Oral prion neuroinvasion occurs independently of PrPC expression in the gut epithelium

The early replication of certain prion strains within Peyer’s patches in the small intestine is essential for the efficient spread of disease to the brain after oral exposure. Our data show that orally acquired prions utilize specialized gut epithelial cells known as M cells to enter Peyer’s patches. M cells express the cellular isoform of the prion protein, PrPC, and this may be exploited by some pathogens as an uptake receptor to enter Peyer’s patches. This suggested that PrPC might also mediate the uptake and transfer of prions across the gut epithelium into Peyer’s patches in order to establish infection. Furthermore, the expression level of PrPC in the gut epithelium could influence the uptake of prions from the lumen of the small intestine. To test this hypothesis, transgenic mice were created in which deficiency in PrPC was specifically restricted to epithelial cells throughout the lining of the small intestine. Our data clearly show that efficient prion neuroinvasion after oral exposure occurred independently of PrPC expression in small intestinal epithelial cells. The specific absence of PrPC in the gut epithelium did not influence the early replication of prions in Peyer’s patches or disease susceptibility. Acute mucosal inflammation can enhance PrPC expression in the intestine, implying the potential to enhance oral prion disease pathogenesis and susceptibility. However, our data suggest that the magnitude of PrPC expression in the epithelium lining the small intestine is unlikely to be an important factor which influences the risk of oral prion disease susceptibility. IMPORTANCE The accumulation of orally acquired prions within Peyer’s patches in the small intestine is essential for the efficient spread of disease to the brain. Little is known of how the prions initially establish infection within Peyer’s patches. Some gastrointestinal pathogens utilize molecules, such as the cellular prion protein PrPC, expressed on gut epithelial cells to enter Peyer’s patches. Acute mucosal inflammation can enhance PrPC expression in the intestine, implying the potential to enhance oral prion disease susceptibility. We used transgenic mice to determine whether the uptake of prions into Peyer’s patches was dependent upon PrPC expression in the gut epithelium. We show that orally acquired prions can establish infection in Peyer’s patches independently of PrPC expression in gut epithelial cells. Our data suggest that the magnitude of PrPC expression in the epithelium lining the small intestine is unlikely to be an important factor which influences oral prion disease susceptibility

The prion 2018 round tables (I): the structure of PrP Sc

Understanding the structure of PrPSc is without doubt a sine qua non to understand not only PrPSc propagation, but also critical features of that process such as the strain phenomenon and transmission barriers. While elucidation of the PrPSc structure has been full of difficulties, we now have a large amount of structural information that allows us to begin to understand it. This commentary article summarizes a round table that took place within the Prion 2018 meeting held in Santiago de Compostela to discuss the state of the art in this matter. Two alternative models of PrPSc exist: the PIRIBS and the 4-rung β-solenoid models. Both of them have relevant features. The 4-rung β-solenoid model agrees with experimental constraints of brain derived PrPSc obtained from cryo-EM and X-ray fiber diffraction studies. Furthermore, it allows facile accommodation of the bulky glycans that decorate brain-derived PrPSc. On the other hand, the infectious PrP23-144 amyloid exhibits a PIRIBS architecture. Perhaps, both types of structure co-exist.Supported by grants BFU2013-48436-C2-1-P and BFU2017- 86692-P from the Spanish Ministries of Economy and Competitiveness and Science, Innovation and Universities, respectively, to JRR and grant 201600029 from the Alberta Prion Research Institute to HW. This work was also supported in part by the Intramural Research Program of the NIAID (BC) and by the National Institute of Health grants R01 NS045585 (IVB), P01 AI106705 (WKS), R01 NS083687 (WKS) and R01 NS103848 (WKS)S

Bank Vole Prion Protein As an Apparently Universal Substrate for RT-QuIC-Based Detection and Discrimination of Prion Strains

Prions propagate as multiple strains in a wide variety of mammalian species. The detection of all such strains by a single ultrasensitive assay such as Real Time Quaking-induced Conversion (RT-QuIC) would facilitate prion disease diagnosis, surveillance and research. Previous studies have shown that bank voles, and transgenic mice expressing bank vole prion protein, are susceptible to most, if not all, types of prions. Here we show that bacterially expressed recombinant bank vole prion protein (residues 23-230) is an effective substrate for the sensitive RT-QuIC detection of all of the different prion types that we have tested so far--a total of 28 from humans, cattle, sheep, cervids and rodents, including several that have previously been undetectable by RT-QuIC or Protein Misfolding Cyclic Amplification. Furthermore, comparison of the relative abilities of different prions to seed positive RT-QuIC reactions with bank vole and not other recombinant prion proteins allowed discrimination of prion strains such as classical and atypical L-type bovine spongiform encephalopathy, classical and atypical Nor98 scrapie in sheep, and sporadic and variant Creutzfeldt-Jakob disease in humans. Comparison of protease-resistant RT-QuIC conversion products also aided strain discrimination and suggested the existence of several distinct classes of prion templates among the many strains tested

