Alpha-Linolenic Acid from Perilla frutescens var. japonica Oil Protects

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Alpha-linolenic acid from Perilla frutescens var. japonica oil protects A#-induced cognitive impairment through regulation of APP processing and A# degradation Ah Young Lee, Myoung Hee Lee, Sanghyun Lee, and Eun Ju Cho J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03941 • Publication Date (Web): 02 Nov 2017 Downloaded from http://pubs.acs.org on November 2, 2017

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Journal of Agricultural and Food Chemistry

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Alpha-linolenic acid from Perilla frutescens var. japonica oil protects Aβ-

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induced cognitive impairment through regulation of APP processing and

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Aβ degradation

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Ah Young Lee1, Myoung Hee Lee2, Sanghyun Lee3, Eun Ju Cho1,*

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University, Busan 46241, Republic of Korea

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Development Administration, Gyeongnam 50424, Republic of Korea.

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3

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Department of Food Science and Nutrition & Kimchi Research Institute, Pusan National

Department of Southern Area Crop Science, National Institute of Crop Science, Rural

Department of Integrative Plant Science, Chung-Ang University, Gyeonggi 17546, Republic

of Korea.

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Corresponding authors: Eun Ju Cho, Department of Food Science and Nutrition, Pusan

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National University, Busan 46241, Republic of Korea. Tel: +82-51-510-2837, Fax: +82-51-

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583-3648. E-mail address: [email protected] (E. J. Cho). 1

ACS Paragon Plus Environment


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ABSTRACT

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Alzheimer’s disease (AD) is characterized by progressive cognitive and memory impairment.

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The major pathological hallmark of AD is the accumulation of amyloid beta (Aβ), which is

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produced from the amyloid precursor protein (APP) through cleavage of β- and γ-secretase.

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Recently, dietary plant oil containing ω-3 polyunsaturated fatty acid has become attractive

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alternative sources to fish oil containing eicosapentaenoic acid or docosahexaenoic acid

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(DHA). We investigated whether ALA isolated from perilla oil has direct effects on

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improvement of cognitive ability and molecular mechanisms in APP processing in

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comparison with DHA. In the present study, ICR mice were treated orally with ALA or DHA

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(100 mg/kg/day) for 14 days after i.c.v. injection of Aβ25-35. Administration of ALA resulted

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in a prevention of learning and memory deficit in Aβ25-35-injected mice compared with the

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control group, observed in T-maze, novel object recognition and Morris water maze tests.

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ALA supplementation also markedly ameliorated the Aβ25-35-induced oxidative stress by

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inhibition of lipid peroxidation and nitric oxide overproduction in the mouse brain, liver and

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kidney, almost down to the levels in DHA-administered group. These effects of ALA on

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protective mechanisms were related to the regulation of APP processing via promoting non-

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amyloidogenic pathway such as up-regulation of soluble APP alpha, C-terminal fragment

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alpha/beta ratio and A disintegrin and metalloprotease10 protein expressions. Furthermore,

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ALA inhibited the amyloidogenic pathway through the down-regulation of β-site APP-

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cleaving enzyme and presenilin2. ALA also enhanced Aβ degradation enzyme, insulin

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degrading enzyme. In conclusion, the present study indicated beneficial effect of ALA in

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improving the cognitive ability against Aβ25-35, and these effects were comparable to those

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exerted by DHA. Its neuroprotective effects are mediated, in part, by regulation of APP

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processing and Aβ degradation, thus ALA might be a potential candidate for prevention or

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treatment of neurodegenerative diseases such as AD.

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KEY WORDS: alpha-linolenic acid, Aβ25-35, Alzheimer’s disease, cognitive ability, perilla

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frutescens

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INTRODUCTION

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Accumulation of amyloid beta (Aβ), which is a neuropathological hallmark of Alzheimer’s

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disease (AD), correlates with cognitive dysfunction.1 Many studies indicated that Aβ plaques

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are accompanied by neurotoxicity and oxidative stress, which generates free radicals with

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lipid peroxidation in the brain.2-6 Amyloid precursor protein (APP) is cleaved by two

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pathways; amyloidogenic and non-amyloidogenic pathways. In the amyloidogenic pathway,

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cleavage by β- and γ-secretase initiates the production of neurotoxic Aβ.7 First, APP is

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cleaved by the β-secretase, also known as β-site APP cleaving enzyme (BACE) and produces

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a transmembrane C-terminal fragment β (CTFβ). CTFβ is then cleaved by γ-secretase, which

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is comprised of four membrane proteins, presenilin1 (PS1), PS2, nicastrin and anterior

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pharynx defective 1.8 On the other hand, the non-amyloidogenic pathway involves α-

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secretase, which cleaves APP to produce non-toxic soluble neurotrophic fragment (sAPPα)

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and CTFα, resulting in decrease the Aβ secretion. A disintegrin and metalloprotease 10

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(ADAM 10), a key protease of α-secretase, has been shown prevention of the excessive Aβ

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accumulation in AD mouse model.9 In the brain, clearance of Aβ involves enzymatic

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degradation by insulin-degrading enzyme (IDE) and neprilysin (NEP).10 In addition, Aβ

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transportation across the blood-brain barrier (BBB) from brain mediated by receptor for

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advanced glycation end products (RAGE) and low-density lipoprotein receptor-related

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protein 1 (LRP-1).11 Therefore, APP processing by α-secretase and regulation of Aβ

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metabolism has been suggested as a potential therapeutic target for AD. Acetylcholinesterase

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inhibitors such as donepezil, rivastigmine and galantamine were used for the symptomatic

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treatment of mild-to-moderate AD patients. However, they have shown limited impact on the

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clinical course of AD, or side effects such as gastrointestinal disturbances or tolerability.12,13

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Several studies demonstrated that plant-derived epigallocatechin gallate, luteolin and

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ginsenoside Rg1 promote the non-amyloidogenic α-secretase activity pathway and reduce the

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generation of Aβ.14-17 Moreover, Grimm et al.18,19 reported that lipids including phospholipids,

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gangliosides and cholesterols exhibit anti-amyloidogenic properties. A number of clinical

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trials targeting the non-amyloidogenic pathway are currently ongoing for application in

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AD.20-22 To date, however, there are no effective therapeutic agents for AD.

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Recently, dietary lipids are major concern in health care. It has been focused not only on the

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amount of dietary lipids but also on the types of fatty acids in the diet.23-25 In general, it was

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recommended that intake of monounsaturated- or polyunsaturated-fatty acids rather than

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saturated fatty acids could reduce the risk of degenerative disease such as cardiovascular

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disease. In particular, there is strong supporting evidences that ω-3 polyunsaturated fatty acid,

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lead to lower risk of certain chronic diseases such as heart disease, arthritis and

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inflammation.26-28 The beneficial effect of ω-3 fatty acid on human health is well established,

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and there is relationship between ω-3 fatty acid uptake and prevention of AD or cognitive

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decline.29-33 In contrast, eicosapentaenoic acid and docosahexaenoic acid (DHA) derived

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from marine products are both associated with toxicological and hormonal effects stimulated

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by environmental contamination such as methyl mercury poisoning. Therefore, the plant-

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derived ω-3 fatty acid, alpha-linolenic acid (ALA), is considered as the best alternative

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source.34 Previous studies showed that consumption of dietary ALA reduced the risk of

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cardiovascular disease35 and stroke.36 In addition, Gao et al.37 demonstrated that long-term

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dietary intake of ALA improved the cognitive function through the activation of extracellular

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signal-regulated kinases (ERK) and Akt signaling in aged-rat model. However, the direct

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effects of ALA on Aβ-injected AD model and its mechanisms have not been completely

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understood yet.

