**2. Biochemistry of transforming growth factor-βs (TGF-βs)**

#### **2.1 Release and activation of TGF-βs**

A characteristic feature in the biology of TGF-βs is that they are usually secreted from cells in latent forms. TGF-βs are synthesized as homodimeric proproteins (proTGF-βs). These

Transforming Growth Factor Beta in the Central Nervous System 131

With regard to mediation of TGF-β actions, different TGF-β receptors have been identified (Massague, 1992). Type I and type II TGF-β receptors bind TGF-βs with high-affinity. Ligand binding induces the assembly of type I and type II receptors into complexes. The exact stoichiometry in the TGF-β-induced heteromeric type I and type II receptor complex is likely to be a heterotetramer comprising two TGF-β type I and two TGF-β type II receptors. Following activation, type II receptors phosphorylate type I receptors in the juxtamembrane region. This phosphorylation is both essential and sufficient for TGF-β signalling (ten Dijke & Hill, 2004). In addition, the type III TGF-β receptor also binds TGF-βs with high affinity. However, it is an extracellular protein, which does not lead to signal transduction. In fact, it

Both type I and type II receptors are single-pass transmembrane proteins with a serine/threonine kinase domain on the cytosolic side of the plasma membrane (Arighi et al., 2009; Attisano & Wrana, 2002). The activated type I kinase propagates the signal inside the cell through the phosphorylation of receptor-regulated Smads (R-Smads: Smad1, Smad2, Smad3, Smad5 and Smad8). Access of the R-Smads to the type I receptors is facilitated by auxiliary proteins such as Smad anchor for receptor activation. Activated R-Smads form heteromeric complexes with Smad4. These complexes accumulate in the nucleus, where they control gene expression in a cell-type-specific and ligand dose-dependent manner. Inhibitory Smads (I-Smads: Smad6 and Smad7) form a distinct subclass of Smads that act in an opposing manner to R-Smads and antagonize signalling (Padgett et al., 1998; Schmierer

TGF-β also uses non-Smad signaling pathways such as the p38 and Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways to convey its signals. Other potential TGF-β-induced non-Smad signaling pathways include the phosphoinositide 3 kinase-Akt-mTOR pathway, the small GTPases Rho, Rac, and Cdc42, and the Ras-Erk-

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

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

**2.4 Receptors and signal transduction pathways** 

& Hill, 2007).

**nervous system** 

MAPK pathway (Mu et al., 2011).

may be a negative regulator of TGF-β function (Chu et al., 2011).

precursor proteins are modified intracellularly prior to secretion. The C-terminal proregions are cleaved from the N-terminal portion of the protein and in the trans Golgi to provide mature TGF-β and the latency-associated protein (LAP) which remain noncovalently associated to form the small latent complex (Clark & Coker, 1998; Khalil, 1999). In turn, disulfide bridges bind the so-called latent TGF-β binding proteins (LTBPs) to this complex to result the TGF-β large latent complex, in which TGF-βs are present in the extracellular space (Koli et al., 2001; Saharinen et al., 1999). LTBPs are large multidomain proteins belonging to the fibrillin-LTBP family of extracellular matrix proteins. LTBPs have a typical repeated domain structure consisting mostly of epidermal growth factor (EGF)-like repeats and characteristic eight cysteine (8-Cys) repeats. They are required for the proper folding and secretion of TGF-βs (Sinha et al., 1998; Todorovic et al., 2005). Following secretion, TGF-β is deposited to the extracellular matrix in the pericellular space covalently via the N-termini of the LTBPs. LTBPs contain multiple proteinase sensitive sites, providing means to solubilize the large latent complex from the extracellular matrix structures (Keski-Oja et al., 2004; Rifkin, 2005).

#### **2.2 Latent TGF-β binding proteins**

There are four mammalian LTBP isoforms encoded by distinct genes, including LTBP-1, -2, - 3, and -4 and different splice variants for each of them (Mangasser-Stephan & Gressner, 1999; Oklu & Hesketh, 2000). The significance of this structural diversity is mostly unclear at present. The potential selective binding of different LTBPs to different proTGF-β types is not well characterized yet. *In vitro* studies suggest that LTBP-1, and -3 can bind to all three types of proTGF-β types efficiently whereas LTBP-2 does not bind to proTGF-βs (Saharinen & Keski-Oja, 2000). TGF-β associated with the large latent complex cannot interact with its receptor and has no biological effect. Therefore, TGF-β activity is regulated by the release of mature TGF-β from the large latent complex. Alternatively, the large latent complex undergoes a conformational change, which exposes the TGF-βs to their receptor binding sites. Many potential activators of the extracellular TGF-βs have been proposed (Annes et al., 2003; Gumienny & Padgett, 2002) including proteases, thrombospondin-1, integrins, reactive oxygen species, and pH. The activation of the 3 different isoforms of TGF-βs may be different. Furthermore, the type of LTBPs may affect the way of activation of TGF-βs (Rifkin, 2005).

#### **2.3 Release of TGF-βs from neurons**

Apart from various peripheral cell types, the model neuron chromaffin cell has also been demonstrated to possess regulated secretion of TGF-β and LTBPs (Krieglstein & Unsicker, 1995). Cholinergic stimulation of bovine chromaffin cells leads to the release of storage vesicles. The released content of the vesicles was shown to contain TGF-β but not other members of the TGF-β superfamily suggesting that TGF-β is stored in chromaffin granules and can be released by exocytosis (Krieglstein & Unsicker, 1995). In addition, the level of active TGF-β has been suggested to be increased by elevated neuronal activity (Lacmann et al., 2007). In primary cell culture of embryonic hippocampal neurons, various treatments leading to increased neuronal activity resulted in tetrodotoxon-dependent elevation of active TGF-β levels (Lacmann et al., 2007).

