Assessment of the zoonotic potential of atypical scrapie prions in humanized mice reveals rare phenotypic convergence but not identity with sporadic CJD prions.

BACKGROUND: Atypical/Nor98 scrapie (AS) is an idiopathic infectious prion disease affecting sheep and goats. Recent findings suggest that zoonotic prions from bovine spongiform encephalopathy (C-BSE) may co-propagate with atypical/Nor98 prions in AS sheep brains. Investigating the risk AS poses to humans is crucial. METHODS: To assess the risk of sheep/goat-to-human transmission of AS, we serially inoculated brain tissue from field and laboratory isolates into transgenic mice overexpressing human prion protein (Met129 allele). We studied clinical outcomes as well as presence of prions in brains and spleens. RESULTS: No transmission occurred on the primary passage, with no clinical disease or pathological prion protein in brains and spleens. On subsequent passages, one isolate gradually adapted, manifesting as prions with a phenotype resembling those causing MM1-type sporadic Creutzfeldt-Jakob disease in humans. However, further characterization using in vivo and in vitro techniques confirmed both prion agents as different strains, revealing a case of phenotypic convergence. Importantly, no C-BSE prions emerged in these mice, especially in the spleen, which is more permissive than the brain for C-BSE cross-species transmission. CONCLUSIONS: The results obtained suggest a low the zoonotic for AS. Rare adaptation may allow the emergence of prions phenotypically resembling those spontaneously forming in humans.

The Smallest Infectious Substructure Encoding the Prion Strain Structural Determinant Revealed by Spontaneous Dissociation of Misfolded Prion Protein Assemblies.

It is commonly accepted that the prion replicative propensity and strain structural determinant (SSD) are encoded in the fold of PrPSc amyloid fibril assemblies. By exploring the quaternary structure dynamicity of several prion strains, we revealed that all mammalian prion assemblies exhibit the generic property of spontaneously generating two sets of discreet infectious tetrameric and dimeric species differing significantly by their specific infectivity. By using perturbation approaches such as dilution and ionic strength variation, we demonstrated that these two oligomeric species were highly dynamic and evolved differently in the presence of chaotropic agents. In general, our observations of seven different prion strains from three distinct species highlight the high dynamicity of PrPSc assemblies as a common and intrinsic property of mammalian prions. The existence of such small infectious PrPSc species harboring the SSD indicates that the prion infectivity and the SSD are not restricted only to the amyloid fold but can also be encoded in other alternative quaternary structures. Such diversity in the quaternary structure of prion assemblies tends to indicate that the structure of PrPSc can be divided into two independent folding domains: a domain encoding the strain structural determinant and a second domain whose fold determines the type of quaternary structure that could adopt PrPSc assemblies.

Prion assemblies: structural heterogeneity, mechanisms of formation, and role in species barrier.

Prions are proteinaceous pathogens responsible for a wide range of neurodegenerative diseases in animal and human. Prions are formed from misfolded, ß-sheet rich, and aggregated conformers (PrPSc) of the host-encoded prion protein (PrPC). Prion replication stems from the capacity of PrPSc to self-replicate by templating PrPC conversion and polymerization. The question then arises about the molecular mechanisms of prion replication, host invasion, and capacity to contaminate other species. Studying these mechanisms has gained in recent years further complexity with evidence that PrPSc is a pleiomorphic protein. There is indeed compelling evidence for PrPSc structural heterogeneity at different scales: (i) within prion susceptible host populations with the existence of different strains with specific biological features due to different PrPSc conformers, (ii) within a single infected host with the co-propagation of different strains, and (iii) within a single strain with evidence for co-propagation of PrPSc assemblies differing in their secondary to quaternary structure. This review summarizes current knowledge of prion assembly heterogeneity, potential mechanisms of formation during the replication process, and importance when crossing the species barrier.

PrP C as a Transducer of Physiological and Pathological Signals.

