GSK-3β pathways

Journal of Ethnopharmacology 178 (2016) 50–57

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Cassia obtusifolia seed ameliorates amyloid β-induced synaptic dysfunction through anti-inflammatory and Akt/GSK-3β pathways Jee Hyun Yi a,1, Hey Jin Park b,c,1, Seungheon Lee d, Ji Wook Jung e, Byeong C. Kim f, Young Choon Lee b,c, Jong Hoon Ryu g,h,n, Dong Hyun Kim b,c,nn a

School of Clinical Sciences, Faculty of Medicine and Dentistry, University of Bristol, Bristol, UK Department of Medicinal Biotechnology, College of Health Sciences, Dong-A University, Busan 604-714, Republic of Korea c Institute of Convergence Bio-Health, Dong-A University, Busan 604-714, Republic of Korea d Department of Aquatic Biomedical Sciences, School of Marine Biomedical Science, College of Ocean Science, Jeju National University, Jeju 690-756, Republic of Korea e Department of Herbal Medicinal Pharmacology, College of Herbal Bio-industry, Daegu Haany University, Kyungsan 712-715, Republic of Korea f Chonnam-Bristol Frontier Laboratory, Biomedical Research Institute, Chonnam National University Hospital, Jebong-ro, Gwangju 501-757, Republic of Korea g Department of Life and Nanopharmaceutical Sciences, College of Pharmacy, Kyung Hee University, Seoul 130-701, Republic of Korea h Department of Oriental Pharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul 130-701, Republic of Korea b

art ic l e i nf o

a b s t r a c t

Article history: Received 29 June 2015 Received in revised form 26 October 2015 Accepted 2 December 2015 Available online 7 December 2015

Ethnopharmacological relevance: Tea infused with the seed of Cassia obtusifolia has been traditionally used as an herbal remedy for liver, eye, and acute inflammatory diseases. Recent pharmacological reports have indicated that Cassiae semen has neuroprotective effects, attributable to its anti-inflammatory actions, in ischemic stroke and Parkinson's disease models. Aim of the study: Previously, the ethanol extract of C. obtusifolia seeds (COE) was reported to have memory enhancing properties. However, the effects of COE in an Alzheimer's disease (AD) model are currently unknown. In this study, we investigated the effect(s) of COE on aberrant synaptic plasticity and memory impairment induced by amyloid β (Aβ), a key toxic component found in the AD brain. Materials and methods: To determine the effect of COE on Aβ-induced aberrant synaptic plasticity, we used acute mouse hippocampal slices and delivered theta burst stimulation to induce long-term potentiation (LTP). Western blots were used to detect Aβ- and/or COE-induced changes in signaling proteins. The novel object location recognition test was conducted to determine the effect of COE on Aβinduced recognition memory impairment. Results: COE was found to ameliorate Aβ-induced LTP impairment in the acute hippocampal slices. Glycogen synthase kinase-3β (GSK-3β), a key molecule in LTP impairment, was activated by Aβ. However, this process was inhibited by COE via Akt signaling. Moreover, COE was found to attenuate Aβ-induced microglia, inducible nitric oxide synthase (iNOS), and cyclooxygenase (COX) activation. In the in vivo studies performed, COE ameliorated the Aβ-induced object recognition memory impairment. Conclusion: These results suggest that COE exhibits neuroprotective activities against Aβ-induced brain disorders. & 2015 Elsevier Ireland Ltd. All rights reserved.

Keywords: Cassia obtusifolia seeds Amyloid beta Long-term potentiation Recognition memory

1. Introduction

n

Corresponding author at: Department of Life and Nanopharmaceutical Sciences, College of Pharmacy, Kyung Hee University, Seoul 130-701, Republic of Korea. nn Corresponding author at: Department of Medicinal Biotechnology, College of Health Sciences, Dong-A University, Busan 604-714, Republic of Korea. E-mail addresses: [email protected] (J.H. Yi), [email protected] (H.J. Park), [email protected] (S. Lee), [email protected] (J.W. Jung), [email protected] (B.C. Kim), [email protected] (Y.C. Lee), [email protected] (J.H. Ryu), [email protected] (D.H. Kim). 1 These authors contributed equally to this study. http://dx.doi.org/10.1016/j.jep.2015.12.007 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.

