**3. Specific cell types and the role of Smad expression for their induction and maintenance**

#### **3.1. Cell type decision, maturation and differentiation**

Both generation and differentiation of neurons in the brain (CNS) comprises several different and complex principles. The initial proliferation of neural precursor cells is followed by the decision to specify neuronal or glial outcome. Afterwards, the primary neurogenesis of neuronal progenitors involves migration, settlement and stopping proliferation by cell cycle exit to enter a terminal differentiation pathway. Many of these basic biological routes as well as further differentiation steps, e.g. dendritic and axonal growth and orientating, synapse formation and myelination depend on Smad proteins. The parallel occurrence of long term and acute Smad-dependent processes require a distinct contextual organisation. For example, in the developing brain an important neural stem cell is provided by radial glia cells which can generate both glial cells and neurons. While TGFβ promoted differentiation of radial glia into astrocytes is mainly regulated by activation of MAPK signalling, neurogenesis is control‐ led by the interplay of Smad2,3 activity and PI3K activity [102].

#### *3.1.1. Glial cells*

Radial glial cells can function as neuronal precursors and also control migration of neurons during cerebral cortex development [103]. Developing neurons are also actively implicated in the radial glial cell fate commitment [104]. Using TGFβ release, neurons can activate Smad signalling in radial glia cells and support their transformation to astrocytes [105] and also induces astrocytic differentiation and GFAP expression [106-109].

Moreover, endothelial cells also promote astrocytic differentiation by BMP-induced Smad signalling, but inhibit oligodendrocyte differentiation of postnatal cortical progenitors [110], and therefore participate in the sequential order of the two macroglial cell gliogenesis. The function of BMP/Smad signalling to drive precursors to astrocytic fate seems a common feature [111,112] which is also involved in CNS injury processes where oligodendrocyte precursors are driven towards type II astrocytes [113]. Accordingly, inhibition of BMP induced nuclear translocation of phospho-Smad1 by FGF2 activated MAPK activity is linked to Smad4 dissociation from Olig2 promoter and results in upregulation of oligodendrogenesis [114]. The very importance of a spatially and temporally regulated BMP induced Smad activation for the fate of neurogenic precursor cells was shown in chick spinal cord. At embryonic day 5, in neuroepithelial progenitors, astrocyte marker expression was inhibited and at embryonic day 6 it was promoted initiating a gliogenic period [115].

#### *3.1.2. Neurons*

activation of GFAP promoter is part of this process [99]. Generally, oligodendroglial differen‐ tiation of precursor cells is actively suppressed by BMPs concomitant with the stimulation of astrocytic differentiation [100]. Accordingly, BMP4-activation directs progenitor cells in vivo

**3. Specific cell types and the role of Smad expression for their induction**

Both generation and differentiation of neurons in the brain (CNS) comprises several different and complex principles. The initial proliferation of neural precursor cells is followed by the decision to specify neuronal or glial outcome. Afterwards, the primary neurogenesis of neuronal progenitors involves migration, settlement and stopping proliferation by cell cycle exit to enter a terminal differentiation pathway. Many of these basic biological routes as well as further differentiation steps, e.g. dendritic and axonal growth and orientating, synapse formation and myelination depend on Smad proteins. The parallel occurrence of long term and acute Smad-dependent processes require a distinct contextual organisation. For example, in the developing brain an important neural stem cell is provided by radial glia cells which can generate both glial cells and neurons. While TGFβ promoted differentiation of radial glia into astrocytes is mainly regulated by activation of MAPK signalling, neurogenesis is control‐

Radial glial cells can function as neuronal precursors and also control migration of neurons during cerebral cortex development [103]. Developing neurons are also actively implicated in the radial glial cell fate commitment [104]. Using TGFβ release, neurons can activate Smad signalling in radial glia cells and support their transformation to astrocytes [105] and also

Moreover, endothelial cells also promote astrocytic differentiation by BMP-induced Smad signalling, but inhibit oligodendrocyte differentiation of postnatal cortical progenitors [110], and therefore participate in the sequential order of the two macroglial cell gliogenesis. The function of BMP/Smad signalling to drive precursors to astrocytic fate seems a common feature [111,112] which is also involved in CNS injury processes where oligodendrocyte precursors are driven towards type II astrocytes [113]. Accordingly, inhibition of BMP induced nuclear translocation of phospho-Smad1 by FGF2 activated MAPK activity is linked to Smad4 dissociation from Olig2 promoter and results in upregulation of oligodendrogenesis [114]. The very importance of a spatially and temporally regulated BMP induced Smad activation for the fate of neurogenic precursor cells was shown in chick spinal cord. At embryonic day 5, in neuroepithelial progenitors, astrocyte marker expression was inhibited and at embryonic day

to commit to the astrocytic rather than the oligodendroglial lineage [101].

**3.1. Cell type decision, maturation and differentiation**

82 Trends in Cell Signaling Pathways in Neuronal Fate Decision

led by the interplay of Smad2,3 activity and PI3K activity [102].

induces astrocytic differentiation and GFAP expression [106-109].

