Protective effect of (−)clausenamide against Aβ-induced neurotoxicity in differentiated PC12 cells

Neuroscience Letters 483 (2010) 78–82

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Protective effect of (−)clausenamide against A␤-induced neurotoxicity in differentiated PC12 cells Jin-Feng Hu, Shi-Feng Chu, Na Ning, Yu-He Yuan, Wei Xue, Nai-Hong Chen ∗ , Jun-Tian Zhang ∗ Key Laboratory of Bioactive Substances and Resources Utilization, Ministry of Education, Department of Pharmacology, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, 1 Xiannongtan street, Xuanwu district, Beijing 100050, China

a r t i c l e

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Article history: Received 8 June 2010 Received in revised form 12 July 2010 Accepted 26 July 2010 Keywords: (−)Clausenamide ␤-amyloid Alzheimer’s disease Apoptosis

a b s t r a c t The neurotoxicity of aggregated ␤-amyloid (A␤) has been implicated as a critical cause in the pathogenesis of Alzheimer’s disease (AD). In the present study, we investigated the effect of (−)clausenamide ((−)Clau), an aqueous extract of leaves of Clausena lassium (lour) skeel, on the neurotoxicity of A␤25–35 . The viability of differentiated PC12 cells was determined by MTT assay. Apoptosis was detected by flow cytometry. DCFH-DA was used for assessment of intracellular ROS generation, JC-1 and Rhodamine 123 for measurement of mitochondrial transmembrane potential (MMP). The intracellular calcium was determined with Fluo-3. The phosphorylation of p38 MAPK and the expression of Bcl-2, Bax, P53, Caspase 3 were examined by Western blot. The results showed that (−)Clau significantly elevated cell viability. Furthermore, (−)Clau arrested the apoptotic cascade by reversing overload of calcium, preventing ROS generation, moderated the dissipation of MMP and the misbalance of Bcl-2 and Bax, inhibiting the activation of p38 MAPK and the expression of P53 and cleaved Caspase 3. Our results suggested that (−)Clau may be a therapeutic agent for AD. © 2010 Elsevier Ireland Ltd. All rights reserved.

Alzheimer’s disease (AD), a progressive neurodegenerative disorder, is the most common form of dementia among the elderly, characterized by a progressive loss of learning and memory and other cognitive function. The parenchyma and cerebrovascular ␤amyloid (A␤) deposits are one of the pathological hallmarkers [21]. Recently, it has become more clearly that deposition of A␤ is considered as a crucial event in initiating the neuronal degeneration in AD. Furthermore, A␤ exhibits neurotoxicity both in vitro and in vivo. Of special interest is observation that intracerebral infusion of the protein causes neurodegeneration, along with impairment of learning and memory. Although the mechanism of A␤-induced cell damage is unclear, the apoptotic cascades activated by A␤ have been demonstrated [11]. Apoptosis is a morphologically and biochemically defined mode of cell death characterized by nuclear and cytoplasmic condensation, DNA fragmentation, formation of apoptotic bodies and so on [19]. In particular, some biomarkers of apoptosis, including the activation of p38 MAPK signal pathway, upregulation of P53 and Bax, and the activation of Caspase 3 were reported in neurons of AD patients [7,8,13]. Clausena lassium (lour) skeel is a Chinese traditional drug which abounds in South China. The chemists in our institute

∗ Corresponding authors. Tel.: +86 10 63165177/9; fax: +86 10 63165177. E-mail addresses: [email protected] (N.-H. Chen), [email protected] (J.-T. Zhang). 0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.07.067

