Journal of Neuroscience Research 85:2500–2511 (2007)
A Novel Compound, Maltolyl p-coumarate, Attenuates Cognitive Deﬁcits and Shows Neuroprotective Effects In Vitro and In Vivo Dementia Models Ki Young Shin,1 Geon Ho Lee,1 Cheol Hyoung Park,1 Hee Jin Kim,1 Soo-Hyun Park,2 Seonghan Kim,1 Hye-Sun Kim,1 Kwan-Sun Lee,3 Beom Young Won,4 Hyung Gun Lee,4 Jin-Ho Choi,2 and Yoo-Hun Suh1,4* 1
Department of Pharmacology, College of Medicine, National Creative Research Initiative Center for Alzheimer’s Dementia and Neuroscience Research Institute, MRC, Seoul National University, Seoul, Korea 2 Faculty of Food Science and Biotechnology, Pukyong National University, Busan, Korea 3 Central Research Institute, Hanmi Pharmaceutical Company, Gyeonggi-do, Korea 4 Braintropia Company, Gyeonggi-do, Korea
To develop a novel and effective drug that could enhance cognitive function and neuroprotection, we newly synthesized maltolyl p-coumarate by the esteriﬁcation of maltol and p-coumaric acid. In the present study, we investigated whether maltolyl p-coumarate could improve cognitive decline in scopolamineinjected rats and in amyloid beta peptide1–42-infused rats. Maltolyl p-coumarate was found to attenuate cognitive deﬁcits in both rat models using passive avoidance test and to reduce apoptotic cell death observed in the hippocampus of the amyloid beta peptide1–42infused rats. We also examined the neuroprotective effects of maltolyl p-coumarate in vitro using SH-SY5Y cells. Cells were pretreated with maltolyl p-coumarate, before exposed to amyloid beta peptide1–42, glutamate or H2O2. We found that maltolyl p-coumarate signiﬁcantly decreased apoptotic cell death and reduced reactive oxygen species, cytochrome c release, and caspase 3 activation. Taking these in vitro and in vivo results together, our study suggests that maltolyl pcoumarate is a potentially effective candidate against Alzheimer’s disease that is characterized by wide spread neuronal death and progressive decline of cognitive function. VC 2007 Wiley-Liss, Inc. Key words: Alzheimer’s disease; apoptosis; cognitive impairment; maltolyl p-coumarate; oxidative stress
et al., 1984; Yankner, 1996; Mattson, 1997; Suh and Checler, 2002). Hippocampal neurons that are related to learning and memory (Karczmar, 1993) are known to be particularly vulnerable in AD (Whitehouse et al., 1982; Dutar et al., 1995; Winkler et al., 1995). Ab deposition may play a fatal role in the pathogenesis of AD by inducing neuronal cell death through the processes, such as glutamate-mediated excitotoxicity, increased production of reactive oxygen species (ROS) or hydrogen peroxide-mediated apoptosis (Behl et al., 1994; Goodman and Mattson, 1994; Mark et al., 1997; Suh and Checler, 2002). Because oxidative stress is one of the major causes of neurodegeneration with aging (Leutner et al., 2001; Floyd and Hensley, 2002) and the subsequent cell death results in cognitive decline, an antioxidant may be an effective candidate that improves impairments of learning and memory processing (Petkov et al., 1993; Wo¨rtwein et al., 1994; D’Hooge and De Deyn, 2001). Thus, a drug that could ameliorate neurotoxicity and cognitive dysfunction should be effective to treat various neurodegenerative diseases including AD. We have reported previously the biologic activities of the root extract of the reed (Phragmites commnis) (Choi et al., 1993, 1997a,b, 1998). p-Coumaric acid, a component of reed root extract, is a phenolic acid that possesses K.Y. Shin and G.H. Lee contributed equally to this work.
Many neurodegenerative diseases including Alzheimer’s disease (AD), Parkinson’s disease, and amyotrophic lateral sclerosis are distinguished by the gradual loss of speciﬁc sets of neurons that results in deﬁcits of movement and cognitive function (Thompson, 1995; Satry and Rao, 2000). AD is especially characterized by the presence of amyloid beta peptide (Ab deposition in neuritic plaques and by neuronal loss in brain regions involved in learning and memory processes (Hyman ' 2007 Wiley-Liss, Inc.
