An α-Cyanostilbene derivative for the enhanced detection and imaging of amyloid fibril aggregates
journal contributionposted on 2021-08-13, 01:45 authored by Nicholas R Marzano, Kelly M Wray, Caitlin L Johnston, Bishnu P Paudel, Yuning HongYuning Hong, Antoine van Oijen, Heath Ecroyd
The aggregation of proteins into amyloid fibrils has been implicated in the pathogenesis of a variety of neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Benzothiazole dyes such as Thioflavin T (ThT) are well-characterized 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 (called ASCP) with aggregation-induced emission properties 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 visualize fibrils via total internal reflection fluorescence 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.
This research performed by N.R.M. and C.L.J. was conducted with the support of the Australian Government Research Training Program Scholarship. A.v.O. is supported by an ARC Laureate Fellowship (FL140100027). We thank Tze Cin Owyong and Anuradha for synthesis of the dye. Finally, we thank staff in Molecular Horizons and the Illawarra Heath and Medical Research Institute for technical and administrative support.
JournalACS Chemical Neuroscience
Pagination12p. (p. 4191-4202)
PublisherAmerican Chemical Society (ACS)
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Science & TechnologyLife Sciences & BiomedicineBiochemistry & Molecular BiologyChemistry, MedicinalNeurosciencesPharmacology & PharmacyNeurosciences & Neurologysingle-laser two-color microscopymolecular chaperonesHspsingle-molecule fluorescencealpha-synucleinamyloid beta (A beta)THIOFLAVIN-T BINDINGSYNUCLEIN AGGREGATIONMOLECULAR-MECHANISMPROTEIN AGGREGATIONHIGH-SENSITIVITYGROWTH-KINETICSINHIBITIONOLIGOMERSELONGATIONNUCLEATIONAcrylonitrileAmyloidFluorescent DyesFluorescencealpha-SynucleinBenzothiazolesamyloid beta (Aβ)α-synuclein