Nobiletin, a citrus flavonoid, improves cognitive impairment and reduces soluble Aβ levels in a triple transgenic mouse model of Alzheimer's disease (3XTg-AD)

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Nobiletin, a citrus flavonoid, improves cognitive impairment and reduces soluble A␤ levels in a triple transgenic mouse model of Alzheimer’s disease (3XTg-AD)

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Akira Nakajima a,b , Yuki Aoyama a , Eun-Joo Shin c , Yunsung Nam c , Hyoung-Chun Kim c , Taku Nagai a , Akihito Yokosuka d , Yoshihiro Mimaki d , Tsuyoshi Yokoi b , Yasushi Ohizumi e,f , Kiyofumi Yamada a,∗ a Department of Neuropsychopharmacology and Hospital Pharmacy, Graduate School of Medicine, Nagoya University, 65 Tsuruma-cho, Showa-ku, Nagoya 466-8560, Japan b Department of Drug Safety Sciences, Graduate School of Medicine, Nagoya University, Tsuruma-cho, Showa-ku, Nagoya 466-8560, Japan c Neuropsychopharmacology and Toxicology Program, College of Pharmacy, Kangwon National University, Chunchon 200-701, Japan d Laboratory of Medicinal Plant Science, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo 192-0392, Japan e Department of Medical Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan f Kansei Fukushi Research Institute, Tohoku Fukushi University, Sendai 989-3201, Japan

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h i g h l i g h t s • • • •

3XTg-AD is a triple transgenic mouse model of Alzheimer’s disease. Nobiletin is a natural compound showing memory-improving effects. Nobiletin improved cognitive impairment in 3XTg-AD mice. Nobiletin reduced soluble A␤ levels in the brain of 3XTg-AD mice.

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Article history: Received 23 March 2015 Received in revised form 13 April 2015 Accepted 16 April 2015 Available online xxx

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Keywords: Memory Alzheimer’s disease 3XTg-AD Oxidative stress Nobiletin

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1. Introduction

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Alzheimer’s disease (AD), the most common form of dementia among the elderly, is characterized by the progressive decline of cognitive function. Increasing evidence indicates that the production and accumulation of amyloid ␤ (A␤), particularly soluble A␤ oligomers, is central to the pathogenesis of AD. Our recent studies have demonstrated that nobiletin, a polymethoxylated flavone from citrus peels, ameliorates learning and memory impairment in olfactory-bulbectomized mice, amyloid precursor protein transgenic mice, NMDA receptor antagonist-treated mice, and senescence-accelerated mouse prone 8. Here, we present evidence that this natural compound improves cognitive impairment and reduces soluble A␤ levels in a triple transgenic mouse model of AD (3XTg-AD) that progressively develops amyloid plaques, neurofibrillary tangles, and cognitive impairments. Treatment with nobiletin (30 mg/kg) for 3 months reversed the impairment of short-term memory and recognition memory in 3XTg-AD mice. Our ELISA analysis also showed that nobiletin reduced the levels of soluble A␤1–40 in the brain of 3XTg-AD mice. Furthermore, nobiletin reduced ROS levels in the hippocampus of 3XTg-AD as well as wild-type mice. These results suggest that this natural compound has potential to become a novel drug for the treatment and prevention of AD. © 2015 Published by Elsevier B.V.

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Alzheimer’s disease (AD), the most common form of dementia among the elderly, is characterized by the progressive decline of ∗ Corresponding author. Tel.: +81 52 744 2674; fax: +81 52 744 2876. E-mail address: [email protected] (K. Yamada).

cognitive function. The neuropathological hallmarks of AD include brain atrophy, amyloid plaques, and neurofibrillary tangles [1,2]. Increasing evidence indicates that the production and accumulation of amyloid ␤ (A␤), particularly soluble A␤ oligomers, cleaved from amyloid precursor protein (APP) is central to the pathogenesis of AD [3–6]. Furthermore, it has been suggested that oxidative stress is induced by soluble A␤ oligomers and contributes to the

http://dx.doi.org/10.1016/j.bbr.2015.04.028 0166-4328/© 2015 Published by Elsevier B.V.

Please cite this article in press as: Nakajima A, et al. Nobiletin, a citrus flavonoid, improves cognitive impairment and reduces soluble A␤ levels in a triple transgenic mouse model of Alzheimer’s disease (3XTg-AD). Behav Brain Res (2015), http://dx.doi.org/10.1016/j.bbr.2015.04.028

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Fig. 1. Chemical structure of nobiletin.

