Neuropharmacology 62 (2012) 997e1003 Contents lists available at SciVerse ScienceDirect Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm Cystamine-tacrine dimer: A new multi-target-directed ligand as potential therapeutic agent for Alzheimer’s disease treatment A. Minarini a, *, A. Milelli a, V. Tumiatti a, M. Rosini a, E. Simoni a, M.L. Bolognesi a, V. Andrisano a, M. Bartolini a, E. Motori b, C. Angeloni b, S. Hrelia b a b Department of Pharmaceutical Sciences, Alma Mater Studiorum, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy Department of Biochemistry “G. Moruzzi”, Alma Mater Studiorum, University of Bologna, Via Irnerio 48, 40126 Bologna, Italy a r t i c l e i n f o a b s t r a c t Article history: Received 5 September 2011 Received in revised form 30 September 2011 Accepted 11 October 2011 Alzheimer’s disease (AD) is the most common cause of dementia, clinically characterized by loss of memory and progressive deficits in different cognitive domains. An emerging disease-modifying approach to face the multifactorial nature of AD may be represented by the development of Multi-Target Directed Ligands (MTDLs), i.e., single compounds which may simultaneously modulate different targets involved in the neurodegenerative AD cascade. The structure of tacrine, an acetylcholinesterase (AChE) inhibitor (AChEI), has been widely used as scaffold to provide new MTDLs. In particular, its homodimer bis(7)tacrine represents an interesting lead compound to design novel MTDLs. Thus, in the search of new rationally designed MTDLs against AD, we replaced the heptamethylene linker of bis(7)tacrine with the structure of cystamine, leading to cystamine-tacrine dimer. In this study we demonstrated that the cystamine-tacrine dimer is endowed with a lower toxicity in comparison to bis(7)tacrine, it is able to inhibit AChE, butyrylcholinesterase (BChE), self- and AChE-induced beta-amyloid aggregation in the same range of the reference compound and exerts a neuroprotective action on SH-SY5Y cell line against H2O2-induced oxidative injury. The investigation of the mechanism of neuroprotection showed that the cystaminetacrine dimer acts by activating kinase 1 and 2 (ERK1/2) and Akt/protein kinase B (PKB) pathways. This article is part of a Special Issue entitled ‘Post-Traumatic Stress Disorder’. Ó 2011 Elsevier Ltd. All rights reserved. Keywords: Tacrine Bis(7)tacrine Alzheimer’s disease MTDL Cystamine-tacrine dimer Oxidative injury 1. Introduction Alzheimer’s disease (AD) is the most common cause of dementia affecting about 6% of the population aged over 65 and its incidence increases with age (Burns and Iliffe, 2009). AD is clinically characterized by memory impairment and progressive deficits in different cognitive domains related to a pronounced degradation of the cholinergic system and to alteration in other neurotransmitter systems such as the glutamatergic and serotoninergic ones (Toledano-Gasca, 1988). From a neuropathological point of view, the hallmarks of AD are represented by formation of senile plaques, which are insoluble deposits mainly of beta-amyloid (Ab) protein derived from the cleavage of a precursor protein APP by enzymes such as b- and g-secretase, and neurofibrillary tangles (NFT) composed of hyperphosphorylated tau protein. Much research effort has been devoted to elucidating the relationships between the hallmarks of this multifactorial disease and the loss of neurons in the hippocampus and nucleus basalis of Maynart (Hardy, 2006). * Corresponding author. Tel.: þ39 512099709; fax: þ39 51247600. E-mail address: anna.minarini@unibo.it (A. Minarini). 0028-3908/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2011.10.007 However, many aspects of etiology and pathological pathways of AD remain unclear and speculated about (Small and Duff, 2008). These pathological lesions have been considered to be the causative features of AD, giving rise to several theories about AD pathogenesis mainly including the cholinergic hypothesis (Terry and Buccafusco, 2003), the amyloid cascade hypothesis (Hardy, 2009), oxidative stress and free radical formation (Gella and Durany, 2009), depicting a more intriguing scenario. Indeed, nowadays the AD therapy is mainly bolstered on acetylcholinesterase (AChE) inhibitors (AChEIs) able to increase the acetylcholine (ACh) levels in the cholinergic synapses (Gura, 2008), and their clinical effectiveness is still under debate (Cummings et al., 2007; Munoz-Torrero, 2008). A more appropriate approach to face the multifactorial nature of AD may be represented by the development of Multi-Target Directed Ligands (MTDLs), which is based on the assumption that a single compound may simultaneously modulate different targets involved in the neurodegenerative AD cascade (Cavalli et al., 2008; Van der Schyf et al., 2007). This strategy led recently to the discovery of several anti-AD drug candidates (Bolognesi et al., 2009; Tumiatti et al., 2008; Van der Schyf and Youdim, 2009). 