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Contrasting neuroprotective and neurotoxic actions of respective metabolites of anti-Parkinson drugs rasagiline and selegiline
Neuroscience Letters 355 (2004) 169–172 www.elsevier.com/locate/neulet
Contrasting neuroprotective and neurotoxic actions of respective metabolites of anti-Parkinson drugs rasagiline and selegiline Orit Bar Ama,b, Tamar Amita,b, Moussa B.H. Youdima,b,* a
Eve Topf and US National Parkinson Foundation Center of Excellence for Neurodegenerative Diseases Research, Technion-Faculty of Medicine, Efron St. PO Box 9697, Haifa 31096, Israel b Department of Pharmacology, Technion-Faculty of Medicine, Efron St. PO Box 9697, Haifa 31096, Israel Received 27 June 2003; received in revised form 17 October 2003; accepted 27 October 2003
Abstract The anti-Parkinson selective irreversible monoamine oxidase B inhibitor drugs, rasagiline and selegiline, have been shown to possess neuroprotective activities in cell culture and in vivo models. While rasagiline is metabolized to its major metabolite aminoindan, selegiline gives rise to L -methamphetamine. Cultured PC-12 cells in absence of serum and nerve growth factor (NGF) die by an apoptotic process. Pretreatment of PC12 cells in absence of serum and NGF for 24 h with either rasagiline (1 mM) or selegiline (1 mM) is neuroprotective and anti-apoptotic as determined by Eliza and MTT tests. However, while aminoindan (1 mM), the major metabolite of rasagiline does not interfere with the neuroprotective activities of rasagiline or selegiline in PC-12 cells deprived of serum and NGF, the major metabolite of selegiline, L -methamphetamine (1 mM), inhibits them. In contrast to L -methamphetamine, aminoindan is itself is neuroprotective in this system. Recently it has been demonstrated that rasagiline directly activates PKC-MAP kinase pathway by a concentration and time dependent phosphorylation of p42 and p44 MAP kinase. In the present studies the neuroprotective activity of rasagiline is blocked by ERK inhibitor, PD98059 (20 mM), suggesting the involvement of PKC-MAP kinase pathway in the neuroprotection. These findings may have implication for the possible disease modifying action of rasagiline in treatment of Parkinson’s disease. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Parkinson’s disease; Neuroprotection; Neurotoxicity rasagiline; Selegiline; Aminoindan; L -Methamphetamine; PKC-MAP kinase pathway
The irreversible selective monoamine oxidase (MAO) B inhibitor Parkinson’s disease (PD) drugs, rasagiline (Npropargyl-1-(R)-aminoindan) [10] and selegiline [3] have been chosen [12] among a handful [12] of other agents as potential neuroprotective compounds for further clinical trials in PD. Rasagiline [20] and selegiline have similar structures and are propargylamine derivatives. However rasagiline is metabolized to its major metabolite aminoindan, while selegiline gives rise to its major metabolite L methamphetamine. Clinically as an anti-Parkinson drug [10] and experimentally as a MAO B inhibitor [18], rasagiline is 10– 20 time more potent. However, both drugs exhibit neuroprotective activities in several neuronal cell culture models and in vivo in response to several neurotoxins [20] and rasagiline is more effective to suppress the cell death in vivo and in vitro experiments [2]. Clinical trials with Abbreviations: PD, Parkinson’s disease; MAO, monoamine oxidase. * Corresponding author. Tel.: þ 972-4-8295-290; fax: 972-4-8513-145. E-mail address: [email protected] (M.B.H. Youdim).