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development and progression of AD pathology before the appearance of amyloid plaques [7,8]. Large numbers of compounds from natural resources have provided novel leading compounds for drug development [9] as well as useful pharmacological tools [10,11]. In the course of our survey of materials from natural resources having anti-dementia drug activity, we identified nobiletin, a polymethoxylated flavone from peels of Citrus depressa (Fig. 1), as a natural compound enhancing protein kinase A (PKA)/extracellular signal-regulated kinase (ERK)/cAMP response element-binding protein (CREB) signaling in PC12D cells and cultured hippocampal neurons [12,13]. We have demonstrated that this natural compound improves impaired memory in olfactory-bulbectomized (OBX) mice, accompanied by restoration of the OBX-induced cholinergic neurodegeneration [14]. In addition, nobiletin reverses learning impairment associated with the blockade of N-methyl-D-aspartate (NMDA) receptor by activating ERK signaling in the hippocampus of mice [15]. We have also demonstrated that nobiletin improves memory deficits in amyloid precursor protein transgenic mice (APP-SL 7-5 Tg mice) and decreases the amyloid burden and plaques in the hippocampus [16]. Furthermore, nobiletin improved brain ischemia-induced learning and memory deficits in mice [17]. Notably, we have recently reported the beneficial effects of nobiletin on age-related cognitive impairment as well as pathological features of AD such as oxidative stress and hyperphosphorylation of tau in senescenceaccelerated mouse prone 8 (SAMP8) [18,19]. These findings from our previous studies raise the possibility that this natural compound may have the potential to become a novel drug for the treatment and prevention of AD. In the present study, we investigated the effects of nobiletin on cognitive impairment and neuropathology in a triple transgenic mouse model of AD (3XTg-AD). The derivation of 3XTg-AD mice has been described previously [20]. Briefly, human APP with the Swedish mutation (KM670/671NL) and human tau with the P301L mutation were co-microinjected into single-cell embryos of homozygous PS1 M146V knock-in mice. These mice progressively develop amyloid plaques, neurofibrillary tangles, and cognitive impairments [20–22]. Moreover, these mice show an early and age-dependent increase in soluble A␤ in the brain [23]. Increased oxidative stress was also observed before the appearance of amyloid plaques and neurofibrillary tangles in these mice [24]. We demonstrate here that nobiletin improves the impairment of short-term memory and recognition memory in 3XTg-AD mice, accompanied by reduction of the soluble A␤1–40 levels in the brain.

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2. Material and methods

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Nobiletin was extracted and isolated as described previously [12], and suspended in saline containing 0.5% Tween 80.

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2.2. Animals Homozygous 3XTg-AD and nontransgenic wild-type mice on the same mixed 129/C57BL6 background as the 3XTg-AD mice were obtained from the Jackson Laboratory (New Harbor, ME, USA). Animals were housed in cages with free access to food and water under conditions of constant temperature (23 ± 1 ◦ C) and humidity (55 ± 5%) and adapted to a standard 12-h light/12-h dark cycle (light cycle: 9:00–21:00). The procedures used in this study were approved by the Institutional Animal Care and Use Committee of Nagoya University in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.

2.3. Experimental schedule Male 3XTg-AD mice aged 6–7 months and age-matched male wild-type mice were used in this study. The experimental schedule is shown in Fig. 2. One group of animals was subjected to various behavioral tests in the following order: open-field test, Y-maze test, elevated-plus maze test, novel-object recognition test, locomotor activity test, water maze test, and passive avoidance test, while a second group of animals was used in the Y-maze test and water maze test. Behavioral testing started at the age of 9–10 months following daily pretreatment with nobiletin (10 or 30 mg/kg, i.p.) or vehicle for 3 months. Nobiletin or vehicle treatment was continued until all behavioral experiments were completed. In the control group, vehicle was administered daily to age-matched wild-type mice. The dose of nobiletin and the administration route were chosen on the basis of our previous study showing the memory-improving effects of nobiletin [14–18,25]. A previous experiment showed that nobiletin crosses the blood–brain barrier [26]. Throughout the course of treatment with nobiletin, the animal’s weight was measured once a week.

2.4. Open-field test The open-field test was carried out as described previously [27]. Mice were placed in the center of the arena and were allowed to explore the open field (diameter: 60 cm, height: 35 cm) for the following 5 min under moderately light conditions (80 lux), while their activity was measured automatically using the Ethovision automated tracking program (Brainscience Idea Co., Ltd., Osaka, Japan). The movement of mice was measured via a camera mounted above the open field.

Please cite this article in press as: Nakajima A, et al. Nobiletin, a citrus flavonoid, improves cognitive impairment and reduces soluble A␤ levels in a triple transgenic mouse model of Alzheimer’s disease (3XTg-AD). Behav Brain Res (2015), http://dx.doi.org/10.1016/j.bbr.2015.04.028

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Locomotor activity test was carried out as described previously [27]. Mice were placed in a standard transparent rectangular rodent cage (25 cm × 30 cm × 18 cm) under moderately light conditions (15 lux). Locomotor activity was then measured for 120 min using an infrared sensor (NS-AS01; Neuroscience, Tokyo, Japan) placed over the cage.

