The role of FGF-9 expression in neuroprotection of melatonin and MPP + -induced parkinsonism model in vivo and in vitro Key words: FGF-9; melatonin;

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2 -9 參 神 MPP + 神 : -9; ; MPP + ; 神 ; 狀 (FGFs)(FGFRs) 理 復 23 FGFs, FGF-2 FGF-9 神 :, 了 FGFs 年來 金, 神 神 (MPP + ) 神 IGFBDNFFGF-9 FGF 神 不 神 FGF-9 量 FGF-9 神 FGF-9 參 神 MPP + 狀 狀 FGF-9 mrna 量 狀 tyrosine hydroxylase (TH, 率 )immunoreactivity 降 TH-positive 神 量流 若 MPP + FGF-9 mrna 量 降 神 流 RT-PCRFGF mrna 量不 易 FGF-20 mrna 量 FGF-20 狀 不 値 離 神 神 神 神 不 FGF 神 FGF-9 神 FGF-2 量 FGF-9 mrna 量 行 FGF-9 MPP + 神 神 1

3 The role of FGF-9 expression in neuroprotection of melatonin and MPP + -induced parkinsonism model in vivo and in vitro Key words: FGF-9; melatonin; MPP + ; parkinsonism; dopamine; glutathione Fibroblast growth factors (FGFs) and their receptors (FGFRs) constitute an elaborate signaling system that participates in many developmental and repair processes. Among the 23 FGF members, FGF-2 and FGF-9 are intimately involved in neuronal protection after ischemic, metabolic, or traumatic brain injury. In addition to FGFs, our previous studies demonstrated that melatonin acts as an antioxidant and free radical scavenger to effectively protect nigrostriatal dopaminergic (DA) neurons from MPP + intoxication. Although previous reports demonstrated that growth factors of IGF and BDNF can be upregulated by melatonin, there is no evidence showing the effect of melatonin on FGFs. Interestingly, our preliminary result showed that melatonin treatment can significantly enhance FGF-9 expression in primary culture of cortical astrocytes. We also detected there is mrna expression of FGF-9 in striatum and SN of adult rats. In the present study, we attempt to investigate the role of FGF-9 in neuroprotection of melatonin and MPP + -induced DA neurodegeneration. We found that the down-regulation of FGF-9 mrna expression was associated with the loss of TH immunoreactivity in doparminergic system of striatum (ST) and substantia nigra (SN) 3 days after unilateral intrastriatal administration of MPP +. Melatonin treatment significantly attenuated the MPP + -induced FGF-9 downregulation and the nigrostriatal dopaminergic neuronal loss. We detected the FGF-20 mrna expression in SN only but not in ST. Melatonin can up-regulate FGF-20 mrna expression in SN whereas MPP + slightly decreases FGF-20 expression. Furthermore, our results from primary cell culture demonstrated that FGF-2 highly expressed in astrocyte and FGF-9 expressed in neurons. Melatonin up-regulates FGF-9 expression in a dose dependent manner in neuron enriched primary culture. Therefore, we continue to investigate the role of FGF-9 in MPP + -induced neurotoxicity and neuroprotection of melatonin. 2

4 老年 老 神 老年 金 金 狀 行 異 行 神 老 流 臨 來 療 金 年來 行 療 刺 年來 神 療 粒 量 力 狀 神 若 粒 來 力 神 療 見 (FGFs)(FGFRs) 理 復 23 FGFs, FGF-2 FGF-9 神 :, 了 FGFs 年來 金, 神 神 (MPP + ) 神 IGFBDNFFGF-9 FGF 神 不 神 FGF-9 量 FGF-9 神 FGF-9 參 神 料 Animal study The MPP + -induced PD model in rats by single intra-striatal injection was described in our previous reports (Chen et al., 2002; Chuang and Chen, 2002). Male rats were intra-striatally infused 1 µl of sterilized saline or 0.1 M MPP +. Rats were sacrificed at 3 days after MPP + treatment. The expression of FGF-9 mrna and protein in substantial nigra (SN) and striatum were determined by RT-PCR and western blot after intrastriatal MPP + infusion. (2) The neuronal survival was evaluated by TH (tyrosin hydroxylase, a marker of DA neurons) immunohistochemistry in striatum and SN after MPP + with or without melatonin treatment. TH immunohistochemistry. After animals were perfused, the brains were dissected and sectioned in a cryostat. The midbrain and striatal sections were reacted immunocytochemically for 3

