Synergistic Inhibitory Effect of GQDsâTramiprosate covalent binding

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Synergistic Inhibitory Effect of GQDs–Tramiprosate covalent binding on amyloid Aggregation Yibiao Liu, Li-Ping Xu, Qiang Wang, Baocheng Yang, and Xueji Zhang ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00439 • Publication Date (Web): 15 Dec 2017 Downloaded from http://pubs.acs.org on December 16, 2017

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ACS Chemical Neuroscience

Synergistic Inhibitory Effect of GQDs–Tramiprosate covalent binding on amyloid Aggregation Yibiao Liu,a, d Li-Ping Xu,*b Qiang Wang,c Baocheng Yang,*a, d and Xueji Zhangb a

Institute of Nanostructured Functional Materials, Huanghe Science & Technology

College, Zhengzhou 450000, P.R. China. b

Research Center for Bioengineering and Sensing Technology, University of Science &

Technology Beijing, Beijing 100083, P.R. China. c

State Key Laboratory of Coal Conversion, Institute of Coal Chemistry, The Chinese

Academy of Sciences, Taiyuan 030001 P.R. China d

Henan Provincial Key Laboratory of Nano-composite Materials and Applications

*

Address correspondence, Tel. & Fax: 86-10-82375840

ABSTRACT: Inhibiting the amyloid aggregation is considered to be an effective strategy to explore possible treatment of amyloid-related diseases including Alzheimer’s disease, Parkinson’s disease, and type II diabetes. Herein, a new high-efficiency and low-cytotoxicity Aβ aggregation inhibitors, GQD-T was designed through the combination of two Aβ aggregation inhibitors，graphene quantum dots (GQDs) and tramiprosate. GQD-T showed the capability of efficiently inhibiting the aggregation of Aβ peptides and rescuing Aβ-induced cytotoxicity due to the synergistic effect of the GQDs and tramiprosate. In addition, the GQD-T has the characteristics of low toxicity and great biocompatibility. It is believed that GQD-T may be a potential candidate of Alzheimer’s drug and this work provides a new strategy for exploring Aβ peptide aggregation inhibitors. KEYWORDS: synergistic inhibitory effect, cytotoxicity, amyloid β (Aβ) peptide, graphene quantum dots (GQDs), tramiprosate

■ INTRODUCTION The cerebral deposition of β-amyloid (Aβ) peptides is a hallmark of Alzheimer’s

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disease (AD).

1-4

Various forms of Aβ peptide aggregates have been identified including

soluble oligomers, protofibrils and matured fibrils and these aggregates of Aβ peptides may be neurotoxic to brain cells.

5-8

Recent researches indicated that inhibiting the Aβ

peptide aggregation is considered to be an effective strategy for the possible treatment of AD. 5, 9 Studies have demonstrated that the formation of Aβ fibrils is related with its specific amino acids sequence. The Aβ peptide is an amphipathic polypeptide consisting of 39–42 amino acids.10 The Aβ peptide includes several different regions including N-terminus11-14, hydrophobic core15, 16, hinge or turn regions,17-19 and C-terminus20, 21. These different regions influence the Aβ aggregation in different way. The His13–Lys16 (HHQK) region of Aβ1-42 at the N-terminus is very important in oligomerization, fibril formation and accounts for the neurotoxicity of Aβ peptide.22-24 This four-residue region is an essential component of the heparin-binding site for glycosaminoglycans (GAGs), which assist the Aβ secondary structure transition from unordered α-helix to stable β-sheet. These β-sheet structures further associate with other monomers to form oligomers, protofibrils and fibrils.22 The formation of β-sheet structure play a key role in nucleation, which is the rate-limiting step in fibril formation.18, 19, 25 Tramiprosate, a mimic of GAGs, could bind to the HHQK subregion specifically, inhibit the aggregation of Aβ peptide, and rescue the Aβ-induced cytotoxicity.

