**5. SOCS molecules and cytokine signalling pathways**

As discussed in the previous section, a diversity of signalling cascades are involved in regu‐ lating neuronal cell proliferation, differentiation and survival. However, JAK-STAT signal‐ ling seems to be one of the central pathways in the regulation of adult neurogenesis. Since its discovery twenty years ago, this pathway has been studied extensively due to its key roles in modulating many different physiological processes through responses to various regulatory molecules [146].

neurogenic niche [50]. Further, it has an important role in the positive regulation of reactive

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249

http://dx.doi.org/10.5772/53268

An important aspect to cytokine signalling via a pathway such as JAK-STAT is the need for its downregulation following activation. Thus far, JAK-STAT signalling is known to be neg‐ atively regulated by protein inhibitors of activated STATs (PIAS), the SH2-containing pro‐ tein tyrosine phosphatases (SHPs) and suppressors of cytokine signalling (SOCS) proteins [160]. In this section, SOCS proteins will be the focus of discussion as the negative regulation of the JAK-STAT signalling pathway by SOCS has several effects on the regulation of neuro‐

The SOCS family consists of eight members, namely, SOCS1-7 and cytokine-inducible Src homology 2 (SH2) protein (CIS). They are characterised by a central SH2 domain, a C-termi‐ nal SOCS box and a variable N-terminal domain. In addition to these, SOCS1 and SOCS3 also contain a small kinase inhibitory domain. CIS was the first member of this protein fami‐ ly to be cloned. It is also unique to the rest of the SOCS family as a result of its SH2 domain

SOCS expression is induced following activation of the JAK-STAT pathway. This initiates a classic negative feedback loop whereby the SOCS proteins activated by JAK-STAT signal‐ ling now go on to inhibit it. SOCS proteins achieve downregulation of signalling by binding to tyrosine phosphorylated proteins via their SH2 domain. The exact mechanism by which signalling inhibition is achieved varies depending on the SOCS protein in question. For ex‐ ample, SOCS1 and SOCS3 both work to block the kinase activity of activated JAK proteins. In the case of SOCS1, this is achieved by directly binding and blocking access to the activat‐ ed JAK. In the case of SOCS3, this is achieved by its binding to the activated gp130 receptor such that STAT proteins can no longer dock onto the phosphorylated tyrosine residues and be activated by JAK. One mechanism of action for SOCS2 is by blocking STAT access to the

SOCS proteins are also able to regulate activity of target proteins, including other SOCS pro‐ teins, through interaction with their SOCS box [163, 164]. Interestingly, SOCS2, SOCS6 and SOCS7 have the potential to interact with all members of the SOCS protein family including themselves [164]. In terms of SOCS2, when expressed at high levels, it is able to inhibit the action of SOCS1 and SOCS3 by targeting them for proteasomal degradation [164]. This has also been proposed as a mechanism for the dual action of SOCS2 on GH signalling as ob‐ served in the overgrowth phenotypes of SOCS2 knockout and overexpressing mice descri‐

Signalling via the JAK-STAT pathway has an important role in neural precursor prolifera‐ tion and differentiation [153, 166-168]. Following the discovery that SOCS proteins regulate the JAK-STAT pathway, the next obvious step was to examine them for possible roles in the nervous system. In doing so, analysis of the SOCS family gene expression in the developing mouse forebrain brought SOCS2 into the spotlight [168]. The genes SOCS1 – SOCS3 and CIS

which differs in a few amino acids from most all other known SH2 domains [161].

astrocytes in the injured CNS [159].

**5.2. The suppressors of cytokine signalling**

genesis and NPC fate.

activated receptor [162].

bed below [165].

#### **5.1. JAK/STAT signalling**

The JAK-STAT pathway can be activated by a range of cytokines, growth factors and hor‐ mones. In the regulation of adult neurogenesis, activation of this pathway is carried out by a group of neuroregulatory cytokines. Members of this cytokine group include CNTF, LIF and cardiotrophin 1 (CT-1), all of which belong to the interleukin 6 family of cytokines. These cyto‐ kines initiate JAK-STAT activation by binding and signalling through the LIF receptor-β (LIFRβ)/ glycoprotein 130 (gp130) receptor complex. The receptor complex bound by CNTF differs slightly in that it has a third extracellular receptor component, the CNTF receptor-α (structurally related to gp130), that is held to the membrane via a glycosylphosphoinositol [147].

