Therapeutic targets and potential of the novel brain- permeable multifunctional iron chelator–monoamine oxidase inhibitor drug, M-30, for the treatment of Alzheimer's disease1

Authors


  • 1

    [Correction added after online publication 15 December 2006; in the title, inhbitor was corrected to inhibitor]

Address correspondence and reprint requests to Professor M. B. H. Youdim, Eve Topf Centers of Excellence and Department of Pharmacology, Technion-Rappaport Family Faculty of Medicine, Efron Street, PO Box 9697, 31096 Haifa, Israel. E-mail: Youdim@tx.technion.ac.il

Abstract

Novel therapeutic approaches for the treatment of neurodegenerative disorders comprise drug candidates designed specifically to act on multiple CNS targets. We have synthesized a multifunctional non-toxic, brain permeable iron chelator drug, M-30, possessing propargyl monoamine oxidase (MAO) inhibitory neuroprotective and iron-chelating moieties, from our prototype iron chelator VK-28. In the present study M-30 was shown to possess a wide range of pharmacological activities, including pro-survival neurorescue effects, induction of neuronal differentiation and regulation of amyloid precursor protein (APP) and β-amyloid (Aβ) levels. M-30 was found to decrease apoptosis of SH-SY5Y neuroblastoma cells in a neurorescue, serum deprivation model, via reduction of the pro-apoptotic proteins Bad and Bax, and inhibition of the apoptosis-associated phosphorylated H2A.X protein (Ser 139) and caspase 3 activation. In addition, M-30 induced the outgrowth of neurites, triggered cell cycle arrest in G0/G1 phase and enhanced the expression of growth associated protein-43. Furthermore, M-30 markedly reduced the levels of cellular APP and β-C-terminal fragment (β-CTF) and the levels of the amyloidogenic Aβ peptide in the medium of SH-SY5Y cells and Chinese hamster ovary cells stably transfected with the APP ‘Swedish’ mutation. Levels of the non-amyloidogenic soluble APPα and α-CTF in the medium and cell lysate respectively were coordinately increased. These properties, together with its brain selective MAO inhibitory and propargylamine- dependent neuroprotective effects, suggest that M-30 might serve as an ideal drug for neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases, in which oxidative stress and iron dysregulation have been implicated.

Abbreviations used

β-amyloid peptide

AD

Alzheimer's disease

APP

amyloid precursor protein

BDNF

brain-derived neurotrophic factor

CHO

Chinese hamster ovary

CTF

C-terminal fragment

DAPI

4,6-diamidino-2-phenylindole

DFO

desferrioxamine

DMEM

Dulbecco's modified Eagle's medium

FCS

fetal calf serum

FullS

full serum medium

GAP-43

growth-associated protein-43

GDNF

glial cell line-derived neurotrophic factor

HIF

hypoxia inducible factor

MAO

monoamine oxidase

MPTP

1-methyl-4-phenyl-1,2,5,6-tetrahydropyridinium

PKC

protein kinase C

ROS

reactive oxygen species

sAPPα

non-amyloidogenic soluble APP

SFree

serum-free medium

TMR

tetre-methyl-rhodamine

TUNEL

terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick end labeling

There is a significant body of evidence to support the hypothesis that brain metal (such as zinc, copper and iron) dysregulation and oxidative stress, resulting in reactive oxygen species (ROS) generation from H2O2 and inflammatory processes, trigger a cascade of events leading to apoptotic/necrotic cell death in neurodegenerative disorders, such as Parkinson's disease, Alzheimer's disease (AD), Huntington's disease and amyotrophic lateral sclerosis (Gerlach et al. 1994; Deibel et al. 1996; Zecca et al. 2004; Squitti et al. 2006). There is also evidence for increased expression of apoptotic proteins (Blum et al. 2001), as well as mitochondria (complex I) and ubiquitin–proteasome system dysfunction, which may lead to breakdown of energy metabolism and subsequent intraneuronal calcium overload (Olanow and Youdim 1996; Mandel et al. 2000; Linazasoro 2002; McNaught et al. 2002). Thus, neurodegenerative diseases appear to be multifactorial, whereby a complex set of reactions act independently or cooperatively, leading eventually to the demise of neurons. This has led to the current notion that drugs directed against a single target will be ineffective and rather a single drug or a cocktail of drugs with pluripharmacological properties may be more suitable (Grunblatt et al. 2004; Youdim and Buccafusco 2005; Van der Schyf et al. 2006). Thus, we have recently designed and synthesized a series of non-toxic multifunctional brain permeable iron chelator drugs derived from our prototype chelator, VK-28; these possess the monoamine oxidase (MAO) A and B inhibitory and neuroprotective properties of the propargyl moiety of rasagiline and have been developed for the treatment of various neurodegenerative disorders (Ben-Shachar et al. 2004; Gal et al. 2005; Zheng et al. 2005a, 2005b). Among these novel multifunctional iron chelators, M-30 and HLA-20 (Fig. 1) were the most effective drugs, with iron chelation potency, radical scavenging and iron-induced membrane lipid peroxidation inhibitory potencies close to those of the prototype iron chelator desferrioxamine (DFO) (Zheng et al. 2005b).

