Effects of Sesaminol Feeding on Brain Aβ Accumulation in a

May 27, 2016 - Furthermore, sesaminol administration increased the gene and protein expression of ADAM10, which is a protease centrally involved in th...
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Effects of sesaminol feeding on brain A# accumulation in a senescence-accelerated mouse-prone 8, SAMP8 Shigeru Katayama, Haruka Sugiyama, Shoko Kushimoto, Yusuke Uchiyama, Masato Hirano, and Soichiro Nakamura J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01237 • Publication Date (Web): 27 May 2016 Downloaded from http://pubs.acs.org on May 31, 2016

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

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Effects of sesaminol feeding on brain Aβ β accumulation in a

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senescence-accelerated mouse-prone 8, SAMP8

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Shigeru Katayama,† Haruka Sugiyama,† Shoko Kushimoto,† Yusuke Uchiyama,§

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Masato Hirano,§ and Soichiro Nakamura*,†

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Minamiminowa, Kamiina, Nagano 399-4598, Japan

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§

Department of Bioscience and Biotechnology, Shinshu University, 8304

Takemoto Oil & Fat Co., Ltd. 11 Hamacho, Gamagori, Aichi 443-0036, Japan

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*

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Soichiro Nakamura, PhD

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Department of Bioscience and Biotechnology, Shinshu University, 8304

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Minamiminowa, Kamiina, Nagano 399-4598, Japan

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Tel&FAX: +81-265-77-1609

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e-mail: [email protected]

Corresponding author:

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Abstract

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Alzheimer’s disease (AD) is characterized by the progressive accumulation of

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extracellular beta-amyloid (Aβ) aggregates. Recently, the senescence-accelerated

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mouse P8 (SAMP8) model was highlighted as useful model of age-related AD.

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Therefore, we used the SAMP8 mouse to investigate the preventive effects of sesame

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lignans on the onset of AD-like pathology. In preliminary in vitro studies, sesaminol

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showed the greatest inhibitory effect on Aβ oligomerization and fibril formation

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relative to sesamin, sesamolin, and sesaminol-triglucoside. Hence, sesaminol was

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selected for further evaluation in vivo. In SAMP8 mice, feed-thorough sesaminol

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(0.05% w/w in standard chow) administered over a 16-week period reduced brain Aβ

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accumulation and decreased serum 8-OHdG, an indicator of oxidative stress.

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Furthermore, sesaminol administration increased the gene and protein expression of

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ADAM10, which is a protease centrally involved in the non-amyloidogenic processing

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of amyloid precursor protein. Taken together, these data suggest that long-term

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consumption of sesaminol may inhibit the accumulation of pathogenic Aβ in the brain.

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Keywords: Aβ, ADAM10, brain, SAMP8, sesaminol

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Introduction

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Alzheimer’s disease (AD) is the most common form of dementia and is

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characterized by cognitive decline and memory impairments that are severe enough to

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interfere with normal activities of daily living.1,

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accumulation of beta-amyloid (Aβ) in extracellular neuritic plaques as well as the

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hyperphosphorylation and aggregation of tau protein in intracellular neurofibrillary

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tangles. The accumulation of these proteins has subtle effects on synaptic efficacy and

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eventually leads to neuronal cell death and the progression of cognitive impairments.3

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Although many researchers have screened for anti-amyloidal agents among naturally

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occurring compounds, an effective and safe preventive therapy for dementia has not

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yet been developed.

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AD is associated with the

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The senescence-accelerated mouse (SAM) model of accelerated aging was

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initially established by Takeda et al.4 and has since been widely utilized for the study

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of aging. SAM prone 8 (SAMP8) mice show spontaneous age-related behavioral

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disorders, cognitive deficits, hair loss, and a shortened life span. Recently, it was

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shown that the SAMP8 mouse also exhibits age-related AD pathology, including

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alterations in secretase-mediated amyloid precursor protein (APP) processing,5,

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Aβ plaque accumulation,7 and hyperphosphorylated tau aggregation.8 Accordingly, the

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SAMP8 mouse has recently been used as an model of age-related AD onset in 3

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Journal of Agricultural and Food Chemistry

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preclinical studies.9, 10

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In Asian countries, sesame (Sesamum indicum L.) has attracted significant

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attention for its potential health benefits. Sesame oil is rich in linoleic and oleic acids

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and has a high content of lignans including sesamin, sesaminol, sesamolinol, and

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sesaminol glucosides.11 Sesame lignans have been reported to have health beneficial

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effects on serum cholesterol, blood pressure, and liver function.12, 13 In the present

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study, we investigated the ability of sesame lignans to inhibit Aβ aggregation in vitro

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and to alleviate age-related oxidative stress, neuroinflammation, amyloidogenic Aβ

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processing, and Aβ accumulation in vivo.

