Tracking Prion Evolution as It Happens

Introduction

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.

Objectives

In this study, we aim to differentiate prion conformers with distinct strain properties, either on a particle-by-particle basis or by capturing the transitional conformers of prion replication.

Methods

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. By combining that with in vitro prion amplification using the Prion Misfolding Shaking Amplification (PMSA) and Prion Misfolding Cyclic Amplification (PMCA) techniques, we gain real time information about the resulting conformers. On a microscopy basis, we apply Total Internal Reflection Fluorescence with a beamsplitter and analyse the fluorescence on two wavelengths on an aggregate-by-aggregate basis.

Results

Spectral Fingerprinting comparison between replication timepoints revealed increasingly different spectra to the starting point in each replication round, which is attributed to the creation of new prion conformers. In microscopy, we could distinguish strains in a statistically significant and semi-automated manner.

Conclusions

Prion conformers reform during in vitro amplification, while prion strains display conformer clouds that can produce distinct fluorescent properties.