After the discovery of prion phenomenon, the physiological role of the cellular prion protein (PrP C ) remained elusive. In the past decades, molecular and cellular analysis has shed some light regarding interactions and functions of PrP C in health and disease. PrP C , which is located mainly at the plasma membrane of neuronal cells attached by a glycosylphosphatidylinositol (GPI) anchor, can act as a receptor or transducer from external signaling. Although the precise role of PrP C remains elusive, a variety of functions have been proposed for this protein, namely, neuronal excitability and viability. Although many issues must be solved to clearly define the role of PrP C , its connection to the central nervous system (CNS) and to several misfolding-associated diseases makes PrP C an interesting pharmacological target. In a physiological context, several reports have proposed that PrP C modulates synaptic transmission, interacting with various proteins, namely, ion pumps, channels, and metabotropic receptors. PrP C has also been implicated in the pathophysiological cell signaling induced by ß-amyloid peptide that leads to synaptic dysfunction in the context of Alzheimer's disease (AD), as a mediator of Aß-induced cell toxicity. Additionally, it has been implicated in other proteinopathies as well. In this review, we aimed to analyze the role of PrP C as a transducer of physiological and pathological signaling.

Prion potentiation after life-long dormancy in mice devoid of PrP.

Prions are neurotropic pathogens composed of misfolded assemblies of the host-encoded prion protein PrPC which replicate by recruitment and conversion of further PrPC by an autocatalytic seeding polymerization process. While it has long been shown that mouse-adapted prions cannot replicate and are rapidly cleared in transgenic PrP0/0 mice invalidated for PrPC, these experiments have not been done with other prions, including from natural resources, and more sensitive methods to detect prion biological activity. Using transgenic mice expressing human PrP to bioassay prion infectivity and RT-QuIC cell-free assay to measure prion seeding activity, we report that prions responsible for the most prevalent form of sporadic Creutzfeldt-Jakob disease in human (MM1-sCJD) can persist indefinitely in the brain of intra-cerebrally inoculated PrP0/0 mice. While low levels of seeding activity were measured by RT-QuIC in the brain of the challenged PrP0/0 mice, the bio-indicator humanized mice succumbed at a high attack rate, suggesting relatively high levels of persistent infectivity. Remarkably, these humanized mice succumbed with delayed kinetics as compared to MM1-sCJD prions directly inoculated at low doses, including the limiting one. Yet, the disease that did occur in the humanized mice on primary and subsequent back-passage from PrP0/0 mice shared the neuropathological and molecular characteristics of MM1-sCJD prions, suggesting no apparent strain evolution during lifelong dormancy in PrP0/0 brain. Thus, MM1-sCJD prions can persist for the entire life in PrP0/0 brain with potential disease potentiation on retrotransmission to susceptible hosts. These findings highlight the capacity of prions to persist and rejuvenate in non-replicative environments, interrogate on the type of prion assemblies at work and alert on the risk of indefinite prion persistence with PrP-lowering therapeutic strategies.

Probing amyloid fibril secondary structures by infrared nanospectroscopy: experimental and theoretical considerations.

Amyloid fibrils are composed of aggregated peptides or proteins in a fibrillary structure with a higher ß-sheet content than their native structure. Attenuated total reflection Fourier transform infrared spectroscopy only provides bulk analysis of a sample therefore it is impossible to discriminate between different aggregated structures. To overcome this limitation, near-field techniques like AFM-IR have emerged in the last twenty years to allow infrared nanospectroscopy. This technique obtains IR spectra with a spatial resolution of ten nanometres, the size of isolated fibrils. Here, we present essential practical considerations to avoid misinterpretations and artefacts during these analyses. Effects of polarization of the incident IR laser, illumination configuration and coating of the AFM probes are discussed, including the advantages and drawbacks of their use. This approach will improve interpretation of AFM-IR spectra especially for the determination of secondary structures of species not accessible using classical ATR-FTIR.

Improving the Predictive Value of Prion Inactivation Validation Methods to Minimize the Risks of Iatrogenic Transmission With Medical Instruments.

Prions are pathogenic infectious agents responsible for fatal, incurable neurodegenerative diseases in animals and humans. Prions are composed exclusively of an aggregated and misfolded form (PrP Sc ) of the cellular prion protein (PrPC). During the propagation of the disease, PrPSc recruits and misfolds PrPC into further PrPSc. In human, iatrogenic prion transmission has occurred with incompletely sterilized medical material because of the unusual resistance of prions to inactivation. Most commercial prion disinfectants validated against the historical, well-characterized laboratory strain of 263K hamster prions were recently shown to be ineffective against variant Creutzfeldt-Jakob disease human prions. These observations and previous reports support the view that any inactivation method must be validated against the prions for which they are intended to be used. Strain-specific variations in PrPSc physico-chemical properties and conformation are likely to explain the strain-specific efficacy of inactivation methods. Animal bioassays have long been used as gold standards to validate prion inactivation methods, by measuring reduction of prion infectivity. Cell-free assays such as the real-time quaking-induced conversion (RT-QuIC) assay and the protein misfolding cyclic amplification (PMCA) assay have emerged as attractive alternatives. They exploit the seeding capacities of PrPSc to exponentially amplify minute amounts of prions in biospecimens. European and certain national medicine agencies recently implemented their guidelines for prion inactivation of non-disposable medical material; they encourage or request the use of human prions and cell-free assays to improve the predictive value of the validation methods. In this review, we discuss the methodological and technical issues regarding the choice of (i) the cell-free assay, (ii) the human prion strain type, (iii) the prion-containing biological material. We also introduce a new optimized substrate for high-throughput PMCA amplification of human prions bound on steel wires, as translational model for prion-contaminated instruments.

