**2. The TGF-β superfamily; cytokines, receptors and signaling**

The TGF-β cytokine superfamily is a large group of proteins comprising 33 different members that include: bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), activins, inhibins, nodal, lefty, mülllerian inhibiting substance (MIS) together with the TGFβ proteins [8, 9]. All members of this cytokine family mediate their effects in a broadly analogous manner, binding specific type I and II transmembrane serine threonine kinase receptors and transducing their signal through similar intracellular Smad proteins [10]. These cytokines are divided into two distinct groups: those of the TGF-β/Activin group which mainly signal through the type I receptors ALK4, -5 and -7 activating Smad2 and -3, and those of the BMP/GDF group [11, 12] which employ ALK1, -2, -3 and -6 to activate Smad1, -5 and -8 [13, 14]. The specificity of Smad activation is therefore mainly determined by the identity of the type I receptor used to transduce the cytokine signal [15] (Figure 1).

TGF-β1, -β2 and -β3 together with some GDFs are unique in that they are synthesized as a large precursor molecule that is cleaved but remains non-covalently linked to its latency associated peptides, in either a small or large complex [18]. The bioavailability of TGF-βs is tightly regulated by the release of active TGF-β from these complexes in the extracellular matrix, so synthesis of TGF-β does not necessarily provide a reliable indication of available cytokine to initiate signaling. Similarly, the bioavailability of BMPs is regulated by binding to secreted extracellular antagonists that prevent BMP (and sometimes Activin) from binding to their receptor [19]. Expression levels of endogenous antagonists, including noggin, chordin, follistatin, gremlin and cerberus, thereby regulate the availability, and therefore, active signaling by their associated ligands [20]. TGF-β signaling is the archetype for signaling by

this cytokine family. TGF-β binds to the constitutively active TGF-β receptor II (TβRII) which can then recruit the type I receptor TGF-β receptor I (TβRI/ALK5). Activation of TβRI by transphosphorylation activates it, initiating downstream signaling [21]. Canonical signaling

and apoptosis. The diagram is adapted from [16] and [17].

**Figure 1. TGF-β superfamily signal transduction.** TGF-β, nodal or activin ligands bind to Type II receptors, which then recruit Type I receptors leading to transphosphorylation of type 1 receptors. Activated type I receptors phosphor‐ ylate Smad 2/3 (*i.e.* R-Smads) which then complex with the co-Smad, Smad4 and translocate to the nucleus to bind DNA at specific DNA motifs. Smad proteins activate or repress transcription through association with various co-activa‐ tor (Co-Act) or co-repressor proteins. This pathway is inhibited by Smad7. BMP signaling operates by a similar para‐ digm. BMP6 and BMP7 bind to their Type II receptor before the complex recruits the Type I receptors, Alk-3 or Alk-6. BMP2 and BMP4, however bind first to their type I receptor before recruiting the type II receptor BMPRII. BMP binding to either receptor can be inhibited by first binding to various extracellular inhibitor proteins, such as noggin. Activa‐ tion of the receptor complex leads to phosphorylation of the receptors and subsequent phosphorylation of Smad1, Smad5, or Smad8, allowing them to form a complex with Smad4. This heteromeric complex translocates to the nu‐ cleus, to target BMP-regulated genes through interaction with co-activators or repressors. Smad 6 and Smad7 may act similarly to inhibit the BMP pathway through interactions with the receptor complex and thus inhibiting R-Smad acti‐ vation. TGF-β and BMP pathways induce the expression of proteins involved in proliferation, differentiation, survival

Role of TGF-β Signaling in Neurogenic Regions After Brain Injury

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5

Role of TGF-β Signaling in Neurogenic Regions After Brain Injury http://dx.doi.org/10.5772/53941 5

in the naïve and injured animals is key to ultimately being able to harness the potential of

There are many different factors important to regulation of neurogenesis, many of which are discussed in other chapters in this book. Here we will focus on the role of the transforming growth factor-β (TGF-β) superfamily and its associated signaling pathways in regulating neurogenesis after brain injury. Members of this family, including the bone morphogenetic proteins (BMPs), Activin, and TGF-β1, -β2 and -β3 have a profound influence on the neuro‐ genic process in naïve animals [7]. Many of these cytokines are induced by injury and play critical roles in many kinds of brain damage related processes around the lesion [3]. We and others recently started to accumulate data on their induction in the neurogenic niches after different types of injury. Here we will focus on the relevance of their induction in these specific brain regions, and the mechanisms through which they may influence the neurogenic response to injury. As there are significant differences between the behavior of cells contributing to neurogenesis during development and in the adult, we will restrict our analysis to that observed in adult animals after injury. Delineation of the specific role of members of the TGFβ superfamily in injury-induced neurogenesis may provide specific therapeutic targets for

**2. The TGF-β superfamily; cytokines, receptors and signaling**

type I receptor used to transduce the cytokine signal [15] (Figure 1).