Contract grant sponsor: Ministry of Science and Technology, Korea; Contract grant sponsor: BK21 Life science. *Correspondence to: Dr. Yoo-Hun Suh, Department of Pharmacology, College of Medicine, Seoul National University, 28 Yeongeon-dong, Jongno-gu, Seoul, 110-799, Korea. E-mail: firstname.lastname@example.org Received 9 February 2007; Revised 6 April 2007; Accepted 7 April 2007 Published online 28 June 2007 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/jnr.21397
Neuroprotective Effects of MPC
Fig. 1. The structure and synthesis of MPC. (A) The structure of MPC was shown. The molecular formula of MPC is as follows: 1HNMR (d, DMSO-d6): 2.26 (s, 3H, CH3C 5 O), 6.42 (d, 1H, maltol, CH 5 CH), 6.58 (d, 1H, CH 5 CH), 6.82 (d, 2H, benzene ring), 7.61 (d, 2H, benzene ring), 7.73 (d, 1H, CH 5 CH), 8.11 (d, 1H, maltol CH 5 CH). Maltolyl p-acetoxycinnamate (B), the precursor component of MPC, was synthesized by the esteriﬁcation between p-acetoxycinnamic acid (C) and maltol (D). Description on the MPC synthesis method was presented in Materials and Methods in detail.
free radical scavenging and antioxidant properties (Hertog et al., 1995; Kato et al., 1997; Lodovici et al., 2001; Niwa et al., 2001; Abdel-Wahab et al., 2003). Additionally, maltol is suggested to be a functional agent that prevents oxidative damage in mice brains (Kim et al., 2004), in horse blood plasma (Lee and Lee, 2005), and in human neuroblastoma cells (Yang et al., 2006). Because both parent compounds, maltol and p-coumarate, are all known compounds, we want to develop new compound having more therapeutic efﬁcacy against Alzheimer’s disease. Based on these previous ﬁndings, we synthesized maltolyl p-coumarate (MPC) by the esteriﬁcation of maltol isolated from Korean ginseng (Panax ginseng C.A. Meyer) and p-coumaric acid. We conﬁrmed that MPC has been successfully synthesized through NMR (nuclear magnetic resonance) and GC/ MS (gas chromatography/mass spectroscopy) method. In the present study, we investigated whether MPC could improve cognitive decline in scopolamineinjected or Ab1–42-infused rats and reduce apoptotic cell death in the brains of the Ab1–42-infused rats. We also checked the neuroprotective effects of MPC in vitro using SH-SY5Y cells. MATERIALS AND METHODS Synthesis of MPC The structure of MPC is shown in Figure 1A. Four grams of p-coumaric acid was added to a mixed solution of 16 ml of pyridine and 8 ml of acetic anhydride. The mixture was stirred overnight at room temperature and vacuum-ﬁltered. The dried solid was added to 15 ml of chloroform, stirred for 10 min, and vacuum-ﬁltered. The product was washed with chloroform to obtain 3.0 g of p-acetoxycinnamic acid (Fig. 1C). 2.8 g of p-acetoxycinnamic acid was dissolved in 40 ml of dimethylformamide. Thionyl chloride (1.05 ml) was added to the solution. The reaction mixture was stirred for 1.5 hr at 2158C to 2208C. Pyridine (2.3 ml) was added followed by the addition of 1.62 g of maltol (Fig. 1D). The temperature Journal of Neuroscience Research DOI 10.1002/jnr
was allowed to rise to room temperature. After stirring overnight, the reaction mixture was vacuum-ﬁltered. The product was washed with water and was vacuum-dried to obtain 3.92 g of maltolyl p-acetoxycinnamate (Fig. 1B). Maltolyl pacetoxycinnamate (6 g) was dissolved in 60 ml of methanol, 30 ml of triethylamine, and 20 ml of water and stirred for 2.5 hr at 20–258C. The reaction mixture was evaporated to dryness under reduced pressure. The residue was taken up in 50 ml of methanol and the suspension was stirred for 10 min and then vacuum-ﬁltered. The product was washed with methanol and vacuum-dried to obtain 4.51 g of MPC. Reagents and Antibodies Ab1–42 peptide was purchased from US Peptide (Rancho Cucamonga, CA) and aged by incubation in 0.1 M PBS (pH 7.4) at 378C for 7 days. Anti-cytochrome c and anti-btubulin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and anti-COX IV and anti-cleaved caspase-3 antibodies were purchased from Cell Signaling (Danvers, MA). All other chemicals and reagents were purchased from Sigma (St. Louis, MO). MPC was dissolved initially in PBS solution containing 2% dimethylsulfoxide (DMSO). All drugs were prepared just before use. Animals Seven-week-old Wistar rats were housed in a speciﬁc pathogen-free room that was automatically maintained on a 12-hr light–dark cycle at 258C and proper humidity. The animals were also given food and water ad lib. All experiments were carried out in accordance with the Guidelines for Animal Experiments of Ethics Committee of Seoul National University. Animal Models Scopolamine Injection. Some researchers have used scopolamine-induced rats to screen the effects of drugs on cognitive enhancement (Park et al., 2000; van der Staay and Bouger, 2005). Rats were given scopolamine (1 mg/kg, i.p.) 1 hr before the acquisition trial. A single dose of MPC (100 mg/kg, p.o.) was given to rats 30 min after scopolamine treatment and after another 30 min. Ab1–42 Infusion. The surgical techniques used in this study were described previously (Itoh et al., 1996; Nitta et al., 1997; Oka et al., 1999, 2000). Rats were anesthetized with sodium pentobarbital (25 mg/kg, i.p.). Continuous infusion of Ab1–42 (600 pmol/day) was maintained for a week by attachment of an infusion kit connected to an osmotic mini-pump (Alzet 1007D; Alza, CA). The infusion kit was implanted into the right ventricle (1.2 mm posterior to the bregma, 1.5 mm lateral to the midline, 4.0 mm ventral to the surface of the skull) according to the brain atlas of Paxinos and Watson (1986). The guide cannula was secured with dental cement and stainless steel skull screws. Rats infused by Ab1–42 for a week were injected with MPC (10 mg/kg, p.o.) or vitamin E (10 mg/kg, p.o.) 2 weeks from the ﬁrst day of Ab1–42 infusion.
