**4. Neurogenesis in GSK3 mutant mice**

that trigger intracellular signalling pathways and changes in gene transcription, although the transcriptional regulators targeted by these signalling pathways and their target genes are yet

In 1980, Hammond and Dale noticed that lithium treatment of grey collie dogs increased their blood cell counts, which they suggested was due to increased proliferation of haematopoetic stem cells [4]. This was confirmed shortly afterwards [5], although the mechanism for lithium's action was not understood. In 1996, GSK3 was identified as a key target of lithium in cells [6], although it was another 8 years before a key role for GSK3 in regulating stem cell pluripotency was elucidated [7, 8]. Pharmacological inhibition of GSK3 activity was shown to maintain the undifferentiated phenotype in mouse and human embryonic stem (ES) cells, while its with‐ drawal promoted differentiation into multiple cell lineages [7]. More recently, it was demon‐ strated that the complex mixture of cytokines, growth factors, hormones, serum and feeder cells traditionally used to maintain self-renewal of ES cells can be replaced with two pharma‐ cological inhibitors; a MAPK inhibitor and a GSK3 inhibitor [8], thus emphasizing the importance of GSK3 for regulating pluripotency. The GSK3 substrates c-myc [9] and Klf5 [10] are among several transcription factors that have been used to induce pluripotency (iPS system). Thus, GSK3 is a key regulator of neurogenesis, although the precise molecular mechanisms are not yet fully understood. This review provides an overview of the extracel‐ lular stimuli and intracellular signalling pathways controlling GSK3 activity, as well as the downstream targets of GSK3 directly linking it to cellular proliferation and differentiation in the brain. GSK3 inhibitors are currently in clinical trials for several neurological disorders associated with impaired neurogenesis, therefore it is timely that cell fate pathways involving

GSK3 is a Ser/Thr kinase of the CMGC family of proline-directed kinases that is highly conserved in all eukaryotes. In mammals, it is ubiquitously expressed in all tissues and subcellular organelles, most highly in the brain [11]. There are 2 isoforms encoded by separate genes (chromosome 19q13.2 for GSK3α and chromosome 3q13.3 for GSK3β) [11]. Their kinase domains are 98% homologous and their substrate specificities are similar, but not identical [12]. A splice variant of GSK3β containing a 13 amino acid insert in the catalytic domain is specif‐ ically expressed in the brain [13], although its function is only just beginning to be investigated [12]. Interestingly, GSK3 is one of the most unusual kinases in the human genome for 3 main reasons; 1) Most (if not all) substrates require 'priming' phosphorylation 4 or 5 residues Cterminal to the GSK3 target site by another kinase *before* they can be efficiently phosphorylated by GSK3 [14]. 2) GSK3 is highly active in cells under basal conditions, opposite to most other kinases. 3) Phosphorylation of GSK3 at an N-terminal serine residue *inhibits* its kinase activity

to be fully clarified.

GSK3 are delineated.

**3. GSK3**

**2. GSK3 and neurogenesis**

154 Trends in Cell Signaling Pathways in Neuronal Fate Decision

Valuable information on the role of GSK3 in cell fate determination has been obtained from mice genetically modified to either increase or decrease expression of GSK3α and β. While GSK3β-knockout mice die in late development due to defects in heart development and/or hepatic apoptosis [22, 23], GSK3β-heterozygous mice and GSK3α-knockout (homozygous) mice are viable and display several behavioural defects, including increased anxiety, decreased aggression and memory defects [24-28]. Also, GSK3α-null mice exhibit decreased numbers and size of Purkinje cells in the cerebellum [24]. Conditional overexpression of GSK3β in the forebrain using the doxycycline/Tet system impaired memory and spatial learning in mice [29]. At the cellular level, GSK3β overexpression increased neuronal cell death, astrocytosis, gliosis and reduced LTP induction. These effects could be restored by reducing GSK3 activity to normal levels by silencing the transgene or by treatment with lithium [30, 31]. In another report, overexpression of GSK3β-S9A in post-natal neurons (Thy-1 promoter) reduced brain size in adult mice, especially in the cerebral cortex, predominantly caused by reduced size of neuronal cell bodies and the somatodendritic compartment [32]. Together, these observations clearly demonstrate that GSK3 is important for healthy development and function of the brain.

In addition to conventional under/over-expression mouse models, GSK3-knockin mice were developed that are insensitive to growth factor inhibition (Ser21/9 mutated to Ala in GSK3α and β, respectively), but remain sensitive to Wnt-induced inhibition [33]. These mice are viable and display no overt developmental or growth defects, but do exhibit increased susceptibility to hyperactivity, stress-induced depression and mild anxiety, as well as abnormal LTP and memory functions [34, 35]. NPC's isolated from GSK3-knockin mice exhibit reduced neuro‐ genesis, despite normal proliferation [36], suggesting defective differentiation/maturation or survival of NPC's. In contrast, mice with double homozygous deletion of GSK3α and β isoforms (i.e. all GSK3 isoforms deleted) display a dramatic increase in proliferation of NPC's and decreased differentiation into post-mitotic neurons [37]. This is accompanied by deregu‐ lation of Wnt, Notch, Hedgehog and FGF signalling pathways. In another mouse model, mice expressing a mutant form of the scaffolding protein Disrupted in Schizophrenia (DISC1), which is mutated in schizophrenia and mood disorder patients, display increased GSK3 activity, causing inhibition of the Wnt signaling pathway and decreased NPC proliferation [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 promotes differentiation of NPC's into post-mitotic neurons.

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

Regulation of Cell Fate in the Brain by GSK3 http://dx.doi.org/10.5772/55180 157

importance of phosphorylation of c-myc by GSK3 in the regulation of cell fate.

NPC's to control cell fate.

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

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.