Alzheimer's disease (AD) is a highly prevalent neurologic disorder that impairs memory and has various other psychological symptoms (Koo et al., 1999). The two major hallmark proteins of AD in the brain are amyloid β (Aβ) and tau (Braak and Braak, 1997; Terry, 1963). Aβ has long been reported as a key component of ADassociated pathologies, including aberrant synaptic plasticity (Selkoe, 2008; Shankar et al., 2008), inflammation (Tuppo and Arias, 2005; Wyss-Coray, 2006), oxidative toxicity (Butterfield, 2002; Varadarajan et al., 2000), excitotoxicity (Mattson et al., 1992), and memory impairment (Lesne et al., 2006; Shankar et al.,

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2008). Therefore, developing drugs that modulate Aβ toxicity has widely been considered a promising approach for treating AD. Aβ exerts detrimental effects on synaptic plasticity and cognition through various pathways. Inflammation and oxidative stress are the two most extensively studied pathways associated with the synaptotoxicity induced by Aβ (Doost Mohammadpour et al., 2015; Hochstrasser et al., 2013). Multiple cellular signaling mechanisms (including glutamate receptor activation (Varga et al., 2015), RAGE receptor signaling (Lv et al., 2015; Origlia et al., 2009), glycogen synthase kinase-3β (GSK-3β) activation (Deng et al., 2014), and caspase activation (D’Amelio et al., 2012; Hu et al., 2015)) are also influenced by Aβ production and accumulation in AD. Anti-inflammatory agents, antioxidants, and intracellular signaling modulators were thought to be beneficial for AD patients (Currais et al., 2014; Dzoyem and Eloff, 2015; Lee et al., 2013; Su et al., 2014). However, no pharmaceutical breakthroughs in the treatment of AD were achieved from the selective manipulation of any of these individual mechanisms. Since the pathology of AD is related to multiple interconnected mechanisms, drugs that target a single pathway may be ineffective in treating this condition. Therefore, the development of multi-target drugs has long been considered an attractive strategy for the treatment of AD. Cassiae semen (the seeds of Cassia obtusifolia L., C. alata L. and C. tora L.) has been used as an herbal remedy for diseases of the liver and eye, as well as inflammatory disorders (Crockett et al., 1992; YP., 1998). The antioxidant activity of the methanolic extracts of Cassiae semen was established (Yen and Chuang, 2000; Yen and Chung, 1999). Previous studies have determined that ethanol extract of the seed of Cassia obtusifolia L. is effective against memory impairment and brain cell damage induced by cholinergic dysfunction (Kim et al., 2007) owing to its anti-inflammatory and antioxidant effects (Kim et al., 2009). However, the effects of COE in Aβ-induced AD models have never been investigated. Since COE has been demonstrated to have neuroprotective activities, we sought to determine if COE influences ADassociated pathologic changes in the brain (i.e., aberrant synaptic plasticity and cognitive impairment). Electrophysiological data and behavioral testing revealed that COE has neuroprotective effects in this Aβ-induced model of synaptic dysfunction.

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University. COE was the same as used in a previous study, where chemical standardization had been performed (Kim et al., 2007). The Complete Protease Inhibitor Cocktail and PhosphoSTOP Phosphatase Inhibitor Cocktail were purchased from Roche (Palo Alto, CA). The rabbit polyclonal anti-pGSK3β, GSK3β, iNOS, COX and β-actin antibodies were purchased from Santa Cruz Biotech (Santa Cruz, CA). AKTi was purchased from Tocris Bioscience (Ellisville, MO). All of the other materials were of the highest grade available and were obtained from normal commercial sources. 2.3. 2.4. Acute hippocampal slice preparation and electrophysiology Mouse hippocampal slices were prepared using micro-vibratome (Lafayette-campden neuroscienceTM). The brain was rapidly removed and placed in ice-cold artificial cerebrospinal fluid (ACSF; bubbled with 95% O2/5% CO2), which comprised: (mM) NaCl, 124; KCl, 3; NaHCO3, 26; NaH2PO4, 1.25; CaCl2, 2; MgSO4, 1; D-glucose, 10. Transverse hippocampal slices (400 μm thick) were prepared. Hippocampal slices were submerged in ACSF (20–25 °C) for 1 h before transfer to the recording chamber (28–30 °C, flow rate ∼3 ml/min) as required. Slices were incubated in Aβ solution (500 nM) for 2 h and then transferred to the recording chamber. For the treatment of COE, slices were incubated in COE solution for 30 min and then the slices were more incubated in COEþAβ solution for 2 h. Minocycline was used as a positive control. AKTi introduced as same condition with COE. Field recordings were made from stratum pyramidale in area CA1. Stimulating electrodes were placed in the Schaffer collateralcommissural pathway. Stimuli (constant voltage) were delivered at 30 s intervals. To induce LTP, theta burst stimulation (5 trains of 4 pulses at 100 Hz) was delivered. The slope of the evoked field potential responses were averaged from four consecutive recordings (EPSPs) evoked at 30 s intervals. 2.4. Western blot analysis

