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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 7, 2005 as doi:10.1096/fj.05-3794fje. |
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Eve Topf and USA National Parkinson Foundation Centers of Excellence for Neurodegenerative Diseases Research, and Department of Pharmacology, Rappaport Family Research Institute, Technion-Faculty of Medicine, Haifa, Israel
2 Correspondence: Department of Pharmacology, Technion-Faculty of Medicine, P.O.B. 9697, Haifa 31096, Israel. E-mail: youdim{at}tx.technion.ac.il
SPECIFIC AIMS
The present study describes new aspects of the prosurvival effects of the propargyl moiety of rasagiline, propargylamine, in a long-term serum deprivation/neurorescue model using human SH-SY5Y neuroblastoma cells. The molecular mechanisms of the neurorescue effects of propargylamine on pro- and anti-apoptotic proteins, neurotrophic factors, and amyloid precursor protein (APP) regulation were investigated.
PRINCIPAL FINDINGS
1. Propargylamine reduced proapoptotic proteins after long-term serum withdrawal
In our neurorescue model, SH-SY5Y cells exposed to long-term serum deprivation for 3 days exhibited a significant increase in levels of the activated form of caspase-3 and in the cleavage of the caspase substrate, poly ADP-ribose polymerase (PARP). Treatment with propargylamine 0.1 and 1 µM significantly reversed the apoptotic effect on activation of caspase-3 and the cleavage of PARP. A propargylamine-induced decrease in the levels of cleaved caspase-3 and PARP exhibited a biphasic dose-response dependency.
Long-term serum deprivation caused an apparent increase in serine-139 phosphorylation of H2A.X, shown to occur early in apoptosis. Consistent with its effect on the apoptotic markers, propargylamine (0.1 and 1 µM) significantly decreased H2A.X levels. Cell viability was markedly reduced (9.48±1.1-fold, P<0.001 vs. control) in these apoptotic conditions. Propargylamine (1 µM) significantly reduced cell death induced by serum deprivation as assessed by an apoptotic cell death detection ELISA (Fig. 1
C).
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2. Effects of propargylamine on antiapoptotic and proapoptotic gene expression and protein level
Considering the importance of Bcl-2 and Bax in the regulation of programmed cell death pathway, the effects of propargylamine on gene expressions as well as protein levels of these proteins were determined. RNA was extracted from SH-SY5Y cells, treated with or without propargylamine (0.1–10 µM). Real-time RT-PCR revealed that the Bcl-2/Bax mRNA expression ratio was markedly reduced after long-term serum withdrawal. Treatment with propargylamine for 2 days significantly increased Bcl-2 mRNA level while markedly reducing Bax gene expression vs. serum-free culture (Fig. 1A
). The Bcl-2/Bax mRNA ratio showed a significant increase after treatment with propargylamine (Fig. 1A
). Consistent with the gene expression results, Western blot analysis showed that in serum-free culture Bcl-2 protein levels were decreased and Bax protein levels were increased (Fig. 1B
, upper panel). The Bcl-2/Bax protein ratio was markedly reduced (Fig. 1B
). Propargylamine increased Bcl-2 levels and reduced Bax levels. The Bcl-2/Bax protein ratio showed a significant increase after treatment with propargylamine compared with serum-free culture (Fig. 1B
). Long-term serum deprivation significantly induced the expression of Bad and Bim levels. Treatment of SH-SY5Y cells with propargylamine (0.1 and 1 µM) markedly reversed these effects.
3. Effects of propargylamine on glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) gene expression
To further evaluate the mechanism of propargylamine-neurorescue activity, the changes in cell survival genes of the neurotrophic factors GDNF and BDNF were determined by real-time RT-PCR in RNA isolated from serum-deprived SH-SY5Y cells (3 days), with propar-gylamine (0.1–10 µM) being introduced into the medium for an additional 2 days. Both GDNF and BDNF mRNA levels were decreased after serum withdrawal, but propargylamine significantly increased mRNA expression levels of GDNF and BDNF.
4. Neurorescue effects of rasagiline against long-term serum withdrawal-induced neuronal death
Treatment of SH-SY5Y cells with rasagiline (1 µM) markedly decreased cleaved caspase-3 and PARP, as well as the levels of apoptosis-associated phosphorylation of H2A.X protein. Rasagiline (1 µM) induced protein levels of Bcl-2 and reduced those of Bax, Bad, and Bim.
