Preparation of plasma and rat brain samples

To measure Hcy, rat brains were isolated and blood samples were collected into EDTA

paraformaldehyde, and stored in 30% sucrose solution for 2 days, until they sank. They were then sectioned on a sliding microtome to obtain a 30-mm-thick coronal section. All sections were stored in tissue stock solution (30% glycerol, 30% ethylene glycol, 30% 3rd D.W., 10%

0.2 m PB; pH 7.2; Sigma , St. Louis, MO, USA ) at 4 ℃ until required. For ELISA, animals were killed after seven weeks, and the SN area was rapidly removed from the brains and fro-zen at 70 ℃.

１０ 4.

Measurements of GSH assay.

For the determination of total GSH, a Bioxytech GSH-420 kit (Oxis Research, Portland, OR, USA) was used. Brain tissues were washed in 0.9% NaCl solution and then homoge-nized in ice-cold precipitation reagent at a ratio 1: 15 (w/v). An aliquot (200 mL) of each brain sample supernatant was transferred to a fresh microcentrifuge tube. The reaction mix-ture (200 mL) and reducing agent were added to each tube and mixed well. Then a chro-mogen or color developer was added. Each reaction was incubated at room temperature (RT) in the dark for at 30 min before the absorbance at 420 nm was measured (Jones, Carlson et al.

1998).

5.

Measurements of Hcy.

Tissue homogenates of striatum were centrifuged (20 min, 14 000 g, 4_C) and the super-natant was transferred to a fresh tube for the dopamine assay using GC-MS. GC–MS analy-ses in both scan and selected ion monitoring modes were performed using an Agilent 6890 gas chromatograph, interfaced to an Agilent 5973 mass-selective detector (70eV, electron impact mode) and installed with an Ultra-2 (5% phenyl–95% methylpolysiloxane bonded phase; 25 m·0.20 mm i.d., 0.11 lm film thickness) cross-linked capillary column (Agilent Technologies, Atlanta, GA, USA). The temperatures of the injector, interface, and ion source were 260, 300, and 230_C, respectively. Helium was used as carrier gas at a flow rate of 0.5–1 mL/min in a constant flow mode. Derivatized extracts were introduced in

split-１１

injection mode (10: 1)and the oven temperature was initially at 100℃ for 2 min, and pro-grammed to rise to 205℃ at 5℃/min and finally to 300℃ (3min) at 20℃/min. The mass range scanned was 50–800 U at a rate of 0.42scan/s. All GC–SIM–MS runs were performed in triplicate. The mass range scanned was 50–800 U at a rate of 0.42scan/s. All GC–SIM–

MS runs were performed in triplicate.

6.

BrdU administration.

To assess ongoing cell proliferation, all animals were assigned to a BrdU injection regime following a modified protocol described by Kralic et al. (Vicario, Tabernero et al. 1993).

Each mouse was injected with BrdU (Sigma, St. Louis, MO, USA) that incorporates into the DNA in the S phase of the cell cycle. BrdU was dissolved in PBS and administered i.p once daily on five subsequent days at a concentration of 50 mg/kg in a 20mg/ml volume.

7. Immunohistochemistry and Immunocytochemistry.

The brain sections and co-cultured cells were rinsed twice in PBS and incubated in 0.2%

Triton X-100 for 30 min at room temperature (RT). For BrdU staining this procedure was added, they were incubated in 50% formamide and 2x SSC buffer at 65℃ for 2 hr. And then they were treated with 2N HCL at 37℃ for 30min. After 2N HCL treat, they were rinsed three times in PBS and incubated hydrogen peroxide blocking solution (Thermo, Fremont, CA USA) for 10min at RT. For immune-staining, they were rinsed and incubated in triton

X-１２

100 for 30 min at RT. They were rinsed three times with 0.5% bovine serum albumin in PBS for blocking. After blocking, they were incubated overnight at 4℃ with primary antibody.

The primary antibodies were used as follows: mouse anti-OX-42 (1: 200 for immunocyto-chemistry; Serotec, Raleigh, NC, USA), mouse anti-GFAP (1: 200 for immunocytochemis-try; Abcam, Cambridge, UK), mouse anti-Ki67 (1: 200 for immunocytochemistry, Chemicon, Billerica, MA, USA), rabbit anti-NMDA2A receptor and NMDA2B receptor (1:200 for im-munocytochemistry, Chemicon, Billerica, MA, USA), mouse anti-BrdU (1: 200 for immu-nohistrochemistry, Roche, IN, USA), mouse anti-tyrosine hydroxylase (TH; 1: 1000 for im-munohistochemistry, Chemicon International, Temecula, CA, USA). After over night, the brain tissues and cells were rinsed three times in 0.5% bovine serum albumin in PBS (10 min/rinse) and incubated with the appropriate biotinylated secondary antibody and avidin–