PiQSARS: A pipeline for quantitative and statistical analyses of ratiometric fluorescent biosensors.

Genetically encoded ratiometric fluorescent probes are cutting-edge tools in biology. They allow precise and dynamic measurement of various physiological parameters within cell compartments. Because data extraction and analysis are time consuming and may lead to inconsistencies between results, we describe here a standardized pipeline for•Semi-automated treatment of time-lapse fluorescence microscopy images.•Quantification of individual cell signal.•Statistical analysis of the data.First, a dedicated macro was developed using the FIJI software to reproducibly quantify the fluorescence ratio as a function of time. Raw data are then exported and analyzed using R and MATLAB softwares. Calculation and statistical analysis of selected graphic parameters are performed. In addition, a functional principal component analysis allows summarizing the dataset. Finally, a principal component analysis is performed to check consistency and final analysis is presented as a visual diagram. The method is adapted to any ratiometric fluorescent probe. As an example, the analysis of the cytoplasmic HyPer probe in response to an acute cell treatment with increasing amounts of hydrogen peroxide is shown. In conclusion, the pipeline allows to save time and analyze a larger amount of samples while reducing manual interventions and consequently increasing the robustness of the analysis.

A seven-residue deletion in PrP leads to generation of a spontaneous prion formed from C-terminal C1 fragment of PrP.

Prions result from a drastic conformational change of the host-encoded cellular prion protein (PrP), leading to the formation of ß-sheet-rich, insoluble, and protease-resistant self-replicating assemblies (PrPSc). The cellular and molecular mechanisms involved in spontaneous prion formation in sporadic and inherited human prion diseases or equivalent animal diseases are poorly understood, in part because cell models of spontaneously forming prions are currently lacking. Here, extending studies on the role of the H2 α-helix C terminus of PrP, we found that deletion of the highly conserved 190HTVTTTT196 segment of ovine PrP led to spontaneous prion formation in the RK13 rabbit kidney cell model. On long-term passage, the mutant cells stably produced proteinase K (PK)-resistant, insoluble, and aggregated assemblies that were infectious for naïve cells expressing either the mutant protein or other PrPs with slightly different deletions in the same area. The electrophoretic pattern of the PK-resistant core of the spontaneous prion (ΔSpont) contained mainly C-terminal polypeptides akin to C1, the cell-surface anchored C-terminal moiety of PrP generated by natural cellular processing. RK13 cells expressing solely the Δ190-196 C1 PrP construct, in the absence of the full-length protein, were susceptible to ΔSpont prions. ΔSpont infection induced the conversion of the mutated C1 into a PK-resistant and infectious form perpetuating the biochemical characteristics of ΔSpont prion. In conclusion, this work provides a unique cell-derived system generating spontaneous prions and provides evidence that the 113 C-terminal residues of PrP are sufficient for a self-propagating prion entity.

Conformation-dependent membrane permeabilization by neurotoxic PrP oligomers: The role of the H2H3 oligomerization domain.

