**5. GSK3 and Wnt signaling**

Wnt signaling is amongst the most important signaling pathways controlling neurogenesis in the developing and adult brain. Several studies have shown that attenuation of this pathway reduces the number of granule neurons and size of the hippocampus, as well as a reduction in the number of radial glial cells due to proliferation and patterning defects [39-41]. It is well accepted that Wnt's help to maintain the proliferative capacity of ES cells and keep them in an undifferentiated state [7], however there is also evidence for Wnt's being required to promote neural differentiation [42, 43]. These seemingly contradictory views are united in a model whereby Wnt signaling promotes proliferation and inhibits differentiation of pluripotent cells. Inhibition of this pathway promotes initial stages of differentiation into neural progenitors, but reactivation of Wnt signaling is required at later stages of the differentiation program to generate mature neurons [44, 45]. Indeed, in adult neurogenesis in the dentate gyrus of the hippocampus, Wnt's are secreted by local astrocytes where they predominantly regulate differentiation into mature neurons (mostly inhibitory GABAergic interneurons), rather than proliferation of NPC's [46-48].

GSK3 is a key component of the Wnt signaling pathway. In unstimulated cells, GSK3 is part of a multi-protein complex with APC, Axin and other proteins that facilitates phosphorylation of β-catenin by CK1 at Ser45, followed by phosphorylation of Ser33/37/41 by GSK3. This creates a recognition site for the E3 ubiquitin ligase βTrCP, which ubiquitinates β-catenin and targets it for degradation via the proteasome. In Wnt-stimulated cells, this multi-protein complex is disrupted, preventing phosphorylation and subsequent ubiquination of β-catenin, thus stabilizing the protein and increasing its cellular abundance. This leads to translocation to the nucleus, where β-catenin binds to several transcription factors facilitating transcription of target genes involved in cell fate regulation, including c-myc [49], NeuroD1 [48, 50, 51], Prox1 [52] and LINE-1 [50]. In adult neurogenesis, Wnt-induced expression of NeuroD1 is required for survival and maturation of adult-born neurons [48, 50, 51]. The prototypcial binding partners of β-catenin in the nucleus are members of the TCF/LEF family, which have been shown to be central mediators of tumourigenesis in the colon, breast and other tissues. In NPC's, activation of the Wnt pathway and elevated transcriptional activity of β-catenin has been shown to promote proliferation and inhibit neuronal differentiation [53, 54], while inhibition of Wnt signaling promotes neuronal differentiation [55, 56]. Accordingly, deletion of GSK3 isoforms promotes proliferation and inhibits differentiation of stem cells [57], consistent with pharmacological GSK3 inhibitors and Wnt-induced inhibition of GSK3 activity [7, 8]. This is dependent on elevated levels of transcriptionally active β-catenin [58], but surprisingly not by members of the TCF/LEF family. Instead, β-catenin was shown to bind to another transcription factor called Oct4, which increased expression of the pluripotency regulator and stem cell marker Nanog [58, 59].

Some transcriptional targets of the Wnt pathway are also targeted by GSK3 post-translation‐ ally. For example, *c-myc* is an established target of the Wnt pathway that promotes cell cycle progression and proliferation [49]. Meanwhile, its protein product is directly phosphorylated by GSK3 at Thr58, which targets it for ubiquitination by the E3 ligase Fbw7, followed by proteasome-mediated degradation [60, 61]. Thus, Wnt-induced inhibition of GSK3 activity could promote high c-myc activity both transcriptionally and post-translationally. However, it has not yet been proven that Wnt-mediated inhibition of GSK3 reduces phosphorylation of c-myc, or any protein other than β-catenin for that matter. Alternatively, simultaneous stimulation of cells by Wnt and growth factors would activate c-myc transcription and reduce its phosphorylation and degradation, respectively, thus combining to increase c-myc abun‐ dance. This would promote proliferation and inhibit differentiation of NPC's. Interestingly, a viral oncogenic form of c-myc is mutated at the GSK3 target site (Thr58) [60]. This mutation prevents phosphorylation of c-myc by GSK3 and subsequent ubiquitination, thus stabilizing the protein and driving uncontrolled proliferation in tumourigenesis. Thus, emphasizing the importance of phosphorylation of c-myc by GSK3 in the regulation of cell fate.

