N-Methyl Mesoporphyrin IX as an Effective Probe for Monitoring

Mar 10, 2017 - Formation of amyloid fibrils by amyloid-β peptide (Aβ) is an important step in Alzheimer's disease (AD) progression. Screening and de...
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N-Methyl mesoporphyrin IX as an effective probe for monitoring Alzheimer’s disease #-amyloid Aggregation in living cells Meng Li, Andong Zhao, Jinsong Ren, and Xiaogang Qu ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00436 • Publication Date (Web): 10 Mar 2017 Downloaded from http://pubs.acs.org on March 12, 2017

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N-Methyl mesoporphyrin IX as an effective probe for monitoring Alzheimer’s disease β-amyloid Aggregation in living cells Meng Li,a,b Andong Zhao,a,b Jinsong Ren,a and Xiaogang Qu*a aLaboratory

of Chemical Biology and State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China. bUniversity of Chinese Academy of Sciences, Beijing 100039, China. ABSTRACT: Formation of amyloid fibrils by amyloid-β peptide (Aβ) is an important step in Alzheimer’s disease (AD) progression. Screening and designing new molecules which can monitor amyloidosis process especially in cells are diagnostically and therapeutically important. Utilizing Thioflavin T (ThT), the commonly used amyloid dye, is the most standardized way to monitor amyloid. However, with the green fluorescence emission and small Stokes shift, the fluorescence of ThT can overlap with that arising from other intrinsic fluorescent components in the cells, making it not suitable for detection of protein aggregates in vivo. Therefore, it is urgent for developing amyloid probes with large Stokes shifts and red-shifted fluorescence emission to detect Aβ aggregates in cells. In this report, we found that NMethyl mesoporphyrin IX (NMM), a widely used G-quadruplex DNA specific fluorescent binder, can be an efficient probe for monitoring Aβ fibrillation in living cells. NMM is non-fluorescent in aqueous solution or monomeric Aβ environments. However, through stacking with the Aβ assemblies, NMM emits strong fluorescence. Furthermore, the large Stokes shift and stable photoluminescence make it an ideal probe for detecting Aβ aggregates in highly fluorescent environments and cell culture. Our results provide a new sight to design and screen new reagents for monitoring the diseases associated with protein conformational disorders. KEYWORDS: N-Methyl mesoporphyrin IX, fluorescence probes, amyloid-β peptide, aggregation, cell culture, Alzheimer’s Disease

INTRODUCTION Alzheimer’s disease (AD) that occurs mostly in older adult population is a kind of progressive neurodegenerative diseases. It has been reported that over 24 million people worldwide are affected by AD. And with an increasing of average life span, the population is expected to double every 20 years.1,2 Although the neurotoxic mechanisms and pathways involved in AD pathogenesis remain unresolved, self-assembly of monomeric amyloid-β peptide (Aβ) into β-sheet-rich fibrillar aggregates is critically important in the pathogenesis.3-5 Methods for highly sensitive detection of the amyloid plaques and understanding the mechanistic details of the Aβ polymerization processes have diagnostic and therapeutic implications. Recent studies demonstrated that early intracellular Aβ accumulation can precede extracellular Aβ deposits formation.6 When examined in human brain tissue, Aβ aggregation is initiated within the cells. Furthermore, the fibrillogenesis of Aβ can be accelerated in the cells due to the presence of lipid membranes, which is associated with a range of cellular properties.7 The intracellular Aβ can cause calcium dyshomeostasis, inhibit proteasome activities, accelerate synaptic dysfunction and even facilitate tau hyperphosphorylation.8,9 Although the critical pathological significance of intracellular Aβ fibrillogenesis has been widely demonstrated, the available tech-