998 A. Minarini et al. / Neuropharmacology 62 (2012) 997e1003 The structure of tacrine, one of the most known AChEIs (Summers, 2006), has been widely used as scaffold to provide new MTDLs endowed with additional biological properties beyond simple AChE inhibition (Munoz-Torrero, 2008; Tumiatti et al., 2010). Its dimer bis(7)tacrine, also called bis(7)cognitin (Pang et al., 1996), exhibited a 1000-time higher AChE inhibition potency, a double interaction with active and peripheral sites of AChE and a better pharmacological profile consisting in the inhibition of the AChE-induced Ab aggregation through the interaction with its peripheral binding site (PAS) (Inestrosa et al., 1996), and in neuroprotective effects related to the interaction with b-secretase enzyme and NMDA and GABAA receptors. (Fang et al., 2010; Fu et al., 2009; Li et al., 2009). Thanks to these findings and to the renewed interest in dual binding AChEIs as modulators of amyloid neuropathology (Castro and Martinez, 2006; Inestrosa et al., 2008; Munoz-Torrero, 2008), bis(7)tacrine may represent an interesting lead compound to design novel MTDLs. In fact, a lot of chemical modifications were performed on bis(7)tacrine structure, in particular on its heptamethylene bridge, aimed at increasing the AChE inhibition (Butini et al., 2008) and/or at widening its biological spectrum activities towards other important AD targets (Bolognesi et al., 2010, 2007b). In this context, we focused on the biological properties of cystamine for its important activities as antioxidant, cyto- and neuroprotective agent (Wood et al., 2007). In particular, systemic administration of cystamine was reported to diminish neural toxicity associated with different toxins (Fox et al., 2004; Stack et al., 2008; Tremblay et al., 2006) and to protect against neurodegeneration (Dedeoglu et al., 2002; Fox et al., 2004). Thus, in the search of new rationally designed MTDLs against AD, we replaced the heptamethylene linker of bis(7) tacrine with the structure of cystamine, leading to cystaminetacrine dimer characterized by a disulfide bridge (Fig. 1). To characterize the “in vitro” biological profile of the cystaminetacrine dimer the ability of this newly synthesized compound to inhibit human AChE and butyrylcholinesterase (BChE), the self- and AChE-induced Ab aggregation was evaluated, using bis(7)tacrine as reference compound. Moreover, the ability of this dimer to counteract the possible damage deriving from oxidative stress was determined. Finally, since kinases have been implicated in the transduction of signal in neurodegenerative disorders (Greggio and Singleton, 2007) the ability of the newly synthesized dimer to modulate pro-survival proteins was also investigated. Our results demonstrated that the cystamine-tacrine dimer is endowed with a lower toxicity in comparison to bis(7)tacrine, it is able to inhibit human AChE, BChE, self- and AChE-induced Ab aggregation in the same extent of the reference compound, to protect the neuroblastoma SH-SY5Y cell line against H2O2-induced oxidative injury by activating the extracellular signal-regulated kinase 1 and 2 (ERK1/2) and Akt/protein kinase B (PKB) pathways. 2. Materials and Methods 2.1. Chemicals and reagents CelLytic M, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 20 ,70 dichlorodihydrofluorescein diacetate (DCFH-DA), H2O2, bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum (FCS), penicillin/streptomycin, LY294002, PD98059, mammalian protease inhibitor mixture, primary antibody to b-actin and all other chemicals of the highest analytical grade were purchased from Sigma Chemical Co. (St. Louis, MO). PhosSTOP was obtained from Roche Diagnostic (Mannheim, Germany). Primary antibodies against phospho-Akt, total Akt, phospho-ERK1/2, total ERK1/2, and horseradish peroxidase-conjugated secondary antibodies (anti-rabbit and anti-mouse) were purchased from Cell Signaling Technologies (Beverly, MA, USA). Human recombinant AChE (E.C.3.1.1.7) lyophilized powder, BChE (E.C.3.1.1.8) from human serum, potassium dihydrogen phosphate, potassium hydrogen phosphate, triton X-100, 5,50 -dithio-bis(2-nitrobenzoic acid), acetylthiocoline iodide Fig. 1. Chemical structures of bis(7)tacrine, cystamine and design strategy of the cystamine-tacrine dimer. and butyrylthiocholine iodide, glycine, 1,1,1,3,3,3,-hexafluoro-2-propanol (HFIP), thioflavin T, DMSO, cystamine, sodium iodide, phenol, silica gel (Silica gel 60, 230e400 mesh) and analytical thin layer chromatography (Silica gel on TLC-PET foils) were purchased by Sigma-Aldrich. Ab(1e40) trifluoroacetate salt and Ab(1e42) were purchased by Bachem AG, Switzerland. 