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

Ribo-STAMPEDE：脑细胞类型分子分析的新工具

• 批准号: 10506300
• 负责人: GIORDANO LIPPI
• 金额: $ 230.8万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10506300
• 关键词: Address; Adopted; Algorithms; Animal Model; Antibodies; Area; Awareness; Axon; BRAIN initiative; Bacterial Artificial Chromosomes; Behavior; Biology; Brain; Cell Separation; Cell physiology; Cells; Censuses; Cognition; Communities; Computational Biology; Cre driver; Cytidine Deaminase; Cytosine; DNA cassette; Data; Data Set; Defect; Dendrites; Development; Disease; Enhancers; Enzymes; FMR1; FMRP; Fragile X Syndrome; Gene Expression; Generations; Genes; Genetic; Genetic Transcription; Genetic Translation; Hippocampus (Brain); Hour; Human; Immunoprecipitation; In Vitro; Individual; Knock-out; Knockout Mice; Libraries; Maps; Measures; Messenger RNA; Methods; Modeling; Molecular; Molecular Profiling; Mus; Mutation; Names; Nature; Nervous system structure; Neuraxis; Neurobiology; Neurons; Neurosciences; Noise; Performance; Physiology; Population; Process; Proteins; Proxy; RNA; RNA Editing; Reagent; Research; Resolution; Resources; Ribosomal Proteins; Sampling; Signal Transduction; Stimulus; Surveys; Synaptic plasticity; System; Technology; Tetanus Helper Peptide; Toxic effect; Transcript; Transgenic Mice; Translating; Translations; Uracil; Viral; Work; adeno-associated viral vector; apoB mRNA editing catalytic subunit; base; brain cell; cell type; improved; in vivo; innovation; innovative technologies; insight; member; migration; molecular scale; new technology; novel; photoactivation; polarized cell; programs; promoter; protein expression; relating to nervous system; response; sequencing platform; single cell technology; single-cell RNA sequencing; tool; transcriptome; transcriptome sequencing; transcriptomics; translatome

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.

项目摘要 中枢神经系统（CNS）细胞类型和状态的详细且全面的人口普查对我们来说至关重要 理解认知和行为的神经底物。由于基因表达程序确定 CNS中细胞的身份和功能，最近开发了单细胞RNA测序和空间 转录组技术已使得对中枢神经系统的细胞和功能多样性有了新的见解。 这些技术依赖于测量转录水平作为基因表达水平的代理。然而， 转录本不均匀地翻译成蛋白质，因为许多转录后机制 调节细胞和亚细胞水平的翻译的位置，特别是在神经元中。克服这些 局限性，我们最近开发了一项创新技术，实现了整个转录组的翻译 在单细胞水平和亚细胞隔室中以高分辨率测量的效率 作为轴突和树突。我们的Ribo-Stamp（通过无抗体突变分析的测量目标）框架 依靠将RNA编辑酶靶向通过融合到核糖体蛋白来翻译mRNA，留下碱 可以通过RNA测序检测到的翻译成绩单的更改。在初步数据中，我们有 证明Ribo-Stamp可用于绘制单个单元的翻译组。在这个项目中，我们扩展 Ribo-Stamp系统用于应用于神经科学中紧急问题的问题。具体来说， 我们开发一个工具包来实现时间和空间控制，从而测量翻译 全面响应单细胞分辨率下的神经元刺激。我们产生一个可诱导的条件 转基因鼠标线，使用可用的CRE驱动程序鼠标启用系统类型的特定表达 线。我们还组装了一个使用细胞类型特异性增强剂来靶向单个神经元亚型的工具箱， 通过与腺相关的病毒载体传递。最后，我们在体内良好的体内验证我们的系统 依靠转化控制的疾病范式。如果成功，我们的工作将产生第一个分子工具包 用于大脑中综合细胞类型分解的分析。我们预计我们的试剂和动物 大脑倡议和神经科学社区的成员实验室将很容易通过模型 大型，将作为促进我们对人脑功能的理解的关键资源。