niques to monitor Aβ aggregation in cells exhibit a number of limitations. Analytical methods, used for detecting Aβ aggregation in vitro, such as nuclear magnetic resonance (NMR)10, atomic force microscopy (AFM) 11,12, scanning electron microscopy (SEM)13, real-time light scattering14,15 and circular dichroism (CD)16, are not suitable for the studies in cell cultures due to the great background signal. Transmission electron microscopy17 and polyacrylamide gel electrophoresis6 followed by western blotting are extremely time-consuming and low throughput. Compared with these techniques, using fluorescent probe possessing high affinity toward amyloid fibrils is superior in terms of simplicity, low cost, high sensitivity and rapidity.18-23 Thioflavin T (ThT)24, the commonly used amyloid dye, is the most standardized way to monitor amyloid. Particularly, ThT has been successfully utilized to investigate the kinetics of Aβ aggregation and to characterize the structure of Aβ fibrils in vitro. However, the poor sensitivity, false-positive response, low specificity, and weak reliability make it unsuitable for kinetic study and quantitative analysis in vivo.23 Especially, with the green fluorescence emission and small Stokes shift, the fluorescence of ThT can overlap with that arising from other intrinsic fluorescent components in the cells, which is the main disadvantages of ThT for in vivo study.25 Therefore, screening sensitive

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probes with red-shifted fluorescence emission and large Stokes shifts would be desirable for detecting Aβ aggregation in cells. As an easily obtained anionic porphyrin, N-Methyl mesoporphyrin IX (NMM) is reported to be sensitive to G-quadruplexes DNA but has no response to duplexes, triplexes and single-stranded forms DNA. Upon binding to quadruplex DNA, for efficient π-π stacking, NMM can adjust its macrocycle geometry to match the terminal face of a G-quadruplex, leading to an enhancement in its fluorescence.26-28 This special property makes it a suitable G-quadruplex specific fluorescent probe.26-28 Although it is widely used for DNA detection, little is known for its specific interactions with amyloid peptide fibrils which have a parallel β-sheets structure.29,30 Herein, we demonstrate NMM can be used to monitor in vitro Aβ fibril formation and detect Aβ aggregates in cells (Scheme 1). Due to the large Stokes shift (210 nm), NMM can be an ideal probe both in vitro and in living cells. To the extent of our knowledge, NMM is the first time to be used as a fluorescent reporter to characterize Aβ40 fibrillization and measure intracellular protein aggregation.

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by the porphyrin ring of NMM, favoring the luminescent state.

Figure 1. (A) NMM fluorescence spectra in the presence of Aβ40 monomers and fibrils. [Aβ40] = 2 µM, [NMM] = 1 µM. (B) Relationship between fluorescence intensity change of NMM at 610 nm and the Aβ40 fibrils concentration. I0 is the fluorescence intensity of NMM without Aβ40 fibrils, c means the concentration of Aβ40 fibrils, and Kd stands for the dissociation constant. Fluorescence measured at [NMM] = 1 µM. The excitation wavelength is 399 nm.

To investigate the importance of β-sheet structure formation in NMM stacking, the experiments were performed at 4 ℃ since the transition of α-helixes to βsheets could be inhibited by low temperature.15 Under this condition, Aβ40 could hardly induce the increase of fluorescence (Figure S2), indicating that the formation of β-sheets was critical for NMM stacking. As the concentration of Aβ40 fibrils increased, the fluorescence of NMM was intensified. The emission intensity enhancement rate (I/I0) was fast at low Aβ40 concentrations and became slower when the concentration was greater than 2 μM. In a low concentration region (c = 0−2 μM), the binding isotherm revealed a linear relationship (Figure 1B). NMM exhibited high binding affinity to Aβ40 fibrils (binding constant ∼4.46×105 M), which was similar to ThT (5.0×105)24 and other commercial stains for amyloidal fibrils.35

Scheme 1 (A) Schematic illustration of NMMbased strategy for sensing Aβ40 aggregates both in vitro and in cells. (B) The structure of NMM. (C) Amino acid sequence of Aβ40.