Analysis of Prion Strains by PrP(Sc) Profiling in Sporadic Creutzfeldt–Jakob Disease

BACKGROUND: Prion diseases are a group of invariably fatal neurodegenerative disorders affecting humans and a wide range of mammals. An essential part of the infectious agent, termed the prion, is composed of an abnormal isoform (PrP(Sc)) of a host-encoded normal cellular protein (PrP(C)). The conversion of PrP(C) to PrP(Sc) is thought to play a crucial role in the development of prion diseases and leads to PrP(Sc) deposition, mainly in the central nervous system. Sporadic Creutzfeldt–Jakob disease (sCJD), the most common form of human prion disease, presents with a marked clinical heterogeneity. This diversity is accompanied by a molecular signature which can be defined by histological, biochemical, and genetic means. The molecular classification of sCJD is an important tool to aid in the understanding of underlying disease mechanisms and the development of therapy protocols. Comparability of classifications is hampered by disparity of applied methods and inter-observer variability. METHODS AND FINDINGS: To overcome these difficulties, we developed a new quantification protocol for PrP(Sc) by using internal standards on each Western blot, which allows for generation and direct comparison of individual PrP(Sc) profiles. By studying PrP(Sc) profiles and PrP(Sc) type expression within nine defined central nervous system areas of 50 patients with sCJD, we were able to show distinct PrP(Sc) distribution patterns in diverse subtypes of sCJD. Furthermore, we were able to demonstrate the co-existence of more than one PrP(Sc) type in individuals with sCJD in about 20% of all patients and in more than 50% of patients heterozygous for a polymorphism on codon 129 of the gene encoding the prion protein (PRNP). CONCLUSION: PrP(Sc) profiling represents a valuable tool for the molecular classification of human prion diseases and has important implications for their diagnosis by brain biopsy. Our results show that the co-existence of more than one PrP(Sc) type might be influenced by genetic and brain region–specific determinants. These findings provide valuable insights into the generation of distinct PrP(Sc) types

Million-fold sensitivity enhancement in proteopathic seed amplification assays for biospecimens by Hofmeister ion comparisons

Recent work with prion diseases and synucleinopathies indicates that accurate diagnostic methods for protein-folding diseases can be based on the ultrasensitive, amplified measurement of pathological aggregates in biospecimens. A better understanding of the physicochemical factors that control the seeded polymerization of such aggregates, and their amplification in vitro, should allow improvements in existing assay platforms, as well as the development of new assays for other proteopathic aggregates. Here, we systematically investigated the effects of the ionic environment on the polymerization of tau, α-synuclein, and the prion protein (PrP) induced by aggregates in biospecimens. We screened salts of the Hofmeister series, a relative ordering of strongly and weakly hydrated salts that tend to precipitate or solubilize proteins. We found that sensitivities of tau-based assays for Alzheimer’s seeds and PrP-based assays for prions were best in weakly hydrated anions. In contrast, we saw an inverse trend with different tau-based assays, improving detection sensitivity for progressive supranuclear palsy seeds by ≈106. Hofmeister analysis also improved detection of sporadic Creutzfeldt–Jakob disease prions in human nasal brushings and chronic wasting disease prions in deer-ear homogenates. Our results demonstrate strong and divergent influences of ionic environments on the amplification and detection of proteopathic seeds as biomarkers for protein-folding diseases

Prion-related peripheral neuropathy in sporadic Creutzfeldt-Jakob disease

OBJECTIVE: To assess whether the involvement of the peripheral nervous system (PNS) belongs to the phenotypic spectrum of sporadic Creutzfeldt-Jakob disease (sCJD). METHODS: We examined medical records of 117 sCJDVV2 (ataxic type), 65 sCJDMV2K (kuru-plaque type) and 121 sCJDMM(V)1 (myoclonic type) subjects for clinical symptoms, objective signs and neurophysiological data. We reviewed two diagnostic nerve biopsies and looked for abnormal prion protein (PrP(Sc)) by western blotting and real-time quaking-induced conversion (RT-QuIC) in postmortem PNS samples from 14 subjects. RESULTS: Seventy-five (41.2%) VV2-MV2K patients, but only 11 (9.1%) MM(V)1, had symptoms or signs suggestive of PNS involvement occurring at onset in 18 cases (17 VV2-MV2K, 9.3%; and 1 MM(V)1, 0.8%) and isolated in 6. Nerve biopsy showed a mixed predominantly axonal and demyelinating neuropathy in two sCJDMV2K. Electromyography showed signs of neuropathy in half of the examined VV2-MV2K patients. Prion RT-QuIC was positive in all CJD PNS samples, whereas western blotting detected PrP(Sc) in the sciatic nerve in one VV2 and one MV2K. CONCLUSIONS: Peripheral neuropathy, likely related to PrP(Sc) deposition, belongs to the phenotypic spectrum of sCJDMV2K and VV2 and may mark the clinical onset. The significantly lower prevalence of PNS involvement in typical sCJDMM(V)1 suggests that the PNS tropism of sCJD prions is strain dependent

