Project Summary/Abstract The endogenous ligands activating ~100 G protein coupled receptors (GPCRs) are still unknown, hence they are named orphans. Considering their understudied biology and demonstrated essential physiological roles, orphan GPCRs represent important, unexploited pharmacological targets. A small family of four evolutionary conserved IDG-eligible orphan GPCRs, Gprc5(a-d), has been linked to the onset of a variety of human diseases, ranging from several cancer types, diabetes, inflammation, renal dysfunction and neuropsychiatric conditions. Nonetheless, Gprc5(a-d) control over intracellular signaling cascades and biological functions are poorly understood. Among them, Gprc5b is also the most expressed orphan GPCR in the central nervous system. Interestingly, members of this class of orphan GPCRs were originally described as retinoic acid- inducible GPCRs. Our working hypothesis is that retinoic acid regulates transcriptional programs in the adult brain by coordinating changes in expression levels of a set of genes that includes Gprc5b. These changes are brain region and cell-type dependent. Considering that Gprc5b expression is enriched in the dentate gyrus of the hippocampus, a region heavily targeted by retinoic acid, we believe that Gprc5b regulation could be prominent here. Furthermore, given that Gprc5b has conserved typical G protein coupling motifs, we hypothesize that changes in Gprc5b levels would alter cell signaling cascades and intracellular levels of second messengers that in turn will modulate cellular functions. A major role of the dentate gyrus consists in maintaining adult neurogenesis, a process that, according to available data, can be possibly regulated by both retinoic acid and Gprc5b. A deficit in adult neurogenesis is one of the main factors in the development of mood disorders. Remarkably, Gprc5b levels were found altered in patients with affective disorders, while retinoic acid has been shown to affect mood-related behaviors both in animal models and patients. Therefore, with this project we will test the hypothesis that, through the described chain of events, retinoic acid controls depressive- like behavior in mice by controlling Gprc5b levels in specific brain areas and cell subpopulations. To this end, we will first investigate the brain region and cell-type specific regulation of Gprc5b by retinoic acid administration in mice. In parallel, we will generate a Gprc5b brain conditional knockout by crossing Gprc5bflx/flx mice with mice expressing Cre recombinase under the control of nestin promoter (Nescre). This novel mouse model will be used to evaluate the role of Gprc5b in depressive-like and anxiety-like behaviors using a battery of tests including sucrose preference test, elevated plus maze, marble burying, tail suspension test, forced swim test, and open field. Similarly, we will test the hypothesis that Gprc5b mediates part of the retinoic acid effect on mood disorders by administering the same sequence of tests to retinoic-acid treated Gprc5bflx/flxNescre and control littermates. Overall, the proposed experiments will illuminate Gprc5b biology and lead to the development of much needed research tools to study the physiological role of this orphan GPCR in vivo.

Project Narrative Gprc5b belongs to the understudied family of orphan receptors Gprc5(a-d) that is widely expressed across human tissues and that has been linked to various diseases like cancer, diabetes, and psychiatric disorders. Originally identified as a retinoic acid induced gene, the interaction between Gprc5b and retinoid signaling in the brain has never been explored. As both retinoic acid and Gprc5b are implicated in affective disorders, our goal is to define how retinoic acid regulates Gprc5b in the brain and to develop research tools to test their crosstalk in vivo in depressive-related behaviors.