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Y-maze test was carried out as described previously [28]. Each arm is 40 cm long, 12 cm high, 3 cm wide at the bottom, and 10 cm wide at the top. The arms converge in an equilateral triangular central area that is 4 cm at its longest axis. Each mouse is placed individually at the center of the apparatus and allowed to move freely through the maze during an 8-min session. The series of arm entries is recorded visually. Alternation is defined as successive entries into the three arms, on overlapping triplet sets. The percent alternation is calculated as the ratio of actual to possible alternations (defined as the total number of arm entries minus 2) multiplied by 100. Spontaneous alternation (%) defined as successive entries into the three arms on overlapping triplet sets is associated with the capacity of short-term memory. Mice that entered arms fewer than 8 times during the test were eliminated because the data obtained from those mice were not considered to reflect precise alternation. 2.7. Novel object recognition test The novel object recognition test was performed as described previously [29]. Mice were individually habituated to an open box (30 cm × 30 cm × 35 [height] cm) for three days. During the training session on the fourth day, two novel objects were placed in the open box, and the animals were allowed to explore for 10 min under moderate light (10 lux). The exploration time for each object was recorded. During the retention session on the fifth day, the animals were placed into the same box (24 h after the training session) and one of the familiar objects, which had been used during training, was replaced with a novel object. The mice were then allowed to explore freely for 15 min. The preference index during the retention session and the ratio of the amount of time spent exploring the novel object to the total time spent exploring both objects was used to measure cognitive function. During the training session, the preference index was calculated as the ratio of time spent exploring the object, which was replaced by a novel object in the retention session, to the total exploration time. 2.8. Elevated-plus maze test

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The elevated-plus maze consisted of two open (25 cm × 8 cm × 0.5 cm) and two closed (25 cm × 8 cm × 20 cm) arms emanating from a common central platform (8 cm × 8 cm) to form a plus shape [30]. The entire apparatus was elevated to 50 cm above floor level under moderate light (10 lux). The test began by placing a mouse on the central platform of the maze facing an open arm. An arm entry was defined as all four paws in the arm. The duration of time spent in an arm and number of arm entries were measured for 5 min.

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The passive avoidance test was performed as described previously [31]. A step-through-type passive avoidance apparatus was used, consisting of two compartments, one light and one dark

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(16 cm × 11 cm × 11 cm), with a grid floor. A guillotine door separated the two compartments of the apparatus. In the training trial, mice were placed individually in the light compartment. The door to the dark compartment was opened 10 s later, and the latency before entering the dark compartment was recorded. When the animal had stepped through the door, the door was closed and a shock (0.03 mA for 1 s) was delivered via the grid floor. Mice that did not enter the dark compartment within 300 s were excluded from experiments. Each mouse was placed again in the light compartment 24 h later, and the step-through latency was recorded until 300 s had elapsed (retention trial). 2.10. Morris water maze test The Morris water maze test was performed as described previously [32] with minor modifications. The apparatus consisted of a circular pool (120 cm in diameter) with white plastic prepared, and the water temperature was maintained at 21–23 ◦ C. In the hiddenplatform training, the platform (7 cm in diameter) was submerged 1 cm below the water surface. Two starting positions were used pseudorandomly, and each mouse was subjected to two trials per day with a 30 s intertrial interval in the hidden-platform training (days 1–8). After reaching the platform, the mouse was allowed to remain on it for 30 s. If the mouse did not find the platform within 60 s, the trial was terminated and the animal was put on the platform for 30 s. The time and distance taken to locate the platform were analyzed in each trial by using the Ethovision system (BrainScience Idea Co. Ltd.). Three and a half hours after the second daily training trial on the 8th day, the mouse was given a probe test (third trial), in which the platform was removed from the pool and the mice were allowed 30 s to search the pool to assess the spatial memory. On the 15th day, to measure swimming ability or motivation, mice were subjected to the visible test in which the platform was marked with a flag that protruded 12 cm above the water surface in order to be highly visible, but in a new location. Two months later, mice were subjected to the hidden-platform training and probe test again as described above. One mouse was excluded from the data analysis because it failed to reach the platform in the visible test. 2.11. Preparation of tissue samples After completion of the behavioral tests, mice were deeply anesthetized with sodium pentobarbital and perfused transcardially with cold saline. The brains were rapidly removed and bisected through the mid-sagittal plane into each hemisphere. After removal of the brainstem and cerebellum, the lateral hemispheres were frozen for subsequent use for sandwich enzyme-linked immunosorbent assays (ELISA). The cerebral cortex, hippocampus, and striatum were isolated from the other hemispheres, and then stored at −80 ◦ C until use. Tissue samples were separately homogenized in 50 mM phosphate-buffered saline (PBS, pH 7.4), and centrifuged at 13,000 × g for 20 min. Supernatant was used for measurements of reactive oxygen species (ROS) level, malondialdehyde (MDA) level, protein carbonyl level, superoxide dismutase (SOD) activity, glutathione peroxidase (GPx) activity, and glutathione reductase (GR) activity. 2.12. Determination of SOD activity SOD activity was determined on the basis of inhibition of superoxide-dependent reactions as described previously [33,34]. The reaction mixture contained 70 mM potassium phosphate buffer (pH 7.8), 30 ␮M cytochrome c, 150 ␮M xanthine, and tissue extract in phosphate buffer diluted 10 times with PBS in a final volume of 3 mL. The reaction was initiated by adding 10 ␮L of 50 units of xanthine oxidase, and the change in absorbance at 550 nm was

