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l -3-n-Butylphthalide regulates amyloid precursor protein processing by PKC and MAPK pathways in SK-N-SH cells over-expressing wild type human APP695
Neuroscience Letters 487 (2011) 211–216
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l-3-n-Butylphthalide regulates amyloid precursor protein processing by PKC and MAPK pathways in SK-N-SH cells over-expressing wild type human APP695 Ying Peng 1 , Yanli Hu 1,2 , Shaofeng Xu, Nan Feng, Ling Wang, Xiaoliang Wang ∗ Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China
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Article history: Received 9 July 2010 Received in revised form 27 September 2010 Accepted 11 October 2010 Keywords: l-3-n-Butylphthalide Amyloid precursor protein A Protein kinase C Mitogen-activated protein kinase
a b s t r a c t Amyloid precursor protein (APP) is cleaved by ␣-secretase, within the amyloid- (A) sequence, resulting in the release of a secreted fragment (␣APPs) and precluding A production. We investigated the effects of a promising anti-AD new drug, l-3-n-butylphthalide (L-NBP), on APP processing and A generation in neuroblastoma SK-N-SH cells overexpressing wild-type human APP695. L-NBP significantly increased ␣APPs release, and reduced A generation. The steady-state full-length APP levels were unaffected by L-NBP. It suggested that L-NBP regulated APP processing towards to the non-amyloidogenic ␣-secretase pathway. Protein kinase C (PKC) and mitogen activated protein (MAP) kinase might be involved in L-NBPinduced ␣APPs secretion. L-NBP significantly increased PKC␣ and activations, lowered PKC␥ activation and increased the phosphorylation of p44/p42 MAPK. Furthermore, PKC and MAPK inhibitors partially reduced L-NBP-induced ␣APPs secretion. The results suggested alternative pharmacological mechanisms of L-NBP regarding the treatment of Alzheimer’s disease (AD). © 2010 Elsevier Ireland Ltd. All rights reserved.
Amyloid precursor protein (APP) proteolysis is the basic process for the generation of amyloid- protein (A) involved in the pathology of Alzheimer’s disease (AD) [15]. APP is cleaved by at least two pathways, amyloidogenic and non-amyloidogenic. In amyloidgenic pathway, APP is cleaved at the A amino-terminus by -secretase and carboxyl-terminus by ␥-secretase; A is then released extracellularly [12]. In the alternative pathway, APP is cleaved by ␣-secretase within the sequence of the A peptide and a secreted form of APP fragment (␣APPs) is released into the extracellular media thereby precluding the formation of A [8]. Under normal metabolic conditions, ␣-secretase processes about 90% of APP by the non-amyloidogenic pathway. The ␣APPs fragment has been reported to have both neurotrophic [35] and neuroprotective activities [19]. Thus, it has been suggested that ␣APPs may serve as an AD therapeutic target [9]. It has been extensively demonstrated that the activation of various second messenger pathways were involved in the cascades that leads to the non-amyloidogenic processing of APP, such as protein kinase C (PKC) and mitogen activated protein (MAP) kinase signal-
∗ Corresponding author at: Pharmacology Department, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, No. 1, Xiannongtan Street, Xicheng District, Beijing 100050, China. Tel.: +86 10 63165173; fax: +86 10 63017757. E-mail address: [email protected] (X. Wang). 1 Equal contribution. 2 Present address: College of Pharmaceutical Sciences, Shihezi University, Shihezi 832002, China. 0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.10.025
ing [4,22]. PKC agonist phorbol esters could increase ␣APPs and decrease A generation [5]. Furthermore, PKC inhibitors reversed PKC agonist-induced ␣APPs release [35]. PKC is a family of at least 12 isoenzymes of serine/threonine protein kinases, central to many signal transduction pathways [24]. The roles of individual PKC isoforms in the regulation of APP proteolytic processing are different [17]. l-3-n-Butylphthalide (L-NBP) was extracted as a pure component from seeds of Apium graveolens Linn, Chinese celery. Previous studies showed that L-NBP significantly reduced the area of cerebral infarct and inhibited platelet aggregation [30,31]. At present, L-NBP has been synthesized and received approval by the State Food and Drug Administration (SFDA) of China for Phase I clinical trial in stroke patients. Recently, we found that L-NBP significantly improved the learning and memory deficits induced by chronic cerebral hypoperfusion and A-intracerebraventricular-infusion in rats [28,29]. In primary neurons and neuroblastoma SH-SY5Y cells, L-NBP attenuated A-induced neuronal apoptosis [27]. Moreover, in the triple transgenic AD mice, L-NBP has been shown to regulate APP processing towards to non-amyloidogenic pathway and reduce A plaque depositions [26]. These results suggested that LNBP might be potential as an AD therapeutic. In the present study, we examined the regulatory effect of L-NBP on APP processing and further elucidated signaling pathways involved in human neuroblastoma SK-N-SH cells overexpressing wild-type APP695 (SK-N-SH APPwt). L-NBP (purity >98%) was synthesized by the Department of Medical Synthetic Chemistry, Institute of Materia Medica. Human
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Fig. 1. L-NBP increased ␣APPs release and had no effect on the steady-state levels of APP in neuroblastoma SK-N-SH APPwt cells. Cells were incubated with L-NBP at each of three concentrations (0.1, 1, or 10 M) or without L-NBP (control) for 24 h. (A) Media were collected, and ␣APPs and steady-state levels of APP were detected with polyclonal antibodies R1736 and C8, respectively, using Western blot. (Band C) Quantitative analysis of the Western blot was expressed as a percentage of ␣APPs (B) and APP (C) from control group. Results are shown as the mean ± S.E.M. and represent six independent experiments. *p < 0.05and **p < 0.01 vs. control group.
neuroblastoma SK-N-SH cells transfected with human wild-type APP695 were a gift from Dr. Dennis J. Selkoe (Center for Neurologic Diseases, Boston, MA). The polyclonal antibodies R1736 that recognizes residues 595–611 of APP695 (␣APPs) and C8 that recognizes the 20 C-terminal residues of APP, were gifts from Dr. Dennis J. Selkoe. A1–42 ELISA kit was purchased from Invitrogen (Carlsbad, CA, USA). Mouse anti-PKC␣, 1, ␥, , and ␦ were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-p44/42 MAP kinase (Thr202/Tyr204) and p44/42 MAP kinase antibody were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). Anti--actin was obtained from Sigma (Saint Louis, MI, USA). Human SK-N-SH APPwt cells were grown in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 100 g/ml penicillin, 100 g/ml streptomycin, and 200 g/ml G418. After incubation with the drugs for the indicated periods, conditioned media were collected and mixed with a complete protease inhibitor cocktail. The media were centrifuged at 3000 × g for 10 min to remove cellular debris, and then were stored at −20 ◦ C. For extracellular signal-regulated kinase (ERK) immunodetection, cells were washed twice with ice-cold PBS buffer and lysated with 20 mmol/L Tris–HCl, pH 8.0, 100 mmol/L NaCl, 0.2 mmol/L EDTA, 3%NP40, 50 mmol/L sodium fluoride, 10 mmol/L sodium pyrophosphate, 2 mmol/L sodium orthovanadate, and complete protease inhibitor cocktail. The lysates were spun at 14,000 rpm for 10 min, and the supernatants were stored at −20 ◦ C. For PKC␣, 1, ␥, , and ␦ assay, cells were mechanically homogenized, and the cellular membrane fractions were isolated from cytoplasmic component with a Membrane and Cytosol Protein Extraction Kit (Beyotime Biotechnology, Wuhan, China). The membrane components were stored at −80 ◦ C. Protein levels were determined using the DC Protein Assay (Bio-Rad, Hercules, CA).
