Neuromol Med (2014) 16:787–798 DOI 10.1007/s12017-014-8328-4

ORIGINAL PAPER

Multiple Mechanisms of Iron-Induced Amyloid Beta-Peptide Accumulation in SHSY5Y Cells: Protective Action of Negletein Priyanjalee Banerjee • Arghyadip Sahoo • Shruti Anand • Anirban Ganguly • Giuliana Righi • Paolo Bovicelli • Luciano Saso • Sasanka Chakrabarti

Received: 23 March 2014 / Accepted: 17 September 2014 / Published online: 24 September 2014 Ó Springer Science+Business Media New York 2014

Abstract The increased accumulation of iron in the brain in Alzheimer’s disease (AD) is well documented, and excess iron is strongly implicated in the pathogenesis of the disease. The adverse effects of accumulated iron in AD brain may include the oxidative stress, altered amyloid beta-metabolism and the augmented toxicity of metalbound amyloid beta 42. In this study, we have shown that exogenously added iron in the form of ferric ammonium citrate (FAC) leads to considerable accumulation of amyloid precursor protein (APP) without a corresponding change in the concerned gene expression in cultured SHSY5Y cells during exposure up to 48 h. This phenomenon is also associated with increased b-secretase activity and augmented release of amyloid beta 42 in the medium. Further, the increase in b-secretase activity, in SHSY5Y cells, upon exposure to iron apparently involves reactive oxygen species (ROS) and NF-jB activation. The synthetic flavone negletein (5,6-dihydroxy-7-methoxyflavone), which is a known chelator for iron, can significantly prevent the effects of FAC on APP metabolism in SHSY5Y cells. Further, this compound inhibits the iron-dependent

P. Banerjee  A. Sahoo  S. Anand  A. Ganguly  S. Chakrabarti (&) Department of Biochemistry, Institute of Postgraduate Medical Education and Research, 244, AJC Bose Road, Kolkata 700020, India e-mail: [email protected]; [email protected] G. Righi  P. Bovicelli CNR ICB-Unity of Rome, c/o Department of Chemistry, Sapienza University of Rome, Rome, Italy L. Saso Department of Physiology and Pharmacology, Vittorio Erspamer, Sapienza University of Rome, Rome, Italy

formation of ROS and also blocks the iron-induced oligomerization of amyloid beta 42 in vitro. In concentrations used in this study, negletein alone appears to have only marginal toxic effects on cell viability, but, on the other hand, the drug is capable of ameliorating the iron-induced loss of cell viability considerably. Our results provide the initial evidence of potential therapeutic effects of negletein, which should be explored in suitable animal models of AD. Keywords Alzheimer’s disease  Amyloid beta 42  b-Secretase  Flavone  Iron

Introduction The pathogenesis of sporadic Alzheimer’s disease (AD) which accounts for the majority cases of progressive dementia in elderly people is uncertain, but some genetic polymorphisms and multiple metabolic and extra-genetic triggers and risk factors are involved in this process (Randall et al. 2009; Sato and Morishita 2013; Khemka et al. 2014). At autopsy, the AD brain shows characteristic lesions of extracellular amyloid plaques, often surrounded by dystrophic neurites, and intra-neuronal neurofibrillary tangles along with widespread loss of neurons, degenerations of dendrites, axons and synapses (Hyman et al. 2012). At the mechanistic level, the toxic actions of accumulated oligomeric amyloid beta-peptides (predominantly Ab42 and Ab40) and hyperphosphorylated tau protein, oxidative stress, transition metal accumulation, mitochondrial dysfunction and inflammatory response work in concert through interdependent damage pathways in the aging brain to trigger and propagate the AD pathology (Swerdlow 2007; Butterfield et al. 2006; Chakrabarti et al. 2013; Reddy and Beal 2008). The effective drug treatment should

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target one or preferably more of these damage pathways operating in AD brain, but at present the therapeutic measures available for this disease are extremely limited. The accumulation of transition metals such as Fe, Cu and Zn have been well documented in AD brain, both in the amyloid plaques and within neurons, and the phenomenon is strongly implicated in AD pathogenic process (Bonda et al. 2011; Lovell et al. 1998; Jomova et al. 2010). For example, the transition metal in a redox-active form binds to Ab42 by co-ordination with three histidine residues and one undetermined residue and generates reactive oxygen species (ROS) with consequent oxidative damage to AD brain (Nakamura et al. 2007; Smith et al. 2007a). Further, it has been shown that the toxic actions of Ab42 oligomers are enhanced when the peptide is in the metalbound form (Smith et al. 2007b; Dai et al. 2010). The oligomerization of Ab42 in vitro is also facilitated by the transition metals (Huang et al. 2004). Apart from these mechanisms of toxicity of the transition metals in AD brain, Fe in particular can impact AD pathology in a radically different manner. The presence of iron-responsive element (IRE) in 50 -UTR of amyloid precursor protein (APP) mRNA implies a direct link between iron metabolism and AD development (Bandyopadhyay et al. 2013). The iron-responsive element binding protein (IREBP) occupies the IRE at the 50 -UTR of APP mRNA preventing the initiation of translation, but in the presence of iron the complex of IREBP–Fe is dissociated from the mRNA leading to an increased synthesis of APP protein (Mills et al. 2010). Thus, an increased intracellular Fe can lead to an accumulation of APP in the brain with a consequent increase in the formation of amyloid beta-peptides including Ab42. It follows that the compounds with ironchelating activities would not only prevent the ironinduced oxidative damage or iron-promoted amyloid betatoxicity in AD brain, but may also substantially diminish the amyloid beta-production in the brain. SHSY5Y cells, human neuroblastoma cell line, have been used widely to study the processing, metabolism and intracellular trafficking of APP and amyloid beta-peptides under different experimental conditions as also to investigate varied cellular effects of Ab42 and related peptides (Prasanthi et al. 2009; Belyaev et al. 2010; Solano et al. 2000; Zheng et al. 2013; Olivieri et al. 2003; Li et al. 1996). In particular, SHSY5Y cells have been used to show the protective effects of several compounds such as coenzyme Q, melatonin, curcumin and N-acetylcysteine on amyloid beta-induced cytotoxicity or oxidative stress-augmented Ab production (Li et al. 2005; Oliveri et al. 2001a, b; Xiong et al. 2011). We have, therefore, thought it prudent to utilize SHSY5Y cells as a good tool to ascertain the mechanisms by which iron exposure leads to alterations of amyloid beta-peptide homeostasis in SHSY5Y cells and

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also to explore if the novel iron-chelating agent negletein, a synthetic flavone, can prevent the phenomenon. The flavones belong to the family of polyphenolic flavonoid compounds, which are widely distributed in vegetables, fruits, cereals, tea, etc., can permeate the cell membrane and have antioxidant and metal-chelating actions (Symonowicz and Kolanek 2012; Kanazawa et al. 2006; Maca´kova´ et al. 2012). The bioavailability of these compounds after dietary intake is low, but epidemiological and experimental studies have implicated these compounds as protective against dementia and AD, but their role in amyloid beta-metabolism is not thoroughly established (Choi et al. 2012; Dragicevic et al. 2011; Baptista et al. 2014; Commenges et al. 2000). The methoxy group containing chemically synthesized flavone negletein has been recently studied for metalchelating, antioxidant and cytoprotective activities (Maca´kova´ et al. 2012; Mladeˇnka et al. 2011; Lombardo et al. 2013). However, the biological potential of negletein is not well tested in different experimental models of disease where metal-dependent toxicity could play a crucial role. Thus, it has been particularly interesting to study the effect of negletein on iron-induced alterations in amyloid betapeptide homeostasis in SHSY5Y cells.

