Huperzine A regulates amyloid precursor protein processing via protein kinase C and mitogen-activated protein kinase pathways in neuroblastoma SK-N-SH cells over-expressing wild type human amyloid precursor protein 695

Neuroscience 150 (2007) 386 –395

HUPERZINE A REGULATES AMYLOID PRECURSOR PROTEIN PROCESSING VIA PROTEIN KINASE C AND MITOGEN-ACTIVATED PROTEIN KINASE PATHWAYS IN NEUROBLASTOMA SK-N-SH CELLS OVER-EXPRESSING WILD TYPE HUMAN AMYLOID PRECURSOR PROTEIN 695 Y. PENG,a D. Y. W. LEE,b,d L. JIANG,a Z. MA,b,d S. C. SCHACHTERc AND C. A. LEMEREa*

the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase, which was blocked by treatment with U0126 and PD98059. In addition, Hup A inhibited acetylcholinesterase activity by 20% in neuroblastoma cells. Our results indicate that the activation of muscarinic acetylcholine receptors, PKC and MAP kinase may be involved in Hup A–induced ␣APPs secretion in neuroblastoma cells and suggest multiple pharmacological mechanisms of Hup A regarding the treatment of Alzheimer’s disease (AD). © 2007 IBRO. Published by Elsevier Ltd. All rights reserved.

a

Center for Neurologic Diseases, Harvard Medical School and Brigham and Women’s Hospital, Harvard New Research Building, Room 636F, 77 Avenue Louis Pasteur, Boston, MA 02115, USA

b

Bio-Organic and Natural Products Laboratory, McLean Hospital, Harvard Medical School, 115 Mill Street, Belmont, MA 02478, USA

c

Department of Neurology and Division of Research and Education in Complementary and Integrative Medical Therapies, Harvard Medical School, Boston, MA 02215, USA

Key words: huperzine A, amyloid precursor protein, A␤, protein kinase C, mitogen-activated protein kinase, muscarinic acetylcholine receptor.

d

Natural Pharmacia International Incorporated, 115 Mill Street, Belmont, MA 02478, USA

Abstract—Alpha-secretase (␣-secretase), cleaves the amyloid precursor protein (APP) within the amyloid-␤ (A␤) sequence, resulting in the release of a secreted fragment of APP (␣APPs) and precluding A␤ generation. We investigated the effects of the acetylcholinesterase inhibitor, huperzine A (Hup A), on APP processing and A␤ generation in human neuroblastoma SK-N-SH cells overexpressing wild-type human APP695. Hup A dose-dependently (0 –10 ␮M) increased ␣APPs release. Therefore, we evaluated two ␣-secretase candidates, a disintegrin and metalloprotease (ADAM) 10 and ADAM17 in Hup A-induced non-amyloidogenic APP metabolism. Hup A enhanced the level of ADAM10, and the inhibitor of tumor necrosis factor-␣ converting enzyme (TACE)/ADAM17 inhibited the Hup A–induced rise in ␣APPs levels, further suggesting Hup A directed APP metabolism toward the non-amyloidogenic ␣-secretase pathway. Hup A had no effect on A␤ generation in this cell line. The steadystate levels of full-length APP and cell viability were unaffected by Hup A. Alpha-APPs release induced by Hup A treatment was significantly reduced by muscarinic acetylcholine receptor antagonists (particularly by an M1 antagonist), protein kinase C (PKC) inhibitors, GF109203X and calphostin C, and the mitogen-activated kinase kinase (MEK) inhibitors, U0126 and PD98059. Furthermore, Hup A markedly increased

Alzheimer’s disease (AD) is pathologically characterized by the presence of extracellular senile plaques and intracellular neurofibrillary tangles in the brain (Selkoe, 1994). Amyloid-␤ peptide (A␤), a 39 – 43 amino acid peptide derived by proteolysis of a large integral membrane protein, amyloid precursor protein (APP), forms the core of senile plaques and congophilic amyloid angiopathy (Kang et al., 1987). APP is cleaved by at least two pathways. In one pathway, APP is cleaved by alpha-secretase (␣-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␤ (Esch et al., 1990; Sisodia et al., 1990). Membrane-bound disintegrin and metalloprotease (ADAMs) family members are candidates of ␣-secretase. ADAM10 and ADAM17 were examined in the current study due to their relevance to AD; ADAM17 is also referred to as TACE (tumor necrosis factor-alpha converting enzyme) (Buxbaum et al., 1998; Lammich et al., 1999). The ␣APPs fragment has been reported to have both neurotrophic (Wallace et al., 1997) and neuroprotective activities (Mattson, 1997). As such, it has been suggested that ␣APPs may serve as an AD therapeutic target (Etcheberrigaray et al., 2004; Small et al., 2005). The alternative pathway results in the generation of A␤ after APP is cleaved at the A␤ amino-terminus by ␤-secretase and carboxyl-terminus by ␥-secretase; A␤ is then released extracellularly (Haass et al., 1992; Shoji et al., 1992; Seubert et al., 1993). Because the proportion of APP processed by each pathway may determine the amount of A␤, the regulation of ␣-, ␤- and ␥-secretase activities may be critically important to the pathogenesis of AD. Previous

