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Investigating Abeta and alpha-synuclein toxicity by analyzing single-cell dynamic

通过分析单细胞动态研究 Abeta 和 α-突触核蛋白毒性

基本信息

批准号：
8837074
负责人：
LUKE J WHITESELL
金额：
$ 29.25万
依托单位：
WHITEHEAD INSTITUTE FOR BIOMEDICAL RES
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-04-15 至 2017-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8837074
关键词：
Affect Alzheimer&apos s Disease Amyloid beta-Protein Behavior Biochemical Pathway Biological Biological Models Biological Process Biology Cell model Cell physiology Cells Collaborations Complex Defect Dementia Development Disease Enhancers Event Gene Expression Regulation Genetic Global Change Grant Health Homeostasis Human Image Image Analysis Individual Investigation Knowledge Lead Life Masks Measurement Measures Mediating Methods Microfluidic Analytical Techniques Microfluidics Microscopy Modeling Molecular Bank Neurodegenerative Disorders Neurons Organelles Parkinson Disease Pathology Patients Pattern Peptides Pharmaceutical Preparations Pharmacology Physiology Poison Population Production Property Proteins Reporter Research Resolution Saccharomyces cerevisiae Saccharomycetales Societies System Therapeutic Time Toxic effect United States National Institutes of Health Work Yeast Model System Yeasts alpha synuclein base combat cost design drug candidate follow-up genome-wide high throughput screening imaging platform induced pluripotent stem cell innovation insight neurotoxicity novel novel strategies protein TDP-43 protein aggregation protein misfolding protein transport research study response screening single cell analysis small molecule tool trafficking

项目摘要

DESCRIPTION (provided by applicant): Neurodegenerative diseases represent a significant and increasing burden on society. In particular, Alzheimer's disease and Parkinson's disease are the most common neurodegenerative disorders in the world and affect 5.4 million and >500,000 people in the USA, respectively. In fact, the global cost of Alzheimer's disease and related dementias was estimated in 2010 to be ~1% of the world's gross domestic product (GDP). Despite the knowledge that virtually all neurodegenerative diseases originate with problems in the folding and trafficking of specific proteins in cells, there are simply no effectiv treatments to halt or cure these diseases. This is at least in part due to the lack of a mechanisti understanding of the disease pathology at the cellular level. The research proposed herein uses the budding yeast, Saccharomyces cerevisiae, as a cellular model system. This system has previously been shown to recapitulate unique features of neuronal toxicity observed in human neurodegenerative diseases. Significantly, all previous analyses in both yeast and neuronal models were limited to steady-state bulk measurements, which average over many cells and mask essentially all intracellular dynamics. These measurements cannot reveal important dynamic properties, which require observation of single cells over time. By contrast, proposed herein is a novel approach to systematically measure the global response of single cells to the expression of disease-relevant proteins, such as α-synuclein (Parkinson's disease) and the Aß peptide (Alzheimer's disease) by following the time-dependent changes of biochemical pathways, protein localization and trafficking, organelles, and metabolites in single cells. Biological circuits function inside cells and hence require a single-cell analysis to uncover all their behaviors. The single-cell resolution will be achieved through the development of an innovative microfluidic analysis platform, which permits the imaging of individual cells over time and allows for the quantification of dynamic responses in single cells. Such systematic single-cell measurements will reveal novel mechanisms relevant to cellular toxicity and will contribute to a systems-level understanding of how Aß and α-synuclein poison cellular physiology. Further, this high-throughput imaging platform will be employed to investigate the cell biological effects of ~100 small- molecule compounds that were previously identified through large-scale small-molecule screening as potential drug candidates and are currently being verified for their efficacy in neurons. Discoveries made with the high- throughput imaging platform described herein will later be validated in mammalian neuronal systems. Significantly, the systems-level, single-cell analysis approach described herein has never before been applied to the investigation of neurodegenerative diseases and has the potential to uncover novel insights about intracellular perturbations on a global scale, which is necessary for identifying effective therapeutics to combat these diseases.

描述（由申请人提供）：神经退行性疾病代表了社会的重大和日益增加的负担。特别地，阿尔茨海默病和帕金森病是世界上最常见的神经退行性疾病，在美国分别影响540万和> 500，000人。事实上，据估计，2010年阿尔茨海默病和相关痴呆症的全球成本约占世界国内生产总值（GDP）的1%。尽管几乎所有的神经退行性疾病都起源于细胞中特定蛋白质的折叠和运输问题，但根本没有有效的治疗方法来阻止或治愈这些疾病。这至少部分是由于缺乏在细胞水平上对疾病病理学的机制理解。本文提出的研究使用芽殖酵母，酿酒酵母，作为细胞模型系统。该系统先前已被证明概括了在人类神经退行性疾病中观察到的神经元毒性的独特特征。值得注意的是，所有以前在酵母和神经元模型中的分析都限于稳态批量测量，其平均超过许多细胞并基本上掩盖了所有细胞内动力学。这些测量不能揭示重要的动态特性，这需要随着时间的推移观察单个细胞。相比之下，本文提出了一种新的方法，通过跟踪单细胞中生化途径、蛋白定位和运输、细胞器和代谢物的时间依赖性变化，系统地测量单细胞对疾病相关蛋白质（例如α-突触核蛋白（帕金森病）和阿尔茨海默病肽（阿尔茨海默病））表达的整体响应。生物回路在细胞内发挥作用，因此需要单细胞分析来揭示它们的所有行为。单细胞分辨率将通过开发创新的微流体分析平台来实现，该平台允许随着时间的推移对单个细胞进行成像，并允许对单细胞中的动态响应进行量化。这种系统性的单细胞测量将揭示与细胞毒性相关的新机制，并将有助于从系统水平了解Ablast和α-突触核蛋白如何毒害细胞生理学。此外，这种高通量成像平台将用于研究约100种小分子化合物的细胞生物学效应，这些化合物先前通过大规模小分子筛选被鉴定为潜在的候选药物，目前正在验证其在神经元中的功效。用本文所述的高通量成像平台进行的发现稍后将在哺乳动物神经元系统中验证。值得注意的是，本文所述的系统水平的单细胞分析方法以前从未应用于神经退行性疾病的研究，并且有可能在全球范围内揭示关于细胞内扰动的新见解，这对于鉴定对抗这些疾病的有效治疗剂是必要的。