RESULTS AND DISCUSSION To demonstrate the sensing ability of NMM for Aβ40 aggregates, the fluorescence spectra of NMM in the absence or presence of Aβ40 were measured (Figure 1). Aβ40 fibrils were obtained by incubation of Aβ40 in aggregation buffer (150 mM NaCl, 10 mM Tris, pH 7.3) for 7 days at 37 ℃.31-33 As shown in Figure 1A and Figure S1, NMM alone showed weak fluorescence. However, remarkably increased fluorescence was observed after Aβ40 fibrils were added. While, in the presence of Aβ40 monomers, no fluorescence change occurred under the same conditions (Figure 1A). The significant increase in its fluorescence intensity could be attributed to a restriction of the torsion oscillations of NMM fragments upon incorporated into the amyloid fibril.34 The fibril framework changed the microenvironment polarity felt

Figure 2. (A) Aβ40 fibrillogenesis process monitored by NMM (solid squares) and ThT (solid circles). Fluorescence measurements were carried at [ThT] = 10 μM, [Aβ40] = 2 μM, [NMM] = 1 μM. (B) Aβ40 fibrillization assay with NMM in rhodamine B fluorescent medium. Fluorescence measurements were carried at [Aβ40] = 10 μM, [NMM] = 1 μM, [RhB] = 0.1 μM.

The different emission behaviors of NMM with Aβ40 monomers and fibrils opened a possibility to utilize it for monitoring the kinetics of Aβ aggregation processes ex situ. After pre-incubation of Aβ40 for different days, NMM was added and the fluorescence intensities were monitored. As depicted in Figure 2A, the fluorescence increased gradually following the increase of incubation time, indicating that fluorescence of NMM was enhanced with an increase in the amount of Aβ40 aggregates. The

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plot of NMM fluorescence intensity versus incubation time had a sigmoidal shape, which was similar to the behavior of ThT (Figure 2A). These results matched the characteristics of a nucleation-dependent polymerization.24 Previous studies showed that some anti-amyloidogenic compounds such as curcumin and quercetin with fluorescence emissions around 460 to 550 nm could significantly interfere with ThT fluorescence readings.21,36 ThT assay was unsuitable for detection of amyloid aggregates in the presence of such compounds. In addition, compared with ThT, NMM exhibited more resistant to photobleaching in the presence of amyloid fibrils (Figure S3). The large Stokes shifts and stable photoluminescence of NMM made it possible to monitor Aβ40 fibrillization in a fluorescent background. To certify whether NMM was capable of detecting Aβ40 fibrillization in fluorescent environment, the experiment was performed in a solution of rhodamine B (RhB), a strong fluorescent probe which has a maximum emission at 575 nm and a long tail extending to 700 nm. As shown in Figure 2B and Figure S4, even in the strong RhB fluorescent medium, NMM could sensitively probe the process of Aβ40 aggregation. To check whether NMM would affect self-assembly of Aβ in situ, NMM (10 μM) was added to Aβ40 solutions (50 μM) followed by incubation for 7 days at 37 ℃. Figure S5 showed the time courses of the Aβ40 fibril formation which monitored by the fluorescence change of NMM. Compared to the fibrillogenesis without NMM, a prolonged nucleation phase and a decelerated elongation rate were induced by NMM. These results indicated that NMM was not only an excellent probe to monitor Aβ40 aggregation process but also an in situ inhibitor for Aβ40 aggregation. In addition to the fluorescence assay results, the effect of NMM on the morphology of Aβ40 aggregates was also monitored by AFM. After 7 days of incubation, control Aβ40 sample without NMM contained typical non-branched amyloid fibrils with lengths of up to 1 μm. However, after co-incubated with NMM, relatively amorphous aggregates with no fibrils were observed (Figure S6), indicating the ability of NMM to inhibit Aβ aggregation in situ. Similar with heme, an iron protoporphyrin IX that was essential for the function of all aerobic cells, the interaction between the porphyrin ring of NMM and the phenylalanine residues in the Cterminal hydrophobic region of Aβ made the NMM a candidate Aβ inhibitor.37

Figure 3. Detection of Aβ aggregates in CFP and ALC bacteria. Fluorescence images of CFP and ALC bacteria untreated or treated with IPTG for 3.5 h. Scale bars: 10 µm. The concentration of NMM was 5 µM.