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We recently reported that supplementation of Perilla frutescens var. japonica oil, which

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contains the highest proportion of ALA among the vegetable oils, ameliorated Aβ-induced

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cognitive deficits evidenced by increased recognition ability and the levels of brain-derived

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neurotrophic factor protein expression, as well as suppressed the inflammatory-related

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protein expressions, such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2

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(COX-2).38 We speculated that ALA from perilla oil may contribute to prevention of Aβ

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neurotoxicity in the brain. There are extensive literatures on the effect of DHA on human

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health, especially in relation to brain development and function.39-42 DHA can be obtained

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from diet, and is commonly found in fish oil or synthesized from ALA in the body. However,

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most studies so far have been shown to limited conversion efficiency of ALA to DHA,43-46

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suggesting that these ω-3 fatty acids that are derived from different sources (plant or marine)

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might have their own comparable specific effects on cognitive improvement. Therefore, this

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study was designed to investigate the direct effect of ALA in comparison with DHA, on the

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cognitive decline induced by Aβ and the associated molecular mechanisms involved in APP

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processing and Aβ metabolism.

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MATERIALS AND METHODS

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Sample Preparation

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P. frutescens was kindly supported by the Southern Area Crop Science, Rural Development

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Administration, Republic of Korea. Perilla oil was treated with urea with cooling, and large 6


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amount of ALA was isolated after gas chromatography-flame ionization detector analysis.47

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DHA ethyl ester was used as a positive control, and was purchased from Cayman Chemical

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(Ann Arbor, MI, USA).

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Animals and experimental protocols

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Male ICR mice (5-weeks-old) weighing between 25 to 27 g were supplied from Orient Inc.

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(Seongnam, Korea). Experimental mice were bred in 4 or 5 per plastic cages in a controlled

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room (temperature, 20 ± 2°C; humidity, 50 ± 10%; 12 h light/dark cycle), and they were

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provided with normal pellet diets and water. Mice were assigned into four groups (n = 6 per

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group) as follows: Normal group = i.c.v. injection of 0.9% NaCl (Bio Basics Inc., Markham,

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Ontario, Canada) + oral administration of water; Control group = i.c.v. injection of Aβ25-35 +

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oral administration of water; ALA group = i.c.v. injection of Aβ25-35 + oral administration of

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alpha-linolenic acid (100 mg/kg/day); DHA group = i.c.v. injection of Aβ25-35 + oral

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administration of docosahexaenoic acid (100 mg/kg/days) for 14 days using a sonde gavage.

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No significant differences in the initial body weights among the groups were observed (Table

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1).

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Aβ25-35-infused mouse model

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To induce aggregation, Aβ25-35 peptide (Sigma Aldrich Co., St. Louis, MO, USA) was

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dissolved in distilled water, and then incubated for 3 days at 37°C.48 The aggregated Aβ25-35

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solution was injected intracerebroventricularly in accordance with method of Laursen and

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Belknap.49 Each mice were lightly anesthetized with zoletil (Virbac Korea, Seoul, Korea) and

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rompun (Bayer Korea, Seoul, Korea). Briefly, 5 µL of 0.9% NaCl or 5 nmol of Aβ25-35 7



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aggregate solution was injected using a in 0.8 mm posterior and 1.5 mm lateral from the

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bregma with depth of 2.2 mm beneath the surface of the brain at a rate of 1 µL/min using a 10

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µL Hamilton microsyringe with 26-gauge stainless-steel needle, which was left with 1 min

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waiting-period in the injection site before removal. All animal experimental procedures were

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approved by the Pusan National University Institutional Animal Core and Use Committee

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(Approval Number: PNU-2015-0888). The behavioral tests started on day 11 after injection at

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the same time, and were performed in accordance with the experimental schedules as shown

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in Figure 1.

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T-maze test

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T-maze test was carried out following the previous reports, which was established by

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Montgomery.50 It consisted of a start box, left and right arms with a door to block the left

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arms. T-maze were constructed using black boards (stems = 50 cm, width = 13 cm, height =

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20 cm). During the training session, the left arm of T-maze was blocked. Each mouse was

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placed at the start box and allowed to explore only the right arm. The number of

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explorations on the right arm of T-maze during 10 min-period was measured. 24 h after the

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training session, the door was removed. They were allowed to freely explore both the right

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and left arms for 10 min and the number of the exploring in each arm was recorded. The

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percentage of space perceptive ability (%) was expressed as a ratio of the number of entries

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either right arm (old route) or left arm (new route) to the number of total arms entries.

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Novel object recognition test

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The novel object recognition test was performed using square-shaped black box (each 40 ×

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30 × 20 cm).51 In the training session, two identical objects (A and A’, plastic bottles) were

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fixed symmetrically at floor of the field. Mouse was placed at squire box for 10 min and the

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number of touches each objects was recorded. After that, the mouse was returned to home

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cage. 24 h after training session, one of the objects was replaced by a new object (B, another

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plastic bottle), which is different shape but in similar size and color. Mouse were located into

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the same field and was allowed to explore familiar object or novel object freely for 10 min.

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The number of touches each objects was measured. The percentage of object perceptive

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ability (%) was expressed a ratio by following equation; [A/(A+A’) X 100 in the training

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session, and B/(A+B) X 100 in the test session].

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Morris water maze test

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The spatial and long-term memory of mice was conducted by the method of Morris.52

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Experimental apparatus consisted of a dark plastic circular pool (diameter = 80 cm, height =

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40 cm) filled with water (22 ± 1°C), and equally divided into four quadrants. Water was made

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opaque by addition with white non-toxic poster color, thereby the platform was invisible. A

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platform (diameter = 8 cm) was placed in the midpoint of one quadrant submerged 1 cm

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beneath the surface of water. Visual cues were presented on the walls of the apparatus for

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navigation. In the training session, each mouse was placed into water pool and allowed to

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swim (maximum 60 s) to find the hidden platform, three trials per day for consecutive 3 days.

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When the mouse that found the hidden platform, it is allowed to stay on the platform for 15 s.

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If the mouse failed to find the hidden platform within 60 s, they were guided to platform and

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allowed to stay there for 15 s to help them remember the platform location. After completion

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of training trial for 3 days, in the primary test of probe trial, the experiment was performed as

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before. The latency time required to reach the hidden platform was recorded. Next, the

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platform was removed and each mouse was allowed to swim freely for 60 s. Then the

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frequency of mice crossing over the target quadrant and the time that mice spent swimming

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in the target quadrant where the platform was previously located was measured. In the tertiary

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test, the water was made transparent and the latency time to reach the visible platform, which

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was positioned 1 cm above the water surface, was recorded.

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Measurement of lipid peroxidation

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The levels of MDA were measured according to previous method.53 After completion of

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behavioral tests, mice were sacrificed under anesthesia with zoletil and rompun. Afterwards,

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the brain, liver, and kidneys of mice were collected immediately and dissected tissues were

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on the ice. Tissues homogenate was prepared, and the supernatant was collected following

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centrifugation at 3000 rpm for 15 min at 4°C. The samples were added to 1% phosphoric acid

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(Samchun Pure Chemical Co., Gyeonggi, Pyeongtaek, Korea) and 0.67% thiobarbituric acid

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(TBA, Lancaster Synthesis, Ward Hill, MA, USA). solution. After boiling at 95°C for 45 min,

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the mixture was cooled in an ice, and 2 mL of 1-butanol (Samchun Pure Chemical Co.) were

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mixed followed by centrifugation at 3,000 rpm for 10 min. The resulting color was detected

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using microplate absorbance reader at 535 and 520 nm. Lipid peroxide levels were expressed

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in terms of MDA equivalents using standard curve.

