Project Summary Normal nervous system function depends on proper communications between neurons and non-neuronal cells. In the human brain, the majority of non-neuronal cells are called glia that consist of nearly half of total brain cells. Despite decades of work focusing on neurons and glia alone, how they communicate with each other remains poorly understood. This is partly due to a dearth of methods to comprehensively profile molecules that are enriched at the neuron-glial interface during dynamic cell-cell interactions. This proposal aims to develop a genetically-guided proteomic toolbox to spatiotemporally profile critical molecules enriched at neuron-glial interface in vivo, allowing for molecular and genetic dissection of neuron-glial interactions. We will first design and generate glial-specific cell surface proximity-labeling probes with the spatiotemporal precision. We will apply this platform in the oligodendrocytes, the sole myelin-producing cells in the central nervous system (CNS), to determine the molecular mechanisms governing the initiation of oligodendrocyte-axon ensheathment. We will leverage this new method, along with single-cell RNA sequencing, genome-wide CRISPR screens, and novel transgenic mouse strains, to interrogate a mysterious cell stage (the pre-myelinating oligodendrocytes) during developmental and adaptive myelination. To extend the glial surface proximity labeling toolkit, we will develop a neuron-glial complementary proximity labeling system allowing for visualization of transient neuron-glial interactions and capture of molecules only enriched at neuron-glial interface in vivo. Using this system we will address the molecular codes governing myelination selectivity between subsets of oligodendrocytes and functionally distinct neuronal subpopulations. This proposed work will fulfill the knowledge gap in myelin biology, and will have broader implications in understanding neuron-glial and glia-glial interaction mechanisms in general. The methods and reagents established by the work will also greatly enrich the methods in studying diverse cell- cell communications, including neuro-immune and cancer-immune interactions.