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(The FASEB Journal. 2006;20:2177-2179.)
© 2006 FASEB

A multifunctional, neuroprotective drug, ladostigil (TV3326), regulates holo-APP translation and processing

Merav Yogev-Falach1, Orit Bar-Am1, Tamar Amit, Orly Weinreb and Moussa B. H. Youdim2

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

ABSTRACT

The recent therapeutic approach in which drug candidates are designed to possess diverse pharmacological properties and act on multiple targets has stimulated the development of the bifunctional drug ladostigil (TV3326) [(N-propargyl-(3R) aminoindan-5yl)-ethyl methyl carbamate]. Ladostigil combines 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. Here, we assessed the dual effects of lodostigil in terms of the molecular mechanism of neuroprotection and amyloid precursor protein (APP) regulation/processing by using an apoptotic model of neuroblastoma SK-N-SH cells. Ladostigil dose-dependently decreased cell death via inhibition of the cleavage and prevention of caspase-3 activation (IC50=1.05 µM) through a mechanism related to regulation of the Bcl-2 family proteins, which resulted in reduced levels of Bad and Bax and induced levels of Bcl-2 gene and protein expression. We have also followed APP regulation/processing and found that ladostigil markedly decreased apoptotic-induced levels of holo-APP protein without altering APP mRNA levels, suggesting a posttranscriptional mechanism. In addition, the drug-elevated phosphorylated protein kinase C (pPKC) levels and stimulated the release of the nonamyloidogenic {alpha}-secretase proteolytic pathway. Similar to ladostigil, its S-isomer, TV3279, which is a ChE inhibitor but lacks MAO inhibitory activity, exerted neuroprotective properties and regulated APP processing, indicating that these effects are independent of MAO inhibition.—Yogev-Falach, M., Bar-Am, O., Amit, T., Weinreb, O., Youdim, M. B. H. The multifunctional neuroprotective anti-Alzheimer/anti-Parkinson drug ladostigil (TV3326) regulates holo-APP translation and processing.


Key Words: amyloid precursor protein • {alpha}-secretase • acetylcholinesterase inhibitor • Alzheimer disease

IN ALZHEIMER’S DISEASE (AD), AMONG SEVERAL pathological features, extracellular deposition of amyloid ß peptid (Aß), neuronal tangles, and marked cholinergic cortical afferent dysfunction are fundamental (1) . Moreover, accumulating evidence indicates that many cytotoxic signals in the AD brain can initiate apoptosis processes, including oxidative stress (OS), inflammation, and accumulation of iron at the sites of neurodegeneration (2 , 3) . Thus, it seems likely reasonable to conclude that AD therapy will require multiple drug therapy to address the varied pathological aspects of the disease.

Recently, we have synthesized a series of bifunctional analogues with a carbamate cholinesterase inhibitory moiety in the aminoindan structure of the MAO-B inhibitor rasagiline (N-propargyl-(1R)-aminoindan), with the purpose of preserving its neuroprotective activity and also to inhibit acetylecholinesterase (AChE) activity to increase the cholinergic transmission. One of these compounds, the R-enantiomer ladostigil (TV3326) [(N-propargyl-(3R) aminoindan-5yl)-ethyl methyl carbamate], has many of the neuroprotective actions of rasagiline (4 5 6 7) , such as prevention of the fall in the mitochondrial potential and cytotoxicity in SH-SY5Y and PC12 cells in response to OS induced by peroxinitrite or glucose (Glc) oxygen deprivation (4 , 7 , 8) . It was also demonstrated to possess neuroprotective activity in vivo, significantly reducing hippocampal cell damage induced by global ischemia in gerbils and the cerebral edema induced in mice by closed head injury (9 10 11) . In addition, we have recently described that ladostigil stimulated the release of the neurotrophic/neuroprotective, nonamyloidogenic-soluble amyloid precursor protein (sAPP{alpha}) via activation of PKC and mitogen-activated protein kinase-(MAPK-) pathways (12) and reduced the levels of APP in mice hippocampus (13) . Here, we describe the mechanism of action of the multifunctional ChE inhibitors (ChEIs) ladostigil and its S-enantiomer TV3279, in terms of neuroprotection and APP processing, by using an apoptotic model of neuroblastoma SK-N-SH cells. Furthermore, studies on structure-activity relationship were conducted to assess whether rasagiline and its propargyl moiety are essential for these effects.

