Phenotype of mice that do not express specific TGF-β ligands, receptors or signaling molecules.
In 1928 Santiago Ramón y Cajal penned what became the accepted view about neurons in the central nervous system; “everything may die, nothing can be regenerated”. He later exhibited his wisdom by adding; “It’s the job of science to rewrite, if possible, this cruel phrase” . Up until 20 years ago, the scientific literature had emphasized that neurogenesis only occurs during development with no new neurons generated in the adult mammalian brain. However, since the discovery of adult neurogenesis, an extensive literature has emerged supporting the constant generation of new neurons in two neurogenic regions of the adult brain: the subventricular zone around the lateral ventricles (SVZ) and the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG) .
The existence of adult neurogenesis gave hope for recovery and regeneration from the many different insults that can damage the brain. After stroke or traumatic brain injury (TBI), immediate massive necrosis occurs followed by a subsequent prolonged period of inflammation and further neuronal death . Although brain injury induces massive cell loss, it also induces an increase in proliferation of NSCs residing in the neurogenic niches . The environment of the neurogenic niche in adult animals is exquisitely regulated, with a finely-tuned balance of soluble and cell-intrinsic factors that regulate the many different processes that are critical to neurogenesis: cell survival, proliferation, differentiation, and migration . Dramatic changes occur in this environment as a consequence of the injury. The careful regulation of neurogenesis is disrupted by the many different cellular, soluble and vascular signals detected by the different cell types in the SVZ and DG. This major environmental alteration leads to increased proliferation of progenitor cells for long periods after the acute injury, yet the ability of the neural progenitor cells to fully differentiate, migrate and integrate into the lesioned area is limited . Understanding the signals that regulate adult neurogenesis in the naïve and injured animals is key to ultimately being able to harness the potential of neuronal replacement and improve stem cell therapy.
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 neurogenic process in naïve animals . Many of these cytokines are induced by injury and play critical roles in many kinds of brain damage related processes around the lesion . 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 TGF-β superfamily in injury-induced neurogenesis may provide specific therapeutic targets for enhancing neurogenesis after trauma.
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 TGF-β 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 . 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  (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 . 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 . Expression levels of endogenous antagonists, including noggin, chordin, follistatin, gremlin and cerberus, thereby regulate the availability, and therefore, active signaling by their associated ligands . 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 . Canonical signaling by these cytokines is through the receptor regulated Smads (R-smads). As previously mentioned, TGF-β and activin signal through activation of Smad2 and Smad3, which are phosphorylated by the Type I receptor, and form a heteromeric complex with the common or co-Smad, Smad4 . 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 TGF-β 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 . Smads can also influence miRNA biogenesis by binding directly to the pri-miRNA to enhance Drosha processing of these molecules to pre-miRNA . 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 .
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.
|Conventional knockout mouse model of TGF-β proteins||Phenotype||References|
|TβRI||Failed angiogenesis, Embryonic lethality (E8)|||
|TβRII||Embryonic lethality (E10.5)|||
|TβRIII||Failed coronary vessel development accompanied by reduced 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|||
|TGFβ-3||Perinatal lethal, delayed lung development|||
|Smad1||Embryonic lethality (E10)||[35, 36]|
|Smad2||Embryonic lethality (E7.5–E12.5)|||
|Smad3||Viable and fertile. Impaired immune function, including defective neutrophil chemotaxis, and impaired mucosal immunity||[38, 39]|
|Smad4||Increased number of Olig2-expressing progeny|||
|Smad5||Embryonic lethality: defective vascular development||[41, 42]|
|Smad7||Significantly smaller than wild-type mice, died within a few|
days of birth
|Smad8||Viable and fertile||[41, 44]|
|BMPRIA||Embryonic lethality (E9.5)|||
|BMPRIB||Viable and exhibit defects in the appendicular skeleton|||
