Introduction
A general characteristic of aggregation is the multiple interaction and cross-feedback among distinct mechanisms
occurring at different hierarchical levels. The comprehension of the different species interconversion during aggregation
is very important since emerging evidences indicate intermediate oligomeric aggregates as primary toxic species. In this
context, A\u3b2 amyloid peptide provides a challenging model for studying aggregation phenomena both for the complexity
of its association process and for the direct implications in Alzheimer\u2019s Disease. Aggregates growth conditions strongly
affect the final morphology, the fibrillar molecular structure as well as the aggregation pathway which is characterized
by the occurrence of multiple transient species.
Methods
The fluorescent dye Thioflavin T (ThT) is widely used to detect amyloid deposits and it is often used in situ to study
aggregation kinetics, under the hypothesis that its presence does not affect the aggregation processes under study. Here
we present an experimental study on A\u3b2(1-40) peptide fibrillation kinetics at pH 7.4. In the observed conditions, A\u3b2(1-
40) undergoes aggregation only if Thioflavin T is present in solution. This phenomenon was analyzed as a function of
temperature, 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 highlight
different sides and critical phases of the aggregation pathway. Circular Dichroism and FTIR measurements are used to
characterize secondary structure of the aggregates.
Results
The selected approach gives detailed information on the time evolution of A\u3b2(1-40) fibrillation process highlighting
structural changes at molecular level, different aggregate species growth and their morphologies. Our data show that
A\u3b2(1-40) fibrillation process occurs only in the presence of ThT and that the observed aggregation involves at least
three different aggregation mechanisms acting in competition. In the first step, small oligomers, which bind ThT, are
formed via non nucleated polymerization mechanism and represent an activated state for following fibrils growth. This
process appear to be a rate limiting step for two distinct fibril nucleation mechanisms probably affected by an high
degree of spatial heterogeneity.
Conclusions
We demonstrated that in the selected experimental conditions ThT triggers the A\u3b2(1 1240) fibrillation process (D\u2019Amico
et. al 2012). Sterical and chemical properties of ThT molecule may modulate the peptide conformation, with similar
mechanisms to the ones that usually drive the binding of this dye to already formed amyloids. So, the presence of ThT
in solution may change the thermodynamic equilibrium trapping specificmore ordered conformations prone to
supramolecular assembly