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TEMPOL protects human neuroblastoma SH-SY5Y cells against ß-amyloid-induced cell toxicity
European Journal of Pharmacology 650 (2011) 544–549
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Neuropharmacology and Analgesia
TEMPOL protects human neuroblastoma SH-SY5Y cells against ß-amyloid-induced cell toxicity Pennapa Chonpathompikunlert a,b,c,d,e, Junkyu Han f, Kazuko Toh a,b,c,d,e, Hiroko Isoda f, Yukio Nagasaki a,b,c,d,e,⁎ a
Department of Materials Science, Graduate School of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8503, Japan Tsukuba Research Center for Interdisciplinary Materials Science (TIMS), University of Tsukuba, Tsukuba, Ibaraki 305-8503, Japan Center for Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Tsukuba, Ibaraki 305-8503, Japan d Master's School of Medical Sciences, Graduate School of Comprehensive Human Science, International Center for Materials Nanoarchitectonics Satellite (MANA), University of Tsukuba, Tsukuba, Ibaraki 305-8503, Japan e National Institute for Materials Science (NIMS), University of Tsukuba, Tsukuba, Ibaraki 305-8503, Japan f Graduate School of Life and Environmental Sciences, The Alliance for Research on North Africa, University of Tsukuba, Tennoudai 1-1-1, Tsukuba, Ibaraki 305-8572, Japan b c
a r t i c l e
i n f o
Article history: Received 31 March 2010 Received in revised form 1 October 2010 Accepted 6 October 2010 Available online 20 October 2010 Keywords: Amyloid-β peptide Neuroblastoma SH-SY5Y cell Superoxide anion generation Hydroxyl radical generation Apoptosis Alzheimer's disease
a b s t r a c t Amyloid-β peptide (Aβ) has been implicated in the pathogenesis of Alzheimer's disease (AD). It can cause cell death in Alzheimer's disease by evoking a cascade of oxidative damage to neurons. Antioxidant compounds may help to elucidate and develop a treatment for Alzheimer's disease. In the present study, we investigated the protective effect of TEMPOL (4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy), a cyclic nitroxide which is particularly effective at reducing oxidative injury, on Aβ1–42-induced SH-SY5Y cell toxicity. Exposure of cells to 20 μM Aβ1–42 for 48 h caused viability loss and apoptotic increase, and pre-treatment with TEMPOL for 24 h significantly reduced the viability loss and apoptotic rate. In addition, TEMPOL inhibited Aβ1–42-induced superoxide anion generation and hydroxyl radical generation to a striking degree. Based on these results, it is concluded that TEMPOL effectively protects SH-SY5Y cells against β-amyloid-induced damage by suppressing the generation of reactive oxygen species especially, superoxide anion. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Alzheimer's disease (AD) is neuropathologically characterized by the deposition of β-amyloid (Aβ) plaques and intracellular neurofibrillary tangles and the loss of neurons in the brain. Although the cause of Alzheimer's disease is not fully understood, several lines of evidence suggest that Aβ-induced oxidative stress plays an important role in the pathogenesis and progression of Alzheimer's disease (Butterfield et al., 2001). For example, it is reported that Aβ induces oxidative stress (Hensley et al., 1994), and that oxidative stress promotes the further production of Aβ by potentiating BACE1 gene expression and Aβ generation (Tamagno et al., 2008). Recent studies have shown that oxidative stress contributes to Aβ accumulation: Aβ, in turn, induces oxidative stress and a lipid peroxidation product, 4hydroxy-2-trans-nonenal (HNE), whose production results in increased levels of β- and γ-secretases, which further enhances Aβ production (Shen et al., 2008; Tamagno et al., 2008), thereby inducing
⁎ Corresponding author. Tsukuba Research Center for Interdisciplinary Materials Science (TIMS), University of Tsukuba, 1-1-1 Ten-noudai, Tsukuba 305-8573, Japan. Tel./fax: +81 29 853 5749. E-mail address: [email protected] (Y. Nagasaki). 0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2010.10.028
a vicious circle of Aβ production/accumulation, oxidative stress generation and the generation of β-/γ-secretase, enzymes that cleaves amyloid precursor protein (APP). Thus, one promising preventive or therapeutic measure in Alzheimer's disease may be to attenuate or suppress the oxidative stress-dependent, Aβ-mediated cytotoxicity. Several studies have shown that 4-hydroxy-2,2,6,6 tetramethylpiperidine-N-oxyl (TEMPOL) is a stable, cell membrane-permeable radical scavenger. As a superoxide dismutase-mimetic compound (Thiemermann, 2003; Yamada et al., 2003), TEMPOL attenuates the effects of superoxide radicals (Krishna et al., 1996), directly reacts with both carbon-centered and peroxy radicals (Chateauneuf et al., 1988), prevents the reduction of hydrogen peroxide to the hydroxyl radical (Samuni et al., 1991), and provides neuroprotection in rat models of transient focal ischemia (Rak et al., 2000), in mouse models of 1-methyl-4-phenylpyridinium (MPP+)-induced Parkinson's disease (Matthews et al., 1999) and in MN9D dopaminergic mesencephalic cell from 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease (Liang et al., 2005). Although TEMPOL is known to exhibit neuroprotective activity, the protective effect of TEMPOL against Aβinduced cytotoxicity has not been reported. In the present study, we investigated the effects of TEMPOL on Aβ-induced neurotoxicity in SH-SY5Y cells. Our study verified that TEMPOL has a neuroprotective effect against Aβ-induced cell toxicity.
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2. Materials and methods
2.4. Measurement of hydroxyl radical generation
Unless otherwise stated, amyloid β-protein(1-42), was purchased from Bachem AG Japan, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1oxyl (TEMPOL) from Sigma Co., USA. and WST-8 from DOJINDO Laboratories, Kumamoto, Japan. The Cell Death Detection ELISA Kits were purchased from Cell Biolabs, Inc., USA. All Detect Lipid Peroxidation Assay Kits and proteins were purchased from Sigma Co., USA. All other chemicals were of analytic grade.