With the large Stokes shift, NMM may be an ideal probe in living bacteria. To determine the applicability of NMM for sensing the Aβ aggregates in bacteria, E. coli cells stably transformed by vector Aβ-linker-ECFP (ALC) were used, and E. coli transformed by the control vector ECFP (CFP) served as controls. This system has been used to screen Aβ inhibitors in our laboratory.32,33 Briefly, ECFP itself can fold into its correct fluorescent structure slowly. While in ALC, Aβ folding controls the fluorescence intensities of the Aβ-ECFP fusion. The misfolding and aggregation of Aβ leads to the entire Aβ-ECFP fusion misfold before formation of the native state of ECFP, resulting in the decrease of ECFP fluorescence. The ability of NMM for detection of Aβ aggregates in ALC was firstly examined under a fluorescence microscopy. When Aβ aggregation occurred, the folding of ECFP was disturbed.32,33 In this condition, the fluorescence of NMM was displayed together with the diminished ECFP fluorescence. On the other hand, in the control CFP cells, the ECFP protein was correctly folded, and the fluorescence of ECFP was obvious (Figure 3). Noteworthy, ECFP fluorescence was not detected in the channel used to measure NMM fluorescence, suggesting that no overlap occurred between the emission spectrum of ECFP and NMM and NMM could be used in the environment contained other green fluorescent probes. Besides, we also quantified NMM luminescence of each samples above using fluorescence spectroscopy. As shown in Figure S7, CFP cells did not display significant NMM fluorescence. While, there was an obvious increase of NMM fluorescence in ALC cells. More importantly, without the addition of IPTG, an inducer of protein expression, both the ALC and CFP cells could not enhance the NMM fluorescence (Figure 3 and Figure S7). This indicated that only binding of NMM to the aggregated fibrils would favour the fluorescent state of the NMM molecules.

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Figure 4. Detection of Aβ aggregates in transfected PC12 cells. The cell nuclei were stained with DAPI. Aβ aggregates were detected using (A) NMM, (B) immunofluorescence assay. [DAPI] = 1 µg/mL, NMM = 3 µM. Scale bars: 10 µm.

For further validating that NMM could detect Aβ aggregates without nonspecific binding in living nerve cells, we used PC12 cells transfected for the overexpression of Aβ (PC12/Aβ cells), and the normal PC12 cells acted as controls. As depicted in Figure 4A, in PC12/Aβ cells, the granular aggregates detected with NMM were dispersed throughout the cell. To exhibit the observed NMM fluorescence in great part arose from NMM binding to Aβ aggregates, immunofluorescence labelling38-40 for Aβ was performed (Figure 4B). Both the direct fluorescence of NMM and the immunofluorescence for Aβ appeared as regions of punctuate fluorescence which most existed in the cytoplasm. However, no NMM fluorescence was detected in the control PC12 cells (Figure S8) and PC12/Aβ cells without staining with NMM or immunofluorescence (Figure S9). These results supported that NMM could probe the protein aggregation not only in vitro but also in cell culture. This property made NMM a more useful probe than ThT, which was not suitable for detecting in vivo protein aggregates due to its green fluorescence emission and small Stokes shift.

CONCLUSION In summary, we have demonstrated the possibility to use NMM as an in situ inhibitor and an ex situ monitor for Aβ amyloidogenesis both in vitro and in cells. Through stacking with Aβ fibrils, NMM emits strongly, resulting in the ability to monitor the kinetic of Aβ fibrillogenesis in vitro. Importantly, compared with ThT, this probe displays large Stokes shift, making it ideal for screening in fluorescent background bio-environment and cell culture. So far, there is no report of using NMM as a strong fluorescent probe for monitoring the protein aggregation both in vitro and in cell culture. Therefore, our results provide a new sight to design and screen new reagents for tracking or treatment of protein misfolding diseases.

