High-Resolution Spatial MIST Technology for Functional Proteomic Study of Neuroinflammation in Alzheimer's Disease

总结 阿尔茨海默病（AD）是最常见的痴呆症，在美国是一个迫在眉睫的危机。尽管 AD研究取得了实质性进展，神经退行性变的分子和细胞过程 仍然没有得到很好的理解，并且AD疗法没有给患者带来显著的益处。传统 AD的特征包括淀粉样蛋白β聚集和神经纤维缠结沉积，而最近 炎症是大脑中的一种先天免疫反应，是第三个标志。尤其是炎症 发生在β淀粉样蛋白瘟疫和神经系统缠结的中心附近， 与神经退行性疾病的进展协同作用。了解分子 细胞在神经炎症中的功能作用机制及其对神经元的影响是研究神经炎症的关键。 寻找有效的治疗靶点。由于高度复杂性和空间异质性的性质， 在神经变性研究中，研究已经极大地转向下一代测序和转录组学工具。 这些关于基因表达的结果仍然需要蛋白质水平的验证，因为蛋白质执行大部分细胞功能。 功能和生化过程。目前对组织样品的多重蛋白质测定要么是人工测定， 密集和低覆盖或低空间分辨率。在这个项目中，我们的目标是开发一个空间蛋白质组学， 技术与蜂窝解决方案，以填补技术空白，及时解决最迫切的问题， AD机制。该技术建立在测量~200 AD的多重原位标记（MIST）阵列上 我们的初步研究中来自单个神经元的相关蛋白质。具有比其他产品高约10- 100倍的多路复用能力 空间蛋白质工具，我们的空间MIST将测量空间中最重要的调控蛋白和标记物， 脑切片的局部细胞。我们提出的两个具体目标包括：（1）优化实验技术 在空间MIST中检测280个在整个小鼠脑切片的信号传导和调节中的关键蛋白，以及（2） 通过空间MIST分析AD期间Aβ积聚和tau富集区附近细胞的分子特征 进展该项目的完成将产生一种广泛使用的使能技术和方法 在AD研究界，从一个新的，临床相关的角度研究AD的发病机制。这 技术将为未来AD发展机制的研究和潜在的 治疗目标

Summary Alzheimer’s disease (AD) is the most common form of dementia and is a looming crisis in the US. Despite substantial progress made in AD research, the molecular and cellular processes governing neurodegeneration are still not well understood, and AD therapies have not resulted in significant benefits to patients. The traditional hallmarks of AD include amyloid beta aggregation and neurofibrillary tangle deposition, while recently inflammation, an innate immune response in the brain, emerges as a third hallmark. Inflammation particularly occurs near epicenters of amyloid beta plagues and neurofibrillary tangles, and it involves complicated cellular interactions that synergize with the progression of neurodegeneration. Understanding the molecular mechanisms of the functional roles of the cells in neuroinflammation and its influences on neurons is the key to searching for effective therapeutic targets. Due to the nature of high complexity and spatial heterogeneity, recent research has vastly turned to next generation sequencing and transcriptomics tools in neurodegeneration studies. These results on gene expression will still need protein-level validation since proteins carry out most of cellular functions and biochemical processes. The current multiplexed protein assays on tissue samples are either labor intensive and low coverage or in low spatial resolution. In this project, we aim to develop a spatial proteomics technology with cellular resolution to fill the technological gap and timely address the most imperative issues in AD mechanisms. This technology is built upon a multiplex in situ tagging (MIST) array that measures ~200 AD relevant proteins from single neurons in our preliminary study. With ~10-100X higher multiplexity than other spatial protein tools, our spatial MIST will measure most important regulatory proteins and markers in spatially localized cells of brain sections. Two specific aims we propose include (1) Optimize the experimental techniques in spatial MIST for detecting 280 key proteins in signaling and regulation of whole mouse brain slices, and (2) Profile the molecular features of cells near Aβ accumulation and tau enriched regions by spatial MIST during AD progression. The completion of this project will generate an enabling technology and method widely accessible in the AD research community to investigate AD pathogenesis from a new, clinically relevant perspective. This technology will lay the foundation for future mechanistic studies of AD development and identification of potential therapeutic targets.