Materials and Methods Cell lines and Reagents Human neuroblastoma cell line SHSY5Y was obtained from the American Type Culture Collection (USA). All common chemicals were of analytical grade and obtained from Sisco Research Laboratory (India). Penicillin–streptomycin, Trypsin–EDTA and fetal bovine serum (FBS) were from Invitrogen (USA). Dulbecco’s modified eagle medium (DMEM), b-mercapto ethanol, agarose, tetramethylethylenediamine (TEMED), triton-9-100, trypan blue, tween 20, ferric ammonium citrate (FAC), mannitol, bovine serum albumin (BSA), glycine, coumarin 3-carboxylic acid, protease inhibitor cocktail, sodium dodecylsulfate (SDS), diethylenetriaminepentaacetic acid (DTPA), thioflavin T, HEPES, N-acetylcysteine (NAC), antimycin A (ANT) and rabbit anti-APP antibody were purchased from Sigma Chemical Co. (USA). Goat anti-rabbit HRP conjugate was purchased from Bangalore Genei (India). RNA extraction kit was obtained from Ambion (USA) while cDNA synthesis kit and SYBR Green master mix were purchased from Bio-Rad (USA). b-Secretase activity assay kit was purchased from Biovision (USA). ECL detection kit was obtained from Pierce (USA) and SN50 from Calbiochem (USA). All the chemicals required for synthesis of negletein were purchased from Sigma-Aldrich (USA).

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Synthesis of Negletein The synthesis of negletein starting from commercially available crysin was carried out by successive methoxylation, bromination, methanolysis and selective demethylation to obtain a mixture of regioisomers (6-bromo-5-hydroxy7-methoxyflavone and 8-bromo-5-hydroxy-7-methoxyflavone) through the intermediate formation of 5-hydroxy-7methoxyflavone. The method utilized tetrabutylammonium tribromide for bromination while methanolysis was carried out for 5 h by adding sodium methoxide and CuBr in N,N dimethylformamide (DMF) to the bromoaromatic substrate (the mixture of regioisomers) in DMF at 120 °C. A Wesley–Moser rearrangement converted the mixture of isomers to mosloflavone, which was selectively demethylated by treatment with a mixture of hydrobromic and acetic acid to obtain negletein (5,6-dihydroxy-7-methoxyflavone). The purity of the compound was more than 95 % as checked by NMR. The detailed procedure was published earlier (Righi et al. 2010). Cell Culture and Treatment Protocol Cells were grown in DMEM medium containing 10 % heat-inactivated fetal bovine serum, 50 units/ml penicillin, 50 lg/ml streptomycin and 2.5 lg/ml amphotericin B in a humidified environment containing 5 % CO2 and 95 % air at 37 °C. Cells were harvested at a density of 5 9 105 cells in 25 cm2 sterile tissue-culture flasks or 1 9 105 cells per well in 12-well cell-culture plates. Cells were treated without (control) or with 66.6–400 lM FAC or negletein (10–20 lM) or negletein (10–20 lM) plus FAC (400 lM) or antimycin A (0.05 lM) for 48 h. For some experiments, the cells were co-exposed to FAC (400 lM) and SN50 (20 lM) or FAC (400 lM) and N-acetylcysteine (2.5 mM). APP Gene Expression Analysis RNA from SHSY5Y cells was extracted using the PureLink RNA minikit (Ambion) according to the manufacturer’s protocol. The extracted RNA was checked for purity by the 260 nm/280 nm ratio as well as by electrophoresis in 2 % agarose gels containing ethidium bromide (1 lg/ml) and visualization on a Syngene Gel Doc XR system. The reverse transcription was performed (80 ng total RNA) as per the manufacturer’s protocol using the iScript cDNA synthesis kit (Bio-Rad). RT-qPCR analysis of the cDNA samples for a fragment (154 bp) of APP gene was performed in triplicate using SYBR Green on a real-time PCR machine (Mini-Opticon, Bio-Rad). The reactions were performed in a 20-ll volume with 5 pmol each of forward and reverse primers for both the target (APP) and the reference b-actin genes. The primers used were APP-

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Forward: 50 AAC CAG TGA CCA TCC AGA 30 ; APPReverse: 50 ACT TGT CAG GAA CGA GAA 30 ; b actinForward: 50 CAG CCA TGT ACG TTG CTA TCC AGG 30 ; and b actin-Reverse: 50 AGC TCC AGA CGC AGG ATG GCA TG 30 . The average quantification cycle (Cq) values from the replicate reactions were calculated for the target and the reference (b-actin) cDNA preparations for each sample. The gene expression was quantified by relative quantitation method following the procedure proposed by Pfaffl (2001). Immunoblotting of APP APP content was measured in membrane-enriched cellular fraction. Briefly, the cells were homogenized in a buffer (225 mM mannitol, 75 mM sucrose, 5 mM HEPES, 1 mM EGTA, 1 mg/ml BSA, pH 7.4) containing protease inhibitor cocktail and centrifuged at 1,0009g for 3 min. The supernatant was re-centrifuged at 1,00,0009g for 1 h at 4 °C, and the pellet re-suspended in lysis buffer (50 mMTris, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1 % SDS, 0.25 % deoxycholate, 0.25 % NP-40) for 5 min keeping on ice. The pellet suspension was subjected to sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and the separated proteins electroblotted on a nitrocellulose membrane using a tank-type electroblotting apparatus. The membrane was then processed for immunostaining by using standard blotting protocols (Page and Thorpe 2002). The primary antibody for APP (polyclonal rabbit anti-APP antibody) was used in 1:2,000 dilution, while that for the c-actin loading control used in 1:5,000 dilution. The secondary antibody–enzyme conjugate (Goat anti-rabbit IgG conjugated to peroxidase) was used in 1:3,500 dilution, and the bands were developed on KodakXAR films by enhanced chemiluminescence technique. The film was digitized and analyzed by Gel Quant imaging and quantitation software. The APP band intensity from each sample was normalized with respect to the corresponding c-actin loading control. Measurement of b-Secretase Activity b-Secretase enzyme activity was measured by fluorometric assay based on the cleavage of a secretase-specific peptide substrate conjugated to two reporter groups EDAN (fluorophore) and DABCYL (quencher). In the uncleaved form, the fluorescence emission from EDANS was quenched by the DABCYL moiety, but the cleavage of the peptide by the secretase separated EDANS from DABCYL allowing for the emission of a fluorescence signal (kex 345 nm/kem 500 nm). Control and FAC-treated cells grown in 12 well plates were dislodged mechanically, collected in tubes and washed thoroughly with PBS by centrifugation at

