A Peptide Binding to the b-Site of APP Improves Spatial Memory and Attenuates Ab Burden in Alzheimer’s Disease Transgenic Mice Shi-gao Yang1., Shao-wei Wang1,2., Min Zhao1, Ran Zhang1, Wei-wei Zhou1, Ya-nan Li1,3, Ya-jing Su1,3, He Zhang1, Xiao-lin Yu2*, Rui-tian Liu1,2* 1 Tsinghua University School of Medicine, Haidian District, Beijing, China, 2 National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, China, 3 School of Life Sciences, Ningxia University, Yinchuan, China Abstract Amyloid precursor protein cleaving enzyme 1 (BACE1), an aspartyl protease, initiates processing of the amyloid precursor protein (APP) into b-amyloid (Ab); the peptide likely contributes to development of Alzheimer’s disease (AD). BACE1 is an attractive therapeutic target for AD treatment, but it exhibits other physiological activities and has many other substrates besides APP. Thus, inhibition of BACE1 function may cause adverse side effects. Here, we present a peptide, S1, isolated from a peptide library that selectively inhibits BACE1 hydrolytic activity by binding to the b-proteolytic site on APP and Ab N-terminal. The S1 peptide significantly reduced Ab levels in vitro and in vivo and inhibited Ab cytotoxicity in SH-SY5Y cells. When applied to APPswe/PS1dE9 double transgenic mice by intracerebroventricular injection, S1 significantly improved the spatial memory as determined by the Morris Water Maze, and also attenuated their Ab burden. These results indicate that the dual-functional peptide S1 may have therapeutic potential for AD by both reducing Ab generation and inhibiting Ab cytotoxicity. Citation: Yang S-g, Wang S-w, Zhao M, Zhang R, Zhou W-w, et al. (2012) A Peptide Binding to the b-Site of APP Improves Spatial Memory and Attenuates Ab Burden in Alzheimer’s Disease Transgenic Mice. PLoS ONE 7(11): e48540. doi:10.1371/journal.pone.0048540 Editor: Zhongcong Xie, Massachusetts General Hospital, United States of America Received March 15, 2012; Accepted September 26, 2012; Published November 1, 2012 Copyright: ß 2012 Yang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the National Natural Science Foundation of China (NSFC) (30971012 and 81171014), the National Science and Technology Major Projects of New Drugs (2012ZX09103301-001). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] (RTL); [email protected] (XLY) . These authors contributed equally to this work. Introduction secretase modulators (GSMs) which selectively lower Ab42 without interfering with the physiological function of c-secretase Accumulation and aggregation of b-amyloid (Ab) likely plays were studied. The results indicate that GSMs may be promising a critical role in AD pathogenesis [1,2]. Inhibition of Ab therapeutics for the treatment of AD [9–11]. Previous reports production and prevention of Ab aggregation, and enhancement demonstrated that BACE1 levels are elevated in postmortem AD of Ab clearance, are appealing strategies to thwart the onset and brains [12–17] and in neurons around amyloid plaques [18]. progression of AD. Ab is produced by sequential cleavage of Ab Moreover, BACE1 levels rise following physiological stress or precursor protein (APP) by b-amyloid precursor protein cleaving injury, such as oxidative stress by Ab, hypoxia [19], and energy enzyme 1 (BACE1) and c-secretase. BACE1 initiates proteolysis of inhibition [20]. Furthermore, overexpression of BACE1 in trans- APP at the N terminus of Ab, forming a large soluble fragment, genic mice accelerates amyloid pathology and neurodegeneration. sAPPb, and the remaining membrane-bound C terminal fragment BACE1 has therefore become an attractive therapeutic target for (C-99). C-99 is then cleaved by c-secretase to form either Ab40 or AD, and many BACE1 inhibitors were reported and showed Ab42 [3,4]. Under normal metabolic conditions, most APP can be potential application in AD treatment [21–23]. However, in processed through an alternative non-amyloidogenic pathway [5]. addition to APP, many substrates, including P-selectin glycopro- Alpha-secretase initiates proteolysis of APP at the peptide bond tein ligand-1 [24], sialyl transferase ST6Gal [25,26], b-subunits of between Lys16 and Leu17 of Ab, producing the soluble sAPPa voltage-gated sodium channels [27], APP-like proteins [28], and fragment and the remaining membrane-bound C terminal the type III isoform of the epidermal growth factor-like factor fragment (C-83). C-83 is then further cleaved by c-secretase to neuregulin 1 (type III-NRG1) [29] are also targets for BACE1 form the p3 peptide instead of Ab. To lower Ab generation, cleavage. Besides, BACE1 plays a role in