Recent advances in understanding mammalian prion structure

Prions are lethal pathogens, which cause fatal neurodegenerative diseases in mammals. They are unique infectious agents and are composed of self-propagating multi-chain assemblies of misfolded host-encoded prion protein (PrP). Understanding prion structure is fundamental to understanding prion disease pathogenesis however to date, the high-resolution structure of authentic ex vivo infectious prions remains unknown. Advances in determining prion structure have been severely impeded by the difficulty in recovering relatively homogeneous prion particles from infected brain and definitively associating infectivity with the PrP assembly state. Recently, however, images of highly infectious ex vivo PrP rods that produce prion-strain specific disease phenotypes in mice have been obtained using cryo-electron microscopy and atomic force microscopy. These images have provided the most detailed description of ex vivo mammalian prions reported to date and have established that prions isolated from multiple strains have a common hierarchical structure. Misfolded PrP is assembled into 20 nm wide rods containing two fibers, each with double helical repeating substructure, separated by a characteristic central gap 8-10 nm in width. Irregularly structured material with adhesive properties distinct to that of the fibers is present within the central gap of the rod. Prions are clearly distinguishable from non-infectious recombinant PrP fibrils generated in vitro and from all other propagating protein structures so far described in other neurodegenerative diseases. The basic architecture of mammalian prions appears to be exceptional and fundamental to their lethal pathogenicity

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

Detection and characterization of proteinase K-sensitive disease-related prion protein with thermolysin

Disease-related PrPSc [pathogenic PrP (prion protein)] is classically distinguished from its normal cellular precursor, PrPC(cellular PrP) by its detergent insolubility and partial resistance to proteolysis. Although molecular diagnosis of prion disease has historically relied upon detection of protease-resistant fragments of PrPSc using PK (proteinase K), it is now apparent that a substantial fraction of disease-related PrP is destroyed by this protease. Recently, thermolysin has been identified as a complementary tool to PK, permitting isolation of PrPSc in its full-length form. In the present study, we show that thermolysin can degrade PrPC while preserving both PK-sensitive and PK-resistant isoforms of disease-related PrP in both rodent and human prion strains. For mouse RML (Rocky Mountain Laboratory) prions, the majority of PK-sensitive disease-related PrP isoforms do not appear to contribute significantly to infectivity. In vCJD (variant Creutzfeldt–Jakob disease), the human counterpart of BSE (bovine spongiform encephalopathy), up to 90% of total PrP present in the brain resists degradation with thermolysin, whereas only ∼15% of this material resists digestion by PK. Detection of PK-sensitive isoforms of disease-related PrP using thermolysin should be useful for improving diagnostic sensitivity in human prion 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

Extension of a Multiscale Particle Scheme to Near-Equilibrium Viscous Flows

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/76880/1/AIAA-40262-641.pd

Amyloid-β nanotubes are associated with prion protein-dependent synaptotoxicity

Growing evidence suggests water-soluble, non-fibrillar forms of amyloid-β protein (Aβ) have important roles in Alzheimer’s disease with toxicities mimicked by synthetic Aβ1–42. However, no defined toxic structures acting via specific receptors have been identified and roles of proposed receptors, such as prion protein (PrP), remain controversial. Here we quantify binding to PrP of Aβ1–42 after different durations of aggregation. We show PrP-binding and PrP-dependent inhibition of long-term potentiation (LTP) correlate with the presence of protofibrils. Globular oligomers bind less avidly to PrP and do not inhibit LTP, whereas fibrils inhibit LTP in a PrP-independent manner. That only certain transient Aβ assemblies cause PrP-dependent toxicity explains conflicting reports regarding the involvement of PrP in Aβ-induced impairments. We show that these protofibrils contain a defined nanotubular structure with a previously unidentified triple helical conformation. Blocking the formation of Aβ nanotubes or their interaction with PrP might have a role in treatment of Alzheimer’s disease

Evaluating the causality of novel sequence variants in the prion protein gene by example

