**3. Biological effects associated with Insulin/IGF-signalling in neural development**

#### **3.1. Insulin/IGF-signalling in ESC**

Insulin and IGF are important factors to keep human ESCs in a proliferative state and to promote self-renewal, where upon IGF-1R has been identified as essential component [35]. Blocking of IGF-signalling results in differentiation, but it is unclear whether a cer‐ tain cell fate is favoured under such condition. Downstream signalling of Insulin and IGF has also been studied in this context and revealed that blocking of this pathway at various points induced differentiation. In this study, blocking is achieved on the level of PI3K through LY294002, of AKT-1, and of mTOR-1 through Rapamycin. Treatment of hu‐ man ESC with LY294002 results in loss of phosphorylation of the downstream molecules AKT, p70S6K, S6 as well as GSK-3β [35-38].

Together with stromal cell-derived factor 1 (SDF-1/CXCL-12), pleiotrophin (PTN), and ephrin-B1 (EFNB1) IGF-2 induces differentiation of human ESC into TH-positive dopaminergic neurons [39].

Interestingly, retinol/vitamin A also induces Nanog transcription and the signal is transduced over the IGF-1R, IRS-1, AKT and both mTOR complexes, mTORC-1 and mTORC-2 [40]. Expression of Nanog is a hallmark of proliferating, pluripotent stem cells. However, Insulin has differentiating capacity into the neuroectodermal lineage when human ESCs are cocul‐ tured with endodermal derived cells. This effect is dependent on PI3K/AKT signalling [41]. Together, these results reflect context-dependent IIS, and possibly cross-talk with other signalling pathways activated e.g. *in vivo* through development or *in vitro* through cocultures.

#### **3.2. Insulin/IGF effects in embryonic neural precursors**

recent studies reveal that Igf-2r is also implicated in specific signal transduction, e.g. in the context of memory enhancement or fear extinction [13,31]. Igf-2r is also expressed in all major neuroanatomical structures, with high expressions in the hippocampus, OB, retina, pituitary, brain stem, and spinal chord. Further it is detected in the choroid plexus, ependymal as well

Detailed analyses of expression of the upstream Igf-signalling members has been reported for the mouse late embryonic and postnatal cerebellum [34]. Igf-1r is ubiquitously expressed, whereas Igf-1 is detected in a subset of Purkinje cells (PC) at E17.5 and in postnatal stages. Igf-2 is confined to the meninges and blood vessels. Both ligands are not detectable in dividing cerebellar granule precursor cells (CGP). Igfbp-1 is not detected, but Igfbp-2 is expressed wide spread in the meninges, PC, internal as well as external granule layer, and choroid plexus. Igfbp-3 is restricted to PC, Igfbp-4 to meninges and choroid plexus, and Igfbp-5 to Calbindinnegative cells of the PC layer. Igfbp-6 is only detected in later stages in a subset of PC.

According to the widespread expression of members of the upstream IIS, their developmental dynamics and cell type specificity, it is conceivable that this signalling pathway exerts important function for development, maintenance as well as function of the various parts of the central nervous system. Some of these functions that are mainly attributable to neuronal

development and fate decision will be highlighted in the following sections.

**3. Biological effects associated with Insulin/IGF-signalling in neural**

Insulin and IGF are important factors to keep human ESCs in a proliferative state and to promote self-renewal, where upon IGF-1R has been identified as essential component [35]. Blocking of IGF-signalling results in differentiation, but it is unclear whether a cer‐ tain cell fate is favoured under such condition. Downstream signalling of Insulin and IGF has also been studied in this context and revealed that blocking of this pathway at various points induced differentiation. In this study, blocking is achieved on the level of PI3K through LY294002, of AKT-1, and of mTOR-1 through Rapamycin. Treatment of hu‐ man ESC with LY294002 results in loss of phosphorylation of the downstream molecules

Together with stromal cell-derived factor 1 (SDF-1/CXCL-12), pleiotrophin (PTN), and ephrin-B1 (EFNB1) IGF-2 induces differentiation of human ESC into TH-positive dopaminergic

Interestingly, retinol/vitamin A also induces Nanog transcription and the signal is transduced over the IGF-1R, IRS-1, AKT and both mTOR complexes, mTORC-1 and mTORC-2 [40]. Expression of Nanog is a hallmark of proliferating, pluripotent stem cells. However, Insulin has differentiating capacity into the neuroectodermal lineage when human ESCs are cocul‐

as endothelial cells [12].

42 Trends in Cell Signaling Pathways in Neuronal Fate Decision

**development**

neurons [39].

**3.1. Insulin/IGF-signalling in ESC**

AKT, p70S6K, S6 as well as GSK-3β [35-38].

The Insulin-receptor (Ir) is expressed in distinct regions of the CNS, including the olfactory bulb (OB), hypothalamus and the pituitary. Accordingly, a Nestin-cre mediated conditional, CNS-specific knock-out of the Ir early during development results in increased Luteinising hormone (LH) release from the hypothalamic-pituitary axis that leads to a deregulation of energy homeostasis and endocrinology of the reproductive system [42].

Neurospheres generated from embryonic striatal precursors in the presence of Egf respond to Igf-1 treatment with increased neuronal differentiation, presumably over an Igf-1r dependent signalling cascade. However, this has not been addressed comprehensively through receptor inhibition but was deduced from lower differentiation upon Igf-2 and Insulin stimuli [16,17,43]. Igf-1 treatment in this setting is not accompanied by increased cell proliferation, but cotreatment with Insulin increases the fraction of dividing cells. It might be that this effect is specific to this combined treatment.

