**2. The Wnt signaling pathway: Canonical and non–canonical signaling cascades**

The binding of Wnt ligands to Fz receptors can trigger the activation of different signaling cascades. In addition to Fz, other proteins have been described as alternative receptors or coreceptors, such as the low-density lipoprotein receptor-related protein 5 (LRP5), LRP6, Ror1, Ror2 and Ryk [3, 33-36], increasing the complexity of the Wnt signaling activation. It has been suggested that the binding of Wnts to specific receptors/co-receptors may selectively activate distinct signaling pathways.

> **Figure 1. Canonical Wnt/β-catenin signaling pathway.** (Left panel) In the absence of a Wnt protein, GSK-3β phos‐ phorylates β-catenin which targets it for ubiquitination by β-TrCP and degradation in the proteasome. (Right panel) Activation of the signaling pathway by the binding of a Wnt ligand to Fz receptor and coreceptors LRP5/6 triggers the association of the destruction complex with phosphorylated LRP. In this condition, the complex may still capture and phosphorylate β-catenin, however the ubiquitination is blocked and it is stabilized in the cytoplasm and enters the

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There are at least two β-catenin-independent pathways: the planar cell polarity (PCP) pathway and the Ca2+ pathway (Figure 2). The PCP pathway was originally identified in *Drosophila* where it regulates tissue polarity and cell migration [10, 57]. This signaling pathway requires Fz receptors and Dvl and activates small GTPases including Rho and Rac and the protein kinase JNK. This pathway is also known as the Wnt/JNK pathway. The activation of the Wnt/Ca2+ pathway triggers the increase in intracellular Ca2+ levels and activates the protein kinases CamKII and protein kinase C (PKC) [10, 58]. It has been suggested that Wnt-mediated Ca2+ release involves heterotrimeric G proteins since it is inhibited by pertussis toxin [59]. As mentioned 10 Fz receptors are known in mammals. Fz receptors are seven-transmembrane-spanning receptors that belong to the G protein-cou‐ pled receptor (GPCR) list as a separate class [60]. Fz receptors have an extracellular aminoterminal region that contains a cysteine-rich domain (CRD) consisting of 120 to 125 residues with 10 conserved cysteines that is relevant for the binding of Wnt proteins [61]. Growing evidence indicate the involvement of G protein in the Wnt/Fz signaling. The first evidence came from inhibition of non-canonical Wnt effects by pertussis toxin [62]. Later on, many reports have indicated that heterotrimeric G protein participates of canonical and non-

canonical Wnt signaling in *Drosophila*, *Xenopus* and mammals [63-69].

nucleus to regulate the transcription of Wnt target genes.

The first Wnt signaling pathway identified was the canonical Wnt/β-catenin pathway (Figure 1). In the absence of Wnt stimulation, the levels of cytoplasmic β-catenin are low since it is ubiquitinated and constantly degraded in the proteasome [37]. β-catenin is phosphorylated by casein kinase 1α (CK1α) and glycogen synthase kinase-3β (GSK-3β) in a multiprotein complex composed also of the scaffold protein axin and adenomatous polyposis coli (APC) [38-42]. Phosphorylated β-catenin is recognized by β-TrCP, which is part of an E3 ubiquitin ligase complex, and is ubiquitinated and subsequently degraded [43]. Activation of the Wnt/β-catenin pathway initiated by the binding of a Wnt ligand to a Fz receptor and coreceptors LRP5/6 activates the protein Dishevelled (Dvl) usually by phosphorylation, and triggers the recruitment of axin to the phosphorylated tail of LRP, inhibiting the degradation pathway consequently inducing the cytoplasmic stabilization of β-catenin which enters the nucleus and regulates the transcription of Wnt target genes [28]. Recently, it was shown that when the destruction complex is associated with phosphorylat‐ ed LRP, it may still capture and phosphorylates β-catenin, but ubiquitination is blocked (Figure 1, right panel) [44].

In the nucleus, β-catenin binds to members of the family of T-cell factor (Tcf) and lymphoid enhancer factor (Lef) [45-47]; this binding displaces Groucho, which is bound to Tcf/Lef and recruits histone deacetylases (HDAC) to repress the transcription of Wnt target genes [48-51]. Several Wnt target genes have been identified including *c-Myc*, *cyclin D1*, *Axin2*, *Calcium/ calmodulin-dependent protein kinase type IV* (CamKIV) [52-55]. In addition, by using an *in silico* analysis based on multiple Classification and Regression Tree (CART), 89 new genes were predicted to be targets of the Wnt/β-catenin pathway [56].

