**5. Insulin/IGF-signalling cross-talk**

#### **5.1. Signalling cross-talk in the central nervous system**

*In vivo*, it is very unlikely that growth factors act alone, but a more likely scenario is the presence of a plethora of signalling molecules and that a phenotypic consequence is a complicated interplay of various signalling pathways. Special emphasise is given to know interactions and cross-talks between such pathways involving IIS, although this is only an emerging field that awaits further studies.

Cultured NPC from the rat adult SVZ respond with neuronal differentiation upon various single stimuli, like Fgf-2, Bdnf, Ngf, and Igf-1. When Fgf-2, Bdnf, and Ngf are applied in combination with Igf-1, significant increases in the number of neurons are observed, indicating synergistic action upon neuronal differentiation [114]. Another study uses immobilised growth factors, alone and in different combinations, on which the authors plated NPC from embryonic rat striatum. Proliferation, neuronal as well as glial differentiation is assessed and reveals that Igf-1 alone mainly drives neuronal differentiation and does not affect proliferation or glial differentiation significantly. In combination with Fgf-2, Igf-1 only slightly increases progenitor states and proliferation, and this effect is even smaller in combination with Egf that also acts mitotic in single treatments. Bdnf increases neuronal differentiation alone, as does Igf-1, however the combination of the two factors results in fewer neurons than single treatments. The differentiation potential of Igf-1 alone is also lowered by presence of the mitotic growth factors Fgf-2 and Egf. Igf-1 alone does not induce glial differentiation. In combination with Cntf that induces astrocytic phenotypes alone, Igf-1 decreases Cntf-mediated differen‐ tiation [115].

The cross-talk of IIS with regard to Fgf-2- as well as Egf-signalling mainly influences stem cell proliferation, but whether it is synergistically is not yet totally clear [47,49,50,51,52]. A putative scenario might be that Igf-1 keeps progenitors in a healthy or fit state in which they are ready for further cell divisions, for which Fgf-2 or Egf are the instructive cues. However, withdrawal of Fgf-2 is a prerequisite for neuronal differentiation upon Igf-1 presence [85]. Synergistically, Igf-1-signalling seems to increase the effects of Fgf-2 on NPC proliferation as has been shown in rat adult hippocampal cells. This effect is dependent on PI3K that is activated upon Fgf-2 treatment [99].

mechanism is therefore deduced to include Igf-2-dependent activation of Gsk-3β that leads to

Igf-2 is further under control of NF-κB transcription factors. Nuclear import of NF-κB is prevented by IκB proteins, which are degraded upon activation of the IκB kinase (IKK) complex. This proteasomal event releases NF-κB into the nucleus. In the adult mouse hippo‐ campus, synaptic IKK/NF-κB signalling is important for function and maturation. Recent data show that Igf-2 is under control of NF-κB-mediated transcriptional regulation [14]. This study shows that Igf-2 increases the number of synapses and of mature spines *in vitro*, while Igf-1 does only have moderate effects in this assay. The Igf-2 effect is predominantly transduced via the Igf-2r and is prevented through MEK inhibitor PD98059, but not through inhibition of Gsk-3β or p38 activity. Corroborating these findings *in vivo*, mice with deficient IKK-2 show

Igf-2-dependent proliferation of mouse CGP is associated with activation of PI3K, but also with mTorc-2 activity that is obvious from increased Akt Ser473 phosphorylation [57]. IIS

*In vivo*, it is very unlikely that growth factors act alone, but a more likely scenario is the presence of a plethora of signalling molecules and that a phenotypic consequence is a complicated interplay of various signalling pathways. Special emphasise is given to know interactions and cross-talks between such pathways involving IIS, although this is only an emerging field that