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1,000g for 5 min. The cell pellet was resuspended in 100-ll b-secretase extraction buffer and sonicated with five bursts (8 s each with 30 s interval) kept in ice. The homogenate was kept in ice for 5 min and then centrifuged for 10 min at 10,000g at 4 °C. The supernatant was used for the enzyme assay. An aliquot of the supernatant was mixed with 100-ll reaction buffer and 2 ll of substrate supplied with the kit, in a total of 200-ll reaction volume. The reaction mixture was incubated for 1 h at 37 °C in dark, and fluorescence intensity was measured at (kex 345 nm/kem 500 nm) keeping appropriate sample and dye blanks. The specific b-secretase activity was expressed as fluorescence unit per 100 lg of protein. The assay produced proportionate increase in fluorescence (enzyme activity) with cell extract containing 10–60 lg of protein. The commercial kit (Biovision, USA) provided a specific inhibitor of b-secretase as well as purified b-secretase, which were used to validate the specificity of b-secretase activity assay in the biological samples. Further, we also examined if negletein (20 lM) or SN50 (20 lM) or NAC (2.5 mM) had any direct quenching effects on the fluorescence-based assay of b-secretase. For this purpose, the b-secretase assay was performed with the purified enzyme and the pure compound (negletein or SN50 or NAC) was added in the assay mixture after completion of 1 h of incubation period and the fluorescence reading recorded and compared with that of an assay mixture containing pure b-secretase only. Measurement of Ab1–42 by Sandwich Elisa Ab42 released in the culture media was analyzed by chemiluminescence-based enzyme-linked immunosorbent assay (ELISA) using a commercial kit (Beta-Mark Abx-42, Covance, USA). An aliquot of the media (400 ll) was vacuum-dried for 3 h, reconstituted in working buffer solution (provided with the kit) and used for Ab42 measurement in duplicate following manufacturer’s protocol. In this method, Ab42 present in the sample or the standard was captured by the antibody-coated wells, and horseradish peroxidase-conjugated detecting antibody was subsequently used followed by the addition of the chemiluminescence substrate. The luminescence signal was measured on a plate luminometer (ELX-800, Biotek). The standard curve was generated by using synthetic Ab42 peptide (0–250 pg/ml) supplied with the kit. Measurement of Hydroxyl Radical Production In Vitro from Fe2? and Ascorbate The generation of hydroxyl radicals from a mixture of iron and ascorbate incubated in vitro was monitored by benzoate hydroxylation assay and coumarin carboxylic acid

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assay as adapted from published methods (Sinha et al. 2013). Sodium benzoate (1 mM) or coumarin3-carboxylic acid (1 mM) was incubated without or with Fe2? (FeSO4, 20 lM) and ascorbate (2 mM) for 1 h in 50-mM phosphate buffer, pH 7.4. The hydroxyl radical scavenger mannitol (20 mM) or the metal chelator such as negletein (5–20 lM) or DTPA (0.1 mM) was present in the incubation mixture in some of the tubes. At the end of the incubation, the fluorescence intensity of the assay mixture was measured after appropriate dilution using kex 305 nm/ kem 407 nm for benzoate hydroxylation assay and kex 388 nm/kem 450 nm for coumarin carboxylic acid assay with necessary blank corrections. Oligomerization of Ab42 The oligomerization of Ab42 was monitored as described earlier (Sinha et al. 2013). An aliquot of Ab42 in HFIP was dried under vacuum (Concentrator Plus; Eppendorf), and the residue re-dissolved in 20 mM NaOH followed by dilution in 50 mM phosphate buffer, pH 7.4. The concentration of Ab42 was calculated from the molar extinction coefficient of the peptide (Barnham et al. 2008). Prepared freshly Ab42 was mostly monomeric and did not give fluorescence with thioflavin T. The oligomerization of Ab42 peptide was induced by incubation at 37 °C for 24 h with or without FAC (400 lM) in the presence or absence of negletein (20 lM) in 50 mM phosphate buffer, pH 7.4. The oligomerized Ab42 after mixing with thioflavin T (5 lM) gave strong fluorescence (kex 435 nm, kem 485 nm) (Sinha et al. 2011). In order to eliminate the direct quenching of thioflavin T fluorescence by negletein, a parallel tube containing Ab42 was incubated for 24 h and 20-lM negletein added to the reaction mixture in this tube at the end of the incubation followed by the measurement of thioflavin T fluorescence. The background fluorescence of a freshly prepared Ab42 (monomeric) was taken after immediately mixing it with thioflavin T, and the fluorescence reading was subtracted from each sample reading. Cell Viability Assay The cell viability was measured by trypan blue exclusion method as published earlier (Jana et al. 2011). The assay by a spectrophotometric method of released LDH in the medium was also performed to estimate the degree of cell death. The spectrophotometric assay for LDH was based on NADH oxidation in the presence of pyruvate, which was monitored by the fall in absorbance at 340 nm (Clark et al. 1997). Enzyme activity in micromoles NADH oxidized per min was calculated using molar extinction coefficient of NADH (6.22 mM-1 cm-1) and expressed as a percentage of LDH released in the media with respect to total LDH content.

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Fig. 1 APP gene expression in SHSY5Y cells treated with ferric ammonium citrate (FAC). Relative gene (APP) expression levels in control (C), 400-lM FAC-treated (FAC) and 400-lM FAC and 20-lM negletein-treated (FAC ? Neg) SHSY5Y cells were determined by RNA extraction followed by RT-qPCR as described in ‘‘Materials and Methods.’’ The results are the mean ± SEM of six experimental observations. Statistically significant, *p \ 0.05 versus C; Dp \ 0.001 versus FAC

Statistical Analysis Statistical significance was calculated by one-way ANOVA followed by Tukey’s post-test to assess differences between groups. A p value B 0.05 was considered to be statistically significant. Each value is expressed as the mean ± SEM of six observations.

Results Effect of Negletein on Iron-Induced Changes in Cellular APP The expression levels of APP gene in control, FAC-treated and FAC plus negletein-treated cells are presented in Fig. 1, which shows statistically significant but mild change in APP mRNA expression in FAC-treated cells compared to that in control. In FAC plus negletein-treated cells, the mRNA expression level of APP gene was marginally decreased with respect to that in FAC-treated cells (Fig. 1). Further, a marginal decrease of APP expression compared to control was also noticed in cells treated with only negletein (data not shown). In contrast, the protein expression level of APP, as evidenced by immunoblotting, was found to increase significantly in FAC-treated cells compared to that in control cells as shown in Fig. 2a, with densitometric analysis confirming almost fivefold increase in the APP band intensity in the FAC (400 lM)-treated cells (Fig. 2b). The phenomenon was however markedly prevented in FAC plus negletein-treated cells (Fig. 2a, b).

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Fig. 2 Immunodetection of APP. Lysate from control (C) or 400-lM FAC-treated (400-lM FAC) or 400-lM FAC and 20-lM negleteintreated (FAC ? Neg) SHSY5Y cells was subjected to SDS-PAGE and immunoblotting as described in the text. a A representative immunoblot of APP. b Densitometric analysis of APP immunoblots. The results are the mean ± SEM of six experimental observations. Statistically significant, hp \ 0.001 versus C; Dp \ 0.001 versus 400-lM FAC

The iron-induced increase in APP level was also observed with lower concentrations of FAC (66.6 and 133.3 lM), while a general cytotoxic agent antimycin A (0.05 lM) treatment decreased the intracellular APP (Fig. 3a, b). The polyclonal APP antibody consistently detected 3 isoforms of APP in SHSY5Y cells with molecular weights 130, 110 and 97 kDa, respectively (data not shown). The prominent APP band (110 kDa) shown in Fig. 2b was densitometrically analyzed for all our experimental purpose. Negletein Prevents Iron-Induced Changes of b-Secretase Activity The results of Fig. 4a show that b-secretase activity in FAC-treated SHSY5Y cells increased in a dose-dependent manner (133.3–400 lM) compared to control. The increase in b-secretase activity was, however, significantly prevented in FAC plus negletein-treated cells (Fig. 4b). Further, the co-treatment with NF-jB translocation inhibitor SN50 or the antioxidant NAC significantly prevented the increase in iron-induced b-secretase activity in SHSY5Y cells (Fig. 4b). The data in Fig. 4c show that negletein (20 lM) or SN50 (20 lM) or NAC (2.5 mM) did not have any direct quenching effect on the fluorescence-based assay of b-secretase. The inhibitor of b-secretase (unspecified concentration, provided with the commercia kit) produced 87 % inhibition of b-secretase activity when assay was performed following manufacturer’s protocol