Cytokine binding results in the dimerization of LIFRβ and gp130 receptors to form a com‐ plex [148]. This initiates autophosphorylation and activation of JAK proteins which are asso‐ ciated with the intracellular domains of the LIFRβ and gp130 receptors [149]. Members of the JAK protein family include JAK1, JAK2, JAK3 and TYK2. Cytokines signalling through the LIFRβ/gp130 pathway have been found to activate at least JAK1, JAK2 and TYK2 [150]. In terms of the CNS, only JAK1 and JAK2 expression has been found at significant levels [151]. JAK2 is highly expressed in the developing brain compared to JAK1, thus, a role for it in the regulation of neurogenesis in the developing brain has been suggested [151].

After activation, JAKs phosphorylate tyrosine residues in the intracellular domains of LIFRβ and gp130. These phosphorylated residues become binding sites for SH2 domain containing proteins such as STAT. STAT proteins are a family of transcription factors comprised of STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b and STAT6 [152]. Upon binding to the ac‐ tivated receptor complex, STAT proteins are phosphorylated by JAKs resulting in their di‐ merization. Dimerized STAT proteins are now able to translocate into the nucleus and induce gene expression of target neural genes such as glial fibrillary acidic protein (GFAP), peripherin and vasoactive intestinal peptide [46]. Other SH2 domain containing proteins can also bind the activated LIFRβ/gp130 receptor complex to activate the Ras/MAPK and PI-3K/Akt signalling pathways [49].

The LIFRβ/gp130 pathway is essential for the regulation of astrogliogenesis in the develop‐ ing and adult brain. In cultured cortical precursors, CNTF, LIF and CT-1 all promote astro‐ cyte formation through LIFRβ/gp130 activation [153-155]. Integral to this pathway is signalling via STAT3, as highlighted by the observation that STAT3 activation in neural stem cells induces glial differentiation, while its inhibition promotes a neuronal fate [156, 157]. Also, in neuroepithelial cells, STAT3 activation promotes astrogliogenisis via LIF in‐ duced bone morphogenetic protein 2 expression [158]. In addition to regulating astroglio‐ genesis, STAT3 induction by CNTF was found to be essential in the maintenance of the SGZ neurogenic niche [50]. Further, it has an important role in the positive regulation of reactive astrocytes in the injured CNS [159].

An important aspect to cytokine signalling via a pathway such as JAK-STAT is the need for its downregulation following activation. Thus far, JAK-STAT signalling is known to be neg‐ atively regulated by protein inhibitors of activated STATs (PIAS), the SH2-containing pro‐ tein tyrosine phosphatases (SHPs) and suppressors of cytokine signalling (SOCS) proteins [160]. In this section, SOCS proteins will be the focus of discussion as the negative regulation of the JAK-STAT signalling pathway by SOCS has several effects on the regulation of neuro‐ genesis and NPC fate.

#### **5.2. The suppressors of cytokine signalling**

ling seems to be one of the central pathways in the regulation of adult neurogenesis. Since its discovery twenty years ago, this pathway has been studied extensively due to its key roles in modulating many different physiological processes through responses to various

The JAK-STAT pathway can be activated by a range of cytokines, growth factors and hor‐ mones. In the regulation of adult neurogenesis, activation of this pathway is carried out by a group of neuroregulatory cytokines. Members of this cytokine group include CNTF, LIF and cardiotrophin 1 (CT-1), all of which belong to the interleukin 6 family of cytokines. These cyto‐ kines initiate JAK-STAT activation by binding and signalling through the LIF receptor-β (LIFRβ)/ glycoprotein 130 (gp130) receptor complex. The receptor complex bound by CNTF differs slightly in that it has a third extracellular receptor component, the CNTF receptor-α (structurally related to gp130), that is held to the membrane via a glycosylphosphoinositol

Cytokine binding results in the dimerization of LIFRβ and gp130 receptors to form a com‐ plex [148]. This initiates autophosphorylation and activation of JAK proteins which are asso‐ ciated with the intracellular domains of the LIFRβ and gp130 receptors [149]. Members of the JAK protein family include JAK1, JAK2, JAK3 and TYK2. Cytokines signalling through the LIFRβ/gp130 pathway have been found to activate at least JAK1, JAK2 and TYK2 [150]. In terms of the CNS, only JAK1 and JAK2 expression has been found at significant levels [151]. JAK2 is highly expressed in the developing brain compared to JAK1, thus, a role for it