In the cerebral cortex, increased Igf-1 expression after Growth hormone (GH) treatment of rat embryonal neural precursors is involved in increased proliferation of early (E14) and late (E17) progenitors and is accompanied by increased neuronal differentiation at both time points. In addition to neuronal differentiation, astrogenesis is also increased but only when late progen‐ itors are exposed to GH. This effect is also blocked in the presence of an Igf-1-blocking antibody and thus illustrates that IIS is transducing GH-induced effects [42,44].

Increased proliferation of E14 rat cortical progenitor upon Igf-1 is observed *in vitro* after treatment of cultured cells, and *in vivo* after intrauterine Igf-1 injection [45].

Igf-1 function in the brain is highly context-dependent and cell-type specific. This interpreta‐ tion is corroborated by the finding that Igf-1 treatment of E19 rat embryonal hippocampal progenitors does influence survival of these cells [46].

In another setting, Igf-1 treatment evokes cell survival of mouse E10 neuroepithelial cells [47] and it is mitogenic for sympathetic neuroblasts [48], showing that progenitor subtypes respond differently to IIS. It is so far ill defined what kind of signalling events, including cross-talks to other pathways, are associated with this differential outcome of Igf-1 stimulation. Interaction with Egf- as well as Fgf-2-signalling and Igf-1 has been studied in striatal-derived neural progenitor cells (NPCs). In this setting, highest numbers of formed spheres are obtained in the presence of Igf-1 and Egf or Igf-1 and Fgf-2. Sphere formation in this context is highly de‐ pendent on presence of Igf-1, since no spheres were observed in the absence of IIS [49]. These data show that Igf-1 is also affecting NPC proliferation in cooperation with other signalling molecules, emphasising the pleiotrophic nature of IIS.

Igf-1-signalling seems to increase the effects of Fgf-2 on NPC proliferation as has been shown in various stem cell populations, like rat adult SVZ neurospheres [50], neurospheres from mouse E13.5 forebrains [51], mouse embryonic OBSC [52], and mouse embryonic striatal NPC [49]. Although not shown in all stem cells investigated so far, it is likely that in one scenario Igf-1 promotes survival and proliferating competence of stem cells but that other mitogens such as Erk-activators are needed to increase proliferation. However, other stem cell popula‐ tions might proliferate upon IIS without further mitogenic instructions as has been observed by Fgf-2-independent Igf-1-mediated proliferation in rat embryonic NPCs from the cerebral cortex and the hippocampus [45,46].

to increased numbers of neurons in the cortical plate. Igf-1 signalling does not affect all neuronal populations in a similar manner but shows regional differences. The neuronal composition of the motor cortex is more increased than the somatosensory cortex of Igf-1 overexpressing mice, which is however also increased compared to wildtype animals. Neurons in the cortical plate are also differently affected. Layer 1 neurons comprise the most increased neuronal fraction in Igf-1-transgenic brains, layer 6 neurons show the smallest increase [63]. It is thus likely that NPC are more sensitive to IIS during early developmental events of the cerebral cortex in which neurogenesis of frontal precedes that of caudal regions and layer 1 is

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

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45

In line with *in vivo* overexpression studies, complete loss of Igf-1 in mice leads to the opposite effect of decreased numbers of specific neuronal populations in the cerebral cortex, e.g. hippocampal granule cells and striatal parvalbumin-positive interneurons. Dopaminergic neurons of the midbrain, basal forebrain cholinergic neurons, as well as motoneurons in the spinal cord do not depend on Igf-1 presence [64]. Further analysis of this Igf-1-deficient mouse revealed Igf-1-dependence of axonal growth and myelination that are strongly impaired. Overall, Igf-1-deficient mice have a decreased number of myelinated axons. This observation goes in line with studies of human IGF-I overexpression driven by a mouse metallothionein promoter, that revealed increased myelin production by an unchanged number of oligoden‐

Overexpression of Igf-1 driven by the Igf-2 promoter results in general brain overgrowth. The greatest effect is seen in the cerebellum, although Igf-1 deletion does not lead to an observable phenotype in this part of the brain, maybe because of compensatory effects from Igf-2 [55,64].

Igf-1r-deficient mice show severe growth retardation and die perinatally. Nestin-cre-mediated conditional knockout of the Igf-1r also results in death shortly after birth but few animals survive up to adulthood [66]. Mice that are heterozygote for the conditional Igf-1r deletion also display severe growth retardation that was studied in detail in the hippocampus. In the postnatal hippocampus all different parts are affected by increased rates of apoptosis rather

Mutations of the type 2 Igf receptor are lethal but are rescued from this perinatal lethality in Igf-2 and Igf-1r null backgrounds [67]. This observation is attributed to increased Igf-2 signalling due to lack of internalisation of Igf-2, which is thought to be a major function of the Igf-2r. Although general hyperplasia is observed including the brain, Igf-2r-signalling was not

Deletion of Igf-2 in mice results in severe growth-deficiency, but it was reported that Igf-2 deficient mice do not show striking morphological aberrations [70,71]. However, one source of Igf-2 during development is the choroid plexus, from which it is released into the cerebro‐ spinal fluid (CSF), and CSF-derived Igf-2 is an important signal for cell proliferation of later embryonic precursors that have contact to the ventricular surface [72]. Progenitors of the SVZ, e.g. Tbr-2-expressing cells, do not have contact to the CSF and thus are not influenced by Igf-2 signalling in their proliferation capacity. Increased proliferation of cortical progenitors is dependent on the apical (ventricular) localisation of the Igf-1r that is mediated through the cell

than by an impaired ability to proliferate through loss of the Igf-1r.

preceding generation of layer 6.

drocytes in adult brains [65].

further investigated in the brain [67-69].