2.2. Synthesis of the cystamine-tacrine dimer The new cystamine-tacrine dimer N,N0 -(2,20 -disulfanediylbis(ethane-2,1-diyl)) bis(1,2,3,4-tetrahydroacridin-9-amine), was synthesized by a biseamination reaction of the 9-chlorotetrahydroacridine (Elsinghorst et al., 2009) with cystamine in phenol (Fig. 2) (Bolognesi et al., 2007b). Briefly, a mixture of 9-chloro-1,2,3,4-tetrahydroacridine (0.200 g, 0.92 mmol, 2 eq.), cystamine (0.07 g, 0.46 mmol, 1 eq), phenol (0.48 g, 5.1 mmol, 11 eq) and sodium iodide (0.03 g, 0.2 mmol, 0.5 eq.) was first heated at 180  C for 2 h and then stirred at room temperature overnight under stream of nitrogen. The mixture was diluted with ethyl acetate and shaken with 10% aqueous KOH. The organic layer was washed with water and brine, dried, filtered and concentrated in vacuo. Purification by flash chromatography (CH2Cl2/MeOH/aqueous 33% ammonia 9:1:0.01) provided cystamine-tacrine dimer as yellow oil (0.11 g, 47% yield). ESI-MS spectra were recorded on Perkin-Elmer 297 and Waters ZQ 4000. 1 H NMR and 13C NMR spectra were recorded on Varian VRX 200 instrument. The elemental analysis was performed with Perkin-Elmer 2400 CHN elemental analyzer. From the new compound satisfactory elemental analyses was obtained, confirming >95% purity. 1 H NMR (free base, 200 MHz, CDCl3): d 1.87e1.90 (m, 8H), 2.71e2.74 (m, 4H), 2.85 (t, J ¼ 6.2 Hz, 4H), 3.06e3.10 (m, 4H), 3.75e3.84 (m, 4H), 4.61 (br s exchangeable with D2O, 2H), 7.37 (t, J ¼ 7.4 Hz, 2H), 7.58 (t, J ¼ 7.0 Hz, 2H), 7.94e8.02 (m, 4H); 13 C NMR (200 MHz, CDCl3): d 22.29, 22.66, 24.77, 33.10, 38.78, 46.57, 116.55, 119.89, 122.58, 124.03, 127.55, 128.69, 146.10, 150.23, 157.74; MS (ESIþ) m/z 515 (M þ H)þ. Anal. calcd for C30H34N4S2: C 70.00 H 6.66 N 10.88. Found: C 69.87 H 6.40 N 11.01. 2.3. Inhibition of human AChE and BChE activities The method of Ellman et al. was followed (Ellman et al., 1961). AChE stock solution was prepared by dissolving human recombinant AChE lyophilized powder in 0.1 M phosphate buffer (pH 8.0) containing 0.1% Triton X-100. Stock solution of BChE from human serum was prepared by dissolving the lyophilized powder in an aqueous solution of 0.1% gelatin. Stock solutions of test compounds (1 mM) were prepared in methanol and diluted in twice-distilled water. The assay solution A. Minarini et al. / Neuropharmacology 62 (2012) 997e1003 999 Fig. 2. Synthesis of the cystamine-tacrine dimer. consisted of 0.1 M phosphate buffer (pH 8.0), with the addition of 340 mM 5,50 -dithio-bis(2-nitrobenzoic acid), 0.02 units of AChE or BChE and 550 mM of substrate (acetylthiocholine iodide or butyrylthiocholine iodide, respectively). Assays were carried out with a blank containing all components except AChE or BChE in order to account for any non-enzymatic reaction. Test compounds were added to the assay solution and preincubated at 37  C with the enzyme for 20 min followed by the addition of substrate. The initial velocity rates were determined at 37  C using a Jasco V-530 double beam spectrophotometer (Jasco Europe, Italy). Absorbance values at 412 nm were recorded for 5 min and enzyme activity was calculated from the slope of the obtained linear trend. The reaction rates obtained in the presence and in the absence of the test compound were compared, and the percent inhibition was calculated. Five different concentrations of each compound were used to obtain inhibition of AChE or BChE activity comprised between 20 and 80%. Each concentration was analyzed in triplicate, and IC50 values were determined graphically from log concentrationeinhibition curves (GraphPad Prism 4.03 software, GraphPad Software Inc.). treated with different concentrations of bis(7)tacrine and cystamine-tacrine dimer (0.005e50 mM) for 24 h. Vehicle controls containing equivalent volumes of DMSO were carried out. Oxidative stress was induced by 200 mM H2O2 for 30 min. When required, cells were pre-treated with specific PI3K/Akt inhibitor LY294002 (LY, 5 mM), and protein kinase ERK1/2 inhibitor PD98059 (PD 10 mM) for 30 min. 2.7. Determination of neuronal viability Neuronal viability in terms of mitochondrial activity was evaluated with the colorimetric MTT assay, as previously described (Angeloni et al., 2008). Briefly, SH-SY5Y cells were washed with HBSS and then incubated with MTT (0.5 mg/mL) for 30 min. After removal of MTT and further washing, the formazan crystals were dissolved in DMSO. The amount of formazan was measured at l ¼ 595 nm with a microplate spectrophotometer (VICTOR3 V Multilabel Counter, Perkin-Elmer Wellesley, MA, USA). Cell viability was expressed as percent of control cells. 