*Corresponding author. Tel: ⫹1-617-525-5214; fax: ⫹1-617-525-5252. E-mail address: [email protected] (C. A. Lemere). Abbreviations: A␤, amyloid-␤ peptide; AChE, acetylcholinesterase; AD, Alzheimer’s disease; ADAM, a disintegrin and metalloprotease; APP, amyloid precursor protein; ␣APPs, soluble ␣-secretase-derived fragment of APP; ERK, extracellular signal-regulated kinase; HEK293, human embryonic kidney 293; HEK293 APPsw, human embryonic kidney 293 cells bearing the Swedish amyloid precursor protein mutation; Hup A, huperzine A; LTP, long-term potentiation; mAChR, muscarinic acetylcholine receptor; MAP, mitogen-activated protein; MEK, mitogen-activated kinase; nAChR, nicotinic acetylcholine receptor; PKC, protein kinase C; SK-N-SH APPwt, SK-N-SH human neuroblastoma cells overexpressing wild type human APP695; TACE, tumor necrosis factor-␣ converting enzyme; TBS-T, Tris–HCl (pH 7.4) containing 150 mmol/l NaCl and 0.05% Tween 20.

0306-4522/07$30.00⫹0.00 © 2007 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2007.09.022

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studies demonstrated that the secretory non-amyloidogenic pathway of APP, driven by ␣-secretase processing, is likely mediated by the activation of the cell-surface muscarinic acetylcholine receptors (mAChR) resulting in the activation of various second messenger cascades (Nitsch et al., 1992; Mills and Reiner, 1999), including protein kinase C (PKC) and mitogen-activated protein (MAP) kinase signaling (Caputi et al., 1997; Mudher et al., 2001). PKC and PKC-coupled receptors are involved in the nonamyloidogenic pathway of APP cleavage both in vitro and in vivo (Caputi et al., 1997; Lin et al., 1999). Huperzine A (Hup A) is a novel plant-derived alkaloid from the Chinese folk medicine “Qian Ceng Ta” (Huperzia serrata). As an acetylcholinesterase (AChE) inhibitor, Hup A has been approved in China to treat AD patients, and has been shown to significantly improve memory in the aged population and patients with AD (Zhang et al., 2002; Zangara, 2003). Compared with tacrine, donepezil, and rivastigmine, Hup A has better penetration through the blood– brain barrier, higher oral bioavailability, and longer duration of AChE inhibitory action and with fewer peripheral cholinergic side effects (Wang et al., 2006). Hup A is currently being tested as a treatment for AD in a phase II clinical trial in the United States (http://www.alzforum.org/ dis/tre/drc/detail.asp? id⫽53). Cholinergic impairment and altered APP processing have been linked as major pathogenic events in AD. Many AChE inhibitors currently in clinical use such as donepezil, rivastigmine and galantamine have been shown to increase ␣APPs release, (Racchi et al., 2004). The effect of AChE inhibitors on stimulating ␣APPs release may involve PKC, MAP kinase and tyrosine kinase– dependent pathways (Yogev-Falach et al., 2002; Racchi et al., 2004; Zhang et al., 2004). We reported previously that in human embryonic kidney 293 cells bearing the Swedish amyloid precursor protein mutation (HEK293 APPsw), Hup A stimulated ␣APPs release and diminished A␤ generation (Peng et al., 2006). However, protein expression, cell function, and cell signaling pathways are quite different between human embryonic kidney 293 (HEK293) cells and neurons. APP processing and A␤ generation are tightly regulated in neurons but HEK293 cells are unable to mimic neuronal function. Thus, we extended our studies to examine the regulatory effects of Hup A on APP processing in human neuroblastoma SK-N-SH cells overexpressing wild-type APP695 (SK-N-SH APPwt). In the present study, our findings demonstrate that Hup A directs APP processing toward the ␣-secretase pathway by mediating the ␣-secretase activity via mAChR activation and PKC and MAP kinase signaling pathways in human neuroblastoma cells.

