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

描述(由申请者提供)：神经退行性疾病给社会带来了巨大且日益沉重的负担。特别是，阿尔茨海默氏症和帕金森氏症是世界上最常见的神经退行性疾病，在美国分别影响540万人和50万人。事实上，据估计，2010年阿尔茨海默病和相关痴呆症造成的全球损失约占全球国内生产总值(GDP)的1%。尽管人们知道，几乎所有的神经退行性疾病都源于细胞内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。这至少在一定程度上是由于在细胞水平上缺乏对疾病病理的机制了解。本文提出的研究使用萌芽酵母酿酒酵母作为细胞模型系统。这个系统以前已经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，之前在酵母和神经元模型中的所有分析都局限于稳态整体测量，这些测量平均在许多细胞上，并基本上掩盖了所有细胞内的动力学。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、蛋白质定位和运输、细胞器和代谢物的随时间的变化，系统地测量单细胞对疾病相关蛋白表达的整体反应，如α-Synuclein(帕金森氏病)和A？多肽(阿尔茨海默病)。生物回路在细胞内运作，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，并允许对单个细胞的动态反应进行量化。这种系统的单细胞测量将揭示与细胞毒性相关的新机制，并将有助于在系统水平上了解Aü和α-Synuclein如何毒害细胞生理。此外，这一高通量成像平台将被用于研究约100种小分子化合物的细胞生物学效应，这些化合物以前通过大规模小分子筛选被确定为潜在的候选药物，目前正在验证其在神经元中的疗效。用这里描述的高通量成像平台所做的发现稍后将在哺乳动物神经系统中得到验证。值得注意的是，本文描述的系统级单细胞分析方法以前从未被应用于神经退行性疾病的研究，并有可能在全球范围内发现关于细胞内扰动的新见解，这对于寻找有效的治疗方法来对抗这些疾病是必要的。

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.