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Materials and Methods

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Materials

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Sesamin, sesaminol, sesamolin, and sesaminol-triglucoside were supplied from

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Takemoto Oil & Fat Co., Ltd. (Aichi, Japan). Human Aβ1-42 was purchased from

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Peptide Institute Inc. (Osaka, Japan). Malondialdehyde (MDA) was purchased from

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Wako Pure Chemical Industries Ltd (Osaka, Japan), and 2-thiobarbituric acid (TBA)

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was purchased from Sigma-Aldrich Japan (Tokyo, Japan). All other chemicals were of

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analytical reagent grade.

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Preparation of Aβ β fibrils

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Human Aβ1-42 was dissolved in 0.1% ammonia solution to yield at a final

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concentration of 250 µM, separated into aliquots, and then stored at −80 °C until use.

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To prepare amyloid fibrils, aliquots of Aβ were diluted to a concentration of 25 µM in

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50 mM phosphate buffer (pH 7.5) containing 100 mM NaCl. The Aβ solution was then

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sealed in a microcentrifuge tube and incubated at 37 °C for 24 h in the presence or

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absence of test compounds (25 µM).

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ThT fluorescence assay

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Amyloid fibril formation was monitored using a Thioflavin T (ThT) assay.

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Briefly, 6 µL of each sample was dissolved in ethanol and added to 1.2 mL of ThT

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solution containing 50 mM glycine-NaOH buffer (pH 8.5). The mixture was briefly

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vortexed and fluorescence was quantified using a FP-6200 spectrofluorometer (JASCO,

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Tokyo, Japan). The excitation and emission wavelengths were 446 nm and 490 nm,

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respectively. The fluorescence signal of each sample was measured 4 times, averaged,

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and the fluorescence value of a ThT blank was subtracted from the result. Ethanol was

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used as a control.

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Electron microscopy

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Transmission electron microscopy (TEM) was used to characterize the structural

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morphology of Aβ fibrils in the presence or absence of test compounds. After

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incubation, 5 µL of each amyloid sample was spotted onto a collodion-coated 400

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mesh copper grid (Nisshin EM Co., Tokyo, Japan). The grids were negatively stained

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with 1% phosphotungstic acid and visualized with a JEM-1400 microscope (JEOL,

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Tokyo, Japan) operated with an accelerating voltage of 80 kV.

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Immuno-dot blotting

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After incubation, 5 µL of each amyloid sample was spotted onto a nitrocellulose

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membrane and dried at room temperature for 30 min. The membrane was then blocked

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at room temperature for 60 min with 3% (w/v) bovine serum albumin (BSA) in

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phosphate buffered saline (PBS) and subsequently washed 3 times with PBS

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containing 0.05% (w/v) Tween-20 (PBST). Next, the membrane was incubated with

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rabbit anti-oligomer antibody A11 (1:2500 dilution; Invitrogen, Camarillo, CA) for 60

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min at room temperature, washed 3 times with PBST, and incubated with anti-rabbit

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IgG-horseradish peroxidase secondary antibody (1:5000 dilution; Invitrogen) for 60

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min at 37 °C. Finally, the membrane was washed with PBST and developed in Pierce

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Western Blotting Substrate (Pierce, Rockford, IL) for 1 min at room temperature.

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Immunoreactive proteins were visualized using an AE-9300 Ez-Capture system (ATTO,

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Tokyo, Japan) using enhanced chemiluminescence.

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Animals

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Male SAMP8 mice aged 15 weeks were purchased from Japan SLC, Inc.

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(Shizuoka, Japan) and allowed to acclimate for 1 week before the experimental period.

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All mice were individually housed in a facility with controlled temperature (20-23 °C)

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and humidity (40-70%) on a 12-h light/dark cycle (light on at 8:00 am). All

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experiments were conducted in accordance with the institutional guidelines established

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by Shinshu University for the care and use of laboratory animals.

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Animal protocol

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Sixteen-week-old SAMP8 mice were divided into a control group and a

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sesaminol group (n = 8). The control group was fed a normal diet of standard chow

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(MF, Oriental Yeast, Tokyo, Japan) and the sesaminol group was fed a diet of standard

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chow containing 0.05% (w/w) freeze-dried sesaminol powder. Food and tap water

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were available ad libitum for a 16-week treatment period. Food intake and body weight

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were measured once per week. After 16 weeks, mice were tested in the Morris water

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maze and then sacrificed for blood and tissue harvesting. Brain and liver tissues were

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quickly excised on ice, weighed, and placed into RNA Later (Sigma) for RNA isolation,

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RIPA lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA) for Western blotting, or

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PBS containing 5 mM BHT for MDA measurement. Tissues were stored at −80 °C

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until use.

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Soluble Aβ β enzyme-linked immunosorbent assay (ELISA) Soluble Aβ was quantified in brain tissue samples using the ELISA method.

