Essential Components of Synthetic Infectious Prion Formation De Novo

Abstract

Prion diseases are a class of neurodegenerative diseases that are uniquely infectious. Whilst their general replication mechanism is well understood, the components required for the formation and propagation of highly infectious prions are poorly characterized. The protein-only hypothesis posits that the prion protein (PrP) is the only component of the prion; however, additional co-factors are required for its assembly into infectious prions. These can be provided by brain homogenate, but synthetic lipids and non-coding RNA have also been used in vitro. Here, we review a range of experimental approaches, which generate PrP amyloid assemblies de novo. These synthetic PrP assemblies share some, but not necessarily all, properties of genuine infectious prions. We will discuss the different experimental approaches, how a prion is defined, the non-protein requirements of a prion, and provide an overview of the current state of prion amplification and generation in vitro.

Keywords: PMCA; RT-QuIC; aggregation; infectivity; prions; synthetic prions.

Figure 1

An outline of the top-down and bottom-up approach for investigating prions. The top-down approach has largely been achieved, with the high resolution structures of two prion strains having been recently solved [17], as well as many more structures of other amyloid classes [18]. The bottom-up approach starts from simplified components and aims to produce a prion with the same structure and biological activity as the brain-derived prions. This approach requires identification of any potential co-factors, and so provides further understanding of the disease process. It allows for modulation of the process by adding potential inhibitors or accelerators, and so contributes to the development of therapeutics.

Figure 2

Whilst the core of the definition of a prion as an infectious protein structure as stated by Prusiner has remained, the boundaries of its definition have since blurred as protein assemblies, which are not infectious in vivo, can possess traits that were thought to be specific for prions. PrP amyloid (orange) is defined by is structure, a PrP protein sequence with a cross-β-sheet structure, whereas a prion (green) is defined by its biological activity (light blue). All solved structures of bona fide prions are PIRIBS amyloids, suggesting that amyloid can contain prion-like traits. However, biochemical properties such as PK resistance (grey) and in vitro replication (dark blue) that are used as proxies for the detection of prions are shared by amyloid assemblies, which are not infectious in vivo. The dashed line (yellow) signifies that much is still unknown about how prions cause disease—the toxicity may be caused by a non-protein species downstream of the prion, as well as by the prion itself.

Figure 3

In vitro prion amplification assays. A direct comparison of the main in vitro aggregation methods, with regard to their respective components, conditions and outputs. Variations with regard to the starting seed and substrate, as well as how the final product is defined, are further discussed in the relevant section, but shown here is the typical set up for each.