selegiline [14] failed to establish its neuroprotective activity because its symptomatic effect could not be distinguished from a possible neuroprotective action. Unlike rasagiline, selegiline is a sympathomimetic amine, which increases blood pressure and heart rate [4] and is metabolized to neurotoxic L -methamphetamine [13]. However, the metabolite of rasagiline is aminoindan and which has been reported to have neuroprotective activity [15]. Tatton et al. [9] have suggested as one reason why neuroprotection was not observed in the clinical trails [14] may be related to L methamphetamine generation, which could in turn interfere with selegiline’s neuroprotective activity. Indeed Abu-Raya et al. [1] have demonstrated that L -methamphetamine interferes with the neuroprotective action of selegiline in oxygen-glucose deprivation induced cell death in nerve growth factor (NGF)-differentiated PC-12 cells. The present study examined the neuroprotective and neurotoxic actions of rasagiline, selegiline and their respective metabolites aminoindan and L -methamphetamine
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2003.10.067
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in PC-12 cells deprived of serum and NGF. Furthermore since rasagiline has been shown to directly activate PKCMAP kinase pathway and Bcl-2 in PC-12 and neuroblastoma SHSY-5Y cells [16,17], the effect of ERK inhibitor, PD98059 on its neuroprotective activity was also determined. Rasagiline, aminoindan, (TVP136), and selegiline were kindly donated by Teva Pharmaceutical Israel, Ltd. L Methamphetamine was a gift from Prof. J. Finberg. PD98059 were obtained from Calbiochem (LaJolla, CA), dissolved (1 £ 100) in dimethyl sulfoxide and stored at 2 20 8C. Tissue culture reagents from Beth-Haemek, (Israel). All other highest-grade chemicals were from Sigma Chemical Co., St. Louis, USA. PC12 cells were placed in microtiter plated (96 wells) at a density of 0.5 – 1 £ 104 cells/well and were allowed to attach for 24 h before treatment containing DMEM (1000 mg/l glucose) and supplemented with 5% FCS, 10% horse serum and a mixture of 1% of pencillin/streptomycin/nystatin. Cell cultures were incubated at 37 8C in humid 5% CO2-95% air environment. After 24 h the medium was replaced with DMEM containing 1% of pencillin/streptomycin/nystatin and 0.1% BSA (serum and NGF free). Methamphetamine and aminoindan were added to the cells and after 1 h incubation – rasagiline and selegiline were added too. The control cell group were supplemented with 5% Fetal Calf Serum (FCS), 10% horse serum and a mixture of 1% of pencillin/streptomycin/nystatin. Cell death detection ELISA kit (Roche Molecular Biochemicals, Indianapolis, IN) was used to detect apoptosis after 24 h treatments [16]. The ELISA was carried out according to the manufacturer’s protocol, based on a quantitative sandwich, using antibodies directed against DNA and histones to detect mono- and oligonucleosomes in the cytoplasm of cells undergoing apoptosis. The absorption was determined in a Tecan Sunrise Eliza-Reader (Switzerland) at l ¼ 405/ 490 nm after automatic subtraction of background readings. MTT was also performed on another sets of experiments. In addition, neuronal cell injury was evaluated by morphological characteristics, visualized by phase-contrast microscope (not presented here) [16]. In the absence of serum and NGF there was a highly significant loss of cell viability within 24 h. However, pretreatment of PC-12 cells with rasagiline and selegiline (1 mM) highly significantly reduce cell death, with rasagiline showing a greater effect both in the Elisa and MTT tests (Figs. 1A,B). Introduction of methamphetamine (1 mM), the metabolite of selegiline, prior to rasagiline or selegiline prevented the neuroprotective action of both drugs. By contrast the metabolite of rasagiline, aminoindan (1 mM) did not interfere with the neuroprotective activity of rasagiline and selegiline (Figs. 1A,B). Furthermore, while methamphetamine (1 mM) did not alter PC-12 cell death initiated by serum and NGF withdrawal, aminoindan induced significant neuroprotection, equivalent to that observed with rasagiline or selegiline. While the ERK
inhibitor, PD98059 (20 mM), had no effect on cell death induce in the serum and NGF system, it prevented the neuroprotection initiated by rasagiline (1 mM) (Fig. 2). In the present study we have shown that rasagiline similar to selegiline protects against serum and NGF withdrawal induced death in partially differentiated PC-12 (and neuroblastoma SHSY-5Y, data not shown). These results are very similar to the neuroprotective actions of
Fig. 1. Neuroprotective-anti-apoptotic effects of rasagiline and its aminoindan metabolite as compared to selegiline and methamphetamine on PC-12 survival in the absence of serum and NGF. PC-12 cells were prepared as described in the text. They were serum and NGF free for 24 h. Individual drugs were introduced at 1 mM final concentrations. (A) Cell death was detected by ELISA. (B) Cell viability was estimated using direct counting cell number by Trypan blue dye exclusion method. The values are expressed as a percentage of viable cells counted in the control. A, aminoindan; and M, methamphetamine. The results are the mean ^ SEM ANOVA (n ¼ 3) in triplicates. *P , 0:01; control vs control serum-NGF free, rasagiline plus M, selegiline plus M and M; **P , 0:05 vs control or serum-NGF free, rasagiline plus M, selegiline plus M and M; #P , 0:01 vs control, rasagiline, selegiline, selegiline plus A.
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Fig. 2. The effect of ERK inhibitor PD98059 on the neuroprotective-antiapoptotic action of rasagiline in serum-NGF free PC-12 cells. The details of the cell preparation and treatments are those described in the text and Fig. 1. The serum-NGF free cells were treated with PD98059 (PD) (20 mM) or rasagiline (1 mM) for 24 h. In the PD98059 (20 mM) ?plus rasagiline (1 mM), PD98059 was added 30 min prior to rasagiline (1 mM) and incubated for 24 h before death was detected by ELISA. The results are the mean ^ SEM ANOVA (n ¼ 4) in triplicates. *P , 0:01 vs serum-NGF free or PD or PD plus rasagiline; **P , 0:05 vs control (full serum) or serum-NGF free or PD or PD plus rasagiline.
rasagiline and selegiline in prevention of PC-12 cells death during oxygen-glucose deprivation [1]. The effects of aminoindan and methamphetamine on neuroprotective activities of selegiline and rasagiline in serum and NGF free cell preparations have not been determined before. Furthermore in these studies we have employed a cell line that does not contain MAO-B, but MAO-A [18] and thus using an MAO-B inhibitor at 1 mM concentration would not inhibit MAO-A. Therefore, MAO inhibition per se does not contribute to rasagiline’s neuroprotective activity. As far as we are aware the action of aminoindan, the major metabolite of rasagiline has not been examined on methamphetamine neurotoxicity or alone on such cell line. The neuroprotective and anti-apoptotic activity of rasagiline in PC-12 and SHSY-5Y cells and in vivo is associated with its induction of anti-apoptotic Bcl-2 and Bcl-xL and activation of PKCa and e dependent MAPK pathway, which results in the prevention of the fall in mitochondrial membrane potential and inhibition of mitochondrial permeability transition [17]. Rasagiline directly activates PKC-MAP kinase pathway by concentration and time dependent phosphorylation of p42 and p44 MAP kinase [16]. For the first time we have shown that ERK1/ ERK2 inhibitor, PD98059, prevented the neuroprotective effect of rasagiline in the serum-NGF free system, with out itself having an effect on cell survival, it suggest the involvement of Bcl-2 and PKC-MAP kinase pathway in neuroprotective activity of rasagiline, with which aminoindan does appear to interfere with. The potent MAO-B inhibitory activity of rasagiline [17], which resides in the interaction of its propargylamine moiety with FAD cofactor of the enzyme [20], is unrelated to its neuroprotective
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activity [2,7,18]. This is substantiated by the similar neuroprotective activity of the S-optical isomer of rasagiline, TVP1022 [2,7,19], which has more than 1000-fold less MOA-B inhibitory activity. Furthermore, PC-12 cells contain only MAO-A [19] and the concentration of rasagiline employed would not be sufficient to inhibit the enzyme. In these studies the neuroprotective activity of rasagiline and TVP 1022 were significantly superior to that of rasagiline confirming those reported by ref. [1]. This may result from the differences between the structure of selegiline and those of rasagiline and its derivatives. Selegiline is a derivative of amphetamine and is metabolized to its major metabolite L -methamphetamine in vivo and similar to amphetamine it is a sympathomimetic compound [4]. L Methamphetamine is known to be neurotoxic to dopamine neurons in vivo and PC-12 and neuroblastoma cells in culture [6]. The present studies not only confirm its neurotoxicity in cell culture, but we have shown that unlike the aminoindan metabolite of rasagiline, L -methamphetamine prevents neuroprotective activity of selegiline as well as rasagiline. However, rasagiline is an aminoindan derivative and neither it, nor aminoindan is vasoactive [5]. Furthermore, both compounds as shown in this study have neuroprotective activity. Maruyama et al. [8] have established that the neuroprotective activity of propargylamines such as rasagiline, its