Equal amounts of protein (40 g) were separated on a 10% SDSpolyacrylamide gel electrophoresis and transferred onto a PVDF membrane, and then blocked with 5% fat-free milk for 2 h. Primary antibodies against: PKC␣, PKC, PKC␦, p44/42 MAP kinase (1:500), PKC1, PKC␥, phospho-p44/42 MAP kinase (1:200), and anti-actin (1:10000) were incubated with the membrane overnight at 4 ◦ C. The membrane was incubated with the horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG for 1 h. The signals were detected using an enhanced chemiluminescence (ECL) kit, scanned using an LAS4000 Fujifilm imaging system (Fujifilm, Tokyo Japan), and analyzed by densitometric evaluation using the Quantity-One software (Bio-Rad, Hercules, CA, USA). A-specific sandwich ELISAs were performed using human A1–42 immunoassay kit (Invitrogen). The absorbance was recorded at a 450 nm wavelength using an uQuant microplate spectrophotometer (Bio-Tek Instruments Inc., Rockville, MD, USA). Cell viability was evaluated using MTT assay. Cells were cultured in 96-well plates. After treatment with L-NBP for 24 h, 15 l of MTT solution was added to each well. The plate was incubated at 37 ◦ C for 4 h. Then, 100 l dimethylsulfoxide was added to each well. The absorbance was recorded at a 570 nm wavelength using a 96-well plate reader. All data were expressed as mean ± S.E.M. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by an LSD post hoc test using SPSS software. A value of p < 0.05 was considered significant. Each experiment was repeated three to six times. Using the polyclonal antibody R1736, specifically recognized residues 595–611 of APP695 and labels ␣APPs, we examined the effect of L-NBP on ␣APPs release in SK-N-SH APPwt cells. Western blotting showed that ␣APPs secretion was at the low level in the control group. L-NBP treatment promoted ␣APPs secretion in a concentration-dependent manner. Compared to the control
Y. Peng et al. / Neuroscience Letters 487 (2011) 211–216
Fig. 2. L-NBP reduced A1–42 levels and had no effect on cell viability in neuroblastoma SK-N-SH APPwt cells. (A) A1–42 levels in media were detected by A sandwich ELISAs. (B) Cell viability was evaluated using MTT analysis. Cells were incubated for 24 h without or with increasing concentrations of L-NBP. Results are shown as mean ± S.E.M. and represent three independent experiments. *p < 0.05 vs. control group.
group, L-NBP increased ␣APPs release by 78.5% (p < 0.05) and 150% (p < 0.01) at the dose of 1 M and 10 M, respectively (Fig. 1A and B). The data suggested that L-NBP might mediate APP processing towards the non-amyloidogenic pathway. Then, we evaluated the steady-stage full length APP levels after 24 hr of L-NBP treatment. L-NBP treatment had no effect on APP levels, further suggesting that L-NBP affected APP processing but not APP synthesis (Fig. 1A and C). ELISA results showed that 10 M L-NBP treatment significantly reduced A1–42 production by 27% (p < 0.05) compared to the control group (Fig. 2A). Cell viabilities of SK-N-SH APPwt cells were determined after 24 h incubation of L-NBP. No changes were observed regarding cell viability in SK-N-SH APPwt cells treated with L-NBP at 1 and 10 M (Fig. 2B). It indicated that A decrease was not due to cell death. In the inactivated form, PKC was localized in the cytosol, but translocated to the membrane after activation. PKC signaling pathway has been wildly demonstrated to involve in APP processing. In this study, we observed PKC isoforms activations in the membrane fraction, including PKC␣, 1, ␥, , and ␦, in the SK-N-SH APPwt cells. Firstly, in the control group, we found PKC␣, 1, ␥, , and ␦ were all expressed in the membrane fractions. 10 M L-NBP treatment for 24 hr significantly elevated membrane-bound PKC␣ (p < 0.05) and PKC (p < 0.01) levels (Fig. 3A–C). PKC1 activation was non-significantly enhanced with L-NBP treatment (Fig. 3A and D). However, L-NBP at concentrations of 1 and 10 M markedly reduced membrane-bound PKC␥ expressions by 34% (p < 0.01) and 52% (p < 0.01) compared to the control group (Fig. 3A and E). In addition, L-NBP had no effect on PKC␦ activation (Fig. 3A and F). Moreover, a specific PKC inhibitor, GF109203X pre-incubated
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with the cells for 30 min prior to L-NBP treatment partially inhibited L-NBP-induced ␣APPs release (Fig. 4B). Taken together, L-NBP might direct APP processing towards non-amyloidogenic pathway by activating specific PKC pathway. Several reports have indicated that MAP kinase could act as a critical mediator in the regulated shedding of different membrane proteins, including APP. Thus, we examined the effect of L-NBP on the MAP kinase pathway. In the control group, MAP kinase was inactivation. While the total MAP kinase protein levels were constant, phosphorylated MAP kinase was elevated in a time-dependent manner. Twenty-four hours after L-NBP treatment, phospho-MAP kinase activation was reached to 7 folds compared to the control group (Fig. 4A). In addition, MEK inhibitor, PD98059 was shown to antagonize L-NBP-induced ␣APPs release (Fig. 4B). The results indicated that MAP kinase might be involved in L-NBP-mediated ␣APPs release. The A cascade hypothesis proposes that cerebral A plays a central role in the pathogenesis of AD [13]. The therapeutics aimed at reducing A production by promoting non-amyloidogenic APP processing is promising in AD treatment [25]. In addition, ␣APPs has been shown neuroprotective and neurotrophic properties [19], such as improving memory in normal and amnesic mice [20], enhancing long-term potentiation (LTP) in hippocampal slices [16], and protecting hippocampal and cortical neurons against the toxic effects of glutamate and A peptide [10]. The present study showed that L-NBP significantly reduced A production and promoted ␣APPs secretion. The A reduction was not due to cell death and A degrading enzyme activation (data not shown). The results were consistent to the previous study in the triple transgenic AD mice. Thus, L-NBP-mediated APP processing towards to non-amyloidogenic pathway and precluding A generation might be the one of the mechanisms of anti-AD treatment. PKC is a multi-gene family and consists of 12 serine/threonine protein kinases originally characterized by their dependency upon lipids for catalytic activity [23,24], and its expression is abundant in neuronal tissues. The roles of individual PKC isoforms in the regulation of APP processing are not yet understood. Recently, PKC␣, 1 and have been reported to involve in APP processing [6,17]. The specific PKC isoforms involved in APP processing probably may vary according to the exact stage of the processing [6]. Cells transfected with antisense PKC␣ and PKC cDNA exhibited reduced ␣APPs release in response to phorbol esters [17,32]. In addition, PKC␣ and PKC1 were demonstrated to be the key regulators of ␣-secretory APP processing in vivo and in vitro study in guinea pigs [33]. In this study, we determined which of the PKC isoforms might be involved in the effect of L-NBP. PKC translocation to the membrane fraction upon activation and membrane localization is often used as a maker for PKC activation. Thus, we observed PKC isoforms activation in the membrane fraction. The results showed that L-NBP markedly increased PKC␣ and PKC activation in the SK-N-SH APPwt cells, whereas PKC1 activation was unchanged. The evidence that PKC inhibitor, GF109203X, partially inhibited LNBP-induced ␣APPs release further confirmed that PKC␣ and PKC might be the targets of L-NBP in regulating non-amyloidogenic APP processing. In addition, PKC␣ is known to phosphorylate Bcl-2 in a site that increases its anti-apoptotic function [34]. Overexpression of PKC results in elevated expression of Bcl-2 [11]. Moreover, suppression of PKC␣ triggers apoptosis by down-regulating Bcl-xL [14]. Our recent study also found that L-NBP protected neuronal cells from H2 O2 -induced apoptosis through activating PKC␣ (data not shown). The effects of PKC␥ and ␦ on APP processing are unclear, but they play the pivotal role in ROS generation, which seems to act upstream of caspase-3 activation. Thus, PKC␥ and ␦ were considered to take part in the apoptosis process [2]. The implication of PKC␥ in PMA-induced apoptosis in U937 has also been described
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Fig. 3. L-NBP regulated PKC isoforms activations in neuroblastoma SK-N-SH APPwt cells. (A) Cells were treated with control alone or 10 M L-NBP for 24 h, and the membrane proteins were extracted for Western blot analysis. Quantitative analysis of the Western blot was expressed as a percentage of PKC␣ (B), PKC (C), PKC1 (D), PKC␥ (E) and PKC␦ (F) from the control group. Results are shown as the mean ± S.E.M. and represent six independent experiments. *p < 0.05and **p < 0.01 vs. control group.