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Nitric oxide (NO) scavenging activity NO concentration in brain, liver and kidney was examined by the method of Schmidt et al.54

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A supernatant of homogenized tissue was mixed with distilled water, and then dilution was

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added to the same amount of Griess reagent (Sigma Aldrich Co.). After incubation in 37°C for

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30 min, the absorbance of each solution was measured using microplate reader at 540 nm.

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The NO levels were calculated based on the NaNO2 (Junsei Chemical Co., Tokyo, Japan)

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standard curve.

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Western blotting

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The mice brains were rapidly removed and homogenized with lysis buffer containing a

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protease inhibitor cocktail. The homogenates were centrifuged at 13,000 rpm for 30 min. The

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protein concentrations were determined in the collected supernatants. Equal amounts of

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proteins were resolved on 10-13% sodium dodecyl sulfate polyacrylamide gel electrophoresis

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(SDS-PAGE). After electrophoresis, proteins were transferred to polyvinylidene fluoride

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membranes (Millipore, MA, USA). Membranes were subsequently incubated with 10% skim

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milk dissolved in phosphate-buffered saline with Tween-20 (PBS-T) for 60 min and further

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incubated with primary antibody in PBS-T overnight at 4°C. The membranes were washed

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with PBS-T for 3 times and incubated with primary antibodies were as follows: [Anti-APP,

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C-terminal (1:1000; Sigma); BACE (1:1000, Cell Signaling, Beverly, MA, USA); PS1

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(1:1000, Cell Signaling); PS2 (1:1000, Cell Signaling); IDE (1:200, Santa Cruz, CA, USA);

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NEP (1:1000, Millipore); RAGE (1:200, Santa Cruz); LRP-1 (1:50000, Abcam, Cambridge,

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MA, USA); ADAM10 (1:200, Santa Cruz); and β-actin (1:200, Santa Cruz)]. After incubation

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with appropriate secondary antibodies (1:1000, Cell Signaling) at room temperature for 1 h,

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the membranes were visualized using pico-enhanced peroxidase detection (ELPIS-Biotech,

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Daejeon, Korea). Western blot bands were assessed with a Davinci-Chemiluminescent

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imaging system (CoreBio, Seoul, Korea).

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Statistical analysis

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Results were expressed as means ± SD. Statistical analysis in this present study was

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performed using IMB SPSS statistics programs 23 (IBM Corporation, NY, USA) and SAS

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9.4 (SAS Institute Inc., Cary, NC, USA). Statistical differences between the normal, control,

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ALA- and DHA-administered groups were determined by one-way ANOVA followed by

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Duncan’s post-hoc test. In the T-maze and novel object recognition test, the perceptive ability

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between training session and test session was compared by student’s t-test. Significance was

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set at P < 0.05.

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RESULTS

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Body weight change in Aβ25-35-inejcted mice model

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Body weight of the mice is summarized in Table 1. During the experiment, there were no

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significant body weight differences among the four groups.

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T-maze test

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The T-maze test, in which a mouse tends to explore an unfamiliar route, is used to assess

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spatial discrimination. We investigated whether the oral administration of ALA reverses

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spatial memory deficit in Aβ25-35-injected mice. In the saline-injected normal group, the

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exploration of the previously unexplored arm was significantly higher than that of the old arm

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(student’s t-test, p = 0.000034). In contrast, the Aβ25-35-injected mice exhibited an impaired

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performance in the T-maze, as they showed no significant preference for the unexplored arm

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as evidenced by the comparable the number of entries to the old arms (student’s t-test, P =

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0.194). However, as represented in Figure 2, Aβ25-35-injected mice treated with ALA

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significantly increased the ratio of new route entries than that of old route (student’s t-test, P

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= 0.042), similarly to the DHA-administered group (student’s t-test, P = 0.016). This finding

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suggested that memory dysfunction induced by Aβ25-35 was ameliorated by the administration

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of ALA.

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Novel object recognition test

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Object perceptible ability, which is a natural tendency of mice to explore a novel object

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instead of a familiar object, was assessed by using novel object recognition test. As shown in

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Figure 3, normal group of mice exhibited the expected differences in the exploration ratio

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between familiar and novel object, showing more touches to the novel object than the

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familiar object during the test session (student’s t-test, P = 0.000298). In contrast, Aβ25-35

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resulted in object recognition dysfunction in mice, which did not discriminate between

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familiar and novel object (student’s t-test, P = 0.403). However, supplementation of Aβ25-35-

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injected mice with ALA or DHA significantly increased their preference for the novel object

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compared with familiar object as evidenced by the increased number of touches to the novel

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object (student’s t-test, ALA: P = 0.011, DHA: P = 0.001). These results indicated that the

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administration of ALA attenuated Aβ25-35-induced object recognition deficits.

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Morris water maze test

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To assess the long-term and spatial memory function, the Morris water maze test was carried

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out. Mice were trained to reach the hidden platform in the water maze over 3 days. Figure 4A

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shows that Aβ25-35-treated mice displayed a significantly longer latency to find the platform

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compared with the normal mice. However, administration of both ALA and DHA reduced the

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time to find the hidden platform as compared with the control group of mice. As shown in

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Figure 4B, the saline-injected normal mice exhibited a significant decrease in time to reach

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the hidden platform in the final test on training day 4, corresponding to 10.2 s. In contrast, the

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Aβ25-35-treated control mice exhibited an impaired spatial memory with a latency

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corresponding to 42 s. Compared with control group, a significant reduction in the escape

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latency was observed in the ALA-administered group (9.6 s), as well as in the DHA-treated

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group (9.2 s). The latency in DHA treated mice was shorter than that of the ALA-

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administered group (F(3,16) = 14.05, P < 0.05). Furthermore, the results of the probe trial were

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expressed as the time spent in the target quadrant where the platform was placed during the

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training session (Fig. 4C). The Aβ25-35-injected control group exhibited a significantly

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decreased time spent in the target quadrant, whereas mice administered with ALA or DHA

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significantly spent more time in the area where the hidden platform had been located,

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indicating improvement of learning and memory ability by ALA and DHA against Aβ25-35

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injection (F(3,16) = 6.21, P < 0.05). In the visible platform, no significant differences were

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observed among the groups to reach the platform (F(3,16) = 0.60, P > 0.1), thus they were not

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different in visual or physical activities (Fig 4D). These results demonstrated that

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supplementation of ALA could ameliorate Aβ25-35-induced disturbances of long-term and

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spatial learning and memory ability.

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Measurement of MDA and NO generation

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The protective effect of ALA treatment on oxidative stress induced by Aβ25-35 was 14


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investigated by measuring the levels of MDA and NO in the brain, liver and kidneys. As

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shown in Table 2, there was a significant rise in the levels of MDA in Aβ25-35-injected brain

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(26.16 nmol/mg protein) compared with non-injected normal brain (17.99 nmol/mg protein).

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However ALA administration decreased the MDA concentration (20.77 nmol/mg protein),

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almost down to the levels observed in DHA-administered mice brain, showing the MDA

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levels of 20.42 nmol/mg protein. (F(3,20) = 5.70, P < 0.05). The MDA levels in the liver of the

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Aβ25-35-injected group were 48.13 nmol/mg protein, which was higher than that of normal

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group (18.71 nmol/mg protein). In contrast, MDA concentration was significantly declined by

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administration of ALA or DHA, showing 27.68 and 20.93 nmol/mg protein, respectively

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(F(3,20) = 190.32, P < 0.05). In addition, Aβ25-35 injection significantly increased MDA

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concentration in the kidney, from 35.52 to 70.52 nmol/mg protein, while administration of

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ALA or DHA decreased the MDA levels with values of 63.66 and 58.01 nmol/mg protein,

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respectively (F(3,20) = 92.17, P < 0.05).