Cultured NPC from the rat adult SVZ respond with neuronal differentiation upon various single stimuli, like Fgf-2, Bdnf, Ngf, and Igf-1. When Fgf-2, Bdnf, and Ngf are applied in combination with Igf-1, significant increases in the number of neurons are observed, indicating synergistic action upon neuronal differentiation [114]. Another study uses immobilised growth factors, alone and in different combinations, on which the authors plated NPC from embryonic rat striatum. Proliferation, neuronal as well as glial differentiation is assessed and reveals that Igf-1 alone mainly drives neuronal differentiation and does not affect proliferation or glial differentiation significantly. In combination with Fgf-2, Igf-1 only slightly increases progenitor states and proliferation, and this effect is even smaller in combination with Egf that also acts mitotic in single treatments. Bdnf increases neuronal differentiation alone, as does Igf-1, however the combination of the two factors results in fewer neurons than single treatments. The differentiation potential of Igf-1 alone is also lowered by presence of the mitotic growth factors Fgf-2 and Egf. Igf-1 alone does not induce glial differentiation. In combination with Cntf that induces astrocytic phenotypes alone, Igf-1 decreases Cntf-mediated differen‐

synaptic mobilisation and expression of the AMPA-receptor GluR1.

downstream signalling is followed up to Gsk-3β in this cellular context.

reduced Igf-2 expression and pErk1/2 levels [14].

58 Trends in Cell Signaling Pathways in Neuronal Fate Decision

**5. Insulin/IGF-signalling cross-talk**

awaits further studies.

tiation [115].

**5.1. Signalling cross-talk in the central nervous system**

Survival of CNS-derived cells is one of the major and comprehensive effects of IIS in the developing and adult organism. IIS is only one of several pathways that influence survival, and thus cross-talk with other pathways might be of principal importance. Bdnf-mediated signalling is one of such pathways that are studied with regard to IIS. No additive function of Bdnf- and Igf-1-mediated survival is reported from different cellular sources [102,116], however other data is contradictory in finding additive affects on prenatal rat hippocampal cell survival [117] that cumulate on increased pGsk-3β. Consistently reported is a stronger survival effect under serum-deprivation for IIS compared to Bdnf. The underlying signalling might again diverge in different cellular systems: hippocampal neurons survive upon Igf-1 and Bdnf through induction of the PI3K-pAkt branch and survival is blocked by LY294002. Activity of the MAPK-pErk1/2 branch does not mediate survival. However, cortical neurons that also depend much stronger on IIS than on Bdnf-signalling for their survival also activate the PI3K-pAkt branch [117], but LY294002 does not completely attribute for survival upon IIS. In addition, Src-kinase is activated in these cortical neurons upon Igf-1, corroborating the finding of an involvement of Pac1-r-signalling in IIS-mediated survival in cortical neurons [91]. Nevertheless, Bdnf and IIS have additive effects in the activation of PI3-K, although this is not detectable on the level of the number of surviving cells. This finding strongly suggests that IIS is overriding the effects of Bdnf in survival of CNS-derived cells. However, one Bdnf effect on hippocampal as well as cortical neurons is to slightly activate Irs-1 and -2 phosphorylation, indicating a potential cross-activation of IIS through Bdnf. Whether this cross-activation is of physiological significance in the presence of Igf-1, remains to be determined.

Studies of neural cell proliferation provide evidence of IIS cross-talk with the Wnt/β-catenin pathway. Nestin-expressing neural progenitors as well as oligodendrocytes express different levels of β-catenin upon Igf-1 deletion or treatment [118,119]. *In vitro*, Igf-1 induces increase of β-catenin as well as CycD1 expression in a PI3K-Akt kinases dependent manner in cultured oligodendrocytes. β-catenin is also known to activate CycD1 and thus defines a point of intersection between IIS and Wnt signalling [118]. Impaired proliferation of NPCs is observed after inactivation of the Igf-1r in Nestin-positive cells. Such NPCs of prenatal mouse brains are dependent on Wnt-signalling when proliferating, and Igf-1r inactivation results in decreased levels of Gsk-3β and β-catenin, but with increased levels of the Wnt-receptor Frizzeled3 and Wnt3a. While the former expression changes are influenced through the Igf-1/PI3K/Aktpathway, increased levels of Frizzeled3 and Wnt3a are thought to reflect a compensatory regulation stimulated by low levels of β-catenin. Inactivation of Gsk-3β in this setting reverts β-catenin levels and overcomes the block of proliferation as consequence of Igf-1r inactivation. Overexpression of Igf-1 increases β-catenin-levels and stimulates progenitor proliferation [119]. These experiments clearly establish an important cross-talk to regulate progenitor proliferation in the developing brain.

