Oleanolic acid ameliorates cognitive dysfunction caused by cholinergic blockade via TrkB-dependent BDNF signaling

Accepted Manuscript Oleanolic acid ameliorates cognitive dysfunction caused by cholinergic blockade via TrkB-dependent BDNF signaling Se Jin Jeon, Hong Ju Lee, Hyung Eun Lee, Se Jin Park, Yubeen Gwon, Haneul Kim, Jiabao Zhang, Chan Young Shin, Dong Hyun Kim, Jong Hoon Ryu PII:

S0028-3908(16)30315-X

DOI:

10.1016/j.neuropharm.2016.07.029

Reference:

NP 6388

To appear in:

Neuropharmacology

Received Date: 21 March 2016 Revised Date:

22 July 2016

Accepted Date: 24 July 2016

Please cite this article as: Jeon, S.J., Lee, H.J., Lee, H.E., Park, S.J., Gwon, Y., Kim, H., Zhang, J., Shin, C.Y., Kim, D.H., Ryu, J.H., Oleanolic acid ameliorates cognitive dysfunction caused by cholinergic blockade via TrkB-dependent BDNF signaling, Neuropharmacology (2016), doi: 10.1016/ j.neuropharm.2016.07.029. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Oleanolic acid ameliorates cognitive dysfunction caused by cholinergic blockade via TrkB-dependent BDNF signaling

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Se Jin Jeon1, Hong Ju Lee3, Hyung Eun Lee1, Se Jin Park1, Yubeen Gwon1, Haneul Kim1, Jiabao Zhang1, Chan Young Shin5, Dong Hyun Kim3,4,* and Jong Hoon Ryu1,2,*

1

Department of Life and Nanopharmaceutical Science, College of Pharmacy, Kyung Hee

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University, Seoul 130-701, Republic of Korea. 2Department of Oriental Pharmaceutical

3

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Science, College of Pharmacy, Kyung Hee University, Seoul 130-701, Republic of Korea. Department of Medicinal Biotechnology, College of Health Sciences and 4Institute of

Convergence Bio-Health, Dong-A University, Busan 604-714, Republic of Korea. 5

Department of Neuroscience, Center for Neuroscience Research, Institute of Biomedical

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Science and Technology, Konkuk University School of Medicine, Seoul, 143-701, Korea.

Number of pages: 28 pages including title and figure legends

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Number of figures: 7, supplementary figure 3 and supplementary table 1

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Running title: Memory-ameliorating effect of oleanolic acid via TrkB

*To whom correspondence should be addressed: Jong Hoon Ryu, Ph.D.

Department of Oriental Pharmaceutical Science, College of Pharmacy, Kyung Hee University, Kyunghee-daero 26, Dongdeamun-gu, Seoul 130-701,Republic of Korea Tel.: +82-2-961-9230; Fax: +82-2-966-3885; E-mail: [email protected] Or Dong Hyun Kim, Ph.D. ([email protected]) 1

ACCEPTED MANUSCRIPT Abstract Oleanolic acid is a naturally occurring triterpenoid and is widely present in food and medicinal plants. To examine the effect of oleanolic acid on memory deficits, we employed a

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cholinergic blockade-induced cognitive deficit mouse model. A single administration of oleanolic acid significantly increased the latency on the passive avoidance task and affected the alternation behavior on the Y-maze task and the exploration time on the novel object

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recognition task, indicating that oleanolic acid reverses the cognitive impairment induced by scopolamine. In accordance with previous reports, oleanolic acid enhanced extracellular-

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signal-regulated kinase 1/2 (ERK1/2) and cAMP response element-binding protein (CREB) phosphorylation and brain-derived neurotrophic factor (BDNF) expression in the hippocampus. Interestingly, ameliorating effect of oleanolic acid on scopolamine-induced memory

impairment

was

abolished

by

(ANA-12),

a

potent

and

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yl)amino]carbonyl}phenyl)benzo[b]thiophene-2-carboxamide

N2-(2-{[(2-oxoazepan-3-

specific inhibitor of tropomyosin receptor kinase B (TrkB), in the passive avoidance task. Similarly, oleanolic acid significantly evoked long-term potentiation in a dose-dependent

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manner, which was diminished by ANA-12 treatment as shown in the electrophysiology study. Together, these results imply that oleanolic acid ameliorates scopolamine-induced memory

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impairment by modulating the BDNF-ERK1/2-CREB pathway through TrkB activation in mice, suggesting that oleanolic acid would be a potential therapeutic agent for the treatment of cognitive deficits.

Keywords: Oleanolic acid; memory improvement; scopolamine model; BDNF signaling; TrkB 2

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1. Introduction The physiological process of aging involves progressive cognitive decline caused by

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the deterioration of brain function, which includes a decrease in learning and memory skills and slower responses to environmental stimuli. Cognitive dysfunction associated with several neurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinson’s disease, and

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Huntington’s disease, is a serious problem. Among these diseases, AD is characterized by progressive neuronal death, neurofibrillary tangles and amyloid plaques, which are associated

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with several behavioral abnormalities, such as dysregulated cholinergic system-induced memory deficit and neuropsychiatric symptoms (Wilcock et al., 1982, Francis et al., 1999, Chen et al., 2008, Dumas and Newhouse, 2011, Anand and Singh, 2013, Rodrigues Simoes et al., 2014). These abnormalities subsequently evoke a large disturbance in daily working

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performance, which may lead to increased social burden. Currently, several agents are used clinically to enhance cholinergic neurotransmission and improve memory performance. For example, donepezil, a widely clinically prescribed drug for AD, has been developed as an

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inhibitor of acetylcholinesterase. However, because donepezil exerts several side effects, such as nausea, diarrhea, anorexia or vomiting, a novel mechanism to improve memory

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impairment should be discovered (Kircher et al., 2005, Kirshner, 2005, Schmitt et al., 2006). Recently, much research interest has been focused on natural product-derived active

compounds for the treatment of several neurodegenerative diseases since the introduction of galantamine, which is obtained synthetically or from the bulb or flowers of Galanthus caucasicus for treatment of AD. Oleanolic acid (3β-hydroxyolean-12-en-28-oic acid), a natural triterpenoid, that is widely present in food and medicinal plants, is one such example. Moreover, oleanolic acid displays several biological activities, including anti-inflammatory 3

ACCEPTED MANUSCRIPT response and anti-cancer properties (Salkovic-Petrisic et al., 2006, Bradley et al., 2012). Most recently, Jung et al., reported that lancemaside A and its metabolite echinocystic acid, oleanane-type triterpenoid saponins, attenuated scopolamine-induced cognitive impairments

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in mice (Jung et al., 2012). In addition, oleanolic acid showed beneficial effect on the central nervous system, especially in ischemic stroke with its anti-oxidative properties (Rong et al., 2011). It has been reported that oleanolic acid up-regulates extracellular-signal-regulated

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kinase 1/2 (ERK1/2) and cAMP response element-binding protein (CREB) phosphorylation and brain-derived neurotrophic factor (BDNF) expression in the hippocampal neurons (Yi et

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al., 2014). Since both the ERK1/2 and CREB pathways are thought to be associated with learning and memory processes, especially, synaptic plasticity (Spencer et al., 2009, Leal et al., 2014), it may be expected that oleanolic acid ameliorates cognitive dysfunction. However, it has not been reported that oleanolic acid has positive effect on cognitive functions.

