Inhibition of Alzheimer\xe2\x80\x99s Amyloid Toxicity with a Tricyclic Pyrone Molecule In Vitro and In Vivo
Small amyloid \xce\xb2 1\xe2\x80\x9342 aggregates are toxic to neurons and may be the primary toxic species in Alzheimer\xe2\x80\x99s disease (AD). Methods to reduce the level of A\xce\xb2, prevent A\xce\xb2 aggregation, and eliminate existing A\xce\xb2 aggregates have been proposed for treatment of AD. A tricyclic pyrone named CP2 is found to prevent cell death associated with A\xce\xb2 oligomers. We studied the possible mechanisms of neuroprotection by CP2. Surface plasmon resonance spectroscopy shows a direct binding of CP2 with A\xce\xb242 oligomer. Circular dichroism spectroscopy reveals monomeric A\xce\xb242 peptide remains as a random coil/\xce\xb1-helix structure in the presence of CP2 over 48 h. Atomic force microscopy (AFM) studies show CP2 exhibits similar ability to inhibit A\xce\xb242 aggregation as that of Congo Red and curcumin. AFM closed-fluid cell study demonstrates that CP2 disaggregates A\xce\xb242 oligomers and protofibrils. CP2 also blocks A\xce\xb2 fibrillations using a protein quantification method. Treatment of 5x FAD mice, a robust A\xce\xb242-producing animal model of AD, with a two-week course of CP2 resulted in 40% and 50% decreases in non-fibrillar and fibrillar A\xce\xb2 species, respectively. Our results suggest that CP2 might be beneficial to AD patients by preventing A\xce\xb2 aggregation and disaggregating existing A\xce\xb2 oligomers and protofibrils.
I. Introduction<!>Materials<!>Surface Plasmon Resonance<!>Circular dichroism<!>Atomic force microscopy<!>Inhibition of A\xce\xb2 aggregation<!>Disaggregation of A\xce\xb2 oligomers and protofibrils<!>Disaggregation of A\xce\xb242 oligomers in solution (a real-time study)<!>Protein quantification method for the study of inhibition of A\xce\xb2 fibrillation<!>Inhibition of fibril formation of A\xce\xb240 and A\xce\xb242 with and without CP2 over an extended period of time<!>Intracerebroventricular infusion of CP2 into 5x FAD mice<!>A\xce\xb242 ELISA<!>Statistics<!>Binding study of CP2 to A\xce\xb2 oligomers via SPR<!>Characterization of A\xce\xb242 conformation in the absence and presence of CP2<!>Comparison of the inhibition of aggregations of A\xce\xb240 and A\xce\xb242 separately with CP2, thioflavin T, Congo Red and curcumin over time (24 and 48 h)<!>Disaggregation of A\xce\xb242 oligomers and protofibrils with CP2, thioflavin T, Congo Red, curcumin, and TP17 (After 24 and 48h)<!>Disaggregation of A\xce\xb242 protofibrils with CP2<!>Disaggregation of A\xce\xb242 oligomers in real-time studies<!>Inhibition of the formation A\xce\xb2 fibrils with CP2<!>Evaluation of CP2 in the Alzheimer model 5x FAD mice<!>V. Conclusion<!>
<p>Extracellular amyloid β (Aβ) peptide plaques, intracellular neurofibrillary tangles, and soluble Aβ oligomers are the neuropathological characteristics of Alzheimer disease (AD; Crowther 1991; De Felice et al. 2008). Many lines of studies strongly support that Aβ aggregates are the neurotoxic species in AD. Importantly, not all Aβ aggregates are equally toxic (Chromy et al. 2003; Liu and Schubert 2006); different Aβ assembly forms may mediate diverse toxic effects at different stages of the disease (Hoozemans et al. 2006). Recent studies suggest that soluble Aβ oligomers may be the primary toxic species in AD (Lambert et al. 1998; Klein et al. 2001; Walsh et al. 2002a; De Felice et al. 2007) and involved in inducing the formation of tangles (De Felice et al. 2008; Hardy and Selkoe 2002). Soluble oligomers made from the 42 amino acid Aβ (Aβ42) are particularly toxic (Walsh et al. 2002a; Walsh et al. 200b; Wang et al. 2002). Aβ dimers recently were linked to impairment of synaptic plasticity and memory (Klyubin et al. 2008; Shankar et al. 2008).</p><p>Methods to reduce the levels of Aβ, prevent Aβ aggregation and eliminate existing Aβ aggregates have been proposed for AD therapies (Selkoe 2004; Talaga 2001; Findeis 2000). Small peptides, called β-blockers, have been used to block the aggregation of Aβ (Tjernberg et al. 1996; Soto et al. 1998; Permanne et al. 2002; Adessi et al. 2003). Since small peptides degrade in vivo by proteases, the half-life in the body is short (Adessi et al. 2003). Agents that reduce Aβ production, such as γ-and β-secretase inhibitors (Walsh et al. 2002b; Josien 2002; Thompson et al. 2006; Ghosh et al. 2008; Olson and Marcin 2007), increase Aβ clearance, such as amyloid vaccines (Youm et al. 2008; Cao et al. 2008), and block Aβ aggregation (Walsh et al. 2005; Blanchard et al. 2004), such as non-steroidal anti-inflammatory agents (Thomas et al. 2001; Lleo et al. 2004), antioxidants (De Felice et al. 2007; Yang et al. 2005; Cheng et al. 2006), and inhibitors of tau protein phosphorylation (Dickey and Petrucelli 2006), have been investigated. Several compounds were evaluated in Alzheimer's transgenic mice models (Cao et al. 2008; Yang et al. 2005; Cheng et al. 2006; Adlard et al. 2008; Quadros et al. 2007; Bales 2007), but the efficacy remains uncertain.