**3. Roles of the Wnt signaling pathway at central synapses**

The Wnt signaling pathway has different roles during development linked to neurite pattern‐ ing and synaptogenesis. Different Wnt ligands have been linked to the presynaptic assembly. In 1997, Salinas and co-workers demonstrated in cerebellar neurons that Wnt-7a increases the levels of synapsin I, a protein associated to synaptic vesicles [70]. Moreover, Wnt-7a mutant mice show a delay in the accumulation of synapsin I [71]. In hippocampal neurons Wnt-7a as well as Wnt-3a and Wnt-7b increases the number of pre-synaptic puncta suggesting a role for these ligands in presynaptic assembly [18, 72, 73]. In addition, Wnt-7a was found to stimulate recycling and endocytosis of synaptic vesicles using FM dyes [74]. In hippocampal neurons, Wnt-7a was also able to increase the expression as well as the clustering of the α7- nicotinic acetylcholine receptor (α7-nAChR), indicating that the Wnt signaling regulates the clustering of presynaptic receptors [75]. Interestingly, all these ligands are able to modulate presynaptic differentiation by activation of the Wnt/β-catenin signaling pathway, suggesting that some of the components associated with this pathway may be involved in the presynaptic effect. On the other hand, the non-canonical ligand Wnt-5a decreases the number of presynaptic 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].

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].

**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‐

The Wnt signaling pathway has different roles during development linked to neurite pattern‐ ing and synaptogenesis. Different Wnt ligands have been linked to the presynaptic assembly. In 1997, Salinas and co-workers demonstrated in cerebellar neurons that Wnt-7a increases the levels of synapsin I, a protein associated to synaptic vesicles [70]. Moreover, Wnt-7a mutant mice show a delay in the accumulation of synapsin I [71]. In hippocampal neurons Wnt-7a as well as Wnt-3a and Wnt-7b increases the number of pre-synaptic puncta suggesting a role for these ligands in presynaptic assembly [18, 72, 73]. In addition, Wnt-7a was found to stimulate recycling and endocytosis of synaptic vesicles using FM dyes [74]. In hippocampal neurons, Wnt-7a was also able to increase the expression as well as the clustering of the α7- nicotinic acetylcholine receptor (α7-nAChR), indicating that the Wnt signaling regulates the clustering of presynaptic receptors [75]. Interestingly, all these ligands are able to modulate presynaptic differentiation by activation of the Wnt/β-catenin signaling pathway, suggesting that some of the components associated with this pathway may be involved in the presynaptic effect. On the other hand, the non-canonical ligand Wnt-5a decreases the number of presynaptic

**3. Roles of the Wnt signaling pathway at central synapses**

vates CamKII and PKC.

118 Trends in Cell Signaling Pathways in Neuronal Fate Decision

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

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 currents facilitating induction of LTP [15, 16]. Interestingly, Wnt-5a produced a two-step increase in the amplitude of NMDAR responses [16]. The mechanisms involved in this twostep effect of Wnt-5a were investigated by the delivery of specific protein kinase inhibitors via the recording pipette. Specifically, the role of PKC and JNK was investigated, since these are two known downstream kinases of the non-canonical pathway. Inhibition of Ca22+-dependent PKC isoforms with Go6976 or the more general PKC inhibitor calphostin C eliminated the first step of potentiation of NMDAR currents and did not affect the second one. On the contrary, the slower developing increase in NMDAR currents was blocked by the JNK inhibitors TI-JIP153-163 and SP600125. This indicate that there are two mechanisms involved in in the potentiation of NMDAR by Wnt-5a. There is a fast PKC-dependent potentiation and a slower JNK-dependent potentiation that does not require previous activation of PKC [16].

adulthood and others during early development [85]. In addition, the distribution of Fzs in hippocampal neurons is also very specific. Some receptors, are located in the synaptic region, while others are mainly located in the soma or in the growth cones of young neurons [85]. These findings suggest that these receptors could be important regulators for the specific activation of the Wnt signaling cascades during the development of hippocampal circuits. In fact, we determined an association of the distribution with specific functions. In hippocampal neurons, Fz1 is located in the synaptic region co-localizing with presynaptic proteins and with active synaptic vesicle recycling sites [82]. Interestingly, overexpression of Fz1 increased the number of clusters of Bassoon, a component of the active zone involved in the structural organization of neurotransmitter release sites that is recruited early during synapse formation [86], suggesting that Fz1 regulates synaptic differentiation. In agreement, treatment with the extracellular CRD of Fz1 decreased Bassoon clustering which was not observed with the CRD of Fz2, indicating a receptor specificity for the synaptic effect [82]. Fz5 also has a role in mature neurons where it modulates the synaptogenic effect of Wnt7a [87]. As well as Fz1, Fz5 is present in synaptosomes and colocalizes with synaptic markers, and changes in the expression of this receptor modulates the density of synaptic sites [87]. In addition to its function in mature neurons, Fz5 was shown to be in high levels in the growth cones of developing hippocampal neurons [85], and we have recently determined that this receptor is involved in neural polarization (unpublished results). We determined that overexpression of Fz5 triggers a mislocalization of axonal proteins such as Tau-1 and phosphorylated MAP1B (MAP1BP), which change their distribution to the whole cell suggesting altered polarization. When the expression of Fz5 is knocked-down by shRNA, MAP1BP is not polarized and is almost completely lost. These findings suggest that in developing hippocampal neurons Fz5 is relevant for neural polarization. These studies indicate that Fz receptors are relevant players in both the developing and the adult nervous system and support the notion that the Wnt signaling pathway is crucial for different aspects of the development and function of the CNS.