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In the present study, we investigated the effects of oleanolic acid on scopolamineinduced memory impairment using several behavioral tasks, such as the passive avoidance, the Y-maze and the novel object recognition. To figure out the mechanism of actions of

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oleanolic acid on cognitive functions, we performed electrophysiological studies and Western blotting under tropomyosin receptor kinase B (TrkB) blockade condition. Here, we report that

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oleanolic acid reverses cognitive dysfunction and such effect would be mediated BDNF signaling and long-term potentiation (LTP) enhancement.

2. Materials and methods

2.1. Animals Male ICR mice (6 weeks old, 25 – 30 g) obtained from the Orient Co., Ltd., a branch 4

ACCEPTED MANUSCRIPT of Charles River Laboratories (Gyeonggi-do, Korea), were housed 5 mice per cage, freely provided with water and food and maintained under constant temperature (23 ± 1 oC) and humidity (60 ± 10%) under a 12-h light/dark cycle (light from 07:30 to 19:30). Pregnant

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Sprague-Dawley female rats were provided from the same company and cared in a different area with the same conditions. Animal maintenance and treatment were performed in accordance with the Animal Care and Use Guidelines issued by Kyung Hee University, Korea.

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All animal experiments were approved by the IACUC (approved NO. KHP-2014-02-03).

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2.2. Materials

Oleanolic acid, donepezil hydrochloride monohydrate, scopolamine hydrobromide, and

N2-(2-{[(2-oxoazepan-3-yl)amino]carbonyl}phenyl)benzo[b]thiophene-2-carboxamide

(ANA-12) were purchased from Sigma-Aldrich (St Louis, MO). The acethylcholine (ACh)

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ELISA assay kit was obtained from Abcam (ab65345, abcam, USA). The antibodies against ERK, CREB, phosphorylated TrkB at tyrosine 706 (pTrkB), TrkB and BDNF were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The antibody against phosphorylated

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ERK at threonine 202/tyrosine 204 (pERK1/2) was purchased from Cell Signaling (Danvers, MA), and the antibody against phosphorylated CREB at serine 133 (pCREB) was obtained

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from Millipore (Temecula, CA). For cell culture, NBM or B27 supplement were obtained from Gibco BRL (Grand Island, NY). All drugs were freshly prepared on the day of testing. Donepezil, ANA-12 and scopolamine were dissolved in a 0.9% saline solution. Oleanolic acid was suspended in 10% Tween 80 solution. TrkB siRNA and control siRNA were obtained from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). All other materials were obtained from standard normal commercial sources and were of the highest grade available.

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ACCEPTED MANUSCRIPT 2.3. Step-through passive avoidance task For the passive avoidance task, male ICR mice were trained (acquisition trial) 24 h prior to a retention trial. The assessment was performed in a box consisting of two identical

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chambers (20 × 20 × 20 cm), in which one was illuminated with a 50 W bulb and the other was non-illuminated, that were separated by a guillotine door (5 × 5 cm), as described previously (Lee et al., 2013). The mice were administered either oleanolic acid (0.625, 1.25,

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2.5, or 5 mg/kg, p.o.), or donepezil (5 mg/kg, p.o) 1 h before the acquisition trial. The control group received the same volume of vehicle (10% Tween 80 solution, p.o.). The mice were

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treated with scopolamine (1 mg/kg, i.p.), or 0.9% saline 30 min before the acquisition trial. The mice were initially placed in the illuminated compartment during the acquisition trial. The door between the two compartments was opened 10 s later. After the mouse entered the non-illuminated compartment, the door automatically closed, and a 3-s electrical foot shock

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(0.5 mA) was delivered via the stainless steel rods. Mice that did not enter the nonilluminated compartment within 60 s after the opening of the door were gently introduced into the dark compartment and recorded 60 s as latency. The retention trial was conducted 24

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h after the acquisition trial by returning individual mice to the illuminated compartment. For both trials, the time for the mouse to enter the dark compartment after opening the door was

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defined as the latency. The latencies were recorded for up to 300 s. For antagonism study, oleanolic acid (2.5 mg/kg, p.o.) was administered 1 h before

the acquisition trial, and sub-effective dose of ANA-12 (0.3 mg/kg, i.p., Supplementary Fig. S2), an inhibitor of the tyrosine protein kinase activity of the trk family of oncogenes and neurotrophin receptors, was treated 30 min after the administration of oleanolic acid. Scopolamine (1 mg/kg, i.p.) was administered 5 min after the treatment with ANA-12. The acquisition trial was conducted at 25 min after the administration of scopolamine. The dose of 6

ACCEPTED MANUSCRIPT ANA-12 in the present study did not impair the passive avoidance task performance, when administered alone. Other procedures were the same as described above.

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2.4. Y-maze task The Y-maze is a horizontal maze with three arms (40 × 3 × 12 cm) that are symmetrically disposed at 120° angles from each other. The Y-maze was composed of dark

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opaque polyvinyl plastic, as described elsewhere (Jung et al., 2014). The mice were initially placed in one arm, and the sequence (i.e., ABCCAB, etc.) and number of arm entries were

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recorded manually for each mouse over an 8 min period. An actual alternation was defined as an entry into all three arms on consecutive choices (i.e., ABC, BAC or CAB but not BCC or CCA). One hour before the test, the mice were orally administered either oleanolic acid (2.5 or 5 mg/kg) or donepezil (5 mg/kg). The control group received 10% Tween 80 solution rather

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than oleanolic acid or donepezil. Scopolamine (1 mg/kg, i.p.) was administered to induce memory impairment 30 min before the test. The Y-maze arms were thoroughly cleaned with water spray between each test to remove residual odors and residues. The alternation score

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(%) for each mouse was defined as the ratio of the number of actual alternations to the possible number (defined as the total number of arm entries minus two) multiplied by 100, as

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shown by the following equation: % alternation = [(number of alternations) / (total arm entry numbers – 2)] × 100.