The production of hydroxyl radicals by SH-SY5Y cells in the absence or presence of Aβ1–42 was evaluated using the deoxyribose assay, based on the principle that the pentose sugar 2-deoxyribose can be cleaved by hydroxyl radicals to release a substance that reacts, on heating, with thiobarbituric acid to produce a pink chromogen (Halliwell et al., 1988; Morel et al., 1990). Briefly, confluent SHSY5Y cells grown in 6-well plates were incubated for 48 h with DMEM containing 3 mM deoxyribose in the presence or absence of 20 μM Aβ1–42. In addition, SH-SY5Y cells were pre-incubated for 24 h with DMEM containing SOD (1000 U/ml), catalase (1,000 U/ml) or TEMPOL (1 and 0.5 mM) prior to exposure to Aβ1–42 for 48 h. At the end of the incubation period, the cell supernatant was removed and assayed for thiobarbituric substances acid-reactive to malondialdehyde (MDA), as described previously (Mihara and Uchiyama, 1978). An aliquot of the cell supernatant was also used for protein quantification by the Lowry method (Lowry et al., 1951) and the hydroxyl radical content of the samples was expressed as nmol/mg protein.
2.1. Cell culturing and treatment with Aß1–42 and/or substances A human SH-SY5Y neuroblastoma cell line was obtained from Isoda Laboratory, University of Tsukuba, Japan. SH-SY5Y neuroblastoma cells were cultured in flasks or on 6-, 24-, 48- or 96-well plates for different purposes, with a 1:1(v/v) mixture of Dulbecco's minimum essential medium (DMEM; Sigma, USA) and Ham's F-12 nutrient mixture (Sigma, USA) supplemented with 15% fetal bovine serum (FBS; Sigma, USA), 1% non-essential MEM amino acid and 1% penicillin (5000 μg/ml)-streptomycin (5000 IU/ml) solution (ICN Biomedicals, Inc.) at 37 °C in a 95% humidified air–5% CO2 incubator. A serum-free modification of Eagle's minimum essential medium (OPTI-MEM Gibco BRL) was used for the cell toxicity assays (Isoda et al., 2002). For this experiment, cell attachment was required; hence, the plates were pretreated overnight with 50 μg/ml fibronectin, which was refined from human plasma (Wako Pure Chemical Industries, Ltd.). In order to achieve aggregation, Aβ1–42 was dissolved in the culture medium and incubated at 37 °C for 72 h prior to use. After 24–48 h of culturing, the cells (which had reached approximately 80% confluence) were washed three times with a culture medium and thereafter incubated for 48 h in Dulbecco's MEM medium without serum in the presence of different concentrations of Aβ1–42. In the experiments designed to examine the protective effect of TEMPOL, the cells were treated overnight with different concentrations of TEMPOL prior to exposure to Aβ1–42 for 48 h.
2.5. Measurement of apoptotic cell death The DNA damage (DNA fragments) of the cell lysates was measured using the Cell Death Detection ELISA Kit according to the manufacturer's protocol (Cell Biolabs, Inc., USA). The cells were treated as described previously prior to ELISA detection, and spectrophotometric data were obtained from measurement at 405 nm against the substrate solution as a blank. 10 μg/ml of camptothecin was employed as a positive control, which is known as a topoisomerase inhibitor that induces apoptosis to cell. 2.6. Statistical analysis All data are presented as means ± S.E.M. Statistical analysis was performed by one-way analysis of variance (ANOVA). A probability value of less than 0.05 was considered significant.
2.2. Measurement of cell viability
3. Results
Cell viability was determined by a quantitative colorimetric assay using WST-8 (DOJINDO Laboratories, Kumamoto, Japan). Briefly, 1 × 104 cells/well were seeded on a 96-well flat-bottom plate at 37 °C overnight. Once the cells were attached, they were washed once with 100 μl of OPTI-MEM medium, followed by the addition of 100 μl of fresh OPTI-MEM medium. The cells were then allowed to grow for 72 h in the desired final concentrations of TEMPOL with 10% (v/v) and 10 μl of 20 μM Aβ1–42 added. After treatment, 10 μl of WST-8 reagent was added to each well containing 100 μl of medium, according to the manufacturer's instructions. The plate was incubated for 1 h at 37 °C and measured at an absorbance of 450 nm by a plate reader.
3.1. Effect of Aß1–42 and TEMPOL on cell viability in SH-SY5Y cells
2.3. Measurement of superoxide anion generation The generation of superoxide anions was determined by means of the nitroblue tetrazolium (NBT) assay described previously (Falasca et al., 1993; Scheid et al., 1996). Briefly, SH-SY5Y cells grown in 24well plates were incubated for 48 h with DMEM containing 25 μg/ml NBT in the presence or absence of 20 μM Aβ1–42. In addition, SH-SY5Y cells were pre-incubated for 24 h with DMEM containing superoxide dismutase (SOD) (EC 1.15.1.1; 1,000 U/ml), catalase (EC 1.11.1.6; 1,000 U/ml) or TEMPOL (1 and 0.5 mM) prior to exposure to Aβ1–42 for 48 h. The concentrations of SOD and catalase were based on the concentrations used in a previous study ranging from 100 to 10,000 U/ ml (Scheid et al., 1996; Thamilselvan et al., 2000), and those of TEMPOL were based on those known to provide significant protection against Aβ1–42 toxicity.
Initial experiments were performed to determine whether Aβ1–42 alone or TEMPOL alone was toxic to human neuroblastoma cells (SHSY5Y) by measuring the WST in the culture medium. The reduction of WST was employed here as an indicator of neural cytotoxicity (Ngamwongsatit et al., 2008). Incubation of confluent SH-SY5Y cells with Aβ1–42 (1.5–20 μM) for 48 h resulted in a significant reduction in cellular viability from 100% for the control (untreated) cells to 30% (P b 0.01) at the highest concentration tested, as well as a dosedependent decline in WST reduction by the cells, as shown in Fig. 1, indicating that Aβ1–42 is strongly cytotoxic to SH-SY5Y cells. In contrast, TEMPOL showed cytotoxicity at a concentration of more than 3 mM, as indicated by the decrease in the measured WST (Fig. 2). Based on the dose–response data from Figs. 1 and 2, the concentration of 20 μM Aβ1–42 was chosen to evaluate the influence of low concentrations of TEMPOL (0.01–3 mM) on Aβ1–42 toxicity in SHSY5Y cells. 3.2. Effect of TEMPOL on ß-amyloid-induced cell toxicity To confirm the protective effect of TEMPOL on Aβ1–42 toxicity, SHSY5Y cells were treated with Aβ1–42 alone or TEMPOL plus Aβ1–42 in a serum-free medium. Aβ1–42 alone significantly decreased cell viability, whereas the combination of Aβ1–42 and TEMPOL showed a unique behavior: the cell viability increased with increasing TEMPOL concentration up to 1.0 mM, then gradually decreased (Fig. 3).
