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

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

**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.

or in the same cells at different developmental time points.

50 Trends in Cell Signaling Pathways in Neuronal Fate Decision

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

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

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

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 Rapamycin with regard to proliferation (Fig. 7).

MTorc-1 inhibition through Rapamycin prevents neuronal differentiation of rat embryonic telencephalon-derived neurospheres that is observed in response to Insulin-signalling [53]. In this setting, Insulin-treatment results in phosporylation of mTorSer2448 and of pS670K, and the former is blocked through addition of Rapamycin, indicating that mTorc-1 is implicated

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MTor-1-dependent neuronal differentiation is also observed in P19 embryonic carcinoma cells that express Pax-6 as well as Hes-5 upon retinoic acid treatment and initiation of neural differentiation. In this setting, Cdc42 is placed as upstream regulator of mTorc-1, and treatment with Rapamycin abolished Cdc42-mediated neural lineage specification as well as terminal

MTorc-1 is also implicated in cell proliferation of rat adult hippocampal progenitors that heavily depend on PI3K-activated Akt-signalling. MTorc-1-dependence is obvious because presence of Rapamycin interfered with increased cell proliferation under Fgf-2-stimulation. Although Igf-1 treatment of these cells did not result in increased proliferation, Igf-1 increases cell division in the presence of Fgf-2 over the rate that is achieved through Fgf-2 alone [99]. Sustained activation of mTor-signalling in mice that are deficient for Tsc-1 results in initially increased proliferation of NPC and premature depletion of the progenitor pool. Self-renewal of Tsc-1-deficient and thus mTor-activated NPC is also dramatically impaired. In addition to increased numbers of cells that differentiate into the neuronal lineage, sustained mTor activity interferes with terminal differentiation of neurons but seems to promote an increase in astrocytes [100]. Increased cell proliferation is also a hallmark of Tsc-2-deficient mouse NPC that are isolated from early (E10.5) developmental time points. This phenotype is also attrib‐

in neuronal differentiation. Apoptosis is not observed under these conditions (Fig. 8).

neuronal differentiation [98].

uted to an activation of mTorc-1-signalling [101].

**Figure 9.** Igf-1-dependent survival of rat hip-pocampal precursors [46].

**Figure 7.** Survival and proliferation upon Igf-1 treat-ment in mouse E13.5 cortical cells [97].

**Figure 8.** Insulin-dependent neuronal differentiation of embryonic neurosheres [50].

MTorc-1 inhibition through Rapamycin prevents neuronal differentiation of rat embryonic telencephalon-derived neurospheres that is observed in response to Insulin-signalling [53]. In this setting, Insulin-treatment results in phosporylation of mTorSer2448 and of pS670K, and the former is blocked through addition of Rapamycin, indicating that mTorc-1 is implicated in neuronal differentiation. Apoptosis is not observed under these conditions (Fig. 8).

MTor-1-dependent neuronal differentiation is also observed in P19 embryonic carcinoma cells that express Pax-6 as well as Hes-5 upon retinoic acid treatment and initiation of neural differentiation. In this setting, Cdc42 is placed as upstream regulator of mTorc-1, and treatment with Rapamycin abolished Cdc42-mediated neural lineage specification as well as terminal neuronal differentiation [98].

MTorc-1 is also implicated in cell proliferation of rat adult hippocampal progenitors that heavily depend on PI3K-activated Akt-signalling. MTorc-1-dependence is obvious because presence of Rapamycin interfered with increased cell proliferation under Fgf-2-stimulation. Although Igf-1 treatment of these cells did not result in increased proliferation, Igf-1 increases cell division in the presence of Fgf-2 over the rate that is achieved through Fgf-2 alone [99]. Sustained activation of mTor-signalling in mice that are deficient for Tsc-1 results in initially increased proliferation of NPC and premature depletion of the progenitor pool. Self-renewal of Tsc-1-deficient and thus mTor-activated NPC is also dramatically impaired. In addition to increased numbers of cells that differentiate into the neuronal lineage, sustained mTor activity interferes with terminal differentiation of neurons but seems to promote an increase in astrocytes [100]. Increased cell proliferation is also a hallmark of Tsc-2-deficient mouse NPC that are isolated from early (E10.5) developmental time points. This phenotype is also attrib‐ uted to an activation of mTorc-1-signalling [101].