2.5. Novel object recognition test The novel object recognition test was performed as previously described (Park et al., 2013). Habituation was conducted by exposing the animal to the experimental apparatus for 10 min per day in the absence of objects for 2 days. On the first training day, the mice were 7

ACCEPTED MANUSCRIPT administered oleanolic acid (2.5 or 5 mg/kg, p.o.) 30 min before scopolamine (1 mg/kg, i.p.) treatment and placed in the presence of two small objects in a black-covered square arena (30 x 30 x 30 cm) for 10 min to observe the behavior. Twenty-four hours after the training trial,

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the test trial was conducted, in which one object was replaced with a novel object, and the mice were allowed to explore each object for 5 min. We measured the time spent by the animal with the new (Tnovel) and the old object (Tfamiliar), respectively, during the observation.

preference, Tnovel / Tnovel

+

+

Tfamiliar), the preference ratio for each object (Novel

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The total exploration time (Tnovel

Tfamiliar; Familiar preference, Tfamiliar / Tnovel

+

Tfamiliar) and the

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discrimination ratio [(Tnovel − Tfamiliar)/(Tnovel + Tfamiliar) × 100] were calculated. The exploration time (%) was calculated as the percentage of time spent exploring either object by the mice. If a mouse remembers its previous exposure to the familiar object, it explores the

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novel object for much longer than the familiar object.

2.6. Measurement of acetylcholine level

Using the ACh assay kit, we evaluated the brain ACh level according to the

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manufacturer’s instructions as follows (ab65345, abcam, USA). Briefly, after 1 h of oleanolic acid (2.5 mg/kg, p.o.) administration, ANA-12 (0.3 mg/kg, i.p.) was injected. Thirty min later,

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mice were sacrificed and brain tissues were removed (n = 10-12 per group). After removal of cerebellum, rest of brain tissue was rapidly homogenized. Each 30 µg protein sample was loaded onto a 96-well reaction plate and incubated with reaction mixture (Choline Probe and Enzyme Mixture) for 40 min at 37 °C protected from light. The fluorescence was measured at 570 nm in a microplate reader. Then data was calculated and expressed in nmol/mg of protein. For choline standards, serial diluted concentration of choline samples (from 5 nmol/mg stock to 0.5 µmol/mg) was used. 8

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2.7. Western blot analysis The mice were sacrificed at indicated time after oleanolic acid administration for

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Western blot. The vehicle-treated group received 10% Tween 80 solution. The isolated hippocampal tissues were homogenized in ice-chilled Tris–HCl buffer (20 mM, pH 7.4) containing 0.32 M sucrose, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 1 mM sodium

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orthovanadate, and one protease inhibitor tablet (cOmplete™, Mini, EDTA-free Protease Inhibitor Cocktail, 04693159001, Roche) per 50 ml of buffer. Then, the homogenates (20 µg

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of total protein) were subjected to SDS-PAGE (10-12% gel) under reducing conditions. The proteins were transferred to PVDF membranes in the transfer buffer [25 mM Tris–HCl (pH 7.4) containing 192 mM glycine and 20% v/v methanol] at 400 mA for 2 h at 4 °C. Then, the membranes were blocked with 5% skim milk and incubated with anti-pTrkB, anti-TrkB, anti-

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CREB, anti-pCREB, anti-BDNF (1:3000 dilution), anti-ERK1/2, anti-pERK1/2 (1:5000 dilution), or anti-β-actin (1:10000) antibodies overnight at 4 °C and then washed with Trisbuffered saline/Tween 20 (TBST). Then, the membranes were incubated with a 1:5000

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dilution of a horseradish peroxidase-conjugated secondary antibody for 2 h and, finally, were developed with enhanced chemiluminescence (Amersham Life Science, Arlington Heights,

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IL). The membranes were analyzed using the LAS-4000 mini bio-imaging program (Fujifilm Lifescience USA, Stamford, CT). In case of cultured cells, after appropriate treatment, cells were washed twice and

harvested by ice-cold homogenized buffer with protease and phosphatase inhibitor cocktails. Then, samples were analyzed the expression of BDNF and phosphorylation of ERK1/2 and CREB by Western blotting with the above mentioned methods.

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ACCEPTED MANUSCRIPT 2.8. Acute hippocampal slice preparation and electrophysiology Mouse hippocampal slices were prepared using micro-vibratome (Lafayettecampden neuroscienceTM). The brain was rapidly removed and placed in ice-cold artificial

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cerebrospinal fluid (aCSF) bubbled with 95% O2/5% CO2 which was comprised of following ingredients (mM): NaCl, 124; KCl, 3; NaHCO3, 26; NaH2PO4, 1.25; CaCl2, 2; MgSO4, 1; Dglucose, 10. Transverse hippocampal slices (400 µm thick) were prepared and submerged in

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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 oleanolic acid solution (1, 10 or 30 µM) for 2 h

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and then transferred to the recording chamber. For the treatment of ANA-12, slices were incubated in ANA-12 solution (100 µM) for 30 min and again incubated in ANA-12 with oleanolic acid solution (30 µM) for 2 h.

Field recordings were made from stratum pyramidale in the area hippocampal CA1.

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Stimulating electrodes were placed in the Schaffer collateral-commissural pathway. Stimuli (constant voltage) were delivered at 30 s intervals. Paired-pulse ratio (PPR) was assessed using a succession of paired pulses separated by time intervals ranging from 25 to 500 ms,

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delivered every 30 s. The degree of facilitation was determined by taking the ratio of the initial slope of the second fEPSP to the initial slope of the first fEPSP. Input/output (I/O)

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curves of the field potentials were constructed using the means of four conditioning stimulus pulses at six stimulation intensities (2, 4, 8, 10, 15, and 20 mV). To induce LTP, one train of high frequency stimulation (100 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.

2.9. Cell culture 10

ACCEPTED MANUSCRIPT Highly purified primary neuron cultures were prepared using pregnant Sprague– Dawley female rats that were killed by decapitation under anesthesia (Jeon et al., 2012). The cerebral cortices of fetus were removed and dissociated with a sterile Pasteur pipette and

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digested with trypsin/EDTA 1X (10,000 unit/ml) for 15 min at 37 °C. The cultures were recovered by Neural basal media (NBM) containing B27 supplement. After that, cells were plated in poly-L-lysine (PLL)-coated 24 well plates with 1x106 cells per well. Cultures were

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kept at 37 °C and 10% CO2 incubator and media was changed every 3–4 days. After 10 days,

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the plates were confluent with neurons and applied to the experiment.

2.10. Blockade of TrkB signaling in culture system

For the inhibition of TrkB-BDNF signaling, we introduced TrkB siRNA in cultured neurons. Briefly, either TrkB-targeted siRNA or control siRNA was transfected

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to the cells by 24 h incubation following the manufacturer's instructions. After transfection, the neurons were supplied with fresh media with or without oleanolic acid (30 µM) treatment for 6 h then harvested to analyze the BDNF expression or its

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2.11. Statistics

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downstream signaling molecules by Western blot.