NPCs from neonatal rat forebrain undergo neuronal differentiation upon Insulin-treatment [53]. The content of GFAP-positive cells in the neurosphere-based assay is unchanged, thus suggesting a preference for neuronal differentiation in this cellular model.

Embryonal stem cells from the olfactory bulb (OBSC) are strongly dependent on IIS as shown *in vitro* as well as *in vivo*. Igf-1 increases OBSC proliferation as well as differentiation without strong lineage restriction, thus affecting neuronal as well as glial differentiation [54]. OBSC proliferation however does not depend strictly on Igf-1, Insulin or pro-Insulin, they rather potentiate the proliferating effect of Fgf-2 and Egf [52]. This study also showed that Igf-1 induces differentiation into the neuronal and astroglial lineages, but oligodendrocyte differ‐ entiation clearly depends on presence of other growth factors.

Several data support the finding that Igf-1 and -2 increase cerebellar granule cell precursor (CGP) proliferation as well as survival of the same [34,55]. Different effects were linked to different concentrations of the cytokine, where lower concentrations favoured survival and higher proliferation [56]. IIS is supporting Sonic hedgehog (Shh) action that is a potent mitogen for CGPs. Blocking of signal transduction through the Igf-1r diminishes Shh-mediated cell proliferation, as well as endogenous supply of Igfbp-5. However, other Igfbps do not interfere with Shh-mediated proliferation, but decrease Igf-1-dependent cell division [34].

Igf-2 treatment of CGP also results in proliferation. However it is unclear whether this occurs over the Igf-1r or 2r [57] but it indicates also important function for Igf-2 in the context of development of the central nervous system.

Igf-1 prevents cell death in primary rat embryonic hippocampal cultures after exposure to glucocorticoids. While increased levels of corticosterone reduce Akt-phosphorylation and lead to cell death, Igf-1 rescues cells from dying by increasing pAkt-levels in a PI3K-dependent manner [58]. The same effect is observed by applying Insulin instead of Igf-1 [59].

Signalling of Insulin/Igf affects NPC proliferation, survival as well as differentiation *in vitro* and this finding is corroborated *in vivo* by studies of knock-out and transgenic mouse models. Transgene-mediated overexpression of Igf-1, driven by the Nestin-promoter, supports *in vitro* finding that Igf-1 influences NPC proliferation as well as neuronal differentiation during development. Cumulative BrdU-labelling shows that Igf-1 decreases the total length of the cell cycle through acceleration of the G1-phase. This higher proliferating activity is paired with a higher rate of cell-cycle re-entry. The overall increased number of progenitors provides a larger pool of NPCs for neuronal differentiation and thus increases numbers of neurons residing in the postnatal cortical plate of Igf-1-overexpressing mice [60]. *In vivo*, Igf-1 also exhibits survival function since apoptosis is reduced upon its overexpression [61,62] which further contributes to increased numbers of neurons in the cortical plate. Igf-1 signalling does not affect all neuronal populations in a similar manner but shows regional differences. The neuronal composition of the motor cortex is more increased than the somatosensory cortex of Igf-1 overexpressing mice, which is however also increased compared to wildtype animals. Neurons in the cortical plate are also differently affected. Layer 1 neurons comprise the most increased neuronal fraction in Igf-1-transgenic brains, layer 6 neurons show the smallest increase [63]. It is thus likely that NPC are more sensitive to IIS during early developmental events of the cerebral cortex in which neurogenesis of frontal precedes that of caudal regions and layer 1 is preceding generation of layer 6.

mouse E13.5 forebrains [51], mouse embryonic OBSC [52], and mouse embryonic striatal NPC [49]. Although not shown in all stem cells investigated so far, it is likely that in one scenario Igf-1 promotes survival and proliferating competence of stem cells but that other mitogens such as Erk-activators are needed to increase proliferation. However, other stem cell popula‐ tions might proliferate upon IIS without further mitogenic instructions as has been observed by Fgf-2-independent Igf-1-mediated proliferation in rat embryonic NPCs from the cerebral

NPCs from neonatal rat forebrain undergo neuronal differentiation upon Insulin-treatment [53]. The content of GFAP-positive cells in the neurosphere-based assay is unchanged, thus

Embryonal stem cells from the olfactory bulb (OBSC) are strongly dependent on IIS as shown *in vitro* as well as *in vivo*. Igf-1 increases OBSC proliferation as well as differentiation without strong lineage restriction, thus affecting neuronal as well as glial differentiation [54]. OBSC proliferation however does not depend strictly on Igf-1, Insulin or pro-Insulin, they rather potentiate the proliferating effect of Fgf-2 and Egf [52]. This study also showed that Igf-1 induces differentiation into the neuronal and astroglial lineages, but oligodendrocyte differ‐

Several data support the finding that Igf-1 and -2 increase cerebellar granule cell precursor (CGP) proliferation as well as survival of the same [34,55]. Different effects were linked to different concentrations of the cytokine, where lower concentrations favoured survival and higher proliferation [56]. IIS is supporting Sonic hedgehog (Shh) action that is a potent mitogen for CGPs. Blocking of signal transduction through the Igf-1r diminishes Shh-mediated cell proliferation, as well as endogenous supply of Igfbp-5. However, other Igfbps do not interfere

Igf-2 treatment of CGP also results in proliferation. However it is unclear whether this occurs over the Igf-1r or 2r [57] but it indicates also important function for Igf-2 in the context of

Igf-1 prevents cell death in primary rat embryonic hippocampal cultures after exposure to glucocorticoids. While increased levels of corticosterone reduce Akt-phosphorylation and lead to cell death, Igf-1 rescues cells from dying by increasing pAkt-levels in a PI3K-dependent