have isolated several compounds from the leaf of this plant. Of which, (−)clausenamide (3-hydroxy-4-phenyl-5a-hydroxybenzylN-methyl-g-lactam, (−)Clau, Fig. 1A) is the aqueous extract of leaves, with a partial chemical structure similar to the pharmacophore of piracetam, a nootropic drug developed in Europe. The compound has been chemically synthesized in our institute, and a US patent has been granted to protect the technology of chemical synthesis (US Patent no. 6787564 B2). After pharmacological study, (−)Clau was demonstrated that it improved the deficiency of learning and memory induced by anisodine, and potentiated synaptic transmission in the dentate gyrus of rats, indicating that (−)Clau could be the promising candidate for AD [6,18,30]. Thus, in this paper, the effect of (−)Clau on the neurotoxicity of A␤ was investigated. (−)Clau was provided by department of medicinal chemistry, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College (Beijing, China) and triturated into fine powder before dissolved in Dimethyl Sulphoxide. Antiphospho-p38 MAPK, anti-Bcl-2, anti-Bax, anti-P53, anti-Caspase 3 subunit, anti-␤-actin primary antibodies and anti-rabbit IgG secondary antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Dimethyl Sulphoxide, A␤25–35 , 3-(3,4dimehylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT), nerve growth factor (NGF) were obtained from Sigma (St Louis, MO, USA). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS) and equine serum were purchased from Gibco BRL (New York, NY, USA). Fluo-3-acetocymethyl ester, JC-1,

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Fig. 1. (A) The chemical structure of (−)Clau. (B) Effect of (−)Clau on cell viability in A␤25–35 -induced cytotoxicity. Differentiated PC12 cells were exposed to different concentrations (0, 1, 10 and 100 ␮M) of (−)Clau and A␤25–35 , the cell viability was evaluated by MTT assay (n = 4, ##P < 0.01 vs. control, *P < 0.05, **P < 0.01 vs. A␤25–35 -treated group).

Rhodamine 123, 2,7-dichlorofluorescein diacetate (DCFH-DA) and FITC-Annexin V/propidium iodide (PI) apoptosis detection kit were purchased from Beyotime (China). Enhanced chemiluminescent (ECL) substrate was from Pierce (Rockford, IL, USA). PC12 cells at a density of 5 × 103 cells per well in 96-well plates were cultured in DMEM media supplemented with 5% FBS and

Fig. 2. Effect of (−)Clau on A␤25–35 -induced apoptosis by flow cytometry. (A) 20 ␮M A␤25–35 induced the increase of both the early apoptosis (Annexin V positive, PI negative) and late apoptosis/necrosis (Annexin V/PI double positive) in PC12 cells for 24 h incubation. However, the apoptosis of PC12 cells was inhibited by (−)Clau. (B) Quantitative analysis of the ratio of early apoptosis and late apoptosis/necrosis (n = 3, ##P < 0.01 vs. control, *P < 0.05, **P < 0.01 vs. A␤25–35 -treated group).

5% equine serum, and l-glutamine (2 mM). Cultures were maintained at 37 ◦ C in 5% CO2 in a humidified incubator. To induce PC12 differentiation, NGF (50 ng/mL) was added to the culture media containing 1% FBS, followed by a 3-day incubation. Then (−)Clau at concentrations of 0, 1, 10, 100 ␮M and 20 ␮M aged A␤25–35 were added to the cells for 24 h, then 5 mg/mL MTT was added and maintained for 4 h. Absorbance was measured at 570 nm using an Ultramark microplate reader. Differentiated PC12 cells in 6-well plate were treated with (−)Clau and A␤25–35 . After 24 h, the cells were collected, washed twice with cold phosphate-buffered saline (PBS, 1.5 mM KH2 PO4 , 8.1 mM Na2 HPO4 , 2.7 mM KCl, 136.7 mM NaCl). Cells were harvested for subsequent usage. The FITC-Annexin V/PI apoptosis detection kit was employed to observe the apoptosis. Approximate 1 × 105 cells were incubated in the dark with Annexin V and PI for 15 min at room temperature. Suspensions were analysed using a FACS scan flow cytometer (Becton Dickinson). The [Ca2+ ]i concentration was determined with Fluo-3acetocymethyl ester. After the cells were treated with (−)Clau and A␤, cells were stained with Fluo-3-acetocymethyl ester at a final concentration of 2.5 mM at 37 ◦ C for 30 min. Next, cells were washed with PBS three times, and the fluorescent intensity was determined by fluorescent microplate reader. The fluorescent probe DCFH-DA was used for assessment of intracellular ROS generation. Cells were loaded with a membranepermeable, nonfluorescent probe, DCFH-DA (5 ␮mol/L) for 20 min at 37 ◦ C under the dark condition, then DCFH-DA-loaded cells were excited at 480 nm and emission was detected at 530 nm. Changes in the inner mitochondrial transmembrane potential (MMP) were determined by incubating with 10 ␮g/mL of Rhodamine 123 for 30 min at 37 ◦ C in the dark. Then the cells were washed with PBS three times, and the fluorescent intensity was determined by flow cytometry. JC-1 was also used to measure the change of inner MMP. Differentiated PC12 cells were incubated with 10 ␮g/mL JC-1 at 37 ◦ C. JC-1 accumulates in mitochondria forming red fluorescent aggregates at high membrane potentials. At low membrane potential, JC-1 exits mainly in the green fluorescent monomeric form. After incubated for 20 min, the cells were washed with PBS for three times and submitted to fluorescence microscopy analysis. JC-1-loaded cells were excited at 488 nm and emission was detected at 590 nm (JC-1 aggregates) and 525 nm (JC-1 monomers). Cells were treated as above describe and washed with PBS and lysed in lysis buffer (50 mmol/L Tris–HCl, 150 mmol/L NaCl, 1% NP-40, 1 mmol/L PMSF, 50 ␮g/mL Leupeptin, 1 ␮g/mL pepstatin A, 20 ␮g/mL aprotinin, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L DTT, 1 mmol/L NaVO3 , 50 mmol/L NaF, 20 mmol/L ␤glycerophosphate Na2 , pH 8.0). Protein concentrations were measured with BCA kit. The cell lysates were solubilized in SDS sample buffer and separated by SDS-PAGE gel, then transferred to