6 it was promoted initiating a gliogenic period [115].

**and maintenance**

*3.1.1. Glial cells*

The commitment of cell fates in the nervous system is strongly dependent on Smad2,3 signalling cascade. However, there have been contradictory results on the role of Smad3 for neuronal precursor proliferation and their differentiation, indicating a complex Smad signal‐ ling network dependent on local, temporal and contextual characteristics.

Some reports show, that TGFβ, which activates Smad3 signalling, diminishes neurogenesis in hippocampus after its chronical increase [116], and in SVZ and DG after intracerebroventric‐ ular TGFβ1 infusion [117] and promotes neuronal differentiation from hippocampal and cortical progenitors [118]. An clear inhibitory function of Smad3 on neural precursor prolif‐ eration was shown in the developing spinal cord [119], where Smad3 also promotes differen‐ tiation and influences the fate of selected neurons. The antiproliferative role of Smad3 and Smad4 plays a role during early-postnatal differentiation of cerebellar neurons into postmitotic neurons, where TGFβ stimulation induces nuclear translocation of phosphorylated Smads and induction of cyclin-dependent kinase inhibitors p21, p27 and markers of neuronal maturity [120].

However, other reports show that TGFβ increased neurogenesis in DG after adrenalectomy [121] or in SVZ after stroke [122].

To clarify the relevance of Smad3 for adult neurogenesis Smad3 null mice were studied [123,124]. These mice show decreased neurogenesis in the DG and the SVZ and exhibit a thinner and more disorganized rostral migratory stream (RMS) of neuronal precursor cells (NPC). Using RMS NPCs migrate from SVZ to reach the olfactory bulb, where they differen‐ tiate into granular and periglomerular neurons. Though a decreased number of proliferating cells demonstrates the requirement of Smad3 for maintaining a proper cell division rate in SVZ, the neuronal fate is not altered by Smad3 deficiency [123].

For differentiation of mesencephalic progenitors into dopaminergic (DA) neurons a concerted interaction of Smad2,3 signalling and p38 MAPK-pathways by TGFβ receptor stimulation is necessary. Accordingly, treatment of ventral mesencephalic neural progenitors with TGFβ increased the number of tyrosine-hydroxylase (TH)-positive cells [125]. Though not required for the neurogenesis, the survival of midbrain dopaminergic neurons depends on function of the homeodomain interacting protein kinase 2 (HIPK2), which interacts with R-Smads to activate TGFβ responsive genes [96]. Loss of HIPK2 increases apoptosis in DA neurons during development. Nevertheless, apoptosis of specific neural progenitors during neural differen‐ tiation also involves Smad phosphorylation as shown for Smad1,5,8 [126,127].

In vitro, treatment of ventral mesencephalic cells with BMP5,6 and 7 also significantly increased the number of TH-positive neurons via Smad phosphorylation and nuclear trans‐ location [128], while the neurotrophin growth/differentiation factor 5 (GDF5) induced Smad pathway promotes neuronal but not dopaminergic differentiation [129]. Remarkably, the in vitro generation of functional dopaminergic substantia nigra neurons for transplantation requires a protocol which uses the temporarily and contextually distinct roles of Smad proteins: Firstly, the dual inhibition of Smad signalling in embryonic stem cells is required to allow induction of floor plate cell state which then passes over to midbrain floor plate cell state. Secondly, an activation of Smad signalling using TGFβ pathway is necessary to finally obtain dopaminergic neurons suitable to threat Parkinson´s like symptoms [130].

neuritic outgrowth is potentiated by BMP4/6 induced phosphorylation of Smad1,5,8 and their

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Otherwise, BMP2 inhibits neuritic outgrowth and differentiation of motor neuron-like NSC-34 cells by activation of Smad1,5,8-dependent signalling and subsequent Id genes activation, which are main targets of Smad signalling and which negatively regulate differentiation of

The axonal morphogenesis is strongly influenced by the TGFβ-regulated signalling protein Smad2. Endogenous Smad2 is constitutively activated and its phosphorylated form is nucle‐ arly localized in human and mouse hippocampal and cortical neurons [148,149] and in primary granule neurons of rat cerebellum [150]. In granule neurons Smad2 can form a physical complex with the endogenous transcriptional modulator SnoN, which is also nuclearly localized and strongly enhances axonal growth [151] and neuronal branching [152] by regulating a large number of neuronal genes [153]. SnoN ubiquitination and degradation is controlled by the nuclear ubiquitin ligase Cdh1-anaphase-promoting complex (Cdh1-APC), which physically interact with nuclear SnoN thus suppressing axonal growth. [151,154]. Obviously Smad2 binding to SnoN facilitates the ability of Cdh1-APC to control SnoN degradation. Consequently, if phosphorylation levels of Smad2 are reduced, SnoN levels increased and axonal growth is stimulated which allow therapeutic potential after brain injury [150]. The constitutive neuronal expression of axonal growth inhibitors e.g. Smad2, Smad3 [148] and Cdh1 [155] and the reduced expression of axon growth promoters (SnoN) in terminally differentiated neurons [150] generate an intrinsic axon growth inhibition control,