MATERIALS AND METHODS

Materials
Ladostigil (TV3326) [(N-propargyl-(3R)aminoindan-5yl)-ethyl methyl carbamate], TV3279 [(N-propargyl-(3S)aminoindan-5yl)-ethyl methyl carbamate], rasagiline (N-propargyl-(1R)-aminoindan), and N-propargylamine were kindly donated by Teva Pharmaceutical (Netanya, Israel). Electrophoresis reagents were obtained from Invitrogen Corp. (Carlsbad, CA). Tissue culture reagents were obtained from Beth-Haemek (Israel). Caspase-3, Bad, Bax, and pPKC(pan) antibodies were purchased from Cell Signaling, (Beverly, MA). Bcl-2 antibody (Ab) was provided from BD, Biosciences Transduction Laboratories (Heidelberg, Germany). Anti-APP N-terminal Ab, MAb 22C11, which recognizes all mature forms of APP, and MAb 6E10, which is directed at amino acid residue 1–17 of Aß within APP, were obtained from Chemicon International (Temecula, CA). ß-Actin Ab, protein G sepharose, and all other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO).

Cell culture and experimental treatments
Human neuroblastoma SK-N-SH and were obtained from American Type Culture Collection (Rockville, MD). Cells were routinely cultured in Dulbecco’s modified Eagle medium (DMEM; 4500 mg/l Glc), containing 10% FCS and a mixture of 1% penicillin/streptomycin/nystatin. Cell cultures were incubated at 37°C in a humid 5% CO2-95% air environment.

The induction of apoptosis of SK-N-SH cells was performed as described previously (15) . The medium was changed 2 d after seeding, and the cells were incubated for another 5 d without medium removal. Drugs were given either daily (chronic treatment) or acutely when the experiments were initiated (acute treatment).

Immunoblotting analyses
Cells were resuspended in radio-immuno-precipitation assay (RIPA) (20 mM Tris–HCl, pH 7.4; 200 mM NaCl; 1% Triton-X 100; 0.1% SDS; 0.2% sodium deoxycholate; 5 mM EDTA) containing cocktail protease inhibitors (Roche). The lysates were precleared by centrifugation. Protein content was determined using the Bradford method. Equal amounts of sample were resolved on sodium dodecyl sulfate-PAGE (SDS-PAGE) and blotted onto polyvinylidene difluoride membranes (Millipore). Membranes were treated with blocking buffer (5% dry milk in PBS or 5% dry milk, 0.05% Tween 20 in TBS). Primary antibodies were diluted in TBS containing 5% BSA and 0.05% Tween 20 and incubated with membranes for 20 h at 4°C, followed by incubation (1 h at room temperature) in dilutions of horseradish peroxidase-conjugated secondary antibodies in the same buffer. Following Ab incubations, membranes were washed in 0.5% Tween 20 TBS. Detection was achieved using Western blotting detection reagent, enhanced chemiluminescence (ECL) (Amersham, Pharmacia, Little Chalfort Buckinghamshire, UK). Quantification of results was accomplished by measuring the optical density (OD) of the labeled bands from the autoradiograms by using the computerized imaging program Bio-1D (Vilber Lourmat, Marne La Vallee, France). The values were normalized to ß-Actin intensity levels.

For the detection of sAPP{alpha}, media samples were immunoprecipitated with the monoclonal antibody (mAb) 6E10, separated on SDS-PAGE gels, and detected with Ab 6E10.

Total RNA extraction, reverse transcription (RT), and quantitative real-time RT-polymerase chain reaction (RT-PCR)
Isolation of total RNA was performed by using Tri ReagentTM Isolation reagent according to the manufacture’s protocol (Sigma). Total RNA was treated with DNase-RNase free (Roche Diagnostics, Mannheim, Germany) for 30 min at 37°C and subsequently extracted by a round of phenol:chlorophorm: isoamylalcohol (25:24:1), followed by one of chloroform. After precipitation with NaOAc (0.3 M) and ethyl alcohol, the RNA pellet was washed with 80% ethyl alcohol (12,000 g) for 10 min and resuspended in 50–100 µl of diethylpyrocarbonate (diethyl pyrocarbonate)-treated water and incubated for 5–10 min at 56°C to facilitate resuspension.