this setting, Igfbp-5 interfered with Shh-mediated cell proliferation. Igf-2-signalling increases levels of Gli-1, a transcription factor that is mainly activated through Shh-signalling. Gli-1 as well as Gsk-3β are regulators of CycD1, which might be another point of intersection between these two signalling pathways besides regulation of Shh-target Nmyc-1 through Gsk-3β [57,126]. Parathath and colleagues established a role for Irs-1 and mTorc-1 upon Shh-mediated CGP proliferation. This study shows specific upregulation of Irs-1 protein upon Shh that does not result in the activation of Akt. Knockdown of Irs-1 abolishes Shh-mediated CGP prolifer‐ ation, which is in turn activated upon Irs-1 overexpression. Irs-1 overexpression increases or maintains expression levels of CycD2 and Nmyc-1, which are implicated in cell cycle control. Irs-1 protein stability in CGP is controlled by the negative feed back loop, in which mTorc-1 activation induces p70S6K that phosphorylates Irs-1 and targets Irs-1 for degradation. Shhdependent increase in Irs-1 is dependent on mTorc-1-inhibition that is followed by suppression of p70S6K-kinase activity [125]. In contrast to this study, Hartmann et al. [57] postulated PI3K/ Akt- and synergistically Shh-dependent transcriptional control of Gli-1 and CycD1 as possible

Insulin/IGF-Signalling in Embryonic and Adult Neural Proliferation and Differentiation in the Mammalian Central...

http://dx.doi.org/10.5772/54946

61

Although these data corroborate tight connections between Shh and IIS, there is need for

As pointed out through the many studies of IIS in the CNS, this signalling pathway has an essential role for development and function of this organ. Albeit studied in many laboratories, our understanding of this central signalling pathway is far from being complete. Still we do not understand how the same biological function is achieved by activating different down‐ stream cascades and why it is necessary to have these multiple pathways that respond to one stimulus. We also do not know why different biological functions are the consequence of IIS at different developmental time points in cells from the same part of the brain. In this light it is of major interest that studies are emerging that focus on cross-talks of various stimuli. Exposure to multiple stimuli at a time probably reflects more the natural environment of a developing and mature neural cell than exposure to one single growth factor. It is also of major interest to dissect different functions during development and to define the molecular players involved into functional switches. In this regard it will be important to dissect different subcomplexes of PI3Ks or Akts that might be implicated in IIS. It will be also needed to carefully analyse the balances between the different branches of the signalling cascades and to identify and close further gaps that might be still in the pathways as they are commonly described today. Given the central role of IIS in body development, growth and maintenance, this knowledge will be of utmost interest and will also be exploited for therapeutic medicine in

further mechanistic investigation to define further knots in the signalling network.

lines of interaction.

various contexts.