Axonal regeneration requires the reversal of an age-dependent loss of intrinsic axonal growth capability [156,157]. In dorsal root ganglion cells (DRG), which possess two branches of a initially unipolar axon, (a) a central branch containing in the spinal cord and (b) a peripheral branch innervating sensory targets, the axotomy of the peripheral branch requires function of transcription factor Smad1 for successful regeneration [158]. While in embryonic DRG neurons during the phase of active axon growth Smad1 RNA and protein were strongly expressed and found abundantly C-terminally phosphorylated in the nuclei (while Smad8 expression was low and Smad5 expression was concentrated to the periventricular zone), in adult DRG neurons Smad1 phosphorylation was diminished [159]. However, after axotomy, the induction and nuclear translocation of Smad1 precedes the onset of axonal extension and are maintained over longer time, demonstrating the importance of Smad1 for the perpetuation of the activated axonal growth program [158]. Interestingly, Smad1-dependent axonal growth program is an intrinsic feature of DRG neurons which functions independently of extracellular BMP. Until now, the detailed underlying mechanism for BMP-independent Smad1 phosphorylation is not completely elucidated. It is possible, that endogenous cytoplasmic kinases or intracellularly available BMP/BMP-receptor complexes might be involved. The ligand-independent C-

various cells including neurogenic precursors and motor-neuron precursors [147].

subsequent inhibition of MEK in chicken neurons [146].

allowing a balance between steady state and neuronal plasticity.

*3.2.2. Subtle control of axonal morphogenesis*

*3.2.3. Axonal regeneration*

In the developing cerebellum, Smad1 is expressed in the external germinal layer and Smad5 is synthesized in newly differentiated granule neurons. In the granule precursor cells, Smad5 overexpression is sufficient to initiate differentiation [131]. In mice lacking Smad4, a decreased number of cerebellar Purkinje cells and parvalbumin-positive interneurons [132] is found while no alterations of proliferation of neuronal precursor cells were detectable. Loss of Smad4 also reduces differentiation of noradrenergic neurons [133].

Basal forebrain cholinergic neurons are essentially involved in the organisation of cortical brain structures, learning and memory. Smad1-5 are expressed in basal forebrain from embryonic day 14 to the adult age [134]. An intact canonical TGFβ/Smad2,3 cascade is important for the function of cholinergic neurons. Smad signalling mediates rapid inhibition of calcium influx in cholinergic basal forebrain neurons [135]. The sleep/wake circadian rhythm controlled by cholinergic neurons of the pedunculopontine nucleus (PPT) is also dependent on phosphor‐ ylation levels of Smad2 and Smad3. Overactivation of Smad2,3 signalling resulting in increased nuclear translocation is linked to sleep/wake circadian rhythm amplitude deficits [136]. BMP9 driven Smad1,5 phosphorylation and their formation of complexes with Smad4 are involved in the induction of the cholinergic phenotype in the basal forebrain [134,137]. Moreover, only in postmitotic cholinergic neurons, this pathway induces NGF expression, an autocrine/ paracrine cholinergic trophic factor, which stimulates ACh production [138]. Accordingly, it is of therapeutic relevance for treatment of Alzheimer´s disease, that BMP9/Smad1,5 signalling can prevent lesion-evoked impairment of the cholinergic septo-hippocampal neurons in adult mice [139]. The basal forebrain cholinergic neuron specific gene expression pattern is also basically linked to an interaction of Smad and Sizn1 (Smad-interacting zinc finger protein), a factor which causes mental retardation if mutated [140,141].

#### **3.2. Axon and synapse formation**

Induction of axons and orienting of axon responses are controlled by several Smad molecules of both the BMP and the TGFβ/activin pathways in a timely and spatially organized manner. Several different neuronal/neural cell types use individual aspects of the Smad machinery.

#### *3.2.1. Basic role of Smads for neuritic outgrowth*

In PC12 cells, which are initially derived from neural crest cells and are used as model system for neuronal differentiation, BMP2 induces neurite outgrowth [142] by activation of TAK1/p38 kinase signalling pathway [143,144], which is in turn tightly controlled by the simultaneous, also BMP2-induced expression of the inhibitory Smad6 and Smad7 [142] performed via activation of Smad1,5,8 pathway. The inhibitory Smads are considered to inversely inhibit BMP signalling in a concerted action by repressing Smad1,5,8, mediating BMP receptor degradation by Smurf1 [16,145], and by physical interaction with TAK1-binding protein, which finally reduces the p38-mediated neuritic outgrowth [143,144]. Neurotrophin 3 induced neuritic outgrowth is potentiated by BMP4/6 induced phosphorylation of Smad1,5,8 and their subsequent inhibition of MEK in chicken neurons [146].

Otherwise, BMP2 inhibits neuritic outgrowth and differentiation of motor neuron-like NSC-34 cells by activation of Smad1,5,8-dependent signalling and subsequent Id genes activation, which are main targets of Smad signalling and which negatively regulate differentiation of various cells including neurogenic precursors and motor-neuron precursors [147].