EXPERIMENTAL PROCEDURES Reagents GF109203X and calphostin C, as well as mitogen-activated kinase (MEK) inhibitors, U0126 and PD98059, were obtained from Calbiochem (La Jolla, CA, USA). The metalloprotease inhibitor TAPI-2 was obtained from Peptides International (Louisville, KY, USA). Each was dissolved (1:100) in dimethyl sulfoxide and

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stored at ⫺20 °C. mAChR and nicotinic acetylcholine receptor (nAChR) antagonists, atropine, mecamylamine, pirenepine dihydrochloride, methoctramine hemihydrate and 4-diphenylacetoxyN-methylpiperidine methiodide were obtained from Sigma (St. Louis, MO, USA). Each was dissolved in distilled water (10 mM) and stored at ⫺20 °C. An Amplex Red AChE assay kit was obtained from Invitrogen (Carlsbad, CA, USA). A Cell Viability Kit (MTT) was obtained from Promega (Madison, WI, USA). Electrophoresis reagents were obtained from Invitrogen Corporation. Amicon Ultra Centrifugal Filter Devices were purchased from Millipore Corporation (Bedford, MA, USA). Human 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, USA). The monoclonal APP antibody, 8E5, recognizes residues 444 –591 of APP, and was a gift from Elan Pharmaceuticals (San Francisco, CA, USA). The polyclonal antibody 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 (Center for Neurologic Diseases). Anti-A␤ monoclonal antibodies for ELISA were kindly provided by Elan Pharmaceuticals. Anti-ADAM10 and anti-TACE/ADAM17 were obtained from ProSci Inc. (Poway, CA, USA). Anti-phosphoMAP kinase and anti-MAP kinase were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). Anti-␤-actin was obtained from Sigma.

Cell culture 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. Cell cultures were incubated at 37 °C in a humid 5% CO2/95% air environment. Cells were grown until nearly confluent, washed with serum-free medium, and incubated in serum-free medium for 18 –24 h. After incubation with the drugs for the indicated periods, conditioned media were collected and mixed with a complete protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA). The media were centrifuged at 3000⫻g for 10 min to remove cellular debris. Supernatants were concentrated with centrifugal filter devices (Amicon Ultra-4). The concentrated conditioned medium was stored at ⫺20 °C. Cells were washed twice with ice-cold PBS and lysed with 500 ␮l of 1% NP-40 in 50 mmol/l Tris–HCl, pH 7.6, 150 mmol/l NaCl, 2 mmol/l EDTA, and complete protease inhibitor mixture. Lysates were spun 14,000 r.p.m. for 10 min, and the supernatants stored at ⫺20 °C. In order to ensure equal loading, protein levels were determined using the DC Protein Assay (Bio-Rad, Hercules, CA).

␣APPs, full-length APP, ADAM10 and TACE/ADAM17 levels After the treatment of the different time points, samples from conditioned media and cell lysates were run on 10 –20% Tricine gels (Invitrogen) and transferred onto 0.2 ␮m nitrocellulose membranes at 400 mA for 2 h and blocked with 5% fat-free milk in 20 mmol/l Tris–HCl (pH 7.4) containing 150 mmol/l NaCl and 0.05% Tween 20 (TBS-T) for 2 h at RT. After washing in TBS-T, the blots were probed with antibodies overnight at 4 °C. Polyclonal antibody R1736 (1:1000) specifically recognizes the ␣- but not the ␤-secretase-generated APP ectodomain fragment. Monoclonal antibody 8E5 (1:1000), which recognizes residues 444 –591 of APP, and polyclonal antibody C8 (1:1000), which recognizes the last 20 C-terminal residues of APP, were used to label the fulllength APP. Polyclonal antibodies against ADAM10 and TACE/ ADAM17 (1:200) recognize the C-terminus of human ADAM10 and TACE, respectively. Anti-␤-actin (1:10,000) was used to detect ␤-actin as a control for gel loading. The membranes were incubated with ECL anti-rabbit or anti-mouse IgG horseradish peroxidase–linked species-specific whole antibody for 2 h at RT.

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The signal was detected using an enhanced chemiluminescence (ECL) kit, scanned, and analyzed by densitometric evaluation using an imaging system and analyzing software (FluorchemTMIS-8800 software, Alpha Innotech, San Leandro, CA, USA).

A␤ levels A␤-specific sandwich ELISAs were performed as described (Seubert et al., 1992; Johnson-Wood et al., 1997). The capture antibody was 266 (to A␤ residues 13–28), and the reporter antibody was biotinylated 3D6 (to A␤ residues 1–5). The absorbance was recorded at a 450 nm wavelength using a 96-well plate reader (Benchmark Plus, Bio-Rad).

Cell viability Cell viability was evaluated using a Cell Viability Kit (MTT: Promega) according to the manufacturer’s instructions. Cells were cultured in 96-well plates. After treatment with Hup A at 6, 12, 18 and 24 h, 15 ␮l of dye solution was added to each well. The plate was incubated at 37 °C for 4 h in a humidified, 5% CO2 atmosphere. Then, 100 ␮l of the solubilization solution was added to each well and the plates were allowed to stand overnight to completely solubilize the formazan crystals. The absorbance was recorded at a 570 nm wavelength using a 96-well plate reader.