After activation, JAKs phosphorylate tyrosine residues in the intracellular domains of LIFRβ and gp130. These phosphorylated residues become binding sites for SH2 domain containing proteins such as STAT. STAT proteins are a family of transcription factors comprised of STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b and STAT6 [152]. Upon binding to the ac‐ tivated receptor complex, STAT proteins are phosphorylated by JAKs resulting in their di‐ merization. Dimerized STAT proteins are now able to translocate into the nucleus and induce gene expression of target neural genes such as glial fibrillary acidic protein (GFAP), peripherin and vasoactive intestinal peptide [46]. Other SH2 domain containing proteins can also bind the activated LIFRβ/gp130 receptor complex to activate the Ras/MAPK and

The LIFRβ/gp130 pathway is essential for the regulation of astrogliogenesis in the develop‐ ing and adult brain. In cultured cortical precursors, CNTF, LIF and CT-1 all promote astro‐ cyte formation through LIFRβ/gp130 activation [153-155]. Integral to this pathway is signalling via STAT3, as highlighted by the observation that STAT3 activation in neural stem cells induces glial differentiation, while its inhibition promotes a neuronal fate [156, 157]. Also, in neuroepithelial cells, STAT3 activation promotes astrogliogenisis via LIF in‐ duced bone morphogenetic protein 2 expression [158]. In addition to regulating astroglio‐ genesis, STAT3 induction by CNTF was found to be essential in the maintenance of the SGZ

in the regulation of neurogenesis in the developing brain has been suggested [151].

regulatory molecules [146].

248 Trends in Cell Signaling Pathways in Neuronal Fate Decision

**5.1. JAK/STAT signalling**

PI-3K/Akt signalling pathways [49].

[147].

The SOCS family consists of eight members, namely, SOCS1-7 and cytokine-inducible Src homology 2 (SH2) protein (CIS). They are characterised by a central SH2 domain, a C-termi‐ nal SOCS box and a variable N-terminal domain. In addition to these, SOCS1 and SOCS3 also contain a small kinase inhibitory domain. CIS was the first member of this protein fami‐ ly to be cloned. It is also unique to the rest of the SOCS family as a result of its SH2 domain which differs in a few amino acids from most all other known SH2 domains [161].

SOCS expression is induced following activation of the JAK-STAT pathway. This initiates a classic negative feedback loop whereby the SOCS proteins activated by JAK-STAT signal‐ ling now go on to inhibit it. SOCS proteins achieve downregulation of signalling by binding to tyrosine phosphorylated proteins via their SH2 domain. The exact mechanism by which signalling inhibition is achieved varies depending on the SOCS protein in question. For ex‐ ample, SOCS1 and SOCS3 both work to block the kinase activity of activated JAK proteins. In the case of SOCS1, this is achieved by directly binding and blocking access to the activat‐ ed JAK. In the case of SOCS3, this is achieved by its binding to the activated gp130 receptor such that STAT proteins can no longer dock onto the phosphorylated tyrosine residues and be activated by JAK. One mechanism of action for SOCS2 is by blocking STAT access to the activated receptor [162].

SOCS proteins are also able to regulate activity of target proteins, including other SOCS pro‐ teins, through interaction with their SOCS box [163, 164]. Interestingly, SOCS2, SOCS6 and SOCS7 have the potential to interact with all members of the SOCS protein family including themselves [164]. In terms of SOCS2, when expressed at high levels, it is able to inhibit the action of SOCS1 and SOCS3 by targeting them for proteasomal degradation [164]. This has also been proposed as a mechanism for the dual action of SOCS2 on GH signalling as ob‐ served in the overgrowth phenotypes of SOCS2 knockout and overexpressing mice descri‐ bed below [165].

