Spatiotemporal Molecular Substrates of TBI at Single Cell Resolution

摘要 创伤性脑损伤（TBI）具有复杂的神经病理学，涉及脑中枢的进行性改变 处理认知和情感行为，由异质细胞群组成。复杂 时空细胞和分子电路潜在的进展性TBI病理，可以演变成其他 诸如慢性创伤性脑病和创伤后应激障碍之类的疾病仍有待于 明白全面了解TBI复杂性的分子机制 由于缺乏有效的方法来检查单个脑细胞中的分子事件， 导致了整体的病理学变化我们最近对海马进行了一项单细胞分辨率研究， 使用单细胞RNA测序（scRNAseq）对TBI的急性期（24小时）进行分析， TBI的通路和调节剂。特别是，我们发现细胞代谢的抑制是一个关键， 脑外伤急性期海马中的致病成分。这一发现表明，跟踪 细胞的代谢状态可用于解决空间和时间依赖性的关键知识缺口， TBI的关键病理驱动因素的进展。在这里，我们提出测试的假设，细胞代谢 调节器通过利用现代生物学的力量来确定TBI的动态和空间致病途径。 高通量技术。我们提出了一个高度整合的团队方法，以从最近的进展中获益， 单细胞RNA测序（scRNAseq）和多重错误稳健荧光原位杂交 （MERFISH）沿着先进的基因-基因和细胞-细胞网络建模，为干预目标提供信息 在特定的时间点或大脑部位，这是TBI领域一个基本的未解决的问题。在目标1中，我们建议 利用scRNAseq、MERFISH和网络建模方法的独特组合来评估和 验证在多个时间多个脑区域中每种细胞类型对TBI的空间和时间脆弱性 以数据驱动的，无偏见的方式，这可以告诉我们TBI发病机制的隐藏调节因子。 我们将重点关注TBI进展过程中细胞代谢途径的时空变化。我们 初步结果支持mt-Rnr 2，编码线粒体肽humanin并参与细胞凋亡， 代谢是TBI的主要部位和时间依赖性驱动因素。在目标2中，我们将从功能上评估 调节mt-Rnr 2（humanin）具有减轻TBI病理和防止进展的治疗潜力。 我们还将探讨细胞类型的具体机制，特别是代谢的作用，潜在的 人性的行动。该提案的总体目标是制定一项创新战略， 全面了解TBI病理学的时空细胞基质的机制， 新的目标和机制，以重新定向TBI的过程，以克服随后的神经系统疾病。

Abstract Traumatic brain injury (TBI) has a complex neuropathology involving progressive alterations in brain centers that process cognitive and emotional behaviors and consist of heterogeneous cell populations. The complex spatiotemporal cell and molecular circuits underlying progressive TBI pathologies that can evolve into other disorders such as chronic traumatic encephalopathy and posttraumatic stress disorder remain to be understood. A comprehensive understanding of the molecular mechanisms underlying the complexity of TBI has been hindered by the lack of effective approaches to examine molecular events in individual brain cells that drive the overall pathology. We recently conducted a single cell resolution study of the hippocampus at the acute phase (24hr) of TBI using single cell RNA sequencing (scRNAseq) and revealed cell-type specific pathways and regulators of TBI. In particular, we found that depression of cell metabolism to be a key pathogenic component in the hippocampus at the acute phase of TBI. This finding suggests that tracking metabolic state of cells can be used to address key knowledge gaps on the spatial and time dependent progression of key pathologic drivers of TBI. Here we propose to test the hypothesis that cell metabolic regulators determine dynamic and spatial pathogenic pathways of TBI by harnessing the power of modern high-throughput technologies. We propose a highly integrative team approach to profit from recent advances in single cell RNA sequencing (scRNAseq) and multiplexed error robust fluorescent in situ hybridization (MERFISH) along with advanced gene-gene and cell-cell network modeling to inform on targets for intervention at specific time points or brain sites, a fundamental unsolved question in the TBI field. In Aim 1, we propose to utilize a unique combination of scRNAseq, MERFISH, and network modeling approaches to assess and validate the spatial and temporal vulnerability of each cell type to TBI in multiple brain regions at multiple time points in a data-driven, unbiased manner, which can inform us about hidden regulators of TBI pathogenesis. We will focus on the spatial and temporal changes in cellular metabolic pathways during TBI progression. Our preliminary results support that mt-Rnr2, encoding a mitochondrial peptide humanin and involved in cell metabolism, is a major site- and time-dependent driver of TBI. In Aim 2, we will functionally assess whether modulating mt-Rnr2 (humanin) has therapeutic potential to mitigate TBI pathology and prevent progression. We will also explore the cell-type specific mechanisms, especially the role of metabolism, underlying the actions of humanin. The overall goal of the proposal is to elaborate on an innovative strategy that can offer a comprehensive mechanistic understanding of the spatiotemporal cell substrates of TBI pathology and uncover novel targets and mechanisms to redirect the courses of TBI to overcome subsequent neurological disorders.