Heterozygosity for cervid S138N polymorphism results in subclinical CWD in gene-targeted mice and progressive inhibition of prion conversion.

Prions are proteinaceous infectious particles that replicate by structural conversion of the host-encoded cellular prion protein (PrPC), causing fatal neurodegenerative diseases in mammals. Species-specific amino acid substitutions (AAS) arising from single nucleotide polymorphisms within the prion protein gene (Prnp) modulate prion disease pathogenesis, and, in several instances, reduce susceptibility of homo- or heterozygous AAS carriers to prion infection. However, a mechanistic understanding of their protective effects against clinical disease is missing. We generated gene-targeted mouse infection models of chronic wasting disease (CWD), a highly contagious prion disease of cervids. These mice express wild-type deer or PrPC harboring the S138N substitution homo- or heterozygously, a polymorphism found exclusively in reindeer (Rangifer tarandus spp.) and fallow deer (Dama dama). The wild-type deer PrP-expressing model recapitulated CWD pathogenesis including fecal shedding. Encoding at least one 138N allele prevented clinical CWD, accumulation of protease-resistant PrP (PrPres) and abnormal PrP deposits in the brain tissue. However, prion seeding activity was detected in spleens, brains, and feces of these mice, suggesting subclinical infection accompanied by prion shedding. 138N-PrPC was less efficiently converted to PrPres in vitro than wild-type deer (138SS) PrPC. Heterozygous coexpression of wild-type deer and 138N-PrPC resulted in dominant-negative inhibition and progressively diminished prion conversion over serial rounds of protein misfolding cyclic amplification. Our study indicates that heterozygosity at a polymorphic Prnp codon can confer the highest protection against clinical CWD and highlights the potential role of subclinical carriers in CWD transmission.

Blood ß-Synuclein and Neurofilament Light Chain During the Course of Prion Disease.

BACKGROUND AND OBJECTIVES: For early diagnosis and disease monitoring of neurodegenerative diseases (NDs), reliable blood biomarkers are needed. Elevated levels of neurofilament light chain protein (NfL), an axonal damage marker, have been described across different NDs, with highest values in prion diseases and amyotrophic lateral sclerosis (ALS). Synaptic degeneration is a common early feature in most NDs and seems to precede neuronal degeneration in prion disease. However, synaptic markers in blood are still missing. Here, we investigated whether the brain-specific protein ß-synuclein might be a suitable blood biomarker for early diagnosis and evaluation of synaptic integrity in prion disease. METHODS: We analyzed blood ß-synuclein with a newly established digital ELISA and NfL with a single-molecule array in samples obtained from human participants and prion and ALS animal models. Furthermore, ß-synuclein was investigated in brain tissue of individuals with Creutzfeldt-Jakob disease (CJD) and controls. RESULTS: We investigated 308 patients, including 129 cases with prion disease, 8 presymptomatic PRNP variation carriers, 60 with ALS, 68 with other ND, and 43 control patients. In CJD symptomatic cases, ß-synuclein and NfL were markedly increased compared to all other diagnostic groups (p < 0.001). In the large majority of presymptomatic PRNP variation carriers, ß-synuclein and NfL levels were within normal ranges. In prion disease animal models, ß-synuclein and NfL displayed normal levels in the presymptomatic phase with a sudden elevation at disease onset and a plateau in the symptomatic phase. In contrast to NfL, ß-synuclein was not elevated in either symptomatic patients with ALS or an ALS animal model. In the discrimination between prion disease and all other groups, ß-synuclein (area under the curve 0.97, 95% CI 0.94-0.99, p < 0.001) was superior to NfL (area under the curve 0.91, 95% CI 0.88-0.94, p < 0.001). In addition, brain tissue ß-synuclein showed significantly reduced levels in patients with CJD compared to control patients (p < 0.001). DISCUSSION: Blood ß-synuclein was significantly elevated in patients with CJD, reflecting ongoing synaptic damage, and showed good discriminative characteristics. We therefore propose it as a candidate blood marker for early diagnosis and monitoring of synaptic integrity in prion disease. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that serum ß-synuclein concentration accurately distinguishes patients with symptomatic CJD from controls.

Primary glia cells from bank vole propagate multiple rodent-adapted scrapie prions.

