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

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 these cases.

### **4.1 Neuronal differentiation and survival**

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-TGFβ 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

Transforming Growth Factor Beta in the Central Nervous System 135

infectious agents and other cell-damaging circumstances (e.g., traumatic or ischemic conditions) can lead to apoptosis. TGF-β1 has been recently characterized as an antiapoptotic factor in a model of staurosporine-induced neuronal death through a mechanism involving activation of the extracellular signal-regulated kinase 1/2 and a concomitant increase phosphorylation of the antiapoptotic protein Bad. 5 (Buisson et al., 2003). This action of TGF-β may be involved in its neuroprotective actions (see below).

TGF-β2 was demonstrated to influence synaptic transmission, rather than synaptogenesis, at some central synapses (Heupel et al., 2008). TGF-β2 was found to be essential for proper synaptic function in the pre-Botzinger complex, a central rhythm organizer located in the brainstem while it was not crucial for the morphology and function of the neuromuscular junction of the diaphragm muscle. Genetic deletion of TGF-β2 in mice strongly impaired both GABA/glycinergic and glutamatergic synaptic transmission in the pre-Botzinger complex area, while numbers and morphology of central synapses of knock-out animals were indistinguishable from their wild-type littermates at embryonic day 18.5 (Heupel et al., 2008). The role of TGF-β in synaptic transmission might be the basis of its proposed function in synaptic facilitation. Prolonged treatment with TGF-β2 induced facilitation of evoked postsynaptic currents in hippocampal neurons suggesting that it may play a role in the cascade of events underlying long-term synaptic facilitation (Fukushima et al., 2007). The long-term electrophysiological changes may be associated with cAMP response elementbinding protein (CREB) because TGF-β2 enhanced the phosphorylation of CREB previously

The effect of TGF-β on synaptogenesis has also been proposed. In particular, TGF-β1 was identified as the molecule responsible for the synaptogenesis promiting effect of Schwann cell-conditioned medium in Xenopus nerve-muscle cocultures (Feng & Ko, 2008). TGF-β1 increased agrin expression and synaptogenesis were along nerve-muscle contacts while immunodepletion of TGF-β1 with a specific antibody abolished the synaptogenic effect of Schwann cell-conditioned medium (Feng & Ko, 2008). These results indicate that TGF-β1 may be a glial signal that instructs neurons to switch from a "growth state" to a

In an induced inflammatory model, the concentration of TGF-β increased in cerebrospinal fluid. This increase occurred earlier than those in the concentrations of other proinflammatory cytokines (Matsumura et al., 2008). In another inflammatory model, systemic injection of complete Freund's adjuvant, TGF-β1 and TGF-β receptor II both markedly increased in the leptomeninges and the parenchymal cells (Wu et al., 2007). Double-staining immunohistochemistry demonstrated TGF-β1 to be induced in both glial cells and cortical neurons, whereas TGF-βRII was induced only in cortical neurons. The intracisternal administration of an anti-TGF-β antibody partially inhibited the resulting fever (Matsumura et al., 2007). Furthermore, intracisternal administration of TGF-β dosedependently raised the body temperature (Matsumura et al., 2008). These findings suggest a novel function of TGF-β as a proinflammatory cytokine in the central nervous system

**4.2 Synaptic transmission and plasticity** 

implicated in long-term potentiation (Fukushima et al., 2007).

**4.3 Involvement in inflammatory and neuroendocrine functions** 

"synaptogenic state".

(Jiang et al., 2000a) Furthermore, TGF-β2 was detected in the synaptic portions of muscle fibres, motoneurons and in injured nerves, indicating that motoneurons may be exposed to multiple and potentially redundant sources of transforming growth factor-beta 2 (Jiang et al., 2000a). In addition, double-ligation experiments were used to demonstrate that motoneurons transport transforming growth factor-beta 2 up and down their axons (Jiang et al., 2000a). To test the effect of TGF-β on motoneuron survival in vivo, TGF-β2 was administered to the hypoglossal nucleus following the avulsion of the hypoglossal nerve in adult rats, which caused a significant attenuation of the motoneuron cell death in a low dose (Jiang et al., 2000b). TGF-β2 was, however, unable to prevent or reduce the axotomyinduced down regulation of choline acetyltransferase suggesting that TGF-β2 is only one of the growth factors regulating the homeostasis of motoneurons (Jiang et al., 2000b).

In addition to motoneurons, TGF-βs may also be required for the differentiation of midbrain dopaminergic neurons influencing motor activity, emotional behavior, and cognition and being involved in the generation of Parkinson's disease, a neurodegenerative disorder of dopaminergic neurons (Markus, 2007). Treatment of cells dissociated from the rat embryonic day 12 midbrain floor with TGF-β significantly increased the number of tyrosine hydroxylase (TH)-positive dopaminergic neurons within 24 h. Neutralization of TGF-β in vitro completely abolished the induction of dopaminergic neurons (Farkas et al., 2003). In addition to the development, the survival of midbrain dopaminergic neurons may also depend on TGF-β. Administration of TGF-β2 and TGF-β3, prevented the death of cultured rat embryonic midbrain dopaminergic neurons at picomolar concentrations (Poulsen et al., 1994). Furthermore, they provided protection against N-methyl-4-phenylpyridinium ion (MPP+) toxicity of dopaminergic neurons (Krieglstein et al., 1995). In contrast to some other cytokines affecting dopaminergic neurons the mechanism of action of the TGF-βs did not involve cell proliferation or delivery of growth factors from astroglial cells (Krieglstein et al., 1995).

Since TGF-β is only one of the factors regulating the differentiation and survival of motoneurons and midbrain dopaminergic cells, their interactions with other regulatory molecules has been examined. For example, glial cell line-derived neurotrophic factor (GDNF) is also a potent survival factor for dopaminergic neurons in culture whose effect can be potentiated by TGF-βs (Poulsen et al., 1994). However, while TGF-β is required for the induction of dopaminergic neurons, GDNF is only required for regulating and/or maintaining a differentiated neuronal phenotype (Roussa et al., 2008). A cooperative role of TGF-β2 and GDNF with regard to promotion of survival has also been demonstrated within the peripheral motor system (Rahhal et al., 2009).

Finally, it has to be emphasized that motoneurons and midbrain dopaminergic cells are 2 neuronal cell types whose development has been demonstrated to be affected by TGF-β. A role of TGF-β in the differentiation and survival of other, as yet unexplored neuronal cell types might also be possible. As far as glial cells, data are available that in the peripheral nervous system, TGF-β regulates the degree of Schwann cell proliferation induced by neuronal contact (Guenard et al., 1995; Parkinson et al., 2001).

TGF-βs are also involved in apoptosis, the genetically regulated form of cell death. Apoptosis enables the balance between growth and elimination of cells and occurs physiologically during the embryonal development or involution processes. Furthermore, infectious agents and other cell-damaging circumstances (e.g., traumatic or ischemic conditions) can lead to apoptosis. TGF-β1 has been recently characterized as an antiapoptotic factor in a model of staurosporine-induced neuronal death through a mechanism involving activation of the extracellular signal-regulated kinase 1/2 and a