Figure 1.

 Chemical structures of the novel iron chelators M-30, HLA-20 and VK-28.

Indeed, chelation has the potential to prevent iron- induced ROS, oxidative stress and aggregation of α-synuclein and β-amyloid peptide (Aβ) (Zecca et al. 2004). The limited neuroprotective studies carried out so far indicate that iron chelation therapy could be a viable neuroprotective approach for neurodegenerative disorders. Thus, the neuroprotective activity of the antibiotic iron chelator 5-chloro-7-iodo-8-hydroxyquinoline (clioquinol) in preventing iron- and 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridinium (MPTP)- induced neurotoxicity in mice has been reported (Kaur et al. 2003). In addition, an early study indicated that sustained intramuscular administration of DFO slowed the clinical progression of AD dementia (Crapper McLachlan et al. 1991). Treatment with clioquinol markedly and rapidly inhibited Aβ accumulation in an AD transgenic mouse model via its action as a metal chelator (Cherny et al. 2001). Unfortunately, clioquinol is highly toxic and DFO has poor penetration across the blood–brain barrier. However, our recent studies have described a significant neuroprotective action of VK-28 against 6- hydroxydopamine lesion in rats (Ben-Shachar et al. 2004) and shown that M-30 has neuroprotective activity against MPTP neurotoxicity in mice (Gal et al. 2005).

The propargylamine moiety of the multifunctional drugs has been found to exert neuroprotective activity against apoptosis induced by serum withdrawal and the endogenous neurotoxin N-methyl(R)salsolinol (Maruyama et al. 2001b; Weinreb et al. 2004). The neuroprotective mechanism involves activation of protein kinase C (PKC) α and ε, and is associated with regulation of the Bcl-2 family of proteins and mitochondrial membrane stabilization (Bar-Am et al. 2004; Youdim et al. 2005a).

Here we have studied the following pharmacological activities of the multifunctional brain permeable iron chelator M-30 and its derivatives: (i) pro-survival neurorescue action, (ii) ability to promote neuronal differentiation and neuronal outgrowth, and (iii) regulatory action on the Alzheimer's amyloid precursor protein (APP) and its toxic amyloidogenic derivative, Aβ, in relation to the non-amyloidogenic soluble APP (sAPPα).

Materials and methods

Materials

Tissue culture reagents were from Beit-Haemek (Kibbutz Beit- Haemek, Israel). Antibodies against Bad, caspase 3 and Bax were from Cell Signaling (Beverly, MA, USA), Bcl-2 antibody was from BD Biosciences Transduction Laboratories (Heidelberg, Germany), and antibody against growth-associated protein-43 (GAP-43) and monoclonal anti-β-amyloid [1–17] (6E10) were purchased from Chemicon (Temecula, CA, USA). Anti-βAPP C-terminal (amino acids 676–695) and β-actin antibody were from Sigma (St Louis, MO, USA). H2A.X was from Upstate (New York, USA) and monoclonal anti-βAPP (1–695) 22C11 was from Boehringer Mannheim (Mannheim, Germany). Donkey anti-rabbit IgG fluorescein-conjugated antibody was from Jackson ImmunoResearch laboratories Inc. (Baltimore, MD, USA). DRAQ5TM was from Alexis (San Diego, CA, USA). Electrophoresis reagents were obtained from Invitrogen (Carlsbad, CA, USA). Other chemicals and reagents were of the highest analytical grade and were purchased from local commercial sources. The iron chelators M-30 (5-[N-methyl-N-propargylaminomethyl]-8-hydroxyquinoline) (MW 299.3), HLA-20 (5-[4-propargylpiperazin-1-ylmethyl]-8-hydroxyquinoline) (MW 390.9) and VK-28 (5-[4-(2-hydroxyethyl) piperazine-1-ylmethyl]-quinoline-8-ol) (MW 396.9) (Fig. 1) were synthesized (Zheng et al. 2005b) and kindly provided by Varinel Inc. (Philadelphia, PA USA). Previous studies demonstrated that the novel chelators had higher binding selectivity for iron over copper (Zheng et al. 2005a, 2005b).

