**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 [103]. Additionally, extracellular TGF-β proteins are activated and released from their latent protein complexes in the brain parenchyma [104]. Local CNS expres‐ sion 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, macrophag‐ es, 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 expressioninoligodendrocytesinthelesionedcortexandcorpuscallosum[113].Ischemiclesions 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,typicallyinthenucleusand/orinthecytoplasmofastrocytes,oligodendrocytesorneurons [86, 108, 115, 116]. We have summarized many studies that have examined changes in the TGFβ superfamily of cytokines after central nervous system injury in Table 2.

and ependymal cells [95]. All three of these receptors are expressed in type A cells of the SVZ, while type B and C cells express BMPRIA and BMPRII [96]. 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 [97]. 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 [97], while in the SVZ, pSmad1/5/8 has been reported in primary NSCs and transit amplifying progenitors, but not in DCX-positive neuroblasts [40]. The soluble BMP inhibitor noggin is

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 neurogene‐ sis [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 [101]. 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 [97]. Decreased BMP signaling in the DG is thought to be responsible for increased neurogenesis driven by exercise [102]. 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 onSVZNSCs,leavingtheirproliferationrateunaffected,butcausinganincreaseinthegeneration of oligodendrocyte precursor cells from primary NSCs at the expense of immature neuro‐ blasts [40].Thisnoggininfusionphenocopies the effectof conditionallydeletingSmad4 inNSCs usingGLAST-cre [40] andis incontrasttothepro-neurogenic effectsofnoggindescribedbyLim et al [96]. 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**

TGF-β family proteins are present in the brain immediately after injury as they are carried into the wound by the blood [103]. Additionally, extracellular TGF-β proteins are activated and released from their latent protein complexes in the brain parenchyma [104]. Local CNS expres‐ sion 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, macrophag‐ es, 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 expressioninoligodendrocytesinthelesionedcortexandcorpuscallosum[113].Ischemiclesions as well as TBI show elevated activin-A mRNA as well as mRNA for the BMPRII receptor [90, 94,

**injury**

also expressed by ependymal cells of the SVZ [96] and by cells of the DG [100].

10 Trends in Cell Signaling Pathways in Neuronal Fate Decision



**TGF-β protein** **Acute brain Insult**

BMP4 Cuprizoneinduced demyelination

BMP7 Traumatic brain injury

Noggin Traumatic brain injury

ActR-1A Traumatic brain injury

in this neurogenic niche.

Activin Ischemia Cerebral Cortex,

Excitotoxicity Amygdala,

**(Animal model)**

Stroke Cerebral cortex,

**Expression in Brain**

corpus callosum

striatum

Hypoxia-ischemia Cerebral Cortex Dentate

Piriform cortex, and thalamus

**Expression in neurogenic niche**

Subventricular

zone

gyrus

gyrus

Dentate gyrus

**Table 2.** TGF-β superfamily cytokine and signaling intermediate expression after different forms of injury.

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 [119] and DG [117, 118, 124] after ischemic injury. Its expression is also induced in neurons of the DG after a demyelinating lesion [131] or after local kainic acid injection [133]. 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 [82]. 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 unpub‐ lished 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

Cerebral cortex Subventricular zone

\_ \_ \_ \_ \_ Dentate

\_ \_ \_ \_ \_ Subventricular zone

**Cell types in which protein is expressed**

Astrocytes and oligodendrocytes

Progenitors cells and

Neurons mRNA,

Neurons, blood vessels mRNA,

Cerebral cortex \_ \_ \_ \_ \_ Astrocytes Protein [144]

neurons

Astrocytes and progenitors cells

Hippocampus Neurons mRNA,

vessels

Microglia and blood

**mRNA and/or protein**

http://dx.doi.org/10.5772/53941

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

mRNA, protein

Protein [145]

Protein [144]

[90]

[89, 146]

[114]

[105, 146-148]

protein

protein

mRNA, protein

protein

**References**

13

[115]