METHODS Materials and measurements. Aβ40 was purchased from American peptide. NMM was synthesized

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by Porphyrin Products Inc (Logan, UT). The concentration of NMM was estimated from its measured absorbance at 379 nm (The extinction coefficient of NMM is 1.45×105 M-1cm-1). Other agents used without further treatment were obtained from Sigma-Aldrich. All the solutions used in the experiments were prepared by deionized water (18.2 MΩ). JASCO V-550 UV/Visible spectrophotometer (JASCO International Co., LTD., Tokyo, Japan) was used to determine the UV-vis absorption spectra. NMM fluorescence and ThT fluorescence were recorded by a JASCO FP-6500 spectrofluorometer. Aβ Preparation. Aβ40 (lot no. U10012) was synthesized by American Peptide. Firstly, 1, 1, 1, 3, 3, 3hexafluoro-2-propanol (HFIP) was added to dissolve the Aβ peptide powder to form 1 mg/mL solution. For further dissolution, the solution in a sealed bottle was shaking at 4° C for 2 hours. Then, the stock solution was stored at -20° C. Before use, a stream of nitrogen was used to evaporate HFIP. After that, water was added to disperse the peptide. Aβ40 fibrils were obtained by incubating the peptides in aggregation buffer (150 mM NaCl, 10 mM Tris, pH 7.3) for 7 days at 37 ° C. ThT fluorescence spectroscopy. The change of ThT fluorescence was used to monitor the kinetics of Aβ aggregation. At different times, aged Aβ samples were diluted into ThT solution (ThT in aggregation buffer). The final concentration of peptide was fixed at 2 μM and the concentration of ThT was 10 μM. The excitation wavelength was 444 nm and the emission intensity at 482 nm. NMM fluorescence spectroscopy. By use of NMM as an ex situ probe, NMM was added into the incubated Aβ40 solution. The concentration of NMM was fixed at 1 μM. To examine the influence of Rhodamine B, 1 μM NMM with 0.1 μM or 0.5 μM Rhodamine B along with 10 μM Aβ40 were measured. In the study of using NMM as an in situ inhibitor, NMM (10 μM) was co-incubated with Aβ40 (50 μM) for 7 days at 37 ° C. Additional NMM was added before PL measurement to make the concentration of NMM constant (1 μM). The fluorescence spectra of NMM were collected from 550 to 700 nm with an excitation wavelength of 399 nm. Atomic Force Microscopy (AFM) assay. For AFM imaging, a 20 μL sample (the final concentration of Aβ40 was 5 μM) was deposited onto freshly cleaved muscovite mica for 15 min, washed twice with 50 μL of deionized water and dried with nitrogen gas. Images were obtained in tapping mode in using Nanoscope V multimode AFM (Veeco Instruments, USA). Imaging of Escherichia coli. The Aβ-ECFP based system was constructed as previously reported method with some modification. In brief, Escherichia coli strain BL21 (DE3) utilized for protein expression was firstly transformed by two different vectors, the ALC vector (Aβ-linker-ECFP) and the CFP vector (linker-ECFP). After that, obtained E.coli was cultured at 37 ° C in lysogeny broth (LB) with ampicillin (50 μgmL-1). To induce protein expression, Isopropyl-β-D-thiogalactopyranoside (IPTG, 1 mM) was added. After induced for 3.5 h, samples were diluted to optical density at 600 nm (OD600) to 0.2. NMM solution was added to the E. coli culture,

