Formation of distinct prion protein amyloid fibrils under identical experimental conditions

Abstract

Protein aggregation into amyloid fibrils is linked to multiple neurodegenerative disorders, such as Alzheimer's, Parkinson's or Creutzfeldt-Jakob disease. A better understanding of the way these aggregates form is vital for the development of drugs. A large detriment to amyloid research is the ability of amyloidogenic proteins to spontaneously aggregate into multiple structurally distinct fibrils (strains) with different stability and seeding properties. In this work we show that prion proteins are capable of forming more than one type of fibril under the exact same conditions by assessing their Thioflavin T (ThT) binding ability, morphology, secondary structure, stability and seeding potential.

Figure 1

Separation of fibrils by ThT fluorescence intensity. (A) ThT fluorescence emission intensities of twenty MoPrP fibril samples prepared under identical conditions. The samples are grouped into three intensity regions, with some samples having low (grey), medium (blue) and high (red) emission intensities. (B) Seeded aggregation kinetics of the three intensity region fibrils using 10% of low, medium and high intensity fibrils. (C) Fluorescence intensity “drops” at the early stages of the aggregation process.

Figure 2

ThT binding to MoPrP amyloid fibrils probed by fluorescence assay. (A) ThT fluorescence emission intensity dependence on the concentration of ThT added to each fibril sample. (B) ThT fluorescence intensity differences between MI and LI samples and HI and MI samples. Hill equation fitting was done to determine the ThT concentration (k) at which the signal intensity midpoint is reached.

Figure 3

Dissociation assay of HI, MI, and LI fibrils and comparison of their secondary structures. (A) Normalized ThT fluorescence intensity values at different GuHCl concentrations, where the grey dotted line represents 50% of normalized fluorescence intensity and coloured lines correspond to each sample’s GuHCl concentration at which the 50% intensity value is reached. (B) FTIR spectra of fibril samples and second order derivative spectra (C), where grey dotted lines show wavenumbers at which the differences between spectra can be observed.

Figure 4

Atomic force microscopy of fibril samples and aggregate size distribution. Images of high intensity (A), medium intensity (B) and low intensity (C) fibrils. Length (D), width (E) and height (F) of single fibrils, where the box plot indicates the interquartile range, error bars are for one standard deviation.