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

点击翻译按钮获取中文摘要

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.