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Passive Avoidance Test A step-through type passive avoidance test apparatus (Model PACS-30, Columbus Instruments Int.) was used to evaluate the effects of MPC on learning and memory, as described previously (Shen et al., 1990). The shuttle box is divided into two chambers of equal size (23.5 3 15.5 3 15.5 cm) separated by a guillotine door (6.5 3 4.5 cm). The light chamber is equipped and rats can enter the dark chamber through the guillotine door. Rats were placed initially in the light chamber with the door open. On entering the dark compartment, the door is closed automatically. Training was repeated until the rats entered the dark compartment within 20 sec (training trial). Rats were placed in the illuminated chamber 24 hr after the training trial. When rats entered the dark chamber, electrical foot shock (0.5 mA) was delivered for 3 sec through the grid ﬂoor and the door was closed automatically (acquisition trial). The rats were again placed in the illuminated chamber 24 hr after the acquisition trial and the latency to enter the dark chamber was measured for 300 sec (retention trial). If a rat did not enter the dark chamber within the cut-off time (300 sec), it was assigned a latency value of 300 sec. In scopolamine-injected group, rats were given scopolamine (1 mg/kg, i.p.), 1 hr before the acquisition trial. A single dose of MPC (100 mg/kg, p.o.) was given to rats 30 min after scopolamine treatment and after another 30 min, rats were placed in the illuminated chamber. In Ab1–42-infusion group, rats were injected with MPC (10 mg/kg, p.o.) or vitamin E (10 mg/kg, p.o.) during 2 weeks before the training trial.
Evaluation of Apoptosis With TUNEL Method Terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick-end labeling (TUNEL) staining was carried out according to the manufacturer’s protocol (In situ cell death detection kit; TMR Red; Roche Diagnostics GmbH, Germany). Apoptotic cell death was visualized by TUNEL method (Gavrieli et al., 1992). For immunohistochemistry assay, brains were removed from animals and immersed in 4% paraformaldehyde. After ﬁxation, the brains were embedded in parafﬁn and cut 7 lm coronal sections. For immunocytochemistry assay, cells were immersed in 4% paraformaldehyde. TUNEL reaction was labeled with TMR red and analyzed by confocal microscopy with appropriate ﬁlters (LSM510, Carl Zeiss, Germany). DAPI (1 lM) staining was carried out for nucleus staining. TUNEL positive cells in three random ﬁelds were counted in two sets of experiments and expressed as percentage of the number of TUNEL positive cells/the total number of cells in each ﬁeld of the hippocampus.
Cell Culture SH-SY5Y cells were maintained in DMEM (Life Technologies, Inc., Grand Island, NY) supplemented with 10% FBS (GIBCO BRL, Gaithersburg, MD) and 0.3% antibiotics at 378C in 5% CO2. SH-SY5Y cells were pretreated with MPC (25, 50 or 100 lM) or vitamin E (50 lM) for 4 hr before the treatment with Ab1–42, glutamate or H2O2.
Cell Viability Test WST-1-metabolizing activity was determined according to the manufacturer’s instructions (Roche, Indianapolis, IN). SH-SY5Y cells were plated in a 96-well plate at a density of 8 3 103 cells/well. As reported previously (Park et al., 2002), cells were allowed to adhere to plates for 24 hr. MPC was introduced into the media of SH-SY5Y cells 4 hr before treatment with 25 lM Ab1–42, 1 mM glutamate, or 200 lM H2O2, which play vital roles in neurodegenerative diseases, such as AD (Behl et al., 1994; Goodman and Mattson, 1994; Mark et al., 1997; Suh and Checler, 2002). This colorimetric assay measures the metabolic activity of viable cells. Brieﬂy, after incubating cells treated with various reagents, 10 ll WST-1 was added to the culture media. The culture was incubated at 378C in a humidiﬁed atmosphere of 95% air and 5% CO2 for 1 hr. The absorbance of the reaction product was measured with an ELISA reader (Bio-Rad, Germany) at a wavelength of 450 nm. Measurement of ROS Generation Intracellular ROS in SH-SY5Y cells were assayed using dye 20 ,70 -dichloroﬂuoroscein diacetate (DCFH-DA; Molecular Probes, Eugene, OR). Cells were washed with HEPES-buffered saline (HBS) and incubated in the dark for 1 hr in HBS containing 200 lM of DCFH-DA. On incubation, DCFHDA is taken up by cells where intracellular esterase cleaves the molecule to DCFH, which is oxidized to DCF in the presence of H2O2. The total ﬂuorescence was measured using a spectroﬂuorometer (Molecular Devices, Sunnyvale, CA) at an emission wavelength of 488 nm and an excitation wavelength of 524 nm (Wang and Zhu, 2003). Western Blotting Cells were lysed in a lysis buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton, 0.5% SDS, 0.5% DOC, and protease inhibitors. Protein was resolved in SDS polyacrylamide gel, electrophoresed at 30–50 lg of protein/lane, and transferred onto a nitrocellulose membrane (Amersham Pharmacia, Buckinghamshire, UK). The protein blot was conﬁrmed with appropriate antibodies and detected using horseradish peroxidase-conjugated secondary antibody (Amersham Pharmacia). Immunoreactive bands were visualized using an ECL enhanced chemiluminescence system (ECL; Amersham Pharmacia). For detection for cytochrome c, the mitochondrial and cytosolic fractions were obtained according to the previous method (Kang et al., 1995). The purity of the fractions was checked with subcellular markers, i.e., anti-cytochrome c oxidase IV for mitochondrial fraction and anti-btubulin for cytosol. Statistical Analysis Data are presented as means 6 SEM. These results were analyzed using one-way analysis of variance (ANOVA) followed by Turkey’s post-hoc test or two-way ANOVA, in which P < 0.05 was considered to be statistically signiﬁcant. Journal of Neuroscience Research DOI 10.1002/jnr
Neuroprotective Effects of MPC
Fig. 2. MPC ameliorates learning and memory impairments both in scopolamine-injected or in Ab1–42-infused rats. A: At 30 min after training trials, scopolamine (1 mg/kg, i.p.) or the same volume of saline was administered to rats. At 30 min after scopolamine injection, the rats were injected with MPC (100 mg/kg, p.o.). Acquisition trials were carried out 30 min after a single MPC treatment. At 24 hr after acquisition trials, the test trials were carried out. B: In Ab1–42-infused rats prepared by Ab1–42 infusion into the lateral ventricle, retention trials were carried out after administrations of MPC (10 mg/kg, p.o.) or vitamin E (10 mg/kg, p.o.) for 14 days. The latency in the passive avoidance test was calculated after 24 hr of foot shock. Data represent mean 6 SEM. #P < 0.05 compared to vehicle control and *P < 0.05 and **P < 0.01 compared to the Ab1–42 peptide-treated group, one-way ANOVA.
RESULTS MPC Ameliorates Learning and Memory Impairments Both in Scopolamine-Injected or Ab1–42-Infused Rats To investigate whether the learning and memory impairments induced by scopolamine or Ab1–42 could be improved by MPC administration, a passive avoidance test was carried out. In scopolamine-injected rats, scopolamine (1 mg/kg, i.p.) was given 1 hr before the acquisition trial of the passive avoidance test. The latency was Journal of Neuroscience Research DOI 10.1002/jnr
measured after 24 hr of foot shock. A single dose of MPC (100 mg/kg, p.o.) was given to rats 30 min after scopolamine treatment (Fig 2A). The latency, which was signiﬁcantly shortened by a single injection of scopolamine (11.26 6 2.3 sec), was recovered almost to that of the vehicle-administered control group (241.4 6 30.94 sec) after a single administration of MPC (215.32 6 53.6 sec). In Ab1–42-infused rats prepared by Ab1–42 infusion into the lateral ventricle, MPC (10 mg/kg, p.o.) or vitamin E (10 mg/kg, p.o.) was administrated for 14 days after surgery (Fig. 2B). The latency was signiﬁcantly higher
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in MPC- (249.73 6 34.72 sec) or vitamin E-administered group (245.6 6 29.02 sec) than in vehicle-administered Ab1–42-infused group (125.69 6 34.64 sec). MPC Reduces Apoptotic Cell Death in CA1 Region of Ab1–42-Infused Rats To examine whether the apoptotic cell death of neurons could be attenuated by MPC, TUNEL staining was carried out using the brain slices of rats. Ab1–42infused rats were administrated with MPC (10 mg/kg, p.o.) or vitamin E (10 mg/kg, p.o.) for 14 days after infusion of Ab1–42 for a week. Figure 3A,B showed that apoptotic cell death of CA1 region was observed much less in MPC- (14.81 6 9.89%) or vitamin E-administered group (13.08 6 5.42%) than in vehicle-administered Ab1–42-infused group (49.52 6 3.22%). MPC Exerts Neuroprotective Effects Against Ab1–42, Glutamate or H2O2 in SH-SY5Y Cells We studied the effects of MPC on neuronal cell death induced by Ab1–42, glutamate, or H2O2. As measured by WST-1 assay, cell viability was signiﬁcantly decreased compared to vehicle-treated control in SHSY5Y cells at 48 and 12 hr after treatment with Ab1–42 (64.61 6 2.92% vs. vehicle treated control cells) or glutamate only (46.14 6 8.11%), respectively (Fig. 4A,B). Pretreatment with MPC, however, signiﬁcantly attenuated the decrease in cell viability in a dose-dependent manner compared to the Ab1–42-treated (25 lM MPC, 70.32 6 3.03; 50 lM MPC, 74.24 6 3.38; 100 lM MPC, 79.17 6 4.19%) or glutamate-treated cells (25 lM MPC, 49.74 6 5.98; 50 lM MPC, 