Cell culture and experimental procedures

Human SH-SY5Y neuroblastoma cells were plated in 100-mm culture dishes and cultured in Dulbecco's modified Eagle's medium (DMEM) (4500 mg/L glucose), containing 10% fetal calf serum (FCS) and 1% of a mixture of penicillin/streptomycin/nystatin. A neurorescue model was set up as described recently (Bar-Am et al. 2005). In brief, when cells reached the required confluence, they were placed into serum-free medium (SFree) for 3 days and then incubated in the absence or presence of the drugs for additional 2 days and assayed as indicated. Cells placed in full serum (FullS) served as control. Rat pheochromocytoma (PC12) cells were maintained in T75 flasks in DMEM (1000 mg/L glucose) and supplemented with 5% fetal calf serum, 10% horse serum and a mixture of 1% penicillin/streptomycin/nystatin. For differentiation studies, PC12 cells were plated in six-well culture dishes or in T25 flasks in DMEM supplemented with 3% growth sera and allowed to attach for 24 h. Subsequently, cells were incubated in the absence or presence of the novel iron chelators for an additional 2 days. Chinese hamster ovary (CHO) cells were stably transfected with human APP695 containing the ‘Swedish’ mutation (APPsw, Lsy670/Asn, Met671/Leu) (CHO/ΔNL) cloned into pcDNA3 by CaPO4 transfection, as described previously (Perez et al. 1996) (generously donated by Todd E. Golde, Mayo Clinic Jacksonville, Jacksonville, FL, USA). CHO/ΔNL cells were cultured in T75 flasks, containing Ham's F12 medium supplemented with 10% FCS and 1% of a mixture of penicillin/streptomycin/nystatin and hygromycin (800 µg/mL) (in order to preserve selection for the transfection). Cell lines were incubated at 37°C in humid 5% CO2−95% air. The dose selection of the drugs was based on our recent study of their various properties related to iron affinity, radical scavenging and cell viability in various cell lines and in vivo (Maruyama et al. 2001b; Youdim et al. 2004, 2005b; Gal et al. 2005; Zheng et al. 2005b).

In situ DNA fragmentation was performed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick end labeling (TUNEL) technique according to the manufacturer's instructions (Roche Molecular Biochemicals, Indianopolis, IN, USA) and as described recently (Reznichenko et al. 2005).

Western blotting and immunoprecipitation

Cells were harvested, and lysed in 1 × non-reducing Laemmli lysis buffer (150 mm NaCl, 50 mm Tris-HCl, pH 8.0, 5 mm EDTA, 1% Triton X-100 and protease- and phosphatase-inhibitor cocktails) for 10 min followed by boiling for 3–5 min. Protein content was determined using the Bradford method (Bio-Rad, Hercules, CA, USA). Equal amounts of protein from cell lysates were electrophoretically separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and processed by standard methods (Yogev-Falach et al. 2003).

β-C-terminal fragment (β-CTF) was detected in cell lysates by immunoprecipitation with monoclonal antibody 6E10 that recognizes an epitope within residues 1–17 of the Aβ domain of APP (a site that constitutes the C-terminus of sAPP cleaved at the α site), followed by western blotting with anti-APP raised against APP C-terminal. Aβ and sAPPα were analyzed in the medium by immnoprecipitation with antibody 6E10 followed by western blotting with antibody 6E10. α-CTF levels were assayed in cell lysate by western blotting with anti-APP C-terminal antibody.

Detection of neurite outgrowth

Cells were plated at low density (0.35 × 104 cells/coverslip) in CC2 four-well tissue chamber slides. The differentiation effect was demonstrated by tracking the expression of a biological cellular marker of differentiation, GAP-43, by immunofluorescence. Cells were visualized using a 60 × objective (NA 1.4) and a Radiance 2000 confocal system as described previously (Reznichenko et al. 2005). The number of differentiated cells was determined by counting cells that had at least one neurite with a length equal to or greater than the diameter of the cell body. Unless otherwise indicated, a minimum of 200 cells were examined in a blinded manner and results are presented as mean ± SEM. Similar results were obtained in two independent experiments.

