**2. Insulin/IGF-expression in the central nervous system**

Insulin, Igf-1 and -2 as well as the three corresponding receptors are widely expressed in the developing and mature central nervous system (CNS) (for a recent review refer to [1]). Expression of Insulin and Igfs is in part under the control of Growth hormone (GH), but action of several tissue- and developmental-specific transcription factors are also involved in Insulin/ Igf expression as is the nutritional status (our own unpublished observations and [12-14]). Coordination and regulation of the biological activity of Igf-1 and -2, but not Insulin, is not only achieved by transcriptional or translational control but also through a set of proteins that have the ability to bind these ligands, namely the Igf-binding proteins (Igfbp). Upto date there

choroid plexus and leptomeninges [22]. Accordingly, IGFBP-2 is found closely associated with human astroglial tumors. In this research field, it serves not only as a staging marker for glioblastoma but is exploited for therapeutic intervention [23]. IGFBP-2 as well as bound IGF-2 are overexpressed in high-grade astrocytomas. MMP-9-mediated proteolysis of IGFBP-2 leads to increased IGF-2 levels in these tumours. This accounts for increased aggressiveness of the tumours through growth- and motility-promoting effects [24]. Expression of Igfbp-3 in the brain is apparently not studied in great detail. However, Igfbp-3 expression increases after brain insults and it has been shown to be upregulated in Alzheimer´s disease brains [25]. Igfbp-3 interacts with retinoic acid and might therefore also be implicated in cell differentiation [26]. Igfbp-4 expression is also detected in most brain regions with declining levels over development [20], being highly expressed in the hippocampus and cerebral cortex in adult stages [27]. A function apart from regulation of Igf action has not been described yet. However, Igfbp-4 expression is decreased in the cerebral cortex, hippocampus and the cerebellum in an *in vivo* model for amyotrophic lateral sclerosis (ALS) [28]. Igfbp-5 is expressed mainly within thalamic nuclei, leptomeninges and the perivascular sheaths in adult rats [27]. Igfbp-6 seems to be expressed in differentiated but not in proliferating cells. Embryonic expression of Igfbp-6 in the CNS seems restricted to the trigeminal ganglia. Igfpb-6 transcription increases postna‐ tally after approx. 21 days in the forebrain and cerebellum, primarily in GABAergic inter‐ neurons. Higher levels are found in the hindbrain, spinal cord and dorsal root ganglia [29]. According to this strong association of Igfbp-6 expression with the cerebellar sensorimotor system, overexpression of Igfbp-6 results in a reduced size of the cerebellum [30]. Igfbp-7 is produced in the adult hippocampus, e.g. in the dentate gyrus (DG) [31]. In the postnatal hippocampus, all components of the extracellular Insulin/Igf-signalling molecules were detected apart from Igfbp-1. Variable amounts of individual components were detectable over two developmental time points studied, as well as in different cellular sources, which comprise neurons as well as glial and endothelial cells, albeit Igf-familiy member expression in the latter

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In rodents, the Insulin-receptor (Ir) is expressed in the olfactory bulb (OB), cerebral cortex, hippocampus, hypothalamus, pituitary, cerebellum, and the choroid plexus [32]. Expression is high in early stages of development and declines in the adult, and is also more enriched in neurons compared to glia. Within mature neurons, enrichment is observed within the postsynaptic density [4,33]. Accordingly, Ir-signalling including downstream mediators like Ras/MAPK and PI3K/Akt/mTor are implicated in synaptic connectivity and dendritic structure [4]. Igf-1r has a similar expression pattern with high expression in the developing cerebellum, midbrain, OB, and hindbrain [12]. However, although expression of Ir and Igf-1r is observed in hippocampus, there are local differences in their distriubtion: Ir is enriched in the CA1 region and Igf-1r is more prominent in the CA3 region. Levels of expression of the Igf-1r are higher during development and decline to adult levels shortly after birth in the brain parenchyma,

while it stays relatively high in the choroid plexus, meninges and vascular sheaths.

