CONICET | Buscador de Institutos y Recursos Humanos

Parkinsons disease is a movement disorder characterized by the presence in the mid-brain of amyloid deposits of the 140-aa protein a-synuclein (AS). Three genetic mutants, A53T, A30P and E46K, have been linked with early onset Parkinsons. The amyloid fibrils consist of interwound protofilaments with a cross b-sheet secondary structure. Although highly conserved among different aggregopathies, mature fibrils exhibit a certain degree of polymorphism, which is poorly characterized and understood. In the present work we used 3-hydroxyflavone derivatives as novel fluorescent probes to distinguish structural features of amyloid fibrils formed by AS and its familial mutants. These dyes exhibit two intensive well-separated emission bands reflecting excited state redistributions. Their intensity ratio constitutes an ultrasensitive and tunable reporter of microenvironment properties such as polarity, hydrogen bonding, and polarizability. The dyes are nonfluorescent in the presence of the wt and mutant monomeric AS proteins but exhibit distinctive fluorescence spectra upon binding to the respective fibrils formed in vitro. The dyes do not simply absorb at the surface but are integrated into the internal fibrillar structures, as indicated by spectral changes corresponding to decreased polarity and partial disruption of hydrogen bonds to water. It is remarkable that each point mutant is clearly distinguishable by the spectral signatures of these external dyes. We anticipate that the 3-hydroxyflavone derivatives may serve as useful tools, including diagnostic, for detecting polymorphism in other amyloid systems as well as for uncovering oligomeric intermediates currently identified as the neurotoxic species.