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).

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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).

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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.

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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).

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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.

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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.

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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).

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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.

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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.

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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.

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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).

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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.

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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).

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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.

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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.

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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- and PI- positive cells, a cluster in the right upper quadrant were significantly increased in L-dopa- treated NPCs compared to control or PPX- treated NPCs (Fig. 9B). Quantitative analy-sis calculated by flow cytogram demonstrated a significant increase of apoptotic cell death in NPCs 72hr after L-dopa treatment than in control or PPX treatment (Fig. 9C).

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３６ Fig. 9 Hcy induce NPCs apoptosis in vitro

caspase-3 activities in NPCs was measured after cocultured with levodopa or PPX treated astrocytes. No change of caspase-3 activity is found in normal NPCs (A). A signifi-cant decrease in caspase-3 occurs at 72h in levodopa- treated NPCs. (n=3/group, *P < 0.005).

Folw cytogram shows ongoing cell death by flow cytometric analysis using annexin V/PI.

Apoptotic cells were significantly increased in levodopa - treated NPCs compared with con-trol and PPX- treated NPCs. But annexin V positive apoptotic cells were decreased after MK-801 treatment (B). Histogram revealed annexin positive cells (C). (n=4/group, *P <

0.005) Values are means ± SD.

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5. Effects of L-dopa treatment on regulation of ERK-MAP kinase signaling

To evaluate whether increased levels of homocysteine may modulate in regulation of ERK-MAP kinase signaling pathways through NMDA receptor, NPCs were co-cultured with L-dopa- or PPX- treated astrocytes for 72 hr. The phosphorylated form of ERK was signifi-cantly increased in L-dopa- treated NPCs compared to controls. However, PPX- treated NPCs did not change significantly the expression of phosphorylated ERK in comparison with controls (Fig. 10A and B). Additionally, MK-801, a NMDA antagonist, administration in dopa- treated NPCs significantly decreased phosphorylated ERK compared to only L-dopa- treated NPCs. It was similar expression of phosphorylated ERK in comparison with controls and PPX- treated NPCs.

３８ Fg. 10 Hcy mediated regulation of ERK-MAP kinase

Hcy mediated phospholylation of ERK was significantly increased in levodopa-treated NPCs compared with control and PPX-treated NPCs. There was no significant change in ERK phosphorylation in PPX-treated NPCs and control Increasing phophorylation of ERK was down-regulated in MK-801 treatment (A). Histogram revealed relative to ERK ratio (B). The results are representative of three replications in each group. *p < 0.02.

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6. L-dopa treatment leads to increase level of homocysteine in both plasma and brain Plasma homocysteine levels did not show a significant change in mice with only MPTP administration or in MPTP- treated mice with PPX administration compared to controls.

However, plasma homocysteine levels were increased significantly increased in MPTP- treated mice with L-dopa treatment compared to control, MPTP- only or MPTP- and PPX- treated mice (Fig. 11A). The level of homocysteine in the brain was increased significantly in MPTP- treated mice with L-dopa administration compared to controls, whereas the con-centration of homocysteine in the brain was decreased significantly in only MPTP- treated mice or in MPTP-treated mice with PPX compared to controls (Fig. 11B).

`

４０ Fig. 11 Measurements of Hcy in plasma and brain.

The concentration of Hcy increases levodopa-treated group. concentration of Hcy in plasma-treated levodopa was significantly increased compared with contorl and MPTP mice(A). Al-so, levodopa-treated mice was significantly increased compared with control and MPTP mice and pramipexole treated mice in brain tissue (B) (*p <0.05 ; **p < 0.01) The data are presented as mean of 5 determinations .+ S.E.

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7. L-dopa treatment leads to decrease neurogenesis in the SVZ zone

To investigate the effect of L-dopa treatment on neurogenesis in mice, NPCs immu-nostained with BrdU were determined in the SVZ. Immunohistochemistry revealed that BrdU-positive NPCs was significantly decreased in MPTP- treated mice compared to controls, whereas L-dopa or PPX treatment in MPTP- treated mice increased BrdU-positive NPCs compared to only MPTP- treated mice (Fig. 7A. a-d). Stereological analysis revealed that decreased number of BrdU-positive cells in the SVZ of MPTP- treated mice compared to controls was more evident, and the number of BrdU positive cells tended to be greater in PPX- treated mice compared to L-dopa- treated mice (Fig. 7B).

Additionally, I analyzed whether MK-801, a NMDA antagonist would lead to modulate de-creased neurogenesis associated with L-dopa treatment. MK-801 administration in L-dopa- treated PD animal model significantly increased the number of BrdU-positive cells in the SVZ compared to L-dopa- treated PD animal model. However, MK801 administration in PPX- treated mice was not changed (Fig. 7A e-f and B).

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Fig. 12 Representative photomicrograph of BrdU+ and Hematoxylin+ after drugs ad-ministration.

Neuroprogenitor cells, BrdU positive cells, in SVZ were decreased in MPTP- treated mince than control (A, a, b). Levodopa –treated mice were increased neuroprogenitor cells than MPTP but more increased in levodopa and MK-801- treated mice (A, c, d). Similary, PPX- and PPX-and MK-801-treated mice were significantly increased neuroprogenitor cells in SVZ (A, e, f) But PPX- and PPX- and MK-801 treated mice had no significant. Graph represents the number of BrdU+ cells in SVZ (B).(*p <0.02 ; **p < 0.002) The data are presented as mean of 5 determinations .+ S.E.

