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.

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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,

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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

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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 severity and functional imaging in L-dopa- treated PD patients compared with placebo-treated patients (Fahn, Oakes et al. 2004), suggesting that the potential long-term effects of L-dopa on PD remain uncertain. Overall, my study suggests that the neuroprotective activity of L-dopa, acting through an anti-apoptotic activity, is comparable to that of PPX. The acti-vation of ERK is widely believed to participate in the survival of dopaminergic neurons (Cavanaugh, Jaumotte et al. 2006; Zigmond 2006) although there is also increasing evidence

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relating its activation with cell death (Canals, Casarejos et al. 2003). There have been few studies regarding the role of ERK signaling in PD models; in vitro studies using MPTP or rotenone have demonstrated that neurotoxins can inhibit ERK activation (De Girolamo and Billett 2006; Chen, Zhang et al. 2008), whereas ERK activation may contribute to dopa-minergic neuronal death in a 6-hydroxydopamine in vitro model (Kulich and Chu 2001). In my study, ERK activation in the midbrain was significantly decreased in MPTP-treated mice compared with control mice, which suggests that ERK activation may be involved in the survival pathway of dopaminergic neurons. Interestingly, ERK activation in MPTP- treated mice was more prominent in L-dopa- treated group compared to MPTP only treatment group as well as PPX- treated group. This is an unexpected finding because dopamine agonists, including PPX, have been known to activate ERK signaling, possibly through the up-regulation of glial cell line-derived neurotrophic factor and brainderived neurotrophic factor (Du, Li et al. 2005; Chen, Zhang et al. 2008). Several in vitro and in vivo studies demon-strated that L-dopa had neurotrophic properties for dopaminergic neurons, thus promoting cell survival and neurite outgrowth, which may be mediated by factors in glial cells that are up-regulated by L-dopa (Han, Mytilineou et al. 1996; Mena, Davila et al. 1997). Mena et al.

reported that L-dopa potentiated the neurotrophic response of nerve growth factor, proposing that subtoxic oxidative stress by L-dopa may provide a trophic effect. In this regard, it is spe-culated that L-dopa or L-dopa metabolite-elicited neurotrophic factors may stimulate cell survival-related trophic factors, thus resulting in the activation of ERK. Of the various

anti-４８

oxidant systems in the brain, the GSH system is particularly important in controlling the cel-lular redox state and is the primary defense against oxidative stress (Cooper and Kristal 1997). In accordance with previous studies, the present study showed that PPX administra-tion significantly increased the level of GSH compared with the level in MPTP- only treated mice. PPX also has an inhibitory effect on ROS production via decreased turnover of dopa-mine metabolism, because PPX acts on dopadopa-mine autoreceptors. In addition, PPX displays anti-oxidant properties through the direct scavenging of free radicals and the stimulation of cellular GSH peroxidase and catalase (Le, Jankovic et al. 2000). In the L-dopa- treated group, the level of GSH did not changed significantly compared with the level in the MPTP- only treated group; this is in contrast to in vitro studies showing detrimental effects of L-dopa on GSH levels(Spencer, Jenner et al. 1995). This discrepancy may be ascribed to differences in the antioxidant defense environment according to experimental designs(Han, Mytilineou et al. 1996) or to a biphasic effect of L-dopa on GSH, in which GSH synthesis is upregulated in response to mild oxidative damage and reduced in response to severe oxidative damage (Mytilineou, Walker et al. 2003).

Overall, the promotion of cell survival signaling by L-dopa and PPX after MPTP treat-ment led to the neuroprotection of dopaminergic neurons, as evidenced by an immunohisto-chemical analysis indicating that TH-ir neuron survival in the SN after MPTP treatment was significantly increased in both the L-dopa and PPX treatment groups, compared with the MPTP only treatment group. Additionally, increased survival of TH-ir cells by L-dopa and

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PPX in MPTP- treated mice was also observed in immobilized groups, suggesting that neu-roprotective effect of L-dopa and PPX would not be resulted from enhanced locomotor activ-ity by these drugs. On direct comparison between L-dopa and PPX, there was no significant difference of neuroprotective effect on dopaminergic neurons in MPTP- treated mice. The similarity between the anti-apoptotic properties of L-dopa and PPX and their comparable neuroprotective effects, through ERK activation for L-dopa and via anti-oxidative effect for PPX, may produce similar increases in the survival of dopaminergic neurons for both agents.