Flow cytometry analysis of nuclear DNA content

Cells were detached with cold phosphate-buffered saline following centrifugation at 200 g for 5 min. Pellets were gently suspended in a hypotonic fluorochrome solution [50 mg/mL propidium iodide in 0.1% (w/v) sodium citrate plus 0.01% (v/v) Nonidet P-40 and 10 mg/mL Dnase-free Rnase in bi-distilled water]. After incubation in the dark at 37°C in 5% CO2−95% air for 30 min, cell samples were stored on ice and analyzed for DNA content on a logarithmic scale by FACScalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) with Cell Quest research software; 3 × 104 events per sample were acquired and the percentage of cells in the G0/G1, S and G2 fractions were determined. (Chelli et al. 2004).

Statistical analysis

All values are expressed as the mean ± SEM. For statistical analysis one-way anova followed by Student's t-test was performed. A value of p < 0.05 was considered significant. Each experiment was repeated three to four times and results from a representative experiment are shown.

Results

Neurorescue effect of the novel iron chelator M-30 against long-term serum withdrawal-induced neuronal death

Serum deprivation significantly increases production of ROS and triggers apoptosis of neuronal cells (Satoh et al. 1996). The present study evaluated the neurorescue effects of M-30 in an extreme apoptotic model of human SH-SY5Y neuroblastoma cells, following prolonged serum deprivation (culture in SFree). In this model system, SH-SY5Y cells exposed to long-term serum deprivation exhibited a significant increase in apoptotic cells compared with cells grown in FullS, as assessed by TUNEL staining. M-30 at a concentration of 5 µm caused a marked decrease in apoptotic cells compared with cells cultured in SFree (Fig. 2a). In addition, an increase in the levels of phosphorylated histone H2A.X, a marker of apoptosis (Paull et al. 2000), was seen in response to long-term serum deprivation (2.17 ± 0.02 fold vs. FullS control; p < 0.001). Treatment of serum-deprived SH-SY5Y cells with increasing concentrations of M-30 resulted in a significant, dose-dependent decrease in the levels of phosphorylated histone H2A.X (Fig. 2b).

Figure 2.

 Neurorescue effect of M-30 against long-term serum deprivation. SH-SY5Y cells were subjected to a prolonged serum deprivation (SFree) stress and then incubated with or without M-30. (a) Apoptotic nuclei were identified by TUNEL analysis. Arrows indicate apoptotic cells. Absolute values of 5–10 separate fields were averaged and apoptotic cells expressed as a mean ± SEM percentage of total cells. (DAPI), 4,6-diamidino-2-phenylindole; TMR, tetra-methyl-rhodamine; †††p < 0.001 versus FullS; ***p < 0.001 versus SFree. (b) Phosphorylated H2A.X levels in cell lysates were examined by immunoblotting analysis. Loading of the lanes was determined by examining levels of β-actin.

To further determine the anti-apoptotic activity of M-30, the levels of activated caspase 3 were quantified by western blotting. Long-term exposure of SH-SY5Y cells to SFree significantly increased the levels of cleaved caspase 3 by ∼ 6 fold compared with levels in cells cultured in FullS (Fig. 3a). In the presence of M-30 (0.01–10 µm), there was a dose-dependent inhibition of cleaved caspase 3 appearance (Fig. 3b).

Figure 3.

 Effect of M-30 on caspase 3 activation. SH-SY5Y cells were placed into SFree for 3 days and then incubated with or without various concentration of M-30 for an additional 2 days. Cells incubated in FullS were used as a control. Levels of cleaved caspase 3 were measured in cell lysates and normalized with respect to levels of β-actin. Quantitative values are mean ± SEM (n = 3). *p < 0.05, **p < 0.01 versus SFree.

Effect of M-30 on Bcl-2 family proteins

Considering the importance of Bcl-2 family proteins in the regulation of the programmed cell death pathway, we examined whether M-30 has any effect on the levels of the ‘BH-3 only’ pro-apoptotic protein Bad, which is particularly associated with the response to withdrawal of survival factors (Ward et al. 2004). Long-term serum deprivation of SH-SY5Y culture resulted in increased levels of Bad (Fig. 4a). M-30 (0.5–10 µm) significantly decreased Bad levels by ∼ 80% compared with levels in SFree cultures (Fig. 4b). In addition, M-30 increased the levels of Bcl-2 and conversely decreased Bax expression (data not shown). Thus, long-term serum deprivation markedly reduced the Bcl-2 to Bax ratio (by 80% of that in FullS culture), whereas treatment with M-30 (5 µm) markedly increased this ratio (∼ 3 fold vs. that in SFree). These results indicate that the neurorescue action of M-30 following long-term serum deprivation-induced apoptosis is associated with a reduction in levels of the pro-apoptotic protein Bad, and modulation of the balance between Bcl-2 and Bax.

