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

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].

maintain basal NMDA receptor synaptic transmission [15, 16].

relevant for neurotransmission.

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synapse numbers in control mice.

nervous system.

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

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

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

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

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

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, BMPs, and Wnts are part of the signaling molecules of the niche that regulate the maintenance, activation and fate specification of neural precursor cells [91, 92].

In Wnt/β-catenin reporter mice (BATGAL) it was shown that this pathway is active in the SGZ and the dentate granule cell layer [23]. In that study, authors determined that Wnt3 is expressed in adult hippocampal astrocytes and that adult hippocampal progenitor (AHP) cells express key components of the Wnt/β-catenin signaling pathway. These findings suggested that the Wnt pathway may be involved in the regulation of adult neurogenesis. *In vitro* analysis in cultured cells revealed that Wnts derived from hippocampal astrocytes stimulate Wnt/βcatenin signaling in isolated AHPs inducing their neuronal commitment [23]. The effect of the Wnt signaling was supported *in vivo* using lentiviral vectors expressing Wnt3a or a secreted mutant Wnt1 protein that blocks Wnt signaling. Lentiviruses were stereotactically injected into the dentate gyrus of rats. As assessed by the incorporation of the nucleotide analog BrdU and immunodetection of the immature neuron protein doublecortin (DCX), blocking the Wnt signaling decreases adult hippocampal neurogenesis while stimulating this pathway has the opposite effect [23]. More recently, and by using the same lentiviral approach to block Wnt signaling in the dentate gyrus of adult rats it was shown that Wnt-mediated adult hippocampal neurogenesis contributes to learning and memory [93]. In the SVZ, β-catenin signaling also plays a role in the proliferation of progenitor cells in the adult mouse brain [94]. Retrovirusmediated expression of a stabilized β-catenin promoted the proliferation of type C cells and inhibited their differentiation into neuroblasts. Also in the SVZ, transduction of the β-catenin inhibitor axin by intracranial lentiviral delivery decreased cell proliferation as revealed by decreased BrdU labeling [95], further supporting a role for Wnt/β-catenin signaling in neural stem cell proliferation in the neurogenic areas of adult brain.

The Wnt-mediated effects in neurogenesis may be caused by the transcriptional activation of NeuroD1 which is dependent on the Wnt/β-catenin signaling activation [25]. NeuroD1 is a basic helix-loop-helix transcription factor important for the generation of granule cell and olfactory neuron in the embryonic and adult brain [96]. NeuroD1 gene promoter has overlap‐ ping DNA-binding site for Sox2 and TCF/LEF, then the activation of this gene implies activation of the canonical Wnt pathway and removal of Sox2 repression from the NeuroD1 gene promoter [25]. More recently, Prox1 was also determined as a target of the Wnt/β-catenin pathway relevant for neurogenesis [22]. Prox1 is expressed in newborn and mature granule cells and is required for the proper differentiation and survival of newborn granule cells, but not for the maintenance of granule cells after they have fully matured [22].

In addition, Wnts could indirectly modulate adult neurogenesis thorough their effects on neuronal activity. As previously described, different Wnts regulate glutamatergic neuro‐ transmission, and evidence indicates that neural progenitor cells respond to neuronal activity as part of their differentiation program [97]. GABA is an important modulator of adult hippocampal neurogenesis being critical for the proper development and matura‐ tion of adult-born neurons [98-100]. Interestingly, Wnt-5a through activation of CaMKII, induces the recycling of functional GABAA receptors on hippocampal neurons and modulates inhibitory synapses [76].

As mentioned, in neurogenic niches Wnts are provided by astrocytes [23], and during aging it was reported that the levels of Wnt3 protein and the number of Wnt3-secreting astrocytes declines [101], which may be one of the factors underlying the impairment of neurogenesis that is observed in aging [102, 103]. On the contrary, running, that is a potent stimulator of adult neurogenesis in the SGZ [104] was found to significantly increase *de novo* expression of Wnt-3 [101], pointing to the Wnt pathway as one of the factors involved in running-mediated increase in neurogenesis. In addition to astrocytes-derived Wnts, an autocrine Wnt signaling activity has been observed in adult hippocampal progenitors (AHPs) derived from adult rat

dentate gyrus. The progression of radial type 1 cells to mature newborn granule neurons is schematized.

