Abstract: Primary cilia are essential organelles that mediate and integrate important signaling pathways. Cilia-based signaling is therefore essential for the development of many tissues, and ciliary dysfunction is linked to hereditary disorders. In spite of growing appreciation for the importance of primary cilia, little is known about the signaling pathways that regulate the assembly and function of these organelles. In previous studies, we identified a kinase, Tau Tubulin Kinase 2 (TTBK2), that is essential for the initiation of ciliogenesis. We subsequently showed that TTBK2 is also required for the stability and maintenance of the cilium, and thus serves as a critical molecular handle to dissect distinct aspects of ciliary regulation. In addition to our work, independent studies also found that Ttbk2 is mutated in a human hereditary ataxia, suggesting that TTBK2 and cilia are important for neuronal function in vivo. The overall objective of this proposal is to define specific cellular requirements for TTBK2 in ciliogenesis, and identify new components of TTBK2-dependent ciliary pathways. Based upon our studies of Ttbk2 mutant mouse embryos and cells, as well as the links between Ttbk2 and a neurodegenerative condition, our central hypothesis is that TTBK2 controls pathways that are critical for both cilium initiation as well as cilium stability, and these pathways are in turn essential to maintain neural function in the adult brain. To test this hypothesis, we propose the following aims: First, we will take advantage of the requirements for TTBK2 at distinct steps of cilia regulation to identify additional components of the networks that control cilium initiation as well as cilium stability. Our previous work demonstrated that TTBK2 is essential for the recruitment of the intraflagellar transport (IFT) machinery that assembles the cilium. We use live imaging to define how TTBK2 mediates IFT recruitment and trafficking and also identify TTBK2-interacting proteins that cooperate with TTBK2 in this process. In addition, we will characterize the role of TTBK2 in mediating cilium stability by suppressing cilium disassembly networks. Second, we will define the mechanisms through which TTBK2 maintains neural function within the adult brain. We find that mice in which Ttbk2 has been removed from adult tissues exhibit a neurodegenerative phenotype. In these experiments we will test a set of TTBK2 truncations that perturb discrete functions of TTBK2 (cilium assembly, cilium stability, or non-ciliary) for their ability to rescue neural phenotypes in Ttbk2 conditional mutant mice. We will also examine whether proteins we identify as components of the distinct TTBK2 regulatory networks are required for neural function. Upon completion of these aims, we will have a greater understanding of the cellular pathways by which Ttbk2 controls cilia formation, and of the links between cilia regulation, neural function, and the biology of neurodegenerative conditions.