The data are expressed as the means ± standard error of the mean (S.E.M.). The data

from the behavioral tests and Western blots were analyzed via one-way analysis of variance (ANOVA) followed by Tukey's post hoc test for multiple comparisons. The interactions between the agonist and the antagonist in the passive avoidance task were analyzed by twoway ANOVA, and Bonferroni’s post-hoc test was used to perform pair-wise comparisons to determine antagonist or agonist effects. For the electrophysiological studies, the data were 11

ACCEPTED MANUSCRIPT analyzed using one-way ANOVA followed by Tukey's post hoc test for multiple comparisons in each point of PPR and I/O curve, and the last five minutes of LTP experiment. The statistical significance was set at P < 0.05. All statistical analyses were performed using Prism

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5.0 software (GraphPad, La Jolla, CA).

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3. Results

3.1. Oleanolic acid attenuates scopolamine-induced cognitive impairment on the step-

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through passive avoidance task

To evaluate the effects of oleanolic acid on the scopolamine-induced contextual long-term memory impairment, we performed the passive avoidance task. As shown in Fig. 1A, one-way ANOVA revealed no significant difference in the latency of the acquisition trial

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between the treated groups [F (6, 52) = 0.3, P > 0.05, Fig. 1A]. Significant step-through latency effects were observed in the retention trial, which was performed at 24 h after the acquisition trial [F (6, 52) = 11.6, P < 0.05, Fig. 1A]. The scopolamine-induced latency

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reduction was significantly ameliorated by a single administration of oleanolic acid (2.5 or 5 mg/kg) in a dose-dependent manner (P < 0.05, Fig. 1A). Additionally, similar effects were

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observed following the administration of donepezil (5 mg/kg, p.o.) as a positive control. Further behavioral tasks and signaling pathways were evaluated using the effective dose range (2.5 – 5 mg/kg) in mice. These results demonstrate that the administration of oleanolic acid effectively alleviated the cognitive dysfunction caused by cholinergic deficits. In addition, we examined whether the effects of oleanolic acid on the cognition were derived from its structural characteristics, pentacyclic triterpenoid. To confirm the effect of pentacyclc triterpenoids on cognition, we selected the ursolic acid whose chemical structure is similar to 12

ACCEPTED MANUSCRIPT oleanolic acid. However, ursolic acid did not ameliorate scopolamine-induced cognitive impairment (Supplemetary Fig. S1).

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3.2. Oleanolic acid alleviates scopolamine-induced memory dysfunction based on the Ymaze task

The Y-maze task was performed to examine the effect of oleanolic acid on

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spontaneous alternation behavior. A significantly different group effect was observed in the spontaneous alternation behavior upon the administration of oleanolic acid [F (4, 35) = 7.1, P

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< 0.05, Fig. 1B]. The percentage of spontaneous alternations in the scopolamine-treated group was significantly lower than that in the vehicle-treated control group (P < 0.05, Fig. 1B), and this reduction in spontaneous alternation was significantly ameliorated by oleanolic acid (2.5 and 5 mg/kg, p.o.) or donepezil (5 mg/kg, p.o.) (P < 0.05, Fig. 1B). However, the total number

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of arm entries was similar across all experimental groups [F (4, 35) = 1.0, P > 0.05, Fig. 1C], suggesting the improvement in the behavior was not resulted from enhanced general

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locomotor behavior.

3.3. Oleanolic acid ameliorates scopolamine-induced memory impairment based on the

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novel object recognition test

Next, to examine whether oleanolic acid improves recognition memory, we

performed the novel object recognition test on the scopolamine model. A significant group effect was observed in the relative preference to each object [F (3, 33) = 46.3, P < 0.05, Fig. 1D] and the discrimination ratio [F (3, 33) = 46.3, P < 0.05, Fig. 1E]. However, the mean total exploration time of all groups to each object were not significantly different (F (3, 33) = 1.8, P > 0.05, Fig. 1F). For the mice injected with scopolamine, a decreased preference for the 13

ACCEPTED MANUSCRIPT novel object and a reduced discrimination ratio between the novel and familiar objects was observed compared to the vehicle-treated controls (P < 0.05), indicating scopolamine-induced recognition memory impairment. This scopolamine-induced memory impairment was

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significantly ameliorated by the administration of oleanolic acid (2.5 and 5 mg/kg) as shown in Fig. 1 (P < 0.05).

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3.4. Oleanolic acid reverses scopolamine-mediated impairment in BDNF signaling and enhances hippocampal LTP

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We examined whether oleanolic acid administration affects the ERK1/2-CREB phosphorylation and/or BDNF expression in the hippocampus of mice. Consistent with our previous studies (Park et al., 2012), we found that the administration of scopolamine significantly changed the phosphorylation levels of ERK1/2 [F (3, 15) = 9.9, P < 0.05, Fig. 2]

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and CREB [F (3, 15) = 6.2, P < 0.05, Fig. 2] or BDNF expression levels [F (3, 15) = 4.6, P < 0.05, Fig. 2]. The scopolamine-induced decrease in both the pERK1/2 and pCREB levels was reversed by the administration of oleanolic acid (2.5 mg/kg) without affecting the total level

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of ERK1/2 or CREB (P < 0.05, Fig. 2), whereas the phosphorylation levels of protein kinase C (PKC) and phosphoinositide 3-kinase (PI3K) were not changed (data not shown). The

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increased phosphorylation of both signals synergistically increased BDNF expression (P < 0.05, Fig. 2).

Since hippocampal BDNF plays various physiological functions such as neuronal

differentiation, synaptic LTP modulation, and learning and memory, we investigated whether oleanolic acid enhances LTP formation. To investigate this, we first evaluated the effect of oleanolic acid on LTP using the hippocampal slice. We measured PPR, I/O curve, and LTP in the hippocampus. Oleanolic acid (1, 10 and 30 µM) did not affect PPR (Fig. 3A) and I/O 14

ACCEPTED MANUSCRIPT curve (Fig. 3B). However, oleanolic acid (10 and 30 µM) facilitated hippocampal LTP [F (3, 24) = 15.5, P < 0.05, n = 7/group, Fig. 3C and D].

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3.5. TrkB activation is required for oleanolic acid-induced cognitive effects To figure out how oleanolic acid activates intracellular signaling pathway(s), we introduced a TrkB specific inhibitor, ANA-12 (Cazorla et al., 2011). If oleanolic acid really

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induces BDNF signaling through TrkB system, the ANA-12 treatment would abolish the behavioral and synaptic effects. The administration of ANA-12 significantly reversed the

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facilitated hippocampal LTP induced by oleanolic acid treatment. Two-way ANOVA also revealed that there is significant interaction between drugs [oleanolic acid treatment, F (1, 36) = 4.7, P < 0.05; ANA-12 treatment, F (1, 36) = 4.4, P < 0.05; interaction oleanolic acid × ANA-12, F (1, 36) = 5.2, P < 0.05, Fig. 4A and B]. Normalized fEPSP was increased by oleanolic acid

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(30 µM) treatment (P < 0.05), and the increased fEPSP was reversed by ANA-12 (100 µM) treatment (P < 0.05). These results suggest that oleanolic acid facilitates hippocampal LTP through TrkB without affecting basal synaptic transmission.