#### *3.2.2. Subtle control of axonal morphogenesis*

Secondly, an activation of Smad signalling using TGFβ pathway is necessary to finally obtain

In the developing cerebellum, Smad1 is expressed in the external germinal layer and Smad5 is synthesized in newly differentiated granule neurons. In the granule precursor cells, Smad5 overexpression is sufficient to initiate differentiation [131]. In mice lacking Smad4, a decreased number of cerebellar Purkinje cells and parvalbumin-positive interneurons [132] is found while no alterations of proliferation of neuronal precursor cells were detectable. Loss of Smad4

Basal forebrain cholinergic neurons are essentially involved in the organisation of cortical brain structures, learning and memory. Smad1-5 are expressed in basal forebrain from embryonic day 14 to the adult age [134]. An intact canonical TGFβ/Smad2,3 cascade is important for the function of cholinergic neurons. Smad signalling mediates rapid inhibition of calcium influx in cholinergic basal forebrain neurons [135]. The sleep/wake circadian rhythm controlled by cholinergic neurons of the pedunculopontine nucleus (PPT) is also dependent on phosphor‐ ylation levels of Smad2 and Smad3. Overactivation of Smad2,3 signalling resulting in increased nuclear translocation is linked to sleep/wake circadian rhythm amplitude deficits [136]. BMP9 driven Smad1,5 phosphorylation and their formation of complexes with Smad4 are involved in the induction of the cholinergic phenotype in the basal forebrain [134,137]. Moreover, only in postmitotic cholinergic neurons, this pathway induces NGF expression, an autocrine/ paracrine cholinergic trophic factor, which stimulates ACh production [138]. Accordingly, it is of therapeutic relevance for treatment of Alzheimer´s disease, that BMP9/Smad1,5 signalling can prevent lesion-evoked impairment of the cholinergic septo-hippocampal neurons in adult mice [139]. The basal forebrain cholinergic neuron specific gene expression pattern is also basically linked to an interaction of Smad and Sizn1 (Smad-interacting zinc finger protein), a

Induction of axons and orienting of axon responses are controlled by several Smad molecules of both the BMP and the TGFβ/activin pathways in a timely and spatially organized manner. Several different neuronal/neural cell types use individual aspects of the Smad machinery.

In PC12 cells, which are initially derived from neural crest cells and are used as model system for neuronal differentiation, BMP2 induces neurite outgrowth [142] by activation of TAK1/p38 kinase signalling pathway [143,144], which is in turn tightly controlled by the simultaneous, also BMP2-induced expression of the inhibitory Smad6 and Smad7 [142] performed via activation of Smad1,5,8 pathway. The inhibitory Smads are considered to inversely inhibit BMP signalling in a concerted action by repressing Smad1,5,8, mediating BMP receptor degradation by Smurf1 [16,145], and by physical interaction with TAK1-binding protein, which finally reduces the p38-mediated neuritic outgrowth [143,144]. Neurotrophin 3 induced

dopaminergic neurons suitable to threat Parkinson´s like symptoms [130].

also reduces differentiation of noradrenergic neurons [133].

84 Trends in Cell Signaling Pathways in Neuronal Fate Decision

factor which causes mental retardation if mutated [140,141].

**3.2. Axon and synapse formation**

*3.2.1. Basic role of Smads for neuritic outgrowth*

The axonal morphogenesis is strongly influenced by the TGFβ-regulated signalling protein Smad2. Endogenous Smad2 is constitutively activated and its phosphorylated form is nucle‐ arly localized in human and mouse hippocampal and cortical neurons [148,149] and in primary granule neurons of rat cerebellum [150]. In granule neurons Smad2 can form a physical complex with the endogenous transcriptional modulator SnoN, which is also nuclearly localized and strongly enhances axonal growth [151] and neuronal branching [152] by regulating a large number of neuronal genes [153]. SnoN ubiquitination and degradation is controlled by the nuclear ubiquitin ligase Cdh1-anaphase-promoting complex (Cdh1-APC), which physically interact with nuclear SnoN thus suppressing axonal growth. [151,154]. Obviously Smad2 binding to SnoN facilitates the ability of Cdh1-APC to control SnoN degradation. Consequently, if phosphorylation levels of Smad2 are reduced, SnoN levels increased and axonal growth is stimulated which allow therapeutic potential after brain injury [150]. The constitutive neuronal expression of axonal growth inhibitors e.g. Smad2, Smad3 [148] and Cdh1 [155] and the reduced expression of axon growth promoters (SnoN) in terminally differentiated neurons [150] generate an intrinsic axon growth inhibition control, allowing a balance between steady state and neuronal plasticity.

