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 antiprolifera-tive effect on NPCs through NMDA receptor mediated apoptosis.
To evaluate blocking effects of NMDA receptor on the neurogenesis of the SVZ, I ex-tended my study into animal model of PD using MPTP. As expected, the neurogenesis in the SVZ measured by BrdU-positive NPCs cells was decreased significantly in MPTP- treated animals, which is in accordance with previous studies demonstrating that dopamine deple-tion led to decrease neurogenesis in the SVZ of postmortem brain of PD patients and animal model of PD (Hoglinger, Rizk et al. 2004). Interestingly, my in vivo data demonstrated that the treatment of NMDA anagonist in L-dopa- treated PD animals significantly increased neu-rogenesis in the SVZ compared to only L-dopa- treated PD animals. Several evidence have suggested that prolonged NMDA receptor activation might decrease the rate of proliferation and the number of newly generated neurons in the SVZ, although the neurogenetic effects of NMDA receptor is depending on exposing time or dose of NMDA agonists/antagonists (Kitayama, Yoneyama et al. 2003). In this regard, NMDA antagonist might attenuate pro-longed NMDA receptor activation induced by increased homocysteine in the brain after L-dopa treatment, and in turn lead to decrease apoptosis of NPCs in the SVZ. This in vivo re-sults further confirm that L-dopa induced hyperhomocysteienemia would modulate NMDA-dependant neurogenesis.
Another interesting finding is comparative analysis of neurogenesis between L-dopa and dopamine agonists. Recent studies reported that NPCs in the SVZ exhibited dopaminergic
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receptors and dopaminergic innervations are important for the proliferation of NPCs in the SVZ(Winner, Desplats et al. 2009). Importantly, the neruogenetic effects are mediated by activation of the D2/D3 dopamine receptors, where dopamine receptor activation induces CNTF release into neurogenic niches (Mori, Jefferson et al. 2008; Yang, Arnold et al. 2008).
Along with augmenting effects of neurogenesis by dopamine agonist in animal model of PD, O'Sullivan et al. demonstrated a positive impact of chronic L-dopa use on the number of neu-ral stem cells in the SVZ of patients with PD, however, there are no available studies dealing with direct comparison of neurogenetic activity between L-dopa and dopamine agonist in the same experimental design until now. In this study, I found that the number of cultured NPCs and BrdU-positive cells in the SVZ was higher significantly in PPX treatment than in L-dopa treatment, showing that the neurogenetic activity of dopamine agonist is superior to that of L-dopa. This difference of neurogenetic activity may be ascribed to detrimental effects of homocysteine on NPCs by L-dopa treatment. In addition, since PPX, as a D2 receptor family, has a more affinity on D3 dopamine receptor relative to L-dopa, this different property may also contribute to difference of neurogenetic activities, being in favor of dopamine agonist.
Indeed, the number of BurU-positive cells in the SVZ tended to be higher in L-dopa treated PD animals compared to only MPTP- treated PD animals, which may imply indirectly that pro-neurogenetic effect through increased dopamine innervations by L-dopa seems to be stronger than anti-neurogenetic effect by L-dopa induced homocysteine. However, further clinical evidence of postmortem study regarding the role of dopamine agonist in modulation
５４ of neurogenesis may clarify this issue.
Regarding the functional impact of adult neurogenesis, Several evidence has suggested that adult neurogenesis has a regulatory role in olfaction, mood, and memory. In the PD ani-mal model, OO reported that an increase of neurogenesis by dopaminergic drug restored the nigrostriatal dopaminergic projection concomitant with function motor recovery, however, there is still debating whether NPCs from the SVZ would transdifferentiate into nigral do-paminergic phenotypes or migrate into the striatum and then incorporate into host neurons.
From a therapeutic perspective, treatment focusing on modulation of adult neurogenesis could be great value in future strategy of PD treatment to repair endogenously the damaged PD brain. Furthermore, neurogenetic activity of dopaminegic drugs commonly used in PD patients is also considering issue in the clinical practice along with perspectives of neuro-portection and dopaminergic medication associated with motor complication of wearing off and dyskinesia. In this regard, my current data may have clinical relevance for human PD treatment in that dopamine agonist has more neurogenetic activity than L-dopa. In addition, my study showed that inhibition of Hcy synthesis is a new therapeutic strategy of PD treatment in terms of neurogenesis. Future study is needed to clarify whether inhibition of Hcy synthesis using clinically available COMT inhibitor lead to protection of NPCs against L-dopa induced hyperhomocystenemia.
In summary, my present study demonstrated that increased homocysteine by L-dopa has a detrimental effect on adult neurogenesis through NMDA receptor-mediated ERK-MAP
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naling pathway, and modulation of L-dopa induced hyperhomocysteinemia by NMDA an-tagonist or dopaminergic agonist has a clinical relevance for human PD treatment in terms of adult neurogenesis. Future study focusing on modulation of adult neurogenesis with small molecule receptor agonists would extend clinical applications.
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