**4.1 GDNF receptors and structures**

#### **4.1.1 GFR**α

196 Etiology and Pathophysiology of Parkinson's Disease

The monomer structure of GDNF includes a central α-spiral and β-fold (A and B). The amino acids of A include two reverse and parallel β-folds. The C-terminal of B contains four β-folds. The dimer, with a reverse and parallel structure, presents a symmetrical receptor

In mammalian brains, GDNF is mainly expressed in the target cells of DA-ergic neurons in the midbrain substantia nigra, such as granular cells in the corpora striatum, nucleus accumbens, thalamic nuclei, tuber olfactorium, hippocampus, cerebellum, callosal gyrus and olfactory bulb, where it acts as the original neurotrophic factor (Sonntag *et al.*, 2005). In the kidneys and gastrointestinal tract, GDNF is expressed in media cells and smooth muscle cells, respectively. Additionally, GDNF induces the differentiation of neural crest cells and Wolffian ducts in intestinal neurons and renal ducts, respectively (Bohn *et al.*, 2000). The peripheral expression of GDNF can also been seen in tissues innervated by sensory neurons (trigeminal ganglion, nodosal ganglion and dorsal root ganglion) and autonomic neurons. The distribution of GDNF in the human brain, which is identical to that in the rat brain, includes high expression in the caudate nucleus (innervated by the substantia nigra), low expression in the dorsal caudate putamen and no expression in the substantia nigra (Schaar *et al.*, 1994). GDNF has also been reported to be expressed in the hippocampus, cortex and spinal cord (Springer *et al.*, 1994). In general, the GDNF expression in peripheral organs is

In the CNS, GFLs maintain the survival and activity of DA-ergic neurons in the midbrain and motor neurons of the cornu anterius medullae spinalis. In addition, GDNF, NRTN and ARTN maintain the differentiation and survival of some peripheral neurons, such as sympathetic neurons, parasympathetic neurons, sensory neurons and neurons in the intestinal wall. Outside of the nervous system, GDNF plays vital roles in the processes of kidney development and spermatic production, along with many other processes in the

In the midbrain of adult animals, GDNF promotes the survival of DA-ergic neurons (Springer *et al.*, 1994). Some scholars have proposed that GDNF plays an essential role in supporting the survival and maintenance of mature DA-ergic neurons (Pascual *et al.*, 2008). Microinjection of 6-OHDA into the corpora striatum leads to the degeneration of DA-ergic neurons in the substantia nigra. This degeneration can be attenuated by pretreatment with GDNF in the substantia nigra, thus abrogating the injury induced by 6-OHDA and hindering the death of DA-ergic neurons. Other evidence has confirmed that GDNF promotes the regeneration of DA-ergic neurons after brain injury (Beck *et al.*, 1995; Tomac *et al.*, 1995; Hou *et al.*, 1996), which correlates with the fact that GDNF promotes the differentiation of DA-ergic neurons after brain injury. For example, GDNF promotes increase of the cell body, prolongation of axons and an increase in dopamine synthesis. Additionally, studies have demonstrated that GDNF can selectively protect DA-ergic neurons against the neurotoxic effects exerted by methylamphetamine (Cass, 1996). Thus far, GDNF, as a potential therapeutic agent for PD, has been studied extensively (Azzouz *et al.*, 2004; Eslamboli *et al.*, 2005). Further research has demonstrated that GDNF can modulate neuronal excitability and transmitter release (Wang *et al.*, 2003; Kobori *et al.*, 2004). For

body (Airaksinen & Saarma, 2002; Manié *et al.*, 2001; Airaksinen *et al.*, 1999).

binding site. A and B are crucial binding sites for GFRα1.

**3.2 GDNF distribution and expression** 

higher than that of neuronal tissues.

**3.3 The functions of GDNF** 

The biological actions exerted by GDNF on cells must be mediated via trans-membrane signal transmission, i.e. membrane receptors, as GDNF is a secreted protein (Josso & di Clemente, 1997). The receptor system for GFLs is divided into two parts. One part, a binding receptor, anchors to the cell surface via GPI and binds to GDNF directly. The identified GFLs include GFRα1, GFRα2, GFRα3 and GFRα4, each with its own binding receptor. For example, GDNF binds to GFRα1, NRTN binds to GFRα2, ARTN binds to GFRα3 and PSPN binds to GFRα4. The other part of the GFLs receptor system consists of the trans-membrane signal transduction receptor, RET, as well as a tyrosine kinase receptor. In the process of signal transduction, the members of the GDNF family share the same RET, but a distinct GFRα (Airaksinen *et al.*, 1999). The homology between GFRα1 and GFRα2 is as high as 48%, and both are expressed in the neurons and tissues of the superior cervical ganglion and dorsal root ganglion (Baloh *et al.*, 1997). The expression of GFRα3 is strictly limited, and is not seen in the CNS (Widenfalk *et al.*, 1998). In contrast, GFRα1 and GFRα2 are widely expressed in the CNS and peripheral organs (Nomoto *et al.* 1998). The expression of GFRα4 has only been observed in avian cells (Saarma & Sariola, 1999). In addition, GFRα contains three corresponding cysteine-rich domains, with different lengths for three structural domains. The second domain binds to GFL, while the functions of the first and third domains are not clear. The first domain is not involved in the binding of RET and GFRα1, and is not expressed in GFRα4.

#### **4.1.2 RET**

RET is a trans-membrane tyrosine kinase receptor encoded by the oncogene *Ret*. *Ret* was first discovered in mice NIH/3T3 cells. Identical to other tyrosine kinase receptors, the RET protein is composed of an extracellular ligand-binding domain, a trans-membrane domain and an intracellular functional tyrosine kinase domain. The RET protein contains 21 exons and many spliceosomes, with four cadherin-like domains and a cysteine-rich domain.