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Fig. 1. Effect of Aβ1–42 on cell viability. SH-SY5Y cells were incubated with increasing concentrations of Aβ1–42 for 48 h. *P b 0.05, **P b 0.01 vs. control (untreated group) analyzed using one-way ANOVA followed by Dunnett's post-hoc test, n = 7.
3.3. Effect of TEMPOL on ß-amyloid-induced reactive oxygen species Exposure of SH-SY5Y cells to 20 μM Aβ1–42 for 48 h caused a significant, 48% increase in superoxide generation (measured as NBT absorbance) compared to the control (untreated) cells (P b 0.01) (Fig. 4A), with a concomitant increase in hydroxyl radical production (P b 0.01) (Fig. 4B). As anticipated, the addition of SOD (1000 U/ml) significantly reduced the Aβ1–42-induced superoxide generation, by 53% (P b 0.01), whereas catalase (1000 U/ml) did not have a significant effect (Fig. 4A). TEMPOL (0.5 and 1 mM) also caused a significant reduction in Aβ1–42-mediated superoxide generation, by 54% (P b 0.01) and 40% (P b 0.05), respectively (Fig. 4A). In contrast, SOD had no effect upon Aβ1–42-mediated hydroxyl radical generation, whereas catalase caused a significant reduction, by 75% (P b 0.01) (Fig. 4B). TEMPOL also caused significant reductions in Aβ1–42mediated hydroxyl radical production, by 35% (P b 0.05) and 60% (P b 0.05), respectively (Fig. 4B). 3.4. Effect of TEMPOL protection against Aß1–42-induced apoptosis in SH-SY5Y cells Biochemically, one hallmark of apoptosis is DNA fragmentation in the nucleus. This parameter was measured to determine if TEMPOL would prevent Aβ1–42-induced apoptosis in SH-SY5Y cells. Aβ1–42 has
Fig. 2. Effect of TEMPOL on cell viability. SH-SY5Y cells were incubated with increasing concentrations of TEMPOL for 24 h. *P b 0.05, **P b 0.01, ***P b 0.001 vs. control (untreated group) analyzed using one-way ANOVA followed by Dunnett's post-hoc test, n = 7.
Fig. 3. Effect of TEMPOL pre-treatment on the viability of SH-SY5Y cells exposed to 20 μM Aβ1–42. SH-SY5Y cells were treated with different concentrations of TEMPOL (0– 3 mM) for 24 h prior to the addition of Aβ1–42 to the culture medium. Forty-eight hours following Aβ1–42 treatment, the cell viability was assessed by WST assay. The data are expressed as means ± S.E.M. *P b 0.05 and **P b 0.01, when the cell viability is compared with that of the cells treated with only 20 μM Aβ1–42 (no TEMPOL pre-treatment); # P b 0.05, when the cell viability is compared with that of the control (untreated group). Analyzed using one-way ANOVA followed by Dunnett's post-hoc test, n = 7.
been reported to induce apoptosis in SH-SY5Y cells, as evidenced by an increases in the number of DNA fragments in the cell lysates (YanPing et al., 1996). Fig. 5 shows the results of our quantitative analysis of DNA fragmentation. It was confirmed that Aβ1–42 significantly increased the number of DNA fragments in SH-SY5Y cells. Pretreatment of the cells with 0.5 and 1 mM TEMPOL markedly decreased the number of DNA fragments in the cell lysates. 4. Discussion Aβ, the major constituent of the senile plaques in Alzheimer's disease, contains a 39–43 amino-acid peptide and is produced by the proteolytic processing of APP, a transmembrane glycoprotein expressed during normal cellular metabolism (Haass, 1996; Selkoe, 1996). Aβ, as well as an 11-amino-acid fragment thereof (i.e., Aβ25–35), can have a neurotoxic effect through a mechanism linked to peptide fibril formation (Kowall et al., 1992; Yankner, 1996) and, consequently, the corresponding synthetic peptides containing residues 25–35, 1–40 or 1–42 are convenient tools for the investigation of Alzheimer's disease. The overwhelming majority of evidence suggests that particularly Aβ1–42 plays a central role in the pathogenesis of Alzheimer's disease. Most of this evidence is genetic: defects in the genes encoding presenilin-1, presenilin-2 and APP are invariably accompanied by elevated levels of Aβ1–42 (Butterfield and Bush, 2004). In the present study, treatment of SH-SY5Y cells with Aβ1–42 clearly decreased cell viability in a dose-dependent manner, thereby providing direct evidence of cellular toxicity induced by Aβ, a result that strongly resembles our previous findings with PC12 cells (Guan et al., 2001, 2003a,b). Recent research has provided convincing evidence that Aβ-associated free-radical damage to neuron is a fundamental process in connection with Alzheimer's disease (Miranda et al., 2000). The implication of reactive oxygen species in the pathogenesis of Alzheimer's disease has led to the notion that exogenous antioxidants or strategies to increase the expression of endogenous antioxidants would constitute useful therapeutic and prophylactic strategies for this disease (Rutten et al., 2002). Several types of exogenously administered medicinal plants (Kim et al., 2007), melatonin (Raghavendra and Kulkarni, 2001) and vitamin E (Boothby and Doering, 2005) have been proposed as drugs for the prophylaxis and therapy of
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Fig. 5. Quantification of nucleosomal DNA fragmentation. The Cell Death Detection ELISA Kit was used to quantify DNA fragmentation in SH-SY5Y cells undergoing apoptosis in the presence of 20 μM Aβ1–42 without or with TEMPOL and camptothecin (10 μg/ml). Cells were grown in 48-well plates and incubated for 48 h. As a control, untreated cells, cells treated with Aβ1–42 (20 μM) only, or cells treated with Aβ1–42 (20 μM) plus TEMPOL (0.5 and 1 mM) were cultured, and positive controls were also cultured with 10 μg/ml of camptothecin, which is a topoisomerase inhibitor that induces apoptosis to cell. The rate of apoptosis is reflected by the enrichment of nucleosomes in the cytoplasm, shown along the y-axis. Experiments were performed in triplicate and repeated three times. #P b 0.001 vs. control (untreated cells), **P b 0.01 vs. 20 μM Aβ1–42 only, analyzed using one-way ANOVA, followed by Dunnett's post-hoc test, n = 5.