previously [1]. L-NBP markedly inhibited PKC␥ activity and nonsignificantly reduced PKC␦ activity, suggesting that L-NBP might possess neuroprotection by antagonizing PKC␥ and ␦ activations. Taken together, L-NBP showed neuroprotective effects in AD treatment by regulating PKC isoforms. MAP kinase serves as an important regulatory pathway in the cleavage of different membrane proteins, including APP [7,21]. There are three pathways in MAP kinase signaling cascades: the extracellular signal-regulated kinase (ERK1/2, p42/44), p38 and c-Jun N-terminal kinase (JNK) pathways. In particular, ERK1/2 pathway has been shown to be involved in APP processing [3,18]. After treatment of L-NBP, ERK1/2 expression was pronouncedly increased. At the meantime, MEK inhibitor, PD98059 partially blocked L-NBP-regulated aAPPs release. These findings indicated that MAP kinase activation might be involved in L-NBP-stimulated ␣APPs release in human neuroblastoma cells.
The relationship of PKC and MAPK signaling pathways on L-NBP-regulated APP processing is unclear. We speculate that L-NBP-mediated APP processing is direct by activation of PKC isozymes ␣ and , and indirect through PKC activation of ERK1/2, but a study is needed to further assess this possibility. L-NBP is on Phase I clinical trial. The acute toxicity test and longterm toxicity test in animal models showed that L-NBP had not the obvious toxicity. L-NBP is liposoluble, and the drug metabolism analysis showed that L-NBP was mainly distributed in the brain after oral administration. In conclusion, our study demonstrated that L-NBP stimulated the non-amyloidogenic cleavage of APP via the PKC and MAP kinase pathways in neuroblastoma SK-N-SH APPwt cells. As a specific PKC activator, the neuroprotective activity of L-NBP, together with the ability to stimulate ␣APPs release, indicates that L-NBP may be beneficial for AD therapy.
Y. Peng et al. / Neuroscience Letters 487 (2011) 211–216
Fig. 4. Effects of L-NBP on PKC and MAP kinase activation in neuroblastoma SKN-SH APPwt cells. (A) 10 M L-NBP treatment increased MAP kinase activation in a time-dependent manner. (B) Specific signaling inhibitors of PKC (GF109203X) and MEK (PD98059) reduced L-NBP-induced ␣APPs release. Results are shown as mean ± S.E.M., and represent three independent experiments. ***p < 0.001 vs. control group, ## p < 0.01 and ### p < 0.001 vs. L-NBP alone.
Acknowledgements This study was supported by the grants from National Natural Sciences Foundation of China (No. 30973511), Natural Science Foundation of Beijing (No. 7093125) and National Science and Technology Major Special Project on Major New Drug Innovation of China (2008ZX09401-004 and 2009ZX09303-003). References [1] M. Abdelhaleem, Differential effect of Bcl-xl over-expression on cell death of the monocytic leukemia cell line U937, Anticancer Res. 22 (2002) 3911–3915. [2] A. Basu, M.D. Woolard, C.L. Johnson, Involvement of protein kinase C-delta in DNA damage-induced apoptosis, Cell Death Differ. 8 (2001) 899–908. [3] J.M. Camden, A.M. Schrader, R.E. Camden, F.A. Gonzalez, L. Erb, C.I. Seye, G.A. Weisman, P2Y2 nucleotide receptors enhance alpha-secretase-dependent amyloid precursor protein processing, J. Biol. Chem. 280 (2005) 18696–18702. [4] A. Caputi, S. Barindelli, L. Pastorino, M. Cimino, J.D. Buxbaum, F. Cattabeni, M. Di Luca, Increased secretion of the amino-terminal fragment of amyloid precursor protein in brains of rats with a constitutive up-regulation of protein kinase C, J. Neurochem. 68 (1997) 2523–2529. [5] F. Checler, Processing of the beta-amyloid precursor protein and its regulation in Alzheimer’s disease, J. Neurochem. 65 (1995) 1431–1444.