Signalling of Insulin/Igf affects NPC proliferation, survival as well as differentiation *in vitro* and this finding is corroborated *in vivo* by studies of knock-out and transgenic mouse models. Transgene-mediated overexpression of Igf-1, driven by the Nestin-promoter, supports *in vitro* finding that Igf-1 influences NPC proliferation as well as neuronal differentiation during development. Cumulative BrdU-labelling shows that Igf-1 decreases the total length of the cell cycle through acceleration of the G1-phase. This higher proliferating activity is paired with a higher rate of cell-cycle re-entry. The overall increased number of progenitors provides a larger pool of NPCs for neuronal differentiation and thus increases numbers of neurons residing in the postnatal cortical plate of Igf-1-overexpressing mice [60]. *In vivo*, Igf-1 also exhibits survival function since apoptosis is reduced upon its overexpression [61,62] which further contributes

with Shh-mediated proliferation, but decrease Igf-1-dependent cell division [34].

manner [58]. The same effect is observed by applying Insulin instead of Igf-1 [59].

suggesting a preference for neuronal differentiation in this cellular model.

entiation clearly depends on presence of other growth factors.

development of the central nervous system.

cortex and the hippocampus [45,46].

44 Trends in Cell Signaling Pathways in Neuronal Fate Decision

In line with *in vivo* overexpression studies, complete loss of Igf-1 in mice leads to the opposite effect of decreased numbers of specific neuronal populations in the cerebral cortex, e.g. hippocampal granule cells and striatal parvalbumin-positive interneurons. Dopaminergic neurons of the midbrain, basal forebrain cholinergic neurons, as well as motoneurons in the spinal cord do not depend on Igf-1 presence [64]. Further analysis of this Igf-1-deficient mouse revealed Igf-1-dependence of axonal growth and myelination that are strongly impaired. Overall, Igf-1-deficient mice have a decreased number of myelinated axons. This observation goes in line with studies of human IGF-I overexpression driven by a mouse metallothionein promoter, that revealed increased myelin production by an unchanged number of oligoden‐ drocytes in adult brains [65].

Overexpression of Igf-1 driven by the Igf-2 promoter results in general brain overgrowth. The greatest effect is seen in the cerebellum, although Igf-1 deletion does not lead to an observable phenotype in this part of the brain, maybe because of compensatory effects from Igf-2 [55,64].

Igf-1r-deficient mice show severe growth retardation and die perinatally. Nestin-cre-mediated conditional knockout of the Igf-1r also results in death shortly after birth but few animals survive up to adulthood [66]. Mice that are heterozygote for the conditional Igf-1r deletion also display severe growth retardation that was studied in detail in the hippocampus. In the postnatal hippocampus all different parts are affected by increased rates of apoptosis rather than by an impaired ability to proliferate through loss of the Igf-1r.

Mutations of the type 2 Igf receptor are lethal but are rescued from this perinatal lethality in Igf-2 and Igf-1r null backgrounds [67]. This observation is attributed to increased Igf-2 signalling due to lack of internalisation of Igf-2, which is thought to be a major function of the Igf-2r. Although general hyperplasia is observed including the brain, Igf-2r-signalling was not further investigated in the brain [67-69].

Deletion of Igf-2 in mice results in severe growth-deficiency, but it was reported that Igf-2 deficient mice do not show striking morphological aberrations [70,71]. However, one source of Igf-2 during development is the choroid plexus, from which it is released into the cerebro‐ spinal fluid (CSF), and CSF-derived Igf-2 is an important signal for cell proliferation of later embryonic precursors that have contact to the ventricular surface [72]. Progenitors of the SVZ, e.g. Tbr-2-expressing cells, do not have contact to the CSF and thus are not influenced by Igf-2 signalling in their proliferation capacity. Increased proliferation of cortical progenitors is dependent on the apical (ventricular) localisation of the Igf-1r that is mediated through the cell polarity protein Pals-1 (Protein Associated with Lin Seven 1) as part of the apical complex. Further support for an Igf-1r-mediated effect of Igf-2-signalling in this context comes from the finding that loss of Pten (Phosphatase and Tensin homolog) opposes the effect of Pals-1 deficiency: Pten-deficient mice have hyperplasic cortices while Pals-1-deficiency results in cortical hypoplasia. The phenotypes are partially reverted through combination of both transgenes [72]. Further downstream signals apart from Igf-1r and PI3K are not analysed in this context.

The function of different Igfbps has been studied through transgenic mouse models. Thereby it turned out, that loss of function mutations do not result in gross morphological changes within the CNS. However, overexpression of Igfbps lead to phenotypes in the CNS, which are exploited to achieve better understanding of IIS within this organ. Overexpression of Igfbp-1 in transgenic mice using either a phosphogylcerate kinase [73] or a methallothionein [74] promoter results in similar phenotypes as observed in adult Igf-1-deficient mice: reduced brain sizes due to loss of mature neurons and decreased progenitor proliferation, impaired myeli‐ nation as well as decreased activation of GFAP-expressing astrocytes in conditions of injury. However, Igfbp-1 is normally not expressed at detectable levels in the CNS and the observed findings are therefore attributed to the lack of Igf-1 as consequence of increased presence of Igfbp-1. Most of the phenotypes observed in mice overexpressing Igfbps resemble those of loss of Igf-1-signalling, which is mainly characterised by reduced organ size. Whether these phenotypes are exclusively caused by impaired Igf-1-signalling or whether Igfbps have also distinct functions, is so far not resolved [75]. Generally, Igfbp-5 is considered to potentiate Igf-1-signalling, while Igfbp-2 mainly opposes Igf-1 and Igf-2 action [55]. This finding indicates that some Igfbps might exert additional functions that might only be apparent in transgenic mice in which the ligands are mutated as well.

