CSF-neurotransmitter-based regulation of the choroid plexus brain barrier

About This Grant

SUMMARY The brain is bathed in cerebrospinal fluid (CSF), a medium rich in health- and growth-promoting factors, metabolites, nucleic acids, and more, whose composition changes profoundly throughout the lifespan and can be used to diagnose certain conditions. Most CSF is produced by the choroid plexus (ChP), a highly vascularized epithelium located in each ventricle of the brain. In addition, the sheet of tight junction-coupled ChP epithelial cells provides a critical blood-CSF barrier that protects the central nervous system from peripheral challenges such as bloodborne pathogens. The ChP detects and responds to changes in CSF composition. Abnormal levels of neurotransmitters in the CSF including glutamate have been reported in a wide range of neurologic and psychiatric disorders that are also characterized by ChP/barrier pathology and inflammation. We hypothesize a causal link between these observations. Specifically, we propose that excessive CSF-glutamate impairs ChP barrier integrity and that it does so through the metabotropic glutamate receptor 8 (mGluR8). Importantly, cAMP has been shown to enhance endothelial tight junctions at the blood-brain barrier (BBB), and experiments in cultured ChP cells suggest that cAMP may similarly regulate blood-CSF barrier permeability. Among the G-Protein Coupled Receptors (GPCRs) that we identified to be predominately and robustly expressed by mouse and human ChP epithelial cells, mGluR8 is coupled to the Gi/o-protein and therefore expected to inhibit cAMP/PKA signaling, consistent with negative regulation of barrier permeability. Our first set of experiments will test our hypothesis by studying cAMP regulation in ChP epithelial cells. Using methods we developed in collaboration with Mark Andermann (BIDMC/Harvard) for in vitro and in vivo two-photon imaging of ChP structure and function at subcellular resolution, we will visualize cAMP with a fluorescent indicator selectively expressed in ChP epithelial cells (cADDis). We will then use a newly generated mGluR8 knockout mouse (shared by Danny Winder, UMass), to test if mGluR8 is necessary and sufficient to mediate glutamate- evoked modulation of cAMP signaling (Aim 1). Next, we will test the hypothesis that CSF glutamate dose- dependently reduces barrier integrity in vivo and thereby causes ChP inflammation (Aim 2). To do so we will leverage technologies we recently developed based on GRAB-sensor technology for real-time tracking of neurotransmitters and neuromodulators in CSF. We will measure CSF glutamate levels with cell-based sensor iGluSnFR and correlate these levels with measurements of barrier permeability and inflammation.