</p><p>Previously, we found that a tricyclic pyrone molecule, CP2 (code name), prevents cell death associated with intracellular Aβ oligomers and inhibits Aβ aggregation (Maezawa et al. 2006; Hong et al. 2007), hence we were curious whether CP2 could disaggregate Aβ oligomers and protofibrils, block Aβ fibrillization, and reduce the levels of Aβ aggregates in Alzheimer's transgenic mice. Surface plasmon resonance (SPR) was used to study the binding of CP2 to Aβ42 oligomers bound onto immobilized anti-Aβ oligomer antibody (known as A11). Circular dichroism (CD) spectroscopy was utilized to monitor monomeric Aβ42 peptide solution structures in the presence and absence of CP2. Atomic force microscopy (AFM) including a real-time study using a closed-fluid cell was used to study the ability of CP2 to inhibit Aβ oligomerization and to disaggregate Aβ oligomers and protofibrils. Protein quantification was conducted to determine the inhibition of fibrillation of Aβ peptides over time with and without CP2. Finally, levels of accumulated Aβ42 in the brains of 5x FAD mice were evaluated by ELISA after two weeks of infusion of CP2. We report herein the effects of CP2 in the inhibition of Aβ aggregation, disaggregation of Aβ oligomers and protofibrils, and reduction of Aβ load in 5x FAD model mice.</p><!><p>The synthesis of CP2 has been reported previously (Hua et al. 2003). Trifluoroacetic acid (TFA) salt of CP2 was prepared by the addition of TFA into a chloroform solution of CP2. The solution was concentrated to dryness under reduced pressure, diluted with deionized water, and lyophilized on a freeze-dry instrument to give CP2•TFA salt. CP2•TFA salt is soluble in water and has a longer shelf-life than the parent compound. Aβ40 and Aβ42 were purchased from Bachem (Torrance, CA) or synthesized using a Discover SPS Microwave Peptide Synthesizer (CEM Co., Matthews, NC), purified through HPLC (acetonitrile and water containing 0.1% TFA were used as solvents), and identified by comparison with the authentic samples purchased from Bachem via HPLC (retention time and co-injection). Fmoc-Val-NovaSynR TGA resin was purchased from EMD Co. (San Diego, CA) and all Fmoc-protected amino acids were purchased from CEM Co. Both commercial Aβ peptides and synthesized peptides were used and similar results were obtained. The anti-amyloid oligomer antibody (catalog No. AB9234), a rabbit polyclonal antibody, was purchased from Chemicon (Temecula, CA). For the immunoglobulin G control in our SPR experiments (see below), we used the rabbit anti-Cdk5 immunoglobulin G (SC-173), which was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).</p><!><p>(Maezawa et al. 2006) A BIAcore 3000 (Biacore Inc., Piscataway, NJ, USA), equipped with four flow cell channels on one Sensor chip, was used for real-time binding studies. Anti-amyloid oligomer antibody was immobilized on the carboxymethylated dextran sensor CM5 chip in the first channel, and the second channel was used as a reference (without immobilization of anti-amyloid oligomer antibody). Anti-Cdk5 antibody, a negative control, was immobilized in the third channel, and the fourth channel had no immobilization (used as a reference). The CM5 sensor chip was activated by the injection of 200 µL of 1:1 mixture of N-ethyl-N'-[(dimethylamino)-propyl]carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in pH 7.4 buffer solution for 30 min, followed by the injection of anti-amyloid oligomer antibody into channel 1 (10 µL of anti-amyloid oligomer antibody was diluted with 600 µL of pH 5.0 sodium acetate buffer, injected at the rate of 10µL per minute) or anti-Cdk5 into channel 3. The resulting anti-amyloid oligomer antibody bound Sensor chip and the anti-Cdk5 antibody bound chip were used in the SPR studies. The reference channels were prepared similarly but without the addition of anti-amyloid oligomer antibody or anti-Cdk5 antibody for correction of background bindings. A running buffer solution consisted of 10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, 0.05% surfactant P20, pH 7.4, was used as a washing solvent between injections of reagents, antibody, Aβ42 oligomers, and CP2. After immobilization of the antibody, a solution of Aβ42 oligomers was injected to the cell, and binding was observed. Aβ42 oligomers