215
[6] O.A. da Cruz e Silva, S. Rebelo, S.I. Vieira, S. Gandy, E.F. da Cruz e Silva, P. Greengard, Enhanced generation of Alzheimer’s amyloid-beta following chronic exposure to phorbol ester correlates with differential effects on alpha and epsilon isozymes of protein kinase C, J. Neurochem. 108 (2009) 319–330. [7] J. Desdouits-Magnen, F. Desdouits, S. Takeda, L.J. Syu, A.R. Saltiel, J.D. Buxbaum, A.J. Czernik, A.C. Nairn, P. Greengard, Regulation of secretion of Alzheimer amyloid precursor protein by the mitogen-activated protein kinase cascade, J. Neurochem. 70 (1998) 524–530. [8] F.S. Esch, P.S. Keim, E.C. Beattie, R.W. Blacher, A.R. Culwell, T. Oltersdorf, D. McClure, P.J. Ward, Cleavage of amyloid beta peptide during constitutive processing of its precursor, Science 248 (1990) 1122–1124. [9] R. Etcheberrigaray, M. Tan, I. Dewachter, C. Kuiperi, I. Van der Auwera, S. Wera, L. Qiao, B. Bank, T.J. Nelson, A.P. Kozikowski, F. Van Leuven, D.L. Alkon, Therapeutic effects of PKC activators in Alzheimer’s disease transgenic mice, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 11141–11146. [10] K. Furukawa, S.W. Barger, E.M. Blalock, M.P. Mattson, Activation of K+ channels and suppression of neuronal activity by secreted beta-amyloid-precursor protein, Nature 379 (1996) 74–78. [11] E. Gubina, M.S. Rinaudo, Z. Szallasi, P.M. Blumberg, R.A. Mufson, Overexpression of protein kinase C isoform epsilon but not delta in human interleukin-3dependent cells suppresses apoptosis and induces bcl-2 expression, Blood 91 (1998) 823–829. [12] C. Haass, M.G. Schlossmacher, A.Y. Hung, C. Vigo-Pelfrey, A. Mellon, B.L. Ostaszewski, I. Lieberburg, E.H. Koo, D. Schenk, D.B. Teplow, D.J. Selkoe, Amyloid beta-peptide is produced by cultured cells during normal metabolism, Nature 359 (1992) 322–325. [13] 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. [14] Y.C. Hsieh, H.C. Jao, R.C. Yang, H.K. Hsu, C. Hsu, Suppression of protein kinase Calpha triggers apoptosis through down-regulation of Bcl-xL in a rat hepatic epithelial cell line, Shock 19 (2003) 582–587. [15] J.T. Huse, R.W. Doms, Closing in on the amyloid cascade: recent insights into the cell biology of Alzheimer’s disease, Mol. Neurobiol. 22 (2000) 81–98; D.J. Selkoe, Normal and abnormal biology of the beta-amyloid precursor protein, Annu. Rev. Neurosci. 17 (1994) 489–517. [16] A. Ishida, K. Furukawa, J.N. Keller, M.P. Mattson, Secreted form of betaamyloid precursor protein shifts the frequency dependency for induction of LTD, and enhances LTP in hippocampal slices, Neuroreport 8 (1997) 2133–2137. [17] C. Lanni, M. Mazzucchelli, E. Porrello, S. Govoni, M. Racchi, Differential involvement of protein kinase C alpha and epsilon in the regulated secretion of soluble amyloid precursor protein, Eur. J. Biochem. 271 (2004) 3068–3075. [18] G. Ma, S. Chen, X. Wang, M. Ba, H. Yang, G. Lu, Short-term interleukin-1(beta) increases the release of secreted APP(alpha) via MEK1/2-dependent and JNKdependent alpha-secretase cleavage in neuroglioma U251 cells, J. Neurosci. Res. 80 (2005) 683–692. [19] M.P. Mattson, Cellular actions of beta-amyloid precursor protein and its soluble and fibrillogenic derivatives, Physiol. Rev. 77 (1997) 1081–1132. [20] H. Meziane, J.C. Dodart, C. Mathis, S. Little, J. Clemens, S.M. Paul, A. Ungerer, Memory-enhancing effects of secreted forms of the beta-amyloid precursor protein in normal and amnestic mice, Proc. Natl. Acad. Sci. U.S.A. 95 (1998) 12683–12688. [21] J. Mills, D. Laurent Charest, F. Lam, K. Beyreuther, N. Ida, S.L. Pelech, P.B. Reiner, Regulation of amyloid precursor protein catabolism involves the mitogenactivated protein kinase signal transduction pathway, J. Neurosci. 17 (1997) 9415–9422. [22] A. Mudher, S. Chapman, J. Richardson, A. Asuni, G. Gibb, C. Pollard, R. Killick, T. Iqbal, L. Raymond, I. Varndell, P. Sheppard, A. Makoff, E. Gower, P.E. Soden, P. Lewis, M. Murphy, T.E. Golde, H.T. Rupniak, B.H. Anderton, S. Lovestone, Dishevelled regulates the metabolism of amyloid precursor protein via protein kinase C/mitogen-activated protein kinase and c-Jun terminal kinase, J. Neurosci. 21 (2001) 4987–4995. [23] Y. Nishizuka, Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C, Science 258 (1992) 607–614. [24] Y. Nishizuka, Protein kinase C and lipid signaling for sustained cellular responses, FASEB J. 9 (1995) 484–496. [25] D.F. Obregon, K. Rezai-Zadeh, Y. Bai, N. Sun, H. Hou, J. Ehrhart, J. Zeng, T. Mori, G.W. Arendash, D. Shytle, T. Town, J. Tan, ADAM10 activation is required for green tea (−)-epigallocatechin-3-gallate-induced alpha-secretase cleavage of amyloid precursor protein, J. Biol. Chem. 281 (2006) 16419–16427. [26] Y. Peng, J. Sun, S. Hon, A.N. Nylander, W. Xia, Y. Feng, X. Wang, C.A. Lemere, l-3-n-Butylphthalide improves cognitive impairment and reduces amyloidbeta in a transgenic model of Alzheimer’s disease, J. Neurosci. 30 (2010) 8180–8189. [27] Y. Peng, C.H. Xing, C.A. Lemere, G.Q. Chen, S.F. Xu, L. Wang, Y.P. Feng, X.L. Wang, l-3-n-Butylphthalide ameliorates beta-amyloid-induced neuronal toxicity in cultured neuronal cells, Neurosci. Lett. 434 (2008) 224–229. [28] Y. Peng, C.H. Xing, S.F. Xu, C.A. Lemere, G.Q. Chen, B. Liu, L. Wang, Y.P. Feng, X.L. Wang, l-3-n-Butylphthalide improves cognitive impairment induced by intracerebroventricular infusion of amyloid- peptide in rats, Eur. J. Pharmacol. 621 (2009) 38–45. [29] Y. Peng, S.F. Xu, G.Q. Chen, L. Wang, Y.P. Feng, X.L. Wang, l-3-n-Butylphthalide improves cognitive impairment induced by chronic cerebral hypoperfusion in rats, J. Pharmacol. Exp. Ther. 321 (2007) 902–910.
216
Y. Peng et al. / Neuroscience Letters 487 (2011) 211–216
[30] Y. Peng, S.F. Xu, L. Wang, Y.P. Feng, X.L. Wang, Effect of l-3-n-utylphthalide on cerebral infarct volume in the transient cerebral ischemia rats, Chin. New Drug 14 (2005) 420–423. [31] Y. Peng, X. Zeng, Y. Feng, X.L. Wang, Antiplatelet and antithrombotic activity of l-3-n-butylphthalide in rats, J. Cardiovasc. Pharmacol. 43 (2004) 876–881. [32] M. Racchi, M. Mazzucchelli, A. Pascale, M. Sironi, S. Govoni, Role of protein kinase Calpha in the regulated secretion of the amyloid precursor protein, Mol. Psychiatry 8 (2003) 209–216.
[33] S. RoBner, K. Mendla, R. Schliebs, V. Bigl, Protein kinase Ca and b1 isoforms are regulators of asecretory proteolytic processing of amyloid precursor protein in vivo, Eur. J. Neurosci. 13 (2001) 1644–1648. [34] P.P. Ruvolo, X. Deng, B.K. Carr, W.S. May, A functional role for mitochondrial protein kinase Calpha in Bcl2 phosphorylation and suppression of apoptosis, J. Biol. Chem. 273 (1998) 25436–25442. [35] W.C. Wallace, C.A. Akar, W.E. Lyons, Amyloid precursor protein potentiates the neurotrophic activity of NGF, Brain Res. Mol. Brain Res. 52 (1997) 201–212.
