工具。 具体目标： 1.建立第一种特异性原位标记溶酶体DNA酶II依赖性细胞死亡的方法。这个 该方法将在固定的组织切片上工作，并选择性地标记正在进行溶核细胞死亡的细胞， 将它们与其他形式的程序性和非程序性死亡区分开来。它将区分和 选择性地标记从溶酶体逃逸的DNase II产生的特征性DNA损伤。 2.扩展新方法，将其与基于FRET的溶酶体DNase II近端共定位相结合 它特有的DNA断裂。这将使对经历DNA酶的细胞的高度特异性检测成为可能 依赖II的细胞死亡。 3.应用最新发展的分子技术对溶酶体核酸酶介导的途径进行成像 广告。验证新方法作为AD细胞死亡综合多标记评估的一部分。 将新的分析方法整合到一套评估细胞死亡的复杂条件下的方法中 神经退行性变。

FRET assay for in situ assessment of nucleolytic lysosomal cell death in Alzheimer's neurodegeneration Abstract The progressive failure of lysosomes is a signature feature of Alzheimer's disease (AD). It is linked to neurodegeneration, as a primary inducer of neuronal cell death through both apoptotic and non-apoptotic mechanisms. Failures to prevent cell death by removing apoptotic effectors expose an underlying layer of non- apoptotic lysosomal death pathways. Among these, the lysosomal nucleolytic pathway remains the most direct and, simultaneously, the least studied. Its key event is the escape of DNase II from lysosomes and its entry into the nucleus resulting in cleavage of nuclear DNA. This rapid mechanism of cell death activates in parallel with other pathways and can have necrotic, apoptotic, or mixed morphology. In result, it is particularly difficult to distinguish in tissue sections. Consequently, in spite of its high importance, this cell death mechanism still lacks specific tools for its selective labeling in situ. In this project we will close this technological gap by developing the first technology for selective labeling of nucleolytic lysosomal cell death (LCD) in tissue sections. The technology will employ a new labeling approach using staggered oligoprobes detecting characteristic DNA damage produced only by lysosomal nuclease. This labeling will combine with FRET co-localization of lysosomal DNase II. DNase II will become fluorescent only in close proximity to its breaks. Together these approaches will co-label both the escaped lysosomal nuclease and the specific DNA damage it produces. The technology will enable detection and evaluation of the major cell death mechanism in tissue sections. We will then expand the new assay by co-labeling the other simultaneously active cell death pathways in AD sections. The project will introduce and comprehensively validate the new technology as the essential molecular tool for the in situ analysis of LCD. Specific aims: 1. To develop the first approach for specific in situ labeling of lysosomal DNase II-dependent cell death. The approach will work in fixed tissue sections and will selectively mark cells undergoing nucleolytic cell death, separating them from other forms of programmed and non-programmed death. It will distinguish and selectively label the characteristic DNA damage produced by DNase II escaped from lysosomes. 2. To expand the new approach by combining it with FRET-based co-localization of lysosomal DNase II near its characteristic DNA breaks. This will make possible the highly specific detection of cells undergoing DNase II-dependent cell death. 3. To apply the newly developed molecular technology to image the lysosomal nuclease-mediated pathway in AD. To validate the new method as part of the comprehensive multi-labeling assessment of cell death in AD. To incorporate the new assay into a set of methods which evaluate cell death in the complex conditions of neurodegeneration.