Total RNA (2 µg) was denatured and reverse-transcribed by using random hexanucleotides (0.5 µg/µl) as described previously (14) .

Quantitative real-time polymerase chain reaction (PCR), using LightCycler and FastStart DNA Master SYBR Green I ready-to-use PCR mix, was performed according to the manufacturer’s protocol (Roche). cDNA (40 ng) was amplified in 20 µl total vol. The sequences of the primers and the size of the products are described in Table 1 . The results are analyzed on the provided program of the LightCycler. The relative expression level of a given mRNA was assessed by normalizing to the housekeeping gene 18S-rRNA and compared with control values.


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  		Table 1. Sequences of primers for quantitative real-time RT-PCR

Statistics
For statistical analysis, one-way ANOVA followed by Student’s t test was performed. P values of P < 0.05 were considered significant. Each experiment was repeated 3–4 times in triplicates, and results from a representative experiment are shown.

RESULTS

Prevention of caspase-3 activation in apoptotic SK-N-SH cells by ladostigil and its S-isomer TV3279
Yang el al. (15) have previously reported that experimental conditions of long-term culture result in apoptotic death of SK-N-SH cells, determined by DNA fragmentation, flow cytometry, and nuclear morphology. In the present study, we have used the apoptotic model of long-term SK-N-SH neuroblastoma culture to examine the potential antiapoptotic activity of the drugs, ladostigil and TV3279, its S-isomer. As expected from previous studies, (15 , 16) , 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). Next, levels of cleaved caspase-3 were quantified by Western blot as a measure of apoptotic activity. Using an anticaspase-3 Ab that detects processed subunits, we could demonstrate that 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 (Fig. 1 A). Treatment of SK-N-SH cells with either ladostigil (1–10 µM) or TV3279 (1–10 µM) dose-dependently reversed the cleavage and activation of caspase-3 (Fig. 1A ) (IC50=1.05 µM and IC50=1.19 µM, respectively). These IC50 values are inconsistent with our previous concentration-dependent studies (7 , 12) . Chronic drug treatment also significantly attenuated the apoptotic effect on the cleavage and activation of caspase-3, compared with the levels in untreated cells (Fig. 1B ); therefore, we decided to use the paradigm of acute drug treatment for our future experiments.


Figure 1
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  		Figure 1. Ladostigil and its S-isomer TV3279 reduced caspase-3 activation. SK-N-SH cells were incubated with full serum for 1 or 5 d and (A) acute or (B) chronically treated without (cont.) or with ladostigil or TV3279, as described in Materials and Methods. Caspase-3 cleavage was examined by immunoblotting analysis. The loading of the lanes was normalized to levels of ß-Actin. Densitometric analysis of Western blot obtained from three independent experiments and data are expressed as mean ± SEM; *P < 0.01, **P < 0.001 vs. control.

Effect of ladostigil on apoptotic-induced Bcl-2 depletion and Bax and Bad accumulation
Considering the roles of the Bcl-2 family members, Bcl-2, Bax, and Bad in the mitochondrial pathway of apoptosis, the effect of ladostigil on these proteins was determined by Western blotting. 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. 2 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. 2A, E ).


Figure 2
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  		Figure 2. Ladostigil regulated Bcl-2 family proteins: Bcl-2, Bax and Bad. SK-N-SH cells were maintained in fibrous sheath (FS) for 1 or 5 d and acute-treated without (cont.) or with increasing concentrations of ladostigil. Bcl-2, Bax (A–D), and Bad (E) levels were examined by immunoblot analysis. The loading of the lanes was normalized to levels of ß-Actin. Quantitated results of (B) Bcl-2 and (C) Bax are presented relative to control. D) Ratios of Bcl-2/Bax protein levels. F) Quantitated results of Bad are presented relative to control. Densitometric analysis of Western blot obtained from three independent experiments, and data are expressed as mean ± SEM; *P h 0.01, **P h 0.001 vs. control.

