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

FRET 测定法原位评估阿尔茨海默病神经变性中溶酶体细胞死亡

• 批准号: 10287444
• 负责人: VLADIMIR V DIDENKO
• 金额: $ 42.5万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-15 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10287444
• 关键词: Aging; Alzheimer' s Disease; Apoptosis; Apoptotic; Area; Biochemical; Biological Assay; Biomedical Technology; Cell Culture Techniques; Cell Death; Cell Nucleus; Cells; Cessation of life; Characteristics; Complex; Conflict (Psychology); Cytopathology; DNA; DNA Damage; Deoxyribonucleases; Detection; Development; Evaluation; Event; Failure; Fingerprint; Fluorescence Resonance Energy Transfer; Goals; Heart Diseases; Histologic; Image; In Situ; Intervention; Label; Legal patent; Link; Lysosomes; Malignant Neoplasms; Manuals; Mediating; Medical; Methods; Modeling; Molecular; Molecular Analysis; Molecular Profiling; Morphology; Nature; Necrosis; Nerve Degeneration; Neurodegenerative Disorders; Nuclear; Pathogenesis; Pathology; Pathway interactions; Permeability; Play; Prevention; Proteins; Research; Resources; Role; Series; Stroke; Technology; Testing; Tissues; Work; amyloidogenesis; base; bioimaging; brain tissue; cell injury; molecular marker; neurodegenerative dementia; neuron loss; new technology; novel; novel strategies; novel therapeutics; nuclease; prevent; stem; tool

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.

FRET测定用于在原位评估细胞核溶解性溶酶体细胞死亡 阿尔茨海默氏神经变性 摘要 溶酶体的进行性衰竭是阿尔茨海默病（AD）的标志性特征。本附件涉及 神经变性，作为通过凋亡和非凋亡的神经元细胞死亡的主要诱导物， 机制等未能通过去除细胞凋亡效应器来防止细胞死亡会暴露出底层的非- 细胞凋亡溶酶体死亡途径。其中，溶酶体溶核途径仍然是最直接的 同时也是研究最少的其关键事件是DNA酶II从溶酶体中逃逸并进入溶酶体， 进入细胞核导致细胞核DNA的分裂。这种快速的细胞死亡机制是平行激活的 与其他途径结合，并且可以具有坏死、凋亡或混合形态。因此， 来区分组织切片。因此，尽管其高度重要性，但这种细胞死亡机制仍然存在。 缺乏用于原位选择性标记的特定工具。 在这个项目中，我们将通过开发第一种选择性标记的技术来缩小这一技术差距。 组织切片中的溶核溶酶体细胞死亡（LCD）。该技术将采用一种新的标签方法 使用交错寡探针检测仅由溶酶体核酸酶产生的特征性DNA损伤。这 标记将联合收割机与溶酶体DNA酶II的FRET共定位结合。DNA酶II仅在 靠近其断裂处。这些方法将共同标记逃逸的溶酶体核酸酶 以及它所造成的特定DNA损伤该技术将使检测和评估的主要 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个来扩展新的检测方法。 同时活跃的细胞死亡途径。该项目将全面介绍和 验证了新技术作为LCD原位分析的必要分子工具。 具体目标： 1.首次建立特异性原位标记溶酶体DNA酶II依赖性细胞死亡的方法。的 该方法将在固定的组织切片中起作用并且将选择性地标记经历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 选择性标记由从溶酶体逃逸的DNase II产生的特征性DNA损伤。 2.通过将其与基于FRET的溶酶体DNA酶II的共定位结合来扩展新方法， 它特有的DNA会断裂这将使高度特异性检测经历DNA酶的细胞成为可能 II依赖性细胞死亡。 3.应用最新发展的分子技术对溶酶体核酸酶介导的通路进行成像， AD.验证新方法作为AD细胞死亡综合多标记评估的一部分。 将新的检测方法纳入一套方法中，以评估在复杂条件下的细胞死亡， 神经变性