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

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 was reported.

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 indicating a remarkable commonness of these diseases.

#### **4.1. Alzheimer´s disease**

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].

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-

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

Smad signalling in both cellular systems.

88 Trends in Cell Signaling Pathways in Neuronal Fate Decision

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

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 activation [189].

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].

A reason for the intraneuronal dislocation of Smads could be due to the disturbed nucleocy‐ toplasmic transport in hippocampal AD neurons [194,195]. The inner nuclear membrane comprises integral proteins, e.g. MAN1, which regulate Smad phosphorylation and nuclear translocation [196], while the outer nuclear membrane is directly continuous with the rough endoplasmic reticulum. Both membranes are separated by nuclear core complexes, which also control nucleocytoplasmic shuttling of Smad2 [197]. Altered nuclear membrane integrity [195] might provoke misrouting of phosphorylated Smads into the associated ER compartment, indicated by the co-localization of phosphorylated Smad2 granules with a luminal marker protein of the ER, protein disulphide isomerase [148,198]. ER containing Smad2 could be secreted and thus explain the extracellular association of pSmad2,3 with amyloid plaques [148,190]. Alternatively, pSmad2 could interact in early endosomes with βA4-peptides which are accumulating in the early stages of AD [199], and there also meet BACE, the β-secretase, generating β-amyloid peptides [200], activated TGFβ receptors type I and II as well as SARA, a Smad anchor for receptor activation [201-203]. It is also possible, that early endosomes can fuse to the ER thus allowing a direct route of Smad proteins to the lumen of the ER [204,205].

Clearance of βA4 peptide was reported by microglia cells [220]. TGFβ released by neurons or astrocytes stimulates βA4 peptide uptake through Smad3-dependent increased scavenger receptor SR-A expression and increased phagocytosis [221]. Though the microglial capacity to remove βA4-plaques is limited [222], peripheral phagocytes, migrating into the brain if their Smad2,3 signalling is blocked at the expense of Smad1,5,8 phosphorylation, might perform

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In other tauopathies, e.g. Pick disease, progressive supranuclear palsy, and corticobasal degeneration, cytoplasmic phospho-tau bound pSmad2,3 was also identified, though reduced nuclear pSmad levels were not found [224] suggesting a different impact on Smad signalling

Parkinson´s disease (PD) is a progressive neurodegenerative disorder which is characterized by dopaminergic neurodegeneration in the substantia nigra (SN) pars compacta. The closely associated loss of dopaminergic inputs into the striatum results in failure of motor programmes (e.g. voluntary movements) and initiates a striatal plastic change [225,226]. The gradual loss of dopaminergic neurons is linked to an increase of dopamine turnover [227,228]. α-synuclein (α-syn) is the major protein component of Lewy bodies, a hallmark of PD. It can aggregate to

It was shown, that the TGFβ3-Smad-HIPK2 pathway is important for the survival of dopami‐ nergic neurons during development [96,230]. However, recently, a very specific and important role of Smad3 for the nigrostriatal system was explored in a Smad3 null mouse model representing an exciting model of PD [231]. These animals show a reduced number of dopaminergic neurons in the rostral SN, resulting from a postnatal neurodegenerative process. The selective pro-survival effect of Smad3 for SN dopaminergic neurons was emphasized by stereological quantifications showing no alterations of the striatal neuronal number. In Smad3 null mice a diminished trophic support provided by Erk1/2 signalling and a reduced astrocytic support to nigral dopaminergic neurons was detected [231]. Though striatal dopamine levels were not changed in Smad3 null mice, a strong increase of DA metabolism, including elevated monoamine-oxidase (MAO) levels were identified, resembling PD findings [232]. Finally, increased expression and aggregation of α-syn in neurites and cell bodies of several telence‐ phalic, mesencephalic and rombencephalic brain regions and in fibres of the primary and secondary motor cortex were found [231]. Two possible functional Smad3-based circuits are suggested. Either Smad3 might directly regulate the α-syn transcription, which further allows modulating DA metabolism, or the Smad3 deficiency caused elevated DA catabolism gener‐ ates oxidative stress which allow toxic aggregation of α-syn. The data qualify Smad3 as a possible target for PD therapy. Interestingly, the currently pursued target to re-implant dopaminergic neurons is also based on modification of Smad signalling during their in vitro

this task successfully [223].

