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The importance of propargylamine moiety in the anti-Parkinson drug rasagiline and its derivatives in MAPK-dependent amyloid precursor protein processing¹

Technion Faculty of Medicine, Eve Topf and NPF Centers for Neurodegenerative Diseases, Department of Pharmacology, Haifa, 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

We have recently demonstrated the involvement of mitogen-activated protein kinase (MAPK) in the regulation of amyloid precursor protein (APP) processing by novel cholinesterase (ChE) inhibitors derived from the monoamine oxidase (MAO)-B inhibitor, anti-Parkinson drug, rasagiline (N-propargyl-1R-aminoindan). The aim of the present study was to investigate the effects of rasagiline and its S-optical isomer TVP1022 on the regulation of APP processing and the signaling pathways involved. Studies of the structure–activity relationship were conducted to assess whether the mechanism of these actions is attributed to the propargylamine moiety on the molecule.

PRINCIPAL FINDINGS

1. Neuroprotective effect of rasagiline against Aß_1-42 toxicity
Previous studies have shown that rasagiline exhibits neuroprotective and antiapoptotic activity against several neurotoxins. PC12 neuronal cells were preincubated for 1 h with rasagiline (1 and 10 µM), followed by 24 h treatment with aggregated Aß_1-42 (10 µM); cell death was assessed by Trypan blue exclusion (Fig. 1 A) and an apoptotic cell death detection ELISA (Fig. 1B ). Figure 1 shows that Aß_1-42 markedly reduced cell viability (66% of control), while rasagiline significantly protected (P<0.05) PC12 cells against Aß_1-42 neurotoxicity. Rasagiline (1 and 10 µM) alone also induced a significant improvement in PC12 cell viability, presumably due to its effect on cell survival after serum withdrawal.

View larger version (25K): [in this window] [in a new window]   Figure 1. Neuroprotective effects of rasagiline against Aß-induced toxicity. PC12 cells were pretreated without or with rasagiline (1 and 10 µM) for 1 h, then incubated without or with Aß1-42 (10 µM) for 24 h. A) Cell viability was estimated using direct counting of cell number by the Trypan blue dye exclusion method. The values are expressed as a percentage of viable cells counted in the control. B) Cell death was detected by ELISA. The values are expressed as a % of the control. The results are the mean ±SE (n=3). *P< 0.05: **P< 0.001 vs. control, untreated cells; #P< 0.05 vs. Aß1-42-induced cell death.

Using the ELISA detection assay, we found that the S-isomer of rasagiline, TVP1022 (1 and 10 µM), which is not an MAO inhibitor, also decreased the percentage of cell death from 142 ± 5% to 108 ± 7%, and 91 ± 11%, respectively, indicating that MAO inhibition is not prerequisite for neuroprotection.

2. Effect of rasagiline and its derivatives on APP processing
The following propargylamine-containing compounds—TV3326 and its S-isomer TV3279, rasagiline and its S-isomer TVP1022, and selegiline—significantly induced the release of the nonamyloidogenic -secretase form of soluble APP. We further characterized in detail the effect of rasagiline on APP processing and the signaling pathways involved. Treatment of PC-12 and SH-SY5Y neuroblastoma cells for 3 h with increasing concentrations of rasagiline resulted in a significant, dose-dependent increase in sAPP released into the medium, compared with the levels in control, untreated cells. The maximal effect was obtained at concentration of 10 µM, resulting in an 2.5-fold increase in sAPP secretion over the basal levels. Pretreatment of PC12 cells with the zinc metalloproteinase Ro31-9790 (100 µM) blocked rasagiline- enhanced cleavage of sAPP. Thus, these findings demonstrate that rasagiline affects APP processing by activating Ro31-9790-sensitive metalloprotease(s), further suggesting that its effect is mediated via -secretase activity. Rasagiline-induced sAPP release was significantly reduced by the MAPK kinase (MEK) inhibitor PD98059 (30 µM) and by the protein kinase C (PKC) inhibitor GF109203X (2.5 µM). These results suggest the involvement of MAPK- and PKC-dependent pathways in rasagiline-stimulated sAPP secretion.

3. Activation of MAPK by rasagiline
Rasagiline dose-dependently increased the immunoreactivity of the phosphorylated MAPK in PC12 cells but had no effect on total levels of MAPK proteins. Activation occurred at doses as low as 0.1 µM of rasagiline and maximal activation at 10 µM. Pretreatment with PD98059 (30 µM) blocked the rasagiline-induced increase of MAPK phosphorylation. Preincubation with GF109203X (2.5 µM) abolished the effect of rasagiline on MAPK activation, suggesting the involvement of PKC in rasagiline-induced MAPK activation.

