Arginase-1 signaling after neonatal stroke

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.