derivative and selegiline resides in the propargylamine moiety. Several studies [1,2] have shown that rasagiline has greater neuroprotective potency in cell culture and in vivo than selegiline. This may be attributed to a combination of the propargylamine moiety and the aminoindan backbone of the drug. Indeed propargylamine itself possess neuroprotective activity almost similar to rasagiline [16]. Rasagiline prevent the neurotoxicity of glutamate in primary cultures of rat hippocampus neurons with equipotent (IC50, 1028 M) that of MK-801 [4] and in vivo, global ischaemia in gerbil [15]. A similar finding has also been reported for 4-hydroxyaminoindan. The metabotropic glutamate 1 (mGlu1) receptor antagonist, 1-aminoindan-1,5-dicaboxylic acid (AIDA), which reduces neuronal death, following oxygen-glucose deprivation in murine cortical cells and rat organotypic hippocampal cultures and in vivo, after global ischaemia in gerbils, possesses an aminoindan to which the dicarboxylic moiety is attached at its one position [11]. The structural similarity in propargylamine attachment to position 1 of aminoindan of rasagiline and its pharmacological neuroprotective activity to AIDA cannot go unnoticed. Our results may point to a possible role for rasagiline as an mGlu1 antagonist and this is presently being investigated. The present study has shown that rasagiline and its major metabolite aminoindan are neuroprotective compound under the condition of serum and NGF withdrawal in partially differentiated PC-12 cells and this may involve the interaction of Bcl-2 with PKC-dependent MAP kinase pathway [16,17]. This is contrast to selegiline, where the latter’s major metabolite L -methamphetamine is neurotoxic.
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The in vivo generation of aminoindan from rasagiline may have an additive effect on the neuroprotective activity of this selective MAO-B inhibitor. Since rasagiline has been chosen as one of few compounds identified for neuroprotective studies in PD [12], it may have superior advantage over selegiline, since, unlike selegiline it does not give rise to neurotoxic L -methamphetamine major metabolite.
Acknowledgements The support of Teva Pharmaceutical Co. (Netanya, Israel), National Parkinson Foundation (Miami, USA) and Technion (Haifa, Israel) are gratefully acknowledged.
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Contrasting neuroprotective and neurotoxic actions of respective metabolites of anti-Parkinson drugs rasagiline and selegiline Orit Bar Ama,b, Tamar Amita,b, Moussa B.H. Youdima,b,* a
Eve Topf and US National Parkinson Foundation Center of Excellence for Neurodegenerative Diseases Research, Technion-Faculty of Medicine, Efron St. PO Box 9697, Haifa 31096, Israel b Department of Pharmacology, Technion-Faculty of Medicine, Efron St. PO Box 9697, Haifa 31096, Israel Received 27 June 2003; received in revised form 17 October 2003; accepted 27 October 2003
Abstract The anti-Parkinson selective irreversible monoamine oxidase B inhibitor drugs, rasagiline and selegiline, have been shown to possess neuroprotective activities in cell culture and in vivo models. While rasagiline is metabolized to its major metabolite aminoindan, selegiline gives rise to L -methamphetamine. Cultured PC-12 cells in absence of serum and nerve growth factor (NGF) die by an apoptotic process. Pretreatment of PC12 cells in absence of serum and NGF for 24 h with either rasagiline (1 mM) or selegiline (1 mM) is neuroprotective and anti-apoptotic as determined by Eliza and MTT tests. However, while aminoindan (1 mM), the major metabolite of rasagiline does not interfere with the neuroprotective activities of rasagiline or selegiline in PC-12 cells deprived of serum and NGF, the major metabolite of selegiline, L -methamphetamine (1 mM), inhibits them. In contrast to L -methamphetamine, aminoindan is itself is neuroprotective in this system. Recently it has been demonstrated that rasagiline directly activates PKC-MAP kinase pathway by a concentration and time dependent phosphorylation of p42 and p44 MAP kinase. In the present studies the neuroprotective activity of rasagiline is blocked by ERK inhibitor, PD98059 (20 mM), suggesting the involvement of PKC-MAP kinase pathway in the neuroprotection. These findings may have implication for the possible disease modifying action of rasagiline in treatment of Parkinson’s disease. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Parkinson’s disease; Neuroprotection; Neurotoxicity rasagiline; Selegiline; Aminoindan; L -Methamphetamine; PKC-MAP kinase pathway
The irreversible selective monoamine oxidase (MAO) B inhibitor Parkinson’s disease (PD) drugs, rasagiline (Npropargyl-1-(R)-aminoindan) [10] and selegiline [3] have been chosen [12] among a handful [12] of other agents as potential neuroprotective compounds for further clinical trials in PD. Rasagiline [20] and selegiline have similar structures and are propargylamine derivatives. However rasagiline is metabolized to its major metabolite aminoindan, while selegiline gives rise to its major metabolite L methamphetamine. Clinically as an anti-Parkinson drug [10] and experimentally as a MAO B inhibitor [18], rasagiline is 10– 20 time more potent. However, both drugs exhibit neuroprotective activities in several neuronal cell culture models and in vivo in response to several neurotoxins [20] and rasagiline is more effective to suppress the cell death in vivo and in vitro experiments [2]. Clinical trials with Abbreviations: PD, Parkinson’s disease; MAO, monoamine oxidase. * Corresponding author. Tel.: þ 972-4-8295-290; fax: 972-4-8513-145. E-mail address: [email protected] (M.B.H. Youdim).
selegiline [14] failed to establish its neuroprotective activity because its symptomatic effect could not be distinguished from a possible neuroprotective action. Unlike rasagiline, selegiline is a sympathomimetic amine, which increases blood pressure and heart rate [4] and is metabolized to neurotoxic L -methamphetamine [13]. However, the metabolite of rasagiline is aminoindan and which has been reported to have neuroprotective activity [15]. Tatton et al. [9] have suggested as one reason why neuroprotection was not observed in the clinical trails [14] may be related to L methamphetamine generation, which could in turn interfere with selegiline’s neuroprotective activity. Indeed Abu-Raya et al. [1] have demonstrated that L -methamphetamine interferes with the neuroprotective action of selegiline in oxygen-glucose deprivation induced cell death in nerve growth factor (NGF)-differentiated PC-12 cells. The present study examined the neuroprotective and neurotoxic actions of rasagiline, selegiline and their respective metabolites aminoindan and L -methamphetamine
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2003.10.067
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in PC-12 cells deprived of serum and NGF. Furthermore since rasagiline has been shown to directly activate PKCMAP kinase pathway and Bcl-2 in PC-12 and neuroblastoma SHSY-5Y cells [16,17], the effect of ERK inhibitor, PD98059 on its neuroprotective activity was also determined. Rasagiline, aminoindan, (TVP136), and selegiline were kindly donated by Teva Pharmaceutical Israel, Ltd. L Methamphetamine was a gift from Prof. J. Finberg. PD98059 were obtained from Calbiochem (LaJolla, CA), dissolved (1 £ 100) in dimethyl sulfoxide and stored at 2 20 8C. Tissue culture reagents from Beth-Haemek, (Israel). All other highest-grade chemicals were from Sigma Chemical Co., St. Louis, USA. PC12 cells were placed in microtiter plated (96 wells) at a density of 0.5 – 1 £ 104 cells/well and were allowed to attach for 24 h before treatment containing DMEM (1000 mg/l glucose) and supplemented with 5% FCS, 10% horse serum and a mixture of 1% of pencillin/streptomycin/nystatin. Cell cultures were incubated at 37 8C in humid 5% CO2-95% air environment. After 24 h the medium was replaced with DMEM