Treatment with ladostigil (1–10 µM) markedly increased the levels of Bcl-2 (Fig. 2A, B ), whereas reduced Bax expression (Fig. 2A, C ) resulted in dose-dependently increased Bcl-2/Bax ratio (Fig. 2D ). In addition, ladostigil treatment almost completely abolished apoptotic-induced protein levels of the proapoptotic regulator of the cell death machinery, Bad (Fig. 2E, F ). Thus, the neuroprotective action of ladostigil against apoptosis in SK-N-SH cells is associated with blockade of the decrease of antiapoptotic protein Bcl-2 and the increase of proapoptotic proteins Bax and Bad. Similarly, the S-isomer TV3279 markedly increased the levels of Bcl-2 while reducing Bax and Bad expression (data not shown).

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), as described in Materials and Methods. Each gene expression was normalized to the housekeeping gene 18S-rRNA, as this transcript was reported to be less susceptible to the influence by external factors (17) . Real-time RT-PCR revealed that ladostigil significantly increased Bcl-2 mRNA levels (Fig. 3 A), while markedly reducing Bax and Bad gene expression vs. control (Fig. 2B and C , respectively).


Figure 3
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  		Figure 3. Ladostigil regulated gene expression of Bcl-2, Bax and Bad. SK-N-SH cells were maintained in FS for 1 or 5 d and acute-treated without (cont.) or with increasing concentrations of ladostigil. Bcl-2 (A), Bax (B) and Bad (C) gene expression levels were measured by quantitative real-time RT-PCR, The amount of each product was normalized to the housekeeping gene 18S-rRNA. Data are representative of 3 independent experiments and are expressed as mean ± SEM; *P < 0.01 vs. control.

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 further examined in the apoptotic model of SK-N-SH cells in long-term culture. As shown in Fig. 4 , 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.


Figure 4
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  		Figure 4. Effects of rasagiline and N-propargylamine on caspase-3 activation induced by high-density SK-N-SH culture. SK-N-SH cells were incubated with FS for 1 or 5 d and treated without or with (A) rasagiline or (B) N-propargylamine. Caspase-3 cleavage was examined by immunoblotting analysis. The loading of the lanes was normalized to levels of ß-Actin. Data are representative of three independent experiments.

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 level (2.39±0.45-fold; P<0.001) vs. 1 d cultured cells (Fig. 5 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. 5A ). However, ladostigil did not affect holo-APP mRNA levels (Fig. 5B ), indicating posttranscriptional regulatory mechanism, as has been previously described (18 , 19) . Similar to ladostigil, rasagiline and N-propargylamine significantly reduced the levels of cellular holo-APP (Fig. 6 ).


Figure 5
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  		Figure 5. Ladostigil and TV3279 decreased holo-APP levels. A) SK-N-SH cells were incubated with FS for 1 or 5 d and treated without (cont.) or with increasing concentration of ladostigil or TV3279. Protein levels of holo-APP in cell lysates were examined by immunoblotting analysis using mAb 22C11. The loading of the lanes was normalized to levels of ß-Actin. Densitometric analysis of Western blot obtained from three independent experiments and data are expressed as mean ± SEM; *P < 0.01, **P < 0.001 vs. control. B) Gene expression of holo-APP was measured by quantitative real-time RT-PCR. The amount of each product was normalized to the housekeeping gene 18S-rRNA. Data are representative of three independent experiments.


Figure 6
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  		Figure 6. Rasagiline and N-propargylamine decreased holo-APP levels. SK-N-SH cells were incubated with FS for 1 or 5 d and treated without or with (A) rasagiline or (B) N-propargylamine. Levels of holo-APP in cell lysates were examined by immunoblotting analysis. The loading of the lanes was normalized to levels of ß-Actin. Data are representative of three independent experiments.