Since the beginning prion research has been largely dependent on animal models for deciphering the disease, drug development or prion detection and quantification. Thereby, ethical as well as cost and labour-saving aspects call for alternatives in vitro. Cell models can replace or at least complement animal studies, but their number is still limited and the application usually restricted to certain strains and host species due to often strong transmission barriers. Bank voles promise to be an exception as they or materials prepared from them are uniquely susceptible to prions from various species in vivo, in vitro and in cell-free applications. Here we present a mainly astrocyte-based primary glia cell assay from bank vole, which is infectible with scrapie strains from bank vole, mouse and hamster. Stable propagation of bank vole-adapted RML, murine 22L and RML, and hamster 263K scrapie is detectable from 20 or 30 days post exposure onwards. Thereby, the infected bank vole glia cells show similar or even faster prion propagation than likewise infected glia cells of the corresponding murine or hamster hosts. We propose that our bank vole glia cell assay could be a versatile tool for studying and comparing multiple prion strains with different species backgrounds combined in one cell assay.

The Neural Gut-Brain Axis of Pathological Protein Aggregation in Parkinson's Disease and Its Counterpart in Peroral Prion Infections.

A neuropathological hallmark of Parkinson's disease (PD) is the cerebral deposition of abnormally aggregated α-synuclein (αSyn). PD-associated αSyn (αSynPD) aggregates are assumed to act, in a prion-like manner, as proteinaceous nuclei ("seeds") capable of self-templated propagation. Braak and colleagues put forward the idea of a neural gut-brain axis mediating the centripetal spread of αSynPD pathology from the enteric nervous system (ENS) to the brain in PD. This has sparked great interest and initiated passionate discussions both in support of and opposing the suggested hypothesis. A precedent for the spread of protein seeds or seeding from the gastro-intestinal (GI) tract to the central nervous system (CNS) had been previously revealed for pathological prion protein in peroral prion infections. This article scrutinizes the similarities and dissimilarities between the pathophysiological spread of disease-associated protein aggregation along the neural gut-brain axis in peroral prion infections and PD. On this basis, evidence supporting the proposed neural gut-brain axis in PD is concluded to be not as robust as that established for peroral prion infections. New tools for the ultrasensitive detection of αSynPD-associated seeding activity in archived or fresh human tissue samples such as real-time quaking induced conversion (RT-QuIC) or protein misfolding cyclic amplification (PMCA) assays can possibly help to address this deficit in the future.

Propagation of CJD Prions in Primary Murine Glia Cells Expressing Human PrPc.

There are various existing cell models for the propagation of animal prions. However, in vitro propagation of human prions has been a long-standing challenge. This study presents the establishment of a long-term primary murine glia culture expressing the human prion protein homozygous for methionine at codon 129, which allows in vitro propagation of Creutzfeldt-Jakob disease (CJD) prions (variant CJD (vCJD) and sporadic CJD (sCJD) type MM2). Prion propagation could be detected by Western blotting of pathological proteinase K-resistant prion protein (PrPSc) from 120 days post exposure. The accumulation of PrPSc could be intensified by adding a cationic lipid mixture to the infectious brain homogenate at the time of infection. Stable propagation of human prions in a long-term murine glia cell culture represents a new tool for future drug development and for mechanistic studies in the field of human prion biology. In addition, our cell model can reduce the need for bioassays with human prions and thereby contributes to further implementation of the 3R principles aiming at replacement, reduction and refinement of animal experiments.

Transmissible α-synuclein seeding activity in brain and stomach of patients with Parkinson's disease.

Cerebral deposition of abnormally aggregated α-synuclein (αSyn) is a neuropathological hallmark of Parkinson's disease (PD). PD-associated αSyn (αSynPD) aggregates can act as proteinaceous nuclei ("seeds") able of self-templated propagation. Since this is strikingly reminiscent to properties of proteinaceous infectious particles (prions), lessons learned from prion diseases suggest to test whether transferred αSynPD can propagate and induce neurological impairments or disease in a new host. Two studies that addressed this question provided divergent results. Intracerebral (i.c.) injection of Lewy body extracts from PD patients caused cerebral αSyn pathology, as well as nigrostriatal neurodegeneration, of wild-type mice and macaques, with the mice also showing motor impairments (Recasens et al. 2014, Ann Neurol 75:351-362). In contrast, i.c. transmission of homogenates from PD brains did not stimulate, after "> 360" days post-injection (dpi), pathological αSyn conversion or clinical symptoms in transgenic TgM83+/- mice hemizygously expressing mutated (A53T) human αSyn (Prusiner et al. 2015, PNAS 112:E5308-E5317). To advance the assessment of possible αSynPD hazards by providing further data, we examined neuropathological and clinical effects upon i.c. transmission of brain, stomach wall and muscle tissue as well as blood from PD patients in TgM83+/- mice up to 612 dpi. This revealed a subtle, yet distinctive stimulation of localized αSyn aggregation in the somatodendritic compartment and dystrophic neurites of individual or focally clustered cerebral neurons after challenge with brain and stomach wall homogenates. No such effect was observed with transmitted blood or homogenized muscle tissue. The detected stimulation of αSyn aggregation was not accompanied by apparent motor impairments or overt neurological disease in TgM83+/- mice. Our study substantiated that transmitted αSynPD seeds, including those from the stomach wall, are able to propagate in new mammalian hosts. The consequences of such propagation and potential safeguards need to be further investigated.