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Fig. 3. Effect of (−)Clau on intracellular calcium in PC12 cells exposed to 20 ␮M A␤25–35 . (A) (−)Clau inhibited the overload of intracellular calcium induced by A␤25–35 . (B) DCFH-DA was used for assessment of intracellular ROS generation, and (−)Clau inhibited the increase of fluorescent density. (C) (−)Clau prevented the decrease of mitochondrial membrane potential by Rhodamine 123 staining, and the same effect was also demonstrated by JC-1 staining (D). (n = 3, ##P < 0.01 vs. control, *P < 0.05, **P < 0.01 vs. A␤25–35 -treated group).

a PVDF membrane (Millipore). The membrane was blocked with 3% BSA and incubated with primary antibody: anti-Bax, anti-Bcl2, anti-P53, anti-p-p38 MAPK or anti-Caspase-3, respectively, then followed by horseradish peroxidase (HRP)-conjugated secondary antibody and detected with the enhanced chemiluminescence plus detection system (Molecular Device, Lmax) and analysed by Science Lab 2005 Image Gauge software. Data were expressed as the mean ± standard deviation (SD). One-way ANOVA followed by Student’s t-test were performed to statistical analysis. P < 0.05 was considered statistically significant. It is well known that A␤ possesses neurotoxicity and the blockade of A␤ toxicity is viewed as a reasonable strategy to treat AD [1,25]. The effect of (−)Clau on neurotoxicity induced by A␤25–35 was evaluated in vitro. In differentiated PC12 cells, treatment with 20 ␮M A␤25–35 resulted in a significant inhibition of MTT reduction. However, (−)Clau remarkably attenuated A␤25–35 -induced damage at dose-dependent manner, and this maximal inhibitory effect of (−)Clau was observed at 100 ␮M (Fig. 1B). Thus, 10, 100 ␮M (−)Clau was selected for the further mechanism study. Recent evidence has highlighted that apoptosis exists in several neuronal cell types exposed to A␤ [9,12,17]. Accordingly, we additionally investigated the effect of (−)Clau on apoptosis induced by A␤25–35 . The early apoptotic cells (Annexin V positive, PI negative), late apoptotic/necrotic cells (Annexin V positive, PI positive) were detected by flow cytometry. The percentage of both Annexin V+/PI− cells and Annexin V+/PI+ cells of A␤-treated group were 26.8 ± 4.2% and 10.7 ± 1.8%. However, the percentage of (−)Clau-treated groups were 11.2 ± 1.9% and 8.7 ± 1.3% of 10 ␮M, 11.0 ± 1.9% and 6.2 ± 1.5% of 100 ␮M, respectively (Fig. 2). The results showed that (−)Clau can reduce A␤-mediated cell apoptosis.