Regulation of sAPP{alpha} release by ladostigil and TV3279
Recently, it was demonstrated that ladostigil regulates acute secretory processing of nonamyloidogenic APP via PKC- MAPK-dependent pathway (12) . The effects of ladostigil on sAPP{alpha} release and on the ratio sAPP{alpha}/holo-APP under the present long-term culture conditions were determinded. Figure 7 A demonstrates that ladostigil and TV3279 significantly induced sAPP{alpha} release into the medium vs. the levels detected in control untreated cells. Also, the ratio sAPP{alpha}/holo-APP progressively increased along the drugs’ concentration range (1–10 µM), further indicating that both ladostigil and TV3279 promote the nonamyloidogenic pathway of APP processing. Fig. 7B shows that this effect is correlated with increased expression of several phosphorylated-active PKC isoforms. Similar to ladostigil, the S-isomer TV3279 (0.1–10 µM) dose-dependently increased the levels of pPKC(pan) (data not shown).


Figure 7
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  		Figure 7. Ladostigil elevated sAPP{alpha} secretion and PKC phosphorylation. A) SK-N-SH cells were incubated for 5 d and treated without or with increasing concentration of ladostigil or its S-isomer, TV3279. Levels of sAPP{alpha} released into the conditioned media were examined by immunoprecipitation followed by immunoblotting with mAb 6E10, as described in Material and Method. B) Phosphorylation of PKC was analyzed in cell lysates and detected with pPKC(pan) Ab. The loading of the lanes was normalized to levels of ß-Actin. Densitometric analysis of Western blot obtained from three independent experiments and data are expressed as mean ± SEM; *P < 0.01, **P < 0.001 vs. control.

DISCUSSION

Numerous clinical trials have demonstrated the safety and efficacy of acetylcholinesterase inhibitors in the treatment of AD. Yet, their benefits in AD are likely to be more complex than simply replacement of lost acetylcholine (Ach) (20 21 22 23) . As reviewed recently, growing preclinical evidence indicates that AChEIs block some of the fundamental neurodegenerative processes involved in AD (24) . AChEIs such as tacrine, donepezil, galantamine, huperazine A, and ganstigmine were reported to protect neurons from death in various cell culture models of neurodegenerative diseases (24) . In addition, evidence indicates that several ChEIs also affect various neuropathological markers of AD and modulate the nonamyloidogenic APP processing (23) . Therefore, it is suggested that AChEIs possessing properties of neuroprotection and/or APP processing might be more beneficial than those that inhibit only AChE to treat AD and in particular to prevent the pathogenesis of AD.

The bifunctional anti-AD drug ladostigil (TV3326) was recently developed by us in an attempt to combine the pharmacophore neuroprotective properties of the anti-PD MAO-B inhibitor drug rasagiline with the ChE inhibitory moiety of rivastigmine, a drug with proven efficacy in AD (4 , 9 , 25 , 26) . Here, we assessed the dual effects of lodostigil in terms of neuroprotection and APP processing (Fig. 8 ). 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 (27 , 28) . This apoptotic model is a reliable cytotoxic system for screening potential neuroprotective agents because it mimics certain aspects of neurodegeneration, including nutrient deprivation, failure to remove metabolic waste, energy depletion, and oxygen insufficiency (16) . Although the exact cellular mechanism of this model is not yet fully understood, the observation of severe cell viability loss and the involvement of PKC regulation implies that cell death in this model could involve oxidative stress and mitochondrial dysfunction (16) .


Figure 8
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  		Figure 8. Proposed schematic model for the dual effects of ladostigil: neuroprotection and regulation/processing of holo-APP.

Despite the extremity of the neurotoxic model 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 recent study, which demonstrated that activation/regulation of PKC in association with Bcl-2 protein family promotes neuronal survival by rasagiline, as well as its propargyl moiety (14 , 29) .