RET can be expressed as many sub-types based on the selective splicing of the primary *Ret* transcript (Tahira *et al.*, 1990; Lorenzo *et al.*, 1995). Two subtypes, RET9 and RET51, have been further studied, and the functional difference between the two is ascribed mainly to structural differences in the tails. The C-terminal end of RET9 contains nine amino acids, and the entire protein consists of 1072 amino acids. RET51 is longer than RET9, with a total of 1114 amino acids, including 51 amino acids in the tail. Both RET9 and RET51 have Tyr1062 in their C-terminal. Tyr1062 is phosphorylated during RET activation and anchoring of the receptor protein. Some researchers have demonstrated the regulation of lateral sequences in the C-terminal of Tyr 1062, which leads to different activities of the two

Actions of GDNF on Midbrain Dopaminergic Neurons: The Signaling Pathway 199

kinase, plays an important role in this process. First, FAK binds to corresponding proteins in the C terminus and phosphorylates itself. The phosphorylated tyrosine then binds to an SH2 domain in Src. Next, many amino acids sites in FAK are phosphorylated by Src which initiates a series of signals. The extracellular segment of the α subunit can bind to other proteins on the membrane, which would, in turn, bind to other SHCs. The phosphorylated tyrosines in SHC then bring the Grb2-SOS complex to the cell membrane and activate Ras. Integrins can bind to many extracellular matrix proteins, including laminin and fibronectin, which play vital roles in cell movement, adherence, synaptogenesis, proliferation, apoptosis, neural development and the inflammatory response (Chao *et al.*, 2003; Anton *et al.*, 1999). Some studies have indicated that GDNF is effective at increasing the expression of integrin αV, and the trans-membrane signal may include integrin β1. Hence, integrin αV and integrin β1 are thought to be candidate

N-cadherin, a member of the cadherin (cell adhesion molecule) super-family, is widely distributed in the CNS, and has a molecular weight of 130 kD. Cadherin super-family molecules are cell surface glucoproteins whose main functions include the regulation of calcium-mediated cell adhesion, cell polarity and morphogenesis, and involvement in the mechanisms underlying intercellular recognition and signal transduction. The recognition of N-cadherin is derived from its actions on cellular differentiation and growth in the CNS. Ncadherin is a canonical trans-membrane protein, with five extracellular segments (cadherin domains) composed of 110 amino acid residues. Research has demonstrated that the entire extracellular segment of N-cadherin is very similar to that of RET (Anders *et al.*, 2001) (Figure 2). The extracellular segments of N-cadherin can interact with catenin and other proteins, so as to regulate intracellular adhesion and recombination, and promote the

Accumulating evidence has demonstrated that both fibroblast growth factor (FGF) and epidermal growth factor (EGF) can exert their actions by binding to N-cadherin via their receptors. Recently, our studies (using plasmid construction and cell transfection) have demonstrated that inhibition of the biological functions of N-cadherin may influence the protective effects exerted by GDNF upon DA-ergic neurons. At the same time, we discovered that GDNF can bind to N-cadherin or RET directly (data not shown). Further studies should be undertaken to determine whether GDNF activation of downstream signal pathways is dependent on the interaction of its receptor and N-cadherin (a process that is

The distance from the neuronal adherent proteins' domain to the cell membrane is similar to that of RET. The encoding of the RET residues is consistent with the naïve RET molecule. The black solid line indicates the inserted sequence for the RET extracellular segment. The black solid circle indicates coherent calcium binding sites. CD = binding site for cadherin, TK = tyrosine kinase, Cys = cysteine-rich domain, Cyt = cytoplasmic domain (adapted from

Aside from RET and GFRα1, heparan sulfate proteoglycans, including syndecan and glypican, are essential in mediating the signal pathway of GDNF (Barnett *et al.*, 2002). Bespalov *et al.* demonstrated that syndecan, exerts its function via binding to heparan sulfate

receptors for GDNF (Cao *et al.*, 2008).

survival and migration of cells.

identical to FGF and EGF).

**4.1.5 Heparan sulfate glycosaminoglycans** 

with high affinity (Bespalov *et al.*, 2011).

Anders *et al.*,*,*2001)

subtypes (Wong *et al.*, 2005). Moreover, RET51 contains two tyrosine residues, Tyr 1090 and Tyr1096, which interact with SHC and GRB2, respectively (Lorenzo *et al.*, 1997; Alberti *et al.*, 1998; Borrello *et al.*, 2002).

RET, a common signal transduction receptor for GFLs, mediates many signal pathways for GDNF and induces apoptosis (Bordeaux *et al.*, 2000), and also inhibits tumor growth (Cañibano *et al.*, 2007) when GDNF is absent. The mutation of *Ret* may lead to diseases with functional depletion. Activating mutations correlate with thyroid cancer and type-II familial polyendocrine adenomatosis. A deactivating mutation of *Ret* is the cause of Hirchsprung disease. In adult animals, the depletion of RET leads to the depletion of DA-ergic cells in the nigrostriatal system. This indicates that RET is a key factor in maintaining the nigrostriatal system. The degradation of RET occurs through Cb-1 ubiquitin ligase mediated receptor recruitment and ubiquitination in the proteasome pathway.

#### **4.1.3 NCAM**

In the CNS, the regions of expression of GFRα1 and RET are not identical, e.g. GFRα1, but not RET, is expressed in the cortex and hippocampus (Trupp *et al.*, 1997; Glazner *et al.*, 1998; Golden *et al.*, 1998; Burazin & Gundlach, 1999; Golden *et al.*, 1999). The absence of RET expression in these regions indicates the existence of RET-independent GDNF signal pathways. In 2003, Paratcha *et al.* demonstrated that GDNF has the ability to transmit signals through directly binding to NCAM (Paratcha *et al.*, 2003). NCAM belongs to the cell adhesion molecule super family, and is encoded by a singular gene consisting of 26 exons (Lin *et al.*, 1993). The selective splicing of the gene can produce almost 120 NCAM subtypes, three of which are named NCAM-120, NCAM-140 and NCAM-180. These three subtypes possess an identical extracellular N-terminus, which contains five immunoglobulin-like domains and two domains located on the cell surface that are homologous with type-III fibronectin. Both NCAM-140 and NCAM-180 are trans-membrane proteins possessing the same trans-membrane domains but different intracellular domains. NCAM-120 has no trans-membrane or intracellular domains, and anchors to the extracellular surface via GPI. NCAM-180 and NCAM-120 are expressed predominantly in fetal neurons and neuroglia, respectively, while NCAM-140 is expressed in both (Noble *et al.*, 1985). Further research has demonstrated that the NCAM-140 subtype mediates the biological activities of GDNF. NCAM is not only involved in the adherence of cells, but also induces the intracellular signal transduction, and thus promotes axon growth and neuronal survival (Ditlevsen *et al.*, 2008) and inhibits the proliferation of astrocytes (Krushel *et al.*, 1998). The action of GDNF on DA-ergic neurons in the midbrain can be attenuated by blocking the function of NACM using anti-NACM antibodies, indicating that GDNF is activated via an NACM-transmitted signaling pathway, even in brain regions that express RET (Chao *et al.*, 2003).