26-28

This results showed that inhibitors targeting specifc Aβ

subregions will be a viable approach for AD treatment.29 The hydrophobic core subregion is another crucial region for controlling the Aβ peptide aggregation. Hydrophobic core region is the main core of Aβ aggregates, and hydrophobic interaction is an important driving force in Aβ aggregation. 30, 31 Some studies have proved that the modifications of hydrophobic regions can accelerate or inhibit fibril formation and promote/induce the disassembly of Aβ fibrils. 32-35 Till now, several different inhibitors have been developed for their specific regions. For example, Aβ antibody and the GAGs inhibits the aggregation of Aβ peptide through combining with the N-terminus region.13, 14 Peptides or peptide mimetics target the hydrophobic core and break the β-sheet structure.36-39 Polyphenol compounds (tannic acid, Congo red and curcumin) could bind with the C-terminus region and inhibit the Aβ aggregation.40-44 Currently, most inhibitors11, 12, 36, 45, 46 focus on only one subregion of Aβ peptide. If


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the inhibitors could target multiple specific subregions simultaneously, the inhibitory effect may be improved due to their synergistic effect. 47 Graphene quantum dots (GQDs), are a single or few-layer graphene with a tiny size less than 100 nm. Due to its photoluminescence properties, low cytotoxicity and great biocompatibility, GQDs have been widely used in biological research, particular in nanobiomedicine48 and cell imaging49, 50 Recently, our group reported graphene quantum dots (GQDs) could inhibit the Aβ peptide aggregation by targeting the hydrophobic region of Aβ peptide.

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Tramiprosate is also proved to be a great inhibitor toward the HHQK subregion of Aβ1-42 peptide. Employing a combination of GQDs and tramiprosate could develop a new effective inhibitor towards the exploration of Alzheimer’s drug. In this work, GQDs and tramiprosate were utilized to construct a new inhibitor (GQD-T) by covalent binding. The sequence and special subregion of Aβ1-42 and the structure of tramiprosate were shown in Fig.1. Inhibitory effects of the GQD-T on Aβ1-42 aggregation were investigated and the results demonstrate that GQD-T inhibit the aggregation of Aβ peptide through breaking the β-sheet structure. And the GQD-T shows greater inhibition efficiency due to the synergistic effect of the GQDs and tramiprosate. The GQD-T is almost nontoxic to the PC-12 cells. This study provides a new strategy for exploring Aβ peptide aggregation inhibitors through targeting multiple specific subregions.

Fig. 1 a) Special subregion in Aβ1-42 peptide, b) The structure of Tramiprosate.

■ RESULTS AND DISCUSSION The Synthesis and and Characterization of GQD-T. GQD-T was synthesized by combining GQDs with tramiprosate using EDC/NHS reaction. The synthetic method is



shown in Fig.S1. The UV−vis absorption, fourier transform infrared (FT-IR) spectra analysis and zeta potential measurement analyser were used to characterize the formation of the GQD-T. Fig. 2 a showed that the change of UV−vis absorption of GQDs before or after the modified with tramiprosate. The maximum absorption peak of GQDs was located at 290 nm (Fig. 2a, black line), which appeared to red shift to 298 nm (Fig. 2a, red line), due to the introduction of tramiprosate. The FT-IR spectrum of GQD-T (Fig. 2b, red line) presented the characteristic peaks of GQDs (Fig. 2b, blue line) and tramiprosate (Fig. 2b, black line), indicating the successful conjugation of GQDs and tramiprosate. The obvious absorption peaks corresponded to the vc=o (1638 cm−1), δN-H(1578 cm−1) and vC-N (1290 cm−1) (Fig. 2 d, red line) of −CONH− groups in the GQD-T. Fig. 2d provides a comparison among the zeta potential value of GQDs, GQD-T and tramiprosate in aqueous solutions. Compared to GQDs with a zeta potential value of -15.8 mV, the GQD-T showed a higher zeta potential value of about -24.2 mV. The increase of zeta potential value of GQD-T was due to the negative charge of tramiprosate transferring to the GQDs after the combining the GQDs and tramiprosate. The zeta potential value of GQD-T is significantly higher than the GQDs, which confirmed the successful assembly of GQD-T in some ways. According to the analysis above, it is confirmed that the combination between GQDs and tramiprosate is successful. In addition, the morphological information on GQDs and GQD-T were also characterized by atomic force microscopy (AFM). As shown in Fig.2 c, the size of GQD-T is about 15-20 nm and the average height is about 3.3 nm. Compared to the GQDs, the sizes of GQD-T increased.


