**3. Distribution of TGF-βs, their binding proteins and receptors in the central nervous system**

The distribution pattern of TGF-βs established using immunohistochemistry at the protein level (Unsicker et al., 1991) and by means of in situ hybridization histochemistry at the mRNA level (Vincze et al., 2010) was similar in several brain regions. TGF-β2 and β3 immunoreactivities were present constitutively in cerebral cortical layers II, III and V and their expression depended on the cortical layer rather than the areas within the cerebral cortex. Furthermore, different regions of hippocampus, as well as widely distributed cells in the hypothalamus and amygdala contained both TGF-ß2 and ß3. Intense labeling of these isoforms was also described in brainstem monoaminergic neurons, and motor nuclei (Unsicker et al., 1991; Vincze et al., 2010). In turn, the striatum, most thalamic nuclei, and the superior colliculus were almost devoid of TGF-β2 and ß3 mRNA and immunoreactivities. However, considerable differences between the distribution of mRNAs and immunoreactivities of TGF-βs have also been reported. Most importantly, TGF-β1 immunoreactivity was reported to be constitutively present only in meninges and the choroid plexus in the brain (Komuta et al., 2009 ; Unsicker et al., 1991) while a more

Transforming Growth Factor Beta in the Central Nervous System 133

Although the topographical distribution of TGF-β receptors in the central nervous system has not been systematically described, the available data suggest widespread localization. When TGF-β receptor mRNA was detected by RT-PCR in rats at different stages of development similar levels were found in several regions of the CNS, including cortex,

Many of the investigations of TGF-β functions did not differentiate between the isoforms of TGF-βs. In many cases, TGF-β1 was applied, which, when exogenously applied, can mimick the effects of other endogenous TGF-β isoforms. Therefore, we will only mention TGF-β in

Distributional data were the first to suggest a role of TGF-βs in the regulation of neuronal differentiation. During the development of the central nervous system, TGF-β immunostaining was most prominent in zones where neuronal differentiation occurs and less intense in zones of active proliferation (Flanders et al., 1991). Subsequent in vitro experiments using quail neural crest cell demonstrated that TGF-β inhibits proliferation of neural crest cells while neurogenesis increased significantly in the presence of TGF-β (Zhang et al., 1997). Subsequent experiments using brains supported an inhibitory role of TGF-βs on neuronal stem cell proliferation (Aigner & Bogdahn, 2008). TGF-β had an antimitotic effect on progenitors and increased expression of neuronal markers in hippocampal and cortical primary cell cultures of developing mouse (Vogel et al., 2010). These effects were dependent upon Smad4. Furthermore, in vivo loss-of-function analyses using TGF-β2(-/-)/TGF-β3(-/-) double mutant mice showed the opposite effect of increased cell proliferation and fewer neurons in the cerebral cortex and hippocampus (Vogel et al., 2010). TGF-β may also play a role in the regulation of adult neurogenesis as it had a pro-neurogenic effect in the dentate gyrus in a model of increased neurogenesis by adrenalectomy as well as in the subventricular zone when administered chronically with adenoviral vectors expressing TGF-β (Mathieu et al., 2011). Furthermore, adrenalectomy increased TGF-β levels in the dentate gyrus while blockade of TGF-β biological activity by administration of an anti-TGF-

midbrain, cerebellum, brain stem and hippocampus (Bottner et al., 1996).

β type II receptor antibody diminished neurogenesis (Battista et al., 2006).

Apart from playing a role in the adoption of neuronal cell fate, TGF-β may also be involved in the differentiation of selected neuronal isoforms at the expense of other isoforms. Within the intermediate and ventral domains, Smad3 promoted differentiation of ventral interneurons at the expense of motoneuron generation. Consequently, the absence of Smad3 expression from the motoneuron progenitor domain during pattern formation of the neural tube was a prerequisite for the correct generation of spinal motoneurons (Garcia-Campmany & Marti, 2007). In turn, the survival of motoneurons may also depend on TGFβs as a potentially continous trophic support factor from muscle fibres or other cell types. Using cultures of purified chick embryonic motoneurons, TGF-βs acted synergistically with basic fibroblast growth factor to keep motoneurons alive (Gouin et al., 1996). Indeed, motoneurons were shown to synthesize TGF-β receptors and to transport them anterogradely, where they were inserted into the axonal membrane and nerve terminal