Wnt Signaling Roles on the Structure and Function of the Central Synapses: Involvement in Alzheimer's Disease

http://dx.doi.org/10.5772/54606

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**4. Role of Wnt signaling in adult neurogenesis**

In the adult brain, there are two regions where there is a continuous generation of new neurons (Figure 3A), the subventricular zone (SVZ) of the lateral ventricles [88] and the subgranular zone (SGZ) in the hippocampal dentate gyrus [89]. In the SVZ, astrocyte-like neural stem cells (NSCs), called type B1 cells, generate type C cells that rapidly proliferate and give rise to type A neuroblasts (Figure 3B). These cells migrate through the rostral migratory stream to the olfactory bulb where they became interneurons [88] (Figure 3A). In the SGZ, radial and nonradial neural precursor cells give rise to transient amplifying progenitors that generate neuroblasts and then became immature neurons that extend dendrites toward the molecular layer and project their axons through the hilus toward the CA3 region [90] (Figure 3C). Newborn neurons then mature and fully integrate into the preexisting hippocampal circuitry.

Adult neurogenesis is highly regulated by intrinsic and extrinsic mechanisms. Many signaling pathways have been identified as regulators of different aspects of neurogenesis. Notch, Shh,

Wnt-5a also regulates postsynaptically the hippocampal inhibitory synapses [76]. *Wnt-5a* induces surface expression and maintenance of GABAA receptor in the membrane of hippo‐ campal neurons, increases the amplitude of GABA-currents due to a postsynaptic mechanisms, and induces the recycling of functional GABAA receptors through activation of CaMKII [76]. Therefore Wnt-5a is able to modulate both, excitatory and inhibitory synapses which must be relevant for neurotransmission.

The novel role for Wnt ligands in synaptic transmission provides a mechanism for Wnt signaling to acutely modulate synaptic plasticity and brain function in later stages of devel‐ opment and in the mature organism. Importantly, neuronal activity modulates the release and expression of Wnt ligands which may be relevant for the function of these ligands during neurotransmission. Activation of NMDA receptors increases the expression of Wnt-2 in hippocampal neurons which then stimulates dendritic arborization [83]. On the other hand, tetanic stimulation induce NMDA receptor-dependent synaptic Wnt3a release [17]. The role for endogenous Wnts was supported by incubation of hippocampal slices with secreted Wnt inhibitors, such as secreted Frizzled-related protein-2 (sFRP-2), which showed that endoge‐ nous Wnt ligands are modulators of glutamatergic neurotransmission being necessary to maintain basal NMDA receptor synaptic transmission [15, 16].

The *in vivo* relevance for the role of Wnt signaling in activity-mediated synaptic connectivity was revealed in mice exposed to an enriched environment (EE). These animals showed increased complexity and number of large mossy fiber terminals in the CA3 region [84]. EE increased Wnt7a/b levels in CA3 pyramidal neurons and inhibiting Wnt signaling through locally applied sFRP-1, suppressed the effects of EE on synapse numbers and further reduced synapse numbers in control mice.

These findings show that Wnt ligands are important regulators of the synaptic structure during development and in adult neurons, and that the Wnt pathway is one of the signaling cascades regulated by neuronal activity that is involved in the regulation of neurotransmission in adult nervous system.

In addition to the role of Wnts, Fz receptor have also been involved in synaptic structure and function. In the hippocampus, we have determined that different Fz receptors have very different patterns of expression during development, being some of them highly expressed in adulthood and others during early development [85]. In addition, the distribution of Fzs in hippocampal neurons is also very specific. Some receptors, are located in the synaptic region, while others are mainly located in the soma or in the growth cones of young neurons [85]. These findings suggest that these receptors could be important regulators for the specific activation of the Wnt signaling cascades during the development of hippocampal circuits. In fact, we determined an association of the distribution with specific functions. In hippocampal neurons, Fz1 is located in the synaptic region co-localizing with presynaptic proteins and with active synaptic vesicle recycling sites [82]. Interestingly, overexpression of Fz1 increased the number of clusters of Bassoon, a component of the active zone involved in the structural organization of neurotransmitter release sites that is recruited early during synapse formation [86], suggesting that Fz1 regulates synaptic differentiation. In agreement, treatment with the extracellular CRD of Fz1 decreased Bassoon clustering which was not observed with the CRD of Fz2, indicating a receptor specificity for the synaptic effect [82]. Fz5 also has a role in mature neurons where it modulates the synaptogenic effect of Wnt7a [87]. As well as Fz1, Fz5 is present in synaptosomes and colocalizes with synaptic markers, and changes in the expression of this receptor modulates the density of synaptic sites [87]. In addition to its function in mature neurons, Fz5 was shown to be in high levels in the growth cones of developing hippocampal neurons [85], and we have recently determined that this receptor is involved in neural polarization (unpublished results). We determined that overexpression of Fz5 triggers a mislocalization of axonal proteins such as Tau-1 and phosphorylated MAP1B (MAP1BP), which change their distribution to the whole cell suggesting altered polarization. When the expression of Fz5 is knocked-down by shRNA, MAP1BP is not polarized and is almost completely lost. These findings suggest that in developing hippocampal neurons Fz5 is relevant for neural polarization. These studies indicate that Fz receptors are relevant players in both the developing and the adult nervous system and support the notion that the Wnt signaling pathway is crucial for different aspects of the development and function of the CNS.