Fig. 4. Effect of SOD, catalase and TEMPOL on the Aβ1–42-mediated generation of (A) superoxide anions and (B) hydroxyl radicals. SH-SY5Y cells were incubated for 48 h with 20 μM Aβ1–42 in the absence or presence of TEMPOL, SOD (1,000 U/ml) and catalase (CAT: 1,000 U/ml). #P b 0.01 vs. control (untreated cells), *P b 0.05 and **P b 0.01 vs. 20 μM Aβ1–42, analyzed using one-way ANOVA, followed by Dunnett's post-hoc test, n = 5.
Alzheimer's disease. The present study adds the stable nitroxide antioxidant, TEMPOL, to this list. Unlike the other agents proposed to date, the antioxidant activity of TEMPOL is mediated by several simultaneously operational mechanisms. TEMPOL works as a superoxide dismutase mimic (Samuni et al., 1991) and directly reacts with both carbon-centered and peroxy radicals (Chateauneuf et al., 1988). The results obtained in this investigation indicate that Aβ1–42 exerts a concentration-dependent toxic effect on confluent neuroblastoma SH-SY5Y cells. This neurotoxic action was significantly reduced by TEMPOL, though TEMPOL itself was found to be neurotoxic at higher concentrations. The role of superoxide generation and subsequent hydroxyl radical production by Aβ1–42 was confirmed, as were the SOD properties of TEMPOL. The ability of TEMPOL to reduce hydroxyl radical generation was also demonstrated. In this investigation, TEMPOL, a superoxide dismutase mimic (SODm) with additional reactive oxygen species-scavenging activities (Thiemermann, 2003; Liang et al., 2005), was able to provide significant protection against the cellular injury and cell death caused by Aβ1–42 at a concentration range of 10 to 20 μM via the reduction of superoxide anion and hydroxyl radical generation. TEMPOL is a stable piperidine nitroxide, which is a water-soluble analogue of the spin label TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) and an active
metabolite of TEMPONE (4-oxo-2,2,6,6-tetramethylpiperidine-Noxyl) in vivo (Chatterjee et al., 2000; Patel et al., 2002; Thiemermann, 2003). It has been shown to reduce oxidative stress and reactive oxygen species-mediated injury both in vivo and in vitro (Thiemermann, 2003; Liang et al., 2005) via a variety of mechanisms, ranging from SODm activities to the scavenging of reactive oxygen species by the oxidation of Fe2+ to Fe3+, thereby reducing the availability of ferric iron to participate in the Fenton or Haber Weiss reaction and, consequently, inhibiting the generation of hydroxyl radicals from superoxide anions and hydrogen peroxide (Mitchell et al., 1990; Krishna et al., 1996; Laight et al., 1997; Glebska et al., 2001). At higher concentrations, the protection afforded by TEMPOL against Aβ1–42-induced neurotoxicity was reduced, and, at a concentration of 3 mM, TEMPOL even seemed to exacerbate the cell dysfunction or injury caused by Aβ1–42. At this concentration, TEMPOL itself displayed toxicity to neural cells. Superoxide can both initiate and terminate lipid peroxidation (Nelson et al., 1994) and thus the activities of SOD are limited to a narrow concentration range in which superoxide cytotoxicity is prevented, while superoxide-dependent termination events are allowed to occur (McCord and Edeas, 2005). There is also some evidence that Cu2+ derived from Cu/ZnSOD can facilitate oxidative stress in the presence of glutathione (Paller and Eaton, 1995). Furthermore, as TEMPOL rapidly dismutates superoxide into hydrogen peroxide, it promotes the generation of hydroxyl radicals via the Fenton or Haber–Weiss reaction, especially if glutathione peroxidase levels or activity are reduced, as has been reported after exposure to paraquat (Takizawa et al., 2007) and Aβ (Behl et al., 1994). There is also some evidence that TEMPOL itself may act as a free radical or stimulate further production of free radicals and promote cell death when present in excess of other intracellular free radicals (Gariboldi et al., 1998; Goralska et al., 2000). For example, in the absence of additional oxidative stress, high doses of TEMPOL can alter the cellular iron metabolism, resulting in altered synthesis of essential iron-dependent proteins, causing an increase in the lowmolecular-weight pool of iron which then becomes available to
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catalyze hydroxyl radical generation (Gariboldi et al., 1998; Goralska et al., 2000). This study also demonstrated that Aβ produces a significant increase in the generation of superoxide anions and hydroxyl radicals in neuroblastoma SH-SY5Y cells and confirmed the notion that reactive oxygen species generation plays a major part in the Aβinduced damage to the neural cells. As anticipated, the Aβ-induced increase in the generation of superoxide anions and hydroxyl radicals was significantly reduced by SOD and catalase, respectively. Moreover, the SODm TEMPOL (0.5 and 1 mM) significantly reduced the Aβinduced increase in the production of superoxide and hydroxyl radicals. Thus, by reducing the production of reactive oxygen species, these agents reduce oxidative stress injury, accounting in part for their cytoprotective action against Aβ-induced neurotoxicity. Moreover, it is well known that reactive oxygen species-induced oxidative DNA damage can cause cell apoptosis (Datta et al., 2002). We next examined the effect of TEMPOL on the Aβ1–42-induced DNA fragmentation in SH-SY5Y cultured cells by ELISA, and the results show that TEMPOL produces a significant decrease in the DNA fragmentation level. Taken together, these findings are consistent with several reports in which TEMPOL has been documented to be useful against oxidative stress toxicity in several models (Chatterjee et al., 2004; Liang et al., 2005; Lipman et al., 2006; Thaler et al., 2010). In conclusion, the results of our study indicate that TEMPOL confers neuroprotection in SH-SY5Y cells against Aβ1–42-induced Alzheimer's disease model. This finding agrees with the previously observed in vivo neuroprotective effects of TEMPOL. Clinical studies are required to establish TEMPOL as a potential anti-Alzheimer's drug.