**TGF-β protein** **Acute brain Insult**

**(Animal model)**

12 Trends in Cell Signaling Pathways in Neuronal Fate Decision

TGF-β2 Ischemia Cerebral cortex,

Traumatic brain

TβRII Ischemia Cerebral cortex,

Traumatic brain

injury

Cuprizoneinduced demyelination

Bilateral cerebral ischemia

Traumatic brain

injury

BMPRII Traumatic brain injury

pSmad 1,5,8

BMPs and receptors

injury

ischemia

TβRI Permanent

Excitotoxicity with kainic acid **Expression in Brain**

cerebellum, striatum

midbrain, cerebellum, and brainstem

**Expression in neurogenic niche**

TGF-β3 Ischemia Cerebral cortex Dentate gyrus Neurons mRNA,

Smad2 Excitotoxicity Cerebral Cortex Hippocampus Neurons, astrocytes and

pSmad2 Stroke Cerebral Cortex \_ \_ \_ \_ \_ Astrocytes, activated

\_ \_ \_ \_ \_ Dentate

Ischemia Cerebral Cortex,

cerebellum

Cerebral cortex, cerebellum

\_ \_ \_ \_ \_ Subventricular zone

Cerebral cortex Subventricular zone

gyrus

Subventricular zone, dentate gyrus

Cerebral cortex Hippocampus Microglia/macrophages,

Stab wound Cerebral cortex \_ \_ \_ \_ \_ Astrocytes Protein [138]

Hippocampus Neurons and

Cerebral cortex \_ \_ \_ \_ \_ Astrocytes and neurons mRNA,

\_ \_ \_ \_ \_ Neurons, astrocytes,

Cerebral Cortex \_ \_ \_ \_ \_ Endothelial cells Protein [141]

microglia

microglia

Hippocampus Neurons mRNA,

Oligodendrocytes mRNA,

Neurons mRNA,

Neurons mRNA,

Astrocytes mRNA,

**Cell types in which protein is expressed**

neurons and astrocytes

endothelial cells, microglia and astrocytes

microglia, endothelial cells, and other nonneuronal cells found in the choroid plexus

Cerebral cortex Hippocampus Astrocytes Protein [112]

**mRNA and/or protein**

mRNA, protein

mRNA, protein

protein

protein

mRNA, protein

Protein [86]

Protein [108]

[115]

[90]

[94]

[144]

[124, 142, 143]

protein

protein

protein

protein

protein

**References**

[86, 135-137]

[108, 109, 111]

[111]

[122]

[122, 139, 140]

**Table 2.** TGF-β superfamily cytokine and signaling intermediate expression after different forms of injury.

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 [119] and DG [117, 118, 124] after ischemic injury. Its expression is also induced in neurons of the DG after a demyelinating lesion [131] or after local kainic acid injection [133]. 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 [82]. 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 unpub‐ lished 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.

the subventricular zone (SVZ) at 3 (a-g) and 7 (h and i) days after traumatic brain injury (TBI). TβRII (a, red) is expressed in Nestin positive (b, green) neural stem cells (NSCs) in the SVZ, and also in ependymal cells (d), lining the walls of the lateral ventricle (LV). Light TGFβ−1 (green) and predominant TGFβ−3 (red) expression is also found in the walls of the LV where the adult NSCs reside (e). (f) Neurons (NeuN, green) are co-localized with TGFβ−2 (red) in the damaged stria‐ tum. (h) The majority of Smad 1,5,8 proteins (red) are co-expressed with Nestin (green). (i) pSmad3 (red) colocalizes with GFAP (green) in the dorsolateral corner of the SVZ. The right column shows coronal sections within the dentate gyrus (DG) of the hippocampus at 3 (j-q) and 7 (r) days after TBI. (j-m) TGFβ−1 (red, j) and TβRII (green) are colocalized in astrocytes (GFAP, blue) in the hilus and GCL (granule cell layer) of the hippocampus (n) TGFβ−1 (red) is co-localized with astrocytes (GFAP positive cells) located in the subgranular zone (SGZ) of the hippocampus. In (o) TGFβ−2 (red) is co-localized with NeuN (green) positive neurons in the hilus of the dentate gyrus. (p) TGFβ−3 (red) is co-localized with GFAP positive (blue) immature progenitors in the SGZ but not with DCX (green) positive neuroblasts. (q) Immunos‐ taining with TGFβ−1 (green) and TGFβ−3 (red) show they are almost entirely colocalized in the SGZ. (r) pSmad3 stain‐ ing in the nuclei of GFAP positive progenitor cells in the SGZ and hilus of the hippocampus. Scale bars: (c, d, f, (inset in