2.8. Intracellular ROS formation 2.4. Inhibition of Ab aggregation induced by human AChE Ab(1e40) lyophilised from a 2.0 mg mL1 HFIP solution, was dissolved in DMSO to obtain a 2.3 mM Ab(1e40) solution. Aliquots (2 mL) of Ab(1e40) in DMSO were then diluted in 0.215 M sodium phosphate buffer (pH 8.0) to a final concentration of 230 mM and incubated for 24 h at room temperature. For co-incubation experiments, aliquots of human recombinant AChE (2.30 mM, ratio 100:1) and AChE in the presence of the tested compound (100 mM) were added. Blanks containing Ab, AChE, Ab plus the tested compound, and AChE plus the tested compound in 0.215 M sodium phosphate buffer (pH 8.0) were prepared. The final volume of each vial was 20 mL (Bartolini et al., 2003). To quantify amyloid fibril formation, the thioflavin T fluorescence method was used (Levine, 1993; Naiki et al., 1991). Thioflavin T binds to amyloid fibrils giving rise to an intense specific emission band at 490 nm in the fluorescence emission spectrum. Therefore, after incubation, samples were diluted to a final volume of 2.0 mL with 50 mM glycine/NaOH buffer (pH 8.5) containing 1.5 mM thioflavin T. Studies were performed on a FP6200 spectrofluorometer (Jasco Europe, Italy). A 300 s time scan of fluorescence intensity was carried out (lexc ¼ 446 nm; lem ¼ 490 nm), and values at plateau were averaged after subtracting the background fluorescence of 1.5 mM thioflavin T solution. Inhibition values (%) were calculated from the corrected IF values obtained in the absence and in the presence of the tested compound. 2.5. Inhibitory potency on Ab(1e42) self-aggregation In order to investigate the Ab(1e42) self-aggregation, a thioflavin T-based fluorometric assay was performed. HFIP-pretreated Ab(1e42) samples (Bachem AG, Switzerland) were resolubilized with a CH3CN/0.3 mM Na2CO3/250 mM NaOH (48.4:48.4:3.2) mixture to obtain a stable stock solution ([Ab(1e42)] ¼ 500 mM) (Bartolini et al., 2007). Experiments were performed by diluting and incubating the peptide in 10 mM phosphate buffer (pH 8.0) containing 10 mM NaCl, at 30  C for 24 h (final Ab concentration ¼ 50 mM) in the absence or presence of inhibitor. Blanks containing the test inhibitors were also prepared and tested. To quantify amyloid fibril formation, the thioflavin T fluorescence method was used (Levine, 1993; Naiki et al., 1991). After incubation, samples were diluted to a final volume of 2.0 mL with 50 mM glycine/NaOH buffer (pH 8.5) containing 1.5 mM thioflavin T. A 300 s time scan of fluorescence intensity was carried out (lexc ¼ 446 nm; lem ¼ 490 nm), and values at plateau were averaged after subtracting the background fluorescence of 1.5 mM thioflavin T solution. The fluorescence intensities were compared and the percent inhibition due to the presence of the tested inhibitor was calculated. IC50 value was obtained from the % inhibition vs log[inhibitor] plot. 2.6. Cell culture and treatments Human neuroblastoma SH-SY5Y cell line was routinely grown at 37  C in a humidified incubator with 5% CO2 in DMEM supplemented with 10% FCS, 2 mM glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin as previously reported (Tarozzi et al., 2009). To evaluate neuronal viability and ROS formation, the SH-SY5Y cells were seeded in 96-well plates at 2  104 cells/well. To evaluate protein expression, SH-SY5Y cells were seeded in 100 mm culture dishes at 4 x 106 cells/ dish. Experiments were carried out 24 h after cells were seeded. SH-SY5Y cells were Intracellular ROS measurement was quantified with the fluorescent probe DCFH-DA as described by Wang and Joseph (Wang and Joseph, 1999). Cells were incubated with 5 mM DCFH-DA in HBSS for 30 min in the dark. After DCFH-DA removal and further washing, the cells were treated with H2O2 200 mM for 30 min and fluorescence was measured using a microplate spectrofluorometer (VICTOR3 V Multilabel Counter, Perkin-Elmer) (lexc ¼ 485 nm and lem ¼ 535 nm). Data are expressed as percent of cells exposed only to H2O2. 