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recorded. One unit of SOD is defined as the quantity required to inhibit the rate of cytochrome c reduction by 50%. For estimating total SOD, 10 ␮M potassium cyanide (KCN) was added to the medium to inhibit cytochrome oxidase activity [35].

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GPx activity was analyzed by a spectrophotometric assay described by Lawrence and Burk [36], using 2.0 mM reduced glutathione and 0.25 mM cumene hydroperoxide as substrates [33,34]. The reaction rate at 340 nm was determined using the NADPH extinction coefficient (6.22 mM−1 cm−1 ) at 25 ◦ C. 1 U of GPx activity was defined as the amount required to oxidize 1 ␮mol NADPH/min. 2.14. Determination of GR activity

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Brain hemispheres from wild-type and 3XTg-AD mice were sonicated in RIPA buffer containing protease inhibitors and centrifuged at 100,000 × g for 30 min at 4 ◦ C. The supernatant was removed and stored as the soluble fraction. The pellets were sonicated in 70% formic acid and centrifuged at 100,000 × g for 30 min at 4 ◦ C. The supernatant was removed and stored as the insoluble fraction. A␤1–40 and A␤1–42 levels were measured using Human A␤ ELISA kits (kit II and kit high-sensitive, respectively) from WAKO (Osaka, Japan). Proteins from the soluble fraction were loaded directly onto ELISA plates, and insoluble fractions were diluted 1:20 in neutralization buffer (1 M Tris base) prior to loading. The protein concentration was determined using a DC Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA).

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GR activity was measured as described previously [34,37]. The reaction mixture contained 1 mM oxidized glutathione and 100 ␮L of sample in phosphate buffer (pH, 7.8) containing 1 mM EDTA. The reaction was initiated by adding NADPH (final concentration, 0.11 mM) and the decrease in absorbance of NADPH at 340 nm was measured at 25 ◦ C. The reaction rate was determined using the NADPH extinction coefficient (6.22 mM−1 cm−1 ). 1 U of GR activity was defined as the amount required to oxidize 1 ␮mol NADPH/min.

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The ROS formation in the hippocampus was assessed by measuring the conversion from 2 ,7 -dichlorofluorescin diacetate (DCFH-DA) to dichlorofluorescein (DCF) as described by Bourré et al. [38]. Brain homogenates were added to a tube containing 2 mL of PBS with 10 nmol DCFH-DA, dissolved in methanol. The mixture was incubated at 37 ◦ C for 3 h and then fluorescence was measured at 480 nm excitation and 525 nm emission. DCF was used as a standard.

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The amount of lipid peroxidation was determined by measuring the accumulation of thiobarbituric acid-reactive substances in tissue homogenates and is expressed in terms of MDA content [39–41]. In brief, 0.1 mL of the homogenate (or standard solutions prepared daily from 1,1,3,3-tetra-methoxypropane) was diluted 10 times with phosphate-buffered saline (PBS) and then mixed with 0.75 mL of working solution (0.37% thiobarbituric acid and 6.4% perchloric acid, 2:1, v/v) and heated to 95 ◦ C for 1 h. After cooling (10 min in an ice water bath), the flocculent precipitate was removed by centrifugation at 3,200 × g for 10 min. The supernatant was neutralized and filtered prior to injection into an octadecylsilane 5 ␮m column. The mobile phase consisted of 50 mM PBS (pH 6.0):methanol (58:42, v/v). Isocratic separation was performed with a flow rate of 1.0 mL/min and detection at 532 nm using a UV–vis high-performance liquid chromatography detector (model LC-20AT and SPD-20A, Shimadzu, Kyoto, Japan). 2.17. Determination of protein carbonyl The extent of protein oxidation was assessed by measuring the content of protein carbonyl groups, which was determined spectrophotometrically by the 2,4-dinitrophenylhydrazine (DNPH)-labeling procedure [39–41] as described by Oliver et al. [42]. The results are expressed as nmol DNPH incorporated per mg of protein based on the extinction coefficient for aliphatic hydrazones of 21 mM−1 cm−1 . Protein was measured using the BCA protein assay reagent (Pierce, Rockford, IL, USA).