Even though the mechanisms of apoptosis induced by A␤ are not well known, nevertheless, the impairment of [Ca2+ ]i homeostasis was reported to play very important roles in A␤-induced apoptotic cascade [2]. Thus, the [Ca2+ ]i level concentration was assayed. Treatment with 20 ␮M A␤25–35 for 24 h resulted in a remarkable increase of [Ca2+ ]i level in PC12 cells, the fluorescent density increased from 2.41 ± 0.39 to 5.48 ± 0.18. However, 10, 100 ␮M (−)Clau reversed the overload in [Ca2+ ]i level stimulated by A␤25–35 , the fluorescent density were 3.83 ± 0.70, 3.64 ± 0.21, respectively (Fig. 3A). Besides altering [Ca2+ ]i homeostasis, A␤ also induces the generation and accumulation of ROS and thus leading to apoptosis of neurons [17,24]. Here, we observed the production of ROS by DCFH-DA. Fig. 3B shows that A␤25–35 elevated the production of ROS (from 42.54 ± 7.05 to 140.48 ± 15.32). However, the fluorescence intensity in (−)Clau-treated group decreased significantly (100.86 ± 11.22, 84.67 ± 10.58, respectively). While mitochondria have been shown to play a crucial role in sensing and propagating apoptosis signals, collapse of MMP is generally related to ROS in the early phase of the A␤-induced apoptosis pathway [7,17,29]. The results of flow cytometry analysis showed A␤25–35 remarkably decreased MMP, the percentage of low MMP cells in all cells was increased from 3.3 ± 0.9% to 65.2 ± 12.6%. However, exposure of differentiated PC12 cells to (−)Clau remarkably prevented the reduction of MMP induced by A␤25–35 (42.4 ± 11.5%, 27.7 ± 6.3%, respectively, Fig. 3C). JC-1 could aggregate in normal mitochondria and present red fluorescence. Exposure of differentiated PC12 cells to A␤25–35 resulted in dissipation of MMP, which was shown as increased green fluorescence. However, (−)Clau could moderate the dissipation of MMP (Fig. 3D).

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Fig. 4. Effect of (−)Clau on the phosphorylation of p38 MAPK, the expression of Bax, Bcl-2, P53 and Caspase 3 in PC12 cells exposed to 20 ␮M A␤25–35 . (A) PC12 cells were lysed after treated with (−)Clau and A␤25–35 , the protein was detected by Western blot. (B) Quantitative analysis of the phosphorylation of p38 MAPK, the expression of Bax, Bcl-2, P53 and Caspase 3 by Science Lab 2005 Image Gauge software (n = 3, ##P < 0.01 vs. control, *P < 0.05, **P < 0.01 vs. A␤25–35 -treated group).

p38 MAPK involves in apoptosis, inflammation and responses to environmental stress [16]. Thus, the effect of (−)Clau on the phosphorylation of p38 was detected. The results of Western blot analysis showed that p38 MAPK was activated by A␤25–35 , but (−)Clau at doses of 10 and 100 ␮M inhibited the phosphorylation of p38 MAPK (Fig. 4). Activation of p38 MAPK has been linked to increase activity of P53 and Caspase-3 [14]. The P53 promoter is thought to be one of the targets of nuclear A␤, because it may be linked to the induction of neuronal apoptosis in AD [5,13], P53 subsequently up-regulates its target gene bax. Bax has been identified in various cell types as an apoptotic member that hinders the caspase cascade and Caspase 3 is a major executioner of apoptotic signals [7,13]. Accordingly, we evaluated the effects of (−)Clau on both the expression of P53, Bax, Bcl-2 and cleaved Caspase 3 in neurons stimulated by A␤25–35 . Treatment with 20 ␮M A␤25–35 for 24 h led to a dramatic increase of P53 and cleaved Caspase 3 expression and the ratio of Bax/Bcl2. However, 10, 100 ␮M (−)Clau down-regulated the expression of P53 and cleaved Caspase-3 and the ratio of Bax/Bcl-2 (Fig. 4), which gave us a good understanding of the molecular mechanisms of anti-apoptotic action of (−)Clau. It has been demonstrated that the neurotoxicity of A␤ plays an important role in AD (1). The protective effect of (−)Clau on A␤25–35 neurotoxicity was investigated in vitro. A␤25–35 resulted in the neurotoxic effects in differentiated PC12 cells. Most importantly, (−)Clau exerted a significant neuroprotective activity against A␤25–35 . It is well-known that neuronal apoptosis is central to neurotoxicity induced by A␤. Prevention of A␤-triggered apoptosis is an optimal cure of AD [1,10]. Apoptosis is characterized by condensation of nuclear chromatin, cytoplasmic blebbing, and exposure of phosphatidylserine residues on the outside of the plasma membrane. Necrosis is accidental cell death and characterized by mitochondrial swelling, rupture of the plasma membrane, and the release of cytoplasmic constituents [19]. Thus, the antiapoptotic effect of (−)Clau was examined by Annexin V/PI kit. After addition of (−)Clau, both of early apoptosis and late apoptosis/necrosis populations decreased significantly, indicated that (−)Clau protect PC12 cells against A␤-induced apoptosis and necrosis. Although the exact mechanisms underlying A␤-induced apoptosis are not well understood, it is proposed that disruption of calcium homeostasis and the oxidative stress have been mechanistically linked to this process [1,15,17,29]. Therefore, inhibiting [Ca2+ ]i overload and the generation of ROS are promising approaches in combating A␤-induced apoptosis. Studies have demonstrated that (−)Clau potently modulated [Ca2+ ]i homeosta-

