Prion strains viewed through the lens of cryo-EM

Mammalian prions are lethal transmissible pathogens that cause fatal neurodegenerative diseases in humans and animals. They consist of fibrils of misfolded, host-encoded prion protein (PrP) which propagate through templated protein polymerisation. Prion strains produce distinct clinicopathological phenotypes in the same host and appear to be encoded by distinct misfolded PrP conformations and assembly states. Despite fundamental advances in our understanding of prion biology, key knowledge gaps remain. These include precise delineation of prion replication mechanisms, detailed explanation of the molecular basis of prion strains and inter-species transmission barriers, and the structural definition of neurotoxic PrP species. Central to addressing these questions is the determination of prion structure. While high-resolution definition of ex vivo prion fibrils once seemed unlikely, recent advances in cryo-electron microscopy (cryo-EM) and computational methods for 3D reconstruction of amyloids have now made this possible. Recently, near-atomic resolution structures of highly infectious, ex vivo prion fibrils from hamster 263K and mouse RML prion strains were reported. The fibrils have a comparable parallel in-register intermolecular β-sheet (PIRIBS) architecture that now provides a structural foundation for understanding prion strain diversity in mammals. Here, we review these new findings and discuss directions for future research

A structural basis for prion strain diversity

Recent cryogenic electron microscopy (cryo-EM) studies of infectious, ex vivo, prion fibrils from hamster 263K and mouse RML prion strains revealed a similar, parallel in-register intermolecular β-sheet (PIRIBS) amyloid architecture. Rungs of the fibrils are composed of individual prion protein (PrP) monomers that fold to create distinct N-terminal and C-terminal lobes. However, disparity in the hamster/mouse PrP sequence precludes understanding of how divergent prion strains emerge from an identical PrP substrate. In this study, we determined the near-atomic resolution cryo-EM structure of infectious, ex vivo mouse prion fibrils from the ME7 prion strain and compared this with the RML fibril structure. This structural comparison of two biologically distinct mouse-adapted prion strains suggests defined folding subdomains of PrP rungs and the way in which they are interrelated, providing a structural definition of intra-species prion strain-specific conformations

Structural features distinguishing infectious ex vivo mammalian prions from non-infectious fibrillar assemblies generated in vitro

Seeded polymerisation of proteins forming amyloid fibres and their spread in tissues has been implicated in the pathogenesis of multiple neurodegenerative diseases: so called "prion-like" mechanisms. While ex vivo mammalian prions, composed of multichain assemblies of misfolded host-encoded prion protein (PrP), act as lethal infectious agents, PrP amyloid fibrils produced in vitro generally do not. The high-resolution structure of authentic infectious prions and the structural basis of prion strain diversity remain unknown. Here we use cryo-electron microscopy and atomic force microscopy to examine the structure of highly infectious PrP rods isolated from mouse brain in comparison to non-infectious recombinant PrP fibrils generated in vitro. Non-infectious recombinant PrP fibrils are 10 nm wide single fibres, with a double helical repeating substructure displaying small variations in adhesive force interactions across their width. In contrast, infectious PrP rods are 20 nm wide and contain two fibres, each with a double helical repeating substructure, separated by a central gap of 8-10 nm in width. This gap contains an irregularly structured material whose adhesive force properties are strikingly different to that of the fibres, suggestive of a distinct composition. The structure of the infectious PrP rods, which cause lethal neurodegeneration, readily differentiates them from all other protein assemblies so far characterised in other neurodegenerative diseases

Prion 2016 poster abstracts

Until now, the 3-dimensional structure of infectious mammalian prions and how this differs from non-infectious amyloid fibrils remained unknown. Mammalian prions are hypothesized to be fibrillar or amyloid forms of prion protein (PrP), but structures observed to date have not been definitively correlated with infectivity. One of the major challenges has been the production of highly homogeneous material of demonstrable high specific infectivity to allow direct correlation of particle structure with infectivity. We have recently developed novel methods to obtain exceptionally pure preparations of prions from prion-infected murine brain and have shown that pathogenic PrP in these high-titer preparations is assembled into rod-like assemblies (Wenborn et al. 2015. Sci. Rep. 10062). Our preparations contain very high titres of infectious prions which faithfully transmit prion strain-specific phenotypes when inoculated into mice making them eminently suitable for detailed structural analysis. We are now undertaking structural characterization of prion assemblies and comparing these to the structure of non-infectious PrP fibrils generated from recombinant Pr