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Aβ25-35 injection significantly elevated the NO levels in the brain (19.41 nmol/mg protein),

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while levels of NO were significantly reduced in both ALA- and DHA-administered groups

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with 16.48 and 16.90 nmol/mg protein, respectively (F(3,20) = 4.15, P < 0.05). The levels of

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NO in the liver tissues of normal group were 17.30 nmol/mg protein compared with Aβ25-35-

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injected control group (49.29 nmol/mg protein). Supplementation of ALA and DHA group

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significantly attenuated NO production, with NO levels of 27.17 and 26.87 nmol/mg protein,

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respectively (F(3,20) = 139.95, P < 0.05). Elevated NO levels in the kidneys were also observed

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after Aβ25-35 injection (28.09 nmol/mg protein). However, ALA and DHA administration

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significantly lowered NO levels: 24.99 and 24.79 nmol/mg protein, respectively (F(3,20) = 9.25,

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P < 0.05). Therefore, significantly lower levels of MDA and NO in the brain, liver and

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kidneys of the ALA-administered mice indicated that the ALA could attenuate oxidative

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stress induced by Aβ25-35.

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Effect of ALA on the amyloidogenic pathway

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In order to investigate whether ALA influences on the amyloidogenic pathway in Aβ25-35-

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injected mice, BACE, PS1, and PS2 protein expressions were measured by Western blotting.

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As shown in Figure 5, significantly higher levels of BACE expression after Aβ25-35 injection

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were apparently inhibited by ALA and DHA administration. To investigate the relationship

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between ALA and γ-secretase activity, we measured the expression levels of PS1 and PS2, the

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components of γ-secretase. Our results indicated significantly increased levels of both PS1

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and PS2 in the Aβ25-35-injected mouse brain compared with normal brain. Although it did not

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reach statistical significance in PS1 levels compared to control group, the PS2 expression was

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noticeably decreased in the ALA-fed group of mice brain. Taken together, these data

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suggested that ALA might contribute to the suppression of Aβ production by modulating the

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activity of β- and γ-secretases.

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Effect of ALA on the non-amyloidogenic pathway

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To study the mechanisms related to the non-amyloidogenic pathway in Aβ25-35-injceted AD

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mice, the protein expressions of ADAM10, sAPPα and CTFs were determined. The results

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indicated that the expression of ADAM 10 was significantly inhibited after Aβ25-35 injection,

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compared with the non-injected normal mice (Fig. 6). However, compared with the after

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Aβ25-35-injected control group, the expression of ADAM10 was significantly up-regulated in 16


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the ALA-administered group. Subsequently, the sAPPα and the ratio of C83/C99 protein

347

expressions in the normal group of mice were significantly elevated compared with the Aβ25-

348

35-injected

349

and C83/C99 expressions. It indicated that ALA regulated the non-amyloidogenic pathway by

350

elevating the expression levels of ADAM10, sAPPα and C83/C99 more effectively than DHA.

control group. In contrast, ALA supplementation increased the levels of sAPPα

351

352

Effect of ALA on Aβ degradation and clearance

353

Figure 7 showed the effect of ALA and DHA on protein expressions related to clearance and

354

degradation of Aβ. Following Aβ25-35 injection, the levels of NEP and IDE, which are

355

responsible for the degradation of Aβ, were decreased. However, supplementation of ALA

356

resulted in a significant up-regulation of IDE expression, but not NEP, suggesting that ALA

357

may promote Aβ degradation in the mice brain via regulation of IDE enzyme activity. We

358

also demonstrated that Aβ caused increase of the levels of RAGE (Fig. 8). However, ALA did

359

not show the effect on the level of RAGE, and LRP-1 expression was no significant

360

differences among the all groups.

361

362

DISCUSSION

363

The regulation of Aβ metabolism can be useful in inhibiting the progression of AD.

364

Recently, studies on the prevention or treatment of AD have been focused on modulating β-

365

and γ-secretases, or activating α-secretase in APP processing.55,56 Aβ25-35, a stretch of 11-

366

amino acid long residues from position 25 to 35, is produced from full-length Aβ. It has been 17



367

demonstrated that intracerebroventricular injection of this neurotoxic short form of Aβ could

368

induce learning and memory dysfunction.57,58 In agreement with this, our present results also

369

showed that injection of Aβ25-35-induced memory and cognitive impairments as well as

370

oxidative stress through amyloidogenesis. Therefore, we investigated the protective activity

371

and its mechanisms of ALA against Aβ25-35.

372

ALA is the mainly plant-derived essential ω-3 fatty acid. It is the precursor of DHA, and

373

considered to play a crucial role in brain development and function. However, there is still

374

limited and conflicting evidence on the function of ALA because of the low efficiency of

375

conversion of ALA to DHA, particularly in humans. Some clinical and experimental studies

376

reported that ALA-enriched diets exert beneficial effects on ischemia and stroked damage in

377

the brain.59-61 Our previous results also indicated that ALA-enriched perilla oil administration

378

effectively improved the cognitive ability in Aβ-injected mice model.38 Therefore, we

379

speculated that ALA itself may be an independent determinant of neuroprotection against Aβ-

380

induced cognitive decline. It is well established that DHA not only inhibited the Aβ

381

fibrillation but also improved neurobehavioral complications in an AD animal model.62,63

382

Therefore, we used DHA as a positive control for neuroprotection.

383

The T-maze and novel object recognition tests are based on the evidence that normal mice

384

spend longer time exploring novel route/object than familiar ones.64,65 In agreement with

385

previous studies,66,67 our results showed that Aβ25-35-injected control mice did not show

386

significant preference to the newly-introduced route/object compared with the familiar (old)

387

route/object. Interestingly, we found that both ALA- and DHA-administered mice exhibited

388

improvement of cognitive performances and memory ability in both the T-maze and novel

389

object recognition tests as evidenced by the high exploring rate of the unfamiliar route/object. 18


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390

Furthermore, we performed Morris water maze test to determine whether ALA improved

391

spatial learning and long-term memory function in the Aβ25-35-injected mice. The result of

392

present study revealed that memory dysfunction was observed in the mice injected with Aβ25-

393

35,

394

quadrant compared with normal group. However, ALA administration reversed these effects

395

and resulted in significantly shorter escape latencies and prolonged time in the target quadrant

396

than in Aβ25-35-injected control mice. These behavioral neuroprotective effects of ALA in line

397

with recent findings that perilla or ALA diet reversed the impairments in the Morris water

398

maze or T-maze tests after stroke.68,69 Our present findings also provided further evidence on

399

the neuroprotective role of ALA against the Aβ25-35 associated learning and memory

400

dysfunction.

as shown by the increased escape latency and decreased time to spent in the target

401

Brain consists largely of lipids and is easily damaged by free radicals compared to other

402

tissues under physiological conditions. Aβ is neurotoxic and induces oxidative stress in the

403

brain. During this process, MDA, which is the final product of membrane lipid peroxidation,

404

is an important index for determining the level of oxidative stress. In addition, NO has been

405

recognized as a toxic reactive free-radical in the central nervous system. According to this

406

evidence, it has been suggested that the production of NO could influence the cognitive

407

deficits.70 In the current study, we investigated the protective effect of ALA against lipid

408

peroxidation and NO production in the brain, liver and kidneys of Aβ25-35–injected mice. Our

409

results indicated that Aβ25-35 produced high concentrations of NO in the brain, liver and

410

kidneys, whereas ALA or DHA supplemented group prevented excessive NO production.