containing 1% of pencillin/streptomycin/nystatin and 0.1% BSA (serum and NGF free). Methamphetamine and aminoindan were added to the cells and after 1 h incubation – rasagiline and selegiline were added too. The control cell group were supplemented with 5% Fetal Calf Serum (FCS), 10% horse serum and a mixture of 1% of pencillin/streptomycin/nystatin. Cell death detection ELISA kit (Roche Molecular Biochemicals, Indianapolis, IN) was used to detect apoptosis after 24 h treatments [16]. The ELISA was carried out according to the manufacturer’s protocol, based on a quantitative sandwich, using antibodies directed against DNA and histones to detect mono- and oligonucleosomes in the cytoplasm of cells undergoing apoptosis. The absorption was determined in a Tecan Sunrise Eliza-Reader (Switzerland) at l ¼ 405/ 490 nm after automatic subtraction of background readings. MTT was also performed on another sets of experiments. In addition, neuronal cell injury was evaluated by morphological characteristics, visualized by phase-contrast microscope (not presented here) [16]. In the absence of serum and NGF there was a highly significant loss of cell viability within 24 h. However, pretreatment of PC-12 cells with rasagiline and selegiline (1 mM) highly significantly reduce cell death, with rasagiline showing a greater effect both in the Elisa and MTT tests (Figs. 1A,B). Introduction of methamphetamine (1 mM), the metabolite of selegiline, prior to rasagiline or selegiline prevented the neuroprotective action of both drugs. By contrast the metabolite of rasagiline, aminoindan (1 mM) did not interfere with the neuroprotective activity of rasagiline and selegiline (Figs. 1A,B). Furthermore, while methamphetamine (1 mM) did not alter PC-12 cell death initiated by serum and NGF withdrawal, aminoindan induced significant neuroprotection, equivalent to that observed with rasagiline or selegiline. While the ERK
inhibitor, PD98059 (20 mM), had no effect on cell death induce in the serum and NGF system, it prevented the neuroprotection initiated by rasagiline (1 mM) (Fig. 2). In the present study we have shown that rasagiline similar to selegiline protects against serum and NGF withdrawal induced death in partially differentiated PC-12 (and neuroblastoma SHSY-5Y, data not shown). These results are very similar to the neuroprotective actions of
Fig. 1. Neuroprotective-anti-apoptotic effects of rasagiline and its aminoindan metabolite as compared to selegiline and methamphetamine on PC-12 survival in the absence of serum and NGF. PC-12 cells were prepared as described in the text. They were serum and NGF free for 24 h. Individual drugs were introduced at 1 mM final concentrations. (A) Cell death was detected by ELISA. (B) Cell viability was estimated using direct counting cell number by Trypan blue dye exclusion method. The values are expressed as a percentage of viable cells counted in the control. A, aminoindan; and M, methamphetamine. The results are the mean ^ SEM ANOVA (n ¼ 3) in triplicates. *P , 0:01; control vs control serum-NGF free, rasagiline plus M, selegiline plus M and M; **P , 0:05 vs control or serum-NGF free, rasagiline plus M, selegiline plus M and M; #P , 0:01 vs control, rasagiline, selegiline, selegiline plus A.
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Fig. 2. The effect of ERK inhibitor PD98059 on the neuroprotective-antiapoptotic action of rasagiline in serum-NGF free PC-12 cells. The details of the cell preparation and treatments are those described in the text and Fig. 1. The serum-NGF free cells were treated with PD98059 (PD) (20 mM) or rasagiline (1 mM) for 24 h. In the PD98059 (20 mM) ?plus rasagiline (1 mM), PD98059 was added 30 min prior to rasagiline (1 mM) and incubated for 24 h before death was detected by ELISA. The results are the mean ^ SEM ANOVA (n ¼ 4) in triplicates. *P , 0:01 vs serum-NGF free or PD or PD plus rasagiline; **P , 0:05 vs control (full serum) or serum-NGF free or PD or PD plus rasagiline.
