Defining the temporal and spatial CSF secretome by TurboID labeling

The cerebrospinal fluid (CSF) is the extracellular environment of the brain and serves as its stage-specific stem cell niche. It contains proteins that can arise from multiple sources, but the functions of these proteins are understudied due to their heterogeneous origins. To investigate CSF protein functions, we must identify their sources. Our collaborative proposal probes source tissues of the CSF proteome by first interrogating the choroid plexus (ChP), a key tissue that contributes to CSF composition. Leveraging AAV2/5 tropism to drive TurboID expression and induce biotinylation of ChP proteins, we will identify post-secretion ChP proteins in CSF by streptavidin enrichment and mass spectrometry. To capture the developmental trajectory of the ChP secretome, we will administer AAV2/5-TurboID intracerebroventricularly in embryonic, neonatal, and adult mice. Our preliminary results in adult mice validate this approach, with significant protein biotinylation observed in ChP and CSF. Following proteomic analysis, we will detail age-specific ChP-secreted protein profiles (Aim 1). We then extend this work to map the brain-wide destination of ChP-secreted CSF proteins (Aim 2). This project will significantly advance our understanding of CSF origins throughout development, and provide a synthetic biology approach to investigate the implications of CSF in conditions across health, age, and disease.