|BMPRII||Embryonic lethality (E9.5), arrest at gastrulation|||
|BMP2||Embryonic lethality (E7.5-10.5), defective cardiac development|
and have defects in cardiac development
|BMP3||Increased bone density in adult|||
|BMP4||Embryonic lethality (E6.5-E9.5), no mesoderm differentiation|
and show little or no mesodermal differentiation
|BMP5||Viable, skeletal and cartilage abnormalities|||
|BMP6||Viable and fertile; slight delay in ossification.|||
|BMP7||Perinatal lethal because of poor kidney development, eye defects that appear to originate during lens induction.||[53-56]|
|BMP8A||Viable: male infertility due to germ cell degeneration|||
|BMP8B||Viable: male infertility due to germ cell depletion|||
|BMP15||Viable: female subfertility|||
|Endoglin||Embryonic lethality (E11.5)||[60, 61]|
|Activin receptor IA (ALK2)||Embryonic lethality (E9.5)|||
|Activin receptor IIB (ActR2B)||Perinatal lethal|||
|Activin-βA||Neonatal lethal, craniofacial defects (cleft palate and loss of whiskers, upper incisors, lower incisors and molars)|||
|Activin-βB||Large litters but delayed parturition; nursing defects;|
Eye lid closure defects at birth
|Noggin||Perinatal lethal, cartilage hyperplasia|||
|Follistatin||Neonatal lethal, craniofacial defects, growth retardation and skin defects retardation and skin defects|||
3. TGF-β superfamily expression and function in normal adult brain: Role in neurogenesis
Adult neurogenesis involves proliferation of neural stem cells (NSCs), cell cycle exit, differentiation, maturation, and integration into the neural circuits, in a process that is involved in learning and memory in the normal adult brain . The neurogenic niche of the adult forebrain subventricular zone (SVZ) is comprised of three major proliferative cell types; A, B and C. Multipotent, self-renewing type B cells occur earliest in the neurogenic lineage of the SVZ and give rise to the rapidly dividing type C cells, or transit amplifying progenitors. Type A cells or neuroblasts differentiate from Type C cells and are migratory neuronal progenitors with proliferative capacity, which migrate to the olfactory bulb where they differentiate into interneurons (reviewed in [69-71]. In the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG), type 1 and type 2 slowly-dividing progenitors give rise to more rapidly dividing intermediate progenitor cells, and these in turn differentiate into immature neuroblasts, which migrate into the granule cell layer, then differentiate into mature neurons and integrate with the existing hippocampal circuitry .
Within the CNS, all three isoforms of TGF-β are produced by both glial and neuronal cells . Immunohistochemical studies show widespread expression of TGF-β2 and -β3 in the developing CNS, and these proteins play a role in regulation of neuronal migration, glial proliferation and differentiation [73-76]. In adult brain, TGF-β receptors are found in all areas of the CNS including the cortex, hippocampus, striatum, brainstem and cerebellum [77, 78]. Immunoreactivity for TβRI and TβRII is detected on neurons, astrocytes and microglia and endothelial cells located in the cortical gray matter, suggesting that almost every cell type in the CNS is a potential target for TGF-β signaling .
The TGF-β superfamily and its downstream targets are capable of controlling proliferation, differentiation, maturation and survival of stem cells and precursors in the neurogenic niches of adult brain . TβRI and TβRII are expressed by Nestin-positive type B and C cells in the SVZ [80, 81]. Our data show mRNA expression of TGF-β1, -β2, and -β3 in both the adult SVZ and DG . 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 . As neurogenesis declines with age , it has been suggested that TGF-β is a possible regulator of this age-related decline . 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 . 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 . 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, endogenous 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 . Thus, TGF-β potentially has opposing roles at different stages of neurogenesis, providing an additional example of the contextual nature of TGF-β action.
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  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 , although short-term infusion of follistatin does not affect neurogenesis in uninjured animals . Infusion of activin to the lateral ventricle of uninjured mice mildly increases the rate of NSC proliferation and neuron generation 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 . 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 . 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 . However, Smad3 knockout mice also have reduced proliferation in the DG potentially pointing to a different role for Smad2 and Smad3 in the DG .