#### **4.1.4 Integrins and N-cadherin**

Integrins are a family of cell surface receptors of cell adhesion molecules (CAMs). As a transmembrane glycoprotein, integrin is distributed on many types of cells, and can interact with extracellular ligands, the intracellular cystoskeleton and signal molecules to integrate the intra-with the extracellular environmental signals. Integrin is a heterodimer composed of an α and a β subunit, coupled by non-covalent bonds. Studies have indicated that integrin assembles some protein complexes via its β subunit, which is responsible for the specificity of those proteins and the subsequent signals. FAK, a non-receptor tyrosine

subtypes (Wong *et al.*, 2005). Moreover, RET51 contains two tyrosine residues, Tyr 1090 and Tyr1096, which interact with SHC and GRB2, respectively (Lorenzo *et al.*, 1997; Alberti *et al.*,

RET, a common signal transduction receptor for GFLs, mediates many signal pathways for GDNF and induces apoptosis (Bordeaux *et al.*, 2000), and also inhibits tumor growth (Cañibano *et al.*, 2007) when GDNF is absent. The mutation of *Ret* may lead to diseases with functional depletion. Activating mutations correlate with thyroid cancer and type-II familial polyendocrine adenomatosis. A deactivating mutation of *Ret* is the cause of Hirchsprung disease. In adult animals, the depletion of RET leads to the depletion of DA-ergic cells in the nigrostriatal system. This indicates that RET is a key factor in maintaining the nigrostriatal system. The degradation of RET occurs through Cb-1 ubiquitin ligase mediated receptor

In the CNS, the regions of expression of GFRα1 and RET are not identical, e.g. GFRα1, but not RET, is expressed in the cortex and hippocampus (Trupp *et al.*, 1997; Glazner *et al.*, 1998; Golden *et al.*, 1998; Burazin & Gundlach, 1999; Golden *et al.*, 1999). The absence of RET expression in these regions indicates the existence of RET-independent GDNF signal pathways. In 2003, Paratcha *et al.* demonstrated that GDNF has the ability to transmit signals through directly binding to NCAM (Paratcha *et al.*, 2003). NCAM belongs to the cell adhesion molecule super family, and is encoded by a singular gene consisting of 26 exons (Lin *et al.*, 1993). The selective splicing of the gene can produce almost 120 NCAM subtypes, three of which are named NCAM-120, NCAM-140 and NCAM-180. These three subtypes possess an identical extracellular N-terminus, which contains five immunoglobulin-like domains and two domains located on the cell surface that are homologous with type-III fibronectin. Both NCAM-140 and NCAM-180 are trans-membrane proteins possessing the same trans-membrane domains but different intracellular domains. NCAM-120 has no trans-membrane or intracellular domains, and anchors to the extracellular surface via GPI. NCAM-180 and NCAM-120 are expressed predominantly in fetal neurons and neuroglia, respectively, while NCAM-140 is expressed in both (Noble *et al.*, 1985). Further research has demonstrated that the NCAM-140 subtype mediates the biological activities of GDNF. NCAM is not only involved in the adherence of cells, but also induces the intracellular signal transduction, and thus promotes axon growth and neuronal survival (Ditlevsen *et al.*, 2008) and inhibits the proliferation of astrocytes (Krushel *et al.*, 1998). The action of GDNF on DA-ergic neurons in the midbrain can be attenuated by blocking the function of NACM using anti-NACM antibodies, indicating that GDNF is activated via an NACM-transmitted

signaling pathway, even in brain regions that express RET (Chao *et al.*, 2003).

Integrins are a family of cell surface receptors of cell adhesion molecules (CAMs). As a transmembrane glycoprotein, integrin is distributed on many types of cells, and can interact with extracellular ligands, the intracellular cystoskeleton and signal molecules to integrate the intra-with the extracellular environmental signals. Integrin is a heterodimer composed of an α and a β subunit, coupled by non-covalent bonds. Studies have indicated that integrin assembles some protein complexes via its β subunit, which is responsible for the specificity of those proteins and the subsequent signals. FAK, a non-receptor tyrosine

**4.1.4 Integrins and N-cadherin** 

recruitment and ubiquitination in the proteasome pathway.

1998; Borrello *et al.*, 2002).

**4.1.3 NCAM** 

kinase, plays an important role in this process. First, FAK binds to corresponding proteins in the C terminus and phosphorylates itself. The phosphorylated tyrosine then binds to an SH2 domain in Src. Next, many amino acids sites in FAK are phosphorylated by Src which initiates a series of signals. The extracellular segment of the α subunit can bind to other proteins on the membrane, which would, in turn, bind to other SHCs. The phosphorylated tyrosines in SHC then bring the Grb2-SOS complex to the cell membrane and activate Ras. Integrins can bind to many extracellular matrix proteins, including laminin and fibronectin, which play vital roles in cell movement, adherence, synaptogenesis, proliferation, apoptosis, neural development and the inflammatory response (Chao *et al.*, 2003; Anton *et al.*, 1999). Some studies have indicated that GDNF is effective at increasing the expression of integrin αV, and the trans-membrane signal may include integrin β1. Hence, integrin αV and integrin β1 are thought to be candidate receptors for GDNF (Cao *et al.*, 2008).

N-cadherin, a member of the cadherin (cell adhesion molecule) super-family, is widely distributed in the CNS, and has a molecular weight of 130 kD. Cadherin super-family molecules are cell surface glucoproteins whose main functions include the regulation of calcium-mediated cell adhesion, cell polarity and morphogenesis, and involvement in the mechanisms underlying intercellular recognition and signal transduction. The recognition of N-cadherin is derived from its actions on cellular differentiation and growth in the CNS. Ncadherin is a canonical trans-membrane protein, with five extracellular segments (cadherin domains) composed of 110 amino acid residues. Research has demonstrated that the entire extracellular segment of N-cadherin is very similar to that of RET (Anders *et al.*, 2001) (Figure 2). The extracellular segments of N-cadherin can interact with catenin and other proteins, so as to regulate intracellular adhesion and recombination, and promote the survival and migration of cells.