Acknowledgments A part of this study was supported by a Grant-in-Aid for Scientific Research (#21240050) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT).
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Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Neuropharmacology and Analgesia
TEMPOL protects human neuroblastoma SH-SY5Y cells against ß-amyloid-induced cell toxicity Pennapa Chonpathompikunlert a,b,c,d,e, Junkyu Han f, Kazuko Toh a,b,c,d,e, Hiroko Isoda f, Yukio Nagasaki a,b,c,d,e,⁎ a
Department of Materials Science, Graduate School of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8503, Japan Tsukuba Research Center for Interdisciplinary Materials Science (TIMS), University of Tsukuba, Tsukuba, Ibaraki 305-8503, Japan Center for Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Tsukuba, Ibaraki 305-8503, Japan d Master's School of Medical Sciences, Graduate School of Comprehensive Human Science, International Center for Materials Nanoarchitectonics Satellite (MANA), University of Tsukuba, Tsukuba, Ibaraki 305-8503, Japan e National Institute for Materials Science (NIMS), University of Tsukuba, Tsukuba, Ibaraki 305-8503, Japan f Graduate School of Life and Environmental Sciences, The Alliance for Research on North Africa, University of Tsukuba, Tennoudai 1-1-1, Tsukuba, Ibaraki 305-8572, Japan b c
a r t i c l e
i n f o
Article history: Received 31 March 2010 Received in revised form 1 October 2010 Accepted 6 October 2010 Available online 20 October 2010 Keywords: Amyloid-β peptide Neuroblastoma SH-SY5Y cell Superoxide anion generation Hydroxyl radical generation Apoptosis Alzheimer's disease
a b s t r a c t Amyloid-β peptide (Aβ) has been implicated in the pathogenesis of Alzheimer's disease (AD). It can cause cell death in Alzheimer's disease by evoking a cascade of oxidative damage to neurons. Antioxidant compounds may help to elucidate and develop a treatment for Alzheimer's disease. In the present study, we investigated the protective effect of TEMPOL (4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy), a cyclic nitroxide which is particularly effective at reducing oxidative injury, on Aβ1–42-induced SH-SY5Y cell toxicity. Exposure of cells to 20 μM Aβ1–42 for 48 h caused viability loss and apoptotic increase, and pre-treatment with TEMPOL for 24 h significantly reduced the viability loss and apoptotic rate. In addition, TEMPOL inhibited Aβ1–42-induced superoxide anion generation and hydroxyl radical generation to a striking degree. Based on these results, it is concluded that TEMPOL effectively protects SH-SY5Y cells against β-amyloid-induced damage by suppressing the generation of reactive oxygen species especially, superoxide anion. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Alzheimer's disease (AD) is neuropathologically characterized by the deposition of β-amyloid (Aβ) plaques and intracellular neurofibrillary tangles and the loss of neurons in the brain. Although the cause of Alzheimer's disease is not fully understood, several lines of evidence suggest that Aβ-induced oxidative stress plays an important role in the pathogenesis and progression of Alzheimer's disease (Butterfield et al., 2001). For example, it is reported that Aβ induces oxidative stress (Hensley et al., 1994), and that oxidative stress promotes the further production of Aβ by potentiating BACE1 gene expression and Aβ generation (Tamagno et al., 2008). Recent studies have shown that oxidative stress contributes to Aβ accumulation: Aβ, in turn, induces oxidative stress and a lipid peroxidation product, 4hydroxy-2-trans-nonenal (HNE), whose production results in increased levels of β- and γ-secretases, which further enhances Aβ production (Shen et al., 2008; Tamagno et al., 2008), thereby inducing
⁎ Corresponding author. Tsukuba Research Center for Interdisciplinary Materials Science (TIMS), University of Tsukuba, 1-1-1 Ten-noudai, Tsukuba 305-8573, Japan. Tel./fax: +81 29 853 5749. E-mail address: [email protected] (Y. Nagasaki). 0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2010.10.028
a vicious circle of Aβ production/accumulation, oxidative stress generation and the generation of β-/γ-secretase, enzymes that cleaves amyloid precursor protein (APP). Thus, one promising preventive or therapeutic measure in Alzheimer's disease may be to attenuate or suppress the oxidative stress-dependent, Aβ-mediated cytotoxicity. Several studies have shown that 4-hydroxy-2,2,6,6 tetramethylpiperidine-N-oxyl (TEMPOL) is a stable, cell membrane-permeable radical scavenger. As a superoxide dismutase-mimetic compound (Thiemermann, 2003; Yamada et al., 2003), TEMPOL attenuates the effects of superoxide radicals (Krishna et al., 1996), directly reacts with both carbon-centered and peroxy radicals (Chateauneuf et al., 1988), prevents the reduction of hydrogen peroxide to the hydroxyl radical (Samuni et al., 1991), and provides neuroprotection in rat models of transient focal ischemia (Rak et al., 2000), in mouse models of 1-methyl-4-phenylpyridinium (MPP+)-induced Parkinson's disease (Matthews et al., 1999) and in MN9D dopaminergic mesencephalic cell from 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease (Liang et al., 2005). Although TEMPOL is known to exhibit neuroprotective activity, the protective effect of TEMPOL against Aβinduced cytotoxicity has not been reported. In the present study, we investigated the effects of TEMPOL on Aβ-induced neurotoxicity in SH-SY5Y cells. Our study verified that TEMPOL has a neuroprotective effect against Aβ-induced cell toxicity.
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2. Materials and methods
2.4. Measurement of hydroxyl radical generation
Unless otherwise stated, amyloid β-protein(1-42), was purchased from Bachem AG Japan, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1oxyl (TEMPOL) from Sigma Co., USA. and WST-8 from DOJINDO Laboratories, Kumamoto, Japan. The Cell Death Detection ELISA Kits were purchased from Cell Biolabs, Inc., USA. All Detect Lipid Peroxidation Assay Kits and proteins were purchased from Sigma Co., USA. All other chemicals were of analytic grade.