**Figure 3. Neurogenesis in the adult brain.** (A) Schematic representation of adult rodent brain highlighting the two neurogenic regions. The hippocampus and the SVZ (boxed). (B) Schematic of the SVZ in the wall of the lateral ventri‐ cles. Distinct stem/progenitor cell types (types B, C, and A) are shown. (C) Neurogenesis in the SGZ of the hippocampal

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BMPs, and Wnts are part of the signaling molecules of the niche that regulate the maintenance,

In Wnt/β-catenin reporter mice (BATGAL) it was shown that this pathway is active in the SGZ and the dentate granule cell layer [23]. In that study, authors determined that Wnt3 is expressed in adult hippocampal astrocytes and that adult hippocampal progenitor (AHP) cells express key components of the Wnt/β-catenin signaling pathway. These findings suggested that the Wnt pathway may be involved in the regulation of adult neurogenesis. *In vitro* analysis in cultured cells revealed that Wnts derived from hippocampal astrocytes stimulate Wnt/βcatenin signaling in isolated AHPs inducing their neuronal commitment [23]. The effect of the Wnt signaling was supported *in vivo* using lentiviral vectors expressing Wnt3a or a secreted mutant Wnt1 protein that blocks Wnt signaling. Lentiviruses were stereotactically injected into the dentate gyrus of rats. As assessed by the incorporation of the nucleotide analog BrdU and immunodetection of the immature neuron protein doublecortin (DCX), blocking the Wnt signaling decreases adult hippocampal neurogenesis while stimulating this pathway has the opposite effect [23]. More recently, and by using the same lentiviral approach to block Wnt signaling in the dentate gyrus of adult rats it was shown that Wnt-mediated adult hippocampal neurogenesis contributes to learning and memory [93]. In the SVZ, β-catenin signaling also plays a role in the proliferation of progenitor cells in the adult mouse brain [94]. Retrovirusmediated expression of a stabilized β-catenin promoted the proliferation of type C cells and inhibited their differentiation into neuroblasts. Also in the SVZ, transduction of the β-catenin inhibitor axin by intracranial lentiviral delivery decreased cell proliferation as revealed by decreased BrdU labeling [95], further supporting a role for Wnt/β-catenin signaling in neural

The Wnt-mediated effects in neurogenesis may be caused by the transcriptional activation of NeuroD1 which is dependent on the Wnt/β-catenin signaling activation [25]. NeuroD1 is a basic helix-loop-helix transcription factor important for the generation of granule cell and olfactory neuron in the embryonic and adult brain [96]. NeuroD1 gene promoter has overlap‐ ping DNA-binding site for Sox2 and TCF/LEF, then the activation of this gene implies activation of the canonical Wnt pathway and removal of Sox2 repression from the NeuroD1 gene promoter [25]. More recently, Prox1 was also determined as a target of the Wnt/β-catenin pathway relevant for neurogenesis [22]. Prox1 is expressed in newborn and mature granule cells and is required for the proper differentiation and survival of newborn granule cells, but

In addition, Wnts could indirectly modulate adult neurogenesis thorough their effects on neuronal activity. As previously described, different Wnts regulate glutamatergic neuro‐ transmission, and evidence indicates that neural progenitor cells respond to neuronal activity as part of their differentiation program [97]. GABA is an important modulator of adult hippocampal neurogenesis being critical for the proper development and matura‐ tion of adult-born neurons [98-100]. Interestingly, Wnt-5a through activation of CaMKII, induces the recycling of functional GABAA receptors on hippocampal neurons and

activation and fate specification of neural precursor cells [91, 92].

122 Trends in Cell Signaling Pathways in Neuronal Fate Decision

stem cell proliferation in the neurogenic areas of adult brain.

modulates inhibitory synapses [76].

not for the maintenance of granule cells after they have fully matured [22].

**Figure 3. Neurogenesis in the adult brain.** (A) Schematic representation of adult rodent brain highlighting the two neurogenic regions. The hippocampus and the SVZ (boxed). (B) Schematic of the SVZ in the wall of the lateral ventri‐ cles. Distinct stem/progenitor cell types (types B, C, and A) are shown. (C) Neurogenesis in the SGZ of the hippocampal dentate gyrus. The progression of radial type 1 cells to mature newborn granule neurons is schematized.

As mentioned, in neurogenic niches Wnts are provided by astrocytes [23], and during aging it was reported that the levels of Wnt3 protein and the number of Wnt3-secreting astrocytes declines [101], which may be one of the factors underlying the impairment of neurogenesis that is observed in aging [102, 103]. On the contrary, running, that is a potent stimulator of adult neurogenesis in the SGZ [104] was found to significantly increase *de novo* expression of Wnt-3 [101], pointing to the Wnt pathway as one of the factors involved in running-mediated increase in neurogenesis. In addition to astrocytes-derived Wnts, an autocrine Wnt signaling activity has been observed in adult hippocampal progenitors (AHPs) derived from adult rat brains. Inhibiting this autocrine Wnt signaling increases the number of neurons formed and leads to a loss of multipotency among AHPs indicating that this autocrine pathway may preserve the balance between neural stem cell maintenance and differentiation [105].