tiation upon Igf-1. IIS in this setting induces expression of Bmp-antagonists Noggin and Smad6, which are involved in the inhibition of an astrocytic cell fate and in favouring neuronal, but more efficiently oligodendroglial differentiation. Further, this study showed again that

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

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

47

Overexpression of Igf-1 through an Igf-2 promoter element results in increased numbers of neurons in the postnatal mouse hippocampal dentate gyrus. This increase in neurogenesis is transiently accompanied by increased numbers of synapses, which makes it possible that IIS

Adult rat NPC derived from the subventricular zone proliferate stronger upon an Igf-1 stimulus. This effect is blocked by the presence of Igfbp-3 that also decreases phosphorylation of Akt [83]. Igf-1 signalling under Fgf-2-free conditions in adult subventricular stem cells is also linked to differentiation [84]. Adult mouse NPC are initially cultivated in the presence of Fgf-2. Subsequent withdrawal of Fgf-2 results in neuronal differentiation that is increased by complementation with Igf-1. Blocking of the Igf-1 signal with an antibody reverts the effect [85]. In this setting, the authors also explored the capacity of Igfbp-2 to influence the neurogenic potential of the NPC, since Igfbp-2 complexes with Igf-1 and proteoglycans like heparin in the rat OB [86]. Low doses of exogenous Igfbp-2 increase neuronal differentiation, while applica‐ tion of a blocking Igfbp-2 antibody interferes with this effect. These experiments indicate that Igfbp-2 does not act by sequestering Igf-1 and by limiting its bioavailability, in which scenario Igfbp-2 would have an opposing effect to Igf-1. Instead, both molecules exert the same phenotypic differentiation and this indicates that Igfbp-2 function in this context is more likely a protection of Igf-1 from degradation. It is conceivable that simultaneous interaction of Igfbp-2

Igf-1 has a major impact on cell survival of adult NPCs.

**Figure 3.** Igf-1 signalling in adult rat hippocampal neurospheres [81].

also influences synaptogenesis [82].

#### **3.3. Insulin/IGF effects in adult neural precursors**

Igf-1, though not produced in high amounts in adult rodent brains, is required for normal neuronal functions throughout life. As different source, peripheral Igf-1 is transported to neuronal cells through an uptake from the blood stream (reviewed in [1]). Neural stem cells of the adult hippocampus respond to peripheral administered Igf-1 with increased prolifera‐ tion as well as neurogenesis but not with astrogenesis [76]. These data were also confirmed *in vitro* using neurospheres generated from the hippocampal dentate gyrus. In this setting, low doses of Igf-1 mediate, and high doses interfere with neuronal differentiation [77]. Igf-1 mediated cell proliferation of adult neuronal progenitor from the hippocampal subgranular zone (SGZ) was also shown in a model of epilepsy [78] as well as in response to physical excercise [79].

Further in vivo evidence for importance of Igf-1-signalling in the adult hippocampus comes from a study of Igf-1-deficient rats. In this context Igf-1 supports survival of newly generated neurons and possibly the maturation of the neuronal phenotype. However, cell proliferation is not affected in this *in vivo* model [80]. Igf-1-instructed differentiation mainly into the oligodendroglial lineage has been shown in rat adult hippocampal NPC [81] (Fig. 3). In this study, withdrawal of proliferation-promoting Fgf-2 is essential to drive NPCs into differen‐ Insulin/IGF-Signalling in Embryonic and Adult Neural Proliferation and Differentiation in the Mammalian Central... http://dx.doi.org/10.5772/54946 47

**Figure 3.** Igf-1 signalling in adult rat hippocampal neurospheres [81].

polarity protein Pals-1 (Protein Associated with Lin Seven 1) as part of the apical complex. Further support for an Igf-1r-mediated effect of Igf-2-signalling in this context comes from the finding that loss of Pten (Phosphatase and Tensin homolog) opposes the effect of Pals-1 deficiency: Pten-deficient mice have hyperplasic cortices while Pals-1-deficiency results in cortical hypoplasia. The phenotypes are partially reverted through combination of both transgenes [72]. Further downstream signals apart from Igf-1r and PI3K are not analysed in

The function of different Igfbps has been studied through transgenic mouse models. Thereby it turned out, that loss of function mutations do not result in gross morphological changes within the CNS. However, overexpression of Igfbps lead to phenotypes in the CNS, which are exploited to achieve better understanding of IIS within this organ. Overexpression of Igfbp-1 in transgenic mice using either a phosphogylcerate kinase [73] or a methallothionein [74] promoter results in similar phenotypes as observed in adult Igf-1-deficient mice: reduced brain sizes due to loss of mature neurons and decreased progenitor proliferation, impaired myeli‐ nation as well as decreased activation of GFAP-expressing astrocytes in conditions of injury. However, Igfbp-1 is normally not expressed at detectable levels in the CNS and the observed findings are therefore attributed to the lack of Igf-1 as consequence of increased presence of Igfbp-1. Most of the phenotypes observed in mice overexpressing Igfbps resemble those of loss of Igf-1-signalling, which is mainly characterised by reduced organ size. Whether these phenotypes are exclusively caused by impaired Igf-1-signalling or whether Igfbps have also distinct functions, is so far not resolved [75]. Generally, Igfbp-5 is considered to potentiate Igf-1-signalling, while Igfbp-2 mainly opposes Igf-1 and Igf-2 action [55]. This finding indicates that some Igfbps might exert additional functions that might only be apparent in transgenic