411

These results can be possible due to our previous result that administration of perilla oil

412

markedly inhibited Aβ25-35-induced NO production via suppression of iNOS and COX-2 19



413

protein expression.38 In addition, several studies have also shown the protective role of ALA

414

on NO generation by blocking iNOS, COX-2 or NF-κB activation in inflammation models.71-

415

72

416

kidneys. In contrast, ALA or DHA administration suppressed the generation of MDA, which

417

suggest that ALA or DHA administration exerts protective effects against lipid peroxidation

418

against Aβ25-35. It has been reported that dietary DHA exerts a protective role from AD by

419

acting as a precursor for neuroprotection against oxidative stress; its mechanism may be

420

attributed to the promotion of antioxidant enzymes.73-75 Moreover, ω-3 fatty acids decreased

421

production of superoxide anion, lactate dehydrogenase, and thiobarbituric acid reactive

422

substances (TBARS) as compared with ω-6 fatty acids.76 From this point of view, the

423

correlation between NO/TBARS and ALA in the present results suggested that the

424

antioxidant effect of ALA may contribute to reduction of cognitive dysfunction in Aβ25-35-

425

infused AD mouse. Indeed, ALA efficacy was confirmed to be at the level of DHA, which

426

was used as a positive control for neuroprotection.

After Aβ25-35 injection, the level of MDA significantly increased in the mice brain, liver and

427

Since our results demonstrated a beneficial role of ALA in the cognitive improvement

428

against Aβ25-35, we assessed the underlying mechanisms of ALA protective actions. The

429

injection of Aβ could induce the amyloidogenic pathway involving the cleavage of BACE

430

and further γ-secretase processing.77 However, administration of ALA resulted in the down-

431

regulation of Aβ25-35-induced BACE and PS2 overexpressions. Similar changes were also

432

observed after DHA administration. Grimm et al.78 also demonstrated that DHA suppressed

433

β- or γ-secretase activity both in SH-SY5Y cells and mice brains. These results suggested that

434

both ALA and DHA inhibited the amyloidogenic pathway by decreasing the activity of β- and

435

γ-secretases. The modulation of α-secretase can be a potential candidate for treating AD.79 20


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436

Although Aβ is toxic, both sAPPα and C83 exert neuro-protective effects through the

437

stimulation of neurite outgrowth and neuronal survival.80 We measured the levels of sAPPα,

438

CTFs, and ADAM10 expressions, which represent alterations in APP processing to the non-

439

amyloidogenic pathway. As compared with control mice, ALA and DHA administration

440

enhanced the expression levels of sAPPα. Furthermore, ALA also significantly increased the

441

levels of C83/C99 and ADAM10, while DHA did not so to a lesser extent. Our findings

442

revealed that ALA could suppress the activity of β- and γ-secretases and it could also be

443

involved in the enhancement of α-secretase activity.

444

Recent studies have highlighted not only the link between Aβ and AD pathology but also

445

the involvement of Aβ production, clearance, and efflux. The reduction of Aβ accumulation

446

occurs through Aβ clearance mechanisms, such as up-regulating Aβ cleaving enzymes, IDE

447

and NEP.81,82 We examined the effect of ALA on the degradation and clearance of Aβ. Lim et

448

al.83 demonstrated that DHA-fed Tg2576 mice exhibited up-regulation of IDE. In our results,

449

Aβ-injected mice showed a decreased IDE and NEP protein expressions compared with

450

normal mice. This tendency was consistent with previous findings, in which the levels of IDE

451

and NEP continually decreased during the progression of AD.84,85 However, ALA- and DHA-

452

administered groups exhibited a reduced inhibition of IDE, thereby lowering Aβ deposition in

453

the mouse brain. On the other hand, the differences in NEP expression were not significant

454

both after ALA- and DHA-administration, compared with the control mice. These results

455

suggested that ALA may inhibit Aβ accumulation, mediated by down-regulation of IDE

456

expression in the brain.

457

RAGE and LRP-1 perform opposite functions into Aβ transportation in the brain. RAGE,

458

which plays a key role in Aβ influx, promotes Aβ transcytosis into the BBB and contributes 21



459

to its transport into the central nervous system.86 On the other hand, LRP-1 clears Aβ from

460

the brain to the blood across the BBB.87 Our results showed that the expression of RAGE was

461

up-regulated in Aβ25-35-injected mice, compared with the normal group. RAGE expression

462

was reduced in mice administered with DHA, but not in those treated with ALA. We did not

463

find any significant changes in level of LRP-1 among all groups.

464

Aβ25-35 is a fragment of full-length Aβ1-42. It has been shown that injection of this peptide

465

into the cerebral ventricle induces neurotoxic effects similar to Aβ1-42 or Aβ1-40. Previous

466

evidences provided that injection of Aβ25-35 is able to elicit the up-regulation of APP and

467

endogenous production of Aβ.88,89 According to Tsuruma et al.,90 Aβ25-35 increased

468

intracellular Aβ1-42 or Aβ1-40, and β- and γ-secretase were activated or up-regulated caused by

469

Aβ stimulation. In addition, injection of Aβ25-35 results in behavioral alterations and modified

470

APP processing through activation of amyloidogenic pathway (up-regulation of C99 levels).91

471

Further study has to be conducted to determine whether ALA shows same biophysiological

472

changes on APP processing and Aβ metabolism using Aβ1-40 or Aβ1-42 peptide.

473

Our study demonstrated that administration of ALA prevented learning and memory

474

impairments in the AD mouse model as evidenced by their performance on the T-maze, novel

475

object recognition, and Morris water maze test. ALA attenuated Aβ25-35–induced oxidative

476

stress by inhibiting the overproduction of MDA and NO levels in the brain, liver and kidneys.

477

These findings provided evidence that ALA may be a potentially valuable source for the

478

treatment of AD. Furthermore, these protective effects of ALA are related to the regulation of

479

APP processing by non-amyloidogenic pathway (down-regulation of BACE, PS2; up-

480

regulation of sAPPα, C83/C99 and ADAM10 protein expressions), as well as increase

481

expression of degrading Aβ enzyme, IDE. In conclusion, the present study indicated that 22


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482

ALA could protect from cognitive dysfunction and memory impairment against Aβ25-35, and

483

these beneficial effects are comparable to those of DHA.

484

485

ABBREVIATIONS USED

486

Aβ, amyloid beta; AD, Alzheimer’s disease; ADAM, A distintegrin and metalloprotease; ALA,

487

alpha-linolenic acid; APP, amyloid precursor protein; BACE, β-site APP cleaving enzyme;

488

BBB, blood-brain-barrier; C83, C-terminal fragment alpha; C99, C-terminal fragment beta;

489

DHA, docosahexahenoic acid; IDE, insulin-degrading enzyme; LRP-1, low-density

490

lipoprotein receptor-related protein-1; MDA, malondialdehyde; NaCl, Sodium chloride;

491

NaNO2, sodium nitrite; NEP, neprilysin; NO, nitric oxide; PBS-T, phosphate-buffered saline

492

with Tween-20; PS, presenilin; RAGE, receptor for advanced end glycation; sAPPα, soluble

493

neurotrophic fragment; SDS-PAGE, sulfate polyacrylamide gel electrophoresis; TBA,

494

thiobarbituric acid; TBARS, thiobarbituric reactive substances

495

496

ACKNOWLEDGMENTS

497

This work was carried out with the support of the “Cooperative Research Program for

498

Agriculture Science & Technology Development (PJ01015603),” Rural Development

499

Administration, Republic of Korea.

23



500

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amelioration on impairment of memory learning in amyloid β-infused rats relates to the

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decreases of amyloid β and cholesterol levels in detergent-insoluble membrane fractions.

