Brain-derived Prion Research Articles

Characterization of the 263K-derived microsomal fraction: a source of prions for nanofiltration validation studies.

The manufacturing processes of plasma products include steps that can remove prions. The efficacy of these steps is measured in validation studies using animal brain-derived prion materials called spikes. Because the nature of the prion agent in blood is not known, the relevance of these spikes, particularly with steps that are based on retention mechanisms such as nanofiltration, is important to investigate. The aggregation and sizes of PrPres assemblies of microsomal fractions (MFs) extracted from 263K-infected hamster brains were analyzed using velocity gradients. The separated gradient fractions were either inoculated to Tg7 mice expressing hamster-PrPc to measure infectivity or used in Protein Misfolding Cyclic Amplification for measuring seeding activity. The collected data allowed for reanalyzing results from previous nanofiltration validation studies. A significant portion of MFs was found to be composed of small PrPres assemblies, estimated to have a size ≤24 mers (~22-528 kDa), and to contain a minimum of 20% of total prion infectivity. With this data we could calculate reductions of 4.10 log (15 N), 2.53 log (35 N), and 1.77 log (35 N) from validation studies specifically for these small PrPres objects. Our gradient data provided evidence that nanofilters can remove the majority of the smallest PrPres entities within microsomes spikes, estimated to be in a size below 24 mers, giving insight about the fact that, in our conditions, size exclusion may not be the only mechanism for retention nanofiltration.

Development of a methodology for large-scale production of prions for biological and structural studies.

Prion diseases are a group of infectious neurodegenerative diseases produced by the conversion of the normal prion protein (PrPC) into the disease-associated form (PrPSc). Extensive evidence indicate that the main or sole component of the infectious agent is PrPSc, which can replicate in affected individuals in the absence of nucleic acids. However, the mechanism of PrPC-to-PrPSc conversion remains elusive, which has been attributed to the lack of sufficient structural information of infectious PrPSc and a reliable system to study prion replication in vitro. In this article we adapted the Protein Misfolding Cyclic Amplification (PMCA) technology for rapid and efficient generation of highly infectious prions in large-scale. Murine prions of the RML strain were efficiently propagated in volumes up to 1,000-fold larger than conventional PMCA. The large-scale PMCA (LS-PMCA) procedure enabled to produce highly infectious prions, which maintain the strain properties of the seed used to begin the reaction. LS-PMCA was shown to work with various species and strains of prions, including mouse RML and 301C strains, hamster Hyper prion, cervid CWD prions, including a rare Norwegian CWD prion, and human CJD prions. We further improved the LS-PMCA into a bioreactor format that can operate under industry-mimicking conditions for continuous and unlimited production of PrPSc without the need to keep adding brain-derived prions. In our estimation, this bioreactor can produce in 1d an amount of prions equivalent to that present in 25 infected animals at the terminal stage of the disease. Our LS-PMCA technology may provide a valuable tool to produce large quantities of well-defined and homogeneous infectious prions for biological and structural studies.

Stilbene Compounds Inhibit the Replications of Various Strains of Prions in the Levels of Cell Culture, PMCA, and RT-QuIC Possibly via Molecular Binding.

Resveratrol shows the ability to block prion replication in a scrapie-infected cell line, SMB-S15, and remove the infectivity of the treated cell lysates in an experimental bioassay. In this study, we compared the effectiveness of three stilbene compounds, resveratrol (Res), pterostilbene (Pte), and piceatannol (Pic), on inhibiting prion propagations in the levels of cell culture, PMCA, and RT-QuIC. All three chemicals showed active suppressions on PrPSc replication in SMB-S15 cells, in which Res seemed to be the most active one, followed by Pic and Pte. Mouse PrP-based PMCA tests using the lysates of SMB-S15 cells and brain homogenates of scrapie agents S15-, 139A-, or ME7-infected mice verified that Res, Pte, and Pic inhibited the amplifications of PK-resistant signals. Res was also the most effective one. Mouse PrP-based RT-QuIC using the above seeds demonstrated that three stilbenes efficiently inhibited the fibril formation. However, Pic was the most effective one, followed by Res and Pte. Furthermore, the inhibition activities of the three stilbenes on the brain-derived prion from a 263K-infected hamster were tested with hamster PrP-based PMCA and RT-QuIC. The results indicated that Pic was the most effective one apparently, followed by Res and Pte. According to the results of Biacore, Res showed binding affinities much stronger than those of Pte, whereas both revealed markedly stronger binding affinities with mouse PrP. Our data here indicate that different stilbenes have the ability to block PrPSc replication in vitro with different prion species. The suppressive effects of stilbene compounds are likely associated with their molecular binding activities with PrPs.