**Figure 9.** Igf-1-dependent survival of rat hip-pocampal precursors [46].

**Figure 8.** Insulin-dependent neuronal differentiation of embryonic neurosheres [50].

**Figure 7.** Survival and proliferation upon Igf-1 treat-ment in mouse E13.5 cortical cells [97].

52 Trends in Cell Signaling Pathways in Neuronal Fate Decision

Igf-1-initiated survival of E19 rat embryonal hippocampal cells is dependent on PI3K-mediated Akt-phosphorylation and subsequent phosphorylation of FoxO3. Phosphorylation of FoxO3 is blocked by LY294002 and partially by Rapamycin, and results in increased survival of hippocampal progenitors. Blocking of MAPK/Erk pathway with PD98059 does not influence survival of these progenitors [46,102] (Fig. 9).

cortical progenitors in an Erk-independent manner, while Igf-1 signalling induces proliferation of adult hippocampal progenitors via Erk-dependent cytoplasmic downstream signals. On the other hand, IIS mediates different biological effects in similar cells through similar downstream molecules, but at different developmental time points: adult hippocampal neurons respond with proliferation to Igf-1 compared to embryonic hippocampal progenitors that need IIS for their survival. This summary indicates that we still need comprehensive analyses of overlap‐ ping or distinct target genes activated through overlapping or distinct signalling pathways

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The study of Otaegi et al. of IIS in OBSC also elaborates on downstream signalling by showing that Igf-1 treatment increases phosphorylation of Akt at Ser473 as well as Thr308 which is associated with increased proliferation and neural differentiation [54] (Fig. 11). Pten, that antagonises PI3-kinase induced phosphorylation of phosphatidylinositol (3,4,5)trisphosphate (PtdIns(3,4,5)P3), reduces basal levels of the activating posttranslational modifications of Akt, which are however overridden by presence of Insulin/Igf-1. Nevertheless, overexpression of Pten impaires neural differentiation without affecting progenitor proliferation, and overex‐ pression of a mutant Pten increases OBSC differentiation. Direct inhibition of PI3-kinase through LY294002 interferes with Igf-1-stimulated Akt-phosphorylation and resultes in impaired proliferation as well as neural differentiation. Thus, OBSC differentiation is under control of basic levels of pAkt while increased pAkt-levels through IIS activation affect

during development and in the various cell types of the CNS.

**Figure 11.** IIS affects proliferation and differentiation in adult OBSC [51].

Early postnatal olfactory bulb neurons do survive upon Igf-1-signalling. In this context, a positive feedback has been proposed between Igf-1 and Bcl-2. Igf-1 increases expression

additionally proliferation.

**Figure 10.** IIS in regulation of proliferation of adult hippocampal progenitors [77, 78].

Igf-1-mediated proliferation of neural progenitors of the adult DG is mainly but not exclusively mediated by MAPK/Erk-signalling, since inhibition of MAPKK/Mek-1/2 through a chemical inhibitor or dominant negative kinases interferes with cell division after Igf-1 treatment. Blocking of PI3 kinase through LY294002 interferes with proliferation even without addition of Igf-1. However, blocking of the Akt-downstream signal through use of a dominant negative kinase also reduces Igf-1-induced mitosis. This finding makes it likely that LY294002-inde‐ pendent activation of Akt might be important for Igf-1-mediated proliferation. Rapamycin does not have significant effects in this setting, making it likely that mTor-complexes are not involved in Igf-1-mediated proliferation of adult hippocampal progenitors [77] (Fig. 10). Further evidence for the implication of Igf-1/MAPK/Erk- signalling in proliferation of adult hippocampal progenitors comes from the study of increased mitosis after a status epilepticus. Here, this lesion activates Igf-1-release from microglia. This increase in Igf-1 results in the activation of the MAPK/Erk-pathway and subsequent cell division. This response is blocked through inhibition of the Igf-1r as well as through U0129-mediated inhibition of the MAPKpathway [78].

It is interesting to point out that the same biological effect can be achieved through Igf-1 by activating different pathways in different cells: IIS induces proliferation in embryonic rat cortical progenitors in an Erk-independent manner, while Igf-1 signalling induces proliferation of adult hippocampal progenitors via Erk-dependent cytoplasmic downstream signals. On the other hand, IIS mediates different biological effects in similar cells through similar downstream molecules, but at different developmental time points: adult hippocampal neurons respond with proliferation to Igf-1 compared to embryonic hippocampal progenitors that need IIS for their survival. This summary indicates that we still need comprehensive analyses of overlap‐ ping or distinct target genes activated through overlapping or distinct signalling pathways during development and in the various cell types of the CNS.

