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

Abstract

In Alzheimer’s disease (AD) and other neurodegenerative disorders, proteins accumulate into ordered aggregates, called amyloids. Recent evidence suggests that these structures include both large, insoluble fibrils and smaller, prefibrillar structures, such as dimers, oligomers, and protofibrils. Recently, focus has shifted to the prefibrillar aggregates because they are highly neurotoxic and their levels appear to correlate with cognitive impairment. Thus, there is interest in finding methods for specifically quantifying these structures. One of the classic ways of detecting amyloid formation is through the fluorescence of the benzothiazole dye, thioflavin T (ThT). This reagent has been a “workhorse” of the amyloid field because it is robust and inexpensive. However, one of its limitations is that it does not distinguish between prefibrillar and fibrillar aggregates. We screened a library of 37 indoles for those that selectively change fluorescence in the presence of prefibrillar amyloid-β(Aβ). From this process, we selected the most promising example, tryptophanol (TROL), to use in a quantitative “thioflavin-like” assay. Using this probe in combination with electron microscopy, we found that prefibrils are largely depleted during Aβ aggregation in vitro but that they remain present after the apparent saturation of the ThT signal. These results suggest that a combination of TROL and ThT provides greater insight into the process of amyloid formation by Aβ. In addition, we found that TROL also recognizes other amyloid-prone proteins, including ataxin-3, amylin, and CsgA. Thus, this assay might be an inexpensive spectroscopic method for quantifying amyloid prefibrils in vitro.

Insight into Amyloid Structure Using Chemical Probes

“…We recently reported an unbiased screening approach to identify compounds that interact with prefibrillar, but not fibrillar, amyloids (105,115). These efforts identified indole‐based compounds that only undergo a change in fluorescence in the presence of prefibrillar structures, with a selectivity coefficient nearly 20‐fold greater than ThT (Figure 5B).…”

“…By optimizing the chemical structure of the indole and the reaction conditions (e.g. buffer and time), one promising probe, tryptophanol (TROL), was developed into a ‘ThT‐like’ spectroscopic assay for prefibrillar amyloids (115). These findings suggest that some of the indoles access a site on prefibrillar Aβ that likely becomes buried or otherwise inaccessible upon fibril formation.…”

Common Benzothiazole and Benzoxazole Fluorescent DNA Intercalators for Studying Alzheimer Aβ 1-42 and Prion Amyloid Peptides

“…This is in part due to the optical properties of many amyloid binding compounds. Most ThT/Congo red analogs and related compounds absorb light at wavelengths that overlap with endogenous protein absorption and fluorescence (15,16). Since many of these compounds have nondistinct optical properties, in vivo and in vitro biochemical studies rely heavily on radiolabeled compounds (1720).…”

Rapid amyloid fibril formation by a winter flounder antifreeze protein requires specific interaction with ice

“…Thioflavin T dye‐binding assays were carried out largely as described previously . Work using ThT was performed in low light conditions.…”