Extracellular signal-regulated kinase (ERK) activity Cells were grown in 12-well plates. Eighteen hours before the experiments, the medium was changed to serum-free medium. After drug treatment, the cells were placed on ice to stop the drug reactions. The medium was aspirated. Cells were washed twice with ice-cold PBS buffer and harvested in 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. Protein concentration was detected by the Bio-Rad method. Each cell lysate, containing 20 ␮g protein, was separated on 10 –20% Tricine gels and identified using phospho-p44/42 MAP kinase (Thr202/Tyr204) antibody or p44/42 MAP kinase antibody. Western blot method was same as above. Data represent three independent experiments.

Statistical analysis All data were expressed as mean⫾the standard error of the mean (S.E.M). Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by an LSD post hoc test using Prism software (GraphPad Inc., San Diego, CA, USA). A value of P⬍0.05 was considered significant. Each experiment was repeated three to six times.

RESULTS Hup A increased ␣APPs secretion, but had no effect on cellular APP or A␤ levels To determine whether Hup A regulates APP processing, we examined its effect on the release of ␣APPs into the conditioned media in SK-N-SH APPwt cells. We chose the polyclonal antibody, R1736, which specifically recognizes residues 595– 611 of APP695 and labels ␣APPs. Western blotting showed that after treatment for 6 and 12 h, the ␣APPs levels were not markedly changed. However, after 18 h treatment, Hup A stimulated the release of ␣APPs in a concentration-dependent manner. The maximal effect of Hup A was observed at a concentration of 10 ␮M, which resulted in a 40% increase in ␣APPs levels compared with

Fig. 1. Hup A increased ␣APPs release in neuroblastoma SK-N-SH APPwt cells. Cells were incubated with Hup A at each of three concentrations (0.1, 1 or 10 ␮M) or without Hup A (controls) for 6, 12, 18 and 24 h. Medium was collected and ␣APPs was detected with polyclonal antibody R1736 using Western blot (A). The results showed that Hup A significantly dose-dependently increased ␣APPs release after 18 h treatment. Densitometric analysis of the Western blot was expressed as a percentage of ␣APPs release from control cells (B). Results are shown as the mean⫾S.E.M. and represent six independent experiments. * P⬍0.05 vs. control.

the control group (Fig. 1, P⬍0.05). The data suggest that Hup A mediated APP processing toward the non-amyloidogenic pathway. The elevated ␣APPs levels returned to the baseline 24 h after incubation with Hup A. It has been reported that increased APP synthesis may lead to elevated APP secretion, therefore, we next evaluated the effect of Hup A on cellular APP levels after treatment of 6, 12, 18 and 24 h. Whole-cell lysates were analyzed by Western blot using the APP antibodies, 8E5 and C8 (data not shown). Hup A treatment had no effect on APP steady-state levels, further suggesting that Hup A affected APP processing but not APP synthesis. The results are shown in Fig. 2. To ensure equal sample loading, we included anti-␤-actin as a control. ELISA measurements showed that A␤ levels in the conditioned media were increased time-dependently. At the 6 h time point, A␤ amounts were 211.5⫾11.9 pmol/l, then gradually increased to 256.6⫾28.5, 285.1⫾20.5 and 322.1⫾18.7 pmol/l at the 12, 18 and 24 h time points, respectively. However, at the same incubation times, Hup A treatment did not affect A␤ levels (Fig. 3A). This result suggests that there is no obvious relationship between the increase of ␣APPs release and A␤ secretion in Hup A–treated SK-N-SH APPwt cells. In addition, the cell viability of SK-N-SH APPwt cells was determined after 6, 12, 18 and 24 h incubation of Hup

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TACE/ADAM17 (Fig. 5). These results further confirmed that the effect of Hup A on APP processing was regulated via ␣-secretase activity, and that TACE/ADAM17 was likely involved. Hup A inhibited AChE activity in the neuroblastoma SK-N-SH APPwt cells In the study, we further confirmed Hup A inhibited AChE activity in the neuroblastoma cell line. After incubation with Hup A 10 ␮M for 18 h, SK-N-SH cells were lysed, and the lysates’ AChE activity was measured. Hup A significantly inhibited AChE activity by 20% compared with the control groups (Fig. 6, P⬍0.01). Inhibition of Hup A–induced ␣APPs release by mAChR antagonists and the inhibitors of PKC and MEK kinase In order to further determine the mechanism of Hup A–induced ␣APPs release, we chose mAChR and nAChR antagonists atropine and mecamylamine hydrochloride, for

Fig. 2. Hup A had no effect on the steady-state levels of APP in neuroblastoma SK-N-SH APPwt cells. Cells were incubated with Hup A at each of three concentrations (0.1, 1 or 10 ␮M) or without Hup A (controls) for 6, 12, 18 and 24 h. Full-length APP, mature and immature (mAPP and imAPP, respectively) in the cell lysates was detected by APP monoclonal antibody, 8E5 (A). The results showed that APP levels were unchanged after Hup A treatment. Densitometric analysis of the Western blot was expressed as a percentage of control (B). Results are shown as the mean⫾S.E.M. and represent six independent experiments.