were prepared by incubation of a 10-µM solution of Aβ42 monomer in pH 7.4 PBS buffer for 24 h at 4°C, and the formation of small Aβ42 oligomers was verified by AFM. To the bound anti-amyloid oligomer antibody-Aβ42 oligomer, a solution of CP2 (40 µM in the aforementioned running buffer) was injected followed by washing with the running buffer solution. The amount of immobilized antibody, bound Aβ42 oligomers and CP2 were determined based on the amount of response units (RU) obtained from the sensograms. Each binding experiment was repeated three times, and similar results were obtained. The amount of immobilization and bindings were calculated by the ΔRU value, obtained by subtracting the RU at the time before compound injection from the RU at the time just after injection of running buffer. The ΔRU obtained from the reference cell was considered as the background binding and was subtracted before analysis. One RU is defined to be 1 pgram/mm2 of bound protein molecules and can be converted into pmol/mm2 (Van Wiggeren et al. 2007).</p><!><p>A Jasco J-815 Spectropolarimeter CD instrument (JASCO; Easton, MD) and a quartz cell (0.1 cm length; 200 µL internal volume) were used to record CD spectra in a spectral range of 190 – 260 nm, 1 nm bandwidth, and 50 nm/min scan speed. A solution of Aβ42 (40 µM) in phosphate buffer solution containing NaCl, Na2CO3, NaOH, and acetonitrile was prepared according to the reported method (Bartolini et al. 2007) to monitor Aβ42 solution structure in the absence and presence of 1.25 equivalents of CP2 over 48 h at 25°C in a time-course manner. Aβ42 (0.12 mg) was dissolved in hexafluoroisopropanol (HFIP; 200 µL) by brief sonication and vortexing, kept at 25°C for 48 h, and HFIP was evaporated at 25°C. The resulting Aβ42 was re-dissolved in a freshly prepared mixture (70 µL) of CH3CN/Na2CO3 (0.3 mM)/NaOH (0.25 M) (48.3:48.3:3.4, v/v/v) by brief sonication and vortexing providing an alkaline solution of Aβ42 (0.38 mM). An aliquot (30 µL) of the Aβ42 solution was diluted with phosphate buffer saline (PBS; 0.25 mL) solution to give a 40-µM solution of Aβ42 in phosphate buffer (7.7 mM) that contained NaCl (8.9 mM), Na2CO3 (15.5 mM), NaOH (0.91 mM), and 5.1% acetonitrile. CP2 (1.25 equivalents; 10 µL of 1.5 mM in PBS-ethanol, 9:1) was added to 280 µL of the above Aβ42 (40 µM) solution, and CD spectra were recorded over 48 h. The control solutions that contained the above incubation buffer with and without CP2 were prepared and their contributions were subtracted in the CD spectra of Aβ42 with and without CP2, respectively. The CD spectrum of CP2 (1.25 equivalents) showed only a small absorption and did not change the overall shape of Aβ42 CD graph except to reduce slightly Aβ42 signal intensity (after subtraction). Similar to that reported study (Bartolini et al. 2007), a plotting of the CD values at 215 nm (β-sheet structure) versus time of incubation of Aβ42 peptide in the presence and absence of CP2 was made to study the conformational changes of the peptide (vide infra).</p><!><p>Either a Nanoscope IIIa SPM (Digital Instruments, Inc. Santa Barbara, CA) or an MFP-3D (Assylum Research, Santa Barbara, CA) atomic force microscope was used to measure the oligomerization of Aβ40 and Aβ42 (Ding and Harper 1999; Goldsbury et al. 2005), and inhibition of aggregation and disaggregation of oligomers and protofibrils of Aβ40 and Aβ42 with CP2. Each experiment was repeated three times and an average value from three experiments was obtained. Quantitative analysis of Aβ oligomeric particle density was carried out from three representative 25-µm2 AFM scans. Image J program (http://rsb.info.nih.gov/)was used to quantify the oligomer densities.</p><!><p>Aβ40-TFA and Aβ42-TFA salts were used separately to prepare stock solutions by the following protocol. Aβ42 (0.25 mg; 0.055 µmol) was dissolved in 150 µL of filtered DMSO (the unused sample was kept at −78°C). From this stock solution, 30 µL (0.011 µmol) of Aβ42 was removed and diluted with 120 µL of phosphate-buffered saline (PBS) solution (derived from 10 mL each of 50 mM of Na2HPO4 and 100 mM of NaCl; pH 7.4), and 150 µL of deionized water [both PBS and water solutions were filtered through a PTFE filter (0.2 µm pore size; Fischer) before usage]. The resulting solution was filtered through a 10,000 MW cutoff filter (Microcon filter; Millipore Co., Bedford, MA) under centrifugation at 21,000 g, 4°C for 15 min on a centrifugator (IEC Micromax RF, Thermo Electron Co., Waltham, MA) to remove any undissolved aggregates. A stock solution of Aβ40 was prepared similarly. For each drug, 30 µL of the Aβ42 solution (37 µM; 1.1 nmol) were used. To Aβ42 solutions, different concentrations of drugs including CP2 [dissolved in deionized water and ethanol (96:4)], TP17 (an inactive tricyclic pyrone; dissolved in DMSO), Congo Red (dissolved in water), thioflavin T (dissolved in water), and curcumin (dissolved in DMSO) (see Figure 1 for structures), were added separately and incubated at 4°C. Aliquots (10 µL each) were removed after the indicated times (such as 1 day, 2 days, etc.), placed onto freshly cleaved micas, washed with water twice, dried with argon, and AFM images were then obtained using a tapping mode with a high aspect ration tip (Veeco Nanoprobe TM tips, Model TESP-HAR).