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Next, we conducted behavioral studies to confirm the in vitro results that memory ameliorating effects of oleanolic acid are mediated through TrkB. After oleanolic acid

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administration (2.5 mg/kg), a sub-effective dose of ANA-12 (0.3 mg/kg, Supplementary Fig. S2) was administered coincidentally with scopolamine, and the latency to enter was measured in the passive avoidance task (Fig. 4C). Scopolamine-induced memory deficit was ameliorated by oleanolic acid treatment [F (5, 52) = 38.3, P < 0.05, Fig. 4C], and this phenomenon was reversed in the presence of ANA-12 (oleanolic acid treatment, F (1, 34) = 39.8, P < 0.001; ANA-12 treatment, F (1, 34) = 160.4, P < 0.001; interaction oleanolic acid × ANA12,

F (1, 34) = 87.0, P < 0.001). Single ANA-12 treatment did not induce any behavioral 15

ACCEPTED MANUSCRIPT alteration in mice as shown in the either ANA-12 alone or scopolamine co-treatment.

3.6. Oleanolic acid activates BDNF-ERK1/2-CREB signaling pathways via TrkB

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activation To further test whether oleanolic acid facilitates hippocampal LTP and ameliorates scopolamine-induced memory impairment through TrkB-BDNF signaling, TrkB siRNA

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system was adopted. After transient transfection, we treated the neurons with oleanolic acid (30 µM). After 6 h of treatment, cells were harvested and Western blot was performed. As

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shown in Fig. 5 and confirmed by one-way ANOVA, there was a significant change in BDNF expression [F (5, 18) = 2.5, P = 0.047], ERK1/2 phosphorylation [F (5, 18) = 4.0, P = 0.042], or CREB phosphorylation [F (5, 18) = 7.4, P = 0.011] between groups. Control cells which were not transfected significantly increased BDNF expression and phosphorylated ERK1/2

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and CREB levels by oleanolic acid treatment (BDNF, P = 0.032; pERK1/2, P = 0.044; pCREB, P = 0.016; Fig. 5). In addition, the levels of BDNF, pERK1/2 or pCREB in case of control siRNA transfected cells, were significantly increased by oleanolic acid treatment

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similar to the results of control cells (BDNF, P = 0.032; pERK1/2, P = 0.044; pCREB, P = 0.016; Fig. 5). However, TrkB siRNA transfected cells did not respond to oleanolic acid

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treatment, unlike the control or control siRNA treated cells (BDNF, P = 0.558; pERK1/2, P = 0.914; pCREB, P = 0.494; Fig. 5). TrkB siRNA transfected cells did not show TrkB expression compared to the control or control siRNA cells (Supplementary Fig. S3). Next, we examined whether TrkB blockade inhibits its downstream signaling in vivo. As shown in Fig. 6 and confirmed by one-way ANOVA, there was a significant change in TrkB phosphorylation [F (3, 20) = 6.2, P= 0.0039], BDNF expression [F (3, 20) = 8.4, P = 0.0008], ERK1/2 phosphorylation [F (3, 20) = 4.9, P = 0.01], or CREB phosphorylation [F (3, 20) = 16

ACCEPTED MANUSCRIPT 11.7, P = 0.0001] between groups. The decreased levels of pTrkB, BDNF, pERK1/2 and pCREB by the administration of scopolamine were reversed by oleanolic acid treatment (pTrkB, P = 0.032; BDNF, P = 0.003; pERK1/2, P = 0.016; pCREB, P = 0.001; Fig. 6).

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Furthermore, ANA-12 treatment completely blocked the effects of oleanolic acid on these molecules (pTrkB, P = 0.028; BDNF, P = 0.002; pERK1/2, P = 0.022; pCREB, P = 0.0001; Fig. 6).

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Previously, it has been reported that oleanolic acid displays binding affinity to the sigma-1 receptor (from Japanese patent, PJ12247993). However, unfortunately, we did not

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obtain any significant results including sigma-1 receptor via receptor binding assay (data not shown). Therefore, we finally wanted to test whether oleanolic acid directly binds to the TrkB receptor. However, a receptor binding assay revealed no specific binding in the range of 1 – 300 µM (Supplementary Table 1). Therefore, the modulation of BDNF signaling through the

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TrkB receptor by oleanolic acid likely involves an indirect mechanism.

3.7. Oleanolic acid increases brain acetylcholine levels

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To figure out whether oleanolic acid affects ACh level in the brain, we performed the ACh ELISA assay (ab65345, Abcam, USA) in whole brain tissue (Fig. 7). There were

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significant changes in the ACh levels between groups [F (3, 44) = 13.8, P < 0.0001]. Oleanolic acid administration increased ACh levels (P < 0.05), and increased levels were reversed by ANA-12 (P < 0.05). However, ANA-12 single treatment did not alter the ACh content. Meanwhile, oleanolic acid did not exert any inhibitory effect on acetylcholinesterase activity (data not shown). These results suggest that the increase of ACh content may be derived from the enhancement of its synthetic or release mechanism.

17

ACCEPTED MANUSCRIPT 4. Discussion In the present study, we investigated the effect of oleanolic acid on a scopolamineinduced memory impairment model and its mode of actions. Oleanolic acid reversed the

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scopolamine-induced cognitive dysfunction, as demonstrated by the passive avoidance, Ymaze spontaneous alternation, and novel object recognition tasks. In addition, oleanolic acid facilitated LTP induction in the slice culture system and this effect was abolished by the

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BDNF receptor blockade. Moreover, we observed that oleanolic acid activated TrkB receptor and its downstream signaling, resulting in the cognitive amelioration in mice.