Accumulating evidence has demonstrated that both fibroblast growth factor (FGF) and epidermal growth factor (EGF) can exert their actions by binding to N-cadherin via their receptors. Recently, our studies (using plasmid construction and cell transfection) have demonstrated that inhibition of the biological functions of N-cadherin may influence the protective effects exerted by GDNF upon DA-ergic neurons. At the same time, we discovered that GDNF can bind to N-cadherin or RET directly (data not shown). Further studies should be undertaken to determine whether GDNF activation of downstream signal pathways is dependent on the interaction of its receptor and N-cadherin (a process that is identical to FGF and EGF).

The distance from the neuronal adherent proteins' domain to the cell membrane is similar to that of RET. The encoding of the RET residues is consistent with the naïve RET molecule. The black solid line indicates the inserted sequence for the RET extracellular segment. The black solid circle indicates coherent calcium binding sites. CD = binding site for cadherin, TK = tyrosine kinase, Cys = cysteine-rich domain, Cyt = cytoplasmic domain (adapted from Anders *et al.*,*,*2001)

#### **4.1.5 Heparan sulfate glycosaminoglycans**

Aside from RET and GFRα1, heparan sulfate proteoglycans, including syndecan and glypican, are essential in mediating the signal pathway of GDNF (Barnett *et al.*, 2002). Bespalov *et al.* demonstrated that syndecan, exerts its function via binding to heparan sulfate with high affinity (Bespalov *et al.*, 2011).

Actions of GDNF on Midbrain Dopaminergic Neurons: The Signaling Pathway 201

GDNF mediates axonal growth and promotes neuronal migration via binding to syndecan 3. Of course, syndecan 3 may mediate the GFL signaling pathway, or may submit GFLs to the RET receptor pathway. With regard to other receptors, activation of the MET tyrosine kinase receptor by heparan sulfate proteoglycans is essential for the NCAM mediated GDNF pathway (Sariola & Saarma, 2003). The effects of heparan sulfate proteoglycans on the integrin αV/β1 and N-cadherin mediated GDNF signal pathway is under

The paucity of heparan sulfate proteoglycans may inhibit GDNF-dependent RET phosphorylation, GDNF mediated axon growth and differentiation of endothelial cells (Barnett *et al.*, 2002). Substantial evidence in mice indicates that a lack of syndecan or GDNF may lead to a decrease in GABA-releasing neurons, which implies that the two molecules

Heat shock protein 27 (HSP27) is a protein with high conservation that is selectively synthesized after being stimulated. It resides ubiquitously in the cell membrane, cytoplasm and nucleus of all prokaryotes and eukaryotes. HSP27 is involved in maintaining microfilaments, signal transduction of cytokines, maintaining the integrity of the cell membrane under stimuli and protection of cells against some stress injuries (Nakamoto & Vígh L, 2007). In 2009, Hong Z *et al.* discovered that 92 proteins were altered under the action of GDNF; among the altered proteins, the phosphorylation of HSP27 significantly increased, accompanied by nuclear translocation. The GDNF induced axonal growth of PC 12 cells is significantly inhibited by interference of HSP27 mRNA, which implies that many proteins are involved in GDNF-mediated neuronal differentiation of DA-ergic neurons. HSP27 is a novel signaling molecule involved in GDNF-mediated axon growth of DA-ergic

GFRα1 and RET are expressed in the DA-ergic neurons of the substantia nigra and ventral tegmental area, as well as in the α-motor neurons of the ventral spine and motor nuclei in the brain stem, such as the hypoglossal nucleus, facial nucleus, nuclei of the trigeminal nerve and the nucleus nervi abducentis (Glazner *et al.*, 1998). RET is mainly located in non-

GDNF activates RET via two pathways, in cis and in trans. When expressed in the same cell, the combination of GFRα1-RET is mediated by a cis signaling system (Yu *et al.*, 1998; Paratcha *et al.*, 2001), delineated as: (1) activation of the cis system, (2) GDNF binds to GPIanchored GFRα1 on the membrane, (3) c-RET is recruited and activated, which is independent of tyrosine kinase activity. On the other hand, when GFRα1 is dissoluble, its binding to GFRα1-RET via a trans signaling system is delineated as: (1) GDNF and dissoluble GFRα1 (sGFRα1) are released from consecutive cells, (2) activation of extracellular c-RET, (3) the activated receptors couple and phosphorylate SHC, (4) c-RET is recruited to the membrane via activation of its tyrosine kinase and phosphorylation of Tyr-1062. Compared with the cis system, the recruitment of RET to the membrane mediated by dissoluble GFRα1 is delayed and persistent, and is dependent on the activation of domains

investigation.

are involved in cortex development.

**4.1.6 Heat shock protein 27** 

neurons (Hong *et al.*, 2009).

**4.2 RET-dependent signaling pathway** 

lipid regions where GDNF action is absent (Paratcha *et al.*, 2001).

of the RET self-kinase (Yu *et al.*, 1998; Paratcha *et al.*, 2001).

Fig. 2. The comparison of human neuronal adherent proteins and the RET extracellular segment.

GDNF mediates axonal growth and promotes neuronal migration via binding to syndecan 3. Of course, syndecan 3 may mediate the GFL signaling pathway, or may submit GFLs to the RET receptor pathway. With regard to other receptors, activation of the MET tyrosine kinase receptor by heparan sulfate proteoglycans is essential for the NCAM mediated GDNF pathway (Sariola & Saarma, 2003). The effects of heparan sulfate proteoglycans on the integrin αV/β1 and N-cadherin mediated GDNF signal pathway is under investigation.

The paucity of heparan sulfate proteoglycans may inhibit GDNF-dependent RET phosphorylation, GDNF mediated axon growth and differentiation of endothelial cells (Barnett *et al.*, 2002). Substantial evidence in mice indicates that a lack of syndecan or GDNF may lead to a decrease in GABA-releasing neurons, which implies that the two molecules are involved in cortex development.

#### **4.1.6 Heat shock protein 27**

200 Etiology and Pathophysiology of Parkinson's Disease

Fig. 2. The comparison of human neuronal adherent proteins and the RET extracellular

segment.