Igf-2 receptor (Igf-2r) is also known as the mannose-6-phosphate (M6P) receptor, which has a role in lysosomal enzyme trafficking. Accordingly, the main function described so far is to internalise the Igf-2 ligand through endocytosis and to mediate degradation of Igf-2. However,

two cell types were not studied in comprehensive detail.

**Figure 2.** General overview of IIS activating ERK in RAS/MAPK-dependent and JNK in CDC42-dependent manner.

are several Igf-1 and -2 binding proteins as well as Igfbp-related (or -like) proteins that bind to the ligands with varying affinities. Igfbps serve different functions, such as stabilisation and regulation of the concentration of diffusible Igfs, as well as facilitating receptor binding and modulation of Igf-bioavailability in the extracellular space [12]. Altogether, the various subtypes of Igfbps bind 99% of circulating Igf-1 and -2. Within this fraction, 75% are bound to Igfbp-3. Only 1% of Igf-1 and -2 is freely available in the extracellular space [15].

Igf-1 and -2 as well as the Igfbps are widely expressed in the brain [16,17]. Their expression patterns have been described in multiple studies, mainly based on mRNA-detection methods (summarised in [12]). Accordingly, Igf-1 is strongly expressed in the spinal cord, midbrain, cerebral cortex, hippocampus and olfactory bulb (OB) during development. Igf-1 transcription decreases postnatally as cell maturation advances to reach low levels in the adult central nervous system. In the OB, Igf-1 expression persists at high levels in cells that are constantly renewed even in the adult organism [18]. Igf-2 is also expressed in various brain regions and also declines over development. However, it is considered to be the most abundantly expressed Igf in the adult brain.

Igfbp-1 seems generally not to be present in sufficient amounts for detection in the central nervous system [1,19]. Expression of Igfbp-2 is found over all gross neuroanatomical structures of the rat brain, increasing from E15 until adulthood [20]. In preneurogenic stages (E10], rat Igfbp-2 is strongly expressed in neuroectodermal structures of the neural tube and the neuroepithelium [21]. In the postnatal brain it is mainly confined to astroglia as well as to the choroid plexus and leptomeninges [22]. Accordingly, IGFBP-2 is found closely associated with human astroglial tumors. In this research field, it serves not only as a staging marker for glioblastoma but is exploited for therapeutic intervention [23]. IGFBP-2 as well as bound IGF-2 are overexpressed in high-grade astrocytomas. MMP-9-mediated proteolysis of IGFBP-2 leads to increased IGF-2 levels in these tumours. This accounts for increased aggressiveness of the tumours through growth- and motility-promoting effects [24]. Expression of Igfbp-3 in the brain is apparently not studied in great detail. However, Igfbp-3 expression increases after brain insults and it has been shown to be upregulated in Alzheimer´s disease brains [25]. Igfbp-3 interacts with retinoic acid and might therefore also be implicated in cell differentiation [26]. Igfbp-4 expression is also detected in most brain regions with declining levels over development [20], being highly expressed in the hippocampus and cerebral cortex in adult stages [27]. A function apart from regulation of Igf action has not been described yet. However, Igfbp-4 expression is decreased in the cerebral cortex, hippocampus and the cerebellum in an *in vivo* model for amyotrophic lateral sclerosis (ALS) [28]. Igfbp-5 is expressed mainly within thalamic nuclei, leptomeninges and the perivascular sheaths in adult rats [27]. Igfbp-6 seems to be expressed in differentiated but not in proliferating cells. Embryonic expression of Igfbp-6 in the CNS seems restricted to the trigeminal ganglia. Igfpb-6 transcription increases postna‐ tally after approx. 21 days in the forebrain and cerebellum, primarily in GABAergic inter‐ neurons. Higher levels are found in the hindbrain, spinal cord and dorsal root ganglia [29]. According to this strong association of Igfbp-6 expression with the cerebellar sensorimotor system, overexpression of Igfbp-6 results in a reduced size of the cerebellum [30]. Igfbp-7 is produced in the adult hippocampus, e.g. in the dentate gyrus (DG) [31]. In the postnatal hippocampus, all components of the extracellular Insulin/Igf-signalling molecules were detected apart from Igfbp-1. Variable amounts of individual components were detectable over two developmental time points studied, as well as in different cellular sources, which comprise neurons as well as glial and endothelial cells, albeit Igf-familiy member expression in the latter two cell types were not studied in comprehensive detail.

