A Microliter-Scale High-throughput Screening System with Quantum-Dot Nanoprobes for Amyloid-β Aggregation Inhibitors

The aggregation of amyloid β protein (Aβ) is a key step in the pathogenesis of Alzheimer’s disease (AD), and therefore inhibitory substances for Aβ aggregation may have preventive and/or therapeutic potential for AD. Here we report a novel microliter-scale high-throughput screening system for Aβ aggregation inhibitors based on fluorescence microscopy-imaging technology with quantum-dot Nanoprobes. This screening system could be analyzed with a 5-µl sample volume when a 1536-well plate was used, and the inhibitory activity could be estimated as half-maximal effective concentrations (EC50). We attempted to comprehensively screen Aβ aggregation inhibitors from 52 spices using this system to assess whether this novel screening system is actually useful for screening inhibitors. Screening results indicate that approximately 90% of the ethanolic extracts from the spices showed inhibitory activity for Aβ aggregation. Interestingly, spices belonging to the Lamiaceae, the mint family, showed significantly higher activity than the average of tested spices. Furthermore, we tried to isolate the main inhibitory compound from Satureja hortensis, summer savory, a member of the Lamiaceae, using this system, and revealed that the main active compound was rosmarinic acid. These results demonstrate that this novel microliter-scale high-throughput screening system could be applied to the actual screening of Aβ aggregation inhibitors. Since this system can analyze at a microscopic scale, it is likely that further minimization of the system would easily be possible such as protein microarray technology

A microliter-scale high-throughput screening system with quantum-dot nanoprobes for amyloid-β aggregation inhibitors.

The aggregation of amyloid β protein (Aβ) is a key step in the pathogenesis of Alzheimer's disease (AD), and therefore inhibitory substances for Aβ aggregation may have preventive and/or therapeutic potential for AD. Here we report a novel microliter-scale high-throughput screening system for Aβ aggregation inhibitors based on fluorescence microscopy-imaging technology with quantum-dot Nanoprobes. This screening system could be analyzed with a 5-µl sample volume when a 1536-well plate was used, and the inhibitory activity could be estimated as half-maximal effective concentrations (EC50). We attempted to comprehensively screen Aβ aggregation inhibitors from 52 spices using this system to assess whether this novel screening system is actually useful for screening inhibitors. Screening results indicate that approximately 90% of the ethanolic extracts from the spices showed inhibitory activity for Aβ aggregation. Interestingly, spices belonging to the Lamiaceae, the mint family, showed significantly higher activity than the average of tested spices. Furthermore, we tried to isolate the main inhibitory compound from Saturejahortensis, summer savory, a member of the Lamiaceae, using this system, and revealed that the main active compound was rosmarinic acid. These results demonstrate that this novel microliter-scale high-throughput screening system could be applied to the actual screening of Aβ aggregation inhibitors. Since this system can analyze at a microscopic scale, it is likely that further minimization of the system would easily be possible such as protein microarray technology

Isolation and identification of active compound from EtOH extract of summer savory.

<p>(A) A flow diagram of isolation steps. (B) Inhibition curves of isolated RA from summer savory (squares) and standard RA (triangles) were determined by the microliter-scale high-throughput screening (MHS) system (B, top) and the ThT assay (B, bottom). Vertical axes of the MHS system and the ThT assay are the percentage of average SD values and the percentage of average fluorescence intensity (FI) values, respectively. The EC₅₀ values of isolated RA and standard RA determined by the MHS system were 9.6 ± 0.1 and 11 ± 2 µM, respectively. In contrast to that, the EC₅₀ values of isolated RA and standard RA determined by ThT assay were 8.6 ± 0.8 and 6.3 ± 1.5 µM, respectively. Error bars represent ±SDs (n=3 separate experiments).</p

Estimation of EC₅₀ values of EtOH extracts from 52 spices using the microliter-scale high-throughput screening system.

<p>Screening was applied to examine the EtOH extracts of dried spices. The ‘black’, ‘gray’, and ‘white’ cells indicate ‘not inhibited (SD ≥ 80%)’, ‘partially inhibited (80% > SD > 20%)’, and ‘completely inhibited (20% ≥ SD)’ wells, respectively. The percentage of SD was defined as SD values before and after incubation of control samples (0% and 100%, respectively). EC₅₀ values were estimated from dose-dependent inhibition curves (n=3 separate experiments). Spices were aligned using the Angiosperm Phylogeny Group classification (APG III) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072992#B25" target="_blank">25</a>].</p

Effect of EtOH on Aβ₄₂ aggregation.