Heat shock protein 27 (HSP27) is a protein with high conservation that is selectively synthesized after being stimulated. It resides ubiquitously in the cell membrane, cytoplasm and nucleus of all prokaryotes and eukaryotes. HSP27 is involved in maintaining microfilaments, signal transduction of cytokines, maintaining the integrity of the cell membrane under stimuli and protection of cells against some stress injuries (Nakamoto & Vígh L, 2007). In 2009, Hong Z *et al.* discovered that 92 proteins were altered under the action of GDNF; among the altered proteins, the phosphorylation of HSP27 significantly increased, accompanied by nuclear translocation. The GDNF induced axonal growth of PC 12 cells is significantly inhibited by interference of HSP27 mRNA, which implies that many proteins are involved in GDNF-mediated neuronal differentiation of DA-ergic neurons. HSP27 is a novel signaling molecule involved in GDNF-mediated axon growth of DA-ergic neurons (Hong *et al.*, 2009).

#### **4.2 RET-dependent signaling pathway**

GFRα1 and RET are expressed in the DA-ergic neurons of the substantia nigra and ventral tegmental area, as well as in the α-motor neurons of the ventral spine and motor nuclei in the brain stem, such as the hypoglossal nucleus, facial nucleus, nuclei of the trigeminal nerve and the nucleus nervi abducentis (Glazner *et al.*, 1998). RET is mainly located in nonlipid regions where GDNF action is absent (Paratcha *et al.*, 2001).

GDNF activates RET via two pathways, in cis and in trans. When expressed in the same cell, the combination of GFRα1-RET is mediated by a cis signaling system (Yu *et al.*, 1998; Paratcha *et al.*, 2001), delineated as: (1) activation of the cis system, (2) GDNF binds to GPIanchored GFRα1 on the membrane, (3) c-RET is recruited and activated, which is independent of tyrosine kinase activity. On the other hand, when GFRα1 is dissoluble, its binding to GFRα1-RET via a trans signaling system is delineated as: (1) GDNF and dissoluble GFRα1 (sGFRα1) are released from consecutive cells, (2) activation of extracellular c-RET, (3) the activated receptors couple and phosphorylate SHC, (4) c-RET is recruited to the membrane via activation of its tyrosine kinase and phosphorylation of Tyr-1062. Compared with the cis system, the recruitment of RET to the membrane mediated by dissoluble GFRα1 is delayed and persistent, and is dependent on the activation of domains of the RET self-kinase (Yu *et al.*, 1998; Paratcha *et al.*, 2001).

Actions of GDNF on Midbrain Dopaminergic Neurons: The Signaling Pathway 203

Fig. 3. The structure of RET and its signal pathway mediation.

RET is recruited to the membrane and binds to GDNF-GFRα1 via the two pathways mentioned above. After recruitment to the membrane, a dimer is formed, which is followed by self-phosphorylation of RET intracellular segments (rendered as tyrosine kinase activity), recruitment of downstream signal molecules and activation of several intracellular signal pathways via a series of enzymatic reactions (Airaksinen *et al.*, 1999). In this way, cell survival, differentiation, proliferation, migration and chemotaxis are regulated. The binding site of SHC receptor proteins, tyrosine 1062, plays vital roles in the activated intracellular signal cascade reactions. After binding to the intracellular tyrosine of RET, SHC forms a complex with the GAB1/2 receptor protein and GRB2/SOS, and induces activation of the PI3K/Akt and RAS/ERK signaling pathways, respectively. After combination of tyrosine 1096 and GRB2, GAB1/2 binds to the p85 subunit of PI3K, causing the activation of PI3K (Airaksinen & Saarma, 2002). GDNF induces the formation of neuronal lamellar parapodia, which is related to the formation of the neural axis (Van Weering & Bos, 1997) and DA-ergic differentiation *in vitro* (Pong *et al.*, 1998). Jun N-terminal Kinase (JNK) is activated by Rhc/Ras-related small molecules (Chiariello *et al.*, 1998), such as GTPase and CDC42. As key regulatory factors for survival of neurotrophin-dependent neurons, JNK and PI3K/Akt may be involved in the trophic action of GDNF (Kaplan & Miller, 1997). Tyrosine 981 binds to Src, and activated p60Src is a key factor for GFL-induced signal cascade reactions. The GDNF signaling pathway regulates axonal epitaxy, distribution of ureters and neural survival (Airaksinen & Saarma, 2002). After binding to tyrosine 1015, PLC is activated and hydrolyzes the second messenger produced by IP3, which increases intracellular calcium and activates multi-signal transmissions such as gene expression. The Ras-MAPK and PI3K/Akt signal pathways are involved in DA-ergic neurons, and GDNF activates cAMP/PKA/CREB in brain neurons in the fetus. The activation of Src kinase and PLC is involved in sensory neurons (Sariola & Saarma, 2003) (Figure 3). RET possesses other tyrosine residues, including Tyr687, Tyr826 and Tyr 1029, which correlate with GFL binding. The mechanisms involved in signal transmission, however, are still unclear.

RET, but not GFR, is expressed in the cerebellum, olfactory tubercles and nuclei in the hypothalamus. This could be explained by the hypothesis that the actions of GFR are independent on RET, and GFR is only active in trans-formation (Saarma & Sariola, 1999).

The extracellular domain of RET contains four cadherin-like structures and a cysteine-rich region. Phosphorylated Y1062 is the binding site for several proteins, which subsequently activates the RAS/ERK, JNK and PI3K/Akt signal pathways via binding to SHC, FRS2, IRS1/2, Dok1-5 and Enigma. Phosphorylated Y905, Y981, Y1015 and 1096 bind to GRB7/10, Src, PLCγ and GRB2, respectively, and mediate a series of signal pathways. (Adapted from Wells & Santoro, 2009).

#### **4.3 RET-independent signaling pathway**

In spite of the vital roles played by RET-mediated signaling pathways, the GFRα1-RET signal system is not an essential pathway for neuronal development (demonstrated in knockout mice). Recently, a series of studies has demonstrated that GDNF may exert its biological actions via RET-independent signaling pathways.

