Effects of docosahexaenoic acid on in vitro amyloid beta peptide 25–35 fibrillation Michio Hashimoto a, ⁎, Hossain Md Shahdat a,b , Masanori Katakura a , Yoko Tanabe a , Shuji Gamoh a , Koji Miwa a , Toshio Shimada c , Osamu Shido a a Department of Environmental Physiology, Shimane University Faculty of Medicine, Izumo 693-8501, Shimane, Japan b Department of Biochemistry and Molecular Biology, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh c Department of Internal Medicine, Shimane University Faculty of Medicine, Izumo 693-8501, Shimane, Japan abstract article info Article history: Received 9 October 2008 Received in revised form 22 December 2008 Accepted 16 January 2009 Available online 29 January 2009 Keywords: Docosahexaenoic acid Alzheimer's disease Aβ 25–35 fibrillation Amyloid β peptide 25–35 (Aβ 25–35 ) encompasses one of the neurotoxic domains of full length Aβ 1–40/42 , the major proteinaceous component of amyloid deposits in Alzheimer's disease (AD). We investigated the effect of docosahexaenoic acid (DHA, 22:6, n-3), an essential brain polyunsaturated fatty acid, on the in vitro fibrillation of Aβ 25–35 and found that it significantly reduced the degree of fibrillation, as shown by a decrease in the intensity of both the thioflavin T and green fluorescence in confocal microscopy. Transmission electron microscopy revealed that DHA-incubated samples were virtually devoid of structured fibrils but had an amorphous-like consistency, whereas the controls contained structured fibers of various widths and lengths. The in vitro fibrillation of Aβ 25–35 appeared to be pH-dependent, with the strongest effect seen at pH 5.0. DHA inhibited fibrillation at all pHs, with the strongest effect at pH 7.4. It also significantly decreased the levels of Aβ 25–35 oligomers. Nonreductive gradient gel electrophoresis revealed that the molecular size of the oligomers of Aβ 25–35 was 10 kDa (equivalent to decamers of Aβ 25–35 ) and that DHA dose-dependently reduced these decamers. These results suggest that DHA decreases the in vitro fibrillation of Aβ 25–35 by inhibiting the oligomeric amyloid species and, therefore, Aβ 25–35 -related neurotoxicity or behavioral impairment could be restrained by DHA. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Deposition of insoluble neuritic plaques and neurofibrillar tangles of amyloid β peptides (Aβ) in the brain is the neuropathological stamp of Alzheimer's disease (AD), characterized by progressive loss of neurons and the deterioration of memory-related learning ability [1]. The major components of neuritic plaques are the 40–42-amino acid- residue-long Aβs that are proteolytically released from membrane- bound amyloid precursor proteins (APP) [2]. After intersecting with the environment, these Aβs self-transform from their native coiled α- helical structures into insoluble fibrillar forms [3]. Although Aβ 1–40 and Aβ 1–42 are the predominant forms, other fragments can be present as well: in the brains of aged patients, Aβ 25–35 , a stretch of 11 amino acid- long residues of the full length from position 25 to 35, is produced by the proteolysis of full-length Aβ 1–40 [4]. The Aβ 25–35 fragment is biologically active and analogous to its full length Aβs [5]; it is thus conceivable that learning-related memory impairment could also be induced by the infusion of this short amyloid into the cerebral ventricle of rats. The hypothesis that Aβ 25–35 forms fibrils and confers toxicity analogous to that of the full-length peptide has been proved by in vitro studies with neurons [6]. These findings are further supported by in vivo studies demonstrating that direct cerebral infusion of Aβ 25–35 into the ventricle impairs memory in mice [7,8]. We have previously reported that the memory impairment of Aβ 1–40 -infused AD model rats is ameliorated and/or prevented by the dietary administration of docosahexaenoic acid (DHA, C22:6, n-3) [9,10]. The brain utilizes large amounts of DHA before birth, during growth spurts and throughout the growing periods [11–12]. The level of DHA decreases in the hippocampus of AD patients [13]. Accordingly, dietary intervention with DHA has been shown to improve AD-related symptoms [14], and supplementation with DHA has been associated with the improve- ment of neurobehavioral complications in AD model animals [15]. Recently, it has been shown that DHA significantly decreases the cerebral cortical levels of Aβ peptides with concomitant ameliorative effects on memory-related learning ability [9,10,15]. Though the amyloid deposition is considered as one of the key steps in the pathogenesis of AD and DHA could be targeted as one of the potential therapies to clear up the amyloid deposits [9,10,15], the mechanism of the action of DHA on the in vitro fibrillation is scarcely reported. In vitro polymerization is postulated to be a model that explains well the mechanistic milieu of fibrillation of Aβ proteins in AD. Thus, the direct interruption of amyloid fibril formation by DHA would be predicted to relate its Aβ-clearance from the in vivo scenario of AD brain. The effect of DHA on neurodegenerative diseases is very significant in that DHA benefits brain functions. Numerous structural and functional studies on full-length Aβ s have also stressed the role of Aβ 25–35 in neurotoxicity: Aβ 25–35 induces neuronal cell death [16], Biochimica et Biophysica Acta 1791 (2009) 289–296 ⁎ Corresponding author. Tel.: +81853 20 2110; fax: +81853 20 2110. E-mail address: michio1@med.shimane-u.ac.jp (M. Hashimoto). 1388-1981/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2009.01.012 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbalip