Elucidating the mechanisms of ependymal ciliary signaling

ABSTRACT Ependymal motile cilia are responsible for the coordinated movement of cerebrospinal fluid (CSF) along the ventricle wall. The synchronized activity of ependymal cilia creates specific flow networks that are regulated by behavioral state. Ependymal cilia motility can be controlled by specific compounds in the CSF to adjust fluid flow, but little is known about the molecular and cellular mechanisms of this regulation. We propose the hypothesis that G protein-coupled receptor (GPCR) signaling pathways are expressed and function within ependymal cilia to modulate ciliary beat frequency. The focus of this proposal is to define the complement of signaling receptors that localize to ependymal cilia and determine how GPCR activation impacts cilia beating and activity-dependent ciliary localization of regulatory signaling proteins. As proof of principle, the GPCR melanin-concentrating hormone (MCH) receptor 1 localizes to ependymal motile cilia and we find that the GPCR signaling protein β-arrestin moves into ependymal cilia in response to MCH treatment. To assess which other GPCRs and signaling proteins are localized to motile cilia in ependymal cells, we will perform proximity labeling in normal and Bardet-Biedl syndrome protein deficient ependymal cells to identify low abundance and transient ciliary signaling proteins. We will also employ pharmacological approaches to evaluate the role of potential ciliary GPCRs in the regulation of ependymal cilia beating. Finally, we will assess potential ciliary GPCR activation and signaling by defining the spatial organization of GPCRs, β-arrestin, and the signaling chaperone Bbs4 within and proximal to the ciliary compartment prior to and after GPCR agonist addition. In order to provide the necessary resolution to define the precise spatial organization, we will use expansion microscopy, which is a cutting-edge imaging technique. The outcomes of this work will provide critical insight into the sensory and signaling functions of ependymal motile cilia and lay the foundation to create new tools to control ependymal cilia activity and discern the role of ependymal cilia in the mature brain.