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In the first part of this chapter we will address what is currently known about the signaling cascades of canonical and non-canonical pathways. Then, we will review recent findings from our and other labs on the specific effects of different Wnt ligands on the structure of pre- and postsynaptic regions and on glutamatergic neurotransmission in hippocampal neurons. The synaptic role of some Fz receptors will also be reviewed. Finally, the neuroprotective effect of the Wnt signaling activation will be discussed mainly focused on the protection against the toxicity of Aβ-peptide aggregates associated to the pathogene‐

**2. The Wnt signaling pathway: Canonical and non–canonical signaling**

The binding of Wnt ligands to Fz receptors can trigger the activation of different signaling cascades. In addition to Fz, other proteins have been described as alternative receptors or coreceptors, such as the low-density lipoprotein receptor-related protein 5 (LRP5), LRP6, Ror1, Ror2 and Ryk [3, 33-36], increasing the complexity of the Wnt signaling activation. It has been suggested that the binding of Wnts to specific receptors/co-receptors may selectively activate

The first Wnt signaling pathway identified was the canonical Wnt/β-catenin pathway (Figure 1). In the absence of Wnt stimulation, the levels of cytoplasmic β-catenin are low since it is ubiquitinated and constantly degraded in the proteasome [37]. β-catenin is phosphorylated by casein kinase 1α (CK1α) and glycogen synthase kinase-3β (GSK-3β) in a multiprotein complex composed also of the scaffold protein axin and adenomatous polyposis coli (APC) [38-42]. Phosphorylated β-catenin is recognized by β-TrCP, which is part of an E3 ubiquitin ligase complex, and is ubiquitinated and subsequently degraded [43]. Activation of the Wnt/β-catenin pathway initiated by the binding of a Wnt ligand to a Fz receptor and coreceptors LRP5/6 activates the protein Dishevelled (Dvl) usually by phosphorylation, and triggers the recruitment of axin to the phosphorylated tail of LRP, inhibiting the degradation pathway consequently inducing the cytoplasmic stabilization of β-catenin which enters the nucleus and regulates the transcription of Wnt target genes [28]. Recently, it was shown that when the destruction complex is associated with phosphorylat‐ ed LRP, it may still capture and phosphorylates β-catenin, but ubiquitination is blocked

In the nucleus, β-catenin binds to members of the family of T-cell factor (Tcf) and lymphoid enhancer factor (Lef) [45-47]; this binding displaces Groucho, which is bound to Tcf/Lef and recruits histone deacetylases (HDAC) to repress the transcription of Wnt target genes [48-51]. Several Wnt target genes have been identified including *c-Myc*, *cyclin D1*, *Axin2*, *Calcium/ calmodulin-dependent protein kinase type IV* (CamKIV) [52-55]. In addition, by using an *in silico* analysis based on multiple Classification and Regression Tree (CART), 89 new genes were

sis of Alzheimer's disease.

116 Trends in Cell Signaling Pathways in Neuronal Fate Decision

distinct signaling pathways.

(Figure 1, right panel) [44].

predicted to be targets of the Wnt/β-catenin pathway [56].

**cascades**

**Figure 1. Canonical Wnt/β-catenin signaling pathway.** (Left panel) In the absence of a Wnt protein, GSK-3β phos‐ phorylates β-catenin which targets it for ubiquitination by β-TrCP and degradation in the proteasome. (Right panel) Activation of the signaling pathway by the binding of a Wnt ligand to Fz receptor and coreceptors LRP5/6 triggers the association of the destruction complex with phosphorylated LRP. In this condition, the complex may still capture and phosphorylate β-catenin, however the ubiquitination is blocked and it is stabilized in the cytoplasm and enters the nucleus to regulate the transcription of Wnt target genes.

There are at least two β-catenin-independent pathways: the planar cell polarity (PCP) pathway and the Ca2+ pathway (Figure 2). The PCP pathway was originally identified in *Drosophila* where it regulates tissue polarity and cell migration [10, 57]. This signaling pathway requires Fz receptors and Dvl and activates small GTPases including Rho and Rac and the protein kinase JNK. This pathway is also known as the Wnt/JNK pathway. The activation of the Wnt/Ca2+ pathway triggers the increase in intracellular Ca2+ levels and activates the protein kinases CamKII and protein kinase C (PKC) [10, 58]. It has been suggested that Wnt-mediated Ca2+ release involves heterotrimeric G proteins since it is inhibited by pertussis toxin [59]. As mentioned 10 Fz receptors are known in mammals. Fz receptors are seven-transmembrane-spanning receptors that belong to the G protein-cou‐ pled receptor (GPCR) list as a separate class [60]. Fz receptors have an extracellular aminoterminal region that contains a cysteine-rich domain (CRD) consisting of 120 to 125 residues with 10 conserved cysteines that is relevant for the binding of Wnt proteins [61]. Growing evidence indicate the involvement of G protein in the Wnt/Fz signaling. The first evidence came from inhibition of non-canonical Wnt effects by pertussis toxin [62]. Later on, many reports have indicated that heterotrimeric G protein participates of canonical and noncanonical Wnt signaling in *Drosophila*, *Xenopus* and mammals [63-69].

