Infectious Prion Protein Research Articles

Microarrays and Deep Sequencing in Clinical Microbiology

Nucleic acid detection technology is revolutionizing microbiological diagnostic testing. With the exception of infectious prion proteins, all pathogenic microbes contain DNA and / or RNA and are thus targets for nucleic acid-based testing. Some clinical microbiology laboratories are largely shifting to nucleic acid testing, which is particularly useful for detecting slow-growing or unculturable organisms such as viruses, mycobacteria, and fastidious bacterial pathogens. However, significant challenges remain before this technology can be applied universally in clinical laboratories.

Enhanced enteric invasion of scrapie agents into the villous columnar epithelium via maternal immunoglobulin

Transmissible spongiform encephalopathies (TSE) are caused by dietary oral exposure to infectious prion proteins (PrPSc); however, the mechanism behind the uptake of PrPSc in the intestines is poorly understood. In addition, epidemiological studies of BSE showed that most cattle are exposed to the agents in the first 6 months of life, during the suckling and weaning periods. In the present study, to elucidate the enteric invasion mechanism of prions and to investigate the age-dependent transmission mechanism suggested by epidemiological studies, wild-type and SCID mice were orally administered brain homogenate from scrapie (Tsukuba 1)-infected mice during the suckling and weaning stages, before being analyzed histopathologically. PrPSc was found to be incorporated into the villous columnar epithelial cells and was also detected in the villous lacteal of 15-day-old suckling mice. However, no such uptake of PrPSc was observed in the weaned mice at 25-days-old. Four different strains of mice were tested. There was no mouse strain difference in the frequency of PrPSc positive columnar epithelial cells. In addition, the uptake of PrPSc in suckling SCID mice lacking maternal antibodies was significantly lower than that in the wild-type suckling mice, and the uptake of PrPSc was enhanced by dilution with purified IgG. In the present study, it was suggested that the weaning period and maternal immunoglobulin are important risk factors for the oral transmission of PrPSc.

Transmission of circulating cell-free AA amyloid oligomers in exosomes vectors via a prion-like mechanism

Recent studies clearly demonstrated that several types of pathogenic amyloid proteins acted as agents that could transmit amyloidosis by means of a prion-like mechanism. Systemic AA amyloidosis is one of the most severe complications of chronic inflammatory disorders, particularly rheumatoid arthritis. It is well known that, similar to an infectious prion protein, amyloid-enhancing factor (AEF) acts as a transmissible agent in AA amyloidosis. However, how AEF transmits AA amyloidosis in vivo remained to be fully elucidated. In the present study, we focused on finding cell-free forms of AEF and its carriers in circulation by using the murine transfer model of AA amyloidosis. We first determined that circulating cell-free AEF existed in blood and plasma in mice with systemic AA amyloidosis. Second, we established that plasma exosomes containing AA amyloid oligomers derived from serum amyloid A had AEF activity and could transmit systemic AA amyloidosis via a prion-like mechanism. These novel findings should provide insights into the transmission mechanism of systemic amyloidoses.

Managing the risks of prion disease transmission through flexible endoscopy

Creutzfeldt-Jakob disease (CJD) and a variant form, vCJD, are types of transmissible spongiform encephalopathies which are caused by abnormal, infectious prion proteins and lead to the degeneration of brain tissue. As vCJD is linked to the lymphoreticular system which includes the gastrointestinal tract, there is a possibility of transmission via a contaminated endoscope. Annette Puzey reviews the most recent advice on how to reduce the risk of transmitting prions to patients undergoing flexible endoscopy, considering patient assessment, the level of tissue infectivity, and the risk of invasive procedures.