The production of hydroxyl radicals by SH-SY5Y cells in the absence or presence of Aβ1–42 was evaluated using the deoxyribose assay, based on the principle that the pentose sugar 2-deoxyribose can be cleaved by hydroxyl radicals to release a substance that reacts, on heating, with thiobarbituric acid to produce a pink chromogen (Halliwell et al., 1988; Morel et al., 1990). Briefly, confluent SHSY5Y cells grown in 6-well plates were incubated for 48 h with DMEM containing 3 mM deoxyribose in the presence or absence of 20 μM Aβ1–42. In addition, SH-SY5Y cells were pre-incubated for 24 h with DMEM containing SOD (1000 U/ml), catalase (1,000 U/ml) or TEMPOL (1 and 0.5 mM) prior to exposure to Aβ1–42 for 48 h. At the end of the incubation period, the cell supernatant was removed and assayed for thiobarbituric substances acid-reactive to malondialdehyde (MDA), as described previously (Mihara and Uchiyama, 1978). An aliquot of the cell supernatant was also used for protein quantification by the Lowry method (Lowry et al., 1951) and the hydroxyl radical content of the samples was expressed as nmol/mg protein.
2.1. Cell culturing and treatment with Aß1–42 and/or substances A human SH-SY5Y neuroblastoma cell line was obtained from Isoda Laboratory, University of Tsukuba, Japan. SH-SY5Y neuroblastoma cells were cultured in flasks or on 6-, 24-, 48- or 96-well plates for different purposes, with a 1:1(v/v) mixture of Dulbecco's minimum essential medium (DMEM; Sigma, USA) and Ham's F-12 nutrient mixture (Sigma, USA) supplemented with 15% fetal bovine serum (FBS; Sigma, USA), 1% non-essential MEM amino acid and 1% penicillin (5000 μg/ml)-streptomycin (5000 IU/ml) solution (ICN Biomedicals, Inc.) at 37 °C in a 95% humidified air–5% CO2 incubator. A serum-free modification of Eagle's minimum essential medium (OPTI-MEM Gibco BRL) was used for the cell toxicity assays (Isoda et al., 2002). For this experiment, cell attachment was required; hence, the plates were pretreated overnight with 50 μg/ml fibronectin, which was refined from human plasma (Wako Pure Chemical Industries, Ltd.). In order to achieve aggregation, Aβ1–42 was dissolved in the culture medium and incubated at 37 °C for 72 h prior to use. After 24–48 h of culturing, the cells (which had reached approximately 80% confluence) were washed three times with a culture medium and thereafter incubated for 48 h in Dulbecco's MEM medium without serum in the presence of different concentrations of Aβ1–42. In the experiments designed to examine the protective effect of TEMPOL, the cells were treated overnight with different concentrations of TEMPOL prior to exposure to Aβ1–42 for 48 h.
2.5. Measurement of apoptotic cell death The DNA damage (DNA fragments) of the cell lysates was measured using the Cell Death Detection ELISA Kit according to the manufacturer's protocol (Cell Biolabs, Inc., USA). The cells were treated as described previously prior to ELISA detection, and spectrophotometric data were obtained from measurement at 405 nm against the substrate solution as a blank. 10 μg/ml of camptothecin was employed as a positive control, which is known as a topoisomerase inhibitor that induces apoptosis to cell. 2.6. Statistical analysis All data are presented as means ± S.E.M. Statistical analysis was performed by one-way analysis of variance (ANOVA). A probability value of less than 0.05 was considered significant.
2.2. Measurement of cell viability
3. Results
Cell viability was determined by a quantitative colorimetric assay using WST-8 (DOJINDO Laboratories, Kumamoto, Japan). Briefly, 1 × 104 cells/well were seeded on a 96-well flat-bottom plate at 37 °C overnight. Once the cells were attached, they were washed once with 100 μl of OPTI-MEM medium, followed by the addition of 100 μl of fresh OPTI-MEM medium. The cells were then allowed to grow for 72 h in the desired final concentrations of TEMPOL with 10% (v/v) and 10 μl of 20 μM Aβ1–42 added. After treatment, 10 μl of WST-8 reagent was added to each well containing 100 μl of medium, according to the manufacturer's instructions. The plate was incubated for 1 h at 37 °C and measured at an absorbance of 450 nm by a plate reader.
3.1. Effect of Aß1–42 and TEMPOL on cell viability in SH-SY5Y cells
2.3. Measurement of superoxide anion generation The generation of superoxide anions was determined by means of the nitroblue tetrazolium (NBT) assay described previously (Falasca et al., 1993; Scheid et al., 1996). Briefly, SH-SY5Y cells grown in 24well plates were incubated for 48 h with DMEM containing 25 μg/ml NBT in the presence or absence of 20 μM Aβ1–42. In addition, SH-SY5Y cells were pre-incubated for 24 h with DMEM containing superoxide dismutase (SOD) (EC 1.15.1.1; 1,000 U/ml), catalase (EC 1.11.1.6; 1,000 U/ml) or TEMPOL (1 and 0.5 mM) prior to exposure to Aβ1–42 for 48 h. The concentrations of SOD and catalase were based on the concentrations used in a previous study ranging from 100 to 10,000 U/ ml (Scheid et al., 1996; Thamilselvan et al., 2000), and those of TEMPOL were based on those known to provide significant protection against Aβ1–42 toxicity.
Initial experiments were performed to determine whether Aβ1–42 alone or TEMPOL alone was toxic to human neuroblastoma cells (SHSY5Y) by measuring the WST in the culture medium. The reduction of WST was employed here as an indicator of neural cytotoxicity (Ngamwongsatit et al., 2008). Incubation of confluent SH-SY5Y cells with Aβ1–42 (1.5–20 μM) for 48 h resulted in a significant reduction in cellular viability from 100% for the control (untreated) cells to 30% (P b 0.01) at the highest concentration tested, as well as a dosedependent decline in WST reduction by the cells, as shown in Fig. 1, indicating that Aβ1–42 is strongly cytotoxic to SH-SY5Y cells. In contrast, TEMPOL showed cytotoxicity at a concentration of more than 3 mM, as indicated by the decrease in the measured WST (Fig. 2). Based on the dose–response data from Figs. 1 and 2, the concentration of 20 μM Aβ1–42 was chosen to evaluate the influence of low concentrations of TEMPOL (0.01–3 mM) on Aβ1–42 toxicity in SHSY5Y cells. 3.2. Effect of TEMPOL on ß-amyloid-induced cell toxicity To confirm the protective effect of TEMPOL on Aβ1–42 toxicity, SHSY5Y cells were treated with Aβ1–42 alone or TEMPOL plus Aβ1–42 in a serum-free medium. Aβ1–42 alone significantly decreased cell viability, whereas the combination of Aβ1–42 and TEMPOL showed a unique behavior: the cell viability increased with increasing TEMPOL concentration up to 1.0 mM, then gradually decreased (Fig. 3).