Figure 4.

 M-30 reduced Bad levels. Protein levels of Bad were analyzed by western blotting in the neurorescue model of SH-SY5Y neuroblastoma cells following M-30 treatment, as described in Fig. 3. Levels of Bad were normalized with respect to levels of β-actin. Quantitative values are mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 versus SFree.

M-30, HLA-20 and VK-28 induce neurite outgrowth and regulate the cell cycle

As iron is essential for cell differentiation (Thelander and Reichard 1979; Witt et al. 1979; Takeda and Weber 1981; Crichton and Ward 1992), we investigated whether the iron chelators M-30 and HLA-20 (Fig. 1) have ability to induce neurite outgrowth in SH-SY5Y and PC12 cell lines, which have been extensively used as models for neuronal differentiation. M-30 treatment (1–10 µm) induced neuronal differentiation of SH-SY5Y cells, accompanied by a significant increase in the expression of the neuronal specific axonal marker of differentiation, GAP-43, as determined by immunofluorescence (Fig. 5a) and western blot analysis (3.04 ± 0.42 fold at 5 µm M-30; p < 0.05 vs. control) (Fig. 5b).

Figure 5.

 Effect of M-30 on SH-SY5Y cell differentiation. SH-SY5Y cells were incubated without (control) or with various concentrations of M-30 for 2 days. (a) After cell fixation and permeabilization, GAP-43 was detected by confocal microscopy, using specific primary antibody and FITC-conjugated secondary antibody (green). The cell- permeant DNA probe DRAQ5 stains nuclei (blue) (Bio-Rad Radiance 2000 confocal system using a 40 × objective, supported with Laser-Sharp 2000 software) (Smith et al. 2000). (b) GAP-43 expression in cell lysates was examined by immunoblotting analysis. Loading of the lanes was determined by examining levels of β-actin.

Similarly, as demonstrated in Figs 6(a) and (b), both M-30 (5 and 10 µm) and HLA-20 (5 and 10 µm) stimulated neurite extension in PC12 cells. Immunocytochemistry showed that GAP-43 immunoreactivity was much stronger in drug-differentiated PC12 cells than in untreated control PC12 cells (Fig. 6b), in accordance with the results of the western blot analysis of GAP-43 (Fig. 6c). GAP-43 protein in M-30- and HLA-20- differentiated PC12 cells was intensely distributed in the neuritic processes (Fig. 6b).

Figure 6.

 Effect of M-30 and HLA-20 on PC12 cell differentiation. PC12 cells were incubated without (control) or with M-30 (1–10 µm) or HLA-20 (1–10 µm) for 2 days. (a) Micrographs showing the overgrowth of neurites capture by an inverted microscope connected to a digital camera (20 × objective). (b) GAP-43 was detected by confocal microscopy, as described in Fig. 5. The number of differentiated cells was determined by counting cells with at least one neurite with a length equal to or greater than the diameter of the cell body; 6–12 fields were counted for each treatment sample. Results are expressed as the percentage of differentiated cells detected per sample in three independent experiments mean ± SEM. *p < 0.05 versus control; †p < 0.05 versus 5 µm M-30. (c) GAP-43 expression in PC12 cell lysates was examined by immunoblotting analysis. Loading of the lanes was determined by examining levels of β-actin.

We next examined the effect of M-30 and HLA-20 on the PC12 cell cycle by flow cytometry. M-30 (5 and 10 µm) increased the number of cells in G0/G1 from 50.7 to 58.7 and 74.6% respectively, and decreased the number in S phase from 26.4 to 22.6 and 12.2% respectively. The proportion of cells in the G2 phase was decreased from 16.4 to 12.9 and 10.8% respectively (Fig. 7 and Table 1). Treatment with HLA-20 (5 and 10 µm) had similar effects on the PC12 cell cycle (Fig. 7 and Table 1), indicating that both M-30 and HLA-20 inhibited the progress of PC12 beyond the G0/G1 phase.

