How do astrocytes remodel neural circuits?

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PROJECT SUMMARY Pruning neuronal connections and eliminating superfluous neurons is required to generate optimized neuronal circuits in the mature brain. Although it is well established that neurons and glia coordinate the refinement of neural circuits, the molecular mechanisms underlying this process remain poorly defined. This K99/R00 proposal will help me advance my career as I investigate the cellular mechanisms by which glia help refine neuronal circuits during development. To generate a deep molecular understanding of neuronal remodeling, I will use the Drosophila larval nervous system, which goes through extensive neuronal remodeling during metamorphosis. In a preliminary screen for new model cells to study neuronal remodeling in vivo, I discovered several new markers for cells that exhibit novel types of remodeling events. In addition, I conducted two large-scale screens to identify new glial pathways that assist in pruning and the elimination of neuronal debris. First, using a single neuronal lineage, I performed an in vivo RNAi screen for glial genes required for glial pruning of neurons. Second, I transcriptionally profiled glia during pruning, identifying upregulated genes in glia, and also screened them for regulators of neuronal remodeling. These genes will serve as a molecular entry point for me to define how glia refine neural networks during development. During the mentored award phase, I will build a system that will allow me to monitor the dynamic cell biological changes that occur during glial phagocytosis and will use this new system to test novel molecules for their role remodeling. In Aim1, I will study the specific process of glial phagocytosis using genetically encoded cellular markers and explant live imaging. These assays will serve as the foundation for understanding how the molecules I identified play a role in glial phagocytosis. In Aim 2, I will use this new system to understand how Tweek, a highly conserved molecule, functions during glial phagocytosis of pruned neurons. Surprisingly, Tweek has no known protein domains or molecular function. I will use CRISPR/Cas9 genomic engineering to create human disease-associated mutations in the fly. This will potentially allow me to understand how mutations in Tweek's human homolog KIAA1109 cause a rare autosomal neurological disease. Finally, in Aim 3, which will be mostly carried out in my own lab, I will use the tools I build in this proposal to examine how a collection of newly identified phagocytic receptors drive neuronal remodeling of synapses, neurites, and in multiple types of lineages. I outlined a series of research and career development milestones that will be met during this award and allow me to grow as a scientist. To strengthen areas needed for a successful research career, the aims are combined with neuroscience coursework and training in areas such as scientific writing, mentoring, and project design. I have a committee made up of an excellent group of scientists who are dedicated to my success and eager to assist me in my professional development. My long- term career goal as an independent investigator is to understand the molecular mechanisms of neuronal remodeling and how abnormalities in this process are associated with neurodevelopmental disorders.