A. No changes were observed regarding cell viability in SK-N-SH APPwt cells treated with Hup A at 0 –10 ␮M (Fig. 3B). Thus, Hup A did not appear to be toxic to SK-N-SH APPwt cells. ADAM10 and TACE/ADAM17 are involved in Hup A–mediated increases in ␣APPs secretion Disintegrin metalloproteases catalyze the shedding of the ectodomain of APPs and other membrane proteins (Allinson et al., 2003). First, we chose C-terminal polyclonal antibodies to detect ADAM10 and TACE/ADAM17 expression at the cellular membrane. After 18 h incubation, Hup A significantly increased the ADAM10 levels in a dosedependent fashion. The maximal effect of Hup A was obtained at a concentration of 10 ␮M, which resulted in a 43% increase in ADAM10 levels compared with the control group (Fig. 4, P⬍0.05). An increase in TACE/ADAM17 levels was also observed in the cellular membrane of SK-N-SH APPwt cells, but compared with the control cells, the difference did not reach significance (data not shown). Furthermore, Hup A–induced ␣APPs release was inhibited by TAPI-2, a selective, competitive inhibitor of

Fig. 3. Hup A had no effect on A␤ levels or cell viability in neuroblastoma SK-N-SH APPwt cells. Cells were incubated for 6, 12, 18 and 24 h without or with increasing concentrations of Hup A. (A) A␤ levels in media were detected by A␤ sandwich ELISAs. A␤ levels were increased in a time-dependent manner. Hup A had no effect on A␤ levels at the same incubation times. (B) Cell viability was evaluated using a Cell Viability Kit (MTT analysis), and the result showed that the cell viability of neuroblastoma cells was unchanged after Hup A incubation. Thus, Hup A was not toxic to neuroblastoma SK-N-SH APPwt cells. Results are shown as mean⫾S.E.M. and represent three independent experiments.

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stimulated the MAP kinase cascade. Hup A (10 ␮M) rapidly activated phosphorylation of MAP kinase as detected using anti-phospho-44/42 MAP kinase (Thr202/Tyr204). While the total MAP kinase protein level was constant (Fig. 8), phosphorylated MAP kinase was elevated in a timedependent manner, which peaked 5–15 min after Hup A treatment. After 30 and 60 min, phospho-MAP kinase activation was reduced and returned to basal levels (Fig. 8A). The phospho-MAP kinase activation induced by Hup A occurred at doses as low as 0.1 ␮M, but had no effect on total MAP kinase levels (Fig. 8B, 8D). Inhibitors of MEK phosphorylation and activation, U0126 and PD 98059, were used to determine the effects of MEK on Hup A–induced phosphorylation of MAP kinase. Pretreatment with U0126 and PD98059 blocked the effect of Hup A on MAP kinase activation (Fig. 8C). Our results indicate that MAP kinase may be involved in Hup A regulation of ␣APPs release.

DISCUSSION Fig. 4. Hup A dose-dependently enhanced ADAM10 protein levels in neuroblastoma SK-N-SH APPwt cells. Cells were incubated with Hup A at each of three concentrations or without Hup A (controls) for 18 h. ADAM10 levels on cellular membranes were detected by polyclonal antibody using Western blot (A). Densitometric analysis of the Western blot was expressed as a percentage of control (B). Results are shown as the mean⫾S.E.M. and represent four independent experiments. * P⬍0.05 vs. control.

co-incubation with Hup A in neuroblastoma cell cultures. The Hup A–induced ␣APPs increase was significantly attenuated by 10 ␮M atropine (Fig. 7A, P⬍0.05). However, nAChR antagonist induced only a small reduction in ␣APPs release. In addition, to ensure which subtype of mAChR activation mainly mediated aAPPs release in the SK-N-SH APPwt cells, M1-, M2- and M3-mAChR antagonists were applied to the cell cultures. The Hup A–mediated increase in ␣APPs secretion was significantly inhibited by 10 ␮M pirenzepine dihydrochloride (M1-mAChR antagonist) (Fig. 7B, P⬍0.05). However, treatment with 10 ␮M methoctramine hemihydrate (M2-mAChR antagonist) or 10 ␮M 4-diphenylacetoxy-N-methylpiperidine methiodide (M3-mAChR antagonist) did not significantly inhibit the Hup A–mediated increase in ␣APPs secretion. This result suggests that mAChR, and M1-mAChR in particular, may be involved in the regulation of Hup A in APP processing in the SK-N-SH APPwt cells. In addition, the cells were pre-incubated with inhibitors of diverse downstream signaling molecules for 30 min prior to Hup A treatment. Alpha-APPs release induced by Hup A was markedly inhibited by the PKC inhibitors, calphostin C (1 ␮M) and GF109203X (2.5 ␮M), and the MEK inhibitors, U0126 (5 ␮M) and PD98059 (30 ␮M), indicating that PKCand MAP kinase– dependent signaling pathways may be involved in Hup A–induced ␣APPs release (Fig. 5). Effect of Hup A on MAP kinase activation In order to investigate further the observation that Hup A–induced ␣APPs release was regulated by the MAP kinase signaling pathway, we investigated whether Hup A

