**1. Introduction**

The development of the nervous system, neuralization of ectodermal cells, specification of cell types as well as generation of neurological diseases are closely linked to Smad proteins, which play a central role by integrating TGFβ and BMP signalling with other essential pathways. Due to new findings on Smad activity in neurons and the nervous system, which comprises new roles for brain plasticity and functions, independent of the canonical signalling pathways, we reconsider their relevance for neuronal differentiation and dedifferentiation processes and as therapeutic targets for treatment of neurological diseases. In this chapter we develop a view at Smad molecules, which attributes them a basic significance and allow proving their specific contextual molecular, cellular and tissue relationships. In order to facilitate the understanding of the complex Smad network in the nervous system an overview of the canonical Smad signalling pathway is briefly summarized in the following paragraph.

Smads are phylogenetic old proteins, which are mediating intracellular signalling of the large group of solube TGFβ ligands (Figure 1), containing transforming growth factor βs (TGFβs), bone morphogentic proteins (BMPs), growth and differentiation factors (GDFs), Müllerian inhibitory factors (MISs), activins and inhibins [4]. Ligand binding to activated heteromeric receptor complexes, recruited from seven type I and five type II serine/threonine receptors, results in the specific phosphorylation of receptor-associated Smads (R-Smads) at two Cterminal serine residues.

Activin, nodal and TGFβ activate R-Smad2 and 3, while BMP acts by R-Smad1,5 and 8 phosphorylation. In the cytoplasm non-phosphorylated R-Smads are sequestrated by inter‐ acting with specific retention proteins e.g. SARA (Smad anchor for receptor activation) [5], endofin [8], tubulin [3], actin, myosin [11] or filamin [12]. Inhibitory Smad(I-Smad)6 and 7 negatively regulate R-Smad signalling by competing for binding to activated type I receptor and inhibiting R-Smad phosphorylation. I-Smads can also prevent R-Smad complexing to the

© 2013 Ueberham and Arendt; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Ueberham and Arendt; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

co-operating Smad(Co-Smad)4, which is required for the nuclear translocation of activated R-Smads to subsequently control Smad sensitive promoter activity. Moreover, I-Smads support recruitment of HECT-type E3-ubiquitin ligases Smurf1/2, which allow type I receptor ubiqui‐ tination and its degradation. Members of HECT-type and RING-type E3 ubiquitin ligases have also been implicated in Smad degradation [13]. Regulating Smad activity allows the control of highly complex developmental networks, e.g. the patterning of ventro-lateral mesoderm, the decisive development of epidermal/neural cell lineages including the induction and estab‐ lishment of neural plate border, the dorso-ventral patterning of the neural tube, or the migration of neural crest cells [18-21].

In R-Smads a linker region, located between the highly conserved N-terminal MH1- and the C-terminal MH2 region (MH represents Mad homology), is rich in potential serine/threo‐ nine phosphorylation sites. Several kinases (e.g. MAPK, GSK, CDKs, CamKII, SGK1) can phosphorylate the linker region and determine cellular distribution or protein stability of Smads [14,23-26]. Linker phosphorylation by mitogen-activated protein kinases (MAPKs) re‐ stricts Smad1 activity by enabling Smurf1 binding, causing polyubiquitination and inhibit‐ ing interaction of Smad1 with nuclear transclocation factor Nup214, thus leading to Smad1

The Role of Smad Proteins for Development, Differentiation and Dedifferentiation of Neurons

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A similar mechanism acts on Smad2 and 3, where ubiquitination is controlled by ubiquitin ligase Nedd4L [17]. A sustained MAPK activation, as reported from dissociated embryonic ectodermal cells, phosphorylates Smad linker and interrupt a continuing BMP mediated Smad1 signalling finally resulting in neuralisation of dissociated Xenopus [27]. Remarably, if linker phosphorylation is performed by extracellular signal regulated kinase (Erk) at nuclearly localized phosphorylated Smad, the duration of Smad targeted gene transcription can be increased [28]. Linker phosphorylation by CDK8 and CDK9 promotes both, the Smad tran‐ scriptional action, and the cooperation with YAP, an effector of the Hippo organ size control pathway, to finally suppress neural differentiation of stem cells [14]. The data demonstrate the

Numerous Smad binding proteins (e.g. Ski, SnoN) effectively influence essential steps during neural development mostly by repression of Smad activity [29-33]. The group of Smad interacting proteins (SIP), containing several zinc finger proteins, complex to Smad and can directly or indirectly regulate its transcription efficiency [34]. For example, the induction of neuroectoderm is dependent on SIP1 [35-37], which is also involved in the myelination process

