Ultra-Multiplexed Nanoscale In Situ Proteomics for Understanding Synapse Types

 描述（由申请人提供）：正在进行重要的工作，以揭示大脑中不同类型的细胞如何影响行为和病理，以及它们如何通过新的遗传，光学和生理工具改变可塑性和疾病。然而，神经元回路的功能活动和失调严重依赖于神经元突触的原位分子组成。虽然很明显，一个给定突触的特性是由这样连接的特定类型的细胞决定的，但对大脑中突触类型的多样性的了解远远少于细胞类型，这可能是因为这是一个本质上的蛋白质组学问题：一个给定的神经元可能与不同的目标形成许多不同种类的突触，因此转录组学（其作为细胞类型分析的流行方法）可能不足以用于突触分型。需要高通量原位蛋白质组学工具来表征在整个脑或脑区域的背景下在单细胞水平上的突触分子组成，并且因此连接与疾病病理学相关的神经元活动和遗传畸变的当前遥远的主题。在这里，我们提出了一个高风险，高回报，据我们所知完全新颖的议程：开发能够解析突触类型的分子蛋白质组组成的工具，在培养的神经元和完整的脑组织中测试它们。为了实现建立一个广泛有用的工具，在原位突触蛋白质组学这一变革性的目标，我们将建立在我们最近的突破，在开发基于DNA的高度多路复用，定量超分辨率成像方法DNA-PAINT（点积累成像在纳米尺度地形）。DNA-PAINT利用短荧光标记的DNA探针的瞬时结合，用于简单且易于实施的定量、高度多路复用、分辨率低于10 nm的超分辨率成像。在本申请中，我们计划开发和应用DNA-PAINT，以实现神经元突触蛋白的定量，超多路复用，原位表征，以了解突触类型和研究细胞类型特异性突触功能。我们的工作成果将是一个广泛有用的原位蛋白质组学工具，用于定量神经元突触的组成，可用于不同的神经生物学实验室研究单细胞水平的突触特性，在固定的组织从整个大脑或细胞培养测定。

 DESCRIPTION (provided by applicant): Significant work is ongoing to reveal how different cell types in the brain contribute to behavior and pathology, and how they change in plasticity and disease, empowered by new genetic, optical, and physiological tools. However, the functional activity and dysregulation of neuronal circuits relies critically on the in situ molecular composition of neuronal synapses. Although it is clear that the properties of a given synapse are determined by, amongst other things, the specific types of cells that are thus connected, far less is known about the diversity of synapse types in the brain than cell types, perhaps because this is an intrinsically proteomic problem: a given neuron might make many different kinds of synapse with different targets, and thus transcriptomics (which is prevailing as a method for cell type analysis) may not suffice for synapse typing. High- throughput in situ proteomic tools are needed to characterize synapse molecular composition at the single-cell level in the context of whole brains or brain regions, and thus to connect the currently distant topics of neuronal activity and genetic aberrations associated with disease pathology. Here, we propose a high-risk, high-payoff, and as far as we know entirely novel agenda: to develop tools capable of resolving the molecular proteomic composition of synapse types, testing them in cultured neurons and intact brain tissues. To achieve this transformative goal of establishing a broadly useful tool for in situ synapse proteomics, we will build on our recent breakthrough in developing the DNA-based highly multiplexed, quantitative super-resolution imaging method DNA- PAINT (Points Accumulation for Imaging in Nanoscale Topography). DNA-PAINT exploits the transient binding of short fluorescently labeled DNA-probes for simple and easy-to-implement quantitative, highly multiplexed, super-resolution imaging with sub-10 nm resolution. In this application, we plan to develop and apply DNA-PAINT to enable quantitative, ultra-multiplexed, in situ characterization of neuronal synapse proteins for understanding synaptic types and studying cell type-specific synaptic functions. The outcome of our work will be a broadly useful in situ proteomic tool for quantification of neuronal synapse composition that can be used by diverse neurobiology laboratories to study single cell-level synapse properties in fixed tissues from whole brains or cell culture assays.