Igf-1, though not produced in high amounts in adult rodent brains, is required for normal neuronal functions throughout life. As different source, peripheral Igf-1 is transported to neuronal cells through an uptake from the blood stream (reviewed in [1]). Neural stem cells of the adult hippocampus respond to peripheral administered Igf-1 with increased prolifera‐ tion as well as neurogenesis but not with astrogenesis [76]. These data were also confirmed *in vitro* using neurospheres generated from the hippocampal dentate gyrus. In this setting, low doses of Igf-1 mediate, and high doses interfere with neuronal differentiation [77]. Igf-1 mediated cell proliferation of adult neuronal progenitor from the hippocampal subgranular zone (SGZ) was also shown in a model of epilepsy [78] as well as in response to physical

Further in vivo evidence for importance of Igf-1-signalling in the adult hippocampus comes from a study of Igf-1-deficient rats. In this context Igf-1 supports survival of newly generated neurons and possibly the maturation of the neuronal phenotype. However, cell proliferation is not affected in this *in vivo* model [80]. Igf-1-instructed differentiation mainly into the oligodendroglial lineage has been shown in rat adult hippocampal NPC [81] (Fig. 3). In this study, withdrawal of proliferation-promoting Fgf-2 is essential to drive NPCs into differen‐

this context.

excercise [79].

mice in which the ligands are mutated as well.

46 Trends in Cell Signaling Pathways in Neuronal Fate Decision

**3.3. Insulin/IGF effects in adult neural precursors**

tiation upon Igf-1. IIS in this setting induces expression of Bmp-antagonists Noggin and Smad6, which are involved in the inhibition of an astrocytic cell fate and in favouring neuronal, but more efficiently oligodendroglial differentiation. Further, this study showed again that Igf-1 has a major impact on cell survival of adult NPCs.

Overexpression of Igf-1 through an Igf-2 promoter element results in increased numbers of neurons in the postnatal mouse hippocampal dentate gyrus. This increase in neurogenesis is transiently accompanied by increased numbers of synapses, which makes it possible that IIS also influences synaptogenesis [82].

Adult rat NPC derived from the subventricular zone proliferate stronger upon an Igf-1 stimulus. This effect is blocked by the presence of Igfbp-3 that also decreases phosphorylation of Akt [83]. Igf-1 signalling under Fgf-2-free conditions in adult subventricular stem cells is also linked to differentiation [84]. Adult mouse NPC are initially cultivated in the presence of Fgf-2. Subsequent withdrawal of Fgf-2 results in neuronal differentiation that is increased by complementation with Igf-1. Blocking of the Igf-1 signal with an antibody reverts the effect [85]. In this setting, the authors also explored the capacity of Igfbp-2 to influence the neurogenic potential of the NPC, since Igfbp-2 complexes with Igf-1 and proteoglycans like heparin in the rat OB [86]. Low doses of exogenous Igfbp-2 increase neuronal differentiation, while applica‐ tion of a blocking Igfbp-2 antibody interferes with this effect. These experiments indicate that Igfbp-2 does not act by sequestering Igf-1 and by limiting its bioavailability, in which scenario Igfbp-2 would have an opposing effect to Igf-1. Instead, both molecules exert the same phenotypic differentiation and this indicates that Igfbp-2 function in this context is more likely a protection of Igf-1 from degradation. It is conceivable that simultaneous interaction of Igfbp-2 with proteoglycans decreases its affinity to Igf-1. The net outcome might be a locally increased amount of free Igf-1 that drives neuronal differentiation of NPC [85]. In another study, differentiation of SVZ NPC was deduced from reduced expression of the stem cell marker Nestin after Igf-1 treatment, but it was not specified whether differentiation of a specific cell lineage was favoured [50].

Further studies in adult mice show that lack of Igf-1 results in disturbed neuroblast exit from the subventricular zone and their subsequent migration to the olfactory bulb through the rostral migratory stream. This Igf-1 function is dependent on PI3-kinase activity and Disa‐ bled-1 (Dab-1) phosphorylation via Src-family kinase [87].

Igf-2 is not only provided from the CSF and important for early development of the brain. In addition, adult mouse hippocampal stem cells from the DG also produce Igf-2, and to a much lesser extend Igf-2 is also detected in adult SVZ-derived stem cells [19,88]. Challenging Igf-2 expression through siRNA-mediated approaches as well as treatment of DG-derived NPCs reveals that Igf-2 also influences proliferation. However, SVZ-derived NPCs do not respond with changed proliferation upon Igf-2 knockdown [88]. Igf-2 but not Igf-1 is involved in neuronal survival of newly generated neurons in the adult DG in a model of fear extinction, that is strongly associated with neurogenesis in the hippocampus [31]. Neuronal survival and fear extinction depend on Igf-1r activity and are opposed by increased levels of Igfbp-7. However, Igf-1 does not exert any observable effect in this setting.

**Figure 4.** IIS mediated sur-vival of cortical neurons through activation of Akt and PAC-1r [91].

normally takes place during early postnatal cerebellar development.

required for axonal growth.

consolidation paradigm.

