Arginase-1 signaling after neonatal stroke

项目总结 这项研究计划是基于一个强有力的科学前提，即缺氧缺血(HI)脑损伤导致脑 精氨酸酶-1(ARG-1)表现出特定的神经保护功能，如胞吐和再生瘢痕 队形。在我们的初步研究中，我们检测到ARG1表达和活性的时空变化 这是新生儿HI的结果。我们发现ARG1主要定位于损伤部位早期的小胶质细胞。 执行胞吐作用，并在稍后的时间点在组织疤痕区域内持续存在于损伤核心。ARG1 抑制会降低ARG1泡出细胞的功能，并恶化组织学结果。虽然ARG1参与了 在其他器官的胞吐和疤痕形成中有很好的文献记载，ARG1依赖的机制 新生儿缺氧缺血性脑病后的胞吐和疤痕形成尚不清楚。我假设ARG-1调节 通过为细胞骨架组装提供多胺来实现胞吐作用，而有效的胞吐作用是一个至关重要的过程 用于再生瘢痕的形成，其中ARG1为细胞外基质的形成提供脯氨酸。在体内使用 Vannucci手术(凝血P9小鼠颈总动脉，然后暴露于低氧)诱导 嗨，我将在以下具体目标上检验我的假设：我将定义HI是否在脑内诱导多胺通路 ARG1小胶质细胞，以及ARG1抑制是否转化为细胞骨架组装和性能缺陷 泡沫化(目标1)。我将描述ARG1信号如何改变SCAR中的细胞组成和产物 细胞外基质(目标2)。我会确定泡沫化是否对疤痕形成是必要的，如果 ARG1信号的变化改变了损伤核心的局部免疫反应，以及这是否影响了 促进瘢痕和组织重塑的祖细胞(目标3)。这些目标将使用新的技术进行， 如TRAPSEQ和SPACE SEQFISH，将显著提高我们对个体过程理解 细胞和细胞转录组的3D图像。拟议的项目将大大提高我们对 精氨酸酶-1途径、胞吐作用和组织再生作为脑缺氧缺血损伤的调节剂。这 该项目还将通过学习以下基础知识显著促进我的科学成长 神经免疫学，多组学方法和高级数据分析对我的独立 研究生涯。费里罗博士和我挑选了杰出的导师，再加上课程和研究 计划是一致的，以解决我的具体知识差距，以确保我的职业发展作为一个独立的 资助内科科学家，并在本提案结束时申请R01。建议的实验和 时间表在我的能力范围内，以及实验室、动物护理和加州大学旧金山分校设施的能力范围内。

PROJECT SUMMARY This research plan is based on a strong scientific premise that hypoxic-ischemic (HI) brain injury induces brain arginase-1 (ARG-1) to exhibit selected neuroprotective functions, such as efferocytosis and regenerative scar formation. In our preliminary studies, we detected spatiotemporal changes in ARG1 expression and activity as a result of neonatal HI. We have shown ARG1 localized mostly in microglia at the injury site early after injury performing efferocytosis and persisted in the injury core at later timepoints in the area of the tissue scar. ARG1 inhibition decreased ARG1 efferocytic function and worsened histological outcomes. While ARG1 involvement in efferocytosis and scar formation in other organs is well documented, ARG1-dependent mechanisms of efferocytosis and scar formation in the neonatal brain after HI are unknown. I hypothesize that ARG-1 regulates efferocytosis by providing polyamines for cytoskeleton assembly, and efficient efferocytosis is a crucial process for regenerative scar formation where ARG1 provides proline for extracellular matrix formation. Using in vivo the Vannucci procedure (common carotid artery coagulation followed by exposure to hypoxia in P9 mice) to induce HI, I will test my hypothesis in the following Specific aims: I will define whether HI induces polyamine pathway in ARG1 microglia, and whether ARG1 inhibition translates to defects in cytoskeleton assembly and performance of efferocytosis (Aim 1). I will characterize how ARG1 signaling alters cell composition in the scar and production of the extracellular matrix (Aim 2). I will determine whether efferocytosis is necessary for scar formation, if changes in ARG1 signaling alter local immune response in the injury core and whether this impacts migration of progenitor cells to the scar and tissue remodeling (Aim 3). The aims will be conducted using novel techniques, such as TRAPseq and spatial seqFISH that will significantly improve our understanding of processes in individual cells and cellular transcriptome in 3D. The proposed project will significantly improve our understanding of arginase-1 pathway, efferocytosis and tissue regeneration as a modifier of brain hypoxic-ischemic injury. This project will also significantly advance my scientific growth through learning besides the basics of neuroimmunology, the multiomics approaches and advanced data analysis necessary for my independent research career. Dr. Ferriero and I selected outstanding mentors, that together with coursework and research plans are aligned to address my specific knowledge gaps to ensure my career development as an independently funded physician scientist and to apply for an R01 at the end of this proposal. The proposed experiments and timeline are within my capabilities and the capabilities of the laboratory, animal care, and UCSF facilities.