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Fig. 2 a) UV−vis absorption, b) FT-IR spectra characterization, c) AFM, and d) Zeta potential characterization of GQDs, Tramiprosate and GQD-T. Synergistic inhibitory effect of GQD–T on Aβ peptide aggregation. After synthesizing the GQD-T, the capability of GQD-T in inhibiting Aβ42 aggregation was investigated. The effect of GQD-T on the assembly of Aβ1-42 peptides was employed by thioflavin T (ThT) fluorescence assay. ThT, an extrinsic fluorescent dye, can combine with amyloid fibrils and fluoresce at excitation wavelength of 450 nm. Its fluorescence intensity increases with the augment of aggregates.52 The fibrillation process was monitored by measuring the fluorescence intensity of ThT at 490 nm upon excitation at 450 nm. When fresh Aβ1–42 peptide alone was incubated in PBS buffer at 37℃, ThT fluorescence intensity as a function of incubation time demonstrated a sigmoidal shape (Fig. 3a), which was consistent with the nucleation-dependent polymerization model. As shown in Fig. 3a, the process of Aβ1-42 peptide aggregation illustrates three stages of a typical protein fibrillation, that is, the lag phase, elongation phase, and steady state. The process of Aβ aggregation is mainly related to lag time (tlag) and the rate of aggregation (k),



which was fitted the function: y=A+B*exp(-kx) where A, B is constant, k is the rate of aggregation (k). The rate of aggregation (k) was obtained by fitting a single exponential function to the growth phase of the aggregation curve, and lag time (tlag) was calculated as the intersection of the linear part of the initial growth phase with the baseline. 55 (Fig. 4a) The lag phase is also called nucleation phase, which is a rate-limiting step in the formation of fibrils.

18, 19, 53

In this phase, the monomeric Aβ aggregated into small

clusters and the ThT fluorescence intensity increased slowly for the first few hours. At the end of the lag phase, a lot of clusters aggregated into amyloid fibrils and the ThT fluorescence intensity started to rise rapidly (Fig. 3a, the black line). However, the ThT fluorescence intensity significantly decreased in the presence of GQDs, tramiprosate or GQD-T, indicating that the formation of Aβ1-42 amyloid fibrils was suppressed (Fig. 3a). It was obvious that the ThT fluorescence intensity is the smallest in the presence of GQD-T compare to several other inhibitors in the steady state. These results indicated that the inhibiting effect of GQD-T is better than GQDs, tramiprosate and GQDs+tramiprosate. And The inhibitory effect followed the order GQD-T > GQDs+Tramiprosate > Tramiprosate > GQDs. We further calculated the lag time (tlag) of Aβ1-42 aggregation in the absence or presence of GQD-T or other two inhibitors. As shown in Fig. 4b, in the presence of GQD-T, the lag time is longest, and the lag time (tlag) also followed the order GQD-T > GQDs+Tramiprosate > Tramiprosate > GQDs. This result proved that the GQD–T had a better inhibitory effect in some ways. In addition, we also investigated the effects of GQD-T on the morphology of Aβ1-42 aggregates using AFM. Forty micromolar Aβ1-42 peptide was incubated in the absence or presence of inhibitors in PBS buffer (10 mM, pH 7.4) at 37°C for 5 days. As shown in Fig. 3b, Aβ1-42 formed a typical structure for amyloid fibrils. In contrast, in the presence of GQDs, tramiprosate or GQD-T (1µM), spherical structures or some very small fibrils were formed. (Fig. 3b-f) In the presence of GQDs or tramiprosate, the fibrils were shortened and small fibrils were observed. In the presence of GQD-T, the fibrils were disaggregated and spherical structures were observed. These results further supported the ThT experiments and indicated that covalent combination between GQDs and


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tramiprosate play synergistic inhibitory action amyloid fibril formation.

Fig. 3 Inhibition of Aβ1-42 peptide aggregation by GQD-T. (a) The kinetics of Aβ1-42 aggregation as monitored by the thioflavin T fluorescence in the absence of inhibitors (■) or the presence of GQDs (●), Tramiprosate (▲), GQDs+Tramiprosate (▼) or GQD-T (◆). The data represented the means of three independent experiments. Error bars indicate ±s.d. The morphology of Aβ1-42 in 10 mM PBS buffer (pH 7.4) in the absence of inhibitors (b) or the presence of GQDs (c), Tramiprosate (d), GQDs+Tramiprosate (e) or GQD-T (f). with GQDs, tramiprosate or GQD-T after incubation at 37 °C 5 days. All the concentration of inhibitors is 1µM. (AFM: tapping mode, size: 1.5 µm × 1.5 µm)