5 specific antibody of tyrosine hydroxylase (TH, a rate-limiting enzyme for DA systesis and marker of DA neurons). Avidin-biotin-peroxidase techniques and glucose oxidase-nickel-diamino-benzidine enhancement were used to detect antigen-antibody. TH-immunoreactive cells represent dopaminergic neurons. Essential controls were conclude a), sections reacted with non-immune sera as the first phase or omitting the first phase; b), the antibodies pre-adsorbed with a specific antigen ( nmole/ml) or adsorbed with a structurally related antigen. Moreover, some of adjacent sections were treated with cresyl violet (Nissl stain) to study the cytoarchitectonics studies. For the quantitative analysis of surviving dopaminergic neurons, TH-positive cells from 8 sections at different levels extending caudally from the anterior midbrain were estimated and summed by counting cells at high magnification (400 ), by an observer who is blind to the experimental condition (Chen and Chuang et al., 2002). The immunoreactivity of TH in striatum or SN was estimated by a UTHSCSA image tool. RNA extraction and reverse transcription-pcr Total RNA was extracted from striatum, SN, or cortex of brain of rats treated with MPP + or melatonin or the combination using Ultraspect TM - RNA isolation system according to the manufacturer s instructions (Biotecx). The primers of FGF-9 reverse transcription (RT)-PCR were as follows: forward, 5 - AGGTGAAGTTGGGAGCTATT-3. The primers for GAPDH were: 5 - AGGTCGGTGTCAACGGATTT-3. PCR conditions were 35 cycles of denaturation at 94 C for 30 sec, annealing at 55 C for 30 sec, and extension at 72 C for 35 sec. PCR products were separated by electrophoresis through 5% acylamide gel and then stained with ethidium bromide and placed on UV illuminator equipped with a camera connected to a computer. The gel image was analyzed using AlphaImager software (Alpha Innotech Corp., San Leando CA). 論 1. MPP + 狀 狀 FGF-9 mrna 量 狀 tyrosine hydroxylase (TH, 率 )immunoreactivity 降 若 MPP + FGF-9 mrna 量 降 TH immunoreactivity 降 2. MPP + 神 流 FGF-9 TH mrna 量 MPP + 更 FGF-20 RT-PCR 不易 FGF-20 mrna 量 値 4

6 3. 離 神 神 神 神 不 FGF 神 FGF-9 神 FGF-2 量 FGF-9 mrna 量 行 FGF-9 MPP + 神 神 5

7 Sham MPP + M+M Sham MPP + M+M L R L R L R L R L R L R 150, *SUM_FGF9/GAPDH /GAPDH ntraleteal side) FGF-9 (100% of co sham- L sham- R MPP+-L MPP+-R M+M-L M+M_R (n=4) (n=8) (n=8) Figure 1. Melatonin prevents the down-regulation of FGF-9 mrna expression induced by MPP + treatment. The upper panel represents the FGF-9 and GAPDH mrna expression in striatum 3 days after MPP + with or without melatonin treatment. L, controlateral side; R, injected side of striatum. The quantitative data of FGF-9 mrna expression normalized with GAPDH was shown in lower panel by densitometry. 6

8 (A) (B) (C) Sham MPP + -injection MPP + + melatonin (D) (E) (F) SNpc SNr + Sham MPP MPP + + melatonin Figure 2. Melatonin prevents the loss of TH immunoreactivity in nigrostriatal dopaminergic system caused by MPP +. Coronal brain sections from sham (A), MPP + -treated (B), and melatonin + MPP + -cotreated rats (C) were immunostained with specific TH antibody in striatum. A significant decrease of TH immunoreactivity was indicated by white arrow in B after MPP + treatment. The arrows in the higher microphotographs shown in D-F (100 ) demonstrated the TH-positive dopaminergic neurons in the SNpc. Notice that melatonin attenuates the MPP + -induced a significant loss of TH-positive neurons in SN. SNpc, subtantia nigra pars compacta; SNr, substantia reticular. 7

9 FGF9 5 FGF-9/GAPDH L R L R MPP + MPP + +Mel FGF FGF-20/GAPDH L R L R MPP + MPP + +Mel TH 5 TH/GAPDH L R L R MPP + MPP + +Mel Figure 3. The FGF-9, FGF-20, and TH mrna expression in substantia nigra after MPP + with or without melatonin treatment. L, controlateral side; R, injected side. 8

10 melatonin M SF FGF9 GAPDH Figure 4. Melatonin up-regulates FGF-9 mrna expression in a dose-dependent manner in primary cortical neuronal culture. Astrocyte neuron P FGF-2 FGF-9 GAPDH Figure 5. The FGF-2, FGF-9, and GAPDH mrna expression in primary astrocyte and neuron culture. P, positive control. 9