RET is recruited to the membrane and binds to GDNF-GFRα1 via the two pathways mentioned above. After recruitment to the membrane, a dimer is formed, which is followed by self-phosphorylation of RET intracellular segments (rendered as tyrosine kinase activity), recruitment of downstream signal molecules and activation of several intracellular signal pathways via a series of enzymatic reactions (Airaksinen *et al.*, 1999). In this way, cell survival, differentiation, proliferation, migration and chemotaxis are regulated. The binding site of SHC receptor proteins, tyrosine 1062, plays vital roles in the activated intracellular signal cascade reactions. After binding to the intracellular tyrosine of RET, SHC forms a complex with the GAB1/2 receptor protein and GRB2/SOS, and induces activation of the PI3K/Akt and RAS/ERK signaling pathways, respectively. After combination of tyrosine 1096 and GRB2, GAB1/2 binds to the p85 subunit of PI3K, causing the activation of PI3K (Airaksinen & Saarma, 2002). GDNF induces the formation of neuronal lamellar parapodia, which is related to the formation of the neural axis (Van Weering & Bos, 1997) and DA-ergic differentiation *in vitro* (Pong *et al.*, 1998). Jun N-terminal Kinase (JNK) is activated by Rhc/Ras-related small molecules (Chiariello *et al.*, 1998), such as GTPase and CDC42. As key regulatory factors for survival of neurotrophin-dependent neurons, JNK and PI3K/Akt may be involved in the trophic action of GDNF (Kaplan & Miller, 1997). Tyrosine 981 binds to Src, and activated p60Src is a key factor for GFL-induced signal cascade reactions. The GDNF signaling pathway regulates axonal epitaxy, distribution of ureters and neural survival (Airaksinen & Saarma, 2002). After binding to tyrosine 1015, PLC is activated and hydrolyzes the second messenger produced by IP3, which increases intracellular calcium and activates multi-signal transmissions such as gene expression. The Ras-MAPK and PI3K/Akt signal pathways are involved in DA-ergic neurons, and GDNF activates cAMP/PKA/CREB in brain neurons in the fetus. The activation of Src kinase and PLC is involved in sensory neurons (Sariola & Saarma, 2003) (Figure 3). RET possesses other tyrosine residues, including Tyr687, Tyr826 and Tyr 1029, which correlate with GFL binding. The mechanisms involved in signal transmission, however, are still

RET, but not GFR, is expressed in the cerebellum, olfactory tubercles and nuclei in the hypothalamus. This could be explained by the hypothesis that the actions of GFR are independent on RET, and GFR is only active in trans-formation (Saarma & Sariola,

The extracellular domain of RET contains four cadherin-like structures and a cysteine-rich region. Phosphorylated Y1062 is the binding site for several proteins, which subsequently activates the RAS/ERK, JNK and PI3K/Akt signal pathways via binding to SHC, FRS2, IRS1/2, Dok1-5 and Enigma. Phosphorylated Y905, Y981, Y1015 and 1096 bind to GRB7/10, Src, PLCγ and GRB2, respectively, and mediate a series of signal pathways. (Adapted from

In spite of the vital roles played by RET-mediated signaling pathways, the GFRα1-RET signal system is not an essential pathway for neuronal development (demonstrated in knockout mice). Recently, a series of studies has demonstrated that GDNF may exert its

unclear.

1999).

Wells & Santoro, 2009).

**4.3 RET-independent signaling pathway** 

biological actions via RET-independent signaling pathways.

Fig. 3. The structure of RET and its signal pathway mediation.

Actions of GDNF on Midbrain Dopaminergic Neurons: The Signaling Pathway 205

and NCAM is low. In contrast, GDNF binds to NCAM140 closely when GFRα1 binds to NCAM; this leads to the phosphorylation of Fyn, a molecule conjugated to the intracellular segments of NCAM, followed by phosphorylation of FAK, and eventually activation of the MAPK signal pathway via normal signal transduction. Interestingly, the combination of GFRα1 and NCAM may downregulate NCAM-mediated cellular adherence when GDNF is absent (Cao *et al.*, 2008). This regulation is indicative of the independent roles played by GFRα1-NCAM and the GDNF-GFRα1-NCAM signaling pathway. In the RET-independent model, GDNF stimulates the migration of neurilemma cells and the growth of synapses. This finding indicates that the GFRα protein, GFLs, or their combination with NCAM,

GDNF is a crucial neurotrophic factor for DA-ergic neurons. Transmembrane signal transduction is mediated by a special receptor system, which includes GFRα, RET, and NCAM140. Thus far, we have identified that another transmembrane cell adhesion molecule, integrin (a heterodimer consisting of α and β subunits), can regulate the signal transduction of GDNF. Some studies have demonstrated that chronic injection of GDNF into the substantia nigra increases the expression of integrin αV and NCAM (Chao *et al.*, 2003). This implies that GDNF exerts special effects on the increase of integrin αV expression, and

Under the influence of GDNF, the combination of phosphorylated SHC and intracellular integrin β1 increases. Data from co-immunoprecipitation demonstrate that integrin β1 and GFRα1 form a complex. Additionally, phosphorylation of SHC in the cytoplasmic domain of integrin β1 was shown to increase after stimulation with GDNF. Other data from molecular models demonstrated the presence of four hydrogen bonds between integrin β1 and GFRα1. These data indicate that integrin β1 may be involved in the transmembrane signaling of GDNF, and that integrin β1 may even be a selective signal receptor for GDNF (Cao *et al.*, 2008).

N-cadherin is a transmembrane adhesion protein, whose cytoplasmic region can interact with various intracellular proteins (Drees *et al.*, 2005; Reynolds, 2007). When its extracellular domain binds a ligand, the intracellular domain of N-cadherin can activate the PI3K/Akt and Ras/Raf/MAPK signaling pathways (Hulit *et al.*, 2007). It is well-known that the structural and functional characteristics of N-cadherin are somewhat similar to that of NCAM and integrin β1, and that it mediates not only adherence, but also signal transduction. In addition, the binding site for the extracellular domains of RET and the GDNF/GFRα1 complex, is a cadherin-like domain. Our previous studies have demonstrated that N-cadherin can also bind to GDNF, and the phosphorylation of the N-cadherin intracellular domain (Tyr860) is mediated by GDNF. Further studies, using gene silencing and immunoblotting, have demonstrated that GDNF activates the intracellular PI3K/Akt signaling pathway via Ncadherin, thus protecting DA-ergic neurons. Results from studies, using flow cytometry and Hoechst 33258 staining, indicate that GDNF interferes with the expression of N-cadherin, and that the apoptosis of injured DA-ergic neurons increases. Additional results from immunoblotting indicate, under the same conditions, that phosphorylated Akt, but not total Akt, decreases in the cytoplasm. Results from immunohistochemistry indicate a decrease in total N-cadherin, phosphorylated N-cadherin (Tyr860) and phosphorylated Akt, however,

activates distinct pathways to regulate differential signal pathways.

that integrin αV may be a selective receptor for functional GDNF.