Neuronal low-density lipoprotein receptor-related protein 1 binds and endocytoses prion fibrils via receptor cluster 4

For infectious prion protein (designated PrP(Sc)) to act as a template to convert normal cellular protein (PrP(C)) to its distinctive pathogenic conformation, the two forms of prion protein (PrP) must interact closely. The neuronal receptor that rapidly endocytoses PrP(C) is the low-density lipoprotein receptor-related protein 1 (LRP1). We show here that on sensory neurons LRP1 is also the receptor that binds and rapidly endocytoses smaller oligomeric forms of infectious prion fibrils, and recombinant PrP fibrils. Although LRP1 binds two molecules of most ligands independently to its receptor clusters 2 and 4, PrP(C) and PrP(Sc) fibrils bind only to receptor cluster 4. PrP(Sc) fibrils out-compete PrP(C) for internalization. When endocytosed, PrP(Sc) fibrils are routed to lysosomes, rather than recycled to the cell surface with PrP(C). Thus, although LRP1 binds both forms of PrP, it traffics them to separate fates within sensory neurons. The binding of both to ligand cluster 4 should enable genetic modification of PrP binding without disrupting other roles of LRP1 essential to neuronal viability and function, thereby enabling in vivo analysis of the role of this interaction in controlling both prion and LRP1 biology.

Infectious prion protein in the filtrate even after 15nm filtration

The evaluation of the removal efficacy during manufacturing is important for the risk assessment of plasma products with respect to possible contamination by infectious prions, as recently reported in several papers on the potential for prion transmission through plasma products. Here, we evaluated a virus removal filter which has 15 nm pores. An antithrombin sample immediately prior to nano-filtration was spiked with prion material prepared in two different ways. The removal (log reduction factor) of prion infectivity using animal bioassays was ≥4.72 and 4.00 in two independent filtrations. However, infectivity was detected in both the pellet and supernatant following ultracentrifugation of the 15 nm filtered samples, indicating difficulty in complete removal. The data supports the conclusion that a certain amount of infectious prion protein is present as a smaller and/or soluble form (less than ∼15 nm in diameter).

Immune cell types involved in early uptake and transport of recombinant mouse prion protein in Peyer’s patches of calves

We have previously reported the early uptake and transport of foreign particles into Peyer's patches (PPs) of newborn and 2-month-old calves and shown that the peak uptake of particles occurs 6 h after inoculation, in addition to site- and size-related effects on particle uptake. We now report the distribution of immune cells within PPs of the distal ileum in newborn and 2-month-old calves inoculated with carbon black. The types of immune cells involved in the early uptake and transport of recombinant mouse prion protein (rMPrP) within PPs of newborn calf were investigated by using monoclonal antibodies CD11c, CD14, CD68, CD172a, and CD21. CD11c(+), CD14(+), CD68(+), CD172a(+), and CD21(+) immune cells were widely distributed in four tissue compartments (villi, dome, interfollicular region, and follicles) of PPs in the distal ileum of newborn and 2-month-old calves, whereas CD11c(+), CD14(+), CD172a(+), and CD21(+) immune cells were more prominently distributed in the dome areas of newborn calves than in 2-month-old calves. Moreover, CD11c(+) and CD14(+) dendritic cells, CD172a(+) and CD68(+) macrophages, and CD21(+) follicular dendritic cells containing rMPrP were primarily observed in the dome and inner follicular regions. The deposition of rMPrP within CD11c(+), CD14(+), CD172a(+), and CD68(+) cells, but not CD21(+) cells, was detected in villous regions. rMPrP-positive immune cells within the interfollicular regions included only CD11c(+) and CD172(+) cells. Although the particles used in this investigation do not include the infectious prion protein, PrP(Sc), our experimental setup provides a useful model for studying immune cells involved in the early uptake and transport of PrP(Sc).

Prion Neurotoxicity: Insights from Prion Protein Mutants

The chemical nature of prions and the mechanism by which they propagate are now reasonably well understood. In contrast, much less is known about the identity of the toxic prion protein (PrP) species that are responsible for neuronal death, and the cellular pathways that these forms activate. In addition, the normal, physiological function of cellular PrP (PrP(C)) has remained mysterious, hampering efforts to determine whether loss or alteration of this function contributes to the disease phenotype. Considerable evidence now suggests that aggregation, toxicity, and infectivity are distinct properties of PrP that do no necessarily coincide. In this review, we will discuss several mutant forms of PrP that produce spontaneous neurodegeneration in humans and/or transgenic mice without the formation of infectious PrP(Sc). These include an octapeptide insertional mutation, point mutations that favor synthesis of transmembrane forms of PrP, and deletions encompassing the central domain whose neurotoxicity is antagonized by the presence of wild-type PrP. By isolating the neurotoxic effects of PrP from the formation of infectious prions, these mutants have provided important insights into possible pathogenic mechanisms. These studies suggest that prion neurotoxicity may involve subversion of a cytoprotective activity of PrP(C) via altered signaling events at the plasma membrane.