Studies using the apoptotic model of long-term SK-N-SH culture demonstrated that AChE expression simultaneously increased with its aggregation in the nuclei of apoptotic cells (15) . Indeed, increasing evidence indicates that AChE might be involved in apoptosis. Transfection with AChE leads to an increase of apoptosis in retinal cells, and highly purified AChE has been shown to have toxic effects in both neuron- and glial-like cell lines via the apoptotic mechanism (30) . AChE was also found to have additional effects that could result in increased APP expression (31) . In this context, increased expression of APP and generation of ß-amyloid peptides may play a central role in the amyloidogenesis process in AD (32) . Thus, the present findings demonstrating that ladostigil markedly suppressed holo-APP protein levels and elevated sAPP{alpha} could be of clinical value toward accelerating nonamyloidogenic APP processing, thereby reducing the possibility of generation of the toxic ß-amyloid peptides. Consistent with this, 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 (13) . Moreover, the observation that ladostigil did not alter APP mRNA levels may suggest that the decrease in APP protein levels can be attributed to suppression of APP translation. Earlier reports have shown the ability of another ChEI, phenserine, to reduce the levels of APP and secreted Aß (33) . In a recent study, selective butyrylcholinesterase inhibition was found to reduce APP and Aß levels in vitro and in vivo (34) . The mechanism underlying these effects may involve both cholinergic and noncholinergic actions regulating APP synthesis and processing, as was characterized with phenserine, which reduced APP levels via lowering the translational efficiency of APP mRNA (33) . In addition, the current study demonstrates that ladostigil elevated the levels of the nonamyloidogenic sAPP{alpha}, as was shown previously in another cellular model system. This mechanism involved PKC- and mitogen-activated protein kinase (MAPK)-dependent pathways (12) . However, the effect does not appear to result from ChE inhibition activity, as rasagiline and N-propargylamine could also induce PKC and ERK activation and promote sAPP{alpha} release (35) . Several ChEIs, such as tacrine (36) , physostigmine (37) , metrifonate (38) , ganstigmine (39) , and donepezil (40) , increased sAPP{alpha} release in cell culture. However, the observations that phenserine (33) and tacrine, at high concentration (37) , decreased sAPP{alpha} release suggest that the regulation of APP processing by ChEIs is not simply associated to AChE inhibition. It is likely that several different mechanisms are in operation. For example, ChEIs increased PKC levels in vitro (41) and attenuated the Aß1–40–induced down-regulation of PKC in rats (42) . Moreover, donepezil promoted the trafficking of {alpha}-secretase to the membrane, thus enhancing {alpha}-secretase activity (40) .

Similar to ladostigil, its S-isomer TV3279, which is a ChEI but lacks MAO inhibitory activity, exerted pronounced neuroprotective properties and APP processing, which suggests that the mode of action is independent of MAO inhibition. These results are consistent with the current and previous data, providing clear evidence that the neuroprotection by ladostigil and rasagiline does not depend on inhibition of MAO-B but rather is associated with some intrinsic pharmacological action of the propargyl moiety on the mitochondrial cell-survival proteins (43 44 45) . N-propargylamine itself has been demonstrated to have a neuroprotective function, with similar potency to that of rasagiline (14 , 29) . It is now apparent that the neuroprotection effect of N-propargylamine is mediated by PKC-, MAPK-dependent activation associated with Bcl-2 family members (14) and mitochondrial membrane stabilization (29) . In addition, we have found recently that the MAO inhibitory potency of N-propargylamine was significantly lower than its ability to induce neuroprotection, further establishing that MAO inhibition is not prerequisite for neuroprotection by propargyl-related compounds (46) .

In conclusion, we have demonstrated the dual actions of the novel drug ladostigil in terms of neuroprotection and modulation of holo-APP and sAPP{alpha} levels, hence reducing the possibility of generating amyloidogenic derivatives. The several targets and diverse pharmacological properties of ladostigil make the drug a potentially valuable for the treatment of AD.

ACKNOWLEDGMENTS

The authors are grateful to Teva Pharmaceutical Co. (Netanya, Israel) and National Parkinson Foundation (Miami, FL), Friedman Foundations (Philadelphia, PA) and Technion-Israel Institute of Technology Research and Development (Haifa, Israel) for their generous support of this work. Friedman and Golding Funds (Technion) are acknowledged. This study is in partial fulfillment of the requirements of the Technion-ISRAEL Institute of Technology for a Ph.D. by M.Y-F.

FOOTNOTES

1 These authors contributed equally to this work.

Received for publication January 19, 2006. Accepted for publication May 25, 2006.

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O. WEINREB, T. AMIT, O. BAR-AM, and M. B.H. YOUDIM
Induction of Neurotrophic Factors GDNF and BDNF Associated with the Mechanism of Neurorescue Action of Rasagiline and Ladostigil: New Insights and Implications for Therapy
Ann. N.Y. Acad. Sci., December 1, 2007; 1122(1): 155 - 168.
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