The BMP family of proteins regulates cell proliferation and fate commitment throughout development and within the adult neurogenic niches . Expression of BMP2, -4 and -7 mRNAs have been reported in neurogenic regions of adult rodent brain , and the BMP receptors BMPRIA, -IB and -II are expressed abundantly in neurons, as well as in astrocytes and ependymal cells . All three of these receptors are expressed in type A cells of the SVZ, while type B and C cells express BMPRIA and BMPRII . In the DG, radial stem cells of the SGZ marked with glial fibrillary acidic protein (GFAP) and Nestin or Sox2 primarily express BMPRIA but not BMPRIB, while mature neurons express only BMPRIB . BMP ligands are also expressed in the adult rat brain [98, 99]. BMP2, -4, -6, and -7 are expressed by cells of the SVZ and DG [96, 97]. In the DG, the BMP signal transducer pSmad1 is strongly expressed in non-dividing primary NSCs and neuroblasts, but is absent in dividing primary NSCs , while in the SVZ, pSmad1/5/8 has been reported in primary NSCs and transit amplifying progenitors, but not in DCX-positive neuroblasts . The soluble BMP inhibitor noggin is also expressed by ependymal cells of the SVZ  and by cells of the DG .
Changing the ratio of BMP to noggin alters the rates of NSC proliferation and neurogenesis in adult animals, indicating that these proteins are primary regulators of basal adult neurogenesis [96, 97, 100]. Administration of exogenous BMP4 or BMP7 potently inhibits the division of NSCs and generation of new neurons in vivo and in vitro [96, 97], as does inhibition of noggin expression . Conversely, infusion of noggin or genetic deletion of the BMPRIA receptor causes an increase in NSC proliferation and generation of NeuN-expressing neurons in the DG [96, 97]. However this increase is transient, there is an eventual depletion of the primary NSC pool and a drastically reduced level of neurogenesis . Decreased BMP signaling in the DG is thought to be responsible for increased neurogenesis driven by exercise . It has been proposed that secretion of noggin from ependymal cells inhibits BMP signaling allowing a low level of basal neurogenesis to occur, while BMP signaling maintains the overall quiescence of the primary NSC pool [96, 97, 100]. Exogenous noggin infusion potentially has a different effect on SVZ NSCs, leaving their proliferation rate unaffected, but causing an increase in the generation of oligodendrocyte precursor cells from primary NSCs at the expense of immature neuroblasts . This noggin infusion phenocopies the effect of conditionally deleting Smad4 in NSCs using GLAST-cre  and is in contrast to the pro-neurogenic effects of noggin described by Lim et al . Thus, although there is still some controversy in the field it its clear that the balance between BMP and noggin is critical to proper maintenance of the adult NSC population.
4. Expression of TGF-β related cytokines in the adult rodent brain after injury
TGF-β family proteins are present in the brain immediately after injury as they are carried into the wound by the blood . Additionally, extracellular TGF-β proteins are activated and released from their latent protein complexes in the brain parenchyma . Local CNS expression of TGF-β, activin, and BMP proteins is increased after many different injuries [72, 105, 106]. Following acute brain injury, TGF-β1 levels are elevated in astrocytes, microglia, macrophages, neurons, ependymal cells and choroid plexus cells with peak expression around 3 days [107-110]. TGF-β2 and -β3 expression has also been found in astrocytes, microglia, endothelial cells and neurons after both ischemic and TBI [111, 112]. We have recently found TGF-β2 expression in oligodendrocytes in the lesioned cortex and corpus callosum . Ischemic lesions as well as TBI show elevated activin-A mRNA as well as mRNA for the BMPRII receptor [90, 94, 114]. Smad proteins are also upregulated after injury and were mainly located in the cerebral cortex, typically in the nucleus and/or in the cytoplasm of astrocytes, oligodendrocytes or neurons [86, 108, 115, 116]. We have summarized many studies that have examined changes in the TGF-β superfamily of cytokines after central nervous system injury in Table 2.
