Quantifying prefibrillar amyloids in vitro by using a "thioflavin-like" spectroscopic method.
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-beta (Abeta). 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 Abeta 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 Abeta. 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.
Reinke
Ashley A
AA
Department of Pathology, University of Michigan, 4000 Life Sciences Institute, 210 Washtenaw Avenue, Ann Arbor, MI 48109-2216, USA.
Abulwerdi
Gelareh A
GA
Gestwicki
Jason E
JE
D016229:Amyloid beta-Peptides / Q000032:analysis*; D052160:Benzothiazoles; D005456:Fluorescent Dyes / Q000737:chemistry*; D008958:Models, Molecular; D013050:Spectrometry, Fluorescence; D013844:Thiazoles / Q000737:chemistry; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000737:chemistry
D016229:Amyloid beta-Peptides; D052160:Benzothiazoles; D005456:Fluorescent Dyes; D013844:Thiazoles; C009462:thioflavin T; C069544:tryptophanol; D014364:Tryptophan