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The results are expressed as the mean ± SE. The data for escape latency and distance moved during training of the Morris water maze test were analyzed by repeated measures analysis of variance (ANOVA). Other data were analyzed by one-way ANOVA, followed by the Tukey–Kramer test. A level of p < 0.05 was considered statistically significant.

We first examined the influence of nobiletin on body weight in 3XTg-AD mice. Body weights of 3XTg-AD mice chronically treated with nobiletin (10 or 30 mg/kg) were similar to those of vehicletreated 3XTg-AD mice throughout the study period (data not shown). 3.2. Effects of nobiletin on performance in the open-field test To investigate the effects of nobiletin on exploratory behavior in 3XTg-AD mice, the open-field test was carried out. The total distance traveled was decreased in 3XTg-AD mice compared with that in wild-type mice (F(3,48) = 17.471, p < 0.001) (Fig. 3). There was no significant difference among vehicle-treated and nobiletin (10 or 30 mg/kg)-treated 3XTg-AD mice in terms of the total distance traveled, suggesting that treatment with nobiletin did not affect motor activity in 3XTg-AD mice (p > 0.05) (Fig. 3).

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Please cite this article in press as: Nakajima A, et al. Nobiletin, a citrus flavonoid, improves cognitive impairment and reduces soluble Aˇ levels in a triple transgenic mouse model of Alzheimer’s disease (3XTg-AD). Behav Brain Res (2015), http://dx.doi.org/10.1016/j.bbr.2015.04.028

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Fig. 4. Effects of nobiletin on locomotor activity in 3XTg-AD mice. Individual mice were placed in a standard transparent rectangular rodent cage and the locomotor activity was recorded using an infrared sensor for a 120 min observation period. Values are shown as the mean ± SE (n = 11–15). **p < 0.01, ***p < 0.001 vs. wild-type mice.

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3.3. Effects of nobiletin on performance in the locomotor activity test We next assessed the effects of nobiletin on locomotor activity in a novel environment. The locomotor activity of 3XTg-AD mice was recorded using an infrared sensor for a 120 min observation period (Fig. 4). During the first 60 min of the observation period, 3XTg-AD mice showed a decrease in locomotor activity compared with wild-type mice (F(3,47) = 7.9299, p < 0.001) (Fig. 4). There was no difference in locomotor activity among vehicle-treated and nobiletin-treated 3XTg-AD mice (Fig. 4). Furthermore, there was no significant difference in locomotor activity among the 4 groups during the next 60 min period (F(3,47) = 2.0056, p = 0.1260) (Fig. 4). These results suggest that treatment with nobiletin did not affect locomotor activity in a novel environment in 3XTg-AD mice. 3.4. Effects of nobiletin on performance in the Y-maze test We next examined the effects of nobiletin on short-term memory in 3XTg-AD mice in the Y-maze test. 3XTg-AD mice have shown impaired short-term memory [43–45]. Consistent with previous studies, 3XTg-AD mice showed a decrease in spontaneous alternation behavior compared with wild-type mice (F(3,45) = 7.9331, p < 0.001) (Fig. 5A). Nobiletin treatment at 30 mg/kg, but not 10 mg/kg, significantly reversed the decrease in spontaneous alternation behavior in 3XTg-AD mice (Fig. 5A). The number of arm entries was significantly decreased in 3XTg-AD mice compared with that in wild-type mice (F(3,45) = 22.468, p < 0.001) (Fig. 5B), but there was no difference in the number of arm entries among the vehicle-treated and nobiletin-treated 3XTg-AD mice (p > 0.05) (Fig. 5B). These results suggest that nobiletin improves short-term memory impairment in 3XTg-AD mice without affecting motor activity.

Fig. 5. Effects of nobiletin on short-term memory impairment in 3XTg-AD mice. (A) Alternation behavior (%). (B) Number of arm entries. Short-term memory was evaluated by recording spontaneous alternation behavior during an 8-min session in the Y-maze test. Values are shown as the mean ± SE (n = 5–20). ***p < 0.001 vs. wild-type mice. #p < 0.05 vs. vehicle-treated 3XTg-AD mice.

