Platform technologies for scalable highly multiplexed proteomic phenotyping of the brain

哺乳动物大脑的复杂性是任何其他器官都无法比拟的，而且了解它的细胞 它们的组成及其全脑组织对于了解大脑的功能和功能障碍是必不可少的。 已经做出了广泛的努力，通过各种透镜绘制脑细胞图，并建立了 产生新见解的无价数据库。然而，每个大脑只有几个分子、细胞类型或区域 已经在非人类灵长类动物和人类大脑中进行了映射，但未能捕捉到全脑范围的复杂细胞间 个人内部的关系。我们在这里的目标是开发通用的、易于采用的平台技术 从小鼠、绒猴到人类的哺乳动物大脑的高度多元化、快速、可扩展的蛋白质组学图谱。 我们建议(目标1)开发分子工具包和平台技术(Elast-saber)以实现高度 小鼠、绒猴、人脑的多路可伸缩蛋白质组学表型。我们将开发一个图书馆， 以100多种规范细胞类型标记为靶点的正交型DNA条形码抗体，与军刀兼容 扩增(由皮鹏音小组开发)和最后运输(由皮彭音小组开发 广浑涌)。我们还将(目标2)验证技术平台，并展示其广泛的实用性和 通过绘制小鼠、绒猴、人类初级运动皮质(M1)的细胞景观图来实现可扩展性 大脑，包括肌萎缩侧索硬化症(ALS)病理组织。我们将交叉验证我们的方法 使用发表于BICCN的MerFish和单细胞测序数据。我们将进行跨物种表演 比较分析，以了解保守的或进化上不同的M1的细胞特征。最后，我们会 (目标3)建立以网络为基础的互动平台，用于数据传播和社区使用。我们将建造 多模式脑图谱允许跨物种比较，并开发小鼠的神经本体论， 绒猴和人脑。重要的是，我们将开发一个以信息学和在线为基础的数据库 可视化工具，允许人们查看、比较和分析多路传输的可伸缩蛋白质组成像数据 跨越三个物种。这些信息学工具将有能力标准化和适应组织学， 连接性、基因表达和活体成像。用户将能够比较多个医疗设备的 根据神经解剖学命名法、本体关系、相似性 他们的蛋白质标记物，或神经连接。这些工具将对社区开放使用。我们的 技术平台将使我们能够建立最全面的初级运动皮质(M1)的3D地图 绒猴和人类大脑，包括迄今患有肌萎缩侧索硬化症(ALS)病理的组织- 为将来绘制全脑细胞类型图和研究脑血管病变提供了基础 大脑紊乱。我们设想，这些可扩展的工具将使研究团体能够审问大脑 结构和功能，包括在多个尺度上复杂的细胞间关系。

The complexity of the mammalian brain is unparalleled by any other organs, and understanding its cellular composition and their brain-wide organization is essential to understand the brain functions and dysfunctions. Extensive efforts have been made toward mapping brain cells through various lenses, and have established invaluable databases yielding new insights. However, only a few molecules, or cell-types, or regions per brain have been mapped in non-human primate and human brains, and fail to capture brain-wide complex intercellular relationships within individuals. We here aim to develop versatile and easy-to-adopt platform technologies for highly multiplexed, rapid, scalable proteomic mapping of mammalian brains from mouse, marmoset, to human. We propose to (Aim 1) develop molecular toolkits and platform technologies (ELAST-SABER) for highly multiplexed scalable proteomic phenotyping of mouse, marmoset, human brains. We will develop a library of orthogonal DNA-barcoded antibodies targeting over 100 canonical cell-type markers, compatible with SABER amplification (developed by the group of PI Peng Yin) and ELAST transport (developed by the group of PI Kwanghun Chung). We will also (Aim 2) validate the technology platform and demonstrate its broad utility and scalability by mapping the cellular landscape in the primary motor cortex (M1) of mouse, marmoset, human brains including tissues with amyotrophic lateral sclerosis (ALS) pathology. We will cross-validate our method using the published MERFISH and single-cell sequencing data from BICCN. We will perform cross-species comparative analysis to understand conserved or evolutionarily distinct cellular features of M1. Finally, we will (Aim 3) establish web-based interactive platforms for data dissemination and community use. We will build multimodal brain atlases allowing cross species comparison, and develop neuro-ontology of the mouse, marmoset and human brain. Importantly, we will develop a database built with informatics and online visualization tools that allow people to view, compare, and analyze multiplexed scalable proteomic imaging data across three species. These informatics tools will have the capacity to standardize and accommodate histological, connectivity, gene expression, and in vivo imaging. Users will be able to compare multi-modality data for the “most comparable structures” either based on their neuroanatomic nomenclature, ontology relationship, similarity of their protein markers, or neural connectivity. These tools will become openly available to the community. Our technology platform will enable us to establish the most comprehensive 3D map of the primary motor cortex (M1) of marmoset and human brains including tissues with amyotrophic lateral sclerosis (ALS) pathology to date— providing a foundation for mapping cell types brain-wide in the future and for studying pathological changes in brain disorders. We envision that these scalable tools will empower the research community to interrogate brain structure and function, including complex intercellular relationships at multiple scales.