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Fig. 3 Effects of iron and antimycin A on APP accumulation in SHSY5Y cells. SHSY5Y cells were treated without (control C) or with 66.6- or 133.3-lM FAC or 0.05-lM antimycin A (ANT) for 48 h, and cell lysate samples were subjected to SDS-PAGE and immunoblotting as described in the ‘‘Materials and Methods.’’ a APP immunoblot (representative). b Densitometric analysis. The results are the mean ± SEM of six experimental observations. Statistically significant, *p \ 0.001 versus C

with 2-ll inhibitor added to reaction mixture containing 2-ll active b-secretase (supplied with the kit) (Fig. 4c). The inhibitor produced a similar degree of inhibition of b-secretase in biological samples (data not shown). Negletein Diminishes Iron-Induced Release of Ab42 A marked increase (more than twofold) in the level of released Ab42 in culture supernatant from FAC-treated SHSY5Y cells was observed in comparison to that from control cells, which was prevented in FAC plus negleteintreated cells. (Fig. 5). Negletein Inhibits Hydroxyl Radical Formation from Iron-Ascorbate Combination The results shown in Table 1 show that the iron–ascorbateinduced increase in hydroxyl radical formation, measured by the enhanced fluorescence emission intensity of the incubation mixture in both benzoate hydroxylation and coumarin carboxylic acid assay, was markedly prevented by negletein in a dose-dependent manner. Typical hydroxyl

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radical scavengers such as mannitol (20 mM) or the metal chelator DTPA (0.1 mM) also strongly inhibited the phenomenon. Negletein at 10 lM concentration was equipotent with 20 mM mannitol in preventing hydroxyl radical formation (Table 1). Negletein Inhibits Amyloid Beta-Oligomerization During an incubation for 24 h, a significant degree of oligomerization of amyloid beta 42 took place as detected

Neuromol Med (2014) 16:787–798 b Fig. 4 b-Secretase activity in FAC-treated SHSY5Y cells. SHSY5Y

cells were treated without (control C) or with varying concentration of FAC (133.3–400 lM) in the absence or presence of other additions such as negletein (20 lM), SN50 (20 lM) or NAC (2.5 mM) for 48 h followed by the measurement of b-secretase activity as described in ‘‘Materials and Methods.’’ In another set of experiments, b-secretase assay was performed with purified b-secretase (provided with the kit) without (P) or with specific inhibitor (I). The direct quenching effect of negletein (20 lM, Neg#) or SN50 (20 lM, SN50#) or NAC (2.5 mM, NAC#) was measured by adding the respective compound in the assay mixture containing active purified b-secretase (P) at the end of the incubation as described in the text. a Dose-dependent effect of FAC on b-secretase activity. The results are the mean ± SEM of six experimental observations. Statistically significant, *p \ 0.01 versus C; Dp \ 0.001 versus C. b Effect of co-treatment with negletein (10/20 lM) or SN50 (20 lM) or NAC (2.5 mM) on bsecretase activity in SHSY5Y cells treated with 400-lM FAC. The activity of b-secretase in treated cells is expressed as the percentage increase over the enzyme activity of control cells. The results are the mean ± SEM of six experimental observations. Statistically significant, Dp \ 0.001 versus FAC. c Direct effect of negletein or SN50 or NAC on fluorescence-based assay of b-secretase. The activity of purified active b-secretase was expressed in arbitrary fluorescence units. Values are the mean ± SEM of three observations. Statistically significant, ***p \ 0.001 versus P

793 Table 1 Negletein prevents hydroxyl radical production in vitro from iron–ascorbate Incubation mixture (additions only)

Benzoate hydroxylation assay Fluorescence intensity (arbitrary units)

Coumarin carboxylic acid assay Fluorescence intensity (arbitrary units)

Fe2? ? Asc

257.95 ± 11.56

402.1 ± 6.3

Fe2? ? Asc ? Negletein (5 lM)

224.96 ± 4.47*

194.5 ± 6.2D

Fe2? ? Asc ? Negletein (10 lM)

138.84 ± 6.92D

109.5 ± 6.8D

Fe2? ? Asc ? Negletein (20 lM)

52.17 ± 5.49D

46.1 ± 7.2D

Fe2? ? Asc ? Mannitol (20 mM)

140.25 ± 4.54D

101.6 ± 3.5D

Fe2? ? Asc ? DTPA (100 lM)

182.12 ± 1.49D

289.3 ± 7.0D

The generation of hydroxyl radicals from a mixture of FeSO4 (20 lM, Fe2?) and ascorbate (2 mM, Asc) in the absence or presence of negletein (5–20 lM), mannitol (20 mM) and DTPA (100 lM) was measured by fluorescence-based benzoate hydroxylation and coumarin carboxylic acid assays as described in ‘‘Materials and Methods.’’ The results are the mean ± SEM of six observations Statistically significant, *p \ 0.05 versus Fe2? ? Asc; versus Fe2? ? Asc

D

p \ 0.001

Fig. 5 Immunoassay of amyloid beta 42 released in culture supernatant. Amyloid beta 42 released into the culture supernatant was measured as described in ‘‘Materials and Methods’’ from control (C), 400-lM FAC-treated (FAC) and 400-lM FAC and 20-lM negleteintreated (FAC ? Neg) SHSY5Y cells. The results are the mean ± SEM of six experimental observations. Statistically significant, h p \ 0.001 versus C; Dp \ 0.001 versus FAC

by thioflavin T assay (Fig. 6). In the presence of FAC (400 lM), the oligomerization was significantly increased, which again was strikingly inhibited by negletein (20 lM). When negletein (20 lM) was added to the reaction mixture containing Ab42 at the end of 24-h incubation period, no inhibition thioflavin T fluorescence was noticed (Fig. 6), indicating that negletein did not have any direct quenching effect on the fluorescence-based assay.