Male CD-1 mice (30–34 g, 10 weeks) were purchased from the SAMTAKO biokorea (Osansi, Korea), and kept in the University Animal Care Unit for 2 weeks prior to the experiments. The animals were housed 4 per cage, allowed access to water and food ad libitum; the environment was maintained at a constant temperature (23 7 1 °C) and humidity (60 710%) under a 12-h light/dark cycle (the lights were on from 07.30 to 19.30 h). The treatment and maintenance of the animals were carried out in accordance with the Animal Care and Use Guidelines Dong-A University, Korea. All of the experimental protocols using animals were approved by the Institutional Animal Care and Use Committee of Dong-A University, Korea.

For the preparation of Western blot samples, coronal-sliced hippocampal tissues were homogenized in an ice-cold Tris–HCl buffer (20 mM, pH 7.4) containing 0.32 M sucrose, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, a Complete Protease Inhibitor Cocktail (1 tablet/50 ml) and a PhosSTOP Phosphatase Inhibitor Cocktail (1 tablet/10 ml). Samples of the homogenates (20 μg of protein) were then subjected to SDS-PAGE (8%) under reducing conditions. The proteins were transferred to PVDF membranes in a transfer buffer [25 mM Tris–HCl buffer (pH 7.4) containing 192 mM glycine and 20% v/v methanol] and further separated at 400 mA for 2 h at 4 °C. The Western blots were then incubated for 1 h with a blocking solution (2% BSA or 5% skim milk), then with rabbit anti-pGSK3β (1:1000), rabbit anti-iNOS (1:1000), or rabbit anti-COX (1:1000) antibody overnight at 4 °C, washed ten times with Tween 20/Trisbuffered saline (TTBS), incubated with a 1:2000 dilution of horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature, washed ten times with TTBS, and finally developed by enhanced chemiluminescence (Amersham Life Science, Arlington Heights, IL). The blots were then stripped and incubated with a rabbit anti-GSK3β (1:2000) or rabbit anti-β-actin antibody (1:2000).

2.2. Materials

2.5. Tissue preparations and immunohistochemistry

The seed of Cassia obtusifolia L. (Chinese senna, American sicklepod or sicklepod) were purchased from the Kyungdong oriental drug store (Seoul, Korea) and identified by emeritus professor Chang Soo Yook, College of Pharmacy, Kyung Hee University and voucher specimens (Cassiae semen KHUOPS-04-31) were deposited at the herbarium of the College of Pharmacy, Kyung Hee

Immediately after the recording, hippocampal slices were fixed with phosphate buffer (0.05 M, pH 7.4) containing 4% paraformaldehyde and then immersed in a 30% sucrose solution in 0.05 M phosphate-buffered saline (PBS), and stored at 4 °C until sectioned. Frozen slices were coronally sectioned on a cryostat at 30 μm using cryostat microtome (Leica CM1850, Leica Biosystems,