In summary, my study demonstrated that both L-dopa and PPX had comparable neuroprotec-tive properties for dopaminergic neurons in MPTP- treated PD animal models, through mod-ulation of cell survival and apoptotic pathways. These data may provide in vivo evidence that L-dopa is not toxic but is neurotrophic to dopaminergic neurons in PD. Nevertheless, my data should be interpreted cautiously in clinical implications for L-dopa therapy because the daily dose of L-dopa used in this study is higher as compared with that normally used in PD patients. Future study with the daily dose of L-dopa commonly used in clinical practice would helpful to resolve this issue.

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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.

This is the first study evaluating the effect of L-dopa induced hyperhomocysteinemia on neurogenesis of in vitro and in vivo system with comparative analysis of dopamine agonist.

The major findings were (1) hyperhomocysteinemia associated with L-dopa treatment exerts an antiproliferative effect on NPCs in the SVZ, (2) L-dopa treatment induced apoptosis of NPCs is mediated by ERK-MAP kinase signaling pathways through NMDA receptor, and (3) dopamine agonist has more augmenting effects of neurogenesis compared to L-dopa.

There is accumulating clinical evidence that chronic administration of L-dopa in patients with PD lead to increase the homocysteine synthesis (Blandini, Fancellu et al. 2001; Miller, Selhub et al. 2003). Similarly, my data showed that L-dopa treatment increased release of homocysteine from cultured astrocytes as well as concentration of homocysteine in both plasma and brain in MPTP- treated PD animals. Experimental studies indicated that homo-cysteine acts as an excitatory aminoacid by activating NMDA receptors (Lipton, Kim et al.

1997; Poddar and Paul 2009) and thus induce mitochondrial dysfunction, free radicals(Jara-Prado, Ortega-Vazquez et al. 2003) and cytosolic calcium accumulation (Kruman, Culmsee et al. 2000), and apoptotic pathways (Jiang, Gu et al. 2000). Accordingly, preclinical evidence has suggested that L-dopa treatment associated with hyperhomocysteinemia may

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lead to detrimental effects on dopaminergic neurons as well as on non-dopaminergic neurons in PD models (Huang, Dragan et al. 2005; Imamura, Takeshima et al. 2007). However, whether L-dopa induced hyperhomocysteinemia may contribute to accelerate progression of nigal motor dysfunction and risk of extra-nigal non-motor features in patients with PD is controversial and remains to be determined. In this regard, scientific evidence addressing metabolic consequences of L-dopa treatment on other non-dopaminergic systems, such as neurogenetic system evaluated in the present study, are of great importance to determine so-phisticated therapeutic strategies for patients with PD.

My current in vitro data demonstrated that increased release of homocysteine from L-dopa treated astrocytes had a neurotoxic peroperty on NPCs of the SVZ, and phosphorylation of ERK through NMDA receptor led to induction of apoptosis in NPCs. In my study, NPCs iso-lated from the SVZ express NMDA receptor subunits 2Aand 2B as well as NR1, where the NR2A subunit is known to conveys high affinity for glutamatergic agonists. The role of NMDA receptor in regulating an upstream MAPK superfamily and ERK mediated proapop-totic signals has been extensively investigated. In NMDA receptor mediated neuronal toxic-ity, largely via the NMDAR-mediated influx of extracellular Ca2+, MAPKERK1/2 is known to be rapidly and transiently activated, and be involved in glutamate-induced apoptosis (Jiang, Gu et al. 2000; Haddad 2005). Additionally, my vitro study showed that a NMDA antagonist (MK-801) treatment significantly attenuated L-dopa induced activation of ERK kinase signaling pathways and apoptotic cell death in the NPCs. This result might further

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support that L-dopa induced hyperhomocysteienemia has an important role in

support that L-dopa induced hyperhomocysteienemia has an important role in