Fig. 6 Negletein prevents oligomerization of amyloid beta 42 in vitro. The oligomerization of amyloid beta 42 (Ab42) in vitro was allowed for a period of 24 h in the absence or presence of 400-lM FAC or 400-lM FAC and 20-lM negletein (Neg) and detected by thioflavin T assay as described in the ‘‘Materials and Methods.’’ The direct quenching effect of 20-lM negletein (Neg#) on thioflavin T fluorescence of Ab42 oligomers was checked by adding the compound after the incubation period as described in the text. The results are the mean ± SEM of four observations. Statistically significant, hp \ 0.001 versus Ab42; Dp \ 0.001 versus Ab42 ? FAC

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Fig. 7 Cell death assessment. a Cell death was assessed as described in ‘‘Materials and Methods’’ in control (C) and 66.6–400-lM FACtreated (FAC) SHSY5Y cells. b The viability of SHSY5Y cells was checked as mentioned in the text in control (C) or after exposure to 20-lM negletein (Neg) or 400-lM FAC (FAC) or 400-lM FAC and

20-lM negletein (FAC ? Neg) for 48 h. The results are the mean ± SEM of six experimental observations. Statistically significant, a #p \ 0.01 versus C; **p \ 0.001 versus C; b *p \ 0.01 versus C; hp \ 0.001 versus C; Dp \ 0.001 versus FAC. I Trypan blue exclusion assay. II LDH release assay

Dose-Dependent Cytotoxic Effect of FAC on SHSY5Y Cells: Prevention by Negletein

Discussion

The results in Fig. 7a demonstrate that the exposure of SHSY5Y cells to varying concentrations of FAC (66.6–400 lM) for 48 h led to statistically significant increase in cell death only with 266.6 lM (10 % increase) or 400 lM FAC (28 % increase), but not with lower concentrations of FAC exposure. A marginal loss of cell viability was noticed in 20-lM negletein-treated cells with respect to control as measured both by trypan blue exclusion technique and released LDH assay (Fig. 7b). However, in 400-lM FAC-treated cells, more extensive cell death was observed, which was markedly ameliorated by 20-lM negletein (Fig. 7b). In other experiments, antimycin A (0.05 lM) caused a comparable degree of cell death as that seen with 400-lM FAC treatment (data not shown).

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The total iron content of brain is high (0.7–3.6 mM varying from region to region), and significant amount of this iron remains as chelatable low-molecular mass form which helps in normal brain functions (Hallgren and Sourander 1958; Gutteridge 1992). In several pathological conditions, the brain content of iron is markedly elevated (Gutteridge 1992). Both postmortem examination and in vivo imaging have revealed significant accumulations of Fe in AD brains, and iron-induced oxidative damage has been widely implicated in AD (Vanhoutte et al. 2005; Smith et al. 1997). However, the role of this metal in regulating different steps of amyloid beta-metabolism has been less clearly defined, though significant work has been done on iron-mediated translational regulation of APP expression (Rogers et al. 2002; Beaudoin et al. 2008). In our present study, we have exposed SHSY5Y cells to FAC to induce

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the iron stress and measured different parameters of amyloid beta-metabolism. Our results show that Fe exposure marginally decreases the APP mRNA expression in SHSY5Y cells, which is unlikely to be biologically significant (Fig. 1). However, as observed by immunoblotting, a profound increase in the intracellular APP content occurs following treatment with varying concentrations of iron (Figs. 2, 3). This may be accounted by the known action of iron on translational upregulation of APP through removal of the iron-response element binding protein from the IRE site in the 50 -UTR of APP mRNA (Rogers et al. 2002; Beaudoin et al. 2008; Bandyopadhyay et al. 2013). More interestingly, however, our data show that the beta-secretase activity is also significantly increased by Fe treatment (Fig. 4a, b) which apparently is mediated by ROS-dependent mechanism as the process is markedly inhibited by Nacetylcysteine, which is a potent scavenger of oxygen free radicals and can also upregulate the intracellular level of reduced glutathione (Thakurta et al. 2012). The ROS can modulate the expression levels of diverse groups of genes such as antioxidant enzymes, cytoprotective and also proteins related to cell growth, differentiation and apoptosis through activation of redox-responsive kinases and transcription factors (Morel and Barouki 1999; Chakrabarti et al. 2013). In particular, ROS can activate the redoxsensitive transcription factor NF-kB, especially the heterodimer of p50/p65 (RelA), in multiple ways, allowing it to translocate in the nucleus and to upregulate the expression levels of a vast array of genes (Morgan and Liu 2011; Hayden and Ghosh 2004). The translocation of NF-kB to nucleus can be blocked by a cell membrane-permeable peptide SN50, and it is interesting to observe that this peptide significantly prevents the iron-induced increase in b-secretase activity (Fig. 4b) in our experimental system (Lin et al. 1995). Taken together, the effects of N-acetylcysteine and SN50 imply that FAC-induced increase in bsecretase activity is mediated by ROS-dependent activation of NF-kB and subsequent upregulation of b-secretase gene. This possibility is supported by the finding that NF-kB response element is present in the b-secretase promoter region and activation of this transcription factor leads to an over-expression of the b-secretase gene, while the p65 (RelA) knockout cells exhibit decreased b-secretase gene expression (Chami and Checler 2012; Sambamurti et al. 2004; Chen et al. 2012). Thus, in iron-treated SHSY5Y cells, the increased accumulation of APP with a concomitant rise in b-secretase activity is likely to increase the Ab42 production and its subsequent release in the medium, which is also verified from our results (Fig. 5). These effects of iron, as explained already, involve both a direct action of the metal and through ROS. In cells co-treated with FAC and negletein (10 or 20 lM), the effects of iron on amyloid beta-metabolism as observed here are markedly

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prevented leading to a decreased release of Ab42 in the medium, which is in conformity with the radical scavenging and iron-chelating effects of the flavone negletein. The flavonoid compounds including the flavones produce a variety of cellular effects in different experimental models when used in micromolar concentrations, and many of them are related to their metal-chelating and anti-oxidant effects (Middleton et al. 2000). The iron-chelating action of negletein is also well documented, and in physiological pH, its potency is comparable to a typical iron chelator such as desferrioxamine when the ratio of the concentrations of the flavone and metal varies from 10:1 to 1:1 (Mladeˇnka et al. 2011). Our results further confirm that negletein (10 or 20 lM) prevents iron-induced oxygen radical formation more potently than a strong metal chelator such as DTPA or a typical hydroxyl radical scavenger such as mannitol (Table 1). It is presumable that in our experimental system (Table 1), negletein prevents iron-induced hydroxyl radical formation by chelating iron as well as scavenging the radical directly like other flavones (Mladenka et al. 2011; Halliwell and Gutteridge 1998). Iron-dependent oligomerization and/or aggregation of amyloid beta-peptide which facilitates the toxic action of the peptide has been extensively studied in different systems (Huang et al. 2004). Negletein prevents the catalytic effects of iron on amyloid beta-oligomerization (Fig. 6), which would reinforce its beneficial effects on amyloid beta-toxicity. However, many flavonoid compounds have cytotoxic actions, which can offset some of their beneficial effects (Lombardo et al. 2013; Procha´zkova´ et al. 2011). We, therefore, examined the cytotoxic effects of negletein on SHSY5Y cells by cell death assays in concentrations at which it attenuates ironinduced APP and Ab42 accumulation. It is interesting that beneficial effects of negletein in our experimental system are observed at a concentration at which negletein is only mildly toxic to the cells (Fig. 7bI, bII). Further, ironmediated loss of cell viability is also prevented by negletein in agreement with its known ability to chelate iron (Fig. 7bI, bII). In this study, we have used wild-type SHSY5Y cells carrying normal APP gene instead of a cell line stably transfected with mutant APP gene and overexpressing the protein. The simple reasoning is that we are interested to find out how altered metabolic milieu of the cell could lead to transcriptional or translational alterations in the expressions of an otherwise normal APP gene or other genes linked to amyloid beta-peptide metabolism. Such an experimental system should be a good tool to study the pathogenesis of sporadic AD where familial AD mutations are absent. Further, for our experiments to elicit changes in amyloid beta-metabolism by iron, we have used FAC in the concentrations of 66.6–400 lM (Fig. 7a). Under exposure to higher concentrations of iron (400 lM), considerable