Figure 7.

 Effect of M-30 and HLA-20 on the cell cycle of PC12 cells. PC12 cells were incubated without (control) or with M-30 (5 and 10 µm) or HLA-20 (5 and 10 µm) for 2 days and analyzed by fluorescence-activated flow cytometry. Results show representative histograms and the percentage of cells at different phases of the cell cycle are shown in Table 1. Values are mean ± SEM from three independent experiments. *p < 0.05, **p < 0.01 versus control.

Table 1. 
G2 (%)S (%)G0/G1 (%) 
16.4 ± 1.126.4 ± 0.950.7 ± 1.5Control
12.9 ± 0.4**22.6 ± 0.9**58.7 ± 2.1*M-30 (5 μM)
10.8 ± 1.2*12.2 ± 0.6**74.6 ± 1.4*M-30 (10 μM)
10.7 ± 0.7**15.8 ± 1.0**68.7 ± 0.9**HLA-20 (5 μM)
11.3 ± 1.5*13.8 ± 0.7**73.1 ± 2.2*HLA-20 (10 μM)

Based on these findings, we next focused on the effects of the prototype iron chelator, VK-28, a derivative of the drugs M-30 and HLA-20 (Fig. 1). Figure 8(a) demonstrates that, similar to M-30 and HLA-20, VK-28 (5 and 10 µm) induced the differentiation of PC12 cells, and triggered cell cycle arrest in G0/G1 (Fig. 8b and Table 2).

Figure 8.

 Effect of VK-28 on PC12 differentiation and the cell cycle. PC12 cells were incubated without (control) or with VK-28 (5 and 10 µm) for 2 days. (a) Micrographs showing the outgrowth of neurites from PC12 cells, using an inverted microscope connected to a digital camera (20 × objective). (b) Flow cytometric analysis of untreated PC12 cells and PC12 cells treated with VK-28 (5 and 10 µm). Representative histograms are shown and the percentage of cells at different phases of the cell cycle is given in Table 2. Values are mean ± SEM from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 versus control.

Table 2. 
G2 (%)S (%)G0/G1 (%) 
17.2 ± 1.424.6 ± 1.149.7 ± 0.7Control
14.5 ± 0.623.2 ± 0.454.1 ± 0.7*VK-28 (5 μM)
10.8 ± 0.5*13.6 ± 0.5**72.7 ± 0.2***VK-28 (10 μM)

Effect of M-30 on APP regulation/processing

Because the 5′ untranslated region of APP mRNA has been shown to have a functional iron-responsive element (Rogers et al. 2002a), we examined whether M-30 has a regulatory effect on holo-APP levels. Holo-APP levels were evaluated by western blot analysis, using the 22C11 antibody that recognizes an epitope located between amino acids 60 and 100 in the N- terminal part of the ectodomain of human APP. Figure 9 shows that M-30 (5 and 10 µm) decreased holo-APP levels in cell lysates of SH-SY5Y cells compared with levels in untreated controls. A similar response was obtained with HLA-20 and VK-28 (data not shown).

Figure 9.

 M-30 decreased holo-APP levels. SH-SY5Y cells were incubated without (control) or with M-30 for 2 days. Cellular holo- APP was measured in cell lysates by immunoblotting, using monoclonal antibody 22C11. Levels of holo-APP were normalized with respect to levels of β-actin. Values are mean ± SEM (n = 3). *p < 0.05 versus control.

The regulatory effect of M-30 on amyloidogenic peptides was further analyzed in well characterized CHO cells, stably transfected with the APP ‘Swedish’ mutation (CHO/ΔNL), as Aβ levels in the medium of SH-SY5Y cells were undetectable whereas CHO/ΔNL cells generate high basal levels of Aβ (Citron et al. 1992; Cai et al. 1993; Shaw et al. 2001). M-30 reduced Aβ generation in the medium of CHO/ΔNL cells (Fig. 10a). Consistent with the decrease in Aβ levels, there was a significant decrease in the cellular β-CTF level (∼12 kDa) upon treatment of CHO/ΔNL cells with M-30, compared with the control level (Fig. 10b). These results complemented the decrease in Aβ levels in the medium.

Figure 10.

 M-30 reduced Aβ secretion and cellular β-CTF levels. CHO/ΔNL cells were incubated without (control) or with M-30 for 2 days. (a) Aβ in the medium was detected by immunoprecipitation and western blotting with monoclonal antibody 6E10. (b) The levels of cellular β-CTF were examined by immunoprecipitation with monoclonal antibody 6E10 followed by western blotting with APP C-terminal antibody. Values are mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 versus control.