sis and inhibited ROS generation, which effectively explains its anti-apoptotic mechanisms. ROS and Ca2+ are two highly correlative key factors in A␤-induced apoptosis. Increase of [Ca2+ ]i level could induce alteration of energy metabolism and promote ROS generation [22]. Whether (−)Clau prevented ROS generation by inhibiting the increase of [Ca2+ ]i level was not clear. Furthermore, A␤ leads to the dissipation of MMP by promoting the accumulation of ROS in mitochondria, directly activating of the MPTP, cytochrome c release followed by Caspase 3 activation. In our present data by JC-1 and Rhodamine 123 staining, it showed that ROS dramatically mediated A␤-induced apoptosis. However, (−)Clau suppressed the reduction in MMP induced by A␤25–35 . So anti-oxidative action of (−)Clau played the important role in its anti-apoptotic pathways, though other possibilities could not be excluded out. Studies have demonstrated that under oxidative stress, reactive oxygen species (ROS) including free radicals such as superoxide, hydroxyl radical and hydrogen peroxide are generated at high levels inducing cellular damage and even cell death. The oxidative stress is the initiating factor and upstream of the activation of MAPKs in many kinds of cells [3,26]. And mitogen-activated protein kinases (MAPKs), including p38 MAPK, are important in cell survival, proliferation, differentiation, and also involved in apoptosis and may play a pivotal role in neurodegeneration [4,20,27]. In our study, it also has been demonstrated that p38 was activated by A␤25–35 in PC12 cells. However, the inhibition of p38 signal pathway by (−)Clau was related to the inhibitory effect of ROS generation. Activation of p38 MAPK is also related to increased activity of classical apoptotic mediators such as P53 and Caspase 3 in different cells [14,23,28]. It has been suggested that P53 is a substrate for p38 MAPK. Our study showed that A␤25–35 promoted the increase of P53 expression in PC12 cells, suggesting that the effect of A␤25–35 on P53 may be related to the activation of p38 MAPK. In general, various cellular stresses increase and activate P53 in the cytoplasm, which plays an important role in apoptosis acting as a transcriptional activator to up-regulate its target gene bax and down-regulate anti-apoptotic protein Bcl-2 [7]. Bax and Bcl-2 are believed to be implicated in the process of apoptotic cell death induced by A␤25–35 . The pro-apoptotic protein Bax in turn induces the activation of Caspase 3, a major executioner of apoptotic signals [7,23]. Furthermore, some studies suggest that Caspase 3 is required for activation of p38 MAPK [8,14]. Significantly, (−)Clau had the distinct impact on the expression of Bcl-2, Bax and cleaved Caspase 3, putatively supporting the theory of anti-apoptotic cascade and thus in vivo cognition-improving functions of (−)Clau. This is the first study to identify that (−)Clau, a chemical structure analogue of piracetam, significantly arrested apoptotic cascade by reversing overload of [Ca2+ ]i , preventing ROS generation, alle-