2. Materials and methods 2.1. Animals

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Korea) and then stored in storage solution at 4 °C. Free floating sections were incubated in blocking solution (2% rabbit serum in PBS) for 2 h, then incubated in PBS (4 °C) containing monoclonal rat anti-CD11b antibody (OX-42, 1:1000 dilution), 0.3% Triton X-100, and 0.5 mg/ml normal rabbit serum (Vector, Burlingame, CA) for overnight. The sections were then incubated in secondary antibody solution containing biotinylated anti-rat IgG (1:200 dilution, Vector, Burlingame, CA) followed by ABC complex (1:100 dilution, Vector, Burlingame, CA) for 1 h at room temperature, and reacted with 0.02% 3,3′-diaminobenzidine and 0.01% H2O2 for about 3 min. After finishing every steps, slices were mounted on gelatin-coated slides, dehydrated in an ascending alcohol series, and cleared in xylene. 2.6. Intracerebroventricular drug injection and object recognition memory test To test the effect of COE on Aβ-induced AD mice model, mice were treated with oligomeric Aβ (10 μM, i.c.v.) or vehicle (5 μl, third ventricle, i.c.v.) 24 h before habituation of object recognition test under mild anesthesia (a mixture of N2O and O2 (70:30) containing 2.5% isoflurane) and then returned to their home cages. Aβ1–42 peptide was dissolved in sterile saline (1 mM) in tubes, which were then sealed and incubated for 24 h at 4 °C for oligomerization (Jo et al., 2011). Mice were habituated to the open field (25 cm  25 cm  25 cm) with an internal cue on one of the four walls for 10 min. Thirty minutes after the habituation; mice were

re-placed in the same box with two distinct objects. The objects consisted of a glass box and plastic cylinder. Mice were allowed to freely explore the objects for 10 min. After 2 h, mice were placed back in the same box for the testing phase. The two objects were again present, but one object was now displaced to a novel spatial location. Mice were allowed to freely explore the environment and the objects for 5 min. Time spent exploring the displaced and nondisplaced objects were measured. Recognition index was calculated by following fomular: (Tdisplaced–Tnon-displaced)/(Tdisplaced þTnon-displaced)*100. Tdisplaced, time spent exploring displaced object. Tnon-displaced, time spent exploring non-displaced object. 2.6.1. Statistical analysis The values are expressed as the means 7S.E.M. The data were analyzed using one-way analysis of variance (ANOVA) followed by Student–Newman–Keuls test for multiple comparisons. The statistical significance was set at P o0.05.

3. Results 3.1. COE prevents Aβ-induced synaptic dysfunction in the hippocampus Synaptic dysfunction induced by oligomeric Aβ is believed to contribute to cellular pathology in early AD. Therefore, we initiated our investigations by evaluating the effect of COE on

Fig. 1. Cassia obtusifolia (COE) prevents Aβ-induced synaptic dysfunction. (A) Extracellular field EPSPs (fEPSP) were evoked by stimulating Schaffer collateral CA1 synapse. (B) Acute hippocampal slices (12-week-old male mice) were incubated with COE (1 and 10 μg/ml) for 30 min and then more incubated with Aβ (500 nM) and COE for 2 h. (C) LTP was evoked by theta burst stimulation (TBS, 5 trains of 4 pulses at 100 Hz). TBS readily induced LTP in control slices (n¼ 7). Pre-incubation of acute slice with Aβ (n¼7) blocked LTP. COE blocked Aβ-induced LTP impairment (1 μg/ml, n ¼7; 10 μg/ml, n ¼7) in a concentration-dependent manner. Minocycline (Min, 20 μM, n¼7) blocked Aβ-induced LTP impairment. (D) Bar chart of normalized fEPSP at 80 min time points of each group in C. Data were represented as mean7 SEM. n¼ 7–8/group. *Po 0.05 compared to control group. #Po 0.05 compared to control Aβ-treated group.

