Ribo-STAMPEDE: novel tools for molecular profiling of brain cell types

项目总结 对中枢神经系统(CNS)细胞类型和状态进行详细和全面的普查对于我们的 对认知和行为的神经基础的理解。当基因表达程序确定 新近发展起来的单细胞RNA测序和空间分析 转录学技术使人们能够对中枢神经系统的细胞和功能多样性有新的见解。 这些技术依赖于测量转录水平作为基因表达水平的替代；然而， 转录本并不统一翻译成蛋白质，因为有许多转录后机制在 在细胞和亚细胞水平上调节翻译的位置，特别是在神经元中。要克服这些障碍 限制，我们最近开发了一种创新技术，使转录组范围的翻译 效率将在高分辨率下测量，既在单细胞水平上，也在亚细胞隔间中，如 作为轴突和树突。我们的Ribo-STAMP(通过无抗体突变谱检测靶点)框架 依赖于靶向RNA编辑酶，通过融合到核糖体蛋白来翻译mRNAs，离开碱基 通过RNA测序可以检测到的翻译转录本的变化。在初步数据中，我们有 证明了Ribo-STAMP可以用于从单个细胞中定位翻译组。在这个项目中，我们扩展了 Ribo-Stamp系统适用于神经科学中特定于紧急问题的问题。具体来说， 我们开发了一个工具包来实现时间和空间控制，使翻译能够被测量 在单细胞分辨率下对神经元刺激作出综合反应。我们生成一个可诱导的、有条件的 利用可用的Cre驱动鼠实现系统的细胞类型特异性表达的转基因小鼠系 台词。我们还组装了一个工具箱，它使用细胞类型特定的增强子来靶向单个神经元亚型， 通过腺相关病毒载体传递。最后，我们在一个具有良好特性的活体实验中验证了我们的系统 依赖于翻译控制的疾病范式。如果成功，我们的工作将产生第一个分子工具包 用于在大脑中大规模地进行全面的细胞类型解析的分析。我们预计我们的试剂和动物 模型将很容易被大脑倡议的成员实验室和神经科学界采用，网址为： 庞大，并将成为促进我们对人脑功能的理解的关键资源。

PROJECT SUMMARY A detailed and comprehensive census of central nervous system (CNS) cell types and states is essential to our understanding of the neural substrates of cognition and behavior. As gene expression programs determine the identity and function of cells in the CNS, recently developed single-cell RNA-sequencing and spatial transcriptomics technologies have enabled new insights into the cellular and functional diversity of the CNS. These technologies rely on measuring transcript levels as a proxy for gene expression levels; however, transcripts are not uniformly translated into protein because numerous post-transcriptional mechanisms are in place to regulate translation at the cellular and sub-cellular levels, particularly in neurons. To overcome these limitations, we have recently developed an innovative technology enabling transcriptome-wide translational efficiency to be measured at high resolution, at both the single cell level and in subcellular compartments such as axons and dendrites. Our Ribo-STAMP (Surveying Targets by Antibody-free Mutation Profiling) framework relies on targeting an RNA editing enzyme to translated mRNAs via fusion to ribosomal proteins, leaving base changes on translated transcripts that can be detected by RNA-sequencing. In preliminary data we have demonstrated that Ribo-STAMP can be used to map the translatome from single cells. In this project, we expand the Ribo-STAMP system for application to questions specific to urgent questions in neuroscience. Specifically, we develop a toolkit to achieve temporal and spatial control, enabling translation to be measured comprehensively in response to neuronal stimuli at single cell resolution. We generate an inducible, conditional transgenic mouse line enabling cell type-specific expression of the system using available Cre driver mouse lines. We also assemble a toolbox that uses cell type-specific enhancers to target individual neuronal subtypes, delivered via adeno-associated viral vectors. Lastly, we validate our system in a well-characterized in vivo disease paradigm that rely on translational control. If successful, our work will generate the first molecular toolkit for comprehensive cell type-resolved profiling in the brain at scale. We anticipate that our reagents and animal model will be readily adopted by the member labs of the BRAIN Initiative and the neuroscience community at large, and will serve as a critical resource for advancing our understanding of human brain function.