Because the non-amyloidogenic processing of APP can preclude the production of Aβ (De Strooper and Annaert 2000) we examined whether the decrease in the levels of Aβ following M-30 treatment was a result of increased sAPPα. In keeping with reduced Aβ generation, treatment of CHO/ΔNL cells with M-30 (5 and 10 µm) resulted in increased sAPPα release into the conditioned medium (Fig. 11a). Moreover, M-30 induced cellular α-CTF levels (∼ 2-fold at 10 µm M-30 vs. control) (Fig. 11b), complementary to the increase in sAPPα.

Figure 11.

 M-30 increased sAPPα secretion and cellular α-CTF levels. CHO/ΔNL cells were incubated without (control) or with M-30 for 2 days. (a) sAPPα levels in the medium were analyzed by immunoprecipitation and western blotting with 6E10.  (b) Lysates were analyzed for α-CTF generation by immunoblotting with APP C-terminal antibody. Values are mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 versus control.

Discussion

New therapeutic strategies for the treatment of neurodegenerative disorders comprise multifunctional drug candidates designed specifically to act on multiple CNS targets (Youdim and Buccafusco 2005; Van der Schyf et al. 2006). We have recently developed several non-toxic, lipophilic brain permeable multifunctional drugs from the pharmacophore of the iron chelator, VK-28. Among these, M-30, which possesses propargyl, has been chosen as a potential therapeutic drug for neurodegenerative diseases (Gal et al. 2005). The present study demonstrates that M-30 has a wide range of pharmacological activities, including a neurorescue response, and effects on neuronal differentiation and APP/Aβ regulation.

The neurorescue potency of M-30 was identified by the ability of the drug to augment SH-SY5Y cell viability even though it was administered following an extreme apoptotic damage of 3 days' serum deprivation. These results are consistent with previous reports indicating that capture of the iron labile pool, either by chelators such as DFO or by enhanced levels of the iron storage protein ferritin, significantly decreases cell death arising from several types of oxidant challenge (Balla et al. 1992; Zdolsek et al. 1993; Ollinger and Brunk 1995; Persson et al. 2001). Thus, the neurorescue effect of M-30 may be attributed to a decrease in intracellular iron concentration potentially associated with a reduction in ROS production, as increased ROS generation was found to mediate serum withdrawal-induced neuronal death by regulating cellular iron homeostasis (Pantopoulos and Hentze 1995; Pantopoulos et al. 1997; Gehring et al. 1999). However, previous findings have supported the hypothesis that iron chelators not only prevent neuronal injury by inhibiting hydroxyl radical formation via the Fenton reaction, but also by inhibition of the iron-dependent hypoxia inducible factor (HIF) prolyl 4-hydroxylase that regulates HIF stability. In this scheme, iron chelators would stabilize HIF-α and transactivate the expression of established protective genes including those coding for vascular endothelial growth factor and erythropoietin (Zaman et al. 1999; Siddiq et al. 2005).

Here we have shown that M-30 decreases apoptosis via multiple protection mechanisms, including inhibition of caspase 3 cleavage and prevention of Ser139 phosphorylation of H2A.X. Moreover, the mechanism of neurorescue associated with M-30 involved induction of the pro-survival protein Bcl-2 and reduction of the levels of the pro-apoptotic members Bad and Bax. Consistent with the neurorescue effects of M-30, our previous studies showed the neurorescue/neuroprotective activity of the N-propargylamine moiety and related propargylamines, such as rasagiline and ladostigil, against cell death induced by a variety of insults [e.g. serum withdrawal and the neurotoxins N-morpholino sydnonimine and N-methyl(R)salsolinol] (Maruyama et al. 2002; Bar-Am et al. 2004; Weinreb et al. 2004; Youdim et al. 2005a). The neuroprotection by propargylamine derivatives was attributed to: (i) stabilization of the mitochondrial membrane potential (ΔΨm) and prevention of permeability transition pore (PTP) (Maruyama et al. 2001a); (ii) induction of anti-apoptotic Bcl-2 protein regulating permeability transition (Akao et al. 2002); (iii) the increase in brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) (Weinreb et al. 2004; Bar-Am et al. 2005); and (iv) activation of antioxidant enzymes, such as superoxide dismutase and catalase (Carrillo et al. 2000). It was shown recently that propargylamines induced neuroprotection via regulation of the pro-survival PKC pathway in association with gene regulation of the Bcl-2-related protein family (Weinreb et al. 2004).

