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Previous studies have revealed that the infectious scrapie isoform of prion protein (PrPSc) harbored in the skin tissue of patients or animals with prion diseases can be amplified and detected through the serial protein misfolding cyclic amplification (sPMCA) or real-time quaking-induced conversion (RT-QuIC) assays. These findings suggest that skin PrPSc-seeding activity may serve as a biomarker for the diagnosis of prion diseases; however, its utility as a biomarker for prion therapeutics remains largely unknown. Cellulose ethers (CEs, such as TC-5RW), widely used as food and pharmaceutical additives, have recently been shown to prolong the lifespan of prion-infected mice and hamsters. Here we report that in transgenic (Tg) mice expressing hamster cellular prion protein (PrPC) infected with the 263K prion, the prion-seeding activity becomes undetectable in the skin tissues of TC-5RW-treated Tg mice by both sPMCA and RT-QuIC assays, whereas such prion-seeding activity is readily detectable in the skin of untreated mice. Notably, TC-5RW exhibits an inhibitory effect on the in vitro amplification of PrPSc in both skin and brain tissues by sPMCA and RT-QuIC. Moreover, we reveal that TC-5RW is able to directly decrease protease-resistant PrPSc and inhibit the seeding activity of PrPSc from chronic wasting disease and various human prion diseases. Our results suggest that the level of prion-seeding activity in the skin may serve as a useful biomarker for assessing the therapeutic efficacy of compounds in a clinical trial of prion diseases and that TC-5RW may have the potential for the prevention/treatment of human prion diseases.

Chronic wasting disease (CWD) is a cervid prion disease caused by the accumulation of an infectious misfolded conformer (PrPSc) of cellular prion protein (PrPC). It has been spreading rapidly in North America and also found in Asia and Europe. Although bovine spongiform encephalopathy (i.e. mad cow disease) is the only animal prion disease known to be zoonotic, the transmissibility of CWD to humans remains uncertain. Here we report the generation of the first CWD-derived infectious human PrPSc by elk CWD PrPSc-seeded conversion of PrPC in normal human brain homogenates using in vitro protein misfolding cyclic amplification (PMCA). Western blotting with human PrP selective antibody confirmed that the PMCA-generated protease-resistant PrPSc was derived from the human PrPC substrate. Two lines of humanized transgenic mice expressing human PrP with either Val or Met at the polymorphic codon 129 developed clinical prion disease following intracerebral inoculation with the PMCA-generated CWD-derived human PrPSc. Diseased mice exhibited distinct PrPSc patterns and neuropathological changes in the brain. Our study, using PMCA and animal bioassays, provides the first evidence that CWD PrPSc can cross the species barrier to convert human PrPC into infectious PrPSc that can produce bona fide prion disease when inoculated into humanized transgenic mice.

The process of neuroinflammation contributes to the pathogenic mechanism of many neurodegenerative diseases. The deleterious attributes of neuroinflammation involve aberrant and uncontrolled activation of glia, which can result in damage to proximal brain parenchyma. Failure to distinguish self from non-self, as well as leukocyte reaction to aggregation and accumulation of proteins in the CNS, are the primary mechanisms by which neuroinflammation is initiated. While processes local to the CNS may instigate neurodegenerative disease, the existence or dysregulation of systemic homeostasis can also serve to improve or worsen CNS pathologies, respectively. One fundamental component of systemic homeostasis is the gut microbiota, which communicates with the CNS via microbial metabolite production, the peripheral nervous system, and regulation of tryptophan metabolism. Over the past 10–15 years, research focused on the microbiota–gut–brain axis has culminated in the discovery that dysbiosis, or an imbalance between commensal and pathogenic gut bacteria, can promote CNS pathologies. Conversely, a properly regulated and well-balanced microbiome supports CNS homeostasis and reduces the incidence and extent of pathogenic neuroinflammation. This review will discuss the role of the gut microbiota in exacerbating or alleviating neuroinflammation in neurodegenerative diseases, and potential microbiota-based therapeutic approaches to reduce pathology in diseased states.

Definitive diagnosis of Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) relies on postmortem finding of disease-associated alpha-synuclein (αSynD) as misfolded protein aggregates in the central nervous system (CNS). The recent development of the real-time quaking induced conversion (RT-QuIC) assay for ultrasensitive detection of αSynD aggregates has revitalized the diagnostic values of clinically accessible biospecimens, including cerebrospinal fluid (CSF) and peripheral tissues. However, the current αSyn RT-QuIC assay platforms vary widely and are thus challenging to implement and standardize the measurements of αSynD across a wide range of biospecimens and in different laboratories. We have streamlined αSyn RT-QuIC assay based on a second generation assay platform that was assembled entirely with commercial reagents. The streamlined RT-QuIC method consisted of a simplified protocol requiring minimal hands-on time, and allowing for a uniform analysis of αSynD in different types of biospecimens from PD and DLB. Ultrasensitive and specific RT-QuIC detection of αSynD aggregates was achieved in million-fold diluted brain homogenates and in nanoliters of CSF from PD and DLB cases but not from controls. Comparative analysis revealed higher seeding activity of αSynD in DLB than PD in both brain homogenates and CSF. Our assay was further validated with CSF samples of 214 neuropathologically confirmed cases from tissue repositories (88 PD, 58 DLB, and 68 controls), yielding a sensitivity of 98% and a specificity of 100%. Finally, a single RT-QuIC assay protocol was employed uniformly to detect seeding activity of αSynD in PD samples across different types of tissues including the brain, skin, salivary gland, and colon. We anticipate that our streamlined protocol will enable interested laboratories to easily and rapidly implement the αSyn RT-QuIC assay for various clinical specimens from PD and DLB. The utilization of commercial products for all assay components will improve the robustness and standardization of the RT-QuIC assay for diagnostic applications across different sites. Due to ultralow sample consumption, the ultrasensitive RT-QuIC assay will facilitate efficient use and sharing of scarce resources of biospecimens. Our streamlined RT-QuIC assay is suitable to track the distribution of αSynD in CNS and peripheral tissues of affected patients. The ongoing evaluation of RT-QuIC assay of αSynD as a potential biomarker for PD and DLB in clinically accessible biospecimens has broad implications for understanding disease pathogenesis, improving early and differential diagnosis, and monitoring therapeutic efficacies in clinical trials.