2.9. Western blot analysis Cells were washed with ice-cold PBS and lysed on ice using CelLytic M containing mammalian protease and phosphatase inhibitor mixture. The resulting lysed cells were left on ice to solubilize for 45 min. The lysates were centrifuged at 5000 g for 5 min at 4  C to remove unbroken cell debris and nuclei. Cell lysate protein concentration was determined by the Bio-Rad Bradford protein assay (Bio-Rad Laboratories, Hercules, CA). Samples were boiled for 5 min prior to separation on 10% SDS-PAGE. The proteins were transferred to a nitrocellulose membrane (Hybond-C; GE Healthcare, Buckinghamshire, UK) in Tris-glycine buffer at 110 V for 90 min. Membranes were then incubated in a blocking buffer containing 5% (w/v) skimmed milk and incubated with either anti-phospho-Akt, anti-total Akt, anti-phosphoERK1/2, anti-total ERK1/2 and anti-b-actin as internal normalizer, overnight at 4  C. The blots were then incubated with secondary antibodies for 60 min at room temperature. The results were visualized by chemiluminescence using ECLÒ Advance reagent according to the manufacturer’s protocol (GE Healthcare). Semiquantitative analysis of specific immunolabeled bands was performed using a Fluor S image analyzer (Bio-Rad, Hercules, CA, USA). 2.10. Statistics Each experiment was performed at least three times, and all values are represented as means  SEM. One-way analysis of variance (ANOVA) was used to compare differences among groups followed by Dunnett’s or Bonferroni’s test (Prism 5, GraphPad Software Inc., San Diego, CA). Values of p < 0.05 were considered to be statistically significant. 3. Results 3.1. Cystamine-tacrine dimer inhibited AChE and BChE In the ChE inhibition assays cystamine-tacrine dimer displayed the ability to act as AChE inhibitor in the nanomolar range with an inhibition potency slightly lower than that displayed by the reference compound bis(7)tacrine. On the other hand, the introduction of the cystamine chain did not affect the inhibitory potency against BChE (Table 1). The cystamine-tacrine dimer resulted equipotent on AChE and BChE. 1000 A. Minarini et al. / Neuropharmacology 62 (2012) 997e1003 Table 1 Inhibition of AChE and BChE activities and of AChE-mediated and self-induced Ab aggregation by bis(7)tacrine and cystamine-tacrine dimer. Assay Bis(7)tacrine AChE (IC50, nM) BChE (IC50, nM) Ab aggregation, self (IC50, mM)b Ab aggregation, AChE-inducedc (% inhibition) 0.81 5.66 8.4 68.0     0.09a 0.15a 1.4 3.5a Cystamine-tacrine dimer 5.04 4.23 24.2 52.6     0.48 0.25 0.8 1.6 a Data from reference (Bolognesi et al., 2007a). % Inhibition of Ab(1e42) self-aggregation at [I] ¼ 10 mM. The [Ab(1e42)]/[I] ratio was equal to 5/1. Values are the mean of two independent experiments in duplicate  SEM. c % inhibition of AChE-induced Ab(1e40) aggregation at [I] ¼ 100 mM. The Ab(1e40)/AChE ratio was equal to 100/1. Values are the mean of three experiments  SEM. b 3.2. Ab aggregation assays As reported in literature, bis(7)tacrine is able to interact with AChE PAS (Bolognesi et al., 2007b) which is involved in Ab-fibril formation (Inestrosa et al., 1996) and is a weak inhibitor of amyloid self-aggregation (Bolognesi et al., 2010). To verify if cystamine-tacrine dimer is endowed with the same activity profile on these two key targets for AD, Ab aggregation experiments were performed in comparison with bis(7)tacrine and data are reported in Table 1. A similar pattern was observed for the two compounds in the aggregation assays, both self- and AChEinduced. 3.3. Effect of cystamine-tacrine dimer on neuronal viability SH-SY5Y cells were treated with increasing concentrations of cystamine-tacrine dimer and bis(7)tacrine (0.005e50 mM) for 24 h to investigate their effect on neuronal viability. In Fig. 3 data obtained by the MTT viability test are reported. Cystamine-tacrine dimer treatment up to 10 mM did not affect neuronal viability. On the other hand, bis(7)tacrine exerted cytotoxic effects on SH-SY5Y at 10 mM, with a significant decrease in cell viability up to 30%. Both compounds significantly reduced cell viability at the highest tested concentration (50 mM). Fig. 3. Effects of cystamine-tacrine dimer and bis(7)tacrine on SH-SY5Y cell viability. Cells were incubated with different concentrations of the compounds (0.005e50 mM) for 24 h. Cell viability was determined by measuring the MTT reduction as reported in Materials and Methods. Data are means  SEM of four independent experiments and expressed as percent of control value. Statistical analysis was performed by the one-way ANOVA followed by Dunnett’s test. *p < 0.05 vs. control cells. 