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Many studies, including ours (Lana et al., 2013, Yakel, 2013, Park et al., 2014), suggest that the activation of cholinergic neurotransmitter system in the hippocampus is essential for learning and memory. Consistent with the cholinergic hypothesis, it has been suggested that the blockade of muscarinic receptors by scopolamine decreases cognitive

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performance, such as working memory, which is relevant to the characteristics of AD (Chambon et al., 2012). In the present study, we found that oleanolic acid reversed the scopolamine-induced deficits in several types of memory, such as contextual long-term

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memory, short-term working memory, or object recognition memory. Thereafter, we first focused on the structural characteristics of oleanolic acid, pentacyclic triterpenoid, which

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displays many biological activities, including anti-inflammation (Kang et al., 2015, Theoduloz et al., 2015). Notably, we found that ursolic acid did not significantly alter cognition (Supplementary Fig. S1) or LTP formation (data not shown), indicating that not all natural pentacyclic triterpenoids exert a similar effect to that of oleanolic acid. Thus, these results suggest that the cognitive effects of oleanolic acid are derived from its unique structure or molecular characteristics and that this compound is a candidate agent for treatment of the memory deficits observed in AD. 18

ACCEPTED MANUSCRIPT In the present study, we also observed that a single administration of oleanolic acid increased the phosphorylation of ERK1/2 in the hippocampus. When a neuron is exposed to synaptic activation, the intracellular calcium level becomes elevated, and several kinases,

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including ERK1/2 become activated (Sutton and Chandler, 2002, Cohen-Matsliah et al., 2007, Zheng et al., 2009). It has been suggested that the activity of ERK1/2, a member of mitogenactivated protein kinases (MAPKs), is required for the establishment of synaptic activity and the development of several forms of memory (Revest et al., 2014). Moreover, CREB, a

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downstream effector of cAMP- and Ca2+-mediated signal transduction pathways, is activated

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by several kinases, such as ERK1/2, Ca2+/calmodulin-dependent kinases, or cAMP-dependent protein kinase A (Williams et al., 2008, Chen et al., 2012, Leal et al., 2014). In addition, it is well known that activated CREB enhances memory by increasing the transcription of memory-related genes, such as c-Fos, activity-regulated cytoskeleton-associated protein (Arc)

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or BDNF (Rapanelli et al., 2010, Leal et al., 2014). As observed, oleanolic acid enhanced ERK1/2 or CREB phosphorylation and BDNF expression levels, which suggests that oleanolic acid may activate MAPK pathways, ERK1/2-CREB-BDNF pathway, and enhance

et al., 2014).

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learning and memory process (Mizuno et al., 2002, Chen et al., 2012, Leal et al., 2014, Revest

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Previously, Yi et al., reported that the anti-depressant-like activity of oleanolic acid may be related to its effect on serotonin, noradrenaline or BDNF (Yi et al., 2013). Fajemiroye et al., suggested that chronic administration of oleanolic acid augmented hippocampal BDNF levels but not acute administration (Fajemiroye et al., 2014), suggesting that BDNF may be a crucial role in the cognition function of oleanolic acid. BDNF is known to be essentially involved in learning and memory, especially, in the LTP formation (Leal et al., 2014) and its receptor, TrkB, activation also affects ERK1/2 MAP kinase pathway (Leal et al., 2014, Revest 19

ACCEPTED MANUSCRIPT et al., 2014). To elucidate memory ameliorating effect of oleanolic acid was from BDNF signaling, we introduced the ANA-12 compound, which specifically blocks the TrkB-BDNF signaling in vivo as well as in vitro. ANA-12 treatment significantly abolished the oleanolic

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acid-induced effects in mice behavior as well as in the hippocampal slice culture, showing that TrkB-BDNF signaling would be directly involved in the effects of oleanolic acid on cognitive function. Meanwhile, the ACh concentration in whole brain tissue was increased

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after the administration of oleanolic acid, suggesting that the increase of ACh content may be derived from the enhancement of its synthetic or release mechanism. Many groups suggest

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that BDNF enhances ACh release in nerve terminal (Knipper et al., 1994, Auld et al., 2001, Garcia et al., 2011). Thus, oleanolic acid may mediate ACh release via TrkB-BDNF signaling, in part, but still needs to further researches.

Next, to support the TrkB-mediated oleanolic acid effects on BDNF signaling

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cascades, we employed siRNA techniques to block TrkB-BDNF induction (Supplementary Fig. S3), then analyzed further signaling cascade. As expected, oleanolic acid could not increase BDNF expression and ERK1/2 and CREB phosphorylayion with TrkB blockade-

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induced by TrkB siRNA introduction compared to the control siRNA or non-treated groups in culture system. In addition, the reversal of scopolamine-induced decreased TrkB activation

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and its downstream signal molecules by oleanolic acid treatment were completely blocked by TrkB blockade in the hippocampus. These results suggest that oleanolic acid-mediated BDNF and ERK1/2-CREB pathway induction was from TrkB receptor activation. Similarly, oleanolic acid-induced hippocampal LTP was blocked by ANA-12 treatment in the electrophysiological studies. Taken together, these results imply that oleanolic acid-induced facilitation of LTP induction and reversal of scopolamine-induced cognitive impairment may be mediated by TrkB activation. 20

ACCEPTED MANUSCRIPT Oleanolic acid is contained in a variety of herbs and vegetables, especially in the Mediterranean food. Until now, several reports have suggested the beneficial effects of oleanolic acid against several disease states, such as rheumatoid arthritis (Lukaczer et al.,

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2005, Choi et al., 2015), or cancer (Parikh et al., 2014, Li et al., 2015). Although various studies have suggested the positive effects of oleanolic acid on brain function (Tsai and Yin, 2012), its effects on the cognition, learning and memory as well as its mechanism of action

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have not yet been studied. Here, we report that oleanolic acid ameliorates cognitive dysfunction caused by cholinergic blockade via the activation of TrkB/BDNF signaling

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confirmed by molecular tools, electrophysiological data, and behavioral studies. In addition, our works strongly imply that oleanolic acid would be a novel candidate for cognitive dysfunction in respect of its mode of action, TrkB-BDNF signaling.

This

work

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Acknowledgements was

supported

by

the

Bio-Synergy

Research

Project

(2014M3A9C4066465) of the Ministry of Science, ICT and Future Planning through the

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National Research Foundation, the Basic Science Research Program through the NRF funded by the Ministry of Education (NRF-2014R1A1A2059179), and the Mid-career Researcher

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Program through NRF grant funded by the Ministry of Education, Science and Technology (MEST) (NRF-2015R1A2A2A01007838).

