**1. Introduction**

192 Etiology and Pathophysiology of Parkinson's Disease

Zhang, W., Dallas, S., Zhang, D., Guo, J. P., Pang, H., Wilson, B., et al. (2007). Microglial

elicited by A30P and A53T mutant alpha-synuclein. *Glia, 55*(11), 1178-1188. Zhang, W., Wang, T., Pei, Z., Miller, D. S., Wu, X., Block, M. L., et al. (2005). Aggregated

Zhu, M., Li, J., & Fink, A. L. (2003). The association of alpha-synuclein with membranes

Parkinson's disease. *FASEB J, 19*(6), 533-542.

40197.

PHOX and Mac-1 are essential to the enhanced dopaminergic neurodegeneration

alpha-synuclein activates microglia: a process leading to disease progression in

affects bilayer structure, stability, and fibril formation. *J Biol Chem, 278*(41), 40186-

Parkinson's disease (PD) is a degenerative disease of the central nervous system (CNS) predominantly occurring in older adults, and is particularly common after the age of 60. The primary pathologic characteristics of PD are neuronal degeneration and cell death of dopaminergic (DA-ergic) neurons, leading to a decrease in the dopamine (DA) level in the striatum. Originally extracted and purified in 1993, glial cell line-derived neurotrophic factor (GDNF) is a novel neurotrophic factor which exerts neurotrophic effects upon DAergic neurons in the midbrain, sympathetic neurons in the superior cervical ganglion and motor neurons in the spinal cord. Specially, GDNF protects midbrain DA-ergic neurons, and promotes their differentiation and survival (Lin *et al.*, 1993). These studies provide impetus to delve more deeply into the underlying mechanisms of GDNF, and suggest potential future applications of GDNF. The present review focuses on the correlates of neurotrophic factors and PD treatments, and the mechanisms of action of GDNF.

#### **2. Parkinson's disease and neurotrophic factors**

#### **2.1 Parkinson's disease**

Parkinson's disease (PD) is also known as paralysis agitans. As a degenerative disease of the central nervous system (CNS), PD is characterized by static tremors, bradykinesia, myotonia and unstable posture. It is a disease normally occurring in older adults, especially after the age of 60. Thus far, the etiology and mechanisms underlying this disease have not been fully elucidated (Daly *et al.*, 1999), though most scholars support the dopamine hypothesis and the oxidative stress hypothesis of PD. According to the dopamine hypothesis, dopamine synthesis decreases following the pathologic degeneration of DA-ergic neurons in the substantia nigra pars compacta (SNpc), leading to a decrease in inhibition of acetylcholine in the corpus striatum and a relative increasing excitability of acetylcholine. The disequilibrium between dopamine and acetylcholine results in the hyper-functioning of the extrapyramidal system, thus leading to paralysis agitans. The oxidative stress hypothesis explains the causes of degeneration of DA-ergic neurons in the SNpc. In PD, hydrogen peroxide (H2O2) and superoxide anions, produced during the process of oxidative metabolism under oxidative stress, are catalyzed by Fe2+ in the SNpc, and then formed into hydroxy radicals with more significant noxious properties. Meanwhile, the activity of complex I of the mitochondrial respiratory chain in DA-ergic neurons decreases, causing

Actions of GDNF on Midbrain Dopaminergic Neurons: The Signaling Pathway 195

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

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.

ligands, are major areas of interest to current scholars.

These lines of evidence indicate that GDNF has cross-species activity.

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

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 degeneration and death of the neurons.

#### **2.2 Neurotrophic factors**

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 shape and function in postsynaptic neurons.

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.