**4. The role of TGF-βs in neural functions** 

**4.1 Neuronal differentiation and survival** 

these cases.

widespread expression of the mRNA of this isoform was described including intense labeling in some cortical and hippocampal cells, the medial preoptic area, the paraventricular hypothalamic nucleus, the central amygdaloid nucleus, and the superior olive. Furthermore, TGF-β2 and β3 immunoreactivities entirely overlapped and, in general, were found in large multipolar neurons (Unsicker et al., 1991) with the level of TGF-ß2 being considerably higher (Bottner et al., 2000). In some areas, including brainstem motoneurons and the area postrema, the 2 isoforms had similar mRNA expression patterns with high intensity labeling suggesting that different isoforms of TGF-βs may be co-expressed in the same cell. In most brain areas, however, the distributions of TGF-ß2 and-ß3 mRNAs were markedly different. In the cerebral cortex, TGF-βs were expressed in different layers. In the hippocampus, TGF-β2 was abundantly expressed only in the dentate gyrus while TGF-β3 in the CA2 region and the dentate gyrus. In the cerebellum, TGF-β2 was present in the Purkinje cell layer while TGF-β3 mRNA was absent in the cerebellum. In addition, the medial mamillary nucleus, the parafascicular thalamic nucleus and the choroid plexus expressed predominantly TGF-β2 while the reticular thalamic nucleus, the superior colliculus, and the inferior olive contained almost exclusively TGF-β3 mRNA (Vincze et al., 2010). An important future question is the type of cells that express TGF-β in the central nervous system. Most previous studies examined the cell type of TGF-β expression following some type of induction. TGF-β1 upregulation in astrocytes and microglia has been reported to be a predominant response to lesion and during pathology (Krohn, 1999; Wu et al., 2007; Wu et al., 2008) that results in the induction of reactive phenotypes (Flanders et al., 1998; Morgan et al., 1993). Under basal conditions, astrocytes were also shown to express TGF-β1 in the preoptic area (Bouret et al., 2004; Dhandapani & Brann, 2003). However, neuronal expression of TGF-β1 has also been reported (Battaglia et al., 2011; Lacmann et al., 2007; Wu et al., 2007). The available data on the cell type specific expression of other TGF-β isoforms is scarce. However, their distributions suggest a dominant neuronal expression (Unsicker et al., 1991; Vincze et al., 2010).

The four types of LTBPs also had distinct distribution patterns in the brain based on the localization of their mRNAs (Dobolyi & Palkovits, 2008). The dominant form in the brain was LTBP3 while LTBP4 also had high level of expression in a variety of forebrain areas. LTBP1 had considerable level of expression in only some brain regions including the choroid plexus, the cerebral cortex, the medial amygdaloid nucleus, the anteromedial and midline thalamic nuclei, the medial preoptic area, the arcuate and dorsomedial hypothalamic nuclei, the superior olive and the area postrema. LTBP2 expression was restricted to the cerebral cortex, the hippocampus, and the lateral hypothalamus (Dobolyi & Palkovits, 2008). Comparison of the distribution of TGF-β and LTBP subtypes suggested that all 3 isoforms of TGF-βs are co-expressed with LTBP3 in the brain. In addition, TGF-βs might also bind to other types of LTBPs in certain brain regions. For example, the distribution of TGF-β1 and LTBP-4 is similar in the supraoptic nucleus and the central nucleus of the amygdala. The choroid plexus, where TGF-β2 expression is dominant contains LTBP1 and 3. The inferior olive and the arcuate nucleus, brain areas with dominant TGF-β3 expression contain large amount of LTBP4 and LTBP1, respectively (Dobolyi & Palkovits, 2008). Nevertheless, further double labeling studies are needed to actually establish co-expression of different isoforms of TGF-βs and LTBPs in single cells of the nervous system.