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and then incubated for another 10 minutes in dark. Phosphate buffered saline (PBS) was used to wash the bacteria twice. The fluorescence images were acquired using an Olympus BX-51 microscope with a 100W mercury arc lamp (Tokyo, Japan) at 400 ×magnification and recorded by the Olympus DP72 CCD camera. In the study of fluorescence measurement, each sample (OD600=0.2) was measured at 610 nm (excitation 399 nm). Cell Fluorescence Staining. Assembly PCR was used to generate the cDNA which can encode human wild-type Aβ. The PCR product was transferred into pcDNA3.1-Aβ. Iscove modified Dulbecco medium (IMDM, Gibco BRL) containing 10% horse serum and 5% fetal bovine serum was used to culture rat pheochromocytoma (PC12) cells (American Type Culture Collection) in a 5% CO2 environment at 37° C. According to the manufacturer’s instructions, PC12 cells were transfected with pcDNA3.1-Aβ using lipofectamine 2000 (Invitrogen) to obtain PC12/Aβ cells. PC12 and PC12/Aβ cells were cultured for 24h at 37° C on glass coverslips. Then, PBS containing 2% glutaraldehyde and 2% paraformaldehyde were used to fix the cells for 30 min at 4° C. After that, NMM solution was added to the cell culture, and incubated for another 10 minutes in dark. These cells were then washed twice with PBS, and viewed with an Olympus BX-51 microscope at 400× magnification. For immunofluorescence assay, cells were firstly blocked for 15min with 3% BSA (w/v) and then incubated overnight with anti-Aβ1-42 antibody at 4° C which followed by incubating with DylightTM594-conjugated affinipure goat anti-rabbit IgG at room temperature for 1h.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Effect of NMM on Aβ40 aggregation; photostability of NMM and ThT; results for control experiments (PDF)

AUTHOR INFORMATION Corresponding Author * Email: [email protected]; Fax: 86-431-85262656

Author Contributions J.R. and X.Q. designed the research, M.L. performed the experiments and analyzed the data; A.Z. collected the AFM images and analyzed the data; M.L., J.R. and X.Q. wrote the manuscript.

Funding This work was supported by 973 Project (2012CB720602) and National Natural Science Foundation of China (Grants 21210002, 21431007, 21533008).

Notes The authors declare no competing financial interest.