**4.2. Parkinson´s disease**

establishment [130,233].

in these diseases compared to AD.

form toxic oligomers and fibrillar structures [226,229].

However, activation of cell cycle is a hallmark of AD and (1) probably supports hyperphos‐ phorylation of tau protein which leads to generation of neurofibrillary tangles and (2) increases neuronal apoptosis by phosphorylation of retinoblastoma protein and activation of E2F based apoptotic impacts (for review see [206]. Hyperphosphorylation of tau affects the neuronal Smad2,3 localization, diminishes its nuclear concentration and thus impedes with transcrip‐ tional Smad functions as shown after βA4 treatment of primary neurons [207]. NFT formation in AD brain disturbs common intra-neuronal transport mechanisms [208]. This disturbance could also interfere with retrograde Smad signalling which could be suggested for hippocam‐ pal neurons but so far has only demonstrated for Drosophila motor neurons [173]. Moreover, we also suggest, that the principle of the two-target-derived signalling integrating neurotro‐ phin (BDNF) and Smad pathway found in rat trigeminal neurons [166,167] might also play a role for AD progression, because BDNF is deficient in entorhinal cortex and hippocampus in AD [209,210]. Finally, a disturbed nuclear Smad localization might influence competence of neurons to express synaptic homeostasis and plasticity, both processes are well investigated for Drosophila motor neurons.

Pathogenic reduction of neuronal Smads can also support activation of neuronal cell cycle, resulting in neuronal apoptosis [211] e.g. by repression of cyclin-dependent kinase inhibitors (CDIs) p15Ink4B, p16Ink4A, or p21Cip, which expression is controlled by TGFβ/Smad signalling [212-214]. Though the increased levels of TGFβ1 in AD [215,216] does not seem to be able to compensate for the compromised canonical neuronal Smad pathway [148,190], astrocytes were shown responsive to this growth factor, which induces amyloid precursor protein (APP) expression in cooperation with Sp1 [217] and CTCF [218]. Altered APP cleavage by the concerted action of α-, β- and γ-secretases is a main aspect of AD pathology and results in the generation of the pathogenetic βA4 peptide 1-42, which is neurotoxic and leads to βA4-amyloid plaques. Whether βA4 peptide 1-42 can further suppress neuronal TGFβ1/Smad2,3 signalling by activation of the inhibitory Smad7 in neurons as already shown for human monocytes cannot be excluded [219].

Clearance of βA4 peptide was reported by microglia cells [220]. TGFβ released by neurons or astrocytes stimulates βA4 peptide uptake through Smad3-dependent increased scavenger receptor SR-A expression and increased phagocytosis [221]. Though the microglial capacity to remove βA4-plaques is limited [222], peripheral phagocytes, migrating into the brain if their Smad2,3 signalling is blocked at the expense of Smad1,5,8 phosphorylation, might perform this task successfully [223].

In other tauopathies, e.g. Pick disease, progressive supranuclear palsy, and corticobasal degeneration, cytoplasmic phospho-tau bound pSmad2,3 was also identified, though reduced nuclear pSmad levels were not found [224] suggesting a different impact on Smad signalling in these diseases compared to AD.