Stability of BSE infectivity towards heat treatment even after proteolytic removal of prion protein.

The unconventional infectious agents of transmissible spongiform encephalopathies (TSEs) are prions. Their infectivity co-appears with PrPSc, aberrant depositions of the host's cellular prion protein (PrPC). Successive heat treatment in the presence of detergent and proteolysis by a keratinase from Bacillus licheniformis PWD-1 was shown before to destroy PrPSc from bovine TSE (BSE) and sheep scrapie diseased brain, however data regarding expected reduction of infectivity were still lacking. Therefore, transgenic Tgbov XV mice which are highly BSE susceptible were used to quantify infectivity before and after the bovine brain treatment procedure. Also four immunochemical analyses were applied to compare the levels of PrPSc. After heating at 115 °C with or without subsequent proteolysis, the original BSE infectivity of 106.2-6.4 ID50 g-1 was reduced to a remaining infectivity of 104.6-5.7 ID50 g-1 while strain characteristics were unaltered, even after precipitation with methanol. Surprisingly, PrPSc depletion was 5-800 times higher than the loss of infectivity. Similar treatment was applied on other prion strains, which were CWD1 in bank voles, 263 K scrapie in hamsters and sheep PG127 scrapie in tg338 ovinized mice. In these strains however, infectivity was already destroyed by heat only. These findings show the unusual heat resistance of BSE and support a role for an additional factor in prion formation as suggested elsewhere when producing prions from PrPC. Leftover material in the remaining PrPSc depleted BSE preparation offers a unique substrate for searching additional elements for prion infectivity and improving our concept about the nature of prions.

Preserving prion strain identity upon replication of prions in vitro using recombinant prion protein.

Last decade witnessed an enormous progress in generating authentic infectious prions or PrPSc in vitro using recombinant prion protein (rPrP). Previous work established that rPrP that lacks posttranslational modification is able to support replication of highly infectious PrPSc with assistance of cofactors of polyanionic nature and/or lipids. Unexpectedly, previous studies also revealed that seeding of rPrP by brain-derived PrPSc gave rise to new prion strains with new disease phenotypes documenting loss of a strain identity upon replication in rPrP substrate. Up to now, it remains unclear whether prion strain identity can be preserved upon replication in rPrP. The current study reports that faithful replication of hamster strain SSLOW could be achieved in vitro using rPrP as a substrate. We found that a mixture of phosphatidylethanolamine (PE) and synthetic nucleic acid polyA was sufficient for stable replication of hamster brain-derived SSLOW PrPSc in serial Protein Misfolding Cyclic Amplification (sPMCA) that uses hamster rPrP as a substrate. The disease phenotype generated in hamsters upon transmission of recombinant PrPSc produced in vitro was strikingly similar to the original SSLOW diseases phenotype with respect to the incubation time to disease, as well as clinical, neuropathological and biochemical features. Infrared microspectroscopy (IR-MSP) indicated that PrPSc produced in animals upon transmission of recombinant PrPSc is structurally similar if not identical to the original SSLOW PrPSc. The current study is the first to demonstrate that rPrP can support replication of brain-derived PrPSc while preserving its strain identity. In addition, the current work is the first to document that successful propagation of a hamster strain could be achieved in vitro using hamster rPrP.

Correction to: preserving prion strain identity upon replication of prions in vitro using recombinant prion protein.

Figure 6 of the original publication [1] contained an error in the Wavenumber in panels B and C. The wavenumbers 1616 (Cm-1) in panels B and C should have been 1516 (cm-1). The updated figure has been published in this correction article; the original article has been updated.

Sialylation Controls Prion Fate in Vivo.