78.49 6 5.32; 100 lM MPC, 91.56 6 5.78%). Pretreatment with 50 lM vitamin E also signiﬁcantly attenuated the decrease in cell viability induced by Ab1–42 (80.51 6 1.85%). MPC Reduces the ROS Accumulation by H2O2 Treatment in SH-SY5Y Cells We checked the effects of MPC on ROS at 24 hr after treatment with H2O2 (200 lM) for 1hr (Fig. 4C,D). Pretreatment with MPC (25, 50, or 100 lM) for 4 hr before treatment with H2O2 ameliorated the decreases in cell viability (25 lM MPC, 59.3 6 4.46; 50 lM MPC, 72.45 6 3.55; 100 lM MPC, 75.76 6 7.74% vs. vehicle treated control) and signiﬁcantly decreased ROS production (25 lM MPC, 79.66 6 6.51; 50 lM MPC, 73.17 6 5.01; 100 lM MPC, 69.21 6 6.44% vs. vehicle-treated H2O2 treated cells) in a dose-dependent manner, as compared to vehicle-treated H2O2-group. Pretreatment with 50 lM vitamin E signiﬁcantly recovered cell viability (71.49 6 3.75%) and ROS production (69.67 6 4.27%). Pretreatment of MPC Attenuates Apoptotic Cell Death by H2O2 in SH-SY5Y Cells To examine whether apoptotic cell death by oxidative stress could be protected by MPC, TUNEL staining was carried out using the SH-SY5Y cells 24 hr after
being treated with H2O2 (200 lM) for 1hr (Fig. 5A,B). TUNEL stained cells were observed much less in the 50 lM MPC-treated group (31.32 6 6.43% vs. total cell numbers) or 50 lM vitamin E-treated group (39.98 6 3.33% vs. total cell numbers) for 4 hr before treatment with H2O2 than in H2O2-treated group (63.71 6 3.32% vs. total cell numbers). Pretreatment With MPC Reduces the Release of Cytochrome c and the Activation of Caspase 3 Induced by H2O2 in Neuronal Cells Cytochrome c release into cytosol and caspase 3 activation were measured at 24 hr after treatment with H2O2 (200 lM) in SH-SY5Y cells by Western blotting. Pretreatment with MPC (25, 50, or 100 lM) for 4 hr before treatment with H2O2 decreased the release of cytochrome c into cytosolic fraction and the activation of caspase 3 in a dose-dependent manner, compared to vehicle treated H2O2-treated cells (Fig. 6A,C). DISCUSSION In the present study, we report that a newly synthesized compound, MPC, improves learning and memory deﬁcits in two dementia animal models, i.e., scopolamine-injected or Ab1–42-infused rats. In addition, MPC protects SH-SY5Y cells against Ab1–42, glutamate, or H2O2. The experimental design used in this study was prepared as previous reports on drug screening (Nitta et al., 1997; Yamada et al., 1999; Park et al., 2000, 2002; Nakamura et al., 2001; Jhoo et al., 2004; Kim et al., 2004). MPC was newly synthesized by the esteriﬁcation of maltol isolated from Korean ginseng (Panax ginseng C.A. Meyer) and p-coumaric acid extracted from reed (Phragmites commnis). Because both parent compounds, maltol and p-coumaric acid are known compounds, we want to develop a new compound having better therapeutic efﬁcacy against Alzheimer’s disease. Several scientists have already reported that the antioxidant effects of the parent compounds were less active than vitamin E or other compounds (Lee and Shibamoto, 2000; Wei et al., 2001; Etoh et al., 2004). We found, however, that MPC had similar antioxidant effects to vitamin E, suggesting that MPC has stronger antioxidant effects than the parent compounds. We reported previously that maltol protected hippocampal neurons against oxidative damage in the brains of mice treated with kainic acid. Administration with maltol (100 mg/kg) remarkably increased the total glutathione level and glutathione peroxidase activity, attenuating kainic acid-induced neuronal loss in the hippocampus (Kim et al., 2004). Maltol of aroma extracts, which was isolated from beans, also inhibited malonaldehyde formation from horse blood plasma oxidized with Fenton’s reagent (Lee and Lee, 2005). Additionally, p-coumaric acid isolated from the root extract of reed is a phenolic acid that is widely distributed in plants. It constitutes a part of the human diet (Scalbert and Williamson, 2000) and possesses free radical scavenging and antioxidant properties Journal of Neuroscience Research DOI 10.1002/jnr
Fig. 3. Apoptotic cells in CA1 region were reduced by MPC administration in Ab1–42-infused rats. Rat brains from Ab1–42-infused rats administered with vehicle, MPC or vitamin E were embedded in parafﬁn and sections were cut at 7 lm thick. Then, brain slices were immunostained with TUNEL staining (A). The percentage of
TUNEL-positive neurons is shown in the graph (B). The results were normalized as percentage ratios compared to the total cell numbers. #P < 0.05 compared to the vehicle and *P < compared to Ab1–42 peptide-treated group, one-way ANOVA. DIC, differential interference contrast. Scale bar 5 50 lm.