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oligomeric Aβ-induced synaptic dysfunction. For these studies, we measured long-term potentiation (LTP) levels in the Shaffer-collateral pathway of the hippocampal slices (Fig. 1A) treated with Aβ (500 nM) and/or COE (1 and 10 μg/mL) (Fig. 1B). There was a significant group effect in this experiment (F4, 30 ¼4.590, P o0.05, Fig. 1D). While theta burst stimulation (TBS), a well-known LTPinducing protocol (5 trains of 4 pulses at 100 Hz), induced LTP in the control slices (144 711% of baseline, Fig. 1C and D), LTP was not induced by TBS in Aβ-treated slices (10974% of baseline, Fig. 1C and D). Interestingly, TBS induced LTP (13277% of baseline, Fig. 1C and D) in slices co-administered Aβ and COE (10 μg/mL) comparable to control levels. However, low concentrations of COE failed to prevent Aβ-induced LTP impairment (1037 9% of baseline, Fig. 1C and D), which suggests that its effects in this model are concentration-dependent. Minocycline (20 μM), as like previous report (Wang et al., 2004), also prevented Aβ-induced LTP impairment (1357 9% of baseline, Fig. 1C and D). 3.2. The effect of COE and Aβ on basal synaptic transmission in the hippocampus Basal synaptic transmission is primarily mediated via AMPA receptors. Changes in AMPA receptor activity can trigger alterations in various synaptic activities, including synaptic plasticity. Therefore, we set out to determine the influence of COE and Aβ on basal synaptic transmission by conducting input-output and paired pulse facilitation tests. Aβ was not found to affect basal synaptic transmission. Moreover, co-administration of COE and Aβ also displayed no influence on the level of basal synaptic transmission (Fig. 2A and B). These results suggest that COE may modulate intracellular signaling pathways involving actions of Aβ, but not those that directly regulate basal synaptic transmissions. 3.3. COE does not rescue Aβ-induced synaptic dysfunction Drug therapy is typically initiated in patients after they have been diagnosed with AD. Therefore, to determine the feasibility of COE as a potential therapeutic agent, its rescue effect on Aβ-induced synaptic dysfunction was evaluated. These studies were performed by incubating hippocampal slices with Aβ solution for 2 h, and then measuring the LTP levels before and after COE infusion (Fig. 3A). As shown in Fig. 3B, Aβ blocked LTP induction in the hippocampal slices (9879% of baseline), but post-infusion with COE failed to reestablish LTP levels. These results suggest that COE cannot rescue Aβ-induced LTP impairment, which was previously established.

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3.4. COE inhibits Aβ-INDUCED GSK-3β activation GSK-3β has been implicated in the aberrant synaptic plasticity induced by Aβ (Jo et al., 2011). As such, we set out to determine if COE plays a role in Aβ-induced GSK-3β signaling. Aβ significantly decreased pGSK-3β levels in hippocampal slices, suggesting that Aβ activates pGSK-3β. This, however, was prevented by COE in a concentration-dependent manner (F4, 20 ¼ 3.501, P o0.05, Fig. 4A). Minocycline was also found to inhibit the Aβ-induced reduction of pGSK-3β levels (Fig. 4A). Interestingly, Akt inhibition blocked the effect of COE on GSK-3β (F3, 16 ¼5.261, P o0.05, Fig. 4B). Moreover, Akt inhibition also blocked the preventive effect of COE on Aβinduced synaptic dysfunction (F2, 12 ¼5.399, P o0.05, Fig. 4C). Taken together, these results suggest that COE regulates Akt signaling, and this might be involved in the effect of COE on Aβ toxicity. 3.5. COE inhibits Aβ-induced inflammation in the hippocampus To determine the mechanism by which COE affects Aβ-induced synaptic dysfunction, we detected and measured markers of inflammation in the hippocampus. Treatment with Aβ increased the number of activated microglia in the hippocampus, which was detected using OX-42 antibodies. When COE was co-administered with Aβ, the number of activated microglia decreased (Fig. 5A). In addition, COE was found to inhibit Aβ-induced increases in iNOS and COX levels in a concentration-dependent manner (iNOS, F3, 16 ¼7.935, P o0.05; COX, F3, 16 ¼4.381, P o0.05, Fig. 5B and C). These results suggest that COE prevents Aβ-induced inflammation in the hippocampus 3.6. Orally administered COE prevents Aβ-induced recognition memory impairment We conducted object location recognition test to determine the effect of orally administered COE on Aβ-induced memory impairment. All groups spent similar amounts of time exploring objects in the sample and test sessions, suggesting that the treatments did not alter motor activity or exploring motivation (sample session, F3, 25 ¼0.469, P 40.05, Fig. 6A; test session, F3, 25 ¼ 0.081, P 40.05, Fig. 6B). However, the Aβ-treated group displayed a significantly lower recognition index than did the groups not treated with Aβ. Further, this decline was prevented by treatment with either COE (50 mg/kg, p.o.), or minocycline (100 mg/kg, p.o.) in the test session (F3, 25 ¼ 3.129, P o0.05, Fig. 6C).

