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

项目摘要 详细而全面的中枢神经系统（CNS）细胞类型和状态普查对于我们的研究至关重要 理解认知和行为的神经基础。由于基因表达程序决定了 身份和功能的细胞在中枢神经系统，最近开发的单细胞RNA测序和空间 转录组学技术使人们对CNS的细胞和功能多样性有了新的认识。 这些技术依赖于测量转录水平作为基因表达水平的代表;然而， 转录物并不均匀地翻译成蛋白质，因为许多转录后机制在转录过程中起作用。 在细胞和亚细胞水平上调节翻译，特别是在神经元中。克服这些 局限性，我们最近开发了一种创新技术， 在单细胞水平和亚细胞区室中， 轴突和树突。我们的Ribo-STAMP（通过无抗体突变分析来测量目标）框架 依赖于通过融合到核糖体蛋白质，将RNA编辑酶靶向到翻译的mRNA， 可以通过RNA测序检测的翻译转录物的变化。在初步数据中， 证明Ribo-STAMP可用于从单细胞定位翻译组。在这个项目中，我们扩大 Ribo-STAMP系统应用于神经科学中的紧急问题。具体地说， 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， 在单细胞分辨率下全面响应神经元刺激。我们生成一个诱导的，有条件的 使用可用的Cre驱动小鼠实现系统的细胞类型特异性表达的转基因小鼠系 线我们还组装了一个工具箱，使用细胞类型特异性增强子来靶向单个神经元亚型， 通过腺相关病毒载体递送。最后，我们验证了我们的系统在一个良好的特点，在体内 疾病模式依赖于翻译控制。如果成功，我们的工作将产生第一个分子工具包 在大脑中进行全面的细胞类型分辨分析。我们希望我们的试剂和动物 该模型将被BRAIN Initiative的成员实验室和神经科学社区采用， 大，并将作为一个重要的资源，促进我们对人类大脑功能的理解。

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.