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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 5, 2003 as doi:10.1096/fj.02-0712fje. |
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Eve Topf and USA National Parkinson Foundation Centers of Excellence for Neurodegenerative Diseases Research and Department of Pharmacology and Rappaport Family Research Institute Technion, Faculty of Medicine, Haifa, 31096, Israel
2Correspondence: Department of Pharmacology, Technion- Faculty of Medicine, P.O.B. 9697, 31096 Haifa, Israel. E-mail: Youdim{at}tx.technion.ac.il
SPECIFIC AIMS
Iron chelators and radical scavengers such as melatonin, R-apomorphine (R-APO), dopamine (DA), and the green tea polyphenol (-)-epigallocatechine-3-gallate (EGCG) possess concentration-dependent biphasic actions in preventing and promoting neuronal cell death in models of Parkinson’s diseases (PD). No adequate explanation has been given so far how or why these compounds, having different structures, behave in a similar manner. Using cDNA microarray, real-time RT-PCR, and protein analysis, we studied the molecular mechanisms and the gene-protein pathways involved in their cell survival and cell death action.
PRINCIPAL FINDINGS
1. Cellular viability in response to antioxidants
The compounds DA, R-APO, EGCG, and melatonin demonstrated a dose-dependent effect on the viability of human neuroblastoma (NB) SH-SY5Y cells. At low concentration (1–10 µM) they had no effect on cell viability, whereas at higher concentrations (>10 µM) they induced a gradual decrease of cell survival, with rank order being R-APO > EGCG > DA > melatonin.
2. cDNA microarray analysis of DA-, R-APO-, and melatonin-treated cells
Using a customized cDNA array containing 25 human cDNA fragments of genes coding for protein related to apoptosis and cell survival pathways, we observed that low concentrations of DA (10 µM), R-APO (1 µM), and melatonin (1 µM) had no effect on gene expression. However, a significant homology in the induction of mRNAs coding for proapoptotic proteins was observed (Table 1
).This included caspase-3, caspase-10, nuclear factor kappa B (NF-
B p105 subunit), tumor suppressor protein p53, and the tumor necrosis factor receptor members fas and fas ligand. DA and R-APO, but not melatonin, increased the expression of Bcl-2 family members bad, bax, tumor necrosis factor ligand member 10 (TRAIL), TRAIL receptor DR5, and DNA damage-inducible transcript gadd45. These data support the cell viability results showing that R-APO is more potent than DA, melatonin, and EGCG in its neurotoxicity effect.
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3. Quantitative real-time PCR and Western blot analysis of antioxidant-treated cells
Real-time RT-PCR of RNA samples isolated from NB SH-SY5Y cells treated with the above concentrations of antioxidants at two incubation times (1.5 and 6 h) was conducted to verify apoptosis-related gene changes. Low neuroprotective concentrations of DA and R-APO (10 and 1 µM, respectively), induced an acute decrease of proapoptotic bax mRNA, evident as early as 1.5 h. A delayed and less marked reduction was observed with melatonin (1 µM), being induced at 6 h of incubation. In parallel, an immediate substantial reduction in Bax protein content was induced by the low concentrations of the three compounds after 1.5 h of exposure; unlike R-APO, it subsequently declined to control values (Fig. 1
).R-APO and DA reduced the expression of the cell cycle inhibitor gadd45 and fas ligand at 1.5 h, accompanied by an increase in the expression of antiapoptotic bcl-2 and bcl-xL mRNAs. This effect was transiently manifested and returned to control levels after 6 h.
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High toxic concentrations of DA (500 µM) and R-APO (50 µM) caused a sustained increase in the expression of bax, gadd45, and fas ligand for up to 6 h. The induction in bax mRNA was accompanied by an increase in Bax protein content (Fig. 1)
after 3 h exposure to both moderate (20 and 200 µM) and high (50 and 500 µM) concentrations of R-APO and DA, respectively. The 50 µM concentration of melatonin also increased bax, gadd45, and fas ligand mRNA expression but to a lower extent. Similarly, the Bax protein content was affected by melatonin (Fig. 1)
. Moreover, the three antioxidants increased significantly caspase-6 expression at 6 h. DA and R-APO reduced markedly bcl-2 and bcl-xL mRNA levels, but no effect was obtained with melatonin.
4. Quantitative real-time PCR of EGCG-treated cells
EGCG is a highly potent neuroprotective compound in cell culture and an in vivo model of PD that decreases the proapoptotic/cell cycle gene expression and increases the cell survival genes. To compare and confirm the effects of EGCG with those of DA, R-APO, and melatonin, real-time PCR analysis was performed. Indeed, a low concentration (1 µM) of EGCG decreased the expression of bax and caspase-6 mRNA. By contrast, a high concentration (50 µM) increased the expression of bax and gadd45 and decreased bcl-2 and bcl-xL mRNA levels.
