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Eve Topf Center of Excellence for Neurodegenerative Diseases Research and Department of Pharmacology, Rappaport Family Research Institute, Technion-Faculty of Medicine, Haifa, Israel
2Correspondence: Dept. of Pharmacology, Technion-Faculty of Medicine P.O.B. 9697, 31096 Haifa, Israel. E-mail: Youdim{at}tx.technion.ac.il
SPECIFIC AIMS
The bifunctional drug ladostigil (TV3326) [(N-propargyl-(3R) aminoindan-5yl)-ethyl methyl carbamate] was developed to combine the neuroprotective effects of the antiparkinson drug rasagiline, a selective monoamine oxidase (MAO)-B inhibitor, with the cholinesterase (ChE) inhibitory activity of rivastigmine in a single molecule, as a potential treatment for Alzheimer’s disease (AD) and Lewy Body disease. We assessed the dual effects of lodostigil in terms of molecular mechanism of neuroprotection and amyloid precursor protein (APP) regulation/processing by using an apoptotic model of neuroblastoma SK-N-SH cells.
PRINCIPAL FINDINGS
1. Prevention of caspase-3 activation in apoptotic SK-N-SH cells by ladostigil and its S-isomer TV3279
Experimental conditions of long-term SK-N-SH neuroblastoma culture, resulting in neuronal death, were characterized by DNA fragmentation, flow cytometry, and nuclear morphology where the potential antiapoptotic activity of the drugs ladostigil and its S-isomer TV3279 were determined. Cell viability was markedly reduced by 81.3%±0.65-fold; P < 0.001 vs. 1 d culture cells, as assessed by an apoptotic cell death detection ELISA kit (Roche Molecular Biochemicals, Indianapolis, IN). Levels of cleaved caspase-3 were quantified by Western blot as a measure of apoptotic activity by using an anticaspase-3 antibody (Ab). In SK-N-SH cells cultured for 5 d, the levels of cleaved caspase-3 were significantly increased (4.98±0.65-fold; P<0.001) vs. 1 d cultured cells. Ladostigil (1–10 µM) or TV3279 (1–10 µM) dose-dependently reversed the cleavage and activation of caspase-3 (IC50=1.05±0.02 µM and IC50=1.19±0.04 µM, P<0.01, respectively). Chronic drug treatment also significantly attenuated the apoptotic effect on the cleavage and activation of caspase-3, compared with the levels in untreated cells, thus the paradigm of acute drug treatment was used for other experiments.
2. Effect of ladostigil on apoptotic-induced Bcl-2 depletion and Bax and Bad accumulation
In the long-term culture SK-N-SH cells, the levels of the prosurvival protein Bcl-2 were significantly reduced (2.50±0.04-fold; P<0.01) vs. 1 d cultured cells (Fig. 1
A), whereas those of the proapoptotic proteins Bax and Bad were markedly increased (4.56±0.34-fold; P<0.01 and 3.25±0.23-fold; P<0.01, respectively) vs. 1 d cultured cells (Fig. 1 A, E
). Treatment with ladostigil (1–10 µM) markedly increased the levels of Bcl-2 (Fig. 1A, B
), while reducing, Bax expression (Fig. 1A, C
), resulting in dose-dependently increased Bcl-2/Bax ratio (Fig. 1D
). In addition, ladostigil abolished apoptotic-induced protein levels of the proapoptotic regulator of the cell death machinery, Bad (Fig. 1E, F
). The S-isomer of ladostigil, TV3279, exhibited similar activity.
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Consistent with the results of protein levels, the effects of ladostigil on gene expression were determined. RNA was extracted from SK-N-SH cells, treated without (control) or with ladostigil (1–10 µM). Each gene expression was normalized to the housekeeping gene 18S-rRNA, since this transcript was reported to be less susceptible to the influence by external factors. Real-time RT-polymerase chain reaction (RT-PCR) revealed that ladostigil significantly increased Bcl-2 mRNA level, while markedly reducing Bax and Bad gene expression vs. control.
3. Effects of rasagiline and N-propargylamine on caspase-3 activation
Because ladostigil and TV3279 were synthesized by introducing a carbamate moiety into various positions in the rasagiline molecule, the neuroprotective effects of rasagiline and its propargyl moiety were examined further in the apoptotic model of SK-N-SH cells in long-term culture. Treatment of SK-N-SH cells with either rasagiline (1 and 10 µM) or N-propargylamine (1 and 10 µM) almost completely inhibited apoptotic-induced caspase-3 cleavage and activation.