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2-Cyanoacrylate (ANCA) Probes Fluorescently Discriminate between Amyloid-β and Prion Plaques in Brain. J. Am. Chem. Soc. 2012, 134, 17338-17341. (21) Cook, N. P.; Torres, V.; Jain, D.; Martí , A. A. Sensing Amyloid-β Aggregation Using Luminescent Dipyridophenazine Ruthenium(II) Complexes. J. Am. Chem. Soc. 2011, 133, 1112111123. (22) Li, M.; Zhao, C.; Yang, X.; Ren, J.; Xu, C.; Qu, X. In situ monitoring Alzheimer's disease β-amyloid aggregation and screening of Aβ inhibitors using a perylene probe. Small 2013, 9, 52-55. (23) Hong, Y.; Meng, L.; Chen, S.; Leung, C. W.; Da, L. T.; Faisal, M.; Silva, D.-A.; Liu, J.; Lam, J. W.; Huang, X.; Tang, B. Z. Monitoring and Inhibition of Insulin Fibrillation by a Small Organic Fluorogen with Aggregation-Induced Emission Characteristics. J. Am. Chem. Soc. 2012, 134, 1680-1689. (24) Levine, H. Thioflavine T interaction with synthetic Alzheimer's disease beta-amyloid peptides: detection of amyloid aggregation in solution. Protein Sci. 1993, 2, 404-410. (25) Cook, N. P.; Kilpatrick, K.; Segatori, L.; Martí , A. A. Detection of α-Synuclein Amyloidogenic Aggregates in Vitro and in Cells using Light-Switching Dipyridophenazine Ruthenium(II) Complexes. J. Am. Chem. Soc. 2012, 134, 2077620782. (26) Hu, D.; Pu, F.; Huang, Z.; Ren, J.; Qu, X. A Quadruplex-Based, Label-Free, and Real-Time Fluorescence Assay for RNase H Activity and Inhibition. Chem. Eur. J. 2010, 16, 26052610. (27) Hu, D.; Huang, Z.; Pu, F.; Ren, J.; Qu, X. A Label-Free, Quadruplex-Based Functional Molecular Beacon (LFG4-MB) for Fluorescence Turn-On Detection of DNA and Nuclease. Chem. Eur. J. 2011, 17, 1635-1641. (28) Zhao, C.; Wu, L.; Ren, J.; Qu, X. A label-free fluorescent turn-on enzymatic amplification assay for DNA detection using ligand-responsive G-quadruplex formation. Chem. Commun. 2011, 47, 5461-5463. (29) Cerf, E.; Sarroukh, R.; Tamamizu-Kato, S.; Breydo, L.; Derclaye, S.; Dufrê ne, Y. F.; Narayanaswami, V.; Goormaghtigh, E.; Ruysschaert, J.-M.; Raussens, V. Antiparallel β-sheet: a signature structure of the oligomeric amyloid β-peptide. Biochem. J. 2009, 421, 415-423. (30) Yu, L.; Edalji, R.; Harlan, J. E.; Holzman, T. F.; Lopez, A. P.; Labkovsky, B.; Hillen, H.; Barghorn, S.; Ebert, U.; Richardson, P. L.; Miesbauer, L.; Solomon, L.; Bartley, D.; Walter, K.; Johnson, R. W.; Hajduk, P. J.; Olejniczak, E. T. Structural Characterization of a Soluble Amyloid β-Peptide Oligomer. Biochemistry 2009, 48, 1870-1877. (31) Li, M.; Yang, X.; Ren, J.; Qu, K.; Qu, X. Using Graphene Oxide High Near-Infrared Absorbance for Photothermal Treatment of Alzheimer's Disease. Adv. Mater. 2012, 24, 17221728. (32) Yu, H.; Li, M.; Liu, G.; Geng, J.; Wang, J.; Ren, J.; Zhao, C.; Qu, X. Metallosupramolecular complex targeting an α/β discordant stretch of amyloid β peptide. Chem. Sci. 2012, 3, 3145-3153. (33) Geng, J.; Li, M.; Ren, J.; Wang, E.; Qu, X. Polyoxometalates as inhibitors of the aggregation of amyloid β peptides associated with Alzheimer's disease. Angew. Chem. Int. Ed. 2011, 50, 4184-4188. (34) Pispisa, B.; Stella, L.; Venanzi, M.; Palleschi, A.; Polese, A.; Formaggio, F.; Toniolo, C. Structural features of linear, homo-Aib-based peptides in solution: a spectroscopic and molecular mechanics investigation. J. Pept. Res. 2000, 56, 298-306. (35) Groenning, M. Binding mode of Thioflavin T and other molecular probes in the context of amyloid fibrils—current status. J. Chem. Biol. 2010, 3, 1. (36) Hudson, S. A.; Ecroyd, H.; Kee, T. W.; Carver, J. A. The thioflavin T fluorescence assay for amyloid fibril detection can be biased by the presence of exogenous compounds. FEBS J. 2009, 276, 5960-5972.

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(37) Yuan, C.; Gao, Z. Aβ interacts with both the iron center and the porphyrin ring of heme: mechanism of heme's action on Aβ aggregation and disaggregation. Chem. Res. Toxicol. 2013, 26, 262-269. (38) Jungbauer, L. M.; Yu, C.; Laxton, K. J.; LaDu, M. J. Preparation of fluorescently-labeled amyloid-beta peptide assemblies: the effect of fluorophore conjugation on structure and function. J. Mol. Recognit. 2009, 22, 403-413. (39) Jimenez, S.; Baglietto-Vargas, D.; Caballero, C.; Moreno-Gonzalez, I.; Torres, M.; Sanchez-Varo, R.; Ruano, D.; Vizuete, M.; Gutierrez, A.; Vitorica, J. Inflammatory response in the hippocampus of PS1M146L/APP751SL mouse model of Alzheimer's disease: age-dependent switch in the microglial phenotype from alternative to classic. J. Neurosci. 2008, 28, 11650-11661. (40) Cattepoel, S.; Hanenberg, M.; Kulic, L.; Nitsch, R. M. Chronic intranasal treatment with an anti-Aβ(30-42) scFv antibody ameliorates amyloid pathology in a transgenic mouse model of Alzheimer's disease. PLoS One 2011, 6, e18296.

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