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

http://dx.doi.org/10.5772/53941

15

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 [114]. Activin expres‐ sion 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 [90]. BMPRII expression is also elevated in the DG after global cerebral ischemia [94], and BMP4 levels

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

**5. Injury-induced neurogenesis and its regulation by TGF-β family**

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 [154]. 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 [155]. Additionally, the actions of TGF-β cytokines are highly context dependent, and they can have very different

i), m, (inset in n), o, (inset in o), p, (inset in r)) 20 µm; (e, g, h, i, q, r) 50 µm.

increase in the SVZ after a demyelinating lesion [115].

effects in the injured as compared to the uninjured brain.

these changes alter post-injury neurogenesis.

**proteins**

**Figure 2. Confocal images of the TGF-β ligands, receptors and signaling proteins in the SVZ and DG in the in‐ jured adult mice brain.** Double and triple labelled inmmunofluorescence staining for TGF-β proteins and receptors, with the following cell-type specific markers: Nestin (for undifferentiated neuronal precursors), NeuN (for mature neu‐ rons), GFAP (for progenitor and astroglial cells), DCX (for neuroblasts). The left column shows coronal sections within

the subventricular zone (SVZ) at 3 (a-g) and 7 (h and i) days after traumatic brain injury (TBI). TβRII (a, red) is expressed in Nestin positive (b, green) neural stem cells (NSCs) in the SVZ, and also in ependymal cells (d), lining the walls of the lateral ventricle (LV). Light TGFβ−1 (green) and predominant TGFβ−3 (red) expression is also found in the walls of the LV where the adult NSCs reside (e). (f) Neurons (NeuN, green) are co-localized with TGFβ−2 (red) in the damaged stria‐ tum. (h) The majority of Smad 1,5,8 proteins (red) are co-expressed with Nestin (green). (i) pSmad3 (red) colocalizes with GFAP (green) in the dorsolateral corner of the SVZ. The right column shows coronal sections within the dentate gyrus (DG) of the hippocampus at 3 (j-q) and 7 (r) days after TBI. (j-m) TGFβ−1 (red, j) and TβRII (green) are colocalized in astrocytes (GFAP, blue) in the hilus and GCL (granule cell layer) of the hippocampus (n) TGFβ−1 (red) is co-localized with astrocytes (GFAP positive cells) located in the subgranular zone (SGZ) of the hippocampus. In (o) TGFβ−2 (red) is co-localized with NeuN (green) positive neurons in the hilus of the dentate gyrus. (p) TGFβ−3 (red) is co-localized with GFAP positive (blue) immature progenitors in the SGZ but not with DCX (green) positive neuroblasts. (q) Immunos‐ taining with TGFβ−1 (green) and TGFβ−3 (red) show they are almost entirely colocalized in the SGZ. (r) pSmad3 stain‐ ing in the nuclei of GFAP positive progenitor cells in the SGZ and hilus of the hippocampus. Scale bars: (c, d, f, (inset in i), m, (inset in n), o, (inset in o), p, (inset in r)) 20 µm; (e, g, h, i, q, r) 50 µm.

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 [114]. Activin expres‐ sion 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 [90]. BMPRII expression is also elevated in the DG after global cerebral ischemia [94], and BMP4 levels increase in the SVZ after a demyelinating lesion [115].

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.