Fig. 4 The kinetic analysis of Aβ1-42 peptide aggregation. a) kinetic analysis of the aggregation curves. Straight line b was fitted to the baseline, and duration of nucleation or lag phase (tlag) was determined as the time point where line b intersected straight line a, a tangent to the steepest region of the elongation curve.55 b) change in the nucleation phase duration (tlag) in the mixtures of the Aβ1-42 peptide with GQD-T different inhibitors. M±S.E., n =3. Significance difference between GQD-T and other two inhibitors was evaluated in unpaired t test and is indicated by *, p < 0.01. In addition，the binding energy of GQD-T or GQDs with Aβ1-42 were calculated separately by Molecular Mechanics Poisson-Boltzmann/surface area (MMPBSA). The conformational changes of Aβ1-42 monomer with GQD-T or GQDs at initial (0 ns) and final state (100 ns) are shown in Fig. S2. And the results showed that the binding energy of GQD-T with Aβ1-42 (-26.7998 kcal/mol) is lower than that of GQDs with Aβ1-42 (-24.0837 kcal/mol) (Table. S1). Therefore, the formation of Aβ fibrils from GQD-T/Aβ complex need a higher activation energy Ea than that from GQDs/Aβ complex (Fig. S3), which from another side proved that the GQDs–tramiprosate covalent binding act synergistically to effectively inhibit Aβ aggregation. To investigate the inhibitory mechanism of GQD-T, circular dichroism was used to confirm the secondary structures of Aβ42 aggregates in the presence of GQD-T. Initially, the CD spectrum of Aβ42 (40 µM) monomer typically displays a curve with a negative peak at 198 nm, which is characteristic of random coils. As Aβ42 continues to aggregate, the negative peak at 198 nm was converted to the positive peak around 196 nm and a


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negative peak around 219 nm. (Fig. 5a) The change of the CD spectra indicates the conformational conversion of Aβ42 from random coils to β-sheets, and thus suggests the formation of Aβ42 fibrils. GQD-T was added to Aβ42 monomer solution and coincubated for 5 days at 37℃ before the structure characterization by CD spectra. The CD studies showed that the negative peak around 198 nm stayed unchanged, which demonstrated that β-sheets structures didn’t appears during the process. (Fig. 5b) The result indicated that the GQD-T inhibit the Aβ42 aggregation through breaking the β-sheet structural formation.

Fig.5. CD spectra. Secondary structures of freshly prepared Aβ1-42 (40 µM) incubation without (a) or with GQD-T (1 µM) (b) for 5 days at 37℃. GQD-T rescue Aβ-induced cytotoxicity. Aβ-induced cytotoxicity is an important factor related to AD. The GQD-T might be useful in blocking Aβ-mediated cellular toxicity on account of excellent capability of inhibiting Aβ1-42 aggregation. To explore the effect of GQD-T on Aβ1-42-mediated cytotoxicity, we used PC12 cells by employing 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay to explore the cellular viability. As can be seen in Fig. 6, the aggregates of Aβ1-42 peptides alone are detected to have obvious cytotoxicity at a concentration of 40 µM and bring about a decrease of 41% in cellular viability (Fig. 6, lane 2). The cell viability increased to about 70% in the presence of the inhibitor GQDs or tramiprosate (Fig. 6, lane3, lane4). However, treatment of the cells with Aβ1-42 peptides in the presence of GQDs/tramiprosate increases the survival of the cells to about 73% (Fig. 6, lane 5). Amazingly, the GQD–T reduce significantly the Aβ-induced cytotoxicity compared to GQDs, tramiprosate or GQDs/tramiprosate and the cell viability increased by 80%, which



demonstrated that GQDs–Tramiprosate covalent binding could result in a synergistic effect on reducing Aβ-induced cytotoxicity. In addition, as depicted by the lane 7 in Fig. 6, the cell survival is over 90% in the presence of GQD-T alone, which indicates that GQD-T is nearly nontoxic under the same conditions.