21

ACCEPTED MANUSCRIPT References

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Leal G, Comprido D, Duarte CB (2014) BDNF-induced local protein synthesis and synaptic plasticity. Neuropharmacology 76 Pt C:639-656. Lee HE, Lee SY, Kim JS, Park SJ, Kim JM, Lee YW, Jung JM, Kim DH, Shin BY, Jang DS, Kang SS, Ryu JH (2013) Ethanolic Extract of the Seed of Zizyphus jujuba var. spinosa Ameliorates Cognitive Impairment Induced by Cholinergic Blockade in Mice. Biomol Ther (Seoul) 21:299-306. Li L, Wei L, Shen A, Chu J, Lin J, Peng J (2015) Oleanolic acid modulates multiple intracellular targets to inhibit colorectal cancer growth. Int J Oncol 47:2247-2254. Lukaczer D, Darland G, Tripp M, Liska D, Lerman RH, Schiltz B, Bland JS (2005) A pilot trial evaluating Meta050, a proprietary combination of reduced iso-alpha acids, rosemary extract and oleanolic acid in patients with arthritis and fibromyalgia. Phytother Res 19:864-869. Mizuno M, Yamada K, Maekawa N, Saito K, Seishima M, Nabeshima T (2002) CREB phosphorylation as a molecular marker of memory processing in the hippocampus for spatial learning. Behav Brain Res 133:135-141. Parikh NR, Mandal A, Bhatia D, Siveen KS, Sethi G, Bishayee A (2014) Oleanane triterpenoids in the prevention and therapy of breast cancer: current evidence and future perspectives. Phytochem Rev 13:793-810. Park SJ, Ahn YJ, Oh SR, Lee Y, Kwon G, Woo H, Lee HE, Jang DS, Jung JW, Ryu JH (2014) Amyrin attenuates scopolamine-induced cognitive impairment in mice. Biol Pharm Bull 37:1207-1213. Park SJ, Kim DH, Jung JM, Kim JM, Cai M, Liu X, Hong JG, Lee CH, Lee KR, Ryu JH (2012) The ameliorating effects of stigmasterol on scopolamine-induced memory impairments in mice. Eur J Pharmacol 676:64-70. Rapanelli M, Lew SE, Frick LR, Zanutto BS (2010) Plasticity in the rat prefrontal cortex: linking gene expression and an operant learning with a computational theory. PLoS One 5:e8656. Revest JM, Le Roux A, Roullot-Lacarriere V, Kaouane N, Vallee M, Kasanetz F, Rouge-Pont F, Tronche F, Desmedt A, Piazza PV (2014) BDNF-TrkB signaling through Erk1/2 MAPK phosphorylation mediates the enhancement of fear memory induced by glucocorticoids. Mol Psychiatry 19:1001-1009. Rodrigues Simoes MC, Dias Viegas FP, Moreira MS, de Freitas Silva M, Riquiel MM, da Rosa PM, Castelli MR, dos Santos MH, Soares MG, Viegas C, Jr. (2014) Donepezil: an important prototype to the design of new drug candidates for Alzheimer's disease. Mini Rev Med Chem 14:2-19. Rong ZT, Gong XJ, Sun HB, Li YM, Ji H (2011) Protective effects of oleanolic acid on cerebral ischemic damage in vivo and H(2)O(2)-induced injury in vitro. Pharm Biol 49:78-85. Salkovic-Petrisic M, Tribl F, Schmidt M, Hoyer S, Riederer P (2006) Alzheimer-like changes in protein kinase B and glycogen synthase kinase-3 in rat frontal cortex and hippocampus after damage to the insulin signalling pathway. J Neurochem 96:1005-1015. Schmitt FA, van Dyck CH, Wichems CH, Olin JT (2006) Cognitive response to memantine in moderate to severe Alzheimer disease patients already receiving donepezil: an exploratory reanalysis. Alzheimer Dis Assoc Disord 20:255-262. Spencer JP, Vauzour D, Rendeiro C (2009) Flavonoids and cognition: the molecular mechanisms underlying their behavioural effects. Arch Biochem Biophys 492:1-9. Sutton G, Chandler LJ (2002) Activity-dependent NMDA receptor-mediated activation of protein kinase B/Akt in cortical neuronal cultures. J Neurochem 82:1097-1105. Tsai SJ, Yin MC (2012) Anti-oxidative, anti-glycative and anti-apoptotic effects of oleanolic acid in brain of mice treated by D-galactose. Eur J Pharmacol 689:81-88. Wilcock GK, Esiri MM, Bowen DM, Smith CC (1982) Alzheimer's disease. Correlation of cortical choline acetyltransferase activity with the severity of dementia and histological abnormalities. J Neurol Sci 57:407-417. Williams CM, El Mohsen MA, Vauzour D, Rendeiro C, Butler LT, Ellis JA, Whiteman M, Spencer JP (2008) Blueberry-induced changes in spatial working memory correlate with changes in hippocampal CREB phosphorylation and brain-derived neurotrophic factor (BDNF) levels. Free Radic Biol Med 45:295305. 23

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Yakel JL (2013) Cholinergic receptors: functional role of nicotinic ACh receptors in brain circuits and disease. Pflugers Arch 465:441-450. Yi LT, Li J, Liu BB, Luo L, Liu Q, Geng D (2014) BDNF-ERK-CREB signalling mediates the role of miR-132 in the regulation of the effects of oleanolic acid in male mice. J Psychiatry Neurosci 39:130169. Yi LT, Li J, Liu Q, Geng D, Zhou YF, Ke XQ, Chen H, Weng LJ (2013) Antidepressant-like effect of oleanolic acid in mice exposed to the repeated forced swimming test. J Psychopharmacol 27:459-468. Zheng F, Luo Y, Wang H (2009) Regulation of brain-derived neurotrophic factor-mediated transcription of the immediate early gene Arc by intracellular calcium and calmodulin. J Neurosci Res 87:380-392.

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ACCEPTED MANUSCRIPT Figure legends

Figure 1. Effects of oleanolic acid on a scopolamine-induced cognitive impairment

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model. (A) Mice were orally administered either oleanolic acid (0.625, 1.25, 2.5, or 5 mg/kg) or donepezil (5 mg/kg, p.o.) 1 h before the acquisition trial. Memory impairment was induced using scopolamine (1 mg/kg, i.p.) 30 min before the acquisition trial. Twenty-four hours after

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the acquisition trial, the retention trial was performed for 300 s. Then, the latency was measured. (B-E) Mice were administered oleanolic acid (2.5 or 5 mg/kg, p.o.), donepezil (5

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mg/kg, p.o.), or the same volume of vehicle (10% Tween 80 solution) 1 h before the Y-maze test or novel object recognition task. Memory impairment was induced using scopolamine (1 mg/kg, i.p.) 30 min before the Y-maze test. Spontaneous alternation behavior (B) and the number of arm entries (C) during an 8 min session were recorded. The percentage of

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exploration time spent on the novel or familiar object (D), the discrimination index (E), and the total exploration time (F) in the novel object recognition test are presented. The data represent the means ± S.E.M. (*P < 0.050 compared to the vehicle-treated control group; # P <

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donepezil.

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0.050 compared to the scopolamine-treated group; n = 8-10 per group). Con, control. DNZ,

Figure 2. Effect of oleanolic acid on the phosphorylation levels of ERK1/2 and CREB and BDNF expression in the hippocampus. Oleanolic acid (2.5 or 5 mg/kg) was administered 30 min before scopolamine (1 mg/kg, i.p.). Control group was treated with the same volume of vehicle solution. Bar graphs demonstrate the quantitative mean value of four different bands. The graphs represent the densitometry analyses of the ratios of pERK/ERK, pCREB/CREB, BDNF/β-actin normalized to the control (taken as 1.0). Data represent means 25

ACCEPTED MANUSCRIPT ± S.E.M. (* P < 0.050 compared to the vehicle-treated control group; # P < 0.050 compared to the scopolamine-treated group; n = 4 per group). OA, oleanolic acid.