The infectious nature of protein misfolding disorders

Compelling evidence indicate that a variety of human diseases arise from the misfolding and oligomerization of an otherwise normal protein. These diseases include some highly prevalent disorders such as Alzheimer's, Parkinson disease and diabetes type 2. These and more than 25 other diseases share as a hallmark event the misfolding of a protein to form ß‐sheet rich aggregates that deposit in diverse organs inducing cell death and tissue damage. The process of misfolding and aggregation follows a seeding‐nucleation mechanism in which small oligomers act as seeds to trigger an uncontrolled aggregation process. The seeding mechanism appears to be the basis by which misfolded prion protein (PrPSc) propagates prion disease in an infectious manner. We have been able to create infectious prion protein in the test tube and produce a variety of novel diseases. So far, prion disease is the only member of the group of protein misfolding disorders that has an infectious origin. However, the inherent infectious nature of misfolded aggregates propagating by a seeding mechanism suggests that other diseases of the group may be transmissible in the same manner as prion disorders. In this presentation, we will show evidences that Alzheimer's disease pathology can be experimentally transmitted in vivo.

Uptake and Dynamics of Infectious Prion Protein in the Intestine

Transmissible spongiform encephalopathies (TSEs) are characterized by the accumulation of a protease-resistant abnormal isoform of the prion protein (PrPSc), which is converted from the cellular isoform of the prion protein (PrPC). In the oral transmission of prion protein, PrPSc can invade a host body through the intestinal tract. There is only limited information available on how the infectious agent passes through one or several biological barriers before it can finally reach the brain. After oral administration, PrPSc withstands the digestive process and may be incorporated by microfold (M) cells or villous columnar epithelial cells in the intestine. After entry into the intestinal epithelium, PrPSc accumulates and is amplified in follicular dendritic cells (FDCs) within Peyer's patches and other isolated lymphoid follicles possibly by an interaction with dendritic cells or macrophages. Following accumulation in gut-associated lymphoid tissues, PrPSc is thought to move to the enteric nervous systems (ENS) by an interaction with FDCs or dendritic cells. As a result of neuroinvasion into the ENS, PrPSc spreads to the central nervous system. In addition, an epidemiological study suggested that most bovine spongiform encephalopathy cases had been exposed to the agent in the first 6 months of life. Developments of the intestinal defense and immune system may be involved in the susceptibility to infection.

Constraining the loop, releasing prion infectivity

Prions are the heretical protein-only agents that propagate biological information in the absence of nucleic acids. In mammals, prions are the infectious agents responsible for transmissible spongiform encephalopathies (TSEs), a group of fatal neurodegenerative diseases, including Creutzfeldt–Jakob disease in humans and bovine spongiform encephalopathy, scrapie, and chronic wasting disease in animals. Prions replicate by propagating the misfolding of the infectious prion protein (termed PrPSc) to the homologous, but natively-folded cellular version of the prion protein (termed PrPC) (1). Despite considerable recent advance in knowledge of the infectious agent's nature and its mechanism of replication, we are still far from understanding the structural requirements for the infectious folding of the prion protein. In this issue of PNAS, Sigurdsson et al. (2) provide an important step forward in elucidating the role of a specific domain of the prion protein in the generation of infectivity.