**6. Concluding remarks**

IIS has also been described to cross-talk with the Tgfβ-pathway in numerous systems [120]. However, a comprehensive analysis of the multiple points of intersections was not published yet for the nervous system. In a general view, Tgfβ-ligands 1, 2, and 3 exert opposing effects compared to IIS. This has been shown for the potential of NPC proliferation and differentiation [121] upon Tgfβ-treatment, since these cells stop proliferating and initiate neuronal differen‐ tiation in mouse E16.5 hippocampus and cerebral cortex, *in vitro* and *in vivo*. Proliferation and differentiation of ESCs are also influenced in opposite ways through IIS or Tgfβ-signalling. Smad4-deficient ESC show increased neuronal differentiation, and hereby preferentially in neuronal lineages of the mid- and anterior hindbrain [122]. Further, more serotonergic neurons of the dorsal midbrain are observed in *in vitro* differentiated Smad4-deficient cultures. However, Smad4 is a transcriptional mediator of several members of the Tgfβ-superfamiliy, including Activin-, Gdf-, Nodal-, Tgfβ- as well as Bmp-signalling. Thus, direct correlation to one specific signalling pathway is not achieved in this study. Neural induction of human ESCs is also increased in the presence of the Alk4,5,7 inhibitor SB431542 that interferes with Activin-, Gdf- and Nodal-signalling apart from Tgfβ. Increased differentiation into the neural lineage is assessed by Pax-6 expression which is accompanied by decreased Bmp-4 and increased Noggin expression. These dynamics point to a contribution of several members of the Tgfβsuperfamily of which the Bmp branch has a major contribution [123], but certainly does not act alone. Of further note, neuronal differentiation of ESC cells is not solely dependent on signalling of the above mentioned members of the Tgfβ-superfamily, since deletion of the common effector Smad4 in ESC does not completely abolish the potential for neuronal differentiation [122]. In contrast, IIS keeps ESCs in a proliferative state and is necessary for selfrenewal. Whether both pathways cross-talk to suppress the respective other one is not investigated so far, but might be a likely scenario, given the connections described in other cellular lineages.

FoxO and Smad proteins interact and the FoxO/Smad complex is implicated in transcriptional control in early mouse cortical NPCs, thereby defining one point of intersection of IIS and Tgfβ. Smad2 and 3 transcriptions factors are part of the canonical Tgfβ-signalling pathway and are phosphorylated upon binding of Tgfβ-ligands to the Tgfβ-receptor 1 and 2. The phos‐ phorylated proteins bind to Smad4 and together translocate to the nucleus. This Smad complex interacts with FoxO1, 3a, and 4, three TF of the forkhead family that are under influence of IIS. Upon activation of the Igf-1r, FoxO TF are phosphorylated in a PI3K-Akt-dependent manner. This phosphorylation leads to retention of FoxO proteins in the cytoplasm. Thus, IIS opposes transcriptional control through the Tgfβ-pathway by interfering with translocation of cofactors into the nucleus [124].

While survival of cerebellar granule precursors depends on IIS, control of CGP proliferation indicates cross-talk of IIS and Shh-signalling [34,55,125]. Igf/Shh interaction is reported to occur on various levels. The study of Fernandez et al. (2010) suggests that IIS via Igf-1r increases Shhmediated proliferation, but IIS-mediated proliferation is independent on Shh-signalling. In this setting, Igfbp-5 interfered with Shh-mediated cell proliferation. Igf-2-signalling increases levels of Gli-1, a transcription factor that is mainly activated through Shh-signalling. Gli-1 as well as Gsk-3β are regulators of CycD1, which might be another point of intersection between these two signalling pathways besides regulation of Shh-target Nmyc-1 through Gsk-3β [57,126]. Parathath and colleagues established a role for Irs-1 and mTorc-1 upon Shh-mediated CGP proliferation. This study shows specific upregulation of Irs-1 protein upon Shh that does not result in the activation of Akt. Knockdown of Irs-1 abolishes Shh-mediated CGP prolifer‐ ation, which is in turn activated upon Irs-1 overexpression. Irs-1 overexpression increases or maintains expression levels of CycD2 and Nmyc-1, which are implicated in cell cycle control. Irs-1 protein stability in CGP is controlled by the negative feed back loop, in which mTorc-1 activation induces p70S6K that phosphorylates Irs-1 and targets Irs-1 for degradation. Shhdependent increase in Irs-1 is dependent on mTorc-1-inhibition that is followed by suppression of p70S6K-kinase activity [125]. In contrast to this study, Hartmann et al. [57] postulated PI3K/ Akt- and synergistically Shh-dependent transcriptional control of Gli-1 and CycD1 as possible lines of interaction.

Although these data corroborate tight connections between Shh and IIS, there is need for further mechanistic investigation to define further knots in the signalling network.