Data from adult Igf-1-deficient mice show however that cerebellar neurons are not affected by the loss of this trophic factor *in vivo* [64]. Contrasting this finding is data showing overexpres‐ sion of human IGF-1 in mouse through a transgene driven by the mouse Igf-2 promoter [92] that results in cerebellar overgrowth through blockage of the ontogenetic cell death that

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Blocking Igf-1 uptake through systemic application of an Igf-1 antibody not only reduces cell proliferation in the adult rat and mouse hippocampus, but is also implicated in synaptic remodelling and interferes with exercise-induced increase of dendritic spines [93]. IIS is also implicated in neuronal maturation and stimulates neurite outgrowth. In postnatal rat slice cultures of the somatosensory cortex, Igf-1-treatment leads to increased apical and basic dendritic branching of pyramidal neurons [94]. As such, Igf-1 is involved in forming mature neuronal networks and ensuring proper information processing. Further data show that Igf-1 mediates axonal growth by inducing plasmalemmal expansions by incorporation of vesicles in the growth cone [95]. This Igf-1-, but not Insulin-dependent effect is mediated by signalling via a growth-cone-specific Igf-1 receptor (βgc), Irs-2, Shp-2, PI3K and Akt. This cascade might also influence dynamics of the cytoskeleton, namely microtubules and actin, both of which are

Igf-2 has recently been shown to control the number of synaptic connections and plasticity in mature neurons of the hippocampus [13,14]. Memory enhancement is specifically associated with Igf-2 function. In this context Igf-2 is regulated by CCAAT enhancer binding protein β (C/EBP) in adult rat hippocampus. C/EBP is one of the transcription factors necessary for memory consolidation. In the study of Chen et al. rats are exposed to increased Igf-2 levels in the hippocampus during memory training which results in improved learning [13]. This effect is specific to Igf-2, since injection of Igf-1 did not have any observable effect in this memory

FoxO proteins are under the control of IIS and therefore a downstream read-out of this important signalling pathway. GFAP-cre was used to generate a triple allelic knockout of FoxO1, 3, and 4 in adult NPCs. The observed effect on cell proliferation is dependent on the age of the animal or passage of cells in culture. NPCs from young (P8) brains displayed hyperproliferation upon loss of FoxO-proteins and the same was observed for early passages of NPCs in culture. This initial hyperproliferation is followed by decreased doubling times and the depleted progenitor pool results in declining neuronal differentiation. FoxOs balance NPC proliferation and prevent premature loss of NPCs. Microarray studies revealed several cell-cycle regulating genes that are affected in their expression by the loss of FoxO. Further‐ more, Aspm (asp (abnormal spindle)-like, microcephaly associated) and anatgonists of the Wnt-signalling cascade, Sfrp-1 (secreted frizzled-related protein) and -2, as well as Sost (sclerostin), are involved in the FoxO-dependent regulation. In this context FoxO might control Wnt-induced cell proliferation by expression of Wnt-antagonists [89].

#### **3.4. Insulin/IGF effects in mature neurons**

Igf-1 applied to postnatal cerebellar granule neurons (CGN) prevents cell death, thus es‐ tablishing IIS as survival factor in mature neurons [90]. Another study described a link of Igf-1-signalling in mature neurons to survival of CGN as well as of cortical neurons. Here, Igf-1 prevents apoptosis through activation of PI3K/Akt, but also through Igf-1r-de‐ pendend transactivation of unliganded Pac-1r (PACAP type 1 receptor) via Src-kinase ac‐ tivity [91] (Fig. 4).

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**Figure 4.** IIS mediated sur-vival of cortical neurons through activation of Akt and PAC-1r [91].

with proteoglycans decreases its affinity to Igf-1. The net outcome might be a locally increased amount of free Igf-1 that drives neuronal differentiation of NPC [85]. In another study, differentiation of SVZ NPC was deduced from reduced expression of the stem cell marker Nestin after Igf-1 treatment, but it was not specified whether differentiation of a specific cell

Further studies in adult mice show that lack of Igf-1 results in disturbed neuroblast exit from the subventricular zone and their subsequent migration to the olfactory bulb through the rostral migratory stream. This Igf-1 function is dependent on PI3-kinase activity and Disa‐

Igf-2 is not only provided from the CSF and important for early development of the brain. In addition, adult mouse hippocampal stem cells from the DG also produce Igf-2, and to a much lesser extend Igf-2 is also detected in adult SVZ-derived stem cells [19,88]. Challenging Igf-2 expression through siRNA-mediated approaches as well as treatment of DG-derived NPCs reveals that Igf-2 also influences proliferation. However, SVZ-derived NPCs do not respond with changed proliferation upon Igf-2 knockdown [88]. Igf-2 but not Igf-1 is involved in neuronal survival of newly generated neurons in the adult DG in a model of fear extinction, that is strongly associated with neurogenesis in the hippocampus [31]. Neuronal survival and fear extinction depend on Igf-1r activity and are opposed by increased levels of Igfbp-7.

FoxO proteins are under the control of IIS and therefore a downstream read-out of this important signalling pathway. GFAP-cre was used to generate a triple allelic knockout of FoxO1, 3, and 4 in adult NPCs. The observed effect on cell proliferation is dependent on the age of the animal or passage of cells in culture. NPCs from young (P8) brains displayed hyperproliferation upon loss of FoxO-proteins and the same was observed for early passages of NPCs in culture. This initial hyperproliferation is followed by decreased doubling times and the depleted progenitor pool results in declining neuronal differentiation. FoxOs balance NPC proliferation and prevent premature loss of NPCs. Microarray studies revealed several cell-cycle regulating genes that are affected in their expression by the loss of FoxO. Further‐ more, Aspm (asp (abnormal spindle)-like, microcephaly associated) and anatgonists of the Wnt-signalling cascade, Sfrp-1 (secreted frizzled-related protein) and -2, as well as Sost (sclerostin), are involved in the FoxO-dependent regulation. In this context FoxO might control

Igf-1 applied to postnatal cerebellar granule neurons (CGN) prevents cell death, thus es‐ tablishing IIS as survival factor in mature neurons [90]. Another study described a link of Igf-1-signalling in mature neurons to survival of CGN as well as of cortical neurons. Here, Igf-1 prevents apoptosis through activation of PI3K/Akt, but also through Igf-1r-de‐ pendend transactivation of unliganded Pac-1r (PACAP type 1 receptor) via Src-kinase ac‐

lineage was favoured [50].

