Prions are unique agents causing spongiform encephalopathies. They are self-replicating molecules, showing mutation-like events and natural selection regarding their environment. Although they do not contain any genetic material, they display strain variation, which is described by conformational differences and variability on both phenotype and zoonotic potential. We aim to investigate prion evolution by examining the distinct prion conformers present within prion preparations, either ex vivo or in vitro generated, and capture the transitional conformers of prion replication. We employ the luminescent conjugated oligothiophene Amytracker 540, which has a flexible backbone that allows it to produce unique spectral fingerprints from prion conformers that correspond to distinct strains. With this dye, we apply the spectral fingerprinting method, which was developed into our lab, to discriminate prion conformers based on a three-dimensional excitation, emission and fluorescence spectrum. In vitro prion amplification is achieved with the established prion misfolding shaking amplification (PMSA) and prion misfolding cyclic amplification (PMCA) techniques. On a microscopy basis, we apply total internal reflection fluorescence with a dichroic emission beamsplitter and analyze the fluorescence on an aggregate-by-aggregate basis. A computer vision algorithm was developed to discriminate aggregates, along with a script for the fluorescence ratio calculation and comparison. Spectral Fingerprinting comparison between replication time points revealed quantifiable differences between replication rounds, which is attributed to seed mutation and conformer selection. In vitro generated prions also clearly differ from the ex vivo seed that they were produced from. The results were confirmed by microscopy, where we could distinguish strains in a statistically significant and semi-automated manner. We conclude that prion conformers do not faithfully replicate during in vitro amplification. Purified prion preparations include distinct but overlapping conformer distributions, in regards to their spectral characteristics.
Chatzitheodosiou et al. (Sun,) studied this question.