The estimation of pathogenicity and penetrance of novel prion protein gene (PRNP) variants presents significant challenges, particularly in the absence of family history, which precludes the application of Mendelian segregation. Moreover, the ambiguities of prion disease pathophysiology renders conventional in silico predictions inconclusive. Here, we describe 2 patients with rapid cognitive decline progressing to akinetic mutism and death within 10 weeks of symptom onset, both of whom possessed the novel T201S variant in PRNP. Clinically, both satisfied diagnostic criteria for probable sporadic Creutzfeldt-Jakob disease and in one, the diagnosis was confirmed by neuropathology. While computational analyses predicted that T201S was possibly deleterious, molecular strain typing, prion protein structural considerations, and calculations leveraging large-scale population data (gnomAD) indicate that T201S is at best either of low penetrance or nonpathogenic. Thus, we illustrate the utility of harnessing multiple lines of prion disease-specific evidence in the evaluation of the T201S variant, which may be similarly applied to assess other novel variants in PRNP

Absence of spontaneous disease and comparative prion susceptibility of transgenic mice expressing mutant human prion proteins

Approximately 15 % of human prion disease is associated with autosomal-dominant pathogenic mutations in the prion protein (PrP) gene. Previous attempts to model these diseases in mice have expressed human PrP mutations in murine PrP, but this may have different structural consequences. Here, we describe transgenic mice expressing human PrP with P102L or E200K mutations and methionine (M) at the polymorphic residue 129. Although no spontaneous disease developed in aged animals, these mice were readily susceptible to prion infection from patients with the homotypic pathogenic mutation. However, while variant Creutzfeldt–Jakob disease (CJD) prions transmitted infection efficiently to both lines of mice, markedly different susceptibilities to classical (sporadic and iatrogenic) CJD prions were observed. Prions from E200K and classical CJD M129 homozygous patients, transmitted disease with equivalent efficiencies and short incubation periods in human PrP 200K, 129M transgenic mice. However, mismatch at residue 129 between inoculum and host dramatically increased the incubation period. In human PrP 102L, 129M transgenic mice, short disease incubation periods were only observed with transmissions of prions from P102L patients, whereas classical CJD prions showed prolonged and variable incubation periods irrespective of the codon 129 genotype. Analysis of disease-related PrP (PrPSc) showed marked alteration in the PrPSc glycoform ratio propagated after transmission of classical CJD prions, consistent with the PrP point mutations directly influencing PrPSc assembly. These data indicate that P102L or E200K mutations of human PrP have differing effects on prion propagation that depend upon prion strain type and can be significantly influenced by mismatch at the polymorphic residue 129

Soluble Aβ aggregates can inhibit prion propagation

Mammalian prions cause lethal neurodegenerative diseases such as Creutzfeldt–Jakob disease (CJD) and consist of multi-chain assemblies of misfolded cellular prion protein (PrPC). Ligands that bind to PrPC can inhibit prion propagation and neurotoxicity. Extensive prior work established that certain soluble assemblies of the Alzheimer's disease (AD)-associated amyloid β-protein (Aβ) can tightly bind to PrPC, and that this interaction may be relevant to their toxicity in AD. Here, we investigated whether such soluble Aβ assemblies might, conversely, have an inhibitory effect on prion propagation. Using cellular models of prion infection and propagation and distinct Aβ preparations, we found that the form of Aβ assemblies which most avidly bound to PrP in vitro also inhibited prion infection and propagation. By contrast, forms of Aβ which exhibit little or no binding to PrP were unable to attenuate prion propagation. These data suggest that soluble aggregates of Aβ can compete with prions for binding to PrPC and emphasize the bidirectional nature of the interplay between Aβ and PrPC in Alzheimer's and prion diseases. Such inhibitory effects of Aβ on prion propagation may contribute to the apparent fall-off in the incidence of sporadic CJD at advanced age where cerebral Aβ deposition is common

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