Fig. 6. The effect of GQD-T on the cell toxicity of Aβ1-42 peptide. Cell viability was determined using the MTT method. Significance difference between GQD-T and other two inhibitors was evaluated in unpaired t test and is indicated by *, p < 0.01. According to the above analysis, GQDs–Tramiprosate covalent binding have a synergistic inhibitory effect on both the amyloid aggregation and the cytotoxicity of Aβ1-42. Possible mechanisms could be connected with the increased binding sites of crucial peptide motifs and the enlarged steric hindrance between neighboring Aβ1-42 peptides because of the GQDs–Tramiprosate covalent binding. Furthermore, the inhibiting affectivity of GQD-T is also excellent. The mean inhibitory constant (IC50) was obtained by ThT fluorescence intensity as shown in Fig. S4. GQD-T show a lower


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IC50 values about 2 µM. It is expected that GQD-T may be a potential therapeutic candidate for AD.

■ CONCLUSIONS In conclusion, the inhibitory effect on Aβ aggregation may be associated with the ability of combining with the key subregions of Aβ1-42 and the number of binding sites. In this work, an inhibitor GQD-T containing multiple binding sites was synthesized by covalent binding GQDs with tramiprosate, which exhibited a synergistic inhibitory effect on both the amyloid aggregation and the cytotoxicity of Aβ1-42. We inferred that GQDs–Tramiprosate covalent binding increased the binding sites and enlarged steric hindrance between neighboring Aβ1-42 peptides. This study provides a new strategy to explore Aβ aggregation inhibitors and may significantly promote the development of therapeutic drugs for AD.

■ EXPERIMENTAL SECTION Materials and Reagents. Aβ1-42 peptides were purchased from Beijing SBS Genetech Co. Ltd. Graphite nanoparticles, tramiprosate, 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC) and N-hydroxy-succinimide (NHS) were purchased from Alfa Aesar China. The GQDs were synthesized by hydrothermal methods according to ref.54 All other reagents were of analytical reagent grade and commercially available. All solutions were prepared with ultrapure water in this work (Milli-Q, 18.2 MΩ﹒cm). The synthesis and characterization of GQD-T. Firstly, graphene sheets (GSs, 0.05 g) were oxidized in concentrated H2SO4 (10 mL) and HNO3 (30 mL) for 15 h under mild ultrasonication (500 W, 40 kHz). Secondly, the mixture was diluted with ultrapure water (250 mL) and filtered through a 0.22 µm microporous membrane to remove the acids. Purified oxidized grapheme nanoparticles were re-dispersed in 75 mL ultrapure water/ethanol

(1

:

1,

v/v

ratio).

The

suspension

was

transferred

to

a

poly(tetrafluoroethylene) (Teflon)-lined autoclave (100 mL) and heated at 200 °C for 10 h. After cooling to room temperature, the resulting suspension was filtered through a 0.22



µm microporous membrane. The GQDs was obtained in the filtrate. The GQD-T was synthesized by EDC/NHS reaction between GQDs and tramiprosate (Fig. S1). In order to conjugate the tramiprosate to GQDs via EDC and NHS coupling, the solution of GQDs (280 µg/mL) with EDC (40 mg, 10.5 mM) and NHS (8.6 mg, 3.5 mM) was reacted for 15 min, respectively, to activate the carboxyl group. Tramiprosate (1.25 mM) was then added to the activated carboxyl GQDs solution, and the solution was kept in the dark overnight. The resulting solution was dialyzed in a 3500 Da dialysis membrane for 2 days to remove the unreacted tramiprosate. The obtained GQD-T were characterized by AFM, UV−vis absorption, Fourier transform infrared (FT-IR) spectra and zeta potential measurement analyser (Nano-ZS90). Thioflavin T fluorescence assay. The kinetics of Aβ1-42 peptide aggregation with or without inhibitors including GQD-T, GQDs and tramiprosate was monitored by using the dye ThT, the fluorescence of which was dependent on the formation of amyloid fibrils. Fluorescence measurements were carried out using a JASCO FP6500 spectrofluorometer. The fluorescence signal (excitation at 450 nm) was recorded between 460 and 600 nm; 10 nm slits were used for both emission and excitation measurements. The Aβ1-42 peptide concentration was 40 µM, and the ThT concentration was 10 µM. At different times, aliquots of the Aβ1-42 peptide solution with or without GQD-T were taken for fluorescence measurements. Morphological characterization. The visualization of Aβ1-42 peptide aggregation with or without GQD-T was performed using a Nanoscope IIIa Multimode AFM (Bruker Inc.) in the tapping mode. 40 µM Aβ1-42 and 40 µM Aβ1-42 in the presence of GQD-T, GQDs or tramiprosate were incubated for 5 days at 37 °C. Briefly, aliquots of 20 µL of each sample were placed on a freshly cleaved substrate. After incubation for 10 min, the substrate was rinsed twice with water and dried before measurement. All the tips (RTESP, 318–384 kHz) were obtained from Bruker Inc. The images presented here were non-filtered and composed of 512 × 512 pixels with scanning areas of 1.5 µm × 1.5 µm. All AFM images were processed using nanoscope analysis software. Circular Dichroism (CD) spectroscopy. CD spectra of Aβ1-42 peptide incubated with or without GQD-T (1µM) were recorded in a Chirascan Spectrometer (Applied Photophysics Co., England), using a quartz cuvette (1 mm path length). The