3.5. Effects of nobiletin on performance in the novel object recognition test To examine the effects of nobiletin on object recognition memory in 3XTg-AD mice, we used the novel object recognition test. No biased exploratory preference to either object was observed among the four groups of mice in the training session, suggesting that there was no difference in motivation and curiosity about novel objects among the groups (F(3,47) = 0.5923, p = 0.6232) (Fig. 6A). In the retention session performed 24 h after training, a marked decrease in the exploratory preference for novel objects was evident in vehicle-treated 3XTg-AD mice compared with that in wild-type mice, indicating impaired discrimination of a novel object from a familiar one (F(3,47) = 13.316, p < 0.001) (Fig. 6A). Treatment with 30 mg/kg nobiletin significantly reversed the recognition memory impairment in 3XTg-AD mice (Fig. 6A). Total time taken to explore two objects during the retention session in 3XTg-AD mice was decreased compared with that in wild-type mice (F(3,47) = 8.2190, p < 0.001) (Fig. 6B), whereas there was no difference in total exploration time among vehicle-treated and nobiletin-treated 3XTg-AD mice (p > 0.05). These results suggest that nobiletin improves recognition memory impairment in 3XTg-AD mice.

3.6. Effects of nobiletin on performance in the elevated-plus maze test The elevated-plus maze test was performed to evaluate the effects of nobiletin on the emotional reactivity of 3XTg-AD mice. There were no significant differences in the times spent in open arms and closed arms among the 4 groups (F(3,47) = 1.1905, p = 0.3236; F(3,47) = 0.6612, p = 0.5800, respectively) (Supplementary Fig. 1A, B). The number of closed-arm entries was significantly decreased in 3XTg-AD mice compared with that in wild-type mice (F(3,47) = 5.3994, p < 0.01) (Supplementary Fig. 1D). There was no significant difference in the number of closed-arm entries among

Please cite this article in press as: Nakajima A, et al. Nobiletin, a citrus flavonoid, improves cognitive impairment and reduces soluble A␤ levels in a triple transgenic mouse model of Alzheimer’s disease (3XTg-AD). Behav Brain Res (2015), http://dx.doi.org/10.1016/j.bbr.2015.04.028

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Fig. 6. Effects of nobiletin on recognition memory impairment in 3XTg-AD mice. (A) Exploratory preference. (B) Total exploration time. The retention session of the novel object recognition test was carried out 24 h after the training session. Values are shown as the mean ± SE (n = 11–15). **p < 0.01, ***p < 0.001 vs. wild-type mice. ##p < 0.01 vs. vehicle-treated 3XTg-AD mice.

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the vehicle-treated and nobiletin-treated 3XTg-AD mice (p > 0.05) (Supplementary Fig. 1D). 3.7. Effects of nobiletin on performance in the passive avoidance test

Fig. 7. Effects of nobiletin on A␤ levels in the brain of 3XTg-AD mice. (A) Levels of soluble A␤1–40 . (B) Levels of soluble A␤1–42 . (C) Levels of insoluble A␤1–42 . A␤ levels were measured using a sandwich ELISA system. Values are shown as the mean ± SE (n = 6–9). ##p < 0.01 vs. vehicle-treated 3XTg-AD mice.

quadrant among the 4 groups (F(3,60) = 1.4503, p = 0.2372) (Supplementary Fig. 3C). In the visible-platform test, all groups had similar escape latencies, and there was no difference in swimming speeds (data not shown), suggesting no difference in motivation or sensory/motor function among the 4 groups. Taken together, these results suggest that spatial learning and memory in 3XTg-AD mice were not significantly impaired at the age of 10–11 months in this study. We further repeated the Morris water maze test at the age of 12–13 months, yielding similar results with no significant spatial learning and memory deficits in 3XTg-AD mice (data not shown). 3.9. Effects of nobiletin on Aˇ levels in the brain of 3XTg-AD mice

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To examine the effects of nobiletin on associative memory in 3XTg-AD mice, we used the passive avoidance test, in which animals learn to associate a location with aversive foot-shocks. There was no significant difference in the step-through latency in the retention test performed 24 h after the training trial among the 4 groups (F(3,39) = 1.6287, p = 0.1984) (Supplementary Fig. 2).

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To examine the effects of nobiletin on spatial learning and memory in 3XTg-AD mice, we used the Morris water maze. Hiddenplatform training was performed daily (2 trials per day) for 8 days. All groups of mice learned the location of the hidden platform as indicated by a reduction of escape latency and distance moved over the 8-day training period (Supplementary Fig. 3A, B). Repeated measures ANOVA revealed no significant difference in acquisition among the 4 groups (escape latency, treatment × day interaction F(3,180) = 1.505, p = 0.1492; distance moved, treatment × day interaction F(3,180) = 1.443, p = 0.1729). On the 8th day, the probe test, in which the platform was removed from the pool, was performed 3.5 h after the second daily training trial. All groups of mice spent more time in the target quadrant where the platform had been placed during the training trials than in any of the other quadrants. There was no significant difference in the time spent in the target