|TGF-β protein||Acute brain Insult(Animal model)||Expression in Brain||Expression in neurogenicniche||Cell types in which protein is expressed||mRNA and/or protein||References|
|TGF-β1||Ischemia||Cerebral cortex||_ _ _ _ _||Microglia, neurons, oligodendrocytes, endothelial cells, astrocytes, macrophages, and ependymal cells||mRNA, protein||[107-110]|
|Transient ischemia||Cerebellum, Cerebral cortex||Hippocampus, Subventricular zone||Microglia, T cells, neuroblasts and neurons||mRNA, protein||[117-120]|
|Permanent ischemia||Cerebral cortex, Striatum||_ _ _ _ _||Neurons, neuroblasts||mRNA, protein||[121-123]|
|Bilateral cerebral ischemia||Cerebellum, Cerebral cortex||Dentate gyrus||Neurons, vessels||Protein||[124, 125]|
|Hypoxic-ischemic||Cerebral cortex, Corpus callosum||_ _ _ _ _||Astrocytes, Microglia and blood vessels||Protein|||
|Stab wound||Cerebral cortex||_ _ _ _ _||Neurons||Protein|||
|Traumatic brain injury||Cerebral cortex||Hippocampus, Subventricular zone||Microglia, astrocytes and neurons||mRNA, protein||[82, 112, 127, 128]|
|Excitotoxic lesion (NMDA)||Gray matter surrounding the lesion||_ _ _ _ _||Astrocytes, neurons||Protein|||
|Triethyltin exposure||Cerebral cortex||Hippocampus||Neurons||mRNA, protein||[130, 131]|
|Cerebral cortex||_ _ _ _ _||Activated glia, meningeal|
cells, choroid plexus
|Excitotoxic Injury||_ _ _ _ _||Hippocampus||Neurons||Protein|||
|Irradiation||Cerebral cortex||_ _ _ _ _||Macrophages and astrocytes||Protein|||
|Excitotoxicity with kainic acid||Cerebral cortex||Hippocampus||Microglia/macrophages, neurons and astrocytes||mRNA, protein||[86, 135-137]|
|Stab wound||Cerebral cortex||_ _ _ _ _||Astrocytes||Protein|||
|TGF-β2||Ischemia||Cerebral cortex, cerebellum, striatum||Hippocampus||Neurons and endothelial cells, microglia and astrocytes||mRNA, protein||[108, 109, 111]|
|TGF-β3||Ischemia||Cerebral cortex||Dentate gyrus||Neurons||mRNA, protein|||
|Traumatic brain injury||Cerebral cortex||Hippocampus||Astrocytes||Protein|||
|TβRI||Permanent ischemia||Cerebral cortex||_ _ _ _ _||Astrocytes and neurons||mRNA, protein|||
|TβRII||Ischemia||Cerebral cortex, midbrain, cerebellum, and brainstem||_ _ _ _ _||Neurons, astrocytes, microglia, endothelial cells, and other non-neuronal cells found in the choroid plexus||mRNA, protein||[122, 139, 140]|
|Traumatic brain injury||Cerebral Cortex||_ _ _ _ _||Endothelial cells||Protein|||
|Smad2||Excitotoxicity||Cerebral Cortex||Hippocampus||Neurons, astrocytes and microglia||Protein|||
|pSmad2||Stroke||Cerebral Cortex||_ _ _ _ _||Astrocytes, activated microglia||Protein|||
|pSmad 1,5,8||Cuprizone-induced demyelination||_ _ _ _ _||Subventricular zone||Oligodendrocytes||mRNA, protein|||
|BMPRII||Traumatic brain injury||_ _ _ _ _||Dentate|
|BMPs and receptors||Ischemia||Cerebral Cortex, cerebellum||Hippocampus||Neurons||mRNA, protein||[124, 142, 143]|
|Bilateral cerebral ischemia||Cerebral cortex, cerebellum||Subventricular zone, dentate gyrus||Neurons||mRNA, protein|||
|Traumatic brain injury||Cerebral cortex||Subventricular zone||Astrocytes||mRNA, protein|||
|BMP4||Cuprizone-induced demyelination||_ _ _ _ _||Subventricular zone||Astrocytes and oligodendrocytes||mRNA, protein|||
|BMP7||Traumatic brain injury||Cerebral cortex||_ _ _ _ _||Astrocytes||Protein|||
|Stroke||Cerebral cortex, corpus callosum||Subventricular zone||Progenitors cells and neurons||Protein|||
|Noggin||Traumatic brain injury||Cerebral cortex||Subventricular zone||Astrocytes and progenitors cells||Protein|||
|ActR-1A||Traumatic brain injury||_ _ _ _ _||Dentate|
|Activin||Ischemia||Cerebral Cortex, striatum||Hippocampus||Neurons||mRNA, protein||[89, 146]|
|Microglia and blood vessels||mRNA, protein|||
|Excitotoxicity||Amygdala, Piriform cortex, and thalamus||Dentate|