#### **2.4 Receptors and signal transduction pathways**

130 Neuroscience – Dealing with Frontiers

precursor proteins are modified intracellularly prior to secretion. The C-terminal proregions are cleaved from the N-terminal portion of the protein and in the trans Golgi to provide mature TGF-β and the latency-associated protein (LAP) which remain noncovalently associated to form the small latent complex (Clark & Coker, 1998; Khalil, 1999). In turn, disulfide bridges bind the so-called latent TGF-β binding proteins (LTBPs) to this complex to result the TGF-β large latent complex, in which TGF-βs are present in the extracellular space (Koli et al., 2001; Saharinen et al., 1999). LTBPs are large multidomain proteins belonging to the fibrillin-LTBP family of extracellular matrix proteins. LTBPs have a typical repeated domain structure consisting mostly of epidermal growth factor (EGF)-like repeats and characteristic eight cysteine (8-Cys) repeats. They are required for the proper folding and secretion of TGF-βs (Sinha et al., 1998; Todorovic et al., 2005). Following secretion, TGF-β is deposited to the extracellular matrix in the pericellular space covalently via the N-termini of the LTBPs. LTBPs contain multiple proteinase sensitive sites, providing means to solubilize the large latent complex from the extracellular matrix structures (Keski-

There are four mammalian LTBP isoforms encoded by distinct genes, including LTBP-1, -2, - 3, and -4 and different splice variants for each of them (Mangasser-Stephan & Gressner, 1999; Oklu & Hesketh, 2000). The significance of this structural diversity is mostly unclear at present. The potential selective binding of different LTBPs to different proTGF-β types is not well characterized yet. *In vitro* studies suggest that LTBP-1, and -3 can bind to all three types of proTGF-β types efficiently whereas LTBP-2 does not bind to proTGF-βs (Saharinen & Keski-Oja, 2000). TGF-β associated with the large latent complex cannot interact with its receptor and has no biological effect. Therefore, TGF-β activity is regulated by the release of mature TGF-β from the large latent complex. Alternatively, the large latent complex undergoes a conformational change, which exposes the TGF-βs to their receptor binding sites. Many potential activators of the extracellular TGF-βs have been proposed (Annes et al., 2003; Gumienny & Padgett, 2002) including proteases, thrombospondin-1, integrins, reactive oxygen species, and pH. The activation of the 3 different isoforms of TGF-βs may be different. Furthermore, the type of LTBPs may affect the way of activation of TGF-βs (Rifkin,

Apart from various peripheral cell types, the model neuron chromaffin cell has also been demonstrated to possess regulated secretion of TGF-β and LTBPs (Krieglstein & Unsicker, 1995). Cholinergic stimulation of bovine chromaffin cells leads to the release of storage vesicles. The released content of the vesicles was shown to contain TGF-β but not other members of the TGF-β superfamily suggesting that TGF-β is stored in chromaffin granules and can be released by exocytosis (Krieglstein & Unsicker, 1995). In addition, the level of active TGF-β has been suggested to be increased by elevated neuronal activity (Lacmann et al., 2007). In primary cell culture of embryonic hippocampal neurons, various treatments leading to increased neuronal activity resulted in tetrodotoxon-dependent elevation of

Oja et al., 2004; Rifkin, 2005).

2005).

**2.2 Latent TGF-β binding proteins** 

**2.3 Release of TGF-βs from neurons** 

active TGF-β levels (Lacmann et al., 2007).

With regard to mediation of TGF-β actions, different TGF-β receptors have been identified (Massague, 1992). Type I and type II TGF-β receptors bind TGF-βs with high-affinity. Ligand binding induces the assembly of type I and type II receptors into complexes. The exact stoichiometry in the TGF-β-induced heteromeric type I and type II receptor complex is likely to be a heterotetramer comprising two TGF-β type I and two TGF-β type II receptors. Following activation, type II receptors phosphorylate type I receptors in the juxtamembrane region. This phosphorylation is both essential and sufficient for TGF-β signalling (ten Dijke & Hill, 2004). In addition, the type III TGF-β receptor also binds TGF-βs with high affinity. However, it is an extracellular protein, which does not lead to signal transduction. In fact, it may be a negative regulator of TGF-β function (Chu et al., 2011).

Both type I and type II receptors are single-pass transmembrane proteins with a serine/threonine kinase domain on the cytosolic side of the plasma membrane (Arighi et al., 2009; Attisano & Wrana, 2002). The activated type I kinase propagates the signal inside the cell through the phosphorylation of receptor-regulated Smads (R-Smads: Smad1, Smad2, Smad3, Smad5 and Smad8). Access of the R-Smads to the type I receptors is facilitated by auxiliary proteins such as Smad anchor for receptor activation. Activated R-Smads form heteromeric complexes with Smad4. These complexes accumulate in the nucleus, where they control gene expression in a cell-type-specific and ligand dose-dependent manner. Inhibitory Smads (I-Smads: Smad6 and Smad7) form a distinct subclass of Smads that act in an opposing manner to R-Smads and antagonize signalling (Padgett et al., 1998; Schmierer & Hill, 2007).

TGF-β also uses non-Smad signaling pathways such as the p38 and Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways to convey its signals. Other potential TGF-β-induced non-Smad signaling pathways include the phosphoinositide 3 kinase-Akt-mTOR pathway, the small GTPases Rho, Rac, and Cdc42, and the Ras-Erk-MAPK pathway (Mu et al., 2011).