</p><!><p>The above monomeric Aβ42 solution (37 µM) was kept at 4°C for 24 h, an aliquot was removed, and the presence of Aβ oligomers was confirmed by AFM. To Aβ42 (60 µL; 2.2 nmol) solution, different concentrations of drugs including CP2, TP17 (an inactive tricyclic pyrone), Congo Red, Thioflavin T, and curcumin, were added separately and incubated at 4°C. Aliquots (10 µL) were removed after the indicated times (such as 1 day, 2 days, etc.), placed onto freshly cleaved micas, washed with water twice, dried with argon, and AFM images were obtained.</p><!><p>A closed-fluid cell (Asylum Research), and a Bio-lever tip (Olympus Cantilevers, AtomicForce, Mannheim, Germany; which are small, soft, low-noise Si3N4 probe for the tapping mode in fluid and force measurements) were used for the real-time study. The AFM tip was cleaned by UV light prior to the in-situ imaging, and the silicon wafer was pre-treated with a mixture of 98% H2SO4 and 30% H2O2 (3:1) for 15 min at 65°C. The two-dimensional closed loop scanner provides images without distortion over a large area. The instrument also allows a continuing scanning of small areas (500 nm × 500 nm) with minimal 2D lateral drift among the scans over time (> 150 h). The Z-closed loop sensors minimize the height distortion of images. Silicon wafers were treated with Ni2+ to render the surface positively charged. Although the negatively charged residues of Aβ42 can interact electrostatically with the surface, it is more likely that the histidine residues in the hydrophilic domain of Aβ (residues 1–16) helps attach Aβ42 onto the surface. Since the hydrophilic domain is believed to not be involved in Aβ42 aggregation/fibrillation and protrudes out of oligomers/fibrils (Chan et al. 1996), the immobilization does not affect the in-situ disaggregation of Aβ42 oligomers/fibrils by CP2 and the AFM observation in solution. Aβ42 (0.1 mg) was dissolved in 60 µL of DMSO as the stock solution, and the solution was kept at − 78°C to prevent Aβ oligomerization. An aliquot of the Aβ42 stock solution (10 µL) was removed and diluted with 90 µL of PBS buffer (pH 7.4) yielding a 37 µM solution of Aβ42 in PBS. The pretreated silicon wafer was glued to a glass cover slip at the bottom of the AFM liquid cell, and 1.08 mL of PBS solution and 30 µL of the above Aβ42 solution were subsequently added via micro-syringes providing a 1 µM of Aβ42 solution. The solution was incubated for 48 h at 25°C, and the presence of Aβ42 oligomers was confirmed by AFM. CP2•TFA (1 mg) was dissolved in 40 µL ethanol and diluted with 960 µL of deionized water (yielding a final concentration of 1.6 mM of CP2), and 50 µL (~10 equivalents) of the CP2•TFA solution was slowly introduced to the fluid cell with a syringe. The addition of CP2 solution was carefully conducted to ensure that the Aβ42 oligomers were not disrupted. A control experiment was conducted to determine that the AFM tip did not scrap off Aβ aggregates from the surface. Briefly, a silicon wafer covered with Aβ42 aggregates was placed into the AFM liquid cell, and 2 mL of PBS buffer (pH 7.4) were injected into the cell. AFM images were observed from a specific location covered with aggregates. Scanning using tapping mode on this location was continuously performed over an extended period of time (24 h), and AFM images showed no loss of aggregates. Therefore, we conclude that continuous scanning using the tapping mode does not result in discernable loss of aggregates.</p><!><p>A Micromax RF refrigerated microcentrifuge (Thermo Electron Co., Fisher Scientific, Pittsburgh, PA) and a UV-vis spectrometer (Shimadzu UV-120-02; Columbia, MD) were used. Four different concentrations of Aβ peptides, 30, 40, 50, 60 µM for Aβ40, and 10, 20, 30, 40 µM for Aβ42 separately in pH 7.4 PBS buffer (500 µL) were freshly prepared (used within a period of 30 minutes to avoid aggregation) and each treated each with 500 µL of freshly prepared Micro BCA™ Working Reagent (Pierce Biotechnology, Rockford, IL) (Smith et al. 1985). The solutions were incubated at 60°C for 30 min. and cooled to 25°C. Absorbance was measured at 569 nm wavelength (a BCA reagent was used as a reference) within 10 min. Linear correlations (Aβ40 & 42 with and without CP2; 2 equivalents) between the absorbance values and concentrations were obtained (Figure 2). The amounts of Aβ can be obtained from the measured UV absorbance using the correlation graphs.