Lymphotoxin-Dependent Prion Replication in Inflammatory Stromal Cells of Granulomas

Prior to invading the nervous system, prions frequently colonize lymphoid organs and sites of inflammatory lymphoneogenesis, where they colocalize with Mfge8+ follicular dendritic cells (FDCs). Here, we report that soft-tissue granulomas, a frequent feature of chronic inflammation, expressed the cellular prion protein (PrPC, encoded by Prnp) and the lymphotoxin receptor (LTbetaR), even though they lacked FDCs and did not display lymphoneogenesis. After intraperitoneal prion inoculation, granulomas of Prnp(+/+) mice, but not Prnp(-/-) granulomas or unaffected Prnp(+/+) skin, accumulated prion infectivity and disease-associated prion protein. Bone-marrow transfers between Prnp(+/+) and Prnp(-/-) mice and administration of lymphotoxin signaling antagonists indicated that prion replication required radioresistant PrPC-expressing cells and LTbetaR signaling. Granulomatous PrPC was mainly expressed by stromal LTbetaR+ mesenchymal cells that were absent from unaffected subcutis. Hence, granulomas can act as clinically silent reservoirs of prion infectivity. Furthermore, lymphotoxin-dependent prion replication can occur in inflammatory stromal cells that are distinct from FDCs.

Scrapie-Induced Defects in Learning and Memory of Transgenic Mice Expressing Anchorless Prion Protein Are Associated with Alterations in the Gamma Aminobutyric Acid-Ergic Pathway

After infection with RML murine scrapie agent, transgenic (tg) mice expressing prion protein (PrP) without its glycophosphatidylinositol (GPI) membrane anchor (GPI(-/-) PrP tg mice) continue to make abundant amounts of the abnormally folded disease-associated PrPres but have a normal life span. In contrast, all age-, sex-, and genetically matched mice with a GPI-anchored PrP become moribund and die due to a chronic progressive neurodegenerative disease by 160 days after RML scrapie agent infection. We report here that infected GPI(-/-) PrP tg mice, although free from progressive neurodegenerative disease of the cerebellum and extrapyramidal and pyramidal systems, nevertheless suffer defects in learning and memory, long-term potentiation, and neuronal excitability. Such dysfunction increases over time and is associated with an increase in gamma aminobutyric acid (GABA) inhibition but not loss of excitatory glutamate/N-methyl-d-aspartic acid. Enhanced deposition of abnormally folded infectious PrP (PrPsc or PrPres) in the central nervous system (CNS) localizes with GABAA receptors. This occurs with minimal evidence of CNS spongiosis or apoptosis of neurons. The use of monoclonal antibodies reveals an association of PrPres with GABAA receptors. Thus, the clinical defects of learning and memory loss in vivo in GPI(-/-) PrP tg mice infected with scrapie agent may likely involve the GABAergic pathway.

Strain‐Associated Variations in Abnormal PrP Trafficking of Sheep Scrapie

Prion diseases are associated with the accumulation of an abnormal form of the host‐coded prion protein (PrP). It is postulated that different tertiary or quaternary structures of infectious PrP provide the information necessary to code for strain properties. We show here that different light microscopic types of abnormal PrP (PrPd) accumulation found in each of 10 sheep scrapie cases correspond ultrastructurally with abnormal endocytosis, increased endo‐lysosomes, microfolding of plasma membranes, extracellular PrPd release and intercellular PrPd transfer of neurons and/or glia. The same accumulation patterns of PrPd and associated subcellular lesions were present in each of two scrapie strains present, but they were present in different proportions. The observations suggest that different trafficking pathways of PrPd are influenced by strain and cell type and that a single prion strain causes several PrPd–protein interactions at the cell membrane. These results imply that strains may contain or result in production of multiple isoforms of PrPd.