Igf-1-initiated survival of E19 rat embryonal hippocampal cells is dependent on PI3K-mediated Akt-phosphorylation and subsequent phosphorylation of FoxO3. Phosphorylation of FoxO3 is blocked by LY294002 and partially by Rapamycin, and results in increased survival of hippocampal progenitors. Blocking of MAPK/Erk pathway with PD98059 does not influence

survival of these progenitors [46,102] (Fig. 9).

54 Trends in Cell Signaling Pathways in Neuronal Fate Decision

**Figure 10.** IIS in regulation of proliferation of adult hippocampal progenitors [77, 78].

pathway [78].

Igf-1-mediated proliferation of neural progenitors of the adult DG is mainly but not exclusively mediated by MAPK/Erk-signalling, since inhibition of MAPKK/Mek-1/2 through a chemical inhibitor or dominant negative kinases interferes with cell division after Igf-1 treatment. Blocking of PI3 kinase through LY294002 interferes with proliferation even without addition of Igf-1. However, blocking of the Akt-downstream signal through use of a dominant negative kinase also reduces Igf-1-induced mitosis. This finding makes it likely that LY294002-inde‐ pendent activation of Akt might be important for Igf-1-mediated proliferation. Rapamycin does not have significant effects in this setting, making it likely that mTor-complexes are not involved in Igf-1-mediated proliferation of adult hippocampal progenitors [77] (Fig. 10). Further evidence for the implication of Igf-1/MAPK/Erk- signalling in proliferation of adult hippocampal progenitors comes from the study of increased mitosis after a status epilepticus. Here, this lesion activates Igf-1-release from microglia. This increase in Igf-1 results in the activation of the MAPK/Erk-pathway and subsequent cell division. This response is blocked through inhibition of the Igf-1r as well as through U0129-mediated inhibition of the MAPK-

It is interesting to point out that the same biological effect can be achieved through Igf-1 by activating different pathways in different cells: IIS induces proliferation in embryonic rat The study of Otaegi et al. of IIS in OBSC also elaborates on downstream signalling by showing that Igf-1 treatment increases phosphorylation of Akt at Ser473 as well as Thr308 which is associated with increased proliferation and neural differentiation [54] (Fig. 11). Pten, that antagonises PI3-kinase induced phosphorylation of phosphatidylinositol (3,4,5)trisphosphate (PtdIns(3,4,5)P3), reduces basal levels of the activating posttranslational modifications of Akt, which are however overridden by presence of Insulin/Igf-1. Nevertheless, overexpression of Pten impaires neural differentiation without affecting progenitor proliferation, and overex‐ pression of a mutant Pten increases OBSC differentiation. Direct inhibition of PI3-kinase through LY294002 interferes with Igf-1-stimulated Akt-phosphorylation and resultes in impaired proliferation as well as neural differentiation. Thus, OBSC differentiation is under control of basic levels of pAkt while increased pAkt-levels through IIS activation affect additionally proliferation.

**Figure 11.** IIS affects proliferation and differentiation in adult OBSC [51].

Early postnatal olfactory bulb neurons do survive upon Igf-1-signalling. In this context, a positive feedback has been proposed between Igf-1 and Bcl-2. Igf-1 increases expression of anti-apoptotic Bcl-2, and overexpression of Bcl-2 *in vivo* results in increased Igf-1 and Igfbp2 [103].

deficient mice show retarded growth and initiation of myelination [110]. This phenotype is not observed in IR-deficient mice and is thus mediated through Igf-1r-signalling [111]. IIS depends on binding of insulin-response substrate (Irs1-4) to the receptors, which results in their

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Proliferation of oligodendroglial rat precursors increases in response to Igf-1 and is mediated over a prolonged time period by Pdk-1, Akt Thr308 as well as Ser473 phosphorylation, and pGsk-3β. Igf-1 treatment also activated the Ras/MAPK-pathway as evidenced by increased pErk-1/-2, following a fast kinetic over a short period of time [112]. Inhibition of upstream signalling molecules revealed concerted involvement of PI3K, MAPK as well as Src-kinase in cell cycle progression, and predominance of Akt-mediated signalling. Although so far ill defined, cross-talk among the different signalling pathways activated through Igf-1 is a likely scenario resulting from this study. In continuation of this study it was observed that Igf-1 increases protein synthesis by signalling through PI3K-Akt-mTor-p70S6K-4E-BP1 as well as by activating Ras/MAPK [113]. Transcription was a prerequisite for mTor-1-dependent protein synthesis upon Igf-1 in proliferating oligodendroglial precursors. Early differentiation of the precursors upon Igf-1 also involves transcriptional processes, however subsequent terminal differentiation is independent. This finding indicates different roles for Igf-1 during oligoden‐