|Neurons, blood vessels||mRNA, protein||[105, 146-148]|
Relatively few studies have examined changes in expression of the TGF-β superfamily of cytokines specifically within the neurogenic regions after brain injury. TGF-β1 expression increases in the SVZ  and DG [117, 118, 124] after ischemic injury. Its expression is also induced in neurons of the DG after a demyelinating lesion  or after local kainic acid injection . Our group recently found that controlled cortical impact injury increased mRNA expression of many TGF-β cytokines, including TGF-β1 and -β2, activin-A, and BMPs -4, -5, -6, and -7 in the DG and SVZ, demonstrating that a distal injury can alter TGF-β signaling pathways in the neurogenic regions . We have observed upregulation of TGF-β1 and -β3 in GFAP and Nestin positive progenitors in the SVZ and DG after TBI (Figure 2 and unpublished data). TβRII is expressed in these Nestin positive progenitors in the lateral SVZ (Figure 2d). Phospho-Smad3 (pSmad3) shows strong nuclear localization in these cells as well (Figure 2i and unpublished data) suggesting a role for TGF-β/activin signaling in the regulation of post-injury neurogenesis. In the DG, TβRII is expressed in GFAP-positive precursors with strong pSmad3 nuclear staining (Figure 2m, 2r) suggesting a similar role for TGF-β cytokines in this neurogenic niche.
Local injury to the hippocampus via saline injection produces a strong induction of activin-βA mRNA in the DG, which can be blocked by inhibiting NMDA receptors . Activin expression in the DG is potently induced by seizures, local excitotoxic lesions, hypoxia/ischemia, TBI or permanent MCAO [89, 114, 146, 148, 149]. Cortical weight drop injury also elevates the expression of the activin receptor ActR-I and the BMP receptor BMPRII in the DG . BMPRII expression is also elevated in the DG after global cerebral ischemia , and BMP4 levels increase in the SVZ after a demyelinating lesion .
The limited studies available indicate that TGF-β, BMP, and activin signaling may all be active in the neurogenic regions after injury. However, it is currently unclear the manner in which they affect the behavior of neural stem cells. Given that these cytokines clearly regulate adult neurogenesis in the uninjured adult, more research in this area is necessary to fully elucidate the effect of brain injury on these signaling pathways, and the mechanisms through which these changes alter post-injury neurogenesis.
5. Injury-induced neurogenesis and its regulation by TGF-β family proteins
We have described the role of TGF-β proteins in the regulation of neurogenesis under basal conditions. In response to various injuries, the rate of neurogenesis is increased and the fate and migration of the neural progenitors is changed. Cerebral ischemia, excitotoxicity and TBI can all promote neurogenesis in the adult DG and SVZ [88, 150-153]. After injury, the altered environment changes the basic processes of proliferation, differentiation, migration and integration. TGF-β related cytokines have the potential to regulate many of these processes. Alteration in the destination of progenitor cells means that many of the neuroblasts change their usual trajectory and migrate towards and into the lesion . The cell fate of progenitor cells can be altered by the changed environment of the injured brain, in both the neurogenic niche and at the lesion site to which the progenitor cells migrate. The environment around the lesion is now very different than the normal location of these progenitors and thus further differentiation and integration occurs in an entirely unique environment . Additionally, the actions of TGF-β cytokines are highly context dependent, and they can have very different effects in the injured as compared to the uninjured brain.