Non‐infectious aggregates of the prion protein react with several PrPSc‐directed antibodies

The key event in the pathogenesis of prion diseases is the conformational conversion of the normal prion protein (PrP) (PrP(C)) into an infectious, aggregated isoform (PrP(Sc)) that has a high content of beta-sheet. Historically, a great deal of effort has been devoted to developing antibodies that specifically recognize PrP(Sc) but not PrP(C), as such antibodies would have enormous diagnostic and experimental value. A mouse monoclonal IgM antibody (designated 15B3) and three PrP motif-grafted monoclonal antibodies (referred to as IgG 19-33, 89-112, and 136-158) have been previously reported to react specifically with infectious PrP(Sc) but not PrP(C). In this study, we extend the characterization of these four antibodies by testing their ability to immunoprecipitate and immunostain infectious and non-infectious aggregates of wild-type, mutant, and recombinant PrP. We find that 15B3 as well as the motif-grafted antibodies recognize multiple types of aggregated PrP, both infectious and non-infectious, including forms found in brain, in transfected cells, and induced in vitro from purified recombinant protein. These antibodies are exquisitely selective for aggregated PrP, and do not react with soluble PrP even when present in vast excess. Our results suggest that 15B3 and the motif-grafted antibodies recognize structural features common to both infectious and non-infectious aggregates of PrP. Our study extends the utility of these antibodies for diagnostic and experimental purposes, and it provides new insight into the structural changes that accompany PrP oligomerization and prion propagation.

Reduction of Prion Infectivity in Packed Red Cells by Separation of Whole Blood and Washing of the Red Cell Fraction.

Although prion diseases are rare in humans, the established link between a new variant form of CJD (vCJD) and the consumption of cattle meat contaminated by BSE have raised concerns about a possible outbreak of a large epidemic in the human population. Over the past few years, BSE has become a significant health concern in several countries, and it now seems apparent that vCJD can also be iatrogenically transmitted from human to human by blood transfusion. Exacerbating this state of affairs is the lack of a reliable test to identify individuals incubating the disease during the long and silent period from the onset of infection to the appearance of clinical symptoms. The purpose of this research study was to evaluate the effectiveness of separation of whole blood and washing of the red cell fraction for the removal of infectious scrapie prion protein (PrPSc) from red blood cell (RBC) suspensions. Samples of human, whole blood were spiked with 5 × 106 LD50 263K PrPSc. Analysis of the treated sample supernatants by Western blot revealed that approximately >88% of the PrPSc was removed with the initial plasma expression and the equivalent of 6% was detected in a saline wash (300 mL; 0.9% saline). The final sample of RBCs revealed no detectable levels of PrPSc by Western blots. Further analysis of the treated RBCs using the PMCA assay indicated detectable amounts of PrPSc only after 2 consecutive amplification rounds. Semi-quantitative analysis of PMCA amplification enabled us to estimate that the treated RBCs contained less than 1 × 104 LD50 PrPSc. This corresponded to removal levels exceeding ≥99% of spiked material in whole blood. These in vitro estimations were confirmed by in vivo infectivity studies in a hamster model of disease transmission.Results from in vivo studies displayed significant differences in the incubation periods of the spiked blood inoculated hamsters (100.1 ± 1.7) versus washed RBCs (135.8 ± 6.7). Moreover, a substantial difference in the attack rate (6/15: 40% in washed RBC, versus 13/13: 100% in spiked blood) further indicated a substantial removal of infectious prions. Comparison of this data with results of animals inoculated with different dilutions of infectious material, indicated a >99.94% reduction of infectivity. Washed, packed human red cells produced by this procedure were able to be stored in standard additive solutions (AS-3) for 42 days while still meeting all in vitro blood bank standards for acceptable red cell quality.Conclusion This data suggests that separation of plasma coupled with a simple, low volume wash of red cells may represent an efficient method to remove prions from red blood cell fractions, thus reducing possible infectivity of these products.

Multiple biochemical similarities between infectious and non‐infectious aggregates of a prion protein carrying an octapeptide insertion

