Single-Cell Functional Proteomics Study of Neurodegeneration in Alzheimer's Disease

摘要 阿尔茨海默病(AD)和其他痴呆症是美国迫在眉睫的公共卫生危机，预计 在未来几十年产生灾难性的医疗保健和经济影响。尽管付出了巨大的努力 在AD研究中，目前的治疗方法在临床上只提供了微乎其微的好处。许多关于AD早期的研究都有 发现大脑的某些区域比其他区域更容易退化。但对这起事件的调查 这种选择性退化背后的分子机制是具有挑战性的，并受到以下复杂性的阻碍 细胞机械和异质性。尽管基于测序的单细胞转录组学可以解决 这一挑战在一定程度上，结论通常还需要在蛋白质水平上进行验证。因为大多数人 细胞功能、药物靶点和临床诊断是基于蛋白质信号和生物标志物的 开发与之相对应的功能蛋白质组学技术将有利于提供另一种 与治疗学更直接相关的观点。此外，在组学水平上调查一大组蛋白质 这是必要的，因为AD发病机制的关键蛋白质队列仍然难以捉摸。在这个项目中，我们将 开发一种单细胞功能蛋白质组学工具，可以通过测量300次来补充单细胞测序 与神经退行性变相关的关键蛋白质。这项新技术将增加功能的覆盖范围 蛋白质组比主流技术提高了10-100倍，这将把生物医学研究带入一个新的领域 水平。这一工具与体内microPET成像和体外标本成像相结合，将有助于识别 与易受伤害的脑细胞亚群相关的生物标志物和调控网络 抗神经退行性变和淀粉样β蛋白毒性。提出的目标是：(1)建立单细胞 叠加式雾化技术分析300种细胞内和表面蛋白，并优化检测方法 通过在小鼠细胞系上进行测试来确定条件，以及(2)确定 使用单细胞叠加式MIST技术、microPET和变性技术的脆弱与耐药脑细胞 在AD小鼠模型上进行成像。该项目的成功将产生创新的技术和方法 这为从新的临床重要角度深入研究AD的发病机制奠定了基础 为进一步研究阿尔茨海默病的发展和药物靶点的全脑研究奠定基础。

Summary Alzheimer’s disease (AD) and other dementias are a looming public health crisis in the US which is expected to generate catastrophic healthcare and economic impacts over the next decades. Despite enormous efforts made in AD research, current treatments provide only marginal benefits in the clinic. Many studies on early AD have found certain regions of the brain are more vulnerable to degeneration than others. But the investigation into the molecular mechanisms behind such selective degeneration is challenging and is hampered by the complexity of cellular machinery and heterogeneity. Although the sequencing based single-cell transcriptomics can address this challenge to certain degree, the conclusions usually still need validation at the protein level. Since most cellular functions, drug targets, and clinical diagnosis are based on protein signaling and biomarkers, the development of a counterpart functional proteomics technology would be beneficial to provide another perspective more directly relevant to therapeutics. Besides, surveying a large panel of proteins at the omics level is necessary since the cohort of proteins critical to AD pathogenesis remains elusive. In this project, we will develop a single-cell functional proteomics tool that could complement single-cell sequencing by measuring 300 critical proteins relevant to neurodegeneration. This novel technology will increase the coverage of functional proteome by 10-100 times over prevailing technologies, and it will take biomedical research broadly to a new level. This tool, in tandem with in vivo microPET imaging and in vitro specimen imaging, will facilitate identification of the biomarkers and regulatory networks pertinent to the subpopulations of brain cells that are vulnerable or resistant to neurodegeneration and amyloid beta toxicity. The proposed aims are (1) Establish single-cell reiterative MIST technology for analyzing 300 intracellular and surface proteins, and optimize the assay conditions by testing on a mouse cell line, and (2) Determine the molecular markers and regulatory networks of vulnerable versus resistant brain cells using single-cell reiterative MIST technology, microPET and degeneration imaging on an AD mouse model. The success of this project will generate innovative technology and methods that enable deep investigation of AD pathogenesis from a new, clinically important perspective, and it will lay the foundation for further brain-wide study of AD development and identification of drug targets.