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Oligonol improves memory and cognition under an amyloid β25-35-induced Alzheimer’s

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mouse model. Nutr. Res. 2014, 34, 595-603.

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(67)Choi, J. Y.; Cho, E. J.; Lee, H. S.; Lee, J. M.; Yoon, Y. H.; Lee, S. Tartary buckwheat

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parenteral nutritional intervention against sensorimotor and cognitive deficits in a mouse

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(69)Lee, J.; Park, S.; Lee, J. Y.; Yeo, Y. K.; Kim, J. S.; Lim, J. Improved spatial learning and

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synuclein in hilus of dentate gyrus. Proteome Sci. 2012, 10, 1-12.

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is an anti-inflammatory agent in inflammatory bowel disease. J. Nutr. Biochem. 2005, 26,

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(72)Ren, J.; Chung, S. H. Anti-inflammatory effect of α-linolenic acid and its mode of action

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through the inhibition of nitric oxide production and inducible nitric oxide synthase gene

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expression via NF-κB and mitogen-activated protein kinase pathways. J. Agric. Food

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Chem. 2007, 55, 5073-5080.

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(73)Hossain, M. S.; Hashimoto, M.; Gamoh, S.; Masumura, S. Antioxidative effects of

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docosahexaenoic acid in the cerebrum versus cerebellum and brainstem of aged

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hypercholesterolemic rats. J. Neurochem. 1999, 72, 1133-1138.

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(74)Bazan, N. G. Cell survival matters: docosahexaenoic acid signaling, neuroprotection and photoreceptors. Trends Nerusci. 2006, 29, 263-271.

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Serhan, C. N.; Bazan, N. G. A role for docosahexaenoic acid-derived neuroprotectin D1

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in neural cell survival and Alzheimer disease. J. Clin. Invest. 2005, 115, 2774.

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B.; Han, S. B.; Chung, H. M.; Hong, J. T. Placenta-derived mesechymal stem cells

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improve memory dysfunction in an Aβ1-42-infused mouse model of Alzheimer’s disease. 33



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L.; Friess, P.; de Wilde, M. C.; Broersen, L. M.; Penke, B.; Péter, M.; Vigh, L.; Grimm, H.

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S.; Hartmann, T. Docosahexaenoic acid reduces amyloid β production via multiple

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pleiotropic mechanisms. J. Biol. Chem. 2011, 286, 14028-14039.

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(79)Nunan, J.; Small, D. H. Regulation of APP cleavage by α-, β-, and γ-secretases. FEBS Lett. 2000, 483, 6-10.

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Hof, P. R.; Koo, E. H.; Dickstein, D. L. Amyloid precursor protein (APP) regulates

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synaptic structure and function. Mol. Cell. Neurosci. 2012, 51, 43-52.

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(81)Vekrellis, K.; Ye, Z.; Qiu, W. Q.; Walsh, D.; Hartley, D.; Chesneau, V.; Rosner, M. R.;

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Selkoe, D. J. Neurons regulate extracellular levels of amyloid β-protein via proteolysis by

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insulin-degrading enzyme. J. Neurosci. 2000, 20, 1657-1665.

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(82)Iwata, N.; Tsubuki, S.; Takaki, Y.; Shirotani, K.; Lu, B.; Gerard, N. P.; Gerard, C.; Hama,

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(83)Lim, G. P.; Calon, F.; Morihara, T.; Yang, F.; Teter, B.; Ubeda, O.; Salem, N.; Frautschy,

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S. A.; Cole, G. M. A diet enriched with the Omega-3 fatty acid docosahexaenoic acid

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reduces amyloid burden in an aged Alzheimer mouse model. J. Neurosci. 2005, 25, 3032-

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Hersh, L. B. Neprilysin regulates amyloid beta peptide levels. J. Mol. Neurosci. 2004, 22,

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5-11.

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(85)Bulloj, A.; Leal, M. C.; Xu, H.; Castaño, E. M.; Morelli, L. Insulin-degrading enzyme

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sorting in exosomes: a secretory pathway for a key brain amyloid-beta degrading

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protease. J. Alzheimer’s Dis. 2010, 19, 79-95.

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(86)Deane, R.; Du Yan, S.; Submamaryan, R. K.; LaRue, B.; Jovanovic, S.; Hogg, E.; Welch,

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D.; Manness, L.; Lin, C.; Yu, J.; Zhu, H.; Ghiso, J.; Frangione, B.; Stern, A.; Marie

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Schmidt, A.; Armstrong, D. L.; Arnold, B.; Liliensiek, B.; Nawroth, P.; Hofman, F.;

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Kindy, M.; Stern, D.; Zlokovic, B. RAGE mediates amyloid-β peptide transport across

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the blood-brain barrier and accumulation in brain. Nat. Med. 2003, 9, 907-913.

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(87)Shibata, M.; Yamada, S.; Ram Kumar, S.; Calero, M.; Badding, J.; Frangione, B.;

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Holtzman, D. M.; Miller, C. A.; Strickland, D. K.; Ghiso, J.; Zlokovic, B. V. Clearance of

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Alzheimer’s amyloid-β1-40 peptide from brain by LDL receptor-related protein-1 at the

741

blood-brain barrier. J. Clin. Invest. 2000, 106, 489-499.

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(88)Whitehead, S. N.; Hachinski, V. C.; Cechetto, D. F. Interaction between a rat model of

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cerebral ischemia and beta-amyloid toxicity: inflammatory responses. Stroke 2005, 36,

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107-112.

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(89)Cheng, G.; Whitehead, S. N.; Hachinski, V.; Cechetto, D. F. Effects of pyrrolidine

746

dithiocarbamate on beta-amyloid (25-35)-induced inflammatory responses and memory

747

deficits in the rat. Neurobiol. Dis. 2006, 23, 140-151.

748

(90)Tsuruma, k.; Tanaka, Y.; Shimazawa, M.; Hara, H. Inudction of amyloid precursor 35



749

protein by the neurotoxic peptide, amyloid beta-25-35, causes retinal ganglion cell death.

750

J. Neurochem. 2010, 113, 1545-1554.

751

(91)Zussy, C.; Brureau, A.; Delair, B.; Marchal, S.; keller, E.; Lxart, G.; Naert, G.; Meunier,

752

J.; Chevallier, N.; Maurice, T.; Givalois, L. Time-course and regional analyses of the

753

physiopathological changes induced after cerebral injection of an amyloid β fragment in

754

rats. Am. J. Pathol. 2011, 179, 315-334.

755

36


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Page 37 of 60


756

FIGURE CAPTIONS

757

Figure 1. Behavioral experimental schedule for mice injected with Aβ25-35.

758

759

Figure 2. Effects of alpha-linolenic acid on the spatial perceptive ability in the T-maze test in

760

Aβ25-35-injected mice. Data are presented as mean ± SD (n=6). *The space perceptive abilities

761

for old and new routes are significantly different as determined by Student’s t-test (P < 0.05).

762

ALA: Oral administration of alpha-linolenic acid (100 mg/kg/day) DHA: Oral administration

763

of docosahexaenoic acid (100 mg/kg/day).

764

765

Figure 3. Effects of alpha-linolenic acid on recognition memory in the novel object

766

recognition test in Aβ25-35-injected mice. Data are presented as mean ± SD (n=6). *The object

767

perceptive abilities for familiar and novel objects are significantly different as determined by

768

student’s t-test (P < 0.05). ALA: Oral administration of alpha-linolenic acid (100 mg/kg/day)

769

DHA: Oral administration of docosahexaenoic acid (100 mg/kg/day).