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viating the dissipation of MMP and the misbalance of Bcl-2 and Bax, inhibiting the activation of p38 MAPK and downregulating the expression of P53 and cleaved Caspase 3, which provided the evidences for (-)Clau as a promising therapeutic agent for Azheimer’s diasease. Acknowledgements This work was supported by National Natural Science Foundation of China Grants (Nos. 30801527, 30973887, Key Program No. U832008), Research Fund for the Doctoral Program of Higher Education (No. 20070023075), and National Key Sci-Tech Major Special Item (Nos. 2008ZX09101 and 2009ZX09303-003). References [1] S.O. Bachurin, Medicinal chemistry approaches for the treatment and prevention of Alzheimer’s disease, Med. Res. Rev. 23 (2003) 48–88. [2] L.M. Canzoniero, B.J. Snider, Calcium in Alzheimer’s disease pathogenesis: too much, too little or in the wrong place? J. Alzheimers Dis. 8 (2005) 147–154 (discussion 209–215). [3] L. Chen, L. Liu, S. Huang, Cadmium activates MAPK pathway via induction of reactive oxygen species and inhibition of protein phosphatase 2A and 5, Free Radical Biol. Med. 45 (2008) 1035–1044. [4] K.J. Cowan, K.B. Storey, Mitogen-activated protein kinases: new signaling pathways functioning in cellular responses to environmental stress, J. Exp. Biol. 206 (2003) 1107–1115. [5] S.M. de la Monte, Y.K. Sohn, J.R. Wands, Correlates of p53- and Fas (CD95)mediated apoptosis in Alzheimer’s disease, J. Neurol. Sci. 152 (1997) 73–83. [6] W.Z. Duan, J.T. Zhang, Effects of (−), (+)clausenamide on anisodine-induced acetylcholine decrease and associated memory deficits in the mouse brain, Acta Pharm. Sin. 33 (1998) 259–263. [7] A. Eckert, U. Keil, C.A. Marques, A. Bonert, C. Frev, K. Schüssel, W.E. Müller, Mitochondrial dysfunction, apoptotic cell death, and Alzheimer’s disease, Biochem. Pharmacol. 66 (2003) 1627–1634. [8] A. Giovanni, E. Keramaris, E.J. Morris, S.T. Hou, M. O’Hare, N. Dyson, G.S. Robertson, R.S. Slack, D.S. Park, E2F1 mediates death of ␤-amyloid-treated cortical neurons in a manner independent of p53 and dependent on Bax and caspase 3, J. Biol. Chem. 275 (2000) 11553–11560. [9] J. Götz, D. David, F. Hoerndli, Y.D. Ke, N. Schonrock, A. Wiesner, T. Fath, L. Bokhari, Y.A. Lim, N. Deters, L.M. Ittner, Functional genomics dissects pathomechanisms in tauopathies: mitosis failure and unfolded protein response, Neurodegen. Dis. 5 (2008) 179–181. [10] J. Hardy, D.J. Selkoe, The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics, Science 297 (2002) 353– 356. [11] T. Harkany, I. Abrahám, C. Kónya, C. Nyakas, M. Zarándi, B. Penke, P.G. Luiten, Mechanisms of beta-amyloid neurotoxicity: perspectives of pharmacotherapy, Rev. Neurosci. 11 (2000) 329–382. [12] K.J. Ivins, J.K. Ivins, J.P. Sharp, C.W. Cotman, Multiple pathways of apoptosis in PC12 cells. CrmA inhibits apoptosis induced by beta-amyloid, J. Biol. Chem. 274 (1999) 2107–2112.