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l-3-n-Butylphthalide regulates amyloid precursor protein processing by PKC and MAPK pathways in SK-N-SH cells over-expressing wild type human APP695 Ying Peng 1 , Yanli Hu 1,2 , Shaofeng Xu, Nan Feng, Ling Wang, Xiaoliang Wang ∗ Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China
a r t i c l e
i n f o
Article history: Received 9 July 2010 Received in revised form 27 September 2010 Accepted 11 October 2010 Keywords: l-3-n-Butylphthalide Amyloid precursor protein A Protein kinase C Mitogen-activated protein kinase
a b s t r a c t Amyloid precursor protein (APP) is cleaved by ␣-secretase, within the amyloid- (A) sequence, resulting in the release of a secreted fragment (␣APPs) and precluding A production. We investigated the effects of a promising anti-AD new drug, l-3-n-butylphthalide (L-NBP), on APP processing and A generation in neuroblastoma SK-N-SH cells overexpressing wild-type human APP695. L-NBP significantly increased ␣APPs release, and reduced A generation. The steady-state full-length APP levels were unaffected by L-NBP. It suggested that L-NBP regulated APP processing towards to the non-amyloidogenic ␣-secretase pathway. Protein kinase C (PKC) and mitogen activated protein (MAP) kinase might be involved in L-NBPinduced ␣APPs secretion. L-NBP significantly increased PKC␣ and activations, lowered PKC␥ activation and increased the phosphorylation of p44/p42 MAPK. Furthermore, PKC and MAPK inhibitors partially reduced L-NBP-induced ␣APPs secretion. The results suggested alternative pharmacological mechanisms of L-NBP regarding the treatment of Alzheimer’s disease (AD). © 2010 Elsevier Ireland Ltd. All rights reserved.
Amyloid precursor protein (APP) proteolysis is the basic process for the generation of amyloid- protein (A) involved in the pathology of Alzheimer’s disease (AD) [15]. APP is cleaved by at least two pathways, amyloidogenic and non-amyloidogenic. In amyloidgenic pathway, APP is cleaved at the A amino-terminus by -secretase and carboxyl-terminus by ␥-secretase; A is then released extracellularly [12]. In the alternative pathway, APP is cleaved by ␣-secretase within the sequence of the A peptide and a secreted form of APP fragment (␣APPs) is released into the extracellular media thereby precluding the formation of A [8]. Under normal metabolic conditions, ␣-secretase processes about 90% of APP by the non-amyloidogenic pathway. The ␣APPs fragment has been reported to have both neurotrophic [35] and neuroprotective activities [19]. Thus, it has been suggested that ␣APPs may serve as an AD therapeutic target [9]. It has been extensively demonstrated that the activation of various second messenger pathways were involved in the cascades that leads to the non-amyloidogenic processing of APP, such as protein kinase C (PKC) and mitogen activated protein (MAP) kinase signal-
∗ Corresponding author at: Pharmacology Department, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, No. 1, Xiannongtan Street, Xicheng District, Beijing 100050, China. Tel.: +86 10 63165173; fax: +86 10 63017757. E-mail address: [email protected] (X. Wang). 1 Equal contribution. 2 Present address: College of Pharmaceutical Sciences, Shihezi University, Shihezi 832002, China. 0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.10.025
ing [4,22]. PKC agonist phorbol esters could increase ␣APPs and decrease A generation [5]. Furthermore, PKC inhibitors reversed PKC agonist-induced ␣APPs release [35]. PKC is a family of at least 12 isoenzymes of serine/threonine protein kinases, central to many signal transduction pathways [24]. The roles of individual PKC isoforms in the regulation of APP proteolytic processing are different [17]. l-3-n-Butylphthalide (L-NBP) was extracted as a pure component from seeds of Apium graveolens Linn, Chinese celery. Previous studies showed that L-NBP significantly reduced the area of cerebral infarct and inhibited platelet aggregation [30,31]. At present, L-NBP has been synthesized and received approval by the State Food and Drug Administration (SFDA) of China for Phase I clinical trial in stroke patients. Recently, we found that L-NBP significantly improved the learning and memory deficits induced by chronic cerebral hypoperfusion and A-intracerebraventricular-infusion in rats [28,29]. In primary neurons and neuroblastoma SH-SY5Y cells, L-NBP attenuated A-induced neuronal apoptosis [27]. Moreover, in the triple transgenic AD mice, L-NBP has been shown to regulate APP processing towards to non-amyloidogenic pathway and reduce A plaque depositions [26]. These results suggested that LNBP might be potential as an AD therapeutic. In the present study, we examined the regulatory effect of L-NBP on APP processing and further elucidated signaling pathways involved in human neuroblastoma SK-N-SH cells overexpressing wild-type APP695 (SK-N-SH APPwt). L-NBP (purity >98%) was synthesized by the Department of Medical Synthetic Chemistry, Institute of Materia Medica. Human
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Fig. 1. L-NBP increased ␣APPs release and had no effect on the steady-state levels of APP in neuroblastoma SK-N-SH APPwt cells. Cells were incubated with L-NBP at each of three concentrations (0.1, 1, or 10 M) or without L-NBP (control) for 24 h. (A) Media were collected, and ␣APPs and steady-state levels of APP were detected with polyclonal antibodies R1736 and C8, respectively, using Western blot. (Band C) Quantitative analysis of the Western blot was expressed as a percentage of ␣APPs (B) and APP (C) from control group. Results are shown as the mean ± S.E.M. and represent six independent experiments. *p < 0.05and **p < 0.01 vs. control group.
neuroblastoma SK-N-SH cells transfected with human wild-type APP695 were a gift from Dr. Dennis J. Selkoe (Center for Neurologic Diseases, Boston, MA). The polyclonal antibodies R1736 that recognizes residues 595–611 of APP695 (␣APPs) and C8 that recognizes the 20 C-terminal residues of APP, were gifts from Dr. Dennis J. Selkoe. A1–42 ELISA kit was purchased from Invitrogen (Carlsbad, CA, USA). Mouse anti-PKC␣, 1, ␥, , and ␦ were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-p44/42 MAP kinase (Thr202/Tyr204) and p44/42 MAP kinase antibody were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). Anti--actin was obtained from Sigma (Saint Louis, MI, USA). Human SK-N-SH APPwt cells were grown in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 100 g/ml penicillin, 100 g/ml streptomycin, and 200 g/ml G418. After incubation with the drugs for the indicated periods, conditioned media were collected and mixed with a complete protease inhibitor cocktail. The media were centrifuged at 3000 × g for 10 min to remove cellular debris, and then were stored at −20 ◦ C. For extracellular signal-regulated kinase (ERK) immunodetection, cells were washed twice with ice-cold PBS buffer and lysated with 20 mmol/L Tris–HCl, pH 8.0, 100 mmol/L NaCl, 0.2 mmol/L EDTA, 3%NP40, 50 mmol/L sodium fluoride, 10 mmol/L sodium pyrophosphate, 2 mmol/L sodium orthovanadate, and complete protease inhibitor cocktail. The lysates were spun at 14,000 rpm for 10 min, and the supernatants were stored at −20 ◦ C. For PKC␣, 1, ␥, , and ␦ assay, cells were mechanically homogenized, and the cellular membrane fractions were isolated from cytoplasmic component with a Membrane and Cytosol Protein Extraction Kit (Beyotime Biotechnology, Wuhan, China). The membrane components were stored at −80 ◦ C. Protein levels were determined using the DC Protein Assay (Bio-Rad, Hercules, CA).