#### **4.2. Parkinson´s disease**

A reason for the intraneuronal dislocation of Smads could be due to the disturbed nucleocy‐ toplasmic transport in hippocampal AD neurons [194,195]. The inner nuclear membrane comprises integral proteins, e.g. MAN1, which regulate Smad phosphorylation and nuclear translocation [196], while the outer nuclear membrane is directly continuous with the rough endoplasmic reticulum. Both membranes are separated by nuclear core complexes, which also control nucleocytoplasmic shuttling of Smad2 [197]. Altered nuclear membrane integrity [195] might provoke misrouting of phosphorylated Smads into the associated ER compartment, indicated by the co-localization of phosphorylated Smad2 granules with a luminal marker protein of the ER, protein disulphide isomerase [148,198]. ER containing Smad2 could be secreted and thus explain the extracellular association of pSmad2,3 with amyloid plaques [148,190]. Alternatively, pSmad2 could interact in early endosomes with βA4-peptides which are accumulating in the early stages of AD [199], and there also meet BACE, the β-secretase, generating β-amyloid peptides [200], activated TGFβ receptors type I and II as well as SARA, a Smad anchor for receptor activation [201-203]. It is also possible, that early endosomes can fuse to the ER thus allowing a direct route of Smad proteins to the lumen of the ER [204,205].

However, activation of cell cycle is a hallmark of AD and (1) probably supports hyperphos‐ phorylation of tau protein which leads to generation of neurofibrillary tangles and (2) increases neuronal apoptosis by phosphorylation of retinoblastoma protein and activation of E2F based apoptotic impacts (for review see [206]. Hyperphosphorylation of tau affects the neuronal Smad2,3 localization, diminishes its nuclear concentration and thus impedes with transcrip‐ tional Smad functions as shown after βA4 treatment of primary neurons [207]. NFT formation in AD brain disturbs common intra-neuronal transport mechanisms [208]. This disturbance could also interfere with retrograde Smad signalling which could be suggested for hippocam‐ pal neurons but so far has only demonstrated for Drosophila motor neurons [173]. Moreover, we also suggest, that the principle of the two-target-derived signalling integrating neurotro‐ phin (BDNF) and Smad pathway found in rat trigeminal neurons [166,167] might also play a role for AD progression, because BDNF is deficient in entorhinal cortex and hippocampus in AD [209,210]. Finally, a disturbed nuclear Smad localization might influence competence of neurons to express synaptic homeostasis and plasticity, both processes are well investigated

Pathogenic reduction of neuronal Smads can also support activation of neuronal cell cycle, resulting in neuronal apoptosis [211] e.g. by repression of cyclin-dependent kinase inhibitors (CDIs) p15Ink4B, p16Ink4A, or p21Cip, which expression is controlled by TGFβ/Smad signalling [212-214]. Though the increased levels of TGFβ1 in AD [215,216] does not seem to be able to compensate for the compromised canonical neuronal Smad pathway [148,190], astrocytes were shown responsive to this growth factor, which induces amyloid precursor protein (APP) expression in cooperation with Sp1 [217] and CTCF [218]. Altered APP cleavage by the concerted action of α-, β- and γ-secretases is a main aspect of AD pathology and results in the generation of the pathogenetic βA4 peptide 1-42, which is neurotoxic and leads to βA4-amyloid plaques. Whether βA4 peptide 1-42 can further suppress neuronal TGFβ1/Smad2,3 signalling by activation of the inhibitory Smad7 in neurons as already shown for human monocytes

for Drosophila motor neurons.

90 Trends in Cell Signaling Pathways in Neuronal Fate Decision

cannot be excluded [219].

Parkinson´s disease (PD) is a progressive neurodegenerative disorder which is characterized by dopaminergic neurodegeneration in the substantia nigra (SN) pars compacta. The closely associated loss of dopaminergic inputs into the striatum results in failure of motor programmes (e.g. voluntary movements) and initiates a striatal plastic change [225,226]. The gradual loss of dopaminergic neurons is linked to an increase of dopamine turnover [227,228]. α-synuclein (α-syn) is the major protein component of Lewy bodies, a hallmark of PD. It can aggregate to form toxic oligomers and fibrillar structures [226,229].