Removal of exogenous prion infectivity in leukoreduced red blood cells unit by a specific filter designed for human transfusion

Five cases of variant Creutzfeldt-Jakob disease (vCJD) infections were attributed to infusion of contaminated blood components, turning to real the interhuman transmissibility of this prion disease from asymptomatic carriers. Preventive policies rely on exclusion from blood donation and benefit of leukoreduction initially implemented against leukotropic viruses. In the absence of available antemortem diagnostic tests, the updated prevalence of silent vCJD infections (1/2000 in the United Kingdom) urges the necessity to enforce blood safety with more efficient active measures able to remove the remaining infectivity. Several affinity resins were demonstrated to reduce high levels of brain-spiked infectivity from human leukoreduced red blood cells (L-RBCs). One was integrated in a device adapted to field constraints (volumes, duration) of human transfusion. We assessed here the ability of the resulting removal filter, termed P-Capt, to remove infectivity from human L-RBC units spiked with scrapie-infected hamster brain (≥10,000 infectious units/mL), through inoculation of hamsters with pre- and post-blood filtration samples. Incubation periods of recipient animals suggest around a 3-log removal of brain-derived prion infectivity by filtration through the P-Capt. On brain-derived spiked infectivity, the P-Capt filter provided a performance similar to the resin packed in columns used for initial proof-of-concept studies, suggesting an appropriate scale-up to efficiently remove infectivity from an individual human blood bag. According to the ability of resin to completely remove apparent endogenous infectivity from hamster leukoreduced blood, the implementation of such a filter, now commercially available, might seriously improve blood safety toward prions.

X-Ray Fiber Diffraction Reveals Major Structural Differences Between Brain-Derived Prions and Recombinant Prion Protein Amyloid

X-ray fiber diffraction was used to study the structure of brain-derived prions and recombinant protein amyloid. Partially oriented, dried fibers were prepared from brain-derived PrP 27-30 and recombinant PrP amyloid. Fiber diffraction patterns were analyzed and used to interrogate models for the structure of the infectious prion.Fiber diffraction patterns of recombinant PrP amyloid displayed characteristic, meridional reflections at ∼4.8 A and equatorial reflections at ∼10 A. These patterns were similar to those of other amyloids and are consistent with a basic cross-β architecture. In contrast, diffraction patterns from brain-derived PrP 27-30 displayed meridional reflections at ∼9.6, ∼6.4, and ∼4.8 A, which correspond to the 2nd, 3rd, and 4th order of a ∼19.2-A repeating unit, suggesting that PrP 27-30 contains four β-strands in a cross-β architecture. Furthermore, PrP 27-30 lacked the typical, equatorial reflection at ∼10 A, but instead produced equatorial reflections characterizing the diameter of the amyloid fiber and of individual protofilaments. Therefore, PrP 27-30 seems to have a structure consistent with a β-helix or β-solenoid, not unlike the model that was proposed earlier (Govaerts et al., 2004). This interpretation was also supported by extensive modeling, simulation of diffraction, electron microscopy, and FTIR.In a previous study, recombinant PrP amyloid induced a transmissible disease in transgenic mice overexpressing PrP, and was thus termed a prion (Legname et al., 2004). Serially transmitted, synthetic prions were purified from mouse brains and analyzed by fiber diffraction. These brain-derived, synthetic prions showed the same structural characteristics as natural isolates and not those of its recombinant protein precursor. The relationship between structural differences and infectivity can be explained by several hypotheses. It remains to be determined which one, if any, is correct.

Natural and synthetic prion structure from X-ray fiber diffraction

A conformational isoform of the mammalian prion protein (PrP(Sc)) is the sole component of the infectious pathogen that causes the prion diseases. We have obtained X-ray fiber diffraction patterns from infectious prions that show cross-beta diffraction: meridional intensity at 4.8 A resolution, indicating the presence of beta strands running approximately at right angles to the filament axis and characteristic of amyloid structure. Some of the patterns also indicated the presence of a repeating unit along the fiber axis, corresponding to four beta-strands. We found that recombinant (rec) PrP amyloid differs substantially from highly infectious brain-derived prions, both in structure as demonstrated by the diffraction data, and in heterogeneity as shown by electron microscopy. In addition to the strong 4.8 A meridional reflection, the recPrP amyloid diffraction is characterized by strong equatorial intensity at approximately 10.5 A, absent from brain-derived prions, and indicating the presence of stacked beta-sheets. Synthetic prions recovered from transgenic mice inoculated with recPrP amyloid displayed structural characteristics and homogeneity similar to those of naturally occurring prions. The relationship between the structural differences and prion infectivity is uncertain, but might be explained by any of several hypotheses: only a minority of recPrP amyloid possesses a replication-competent conformation, the majority of recPrP amyloid has to undergo a conformational maturation to acquire replication competency, or inhibitory forms of recPrP amyloid interfere with replication during the initial transmission.

