Multiparametric Fluorescence Detection of Early Stages in the Amyloid Protein Aggregation of Pyrene-labeled α-Synuclein

The aggregation of α-synuclein, a presynaptic protein, has an important role in the etiology of Parkinson’s disease. Oligomers or protofibrils adopting the cross-β-sheet structure characteristic of fibrillating amyloid proteins are presumed to be the primary cytotoxic species. Current techniques for monitoring the kinetics of α-synuclein aggregation based on fluorescent dyes such as Thioflavin-T and Congo red detect only the terminal fibrillar species, are discontinuous and notoriously irreproducible. We have devised a new fluorescence aggregation assay that is continuous and provides a large set of fluorescence parameters sensitive to the presence of oligomeric intermediates as well as fibrils. The approach involves tagging functionally neutral Ala-to-Cys variants of α-synuclein with the long-lifetime fluorophore pyrene. Upon induction of aggregation at 37 °C, the entire family of steady-state descriptors of pyrene emission (monomer intensity, solvent polarity ratio (II/IIII), and anisotropy; and excimer intensity) change dramatically, particularly during the early stages in which oligomeric intermediates form and evolve. The pyrene probe senses a progressive decrease in polarity, an increase in molecular mass and close intermolecular association in a manner dependent on position in the sequence and the presence of point mutations. The time-resolved decays (0–160 ns) of intensity and anisotropy exhibited complex, characteristic features. The new assay constitutes a convenient platform for the high-throughput screening of agents useful in the diagnosis and therapy of Parkinson’s disease as well as in basic investigations.