**3.1 The structure of GDNF**

The GDNF family ligands (GFLs) belong to the transforming growth factor-β (TGF-β) superfamily, and include four neurotrophic factors: glial cell line-derived neurotrophic factor (GDNF), neurturin (NRTN), artemin (ARTN) and persephin (PSPN). These factors possess structures similar to other factors in the TGF-β super-family, and function as homodimers (Ibáñez, 1998). GFLs transduce signals through a receptor system, which is a protein complex consisting of RET tyrosine protein kinase and the GFRα family of proteins, playing an important role in the biological processes of cell survival, differentiation and migration.

The GFLs are secretory proteins, derived from the precursor preproGFL. ProGFLs can be activated by proteolytic cleavage. The diffusion and local concentration of proGFLs are influenced by the combination of GFLs and the lateral chain of heparan sulfate in the extracellular matrix (Hamilton *et al.*, 1998). Despite the fact that the binding sites for the GFLs have been extensively studied, the gene regulation, mechanisms of secretion and the precursors leading to the activation of GFLs have not been fully elucidated (Golden *et al.*,

antioxidants (esp. glutathione) to disappear. At this point, the free radicals can no longer be removed. Thus, by oxidizing the lipoids of neurilemma, destroying the functions of the membranes and directly demolishing DNA in DA-ergic neurons, the free radicals cause the

Neurotrophic factors (NTFs), discovered at the end of the 20th century, are dissoluble polypeptides derived from tissues (e.g. muscles) which are innervated by nerves, or from astrocytes. They can promote and maintain the growth, survival and differentiation of specific neurons, as well as influence synaptic plasticity. More than twenty NTFs are known to exist, including brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF). Sharing as much as 50 - 60% homology in amino acid sequence, NTFs are quite congenerous. In general, the NTFs enter the nerve terminal through receptor-mediated endocytosis, then transport to the cell soma by retrograde axoplasmic transport. There, they promote the synthesis of numerous proteins supporting the growth, development and functional integrity of neurons. Recently, some NTFs were found to be produced by neurons and transported to nerve endings by anterograde axoplasmic transport, where they play a role in supporting the integrity of

Neurotrophic factors, especially GDNF, exert protective effects on DA-ergic neurons, correlating with the etiology of PD. Some studies have demonstrated that the amount of NTFs is decreased in PD patients, compared with control. It is highly likely that this decrease could lead to the degeneration and death of DA-ergic neurons and induce the clinical symptoms of PD. In addition, some scholars propose that the apoptosis of numerous neurons during the process of development may be related to a decrease in NTFs. Studies based on animal models have demonstrated that the application of NTFs-medium can increase the survival rate of fetal DA-ergic neurons and reduce apoptosis *in vitro* (Siegel & Chauhan, 2000). Currently, no evidence exists addressing whether defects of NTFs or decreased expression of receptors for NTFs occur during the pathogenic progress of PD.

**3. The structure, expression and basic physiological properties of GDNF** 

The GDNF family ligands (GFLs) belong to the transforming growth factor-β (TGF-β) superfamily, and include four neurotrophic factors: glial cell line-derived neurotrophic factor (GDNF), neurturin (NRTN), artemin (ARTN) and persephin (PSPN). These factors possess structures similar to other factors in the TGF-β super-family, and function as homodimers (Ibáñez, 1998). GFLs transduce signals through a receptor system, which is a protein complex consisting of RET tyrosine protein kinase and the GFRα family of proteins, playing an important role in the biological processes of cell survival, differentiation and migration. The GFLs are secretory proteins, derived from the precursor preproGFL. ProGFLs can be activated by proteolytic cleavage. The diffusion and local concentration of proGFLs are influenced by the combination of GFLs and the lateral chain of heparan sulfate in the extracellular matrix (Hamilton *et al.*, 1998). Despite the fact that the binding sites for the GFLs have been extensively studied, the gene regulation, mechanisms of secretion and the precursors leading to the activation of GFLs have not been fully elucidated (Golden *et al.*,

degeneration and death of the neurons.

shape and function in postsynaptic neurons.

