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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 9, 2004 as doi:10.1096/fj.04-1916fje. |
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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, Israel
2 Correspondence: Department of Pharmacology, Technion-Faculty of Medicine, P.O.B. 9697, 31096 Haifa, Israel. E-mail: Youdim{at}tx.technion.ac.il
SPECIFIC AIMS
Using a model of serum deprivation, we investigated the molecular mechanism by which the anti-Parkinson/monoamine oxidase B (MAO-B) inhibitor drug rasagiline exerts its neuroprotective effect in rat pheochromocytoma PC12 cells. Studies of structure-activity relationship were conducted to assess whether the propargyl moiety of rasagiline is essential for the novel neuroprotective mechanism involving interaction of protein kinase C (PKC) pathway with the Bcl-2 family proteins.
PRINCIPAL FINDINGS
1. Prevention of serum deprivation-induced apoptosis by rasagiline
To determine whether rasagiline could act as a survival factor, partially differentiated PC12 cells were placed in fresh serum-free medium in the presence or absence of rasagiline. Cells placed in fresh complete medium served as full-serum control. PC12 cell viability was markedly reduced by 24 h serum withdrawal (75.8±6% of full-serum control). Figure 1
A shows that rasagiline significantly reduced cell death induced by serum deprivation, as assessed by an apoptotic cell death detection ELISA. Similar results were obtained by the MTT reduction analysis. Consistent with its effect on apoptosis, rasagiline prevented the appearance of the cleaved, activated form of caspase-3 and cleavage of the caspase substrate poly (ADP-ribose) polymerase (PARP) (Fig. 1B
). The PKC pathway has been described to function as a main neuronal survival pathway. We therefore tested whether the activation of PKC is crucial for rasagiline-mediated neuroprotection, using the specific broad-spectrum PKC inhibitor GF109203X, which exhibits high affinity for the conventional PKCs (
, ß, 
), and the novel isoenzyme PKC
and PKC
. These experiments show that GF109203X markedly reversed rasagiline-suppressive effects on the cleavage and activation of caspase-3 and PARP in serum withdrawal-induced programmed cell death (Fig. 1B
). Rasagiline decreased serum-free induced protein level of the proapoptotic regulator Bad; this effect was blocked by GF109203X (Fig. 1B
). These data suggest that rasagiline neuroprotection against serum withdrawal is mediated through PKC signaling.
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2. The effect of rasagiline on PKC
To further determine the role of PKC in rasagiline neuroprotection, the effects of rasagiline on PKC activation were investigated. Short-term treatment of PC12 cells with rasagiline (1 h) dose-dependently induced PKC phosphorylation. The maximal effect was observed at 1 µM (2.1±0.7-fold, n=3, P<0.05 vs. control). Pretreatment with GF109203X (2.5 µM) significantly reduced rasagiline-induced PKC phosphorylation. Next, we analyzed the effects of rasagiline treatment on the subcellular redistribution of p-PKC (pan), PKC, and PKC to establish the activation of PKC and obtain data on the isoforms possibly involved in the effect of rasagiline. Stimulation with rasagiline (1 µM) led to p-PKC (pan), PKC, and PKC translocation to the membrane fractions (
40%), suggesting that these isoforms are activated by rasagiline.
3. Modification of mRNA levels of Bcl-2 family members, PKC isoforms, and brain-derived neurotrophic factor BDNF by rasagiline
We examined the changes in cell survival/death-related genes, including selected Bcl-2 and PKC family members. Real-time RT-PCR consists of RNA samples isolated from PC-12 cells maintained in full-serum or serum-free media and treated without or with rasagiline for 24 h. Real-time RT-PCR shows that in serum-free culture the expression level of Bcl-xL was significantly reduced (60% of full-serum culture) whereas Bad and Bax mRNA levels were markedly increased (1.8- and 2.5-fold, respectively, vs. full-serum culture) (Fig. 2
). Rasagiline treatment for 24 h significantly induced Bcl-xL and Bcl-w mRNA expression (at 1 µM, 2- and 1.4-fold, respectively, vs. serum-free culture). Rasagiline markedly reduced both mRNAs of Bad and Bax expression (at 1 µM, 70% of serum-free culture). The effect of rasagiline on PKC gene expression was examined by real-time RT-PCR analysis using PKC-, PKC-, and PKC-specific RT primers. Treatment of serum-free PC12 cells with rasagiline for 24 h significantly up-regulated PKC and PKC mRNA levels compared with the decreased expression detected in serum-free culture alone. Rasagiline reversed the increased level of PKC mRNA observed in serum-deprived cells. Moreover, quantitative real-time RT-PCR uncovered a possible association between rasagiline mechanism of action and BDNF gene, showing that serum-deprived PC12 cells treated with 1 and 10 µM of rasagiline overexpressed BDNF mRNA by 3.5-fold vs. serum-free treated cells alone.