schizophrenia [30, 125]. Among the Wnt components that are affected in AD, it was shown that β-catenin levels are reduced in AD patients carrying presenilin-1 (PS-1)-inherited mutations [124], while the secreted Wnt antagonist Dickkopf-1 (Dkk1) is elevated in postmor‐ tem AD brains and brains from transgenic mouse models for AD [121, 126]. A variant of the LRP6 has been associated with late-onset AD, which confers low levels of Wnt signaling [119]. In addition, genetic studies show a link between Wnt signaling and AD. Epidemiological data show an increased risk for AD in populations where the allele 4 of apo-lipoprotein E (apoE4) is present. Interestingly apoE4 causes inhibition of the canonical Wnt signaling in PC12 cells upon stimulation with Wnt-7a as determined by luciferase activities and nuclear β-catenin levels [127]. Aβ directly binds to the extracellular CRD of Fz5 at or in close proximity to the Wnt-binding site inhibiting the canonical Wnt signaling pathway [128], linking directly Aβ to Wnt impairment. Moreover, the exposure of cultured rat hippocampal neurons to Aβ results in inhibition of canonical Wnt signaling as determined by destabilization of endogenous levels of β-catenin, increase in GSK-3β activity, and a decrease in the expression of some Wnt target genes [129]. Moreover, acute exposure to Aβ increases Dkk1 mRNA levels in hippocampal

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brain slices, which seems to be associated to synaptic loss induced by Aβ [130].

of β-catenin as a survival element in AD.

neuroprotive effect elicited by this Wnt ligand [14].

As mentioned, one of the hallmarks of AD brains is the abnormal phosphorylation of the tau protein which accumulates as intraneuronal neurofibrillary tangles [131]. Several kinases can phosphorylate tau *in vitro*; however, the bulk of the information supports that Cdk5, extrac‐ ellular signal-related kinase 2, microtubule affinity-regulating kinase and GSK-3β, a key component of the Wnt cascade, are the most relevant kinases for tau phosphorylation *in vivo* [132, 133]. Cultured neurons exposed to Aβ show an increased GSK-3β activity [134, 135], and active GSK-3β has been found in brains staged for AD neurofibrillary changes, with a con‐ comitant decrease in β-catenin levels and an increase in *tau* hyperphosphorylation [136]. Also, neurodegeneration and spatial learning deficits have been observed in GSK-3β conditional transgenic mice [137, 138]. Interestingly, a study shows that the phosphorylation of tau antagonizes apoptosis by stabilizing β-catenin; therefore, up-regulation of β-catenin during tau phosphorylation prevents the cell from going into apoptosis. Increasing levels of phos‐ phorylated tau was correlated with increased levels of nuclear β-catenin, and the knockdown of β-catenin antagonizes the anti-apoptotic effects of *tau* [139]. These findings support a role

Several studies have shown neuroprotective properties of the Wnt signaling activation against the toxicity of Aβ peptide. In cultured hippocampal neurons, exposure to Aβ aggregates causes a decrease in endogenous β-catenin levels, and this effect was overcome by direct activation of the pathway with Wnt-3a conditioned media [117, 129]. The protective effect of Wnt-3a against the toxicity of Aβ oligomers was shown to be mediated by Fz1 receptor, since this effect is modulated by the expression levels of Fz1 in both, PC12 cells and hippocampal neurons [14]. Overexpression of Fz1 significantly increased cell survival induced by Wnt-3a and diminished caspase-3 activation, while knocking-down the expression of the receptor by antisense oligonucleotides decreased the stabilization of β-catenin induced by Wnt-3a and decreased the

The Wnt signaling has also been involved in the mechanism of the orphan nuclear receptor TLX (also known as NR2E1), which is an important regulator of neural stem cell maintenance and self-renewal in embryonic and adult brains [106, 107] and is involved in neurogenesis in the SVZ [108] and hippocampus [109]. To stimulate neural stem cell proliferation and selfrenewal TLX activates the Wnt/β-catenin pathway in adult mouse neural stem cells by activating the expression of Wnt-7a, which expression was found to be downregulated in TLXnull mice, through binding to two TLX binding sites present in the Wnt-7a gene promoter [95]. Wnt-7a is important for adult neural stem cell proliferation *in vivo* since there is a decreased BrdU labeling in the SGZ and SVZ of adult Wnt7a knockout mice. In TLX-/- mice, intracranial lentiviral transduction of active β-catenin led to a considerable rescue of cell proliferation in the SVZ, suggesting that Wnt/β-catenin acts downstream of TLX to regulate neural stem cell proliferation *in vivo* [95].

It has been shown that low oxygen is associated with increased levels of β-catenin *in vivo*, and that hypoxia inducible factor-1α (HIF-1α) modulates the Wnt/β-catenin signaling in embryonic stem cells exposed to low oxygen [110]. Recently, we determined *in vivo* that hypoxia stimulates the activation of the Wnt/β-catenin signaling pathway in the hippocampus of adult mice (our unpublished results), and stimulates cell proliferation in the SGZ of 2 month old wild-type mice.

Altogether, these findings indicate that the Wnt pathway is relevant not only for the develop‐ ment of the nervous system but also for the development of new neurons in the adult brain, being important for the maintenance and self-renewal of the stem cell pool and for the commitment and proliferation of new neurons.