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Fig. 1. Effect of Aβ1–42 on cell viability. SH-SY5Y cells were incubated with increasing concentrations of Aβ1–42 for 48 h. *P b 0.05, **P b 0.01 vs. control (untreated group) analyzed using one-way ANOVA followed by Dunnett's post-hoc test, n = 7.
3.3. Effect of TEMPOL on ß-amyloid-induced reactive oxygen species Exposure of SH-SY5Y cells to 20 μM Aβ1–42 for 48 h caused a significant, 48% increase in superoxide generation (measured as NBT absorbance) compared to the control (untreated) cells (P b 0.01) (Fig. 4A), with a concomitant increase in hydroxyl radical production (P b 0.01) (Fig. 4B). As anticipated, the addition of SOD (1000 U/ml) significantly reduced the Aβ1–42-induced superoxide generation, by 53% (P b 0.01), whereas catalase (1000 U/ml) did not have a significant effect (Fig. 4A). TEMPOL (0.5 and 1 mM) also caused a significant reduction in Aβ1–42-mediated superoxide generation, by 54% (P b 0.01) and 40% (P b 0.05), respectively (Fig. 4A). In contrast, SOD had no effect upon Aβ1–42-mediated hydroxyl radical generation, whereas catalase caused a significant reduction, by 75% (P b 0.01) (Fig. 4B). TEMPOL also caused significant reductions in Aβ1–42mediated hydroxyl radical production, by 35% (P b 0.05) and 60% (P b 0.05), respectively (Fig. 4B). 3.4. Effect of TEMPOL protection against Aß1–42-induced apoptosis in SH-SY5Y cells Biochemically, one hallmark of apoptosis is DNA fragmentation in the nucleus. This parameter was measured to determine if TEMPOL would prevent Aβ1–42-induced apoptosis in SH-SY5Y cells. Aβ1–42 has
Fig. 2. Effect of TEMPOL on cell viability. SH-SY5Y cells were incubated with increasing concentrations of TEMPOL for 24 h. *P b 0.05, **P b 0.01, ***P b 0.001 vs. control (untreated group) analyzed using one-way ANOVA followed by Dunnett's post-hoc test, n = 7.
Fig. 3. Effect of TEMPOL pre-treatment on the viability of SH-SY5Y cells exposed to 20 μM Aβ1–42. SH-SY5Y cells were treated with different concentrations of TEMPOL (0– 3 mM) for 24 h prior to the addition of Aβ1–42 to the culture medium. Forty-eight hours following Aβ1–42 treatment, the cell viability was assessed by WST assay. The data are expressed as means ± S.E.M. *P b 0.05 and **P b 0.01, when the cell viability is compared with that of the cells treated with only 20 μM Aβ1–42 (no TEMPOL pre-treatment); # P b 0.05, when the cell viability is compared with that of the control (untreated group). Analyzed using one-way ANOVA followed by Dunnett's post-hoc test, n = 7.
been reported to induce apoptosis in SH-SY5Y cells, as evidenced by an increases in the number of DNA fragments in the cell lysates (YanPing et al., 1996). Fig. 5 shows the results of our quantitative analysis of DNA fragmentation. It was confirmed that Aβ1–42 significantly increased the number of DNA fragments in SH-SY5Y cells. Pretreatment of the cells with 0.5 and 1 mM TEMPOL markedly decreased the number of DNA fragments in the cell lysates. 4. Discussion Aβ, the major constituent of the senile plaques in Alzheimer's disease, contains a 39–43 amino-acid peptide and is produced by the proteolytic processing of APP, a transmembrane glycoprotein expressed during normal cellular metabolism (Haass, 1996; Selkoe, 1996). Aβ, as well as an 11-amino-acid fragment thereof (i.e., Aβ25–35), can have a neurotoxic effect through a mechanism linked to peptide fibril formation (Kowall et al., 1992; Yankner, 1996) and, consequently, the corresponding synthetic peptides containing residues 25–35, 1–40 or 1–42 are convenient tools for the investigation of Alzheimer's disease. The overwhelming majority of evidence suggests that particularly Aβ1–42 plays a central role in the pathogenesis of Alzheimer's disease. Most of this evidence is genetic: defects in the genes encoding presenilin-1, presenilin-2 and APP are invariably accompanied by elevated levels of Aβ1–42 (Butterfield and Bush, 2004). In the present study, treatment of SH-SY5Y cells with Aβ1–42 clearly decreased cell viability in a dose-dependent manner, thereby providing direct evidence of cellular toxicity induced by Aβ, a result that strongly resembles our previous findings with PC12 cells (Guan et al., 2001, 2003a,b). Recent research has provided convincing evidence that Aβ-associated free-radical damage to neuron is a fundamental process in connection with Alzheimer's disease (Miranda et al., 2000). The implication of reactive oxygen species in the pathogenesis of Alzheimer's disease has led to the notion that exogenous antioxidants or strategies to increase the expression of endogenous antioxidants would constitute useful therapeutic and prophylactic strategies for this disease (Rutten et al., 2002). Several types of exogenously administered medicinal plants (Kim et al., 2007), melatonin (Raghavendra and Kulkarni, 2001) and vitamin E (Boothby and Doering, 2005) have been proposed as drugs for the prophylaxis and therapy of
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Fig. 5. Quantification of nucleosomal DNA fragmentation. The Cell Death Detection ELISA Kit was used to quantify DNA fragmentation in SH-SY5Y cells undergoing apoptosis in the presence of 20 μM Aβ1–42 without or with TEMPOL and camptothecin (10 μg/ml). Cells were grown in 48-well plates and incubated for 48 h. As a control, untreated cells, cells treated with Aβ1–42 (20 μM) only, or cells treated with Aβ1–42 (20 μM) plus TEMPOL (0.5 and 1 mM) were cultured, and positive controls were also cultured with 10 μg/ml of camptothecin, which is a topoisomerase inhibitor that induces apoptosis to cell. The rate of apoptosis is reflected by the enrichment of nucleosomes in the cytoplasm, shown along the y-axis. Experiments were performed in triplicate and repeated three times. #P b 0.001 vs. control (untreated cells), **P b 0.01 vs. 20 μM Aβ1–42 only, analyzed using one-way ANOVA, followed by Dunnett's post-hoc test, n = 5.