</p><!><p>Aβ40(60 µM) and Aβ42 (40 µM) in pH 7.4 PBS buffer (200 µL each) were treated separately with and without CP2 (2 equivalents) in water (14 µL) at 25°C. Each day, an aliquot, 30 µL, was removed, centrifuged at 15,000 g, 4°C for 15 min. (to remove Aβ fibrils), and the amount of protein in the supernatant solution was quantified using the aforementioned BCA reagent.</p><!><p>5x FAD mice were obtained from Dr. Robert Vassar, Department of Cell and Molecular Biology, Northwestern University (Oakley et al. 2006). All animal procedures were approved by the UC Davis Animal Care and Use Committee. All surgical procedures were performed with care to minimize pain and discomfort. CP2 or an equivalent amount of DMSO (solvent; mock) was dissolved in 100 µL of artificial cerebrospinal fluid to make a working solution of 100 µM. The solution was transferred into osmotic mini-pumps (Alzet, Cupertino, CA). We followed a reported procedure to implant the pump (Dolev and Michaelson 2004). Three-month-old 5x FAD mice were anesthetized with an intraperitoneal (i.p.) injection of Ketamin (100 mg/Kg body weight) with Xylazine (10 mg/Kg body weight). A brain-infusion cannula (brain infusion kit #3, Alzet) was stereotaxically positioned (antero-posterior, 0.7 mm; mediolateral, 1.5 mm; dorsoventral; 3 mm to Bregma) by cementing with Loctite 454 (Alzet, Cupertino, CA). This cannula was then connected to the subcutaneously implanted osmotic mini-pump (Alzet model 1002). Mice were infused (n = 6 for each group) at a flow rate of 25 µL/h for 2 weeks of CP2 or vehicle. After 2 weeks, the mice were sacrificed and their brains were removed and snap frozen for Aβ42 ELISA.</p><!><p>For Aβ42 ELISA, the corresponding 1 mm-thick fresh frozen mouse brain sections containing hippocampus and subiculum were homogenized in 300 µL of radioimmune precipitation assay (RIPA) buffer containing protease inhibitors. The homogenates were sonicated briefly then incubated for 20 min at 4°C. The RIPA insoluble fraction from brain homogenate was spun down by 100,000 × g centrifugation. The pellet was resuspended in 300 µL of 70% formic acid (FA) and then incubated for 30 min at 4°C. FA soluble fraction was obtained by 100,000 × g centrifugation. The FA extracts were neutralized by diluting 1:20 in 1 mole/L Tris buffer (pH 11). The Aβ42 concentration in each sample was determined by Aβ42 specific sandwich ELISA following the protocol of the manufacturer (IBL, Minneapolis, MN). All readings were in the linear range of the assay. The average of the duplicates was determined, and then the mean and standard error for each group of mice were calculated. The values were expressed in nanograms of Aβ per milligram of protein.</p><!><p>The statistical significance of differences between groups was examined by t-test, using the SigmaStat 3.1 program (Systat Inc. Point Richmond, CA).</p><!><p>SPR has been used extensively to study the interaction of peptides and proteins. For instance, some pentapeptides (Tjernberg et al. 1996; Soto et al. 1998) were found to bind to Aβ40 peptide by SPR. In brief, Aβ peptide was immobilized from a solution of very low concentration (~pico mole/L; to avoid aggregation) on a sensor chip and maintained in a pH 7.4 buffer solution. When a drug molecule flows through the chip and a binding occurs between the drug and peptide, the refractive index of the medium next to the chip will change. This is represented by a response unit that is equivalent to the mass of bound drug in pico gram. To study whether CP2 binds to Aβ oligomers, we immobilized the oligomer-specific antibody, anti-amyloid oligomer antibody (MW ~150 kDa), onto the chip. Based on the sensogram (Figure 3), there were 0.052 pmol of anti-amyloid oligomer antibody immobilized after two injections of the antibody. After eluting the flow cells with running buffer for 20 min to remove all unbound antibody, premade 10 µM Aβ42 oligomers were injected (four injections over 100 min) into the flow cells and 0.426 pmol of Aβ42 oligomers were found to bind to the antibody. After washing the flow cell with running buffer for 100 min to remove any unbound Aβ oligomer, a solution of CP2 (40 µM) was injected and then washed with running buffer, and 0.21 pmol of CP2 was found to bind to the antibody-Aβ oligomer complex. The