770

771

Figure 4. (A) Effects of alpha-linolenic acid on escape latency to the platform in the Morris

772

water maze test in Aβ25-35-injected mice. (B) Effects of alpha-linolenic acid on latency to

773

reach the hidden platform in the Morris water maze test on the final test day in Aβ25-35-

774

injected mice. (C) Effects of alpha-linolenic acid on occupancy time of the target quadrant in

775

the Morris water maze test in Aβ25-35-injected mice. The percentage of time spent in the target

776

quadrant was calculated in the water maze test on the final test day. (D) Effects of alpha37



Page 38 of 60

777

linolenic acid on latency to reach the exposed platform in the Morris water maze test in Aβ25-

778

35-injected

779

significantly different (P < 0.05) from each other. NS: No significance ALA: Oral

780

administration of alpha-linolenic acid (100 mg/kg/day) DHA: Oral administration of

781

docosahexaenoic acid (100 mg/kg/day).

mice. Data represent mean ± SD (n=5).

a-b

Means with different letters are

782

783

Figure 5. Effects of alpha-linolenic acid on BACE, PS1 and PS2 protein expression levels in

784

the Aβ25-35-injected mice brain. Data represent mean ± SD (n=3).

785

letters are significantly different (P < 0.05) from each group. ALA: Oral administration of

786

alpha-linolenic acid (100 mg/kg/day) DHA: Oral administration of docosahexaenoic acid

787

(100 mg/kg/day).ALA: Oral administration of alpha-linolenic acid (100 mg/kg/day) DHA:

788

Oral administration of docosahexaenoic acid (100 mg/kg/day). β-actin was used as a loading

789

control.

a-d

Means with different

790

791

Figure 6. Effects of alpha-linolenic acid on sAPPα, CTFs, and ADAM10 protein expression

792

levels in the Aβ25-35-injected mice brain. Data represent mean ± SD (n=3).

793

different letters are significantly different (P < 0.05) from each group. ALA: Oral

794


795

docosahexaenoic acid (100 mg/kg/day). β-actin was used as a loading control.

796

38


a-d

Means with

Page 39 of 60


797

Figure 7. Effects of alpha-linolenic acid on IDE and NEP protein expression levels in the

798

Aβ25-35-injected mice brain. Data represent mean ± SD (n=3).

799

are significantly different (P < 0.05) from each group. ALA: Oral administration of alpha-

800

linolenic acid (100 mg/kg/day) DHA: Oral administration of docosahexaenoic acid (100

801

mg/kg/day). β-actin was used as a loading control.

a-c

Means with different letters

802

803

Figure 8. Effects of alpha-linolenic acid on RAGE and LRP-1 protein expression levels in

804


805

letters are significantly different (P < 0.05) from each group. NS: No significance. ALA: Oral

806


807


39


a-c



808

Page 40 of 60

Table 1. Change of body weight in Aβ25-35-injected mice Body weight (g) Group Stocked day

NS

Injection

33.82 ± 2.81

NS

Oral

Behavioral

administration

experiment

33.97 ± 2.38

NS

35.50 ± 2.71

Dissection

NS

35.50 ± 2.90

NS

Normal

25.13 ± 1.72

Control

24.83 ± 1.34

33.75 ± 1.34

33.93 ± 1.65

35.80 ± 1.63

36.47 ± 1.07

ALA

25.60 ± 1.07

32.48 ± 0.72

34.15 ± 1.66

35.80 ± 1.89

33.90 ± 2.08

DHA

25.43 ± 1.35

32.87 ± 2.04

34.22 ± 2.45

34.58 ± 2.68

34.47 ± 3.07

809

Data are presented as mean ± SD (n=6). NS: No significance. ALA: Oral administration of

810


811

(100 mg/kg/day).

40


Page 41 of 60


812

Table 2. Effect of alpha-linolenic acid on lipid peroxidation (A) and NO generation (B)

813

in the brain, liver and kidney of Aβ25-35-injected mice. (A) MDA (nmol/mg protein) Group Brain

Liver b

Normal

17.99 ± 5.68

Control

26.16 ± 0.97a

ALA

20.77 ± 3.85

DHA

20.42 ± 1.39b

Kidney c

b

d

18.71 ± 2.05

35.52 ± 3.82

48.13 ± 2.43a

70.52 ± 2.83a

b

b

27.68 ± 1.89

63.66 ± 3.97

20.93 ± 2.99c

58.01 ± 4.63c

(B) NaNO2 (nmol/mg protein) Group Brain

Liver

Kidney

Normal

16.74 ± 1.04b

17.30 ± 0.96c

20.59 ± 0.80c

Control

19.41 ± 1.14

ALA

DHA

a

a

a

49.29 ± 4.83

28.09 ± 0.62

16.48 ± 2.70b

27.17 ± 1.67b

24.99 ± 4.76b

16.90 ± 0.93b

26.87 ± 2.12b

24.79 ± 0.98b

814 a-d

815

Data are presented as mean ± SD (n=6).

816

different (P < 0.05) from each group. ALA: Oral administration of alpha-linolenic acid (100

817

mg/kg/day) DHA: Oral administration of docosahexaenoic acid (100 mg/kg/day).

Means with different letters are significantly

41



818 819

Figure 1. Behavioral experimental schedule for mice injected with Aβ25-35.

42


Page 42 of 60

Page 43 of 60


60

*

*

*

Space perceptive ability (%)

50

40 old route 30

new route

20

10

0 Normal

Control

ALA

DHA

820 821

Figure 2. Effects of alpha-linolenic acid on the spatial perceptive ability in the T-maze test in

822

Aβ25-35-injected mice. Data are presented as mean ± SD (n=6). *The space perceptive abilities

823

for old and new routes are significantly different as determined by Student’s t-test (P < 0.05).

824

ALA: Oral administration of alpha-linolenic acid (100 mg/kg/day) DHA: Oral administration

825

of docosahexaenoic acid (100 mg/kg/day).

43



80

*

*

Page 44 of 60

*

Object perceptive ability (%)

70 60 50 familiar object

40

novel object 30 20 10 0

826

Normal

Control

ALA

DHA

827

Figure 3. Effects of alpha-linolenic acid on recognition memory in the novel object

828

recognition test in Aβ25-35-injected mice. Data are presented as mean ± SD (n=6). *The object

829

perceptive abilities for familiar and novel objects are significantly different as determined by

830

student’s t-test (P < 0.05). ALA: Oral administration of alpha-linolenic acid (100 mg/kg/day)

831

DHA: Oral administration of docosahexaenoic acid (100 mg/kg/day).

44



Control

ALA

DHA

70 60 50 a

40 30

1 day

Tertiary

Secondary

Primary

Tertiary

2 day

3 day

(D)

a

40 b 30 20 10 0 Control

ALA

a 60 50 40 30 20

b

b

b

ALA

DHA

10

Normal

4 day

50

Normal

70

0

a

a

(B)

Latency to reach platform (seconds)

60

Secondary

Primary

Secondary

0

Tertiary

10

Final

b b b

20

(C) Occupancy in target quadrant (%)

Normal

80

Primary

Latency to reach plarform (seconds)

(A)

Latency to reach platform (seconds)

Page 45 of 60

Control

35 NS 30 25 20 15 10 5 0

DHA

Normal

Control

ALA

DHA

832

Figure 4. (A) Effects of alpha-linolenic acid on escape latency to the platform in the Morris

833

water maze test in Aβ25-35-injected mice. (B) Effects of alpha-linolenic acid on latency to

834

reach the hidden platform in the Morris water maze test on the final test day in Aβ25-35-

835

injected mice. (C) Effects of alpha-linolenic acid on occupancy time of the target quadrant in

836

the Morris water maze test in Aβ25-35-injected mice. The percentage of time spent in the target

837

quadrant was calculated in the water maze test on the final test day. (D) Effects of alpha45



838

linolenic acid on latency to reach the exposed platform in the Morris water maze test in Aβ25-

839

35-injected

840

significantly different (P < 0.05) from each other. NS: No significance ALA: Oral

841


842

docosahexaenoic acid (100 mg/kg/day).

mice. Data represent mean ± SD (n=5).

a-b

Means with different letters are

46


Page 46 of 60

Page 47 of 60


Normal Control

ALA

DHA

BACE PS1 PS2 β-actin 843

Protein expression (fold of normal)

1.6

a a

1.4 1.2 d

c

a

b

a

c

a b

b c

1

Normal

0.8

Control ALA

0.6

DHA 0.4 0.2 0 BACE

PS1

PS2

844

Figure 5. Effects of alpha-linolenic acid on BACE, PS1 and PS2 protein expression levels

845

in the Aβ25-35-injected mice brain. Data represent mean ± SD (n=3).