terminals [72], indicating that canonical and non-canonical signaling pathways may have promoting and inhibitory effects on presynaptic differentiation respectively. In accordance, electrophysiological recordings on adult rat hippocampal slices showed that Wnt-7a, but not Wnt-5a, increased neurotransmitter release in CA3-CA1 synapses by decreasing paired pulse facilitation and increasing the frequency of miniature excitatory postsynaptic currents (mEPSC) [73]. Also, Wnt-7a/Dvl1 double mutant mice exhibit decreased mEPSC frequency at the mossy fiber-granule cell synapse revealing a defect in neurotransmitter release [18].

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The Wnt signaling also plays relevant roles in the postsynaptic structure. Wnt-5a, which activates non-canonical Wnt signaling cascades in hippocampal neurons [19, 76], modulates postsynaptic assembly by increasing the clustering of the postsynaptic density protein-95 (PSD-95) and increases spine morphogenesis in cultured hippocampal neurons [15, 19]. PSD-95 is a scaffold protein of the postsynaptic density (PSD), which is a multiprotein complex that interacts with key molecules involved in the regulation of glutamate receptor targeting and trafficking and regulatory proteins relevant for neurotransmission [77, 78]. In hippocampal neurons, Wnt-5a induces a fast increase in the number of clusters of PSD-95 without affecting total levels of PSD-95 protein or presynaptic protein clustering [19]. This postsynaptic effect is dependent on Wnt/JNK signaling pathway as demonstrated by using JNK inhibitors. In longterm experiments, we observed that Wnt-5a is also able to increase the total number of synapses [79]. When hippocampal neurons were incubated with the formylated hexapeptide Foxy-5, which is derived from the sequence of Wnt-5a and mimics the full Wnt-5a molecule action in neurons and other systems [19, 80], there was an increase in PSD-95 since 1 hour, but after 24 hours an increase in the synaptic vesicle protein 2 (SV2) clustering was also observed. In

consequence, there was an increase in the total number of synaptic contacts [79].

tion [15, 81], supporting the activation of this non-canonical Wnt pathway.

Also, we determined that Wnt-5a induced a transient formation of dendrite protrusions that resulted in a net increase of mature dendrite spines. Videomicroscopy revealed that Wnt-5a induced *de novo* formation of dendritic spines and also increased the size of the preexisting ones [15]. Interestingly, treatment with the soluble CRD region of Fz2, acting as a Wnt scavenger, decreased spine density in cultured neurons, supporting the physiological rele‐ vance of this finding and supporting the implication of Wnt ligands in dendrite spine mor‐ phogenesis. Wnt-7a is also able to increase the density and maturity of dendritic spines through a CamKII-dependent mechanism [81]. Wnt-7a rapidly activates CaMKII in spines and inhibition of this kinase abolishes the effects of Wnt-7a on spine growth and excitatory synaptic strength. This finding implicates the Wnt/Ca2+ signaling cascade in synaptic effects of Wnt ligands. Interestingly, Wnt-5a and Wnt-7a induces an increase in intracellular Ca2+ concentra‐

In addition to the structural effects of Wnt ligands at the excitatory synapse, different Wnts have shown modulatory effects on glutamatergic neurotransmission. Wnt-3a modulates the recycling of synaptic vesicles in hippocampal synapses [73, 82] and is able to induce an increase in the frequency of mEPSC [20]. In hippocampal slices, blockade of Wnt signaling impairs longterm potentiation (LTP), whereas activation of Wnt signaling facilitates LTP [17]. In the case of Wnt-5a, acute application of this ligand in hippocampal slices increases the amplitude of field excitatory postsynaptic potentials (fEPSP) and upregulates synaptic NMDA receptor

**Figure 2. β-catenin-independent Wnt signaling pathways.** In the Wnt/JNK pathway or PCP pathway, a Wnt ligand through a Fz receptors and Dvl activates small GTPases including Rho and Rac and JNK, which in turns modulate cytos‐ keletal organization. The activation of the Wnt/Ca2+ pathway triggers an increase in intracellular Ca2+ levels which acti‐ vates CamKII and PKC.
