PROJECT SUMMARY The choroid plexus (ChP) comprises a network of cells that form a critical brain barrier that can mediate secondary damage in certain brain disorders and trauma. The Lehtinen lab has developed a suite of tools to study the ChP across development ex vivo and in vivo. This project applies imaging technology to study blood- CSF barrier permeability regulation at the cellular level. Our overarching hypothesis is that the intracellular messenger cAMP regulates endothelial tight junctions between ChP epithelial cells and thereby blood-CSF barrier integrity as it does in the nearby blood-brain barrier (BBB). The main effectors of cAMP, PKA and Epac, regulate endothelial tight junction redistribution and barrier permeability. Gi/o-linked G-Protein Coupled Receptors (GPCRs) are strong upstream regulators of cAMP. The Lehtinen laboratory's single nucleus sequencing data suggest that ChP epithelial cells selectively and developmentally express mGlur8, which via Gαi, activation in other brain tissues inhibits cAMP production. To elucidate mechanisms of neurotransmitter alteration of the blood-CSF barrier we will study the cellular mechanisms of neurotransmitter-activated GPCR- cAMP signaling in ChP epithelial cells. The Lehtinen lab, in collaboration with co-sponsor Mark Andermann's lab, recently established a protocol for ex vivo and in vivo imaging of ChP structure and function based on using fluorescent reporters of calcium activity. I will apply these techniques to reveal how GPCRs modulate cAMP levels using fluorescent cAMP indicators in ChP explants (Aim 1). In Aim 2 we will use an in vivo preparation to map the populations of receptors that are accessible to central vs peripheral ligands. Those located on the apical membrane are in contact with the CSF and those on the basal surface are exposed to the blood. With functional assays including peripheral delivery of low molecular weight fluorescent dyes, we will assess the effects of central (CSF) vs peripheral (intravenous) delivered ligands such as mGluR8 agonists on cAMP and blood-CSF barrier permeability. Together these studies will reveal mechanisms how neurotransmitters, specifically glutamate, may contribute to blood-CSF barrier integrity in health and disease. The research and training proposed will take place at Boston Children's Hospital, a world-renowned pediatric hospital that offers an exceptional research environment and countless opportunities to carry research from bench to bedside. Importantly, the research proposed will take place under the guidance of Dr. Maria Lehtinen, an expert in the field of choroid plexus and CSF biology. In addition, Dr. Mark Andermann (co-mentor) is a leader in in vivo optical imaging techniques. The results from this proposal will result in first-authored publications and a wealth of preliminary data for a competitive K99/R00 application. This fellowship will provide the candidate with the opportunity to begin training in choroid plexus biology and make significant scientific contributions while also helping launch an independent academic career.

PROJECT NARRATIVE The choroid plexuses (ChP) provide a critical blood-CSF barrier whose breakdown is implicated in several neurological and psychiatric disorders. The goal of this study is to investigate the molecular mechanisms by which GPCR-cAMP signaling regulate permeability of the blood-CSF barrier. Successful completion of this project will provide a roadmap for the molecular mechanisms regulating ChP barrier integrity, targets which can be exploited to improve therapeutic interventions.