Prions or PrPSc are proteinaceous infectious agents that consist of misfolded, self-replicating states of a sialoglycoprotein called the prion protein or PrPC The current work tests a new hypothesis that sialylation determines the fate of prions in an organism. To begin, we produced control PrPSc from PrPC using protein misfolding cyclic amplification with beads (PMCAb), and also generated PrPSc with reduced sialylation levels using the same method but with partially desialylated PrPC as a substrate (dsPMCAb). Syrian hamsters were inoculated intraperitoneally with brain-derived PrPSc or PrPSc produced in PMCAb or dsPMCAb and then monitored for disease. Animals inoculated with brain- or PMCAb-derived PrPSc developed prion disease, whereas administration of dsPMCAb-derived PrPSc with reduced sialylation did not cause prion disease. Animals inoculated with dsPMCAb-derived material were not subclinical carriers of scrapie, as no PrPSc was detected in brains or spleen of these animals by either Western blotting or after amplification by serial PMCAb. In subsequent experiments, trafficking of brain-, PMCAb-, and dsPMCAb-derived PrPSc to secondary lymphoid organs was monitored in wild type mice. PrPSc sialylation was found to be critical for effective trafficking of PrPSc to secondary lymphoid organs. By 6 hours after inoculation, brain- and PMCAb-derived PrPSc were found in spleen and lymph nodes, whereas dsPMCAb-derived PrPSc was found predominantly in liver. This study demonstrates that the outcome of prion transmission to a wild type host is determined by the sialylation status of the inoculated PrPSc Furthermore, this work suggests that the sialylation status of PrPSc plays an important role in prion lymphotropism.

Reversible off and on switching of prion infectivity via removing and reinstalling prion sialylation.

The innate immune system provides the first line of defense against pathogens. To recognize pathogens, this system detects a number of molecular features that discriminate pathogens from host cells, including terminal sialylation of cell surface glycans. Mammalian cell surfaces, but generally not microbial cell surfaces, have sialylated glycans. Prions or PrP(Sc) are proteinaceous pathogens that lack coding nucleic acids but do possess sialylated glycans. We proposed that sialylation of PrP(Sc) is essential for evading innate immunity and infecting a host. In this study, the sialylation status of PrP(Sc) was reduced by replicating PrP(Sc) in serial Protein Misfolding Cyclic Amplification using sialidase-treated PrP(C) substrate and then restored to original levels by replication using non-treated substrate. Upon intracerebral administration, all animals that received PrP(Sc) with original or restored sialylation levels were infected, whereas none of the animals that received PrP(Sc) with reduced sialylation were infected. Moreover, brains and spleens of animals from the latter group were completely cleared of prions. The current work established that the ability of prions to infect the host via intracerebral administration depends on PrP(Sc) sialylation status. Remarkably, PrP(Sc) infectivity could be switched off and on in a reversible manner by first removing and then restoring PrP(Sc) sialylation.

Synthetic scrapie infectivity: interaction between recombinant PrP and scrapie brain-derived RNA.

The key molecular event in human cerebral proteinopathies, which include Alzheimer's, Parkinson's and Huntington's diseases, is the structural conversion of a specific host protein into a ß-sheet-rich conformer. With regards to this common mechanism, it appears difficult to explain the outstanding infectious properties attributed to PrP(Sc), the hallmark of another intriguing family of cerebral proteinopathies known as transmissible spongiform encephalopathies (TSE) or prion diseases. The infectious PrP(Sc) or "prion" is thought to be composed solely of a misfolded form of the otherwise harmless cellular prion protein (PrP(c)). To gain insight into this unique situation, we used the 263K scrapie hamster model to search for a putative PrP(Sc)-associated factor that contributes to the infectivity of PrP(Sc) amyloid. In a rigorously controlled set of experiments that included several bioassays, we showed that originally innocuous recombinant prion protein (recPrP) equivalent to PrP(c) is capable of initiating prion disease in hamsters when it is converted to a prion-like conformation (ß-sheet-rich) in the presence of RNA purified from scrapie-associated fibril (SAF) preparations. Analysis of the recPrP-RNA infectious mixture reveals the presence of 2 populations of small RNAs of approximately 27 and 55 nucleotides. These unprecedented findings are discussed in light of the distinct relationship that may exist between this RNA material and the 2 biological properties, infectivity and strain features, attributed to prion amyloid.

Is there a risk of prion-like disease transmission by Alzheimer- or Parkinson-associated protein particles?