data suggests that for each antibody, there are 8 molecules of Aβ42 (based on MW of the monomer) bound to the antibody, and 4 molecules of CP2 bind to each Aβ42 oligomer-antibody. Similar binding studies were also carried out by immobilizing anti-Cdk5 antibody in one of the flow channels as a negative control, and no bindings of Aβ42 oligomers were found (graph not shown). We also found that CP2 did not bind to anti-amyloid oligomer antibody alone when CP2 was injected to a separate flow cell immobilized with the antibody. Assuming two binding sites of the antibody are fully occupied with Aβ42 oligomers, the oligomer should be a tetramer, and two CP2 molecules bind to each tetrameric Aβ oligomer. The real-time binding study using SPR shows Aβ42 oligomers bind to anti-amyloid oligomer antibody and CP2 binds to the Aβ oligomer-antibody complex.</p><!><p>It is accepted that Aβ undergoes conformational changes or misfolding from random coil/α-helix to β-sheet structure, resulting in oligomerization (Bartolini et al. 2007; Fezoui and Teplow 2002). CD spectroscopy offers a real-time monitoring of protein conformations in aqueous solution (Fezoui and Teplow 2002). A PBS buffer solution containing Na2CO3-NaOH-acetonitrile was established (Bartolini et al. 2007) recently to study Aβ conformational changes in the presence of Aβ42 inhibitors and marketed drugs for AD treatment using CD. Hence, the same buffer solution was used to examine whether CP2 maintains Aβ42 random coil/α-helix structures and blocks β-sheet formation over time using CD spectroscopy. Figure 4, left panel, shows Aβ42 conformational changes in the absence of CP2 over 48 h, in which CD value at 198 nm (random coil structure) gradually decreased over time and 215 nm (β-sheet) value increased. On the other hand, in the presence of 1.25 equivalent of CP2 (Figure 4, middle panel), CD value at 215 nm remains unchanged. A plotting of CD values at 215 nm versus time is shown in Figure 4, right panel. The sigmoid graph of Aβ42 in the absence of CP2 exhibits an exponential growth phase of β-sheet structure (Bartolini et al. 2007), while that of the presence of CP2 shows no exponential growth. The results imply that CP2 blocks β-sheet formation. The intensity at 198 nm of Aβ42 peptide plus CP2 is weaker than that of Aβ42 alone because the solution of Aβ-CP2 is slightly diluted compared with that of Aβ alone (from the addition of a solution of CP2 in PBS) and the contribution of the CD graph of CP2 (control) was subtracted from the observed Aβ-CP2 graph.</p><!><p>We used thioflavin T (LeVine 1997), Congo Red (Findeis 2000; Lorenzo and Yankner 1994), and curcumin (Begum et al. 2008), all of which known to interact with amyloid aggregates, to compare the ability of inhibition of Aβ aggregation with CP2. Compound TP17 is an inactive tricyclic pyrone molecule that fails to protect MC65 cells from toxicity due to Aβ oligomers (Maezawa et al. 2006), and was used as a negative control. We predicted that since thioflavin T and TP17 do not have high affinities to Aβ oligomers (Maezawa et al. 2008), they are less likely to affect the initial Aβaggregation. Aβ40 peptide is less toxic and aggregates much slower when compared with Aβ42; its aggregation was performed at 25°C, while Aβ42 at 4°C. Figure 5 summarizes the results of inhibition of aggregation of Aβ40 peptide with 2 equivalents each of the compounds, in which Image J program was used to quantify the peptide densities. Three representative images of each experiment (treatment with and without drugs) were used in the quantification of peptide densities. CP2, Congo Red, and curcumin inhibit Aβ40 aggregation after 24 and 48 h of incubation, but not TP17 and thioflavin T. The degrees of inhibition of CP2, Congo Red, and curcumin are similar in that about 50% of Aβ40 aggregates were reduced over 48 h compared with that of control (without drug).</p><p>Similarly, Figure 5 summarizes results of the inhibitions of Aβ42 separately with 2 equivalents each of CP2, thioflavin T, Congo Red, curcumin, and TP17 after 24 and 48 h of incubation. CP2, Congo Red and curcumin have similar inhibitory activities, while thioflavin T and TP17 have no inhibitory activity. In the presence of 2 equivalents of the compounds, Aβ40 and Aβ42 aggregates increase over time but at a much slower rate as compared to without the compounds. Hence bioactive compounds, such as Congo Red, curcumin and CP2, inhibit amyloid fibril formation and Aβ aggregation, while inactive compounds such as thioflavin T and TP17 do not.</p><!