The TGF-β cytokine superfamily is a large group of proteins comprising 33 different members that include: bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), activins, inhibins, nodal, lefty, mülllerian inhibiting substance (MIS) together with the TGFβ proteins [8, 9]. All members of this cytokine family mediate their effects in a broadly analogous manner, binding specific type I and II transmembrane serine threonine kinase receptors and transducing their signal through similar intracellular Smad proteins [10]. These cytokines are divided into two distinct groups: those of the TGF-β/Activin group which mainly signal through the type I receptors ALK4, -5 and -7 activating Smad2 and -3, and those of the BMP/GDF group [11, 12] which employ ALK1, -2, -3 and -6 to activate Smad1, -5 and -8 [13, 14]. The specificity of Smad activation is therefore mainly determined by the identity of the

TGF-β1, -β2 and -β3 together with some GDFs are unique in that they are synthesized as a large precursor molecule that is cleaved but remains non-covalently linked to its latency associated peptides, in either a small or large complex [18]. The bioavailability of TGF-βs is tightly regulated by the release of active TGF-β from these complexes in the extracellular matrix, so synthesis of TGF-β does not necessarily provide a reliable indication of available cytokine to initiate signaling. Similarly, the bioavailability of BMPs is regulated by binding to secreted extracellular antagonists that prevent BMP (and sometimes Activin) from binding to their receptor [19]. Expression levels of endogenous antagonists, including noggin, chordin, follistatin, gremlin and cerberus, thereby regulate the availability, and therefore, active signaling by their associated ligands [20]. TGF-β signaling is the archetype for signaling by

neuronal replacement and improve stem cell therapy.

4 Trends in Cell Signaling Pathways in Neuronal Fate Decision

enhancing neurogenesis after trauma.

**Figure 1. TGF-β superfamily signal transduction.** TGF-β, nodal or activin ligands bind to Type II receptors, which then recruit Type I receptors leading to transphosphorylation of type 1 receptors. Activated type I receptors phosphor‐ ylate Smad 2/3 (*i.e.* R-Smads) which then complex with the co-Smad, Smad4 and translocate to the nucleus to bind DNA at specific DNA motifs. Smad proteins activate or repress transcription through association with various co-activa‐ tor (Co-Act) or co-repressor proteins. This pathway is inhibited by Smad7. BMP signaling operates by a similar para‐ digm. BMP6 and BMP7 bind to their Type II receptor before the complex recruits the Type I receptors, Alk-3 or Alk-6. BMP2 and BMP4, however bind first to their type I receptor before recruiting the type II receptor BMPRII. BMP binding to either receptor can be inhibited by first binding to various extracellular inhibitor proteins, such as noggin. Activa‐ tion of the receptor complex leads to phosphorylation of the receptors and subsequent phosphorylation of Smad1, Smad5, or Smad8, allowing them to form a complex with Smad4. This heteromeric complex translocates to the nu‐ cleus, to target BMP-regulated genes through interaction with co-activators or repressors. Smad 6 and Smad7 may act similarly to inhibit the BMP pathway through interactions with the receptor complex and thus inhibiting R-Smad acti‐ vation. TGF-β and BMP pathways induce the expression of proteins involved in proliferation, differentiation, survival and apoptosis. The diagram is adapted from [16] and [17].

this cytokine family. TGF-β binds to the constitutively active TGF-β receptor II (TβRII) which can then recruit the type I receptor TGF-β receptor I (TβRI/ALK5). Activation of TβRI by transphosphorylation activates it, initiating downstream signaling [21]. Canonical signaling by these cytokines is through the receptor regulated Smads (R-smads). As previously men‐ tioned, TGF-β and activin signal through activation of Smad2 and Smad3, which are phos‐ phorylated by the Type I receptor, and form a heteromeric complex with the common or co-Smad, Smad4 [22]. This Smad complex translocates to the nucleus where it regulates the transcription of numerous genes in cooperation with other transcription factors, coactivators and corepressors. Inhibitory Smads, or I-smads, are Smad-activated proteins that provide negative feedback to the Smad pathway through a variety of mechanisms [16, 23]. BMP signaling is similar in form to TGF-β signaling, although the specifics of individual receptors and R-Smads (1, 5, 8/9) involved vary according to the specific cytokine. For a full review of signaling and receptor nomenclature by this cytokine family please refer to some excellent reviews [14, 24]. The Smad pathway is by no means the only mechanism through which TGFβ cytokine signals are transduced from the receptor to the nucleus. Smad-independent pathways include activation of MAPKs, Ras/ERK, JNK, p38, PI3K-Akt, NF-kappaB, JAK/STAT, PP2A/S6 phosphatases and small Rho-related GTPases (16, 25). Some of the non-Smad kinases can influence Smad directed signaling by complexing with, or modifying the Smad proteins directly [16, 25]. Another level of control was found when it was shown that TGF-β/BMP signaling is both regulated by, and can regulate transcription of miRNAs [26]. Smads can also influence miRNA biogenesis by binding directly to the pri-miRNA to enhance Drosha processing of these molecules to pre-miRNA [27]. An intricate balance between Smad and non-Smad signaling superimposed on cell intrinsic and environmental conditions determines the specificity and the ultimate response of each cell to TGF-β signaling. Thus, there is a complexity to TGF-β superfamily signaling that befits cytokines that signal to multiple different cell types, in context dependent manners to influence many different physiologic processes [28].

