Physiological Condition Dependent Changes in Ciliary GPCR Localization in the Brain

Abstract

Primary cilia are small immotile cellular appendages which mediate diverse types of singling and are found on most mammalian cell types including throughout the central nervous system. Cilia are known to localize certain G protein-coupled receptors (GPCRs) and are critical for mediating the signaling of these receptors. Several of these neuronal GPCRs have recognized roles in feeding behavior and energy homeostasis. Heterologous cell line and model systems like C. elegans and Chlamydomonas have implicated both dynamic GPCR cilia localization and cilia length and shape changes as key for signaling. However, it is unclear if mammalian ciliary GPCRs utilize similar mechanisms in vivo and under what physiological conditions these processes may occur. Here, we use the ciliary GPCRs, melanin concentrating hormone receptor 1 (MCHR1) and neuropeptide-Y receptor 2 (NPY2R) as model ciliary receptors to determine if dynamic localization to cilia occurs. We tested physiological conditions in which these GPCRs have been implicated such as feeding behavior, obesity, and circadian rhythm. Cilia were imaged using confocal microscopy and analyzed with a computer assisted approach allowing for unbiased and high throughput analysis of cilia. We analyzed GPCR positive cilia, cilia frequency as well as cilia length and receptor occupancy. Interestingly we observed changes in ciliary length, receptor occupancy, and cilia frequency under different conditions, but no consistent theme across GPCRs or brain nuclei was observed. A better understanding of the subcellular localization dynamics of ciliary GPCRs could reveal unrecognized molecular mechanisms regulating behaviors like feeding. Significance StatementOften, primary cilia localize specific G protein-coupled receptors (GPCRs) for subcellular signaling. Cell lines and model systems have indicated that cilia deploy dynamic GPCR localization and change their shape or length to modulate signaling. We used mice to assess neuronal cilia GPCRs under physiological conditions associated with both the receptors known functions and ciliopathy clinical features like obesity. We show that certain cilia with specific GPCRs appear to dynamically alter their length while others appear relatively stable under these conditions. These results implicate multiple themes across cilia GPCR mediated signaling and indicate that not all cilia modulate GPCR signaling using the same mechanisms. These data will be important for potential pharmacological approaches to target cilia GPCR-mediated signaling.