Further analyses of these potential downstream signalling components reveal that initiation of myelination is coupled to the activity of Irs-2 but not to Irs-1. Irs-2-deficient mice display a defect in timing of myelination and this effect can not be compensated through a simultaneous upregulation of Igf-1r and Irs-1 expression as well as signalling in these mice [111]. Increased

Igf-2 influences proliferation of adult DG progenitors [88]. In this study, knockdown of Igf-2 results in decreased mitosis that is paired with reduced phosphorylation of Irs-1 and Akt. This phenotype is rescued through overexpression of a dominant-active Akt. Blocking of Igf-1rsignalling reduces proliferation that exceeds the drop observed after Igf-2 knockdown. This indicates that NPC proliferation in the adult DG is influenced through Igf-1 and Igf-2 ligands

Signalling of Igf-2 through its cognate Igf-2r has been shown to be important for neuronal maturation with regard to synapse density and spine morphology [14]. Downstream signals included the activation of MAPK/Erk- pathway, since changes at the level of the synapses were

Igf-2 dependent memory enhancement in adult rat hippocampus requires signalling via the Igf-2r [13]. This Igf-2 effect is associated with *de novo* protein synthesis. Further analyses of the downstream signalling cascade reveales involvement of Gsk-3β, since the Igf-2-specific effect is blocked by the presence of the Gsk-3β-inhibitor SB216763. It was shown that this cascade does not act on a widespread cell level but that it mainly regulates molecular events at specific synapses, e.g. synapses that are "transcriptionally marked" through training. Igf-2 results in increased synaptic activity as revealed by stable long-term potentiation (LTP). The underlying

Igf-1r signalling is shown through increased levels of pAkt, pGsk-3β and pErk-1/2.

respective phosphorylation.

droglial cell development.

that signal both through the Igf-1r onto Akt.

inhibited through PD98059.

**Figure 12.** Survival of CGN upon IIS [90, 104, 105].

Igf-1 action as survival factor for postnatal cerebellar granule neurons (CGN) requires different intracellular signalling [90]. In this context, Igf-1 activation of PI3K leads to inhibition of Erk as well as Caspase-3, two pathways exerting different events in cell death, plasma membrane damage and DNA break-down, respectively. Activated PI3K phosphorylates not only Akt but also Protein kinase A (PKA). Whereas pAkt activates Caspase-3 and is leading to DNA breakdown, PKA suppresses sustained Erk activation via phosphorylation of cRaf at Ser259 and prevents plasma membrane damage (Fig. 12).

Further studies of Igf-1-dependent survival of rat CGN links PI3K activation of Akt to phosphorylation of Bad at Ser136 that is responsible for releasing anti-apoptotic Bcl-XL [104,105]. This finding is partially corroborated *in vivo*, where Igf-1 overexpression results in increased levels of Bcl-XL at early time points (P7) and decreased expression of pro-apoptotic Bax and Bad at P14 and P21. However, the investigation of the phosphorylation status of Bad *in vivo* was not conclusive [92].

Oligodendrogenesis and initiation as well as maintenance of myelination are strongly de‐ pendent on IIS. Several mouse models in which normal IIS was manipulated display pheno‐ types affecting oligodendroglial cells: overexpression of Igf-1 driven by diverse promoters or administration of Igf-1 into the CSF result in increased numbers of oligodendrocytes and myelination [106-108], while loss of Igf-1 leads to impaired and lost myelination [109]. IIS in oligodendrocytes and their precursors is dependent on signalling over the Igf-1r, since Igf-1rdeficient mice show retarded growth and initiation of myelination [110]. This phenotype is not observed in IR-deficient mice and is thus mediated through Igf-1r-signalling [111]. IIS depends on binding of insulin-response substrate (Irs1-4) to the receptors, which results in their respective phosphorylation.

of anti-apoptotic Bcl-2, and overexpression of Bcl-2 *in vivo* results in increased Igf-1 and

Igf-1 action as survival factor for postnatal cerebellar granule neurons (CGN) requires different intracellular signalling [90]. In this context, Igf-1 activation of PI3K leads to inhibition of Erk as well as Caspase-3, two pathways exerting different events in cell death, plasma membrane damage and DNA break-down, respectively. Activated PI3K phosphorylates not only Akt but also Protein kinase A (PKA). Whereas pAkt activates Caspase-3 and is leading to DNA breakdown, PKA suppresses sustained Erk activation via phosphorylation of cRaf at Ser259 and