Our results showed that Hup A directed APP metabolism toward the non-amyloidogenic ␣-secretase pathway and stimulated ␣APPs release from human neuroblastoma SK-

Fig. 5. Specific signaling inhibitors of TACE/ADAM17 (TAPI-2), PKC (GF109203X and calphostin C) and MEK (U0126 and PD98059) reduced Hup A–induced ␣APPs release in neuroblastoma SK-N-SH APPwt cells. Cells were pre-incubated for 30 min with vehicle alone or with 1 ␮M calphostin C, 2.5 ␮M GF109203X, 10 ␮M TAPI-2, 5 ␮M U0126, or 30 ␮M PD98059. After pre-incubation, the cells were incubated for 18 h without or with 10 ␮M Hup A. The results showed that the inhibitors of TACE/ADAM17, PKC and MEK partially inhibited Hup A–induced ␣APPs secretion. Alpha-APPs in the media was detected by polyclonal APP antibody R1736 using Western blot. Results are shown as the mean⫾S.E.M. and represent three independent experiments.

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indicate that TACE/ADAM17 may be involved in Hup A–induced APP processing. In addition, it was reported that AChE inhibitors increased TACE/ADAM17 and ADAM10 activity by promoting its trafficking in neuroblastoma cell lines (Zimmermann et al., 2004). The induction of ␣-secretase activity and the increase of non-amyloido-

Fig. 6. Hup A reduced AChE activity in neuroblastoma SK-N-SH APPwt cells. Cells were incubated with or without 10 ␮M Hup A for 18 h. The AChE activity was measured by an Amplex Red AChE assay kit. The results showed that Hup A significantly reduced AChE activity. Results are shown as the mean⫾S.E.M. and represent four independent experiments. ** P⬍0.01 vs. control.

N-SH APPwt cells. The enhancement of ␣APPs release was determined by polyclonal antibody R1736, which specifically recognizes the residues 595– 611 of APP695 (C terminal of ␣APPs). The effective concentration of Hup A is similar to that previously reported to protect cortical neurons and PC12 cells from A␤ injury (Xiao et al., 1999, 2002). We found that Hup A had no effect on the steadystate levels of APP, indicating that Hup A– elevated secretion of ␣APPs was not the result of increased synthesis of APP, but instead was due to the increased cleavage of APP via the ␣-secretase pathway. APP ␣-secretase is a membrane-associated metalloprotease (Sisodia, 1992). Members of the ADAM family have been put forward as candidate ␣-secretases (Buxbaum et al., 1998; Lammich et al., 1999). Putative mediators of ␣-secretase cleavage mainly include three members of ADAM family: kuzbanian/ADAM10 (Artavanis-Tsakonas et al., 1999), TACE/ADAM17 (Black et al., 1997; Buxbaum et al., 1998) and meltrin-␥/ADAM9 (Koike et al., 1999). ADAM10 and TACE/ADAM17 are considered likely candidates for ␣-secretase APP cleavage (Lammich et al., 1999; Nunan and Small, 2000). In our study, we found that the highest elevation in ADAM10 protein levels occurred in SK-N-SH cells after incubation with Hup A for 18 h. TACE/ ADAM17 protein levels were also somewhat elevated. It is possible that the stimulatory effects of Hup A on ADAM10 may be persistent, but the effects on TACE/ADAM17 may be transient and gradually disappear. Our results agree with earlier reports, suggesting TACE/ADAM17 shows a transient “inducible” activity upon PKC activation, however, ADAM10 exhibits “constitutive” and inducible activities for substrate processing (Obregon et al., 2006; Stoeck et al., 2006). In order to understand clearly the role of TACE/ ADAM17 in Hup A–regulated ␣APPs release, we chose a selective, competitive inhibitor of TACE/ADAM17, TAPI-2, and found that the effects of Hup were partially inhibited. Others also found that the inhibition of TACE/ADAM17 expression partially inhibited ␣APPs release (Buxbaum et al., 1998; Camden et al., 2005). Together, these results