Real time quaking-induced conversion analysis of cerebrospinal fluid in sporadic Creutzfeldt-Jakob disease

OBJECTIVE: Current cerebrospinal fluid (CSF) tests for sporadic Creutzfeldt-Jakob disease (sCJD) are based on the detection of surrogate markers of neuronal damage such as CSF 14-3-3 which are not specific for sCJD. A number of prion protein conversion assays have been developed, including real-time quaking induced conversion (RT-QuIC). The objective of this study is to investigate whether CSF RT-QuIC analysis could be used as a diagnostic test in sCJD. METHODS: An exploratory study was undertaken which analysed 108 CSF samples from patients with neuropathologically confirmed sCJD or from control patients. Of the 108 CSF samples 56 were from sCJD patients (30 female, 26 male, aged 31–84 years; 62.3 ± 13.5 years) and 52 were from control patients (26 female, 26 male, aged 43–84 years; 67.8 ± 10.4 years). A confirmatory group of 118 patients were subsequently examined which consisted of 67 cases of neuropathologically confirmed sCJD (33 female, 34 male, aged 39–82 years; 67.5 ± 9.0 years) and 51 control cases (26 female, 25 male, aged 36–87 years; 63.5 ± 11.6 years). RESULTS: The exploratory study showed that RT-QuIC analysis had a sensitivity of 91% and a specificity of 98% for the diagnosis of sCJD. These results were confirmed in the confirmatory study which showed that CSF RT-QuIC analysis had a sensitivity and specificity of 87% and 100% respectively. INTERPRETATION: This study shows that CSF RT-QuIC analysis has the potential to be a more specific diagnostic test for sCJD than current CSF tests

A single ultrasensitive assay for detection and discrimination of tau aggregates of Alzheimer and Pick diseases

Abstract: Multiple neurodegenerative diseases are characterized by aggregation of tau molecules. Adult humans express six isoforms of tau that contain either 3 or 4 microtubule binding repeats (3R or 4R tau). Different diseases involve preferential aggregation of 3R (e.g Pick disease), 4R (e.g. progressive supranuclear palsy), or both 3R and 4R tau molecules [e.g. Alzheimer disease and chronic traumatic encephalopathy]. Three ultrasensitive cell-free seed amplification assays [called tau real-time quaking induced conversion (tau RT-QuIC) assays] have been developed that preferentially detect 3R, 4R, or 3R/4R tau aggregates in biospecimens. In these reactions, low-fg amounts of a given self-propagating protein aggregate (the seed) are incubated with a vast excess of recombinant tau monomers (the substrate) in multi-well plates. Over time, the seeds incorporate the substrate to grow into amyloids that can then be detected using thioflavin T fluorescence. Here we describe a tau RT-QuIC assay (K12 RT-QuIC) that, using a C-terminally extended recombinant 3R tau substrate (K12CFh), enables sensitive detection of Pick disease, Alzheimer disease, and chronic traumatic encephalopathy seeds in brain homogenates. The discrimination of Pick disease from Alzheimer disease and chronic traumatic encephalopathy cases is then achieved through the quantitative differences in K12 RT-QuIC assay thioflavin T responses, which correlate with structural properties of the reaction products. In particular, Fourier transform infrared spectroscopy analysis of the respective K12CFh amyloids showed distinct β-sheet conformations, suggesting at least partial propagation of the original seed conformations in vitro. Thus, K12 RT-QuIC provides a single assay for ultrasensitive detection and discrimination of tau aggregates comprised mainly of 3R, or both 3R and 4R, tau isoforms

α-Synuclein seeding activity in duodenum biopsies from Parkinson's disease patients

Abnormal deposition of α-synuclein is a key feature and biomarker of Parkinson's disease. α-Synuclein aggregates can propagate themselves by a prion-like seeding-based mechanism within and between tissues and are hypothesized to move between the intestine and brain. α-Synuclein RT-QuIC seed amplification assays have detected Parkinson's-associated α-synuclein in multiple biospecimens including post-mortem colon samples. Here we show intra vitam detection of seeds in duodenum biopsies from 22/23 Parkinson's patients, but not in 6 healthy controls by RT-QuICR. In contrast, no tau seeding activity was detected in any of the biopsies. Our seed amplifications provide evidence that the upper intestine contains a form(s) of α-synuclein with self-propagating activity. The diagnostic sensitivity and specificity for PD in this biopsy panel were 95.7% and 100% respectively. End-point dilution analysis indicated up to 106 SD50 seeding units per mg of tissue with positivity in two contemporaneous biopsies from individual patients suggesting widespread distribution within the superior and descending parts of duodenum. Our detection of α-synuclein seeding activity in duodenum biopsies of Parkinson's disease patients suggests not only that such analyses may be useful in ante-mortem diagnosis, but also that the duodenum may be a source or a destination for pathological, self-propagating α-synuclein assemblies