**3.1 The structure of GDNF** 

**2.2 Neurotrophic factors** 

1999). Similarly, the protease responsible for the breakdown and activation of the GFL precursor has yet to be revealed. Recent evidence has uncovered the biological activities for the precursor of secretory neurotrophic factors (Lee *et al.*, 2001). The secreted NGF and BDNF are sheared outside the cell by serine protein kinases and selective matrix metalloproteinases. ProNGF has a high affinity for p75NTR, a receptor responsible for the apoptosis of neurons in culture media, and maintains mild activation of the pathway, accompanied by differentiation and survival of cells mediated by TrkA. Whether the proteolytic cleavage mediates the biological activities of the GFLs, and whether the functional GFLs are different from other ligands, are major areas of interest to current scholars.

Of the 4 members of the GFLs, GDNF and NRTN were the first to be extracted. Subsequently, ARTN and PSPN were identified through basic data analysis and the use of homogeneous clones (Baloh *et al.*, 2000). The GDNF gene for both humans and mice has been cloned and expressed in prokaryotic and eukaryotic vectors. The human GDNF gene is located on chromosome 5p13. 1-13. 3, and is composed of two exons and one intron. In humans and mice, two types of GDNF mRNA with different lengths exist: a large fragment of 633bp and small fragment of 555bp, encoding for polypeptides of GDNF precursors with 211 amino acids and 185 amino acids, respectively. After the removal of 26 amino acids in the N terminal, the large fragment becomes smaller and the two fragments (large and small) are eventually transformed into polypeptides consisting of 134 amino acids (Trupp *et al.*, 1995). The mature protein of GDNF consists of 7 cysteine residues, and as such, 3 intrachain disulfide bonds are formed between the sites of amino acids 41 and 102, 68 and 131 and 72 and 133. The cysteine residues in the side chain of amino acid 101 form interchain disulfide bonds, thus forming the structure of the homodimer of GDNF (Haniu *et al.*, 1996). The structural features are quite similar to that of the TGF-β family members. Two glycosylation sites lie in the polypeptide chain, which has a molecular weight of 20 kD. The molecular weight of the natural GDNF dimer, which contains a heparan biding site, is 40 - 45 kD. X-ray methods have demonstrated that the structure of rat GDNF contains two finger-like structures lying in the rostral and caudal parts. Additionally, the amino acids in the middle form a helical structure; two monomers are connected by a pair of disulfide bonds, and the four finger-like structures form a plane (Eigenbrot *et al.*, 1997) (Figure 1). GDNF has conservation in evolution, as evidenced by the significant similarities between humans and mice in amino acid sequences in the mature proteins of GDNF (as high as 93%). Human GDNF, produced by genetic engineering, exerts activities on murine DA-ergic neurons. These lines of evidence indicate that GDNF has cross-species activity.

Fig. 1. The molecular structure for the crystal monomer of GDNF.

Actions of GDNF on Midbrain Dopaminergic Neurons: The Signaling Pathway 197

example, *in vitro*, GDNF can rapidly activate the MAPK signaling pathway after acting on midbrain DA-ergic neurons, and increase neuronal excitability (Yang *et al.*, 2001). In addition, the GDNF signaling system can mediate the migration and chemotaxis of neurons (Tang *et al.*, 1998). Some researchers have also demonstrated that GDNF can potentiate analgesia in neuropathic pain (Sakai *et al.*, 2008). Further, GDNF plays critical roles in the neuronal development of sympathetic and motor neurons, and synapse formation in the

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

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

hippocampus (Moore *et al.*, 1996; Ledda *et al.*, 2007).

**4. The signal pathway for GDNF activation** 

binding of RET and GFRα1, and is not expressed in GFRα4.

**4.1 GDNF receptors and structures** 

**4.1.1 GFR**α

**4.1.2 RET** 

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

#### **3.2 GDNF distribution and expression**

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 higher than that of neuronal tissues.

#### **3.3 The functions of GDNF**

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

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 example, *in vitro*, GDNF can rapidly activate the MAPK signaling pathway after acting on midbrain DA-ergic neurons, and increase neuronal excitability (Yang *et al.*, 2001). In addition, the GDNF signaling system can mediate the migration and chemotaxis of neurons (Tang *et al.*, 1998). Some researchers have also demonstrated that GDNF can potentiate analgesia in neuropathic pain (Sakai *et al.*, 2008). Further, GDNF plays critical roles in the neuronal development of sympathetic and motor neurons, and synapse formation in the hippocampus (Moore *et al.*, 1996; Ledda *et al.*, 2007).