3.4. Cytoprotective effect of cystamine-tacrine dimer against H2O2-induced cell death To investigate the protective effects of cystamine-tacrine dimer in comparison with bis(7)tacrine, SH-SY5Y cells were pre-treated with different concentrations of the compounds (0.005e0.5 mM) for 24 h prior to the addition of 200 mM H2O2. This H2O2 concentration was chosen as it represents the concentration that evoked an approximate 50% loss in the ability of cells to reduce MTT (data not shown). Fig. 4 shows the concentration-dependent protective effect of the compounds against H2O2-induced cell death. Cystamine-tacrine dimer treatment was able to significantly increase cell viability in respect to H2O2-stressed cells at any tested concentration. Exposure of cells to 0.5 mM cystamine-tacrine dimer evoked a complete protection against peroxide-induced injury. On the contrary, bis(7)tacrine was not able to significantly increase cell viability in comparison to H2O2-stressed cells at any tested concentration. 3.5. Effect of cystamine-tacrine dimer on intracellular ROS production To determine whether the neuroprotective effects of cystaminetacrine dimer could be ascribed to its antioxidant capacity, we evaluated the H2O2-induced intracellular ROS formation in SHSY5Y cells pre-treated with cystamine-tacrine dimer and bis(7) tacrine, using the fluorescent probe DCFH-DA (Fig. 5). Cystaminetacrine dimer was able to significantly decrease ROS production at any tested concentration with the highest effect at 0.5 mM. On the contrary, bis(7)tacrine did not show any antioxidant ability to counteract ROS production. 3.6. Role of ERK1/2 and Akt pathways in cystamine-tacrine dimer neuroprotection In order to clarify the mechanism behind cystamine-tacrine dimer neuroprotection, we investigated the time dependent Fig. 4. Cytoprotective effects of cystamine-tacrine dimer and bis(7)tacrine against H2O2induced cell death. SH-SY5Y cells were treated with different concentrations (0.005e0.5 mM) of the compounds for 24 h and then stressed with 200 mM H2O2 for 30 min. After 24 h, cell viability was determined by measuring the MTT reduction as reported in Materials and Methods. Data are means  SEM of four independent experiments and are expressed as percent of control cells. Statistical analysis was performed by the one-way ANOVA followed by Bonferroni’s test. *p < 0.05 vs. control cells, #p < 0.05 vs. H2O2. A. Minarini et al. / Neuropharmacology 62 (2012) 997e1003 1001 Fig. 5. Inhibition of H2O2-induced ROS production by cystamine-tacrine dimer and bis(7) tacrine. SH-SY5Y cells were incubated with different concentrations (0.005e0.5 mM) of the compounds for 24 h and then exposed to 200 mM H2O2 for 30 min. Intracellular ROS production was quantified using the fluorescent probe DCFH-DA, as described in Materials and Methods. Data are means  SEM of four independent experiments and are expressed as percent of H2O2-treated cells. Statistical analysis was performed by one-way ANOVA followed by Dunnett’s test. *p < 0.05 vs. H2O2-treated cells (indicated as “0”). effect of cystamine-tacrine dimer treatment on two important anti-apoptotic protein kinases: ERK1/2 and Akt. Figs. 6 and 7 show the time-course activation (phosphorylation) of ERK1/2 and Akt in SH-SY5Y cells treated with 0.5 mM cystaminetacrine dimer. Levels of ERK1/2 were found to be rapidly modified, reaching significant activation after 0.5 h treatment. The activation lasted until the longest time exposure. Akt was not activated at 0.5 h of cystamine-tacrine dimer treatment as values were comparable to control cells, while Akt levels reached significant activation after 1 h. Parallel blots were run and probed with antibodies that detected total levels of ERK1/2 and total levels of Akt, demonstrating no modification in the total amount of proteins. On the contrary, 0.5 mM bis(7)tacrine treatment did not significantly influence ERK1/2 and Akt phosphorylation at any considered time (data not shown). Fig. 7. Time-course activation of Akt in SH-SY5Y cells. Proteins were extracted at the indicated time points following cystamine-tacrine dimer (0.5 mM) treatment. Cell lysates were immunoblotted with specific antibodies for phospho-Akt and totalAkt. Representative immunoblots of three different experiments are reported. Results