**Conventional knockout mouse model of TGF-β proteins**

Activin receptor IA

Activin receptor IIB

(ALK2)

(ActR2B)

**Phenotype References**

[38, 39]

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Role of TGF-β Signaling in Neurogenic Regions After Brain Injury

[43]

[48]

[50]

[53-56]

[64]

Smad1 Embryonic lethality (E10) [35, 36] Smad2 Embryonic lethality (E7.5–E12.5) [37]

neutrophil chemotaxis, and impaired mucosal immunity

Smad3 Viable and fertile. Impaired immune function, including defective

Smad7 Significantly smaller than wild-type mice, died within a few

BMP2 Embryonic lethality (E7.5-10.5), defective cardiac development and have defects in cardiac development

BMP4 Embryonic lethality (E6.5-E9.5), no mesoderm differentiation

BMP3 Increased bone density in adult [49]

and show little or no mesodermal differentiation

BMP5 Viable, skeletal and cartilage abnormalities [51] BMP6 Viable and fertile; slight delay in ossification. [52]

BMP8A Viable: male infertility due to germ cell degeneration [57] BMP8B Viable: male infertility due to germ cell depletion [58] BMP15 Viable: female subfertility [59] Endoglin Embryonic lethality (E11.5) [60, 61]

Embryonic lethality (E9.5) [62]

Perinatal lethal [63]

BMP7 Perinatal lethal because of poor kidney development, eye defects that appear to originate during lens induction.

Activin-βA Neonatal lethal, craniofacial defects (cleft palate and loss of

whiskers, upper incisors, lower incisors and molars)

days of birth

Smad4 Increased number of Olig2-expressing progeny [40] Smad5 Embryonic lethality: defective vascular development [41, 42]

Smad8 Viable and fertile [41, 44] BMPRIA Embryonic lethality (E9.5) [45] BMPRIB Viable and exhibit defects in the appendicular skeleton [46] BMPRII Embryonic lethality (E9.5), arrest at gastrulation [47]

Genetic evidence indicates that TGF-β family members regulate embryonic, perinatal or neonatal development of the mouse embryo. Most mice null for one TGF-β superfamily ligand, receptor, protein or signaling protein fail in either gastrulation or mesoderm differentiation. Table 1 lists known phenotypes of mice that are null for specific proteins in the TGF-β superfamily signaling pathways.



by these cytokines is through the receptor regulated Smads (R-smads). As previously men‐ tioned, TGF-β and activin signal through activation of Smad2 and Smad3, which are phos‐ phorylated by the Type I receptor, and form a heteromeric complex with the common or co-Smad, Smad4 [22]. This Smad complex translocates to the nucleus where it regulates the transcription of numerous genes in cooperation with other transcription factors, coactivators and corepressors. Inhibitory Smads, or I-smads, are Smad-activated proteins that provide negative feedback to the Smad pathway through a variety of mechanisms [16, 23]. BMP signaling is similar in form to TGF-β signaling, although the specifics of individual receptors and R-Smads (1, 5, 8/9) involved vary according to the specific cytokine. For a full review of signaling and receptor nomenclature by this cytokine family please refer to some excellent reviews [14, 24]. The Smad pathway is by no means the only mechanism through which TGFβ cytokine signals are transduced from the receptor to the nucleus. Smad-independent pathways include activation of MAPKs, Ras/ERK, JNK, p38, PI3K-Akt, NF-kappaB, JAK/STAT, PP2A/S6 phosphatases and small Rho-related GTPases (16, 25). Some of the non-Smad kinases can influence Smad directed signaling by complexing with, or modifying the Smad proteins directly [16, 25]. Another level of control was found when it was shown that TGF-β/BMP signaling is both regulated by, and can regulate transcription of miRNAs [26]. Smads can also influence miRNA biogenesis by binding directly to the pri-miRNA to enhance Drosha processing of these molecules to pre-miRNA [27]. An intricate balance between Smad and non-Smad signaling superimposed on cell intrinsic and environmental conditions determines the specificity and the ultimate response of each cell to TGF-β signaling. Thus, there is a complexity to TGF-β superfamily signaling that befits cytokines that signal to multiple different cell types,

in context dependent manners to influence many different physiologic processes [28].