><p>Since Aβ oligomers are toxic to cells, we investigated whether CP2, Congo Red, and curcumin would disaggregate Aβ oligomers and protofibrils to the non-toxic monomer. Hence, Aβ42 oligomers were produced from the aggregation of Aβ42 monomer (37 µM) in pH 7.4 PBS solution at 4°C for 24 h, and sizes of the oligomers are 25 – 50 nm in width and 4 – 7 nm in height (see Figure 6, left middle panel). AFM images of the untreated solution and drug-treated solutions by additions of 2 equivalents each of CP2, thioflavin T, Congo Red, curcumin, and TP17 separately after incubation of 24 and 48 h were obtained. It should be noted that Aβ monomer and possibly dimer were too small (< 2 nm of diameter) to be detected by AFM and only oligomers (with n > 2) were detected. Figure 6 (top and middle panels) shows representative AFM images and section analyses of sizes of Aβ42 oligomers and oligomers treated with 2 equivalents of CP2 over 24 and 48 h. Results of Aβ42 particle densities from three representative images are summarized in Figure 6 (lower panel). After 48 h, CP2 showed the greatest disaggregating ability, and curcumin has a greater disaggregation activity than Congo Red. Thioflavin T and TP17 did not show disaggregation activities that are in agreement with their bio-inactivities in protection of MC65 cells.</p><p>Since the disaggregated Aβ42 monomer can re-aggregate to oligomers over time, we studied the amount of CP2 required to maintain Aβ42 peptide in monomeric form (derived from the disaggregation of oligomers) without aggregation over 5 days. Hence, Aβ42 oligomers (37 µM; after incubation for 24 h at 4°C; density is shown in Figure 7, day 1) in pH 7.4 PBS solution were treated with various amounts of CP2 and incubated at 4°C over 5 days (days 2 to 6). Each day, an aliquot was removed and AFM images were taken (in various locations of the mica), and three representative images were used to calculate the peptide particle density. Results are summarized in Figure 7. On day 4, protofibrils were observed in the Aβ42 solution without CP2, and on day 6, protofibrils predominated. Aβ42 solutions incubated with 2 and 5 equivalents of CP2 showed lesser amounts of oligomers after 24 h but the oligomers increased after 5 days. On the other hand, in the presence of 10 equiv. of CP2, ~8% of oligomers were found compared to without CP2 after 5 days.</p><p>The results suggest that disaggregated Aβ42 monomer and smaller oligomers can recombine to form larger oligomers in the presences of 1 – 5 equivalents of CP2. However, in the presence of 10 equivalents of CP2, only a few oligomers were found even after 5 days. It is suggestive that CP2 binds to Aβ oligomers and breaks apart the oligomers. The binding of CP2 with monomeric Aβ is reversible and Aβ42 monomer re-aggregates to form oligomers over time in a closed compartment.</p><!><p>Aβ oligomers aggregate further to form protofibrils, which have been revealed to be toxic to neuronal cells and to affect learning in mice (Walsh et al. 2002a; Hartley et al. 1999; Nilsberth et al. 2001; Martins et al. 2008). Hence, a study of the disaggregation of Aβ42 protofibrils with CP2 was carried out. Aβ42 protofibrils were produced by incubation of Aβ42 monomer in PBS for 4 days and the structures were shown by AFM (Figure 8A). Two equivalents of CP2 were then added to the protofibril solution and incubated for 24 h. AFM images of the incubated sample revealed the presence of small amounts of oligomers and disappearance of protofibrils (Figure 8B). The studies show that CP2 disaggregates soluble protofibrils to small oligomers and subsequently to monomer.</p><!><p>In the above disaggregation studies, an aliquot of Aβ42 solution was removed each day and oligomers were measured. The process involves washing of the sample over mica with water to remove PBS buffer and any unbound surface materials, and examining various locations of the mica to ensure that representative images were used. To eliminate any ambiguity associated with the possible inconsistency in sampling of the incubated Aβ solution and effect of washing of the surface (which could dislodge some aggregates), we conducted an AFM experiment to monitor the disaggregation in real time. Aβ42 oligomers were treated with CP2 (10 equivalents which were found to inhibit Aβ42 oligomerization over 5 days) in a closed AFM cell over time to observe the disaggregation. Figure 9A depicted an AFM image of Aβ42 (1 µM) oligomers formed after incubation at 25°C for 48 h in PBS buffer (pH 7.4). The AFM scan was continuously performed at the same location (the full scan size is 1.6 µm), and after addition of CP2, Aβ42 oligomers gradually disappeared over 50, 120, and 180 min (Figures 9C, 9D, and 9F, respectively). Figures 9B and 9E show the height and width of the oligomers