Equal amounts of protein (40 g) were separated on a 10% SDSpolyacrylamide gel electrophoresis and transferred onto a PVDF membrane, and then blocked with 5% fat-free milk for 2 h. Primary antibodies against: PKC␣, PKC, PKC␦, p44/42 MAP kinase (1:500), PKC1, PKC␥, phospho-p44/42 MAP kinase (1:200), and anti-actin (1:10000) were incubated with the membrane overnight at 4 ◦ C. The membrane was incubated with the horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG for 1 h. The signals were detected using an enhanced chemiluminescence (ECL) kit, scanned using an LAS4000 Fujifilm imaging system (Fujifilm, Tokyo Japan), and analyzed by densitometric evaluation using the Quantity-One software (Bio-Rad, Hercules, CA, USA). A-specific sandwich ELISAs were performed using human A1–42 immunoassay kit (Invitrogen). The absorbance was recorded at a 450 nm wavelength using an uQuant microplate spectrophotometer (Bio-Tek Instruments Inc., Rockville, MD, USA). Cell viability was evaluated using MTT assay. Cells were cultured in 96-well plates. After treatment with L-NBP for 24 h, 15 l of MTT solution was added to each well. The plate was incubated at 37 ◦ C for 4 h. Then, 100 l dimethylsulfoxide was added to each well. The absorbance was recorded at a 570 nm wavelength using a 96-well plate reader. All data were expressed as mean ± S.E.M. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by an LSD post hoc test using SPSS software. A value of p < 0.05 was considered significant. Each experiment was repeated three to six times. Using the polyclonal antibody R1736, specifically recognized residues 595–611 of APP695 and labels ␣APPs, we examined the effect of L-NBP on ␣APPs release in SK-N-SH APPwt cells. Western blotting showed that ␣APPs secretion was at the low level in the control group. L-NBP treatment promoted ␣APPs secretion in a concentration-dependent manner. Compared to the control
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Fig. 2. L-NBP reduced A1–42 levels and had no effect on cell viability in neuroblastoma SK-N-SH APPwt cells. (A) A1–42 levels in media were detected by A sandwich ELISAs. (B) Cell viability was evaluated using MTT analysis. Cells were incubated for 24 h without or with increasing concentrations of L-NBP. Results are shown as mean ± S.E.M. and represent three independent experiments. *p < 0.05 vs. control group.
group, L-NBP increased ␣APPs release by 78.5% (p < 0.05) and 150% (p < 0.01) at the dose of 1 M and 10 M, respectively (Fig. 1A and B). The data suggested that L-NBP might mediate APP processing towards the non-amyloidogenic pathway. Then, we evaluated the steady-stage full length APP levels after 24 hr of L-NBP treatment. L-NBP treatment had no effect on APP levels, further suggesting that L-NBP affected APP processing but not APP synthesis (Fig. 1A and C). ELISA results showed that 10 M L-NBP treatment significantly reduced A1–42 production by 27% (p < 0.05) compared to the control group (Fig. 2A). Cell viabilities of SK-N-SH APPwt cells were determined after 24 h incubation of L-NBP. No changes were observed regarding cell viability in SK-N-SH APPwt cells treated with L-NBP at 1 and 10 M (Fig. 2B). It indicated that A decrease was not due to cell death. In the inactivated form, PKC was localized in the cytosol, but translocated to the membrane after activation. PKC signaling pathway has been wildly demonstrated to involve in APP processing. In this study, we observed PKC isoforms activations in the membrane fraction, including PKC␣, 1, ␥, , and ␦, in the SK-N-SH APPwt cells. Firstly, in the control group, we found PKC␣, 1, ␥, , and ␦ were all expressed in the membrane fractions. 10 M L-NBP treatment for 24 hr significantly elevated membrane-bound PKC␣ (p < 0.05) and PKC (p < 0.01) levels (Fig. 3A–C). PKC1 activation was non-significantly enhanced with L-NBP treatment (Fig. 3A and D). However, L-NBP at concentrations of 1 and 10 M markedly reduced membrane-bound PKC␥ expressions by 34% (p < 0.01) and 52% (p < 0.01) compared to the control group (Fig. 3A and E). In addition, L-NBP had no effect on PKC␦ activation (Fig. 3A and F). Moreover, a specific PKC inhibitor, GF109203X pre-incubated
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with the cells for 30 min prior to L-NBP treatment partially inhibited L-NBP-induced ␣APPs release (Fig. 4B). Taken together, L-NBP might direct APP processing towards non-amyloidogenic pathway by activating specific PKC pathway. Several reports have indicated that MAP kinase could act as a critical mediator in the regulated shedding of different membrane proteins, including APP. Thus, we examined the effect of L-NBP on the MAP kinase pathway. In the control group, MAP kinase was inactivation. While the total MAP kinase protein levels were constant, phosphorylated MAP kinase was elevated in a time-dependent manner. Twenty-four hours after L-NBP treatment, phospho-MAP kinase activation was reached to 7 folds compared to the control group (Fig. 4A). In addition, MEK inhibitor, PD98059 was shown to antagonize L-NBP-induced ␣APPs release (Fig. 4B). The results indicated that MAP kinase might be involved in L-NBP-mediated ␣APPs release. The A cascade hypothesis proposes that cerebral A plays a central role in the pathogenesis of AD [13]. The therapeutics aimed at reducing A production by promoting non-amyloidogenic APP processing is promising in AD treatment [25]. In addition, ␣APPs has been shown neuroprotective and neurotrophic properties [19], such as improving memory in normal and amnesic mice [20], enhancing long-term potentiation (LTP) in hippocampal slices [16], and protecting hippocampal and cortical neurons against the toxic effects of glutamate and A peptide [10]. The present study showed that L-NBP significantly reduced A production and promoted ␣APPs secretion. The A reduction was not due to cell death and A degrading enzyme activation (data not shown). The results were consistent to the previous study in the triple transgenic AD mice. Thus, L-NBP-mediated APP processing towards to non-amyloidogenic pathway and precluding A generation might be the one of the mechanisms of anti-AD treatment. PKC is a multi-gene family and consists of 12 serine/threonine protein kinases originally characterized by their dependency upon lipids for catalytic activity [23,24], and its expression is abundant in neuronal tissues. The roles of individual PKC isoforms in the regulation of APP processing are not yet understood. Recently, PKC␣, 1 and have been reported to involve in APP processing [6,17]. The specific PKC isoforms involved in APP processing probably may vary according to the exact stage of the processing [6]. Cells transfected with antisense PKC␣ and PKC cDNA exhibited reduced ␣APPs release in response to phorbol esters [17,32]. In addition, PKC␣ and PKC1 were demonstrated to be the key regulators of ␣-secretory APP processing in vivo and in vitro study in guinea pigs [33]. In this study, we determined which of the PKC isoforms might be involved in the effect of L-NBP. PKC translocation to the membrane fraction upon activation and membrane localization is often used as a maker for PKC activation. Thus, we observed PKC isoforms activation in the membrane fraction. The results showed that L-NBP markedly increased PKC␣ and PKC activation in the SK-N-SH APPwt cells, whereas PKC1 activation was unchanged. The evidence that PKC inhibitor, GF109203X, partially inhibited LNBP-induced ␣APPs release further confirmed that PKC␣ and PKC might be the targets of L-NBP in regulating non-amyloidogenic APP processing. In addition, PKC␣ is known to phosphorylate Bcl-2 in a site that increases its anti-apoptotic function [34]. Overexpression of PKC results in elevated expression of Bcl-2 [11]. Moreover, suppression of PKC␣ triggers apoptosis by down-regulating Bcl-xL [14]. Our recent study also found that L-NBP protected neuronal cells from H2 O2 -induced apoptosis through activating PKC␣ (data not shown). The effects of PKC␥ and ␦ on APP processing are unclear, but they play the pivotal role in ROS generation, which seems to act upstream of caspase-3 activation. Thus, PKC␥ and ␦ were considered to take part in the apoptosis process [2]. The implication of PKC␥ in PMA-induced apoptosis in U937 has also been described
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Fig. 3. L-NBP regulated PKC isoforms activations in neuroblastoma SK-N-SH APPwt cells. (A) Cells were treated with control alone or 10 M L-NBP for 24 h, and the membrane proteins were extracted for Western blot analysis. Quantitative analysis of the Western blot was expressed as a percentage of PKC␣ (B), PKC (C), PKC1 (D), PKC␥ (E) and PKC␦ (F) from the control group. Results are shown as the mean ± S.E.M. and represent six independent experiments. *p < 0.05and **p < 0.01 vs. control group.