During early ontogenesis, development and aging as well as prior to individual cell death Smad proteins differentially affect cellular function, depending on time and local partners. Already at very early stages of postnatal rat development Smads1-7 were detectable in the ovary [39]. At all stages of follicular development (e.g. in primordial, primary and secondary follicles) Smad proteins are present in oocytes as well as in granulosa cells and theca cells, though with different concentrations [39-41]. While Smad3,6 and 8 content did not change, the expression of Smad1,2,4,5,6 and 7 seems to be regulated in ovary [39]. In mice oocytes a clear and persistent nuclear localization of phosphorylated Smad1,5,9 and Smad2,3 demonstrates activated activin/GDF9/nodal/TGFβ and BMP/MIS pathways, respectively, and confirms oocytes as a major target for Smad signalling pathways [42]. Smad proteins play also important roles already during maturation of human oocytes [43]. In unfertilized oocytes, Smad2 and 3 and TGFβ receptors I and II are present [44]. After fertilization at the 4-cell and 8-cell stages Smad2 and 3 are also present, while neither of the TGFβ receptors is detectable. Later on in

**2. The role of Smads during neural/non-neuronal development**

degradation or cytoplasmic retention [15].

and the oligodendrocyte maturation [38].

particular role of the linker region for distinct Smad functions.

**2.1. Already early development requires Smad expression**

**Figure 1. Synopsis of canonical Smad signalling**. The upper panel shows the binding of ligands belonging to the TGFβ superfamily to their serine/threonine kinase receptors types I and II, leading to intracellular activation of R-Smads by receptor type I induced phosphorylation of the C-terminal Smad motif SSXS. The left and right receptor complexes represent the TGFβ/activin/nodal linked Smad2/3 signalling and the BMP/GDF linked Smad1/5/8 signal‐ ling, respectiviely. The middle panel also displays a set of relevant binding partners of Smad proteins and shows the generation of heterotrimer or heterodimer Smad complexes consisting of C-terminally phosphorylated R-Smads and Co-Smad4, which are transported into the nucleus to excert their transcriptional activity by binding to susceptible pro‐ moter sequences (a). In (b) and (c) examples of the agonist-induced Smad linker phosphorylation mediated by cyclindependent kinases are shown, which increases Smad transcriptional activity prior to signal termination and Smad degradation [14, 17]. In contrast, the MAPK triggered linker phosphorylation of Smads in cytoplasm diminishes the receptor type I responsible Smad phosphorylation and favours the cytoplasmic retention and degradation of Smad. More information is provided in the main text.*Note*: Numbers in square brackets indicate relevant references included in the reference list provided at the chapters end.*Abbreviations*: CDK, cyclin-dependent kinase; dynein, dynein light chain km23-1 or km23-2; MAPK, mitogen activated protein kinase; MT, microtubuli; Nedd4L, neuronal precursor cell expressed developmentally down-regulated 4-like ubiquitin ligase; Nuc214, nucleoporin 214; SARA, Smad achor for receptor activation; Smurf1, HECT-domain ubiquitin ligase Smurf1.

In R-Smads a linker region, located between the highly conserved N-terminal MH1- and the C-terminal MH2 region (MH represents Mad homology), is rich in potential serine/threo‐ nine phosphorylation sites. Several kinases (e.g. MAPK, GSK, CDKs, CamKII, SGK1) can phosphorylate the linker region and determine cellular distribution or protein stability of Smads [14,23-26]. Linker phosphorylation by mitogen-activated protein kinases (MAPKs) re‐ stricts Smad1 activity by enabling Smurf1 binding, causing polyubiquitination and inhibit‐ ing interaction of Smad1 with nuclear transclocation factor Nup214, thus leading to Smad1 degradation or cytoplasmic retention [15].

A similar mechanism acts on Smad2 and 3, where ubiquitination is controlled by ubiquitin ligase Nedd4L [17]. A sustained MAPK activation, as reported from dissociated embryonic ectodermal cells, phosphorylates Smad linker and interrupt a continuing BMP mediated Smad1 signalling finally resulting in neuralisation of dissociated Xenopus [27]. Remarably, if linker phosphorylation is performed by extracellular signal regulated kinase (Erk) at nuclearly localized phosphorylated Smad, the duration of Smad targeted gene transcription can be increased [28]. Linker phosphorylation by CDK8 and CDK9 promotes both, the Smad tran‐ scriptional action, and the cooperation with YAP, an effector of the Hippo organ size control pathway, to finally suppress neural differentiation of stem cells [14]. The data demonstrate the particular role of the linker region for distinct Smad functions.

Numerous Smad binding proteins (e.g. Ski, SnoN) effectively influence essential steps during neural development mostly by repression of Smad activity [29-33]. The group of Smad interacting proteins (SIP), containing several zinc finger proteins, complex to Smad and can directly or indirectly regulate its transcription efficiency [34]. For example, the induction of neuroectoderm is dependent on SIP1 [35-37], which is also involved in the myelination process and the oligodendrocyte maturation [38].
