**8. GSK3 and Hedgehog signalling**

The Hedgehog signaling pathway is a critical regulator of cell fate during development and for maintaining proliferation of cell stem niches in adults, including NPC's in the brain. In mammals, there are 3 Hedgehog proteins, with Sonic hedgehog being the most widely studied. These are secreted proteins that bind to a receptor at the surface of target cells called patched. When Hedgehog binds to patched, patched is released from another receptor protein at the cell surface called smoothened, allowing smoothened levels to increase. Smoothened inhibits the proteolytic cleavage of the zinc-finger proteins Gli (*cubitus interruptus* in flies), which are critical effectors of the Hedgehog pathway. Gli1 and Gli2 are transcriptional activators, while Gli3 is a repressor. These proteins regulate the transcription of cell cycle-related genes, such as cyclins D and E, N-myc, Bcl2, POU3F1, Runx2 and Tbx2, as well as patched in a negative feedback loop. In the absence of Hedgehog ligands, Gli associates with a scaffolding complex containing Cos2 and Fused that facilitates phosphorylation by GSK3 and CK1 (following priming by PKA). This phosphorylation targets Gli for ubiquitination and proteolysis, generating a truncated repressor form lacking the C-terminal activation domains [101-105]. But in the presence of Hedgehog, this signaling complex is disrupted, inhibiting phosphory‐ lation and processing of Gli, leading to accumulation of transcriptionally active full-length protein in the nucleus. Thus, GSK3 antagonises Hedgehog signaling by mediating the degradation of Gli proteins. The scaffolding protein Sufu, a negative regulator of Hedgehog signaling, is also phosphorylated by GSK3, which stabilizes the protein and increases degra‐ dation of Gli proteins [106]. Thus, GSK3 promotes Gli degradation directly and via stabilization of Sufu.

The Hedgehog pathway is similar to the Wnt pathway, in that it utilizes the constitutive activity of GSK3 to negatively regulate the key transcriptional effector of each pathway (β-catenin for Wnt, Gli for Hedgehog). Ligand-mediated activation of both pathways reduces phosphoryla‐ tion of these key effectors by disrupting their respective signaling/scaffolding complexes without directly inhibiting GSK3 activity (i.e. they exclusively reduce phosphorylation of a specific substrate). In contrast, growth factor signaling directly inhibits GSK3 kinase activity

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

The Hedgehog signaling pathway is a critical regulator of cell fate during development and for maintaining proliferation of cell stem niches in adults, including NPC's in the brain. In mammals, there are 3 Hedgehog proteins, with Sonic hedgehog being the most widely studied. These are secreted proteins that bind to a receptor at the surface of target cells called patched. When Hedgehog binds to patched, patched is released from another receptor protein at the cell surface called smoothened, allowing smoothened levels to increase. Smoothened inhibits the proteolytic cleavage of the zinc-finger proteins Gli (*cubitus interruptus* in flies), which are critical effectors of the Hedgehog pathway. Gli1 and Gli2 are transcriptional activators, while Gli3 is a repressor. These proteins regulate the transcription of cell cycle-related genes, such as cyclins D and E, N-myc, Bcl2, POU3F1, Runx2 and Tbx2, as well as patched in a negative feedback loop. In the absence of Hedgehog ligands, Gli associates with a scaffolding complex containing Cos2 and Fused that facilitates phosphorylation by GSK3 and CK1 (following priming by PKA). This phosphorylation targets Gli for ubiquitination and proteolysis, generating a truncated repressor form lacking the C-terminal activation domains [101-105]. But in the presence of Hedgehog, this signaling complex is disrupted, inhibiting phosphory‐ lation and processing of Gli, leading to accumulation of transcriptionally active full-length protein in the nucleus. Thus, GSK3 antagonises Hedgehog signaling by mediating the degradation of Gli proteins. The scaffolding protein Sufu, a negative regulator of Hedgehog signaling, is also phosphorylated by GSK3, which stabilizes the protein and increases degra‐ dation of Gli proteins [106]. Thus, GSK3 promotes Gli degradation directly and via stabilization

The Hedgehog pathway is similar to the Wnt pathway, in that it utilizes the constitutive activity of GSK3 to negatively regulate the key transcriptional effector of each pathway (β-catenin for Wnt, Gli for Hedgehog). Ligand-mediated activation of both pathways reduces phosphoryla‐ tion of these key effectors by disrupting their respective signaling/scaffolding complexes without directly inhibiting GSK3 activity (i.e. they exclusively reduce phosphorylation of a specific substrate). In contrast, growth factor signaling directly inhibits GSK3 kinase activity

mechanisms are yet to be fully clarified.