Fig. 7. Effects of cholinergic receptor antagonists on the Hup A–induced ␣APPs release in neuroblastoma SK-N-SH APPwt cells. Cells were pre-incubated for 30 min with vehicle control alone or with mAChR antagonists, atropine (10 ␮M), nAChR antagonists, mecamylamine hydrochloride (10 ␮M), M1-mAChR antagonist, pirenzepine dihydrochloride (10 ␮M), M2-mAChR antagonist, methoctramine hemihydrate (10 ␮M) and M3-mAChR antagonist and 4-diphenylacetoxy-N-methylpiperidine methiodide (10 ␮M). After pre-incubation, the cells were incubated for 18 h without or with 10 ␮M Hup A. The results showed: (A) mAChR antagonist significantly blocked Hup A–induced ␣APPs release, indicating that mAChR might be involved in Hup A–induced ␣APPs secretion; (B) M1-AChR antagonist inhibited Hup A–induced ␣APPs release in neuroblastoma SK-N-SH APPwt cells. Results are shown as the mean⫾S.E.M. and represent six independent experiments. * P⬍0.05, ** P⬍0.01 vs. control, # P⬍0.05 vs. Hup A group.

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Fig. 8. Effects of Hup A on MAP kinase activation in neuroblastoma SK-N-SH APPwt cells. (A) Cells were treated without or with 10 ␮M Hup A for different times. The results showed that phosphorylated MAP kinase was elevated in a time-dependent manner, which peaked 5–15 min after Hup A treatment. After 30 and 60 min, phospho-MAP kinase activation decreased and returned to the basal level. (B) Cells were incubated 15 min with increasing concentrations of Hup A. The results showed that the phospho-MAP kinase activation induced by Hup A occurred with doses as low as 0.1 ␮M, but had no effect on total MAP kinase levels. (C) Cells were pre-incubated for 30 min with vehicle alone, or with MEK inhibitors U0126 (5 ␮M) or PD98059 (30 ␮M), and then incubated without or with 10 ␮M Hup A for 15 min. The results showed that MEK inhibitors blocked the effect of Hup A on MAP kinase activation. (D) Quantification of the phospho-MAP kinase blot, using the total MAP kinase blot as control values. Cell lysates were analyzed by Western blot using anti-phospho-p44/42 MAPK (top) and anti-p44/42 MAPK (bottom). Results are shown as mean⫾S.E.M. and represent three independent experiments. * P⬍0.05, ** P⬍0.01 vs. control.

genic ␣-secretase cleavage of APP to form ␣APPs by Hup A suggest Hup A may have potential as a treatment for AD. It has been extensively demonstrated that the activation of various second messenger pathways is involved in the cascades that lead to the non-amyloidogenic processing of APP (Buxbaum et al., 1993; Hung et al., 1993), such as PKC and MAP kinase cascades. It is not due to APP phosphorylation (Hung and Selkoe, 1994). Instead, it seems to change secretase activities or APP trafficking by protein phosphorylation (Koo, 1997; Skovronsky et al., 2000). PKC is a multigene family, and its expression is abundant in neuronal tissues. PKC regulates various physical functions, such as neuronal survival and neurotransmitter release (Nishizuka, 1992). The role of PKC in the Hup A–mediated increase in ␣APPs release was demonstrated by the use of two PKC-specific inhibitors, compounds GF109203X and calphostin C. We showed that PKC inhibitors partially inhibited ␣APPs release induced by Hup A, suggesting that the PKC pathway might be involved in Hup A–regulated ␣APPs release. Furthermore, we determined if MAP kinase pathway was involved in Hup A–regulating ␣APPs release. Previous reports showed that MAP kinase serves as an important regulatory pathway in the cleavage of different membrane proteins, including APP (Mills et al., 1997; Desdouits-Magnen et al., 1998). There are three pathways in MAP kinase signaling cascades: the 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 (Camden et al., 2005; Ma et al., 2005). ERK1/2 (p42/44), as one of the MAP kinase pathways, is physiologically regulated by MEK. We used two potent and specific MEK inhibitors, PD98059 and U0126, to determine if p42/44 MAP kinase was involved in ␣APPs processing induced by Hup A. The results indicated that activation of the p42/44 MAP kinase pathway was necessary for nonamyloidogenic ␣APPs processing. Next, we examined the effect of Hup A on p42/44 MAP kinase pathway. We found that Hup A stimulated MAP kinase phosphorylation, and after incubation with MEK inhibitors, PD98059 and U0126, the phosphorylation effects of Hup A were completely blocked in neuroblastoma SK-N-SH APPwt cells, further suggesting that Hup A acts on the p42/44 MAP kinase pathway. Together, these findings indicate that MAP kinase activation is involved in Hup A–stimulated ␣APPs release in human neuroblastoma cells. In contrast, we found that the MAP kinase pathway was not strongly involved in Hup A–regulated APP processing in HEK293 APPsw cells, indicating Hup A might mediate APP processing via different pathways in different cell types (Peng et al., 2006). However, Yan et al. (2007) recently reported the involvement of M1-mAChRs, PKC and MAP kinase in Hup A–regulated APP processing in HEK293 APPsw cells. The differences in our results may be due to differences in the transfected cell lines or protocols used in these studies. The effect of AChE inhibitors on APP processing can be modulated by stimulation of phospholipase C– coupled receptors, such as the muscarinic receptors M1 and M3,