**4.3.4 GDNF and integrins** 

**4.3.5 GDNF and N-cadherin** 

#### **4.3.1 GDNF and Src**

As mentioned previously, the activation of Src family kinases mediated by GDNF is independent of RET. Src family kinases are non-receptor type tyrosine kinases. The Src family is divided into three sub-families: the SrcA sub-family, consisting of Src, Yes, Fyn, and Fgr; the SrcB sub-family, consisting mainly of Lck, Hck, Blk, and Lyn; and another subfamily composed of Frk. SrcA and SrcB sub-families are specific to vertebrates. The activation of Src family kinases is closely related to a series of biological functions, such as cell division, migration, apoptosis and differentiation. Poteryaev *et al.* demonstrated that GDNF activates Src-family tyrosine kinases, after binding to the GFRα1-GPI complex, in mice DRG neurons (RET tyrosine kinase paucity); phosphorylation of MAPK kinase, PLC2γ and CREB ensue, and induce the third messenger IP3 to increase calcium concentration, eventually promoting cell survival and Fos expression (Paratcha & Ibáñez, 2002). Popsueva *et al.* also demonstrated that GDNF can promote the differentiation of MDCK cells (GFRα1 positive and RET negative) via activation of the Src family kinases (Popsueva *et al.*, 2003). Trupp also demonstrated, using an immortalized neuronal precursor cell line, RN33B (high expression of GFRα1 and no expression of RET), that GDNF does not activate the Ras/ERK signaling pathway, in these cells; instead GDNF induces the activation of Src family kinases, rapid phosphorylation of CREB and up-regulation of c-fos mRNA (Trupp *et al.*, 1999). This study provides additional evidence supporting the notion that the functions of GDNF are independent of RET.

#### **4.3.2 GDNF and MET**

The differential expression models for GFR and RET indicate that: (1) GFR functions via signal transduction pathways that do not involve RET, and (2) other GDNF ligands (binding to GFR) are involved. MET is a hepatocyte growth factor receptor (HGFR) that is normally expressed in epithelium-derived cells. The expression of hepatocyte growth factor is limited in desmohemoblast-derived cells. The primary precursor proteins of MET are cleaved to produce α and β subunits. The mature receptor is formed via a disulfide bond, and plays preliminary roles in embryonic development and wound healing. After stimulating hepatocyte growth factor, MET induces several biological reactions. In many RET-absent but GFRα1-rich cells, GDNF induces the phosphorylation of MET, and then activates the Src family kinases. This provides impetus for research on a RET-independent GDNF signaling system. The RETindependent activation of Src and MET, induced by GDNF, may be regulated by heparan sulfate and NCAM. However, *in vivo* immunoprecipitation studies have demonstrated that MET does not bind to GDNF, implying an uncertain role for GDNF-induced MET activation.

#### **4.3.3 GDNF and NCAM**

The GFRα receptor is expressed more widely than RET in many regions of the nervous system, especially the procerebrum, cortex and internal ear (Trupp *et al.*, 1997; Kokaia *et al.*, 1999), indicating that the signal transduction in neurons and glials, and GLF-protein binding may not always be dependent on RET. Other studies demonstrated that GFRα1, but not RET, is expressed in the RN33B cell line, and the signal pathway induced by GDNF overlaps with that induced by NCAM. In 2003, Paratcha *et al.* demonstrated that NCAM may mediate actions via RET-independent trans-membrane signaling. Further studies demonstrated that NCAM is involved in the promotion effects, engendered by GDNF, affecting the survival of DA-ergic neurons (Paratcha *et al.*, 2003). When GFRα is absent, the binding between GFLs and NCAM is low. In contrast, GDNF binds to NCAM140 closely when GFRα1 binds to NCAM; this leads to the phosphorylation of Fyn, a molecule conjugated to the intracellular segments of NCAM, followed by phosphorylation of FAK, and eventually activation of the MAPK signal pathway via normal signal transduction. Interestingly, the combination of GFRα1 and NCAM may downregulate NCAM-mediated cellular adherence when GDNF is absent (Cao *et al.*, 2008). This regulation is indicative of the independent roles played by GFRα1-NCAM and the GDNF-GFRα1-NCAM signaling pathway. In the RET-independent model, GDNF stimulates the migration of neurilemma cells and the growth of synapses. This finding indicates that the GFRα protein, GFLs, or their combination with NCAM, activates distinct pathways to regulate differential signal pathways.

#### **4.3.4 GDNF and integrins**

204 Etiology and Pathophysiology of Parkinson's Disease

As mentioned previously, the activation of Src family kinases mediated by GDNF is independent of RET. Src family kinases are non-receptor type tyrosine kinases. The Src family is divided into three sub-families: the SrcA sub-family, consisting of Src, Yes, Fyn, and Fgr; the SrcB sub-family, consisting mainly of Lck, Hck, Blk, and Lyn; and another subfamily composed of Frk. SrcA and SrcB sub-families are specific to vertebrates. The activation of Src family kinases is closely related to a series of biological functions, such as cell division, migration, apoptosis and differentiation. Poteryaev *et al.* demonstrated that GDNF activates Src-family tyrosine kinases, after binding to the GFRα1-GPI complex, in mice DRG neurons (RET tyrosine kinase paucity); phosphorylation of MAPK kinase, PLC2γ and CREB ensue, and induce the third messenger IP3 to increase calcium concentration, eventually promoting cell survival and Fos expression (Paratcha & Ibáñez, 2002). Popsueva *et al.* also demonstrated that GDNF can promote the differentiation of MDCK cells (GFRα1 positive and RET negative) via activation of the Src family kinases (Popsueva *et al.*, 2003). Trupp also demonstrated, using an immortalized neuronal precursor cell line, RN33B (high expression of GFRα1 and no expression of RET), that GDNF does not activate the Ras/ERK signaling pathway, in these cells; instead GDNF induces the activation of Src family kinases, rapid phosphorylation of CREB and up-regulation of c-fos mRNA (Trupp *et al.*, 1999). This study provides additional evidence supporting the notion that the functions of GDNF are