４４

IV. DISCUSSION

PART. A

Neuroprotective effects of L-dopa on dopaminergic neurons is com-arable to pramipexole in MPTP- treated animal model of Parkinson’s disease.

Until now, there have been no in vivo data that directly compare neuroprotection between L-dopa and PPX. In this study, to closely mimic the neuroprotective strategies in PD in a clinical setting, I used a subchronic model of MPTP, which is suitable for evaluating the apoptotic cell death pathway, and chronically administrated candidate drugs after the devel-opment of nigral pathology. My study demonstrated that both L-dopa and PPX have neuro-protective properties on dopaminergic neurons in the MPTP- treated animal model of PD, acting through the promotion of cell survival signaling and inhibition of apoptotic signaling.

Inhibitory effects on the JNK-related apoptotic pathway were similar between L-dopa and PPX, whereas L-dopa more potently activated ERK, and PPX seemed to exhibit a greater anti-oxidant effect. These results suggest that the neuroprotective effect of L-dopa on dopa-minergic neurons is comparable to that of PPX in an MPTP- treated PD model. MPTP may disrupt the balance between neuronal survival and apoptosis, producing a condition prone to neuronal degeneration. Through activation of the MAPK pathway by production of ROS, MPTP inhibits the activation of the ERK signaling pathway(De Girolamo and Billett 2006) and activates the JNK signaling pathway resulting in the phosphorylation of c-Jun (Saporito,

４５

Thomas et al. 2000). Activated JNK can promote the release of cytochrome c from the mito-chondrial inner membrane through a Bax-dependent mechanism, enhancing the formation of apoptosomes (Vila, Ramonet et al. 2008). Additionally, activated JNK can translocate to mi-tochondria, where it can phosphorylate Bcl-2 proteins, thereby inhibiting the anti-apoptotic activity of Bcl-2 (Dhanasekaran and Reddy 2008). As expected, MPTP treatment in my study markedly activated JNK and increased the expression of related apoptotic proteins such as Bax and cytochrome c, concomitantly with decreased Blc-2 expression and signifi-cant inhibition of ERK activation. Ample evidence exists showing that dopamine agonists such as PPX have neuroprotective effects through an antiapoptotic activity, by decreasing the fall in mitochondrial membrane potential, cytochrome c release, and caspase activation in experimental models generating ROS (Gu, Iravani et al. 2004; Karunakaran, Saeed et al.

2008). This effect would seem to be mediated by a mechanism that is either independent of or dependent on dopamine receptors. As expected, PPX treatment in this study decreased the MPTP- induced activation of JNK-related apoptosis, thereby restoring some of the overall balance between neuronal survival and apoptosis, which was disrupted by MPTP treatment.

Additionally, in contrast that the neuroprotective activity of PPX has been shown to occur in condition of pretreatment or pre-incubation before the introduction of neurotoxins (Anderson, Neavin et al. 2001; Gu, Iravani et al. 2004; Iravani, Haddon et al. 2006), this study demon-strated that PPX can also exert neuroprotective activity when administered after the onset of pathological changes in the SN. Interestingly, L-dopa treatment in this study decreased

sig-４６

nificantly the MPTP- induced activation of JNK-related apoptosis. These results conflict with previous in vitro data demonstrating that L-dopa can be toxic to dopaminergic neurons.

Although the exact mechanism is not fully understood, the capacity of L-dopa to undergo oxidative metabolism and generate ROS has been suggested as a possibility (Melamed, Offen et al. 1998). However, several studies using co-cultured neurons and glial cells have shown that glia can buffer ROS and that L-dopa has protective effects on dopaminergic neu-rons, even in high doses (Han, Mytilineou et al. 1996; Mena, Davila et al. 1998). The results of the in vivo administration of L-dopa to PD animal models have been conflicting; (Blunt, Jenner et al. 1993)) suggested a suppressive effect of L-dopa on dopaminergic neurons in the ventral tegmental area ipsilateral to a 6-hydroxydopamine lesions, whereas (Murer, Dziewczapolski et al. 1998) and (Datla, Blunt et al. 2001) demonstrated that chronic admini-stration of L-dopa increased the density of dopaminergic fibers or neurons without toxic ef-fects to remaining nigral dopaminergic neurons. ELLDOPA, a clinical trial to explore L-dopa toxicity in early stages of PD, showed contradictory findings between progression of clinical

Although the exact mechanism is not fully understood, the capacity of L-dopa to undergo oxidative metabolism and generate ROS has been suggested as a possibility (Melamed, Offen et al. 1998). However, several studies using co-cultured neurons and glial cells have shown that glia can buffer ROS and that L-dopa has protective effects on dopaminergic neu-rons, even in high doses (Han, Mytilineou et al. 1996; Mena, Davila et al. 1998). The results of the in vivo administration of L-dopa to PD animal models have been conflicting; (Blunt, Jenner et al. 1993)) suggested a suppressive effect of L-dopa on dopaminergic neurons in the ventral tegmental area ipsilateral to a 6-hydroxydopamine lesions, whereas (Murer, Dziewczapolski et al. 1998) and (Datla, Blunt et al. 2001) demonstrated that chronic admini-stration of L-dopa increased the density of dopaminergic fibers or neurons without toxic ef-fects to remaining nigral dopaminergic neurons. ELLDOPA, a clinical trial to explore L-dopa toxicity in early stages of PD, showed contradictory findings between progression of clinical