A major component of the brain post-injury in comparison to the uninjured brain is the inflammatory response, both of local CNS cells and invading macrophages. While the majority of studies have indicated that inflammation is detrimental to neurogenesis, it is now appreciated that the effect of inflammation on neurogenesis is multifaceted . Of particular importance is the response of local microglia and astrocytes in the neurogenic regions. Microglia are potent regulators of neurogenesis, and in certain contexts can powerfully inhibit the process . However microglia have also been shown to promote neurogenesis [158, 159], and studies have described differential action of acute vs. chronically activated microglia on NSC division and neurogenesis, as well as for microglia activated by different mechanisms or by different cytokines [160, 161]. As TGF-β proteins are prominent anti-inflammatory molecules , their actions after brain injury can regulate neurogenesis by acting directly on NSCs as well as indirectly through their effects on the glial inflammatory response .
Due to their pleiotropic actions, TGF-β superfamily proteins have been investigated as potential treatments for a variety of CNS injuries, and several studies have demonstrated potential uses for these cytokines as therapeutic molecules (see Table 3). They have also provided insights into the action of these molecules as regulators of neural stem/progenitor cell (NSPC) proliferation and differentiation, with respect to both endogenous and transplanted stem cell populations.
|TGF-β related protein||Animal Model||Mode of administration||Effect on cell proliferation and neurogenesis||BehavioralOutcome||Reference|
|TGFβ-1||Transient ischemia||Intranasal aerosol spray||Decreased NSC proliferation and induce the number of DCX expressing neuronal precursors||Reduced Neurological Severity Score deficits|||
|Adrenalectomy||Intraventricular infusion||Decreased the percentage of dividing cells which co-express PSA-NCAM in the DG||None measured|||
|Adrenalectomy||Adenoviral overexpression||Increased NSC proliferation and neurogenesis in the SVZ||None measured|||
|Prenatal LPS inflammation||Adult adenoviral overexpression||Inhibited chronic microglial activation and restored neurogenesis||None measured|||
|Naïve animals||Injected into the cerebrospinal fluid||Number of proliferating cells in the hippocampus and in the lateral ventricle wall is substantially reduced, fewer|
neuronal precursor cells
|Naïve animals||Transgenic astrocytic overexpression||Decreased DG cell proliferation and generation of neuroblasts and neurons||None measured|||
|Noggin||Permanent MCAO||Transgenic neuronal overexpression||Increased immature oligodendrocyte generation||Reduced motor deficits|||
|Naïve animals||Intraventricular infusion||Promoted neuronal differentiation of SVZ precursor cells transplanted to the striatum||None measured|||
|Cuprizone-induced corpus callosum demyelination||Intraventricular infusion||Decreased astrocyte and increased oligodendrocyte generation from the SVZ||None measured|||
|Spinal cord injury||Overexpression by transplanted NPCs.||Increased neuronal and oligodendroglial differentiation of transplanted NPCs||Improved motor recovery|||
|BMP7||Transient ischemia||Intraventricular infusion||Increased SVZ proliferation and neurogenesis||Reduced motor deficits|||
|Naïve animals||Intraventricular infusion||Inhibited SVZ proliferation||None measured|||
|Chordin||Lysolectithin-induced corpus callosum demyelination||Intraventricular infusion||Increased NPCs migrating to lesion, and increased oligodendrocyte differentiation||None measured|||
|Activin-A||Excitotoxic hippocampal lesion||Continuous intraventricular infusion||Decreased astrocyte and microglial inflammation, and increases neurogenesis||None measured|||