of scanning densitometry analysis performed on three independent autoradiographs are presented. Data were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s test. *p < 0.05 with respect to Control. To investigate the role of these protein kinases in mediating the neuroprotection afforded by cystamine-tacrine dimer, we pre-treated SH-SY5Y cells with specific inhibitors of ERK1/2phosphorylation (PD98059, PD) and Akt phosphorylation (LY294002, LY) for 30 min before the addition of 0.5 mM cystaminetacrine dimer. Cell viability in the absence or presence of the different inhibitors is shown in Fig. 8. The two inhibitors in the presence of H2O2 did not influence cell viability (data not shown). Inhibitor pre-treatment significantly reversed the neuroprotective effects of cystamine-tacrine dimer. In particular, LY was able to inhibit neuroprotection with a higher efficacy in respect to PD. 4. Discussion Fig. 6. Time-course activation of ERK1/2 in SH-SY5Y cells. Proteins were extracted at the indicated time points following cystamine-tacrine dimer (0.5 mM) treatment. Cell lysates were immunoblotted with specific antibodies for phospho-ERK1/2 and totalERK1/2. Representative immunoblots of three different experiments are reported. Results of scanning densitometry analysis performed on three independent autoradiographs are presented. Data were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s test. *p < 0.05 with respect to Control. AD is a multifactorial disease characterized by alterations of different cellular pathways and neuronal loss. In particular, several hypotheses have been formulated to correlate the pathological lesions and neuronal cytopathology and the etiology of this disease. The most important ones are related to the loss of cholinergic neurons, to the role of free radical species and to the presence of the Ab protein. On these bases a drug able to simultaneously modulate these important targets may be considered an efficacious therapeutic agent capable to face the causes of the pathology and not only a simple palliative drug. To fulfil this strategy we have designed a new MTDL for AD treatment starting from the chemical structure of the known AChEI bis(7)tacrine. As reported above, other than with cholinesterase enzymes, this compound demonstrated the ability to interact with several AD targets such as Ab peptide, beta-secretase enzyme, NMDA and GABAA receptors. With the aim to improve its biological profile as radical scavenger we introduced, inside the polymethylene chain, a disulfide bond, deriving from the cystamine structure (Fig. 1). The resulting cystamine-tacrine dimer showed the ability to inhibit AChE and BChE in the same range of bis(7) tacrine, without any selectivity towards these two cholinesterases. 1002 A. Minarini et al. / Neuropharmacology 62 (2012) 997e1003 Fig. 8. Cytoprotective effect of cystamine-tacrine dimer against H2O2-induced cell death in the presence/absence of ERK1/2 and Akt inhibitors. SH-SY5Y cells were incubated with PD or LY for 30 min, treated with cystamine-tacrine dimer for 24 h and then stressed with 200 mM H2O2 for 30 min. After 24 h, cell viability was determined by measuring the MTT reduction as reported in Materials and Methods. Data are means  SEM of four independent experiments and are expressed as percent of control cells. Statistical analysis was performed by the one-way ANOVA followed by Bonferroni’s test. *p < 0.05 vs. control cells, #p < 0.05 vs. H2O2. The inhibition of both cholinesterases might result useful in order to increase the concentration of the neurotransmitter ACh in the neuronal synaptic clef in severe form of AD, in which BChE is thought to play a compensative role in hydrolysing ACh following the massive loss of cholinergic neurons (Greig et al., 2005). An additional therapeutic property of cystamine-tacrine dimer, which contributes to its consideration as MTDL, is represented by the inhibition of Ab aggregation. The same action was also exerted by bis(7)tacrine and the inhibition values of the two compounds are comparable. Ab aggregates represent an important hallmark of AD, and both fibrilar and soluble oligomeric forms of Ab peptides have been related to neurotoxicity and apoptosis in cortical neurons (Chiang et al., 2008; Kawahara, 2010; Kawahara et al., 2009). In this context, the neuroprotective activity of bis(7)tacrine against Abinduced neuronal apoptosis has been related to the regulation of L-type voltage-dependent calcium channels (VDCCs), leading to a decrease of