4. Effects of propargylamine on sAPP processing and MAPK activation
To determine the role of the propargyl moiety in these drugs on APP processing, the effects of propargylamine on sAPP release and MAPK activation were assessed. Figure 2 A, B shows that treatment of SH-SY5Y neuroblastoma and PC12 cells with increasing concentrations of propargylamine resulted in a significant, dose-dependent increase in sAPP into the medium compared with the levels in control, untreated cells. The maximal effect was obtained at a concentration of 10 µM. Based on immunoblot analysis with anti-phospho-p44/p42, propargylamine dose-dependently induced MAPK phosphorylation in PC12 cells. These findings indicate that the effects of propargylamine on both sAPP release and MAPK activation are similar to those of rasagiline, TVP1022, TV3326, and TV3279. However, the metabolite of rasagiline, aminoindan (TVP136), which lacks the propargyl group, showed no such effects.

View larger version (27K): [in this window] [in a new window]   Figure 2. Effect of propargylamine on sAPP release. A) SH-SY5Y neuroblastoma cells or B) PC12 cells were incubated for 3 h without or with increasing concentrations of propargylamine and sAPP was measured in the media. Densitometric analysis of Western blots expressed as % of basal sAPP release. Results are shown as the mean ±SE (3–5) of independent experiments. *P < 0.05; **P < 0.001 vs. control.

CONCLUSIONS AND SIGNIFICANCE

In the present study, we show for the first time that the anti-Parkinson propargylamine-containing MAO-B inhibitor drug rasagiline has neuroprotective properties against Aß-induced cytotoxicity. Our results demonstrate that rasagiline induces sAPP release and ERK activation, and it is suggested that the mechanism of action is attributed to the propargylamine moiety on the molecule.

Several reports have already shown that rasagiline exhibits various neuroprotective activities in neuronal cell cultures and in vivo. Here we demonstrate that rasagiline significantly inhibited Aß_1-42-induced cytotoxicity in PC12 cells. It is unlikely that the neuroprotective effect is related to MAO-B inhibition, since PC12 cells contain MAO type A rather than type B. Moreover, the S-isomer of rasagiline TVP1022, an inhibitor of neither MAO-A nor -B, protected PC12 cells from Aß-evoked apoptosis, suggesting that the mode of action is independent of MAO inhibition. These results are consistent with previous reports providing clear evidence that the neuroprotection by rasagiline and its derivatives does not depend on inhibition of MAO-B, but rather is associated with some intrinsic pharmacological action of the propagyl moiety in these compounds.

We have recently described several bifunctional novel carbamate-containing analogs of rasagiline, TV3326 and TV3279, by combining ChE and MAO inhibitory activities in one molecule as a potential treatment for Alzheimer disease (AD). These drugs potently stimulate the release of the nonamyloidogenic sAPP by PKC- and MAPK-dependent pathways. Thus, a major aim of this study was to determine whether rasagiline can regulate APP processing and to investigate the signaling pathways involved in its action. In our investigation we have observed that short treatment (3 h) with rasagiline can affect APP metabolism by stimulating sAPP release in SH-SY5Y neuroblastoma and PC12 cells. Increased sAPP secretion was detected by the mAb 22C11 as well as by the mAb 6E10, which recognizes -secretase-cleaved APP. The increase was dose dependent, and the stimulatory effect of rasagiline on sAPP secretion was inhibited by the hydroxamic acid-based metalloprotease inhibitor Ro31-9790, indicating that the effect was mediated via -secretase processing. Thus, rasagiline affects APP metabolism by increasing the -secretase processing pathway and so could be beneficial for the treatment of AD by shifting the balance of APP processing toward a presumably nonpathogenic pathway. Additional studies strongly suggest that the nonamyloidogenic sAPP has potent neurotrophic and neuroprotective activities against excitotoxic and oxidative insults in various cellular models. Therefore, it can be suggested that sAPP, derived from rasagiline-mediated APP processing, can serve as a neuroprotective agent against the toxic activity of Aß.

The data presented here demonstrate that sAPP release induced by rasagiline was modulated by inhibitors of PKC and the Erk MAPK signaling pathway. Moreover, in results complementary to the inhibitor studies, we found that rasagiline dose-dependently increased the immunoreactivity of the phosphorylated MAPK in PC12 cells. The MEK inhibitor PD98059 antagonized MAPK activation, indicating that MEK phosphorylates MAPK in the presence of rasagiline. Activation of MAPK was effectively attenuated by GF109203X, indicating dependence on the PKC signaling pathway activity.

In conclusion, by comparing the actions of rasagiline with those of its S-isomer, TVP1022, which is at least 1000-fold weaker as an inhibitor of MAO, we have been able to demonstrate that MAO-B inhibition is not a prerequisite for sAPP-induced release or ERK phosphorylation. The structure–activity relationship among rasagiline-related compounds suggests the crucial role of the propargyl moiety on these molecules for processing of APP, since propargylamine itself induced secretion of the nonamyloidogenic -secretase form of the solubles APP into the conditioned media of SH-SY5Y neuroblastoma and PC12 cells and significantly increased MAPK phosphorylation with a potency similar to that of rasagiline and its derivatives.

View larger version (24K): [in this window] [in a new window]   Figure 3. Importance of the propargylamine moiety in rasagiline and its derivatives in MAPK-dependent APP processing.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0078fje; doi; 10.1096/fj.03-0078fje

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