We used an ELISA system to investigate the effects of nobiletin on A␤ levels in the brain of 3XTg-AD mice. Administration of nobiletin significantly reduced the levels of soluble A␤1–40 in 3XTgAD mice in a dose-dependent manner (F(2,20) = 6.3840, p < 0.01) (Fig. 7A). In contrast, nobiletin treatment had no effect on soluble A␤1–42 levels (F(2,20) = 1.0293, p > 0.05) (Fig. 7B). Furthermore, nobiletin treatment had no effect on insoluble A␤1–42 levels in 3XTg-AD mice (F(2,20) = 2.1102, p > 0.05) (Fig. 7C). Levels of soluble A␤1–40 and A␤1–42 in wild-type mice and insoluble A␤1–40 in all groups were below the limit of detection. The detection limits for soluble A␤1–40 , soluble A␤1–42 , insoluble A␤1–40 , and insoluble A␤1–42 were 0.31, 0.03, 6.12, and 0.64 pg/mg protein, respectively. 3.10. Effects of nobiletin on the levels of oxidative stress markers in the brain of 3XTg-AD mice We next examined the effects of nobiletin on the levels of oxidative stress markers such as ROS, MDA, and protein carbonyl in the cerebral cortex, hippocampus, and striatum of 3XTg-AD mice because our previous report showed that nobiletin has the ability to reduce oxidative stress through modulation of endogenous antioxidant defense systems in the brain of SAMP8 mice [18]. Decreased ROS levels were observed in the hippocampus in 3XTg-AD mice

Please cite this article in press as: Nakajima A, et al. Nobiletin, a citrus flavonoid, improves cognitive impairment and reduces soluble A␤ levels in a triple transgenic mouse model of Alzheimer’s disease (3XTg-AD). Behav Brain Res (2015), http://dx.doi.org/10.1016/j.bbr.2015.04.028

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treated with 30 mg/kg nobiletin compared with those in wild-type mice as well as in vehicle-treated 3XTg-AD mice (F(3,19) = 8.8788, p < 0.001) (Fig. 8A). There were no significant differences in the MDA and protein carbonyl levels among groups in any brain regions examined (p > 0.05) (Fig. 8B, C). We also examined the effects of nobiletin on the enzymatic activities of intracellular antioxidant enzymes such as GPx, GR, and SOD in the cerebral cortex, hippocampus, and striatum of 3XTgAD mice. There was no significant difference in GPx activity among the groups in any of the brain regions examined (p > 0.05) (Fig. 9A). Increased GR activity was observed in the cerebral cortex in 3XTgAD mice treated with 30 mg/kg nobiletin compared with that in wild-type mice (F(3,19) = 5.0637, p < 0.01) (Fig. 9B). Decreased SOD activity was observed in the striatum in 3XTg-AD mice treated with 30 mg/kg nobiletin compared with that in wild-type mice (F(3,19) = 4.4531, p < 0.05) (Fig. 9C). 4. Discussion The present study has demonstrated that nobiletin, a citrus flavonoid, improves the impairment of short-term memory and recognition memory in 3XTg-AD mice. Furthermore, we have shown that treatment with nobiletin reduces the soluble A␤1–40 levels in the brain of 3XTg-AD mice.

Cerebral cortex

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Fig. 9. Effects of nobiletin on the activities of GPx, GR, and SOD in the brain of 3XTgAD mice. (A) GPx activity. (B) GR activity. (C) SOD activity. Values are shown as the mean ± SE (n = 3–10). *p < 0.05, **p < 0.01 vs. wild-type mice.

It has been reported that 3XTg-AD mice show behavioral abnormalities including learning and memory impairment in an age-dependent manner in several behavioral tests [22,46,47]. In the present study, we employed the open-field test, locomotor activity test, Y-maze test, novel object recognition test, elevated-plus maze test, passive avoidance test, and water maze test to examine the beneficial effects of nobiletin on behavioral abnormalities in 3XTg-AD mice. Here, we showed that treatment with nobiletin reversed the decrease in spontaneous alternation behavior in the Y-maze test, a hippocampus-dependent task of short-term memory [44,48], in 3XTg-AD mice. Furthermore, nobiletin improved the impairment of object recognition memory, which is dependent on the cortex and hippocampus [29,49,50], in 3XTg-AD mice. In contrast, nobiletin did not affect the reduced exploratory behavior in 3XTg-AD mice in the open-field test and locomotor activity test. It has been suggested that reduced exploratory behavior in 3XTg-AD mice is associated with an increased anxiety level [51,52]. Considering that nobiletin did not affect exploratory behaviors in the open-field test and locomotor activity test, nor affect the number of arm entries in the Y-maze test or total exploration time in the novel object recognition test, the beneficial effects of nobiletin on memory impairment in 3XTg-AD mice may not be due to alteration of emotional state.