previously [1]. L-NBP markedly inhibited PKC␥ activity and nonsignificantly reduced PKC␦ activity, suggesting that L-NBP might possess neuroprotection by antagonizing PKC␥ and ␦ activations. Taken together, L-NBP showed neuroprotective effects in AD treatment by regulating PKC isoforms. MAP kinase serves as an important regulatory pathway in the cleavage of different membrane proteins, including APP [7,21]. There are three pathways in MAP kinase signaling cascades: the extracellular signal-regulated kinase (ERK1/2, p42/44), p38 and c-Jun N-terminal kinase (JNK) pathways. In particular, ERK1/2 pathway has been shown to be involved in APP processing [3,18]. After treatment of L-NBP, ERK1/2 expression was pronouncedly increased. At the meantime, MEK inhibitor, PD98059 partially blocked L-NBP-regulated aAPPs release. These findings indicated that MAP kinase activation might be involved in L-NBP-stimulated ␣APPs release in human neuroblastoma cells.
The relationship of PKC and MAPK signaling pathways on L-NBP-regulated APP processing is unclear. We speculate that L-NBP-mediated APP processing is direct by activation of PKC isozymes ␣ and , and indirect through PKC activation of ERK1/2, but a study is needed to further assess this possibility. L-NBP is on Phase I clinical trial. The acute toxicity test and longterm toxicity test in animal models showed that L-NBP had not the obvious toxicity. L-NBP is liposoluble, and the drug metabolism analysis showed that L-NBP was mainly distributed in the brain after oral administration. In conclusion, our study demonstrated that L-NBP stimulated the non-amyloidogenic cleavage of APP via the PKC and MAP kinase pathways in neuroblastoma SK-N-SH APPwt cells. As a specific PKC activator, the neuroprotective activity of L-NBP, together with the ability to stimulate ␣APPs release, indicates that L-NBP may be beneficial for AD therapy.
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Fig. 4. Effects of L-NBP on PKC and MAP kinase activation in neuroblastoma SKN-SH APPwt cells. (A) 10 M L-NBP treatment increased MAP kinase activation in a time-dependent manner. (B) Specific signaling inhibitors of PKC (GF109203X) and MEK (PD98059) reduced L-NBP-induced ␣APPs release. Results are shown as mean ± S.E.M., and represent three independent experiments. ***p < 0.001 vs. control group, ## p < 0.01 and ### p < 0.001 vs. L-NBP alone.
Acknowledgements This study was supported by the grants from National Natural Sciences Foundation of China (No. 30973511), Natural Science Foundation of Beijing (No. 7093125) and National Science and Technology Major Special Project on Major New Drug Innovation of China (2008ZX09401-004 and 2009ZX09303-003). References [1] M. Abdelhaleem, Differential effect of Bcl-xl over-expression on cell death of the monocytic leukemia cell line U937, Anticancer Res. 22 (2002) 3911–3915. [2] A. Basu, M.D. Woolard, C.L. Johnson, Involvement of protein kinase C-delta in DNA damage-induced apoptosis, Cell Death Differ. 8 (2001) 899–908. [3] J.M. Camden, A.M. Schrader, R.E. Camden, F.A. Gonzalez, L. Erb, C.I. Seye, G.A. Weisman, P2Y2 nucleotide receptors enhance alpha-secretase-dependent amyloid precursor protein processing, J. Biol. Chem. 280 (2005) 18696–18702. [4] A. Caputi, S. Barindelli, L. Pastorino, M. Cimino, J.D. Buxbaum, F. Cattabeni, M. Di Luca, Increased secretion of the amino-terminal fragment of amyloid precursor protein in brains of rats with a constitutive up-regulation of protein kinase C, J. Neurochem. 68 (1997) 2523–2529. [5] F. Checler, Processing of the beta-amyloid precursor protein and its regulation in Alzheimer’s disease, J. Neurochem. 65 (1995) 1431–1444.