Fig. 2. The effect of Cassia obtusifolia (COE) and Aβ on basal synaptic transmission. (A) Input–output curves were plotted for control (n¼ 10), Aβ-treated (n¼ 10) and Aβ þCOE co-treated (n¼ 10) hippocampal slices. Comparison of the three curves reveals no significant difference in basal synaptic transmission). (B) Paired pulse facilitation is unaffected by Aβ and COE (n¼ 10/groups). Symbols and error bars indicate mean 7 SEM.

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Fig. 3. The effect of Cassia obtusifolia (COE) on Aβ-induced synaptic dysfunction established. (A) To examine the rescuing effect of COE on LTP impairment, which was previously established by Aβ, acute hippocampal slices (12-week-old male mice) were incubated with Aβ (500 nM) for 2 h. And then, LTP was measured with perfusion of COE (10 μg/ml). Aβ blocked LTP in the hippocampal slices (B, n ¼7). However, COE failed to rescue LTP impairment induced by Aβ pretreatment (C, n¼ 7). Symbols and error bars indicate mean 7 SEM. TBS, theta burst stimulation (5 trains of 4 pulses at 100 Hz).

Fig. 4. Cassia obtusifolia (COE) blocks Aβ-induced GSK-3β activation. (A) To examine the effect of COE on Aβ-induced GSK-3β signaling changes, acute hippocampal slices were incubated with Aβ, Aβþ COE (1 and 10 μg/ml), and Aβþ minocycline (20 μM). (B) To test the involvement Akt signaling in the effect of COE on Aβ-induced GSK-3β activation, slices were incubated with Aβ (500 nM), Aβ þ COE (10 μg/ml), and Aβþ COE (10 μg/ml) þ AKTi (50 μM). (C) To test the involvement Akt signaling in the effect of COE on Aβinduced LTP impairment, slices were incubated with Aβ (500 nM), Aβþ COE (10 μg/ml), and Aβ þ COE (10 μg/ml) þ AKTi (50 μM). Data were represented as mean 7SEM. n¼ 5/ group. *P o0.05 compared to control group. #P o0.05 compared to control Aβ-treated group.

4. Discussion Aβ is widely considered a major contributor to the toxicity found in the AD brain (Hardy, 2006). Studies have proven that Aβ has toxic effects in the brain, including abnormal intracellular signaling and neuro-inflammation (Butterfield, 2002; Lesne et al., 2006; Shankar et al., 2008). In the present study, we further confirmed that oligomeric Aβ triggers synaptic transmission dysfunction, neuro-inflammation, and memory impairment. We found that while COE can prevent these Aβ toxicities in some

cases, it could not ameliorate the established synaptic dysfunction. These results suggest that the daily consumption of C. obtusifolia seeds (which is traditionally consumed as a tea) may have preventive effects on AD progression. Synaptic plasticity has been suggested as an underlying cellular and molecular mechanism of learning and memory (Bliss and Collingridge, 1993; Maren and Baudry, 1995). One form of synaptic plasticity, LTP, is a physiological process vital to the controlled facilitation of synaptic efficacy. AD brains display lower LTP levels than healthy brains, which are known to result from the

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Fig. 5. Cassia obtusifolia (COE) blocks Aβ-induced inflammation. To test the effect of COE (1 and 10 μg/ml) on Aβ-induced inflammation, acute hippocampal slices (12-week old) were incubated with Aβ and Aβþ COE (1 and 10 μg/ml). (A) Representatives of immunohistochemistry for OX-42 immunopositive cells. Bar ¼20 μm. (B) Representatives of western blot for iNOS and COX. (C) Quantitative analysis of immunoreactivities of iNOS and COX. n¼ 5/group. Data were represented as mean7 SEM. n¼ 10/group. *Po 0.05 compared to control group. #Po 0.05 compared to control Aβ-treated group.

synaptotoxic effects of Aβ (Oddo et al., 2003; Palop and Mucke, 2010). LTP impairment parallels memory impairment following Aβ injection, and aberrant synaptic plasticity and cognitive dysfunction appear at similar ages in AD models (Giannopoulos et al., 2014; Seo et al., 2014). Thus, the mechanisms by which Aβ promotes synaptotoxicity are analogous to those that cause memory impairment in AD models. In the present study, Aβ triggered impairments in both LTP and recognition memory. Moreover, COE was shown to prevent these neurotoxic effects of Aβ. We therefore suggest that COE could potentially be used as a preventive agent against AD progression. Aβ oligomers are considered to be extremely synaptotoxic (Selkoe, 2008; Shankar et al., 2007). Although the precise mechanisms are still unclear, GSK-3β is believed to play a key role in