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Briefly, whole brain tissue was homogenized in 70% formic acid and the homogenates

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were centrifuged at 100,000g for 60 min at 4 °C. The resulting supernatants were

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neutralized with 1 M Tris and soluble Aβ was measured using a human Aβ1-42 ELISA

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kit (Wako Pure Chemicals) according to manufacturer specifications.

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Determination of serum 8-hydroxydeoxyguanosine (8-OHdG)

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Serum was filtered with an Amicon Ultra-10,000 NMWL centrifuge tube

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(Millipore, Bedford, MA) to remove any high molecular weight impurities that might

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have interfered with spectrophotometric measurement. Serum 8-OHdG was measured

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using a competitive ELISA kit (8-OHdG Check, Nikken Seil, Shizuoka, Japan)

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according to manufacturer specifications.

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Thiobarbituric acid reactive substances (TBARS) assay

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To assess lipid peroxidation, a TBARS assay was performed on brain tissue

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homogenates according to the method of Ohkawa et al.14 with modifications. Briefly,

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100 µl of 8.3% (w/v) tissue homogenate was combined with 0.2 mL of 8.1% sodium

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dodecyl sulfate, followed by the addition of 1.5 mL of 20% acetic acid (pH 3.5) and

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1.5mL of 0.8% TBA. The reaction mixture was vortexed and subsequently incubated at

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90 °C for 1 h. After cooling on ice, 5 mL of a butanol:pyrimidine mixture (15:1) was

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added and the resultant mixture was centrifuged at 1,000g for 15 min. The absorbance

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of the upper layer of the supernatant was measured at 532 nm with a UV-1700

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spectrophotometer (Shimadzu, Kyoto, Japan). MDA was used as standard and the

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results were expressed as µmol of MDA/g of protein measured by the Bradford

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method.

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Morris water maze testing

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Spatial learning and memory was evaluated using the Morris water maze.15 A

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circular plastic pool (diameter, 120 cm; height, 50 cm) was filled with water and the

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water temperature was maintained at 25 ± 1 °C. An escape platform (diameter, 11 cm)

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was hidden 1 cm below the surface of the water in a fixed location. Mice were trained

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to locate the platform in training trials 3 times per day for 4 consecutive days. Each

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trial lasted a maximum of 60 s and the inter-trial interval was approximately 30 s.

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When mice successfully located the platform, they were allowed to remain on the

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platform for 10 s; otherwise, mice that were unable to locate the platform within 60 s

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were placed on the platform for 10 s at the end of trial. One day after the last training

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trial, mice were subjected to a 60 s probe trial in which the platform was removed from

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the pool. Latency to cross the former location of the platform was recorded by an

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overhead camera and the ANY-maze video tracking system (Stoelting Co., Wood Dale,

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IL).

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Quantitative real-time PCR

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Total RNA was extracted from whole brain tissues using the RNAiso Plus kit

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(TaKaRa Bio, Shiga, Japan) according to manufacturer specifications. cDNA was

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synthesized from 0.25 mg of total RNA using a reverse transcription system (High

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capacity RNA-to-cDNA kit, Applied Biosystems). Quantitative real-time PCR was

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performed using the StepOne real-time PCR System (Applied Biosystems) with the

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KAPA SYBR FAST Universal qPCR kit (Kapa Biosystems, Cape Town, South Africa).

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The expression level of each gene was determined using the comparative Ct method

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and normalized to β-actin as an internal standard. The PCR reaction consisted of one

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cycle at 95 °C for 3 min, 40 cycles (95 °C for 3 s and 60 °C for 20 s), and a final

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dissociation cycle (95 °C for 15 s). The primer sequences used in this study are shown

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in Table 1.

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Western blotting

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Whole tissue extracts were homogenized in RIPA lysis buffer and subsequently

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centrifuged at 13,500g for 10 min at 4 °C. Tissue supernatants were collected and

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protein concentrations were determined using the DC protein assay kit (Bio-Rad,

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Hercules, CA) with BSA as a standard. Protein samples (25 µg) were then

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electrophoresed on 15% polyacrylamide gels and electroblotted onto polyvinylidene

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fluoride (PVDF) membranes (Clear Blot Membrane-P; ATTO). PVDF membranes

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were next incubated with antibodies against ADAM10 (1:5000 dilution; Enzo Life

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Sciences, Farmingdale, NY), BACE1 (1:100 dilution; Enzo Life Sciences), or β-actin

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(1:250 dilution; Enzo Life Sciences), followed by incubation with an anti-rabbit IgG

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horseradish peroxidase-conjugated secondary antibody (1:2500 dilution; Anaspec, San

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Jose, CA). Chemiluminescence detection was performed using Pierce Western blotting

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substrate (Thermo Scientific, Rockford, IL) and the AE-9300 Ez-Capture System

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(ATTO).

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Statistical analysis Data are presented as mean ± SE. Statistical analyses were performed by Student’s t-test. A level of p