846

letters are significantly different (P < 0.05) from each group. ALA: Oral administration of

847


848

(100 mg/kg/day).ALA: Oral administration of alpha-linolenic acid (100 mg/kg/day) DHA:

849

Oral administration of docosahexaenoic acid (100 mg/kg/day). β-actin was used as a loading

850

control.

47


a-d



Normal

Control

ALA

Page 48 of 60

DHA

sAPPα CTFβ (C99) CTFα (C83)

ADAM10 β-actin Protein expression (fold of normal)

1.4 1.2

a c

1

a b

a

b

d

c

0.8

Normal Control

0.6

ALA

0.4 c

0.2

DHA

b

d d

0 851

sAPPα

C83/C99

ADAM10

852

Figure 6. Effects of alpha-linolenic acid on sAPPα, CTFs, and ADAM10 protein expression

853

levels in the Aβ25-35-injected mice brain. Data represent mean ± SD (n=3).

854

different letters are significantly different (P < 0.05) from each group. ALA: Oral

855


856


48


a-d

Means with

Page 49 of 60


Normal

Control

ALA

DHA

IDE NEP

Protein expression (fold of normal)

β-actin 1.4 1.2 1

a ab

b

a c

0.8

b

b

b

Normal Control ALA

0.6

DHA

0.4 0.2 0 IDE

NEP

857 858

Figure 7. Effects of alpha-linolenic acid on IDE and NEP protein expression levels in the

859

Aβ25-35-injected mice brain. Data represent mean ± SD (n=3).

860

are significantly different (P < 0.05) from each group. ALA: Oral administration of alpha-

861

linolenic acid (100 mg/kg/day) DHA: Oral administration of docosahexaenoic acid (100

862

mg/kg/day). β-actin was used as a loading control.

a-c

49


Means with different letters


Normal

Control

ALA

Page 50 of 60

DHA

RAGE LRP-1 β-actin

Protein epxressions (fold of normal)

1.4 1.2

a

a

NS

b 1

c Normal

0.8

Control

0.6

ALA

0.4

DHA

0.2 0 RAGE

LRP-1

863

864

Figure 8. Effects of alpha-linolenic acid on RAGE and LRP-1 protein expression levels in

865


866

letters are significantly different (P < 0.05) from each group. NS: No significance. ALA: Oral

867


868


50


a-c


Page 51 of 60

869


TOC Graphics

870

51



Figure 1. Behavioral experimental schedule for mice injected with Aβ25-35. 250x70mm (96 x 96 DPI)


Page 52 of 60

Page 53 of 60


Effects of alpha-linolenic acid on the spatial perceptive ability in the T-maze test in Aβ25-35-injected mice. Data are presented as mean ± SD (n=6). *The space perceptive abilities for old and new routes are significantly different as determined by Student’s t-test (P < 0.05). ALA: Oral administration of alphalinolenic acid (100 mg/kg/day) DHA: Oral administration of docosahexaenoic acid (100 mg/kg/day). 158x105mm (96 x 96 DPI)



Effects of alpha-linolenic acid on recognition memory in the novel object recognition test in Aβ25-35-injected mice. Data are presented as mean ± SD (n=6). *The object perceptive abilities for familiar and novel objects are significantly different as determined by student’s t-test (P < 0.05). ALA: Oral administration of alphalinolenic acid (100 mg/kg/day) DHA: Oral administration of docosahexaenoic acid (100 mg/kg/day). 159x96mm (96 x 96 DPI)


Page 54 of 60

Page 55 of 60


(A) Effects of alpha-linolenic acid on escape latency to the platform in the Morris water maze test in Aβ25-35injected mice. (B) Effects of alpha-linolenic acid on latency to reach the hidden platform in the Morris water maze test on the final test day in Aβ25-35-injected mice. (C) Effects of alpha-linolenic acid on occupancy time of the target quadrant in the Morris water maze test in Aβ25-35-injected mice. The percentage of time spent in the target quadrant was calculated in the water maze test on the final test day. (D) Effects of alphalinolenic acid on latency to reach the exposed platform in the Morris water maze test in Aβ25-35-injected mice. Data represent mean ± SD (n=5). a-bMeans with different letters are significantly different (P < 0.05) from each other. NS: No significance ALA: Oral administration of alpha-linolenic acid (100 mg/kg/day) DHA: Oral administration of docosahexaenoic acid (100 mg/kg/day). 175x165mm (96 x 96 DPI)



Effects of alpha-linolenic acid on BACE, PS1 and PS2 protein expression levels in the Aβ25-35-injected mice brain. Data represent mean ± SD (n=3). a-dMeans with different letters are significantly different (P < 0.05) from each group. ALA: Oral administration of alpha-linolenic acid (100 mg/kg/day) DHA: Oral administration of docosahexaenoic acid (100 mg/kg/day).ALA: Oral administration of alpha-linolenic acid (100 mg/kg/day) DHA: Oral administration of docosahexaenoic acid (100 mg/kg/day). β-actin was used as a loading control. 127x147mm (96 x 96 DPI)


Page 56 of 60

Page 57 of 60


Effects of alpha-linolenic acid on sAPPα, CTFs, and ADAM10 protein expression levels in the Aβ25-35-injected mice brain. Data represent mean ± SD (n=3). a-dMeans with different letters are significantly different (P < 0.05) from each group. ALA: Oral administration of alpha-linolenic acid (100 mg/kg/day) DHA: Oral administration of docosahexaenoic acid (100 mg/kg/day). β-actin was used as a loading control. 127x146mm (96 x 96 DPI)



Effects of alpha-linolenic acid on IDE and NEP protein expression levels in the Aβ25-35-injected mice brain. Data represent mean ± SD (n=3). a-cMeans with different letters are significantly different (P < 0.05) from each group. ALA: Oral administration of alpha-linolenic acid (100 mg/kg/day) DHA: Oral administration of docosahexaenoic acid (100 mg/kg/day). β-actin was used as a loading control. 127x127mm (96 x 96 DPI)


Page 58 of 60

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Effects of alpha-linolenic acid on RAGE and LRP-1 protein expression levels in the Aβ25-35-injected mice brain. Data represent mean ± SD (n=3). a-cMeans with different letters are significantly different (P < 0.05) from each group. NS: No significance. ALA: Oral administration of alpha-linolenic acid (100 mg/kg/day) DHA: Oral administration of docosahexaenoic acid (100 mg/kg/day). β-actin was used as a loading control. 127x129mm (96 x 96 DPI)



TOC Graphics 254x190mm (96 x 96 DPI)


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Alpha-Linolenic Acid from Perilla frutescens var. japonica Oil Protects

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