215
[6] O.A. da Cruz e Silva, S. Rebelo, S.I. Vieira, S. Gandy, E.F. da Cruz e Silva, P. Greengard, Enhanced generation of Alzheimer’s amyloid-beta following chronic exposure to phorbol ester correlates with differential effects on alpha and epsilon isozymes of protein kinase C, J. Neurochem. 108 (2009) 319–330. [7] J. Desdouits-Magnen, F. Desdouits, S. Takeda, L.J. Syu, A.R. Saltiel, J.D. Buxbaum, A.J. Czernik, A.C. Nairn, P. Greengard, Regulation of secretion of Alzheimer amyloid precursor protein by the mitogen-activated protein kinase cascade, J. Neurochem. 70 (1998) 524–530. [8] F.S. Esch, P.S. Keim, E.C. Beattie, R.W. Blacher, A.R. Culwell, T. Oltersdorf, D. McClure, P.J. Ward, Cleavage of amyloid beta peptide during constitutive processing of its precursor, Science 248 (1990) 1122–1124. [9] R. Etcheberrigaray, M. Tan, I. Dewachter, C. Kuiperi, I. Van der Auwera, S. Wera, L. Qiao, B. Bank, T.J. Nelson, A.P. Kozikowski, F. Van Leuven, D.L. Alkon, Therapeutic effects of PKC activators in Alzheimer’s disease transgenic mice, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 11141–11146. [10] K. Furukawa, S.W. Barger, E.M. Blalock, M.P. Mattson, Activation of K+ channels and suppression of neuronal activity by secreted beta-amyloid-precursor protein, Nature 379 (1996) 74–78. [11] E. Gubina, M.S. Rinaudo, Z. Szallasi, P.M. Blumberg, R.A. Mufson, Overexpression of protein kinase C isoform epsilon but not delta in human interleukin-3dependent cells suppresses apoptosis and induces bcl-2 expression, Blood 91 (1998) 823–829. [12] C. Haass, M.G. Schlossmacher, A.Y. Hung, C. Vigo-Pelfrey, A. Mellon, B.L. Ostaszewski, I. Lieberburg, E.H. Koo, D. Schenk, D.B. Teplow, D.J. Selkoe, Amyloid beta-peptide is produced by cultured cells during normal metabolism, Nature 359 (1992) 322–325. [13] 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. [14] Y.C. Hsieh, H.C. Jao, R.C. Yang, H.K. Hsu, C. Hsu, Suppression of protein kinase Calpha triggers apoptosis through down-regulation of Bcl-xL in a rat hepatic epithelial cell line, Shock 19 (2003) 582–587. [15] J.T. Huse, R.W. Doms, Closing in on the amyloid cascade: recent insights into the cell biology of Alzheimer’s disease, Mol. Neurobiol. 22 (2000) 81–98; D.J. Selkoe, Normal and abnormal biology of the beta-amyloid precursor protein, Annu. Rev. Neurosci. 17 (1994) 489–517. [16] A. Ishida, K. Furukawa, J.N. Keller, M.P. Mattson, Secreted form of betaamyloid precursor protein shifts the frequency dependency for induction of LTD, and enhances LTP in hippocampal slices, Neuroreport 8 (1997) 2133–2137. [17] C. Lanni, M. Mazzucchelli, E. Porrello, S. Govoni, M. Racchi, Differential involvement of protein kinase C alpha and epsilon in the regulated secretion of soluble amyloid precursor protein, Eur. J. Biochem. 271 (2004) 3068–3075. [18] G. Ma, S. Chen, X. Wang, M. Ba, H. Yang, G. Lu, Short-term interleukin-1(beta) increases the release of secreted APP(alpha) via MEK1/2-dependent and JNKdependent alpha-secretase cleavage in neuroglioma U251 cells, J. Neurosci. Res. 80 (2005) 683–692. [19] M.P. Mattson, Cellular actions of beta-amyloid precursor protein and its soluble and fibrillogenic derivatives, Physiol. Rev. 77 (1997) 1081–1132. [20] H. Meziane, J.C. Dodart, C. Mathis, S. Little, J. Clemens, S.M. Paul, A. Ungerer, Memory-enhancing effects of secreted forms of the beta-amyloid precursor protein in normal and amnestic mice, Proc. Natl. Acad. Sci. U.S.A. 95 (1998) 12683–12688. [21] J. Mills, D. Laurent Charest, F. Lam, K. Beyreuther, N. Ida, S.L. Pelech, P.B. Reiner, Regulation of amyloid precursor protein catabolism involves the mitogenactivated protein kinase signal transduction pathway, J. Neurosci. 17 (1997) 9415–9422. [22] A. Mudher, S. Chapman, J. Richardson, A. Asuni, G. Gibb, C. Pollard, R. Killick, T. Iqbal, L. Raymond, I. Varndell, P. Sheppard, A. Makoff, E. Gower, P.E. Soden, P. Lewis, M. Murphy, T.E. Golde, H.T. Rupniak, B.H. Anderton, S. Lovestone, Dishevelled regulates the metabolism of amyloid precursor protein via protein kinase C/mitogen-activated protein kinase and c-Jun terminal kinase, J. Neurosci. 21 (2001) 4987–4995. [23] Y. Nishizuka, Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C, Science 258 (1992) 607–614. [24] Y. Nishizuka, Protein kinase C and lipid signaling for sustained cellular responses, FASEB J. 9 (1995) 484–496. [25] D.F. Obregon, K. Rezai-Zadeh, Y. Bai, N. Sun, H. Hou, J. Ehrhart, J. Zeng, T. Mori, G.W. Arendash, D. Shytle, T. Town, J. Tan, ADAM10 activation is required for green tea (−)-epigallocatechin-3-gallate-induced alpha-secretase cleavage of amyloid precursor protein, J. Biol. Chem. 281 (2006) 16419–16427. [26] Y. Peng, J. Sun, S. Hon, A.N. Nylander, W. Xia, Y. Feng, X. Wang, C.A. Lemere, l-3-n-Butylphthalide improves cognitive impairment and reduces amyloidbeta in a transgenic model of Alzheimer’s disease, J. Neurosci. 30 (2010) 8180–8189. [27] Y. Peng, C.H. Xing, C.A. Lemere, G.Q. Chen, S.F. Xu, L. Wang, Y.P. Feng, X.L. Wang, l-3-n-Butylphthalide ameliorates beta-amyloid-induced neuronal toxicity in cultured neuronal cells, Neurosci. Lett. 434 (2008) 224–229. [28] Y. Peng, C.H. Xing, S.F. Xu, C.A. Lemere, G.Q. Chen, B. Liu, L. Wang, Y.P. Feng, X.L. Wang, l-3-n-Butylphthalide improves cognitive impairment induced by intracerebroventricular infusion of amyloid- peptide in rats, Eur. J. Pharmacol. 621 (2009) 38–45. [29] Y. Peng, S.F. Xu, G.Q. Chen, L. Wang, Y.P. Feng, X.L. Wang, l-3-n-Butylphthalide improves cognitive impairment induced by chronic cerebral hypoperfusion in rats, J. Pharmacol. Exp. Ther. 321 (2007) 902–910.
216
Y. Peng et al. / Neuroscience Letters 487 (2011) 211–216
[30] Y. Peng, S.F. Xu, L. Wang, Y.P. Feng, X.L. Wang, Effect of l-3-n-utylphthalide on cerebral infarct volume in the transient cerebral ischemia rats, Chin. New Drug 14 (2005) 420–423. [31] Y. Peng, X. Zeng, Y. Feng, X.L. Wang, Antiplatelet and antithrombotic activity of l-3-n-butylphthalide in rats, J. Cardiovasc. Pharmacol. 43 (2004) 876–881. [32] M. Racchi, M. Mazzucchelli, A. Pascale, M. Sironi, S. Govoni, Role of protein kinase Calpha in the regulated secretion of the amyloid precursor protein, Mol. Psychiatry 8 (2003) 209–216.
[33] S. RoBner, K. Mendla, R. Schliebs, V. Bigl, Protein kinase Ca and b1 isoforms are regulators of asecretory proteolytic processing of amyloid precursor protein in vivo, Eur. J. Neurosci. 13 (2001) 1644–1648. [34] P.P. Ruvolo, X. Deng, B.K. Carr, W.S. May, A functional role for mitochondrial protein kinase Calpha in Bcl2 phosphorylation and suppression of apoptosis, J. Biol. Chem. 273 (1998) 25436–25442. [35] W.C. Wallace, C.A. Akar, W.E. Lyons, Amyloid precursor protein potentiates the neurotrophic activity of NGF, Brain Res. Mol. Brain Res. 52 (1997) 201–212.