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Fig. 4. MPC reduces neurotoxicity induced by Ab1–42, glutamate or H2O2 and the production of ROS induced by H2O2 in SH-SY5Y cells. SH-SY5Y cells were plated in a 96-well plate at a density of 8 3 103 cells/well. MPC was introduced into the media of SH-SY5Y cells 4 hr before treatment with 25 lM Ab1–42 peptide (A), 1 mM glutamate (B), or 200 lM H2O2 (C) for 48, 12, and 1 hr, respectively. WST-1-metabolizing activity was determined according to the
manufacturer’s instructions (Roche). The intracellular level of ROS was checked using DCFH-DA (D). MPC (25, 50, or 100 lM) was pretreated for 4 hr before treatment with H2O2. Data were expressed as the percent of vehicle control value 6 SEM. At least two experiments were carried out in triplicate. #P < 0.05 compared to each vehicle-treated control and *P < 0.05 and **P < 0.01 compared to Ab1–42-, glutamate-, or H2O2-treated group, one-way ANOVA.
(Hertog, 1995; Kato et al., 1997; Lodovici et al., 2001; Abdel-Wahab et al., 2003). Moreover, p-coumaric acid derivatives exhibited inhibitory activity stronger than that of vitamin C or E on peroxynitrite-mediated lipoprotein nitration (Niwa et al., 2001). To characterize a pharmacologic property of MPC, a newly synthesized compound, we investigated whether MPC could improve learning and memory deﬁcits in scopolamine-injected or Ab-infused rats. First, we reported that MPC ameliorated scopolamine-induced learning and memory impairments, using a passive avoidance test. Scopolamine, one of muscarinic receptor antagonists, can induce amnesia in animals by
blocking cholinergic neurotransmission. The effects of scopolamine are generally interpreted within the framework of the cholinergic hypothesis of cognitive dysfunction (Bartus et al., 1982; Bartus, 2000). As a consequence, an animal model with scopolamine-induced amnesia has widely been used as a pharmacological model to test the effectiveness of new cognition-enhancing drugs (Park et al., 2000; van der Staay and Bouger, 2005). It must be sure that learning impaired by scopolamine administration could be studied in a passive avoidance test (Stone et al., 1988). In the recent study, we found that MPC increased activities of choline acetyltransferase (ChAT) in the brains of SAMP8 mice (data Journal of Neuroscience Research DOI 10.1002/jnr
Neuroprotective Effects of MPC
Fig. 5. Apoptotic cell death is attenuated by pretreatment of MPC or vitamin E in SH-SY5Y cells. To examine whether apoptotic cell death by oxidative stress could be protected by MPC, TUNEL staining was carried out using the SH-SY5Y cells 24 hr after being treated with H2O2 (200 lM) for 1 hr. SH-SY5Y cells were ďŹ xed in 4% paraformaldehyde and were immunostained with TUNEL after the
Journal of Neuroscience Research DOI 10.1002/jnr
drug treatment (A). The percentage of TUNEL-positive neurons was shown in the graph (B). The results were normalized as percentage ratios compared to the total cell numbers. #P < 0.05 compared to control and *P < 0.05 compared to H2O2-treated group, one-way ANOVA. DIC, differential interference contrast. Scale bar 5 50 lm.
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Fig. 6. Pretreatment with MPC reduces cytosolic cytochrome c release and the activation of caspase 3 by H2O2 in SH-SY5Y cells. Representative immunoblots for cytochrome c in cytosolic (A) or mitochondrial fractions (B) of vehicle-, H2O2-, MPC- or vitamin E-treated SHSY5Y cells were shown. Cytochrome c release into cytosol was examined at 24 hr after treatment with H2O2 (200 lM) in SH-SY5Y cells by Western blotting. MPC was pretreated (25, 50, or 100 lM) for 4 hr before treatment with H2O2. B: Representative immunoblots for
active caspase 3 were shown. MPC was pretreated (25, 50, or 100 lM) for 4 hr before treatment with H2O2 (200 lM). The purity of the fractions was checked with subcellular markers, i.e., anti-cytochrome c oxidase IV for mitochondrial fraction and anti-b-tubulin for cytosol. Relative ratios of the densitometric values of cytochrome c in cytosolic fraction (D) or mitochondrial fraction (E), and of active caspase 3 (F) were measured using the M4 image analysis program (*P < 0.05, compared to H2O2-treated group, one-way ANOVA). Journal of Neuroscience Research DOI 10.1002/jnr
Neuroprotective Effects of MPC
not shown). Accordingly, MPC can improve scopolamine-induced cognitive impairment through the activation of ChAT. Second, we used Ab1–42 infused rats prepared by Ab1–42 infusion into the cerebral ventricle for 1 week. In the passive avoidance test, we showed that MPC administration (10 mg/kg, p.o.) for 2 weeks improved cognitive deﬁcits induced by Ab1–42 infusion. The Abinfused animal model produces several pathologic hallmarks of AD including amyloid deposition, robust neuroinﬂammation, decrease in synaptic markers, neuronal death, and memory impairments (Nitta et al., 1997; Suh and Checler, 2002; Craft et al., 2004). This model also exhibits impairments of nicotine- and K1-stimulated acetylcholine or dopamine release from the frontal cortex/hippocampus and striatum, respectively (Itoh et al., 1996). In conclusion, the Ab-infused model could be useful as an animal model for evaluating development processes at the early or middle stages of Alzheimer-like dementia (Nakamura et al., 2001). To examine neuroprotective effects of MPC in vitro, we investigated the effects of MPC on the neurotoxicity induced by Ab1–42, glutamate, or H2O2 in SHSY5Y neuroblastoma cells. We show that MPC could reduce Ab1–42-induced toxicity in the cells. Numerous senile plaques have been found in the brains of AD patients at autopsy (Roth et al., 1966; Suh and Checler, 2002) and these senile plaques consist of ﬁbrillar deposits of Ab peptides (Hardy, 1997; Suh and Checler, 2002). Ab peptide deposition precedes the development of neuroﬁbrillary change (Mann, 1985), and high micromolar concentrations of Ab peptides are neurotoxic (Yankner et al., 1989; Suh and Checler, 2002). Mechanistic studies of Ab neurotoxicity in cell culture showed a dependence on glutamate-mediated excitotoxicity, mediation of ROS production, and hydrogen peroxidemediated apoptosis (Behl et al., 1994; Goodman & Mattson, 1994; Mark et al., 1997). Several studies have suggested a strategy that decreases Ab neurotoxicity might be beneﬁcial for AD therapeutics (Behl et al., 1992; Chyan et al., 1999; Luo et al., 2002). We showed that MPC protected cultured neuronal cells against Ab1–42. The Ab1–42 treatment in the cell culture decreased cell death by about 30%. Ab1–42induced cell death was inhibited by pretreatment with MPC. Although the precise mechanism of this reversal is unclear, the protective effect of MPC against Ab1–42induced neurotoxicity is likely to result from the reduction of glutamate excitotoxicity and ROS. In addition, we show that MPC could reduce glutamate-induced toxicity in SH-SY5Y neuroblastoma cells. Glutamate-mediated excitotoxicity plays a vital role in the pathogenesis of neurodegenerative diseases such as AD and brain ischemia (Meldrum et al., 1990). Glutamate is an excitatory neurotransmitter and is related to the mechanisms of learning and memory processing (Greenamyre, 1986; Cotman et al., 1987). However, overstimulation of N-methyl-D-aspartate (NMDA) receptors induced by glutamate can lead to increase in intracelJournal of Neuroscience Research DOI 10.1002/jnr
lular Ca21 level and excitotoxicity, and subsequently results in neuronal death (MacDermott et al., 1986; Nicotera and Orrenius, 1992). Several studies have suggested that a strategy to modulate the excitotoxicity through NMDA receptor antagonists might be beneﬁcial for the treatments of several brain diseases (Simon et al., 1984; Keyser et al., 1999). Here, we report that MPC protected the cultured cells against glutamate-induced neurotoxicity. The treatment of glutamate in the cell culture decreased cell viability by about 50%. Glutamate-induced cell death was reversed by pretreatment with MPC. We also showed that MPC could reduce H2O2induced toxicity, ROS production, cytochrome c release into cytosol, and caspase 3 activation in SH-SY5Y neuroblastoma cells. Because H2O2 may perturb the antioxidant defense system in the cell and result in apoptotic cell death (Chandra et al., 2000; Bilici et al., 2001), an H2O2induced toxicity model has been used for studying oxidative stress-induced neurodegeneration (Richter-Landsberg and Vollgraf, 1998). The accumulation of macromolecular damage induced by ROS is the central causal factor that promotes the process of aging (Sohal, 2002). The oxidative modiﬁcation of proteins by ROS is involved in the pathogenesis of both normal aging and neurodegenerative diseases (Beal, 2002; Suh and Checler, 2002). Moreover, brain tissue is highly vulnerable to oxidative stress because of its oxidative damage potential (Leutner et al., 2001; Floyd and Hensley, 2002). ROS can react with polyunsaturated fatty acids to form lipid peroxides, and the accumulation of end-products of lipid peroxidation with age may contribute to the aging process (Inal et al., 2001; Kasapoglu and Ozben, 2001; Leutner et al., 2001; Wickens, 2001; Montine et al., 2002). More powerful antioxidants such as Egb761, clotrimazole, dehydroepiandrosterone, melatonin, indole propionic acid, and DHED have all been shown to inhibit oxidative stress-induced toxicity in various cell lines (Barlow-Walden et al., 1995; Cazevieille et al., 1997; Chyan et al., 1999; Martin et al., 2000; Okatani et al., 2000; Isaev et al., 2002; Luo et al., 2002; Zhang et al., 2002; Suh et al., 2005). We described that MPC protected the cultured cells against H2O2 by decreasing ROS production, cytochrome c release into cytosol, and caspase 3 activation induced by H2O2. Similar to the effects of vitamin E, pretreatment of MPC reduced ROS level, cytochrome c release into cytosol, and caspase 3 activation and rescued SH-SY5Y cells from the subsequent H2O2-induced apoptotic cell death. Recently, we determined that MPC was not a direct scavenger of hydroxyl radicals (data not shown). Further study on its detailed antioxidant mechanisms remains to be clariﬁed. MPC might exert an neuroprotective effects blocking ROS production, cytochrome c release, or caspase 3 activation. Summarizing our results, newly synthesized compound, MPC, attenuated learning and memory impairments in vivo dementia animal models, scopolamine-injected or Ab1–42-infused rats. In addition, MPC showed neuroprotective effects against Ab1–42, glutamate, or H2O2 in vitro neuronal cells, reducing ROS, cytochrome c re-
Shin et al.