A nine-octapeptide insertion in the prion protein (PrP) gene is associated with an inherited form of human prion disease. Transgenic (Tg) mice that express the mouse homolog of this mutation (designated PG14) spontaneously accumulate in their brains an insoluble and weakly protease-resistant form of the mutant protein. This form (designated PG14(Spon)) is highly neurotoxic, but is not infectious in animal bioassays. In contrast, when Tg(PG14) mice are inoculated with the Rocky Mountain Laboratory (RML) strain of prions, they accumulate a different form of PG14 PrP (designated PG14(RML)) that is highly protease resistant and infectious in animal transmission experiments. We have been interested in characterizing the molecular properties of PG14(Spon) and PG14(RML), with a view to identifying features that determine two, apparently distinct properties of PrP aggregates: their infectivity and their pathogenicity. In this paper, we have subjected PG14(Spon) and PG14(RML) to a panel of assays commonly used to distinguish infectious PrP (PrP(Sc)) from cellular PrP (PrP(C)), including immobilized metal affinity chromatography, precipitation with sodium phosphotungstate, and immunoprecipitation with PrP(C)- and PrP(Sc)-specific antibodies. Surprisingly, we found that aggregates of PG14(Spon) and PG14(RML) behave identically to each other, and to authentic PrP(Sc), in each of these biochemical assays. PG14(Spon) however, in contrast to PG14(RML) and PrP(Sc), was unable to seed the misfolding of PrP(C) in an in vitro protein misfolding cyclic amplification reaction. Collectively, these results suggest that infectious and non-infectious aggregates of PrP share common structural features accounting for their toxicity, and that self-propagation of PrP involves more subtle molecular differences.

Prion Protein α-to-β Transition Monitored by Time-Resolved Fourier Transform Infrared Spectroscopy

The conformational change of the recombinant, murine prion protein (PrP) from an alpha-helical to a beta-sheet enriched state was monitored by time-resolved Fourier transform infrared (FT-IR) spectroscopy. The alpha-to-beta transition is induced by reduction of the single disulfide bond in PrP. This transition is believed to generate the scrapie form PrP(Sc), the supposed infectious agent of transmissible spongiform encephalopathies. We followed the kinetics of this conformational change using a novel method for amide I band analysis of the infrared (IR) spectra. The amide I analysis provides the secondary structure. The amide I decomposition was calibrated with the three dimensional structure of cellular PrP solved by nuclear magnetic resonance (NMR). The novel secondary structure analysis provides a root mean squared deviation (RMSD) of only 3% as compared to the NMR structure. Reduction of alpha-helical PrP caused the transient accumulation of a partially unfolded intermediate, followed by formation of a state with higher beta-sheet than alpha-helical structure contents. The novel approach allows us to now determine the secondary structure of the beta-sheet conformation. This was not determined by either NMR or X-ray. The experiments were performed in a double-sealed security cuvette developed for IR analysis of potentially infectious PrP samples outside the biosafety laboratory.

Modeling of a propagation mechanism of infectious prion protein; a hexamer as the minimum infectious unit

To construct a new model of the propagation mechanism of infectious scrapie-type prion protein (PrP Sc), here we conducted a disruption simulation of a PrP Sc nonamer using structure-based molecular dynamics simulation method based on a hypothetical PrP Sc model structure. The simulation results showed that the nonamer disrupted in cooperative manners into monomers via two significant intermediate states: (1) a nonamer with a partially unfolded surface trimer and (2) a hexamer and three monomers. Dimers and trimers were rarely observed. Then, we propose a new PrP Sc propagation mechanism where a hexamer plays an essential role as a minimum infectious unit.

Anti-LRP/LR Antibody W3 Hampers Peripheral PrPSc Propagation in Scrapie Infected Mice

We identified the 37/67kDa laminin receptor (LRP/LR) as a cell surface receptor for the cellular prion protein (PrPc) and the infectious prion protein (PrPSc). Recently, we showed that anti-LRP/LR antibody W3 cured scrapie infected N2a cells. Here, we demonstrate that W3 delivered by passive immunotransfer into C57BL/6 mice reduced the PrPSc content in the spleen significantly by 66%, demonstrating an impairment of the peripheral PrPSc propagation. In addition, we observed a 1.8 fold increase in survival of anti-LRP/LR antibody W3 treated mice (mean survival of 31 days) compared to preimmune serum treated control animals (mean survival of 17 days). We conclude that the significant effect of anti-LRP/LR antibody W3 on the reduction of peripheral PrPSc propagation might be due to the blockage of the prion receptor LRP/LR which is required, as previously shown in vitro, for PrPSc propagation in vivo.