Real-time analysis of amyloid fibril formation of α-synuclein using a fibrillation-state-specific fluorescent probe of JC-1

Abstract

α-Synuclein is a pathological component of PD (Parkinson's disease) by participating in Lewy body formation. JC-1 (5,5′,6,6′-tetrachloro-1,1,3,3′-tetraethylbenzimidazolyl carbocyanine iodide) has been shown to interact with α-synuclein at the acidic C-terminal region with a Kd of 2.6 μM. JC-1 can discriminated between the fibrillation states of α-synuclein (monomeric, oligomeric intermediate and fibrillar forms) by emitting the enhanced binding fluorescence of different colours at 590, 560 and 538 nm respectively with the common excitation at 490 nm. The fibrillation-state-specific interaction of JC-1 allowed us to perform real-time analyses of the α-synuclein fibrillation in the presence of iron as a fibrillation inducer, rifampicin as a fibrillation inhibitor, baicalein as a defibrillation agent and dequalinium as a protofibril inducer. In addition, various α-synuclein fibrils with different morphologies prepared with specific ligands such as metal ions, glutathione, eosin and lipids were monitored with their characteristic JC-1-binding fluorescence spectra. FRET (fluorescence resonance energy transfer) between thioflavin-T and JC-1 was also employed to specifically identify the amyloid fibrils of α-synuclein. Taken together, we have introduced JC-1 as a powerful and versatile probe to explore the molecular mechanism of the fibrillation process of α-synuclein in vitro. It could be also useful in high-throughput drug screening. The specific α-synuclein interaction of JC-1 would therefore contribute to our complete understanding of the molecular aetiology of PD and eventual development of diagnostic/therapeutic strategies for various α-synucleinopathies.