**7. GSK3 and Notch signaling**

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

GSK3's involvement in the Wnt pathway is also regulated by the scaffolding protein DISC1. This protein directly binds to GSK3 to inhibit phosphorylation of β-catenin, preventing its degradation and activating its transcriptional activity [38]. This promotes NPC proliferation during embryonic development and in the adult brain. Depletion of DISC1 or expression of DISC1 mutants associated with mood disorders and schizophrenia reduced NPC proliferation and induced schizophrenia and depression-like symptoms in mice [38, 73]. These defects were normalized by administration of pharmacological inhibitors of GSK3. These studies demon‐ strate that DISC1 is a negative regulator of the Wnt pathway and NPC proliferation by directly inhibiting GSK3-mediated phosphorylation of β-catenin. Interestingly, one schizophreniaassociated mutant of DISC1 (S704C) did not affect GSK3 activity, Wnt signaling or NPC proliferation, but instead impaired neuronal migration in the developing cortex via reduced binding to cytoskeletal proteins (Dixdc1) [73]. Elsewhere, it was shown that DISC1 acts as a molecular switch between proliferation and migration in NPC's, whereby DISC1 inhibits GSK3-mediated phosphorylation of β-catenin and activates its transcriptional activity to drive proliferation of NPC's, while in committed, post-mitotic neurons, DISC1 regulates neuronal migration via another protein called BBS1 and the centrosome [74]. Together, these studies demonstrate that DISC1 regulates NPC proliferation and neuronal migration through GSK3-

As well as DISC1, other upstream regulators of GSK3 are genetically linked to mood disorders and schizophrenia, including Akt [75], Neuregulin [76] and the dopamine/β-arrestin signaling complex [77, 78]. GSK3 kinase activity is also inhibited by several mood-stabilizers, antidepressants and anti-psychotic drugs [6, 79, 80], while genetic manipulation of GSK3 activity in mice produces behaviours correlating with mood disorders [24-28]. A single nucleotide polymorphism in the promoter region of GSK3β has also been correlated with onset of Bipolar disorder [81]. Together, these observations strongly implicate elevated GSK3 activity in the etiology of mood disorders and schizophrenia. Neurogenesis is decreased in these disorders [82, 83], but is promoted by mood stabilizing drugs, such as lithium [84, 85]. Therefore, moodstabilizing drugs may act (at least in part) by promoting neurogenesis via inhibition of GSK3. If so, promoting neurogenesis using GSK3 inhibitors could also be beneficial to other mental disorders, including neurodegenerative diseases such as Alzheimer's disease and age-related cognitive decline, as well repair following brain injury, such as stroke and spinal chord injuries.

degradation of HIF1α in undifferentiated versus differentiated cells.

**6. GSK3, DISC1 and mood disorders**

158 Trends in Cell Signaling Pathways in Neuronal Fate Decision

dependent and independent pathways, respectively.

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 acquiring the same fate.

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 absence of ligand stimulation, Notch reduces β-catenin levels to antagonize Wnt signaling. Thus, both pathways are suppressed, consistent with a differentiated phenotype. Another report has shown that FGF2-mediated inhibition of GSK3 activity leads to an increase in transcriptionally-active β-catenin in the cell nucleus, which simultaneously activates TCF/LEF and Notch/RBP-Jk transcriptional complexes, promoting proliferation and inhibiting differ‐ entiation, respectively [98]. Although both pathways promote maintenance of stem cells as expected, FGF2-mediated inhibition of GSK3 is unexpected, since it is commonly assumed that growth factors do not influence β-catenin levels in the Wnt pathway [19], although this observation has been confirmed by several other groups [99, 100]. It is possible that FGF signaling is an exception to the growth factor rule. All together, it seems clear that there are interactions between Notch and other cell fate pathways involving GSK3, although the precise mechanisms are yet to be fully clarified.