rasagiline and selegiline in prevention of PC-12 cells death during oxygen-glucose deprivation [1]. The effects of aminoindan and methamphetamine on neuroprotective activities of selegiline and rasagiline in serum and NGF free cell preparations have not been determined before. Furthermore in these studies we have employed a cell line that does not contain MAO-B, but MAO-A [18] and thus using an MAO-B inhibitor at 1 mM concentration would not inhibit MAO-A. Therefore, MAO inhibition per se does not contribute to rasagiline’s neuroprotective activity. As far as we are aware the action of aminoindan, the major metabolite of rasagiline has not been examined on methamphetamine neurotoxicity or alone on such cell line. The neuroprotective and anti-apoptotic activity of rasagiline in PC-12 and SHSY-5Y cells and in vivo is associated with its induction of anti-apoptotic Bcl-2 and Bcl-xL and activation of PKCa and e dependent MAPK pathway, which results in the prevention of the fall in mitochondrial membrane potential and inhibition of mitochondrial permeability transition [17]. Rasagiline directly activates PKC-MAP kinase pathway by concentration and time dependent phosphorylation of p42 and p44 MAP kinase [16]. For the first time we have shown that ERK1/ ERK2 inhibitor, PD98059, prevented the neuroprotective effect of rasagiline in the serum-NGF free system, with out itself having an effect on cell survival, it suggest the involvement of Bcl-2 and PKC-MAP kinase pathway in neuroprotective activity of rasagiline, with which aminoindan does appear to interfere with. The potent MAO-B inhibitory activity of rasagiline [17], which resides in the interaction of its propargylamine moiety with FAD cofactor of the enzyme [20], is unrelated to its neuroprotective
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activity [2,7,18]. This is substantiated by the similar neuroprotective activity of the S-optical isomer of rasagiline, TVP1022 [2,7,19], which has more than 1000-fold less MOA-B inhibitory activity. Furthermore, PC-12 cells contain only MAO-A [19] and the concentration of rasagiline employed would not be sufficient to inhibit the enzyme. In these studies the neuroprotective activity of rasagiline and TVP 1022 were significantly superior to that of rasagiline confirming those reported by ref. [1]. This may result from the differences between the structure of selegiline and those of rasagiline and its derivatives. Selegiline is a derivative of amphetamine and is metabolized to its major metabolite L -methamphetamine in vivo and similar to amphetamine it is a sympathomimetic compound [4]. L Methamphetamine is known to be neurotoxic to dopamine neurons in vivo and PC-12 and neuroblastoma cells in culture [6]. The present studies not only confirm its neurotoxicity in cell culture, but we have shown that unlike the aminoindan metabolite of rasagiline, L -methamphetamine prevents neuroprotective activity of selegiline as well as rasagiline. However, rasagiline is an aminoindan derivative and neither it, nor aminoindan is vasoactive [5]. Furthermore, both compounds as shown in this study have neuroprotective activity. Maruyama et al. [8] have established that the neuroprotective activity of propargylamines such as rasagiline, its derivative and selegiline resides in the propargylamine moiety. Several studies [1,2] have shown that rasagiline has greater neuroprotective potency in cell culture and in vivo than selegiline. This may be attributed to a combination of the propargylamine moiety and the aminoindan backbone of the drug. Indeed propargylamine itself possess neuroprotective activity almost similar to rasagiline [16]. Rasagiline prevent the neurotoxicity of glutamate in primary cultures of rat hippocampus neurons with equipotent (IC50, 1028 M) that of MK-801 [4] and in vivo, global ischaemia in gerbil [15]. A similar finding has also been reported for 4-hydroxyaminoindan. The metabotropic glutamate 1 (mGlu1) receptor antagonist, 1-aminoindan-1,5-dicaboxylic acid (AIDA), which reduces neuronal death, following oxygen-glucose deprivation in murine cortical cells and rat organotypic hippocampal cultures and in vivo, after global ischaemia in gerbils, possesses an aminoindan to which the dicarboxylic moiety is attached at its one position [11]. The structural similarity in propargylamine attachment to position 1 of aminoindan of rasagiline and its pharmacological neuroprotective activity to AIDA cannot go unnoticed. Our results may point to a possible role for rasagiline as an mGlu1 antagonist and this is presently being investigated. The present study has shown that rasagiline and its major metabolite aminoindan are neuroprotective compound under the condition of serum and NGF withdrawal in partially differentiated PC-12 cells and this may involve the interaction of Bcl-2 with PKC-dependent MAP kinase pathway [16,17]. This is contrast to selegiline, where the latter’s major metabolite L -methamphetamine is neurotoxic.
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The in vivo generation of aminoindan from rasagiline may have an additive effect on the neuroprotective activity of this selective MAO-B inhibitor. Since rasagiline has been chosen as one of few compounds identified for neuroprotective studies in PD [12], it may have superior advantage over selegiline, since, unlike selegiline it does not give rise to neurotoxic L -methamphetamine major metabolite.
Acknowledgements The support of Teva Pharmaceutical Co. (Netanya, Israel), National Parkinson Foundation (Miami, USA) and Technion (Haifa, Israel) are gratefully acknowledged.
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