4. Regulation of holo-APP levels
The regulatory effect of ladostigil and TV3279 on cellular holo-APP levels was examined in the experimental conditions of SK-N-SH cells in long-term culture. SK-N-SH cells in long-term culture were found to exhibit a significantly increased cellular holo-APP levels (2.39±0.45-fold; P<0.001) vs. 1 d cultured cells (Fig. 2
A). Treatment of cells with either ladostigil (0.1–10 µM) or TV3279 (0.1–10 µM) resulted in a concentration-dependent decrease in cellular holo-APP levels compared with the levels in control untreated cells (Fig. 2A
). However, ladostigil did not affect holo-APP mRNA levels (Fig. 2B
), indicating posttranscriptional regulatory mechanism, as has been previously described. Similar to ladostigil, rasagiline and N-propargylamine significantly reduced the levels of cellular holo-APP.
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5. Regulation of sAPP
release by ladostigil and TV3279
Ladostigil regulates acute secretory processing of nonamyloidogenic APP via PKC-MAPK-dependent pathway. The effects of ladostigil on sAPP release and on the ratio sAPP/holo-APP under the present long-term culture conditions were determined. Ladostigil and TV3279 significantly induced sAPP release into the medium vs. the levels detected in control untreated cells. Also, the ratio sAPP/holo-APP progressively increased along the concentration range of the drugs (1–10 µM), which further indicated that both ladostigil and TV3279 promote the nonamyloidogenic pathway of APP processing. This effect is correlated with increased expression of several phosphorylated-active PKC isoforms.
CONCLUSIONS AND SIGNIFICANCE
Acetylcholinesterase inhibitors (ChEIs) in the treatment of AD are likely to be more complex than simply increasing lost acetylcholine (Ach). Growing preclinical evidence indicates that AChEIs may have disease-modifying activity in AD. ChEIs, such as, tacrine, donepezil, galantamine, huperazine A, and ganstigmine, were reported to protect neurons from death in various neuronal culture models; affect various neuropathological markers of AD; and modulate the nonamyloidogenic APP processing. Therefore, it is suggested that ChEIs possessing properties of neuroprotection and/or APP processing might be more beneficial than those that inhibit only ChE to treat AD and in particular to prevent the pathogenesis of AD.
The bifunctional anti-AD drug ladostigil was recently developed by us in an attempt to combine the pharmacophore neuroprotective properties of the anti-Parkinson drug rasagline with the ChE carbamate inhibitory moiety of anti AD drug rivastigmine. Thus, we assessed the dual effects of lodostigil in terms of neuroprotection and APP processing (Fig. 3
). The current study demonstrates that ladostigil provided substantial protection from induction of cell death caused by long-term cultured human neuroblastoma SK-N-SH cells, a cell line that has been shown to exhibit a neuronal phenotype and has multiple neurochemical markers. This apoptotic model is a reliable cytotoxic system for screening potential neuroprotective agents, as it mimics certain aspects of neurodegeneration.
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Despite the extremity of the neurotoxic condition used, highly significant neuroprotection was evident on treatment with ladostigil, including inhibition of caspase-3 activation, induction of Bcl-2, and reduction of Bad and Bax gene and protein expression. These findings are consistent with our demonstration that activation/regulation of PKC in association with Bcl-2 protein family promotes neuronal survival via its propargyl moiety.
Apoptotic models of long-term SK-N-SH culture studies have demonstrated that AChE expression simultaneously increases with its aggregation in the nuclei of apoptotic cells. AChE was also reported to induce an increase in APP expression. In this respect, increased expression of APP and generation of ßbeta;-amyloid peptides may play a central role in the amyloidogenesis process in AD. Thus, the present findings demonstrating that ladostigil markedly suppressed holo-APP protein levels and elevated sAPP could be of clinical value toward accelerating nonamyloidogenic APP processing and could thereby reduce the possibility of generation of the toxic ßbeta;-amyloid peptides. Consistent with this finding, our previous study showed that treatment with ladostigil markedly decreased the levels of cell-associated, holo-APP in the mice hippocampus, which indicates that APP expression can also be regulated by ladostigil under in vivo conditions. Moreover, the observation that ladostigil did not alter APP mRNA levels may suggest that the decrease in APP protein levels could be attributed to suppression of APP translation. In addition, ladostigil elevated the levels of the nonamyloidogenic sAPP, which confirms previous results. This mechanism involves PKC- and mitogen-activated protein kinase (MAPK)-dependent pathways and does not appear to result from ChE inhibition because the MAO B inhibitor rasagiline, which does not have cholinergic activity, has the same mechanism of action to promote sAPP release. Similarly the S-isomer of ladostigil, TV3279, which is a ChEI but lacks MAO inhibitory activity, has the same property. Our previous studies have clearly demonstrated that the propargyl moiety of these compounds is responsible for this activity, because propargylamine, which is neither a ChE or MAO inhibitor, has the same effect, with similar potency.
In conclusion, ladostigil has dual actions in terms of neuroprotection and modulation of holo-APP and sAPP levels, hence reducing the possibility of generating amyloidogenic derivatives. The diverse pharmacological properties of multifunctional ladostigil make it potentially valuable for the treatment of AD.
FOOTNOTES
1 These authors contributed equally to this work. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4910fje
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