160 Trends in Cell Signaling Pathways in Neuronal Fate Decision

**8. GSK3 and Hedgehog signalling**

of Sufu.

FGF [109], EGF [110], TGF [110], CNTF [111]. Among the most potent growth factors are the neurotrophins (BDNF, NGF, NT-3, NT-4), whereby direct administration [112] or genetic deletion [113] increases and reduces neurogenesis, respectively. Accordingly, their receptor molecules, called Trk receptors, are predominantly expressed by post-mitotic neurons, but not NPC's or neural crest cells [114]. BDNF signaling, in particular, has been shown to promote survival of newly-generated neurons [115], while defects in BDNF signaling have been linked to decreased neurogenesis and neuronal survival in several neurological disorders, including Alzheimer's disease, age-related cognitive decline, Bipolar disorder and Schizophrenia (for reviews, see [116, 117]).

and GSK3-mediated protection of neurons is via Mcl-1. Indeed, nutrient deprivation correlates with decreased levels of Mcl-1 and induction of apoptosis in neurons, which was dependent on phosphorylation by GSK3 [125, 126]. In contrast, Bax is a pro-apoptotic member of the Bcl2 family. Phosphorylation by GSK3 at Ser163 promotes its translocation to the mitochondrial outer membrane, where it oligomerizes to form a pore promoting cytochrome c release and cell death [127, 128]. Inhibition of GSK3 via growth factor signaling or therapeutics (e.g. lithium) antagonizes Bax translocation and cytochrome c release, promoting cell survival.

Regulation of Cell Fate in the Brain by GSK3 http://dx.doi.org/10.5772/55180 163

Other downstream targets of GSK3 that promote neuronal survival include the transcription factors HIF1α, HSF1 [129], Mef2D [130] and Bcl3 [131] by activating transcription of prosurvival genes. For example, increased activity of GSK3 during potassium withdrawal from cerebellar granule neurons leads to increased phosphorylation and degradation of Mef2D, which could be blocked by a GSK3-resistant form of Mef2D [130]. The neurotrophins NGF and BDNF promote Mef2D stability by reducing GSK3-mediated degradation, leading to increased transcription of its target gene Bcl-w, an anti-apoptotic member of the Bcl-2 family [132].

**10. GSK3, ubiquitin ligases and proteasome-mediated degradation**

tiation and survival of neurons.

It is noticeable that many substrates of GSK3 are targeted for ubiquitination and proteasomemediated degradation following phosphorylation by GSK3. Most of these are transcription factors that are phosphorylated by GSK3 within an [ST]PPx[ST]P motif, including c-myc [61, 133, 134], c-jun [135], Klf5 [136, 137], cyclin E1 [138], Gli3 [101, 102] and snail [139]. These transcription factors have short half-lives, largely due to the actions of GSK3, which is highly active under basal conditions in mature, differentiated cells. However, GSK3 activity levels are comparatively lower in undifferentiated cells, induced by persistent Wnt, Notch, Hedge‐ hog and/or growth factor signaling to maintain the proliferative capacity of these cells [140]. Here, phosphorylation and ubiquitination of transcription factors by GSK3 is reduced, thus stabilizing the proteins (prolonging their half-lives) and contributing to stem/precursor cell proliferation. A common E3 ligase targeting GSK3 substrates is Fbw7. In fact, most of the reported targets of Fbw7 are established GSK3 substrates (see [141]). Genetic studies in mice indicate that Fbw7 is required for differentiation and survival of NPC's. For example, brainspecific deletion of Fbw7 increased apoptosis of NPC's, which could be rescued by concomitant deletion of c-jun [142]. Similar results were observed for cerebellar development in mice [143]. NPC differentiation was also impaired, particularly a decrease in the number of mature neurons and increased glial progenitors (although no change in mature astrocytes). This was alleviated by inhibition of Notch signaling. Supporting this, a separate study showed that brain-specific deletion of Fbw7 skewed the differentiation of NPC's towards astrocytes, rather than neurons, which could be restored with a pharmacological inhibitor of the Notch pathway [144]. Meanwhile, the maintenance/proliferation of the NPC's was not affected. Together, these observations show that GSK3 and Fbw7 share common substrates that regulate the differen‐