48 Trends in Cell Signaling Pathways in Neuronal Fate Decision

bled-1 (Dab-1) phosphorylation via Src-family kinase [87].

However, Igf-1 does not exert any observable effect in this setting.

Wnt-induced cell proliferation by expression of Wnt-antagonists [89].

**3.4. Insulin/IGF effects in mature neurons**

tivity [91] (Fig. 4).

Data from adult Igf-1-deficient mice show however that cerebellar neurons are not affected by the loss of this trophic factor *in vivo* [64]. Contrasting this finding is data showing overexpres‐ sion of human IGF-1 in mouse through a transgene driven by the mouse Igf-2 promoter [92] that results in cerebellar overgrowth through blockage of the ontogenetic cell death that normally takes place during early postnatal cerebellar development.

Blocking Igf-1 uptake through systemic application of an Igf-1 antibody not only reduces cell proliferation in the adult rat and mouse hippocampus, but is also implicated in synaptic remodelling and interferes with exercise-induced increase of dendritic spines [93]. IIS is also implicated in neuronal maturation and stimulates neurite outgrowth. In postnatal rat slice cultures of the somatosensory cortex, Igf-1-treatment leads to increased apical and basic dendritic branching of pyramidal neurons [94]. As such, Igf-1 is involved in forming mature neuronal networks and ensuring proper information processing. Further data show that Igf-1 mediates axonal growth by inducing plasmalemmal expansions by incorporation of vesicles in the growth cone [95]. This Igf-1-, but not Insulin-dependent effect is mediated by signalling via a growth-cone-specific Igf-1 receptor (βgc), Irs-2, Shp-2, PI3K and Akt. This cascade might also influence dynamics of the cytoskeleton, namely microtubules and actin, both of which are required for axonal growth.

Igf-2 has recently been shown to control the number of synaptic connections and plasticity in mature neurons of the hippocampus [13,14]. Memory enhancement is specifically associated with Igf-2 function. In this context Igf-2 is regulated by CCAAT enhancer binding protein β (C/EBP) in adult rat hippocampus. C/EBP is one of the transcription factors necessary for memory consolidation. In the study of Chen et al. rats are exposed to increased Igf-2 levels in the hippocampus during memory training which results in improved learning [13]. This effect is specific to Igf-2, since injection of Igf-1 did not have any observable effect in this memory consolidation paradigm.

Taken together, IIS has different effects in the developing and mature CNS that vary among the different cell types as summarised in Fig. 5. Differences might be mediated through specific downstream cascades or effector molecules present in only one specific cell or at a specific developmental time point. Another scenario is the cross-talk between IIS and other cytokines that are produced by the organism. Posttranslational modification of the receptors might be another general mechanism that specifies different biological responses to common signal inducers (ligand/receptor) and common intracellular signal transducers. The IGF-1r is sumoylated and modulated in its activity. Sehat et al. showed [96] this sumoylation of the IGF-1 receptor that leads to internalisation of the ligand bound protein complex, which is transduced to the nucleus and binds directly to genomic DNA. Since binding occurs predominantly in intergenic regions, the biological relevance for differential gene transcription has still to be shown. But nevertheless this reflects a possible mechanism through which specificity in activation of different target genes upon the same stimulus might be obtained in different cells or in the same cells at different developmental time points.

**4. Insulin/IGF-signalling networks**

**4.1. Signalling networks in neural progenitors and neurons**

accounts for the mitogenic effect of Igf-1 [45] (Fig. 6).

**Figure 6.** Igf-1-dependent embryonal NPC proliferation in rat [45].

Rapamycin with regard to proliferation (Fig. 7).

Igf-1-dependent embryonal NPC proliferation in rat has been shown to be dependent on the PI3K/Akt pathway, but not on the MAPK/Erk branch of signalling. Chemical inhibitors of the latter, U0129 and PD98059, have no effect on NPC proliferation, while PI3K-inhibition through LY294002 interferes with Akt- and downstream Gsk-3β-phosphorylation as well as prolifera‐ tion. Phosphorylated and thus inactivated Gsk-3β leads to stabilisation of CycD1 (Cyclin D1). Together with increased expression of CycD3, and CycE as cell cycle promoters, decreased expression of inhibitory cell cycle proteins p27 and p57 is observed upon Igf-1-treatment and

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Cortical progenitors isolated from mouse E13.5 brains were investigated with regard to increased Akt-1-signalling. Overexpression of Akt-1 results in increased proliferation as well as survival of progenitors *in vivo* and *in vitro*, consistant with effects of increased IIS. This Akt-1 function does not depend on its action upon p21, but at least in part of mTorc-1, because proliferation is partially hampered through Rapamycin. Survival is not affected through mTorc1 inhibition [97]. Differential downstream signalling of Akt in cortical progenitors is indicated by this study, which also reports that Rapamycin does only block proliferation in a subset of cells at E13.5, and progenitors of later developmental stages are also insensitive to

**Figure 5.** Differences in IIS function in different cell types of the CNS and in developmentThe Insulin-Igf Signalling (IIS) pathway has multiple effects on different cells of the CNS. The effects, beginning from cues for ESC proliferation and self-renewal, transcend to those of proliferation, self-renewal, differentiation and fate determination of NPCs. IIS con‐ tributes to either formation of neurons, or glia, oligodendrocytes and myelin.
