IntroductionA general characteristic of aggregation is the multiple interaction and cross-feedback among distinct mechanismsoccurring at different hierarchical levels. The comprehension of the different species interconversion during aggregationis very important since emerging evidences indicate intermediate oligomeric aggregates as primary toxic species. In thiscontext, Aβ amyloid peptide provides a challenging model for studying aggregation phenomena both for the complexityof its association process and for the direct implications in Alzheimer’s Disease. Aggregates growth conditions stronglyaffect the final morphology, the fibrillar molecular structure as well as the aggregation pathway which is characterizedby the occurrence of multiple transient species.MethodsThe fluorescent dye Thioflavin T (ThT) is widely used to detect amyloid deposits and it is often used in situ to studyaggregation kinetics, under the hypothesis that its presence does not affect the aggregation processes under study. Herewe present an experimental study on Aβ(1-40) peptide fibrillation kinetics at pH 7.4. In the observed conditions, Aβ(1-40) undergoes aggregation only if Thioflavin T is present in solution. This phenomenon was analyzed as a function oftemperature, ThT and peptide concentrations in order to explore the underlying fibrillation mechanism. Light scattering,ThT fluorescence emission, two photon excitation fluorescence microscopy, were used in a kinetic fashion to highlightdifferent sides and critical phases of the aggregation pathway. Circular Dichroism and FTIR measurements are used tocharacterize secondary structure of the aggregates.ResultsThe selected approach gives detailed information on the time evolution of Aβ(1-40) fibrillation process highlightingstructural changes at molecular level, different aggregate species growth and their morphologies. Our data show thatAβ(1-40) fibrillation process occurs only in the presence of ThT and that the observed aggregation involves at leastthree different aggregation mechanisms acting in competition. In the first step, small oligomers, which bind ThT, areformed via non nucleated polymerization mechanism and represent an activated state for following fibrils growth. Thisprocess appear to be a rate limiting step for two distinct fibril nucleation mechanisms probably affected by an highdegree of spatial heterogeneity.ConclusionsWe demonstrated that in the selected experimental conditions ThT triggers the Aβ(1−40) fibrillation process (D’Amicoet. al 2012). Sterical and chemical properties of ThT molecule may modulate the peptide conformation, with similarmechanisms to the ones that usually drive the binding of this dye to already formed amyloids. So, the presence of ThTin solution may change the thermodynamic equilibrium trapping specificmore ordered conformations prone tosupramolecular assembly.
|Numero di pagine||1|
|Stato di pubblicazione||Published - 2012|