Anα-cyanostilbene derivative for the enhanced detection and imaging of amyloid fibril aggregates

Abstract

The aggregation of proteins into amyloid fibrils has been implicated in the pathogenesis of a variety of neurodegenerative diseases, including Alzheimer's and Parkinson's disease. Benzothiazole dyes such as Thioflavin T (ThT) are well characterised and widely used fluorescent probes for monitoring amyloid fibril formation. However, existing dyes lack sensitivity and specificity to oligomeric intermediates formed during fibril formation. In this work we describe the use of an α-cyanostilbene derivative with aggregation-induced emission properties (called ASCP) as a fluorescent probe for the detection of amyloid fibrils. Similar to ThT, ASCP is fluorogenic in the presence of amyloid fibrils and upon binding and excitation at 460 nm produces a red-shifted emission with a large Stokes shift of 145 nm. ASCP has a higher binding affinity to fibrillar α-synuclein than ThT and likely shares the same binding sites to amyloid fibrils. Importantly, ASCP was found to also be fluorogenic in the presence of amorphous aggregates and can detect oligomeric species formed early during aggregation. Moreover, ASCP can be used to visualise fibrils via Total Internal Reflection Fluorescence (TIRF) microscopy and, due to its large Stokes shift, simultaneously monitor the fluorescence emission of other labelled proteins following excitation with the same laser used to excite ASCP. Consequently, ASCP possesses enhanced and unique spectral characteristics compared to ThT that make it a promising alternative for the in vitro study of amyloid fibrils and the mechanisms by which they form.

Illuminating amyloid fibrils: Fluorescence-based single-molecule approaches

“…Fluorescence-based TIRF methods, such as FRET (including single-molecule FRET) and photobleaching (to determine stoichiometries of interactions via the stepwise photobleaching of fluorophores) can be exploited to determine the molecular details of protein–protein interactions. Moreover, the use of fluorescent probes, such as aggregation-induced emission (AIE) dyes, in combination with TIRF-based single-molecule approaches has facilitated the visualisation and tracking of protein aggregates without the need for a fluorophore conjugated to the protein of interest [106] , [107] , [108] .…”

“…The relatively small Stokes shift of ThT (the emission photon wavelength is close to the wavelength of the excitation photon [125] ), can cause problems in separating the emission signal from scattered excitation light [126] , [127] . Recently, AIE dyes with larger Stokes shifts that can be used to visualise fibrils have been described [29] , [108] . Apart from the advantage of an increased affinity for amyloid fibrils, another advantage of some of these recently described AIE dyes is that two-colour experiments can be performed using only one excitation laser.…”

“…As an example, the AIE dye α-cyanostilbene derivative ASCP [29] has a very large Stokes shift (145 nm) and can be excited using a 488 nm laser light source which is commonly found in commercial fluorescence microscopes (excitation maximum at 460 nm and emission maximum at 605 nm). Recently, ASCP was used to simultaneously image molecular chaperones (labelled with an AlexaFluor488 fluorophore and emission monitored at 500–550 nm) as they interacted with α-syn fibrils (excited with the 488 nm excitation wavelength and emission monitored at 675 nm and beyond) [108] .…”

“…Conclusive evidence for this mechanistic model could be provided by using TIRF-based experiments, including fluorophore photobleaching to directly observe and quantify molecular crowding, the position on the fibril at which this occurs, and the binding and dissociation rates of Hsp70/40 and NEF Hsp110. Moreover, the discovery of new AIE dyes with large Stokes shifts, such as ASCP [88] , [108] provide the possibility to visualise fibrils and proteins that interact with them using only one excitation source [108] . More complex three-colour fluorescence experiments would enable more components to be observed simultaneously [213] , [214] and thus be invaluable for the study of how chaperone machines, such as how the Hsp70/Hsp40/Hsp110 disaggregase system [66] , [75] , interact with amyloid fibrils.…”