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

总结 阿尔茨海默氏症（AD）和其他痴呆症是美国迫在眉睫的公共卫生危机，预计将 在未来几十年内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，目前的治疗在临床上仅提供边际效益。许多关于早期AD的研究 发现大脑的某些区域比其他区域更容易退化。但是对 这种选择性变性背后的分子机制是具有挑战性的，并且受到以下复杂性的阻碍： 细胞机制和异质性。尽管基于测序的单细胞转录组学可以解决 这在一定程度上是一种挑战，但结论通常仍需要在蛋白质水平上进行验证。由于大多数 细胞功能、药物靶点和临床诊断是基于蛋白质信号传导和生物标志物， 开发对应的功能蛋白质组学技术将有益于提供另一种 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 是必要的，因为对AD发病机制至关重要的蛋白质组仍然难以捉摸。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个 与神经变性相关的关键蛋白质。这项新技术将增加功能的覆盖面， 蛋白质组的10-100倍，它将把生物医学研究广泛地带到一个新的领域。 水平该工具与体内microPET成像和体外标本成像相结合，将有助于识别 的生物标志物和监管网络有关的亚群的脑细胞是脆弱的，或 抗神经变性和淀粉样蛋白β毒性。提出的目标是（1）建立单细胞 MIST技术用于分析300种细胞内和表面蛋白，并优化分析方法 通过在小鼠细胞系上进行测试来确定条件，以及（2）确定以下的分子标记和调控网络： 使用单细胞重复MIST技术、microPET和变性， 在AD小鼠模型上成像。该项目的成功将产生创新的技术和方法 这使得从一个新的，临床上重要的角度深入研究AD发病机制，它将奠定 为进一步研究AD的发展和药物靶点的确定奠定了基础。

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.