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changes in amyloid beta-metabolism occurs concomitant with significant loss of cell viability, which may imply that increased amyloid beta-metabolism is a response to cellular injury. However, 0.05 lM antimycin A, the complex III inhibitor, which causes cell death comparable to 400 lM FAC (data not shown), results in a fall of APP content instead of an increase (Fig. 3a). Further, with exposure to lower iron concentrations where cell death is marginal or similar to control, noticeable changes occur in intracellular APP content and b-secretase activity (Figs. 3a, b, 4a, 7a, b). Thus, cell death or injury per se is not the cause for iron-induced changes in amyloid beta-metabolism, and instead, iron may be playing a physiological role in regulating APP synthesis and processing. However, one valid question that needs to be addressed at this stage is whether the FAC concentrations used by us are comparable to brain iron content in physiological or pathological conditions. FAC has been used extensively in various cell lines in concentrations ranging from \5 lM to more than 500 lM to investigate the regulation of iron uptake, the irondependent toxicity and iron-responsive translational regulation of specific protein expression (Aracena et al. 2009; Aguirre et al. 2005; Hoepken et al. 2004; Wan et al. 2011; Mura et al. 2006; Rogers et al. 2002; Beaudoin et al. 2008; Hickok et al. 2011). However, it is important to emphasize that, though a rise in intracellular iron content occurs in cell lines following FAC treatment, the absolute values of intracellular iron as reported in different studies are not entirely consistent, and thus, it is somewhat difficult to compare these values with brain iron content in physiological and pathological conditions (Aguirre et al. 2005; Riemer et al. 2004; Hoepken et al. 2004; Mura et al. 2006). Various metal-chelating agents such as clioquinol, pyrrolidine dithiocarbamate and deferoxamine have been used in several AD transgenic animals where some improvement of behavioral deficits and decrease in multiple AD-related pathologies such as amyloid beta-peptide load, GSK3b activation and tau phosphorylation have been noted (Guo et al. 2013a, b; Prasanthi et al. 2012; Duce et al. 2011). Clinical trials in AD with several metal-chelating agents such as clioquinol, deferoxamine, d-penicillamine and deferiprone have met with limited success, but the toxicity of these compounds has been a matter of concern (Duce et al. 2011). The results of the present study have highlighted the beneficial effects of negletein in preventing iron-induced accumulation of APP and amyloid beta-peptide in cells of neural origin as well as in attenuating ironcatalyzed oligomerization of Ab42 in vitro. Although the results are obtained in a cell-based model or with in vitro experiments, the implications in AD pathogenesis are obvious, and thus, negletein should be explored further in AD animal models for its potential beneficial effects as a disease-modifying agent.

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Neuromol Med (2014) 16:787–798 Acknowledgments The work was supported by a Grant from Department of Biotechnology, Govt. of India, New Delhi. PB was supported by a Senior Research Fellowship from Department of Science and Technology, Govt. of India, New Delhi. Conflict of interest

The authors have no conflict of interests.

References Aguirre, P., Mena, N., Tapia, V., Arredondo, M., & Nunez, M. T. (2005). Iron homeostasis in neuronal cells: A role for IREG1. BioMed Central Neuroscience. doi:10.1186/1471-2202-6-3. Aracena, P., Aguirre, P., Munoz, P., & Nunez, M. T. (2009). Iron and glutathione at the crossroad of redox metabolism in neurons. Biological Research, 39, 157–165. Bandyopadhyay, S., Cahill, C., Balleidier, A., Huang, C., Lahiri, D. K., Huang, X., et al. (2013). Novel 50 untranslated region directed blockers of iron-regulatory protein-1 dependent amyloid precursor protein translation: Implications for down syndrome and Alzheimer’s disease. PLoS One, 8(7), e65978. Baptista, F. I., Henriques, A. G., Silva, A. M., Wiltfang, J., da Cruz, E., & Silva, O. A. (2014). Flavonoids as therapeutic compounds targeting key proteins involved in Alzheimer’s disease. ACS Chemical Neuroscience, 5(2), 83–92. Barnham, K. J., Kenche, V. B., Ciccotosto, G. D., Smith, D. P., Tew, D. J., Liu, X., et al. (2008). Platinum-based inhibitors of amyloid-b as therapeutic agents for Alzheimer’s disease. Proceedings of the National Academy Sciences of the United States of America, 105(19), 6813–6818. Beaudoin, M. E., Poirel, V.-J., & Krushel, L. A. (2008). Regulating amyloid precursor protein synthesis through an internal ribosomal entry site. Nucleic Acids Research, 36(21), 6835–6847. Belyaev, N. D., Kellett, K. A., Beckett, C., Makova, N. Z., Revett, T. J., & Nalivaeva, N. N. (2010). The transcriptionally active amyloid precursor protein (APP) intracellular domain is preferentially produced from the 695 isoform of APP in a {beta}secretase-dependent pathway. Journal of Biological Chemistry, 285(53), 41443–41454. Bonda, D. J., Lee, H., Blair, J. A., Zhu, X., Perry, G., & Smith, M. A. (2011). Role of metal dyshomeostasis in Alzheimer’s disease. Metallomics, 3(3), 267–270. Butterfield, D. A., Perluigi, M., Sultana, R., et al. (2006). Oxidative stress in Alzheimer’s disease brain: New insight from redox proteomics. European Journal of Pharmacology, 545(1), 39–50. Chakrabarti, S., Sinha, M., Thakurta, I. G., Banerjee, P., & Chattopadhyay, M. (2013). Oxidative stress and amyloid beta toxicity in Alzheimer’s disease: Intervention in a complex relationship by antioxidants. Current Medicinal Chemistry, 20(37), 4648–4664. Chami, L., & Checler, F. (2012). BACE1 is at the crossroad of a toxic vicious cycle involving cellular stress and b-amyloid production in Alzheimer’s disease. Molecular Neurodegeneration, 7, 52. doi:10.1186/1750-1326-7-52. Chen, C. H., Zhou, W., Liu, S., Deng, Y., Cai, F., Tone, M., et al. (2012). Increased NF-jB signalling up-regulates BACE1 expression and its therapeutic potential in Alzheimer’s disease. The International Journal of Neuropsychopharmacology, 15(1), 77–90. Choi, D. Y., Lee, Y. J., Hong, J. T., & Lee, H. J. (2012). Antioxidant properties of natural polyphenols and their therapeutic potentials for Alzheimer’s disease. Brain Research Bulletin, 87(2–3), 144–153. Clark, J. B., Bates, T. E., Boakye, P., Kuimov, A., & Land, J. M. (1997). Investigation of mitochondrial defects in brain and

