Wnt ligands play crucial roles in the development and regulation of synapse structure and function. Specifically, Wnt-5a acts as a secreted growth factor that regulates dendritic spine formation in rodent hippocampal neurons, resulting in postsynaptic development that promotes the clustering of the PSD-95 (postsynaptic density protein 95). Here, we focused on the early events occurring after the interaction between Wnt-5a and its Frizzled receptor at the neuronal cell surface. Additionally, we studied the role of heterotrimeric G proteins in Wnt-5a-dependent synaptic development. We report that FZD9 (Frizzled9), a Wnt receptor related to Williams syndrome, is localized in the postsynaptic region, where it interacts with Wnt-5a. Functionally, FZD9 is required for the Wnt-5a-mediated increase in dendritic spine density. FZD9 forms a precoupled complex with G␣ o under basal conditions that dissociates after Wnt-5a stimulation. Accordingly, we found that G protein inhibition abrogates the Wnt-5a-dependent pathway in hippocampal neurons. In particular, the activation of G␣ o appears to be a key factor controlling the Wnt-5a-induced dendritic spine density. In addition, we found that G␤␥ is required for the Wnt-5a-mediated increase in cytosolic calcium levels and spinogenesis. Our findings reveal that FZD9 and heterotrimeric G proteins regulate Wnt-5a signaling and dendritic spines in cultured hippocampal neurons.The Wnt family proteins are secreted growth factors that regulate developmental processes, such as cell fate and polarity and general cell maintenance events, by modulating homeostasis and the cell cycle (1, 2). Furthermore, Wnt signaling controls various steps in the differentiation of the central nervous system (3-5), including axon guidance, dendrite development, synapse formation and plasticity (6, 7). Certain Wnt ligands, such as Wnt-7a and Wnt-3a, regulate the development and activity of the pre-and postsynaptic regions of glutamatergic synapses (8 -10). Similarly, previous results have shown that Wnt-5a regulates the synaptic structure and function by inducing the clustering of PSD-95 through the activation of the c-Jun N-terminal kinase (JNK) (11) and modulates glutamate receptors through nitric oxide production (12). Additionally, Wnt-5a promotes the de novo formation of dendritic spines in hippocampal neurons (13) through the non-canonical Wnt/Ca 2ϩ pathway.The Frizzled (FZD) 2 family has 10 members in vertebrates and represents unconventional G protein-coupled receptors (GPCRs) (14). FZD proteins have been identified as Wnt receptors, and they can mediate the signaling triggered by several Wnt ligands. Wnt-5a activity has been linked to several FZDs receptors and other co-receptors in different model systems (15-18). However, the FZD receptor mediating Wnt-5a signaling and spine formation in hippocampal neurons has not yet been identified.Several signaling cascades can be triggered upon Wnt binding to a FZD receptor, including canonical (i.e. ␤-catenin-dependent) and non-canonical pathways (19). Co...

The ghrelin receptor [growth hormone secretagogue receptor (GHSR)‐1a] represents a promising pharmacologic target for the treatment of metabolic disorders, including obesity and cachexia, via central appetite modulation. The GHSR‐1a has a complex pharmacology, highlighted by G‐protein–dependent and—independent downstream signaling pathways and high basal constitutive activity. The functional selectivity and signaling bias of many GHSR‐1a–specific ligands has not been fully characterized. In this study, we investigated the pharmacologic properties of ghrelin, MK‐0677, L692,585, and [d‐Lys3]‐growth hormone–releasing peptide‐6 (Dlys), JMV2959, and [d‐Arg(1),d‐Phe(5),d‐Trp(7, 9), Leu(11)]‐substance P (SP‐analog). We investigated their effect on basal GHSR‐1a constitutive signaling, ligand‐directed downstream GHSR‐1a signaling, functional selectivity, and signaling bias. Dlys behaved as a partial antagonist with a strong bias toward GHSR‐1a–β‐arrestin signaling, whereas JMV2959 acted as a full unbiased GHSR‐1a antagonist. Moreover, the SP‐analog behaved as an inverse agonist increasing G‐protein–dependent signaling, but only at high concentrations, whereas, at low concentrations, the SP‐analog attenuated β‐arrestin–dependent signaling. Considering the limited success in the clinical development of GHSR‐1a–targeted drugs so far, these findings provide a novel insight into the pharmacologic characteristics of GHSR‐1a ligands and their signaling bias, which has important implications in the design of novel, more selective GHSR‐1a ligands with predictable functional outcome and selectivity for preclinical and clinical drug development.—Ramirez, V. T., van Oeffelen, W. E. P. A., Torres‐Fuentes, C., Chruścicka, B., Druelle, C., Golubeva, A. V., van de Wouw, M., Dinan, T. G., Cryan, J. F., Schellekens, H. Differential functional selectivity and downstream signaling bias of ghrelin receptor antagonists and inverse agonists. FASEB J. 33, 518–531 (2019). http://www.fasebj.org

The Wnt signaling pathway plays important roles during different stages of neuronal development, including neuronal polarization and dendritic and axonal outgrowth. However, little is known about the identity of the Frizzled receptors mediating these processes. In the present study, we investigated the role of Frizzled-5 (Fzd5) on neuronal development in cultured Sprague-Dawley rat hippocampal neurons. We found that Fzd5 is expressed early in cultured neurons on actin-rich structures localized at minor neurites and axonal growth cones. At 4 DIV, Fzd5 polarizes towards the axon, where its expression is detected mainly at the peripheral zone of axonal growth cones, with no obvious staining at dendrites; suggesting a role of Fzd5 in neuronal polarization. Overexpression of Fzd5 during the acquisition of neuronal polarity induces mislocalization of the receptor and a loss of polarized axonal markers. Fzd5 knock-down leads to loss of axonal proteins, suggesting an impaired neuronal polarity. In contrast, overexpression of Fzd5 in neurons that are already polarized did not alter polarity, but decreased the total length of axons and increased total dendrite length and arborization. Fzd5 activated JNK in HEK293 cells and the effects triggered by Fzd5 overexpression in neurons were partially prevented by inhibition of JNK, suggesting that a non-canonical Wnt signaling mechanism might be involved. Our results suggest that, Fzd5 has a role in the establishment of neuronal polarity, and in the morphogenesis of neuronal processes, in part through the activation of the non-canonical Wnt mechanism involving JNK.

The Wnt signaling pathway has been implicated in several different aspects of neural development and function, including dendrite morphogenesis, axonal growth and guidance, synaptogenesis and synaptic plasticity. Here, we studied several Frizzled Wnt receptors and determined their differential expression during hippocampal development. In cultured hippocampal neurons, the cellular distributions of Frizzleds vary greatly, some of them being localized at neurites, growth cones or synaptic sites. These findings suggest that the Wnt signaling pathway might be temporally and spatially fine tuned during the development of neuronal circuits through specific Frizzled receptors.