Aβ-induced synaptic dysfunction. Aβ activates GSK-3β through various pathways (including caspase-3 activation (Jo et al., 2011) and TrKB inhibition (Hu et al., 2013)), which enhance long-term depression, another form of synaptic plasticity, and depresses LTP (Olsen and Sheng, 2012; Shankar et al., 2008). In the present study, we found that COE prevents Aβ-induced GSK-3β activation. Moreover, this effect was mediated by Akt, an upstream regulator of GSK-3β. Interestingly, we found that Akt inhibition prevented protective effect of COE on Aβ-induced synaptic dysfunction. However, we could not find any effect of Akt inhibition on microglial activation (data not shown). These results suggest that COE blocks Aβ inhibition of LTP through Akt signaling and this is independent mechanism from the effect of COE on neuroinflammation. COE has many functional ingredients including

Fig. 6. Cassia obtusifolia (COE) prevents Aβ-induced recognition memory impairment. To test the effect of COE on Aβ-induced memory impairment, mice (12-week old) were treated with COE (50 mg/kg, p.o.) 1 h before Aβ injection (i.c.v. 10 μM, 5 μl). (A) Sample session was conducted 24 h after Aβ injection. Total exploration time was measured during sample session. (B,C) Test session was conducted 2 h after sample session. Total exploration time (C) and recognition index (D) were measured. Data were represented as mean 7 SEM. n¼ 7–8/group. *P o 0.05 compared to control group. #P o 0.05 compared to control Aβ-treated group.

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obtusifolin, alaternin, and rubrofusarin. Previous report indicated that alaternin exerted neuroprotective effect through its anti-inflammatory effect (Shin et al., 2010). Therefore, we suggest that various effective ingredients of COE might mediate its various effects on Aβ toxicity. Inflammation is another mechanism associated with Aβ-induced synaptic dysfunction. Previous reports have suggested that minocycline, an inhibitor of microglial activation, prevents Aβ toxicity (Choi et al., 2007; Wang et al., 2004). Activation of other inflammatory mediators (e.g., IL-2, iNOS, and COX) were also suggested as potential mechanisms of Aβ-induced synaptic dysfunction (Di Filippo et al., 2008; Ko et al., 2015; Kotilinek et al., 2008; Rowan et al., 2004). In accordance with these reports, the present study suggests that Aβ activates inflammatory responses in the hippocampus along with synaptic dysfunction. We have previously reported that COE does reduce neuro-inflammation in the ischemic brain (Kim et al., 2009). Comparably, the current report determined that COE prevents microglial activation, as well as iNOS and COX activation, induced by Aβ in the hippocampus. Thus, these mechanisms may explain the effect of COE on Aβ-induced synaptic dysfunction. We previously reported that COE and obtusifolin, a chemical component of COE, both inhibit acetylcholinesterase (Kim et al., 2007). Moreover, COE and alaternin, another neuroprotective compound extracted from COE, showed neuroprotective effects in ischemic brain damage models (Kim et al., 2009; Shin et al., 2010). Therefore, the effects of COE on Aβ-induced synaptic toxicity may be attributable to obtusifolin and/or alaternin. Interestingly, studies have shown that acetylcholinesterase inhibitors can rescue Aβ-induced memory impairment (Dong et al., 2005; Mehta et al., 2012). This effect was not observed with COE in the experiments described in this work. However, we administered COE at 10 μg/mL concentrations in the present study, which is less than its IC50 determined from acetylcholinesterase assays. This suggests that COE may have various effects on multiple targets for the treatment of AD.

Conflict of interest The authors declare no conflict of interests.

Author contributions The study was conceived and designed by D.H.K and J.H.R Electrophysiological studies were conducted by J.H.Y, H.J.P, S.L and biochemical assays were conducted by J.H.Y and J.W.J. Behavioral experiment was conducted by H.J.P and Y.C.L. The manuscript was written by B.C.K, Y.C.L, J.H.R, and D.H.K.

Acknowledgment This research was supported by Dong-A University Research Supporting Program.

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