Neuromol Med (2014) 16:787–798 skeletal muscle. In A. J. Turner & H. S. Bachelard (Eds.), Neurochemistry: A practical approach (pp. 151–174). New York: Oxford University Press Inc. Commenges, D., Scotet, V., Renaud, S., Jacqmin-Gadda, H., Barberger-Gateau, P., & Dartigues, J. F. (2000). Intake of flavonoids and risk of dementia. European Journal of Epidemiology, 16(4), 357–363. Dai, X., Sun, Y., Gao, Z., & Jiang, Z. (2010). Copper enhances amyloid-b peptide neuro-toxicity and non b-aggregation: A series of experiments conducted upon copper- bound and copperfree amyloid-b peptide. Journal of Molecular Neuroscience, 41(1), 66–73. Dragicevic, N., Smith, A., Lin, X., Yuan, F., Copes, N., Delic, V., et al. (2011). Green tea epigallocatechin-3-gallate (EGCG) and other flavonoids reduce Alzheimer’s amyloid-induced mitochondrial dysfunction. Journal of Alzheimer’s disease, 26(3), 507–521. Duce, J. A., Bush, A. I., & Adlard, P. A. (2011). Role of amyloidbeta-metal interactions in Alzheimer’s disease. Future Neurology, 6(5), 641–659. Guo, C., Wang, T., Zheng, W., Shan, Z. Y., Teng, W. P., & Wang, Z. Y. (2013a). Intranasal deferoxamine reverses iron-induced memory deficits and inhibits amyloidogenic APP processing in a transgenic mouse model of Alzheimer’s disease. Neurobiology of Aging, 34(2), 562–575. Guo, C., Wang, P., Zhong, M. L., Wang, T., Huang, X. S., Li, J. Y., et al. (2013b). Deferoxamine inhibits iron induced hippocampal tau phosphorylation in the Alzheimer transgenic mouse brain. Neurochemistry International, 62(2), 165–172. Gutteridge, J. M. C. (1992). Iron and oxygen radicals in brain. Annals of Neurology, 32(S1), S16–S21. Hallgren, B., & Sourander, P. (1958). The effect of age on the nonhaemin iron in the human brain. Journal of Neurochemistry, 3(1), 41–51. Halliwell, B., & Gutteridge, J. M. C. (1998). Free radicals in biology and medicine. Oxford: Oxford University Press. Hayden, M. S., & Ghosh, S. (2004). Signaling to NF-kB. Genes and Development, 18(18), 2195–2224. Hickok, J. R., Sahni, S., Mikhed, Y., Bonini, M. G., & Thomas, D. D. (2011). Nitric oxide suppresses tumor cell migration through N-Myc downstream-regulated gene-1 (NDRG1) expression role of chelatable iron. The Journal of Biological Chemistry, 286(48), 41413–41424. Hoepken, H. H., Korten, T., Robinson, S. R., & Dringen, R. (2004). Iron accumulation, iron-mediated toxicity and altered levels of ferritin and transferring receptor in cultured astrocytes during incubation with ferric ammonium citrate. Journal of Neurochemistry, 88, 1194–1202. Huang, X., Atwood, C. S., Moir, R. D., Hartshorn, M. A., Tanzi, R. E., & Bush, A. I. (2004). Trace metal contamination initiates the apparent auto-aggregation, amyloi- dosis, and oligomerization of Alzheimer’s Ab peptides. Journal of Biological Inorganic Chemistry, 9(8), 954–960. Hyman, B. T., Phelps, C. H., Beach, T. G., Bigio, E. H., Cairns, N. J., Carrillo, M. C., et al. (2012). National institute on agingAlzheimer’s association guidelines for the neuropathologic assessment of Alzheimer’s disease. Alzheimers and Dementia, 8(1), 1–13. Jana, S., Sinha, M., Chanda, D., Roy, T., Banerjee, K., Munshi, S., et al. (2011). Mitochondrial dysfunction mediated by quinone oxidation products of dopamine: Implications in dopamine cytotoxicity and pathogenesis of Parkinson’s disease. Biochimica et Biophysica Acta, 1812(6), 663–673. Jomova, K., Vondrakova, D., Lawson, M., & Valko, M. (2010). Metals, oxidative stress and neurodegenerative disorders. Molecular and Cellular Biochemistry, 345(1–2), 91–104.

797 Kanazawa, K., Uehara, M., Yanagitani, H., & Hashimoto, T. (2006). Bioavailable flavonoids to suppress the formation of 8-OHdG in HepG2 cells. Archives of Biochemistry and Biophysics, 455(2), 2197–2203. Khemka, V. K., Bagchi, D., Bandyopadhyay, K., Bir, A., Chattopadhyay, M., Biswas, A., et al. (2014). Altered serum levels of adipokines and insulin in probable Alzheimer’s disease. Journal of Alzheimers Disease. doi:10.3233/JAD-140006. Li, Y. P., Bushnell, A. F., Lee, C. M., Perlmutter, L. S., & Wong, S. K. (1996). Beta-amyloid induces apoptosis in human-derived neurotypic SH-SY5Y cells. Brain Research, 738(2), 196–204. Li, G., Zou, L. Y., Cao, C. M., & Yang, E. S. (2005). Coenzyme Q10 protects SHSY5Y neuronal cells from beta amyloid toxicity and oxygen-glucose deprivation by inhibiting the opening of the mitochondrial permeability transition pore. Biofactors, 25(1–4), 97–107. Lin, Y.-Z., Yao, S. Y., Veach, R. A., Torgerson, T. R., & Hawiger, J. (1995). Inhibition of nuclear translocation of transcription factor NF-jB by a synthetic peptide containing a cell membranepermeable motif and nuclear localization sequence. The Journal of Biological Chemistry, 270(24), 14255–14258. Lombardo, E., Sabellico, C., Ha´jek, J., Stanˇkova´, V., Filipsky´, T., Balducci, V., et al. (2013). Protection of cells against oxidative stress by nanomolar levels of hydroxyflavones indicates a new type of intracellular antioxidant mechanism. PLoS One, 8(4), e60796. Lovell, M. A., Robertson, J. D., Teesdale, W. J., Campbell, J. L., & Markesbery, W. R. (1998). Copper, iron and zinc in Alzheimer’s disease senile plaques. Journal of the Neurological Sciences, 158(1), 47–52. Maca´kova´, K., Mladeˇnka, P., Filipsky´, T., Rˇ´ıha, M., Jahoda´rˇ, L., Trejtnar, F., et al. (2012). Iron reduction potentiates hydroxyl radical formation only in flavonols. Food Chemistry, 135(4), 2584–2592. Middleton, E, Jr, Kandaswami, C., & Theoharides, T. C. (2000). The effects of plant flavonoids on mammalian cells: Implications for inflammation, heart disease, and cancer. Pharmacological Reviews, 52(4), 673–751. Mills, E., Dong, X.-P., Wang, F., & Xu, H. (2010). Mechanisms of brain iron transport: Insight into neurodegeneration and CNS disorders. Future Medicinal Chemistry, 2(1), 51–64. Mladeˇnka, P., Maca´kova´, K., Filipsky´, T., Zatloukalova´, L., Jahoda´rˇ, L., Bovicelli, P., et al. (2011). In vitro analysis of iron chelating activity of flavonoids. Journal of Inorganic Biochemistry, 105(5), 693–701. Morel, Y., & Barouki, R. (1999). Repression of gene expression by oxidative stress. The Biochemical Journal, 342(3), 481–496. Morgan, M. J., & Liu, Z-g. (2011). Crosstalk of reactive oxygen species and NF-jB signaling. Cell Research, 21(1), 103–115. Mura, C. V., Delgado, R., Aguirre, P., Bacigalupo, J., & Nu´n˜ez, M. T. (2006). Quiescence induced by iron challenge protects neuroblastoma cells from oxidative stress. Journal of Neurochemistry, 98(1), 11–19. Nakamura, M., Shishido, N., Nunomura, A., Smith, M. A., Perry, G., Hayashi, Y., et al. (2007). Three histidine residues of amyloidbeta peptide control the redox activity of copper and iron. Biochemistry, 46(44), 12737–12743. Olivieri, G., Baysang, G., Meier, F., Mu¨ller-Spahn, F., Sta¨helin, H. B., Brockhaus, M., et al. (2001a). N-acetyl-L-cysteine protects SHSY5Y neuroblastoma cells from oxidative stress and cell cytotoxicity: Effects on beta-amyloid secretion and tau phosphorylation. Journal of Neurochemistry, 76(1), 224–233. Olivieri, G., Hess, C., Savaskan, E., Ly, C., Meier, F., Baysang, G., et al. (2001b). Melatonin protects SHSY5Y neuroblastoma cells from cobalt-induced oxidative stress, neurotoxicity and increased beta-amyloid secretion. Journal of Pineal Research, 31(4), 320–325.