GSK3 is an established target of many growth factor signaling pathways. Ligand-binding to their respective receptors at the cell surface induces dimerisation/oligomerisation, activating their intrinsic kinase activities and autophosphorylation of key tyrosine residues. This provides binding sites for SH2 and SH3-domain-containing adaptor proteins, such as IRS and Grb proteins, promoting recruitment and activation of phosphatidylinositol 3 kinase (PI3K). This lipid kinase converts phosphatidylinositol 4,5 bisphosphate (PIP2) to PIP3, recruiting members of the AGC family of kinases (e.g. PKB/Akt), where they are phosphorylated and activated by PDK1. These AGC kinases phosphorylate an N-terminal serine on GSK3 (Ser21 on GSK3α, Ser9 on GSK3β) that binds to the substrate binding site on GSK3, thus acting as a pseudo-substrate to inhibit phosphorylation of primed substrates [17].

Numerous studies have demonstrated that pharmacological inhibitors of GSK3 promote survival of neurons subjected to a range of toxic stimuli (for a review, see [118]), while overexpression of GSK3 promotes neuronal apoptosis *in vitro* and *in vivo* [29, 119, 120]. Importantly, GSK3α/β -knockin mice (S21/9A) that are insensitive to growth factor inhibition exhibit reduced neurogenesis, despite normal proliferation of NPC's [36]. As noted previously, this could suggest defective differentiation patterns in the NPC's, but could equally represent decreased survival of newly generated neurons. In addition, several substrates of GSK3 are associated with cell survival (discussed below). Together, these observations strongly suggest that growth factor-mediated inhibition of GSK3 appears to be a key mechanism by which growth factors promote survival of newly generated neurons during neurogenesis.

A key downstream target of GSK3 promoting survival is Mcl1. This anti-apoptotic, prosurvival member of the Bcl2 family is directly phosphorylated by GSK3 at Ser155 and Ser159, targeting it for ubiquitination by E3 ligases (Fbw7, βTrCP and/or Trim17) and degradation by the proteasome [121, 122]. Upon growth factor stimulation, GSK3 is inhibited, reducing GSK3 mediated phosphorylation of Mcl1, thus stabilizing the protein and promoting neuronal survival. The Notch signaling pathway has also been shown to promote survival of neurons via Mcl-1, although the role of GSK3 was not investigated [123]. NPC's and newly-committed neurons in the subventricular zone and surrounding areas of mice express high levels of Mcl-1, while Mcl-1 deficiency caused widespread apoptosis, especially in newly-committed neurons as they migrate away from this region [124]. This suggests that a key target of growth factor and GSK3-mediated protection of neurons is via Mcl-1. Indeed, nutrient deprivation correlates with decreased levels of Mcl-1 and induction of apoptosis in neurons, which was dependent on phosphorylation by GSK3 [125, 126]. In contrast, Bax is a pro-apoptotic member of the Bcl2 family. Phosphorylation by GSK3 at Ser163 promotes its translocation to the mitochondrial outer membrane, where it oligomerizes to form a pore promoting cytochrome c release and cell death [127, 128]. Inhibition of GSK3 via growth factor signaling or therapeutics (e.g. lithium) antagonizes Bax translocation and cytochrome c release, promoting cell survival.

FGF [109], EGF [110], TGF [110], CNTF [111]. Among the most potent growth factors are the neurotrophins (BDNF, NGF, NT-3, NT-4), whereby direct administration [112] or genetic deletion [113] increases and reduces neurogenesis, respectively. Accordingly, their receptor molecules, called Trk receptors, are predominantly expressed by post-mitotic neurons, but not NPC's or neural crest cells [114]. BDNF signaling, in particular, has been shown to promote survival of newly-generated neurons [115], while defects in BDNF signaling have been linked to decreased neurogenesis and neuronal survival in several neurological disorders, including Alzheimer's disease, age-related cognitive decline, Bipolar disorder and Schizophrenia (for

GSK3 is an established target of many growth factor signaling pathways. Ligand-binding to their respective receptors at the cell surface induces dimerisation/oligomerisation, activating their intrinsic kinase activities and autophosphorylation of key tyrosine residues. This provides binding sites for SH2 and SH3-domain-containing adaptor proteins, such as IRS and Grb proteins, promoting recruitment and activation of phosphatidylinositol 3 kinase (PI3K). This lipid kinase converts phosphatidylinositol 4,5 bisphosphate (PIP2) to PIP3, recruiting members of the AGC family of kinases (e.g. PKB/Akt), where they are phosphorylated and activated by PDK1. These AGC kinases phosphorylate an N-terminal serine on GSK3 (Ser21 on GSK3α, Ser9 on GSK3β) that binds to the substrate binding site on GSK3, thus acting as a