in the marked red lines before and after adding CP2 (for 2 h), respectively. The initial heights of the oligomers were ~ 6.0 nm, and after addition of CP2, the heights were reduced to ~ 1.0 nm. Only a few small and thin annular structures remained on the surface after 3 h (Figure 9F). These aggregates appear to be strongly bound to the surface since rinsing of the surface repeatedly with the buffer solution did not change the surface morphology (results not shown). Since Ni2+ ions were initially deposited on the surface (see Method Section), it appears that small amounts of oligomeric aggregates were strongly retained by the Ni2+ ions (on the silicon wafer that were placed for anchoring histidine residues of Aβ). The time-lapse AFM studies clearly demonstrate that CP2 inhibits Aβ peptide aggregation and disaggregates Aβ oligomers and protofibrils.</p><!><p>Although CP2 binds to Aβ oligomers and disaggregates the oligomers, we probed whether CP2 inhibits Aβ fibril formation in PBS solution over time at 25°C. Monomeric Aβ40 and Aβ42 peptides were treated with 2 equivalents of CP2 separately for one week at 25°C, and each day the concentrations of Aβ peptides were quantified using a micro BCA™ protein assay reagent kit and results are summarized in Figure 10. In the absence of CP2, we found ~73% of Aβ40 and ~43% of Aβ42 in the solution after one week. On the other hand, in the presence of CP2, there was ~88% and ~81% of Aβ40 and 42, respectively that remained in the solution after one week of incubation. The data shows that CP2 not only disaggregates Aβ oligomers but also inhibits fibril formation, which is in agreement with the results of the inhibitions of aggregation of Aβ peptides (vide supra), as further aggregation of oligomers leads to protofibrils and fibrils.</p><!><p>To test if CP2 has anti-Aβ actions in vivo, we examined the efficacy of CP2 in 5x FAD mice. Neurons in these mice were engineered to generate a large amount of Aβ42, which results in robust Aβ accumulation and aggregation in the brain. Histologically detectable amyloid depositions begin by 2 months, especially in the hippocampus and subiculum, and increase sharply between 2–4 months. We infused 100 µM of CP2 or a mock solution into cerebral ventricles of 3-month-old 5x FAD mice for two weeks at a speed of 0.25 µL/h (equivalent to approximately 370 ng per day). At the completion of this treatment, the mice were sacrificed and brains removed for quantification of Aβ levels by sandwich ELISA. The brains were extracted sequentially by RIPA and FA. Previously we found that the majority of Aβ in 5xFAD brain resides in the RIPA-soluble and FA-soluble fractions. The RIPA buffer would first solubilize membrane-associated intraneuronal and matrix-bound extracellular Aβ, as well as soluble extracellular Aβ, mostly in non-fibrillar forms. The subsequent FA solubilization of RIPA-insoluble samples would dissolve the fibrillar Aβ that exists in the amyloid plaques (Hong et al. 2008). CP2 treatment resulted in a 40% reduction of RIPA-soluble Aβ and a 50% reduction of FA-soluble Aβ (Figure 11). Thus, in a mouse model with perhaps the most robust Aβ42 production among all AD models, a short 2-week treatment of CP2 showed a promising anti-Aβ effect.</p><!><p>Decreasing the amount of Aβ aggregates in Alzheimer's brain is desirable in that neurotoxic pathogen is diminished. Surface plasmon resonance spectroscopy, atomic force microscopy, and protein quantification studies showed that tricyclic pyrone CP2 binds to Aβ42 oligomers, inhibits Aβ aggregations, and disaggregates Aβ42 oligomers and protofibrils. Circular dichroism studies revealed that CP2 stabilizes the random coil/α-helix structures of monomeric Aβ42 peptide, which leads to the inhibition of fibrillation. Subsequently, treatment of a perhaps most robust Aβ42-producing Alzheimer model called 5x FAD mice with CP2 for two weeks resulted in a 40% reduction of non-fibrillar Aβ species and 50% reduction of fibrillar Aβ species. These results show the promising anti-Aβ effects of CP2 and warrant further investigation of CP2 by testing other treatment modalities and development of CP2 analogs for enhanced bioactivity. Mechanisms of disaggregation of Aβ may be studied using CP2 and its analogs.</p><!><p>Alzheimer's disease</p><p>β-amyloid protein</p><p>atomic force microscopy</p><p>circular dichroism</p><p>dimethyl sulfoxide</p><p>N-ethyl-N'-[(dimethylamino)propyl]carbodiimide</p><p>enzyme-linked immunosorbent assay</p><p>formic acid</p><p>hexafluoroisopropanol</p><p>N-hydroxysuccinimide</p><p>radioimmune precipitation assay</p><p>response unit</p><p>surface plasmon resonance</p><p>trifluoroacetic acid</p>