Wnt signaling acts at various stages during neural development, including neuronal migration, axon and dendrite development, synapse formation/maturation, and neuronal plasticity (2, 6, 35). Wnt pathways are classified into canonical Wnt/β-catenin or noncanonical (β-catenin-independent) pathways. Canonical Wnt/β-catenin signaling is mediated by nuclear translocation of its central effector β-catenin. Noncanonical Wnt signaling occurs independently of β-catenin and is stimulated by Wnt ligands that bind to a receptor complex of Fzd, Ror1/2 or Ryk (1, 13, 36).
A series of small molecule inhibitors specifically target several of the proteins in the Wnt signaling pathway, such as Fzd, DVL, PORCN or Tankyrase, and could also be used as chemical probes to dissect the mechanism(s) of these signaling pathways (37, 38). We therefore used Wnt-C59, a PORCN inhibitor, in young hippocampal neurons, to research polarization, the development of dendritic trees, and maintenance and/or maturation of dendritic spines; these inhibitory molecules have the potential to be developed both as study reagents in cell biology as therapeutic agents (39, 40). Our present results show that the early inhibition of PORCN in young hippocampal neurons, starting at picomolar Wnt-C59 concentration, drastically reduces the length of the neurites by up to 80%, and affects the dendritic arbor complexity and the signaling and contact that could be established between the different cells in culture. Nonneuronal cells are known to show no contact inhibition when in contact with the growth cones or neurites of neurons. These observations on the contact behavior of nonneuronal cells imply a series of signals from both types of cells, which in our experiments are lost with the inhibition of the Wnt pathway (41). Together, these results, show that the endogenous production of the different Wnt ligands plays an early and important role in the growth and architecture of neurites and the signaling between the different cells of the in vitro neuronal culture.
To evaluate the individual activity of Wnt ligands, sister cultures of hippocampal neurons previously treated with Wnt-C59 to shut down Wnt signaling activity were incubated with exogenous Wnts. First, we tested Wnt3a, a ligand that has canonical activity but has also shown some Ca2+ modulation (42, 43). Then, tested a noncanonical ligand Wnt5a which stimulates PSD-95 clustering (44) and finally, Wnt7a, a classic ligand of the Wnt-β-catenin-dependent pathway, which promotes the presynaptic colocalization of several neural receptors (35, 45). Our results indicated that all exogenous Wnt ligands (Wnt3a, 5a and 7a) rescued the changes in neuronal morphology. Specifically, Wnt3a restores the length of neurites to a value similar to that of the control; however, Wnt7a increased the neurite length beyond that of the control. Wnt5a had the same effect but at higher relative Wnt concentration. In addition, all 3 Wnts ligands restored dendritic arbor complexity with the recovery of secondary and tertiary projections.
Previous studies, have indicated that specific Wnt ligands induce neurite extension in various neuronal types: Wnt3 and Wnt3a in cultures of neural precursors of spinal cord cells (46), Wnt7a in cultured mouse cerebellar granule cells (47), Wnt5a and Wnt3b in chick retinal ganglion cell axons (48) and Wnt7b in mouse hippocampal neurons (49). More interestingly, different Wnt ligands have distinct effects on growing axons, and canonical and noncanonical pathways can have opposite effects (2). Adding to the complexity, the canonical ligand Wnt3a can activate both canonical and noncanonical signaling pathways in the same cell type (13, 50). Although the interaction between canonical and noncanonical Wnt signaling is complex, these signaling pathways generally inhibit each, as demonstrated during development (51).
Recent studies in our laboratory with Wnt-C59, a specific PORCN inhibitor in SH-SY5Y cells, indicated that both the canonical Wnt3a ligand and a noncanonical Wnt5a ligand increased the basal expression of Teneurin (52), a new trans-synaptic signal in both the peripheral nervous system and the CNS (53, 54). This work not only demonstrate the involvement of Wnt signaling in regulating Ten-3 expression but also revealed that Wnt3a (a canonical Wnt ligand) increases the expression of Ten-3 through a mechanism dependent on the secretion and activity of Wnt5a (a noncanonical ligand). Although the work raises new questions, the results seem to demonstrate the upregulation of Ten-3 by Wnt signaling and suggest that Ten-3 modulation is possible because of crosstalk between the canonical and noncanonical Wnt pathways (52).
An alternative derived from our results is the possibility of modulating the neuronal morphology in the neurogenic niche and in a determined time frame, after PORCN inhibition by Wnt-C59, using the "replacement" of specific Wnt ligands, and thereby avoiding other secreted endogenous Wnt inhibitors, such as Dickkopf-1 (Dkk-1) or the secreted-frizzled-related protein (sFRP), which would allow the formation of new connections and therefore new neural networks (39, 55, 56). An interesting example is Wnt signaling inhibition by Wnt-C59 which prevents the activity of Wnt3a and Wnt8B, in canonical and noncanonical Wnt-mediated signaling to induce cortical motor neurons or their progenitors from iPSCs in humans (26, 57). This strategy might offer a proof of concept on the preclinical side, where the inhibition of Wnts signaling in mammals can be achieved by inhibiting PORCN with small molecule inhibitors such as Wnt-C59, providing a safe and feasible strategy in vivo. Our results increase the possibility that a specific PORCN inhibitor contributes to regulating the Wnt pathway so that it become a safe and plausible methodology for cell replacement therapy in neurological diseases.