5. Caspase-3 protein level and activity in response to the antioxidants
Caspase-3, a cysteine protease and an important component of the apoptosis machinery, is activated by multiple proteolytic cleavage of its 32 kDa precursor form to generate an enzymatic active p17/p12 complex. With low concentrations of the antioxidants, only the 32 kDa precursor of caspase-3 was detected in NB SH-SY5Y cells. However, high concentrations of DA (200 and 500 µM), R-APO (20 and 50 µM), EGCG (50 µM) or melatonin (50 µM) significantly induced the cleavage of caspase-3 as demonstrated by the appearance of the 17 and 12 kDa fragments or by cleavage of the substrate Ac-DEVD-pNA.
CONCLUSIONS AND SIGNIFICANCE
The present study provides, for the first time new insights into the molecular events involved in the dose-dependent anti- and/or proapoptotic activities of catechol-derived and indole amine compounds at low and high concentrations. For this purpose, low neuroprotective and 50-fold higher concentrations were chosen. cDNA microarray has demonstrated a concentration-dependent homology among antioxidants R-APO, DA, melatonin, and EGCG for modulation of cell survival/cell death-related gene pathways. The gene microarray analysis provides the first evidence for a selective, dose-dependent regulation of a number of mRNAs by these drugs. A schematic overview suggesting the potential gene targets involved in the proapoptotic and antiapoptotic action of DA, R-APO, melatonin, and EGCG is described in Fig. 2
.An important aspect of our study is the significant homology between the catechol-derived compounds and differences observed with the indolamine melatonin.
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No significant gene changes were observed with low concentrations of R-APO, DA, and melatonin previously reported to induce effective neuroprotection in both neuronal primary and cell line cultures. However, when a quantitative real-time PCR method was applied, specific gene expression changes as a function of concentration and time were observed. This discrepancy may result from the sensitivity thresholds of the methods. Low DA, R-APO, and melatonin concentrations induced an immediate expression of antiapoptotic bcl-xL and/or bcl-2 mRNAs whereas bax mRNA was reduced. The gene changes were correlated with alterations in protein levels of both Bcl-2 and Bax. The increased Bcl-2 to Bax ratio as a result of R-APO, DA, and melatonin suggests the involvement of these pathways in their neuroprotective and antiapoptotic actions. Similarly, the potent antioxidant EGCG at low concentration decreased proapoptotic bax and caspase-6, supporting previous findings.
However, a proapoptotic pattern of gene expression was observed at high concentrations of the antioxidants. A similar gene profile was obtained after exposure to R-APO, DA, and melatonin, including up-regulation of caspases-3 and 10, fas and fas ligand, NF-B p105 subunit, and p53 mRNAs. DA and R-APO displayed a greater homology of gene expression than melatonin, suggesting that these drugs may share a similar mechanism in their cell death action. The wide range between the neuroprotective and the toxic concentrations of melatonin may be critical to its pharmacotherapy, as it may provide a safer dosage window than R-APO and DA. A possible explanation for the high cell viability in the presence of high melatonin concentration may be that caspase-3 activation per se or its upstream effectors may not be indispensable for onset of apoptosis as has been suggested.
The neurotransmitter DA has been suggested to serve as an endogenous neurotoxin, thereby participating in neurodegenerative processes in PD. This assumption is based on observations that high concentrations (200–1000 µM) of DA induce apoptosis in neuronal cell cultures. They have implicated endogenous dopamine as a neurotoxic culprit in PD. However, the highly active intraneuronal monoamine oxidase never allows the buildup of such a high nonphysiological concentration of intraneuronal or extraneuronal DA. Only when the enzyme is inhibited does the neuronal DA level increase. Moreover, long-term treatment of PD patients with L-DOPA or anti-Parkinson monoamine oxidase-B inhibitor drug selegiline, which increases brain DA, does not induce the progression of neurodegeneration. The present study has clearly shown that at low concentrations, DA, which is relatively closer to its physiological concentration, does not affect cell viability, but rather activates cell survival genes and their associated proteins. This supports the most recent view that low DA concentration may indeed have a beneficial effect on dopamine neuron plasticity. This assumption may also be relevant for the dopaminergic receptor agonist R-APO, since it may have a neuroprotective action in addition to its favorable symptomatic action in PD patients.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0712fje; to cite this article, use FASEB J. (March 5, 2003) 10.1096/fj.02-0712fje 
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