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both via PKC-dependent and -independent mechanisms (Nitsch et al., 1992). The signaling pathways downstream of the M3 mAChR clearly involve both PKC-dependent and -independent mechanisms coupled to the activation of the MAP kinase pathway (Slack, 2000). M1-mAChR is the predominant subtype in the cortex and hippocampus of the brain, two regions strongly related to cognitive function, resulting in interest as a therapeutic target (Wei et al., 1994). In a triple transgenic AD model bearing mutant APP, presenilin 1 and tau, Caccamo et al. (2006) found that an M1 agonist modulated APP processing through the activation of both ERK1/2 and PKC pathways, providing further evidence of a mechanistic relationship between M1 receptor activation and APP processing. In the present human neuroblastoma SK-N-SH APPwt cells, we found that a mAChR antagonist blocks Hup A–induced ␣APPs release, confirming the important role of mAChR in Hup A–mediated ␣APPs release. In addition, M1-mAChR showed more potent activity in regulating Hup A–induced aAPPs secretion compared with the other mAChR subtypes tested. Thus, Hup A may activate mAChR, thereby initiating downstream PKC/MAPK pathways to mediate non-amyloidogenic APP processing in neuroblastoma SKN-SH APPwt cells. There was dissociation between ␣APPs release and A␤ production in our study. Several studies also showed a similar finding in human primary neurons and neuroblastoma cells (Dyrks et al., 1994; LeBlanc et al., 1998). These results suggest that there may be more complex mechanisms of APP processing. In HEK293 cells that were transfected with APP carrying the Swedish familial AD double mutation, we found that Hup A increased ␣APPs release and reduced A␤ level (Peng et al., 2006). Thus, the regulation of APP metabolism might be cell type-specific and A␤ generation may not correlate well with ␣APPs secretion in some cell types. Alternatively, the presence of a mutant form of human APP, as opposed to the wild type form, may be required to see a change in A␤ levels in neuroblastoma cells. AD-like transgenic mouse models overexpressing human mutant APP generate higher levels of A␤ and deposit plaques earlier than those bearing wild-type APP. It is possible that mutant APP is conformed in a way that is advantageous for cleavage by ␤- and/or ␥-secretases. It is also possible that intracellular trafficking is altered by the presence of the mutation thereby enhancing its potential for processing along the amyloidogenic pathway. Alpha-APPs has various neuroprotective and neurotrophic activities (Mattson, 1997), such as enhancing longterm potentiation (LTP) in hippocampal slices (Ishida et al., 1997), enhancing memory in normal and amnesic mice (Meziane et al., 1998), alleviating impaired LTP and cognitive deficits in ADAM10 and APPV717I double transgenic mice (Postina et al., 2004), stimulating neurite outgrowth (Small et al., 1994), mediating synapse formation (Morimoto et al., 1998), having trophic effects on rat cerebral cortical neurons (Araki et al., 1991), and protecting hippocampal and cortical neurons against the toxic effects of glutamate and A␤ peptide (Furukawa et al., 1996). Hup A exerts potent neuroprotective and anti-apoptotic activities

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against diverse injury in vitro and in vivo, such as reducing glutamate-, A␤-, and H2O2-induced cytotoxicity (Ved et al., 1997; Xiao et al., 1999, 2002), protecting neurons against A␤-, serum deprivation– and staurosporine-induced apoptosis (Xiao et al., 2002; Zhou and Tang, 2002; Zhang and Tang, 2003), inducing nerve growth factor synthesis in cultured astrocytes, and enhancing neurite outgrowth of PC12 cells (Tang et al., 2005). In addition, Hup A alleviated cognitive deficits and protected neurons in various animal models (Wang et al., 2000; Ye et al., 2000; Ou et al., 2001; Xu et al., 2006). Thus, it is possible that ␣APPs derived from Hup A–regulated APP processing serve as a neuroprotective agent and contribute to the treatment of AD patients.

CONCLUSION In conclusion, our study is the first to demonstrate that Hup A can stimulate the non-amyloidogenic cleavage of APP via the mAChR, PKC and MAP kinase pathways in neuroblastoma SK-N-SH APPwt cells. The enhancement of ␣APPs release may be via the mAChR/PKC cascade. The neuroprotective activity of Hup A, together with the ability to stimulate ␣APPs release, indicates that Hup A may be beneficial for AD therapy. Acknowledgments—This work was funded by philanthropic donations to the Foundation for Neurologic Diseases.

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(Accepted 4 October 2007) (Available online 14 September 2007)