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.

PROJECT NARRATIVE The type and state of a cell in the nervous system is determined by its global protein expression profile, yet technologies to comprehensively interrogate the mRNA translatome of individual cells are lacking. In this project, we develop and validate a suite of molecular tools and animal models to map mRNA translation in the nervous system at single-cell resolution and at transcriptome-wide scale. The resources and datasets we generate will enable fundamentally novel insights into brain cellular diversity and functional states.