**4.2 Synaptic transmission and plasticity**

134 Neuroscience – Dealing with Frontiers

(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).

1995).

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.,

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

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

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,

the peripheral motor system (Rahhal et al., 2009).

neuronal contact (Guenard et al., 1995; Parkinson et al., 2001).

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

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).

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 "synaptogenic state".

#### **4.3 Involvement in inflammatory and neuroendocrine functions**

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

Transforming Growth Factor Beta in the Central Nervous System 137

A number of studies have documented that TGF-β1 levels are enhanced in the brain following cerebral ischemia (Dhandapani & Brann, 2003). The expressions of other isoforms are also enhanced albeit with a different pattern around a focal lesion. Following middle cerebral artery occlusion, TGF-β1 expression was elevated in the penumbra around the lesion site while TGF-β2 and 3 expressions showed increases in particular cortical layers

TGF-β1 administered into the brain reduced the infarct size dose dependently in experimental models of ischemia including permanent occlusion of the left middle cerebral artery by microbipolar electrocoagulation in mice (Prehn et al., 1993), autologous clot embolus injection into the right internal carotid artery in rabbit (Gross et al., 1993), and transient middle cerebral artery occlusion in rat (Zhu et al., 2002). In turn, antagonizing the endogenous action of TGF-β1 with a soluble TGF-β type II receptor resulted in a dramatic increase in infarct area (Ruocco et al., 1999). An intracortical injection of the soluble antagonsi in rats subjected to a 30-minute reversible cerebral focal ischemia aggravated the volume of infarction (Ruocco et al., 1999). These results suggest that, in response to an ischemic insult, brain tissue responds by the synthesis of TGF-β1, which is involved in the

Despite the accumulating knowledge on the signal transduction of TGF-βs, the signaling pathway mediating its protective effect is not fully understood. Bad is a proapoptotic member of the Bcl-2 family and is inactivated on phosphorylation via mitogen-activated protein kinase (MAPK). A gradual activation of extracellular signal-regulated kinase 1/2 and MAPK-activated protein kinase-1 and a concomitant increase in Bad phosphorylation in mouse brains after adenovirus-mediated TGF-β1 transduction under nonischemic and ischemic conditions induced by transient middle cerebral artery occlusion (Zhu et al., 2002). Consistent with these effects, the ischemia-induced increases in Bad protein level and caspase-3 activation were suppressed in TGF-β1-transduced brain. Consequently, DNA fragmentation, ischemic lesions, and neurological deficiency were significantly reduced. Furthermore, inhibitors of the MAPK signal transduction pathway abolished the neuroprotective activity of TGF-β1 in staurosporine-induced apoptosis, indicating that activation of MAPK is necessary for the antiapoptotic effect of TGF-β1 (Zhu et al., 2002). These data suggest that TGF-β1 regulates the expression and ratio of apoptotic (Bad) and antiapoptotic proteins, creating an environment favorable for cell survival of death-inducing

The physiological role of astrogliosis remains controversial with respect to the beneficial or detrimental influence of reactive astrocytes on CNS recovery. On the one hand, the very dense network of processes built up in the scar by reactive astrocytes suggests that the scar tissue may fulfill important functions as a barrier isolating and protecting the intact tissue from the lesions, from which toxic molecules could be released. On the other hand, molecules expressed in lesion scars on the astroglial cell surface or secreted molecules render the reactive astrocyte a less favorable substrate, which could be inhibitory to neuritic outgrowth. Nevertheless, scar formation in the nervous system begins within hours after

**5.1 Neuroprotective function in brain ischemia** 

limitation of the extent of the injury.

insults (Dhandapani & Brann, 2003).

**5.2 TGF-β in astrogliosis** 

throughout the ipsilateral cerebral cortex (Vincze et al., 2010).

The potential involvement of TGF-β in central reproductive regulation is also an emerging topic. Gonadotropin-releasing hormone neurons in the preoptic area contain TGF-β receptors as well as SMAD2/3 suggesting that they are fully capable of responding directly to TGF-β1 stimulation (Prevot et al., 2000). Subsequent double-labeling experiments showed that astrocytes in the preoptic area expressed TGF-β1 mRNA and that GnRH perikarya were often found in close association with TGF-β1 mRNA-expressing cells (Bouret et al., 2004). Incubation of preoptic explants with TGF-β1 caused a significant, dose-dependent decrease in GnRH mRNA expression in individual neurons. This effect was inhibited by addition of the soluble form of TGF-β-RII to the incubation medium (Bouret et al., 2004). These results support that astrocyte-derived TGF-β1 may directly influence GnRH expression and/or secretion in vivo by acting on the perikarya of GnRH neurons.

Intracisternal administration of TGF-β induces an increase in fat oxidation while intracisternal administration of anti-TGF-β antibody partially inhibits an increase in fat oxidation during treadmill running in rats indicating a regulatory role of TGF-β in the brain on fat oxidation during exercise (Fujikawa et al., 2007). Since TGF-β3 increased noradrenaline levels in the paraventricular and ventromedial hypothalamic nuclei and chemical lesion of noradrenaline input to these nuclei completely abolished the regulatory effect of TGF-β on fat oxidation it has been suggested that TGF-β in the brain enhances fat oxidation via noradrenergic neurons in the paraventricular and ventromedial hypothalamic nuclei (Fujikawa et al., 2007). An inhibitor of FA oxidation could induce an activation of TGF-β in the CSF suggesting that shortage of energy derived from fatty acids leads to the activation of TGF-β (Fujikawa et al., 2011).

TGF-β1 and 3 also co-localize with arginine vasopressin in magnocellular neurons of the supraoptic and paraventricular nuclei of the hypothalamus suggesting that TGF-β secreted by the neurohypophysis might regulate the proliferation and secretion of certain anterior pituitary cells (Fevre-Montange et al., 2004). So far, the regulatory role of TGF-β on hormone secretion, gene transcription, and cellular growth of prolactin-producing cells has been shown. TGF-β inhibited the transcriptional activity of the estrogen receptor although estrogens had no effect on TGF-β-specific Smad protein transcriptional activity (Giacomini et al., 2009). A diurnal pattern of expression of TGF-β as well as SMAD3 was found in the suprachiasmatic and paraventricular nuclei of young animals, a rhythm that was not onserved in older mice suggesting a diurnal and age-dependent function of the TGF-β system in these nuclei (Beynon et al., 2009). These expressional data indicate numerous yet unexplored functions of TGF-βs in the hypothalamus. Therefore, promising future investigations on the role of TGF-βs in endocrine regulations are eagerly awaited.