The relationship between prion propagation and the generation of neurotoxic species and clinical onset remains unclear. Several converging lines of evidence suggest that interactions with lipids promote various precursors to form aggregation-prone states that are involved in amyloid fibrils. Here, we compared the cytotoxicities of different soluble isolated oligomeric constructs from murine full-length PrP and from the restricted helical H2H3 domain with their effects on lipid vesicles. The helical H2H3 domain is suggested to be the minimal region of PrP involved in the oligomerization process. The discrete PrP oligomers of both the full-length sequence and the H2H3 domain have de novo ß-sheeted structure when interacting with the membrane. They were shown to permeabilize synthetic negatively charged vesicles in a dose-dependent manner. Restricting the polymerization domain of the full-length PrP to the H2H3 helices strongly diminished the ability of the corresponding oligomers to associate with the lipid vesicles. Furthermore, the membrane impairment mechanism occurs differently for the full-length PrP oligomers and the H2H3 helices, as shown by dye-release and black lipid membrane experiments. The membrane damage caused by the full-length PrP oligomers is correlated to their neuronal toxicity at submicromolar concentrations, as shown by cell culture assays. Although oligomers of synthetic H2H3 could compromise in vitro cell homeostasis, they followed a membrane-disruptive pattern that was different from the full-length oligomers, as revealed by the role of PrPC in cell viability assays.

Host prion protein expression levels impact prion tropism for the spleen.

Prions are pathogens formed from abnormal conformers (PrPSc) of the host-encoded cellular prion protein (PrPC). PrPSc conformation to disease phenotype relationships extensively vary among prion strains. In particular, prions exhibit a strain-dependent tropism for lymphoid tissues. Prions can be composed of several substrain components. There is evidence that these substrains can propagate in distinct tissues (e.g. brain and spleen) of a single individual, providing an experimental paradigm to study the cause of prion tissue selectivity. Previously, we showed that PrPC expression levels feature in prion substrain selection in the brain. Transmission of sheep scrapie isolates (termed LAN) to multiple lines of transgenic mice expressing varying levels of ovine PrPC in their brains resulted in the phenotypic expression of the dominant sheep substrain in mice expressing near physiological PrPC levels, whereas a minor substrain replicated preferentially on high expresser mice. Considering that PrPC expression levels are markedly decreased in the spleen compared to the brain, we interrogate whether spleen PrPC dosage could drive prion selectivity. The outcome of the transmission of a large cohort of LAN isolates in the spleen from high expresser mice correlated with the replication rate dependency on PrPC amount. There was a prominent spleen colonization by the substrain preferentially replicating on low expresser mice and a relative incapacity of the substrain with higher-PrPC level need to propagate in the spleen. Early colonization of the spleen after intraperitoneal inoculation allowed neuropathological expression of the lymphoid substrain. In addition, a pair of substrain variants resulting from the adaptation of human prions to ovine high expresser mice, and exhibiting differing brain versus spleen tropism, showed different tropism on transmission to low expresser mice, with the lymphoid substrain colonizing the brain. Overall, these data suggest that PrPC expression levels are instrumental in prion lymphotropism.

Evidence of conformational landscape alteration and macromolecular complex formation in the early stages of in vitro human prion protein oxidation.

Oxidative stress is proposed to be one of the major causes of neurodegenerative diseases. Cellular prion protein (PrP) oxidation has been widely studied using chemical reagents such as hydrogen peroxide. However, the experimental conditions used do not faithfully reflect the physiological environment of the cell. With the goal to explore the conformational landscape of PrP under oxidative stress, we conducted a set of experiments combining the careful control of the nature and the amount of ROS produced by a60Co γ-irradiation source. Characterization of the resulting protein species was achieved using a set of analytical techniques. Under our experimental condition hydroxyl radical are the main reactive species produced. The most important findings are i) the formation of molecular assemblies under oxidative stress, ii) the detection of a majority of unmodified monomer mixed with oxidized monomers in these molecular assemblies at low hydroxyl radical concentration, iii) the absence of significant oxidation on the monomer fraction after irradiation. Molecular assemblies are produced in small amounts and were shown to be an octamer. These results suggest either i) an active recruitment of intact monomers by molecular assemblies' oxidized monomers then inducing a structural change of their intact counterparts or ii) an intrinsic capability of intact monomer conformers to spontaneously associate to form stable molecular assemblies when oxidized monomers are present. Finally, abundances of the intact monomer conformers after irradiation were modified. This suggests that monomers of the molecular assemblies exchange structural information with intact irradiated monomer. All these results shed a new light on structural exchange information between PrP monomers under oxidative stress.

Crossing Species Barriers Relies on Structurally Distinct Prion Assemblies and Their Complementation.