widespread expression of the mRNA of this isoform was described including intense labeling in some cortical and hippocampal cells, the medial preoptic area, the paraventricular hypothalamic nucleus, the central amygdaloid nucleus, and the superior olive. Furthermore, TGF-β2 and β3 immunoreactivities entirely overlapped and, in general, were found in large multipolar neurons (Unsicker et al., 1991) with the level of TGF-ß2 being considerably higher (Bottner et al., 2000). In some areas, including brainstem motoneurons and the area postrema, the 2 isoforms had similar mRNA expression patterns with high intensity labeling suggesting that different isoforms of TGF-βs may be co-expressed in the same cell. In most brain areas, however, the distributions of TGF-ß2 and-ß3 mRNAs were markedly different. In the cerebral cortex, TGF-βs were expressed in different layers. In the hippocampus, TGF-β2 was abundantly expressed only in the dentate gyrus while TGF-β3 in the CA2 region and the dentate gyrus. In the cerebellum, TGF-β2 was present in the Purkinje cell layer while TGF-β3 mRNA was absent in the cerebellum. In addition, the medial mamillary nucleus, the parafascicular thalamic nucleus and the choroid plexus expressed predominantly TGF-β2 while the reticular thalamic nucleus, the superior colliculus, and the inferior olive contained almost exclusively TGF-β3 mRNA (Vincze et al., 2010). An important future question is the type of cells that express TGF-β in the central nervous system. Most previous studies examined the cell type of TGF-β expression following some type of induction. TGF-β1 upregulation in astrocytes and microglia has been reported to be a predominant response to lesion and during pathology (Krohn, 1999; Wu et al., 2007; Wu et al., 2008) that results in the induction of reactive phenotypes (Flanders et al., 1998; Morgan et al., 1993). Under basal conditions, astrocytes were also shown to express TGF-β1 in the preoptic area (Bouret et al., 2004; Dhandapani & Brann, 2003). However, neuronal expression of TGF-β1 has also been reported (Battaglia et al., 2011; Lacmann et al., 2007; Wu et al., 2007). The available data on the cell type specific expression of other TGF-β isoforms is scarce. However, their distributions suggest a dominant neuronal expression

The four types of LTBPs also had distinct distribution patterns in the brain based on the localization of their mRNAs (Dobolyi & Palkovits, 2008). The dominant form in the brain was LTBP3 while LTBP4 also had high level of expression in a variety of forebrain areas. LTBP1 had considerable level of expression in only some brain regions including the choroid plexus, the cerebral cortex, the medial amygdaloid nucleus, the anteromedial and midline thalamic nuclei, the medial preoptic area, the arcuate and dorsomedial hypothalamic nuclei, the superior olive and the area postrema. LTBP2 expression was restricted to the cerebral cortex, the hippocampus, and the lateral hypothalamus (Dobolyi & Palkovits, 2008). Comparison of the distribution of TGF-β and LTBP subtypes suggested that all 3 isoforms of TGF-βs are co-expressed with LTBP3 in the brain. In addition, TGF-βs might also bind to other types of LTBPs in certain brain regions. For example, the distribution of TGF-β1 and LTBP-4 is similar in the supraoptic nucleus and the central nucleus of the amygdala. The choroid plexus, where TGF-β2 expression is dominant contains LTBP1 and 3. The inferior olive and the arcuate nucleus, brain areas with dominant TGF-β3 expression contain large amount of LTBP4 and LTBP1, respectively (Dobolyi & Palkovits, 2008). Nevertheless, further double labeling studies are needed to actually establish co-expression of different isoforms of TGF-βs and LTBPs in single cells of the

(Unsicker et al., 1991; Vincze et al., 2010).

nervous system.

Although the topographical distribution of TGF-β receptors in the central nervous system has not been systematically described, the available data suggest widespread localization. When TGF-β receptor mRNA was detected by RT-PCR in rats at different stages of development similar levels were found in several regions of the CNS, including cortex, midbrain, cerebellum, brain stem and hippocampus (Bottner et al., 1996).