5. Propargylamine and rasagiline regulated holo-APP levels and sAPP
release after long-term serum deprivation
Apoptosis induced by exposure of SH-SY5Y cells to long-term serum withdrawal increased holo-APP gene expression significantly (Fig. 2
A) and protein levels (Fig. 2B
), while propargylamine significantly down-regulated holo-APP using holo-APP N-terminal antibody, 22C11 (Fig. 2B
), and further confirmed using an anti-APP C-terminal antibody (Fig. 2C
). However, propargylamine did not affect holo-APP mRNA levels (Fig. 2A
), indicating post-transcriptional regulatory mechanism. Similarly, rasagiline reduced protein levels of holo-APP. Two-dimensional PAGE and immunoblot assay detected several holo-APP forms in cell lysate, which migrated at 110–130 kDa and at an isoelectric point of 
4.5. The wide range of APP isoelectric forms may result from different post-transcriptional modifications. Long-term serum withdrawal increased the levels of holo-APP forms whereas propargylamine and rasagiline reversed this effect. Both propargylamine (Fig. 2D
) and rasagiline increased the non-amyloidogenic -secretase form of sAPP levels in the medium of serum-deprived SH-SY5Y cells.
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CONCLUSIONS AND SIGNIFICANCE
It is widely thought that apoptosis may be involved in progressive neurodegenerative disorders such as Parkinson’s disease (PD) and Alzheimer’s disease (AD). At the time of clinical diagnosis, patients have already lost a significant percentage of their neurons. Thus, there is an effort to develop drugs with neurorescue activity model (Fig. 3
). In the serum-deprived model used, the severe loss of viability is a consequence of apoptotic-induced cell death, as detected by significant up-regulations of cleaved caspase-3, PARP and H2A.X. Gene and protein expression of the anti-apoptotic Bcl-2 were down-regulated whereas those of the proapoptotic Bax were up-regulated, consistent with the notion that the ratio of Bcl-2 to Bax predetermines the susceptibility of cell survival. The potent proapoptotic "BH3-only" proteins Bad and Bim were also up-regulated, in accordance with the regulatory effect of growth factor withdrawal on these proteins. Thus, serum deprivation-induced cell death may serve as an effective model for screening potential neuronal survival agents in neurorescue research.
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The prosurvival effects of propargylamine were mediated by the Bcl-2 family proteins, increasing the Bcl-2/Bax ratio of mRNA and protein levels, and reducing the "BH3-only" proteins, Bad and Bim. Rasagiline exhibited similar effects. These results are consistent with our published works providing evidence for the neuroprotective mechanism of rasagiline and propar-gylamine mediated by an PKC-MAPK-dependent process, involving up-regulation and translocation of PKC and PKC
with down-regulation of PKC
, which are involved in cell survival pathway. Activation of PKC and PKC is associated with increased expression of the antiapoptotic Bcl-2 family proteins. Propargylamine also up-regulated gene expression levels of GDNF and BDNF after long-term serum deprivation of SH-SY5Y cells, consistent with the ability of rasagiline to increase the protein and mRNA levels of GDNF in SH-SY5Y cells by activation of nuclear factor-
transcription factor. Both neurotrophic factors induce neuroprotective and neurorescue activity in dopaminergic and cholinergic neurons and promote survival of major neuronal types in vitro and in vivo.
In AD, increased expression and/or altered processing of APP is thought to play a central role in amyloidogenesis processes, leading to the formation of senile plaques. Propargylamine and rasagiline reduced the levels of cell-associated holo-APP in mice hippocampus and in the present in vitro study. The regulatory mechanism of propargylamine on holo-APP expression is presumably post-transcriptional, as it suppresses holo-APP protein levels without altering mRNA expression. Several reports support the important role for translational regulatory mechanism to control APP synthesis and probably Aß peptide secretion through the 5'-untranslated region (5'-UTR) of the precursor transcript. Propargylamine and rasagiline stimulated the neuroprotective/neurotrophic fragment, sAPPalpha release into the medium via PKC-MAPK signaling pathway.
Rasagiline and propargylamine have been shown to possess neuroprotective activity, but the present study describes for the first time their neurorescue property and molecular mechanism. These findings may support the possible disease-modifying activity of rasagiline, as suggested in a recent controlled clinical trial of early PD patients.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3794fje;
1 These authors contributed equally to this work. 
This article has been cited by other articles:
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