Prion replication results from the autocatalytic templated assisted conversion of the host-encoded prion protein PrPC into misfolded, polydisperse PrPSc conformers. Structurally distinct PrPSc conformers can give rise to multiple prion strains. Within and between prion strains, the biological activity (replicative efficacy and specific infectivity) of PrPSc assemblies is size dependent and thus reflects an intrinsic structural heterogeneity. The contribution of such PrPSc heterogeneity across species prion adaptation, which is believed to be based on fit adjustment between PrPSc template(s) and host PrPC, has not been explored. To define the structural-to-fitness PrPSc landscape, we measured the relative capacity of size-fractionated PrPSc assemblies from different prion strains to cross mounting species barriers in transgenic mice expressing foreign PrPC. In the absence of a transmission barrier, the relative efficacy of the isolated PrPSc assemblies to induce the disease is like the efficacy observed in the homotypic context. However, in the presence of a transmission barrier, size fractionation overtly delays and even abrogates prion pathogenesis in both the brain and spleen tissues, independently of the infectivity load of the isolated assemblies. Altering by serial dilution PrPSc assembly content of non-fractionated inocula aberrantly reduces their specific infectivity, solely in the presence of a transmission barrier. This suggests that synergy between structurally distinct PrPSc assemblies in the inoculum is requested for crossing the species barrier. Our data support a mechanism whereby overcoming prion species barrier requires complementation between structurally distinct PrPSc assemblies. This work provides key insight into the "quasispecies" concept applied to prions, which would not necessarily rely on prion substrains as constituent but on structural PrPSc heterogeneity within prion population.

Symmetry-breaking transitions in the early steps of protein self-assembly.

Protein misfolding and subsequent self-association are complex, intertwined processes, resulting in development of a heterogeneous population of aggregates closely related to many chronic pathological conditions including Type 2 Diabetes Mellitus and Alzheimer's disease. To address this issue, here, we develop a theoretical model in the general framework of linear stability analysis. According to this model, self-assemblies of peptides with pronounced conformational flexibility may become, under particular conditions, unstable and spontaneously evolve toward an alternating array of partially ordered and disordered monomers. The predictions of the theory were verified by atomistic molecular dynamics (MD) simulations of islet amyloid polypeptide (IAPP) used as a paradigm of aggregation-prone polypeptides (proteins). Simulations of dimeric, tetrameric, and hexameric human-IAPP self-assemblies at physiological electrolyte concentration reveal an alternating distribution of the smallest domains (of the order of the peptide mean length) formed by partially ordered (mainly ß-strands) and disordered (turns and coil) arrays. Periodicity disappears upon weakening of the inter-peptide binding, a result in line with the predictions of the theory. To further probe the general validity of our hypothesis, we extended the simulations to other peptides, the Aß(1-40) amyloid peptide, and the ovine prion peptide as well as to other proteins (SOD1 dimer) that do not belong to the broad class of intrinsically disordered proteins. In all cases, the oligomeric aggregates show an alternate distribution of partially ordered and disordered monomers. We also carried out Surface Enhanced Raman Scattering (SERS) measurements of hIAPP as an experimental validation of both the theory and in silico simulations.

Prion strain-dependent tropism is maintained between spleen and granuloma and relies on lymphofollicular structures.

In peripherally acquired prion diseases, prions move through several tissues of the infected host, notably in the lymphoid tissue, long before the occurrence of neuroinvasion. Accumulation can even be restricted to the lymphoid tissue without neuroinvasion and clinical disease. Several experimental observations indicated that the presence of differentiated follicular dendritic cells (FDCs) in the lymphoid structures and the strain type are critical determinants of prion extraneural replication. In this context, the report that granulomatous structures apparently devoid of FDCs could support prion replication raised the question of the requirements for prion lymphotropism. The report also raised the possibility that nonlymphoid tissue-tropic prions could actually target these inflammatory structures. To investigate these issues, we examined the capacity of closely related prions, albeit with opposite lymphotropism (or FDC dependency), for establishment in experimentally-induced granuloma in ovine PrP transgenic mice. We found a positive correlation between the prion capacity to accumulate in the lymphoid tissue and granuloma, regardless of the prion detection method used. Surprisingly, we also revealed that the accumulation of prions in granulomas involved lymphoid-like structures associated with the granulomas and containing cells that stain positive for PrP, Mfge-8 but not CD45 that strongly suggest FDCs. These results suggest that the FDC requirement for prion replication in lymphoid/inflammatory tissues may be strain-dependent.

Early stage prion assembly involves two subpopulations with different quaternary structures and a secondary templating pathway.