PrP Sc of scrapie 263K propagates efficiently in spleen and muscle tissues with protein misfolding cyclic amplification

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are transmissible neurodegenerative disorders of protein conformation. This group of diseases is caused by infectious agents, termed prions, which can convert normal conformation (PrP C) into misfolded protein (PrP Sc). The infectivity of non-neuronal tissues has been wildly addressed, but the propagating features and the biochemical properties of prion generated from these tissues are only partially settled. In this study, utilizing protein misfolding cyclic amplification (PMCA), the in vitro conversion of PrP C into PrP Sc in spleen and muscle tissues can be induced by PrP Sc produced in vivo. The further propagation of newly formed PrP Sc in normal brain and some of the biochemical properties of new PrP Sc are similar as the brain-derived prions, implying the naturally infectious pathway of prion from peripheral generation to neuro-invasion. However, compared with the brain-derived PrP Sc, the weaker resistance of new PrP Sc to some inactivated agents, i.e. sodium hydroxide and thermal inactivation, are observed. Our data provide the reliable evidence that the brain-derived PrP Sc can utilize the PrP C from non-neuronal tissues for its propagation. Similarity of the replicative ability in PMCA in vitro and the infectivity in vivo highlights the possibility to use PMCA instead of bioassay to investigate the propagation of prion.

Molecular Mechanism for Low pH Triggered Misfolding of the Human Prion Protein

Conformational changes in the prion protein cause transmissible spongiform encephalopathies, also referred to as prion diseases. In its native state, the prion protein is innocuous (PrPC), but it can misfold into a neurotoxic and infectious isoform (PrPSc). The full-length cellular form of the prion protein consists of residues 23-230, with over half of the sequence belonging to the unstructured N-terminal domain and the remaining residues forming a small globular domain. During misfolding and aggregation, portions of both the structured and unstructured domains are incorporated into the aggregates. After limited proteolysis by proteinase K, the most abundant fragment from brain-derived prion fibrils is a 141-residue fragment composed of residues 90-230. Here we describe simulations of this fragment of the human prion protein at low pH, which triggers misfolding, and at neutral pH as a control. The simulations, in agreement with experiment, show that this biologically and pathologically relevant prion construct is stable and native-like at neutral pH. In contrast, at low pH the prion protein is destabilized via disruption of critical long-range salt bridges. In one of the low pH simulations this destabilization resulted in a conformational transition to a PrPSc-like isoform consistent with our previous simulations of a smaller construct.

Removal of Prions by the Manufacturing Process of Haemate® P/Humate-P®.

Prions, the causative agents of Creutzfeldt-Jakob Disease (CJD) and related neurodegenerative disorders in humans including variant CJD (vCJD) have up to now never been demonstrated in human blood although if in UK two probable transmissions of vCJD by blood transfusion were reported (Llewelyn et al., Lancet 2004; 363:417–421 and Peden et al., Lancet 2004; 364:527–529). In order to minimize a theoretical risk of human prion contamination of plasma pools, potential donors at risk of incubating CJD are excluded from donation permanently. Due to the implementation of these precautionary measures, the risk to collect plasma from a donor developing vCJD subsequent to a donation is very low. In addition, the amount of prions present in plasma of a healthy donor (i.e., a donor in the incubation period of (v)CJD) can be considered very low (Comer Risk Assessment of Exposure to vCJD Infectivity in Blood and Blood Products for Department of Health [UK], Revision DE, Det Nordske Veritas, February 2003). Even with this very low risk related to plasma for fractionation, the capacity of selected steps of the manufacturing process of the VWF/FVIII product Haemate® P/Humate-P® to remove prions has been evaluated in prion removal studies. As it has never been possible to isolate infectious prion protein, termed PrPSc, from human blood or plasma, the model prion strain 263K (isolated from brain of deliberately infected hamsters) was spiked to intermediates of scaled-down plasma protein purification processes. As there is no knowledge of the biophysicochemical nature of potential prion contaminants in plasma, we employed two different prion spike preparations in these studies: membrane associated prions in so-called microsomes and purified prion protein PrPSc, an almost native, molecular, non-membrane associated form of prion infectivity. Laboratory studies were performed by spiking either experimental plasma pools or an intermediate at a production stage further downstream with prion preparations and investigating the prion reduction by consecutive scaled-down process steps. These investigational studies resulted in a mean overall prion reduction factor of 5.3 log10 and 6.4 log10, respectively, for the two hamster derived spike preparations.Reduction of prions by selected manufacturing steps for Haemate® P/Humate-P®Manufacturing stagesPrion Reduction Factor [log10]MicrosomesPrPScCombined steps cryoprecipitation, Al(OH)3, glycine and NaCl precipitation3.53.9Combined steps ultracentrifugation/sterile filtration1.82.5Mean Overall Prion Reduction5.36.4Additionally, confirmative data were obtained in studies employing human brain-derived prion preparations (sCJD and vCJD) as spiking material in selected studies of a single manufacturing step. Therefore, it can be concluded that the risk of prion transmission by the VWF/FVIII product Haemate® P/Humate-P® is extremely remote based on complementary safety procedures, i.e., collection of plasma by stringent donor selection and the overall prion reduction factor which clearly exceeds a potential prion load in the manufacturing pool. This high safety margin is confirmed in a probabilistic risk calculation, demonstrating at a probability of 95% a safety factor for each vial of the product of 6.4 log10.