The second finding of this study was the ability of M-30 to induce neuronal differentiation and negatively regulate cell cycle progression. Similar effects were also found for the multifunctional iron chelator drug HLA-20, which acts as a weak MAO inhibitor (Zheng et al. 2005a) and for the iron chelator prototype derivative VK-28. All three drugs were shown to induce the characteristics of neuronal differentiation, including cell body elongation, stimulation of neurite outgrowth, arrest of the cell cycle in G0/G1 and specific up-regulation of the expression of the neuronal marker GAP-43. These findings are in accordance with results of previous studies suggesting that the cell cycle-blocking activity of iron chelators triggers the process of differentiation through various iron-associated biological events (Lederman et al. 1984; Bergeron and Ingeno 1987; Tomoyasu et al. 1993; Renton and Jeitner 1996; Tanaka et al. 1999). Indeed, many cell cycle- regulating factors require iron ions for their function (Rubin et al. 1993; Evans et al. 1995) and it was reported that preincubation of iron chelators with Fe3+ ions abolished their differentiation induction activity (Tanaka et al. 1995; Tanaka et al. 1997). A recent study showed that DFO induced leukemic cell differentiation potentially by HIF-1α (Jiang et al. 2005). In addition, the effect of the multifunctional drugs M-30 and HLA-20 on neuronal differentiation may be associated with the propargyl moiety, because N-propargylamine and rasagiline increase the expression of neuronal growth factor, and induce mRNAs of GDNF and BDNF (Maruyama et al. 2004; Weinreb et al. 2004; Bar-Am et al. 2005). Thus, these multifunctional drugs might be of particular benefit in the treatment of neurological conditions that involve loss of synaptic connections and neuronal function through their neurotrophic activity.

Finally, our data have demonstrated the regulatory effect of M-30 on APP expression/processing, resulting in reduced APP expression and Aβ generation. The finding that M-30 markedly suppressed APP holo-protein levels is consistent with previous studies showing that other metal chelators (such as DFO, dimercaptopropanol or EGCG) significantly reduced APP levels by modulating APP translation via an IRE in the 5′ untranslated region of the APP transcript (Rogers et al. 2002b; Payton et al. 2003; Rogers and Lahiri 2004; Reznichenko et al. 2006). Indeed, M-30 was found to suppress the translation of a luciferase reporter gene fused to the APP mRNA 5′ untranslated region (maximal inhibition of 33.7 ± 8.3% at 100 µm vs. control).

Most importantly, M-30 markedly lowered secreted levels of Aβ peptide in the conditioned medium and cellular β-CTF in CHO/ΔNL cells (stably transfected with the APP ‘Swedish’ mutation). Moreover, M-30 was found to activate the non-amyloidogenic pathway of APP processing, increasing the amount of secreted sAPPα derivative, as shown previously for propargyl-containing compounds (Yogev-Falach et al. 2002). This mechanism involved PKC and a mitogen-activated protein kinase-dependent signaling pathway. Thus, the reduction in Aβ levels in response to M-30 may result from activation of the non-amyloidogenic pathway of APP processing, as up-regulation of α-secretase cleavage not only increases the secretion of the neuroprotective sAPPα (Mattson 1997; De Strooper and Annaert 2000), but also reciprocally decreases the production of the neurotoxic Aβ peptide (Checler 1995). In vivo, a disintegrin metalloproteinase has been reported to prevent amyloid plaque formation and hippocampal defects in an AD mouse model (Postina et al. 2004). Attenuation of Aβ production by the novel iron chelator drugs could be of therapeutic value for AD therapy, as increased generation of Aβ plays a central role in AD plaque formation (Cuajungco et al. 2005).

In summary, the multifunctional brain permeable drug M-30, which has propargyl MAO inhibitory and iron-chelating moieties, was found to induce neurorescue activity, promote neurite outgrowth, and reduce holo-APP expression and Aβ generation. It is apparent that the compound targets a number of pharmacological sites involved in neurodegeneration processes and thus might serve as potential neuroprotective/neurorescue drug for the treatment of various neurodegenerative diseases.

Acknowledgements

We are grateful to Institute for the Study of Aging (New York, USA) and Technion-Research and Development (Haifa, Israel) for the support of this work.

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