The misfolding and aggregation of endogenous proteins in the central nervous system is a neuropathological hallmark of Alzheimer's disease (AD), Parkinson's disease (PD), as well as prion diseases. A molecular mechanism referred to as "nucleation-dependent aggregation" is thought to underlie this neuropathological phenomenon. According to this concept, disease-associated protein particles act as nuclei, or seeds, that recruit cellular proteins and incorporate them, in a misfolded form, into their growing aggregate structure. Experimental studies have shown that the aggregation of the AD-associated proteins amyloid-ß (Aß) and tau, and of the PD-associated protein α-synuclein, can be stimulated in laboratory animal models by intracerebral (i.c.) injection of inocula containing aggregated species of the respective proteins. This has raised the question of whether AD or PD can be transmitted, like certain human prion diseases, between individuals by self-propagating protein particles potentially present on medical instruments or in blood or blood products. While the i.c. injection of inocula containing AD- or PD-associated protein aggregates was found to cause neuronal damage and clinical abnormalities (e.g., motor impairments) in some animal models, none of the studies published so far provided evidence for a transmission of severe or even fatal disease. In addition, available epidemiological data do not indicate a transmissibility of AD or PD between humans. The findings published so far on the effects of experimentally transmitted AD- or PD-associated protein seeds do not suggest specific precautionary measures in the context of hemotherapy, but call for vigilance in transfusion medicine and other medical areas.

Association of prion protein genotype and scrapie prion protein type with cellular prion protein charge isoform profiles in cerebrospinal fluid of humans with sporadic or familial prion diseases.

The present study investigates whether posttranslational modifications of cellular prion protein (PrP(C)) in the cerebrospinal fluid (CSF) of humans with prion diseases are associated with methionine (M) and/or valine (V) polymorphism at codon 129 of the prion protein gene (PRNP), scrapie prion protein (PrP(Sc)) type in sporadic Creutzfeldt-Jakob disease (sCJD), or PRNP mutations in familial Creutzfeldt-Jakob disease (fCJD/E200K), and fatal familial insomnia (FFI). We performed comparative 2-dimensional immunoblotting of PrP(C) charge isoforms in CSF samples from cohorts of diseased and control donors. Mean levels of total PrP(C) were significantly lower in the CSF from fCJD patients than from those with sCJD or FFI. Of the 12 most abundant PrP(C) isoforms in the examined CSF, one (IF12) was relatively decreased in (1) sCJD with VV (vs. MM or MV) at PRNP codon 129; (2) in sCJD with PrP(Sc) type 2 (vs. PrP(Sc) type 1); and (3) in FFI versus sCJD or fCJD. Furthermore, truncated PrP(C) species were detected in sCJD and control samples without discernible differences. Finally, serine 43 of PrP(C) in the CSF and brain tissue from CJD patients showed more pronounced phosphorylation than in control donors.

Infrared microspectroscopy detects protein misfolding cyclic amplification (PMCA)-induced conformational alterations in hamster scrapie progeny seeds.

The self-replicative conformation of misfolded prion proteins (PrP) is considered a major determinant for the seeding activity, infectiousness, and strain characteristics of prions in different host species. Prion-associated seeding activity, which converts cellular prion protein (PrP(C)) into Proteinase K-resistant, infectious PrP particles (PrP(TSE)), can be monitored in vitro by protein misfolding cyclic amplification (PMCA). Thus, PMCA has been established as a valuable analytical tool in prion research. Currently, however, it is under discussion whether prion strain characteristics are preserved during PMCA when parent seeds are amplified in PrP(C) substrate from the identical host species. Here, we report on the comparative structural analysis of parent and progeny (PMCA-derived) PrP seeds by an improved approach of sensitive infrared microspectroscopy. Infrared microspectroscopy revealed that PMCA of native hamster 263K scrapie seeds in hamster PrP(C) substrate caused conformational alterations in progeny seeds that were accompanied by an altered resistance to Proteinase K, higher sedimentation velocities in gradient ultracentrifugations, and a longer incubation time in animal bioassays. When these progeny seeds were propagated in hamsters, misfolded PrP from brain extracts of these animals showed mixed spectroscopic and biochemical properties from both parental and progeny seeds. Thus, strain modifications of 263K prions induced by PMCA seem to have been partially reversed when PMCA products were reinoculated into the original host species.

BSE-associated prion-amyloid cardiomyopathy in primates.

Prion amyloidosis occurred in the heart of 1 of 3 macaques intraperitoneally inoculated with bovine spongiform encephalopathy prions. This macaque had a remarkably long duration of disease and signs of cardiac distress. Variant Creutzfeldt-Jakob disease, caused by transmission of bovine spongiform encephalopathy to humans, may manifest with cardiac symptoms from prion-amyloid cardiomyopathy.