PROJECT SUMMARY (CHEN, JI) Primary cilia are the “cellular antennae” that detect various extracellular signals including hormones. Defects in primary cilia cause a broad spectrum of human diseases including obesity. The type 3 adenylyl cyclase (AC3) is the major adenylyl cyclase and a key enzyme mediating the cAMP signal pathway in olfactory sensory cilia and in primary cilia throughout the central nervous system (CNS). AC3 in olfactory sensory cilia is essential for the olfactory signal transduction and loss of AC3 leads to anosmia (loss of smell). Moreover, multiple genetic evidence has demonstrated that defects in AC3 are associated with obesity and depression. Nevertheless, the functional mechanisms of AC3, and the signaling transduction pathway in primary cilia in the CNS remain to be elucidated. Because olfactory sensory cilia and neuronal primary cilia are structurally related and share a common intraflagellar protein transport system, we hypothesize that the cAMP signaling in the primary cilia of CNS neurons resembles that in olfactory sensory cilia and alteration of this signaling in neuronal primary cilia impacts obesity and depression. To test this hypothesis, we will first use fluorescent nanoparticles in co- immnunostaining experiments and take a mass spectrometry based proteomic approach to identify signal proteins that transduce the cAMP signaling in the primary cilia of CNS neurons. We will next genetically engineer a light-activated adenylyl cyclase into primary cilia to enable optogenetic manipulation of cAMP level in neuronal primary cilia. We will also determine how cAMP in neuronal primary cilia modulate neuronal functions. In addition, we will further identify which neuronal ensembles with loss of AC3 leads to obesity and depression using tissue-specific AC3 knockout mice. Completion of this project will identify molecular components that mediate ciliary cAMP signal transduction, generate useful tools for cilium research, and provide novel insights into the (patho-)physiological roles of AC3 in obesity and depression.