#### *3.2.3. Axonal regeneration*

Axonal regeneration requires the reversal of an age-dependent loss of intrinsic axonal growth capability [156,157]. In dorsal root ganglion cells (DRG), which possess two branches of a initially unipolar axon, (a) a central branch containing in the spinal cord and (b) a peripheral branch innervating sensory targets, the axotomy of the peripheral branch requires function of transcription factor Smad1 for successful regeneration [158]. While in embryonic DRG neurons during the phase of active axon growth Smad1 RNA and protein were strongly expressed and found abundantly C-terminally phosphorylated in the nuclei (while Smad8 expression was low and Smad5 expression was concentrated to the periventricular zone), in adult DRG neurons Smad1 phosphorylation was diminished [159]. However, after axotomy, the induction and nuclear translocation of Smad1 precedes the onset of axonal extension and are maintained over longer time, demonstrating the importance of Smad1 for the perpetuation of the activated axonal growth program [158]. Interestingly, Smad1-dependent axonal growth program is an intrinsic feature of DRG neurons which functions independently of extracellular BMP. Until now, the detailed underlying mechanism for BMP-independent Smad1 phosphorylation is not completely elucidated. It is possible, that endogenous cytoplasmic kinases or intracellularly available BMP/BMP-receptor complexes might be involved. The ligand-independent C- terminal phosphorylation of the SSXS domain of Smad2 and Smad3 was already reported [160] suggesting a possible extracellular ligand-independent universal strategy. Probably, Smad1 signalling is also involved in the axonogenesis of many other classes of neurons, e.g. Purkinje cells, retinal ganglionic, olfactory and motor neurons which were already found positively labelled for pSmad1 immunoreactivity [159]. Due to the possible linker phosphorylation of Smad1 by several kinases (e.g. MAPK, GSK, CDK), the molecule might serve as an integrator of various pathways relevant for axon growth and regeneration [14,23-26]. However, for clinical and therapeutic relevance, the transduction of adeno-associated vector encoded BMP4 allows stimulation of Smad1 phosphorylation and activation of axonal growth independent of axotomy and also promotes sensory axon regeneration after axotomy [159]. At least partly BMP effects are dependent on its binding to repulsive guidance molecules [161].

is involved in the acquisition of positional identity markers during trigeminal ganglia devel‐ opment and selectively works in ophthalmic and maxillary but not in mandibular axons obviously due to the failure of BDNF in the mandibular target field [166]. The underlying mechanism suggests to re-evaluate the role and extent of Smad based transcriptional regula‐ tion. (1) Nearly all cellular surfaces/membranes, including dendrites, axons, growth cones and spines might receive information which could be integrated by Smad signalling. (2) The retrograde transport of other ligands of the TGFβ group was already reported, e.g. TGFβ2 in normal and injured motor neurons [168], implicating the question whether axonal Smad signalling is involved in injury-response. (3) The mechanism might work with all those extracellular and also intracellular factors that affect Smad translation, and might at least partly explain neurodegenerative disorders, which are characterized by disturbed axonal transport

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The specific structure of axons and the axonal transport of Smad signals [166] as well as simple translocation of receptor phosphorylated Smads from cytoplasmic receptor site into the nucleus require intact microtubule network. Phosphorylated Smad2 is transported using microtubules by support of kinesin-1 and kinesinATPase activity [169] and also requires dynein light chain km23-1 [6], whereas pSmad3 transport was dependent on km23-2 function [170]. Additionally retrograde axonal transport of phosphorylated Smads, which is necessary for transition of axonal synaptic BMP-like ligand mediated phosphorylation of Smads in Drosophila, also depends on an intact function of p150Glued protein. P150Glued is a compo‐ nent of the dynactin complex, which is necessarily involved in synapse assembly and stability [171]. However, a p150Glued knockdown induced disruption of synaptic homeostasis can be rescued by activation of Smad signalling at the soma [172,173], confirming the important role

and/or neurotrophin deficiency at neuronal terminals

of nuclear phosphorylated Smad to synaptic homeostasis.

Smad signalling pathways [174].

*3.2.5. Astrocytic and oligodenrocytic Smad signalling influences neuronal axonal growth*

nucleus [177] and improve axon regeneration after spinal cord injury.

Astrocytes surrounding synapses are also target of neuronal activity. Glial metabotropic glutamate 2/3 receptor activation by neuronally released glutamate induces astrocytic TGFβ1 secretion, leading to GFAP gene activation and astrocyte differentiation involving astrocytic

Chondroitin sulphate proteoglycans (CSPG), mainly synthesized by astrocytes, can inhibit axonal growth and regeneration. After traumatic brain injury or disruptions of the blood brain barrier, mature or fibrinogen-coupled latent TGFβ is released into the CNS and increases the CSPG expression by a Smad signalling manner in astrocytes [175,176]. Both astrocytic Smad2 and Smad3 expression and phosphorylation leads to inhibition of neuronal outgrowth and is required for astrocytic neurocan synthesis, whereas phosphacan only requires Smad2 [175,176]. Reduced Smad3 levels selectively reduced induction of chondroitin-4- sulphotrans‐ ferase 1 and the amount of 4-sulfated CSPGs secreted by astrocytes and also promoted axonal growth of neurons which were fed on these astrocytes [175]. Taxol treatment of astrocytes can reduce CSPG expression by interfering with kinesin-1-dependent pSmad transport into the

While Smad1 positively supports axonal growth, the inhibitory Smad6 can block axon outgrowth, as shown for post-mitotic dI1 axons of the spinal cord [82] and therefore exert roles in spatially limiting the influence of BMP signalling on neurons.

