Activity-dependent probes for spatially-defined proteomics

项目摘要 揭示功能定义的神经系综的分子特性对于理解 这些网络如何产生电路功能和动物行为。该提案旨在开发和部署 新的分子技术将使激活的蛋白质亚细胞标记和浓缩成为可能 大脑中的神经元亚群。大范围、无偏见地将蛋白质表达与神经功能联系起来 这在现有技术中是不可能的。因此，这里提出的研究计划将填补一个关键的 神经学家的分子工具箱中尚未满足的差距。虽然现有技术一直专注于获得 基因获取激活神经元的基因组或转录组，这里的方法将识别实际的 在功能相关神经元的特定亚细胞隔间表达的蛋白质。这是一把钥匙 区别，因为单靠基因表达不能揭示 翻译的蛋白质--这是执行特定生物化学功能的终极分子。 细胞。为了实现这一目标，将使用蛋白质开发新的依赖活性的邻近标记探针 工程学。这些探头将通过在以下方面执行的高吞吐量筛选进行改进和优化 培养细胞，然后改装成用于哺乳动物神经元。同时，探测器将进行测试和 其特点是在小鼠的大脑中改善和基准它们的功能。为了演示它们的用途， 活性依赖的探针将被用来标记在经历高级神经细胞的神经元中存在的蛋白质 小鼠对行为药物体验的反应。5-MeO-DMT是一种致幻药物，在 人类一直被认为具有治疗神经精神疾病的潜力。神经元 被5-MeO-DMT激活的DNA将被活性依赖的探针标记，以及它们的空间分布 整个大脑将使用这种新分子酶的荧光读数进行检查。此外, 探针还将标记存在于这些激活神经元中的蛋白质，从而允许浓缩和 使用液相色谱质谱(蛋白质组学)对这些分子进行无偏分析。该小组- 这些神经元的细胞蛋白质组将为了解其发病机制提供必要的生物学见解。 致幻药物，除了为新药发现和开发提供潜在的下游靶点 发展。更广泛地说，新的探测器将免费分发给神经科学界，以使 神经元功能相关群体中蛋白质表达的研究。

Project Summary Uncovering the molecular properties of functionally-defined neural ensembles is essential for understanding how these networks give rise to circuit function and animal behavior. This proposal aims to develop and deploy new molecular technologies that will enable the sub-cellular tagging and enrichment of proteins in activated subgroups of neurons in the brain. Linking protein expression to neural function at a large and unbiased scale is currently not possible with existing technologies. Thus the research program proposed here will fill a critical unmet gap in the molecular toolbox for neuroscientists. While prior technologies have focused on gaining genetic access to the genome or transcriptome of activated neurons, the approach here will identify the actual proteins expressed in specific subcellular compartments of functionally-relevant neurons. This is a key distinction, as gene expression alone cannot reveal to the actual physical location and expression patterns of translated proteins – which are the ultimate molecules that carry out the specific biochemical functions of our cells. To enable this goal, new activity-dependent proximity labeling probes will be developed using protein engineering. These probes will be improved and optimized through high-throughput screens performed in cultured cells, and then adapted for use in mammalian neurons. Concurrently, the probes will be tested and characterized in the mouse brain to improve and benchmark their function. To demonstrate their utility, the activity-dependent probes will be used to tag the proteins that are present in neurons undergoing high neural activity in response to a behavioral drug experience in mice. 5-MeO-DMT is a hallucinogenic drug that in humans has been associated with therapeutic potential for treating neuropsychiatric diseases. Neurons activated by 5-MeO-DMT will be labeled by the activity-dependent probes, and their spatial distribution throughout the brain will be examined using the fluorescent read-out of the new molecular enzyme. In addition, the probes will also tag the proteins that are present in these activated neurons, allowing the enrichment and unbiased profiling of these molecules using liquid chromatography mass spectrometry (proteomics). The sub- cellular proteome of these neurons will provide essential biological insight into the mechanism of hallucinogenic drugs, in addition to providing potential downstream targets for new drug discovery and development. More broadly, the new probes will be distributed freely to the neuroscience community to enable the study of protein expression in functionally-relevant populations of neurons.