Technological advancement has given researchers a growing number of tools for visualizing amyloid-β peptides, including two-photon microscopy for real-time measurement of plaque growth in AD mouse models (Yan et al., 2009 and Meyer-Luehmann et al., 2008). However, Ikezu’s study is the first to dynamically and quantitatively track formation of amyloid-β oligomers, now widely seen as the most neurotoxic Aβ species. Last year, researchers published an enzyme-linked immunosorbent assay (ELISA) specific for Aβ oligomers (Xia et al., 2009 and ARF related news story), but when it comes to live imaging, standard reagents for plaque staining, e.g., thioflavin or Congo red, bind poorly to the β-sheet structure of Aβ oligomers. They also poorly penetrate the brain. Ikezu’s lab found several β-sheet binding fluorescent dyes that do reach the brain, e.g., X-34, FSB, and derivatives (Flaherty et al., 2007), but all these amyloid imaging compounds have a key problem. “Once they bind to the plaque, they inhibit aggregation,” Ikezu said. “They competitively interfere with assembly of the β-sheet structure.” Furthermore, the short half-life of these reagents precludes their use in long-term imaging studies.

Quantum dots (QD), on the other hand, are fluorescent semiconductor nanocrystals that “emit signals for a long time, almost indefinitely,” Ikezu said. “You keep [the QD probe] on ice for one month, and it’s still usable.” A previous report described Aβ imaging using QD, but the labeling relied on non-specific ionic interactions between QD particles and Aβ fibrils (Ji et al., 2006). Ikezu’s team, led by first author Kiyotaka Tokuraku, is the first to make Aβ-specific nanoprobes by directly coupling Aβ peptide to QDs. Tokuraku, a professor at Miyakonojo National College of Technology, Japan, did the work during his recent sabbatical in the Ikezu lab.

Aggregation in Real Time
Quantum-dot Aβ nanoprobes (green) were incubated with free Aβ42 (to enable aggregation) on a 96-well plate and visualized by confocal microscopy every 30 minutes for 20 hours. To see a dynamic rendition of Aβ aggregation, see movie (the movie takes a few seconds to download). Additional movies are also available as supporting information in the paper. Image credit: Tsuneya Ikezu and PLoS ONE

The researchers attached a crosslinker to the QDs before coupling them to Aβ40 peptides. They used QDAβ to measure Aβ oligomerization in vitro, showing that the size of Aβ aggregates could be estimated from relative intensities of spots imaged by fluorescence microscopy. The QD probes could also monitor Aβ fibrillization (see image), as long as non-conjugated Aβ was mixed with the QDAβ. (Because QD particles are quite large relative to Aβ peptides, it is likely that QD steric hindrance inhibits fibril formation.)

Ikezu suggested that the Aβ QD nanoprobes would be good for single-molecule imaging of Aβ in cultured cells—seeing how Aβ oligomers get endocytosed, for example. As described in the current report, his team has used QDAβ to watch microglia gobble up Aβ. The phagocytes seemed to prefer monomeric QDAβ over oligomers, and the ingested QDAβ mainly accumulated in the lysosome.

Another potential application for QDAβ is small-scale drug screening. “You can see how drugs block different kinds of Aβ aggregates,” Ikezu said. He and colleagues showed that Aβ-specific antibodies, but not control antibodies to tubulin, were able to block QDAβ aggregation.—Esther Landhuis.

Reference:
Tokuraku K, Marquardt M, Ikezu T. Real-Time Imaging and Quantification of Amyloid-Beta Peptide Aggregates by Novel Quantum-Dot Nanoprobes. PLoS ONE. Dec 2009;4(12):e8492. Abstract