In Drosophila, Smad2 is involved in motor neuron axon guidance, as null mutants of Smad2 exhibit axon guidance defects [162]. It is suggested, that a chemoattractant signal of TGFβ superfamily members provided by muscle cells might guide motor axons to their appropriate innervation sites.

#### *3.2.4. R-Smads and Co-Smads exert neuronal plasticity*

The development of peripheral tissues is connected to axonal growth of peripheral nerves, linking sensory information to the spinal cord via neuronal cell bodies localized in ganglia. After initial axon extension for trigeminal sensory neurons in mice, a Smad4-dependent retrograde signalling from developing face to the neuronal nuclei was found, which control the expression of genes in neurons of the trigeminal ganglion in dependence on their position. Extrinsic signals released from developing craniofacial tissue, e.g. BMP4, activate phosphor‐ ylation of Smad1,5,8 at the axon terminals and selectively retrogradely signals this information to selected trigeminal neuronal cell bodies thus inducing spacially patterned expression of further transcription factors along the dorso-ventral axis of the trigeminal ganglion [163]. A comparable retrograde mechanism based on dSmad2 was found in R7 axons in the Drosophila visual system, where activin, secreted from the R7 cells growth cone in an autocrine manner, activates its receptor and initiates intracellular Smad2 phosphorylation. The pSmad2 com‐ plexes to the nuclear import adaptor Importin-α3, shuttles to the nucleus and reduces growth cone motility and synaptogenesis via transcriptional regulation of several target genes [164]. Already in Drosophila, a retrograde Smad-based signalling of the BMP homolog Gbb regulates synaptic growth in presynaptic motor neurons [165]. The underlying mechanism for the retrograde Smad-based signalling was recently examined for the BMP4 signalling system. Ji and colleagues (2012) discovered that endosomes carrying the BMP-signalling complex containing phosphorylated Smad1,5,8, are essentially linked to a BDNF-induced axonal or growth cone translation of Smad proteins to retrogradely control transcription in trigeminal neurons [166,167]. This `two-target-derived signalling´ integrates neurotrophin and BMP signals on the level of Smad proteins, which are axonally translated on demand. This process is involved in the acquisition of positional identity markers during trigeminal ganglia devel‐ opment and selectively works in ophthalmic and maxillary but not in mandibular axons obviously due to the failure of BDNF in the mandibular target field [166]. The underlying mechanism suggests to re-evaluate the role and extent of Smad based transcriptional regula‐ tion. (1) Nearly all cellular surfaces/membranes, including dendrites, axons, growth cones and spines might receive information which could be integrated by Smad signalling. (2) The retrograde transport of other ligands of the TGFβ group was already reported, e.g. TGFβ2 in normal and injured motor neurons [168], implicating the question whether axonal Smad signalling is involved in injury-response. (3) The mechanism might work with all those extracellular and also intracellular factors that affect Smad translation, and might at least partly explain neurodegenerative disorders, which are characterized by disturbed axonal transport and/or neurotrophin deficiency at neuronal terminals

terminal phosphorylation of the SSXS domain of Smad2 and Smad3 was already reported [160] suggesting a possible extracellular ligand-independent universal strategy. Probably, Smad1 signalling is also involved in the axonogenesis of many other classes of neurons, e.g. Purkinje cells, retinal ganglionic, olfactory and motor neurons which were already found positively labelled for pSmad1 immunoreactivity [159]. Due to the possible linker phosphorylation of Smad1 by several kinases (e.g. MAPK, GSK, CDK), the molecule might serve as an integrator of various pathways relevant for axon growth and regeneration [14,23-26]. However, for clinical and therapeutic relevance, the transduction of adeno-associated vector encoded BMP4 allows stimulation of Smad1 phosphorylation and activation of axonal growth independent of axotomy and also promotes sensory axon regeneration after axotomy [159]. At least partly

While Smad1 positively supports axonal growth, the inhibitory Smad6 can block axon outgrowth, as shown for post-mitotic dI1 axons of the spinal cord [82] and therefore exert roles

In Drosophila, Smad2 is involved in motor neuron axon guidance, as null mutants of Smad2 exhibit axon guidance defects [162]. It is suggested, that a chemoattractant signal of TGFβ superfamily members provided by muscle cells might guide motor axons to their appropriate