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Figure 3. The effect of oleanolic acid on synaptic transmission in the hippocampus. Acute hippocampal slices (6-week-old male mice) were incubated with oleanolic acid (1, 10 and 30 µM) for 2 h. Extracellular field EPSPs (fEPSP) were evoked by stimulating Schaffer

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collateral CA1 synapse. (A) Paired-pulse ratio. (B) Input/output curve. (C) LTP was evoked by one train of HFS (each 100 Hz, 1 sec, black arrow). (D) Residual potentiation of fEPSPs

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(LTP ratio) was visualized during the last five minutes of the one-hour recording. Data were represented as mean ± SEM. n = 7 per group. *P < 0.05. OA denotes oleanolic acid. The number in parentheses means the concentration of oleanolic acid (µM).

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Figure 4. Hippocampal BDNF induction was essential for the function of oleanolic acid in mice. (A) Hippocampal slices were incubated in ANA-12 solution (100 µM) for 30 min and then the slices were more incubated in ANA-12 + oleanolic acid solution (30 µM) for 2 h.

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LTP was evoked by one train of HFS (each 100 Hz, 1 sec, black arrow). (B) Residual potentiation of fEPSPs (LTP ratio) during the last five minutes of the one-hour recording.

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Data were represented as mean ± SEM. n = 10/group. *P < 0.05, compared to the vehicletreated control group;

#

P < 0.05, compared to the oleanolic acid-treated group. (C) After

oleanoilc acid administration, ANA-12 (0.3 mg/kg) was injected with or without scopolamine treatment. Twenty-four hours after the acquisition trial, the retention trial was performed for 300 s. Then, the latency was measured. The bar represents the mean ± S.E.M. * Significantly different compared to the vehicle-treated control group; # significantly different compared to the scopolamine only-treated groups; $ significantly different compared to the groups with the 26

ACCEPTED MANUSCRIPT co-treatment with oleanolic acid and scopolamine (P < 0.05, n = 8-10 for each group). OA denotes oleanolic acid.

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Figure 5. Oleanolic acid-mediated BDNF and its downstream signaling induction was through TrkB receptor in neurons. Primary neurons were transfected with TrkB siRNA or control siRNA for 24 h and recovered with fresh media another 24 h. Then, 30 µM of oleanoilc acid was treated. After 6 h of treatment, cells were lyzed with sample buffer and

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Western blot was performed. The graphs represent the densitometry analyses of the ratios of

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pERK/ERK, pCREB/CREB, BDNF/β-actin normalized to the control (taken as 1.0). Data represent means ± S.E.M. * Significantly different compared to the control group. (P < 0.05, n = 4). Control, non-transfected cells. SiControl, control siRNA-transfected cells. SiTrkB, TrkB siRNA-transfected cells. OA, oleanolic acid. n.s., no significance.

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Figure 6. Oleanolic acid-induced TrkB activation and downstream BDNF-ERK-CREB induction in hippocampus. One hour after oleanoilc acid administration (2.5 mg/kg), ANA-

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12 (0.3 mg/kg) was concomitantly injected with or without scopolamine treatment. Thirty min later, mice were sacrificed and prepared for Western blot sampling. The graphs represent the

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densitometry analyses of the ratios of pTrkB/TrkB, pERK/ERK, pCREB/CREB, BDNF/βactin normalized to the control (taken as 1.0). Data represent means ± S.E.M. * Significantly different compared to the control group. # significantly different compared to the scopolamine only-treated groups;

$

significantly different compared to the groups with the co-treatment

with oleanolic acid and scopolamine (P < 0.05, n = 6). ANA-12, ANA-12-treated groups. OA, oleanolic acid.

Figure 7. The effects of oleanolic acid on the concentration of acetylcholine (ACh) in the 27

ACCEPTED MANUSCRIPT brain. After 1 h of oleanolic acid (2.5 mg/kg, p.o.) administration, ANA-12 (0.3 mg/kg, i.p.) was injected and thirty min later, mice were sacrificed and brain tissues were removed. After the removal of cerebellum, rest of brain tissue was rapidly homogenized. Each 30 µg protein

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sample was loaded onto a 96-well reaction plate and incubated with reaction mixture. The fluorescence was measured at 570 nm in a microplate reader. Then data was calculated and expressed in nmol/mg of protein.

*

Significantly different compared to the vehicle-treated

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control group; # significantly different compared to the oleanolic acid-treated groups. ANA-12,

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ANA-12-treated groups. OA, oleanolic acid-treated groups.

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Fig.1 (B)

#

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# $

*

*

0

160

120

50

100

180

SC

LTP ration (% of baseline)

250

HFS

Latency (s)

220

Control OA (30) OA + ANA-12 ANA-12 (100 µM)

M AN U

Normalized fEPSP Slope (% of baseline)

300

RI PT

(B)

0

0

0

2.5

2.5

+

+ +

-

+

+

+ +

-

-

OA (mg/kg) Scopolamine ANA-12

*

#

ACCEPTED MANUSCRIPT

Fig.5 BDNF

RI PT

β-actin p-ERK

SC

ERK p-CREB

0

30

0

Control

Si Control

4

*

0

30

3

n.s.

2 1 0

30

AC C

0

Control

0

30

Si Control

OA (µM)

Si TrkB

TE D

*

EP

Relative intensity of protein

30

M AN U

CREB

0

30

Si TrkB

ERK1/2 CREB BDNF

OA (µ µM)

ACCEPTED MANUSCRIPT

Fig.6 P-TrkB

RI PT

TrkB

BDNF

SC

β-actin

M AN U

p-ERK

ERK p-CREB

Control

TE D

CREB

0

2.5

2.5

OA (mg/kg)

0

0

0.3

ANA-12 (mg/kg)

EP

Scopolamine (1 mg/kg)

$

AC C

Relative intensity of protein

1.5

TrkB BDNF ERK1/2 CREB

#

1.0

*

*

0.5

0.0 Control

0

2.5

2.5

+

+ -

+ +

-

OA (mg/kg) Scopolamine (1 mg/kg) ANA-12 (0.3 mg/kg)

ACCEPTED MANUSCRIPT

Fig.7

RI PT

*

1.0

#

0.5

2.5

2.5

0

SC

Acetylcholine (nmol/mg protein)

1.5

-

+

+

ANA-12 (0.3 mg/kg)

AC C

EP

TE D

Control

OA (mg/kg)

M AN U

0.0

ACCEPTED MANUSCRIPT Highlights


Oleanolic acid activated the ERK1/2-CREB-BDNF pathway in the hippocampus via

RI PT

TrkB activation
Scopolamine-induced memory dysfunction was ameliorated by oleanolic acid treatment

SC


Oleanolic acid evoked the long-term potentiation in a dose-dependent manner

AC C

EP

TE D

M AN U


Oleanolic acid would be a potential therapeutic agent for the cognitive problem