Signalling via the JAK-STAT pathway has an important role in neural precursor prolifera‐ tion and differentiation [153, 166-168]. Following the discovery that SOCS proteins regulate the JAK-STAT pathway, the next obvious step was to examine them for possible roles in the nervous system. In doing so, analysis of the SOCS family gene expression in the developing mouse forebrain brought SOCS2 into the spotlight [168]. The genes SOCS1 – SOCS3 and CIS were found to be expressed at all ages (E10 to P25) with a common peak in expression be‐ tween E14 and P8. However, the level of SOCS2 expression was much higher in comparison. The spatial pattern of SOCS2 expression also distinguished it from the other SOCS genes, with moderate to high levels of expression in neurogenic regions and in newborn neurons. In the adult, SOCS2 was maintained in the CA3 region of the hippocampus and at a moder‐ ate level in the dentate gyrus, compared to other SOCS genes whose expression was not lo‐ calized, if expressed at all under basal conditions. SOCS2 expression was also present in the cerebral cortex and other regions such as the olfactory bulb, forebrain and cerebellum. Inter‐ estingly, SOCS2 was first upregulated at the time of neuronal differentiation, which is be‐ tween the developmental stages E10 and E12, suggesting a role for SOCS2 in neural precursor differentiation [168].

induces SOCS gene expression [179]. JAK2 may also activate STAT1 and STAT3, however this can be cell type specific [180, 181]. One mechanism by which SOCS2 may block STAT5 activation is via its binding to phosphorylated tyrosine residues at the STAT5 binding site

Regulation of Basal and Injury-Induced Fate Decisions of Adult Neural Precursor Cells: Focus on SOCS2 and Related

Signalling Pathways

251

http://dx.doi.org/10.5772/53268

SOCS2 can also regulate signalling via the EGF receptor (EGFR) [175, 176]. The main physio‐ logical target for EGFR is EGF. EGFR primarily activates and signals through the Ras/MAPK pathway [183]. In terms of the neuronal effects of EGF, it has been shown to enhance neurite outgrowth and survival of different populations of cultured neurons [175, 183]. Relevant to this review, it also has an important role in neurogenesis. As described above, in the adult SVZ and dentate gyrus, EGF regulates neural precursor cell proliferation [37]. The impor‐ tance of this role is evident when, in response to brain injury, there is an expansion of neural stem cell numbers in the SVZ as a result of an increased responsiveness to EGF due to EGFR upregulation [184]. Important for SOCS2 interaction, EGF also activates STAT5, a process in‐ volving the Src tyrosine kinase [185-187]. Overexpression of SOCS2 in PC12 cells inhibited this EGF induced STAT5 phosphorylation [176]. The EGFR was also constitutively phos‐ phorylated at the Src binding site, Tyr845, in SOCS2 overexpressing PC12 cells. It was there‐ fore proposed that SOCS2 competitively bound to Tyr845 and blocked its dephosphorylation by the phosphatase SHP2 to allow prolonged Src activation and enhancement of neurite out‐

However, while SOCS2 regulated SVZ-derived neurogenesis in a GH dependent manner during development, in the adult SVZ it appears to regulate neurogenesis via regulation of erythropoietin signalling [188]. Further, the mechanism by which SOCS2 regulates adult hippocampal neurogenesis is different and does not appear to involve GH or erythropoietin, although Epo transiently enhanced SGZ NPC proliferation [189, 190]. Hippocampal neuro‐ genesis was studied under control and voluntary exercise conditions (to enhance basal hip‐ pocampal neurogenesis) in wildtype, SOCS2Tg and GHR-/- mice. Mice of all 3 genotypes had similar basal levels of neurogenesis and equivalently increased neurogenesis in response to exercise at early timepoints (8 days) aimed at measuring extent of NPC proliferation. How‐ ever, at later timepoints (35 days) aimed at examining newborn neuron survival, there was a 50% increase in the survival of adult hippocampal neurons in SOCS2Tg mice, under basal conditions and following voluntary exercise. Additionally, SOCS2Tg mice performed better than wildtype animals in the Morris Water Maze which probes hippocampal-dependent cognition [190]. This was an exciting result, as it identified SOCS2 as a potential therapeutic target that could enhance the survival of newly born neurons following brain injury. How‐ ever, given that GHR-/- mice showed no differences in adult hippocampal neurogenesis compared to wildtype, the mechanism by which SOCS2 promotes survival in this case re‐ mains to be determined. One possible explanation for this increase in neuronal survival in SOCS2Tg mice may be that the enhanced neurite outgrowth observed in SOCS2Tg neurons may aid functional integration into existing circuitry and the consequent maturation and

on the GHR [182].

growth [176].

survival of neurons.