|Naïve mice||Transgenic overexpression||Increases new neuron survival||Reduced anxiety-like behavior|||
|Activin-A or Activin-B||Naïve mice||ICV injection||Not examined||Reduced depression-like behavior|||
|Follistatin||Excitotoxic hippocampal lesion||Continuous intraventricular infusion||Increased NSC proliferation and neurogenesis.||None measured|||
|Naïve mice||Transgenic overexpression||Potently inhibited neurogenesis||Increased anxiety-like behavior|||
TGF-β1 treatment improves the outcome in several models of injury as it is strongly neuroprotective [76, 133, 171, 172] and in certain circumstances can promote neurogenesis after injury. After middle cerebral artery occlusion (MCAO) in mice, intranasal treatment with TGF-β1 increases the number of proliferative DCX-positive neural progenitors and the number of new neurons in the SVZ and striatum, while decreasing the fraction of proliferative cells that express GFAP . After adrenalectomy, TGF-β also stimulates neurogenesis. TGF-β1 expression is upregulated and is necessary for the increased rates of neurogenesis in the SVZ and DG caused by adrenalectomy . In this model TGF-β mediated downregulation of microglial activation and proliferation may be partially responsible for the increased neurogenesis [163, 165]. TGF-β1 can also inhibit chronic microglial activation induced by prenatal LPS exposure, and ameliorate the LPS-mediated decrease in neurogenesis  suggesting that the anti-inflammatory action of TGF-β participates in its pro-neurogenic effects. Conversely, in naïve animals intracerebroventricular infusion of TGF-β1 lowered the number of DCX-positive neuronal precursors in the neurogenic niches. This reduced level of proliferation in the TGF-β1 infused brains was strongly correlated with an increased accumulation of pSmad2 in Sox2/GFAP expressing cells of the SGZ . Transgenic overexpression of TGF-β1 in naïve mice also leads to reduce neurogenesis . The opposite effects of TGF-β1 in injured as compared to naïve animals illustrate the difficulty in assigning one specific role to TGF-β1 due to its context-dependent effects. Chronic inflammation, either after lesion or in neurodegenerative disease, provides a different environment for the consequences of TGF-β signaling. The anti-inflammatory actions of TGF-β can have an important role in influencing neurogenic processes, independent of direct effects on neural progenitor cells. Dysregulation of TGF-β signaling is being acknowledged as a potential source for chronic inflammation. Indeed, aberrant TGF-β signaling and consequent accumulation of activated microglia in the neurogenic regions may play an important role in the progression of Alzheimer’s disease [171, 173].
Recent studies have demonstrated a critical role for activin signaling as a modulator of adult neurogenesis  in addition to its well-established role as a neuroprotective molecule [174, 175]. After local excitotoxic injury to the hippocampus, ablating activin signaling by infusion of the activin inhibitor follistatin potently inhibits post-injury neurogenesis and exacerbates the inflammatory response of astrocytes and microglia. Conversely, infusion of activin-A facilitates neurogenesis and represses gliosis . Perhaps related to its effects on neurogenesis, activin can also regulate anxiety and depression-like behavior in rodents, and the activin pathway may be a useful therapeutic target for treating depression. Hippocampal infusion of activin-A or activin-B reduces measures of depression in a forced swim test, with a similar efficacy to that of the antidepressant fluoxetine . Further, transgenic mice which overexpress activin-A, have decreased anxiety measures in spontaneous place preference tests, while mice which overexpress follistatin, display the reverse .