<p>30 nM QDAβ and 30 µM Aβ₄₂ were mixed in 1xPBS, 3% DMSO containing 0, 2.5, 5, 10, 20, or 40% EtOH, each sample was incubated at 37 ^°C for 24 h in a 1536-well plate. The wells were observed using an inverted fluorescence microscope using a 4x objective. The images show 200 × 200 pixels in the center of each well.</p

Time-dependent Aβ aggregation.

<p>(A) 30 nM QDAβ and 30 µM Aβ₄₂ were incubated in a 1536-well plate at 37 ^°C, and observed over time by an inverted fluorescence microscope using a 4x objective. All images show the same field of a well. (B) Variations of fluorescence intensities of 10,000 pixels (100 × 100 pixel) in the center region of micrographs were estimated as SD values, the mean values were plotted against incubation time periods. Error bars represent ±SDs of the mean values of fluorescence intensities (n=3 separate experiments).</p

Estimation of EC₅₀ by the microliter-scale high-throughput screening system.

<p>(A) A schematic illustration of a microliter-scale high-throughput screening system for three samples. (B) The concept of estimation of EC₅₀ values from inhibition curves that are a plotted percentage of SD versus concentrations of inhibitors. The EC₅₀ of sample A is higher than that of sample C, and sample B did not inhibit Aβ aggregation. (C and D) Estimations of EC₅₀ of well-known inhibitors, curcumin (Cur), rosmarinic acid (RA), tannic acid (TA), and myricetin (Myr). 30 nM QDAβ and 30 µM Aβ₄₂ was incubated with various concentrations of the four inhibitors at 37 ^°C for 24 h (C). The SD values from the fluorescence images plotted against several concentrations of inhibitors (D). Error bars represent ±SDs of the mean values from fluorescence intensities (n=3 separate experiments). EC₅₀ values of Cur, RA, TA, and Myr were 31 ± 16, 11 ± 2, 1.8 ± 1.5, and 1.0 ± 0.3 µM, respectively.</p

Imaging of Aβ₄₂ aggregation using a QDAβ nanoprobe.

<p>(A) QDAβ was prepared by crosslinking CysAβ₄₀ and amino (PEG) Qdot655 according to our recent study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072992#B12" target="_blank">12</a>] (left). QDAβ coaggregated with unlabeled Aβ₄₂, and the Aβ₄₂ fibrils that formed could be visualized under fluorescence microscopy (right). (B) 30 nM QDAβ and 30 µM unlabeled Aβ₄₂ was incubated at 37 ^°C for 24 h in a 1536-well plate, and was observed using an inverted fluorescence microscope using a 4x objective. Left and right panels show before and after incubation, respectively. (C) Magnified image of the aggregates observed using a 10× objective.</p

Correlation between Aβ aggregation and variations of fluorescence intensity.

<p>(A) Magnified images of center region (100 × 100 pixel) in fluorescence micrographs of QDAβ- Aβ₄₂ coaggregates before (left) and after (right) incubation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072992#pone-0072992-g001" target="_blank">Figure 1B</a>). (B) Schematic illustrations of the distribution of QDAβ (red) and Aβ₄₂ (gray) molecules before (left) and after (right) incubation of samples. QDAβ molecules are diffused in the sample solution before incubation (left), and QDAβ molecules are inserted in Aβ₄₂ fibrils after incubation (right). (C) The histograms of fluorescence intensities of 10,000 pixels (100 × 100 pixel) before (left) and after (right) incubation of samples.</p

Concentration-dependent Aβ aggregation.

<p>(A) Various concentrations of Aβ₄₂ and 30 nM QDAβ were incubated in a 1536-well plate at 37 ^°C for 24 h. Each well was observed using an inverted fluorescence microscope using a 4x objective. (B) Variations of fluorescence intensities of 10,000 pixels (100 × 100 pixel) in the center region of micrographs were estimated as SD values, the mean values were plotted against the concentrations of added Aβ₄₂. A linear equation and R² in B were determined using the data of less than 30 µM of Aβ concentrations. Error bars represent ±SDs of the mean values of fluorescence intensities (n=3 separate experiments).</p