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concentration of Aβ1-42 solution for CD analysis was 40 µM and the Aβ1-42 solutions incubated with or without GQD-T were prepared at 1.0 equiv (we define equiv as the molar ratio of modulators to Aβ1-42). The spectra were taken as the average of three accumulations from 190 and 280 nm at a speed of 50 nm/min. All of the samples were incubated at 37 °C in 10 mM phosphate buffer solution (PBS) with a continuous agitation speed of 300 rpm. Spectra were calibrated by subtracting the buffer or sample solution baseline. Atomistic MD Simulations. Atomistic MD simulations were performed using the AMBER14 software. In the simulations of coassemblies, the GQD-T or GQDs were placed near the HHQK subregion and hydrophobic core subregion of the Aβ1-42 peptide according to the projected binding sites. The atomistic MD simulations system was solvated with ultrapure water. The initial conformation underwent a 0 ns MD simulation. Then for each system, two 100 ns MD simulations were performed. And the binding energy of GQD-T and GQDs with Aβ1-42 monomer were calculated separately by MMPBSA. Cytoxicity assay. PC12 cells (rat pheochromocytoma, American Type Culture Collection) were cultured in RPMI-1640 supplemented with 10% fetal bovine serum in a humidified 5% CO2 environment at 37 °C. The cells were plated at a density of 10 000 cells per well on 96-well plates in fresh medium. Aβ1-42 peptides (40 µM) that had been incubated with or without GQD-T for 4 days at 37 °C were added, and the PC12 cells were further incubated for 24 h at 37 °C. Cytotoxicity was measured by using a modified MTT assay. Absorbance values of formazan were determined at 490 nm with an automatic plate reader.

■ ASSOCIATED CONTENT Supporting Information The synthetic method of GQD-T, the binding energy of GQD-T with Aβ1-42 peptide and the mean inhibitory constant (IC50) of GQD-T (Fig. S1-S4 and Table S1). This material is available free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATION



Corresponding Author *Li-Ping Xu: Tel & Fax: (+86) 10-8237-6831; e-mail: [email protected]. *Baocheng Yang e-mail: [email protected].

Author Contributions Yibiao Liu designed and done research, Dr. Li-Ping Xu and Qiang Wang helped in analyzing data. Prof. Dr. Baocheng Yang and Prof. Dr. Xueji Zhang guided in designing research and revising manuscript. All authors reviewed the manuscript. Funding The work is supported by National Natural Science Foundation of China (NSFC Grant no. 21475009, 21475008) and Beijing Municipal Science & Technology Commission (Z161100000116037). Notes There is no conflicts of interest among authors.

■ ACKNOWLEDGMENTS The authors thank Dr. Wenhao Dai for advice.

■ REFERENCES 1. Haass, C., and Selkoe, D. J. (2007) Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid [beta]-peptide, Nat. Rev. Mol. Cell. Biol. 8, 101-112. 2. Gotz, J., and Ittner, L. M. (2008) Animal models of Alzheimer's disease and frontotemporal dementia, Nat. Rev. Neurosci. 9, 532-544. 3. Geng, J., Li, M., Wu, L., Ren, J., and Qu, X. (2012) Liberation of copper from amyloid plaques: making a risk factor useful for Alzheimer’s disease treatment, J. Med. Chem. 55, 9146-9155. 4. Sevigny, J., Chiao, P., Bussière, T., Weinreb, P. H., Williams, L., Maier, M., Dunstan, R., Salloway, S., Chen, T., Ling, Y., O’Gorman, J., Qian, F., Arastu, M., Li, M., Chollate, S., Brennan, M. S., Quintero-Monzon, O., Scannevin, R. H., Arnold, H. M., Engber, T.,


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