myelination in the extensive efforts have targeted a, b and c-secretase [4,6,7]. peripheral and central nervous systems during development, and However, c-secretase also cleaves other substrates including may have cognitive and synaptic functions independent of APP Notch, and therapeutic inhibition of c-secretase may lead to toxic processing [29–31]. Some reports have indicated that down- side effects, due to the impact on the important signaling pathways regulation of BACE1 reduces Ab loads effectively and BACE1 and other activities [8]. To avoid these side effects, some c- knockout mice are healthy, fertile and have no histological PLOS ONE | www.plosone.org 1 November 2012 | Volume 7 | Issue 11 | e48540 A Peptide Improves Memory and Reduces Ab Burden pathologies [32–34]. Other studies reported serious morbid effects, a water bath for 10 min, aliquoted into microcentrifuge tubes, like early death, reduced size, and cognitive deficits in BACE1- dried under vacuum, and stored at 220uC. Immediately prior to knockout animals, which suggest the potential liabilities of BACE1 use, the HFIP-treated Ab42 was dissolved in dimethylsulfoxide inhibition [35,36]. Therefore, inhibition of BACE1 activity may (DMSO) to 1 mg/mL and diluted to 10 mM in PBS, pH 7.4. All also block physiological processing, thus leading to various side the other peptides containing the b-secretase site of APP and the effects [25,26,29]. An agent that can bind to the b-cleavage site of N-terminal peptides of Ab, EVKMDAEFRHDS (Ab-4-8), APP may inhibit the production of Ab without the potential EVKMDAEF (Ab-4-4), EVKMDAE (Ab-4-3), DAEFRHDS adverse effects of BACE1 inhibition. Similar approaches were (Ab1-8) and FRHDS (Ab4-8) (figure 1A) were synthesized by demonstrated with a monoclonal antibody and protein that bind GL Biochem Ltd. Co (Shanghai, China). The following antibodies to the b-cleavage site of APP [37–39]. As of yet, only a few b-site- were used: A8717 (rabbit polyclonal raised against C-terminal of directed antibodies and few peptide have been reported to APP, C99 and C83, Sigma-Aldrich Corp. St. Louis, MO, USA), improve cognitive function and reduce neuropathology in vivo HRP-9E10 (HRP-conjugated anti-M13 antibody, Santa Cruz, [37,38,40,41]. Using phage display, we report on a peptide that USA), and HRP-conjugated mouse anti-His tag monoclonal inhibits Ab generation and cytotoxicity, and attenuates memory antibody (Gold Bridge Co., Beijing, China). Both Ab40 and deficits and decreases Ab burden in AD transgenic mice, by Ab42 kits for Ab measurement were purchased from IBL Co. Ltd. binding to the b-secretase cleavage site of APP and to the N- (Gunma, Japan). terminal of Ab. Bio-Panning Using the Phage Display Library Materials and Methods Peptide selection was performed using three rounds of panning, essentially as described by the manufacturer with slight modifica- Materials tions. Amine-binding 96-well microtiter plates were coated with The Ph.D.-C7C disulfide constrained peptide library kit 1 mg/mL of Ab-4-8 in PBS (10 mM phosphate, 150 mM NaCl, 9 encoding 1.2610 random 7-amino acid insertions was obtained pH 9.0) overnight at 4uC. Plates were blocked with 5% BSA for at from New England Biolabs. Ab42 was purchased from the least 2 h at room temperature. An aliquot of 261011 phage units American Peptide Company (Sunnyvale, CA, USA). For Ab42 from the Ph.D.-C7C library was incubated with the peptide. Plates preparation, Ab42 was dissolved in 100% 1,1,1,3,3,3-hexafluoro- were thoroughly washed with TBST (TBS (50 mM Tris, 2-propanal (HFIP) to a concentration of 1 mg/mL, sonicated in 150 mM NaCl, pH7.5)+0.1% Tween-20 [v/v]) solution to remove Figure 1. Binding of isolated peptides to the b-secretase cleavage sites of APP and the N-terminal of Ab. (A) The peptides of the b- secretase cleavage sites of APP and the N-terminal of Ab used in this study. (B) Ab-4-8, Ab-4-4 and Ab1-8 were coated on an amine-binding 96-well ELISA plate. Three of the selected phage clones were added to the wells. The HRP-9E10 antibody was added to the wells, followed by TMB substrate. Absorbance was read at 450 nm. (C) The peptides displaying positive phage clones were synthesized and coated on an amine-binding 96-well ELISA plate. Ab-4-3 and Ab4-8 with His-tags were added to the wells. Mouse anti-His tag antibodies and HRP-conjugated anti-mouse antibodies were sequentially added to the wells and the absorbance was measured as in (B). (D) The chimeric peptides were coated to an amine-binding ELISA plate. Ab-4-3 and Ab4-8 with His-tags were added to the wells.
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