biotin complex (Elite Kit; Vector Laboratories, Burlingame, CA, USA) for 1 h at RT. The horseradish peroxidase reaction was detected with 0.05% diaminobenzidine (DAB, Vector Laboratories, Burlingame, CA, USA) and 0.03% H₂O₂. For Immunofluorescence labeling, cells were incubated with goat anti-mouse IgG (AlexaFluor-488, green) and goat anti-rabbit IgG (Alexa Fluor-594, red) secondary antibodies for 1 h at RT. Cell nuclei were counter-stained with DAPI (1: 2000 dilution, Molecular Probes) for 1 h at RT. Processing was stopped with H₂O, and sections were dehydrated through graded alcohols, cleared in xylene, and overslipped in permanent mounting medium (Vector, Vector Laboratories, Burlingame, CA, USA). For Hematoxylin staining, the brain tissues were dried and stained with Mayer’s

１３

Hematoxylin (MUTO, Tokyo, Japan). The immune-stained cells were analyzed by bright-field microscopy and viewed using an conforcal laser scanning microscope (Olympus, Tokyo, Japan).

8. Stereological cell counts.

Unbiased stereological estimations of the total number of the stained cells in the SVZ and SN were made using an optical fractionator, as previously described with some modifica-tions (Kirik, Rosenblad et al. 1998). This sampling technique is not affected by tissue vol-ume changes and does not require reference volvol-ume determinations. The sections used for counting covered SVZ and the entire SN, from the rostral tip of the pars compacta back to the caudal end of the pars reticulate. This generally yielded 8–12 sections in a series. Sam-pling was performed with the Olympus C.A.S.T.-Grid system (Olympus Denmark A/S, Denmark), using an Olympus BX51 microscope, connected to the stage and feeding the computer with the distance information in the z-axis. The SN was delineated at 1.25 magni-fication. A counting frame (46%, 40, 1699 mm²) was placed randomly on the first counting area and systematically moved though all counting areas until the entire delineated area was sampled. Actual counting was performed using a x100 oil objective. Guard volumes (4 mm from the top and 4–6 mm from the bottom of the section) were excluded from both surfaces to avoid the problem of lost cap, and only the profiles that came into focus within the

count-１４

ing volume (with a depth of 10mm) were counted. The total number of stained cells was cal-culated according to the optical fractionator formula (West, Slomianka et al. 1991).

9. Total RNA Extraction and Reverse Transcriptive PCR (RT-PCR).

Total RNA was extracted from the NPCs using Trizol reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s protocol. An equal amount of RNA (approxi-mately 1mg) in each experiment was reverse transcribed using a amfiRivert cDNA Synthesis Premix (GenDEPOT, Barker, TX, USA). Subsequently, 2ml of cDNA was used as a template for RT- PCR analysis in amfiRivert 1-Step RT-PCR Kit. (GenDEPOT, Barker, TX, USA).

The PCR reaction was performed using 10 pmol each of the primers for NMDA receptor 1 (NR1; forward 5’-CTG CAA CCC TCA CTT TTG AG-3’, reverse5’-TGC AAA AGC CAG CTG CAT CT-3’), NMAD 2A receptor (NR2A; forward 5’-GAC GGT CTT GGG ATC TTA AC-3’, reverse 5’- TGA CCA TGA ATT GGT GCA GG-3’), NMAD 2B receptor (NR2B; forward 5’- CAA GAA CAT GGC CAA CCT GT-3’, reverse 5’- GGT ACA CAT TGC TGT CCT TC-3’), NMAD 2C receptor (NR2C; forward 5’- TGG AAA CTT CGA CAC TCG GT-3’, reverse 5’- TCC AAA GAG CTG CTC ACG TC-3’) (Sun, Shipley et al.

2000). After an initial denaturation at 94℃ for 5 min, 30 cycles of PCR were performed, consisting of denaturation (30s, 94℃), annealing (1 min, 58℃ [NR1], 55℃ [NR2A, NR2B], 57℃ [NR2C]) extension (1 min, 72℃ followed by a final extension (10 min, 72℃). The PCR products were separated by electrophoresis on 2% agarose gel and stained with

ethi-１５

dium bromide. Gels were examined under UV illumination.

10. Flow cytometric measurement of cell death using Annexin-V/PI.

NPCs co-cultured with L-dopa-treated astrocytes were harvested by trypsinization and pel-leted by centrifugation at 1500rpm for 5min. Cell pellets were washed once in ice cold PBS, followed by gentle re-suspension in 100ml of annexin-V binding buffer containing 5ml of FITC-labeled annexin V (Annexin-V; 100mg/ml stock in PBS) and 5ml propidium iodide (PI;

100mg/ml stock in PBS) solution for 15min (BD, San diego, CA, USA). And then add 400ml 1x binding buffer. Samples were immediately kept on ice and analyzed on FACS. Data was acquired and analyzed using Winmdi software. Acquisition gates were wet using the forward and side light scatter of the cells and a minimum of 10,000 events were collected for each sample.

11. Caspase -3 activity assay.

The caspase-3 activity was measured by caspase-3 activity assay kit. (Chemicon, Billerica, MA, USA). Caspase-3 activity was determined by monitoring proteolysis of corresponding colorimeric substrates. NPCs co-cultured with L-dopa-treated astrocytes were collected and washed in ice-cold PBS pH 7.0. NPCs were subsequently lysed in 1x lysis buffer for 10min in ice and the lysates were clarified by centrifugation at 13000rpm. After centrifugation for 10mins, cytosolic extracts of NPCs were transferred to a fresh tube and putted on ice. Then

１６

30mg of the caspase-3-specific colorimetric substrate acetyl-Asp-Glu-Val-Asp-7-p-nitroaniline (Ac-DEVD-pNA) was added in cytosolic extracts. They were incubated for 1hr at 37 . The release of℃ DEVD-pNA were quantified at 405nm by ELISA plate reader.

12. Western blot analysis.

For western blotting, NPCs co-cultured with L-dopa-treated astrocytes were washed in ice-cold and lysis buffer containing protease inhibitor (iNtRON Biotechnology, SeongNam, S.

Korea). Briefly, 50 mg of protein for each specimen were separated by sodium dodecyl sul-fate polyacrylamide gel electrophoresis and transferred to Hybond-ELC C pure nitrocellu-lose membrane (Amersham, Piscataway, NJ, USA). The membranes were blocked in non-fat milk. Membranes were probed with 1 : 1000 dilutions of the following primary antibodies : rabbit polyclonal ERK1/2 and rabbit polyclonal phosphor-ERK1/2 (Cell Signaling, Danvers, MA, USA). As a secondary antibody, a 1: 2000 dilution of horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit antibody (Zymed Laboratories, San Francisco, CA, USA) was used. Antigen–antibody complexes were visualized with a chemiluminescence system (Amersham, Piscataway, NJ, USA), followed by exposure to x-ray film (Kodak X-OMAT, Rochester, NY, USA). For semiquantitative analysis, the densities of the im-munoblot bands were measured by computer imaging (Image J; NIH, Bethesda, MD, USA).

１７ 13.

Statistical analysis.

The group means were compared using the Mann-Whitney U-test for pairs and the Kruskal-Wallis analysis for multiple groups. P values less than 0.05 were considered statisti-cally significant. Statistical analyses were performed using commercially available software (version 12.0; SPSS Inc., Chicago, IL).

１８

III. RESULTS

PART. A

Neuroprotective effects of levodopa on dopaminergic neurons is com-parable to pramipexol in MPTP- treated animal model of Parkinson’s Disease.

1. Effects of L-dopa and PPX administration on GSH levels in MPTP-treated mice.

Beginning on day 43 after the first administration of either L-dopa or PPX in MPTP- treated mice, the midbrain was isolated and assayed for GSH. The level of GSH in MPTP- only treated mice was significantly decreased compared with that in control mice. The level of GSH was significantly increased in MPTP- treated mice after PPX administration com-pared with MPTP- only treatment. L-dopa administration also increased the level of GSH in MPTP- treated mice, although not significantly. The level of GSH was not different between L-dopa- and PPX- and MPTP- treated mice (Fig. 1).

１９

Fig. 1 The level of glutathione (GSH) in the substantia nigra (SN).

Beginning on day 43 after the first administration of either L-dopa- or PPX- in MPTP- treated mice, the midbrain was isolated and assayed for GSH. The level of GSH in MPTP- treated mice was significantly decreased compared with that in control mice. PPX treatment significantly increased the GSH level in MPTP- treated mice (p < 0.05). L-dopa administra-tion also tended to increase the GSH level in MPTP- treated mice, but without statistical sig-nificance. The data are displayed as the mean (column) ± SEM (bar). The results are repre-sentative of five replications in each group. *p < 0.05, **p < 0.01.

２０

2. Effects of L-dopa and PPX administration on ERK phosphorylation in MPTP- treated mice

.