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4. Effects of propargylamine on cell viability and PKC activation
The effects of propargylamine on cell viability were examined to determine the importance of the propargyl moiety in rasagiline. Propargylamine (1 and 10 µM) significantly reduced cell death as induced by serum deprivation in PC12 cells. Similar to rasagiline, treatment of PC12 cells with increasing concentrations of propargylamine resulted in a significant dose-dependent increase in PKC phosphorylation, whereas pretreatment with GF109203X (2.5 µM) blocked the induction of PKC phosphorylation by propargylamine. Real-time RT-PCR analysis revealed that treatment of PC12 cells with propargylamine (10 µM) for 24 h significantly increased Bcl-xL, Bcl-w, PKC, and PKC and decreased Bad and PKC mRNA expression levels vs. levels observed in serum-free culture.
CONCLUSIONS AND SIGNIFICANCE
This study provides evidence whereby activation/regulation of PKC in association with Bcl-2 protein family promotes neuronal survival by the anti-Parkinsonian drug rasagiline, and this property is related to its propargyl moiety (see Fig. 3
). The role for PKC activation in the mechanism of rasagiline neuroprotection is supported by evidence that rasagiline can activate the essential PKC isoforms involved in cell survival pathways PKC and PKC and blocking of its neuroprotective action by inhibition of PKC activity. Real-time PCR analyses revealed that exposure of serum-deprived PC12 cells to rasagiline markedly increased PKC and PKC gene expression. By contrast, rasagiline prevented the increase in PKC mRNA that occurred in serum-deprived PC12 cells. These data complement earlier observations that certain PKC isoforms are intimately involved in cell survival/death signals that protect against cell death. PKC is known to phosphorylate Bcl-2 in a site that increases its anti-apoptotic function and overexpression of PKC results in increased expression of Bcl-2. Suppression of PKC triggers apoptosis through down-regulation of Bcl-xL. MAPK/ERK cascades, which have been shown to inhibit cell death and can be activated by PKC, was recently found to be up-regulated by rasagiline and its other propargylamine derivatives. Recent study of structure-activity relationship among rasagiline-related compounds revealed the importance of the propargyl moiety for neuroprotection. That propargylamine itself significantly augmented the viability of serum-deprived cells, induced PKC activation, increased PKC and PKC, and reduced PKC mRNA expression strengthens its crucial role in the neuroprotective activity of rasagiline.
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Our findings further demonstrate involvement of the PKC pathway in rasagiline-induced inactivation of the BH3-only proapoptotic Bcl-2 family member, Bad. This is consistent with previous reports indicating that the PKC-dependent pathway can promote cell survival via phosphorylation and inactivation of Bad. This study provides further evidence that the neuroprotective effect of rasagiline and propargylamine against serum deprivation is mediated by gene regulation of the Bcl-2-related protein family. Real-time RT-PCR revealed a decrease in mRNA of the proapoptotic members Bax and Bad and an increase in mRNA of the cell survival members Bcl-w and Bcl-xL. These results are relevant to the fact that the Bcl-2-related protein family regulates the mitochondrial membrane permeability transition pore (PTP) and triggers dissipation of mitochondrial membrane potential (
m) and release of cytochrome c inducing downstream events, including caspase-3 activation, leading to cell death. Previous reports have shown that rasagiline suppresses cell death through direct interaction with mitochondrial apoptosis cascade and helps maintain mitochondrial membrane potential (m) and PTP. These results are consistent with reports providing clear evidence that the neuroprotection by rasagiline and its derivatives does not depend on inhibition of MAO-B but is associated with some intrinsic pharmacological action of the propargyl moiety, which acts on the mitochondria cell survival Bcl-2 family proteins and their interaction with PKC signaling pathway.
We show that rasagiline increases expression of BDNF, a neurotrophin found to promote survival of all major neuronal types affected in Alzheimer’s disease and Parkinson’s disease (PD). Reduced expression of BDNF was demonstrated in the substantia nigra of individuals with PD, and BDNF prevented the spontaneous death of dopaminergic neurons in rat primary mesencephalic culture as well as a reduction in striatal dopamine content induced by N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mice. BDNF was also reported to regulate PKC activation and to affect Bcl-w and Bcl-xL expression. Neuronal cell culture studies have described how rasagiline increases the expression of the glial cell line-derived neurotrophic factor (GDNF) and the activities of antioxidant enzymes. A recent proteomic and genomic analysis of midbrain from mice treated with rasagiline shows a highly significant increase in nerve growth factor ß-NGF. These data indicate that rasagiline can suppress the death process and promote survival of dopamine neurons. Further studies are under way to clarify the interrelationship between rasagiline-induced BDNF, GDNF, or other neurotrophic substances, PKC signaling pathway, and the Bcl-2-related protein family resulting in neuroprotection.
FOOTNOTES
1 These authors share equal recognition. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1916fje; doi: 10.1096/fj.04-1916fje
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