Numerous studies have demonstrated that pharmacological inhibitors of GSK3 promote survival of neurons subjected to a range of toxic stimuli (for a review, see [118]), while overexpression of GSK3 promotes neuronal apoptosis *in vitro* and *in vivo* [29, 119, 120]. Importantly, GSK3α/β -knockin mice (S21/9A) that are insensitive to growth factor inhibition exhibit reduced neurogenesis, despite normal proliferation of NPC's [36]. As noted previously, this could suggest defective differentiation patterns in the NPC's, but could equally represent decreased survival of newly generated neurons. In addition, several substrates of GSK3 are associated with cell survival (discussed below). Together, these observations strongly suggest that growth factor-mediated inhibition of GSK3 appears to be a key mechanism by which

growth factors promote survival of newly generated neurons during neurogenesis.

A key downstream target of GSK3 promoting survival is Mcl1. This anti-apoptotic, prosurvival member of the Bcl2 family is directly phosphorylated by GSK3 at Ser155 and Ser159, targeting it for ubiquitination by E3 ligases (Fbw7, βTrCP and/or Trim17) and degradation by the proteasome [121, 122]. Upon growth factor stimulation, GSK3 is inhibited, reducing GSK3 mediated phosphorylation of Mcl1, thus stabilizing the protein and promoting neuronal survival. The Notch signaling pathway has also been shown to promote survival of neurons via Mcl-1, although the role of GSK3 was not investigated [123]. NPC's and newly-committed neurons in the subventricular zone and surrounding areas of mice express high levels of Mcl-1, while Mcl-1 deficiency caused widespread apoptosis, especially in newly-committed neurons as they migrate away from this region [124]. This suggests that a key target of growth factor

pseudo-substrate to inhibit phosphorylation of primed substrates [17].

reviews, see [116, 117]).

162 Trends in Cell Signaling Pathways in Neuronal Fate Decision

Other downstream targets of GSK3 that promote neuronal survival include the transcription factors HIF1α, HSF1 [129], Mef2D [130] and Bcl3 [131] by activating transcription of prosurvival genes. For example, increased activity of GSK3 during potassium withdrawal from cerebellar granule neurons leads to increased phosphorylation and degradation of Mef2D, which could be blocked by a GSK3-resistant form of Mef2D [130]. The neurotrophins NGF and BDNF promote Mef2D stability by reducing GSK3-mediated degradation, leading to increased transcription of its target gene Bcl-w, an anti-apoptotic member of the Bcl-2 family [132].

## **10. GSK3, ubiquitin ligases and proteasome-mediated degradation**

It is noticeable that many substrates of GSK3 are targeted for ubiquitination and proteasomemediated degradation following phosphorylation by GSK3. Most of these are transcription factors that are phosphorylated by GSK3 within an [ST]PPx[ST]P motif, including c-myc [61, 133, 134], c-jun [135], Klf5 [136, 137], cyclin E1 [138], Gli3 [101, 102] and snail [139]. These transcription factors have short half-lives, largely due to the actions of GSK3, which is highly active under basal conditions in mature, differentiated cells. However, GSK3 activity levels are comparatively lower in undifferentiated cells, induced by persistent Wnt, Notch, Hedge‐ hog and/or growth factor signaling to maintain the proliferative capacity of these cells [140]. Here, phosphorylation and ubiquitination of transcription factors by GSK3 is reduced, thus stabilizing the proteins (prolonging their half-lives) and contributing to stem/precursor cell proliferation. A common E3 ligase targeting GSK3 substrates is Fbw7. In fact, most of the reported targets of Fbw7 are established GSK3 substrates (see [141]). Genetic studies in mice indicate that Fbw7 is required for differentiation and survival of NPC's. For example, brainspecific deletion of Fbw7 increased apoptosis of NPC's, which could be rescued by concomitant deletion of c-jun [142]. Similar results were observed for cerebellar development in mice [143]. NPC differentiation was also impaired, particularly a decrease in the number of mature neurons and increased glial progenitors (although no change in mature astrocytes). This was alleviated by inhibition of Notch signaling. Supporting this, a separate study showed that brain-specific deletion of Fbw7 skewed the differentiation of NPC's towards astrocytes, rather than neurons, which could be restored with a pharmacological inhibitor of the Notch pathway [144]. Meanwhile, the maintenance/proliferation of the NPC's was not affected. Together, these observations show that GSK3 and Fbw7 share common substrates that regulate the differen‐ tiation and survival of neurons.