Compared with control mice, MPTP- treated mice exhibited significantly decreased ex-pression of phosphorylated ERK. The exex-pression of phosphorylated ERK was significantly increased in MPTP- treated mice after L-dopa administration compared with MPTP- only treatment. However, PPX administration did not significantly change the expression of phosphorylated ERK in comparison with MPTP- only treatment (Fig. 2).

3. Effects of L-dopa and PPX administration on JNK phosphorylation in MPTP- treated mice.

The phosphorylated form of JNK was significantly increased in MPTP- treated mice compared with control mice. Both L-dopa and PPX administration led to significantly de-creased expression of JNK phosphorylation in MPTP- treated mice. The degree of reduction in JNK phosphorylation did not differ significantly between L-dopa- and PPX- treated groups (Fig. 3).

２１

Fig. 2 Effects of L-dopa and PPX administration on ERK phosphorylation.

The expression of phosphorylated ERK was significantly decreased in MPTP- treated mice compared with control mice. L-dopa administration in MPTP- treated mice signifi-cantly increased ERK phosphorylation compared with the level in MPTP- only treated mice.

There was no significant change in ERK phosphorylation in PPX- treated mice. The results are representative of three replications in each group. *p < 0.01.

２２

Fig. 3 Effects of L-dopa and PPX administration on JNK phosphorylation in MPTP- treated mice.

JNK phosphorylation was significantly increased in MPTP- treated mice compared with sa-line-treated mice. Both L-dopa and PPX administration in MPTP- treated mice led to signifi-cant and similar decreases in the expression of JNK phosphorylation. The results are repre-sentative of three replications in each group. *p < 0.01.

２３

4. Effects of L-dopa and PPX administration on apoptosis related proteins (Bax, cytochrome c, and Bcl-2).

Compared with control mice, MPTP- treated mice showed significantly increased expres-sion of Bax. L-dopa administration significantly decreased Bax expresexpres-sion in MPTP- treated mice compared with the expression in MPTP- only treated mice. PPX administration also significantly reduced Bax expression in MPTP- treated mice. A similar pattern was observed for the expression of cytochrome c in MPTP- treated mice. Both L-dopa and PPX signifi-cantly decreased the expression of cytochrome c, which had been elevated after MPTP treatment. PPX also significantly decreased expression of Bax. There was no statistical dif-ference in the expression level of Bax or cytochrome c between the L-dopa-treated and PPX- treated groups. The expression of Bcl-2 was quite contrast. The administration of L-dopa and PPX significantly increased the expression of Bcl-2, which had been decreased after MPTP treatment. The degree of increased Bcl-2 expression did not differ significantly between the two groups (Fig. 4).

２４

２５

Fig. 4 Effects of L-dopa and PPX administration on apoptosis related proteins.

The expression of Bax was significantly increased in MPTP- treated mice compared with saline-treated mice. Both Ldopa and PPX administration in MPTP-treated mice significantly decreased Bax expression to a similar degree, as compared with the expression in MPTP- only treated mice (a). Both L-dopa and PPX significantly attenuated MPTP- induced in-creases in the expression of cytochrome c (b). The expression of Bcl-2 was quite the reverse:

the MPTP- induced decrease in Bcl-2 expression was significantly increased after L-dopa or PPX administration (c). The degree of the increase in Bcl-2 expression was not significantly different between the two agents. The results are representative of three replications in each group. *p < 0.01.

２６

5. Effects of L-dopa and PPX administration on survival of dopaminergic neurons in MPTP- treated mice.

Brain tissue was harvested from MPTP- treated mice at 2 weeks after 4 weeks of treat-ment with either L-dopa or PPX had been completed. Immunohistochemical analyses showed that both L-dopa and PPX treatment dramatically rescued the decline in the number of tyrosine hydroxylase immunoreactive (TH-ir) and Nissl-stained cells in the SN of MPTP- treated mice (Fig. 5a). Stereological analysis revealed that TH-ir and Nissl-stained cells were significantly decreased in the SN of MPTP- treated mice, which showed an approximately 50% reduction compared with control mice. Both L-dopa and PPX administration in MPTP- treated mice significantly increased the number of TH-ir and Nissl stained cells compared with the number in MPTP- only treated mice (Fig. 5b). The number of TH-ir cells tended to be greater in L-dopa-treated mice than in PPX- treated mice, but the difference was not sta-tistically significant.

２７

２８

Fig. 5 Effects of L-dopa and PPX administration on survival of dopaminergic neurons.