intracellular Ca2þconcentration (Fu et al., 2006). The contribute of the disulfide bridge to radical scavenger activity was investigated in the neuronal cell line SH-SY5Y. This cell line has been widely used as model of neurons since the early 1980’s as these cells possess many biochemical and functional properties of neurons (Xie et al., 2010). Firstly, the overall toxicity of the cystamine-tacrine dimer was assessed by MTT viability test using bis(7)tacrine as reference compound. The results showed that, while both compounds did not show any toxic effect at the lowest concentrations, at 10 and 50 mM bis(7)tacrine was significantly more toxic than the cystaminetacrine dimer. Then we investigated the ability to counteract oxidative stress induced by H2O2. In agreement with the rational design strategy, only cystamine-tacrine dimer was able to protect cells against hydrogen peroxide injury, while bis(7)tacrine did not show any detectable effect. It is worth to mention that these results are in disagreement with those obtained by Xiao et al. (2000) that showed that bis(7)tacrine was able to counteract oxidative stress induced by H2O2 in PC12 cell line (Xiao et al., 2000). The different outcomes might relate to the different behaviour of PC12 cells in respect to SH-SY5Y. In fact, in the study carried out by Xiao et al. bis(7)tacrine did not induce any toxic effect on PC12 cells at any tested concentration. In particular, our results showed that cystamine-tacrine dimer was effective even at the lowest tested concentration (0.005 mM) and was able to totally reverse H2O2-induced damage at 0.5 mM. The data on ROS production measured by the DCFH-DA fluorescent probe demonstrated that protective effect of cystamine-tacrine dimer is strictly linked to its ability to, directly or indirectly, scavenge oxygen peroxide. In agreement with data on cell viability, 0.5 mM cystamine-tacrine dimer had the maximum effect in reducing ROS production, while bis(7)tacrine did not show any significant effect at any tested concentration. This important neuroprotective effect might be related to the activation of two important cell survival pathways: PI3K/Akt and ERK1/2 signaling pathways (Susin et al., 1999). The activation of PI3K/Akt pathway has been demonstrated to protect neurons against apoptosis (Datta et al., 1997) as phosphorylated Akt acts both to stimulate anti-apoptotic factors and to inhibit proapoptotic factors (Yoshimoto et al., 2001). Moreover, the activation of PI3K/Akt signaling pathway inhibits the toxicity of b-amyloid (Yin et al., 2011). The involvement of the Akt survival pathway has been also observed in the case of the huntingtin-induced toxicity in Huntington’s disease (Humbert et al., 2002; Magrane et al., 2005; Nakagami, 2004; Nassif et al., 2007). In neurons, ERK1/2 can function to either support cell survival or promote cell death (Stanciu et al., 2000). Moreover, in neurons a significant cross talk between PI3K and ERK1/2 has been observed (Perkinton et al., 2002, 1999). In agreement with our hypothesis, the highest concentration of cystamine-tacrine dimer was able to phosphorylate (activate) both ERK1/2 and Akt after 0.5 h and 1 h, respectively. On the contrary, bis(7)tacrine treatment did not increase the phosphorylation of any of these two prosurvival kinases. The involvement of ERK1/2 and Akt in the protection elicited by cystamine-tacrine dimer was further confirmed by using selective inhibitors of their phosphorylation. Akt phosphorylation inhibition had a higher effect than ERK1/2 inhibition in reducing cystaminetacrine dimer protective effect against oxidative stress. In conclusion, a new cystamine-tacrine dimer, analogue of bis(7) tacrine, was synthesized and evaluated on different important AD targets. In particular this compound showed the ability i) to inhibit cholinesterases in the nanomolar range; ii) to inhibit self- and AChEinduced Ab aggregation in the same range of reference compound bis(7)tacrine; iii) to be less cell toxic than bis(7)tacrine; iv) to act as radical scavenger in the nanomolar range therefore protecting cells against oxidative stress induced by H2O2; v) to activate the prosurvival ERK1/2 and Akt pathways in neuronal cell lines. As general sum up, all these data allowed us to consider cystamine-tacrine dimer as a new MTDL which may be potentially useful in AD treatment, thanks to its well balanced biological profile as cholinesterases inhibitor and cytoprotective agent. 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