The dynamics of aggregation and structural diversification of misfolded, host-encoded proteins in neurodegenerative diseases are poorly understood. In many of these disorders, including Alzheimer's, Parkinson's and prion diseases, the misfolded proteins are self-organized into conformationally distinct assemblies or strains. The existence of intrastrain structural heterogeneity is increasingly recognized. However, the underlying processes of emergence and coevolution of structurally distinct assemblies are not mechanistically understood. Here, we show that early prion replication generates two subsets of structurally different assemblies by two sequential processes of formation, regardless of the strain considered. The first process corresponds to a quaternary structural convergence, by reducing the parental strain polydispersity to generate small oligomers. The second process transforms these oligomers into larger ones, by a secondary autocatalytic templating pathway requiring the prion protein. This pathway provides mechanistic insights into prion structural diversification, a key determinant for prion adaptation and toxicity.

Causative Links between Protein Aggregation and Oxidative Stress: A Review.

Compelling evidence supports a tight link between oxidative stress and protein aggregation processes, which are noticeably involved in the development of proteinopathies, such as Alzheimer's disease, Parkinson's disease, and prion disease. The literature is tremendously rich in studies that establish a functional link between both processes, revealing that oxidative stress can be either causative, or consecutive, to protein aggregation. Because oxidative stress monitoring is highly challenging and may often lead to artefactual results, cutting-edge technical tools have been developed recently in the redox field, improving the ability to measure oxidative perturbations in biological systems. This review aims at providing an update of the previously known functional links between oxidative stress and protein aggregation, thereby revisiting the long-established relationship between both processes.

Transient multimers modulate conformer abundances of prion protein monomer through conformational selection.

Prions are known to be involved in neurodegenerative pathologies such as Creutzfeld-Jakob disease. Current models point to a molecular event which rely on a transmissible structural change that leads to the production of ß-sheet-rich prion conformer (PrPSc). PrPSc itself has the capability to trigger the structural rearrangement of the ubiquitously present prion (PrPc) substrate in a self-perpetuating cascade. In this article, we demonstrate that recombinant PrPc exists in a conformational equilibrium. The conformers' abundances were shown to be dependent on PrPc concentration through the formation of transient multimers leading to conformational selection. The study of PrPc mutants that follow dedicated oligomerization pathways demonstrated that the conformers' relative abundances are modified, thus reinforcing the assertion that the nature of conformers' interactions orient the oligomerization pathways. Further this result can be viewed as the "signature" of an aborted oligomerization process. This discovery sheds a new light on the possible origin of prion protein diseases, namely that a change in prion protein structure could be transmitted through the formation of transient multimers having different conformer compositions. This could explain the selection of a transient multimeric type that could be viewed as the precursor of PrPSc responsible for structural information transmission, and strain apparition.

A bi-monomeric, nonlinear Becker-Döring-type system to capture oscillatory aggregation kinetics in prion dynamics.

In this article, in order to understand the appearance of oscillations observed in protein aggregation experiments, we propose, motivate and analyse mathematically the differential system describing the kinetics of the following reactions: [Formula: see text] with n finite or infinite. This system may be viewed as a variant of the seminal Becker-Döring system, and is capable of displaying sustained though damped oscillations.

Heterogeneity and Architecture of Pathological Prion Protein Assemblies: Time to Revisit the Molecular Basis of the Prion Replication Process?

Prions are proteinaceous infectious agents responsible for a range of neurodegenerative diseases in animals and humans. Prion particles are assemblies formed from a misfolded, ß-sheet rich, aggregation-prone isoform (PrPSc) of the host-encoded cellular prion protein (PrPC). Prions replicate by recruiting and converting PrPC into PrPSc, by an autocatalytic process. PrPSc is a pleiomorphic protein as different conformations can dictate different disease phenotypes in the same host species. This is the basis of the strain phenomenon in prion diseases. Recent experimental evidence suggests further structural heterogeneity in PrPSc assemblies within specific prion populations and strains. Still, this diversity is rather seen as a size continuum of assemblies with the same core structure, while analysis of the available experimental data points to the existence of structurally distinct arrangements. The atomic structure of PrPSc has not been elucidated so far, making the prion replication process difficult to understand. All currently available models suggest that PrPSc assemblies exhibit a PrPSc subunit as core constituent, which was recently identified. This review summarizes our current knowledge on prion assembly heterogeneity down to the subunit level and will discuss its importance with regard to the current molecular principles of the prion replication process.