Fig. 4. Effect of SOD, catalase and TEMPOL on the Aβ1–42-mediated generation of (A) superoxide anions and (B) hydroxyl radicals. SH-SY5Y cells were incubated for 48 h with 20 μM Aβ1–42 in the absence or presence of TEMPOL, SOD (1,000 U/ml) and catalase (CAT: 1,000 U/ml). #P b 0.01 vs. control (untreated cells), *P b 0.05 and **P b 0.01 vs. 20 μM Aβ1–42, analyzed using one-way ANOVA, followed by Dunnett's post-hoc test, n = 5.
Alzheimer's disease. The present study adds the stable nitroxide antioxidant, TEMPOL, to this list. Unlike the other agents proposed to date, the antioxidant activity of TEMPOL is mediated by several simultaneously operational mechanisms. TEMPOL works as a superoxide dismutase mimic (Samuni et al., 1991) and directly reacts with both carbon-centered and peroxy radicals (Chateauneuf et al., 1988). The results obtained in this investigation indicate that Aβ1–42 exerts a concentration-dependent toxic effect on confluent neuroblastoma SH-SY5Y cells. This neurotoxic action was significantly reduced by TEMPOL, though TEMPOL itself was found to be neurotoxic at higher concentrations. The role of superoxide generation and subsequent hydroxyl radical production by Aβ1–42 was confirmed, as were the SOD properties of TEMPOL. The ability of TEMPOL to reduce hydroxyl radical generation was also demonstrated. In this investigation, TEMPOL, a superoxide dismutase mimic (SODm) with additional reactive oxygen species-scavenging activities (Thiemermann, 2003; Liang et al., 2005), was able to provide significant protection against the cellular injury and cell death caused by Aβ1–42 at a concentration range of 10 to 20 μM via the reduction of superoxide anion and hydroxyl radical generation. TEMPOL is a stable piperidine nitroxide, which is a water-soluble analogue of the spin label TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) and an active
metabolite of TEMPONE (4-oxo-2,2,6,6-tetramethylpiperidine-Noxyl) in vivo (Chatterjee et al., 2000; Patel et al., 2002; Thiemermann, 2003). It has been shown to reduce oxidative stress and reactive oxygen species-mediated injury both in vivo and in vitro (Thiemermann, 2003; Liang et al., 2005) via a variety of mechanisms, ranging from SODm activities to the scavenging of reactive oxygen species by the oxidation of Fe2+ to Fe3+, thereby reducing the availability of ferric iron to participate in the Fenton or Haber Weiss reaction and, consequently, inhibiting the generation of hydroxyl radicals from superoxide anions and hydrogen peroxide (Mitchell et al., 1990; Krishna et al., 1996; Laight et al., 1997; Glebska et al., 2001). At higher concentrations, the protection afforded by TEMPOL against Aβ1–42-induced neurotoxicity was reduced, and, at a concentration of 3 mM, TEMPOL even seemed to exacerbate the cell dysfunction or injury caused by Aβ1–42. At this concentration, TEMPOL itself displayed toxicity to neural cells. Superoxide can both initiate and terminate lipid peroxidation (Nelson et al., 1994) and thus the activities of SOD are limited to a narrow concentration range in which superoxide cytotoxicity is prevented, while superoxide-dependent termination events are allowed to occur (McCord and Edeas, 2005). There is also some evidence that Cu2+ derived from Cu/ZnSOD can facilitate oxidative stress in the presence of glutathione (Paller and Eaton, 1995). Furthermore, as TEMPOL rapidly dismutates superoxide into hydrogen peroxide, it promotes the generation of hydroxyl radicals via the Fenton or Haber–Weiss reaction, especially if glutathione peroxidase levels or activity are reduced, as has been reported after exposure to paraquat (Takizawa et al., 2007) and Aβ (Behl et al., 1994). There is also some evidence that TEMPOL itself may act as a free radical or stimulate further production of free radicals and promote cell death when present in excess of other intracellular free radicals (Gariboldi et al., 1998; Goralska et al., 2000). For example, in the absence of additional oxidative stress, high doses of TEMPOL can alter the cellular iron metabolism, resulting in altered synthesis of essential iron-dependent proteins, causing an increase in the lowmolecular-weight pool of iron which then becomes available to
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catalyze hydroxyl radical generation (Gariboldi et al., 1998; Goralska et al., 2000). This study also demonstrated that Aβ produces a significant increase in the generation of superoxide anions and hydroxyl radicals in neuroblastoma SH-SY5Y cells and confirmed the notion that reactive oxygen species generation plays a major part in the Aβinduced damage to the neural cells. As anticipated, the Aβ-induced increase in the generation of superoxide anions and hydroxyl radicals was significantly reduced by SOD and catalase, respectively. Moreover, the SODm TEMPOL (0.5 and 1 mM) significantly reduced the Aβinduced increase in the production of superoxide and hydroxyl radicals. Thus, by reducing the production of reactive oxygen species, these agents reduce oxidative stress injury, accounting in part for their cytoprotective action against Aβ-induced neurotoxicity. Moreover, it is well known that reactive oxygen species-induced oxidative DNA damage can cause cell apoptosis (Datta et al., 2002). We next examined the effect of TEMPOL on the Aβ1–42-induced DNA fragmentation in SH-SY5Y cultured cells by ELISA, and the results show that TEMPOL produces a significant decrease in the DNA fragmentation level. Taken together, these findings are consistent with several reports in which TEMPOL has been documented to be useful against oxidative stress toxicity in several models (Chatterjee et al., 2004; Liang et al., 2005; Lipman et al., 2006; Thaler et al., 2010). In conclusion, the results of our study indicate that TEMPOL confers neuroprotection in SH-SY5Y cells against Aβ1–42-induced Alzheimer's disease model. This finding agrees with the previously observed in vivo neuroprotective effects of TEMPOL. Clinical studies are required to establish TEMPOL as a potential anti-Alzheimer's drug.
Acknowledgments A part of this study was supported by a Grant-in-Aid for Scientific Research (#21240050) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT).
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