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 propel cerebrospinal fluid (CSF), respectively. Ciliary dysfunction leads to astrocytic overgrowth (astrogliosis) or ependymal cell malfunction and hydrocephalus. It is vital for the function of cilia in cell signaling and motility that cilium number, length, and intraciliary or intraflagellar transport (IFT) of cargo proteins are dynamically regulated. A critical barrier in understanding this regulation is the lack of knowledge on dynamically activated factors in ciliogenesis and cilium function. Although cilia are membrane structures, research so far has focused on the role of proteins in the regulation of cilia, and little is known about the role of lipids in this process. Our research goals are to 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 1) test that cilia are regulated by ceramide-associated protein complexes; 2) define these complexes by using a novel technique to pull down ceramide enriched- and cilium-derived membrane vesicles and covalently crosslink a bifunctional 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 support cilium function, and how the generation of ciliogenic ceramide is regulated; 2) defining a mechanism of cilium extension and IFT regulation by interaction of ceramide with atypical PKC, GSK-3?, and HDAC6; 3) defining SMase activation in 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 and activation of signaling proteins in cilia, in particular of the sonic hedgehog 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.
Cilia are protrusions of the cell membrane that work like antenna or whips. Antenna-like primary cilia sense growth factors that regulate activation and proliferation of astrocytes and other neural cells in the brain. Whip- like motile cilia of ependymal cells move fluids that contain growth factors such as the cerebrospinal fluid. Therefore, primary and motile cilia are vital for brain development and function. The P.I.'s laboratory has found that a fat-like substance (lipid) called ceramide is critical for cilia. In the present study, the P.I. will determine how ceramide regulates cilia in the brain, a fundamental and novel mechanism in basic neuroscience and membrane biology.
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