are several Igf-1 and -2 binding proteins as well as Igfbp-related (or -like) proteins that bind to the ligands with varying affinities. Igfbps serve different functions, such as stabilisation and regulation of the concentration of diffusible Igfs, as well as facilitating receptor binding and modulation of Igf-bioavailability in the extracellular space [12]. Altogether, the various subtypes of Igfbps bind 99% of circulating Igf-1 and -2. Within this fraction, 75% are bound to

**Figure 2.** General overview of IIS activating ERK in RAS/MAPK-dependent and JNK in CDC42-dependent manner.

Igf-1 and -2 as well as the Igfbps are widely expressed in the brain [16,17]. Their expression patterns have been described in multiple studies, mainly based on mRNA-detection methods (summarised in [12]). Accordingly, Igf-1 is strongly expressed in the spinal cord, midbrain, cerebral cortex, hippocampus and olfactory bulb (OB) during development. Igf-1 transcription decreases postnatally as cell maturation advances to reach low levels in the adult central nervous system. In the OB, Igf-1 expression persists at high levels in cells that are constantly renewed even in the adult organism [18]. Igf-2 is also expressed in various brain regions and also declines over development. However, it is considered to be the most abundantly expressed

Igfbp-1 seems generally not to be present in sufficient amounts for detection in the central nervous system [1,19]. Expression of Igfbp-2 is found over all gross neuroanatomical structures of the rat brain, increasing from E15 until adulthood [20]. In preneurogenic stages (E10], rat Igfbp-2 is strongly expressed in neuroectodermal structures of the neural tube and the neuroepithelium [21]. In the postnatal brain it is mainly confined to astroglia as well as to the

Igfbp-3. Only 1% of Igf-1 and -2 is freely available in the extracellular space [15].

Igf in the adult brain.

40 Trends in Cell Signaling Pathways in Neuronal Fate Decision

In rodents, the Insulin-receptor (Ir) is expressed in the olfactory bulb (OB), cerebral cortex, hippocampus, hypothalamus, pituitary, cerebellum, and the choroid plexus [32]. Expression is high in early stages of development and declines in the adult, and is also more enriched in neurons compared to glia. Within mature neurons, enrichment is observed within the postsynaptic density [4,33]. Accordingly, Ir-signalling including downstream mediators like Ras/MAPK and PI3K/Akt/mTor are implicated in synaptic connectivity and dendritic structure [4]. Igf-1r has a similar expression pattern with high expression in the developing cerebellum, midbrain, OB, and hindbrain [12]. However, although expression of Ir and Igf-1r is observed in hippocampus, there are local differences in their distriubtion: Ir is enriched in the CA1 region and Igf-1r is more prominent in the CA3 region. Levels of expression of the Igf-1r are higher during development and decline to adult levels shortly after birth in the brain parenchyma, while it stays relatively high in the choroid plexus, meninges and vascular sheaths.

Igf-2 receptor (Igf-2r) is also known as the mannose-6-phosphate (M6P) receptor, which has a role in lysosomal enzyme trafficking. Accordingly, the main function described so far is to internalise the Igf-2 ligand through endocytosis and to mediate degradation of Igf-2. However, 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 as endothelial cells [12].

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.

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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

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

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

Increased proliferation of E14 rat cortical progenitor upon Igf-1 is observed *in vitro* after

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

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

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

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

specific to this combined treatment.

energy homeostasis and endocrinology of the reproductive system [42].

and thus illustrates that IIS is transducing GH-induced effects [42,44].

progenitors does influence survival of these cells [46].

molecules, emphasising the pleiotrophic nature of IIS.

treatment of cultured cells, and *in vivo* after intrauterine Igf-1 injection [45].

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.