The development of peripheral tissues is connected to axonal growth of peripheral nerves, linking sensory information to the spinal cord via neuronal cell bodies localized in ganglia. After initial axon extension for trigeminal sensory neurons in mice, a Smad4-dependent retrograde signalling from developing face to the neuronal nuclei was found, which control the expression of genes in neurons of the trigeminal ganglion in dependence on their position. Extrinsic signals released from developing craniofacial tissue, e.g. BMP4, activate phosphor‐ ylation of Smad1,5,8 at the axon terminals and selectively retrogradely signals this information to selected trigeminal neuronal cell bodies thus inducing spacially patterned expression of further transcription factors along the dorso-ventral axis of the trigeminal ganglion [163]. A comparable retrograde mechanism based on dSmad2 was found in R7 axons in the Drosophila visual system, where activin, secreted from the R7 cells growth cone in an autocrine manner, activates its receptor and initiates intracellular Smad2 phosphorylation. The pSmad2 com‐ plexes to the nuclear import adaptor Importin-α3, shuttles to the nucleus and reduces growth cone motility and synaptogenesis via transcriptional regulation of several target genes [164]. Already in Drosophila, a retrograde Smad-based signalling of the BMP homolog Gbb regulates synaptic growth in presynaptic motor neurons [165]. The underlying mechanism for the retrograde Smad-based signalling was recently examined for the BMP4 signalling system. Ji and colleagues (2012) discovered that endosomes carrying the BMP-signalling complex containing phosphorylated Smad1,5,8, are essentially linked to a BDNF-induced axonal or growth cone translation of Smad proteins to retrogradely control transcription in trigeminal neurons [166,167]. This `two-target-derived signalling´ integrates neurotrophin and BMP signals on the level of Smad proteins, which are axonally translated on demand. This process

BMP effects are dependent on its binding to repulsive guidance molecules [161].

in spatially limiting the influence of BMP signalling on neurons.

*3.2.4. R-Smads and Co-Smads exert neuronal plasticity*

86 Trends in Cell Signaling Pathways in Neuronal Fate Decision

innervation sites.

The specific structure of axons and the axonal transport of Smad signals [166] as well as simple translocation of receptor phosphorylated Smads from cytoplasmic receptor site into the nucleus require intact microtubule network. Phosphorylated Smad2 is transported using microtubules by support of kinesin-1 and kinesinATPase activity [169] and also requires dynein light chain km23-1 [6], whereas pSmad3 transport was dependent on km23-2 function [170]. Additionally retrograde axonal transport of phosphorylated Smads, which is necessary for transition of axonal synaptic BMP-like ligand mediated phosphorylation of Smads in Drosophila, also depends on an intact function of p150Glued protein. P150Glued is a compo‐ nent of the dynactin complex, which is necessarily involved in synapse assembly and stability [171]. However, a p150Glued knockdown induced disruption of synaptic homeostasis can be rescued by activation of Smad signalling at the soma [172,173], confirming the important role of nuclear phosphorylated Smad to synaptic homeostasis.

#### *3.2.5. Astrocytic and oligodenrocytic Smad signalling influences neuronal axonal growth*

Astrocytes surrounding synapses are also target of neuronal activity. Glial metabotropic glutamate 2/3 receptor activation by neuronally released glutamate induces astrocytic TGFβ1 secretion, leading to GFAP gene activation and astrocyte differentiation involving astrocytic Smad signalling pathways [174].

Chondroitin sulphate proteoglycans (CSPG), mainly synthesized by astrocytes, can inhibit axonal growth and regeneration. After traumatic brain injury or disruptions of the blood brain barrier, mature or fibrinogen-coupled latent TGFβ is released into the CNS and increases the CSPG expression by a Smad signalling manner in astrocytes [175,176]. Both astrocytic Smad2 and Smad3 expression and phosphorylation leads to inhibition of neuronal outgrowth and is required for astrocytic neurocan synthesis, whereas phosphacan only requires Smad2 [175,176]. Reduced Smad3 levels selectively reduced induction of chondroitin-4- sulphotrans‐ ferase 1 and the amount of 4-sulfated CSPGs secreted by astrocytes and also promoted axonal growth of neurons which were fed on these astrocytes [175]. Taxol treatment of astrocytes can reduce CSPG expression by interfering with kinesin-1-dependent pSmad transport into the nucleus [177] and improve axon regeneration after spinal cord injury.

The Smad-interacting-protein 1 (Sip1) is an essential modulator for CNS myelination. In oligodendrocytes, it functions in a dual-mode manner by repressing the differentiation inhibitory signals of the BMP-receptor activated Smad1,5,8 activity [178] and activating oligodendrocytes-promoting factors, thus controlling proper myelination in the CNS [38].

postsynaptically at the NMJs co-regulates the phosphorylation of neuronal Mad levels by

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These data indicate, that both activin and BMP directed R-Smad signalling controls synaptic

Several neurological diseases are characterized by disturbed cellular or subcellular Smad localization and show artificial Smad sequestration or deposition. Especially in neurodege‐ nerative and motor neuron diseases a disruption of Smad controlled transcriptional machinery

Neurodegenerative diseases are characterised by selective and progressive loss of specific populations of neurones. Four disease-classes are proposed based on four major affected proteins, tau, βA4-amyloid (βA4), α-synuclein and TDP-43, and therefore, neurodegenerative diseases can roughly be classified into four main groups: amyloidosis, tauopathies, synuclei‐ nopathies and TDP-43 proteinopathies (for review see [188]). Recent data indicate, that in all four disease groups Smad proteins are essentially involved in the disease progression, finally

