Regulation of cilia by ceramide

 DESCRIPTION (provided by applicant): Cilia are protrusions of the cell membrane with sensory (primary cilia) or motor (motile cilia) function. In astrocytes and ependymal cells, primary and motile cilia regulate cell division and migration, and drive circulation of the cerebrospinal fluid (CSF), respectively. Ciliary dysfunction leads to astrocytic overgrowth (astrogliosis) or ependymal cell malfunction and hydrocephalus. It is vital for the function of cila in cell signaling and motility that cilium number, length, and intraflagellar transport (IFT) of cago proteins are dynamically regulated. A critical barrier in understanding this regulation is the lack
of knowledge on dynamically activated factors for ciliogenesis. Although cilia are lipid membrane structures, research so far has focused on the role of proteins in the regulation of ciliogenesis, and little is known about the role of lipids in this process. Our research goals are o determine how membrane lipids and proteins interact in the regulation of cilia and how modulation of lipid metabolism can be utilized to support the function of cilia in astrocytes and ependymal cells. Our central hypothesis is that the sphingolipid ceramide regulates cilium length and IFT, which is critical for the function of cilia in astrocytes and ependymal cells. Our objectives are to test that cilia are regulated by ceramide-associated protein complexes;2) define these complexes by using a novel technique to pull down ACEC- and cilium-derived membrane vesicles and covalently crosslink a ceramide analog to its interacting proteins to identify ceramide binding domains;3) test that induction of receptors in cilia is regulated by ceramide;and 4) test that astroglial activation and ependymal cell function is regulated by ceramide in vitro and in vivo. Our expected outcomes include 1) determining ceramide species that promote ciliogenesis and how the generation of ciliogenic ceramide is regulated;2) defining a mechanism of cilium extension and IFT regulation by ceramide-aPKC sequestration;3) defining SMase activation in Rab11a vesicle transport pathways and their function for ceramide flux to the cilium;4) identifying proteins and protein domains that associate with ceramide;5) determining that transport of Shh signaling proteins into cilia and activation of the Shh cell signaling pathway are regulated by ceramide;and 6) defining a mechanism by which ceramide regulates astrocyte activation and ependymal cell-driven CSF flow. The impact of this project is on defining a fundamental and novel mechanism in basic neuroscience and membrane biology, which has broad implications for our understanding of the regulation of cilia by lipid-protein interaction and the importance of this regulation for the function of astrocytes and ependymal cells during brain development and aging. Aim 1 will test the hypothesis that ceramide stabilizes cilia in astrocytes and ependymal cells. Aim 2 will test the hypothesis that ceramide regulates IFT and receptor activation in cilia. Aim 3 will test the hypothesis that ciliogenic ceramide regulates astrocyte and ependymal cell function.