Further studies of Igf-1-dependent survival of rat CGN links PI3K activation of Akt to phosphorylation of Bad at Ser136 that is responsible for releasing anti-apoptotic Bcl-XL [104,105]. This finding is partially corroborated *in vivo*, where Igf-1 overexpression results in increased levels of Bcl-XL at early time points (P7) and decreased expression of pro-apoptotic Bax and Bad at P14 and P21. However, the investigation of the phosphorylation status of Bad

Oligodendrogenesis and initiation as well as maintenance of myelination are strongly de‐ pendent on IIS. Several mouse models in which normal IIS was manipulated display pheno‐ types affecting oligodendroglial cells: overexpression of Igf-1 driven by diverse promoters or administration of Igf-1 into the CSF result in increased numbers of oligodendrocytes and myelination [106-108], while loss of Igf-1 leads to impaired and lost myelination [109]. IIS in oligodendrocytes and their precursors is dependent on signalling over the Igf-1r, since Igf-1r-

Igfbp2 [103].

**Figure 12.** Survival of CGN upon IIS [90, 104, 105].

56 Trends in Cell Signaling Pathways in Neuronal Fate Decision

prevents plasma membrane damage (Fig. 12).

*in vivo* was not conclusive [92].

Proliferation of oligodendroglial rat precursors increases in response to Igf-1 and is mediated over a prolonged time period by Pdk-1, Akt Thr308 as well as Ser473 phosphorylation, and pGsk-3β. Igf-1 treatment also activated the Ras/MAPK-pathway as evidenced by increased pErk-1/-2, following a fast kinetic over a short period of time [112]. Inhibition of upstream signalling molecules revealed concerted involvement of PI3K, MAPK as well as Src-kinase in cell cycle progression, and predominance of Akt-mediated signalling. Although so far ill defined, cross-talk among the different signalling pathways activated through Igf-1 is a likely scenario resulting from this study. In continuation of this study it was observed that Igf-1 increases protein synthesis by signalling through PI3K-Akt-mTor-p70S6K-4E-BP1 as well as by activating Ras/MAPK [113]. Transcription was a prerequisite for mTor-1-dependent protein synthesis upon Igf-1 in proliferating oligodendroglial precursors. Early differentiation of the precursors upon Igf-1 also involves transcriptional processes, however subsequent terminal differentiation is independent. This finding indicates different roles for Igf-1 during oligoden‐ droglial cell development.

Further analyses of these potential downstream signalling components reveal that initiation of myelination is coupled to the activity of Irs-2 but not to Irs-1. Irs-2-deficient mice display a defect in timing of myelination and this effect can not be compensated through a simultaneous upregulation of Igf-1r and Irs-1 expression as well as signalling in these mice [111]. Increased Igf-1r signalling is shown through increased levels of pAkt, pGsk-3β and pErk-1/2.

Igf-2 influences proliferation of adult DG progenitors [88]. In this study, knockdown of Igf-2 results in decreased mitosis that is paired with reduced phosphorylation of Irs-1 and Akt. This phenotype is rescued through overexpression of a dominant-active Akt. Blocking of Igf-1rsignalling reduces proliferation that exceeds the drop observed after Igf-2 knockdown. This indicates that NPC proliferation in the adult DG is influenced through Igf-1 and Igf-2 ligands that signal both through the Igf-1r onto Akt.

Signalling of Igf-2 through its cognate Igf-2r has been shown to be important for neuronal maturation with regard to synapse density and spine morphology [14]. Downstream signals included the activation of MAPK/Erk- pathway, since changes at the level of the synapses were inhibited through PD98059.

Igf-2 dependent memory enhancement in adult rat hippocampus requires signalling via the Igf-2r [13]. This Igf-2 effect is associated with *de novo* protein synthesis. Further analyses of the downstream signalling cascade reveales involvement of Gsk-3β, since the Igf-2-specific effect is blocked by the presence of the Gsk-3β-inhibitor SB216763. It was shown that this cascade does not act on a widespread cell level but that it mainly regulates molecular events at specific synapses, e.g. synapses that are "transcriptionally marked" through training. Igf-2 results in increased synaptic activity as revealed by stable long-term potentiation (LTP). The underlying mechanism is therefore deduced to include Igf-2-dependent activation of Gsk-3β that leads to synaptic mobilisation and expression of the AMPA-receptor GluR1.

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

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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.

treatment [99].

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

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