It was shown, that the TGFβ3-Smad-HIPK2 pathway is important for the survival of dopami‐ nergic neurons during development [96,230]. However, recently, a very specific and important role of Smad3 for the nigrostriatal system was explored in a Smad3 null mouse model representing an exciting model of PD [231]. These animals show a reduced number of dopaminergic neurons in the rostral SN, resulting from a postnatal neurodegenerative process. The selective pro-survival effect of Smad3 for SN dopaminergic neurons was emphasized by stereological quantifications showing no alterations of the striatal neuronal number. In Smad3 null mice a diminished trophic support provided by Erk1/2 signalling and a reduced astrocytic support to nigral dopaminergic neurons was detected [231]. Though striatal dopamine levels were not changed in Smad3 null mice, a strong increase of DA metabolism, including elevated monoamine-oxidase (MAO) levels were identified, resembling PD findings [232]. Finally, increased expression and aggregation of α-syn in neurites and cell bodies of several telence‐ phalic, mesencephalic and rombencephalic brain regions and in fibres of the primary and secondary motor cortex were found [231]. Two possible functional Smad3-based circuits are suggested. Either Smad3 might directly regulate the α-syn transcription, which further allows modulating DA metabolism, or the Smad3 deficiency caused elevated DA catabolism gener‐ ates oxidative stress which allow toxic aggregation of α-syn. The data qualify Smad3 as a possible target for PD therapy. Interestingly, the currently pursued target to re-implant dopaminergic neurons is also based on modification of Smad signalling during their in vitro establishment [130,233].

#### **4.3. Motor neuron diseases**

As mentioned above, Smads are essentially controlling the plasticity of NMJ, the intra-axonal transport and affect axonal repair processes. Accordingly, selective motor neuron diseases exhibit disturbances of Smad signalling, e.g. spinal muscular atrophy, [234], spinal bulbar muscular atrophy [235] or amyotrophic lateral sclerosis (ALS).

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ALS is a progressive neurodegenerative disease, which targets upper and lower motor neurons. In the primary motor cortex and the anterior horn of the spinal cord, motor neurons disappear and the pyramidal tract degenerates [236-238]. The remaining motor neurons exhibit inclusion bodies e.g. Bunina bodies, hyaline and skein-like inclusions. The pathological transactive response DNA-binding protein with a molecular weight of 43 kDa (TDP-43) was shown to be the major disease protein in ALS. Recently, increased nuclear immunoreactivity for pSmad2,3 in motor neurons was reported from sporadic ALS patients in spinal cord [239]. Motor neurons, where a colocalization of TDP-43 and pSmad2,3 in skeine-like and round hyaline inclusions was detected [239], show reduced nuclear pSmad2,3 immunoreactivity [240]. Obviously a disruption of Smad signalling by Smad segregation, comparable to AD or several tauopathies, enhances the loss of motor neuron function. Smad4 seems actively involved in the control of motor function as targeted disruption of Smad4 demonstrates [132]. Probably, increased nuclear pSmad2,3 content in the remaining ALS motor neurons indicates an endogenously initiated approach to a functional improvement. Experiments with an ALS mouse model support this hypothesis, because TGFβ2 administration ameliorated the motor performance of the mice [241]. A reduction of TDP-43 containing aggregates by Smad2 overexpression confirms in vitro a protective role of activated Smad signalling in ALS [242].

In summary, initiation and/or progression of many neurological disorders are directly linked to altered Smad signalling, comprising cytoplasmic Smad aggregation/sequestration, and nuclear reduction, disruption of transcriptional machinery and stimulating proapoptotic signalling, therefore disturbing biological processes, which are essential for all phases of nervous system development and homeostasis.

## **Acknowledgements**

This manuscript was supported by the Project BBZ09: 14494 (University Leipzig) and the AFI-Project 984 000-150.