[38]. Together, these observations suggest that inhibition of GSK3 by the Wnt signalling pathway promotes NPC proliferation, while inhibition of GSK3 by growth factor signalling

Wnt signaling is amongst the most important signaling pathways controlling neurogenesis in the developing and adult brain. Several studies have shown that attenuation of this pathway reduces the number of granule neurons and size of the hippocampus, as well as a reduction in the number of radial glial cells due to proliferation and patterning defects [39-41]. It is well accepted that Wnt's help to maintain the proliferative capacity of ES cells and keep them in an undifferentiated state [7], however there is also evidence for Wnt's being required to promote neural differentiation [42, 43]. These seemingly contradictory views are united in a model whereby Wnt signaling promotes proliferation and inhibits differentiation of pluripotent cells. Inhibition of this pathway promotes initial stages of differentiation into neural progenitors, but reactivation of Wnt signaling is required at later stages of the differentiation program to generate mature neurons [44, 45]. Indeed, in adult neurogenesis in the dentate gyrus of the hippocampus, Wnt's are secreted by local astrocytes where they predominantly regulate differentiation into mature neurons (mostly inhibitory GABAergic interneurons), rather than

GSK3 is a key component of the Wnt signaling pathway. In unstimulated cells, GSK3 is part of a multi-protein complex with APC, Axin and other proteins that facilitates phosphorylation of β-catenin by CK1 at Ser45, followed by phosphorylation of Ser33/37/41 by GSK3. This creates a recognition site for the E3 ubiquitin ligase βTrCP, which ubiquitinates β-catenin and targets it for degradation via the proteasome. In Wnt-stimulated cells, this multi-protein complex is disrupted, preventing phosphorylation and subsequent ubiquination of β-catenin, thus stabilizing the protein and increasing its cellular abundance. This leads to translocation to the nucleus, where β-catenin binds to several transcription factors facilitating transcription of target genes involved in cell fate regulation, including c-myc [49], NeuroD1 [48, 50, 51], Prox1 [52] and LINE-1 [50]. In adult neurogenesis, Wnt-induced expression of NeuroD1 is required for survival and maturation of adult-born neurons [48, 50, 51]. The prototypcial binding partners of β-catenin in the nucleus are members of the TCF/LEF family, which have been shown to be central mediators of tumourigenesis in the colon, breast and other tissues. In NPC's, activation of the Wnt pathway and elevated transcriptional activity of β-catenin has been shown to promote proliferation and inhibit neuronal differentiation [53, 54], while inhibition of Wnt signaling promotes neuronal differentiation [55, 56]. Accordingly, deletion of GSK3 isoforms promotes proliferation and inhibits differentiation of stem cells [57], consistent with pharmacological GSK3 inhibitors and Wnt-induced inhibition of GSK3 activity [7, 8]. This is dependent on elevated levels of transcriptionally active β-catenin [58], but surprisingly not by members of the TCF/LEF family. Instead, β-catenin was shown to bind to another transcription factor called Oct4, which increased expression of the pluripotency

promotes differentiation of NPC's into post-mitotic neurons.

**5. GSK3 and Wnt signaling**

156 Trends in Cell Signaling Pathways in Neuronal Fate Decision

proliferation of NPC's [46-48].

regulator and stem cell marker Nanog [58, 59].

Other isoforms of c-myc are also phosphorylated and targeted for degradation by GSK3 (i.e. L-myc, N-myc). In NPC's, deletion of c- and L-myc does not affect proliferation/differentiation, while deletion of N-myc significantly decreases NPC proliferation and impairs differentiation into mature neurons [62], suggesting that N-myc is the critical member of this family regulating neurogenesis and brain development [63, 64]. Like c-myc, GSK3 phosphorylates N-myc at Thr58 to promote ubiquitination by Fbw7 and degradation by the lysosome [65]. This is antagonized by growth factor-mediated inhibition of GSK3 activity (e.g. IGF1). Phosphoryla‐ tion of N-myc by GSK3 requires prior 'priming' phosphorylation at Ser62 by Cdk1, which is increased during mitosis, causing increased N-myc degradation [65]. This was shown to be important for exiting the cell cycle – the first step along the differentiation pathway. Cdk1 activity is dependent on binding to its co-factors cyclin A and B1 [66], whose transcription is controlled by the Hedgehog pathway, as is the transcription of N-myc [67, 68]. Therefore, Nmyc appears to be a point at which multiple signaling pathways involving GSK3 intersect in NPC's to control cell fate.