[13] Y. Koriyama, K. Chiba, T. Mohri, Propentofylline protects ␤-amyloid proteininduced apoptosis in cultured rat hippocampal neurons, Eur. J. Pharmacol. 458 (2003) 235–241. [14] C.J. Lagranha, S.M. Hirabara, R. Curi, T.C. Pithon-Curi, Glutamine supplementation prevents exercise-induced neutrophil apoptosis and reduces p38 MAPK and JNK phosphorylation and p53 and caspase 3 expression, Cell Biochem. Funct. 25 (2007) 563–569. [15] C.S. Lai, M.S. Yu, W.H. Yuen, K.F. So, S.Y. Zee, R.C. Chang, Antagonizing betaamyloid peptide neurotoxicity of the anti-aging fungus Ganoderma lucidum, Brain Res. 1190 (2008) 215–224. [16] S.Y. Lee, J.W. Lee, H. Lee, H.S. Yoo, Y.P. Yun, K.W. Oh, T.Y. Ha, J.T. Hong, Inhibitory effect of green tea extract on beta-amyloid-induced PC12 cell death by inhibition of the activation of NF-kappaB and ERK/p38 MAP kinase pathway through antioxidant mechanisms, Brain Res. Mol. Brain Res. 140 (2005) 45–54. [17] G. Li, R. Ma, C. Huang, Q. Tang, Q. Fu, H. Liu, B. Hu, J. Xiang, Protective effect of erythropoietin on beta-amyloid-induced PC12 cell death through antioxidant mechanisms, Neurosci. Lett. 442 (2008) 143–147. [18] S.L. Liu, J.T. Zhang, Effects of naloxone on l-clausenamide-induced long-term potentiation in dentate gyrus of anesthetized rats, Acta Pharmacol. Sin. 20 (1999) 112–116. [19] G. Majno, I. Joris, Apoptosis, oncosis, and necrosis. An overview of cell death, Am. J. Pathol. 146 (1995) 3–15. [20] Y. Mei, Z. Yuan, B. Song, D. Li, C. Ma, C. Hu, Y.P. Ching, M. Li, Activating transcription factor 3 up-regulated by c-Jun NH(2)-terminal kinase/c-Jun contributes to apoptosis induced by potassium deprivation in cerebellar granule neurons, Neuroscience 151 (2008) 771–779. [21] D.L. Price, S.S. Sisodia, S.E. Gandy, Amyloid ␤ amyloidosis in Alzheimer’s disease, Curr. Opin. Neurol. 8 (1995) 268–274. [22] T. Przygodzki, A. Sokal, M. Bryszewska, Calcium ionophore A23187 action on cardiac myocytes is accompanied by enhanced production of reactive oxygen species, Biochim. Biophys. Acta 1740 (2005) 481–488. [23] X.Y. Qin, Y. Cheng, J. Cui, Y. Zhang, L.C. Yu, Potential protection of curcumin against amyloid beta-induced toxicity on cultured rat prefrontal cortical neurons, Neurosci. Lett. 463 (2009) 158–161. [24] B.J. Tabner, S. Turnbull, O.M.A. El-Agnaf, D. Allsop, Formation of hydrogen peroxide and hydroxyl radicals from A␤ and ␣-synuclein as a possible mechanism of cell death in Alzheimer’s disease and Parkinson’s disease, Free Radical Biol. Med. 32 (2002) 1076–1083. [25] T. Tomiyama, A. Shoji, K. Kataoka, Y. Suwa, S. Asano, H. Kaneko, N. Endo, Inhibition of amyloid beta protein aggregation and neurotoxicity by rifampicin. Its possible function as a hydroxyl radical scavenger, J. Biol. Chem. 271 (1996) 6839–6844. [26] A. Valencia, J. Moran, Reactive oxygen species induce different cell death mechanisms in cultured neurons, Free Radical Biol. Med. 36 (2004) 1112–1125. [27] S. Yamagishi, M. Yamada, Y. Ishikawa, T. Matsumoto, T. Ikeuchi, H. Hatanaka, p38 mitogen-activated protein kinase regulates low potassium-induced c-Jun phosphorylation and apoptosis in cultured cerebellar granule neurons, J. Biol. Chem. 276 (2001) 5129–5133. [28] D.C. Zebrowski, R.R. Alcendor, L.A. Kirshenbaum, J. Sadoshima, Caspase-3 mediated cleavage of MEKK1 promotes p53 transcriptional activity, J. Mol. Cell Cardiol. 40 (2006) 605–618. [29] J.J. Zhang, R.F. Zhang, X.K. Meng, Protective effect of pyrroloquinoline quinone against Abeta-induced neurotoxicity in human neuroblastoma SH-SY5Y cells, Neurosci. Lett. 464 (2009) 165–169. [30] X.Z. Zhu, X.Y. Li, J. Liu, Recent pharmacological studies on natural products in China, Eur. J. Pharmacol. 500 (2004) 221–230.