The differential expression models for GFR and RET indicate that: (1) GFR functions via signal transduction pathways that do not involve RET, and (2) other GDNF ligands (binding to GFR) are involved. MET is a hepatocyte growth factor receptor (HGFR) that is normally expressed in epithelium-derived cells. The expression of hepatocyte growth factor is limited in desmohemoblast-derived cells. The primary precursor proteins of MET are cleaved to produce α and β subunits. The mature receptor is formed via a disulfide bond, and plays preliminary roles in embryonic development and wound healing. After stimulating hepatocyte growth factor, MET induces several biological reactions. In many RET-absent but GFRα1-rich cells, GDNF induces the phosphorylation of MET, and then activates the Src family kinases. This provides impetus for research on a RET-independent GDNF signaling system. The RETindependent activation of Src and MET, induced by GDNF, may be regulated by heparan sulfate and NCAM. However, *in vivo* immunoprecipitation studies have demonstrated that MET does not bind to GDNF, implying an uncertain role for GDNF-induced MET activation.

The GFRα receptor is expressed more widely than RET in many regions of the nervous system, especially the procerebrum, cortex and internal ear (Trupp *et al.*, 1997; Kokaia *et al.*, 1999), indicating that the signal transduction in neurons and glials, and GLF-protein binding may not always be dependent on RET. Other studies demonstrated that GFRα1, but not RET, is expressed in the RN33B cell line, and the signal pathway induced by GDNF overlaps with that induced by NCAM. In 2003, Paratcha *et al.* demonstrated that NCAM may mediate actions via RET-independent trans-membrane signaling. Further studies demonstrated that NCAM is involved in the promotion effects, engendered by GDNF, affecting the survival of DA-ergic neurons (Paratcha *et al.*, 2003). When GFRα is absent, the binding between GFLs

**4.3.1 GDNF and Src** 

independent of RET.

**4.3.2 GDNF and MET** 

**4.3.3 GDNF and NCAM** 

GDNF is a crucial neurotrophic factor for DA-ergic neurons. Transmembrane signal transduction is mediated by a special receptor system, which includes GFRα, RET, and NCAM140. Thus far, we have identified that another transmembrane cell adhesion molecule, integrin (a heterodimer consisting of α and β subunits), can regulate the signal transduction of GDNF. Some studies have demonstrated that chronic injection of GDNF into the substantia nigra increases the expression of integrin αV and NCAM (Chao *et al.*, 2003). This implies that GDNF exerts special effects on the increase of integrin αV expression, and that integrin αV may be a selective receptor for functional GDNF.

Under the influence of GDNF, the combination of phosphorylated SHC and intracellular integrin β1 increases. Data from co-immunoprecipitation demonstrate that integrin β1 and GFRα1 form a complex. Additionally, phosphorylation of SHC in the cytoplasmic domain of integrin β1 was shown to increase after stimulation with GDNF. Other data from molecular models demonstrated the presence of four hydrogen bonds between integrin β1 and GFRα1. These data indicate that integrin β1 may be involved in the transmembrane signaling of GDNF, and that integrin β1 may even be a selective signal receptor for GDNF (Cao *et al.*, 2008).

#### **4.3.5 GDNF and N-cadherin**

N-cadherin is a transmembrane adhesion protein, whose cytoplasmic region can interact with various intracellular proteins (Drees *et al.*, 2005; Reynolds, 2007). When its extracellular domain binds a ligand, the intracellular domain of N-cadherin can activate the PI3K/Akt and Ras/Raf/MAPK signaling pathways (Hulit *et al.*, 2007). It is well-known that the structural and functional characteristics of N-cadherin are somewhat similar to that of NCAM and integrin β1, and that it mediates not only adherence, but also signal transduction. In addition, the binding site for the extracellular domains of RET and the GDNF/GFRα1 complex, is a cadherin-like domain. Our previous studies have demonstrated that N-cadherin can also bind to GDNF, and the phosphorylation of the N-cadherin intracellular domain (Tyr860) is mediated by GDNF. Further studies, using gene silencing and immunoblotting, have demonstrated that GDNF activates the intracellular PI3K/Akt signaling pathway via Ncadherin, thus protecting DA-ergic neurons. Results from studies, using flow cytometry and Hoechst 33258 staining, indicate that GDNF interferes with the expression of N-cadherin, and that the apoptosis of injured DA-ergic neurons increases. Additional results from immunoblotting indicate, under the same conditions, that phosphorylated Akt, but not total Akt, decreases in the cytoplasm. Results from immunohistochemistry indicate a decrease in total N-cadherin, phosphorylated N-cadherin (Tyr860) and phosphorylated Akt, however,

Actions of GDNF on Midbrain Dopaminergic Neurons: The Signaling Pathway 207

Fig. 4. A membrane lipid raft is an ultrastructure rich in cholesterol and

ERK1/2 pathway (Beggs *et al.*, 1997; Niethammer *et al.*, 2002).

NCAM can function as a receptor of GDNF signal transduction (Sjöstrand *et al.*, 2007; Sjöstrand & Ibáñez, 2008). Under the influence of GDNF, NCAM140 binds to Fyn and is recruited to lipid rafts, followed by stimulation of the migration of Schwann cells and promotion of neuronal axonal growth (Paratcha *et al.*, 2003). In membrane lipid rafts, the binding of NCAM and Fyn leads to recruitment of FAK and the activation of the Ras-Raf-

Integrin β1 is a transmembrane cell adhesion molecule, which is expressed in DA-ergic neurons in the substantia nigra of adult mice (Cao *et al.*, 2008). Inhibitory antibodies can counteract the effects of GDNF on the promotion of survival and growth of DA-ergic

phosphosphingolipids.

total Akt does not change. Finally, through immunoblotting it was discovered that the levels of phosphorylated N-cadherin (Tyr860) and phosphorylated Akt are dose-dependent on GDNF, and that the peak levels of both occur at 50 ng/ml (*in vitro*) and 13 ng/μl (*in vivo*) of GDNF. The levels of phosphorylated N-cadherin (Tyr860) and phosphorylated Akt are also timedependent, and the peak levels of both occur at 15 min (*in vitro*) and 30 min (*in vivo*) after GDNF actions. Statistical analyses show that the two phosphorylations are positively related. Thus, it may be concluded that GDNF activates the PI3K/Akt pathway via N-cadherin to protect DA-ergic neurons.