lease into cytosol, and caspase 3 activation induced by H2O2. Taking these in vitro and in vivo results together, our study suggests that MPC is a potentially effective candidate against AD that is characterized by widespread neuronal death and progressive decline of cognitive function. ACKNOWLEDGMENTS This study was supported by a National Creative Research Initiative Grant (2006–2009) from Ministry of Science and Technology and in part by BK21 Human Life Sciences. We thank Dr. Sung Ki Lim, chairman of Hanmi Pharmaceutical Company Limited for very helpful discussion. REFERENCES Abdel-Wahab MH, El-Mahdy MA, Abd-Ellah MF, Helal GK, Khalifa F, Hamada FM. 2003. Inﬂuence of p-coumaric acid on doxorubicininduced oxidative stress in rat’s heart. Pharmacol Res 48:461–465. Barlow-Walden LR, Reiter RJ, Abe M, Pablos M, Menendez-Pelaez A, Chen LD. 1995. Melatonin stimulates brain glutathione peroxidase activity. Neurochem Int 26:497–502. Bartus RT, Dean RL, Beer B, Lippa AS. 1982. The cholinergic hypothesis of geriatric memory dysfunction. Science 217:408–414. Bartus RT. 2000. On neurodegenerative diseases, models, and treatment strategies: lessons learned and lessons forgotten a generation following the cholinergic hypothesis. Exp Neurol 163:495–529. Beal MF. 2002. Oxidatively modiﬁed proteins in aging and disease. Free Radic Biol Med 32:797–803. Behl C, Davis JB, Cole GM, Schubert D. 1992. Vitamin E protects nerve cells from amyloid b protein toxicity. Biochem Biophys Res Commun 186:944–950. Behl C, Davis JB, Lesley R, Schubert D. 1994. Hydrogen peroxide mediates amyloid beta protein toxicity. Cell 77:817–827. Bilici M, Efe H, Koroglu MA, Uydu HA, Bekaroglu M, Deger O. 2001. Antioxidative enzyme activities and lipid peroxidation in major depression: alteration by antidepressant treatments. J Affect Disord 64:43–51. Cazevieille C, Safa R, Osborne NN. 1997. Melatonin protects primary cultures of rat cortical neurons from NMDA excitotoxicity and hypoxia/reoxygenation. Brain Res 768:120–124. Chandra J, Samali A, Orrenius S. 2000. Triggering and modulation of apoptosis by oxidative stress. Free Radic Biol Med 29:323–333. Choi JH, Kim IS, Kim JI, Kim DW, Yoon TH. 1993. Effect of reed root extract (Phragmites communis) on physiological activity of SD rats. Kor J Gerontol 3:109–115. Choi JH, Kim DW, Kim KS, Kim CM, Baek YH. 1997a. Effect of reed root extract (RRE) on learning and memory impairment animal model SAMP8. 2. Feeding effect of RRE on oxygen radicals and their scavenger enzymes in SAMP8 brain. Kor J Gerontol 7:23–28. Choi JH, Kim DW, Choi JS, Han YS, Baek YH. 1997b. Effect of reed root extract (RRE) on learning and memory impairment animal model SAMP8. 3. Feeding effect of RRE on neurotransmitters and their metabolites in SAMP8 brain. Kor J Gerontol 7:29–36. Choi JH, Kim IS, Kim DW, Kim JH, Han YS Kim HS. 1998. Effect of p-coumaric acid on lipid metabolism in SAMP8 serum. Kor J Gerontol 8:99–104. Chyan YJ, Poeggeler B, Omar RA, Chain DG, Frangione B, Ghiso J, Pappolla MA. 1999. Potent neuroprotective properties against the Alzheimer beta-amyloid by an endogenous melatonin-related indole structure, indole-3-propionic acid. J Biol Chem 274:21937–21942. Craft JM, Watterson DM, Frautschy SA, Van Eldik LJ. 2004. Aminopyridazines inhibit b-amyloid induced glial activation and neuronal damage in vivo. Neurobiol Aging 25:1283–1292.
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