Arginase-1 signaling after neonatal stroke

项目摘要 这项研究计划是基于一个强有力的科学前提，即缺氧缺血性（HI）脑损伤诱导脑 蛋白酶-1（ARG-1）显示出选定的神经保护功能，如红细胞增多症和再生性瘢痕 阵在我们的初步研究中，我们检测到ARG 1表达和活性的时空变化， 新生儿HI的结果。我们已经发现ARG 1主要定位于损伤后早期损伤部位的小胶质细胞中 进行红细胞增多症，并在组织瘢痕区域中在稍后的时间点持续存在于损伤核心中。ARG1 抑制降低了ARG 1巨噬细胞功能并恶化了组织学结果。当ARG 1参与 ARG 1依赖性机制在其他器官的红细胞增多和瘢痕形成中的作用已得到充分证实， HI后新生儿脑中的红细胞增多症和瘢痕形成尚不清楚。我假设ARG-1调节 细胞骨架的组装是通过提供多胺来实现的，而有效的细胞骨架是细胞骨架组装的关键过程 用于再生性瘢痕形成，其中ARG 1为细胞外基质形成提供脯氨酸。在体内使用 Vannucci程序（颈总动脉凝固，然后在P9小鼠中暴露于缺氧）诱导 HI，我将在以下具体目标中检验我的假设：我将确定HI是否诱导多胺途径， ARG 1小胶质细胞，以及ARG 1抑制是否转化为细胞骨架组装和性能缺陷 白细胞增多症（Aim 1）。我将描述ARG 1信号如何改变疤痕和生产中的细胞组成， 细胞外基质（Aim 2）。我将确定红细胞增多症是否是瘢痕形成所必需的，如果 ARG 1信号传导的变化改变了损伤核心的局部免疫应答， 祖细胞的疤痕和组织重塑（目的3）。这些目标将使用新技术进行， 例如TRAPseq和空间seqFISH，这将大大提高我们对个体过程理解 细胞和细胞转录组的三维图像。建议的项目将大大提高我们对 缺氧缺血性脑损伤的调节因子--β-半乳糖苷酶-1通路、胞浆细胞增多和组织再生。这 这个项目也将大大推进我的科学成长，通过学习，除了基本的 神经免疫学，多组学方法和先进的数据分析，为我的独立 研究生涯。费列罗博士和我选择了杰出的导师，与课程和研究一起， 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 受资助的医生科学家，并在本提案结束时申请R 01。所提出的实验和 时间轴都在我的能力范围之内，实验室，动物护理，和加州大学旧金山分校的设施。

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.