Some substrates of GSK3 are upstream of the Wnt pathway and can regulate its activity. For example, hypoxia-inducible factor 1 α (HIF1α) is a basic helix-loop-helix (bHLH)-structured transcription factor that is induced by low oxygen conditions to activate transcription of genes that provide protection and adaption of cells to oxidative stress and hypoxic conditions. HIF1α is phosphorylated by GSK3, promoting its degradation by the proteasome [69, 70]. Recently, it was shown that HIF1α promotes Wnt activation and transcription of TCF/LEF members in undifferentiated, but not differentiated cells [71]. Low GSK3 activity in undiffer‐ entiated cells would reduce GSK3-mediated phosphorylation and degradation of HIF1α, thus stabilizing the protein and leading to activation of the Wnt pathway. Simultaneously, low GSK3 activity (downstream of Wnt) would prevent β-catenin phosphorylation/degradation, increasing its transcriptional activity with TCF/LEF. Interestingly, the authors show that the subgranular zone of the dentate gyrus containing NPC's is hypoxic due to fewer blood vessels in the region and contains relatively high levels of HIF1α and transcriptionally active β-catenin. Thus, decreased oxygen levels could be important for maintaining the NPC niche in the hippocampus via GSK3, HIF1α and the Wnt pathway. It be should be noted that other groups have found that prolonged hypoxia increases the activity of GSK3 in cultured cells and *in vivo* [69, 72]. This might reflect differences between GSK3-mediated phosphorylation and degradation of HIF1α in undifferentiated versus differentiated cells.

**7. GSK3 and Notch signaling**

acquiring the same fate.

A role for the Notch signaling pathway in regulating development was discovered almost a century ago by pioneering work in fruit flies by John Dexter and Thomas Hunt Morgan [86]. It was later found that absence of Notch in flies caused neuronal hyperplasia and a decrease in glial cells in the brain [87]. Generally, Notch signaling promotes proliferation of NPCs and inhibits their differentiation, but has also been linked with glial cell specification [88, 89], neurite outgrowth [90] and learning and memory [91]. Notch is a family of 4 single-pass transmembrane proteins (Notch1-4) that are expressed at the cell surface of ES cells and NPCs (especially Notch1). Ligand-mediated activation of Notch leads to its proteolytic cleavage by Presenillin-g, releasing the Notch intracellular domain (NICD) for translocation to the nucleus where it binds with the transcription factor RBP-Jk to activate transcription of target genes including the bHLH transcriptional repressors *Hes1* and *Hes5*. These proteins repress tran‐ scription of pro-neural genes, thus inhibiting differentiation into neurons and maintaining proliferation of NPC's. Ligands that activate Notch, such as Delta-like 1 (Dll1) and Jagged1 (Jag1) are themselves transmembrane proteins. These are typically located at the cell surface of differentiated neurons that bind and stimulate activation of Notch on neighbouring NPC's. Thus, Notch signaling functions in a localized fashion, whereby a differentiating neuron expressing the ligands Dll1 or Jag1 binds Notch on a neighbouring cell to activate downstream signaling that inhibits differentiation and maintains proliferation of NPC's, thus establishing a stem cell niche in the brain. In other words, Notch signaling prevents equipotent cells from

Regulation of Cell Fate in the Brain by GSK3 http://dx.doi.org/10.5772/55180 159

GSK3 was originally found to be associated with the Notch signaling pathway in *Drosophila* development [92]. Since then, it has been shown to bind and phosphorylate the intracellular domain of Notch, stabilizing the protein by reducing its degradation by the proteasome [93]. However, other studies report that phosphorylation *de*stabilizes Notch and promotes its degradation [94, 95]. The reason for these contradictory observations are not clear, although considering that low GSK3 activity and high Notch signaling correlate with the highly proliferative, undifferentiated nature of ES cells/NPC's, it is likely that GSK3-mediated phosphorylation targets Notch for degradation by the proteasome. That is, low GSK3 mediated phosphorylation and degradation of NICD increases its abundance and transcrip‐ tional activity to promote proliferation/suppress differentiation. This is consistent with many other proteins that are destabilized by GSK3 phosphorylation, such as c-myc, HIF1α, β-catenin, etc. It is possible that prior phosphorylation by the cyclin C:Cdk8 complex could prime NICD

for subsequent phosphorylation by GSK3 [96], although this remains to be proven.

Cross-talk between the Notch and Wnt signaling pathways has been suggested by several studies, although the reports are contradictory. One study shows that Notch binds to the unphosphorylated, transcriptionally-active form of β-catenin, targeting it for degradation by the lysosome, thus suppressing expression of Wnt target genes [97]. At first glance, this is surprising, since both pathways are pro-proliferation/anti-differentiation. However, it should be noted that Notch-mediated degradation of β-catenin does not require ligand activation. Therefore, this mechanism might be more relevant to differentiated cells, such that in the