Alzheimer´s disease (AD), representing a mixture of both amyloidosis and tauopathy, is a severe neurodegenerative disorder and the most common cause of dementia in the elderly. Typical clinical symptoms are memory loss, disturbed activities of daily life and deficiency of social competence. Common morphological correlates to the clinical features are extracellular βA4-plaque depositions, intraneuronal tau pathology, neuronal cell death, and cell cycle

In AD, we identified a strong disturbance of the normal constitutive nuclear localization of phosphorylated Smad2 and Smad3 in hippocampal and cortical neurons [148], which subse‐ quently was confirmed by others [190,191]. In AD brain, a strong colocalization of pSmad2,3 with intracellular neurofibrillary tangles (NFTs) in neurons and with βA4-amyloid plaques in addition to a sequestration of pSmad2,3 in cytoplasmic granular vesicles is detectable [148]. Additionally a significant reduction of Smad2,3,4, which are involved in activation of cell cycle proteins was described. Smad4 directly controls cyclin-dependent kinase 4 (CDK4) expression in neuronal cells and is involved in cell cycle activation of neurons in AD brain [192]. It is quite possible, that the recently identified TGFβ1-induced antiapoptotic factor (TIAF1), which can bind and block Smad4-dependent promoter activation [193], participates in the cytoplasmic Smad sequestration in AD neurons and suppresses Smad-regulated promoter activation. Environmental stress or TGFβ1, can induce TIAF aggregation, which in turn removes soluble

Smad4, induces apoptosis and activates βA4 generation and its aggregation [193].

function at NMJ and affects the development of synaptic homeostasis.

inhibiting postsynaptic Gbb secretion [187].

was reported.

**4.1. Alzheimer´s disease**

activation [189].

**4. Role of Smads for neurological disorders**

indicating a remarkable commonness of these diseases.

#### **3.3. Smads contribute to synapse formation and synaptic transmission processes**

Synapse formation and remodelling are results of intrinsic programs and environmental insults. Synapses are characterized by close cell-to-cell communications, which also include synaptic transmission and signalling processes mediated by growth factors e.g. members of the TGFβ superfamily. In spinster a mutation of the inhibitory Smad Dad (Daughters against dpp) causes synaptic overgrowth [179]. A well investigated synaptic system is the neuromus‐ cular junction (NMJ) of Drosophila, showing the involvement of Smad proteins in synapse function both in pre- and in postsynaptic cells. The release of muscular BMP4-type ligand Gbb, which is required for intact function of a large part of postsynaptic muscles [165], allows its binding to a presynaptic receptor consisting of one type I and one type II receptor, Sax or Tkv and Wit, respectively [180,181], and the subsequent phosphorylation of the R-Smad, Mothers against dpp (Mad) [165]. Activated Mad is responsible for presynaptic nuclear propagation of the postsynaptic muscular BMP signals and finally for regulating synapse growth, morphol‐ ogy and function of motor-neurons at least partly by altering transcription [165,180,182]. The retrograde transport of Mad utilizes microtubule motor proteins, e.g. dynein complexes, and is sensitive to disruption of dynein motor function [165]. The Drosophila Co-Smad/Smad4 homolog Medea (Med) is also necessary for presynaptic BMP signalling cascade and essential for synaptic growth [183]. Both, Med and Mad mutants, exhibit defects in neurotransmitter release and synaptic ultrastructure [165,183]. It is suggested that an equilibrium exist between the binding of presynaptic phosphorylated Mad to Med and the binding of Med to the RING finger E3 ubiquitin ligase Hiw [184], which regulate the synaptic growth in NMJ [183].

Additionally to the retrograde neuronal Mad/Med-mediated Gbb signalling, anterogradely released TGFβ type ligands activate Mad phosphorylation at the postsynaptic density (PSD) zone of postsynaptic muscles and NMJ depolarization supports Mad nuclear transition in the muscles [185]. In summary, a mutual information flow of neuron and muscle on each phys‐ iological state and the synaptic cleft situation is essentially controlled by specific R- and Co-Smad signalling in both cellular systems.

Importantly, the activation of Mad phosphorylation and nuclear translocation by the BMP ligand Gbb directs the expression of synaptic homeostasis independently of synaptic growth control in Drosophila [173]. However, retrograde transport of Mad, which was phosphorylated at the periphery of the axon terminal due to synaptic Gbb binding, mediates synaptic homeo‐ stasis. Remarkably, Gbb induced phosphorylation of Mad at the soma site of the neuron compensate for a disturbed retrograde axonal pMad transport completely [173]. The release of muscular Gbb into the synaptic cleft at the periphery to activate neuronal retrograde pMad transport, can be regulated by activin, which controls the muscular Gbb synthesis by recruiting the Drosophila dSmad2 [186]. Cdc42-interacting protein 4 (dCIP4) which is also localized postsynaptically at the NMJs co-regulates the phosphorylation of neuronal Mad levels by inhibiting postsynaptic Gbb secretion [187].

These data indicate, that both activin and BMP directed R-Smad signalling controls synaptic function at NMJ and affects the development of synaptic homeostasis.