Isolation of Proteinase K-Sensitive Prions Using Pronase E and Phosphotungstic Acid

Disease-related prion protein, PrPSc, is classically distinguished from its normal cellular precursor, PrPC, by its detergent insolubility and partial resistance to proteolysis. Molecular diagnosis of prion disease typically relies upon detection of protease-resistant fragments of PrPSc using proteinase K, however it is now apparent that the majority of disease-related PrP and indeed prion infectivity may be destroyed by this treatment. Here we report that digestion of RML prion-infected mouse brain with pronase E, followed by precipitation with sodium phosphotungstic acid, eliminates the large majority of brain proteins, including PrPC, while preserving >70% of infectious prion titre. This procedure now allows characterization of proteinase K-sensitive prions and investigation of their clinical relevance in human and animal prion disease without being confounded by contaminating PrPC

Sex effects in mouse prion disease incubation time.

Prion disease incubation time in mice is determined by many factors including PrP expression level, Prnp alleles, genetic background, prion strain and route of inoculation. Sex differences have been described in age of onset for vCJD and in disease duration for both vCJD and sporadic CJD and have also been shown in experimental models. The sex effects reported for mouse incubation times are often contradictory and detail only one strain of mice or prions, resulting in broad generalisations and a confusing picture. To clarify the effect of sex on prion disease incubation time in mice we have compared male and female transmission data from twelve different inbred lines of mice inoculated with at least two prion strains, representing both mouse-adapted scrapie and BSE. Our data show that sex can have a highly significant difference on incubation time. However, this is limited to particular mouse and prion strain combinations. No sex differences were seen in endogenous PrP(C) levels nor in the neuropathological markers of prion disease: PrP(Sc) distribution, spongiosis, neuronal loss and gliosis. These data suggest that when comparing incubation times between experimental groups, such as testing the effects of modifier genes or therapeutics, single sex groups should be used

RML prion infectivity following digestion with 1 mg/ml pronase E.

<p>10% (w/v) RML brain homogenate was incubated with pronase E (1 mg/ml at 37°C) for varying incubation times. For each time point RML prion infectivity was measured by the Scrapie Cell Assay and expressed as a percentage of total infectivity present in the untreated sample; mean ± S.E.M. (<i>n</i> = 5).</p

Pronase E digested and NaPTA precipitated mouse brain homogenate evaluated by silver stain SDS-PAGE.

<p>10% (w/v) homogenates from normal CD-1 brain (CD-1) or RML prion-infected brain (RML) were either untreated (Pro −, NaPTA −), digested by pronase E at 100 µg/ml, 37°C for 30 min (Pro +, NaPTA −), precipitated by NaPTA (Pro −, NaPTA +) or sequentially digested by pronase E at 100 µg/ml, 37°C for 30 min and precipitated by NaPTA (Pro +, NaPTA +). The equivalent of 2 µl 10% (w/v) brain homogenate was loaded in each lane and the gel was stained with silver nitrate.</p

RML prion infectivity and PrP content following digestion with PK, thermolysin or pronase E.

<p>10% (w/v) RML prion-infected brain homogenate was incubated at 37°C with (A) PK (50 µg/ml); (B) thermolysin (100 µg/ml) or (C) pronase E (100 µg/ml) for varying incubation times. For each time point, PrP (filled circles) was measured by ELISA and expressed as a percentage of total PrP present in the untreated sample; mean ± S.E.M. (<i>n</i> = 3, PK and thermolysin; <i>n</i> = 5, pronase E). RML prion infectivity (open circles) was measured by the Scrapie Cell Assay and expressed as a percentage of total infectivity present in the untreated sample; mean ± S.E.M. (<i>n</i> = 3, PK and thermolysin; <i>n</i> = 5, pronase E). Immunoblots of the corresponding protease and incubation time point are shown in panels to the right. Blots were probed with anti-PrP monoclonal antibody ICSM35. Apparent molecular masses are shown in kDa. Data in panels A and B have been published previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015679#pone.0015679-Cronier1" target="_blank">[25]</a>.</p