TβRI Failed angiogenesis, Embryonic lethality (E8) [29] TβRII Embryonic lethality (E10.5) [30]

epicardial cell invasion. Embryonic lethality (E14.5)

TGFβ-1 Loss of a critical regulator of immune function [32, 33] TGFβ-2 Perinatal lethal, craniofacial defects [34] TGFβ-3 Perinatal lethal, delayed lung development [33]

TβRIII Failed coronary vessel development accompanied by reduced

superfamily signaling pathways.

6 Trends in Cell Signaling Pathways in Neuronal Fate Decision

**Conventional knockout mouse model of TGF-β proteins**

Genetic evidence indicates that TGF-β family members regulate embryonic, perinatal or neonatal development of the mouse embryo. Most mice null for one TGF-β superfamily ligand, receptor, protein or signaling protein fail in either gastrulation or mesoderm differentiation. Table 1 lists known phenotypes of mice that are null for specific proteins in the TGF-β

**Phenotype References**

[31]


and DG [82]. In the adult human brain, TGF-β1 protein expression has been reported in the hippocampus, and the protein levels significantly increased with the age of the individual [83]. As neurogenesis declines with age [84], it has been suggested that TGF-β is a possible regulator of this age-related decline [83]. Signaling by the Smad2/3 pathway is high in the hippocampus and specifically the dentate gyrus, indicating a role for TGF-β and/or activin in regulation of neurogenesis [85, 86]. When TGF-β protein is overexpressed or infused directly into the lateral ventricles of uninjured animals, hippocampal neurogenesis is dramatically inhibited [81, 87]. This may be due to a direct anti-proliferative effect of TGF-β on type 1 and 2 primary NSCs [17]. A direct effect of TGF-β on NSCs is supported by *in vitro* studies showing that TGF-β1 treatment of cultured adult NSCs induces the cyclin-dependent kinase inhibitor (p21) and leads to cell cycle termination, without altering the differentiation choices of the NSCs [81]. Additionally, overexpression studies lead to increased TGF-β signaling in many different cell types within the neurogenic niche, making the exact contribution of more restricted, endoge‐ nous TGF-β difficult to determine. Recent data have suggested that TGF-β signaling at later stages of neurogenesis is critical for newborn neuron survival and maturation in the DG. Conditional deletion of the TβRI (ALK5) gene specifically in immature and mature neurons, leads to decreased neurogenesis and reduced survival of newborn neurons [85]. Thus, TGFβ potentially has opposing roles at different stages of neurogenesis, providing an additional

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Activin receptors are expressed throughout the brain, with strong expression in the neuronal layers of the hippocampus [88-90]. We have found that mRNA for activin-A and for activin's endogenous high affinity inhibitor, follistatin, are expressed in both the SVZ and DG of the adult mouse [82] and several recent reports have demonstrated that activin-A modulates adult neurogenesis [88, 91, 92]. Chronic overexpression of follistatin by neurons of the hippocampus almost entirely ablates adult DG neurogenesis, due to drastically lowered survival of adult-generated neurons [91], although short-term infusion of follistatin does not affect neurogenesis in uninjured animals [88]. Infusion of activin to the lateral ventri‐ cle of uninjured mice mildly increases the rate of NSC proliferation and neuron genera‐ tion in the DG, indicating that activin might stimulate division of NSCs. This effect may be indirect as activin has a potent anti-inflammatory effect in the CNS, and may modulate local microglia to stimulate neurogenesis [88]. Smad3 knockout mice have decreased levels of cell proliferation in the SVZ and along the rostral migratory stream, and decreased levels of olfactory bulb neurogenesis [93]. As these mice have defective signaling by both TGF-β and activin, these data suggest that activin signaling in the SVZ may be the predominant Smad3-utilizing cytokine in defining basal levels of neurogenesis. In the DG pSmad2 is normally absent from Sox2-positive type 1 and 2 primary NSCs in the DG of adult mice [17]. However, Smad3 knockout mice also have reduced proliferation in the DG potential‐

The BMP family of proteins regulates cell proliferation and fate commitment throughout development and within the adult neurogenic niches [19]. Expression of BMP2, -4 and -7 mRNAs have been reported in neurogenic regions of adult rodent brain [94], and the BMP receptors BMPRIA, -IB and -II are expressed abundantly in neurons, as well as in astrocytes

example of the contextual nature of TGF-β action.

ly pointing to a different role for Smad2 and Smad3 in the DG [93].

**Table 1.** Phenotype of mice that do not express specific TGF-β ligands, receptors or signaling molecules.