In the naïve rodent, BMPs usually act to suppress neurogenesis in the SVZ and DG whereas the BMP inhibitor noggin promotes it . In contrast, inhibition of BMP signaling by upregulation of the BMP inhibitor chordin after lysolecithin-induced demyelination of the corpus callosum, led to redirection of SVZ precursors away from a neuronal lineage towards that of oligodendrocytes . This change in differentiation potential was accompanied by a change in the migration pattern of the SVZ precursors, away from the rostral migratory stream, and towards the corpus callosum. Injury-induced changes in expression of regulatory factors often alter the normal pattern of cell differentiation and migration [176, 177]. In a different model of demyelination, cuprizone-induced upregulation of BMP-4 resulted in more SVZ precursors becoming astrocytes, with a concomitant reduction in the number of mature oligodendrocytes . Intraventricular infusion of noggin in this model increased the generation of oligodendrocytes from the SVZ  illustrating that inhibition of BMP signaling has the potential to promote remyelination in models of multiple sclerosis. The astrogliogenic potential of BMP has been demonstrated in multiple studies, where various precursors are pushed towards the astrocytic lineage [168, 178]. This is also true with transplanted neural stem cells or mesenchymal stem cells, where BMPs around the implantation site push the transplanted cells towards astrocytes . If these cells are being used to enhance repair after spinal cord or TBI, inhibition of BMP becomes an attractive option to promote neuronal or oligodendrocyte differentiation rather than that of astrocytes. In contrast to all these studies, one group has shown that BMP-7 has neuroprotective properties which may enhance the survival of immature neurons [142, 180]. In one study, infusion of BMP-7 into the lateral ventricles of rats 24 hours after transient MCAO led to increased numbers of proliferating NSCs and more mature neurons generated in the SVZ while also facilitating behavioral recovery . However, a different group has shown that transgenic expression of the BMP-inhibitor noggin in neurons after permanent MCAO in the mouse enhances functional recovery . These conflicting data illustrate the sometimes confusing nature of the literature whereby BMP effects, similar to those of TGF-β are extremely contextual and are dependent on the exact model used. Overall, although some BMPs may have neuroprotective properties, the vast majority of the literature supports the view that BMP induction after injury is not beneficial for recovery, and that inhibition of BMP signaling may have therapeutic potential.
6. Future therapeutic strategies
In spite of extensive research in the field of brain injury or stroke, there is little effective treatment for these injuries . Many of the neuroprotective treatments that have been successful in rodents have failed in clinical trials . Harnessing the regenerative capacity of the adult brain is one strategy for repairing and replacing injured tissue, together with enhancing neurotrophic support of existing neurons to promote survival [184, 185]. A complementary strategy also under development is transplantation of neural stem cells or committed progenitors into the lesion. However, when multipotent NSCs were implanted directly into non-neurogenic regions in the injured brain, such as in the cortex or striatum, they failed to generate neurons but instead generated glial cells [186, 187]. Endogenous neural progenitors also are limited in their differentiation potential, presumably because the post-lesion environment is one that supports glial differentiation in preference to that of neurons . As TGF-β family members can promote astrogliogenesis [189, 190], it would seem that in some circumstances, inhibition of specific cytokine signals would increase neuronal differentiation. A further consideration for repair and neuronal survival is promotion of oligodendrocyte survival and differentiation, since remyelination is critical to continued survival and function of many neurons. Inhibition of BMP action through infusion of noggin can promote oligodendrocyte differentiation after demyelination . Inflammation after injury is yet one more factor that alters the environment for regeneration. Although often thought of as a short-lived phenomenon, there can be longer lasting inflammatory changes that persist months after injury . One of the major problems with development of members of the TGF-β superfamily or their inhibitors for therapeutic use are the pleiotropic nature of their effects. Thus TGF-β1 itself is neuroprotective and anti-inflammatory, which should promote recovery, but it inhibits proliferation of precursors, and also promotes development of the glial scar through upregulation of many extracellular matrix molecules, and through enhancing the migration of astrocytes [128, 192].
These cytokines act in a context dependent and concentration dependent manner, which adds an additional layer of complexity. To develop better therapeutic strategies we need a deeper understanding of the mechanisms through which the many actions of each cytokine are mediated. We may then be able to target specific molecules in the downstream signaling pathways, to avoid the pleiotropic effects that are emblematic of the activity of this cytokine family.