123

798 Olivieri, G., Otten, U., Meier, F., Baysang, G., Dimitriades-Schmutz, B., Mu¨ller-Spahn, F., et al. (2003). Beta-amyloid modulates tyrosine kinase B receptor expression in SHSY5Y neuroblastoma cells: Influence of the antioxidant melatonin. Neuroscience, 120(3), 659–665. Page, M., & Thorpe, R. (2002). Protein blotting by electroblotting. In J. M. Walker (Ed.), The protein protocols handbook (pp. 317–319). New Jersey: Humana Press. Pfaffl, M. W. (2001). A new mathematical model for relative quantitative real-time RT-PCR. Nucleic Acids Research, 29(9), 2002–2007. Prasanthi, J. R., Huls, A., Thomasson, S., Thompson, A., Schommer, E., & Ghribi, O. (2009). Differential effects of 24-hydroxycholesterol and 27-hydroxycholesterol on b-amyloid precursor protein levels and processing in human neuroblastoma SHSY5Y cells. Molecular Neurodegeneration, 4, 1. doi:10.1186/ 1750-1326-4-1. Prasanthi, J. R., Schrag, M., Dasari, B., Marwarha, G., Dickson, A., Kirsch, W. M., et al. (2012). Deferiprone reduces amyloid-b and tau phosphorylation levels but not reactive oxygen species generation in hippocampus of rabbits fed a cholesterol-enriched diet. Journal of Alzheimer’s Disease, 30(1), 167–182. Procha´zkova´, D., Bousˇova´, I., Wilhelmova´, N., et al. (2011). Antioxidant and prooxidant properties of flavonoids. Fitoterapia, 82(4), 513–523. Randall, C. N., Strasburger, D., Prozonic, J., Morris, S. N., Winkie, A. D., Parker, G. R., et al. (2009). Cluster analysis of risk factor genetic polymorphisms in Alzheimer’s disease. Neurochemical Research, 34(1), 23–28. Reddy, P. H., & Beal, M. F. (2008). Amyloid beta, mitochondrial dysfunction and synaptic damage: Implications for cognitive decline in aging and Alzheimer’s disease. Trends in Molecular Medicine, 14(2), 45–53. Riemer, J., Hoepken, H. H., Czerwinska, H., Robinson, S. R., & Dringen, R. (2004). Colorimetric ferrozine-based assay for the quantitation of iron in cultured cells. Analytical Biochemistry, 331(2), 370–375. Righi, G., Antonioletti, R., Silvestri, I. P., D’Antona, N., Lambusta, D., & Bovicelli, P. (2010). Convergent synthesis of mosloflavone, negletein and baicalein from crysin. Tetrahedron, 66(2010), 1294–1298. Rogers, J. T., Randall, J. D., Cahill, C. M., Eder, P. S., Huang, X., Gunshin, H., et al. (2002). An iron-responsive element type II in the 50 -untranslated region of the Alzheimer’s amyloid precursor protein transcript. The Journal of biological Chemistry, 277(47), 45518–45528. Sambamurti, K., Kinsey, R., Maloney, B., Ge, Y. W., & Lahiri, D. K. (2004). Gene structure and organization of the human betasecretase (BACE) promoter. Federation of American Societies for Experimental Biology Journal, 18, 1034–1036. Sato, N., & Morishita, R. (2013). Roles of vascular and metabolic components in cognitive dysfunction of Alzheimer disease: Short- and long-term modification by non-genetic risk factors. Frontiers in Aging Neuroscience, 5(1), 64. Sinha, M., Behera, P., Bhowmick, P., Banerjee, K., Basu, S., & Chakrabarti, S. (2011). Aging promotes amyloid-b peptide

123

Neuromol Med (2014) 16:787–798 induced mitochondrial dysfunctions in rat brain: A molecular link between aging and Alzheimer’s disease. Journal of Alzheimer’s Disease, 27(4), 753–765. Sinha, M., Bhowmick, P., Banerjee, A., & Chakrabarti, S. (2013). Antioxidant role of amyloid b protein in cell-free and biological systems: Implication for the pathogenesis of Alzheimer disease. Free Radical Biology and Medicine, 56(1), 184–192. Smith, D. G., Cappai, R., & Barnham, K. J. (2007a). The redox chemistry of the Alzheimer’s disease amyloid b peptide. Biochimica et Biophysica Acta, 1768(8), 1976–1990. Smith, D. P., Ciccotosto, G. D., Tew, D. J., Fodero-Tavoletti, M. T., Johanssen, T., & Masters, C. L. (2007b). Concentration dependent Cu2þ induced aggregation and dityrosine formation of the Alzheimer’s disease amyloid-b peptide. Biochemistry, 46(10), 2881–2891. Smith, M. A., Harris, P. L. R., Sayre, L. M., & Perry, G. (1997). Iron accumulation in Alzheimer disease is a source of redoxgenerated free radicals. Proceedings of the National Academy of Sciences of the United States of America, 94(18), 9866–9868. Solano, D. C., Sironi, M., Bonfini, C., Solerte, S. B., Govoni, S., & Racchi, M. (2000). Insulin regulates soluble amyloid precursor protein release via phosphatidyl inositol 3 kinase-dependent pathway. Federation of American Societies for Experimental Biology Journal, 14(7), 1015–1022. Swerdlow, R. H. (2007). Pathogenesis of Alzheimer’s disease. Clinical Interventions in Aging, 2(3), 347–359. Symonowicz, M., & Kolanek, M. (2012). Flavonoids and their properties to form chelate complexes. Biotechnology and Food Science, 76(1), 35–41. Thakurta, I. G., Chattopadhyay, M., Ghosh, A., & Chakrabarti, S. (2012). Dietary supplementation with N-acetyl cysteine, atocopherol and a-lipoic acid reduces the extent of oxidative stress and proinflammatory state in aged rat brain. Biogerontology, 13(5), 479–488. Vanhoutte, G., Dewachter, I., Borghgraef, P., & Van Leuven, A. (2005). Non invasive in vivo MRI detection of neuritic plaques associated with iron in APP[V7171] transgenic mice, a model for Alzheimer’s disease. Magnetic Resonance in Medicine, 53(3), 607–613. Wan, L., Nie, G., Zhang, J., Luo, Y., Zhang, P., & Zhang, Z., et al. (2011). b-Amyloid peptide increaes levels of iron content and oxidative stress in human cell and Caenorhabditis elegans models of Alzheimer disease. Free Radical Biology & Medicine, 50(1), 122–129. Xiong, Z., Hongmei, Z., Lu, S., & Yu, L. (2011). Curcumin mediates presenilin-1 activity to reduce b-amyloid production in a model of Alzheimer’s Disease. Pharmacological Reports, 63(5), 1101–1108. Zheng, L., Calvo-Garrido, J., Hallbeck, M., Hultenby, K., Marcusson, J., & Cedazo-Minguez, A. (2013). Intracellular localization of amyloid-b peptide in SH-SY5Y neuroblastoma cells. Journal of Alzheimer’s Disease, 37(4), 713–733.