Immunohistochemical analysis showed that both L-dopa and PPX dramatically attenuated the decline in the number of TH-ir and Nissl-stained cells in the SN of MPTP- treated mice (a). On stereological analysis, TH-ir and Nissl-stained cells in the SN were significantly de-creased in MPTP- treated mice, showing 50% reduction compared with saline-treated mice.

After treatment with either L-dopa or PPX in MPTP- treated mice, number of TH-ir cells was significantly increased compared with the number in MPTP- only treated mice (b). The number of TH-ir cells tended to be greater in L-dopa- treated mice than in PPX- treated mice; however, the difference was not statistically significant. The data are displayed as the mean (column) ± SEM (bar). The results are representative of five replications in each group.

*p < 0.05, **p < 0.01. Scale bar: 100mm.

２９

PART. B

Increased level of homocysteine induced by levodopa inhibits neurogenesis by mediating NMDA receptor signal cascade in MPTP- treated animal model of Parkinson’s disease: comparison with pramipexol

1. The phenotype and proliferative capacity of cultured NPCs from the SVZ.

To determine the phenotypic properties of NPCs, the neural stem cell marker of nestin (Wiese, Rolletschek et al. 2004) was used to reveal cells expressing a neuronal phenotype, while astrocytes marker of GFAP and microglia marker of OX-42 were used to reveal the presence of glial cells. When the phenotype of the cultured NPCs was examined in vitro, no GFAP-positive and OX-42-positive cells were observed (Fig. 6A and B) and most NPCs showed nestin-positive (Fig. 6C). In addition, the NPCs were immunostained with Ki67, a proliferation marker (Morimoto, Kim et al. 2009) Fig 6D).

３０

Fig. 6 Most NPCs continue to express a neuronal phenotype and ability to proliferate for several days in vitro.

NPCs from SVZ were dissociated, cultured for several days and then double-stained by spe-cific antibody (GFAP; astrocytes marker, OX-42; microglia marker, nestin; neural stem cell marker and Ki67; proliferation marker). They were visualized by a secondary antibody con-jugated to fluorescein with DAPI.

３１

2. mRNA Expression and Immunodetection of NMDA Receptor Subunits in cultured NPCs.

Immucytochemistry revealed that NPCs expressed NMDA receptor subunits 2Aand 2B with a strong expression of NMDA receptor subunit 2A (Fig. 7A and B). These results were confirmed by RT-PCR, showing total RNA isolated from NPCs expressed detectable NR1, NR2A, NR2B and NR2C (Fig. 7C).

3. Increased level of homocysteine in astrocytes culture media after L-dopa treatment.

To evaluate whether L-dopa introduction would increase the level of homocysteine, I de-termined concentration of homocysteine in astrocytes culture media treated with L-dopa. The extracellular concentration of homocysteine increased linearly with time during incubations with L-dopa and reached higher levels at 72hr after L-dopa treatment. Release of homocys-teine by L-dopa treatment was dependant on the number of astrocytes with maximum level in the highest dose of L-dopa (Fig. 8A and B). However, there were no detectable homocysteine in astrocytes culture media after PPX treatment (Fig 8C and D).

３２

Fig. 7 Expression of NMDA Receptor Subunits in cultured NPCs.

Cultured NPCs were expressed NMDA receptor subunits 2A (a), 2B (b). with specific antibodies. And NMDA receptor 1 (NR1; 145bp), 2A (NR2A; 140bp), 2B (NR2B; 229bp), and 2C (NR2C, 220bp) subunits were detected in mRNA levels from NPCs.

３３

Fig. 8 L-dopa stimulates the release of Hcy from astrocytes.

Export of Hcy was studied after astrocyte treated with several dose of levodopa and PPX.

The extracellular Hcy from astrocytes increased linearly with time during 24h and 72h. Hcy export by levodopa was dose-dependent and sensitive to cell number. However, Hcy was not detected in PPX treated astrocyte culture media.

３４

4. Increased apoptosis in NPCs after L-dopa treatment.

To exam whether increased release of homocysteine after L-dopa treatment could induce apoptosis, NPCs were co-cultured with L-dopa- or PPX- treated astrocytes for 24hr and 72hr.

Caspase-3 activity in L-dopa- treated NPCs was increased significantly in a time-dependent manner, and this activity was significantly higher than control or PPX- treated NPCs at 72hr (Fig.9A). In addition, flow cytometric assays using annexin V/PI revealed that Annexin-V-

Caspase-3 activity in L-dopa- treated NPCs was increased significantly in a time-dependent manner, and this activity was significantly higher than control or PPX- treated NPCs at 72hr (Fig.9A). In addition, flow cytometric assays using annexin V/PI revealed that Annexin-V-