A chemical screening approach reveals that indole fluorescence is quenched by pre-fibrillar but not fibrillar amyloid-β

Abstract

Aggregated amyloid-β (Aβ) peptide is implicated in the pathology of Alzheimer's disease. In vitro and in vivo, these aggregates are found in a variety of morphologies, including globular oligomers and linear fibrils, which possess distinct biological activities. However, known chemical probes, including the dyes thioflavin T and Congo Red, appear to lack selectivity for specific amyloid structures. To identify molecules that might differentiate between these architectures, we employed a fluorescence-based interaction assay to screen a collection of 68 known Aβ ligands against preformed oligomers and fibrils. In these studies, we found that the fluorescence of five indole-based compounds was selectively quenched (~15%) in the presence of oligomers, but remained unchanged after addition of fibrils. These results suggest that indoles might be complementary to existing chemical probes for studying amyloid formation in vitro.

Quantifying Prefibrillar Amyloids in vitro by Using a “Thioflavin‐Like” Spectroscopic Method

“…Then we calculated the ratio of the fluorescence change between these samples (Δfluorescence prefibril sample/Δfluorescence fibril sample) and termed this value “prefibrillar selectivity”. This analysis confirmed [23, 25] that ThT and another common probe, bis-ANS, had poor prefibrillar selectivity (values of 0.2 and 1.1, respectively; Figure 1B). However, we identified ten compounds ( 3 , 6 , 12 , 13 , 18 , 19 , 20 , 22 , 24 , and 34 ) with better selectivity than the initial indole (Figure 1B and Table S1).…”

“…As previously reported, the prefibrillar solution contained a mixture of spherical oligomers and smaller aggregates. [23, 24] The fibril sample contained exclusively elongated fibrils from aged Aβ preparation. Following a 60-minute incubation, the fluorescence of each indole-treated sample was recorded and compared to that of the indole alone.…”

“…We followed established protocols to obtain predominantly prefibrils or fibrils. [23, 24] Briefly, to obtain prefibrils, Aβ was suspended in 1 % DMSO followed by DMEM-F12 cell culture medium (Gibco, Carlsbad, CA) to a final concentration of 25 μM, vortexed, and sonicated for 90 s. The sample was then incubated for 2 days at 4 °C without agitation. To obtain fibrils, Aβ was suspended in 1 % DMSO followed by PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.2) to a final concentration of 25 μM, vortexed, sonicated for 90 s, and agitated for 7–10 days at 37 °C.…”

“…[23] Here, we expand on this initial observation by screening a focused collection of 37 indoles. Using the most promising probe (tryptophanol, TROL), we developed an assay that quantifies prefibrillar Aβ, even in complex mixtures.…”

Biochemistry of Amyloid -Protein and Amyloid Deposits in Alzheimer Disease

“…Classical biochemical analyses of the synthetic aggregates have been supplemented with newer biophysical methods such as scanning tunneling microscopy (Liu et al 2009a;Ma et al 2009), atomic force microscopy , quartz crystal microbalance (Ogi et al 2009), hydrogen exchange mass spectrometry ), electron capture dissociation Fourier-transform ion cyclotron resonance mass spectroscopy (Sargaeva et al 2009), single-molecule spectroscopy (Ding et al 2009), fluorescence photobleaching and quenching (Reinke et al 2009;Edwin et al 2010), click peptide technique (Taniguchi et al 2009), and ion mobility coupled with mass spectrometry ). The experimental conditions for assembling synthetic Ab monomers into oligomers vary enormously with regard to the roles of temperature, salts, detergents, lipids, metal ions, fatty acids, and other molecules (Sahoo et al 2009;Yu et al 2009;Ahmed et al 2010;Ladiwala et al 2010;Ryan et al 2010), and each such condition provides constraints on the techniques the can be used to study the synthetic peptide.…”