Please cite this article in press as: Nakajima A, et al. Nobiletin, a citrus flavonoid, improves cognitive impairment and reduces soluble A␤ levels in a triple transgenic mouse model of Alzheimer’s disease (3XTg-AD). Behav Brain Res (2015), http://dx.doi.org/10.1016/j.bbr.2015.04.028

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Regarding the mechanisms by which nobiletin improves memory impairment in 3XTg-AD mice, we showed here that treatment with nobiletin reduces the soluble A␤1–40 levels in the brain of these mice. Given that soluble A␤ has been demonstrated to induce synaptic dysfunction and memory impairment [4–6,22,53], it is possible that nobiletin improves cognitive impairment in 3XTg-AD mice, at least in part, by reducing soluble A␤ levels in the brain. Interestingly, a recent report has shown that nobiletin significantly increases the activity of neprilysin, the dominant A␤ peptidedegrading enzyme, in SK-N-SH cells [54], raising the possibility that this increased neprilysin activity by nobiletin may contribute to the reduced soluble A␤ levels in the brain of nobiletin-treated 3XTgAD mice. Further analysis of the effects of nobiletin on metabolism of A␤ peptide as well as APP processing is necessary to clarify the mechanism by which nobiletin reduces the soluble A␤ levels in the brain of 3XTg-AD mice. It has been reported that memory impairment in 3XTg-AD mice emerges as a long-term retention deficit in the passive avoidance test and water maze test at 4–6 months of age and further develops in an age-dependent manner [22]. Furthermore, in 3XTg-AD mice, intracellular A␤ is detectable between 3 and 6 months of age, and extracellular deposits of A␤ are evident by 12 months of age [20]. In the present study, however, our results showed that the performance of 3XTg-AD mice at 10–11 months of age was not impaired in the passive avoidance test and water maze test. In addition, insoluble A␤1–40 levels in the brain of 13- to 14-month-old 3XTg-AD mice were below the limit of detection, suggesting slow progression of A␤ pathology in the colony of 3XTg-AD mice used in the present study. This slow progression of behavioral deficits and A␤ pathology may have been due to the gender of mice that we used in the present study, namely, only male mice. In fact, it has been shown that female 3XTg-AD mice exhibit significantly greater A␤ burden and larger behavioral deficits than age-matched males [44,46]. Similarly, Hirata-Fukae et al. reported that female 3XTg-AD mice show more extensive A␤ pathology, and that formic acid-extracted total A␤1–40 and A␤1–42 were not detectable until 16 months of age in male 3XTg-AD mice [23]. Consistent with the slow progression of learning and memory deficits and A␤ pathology, the levels of oxidative stress markers such as ROS, MDA, and protein carbonyl were not increased in the brain of male 3XTg-AD mice at the age of 13–14 months in the present study. Nevertheless, we found that nobiletin treatment significantly reduced ROS levels in the hippocampus of 3XTg-AD mice as well as wild-type mice, suggesting its beneficial effects against oxidative stress. To support this idea, recent in vitro and in vivo studies have also shown the antioxidant properties of nobiletin [18,55–58]. In summary, the present study has demonstrated that chronic treatment with nobiletin, a natural compound derived from citrus peels, improves the memory impairment and reduces the soluble A␤ levels in the brain of 3XTg-AD mice, suggesting that this natural compound has potential to become a novel drug for the treatment and prevention of AD. Further work, including clinical trials in patients with AD and/or mild cognitive impairment, will be necessary to determine whether nobiletin can exhibit therapeutic efficacy, as was demonstrated in 3XTg-AD mice in the present study.

Acknowledgments We thank Drs. Y. Ohya and K. Yano, Division for Research of Laboratory Animals, Nagoya University, for their technical assistance. This work was supported in part by Grants for Project Research (Development of Fundamental Technology for Analysis and Evaluation of Functional Agricultural Products and Functional Foods) from the Ministry of Agriculture, Forestry and Fisheries of Japan, Grants-in-aid for Scientific Research 23659135, 24111518,

25116515, and 26670121 from the Japan Society for the Pro- Q3 motion of Science, and grants from the Research Foundation for Oriental Medicine and the Public Foundation of Chubu Science and Technology Center.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bbr.2015.04.028

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Please cite this article in press as: Nakajima A, et al. Nobiletin, a citrus flavonoid, improves cognitive impairment and reduces soluble A␤ levels in a triple transgenic mouse model of Alzheimer’s disease (3XTg-AD). Behav Brain Res (2015), http://dx.doi.org/10.1016/j.bbr.2015.04.028

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