Importance Deposition of the pathological α-synuclein (αSynP) in the brain is the hallmark of synucleinopathies, including Parkinson disease (PD), Lewy body dementia (LBD), and multiple system atrophy (MSA). Whether real-time quaking-induced conversion (RT-QuIC) and protein misfolding cyclic amplification (PMCA) assays can sensitively detect skin biomarkers for PD and non-PD synucleinopathies remains unknown. Objective To develop sensitive and specific skin biomarkers for antemortem diagnosis of PD and other synucleinopathies. Design, Setting, and Participants This retrospective and prospective diagnostic study evaluated autopsy and biopsy skin samples from neuropathologically and clinically diagnosed patients with PD and controls without PD. Autopsy skin samples were obtained at 3 medical centers from August 2016 to September 2019, and biopsy samples were collected from 3 institutions from August 2018 to November 2019. Based on neuropathological and clinical diagnoses, 57 cadavers with synucleinopathies and 73 cadavers with nonsynucleinopathies as well as 20 living patients with PD and 21 living controls without PD were included. Specifically, cadavers and participants had PD, LBD, MSA, Alzheimer disease, progressive supranuclear palsy, or corticobasal degeneration or were nonneurodegenerative controls (NNCs). A total of 8 approached biopsy participants either refused to participate in or were excluded from this study due to uncertain clinical diagnosis. Data were analyzed from September 2019 to April 2020. Main Outcomes and Measures Skin αSynPseeding activity was analyzed by RT-QuIC and PMCA assays. Results A total of 160 autopsied skin specimens from 140 cadavers (85 male cadavers [60.7%]; mean [SD] age at death, 76.8 [10.1] years) and 41 antemortem skin biopsies (27 male participants [66%]; mean [SD] age at time of biopsy, 65.3 [9.2] years) were analyzed. RT-QuIC analysis of αSynPseeding activity in autopsy abdominal skin samples from 47 PD cadavers and 43 NNCs revealed 94% sensitivity (95% CI, 85-99) and 98% specificity (95% CI, 89-100). As groups, RT-QuIC also yielded 93% sensitivity (95% CI, 85-97) and 93% specificity (95% CI, 83-97) among 57 cadavers with synucleinopathies (PD, LBD, and MSA) and 73 cadavers without synucleinopathies (Alzheimer disease, progressive supranuclear palsy, corticobasal degeneration, and NNCs). PMCA showed 82% sensitivity (95% CI, 76-88) and 96% specificity (95% CI, 85-100) with autopsy abdominal skin samples from PD cadavers. From posterior cervical and leg skin biopsy tissues from patients with PD and controls without PD, the sensitivity and specificity were 95% (95% CI, 77-100) and 100% (95% CI, 84-100), respectively, for RT-QuIC and 80% (95% CI, 49-96) and 90% (95% CI, 60-100) for PMCA. Conclusions and Relevance This study provides proof-of-concept that skin αSynPseeding activity may serve as a novel biomarker for antemortem diagnoses of PD and other synucleinopathies.

Alteration in cellular prion protein (PrPC) localization on the cell surface through mediation of the glycosylphosphatidylinositol (GPI) anchor has been reported to dramatically affect the formation and infectivity of its pathological isoform (PrPSc). A patient with Gerstmann-Sträussler-Scheinker (GSS) syndrome was previously found to have a nonsense heterozygous PrP-Q227X mutation resulting in an anchorless PrP. However, the allelic origin of this anchorless PrPSc and cellular trafficking of PrPQ227X remain to be determined. Here, we show that PrPSc in the brain of this GSS patient is mainly composed of the mutant but not wild-type PrP (PrPWt), suggesting pathological PrPQ227X is incapable of recruiting PrPWt in vivo. This mutant anchorless protein, however, is able to recruit PrPWt from humanized transgenic mouse brain but not from autopsied human brain homogenates to produce a protease-resistant PrPSc-like form in vitro by protein misfolding cyclic amplification (PMCA). To further investigate the characteristics of this mutation, constructs expressing human PrPQ227X or PrPWt were transfected into neuroblastoma cells (M17). Fractionation of the M17 cells demonstrated that most PrPWt is recovered in the cell lysate fraction, while most of the mutant PrPQ227X is recovered in the medium fraction, consistent with the results obtained by immunofluorescence microscopy. Two-dimensional gel-electrophoresis and Western blotting showed that cellular PrPQ227X spots clustered at molecular weights of 22–25 kDa with an isoelectric point (pI) of 3.5–5.5, whereas protein spots from the medium are at 18–26 kDa with a pI of 7–10. Our findings suggest that the role of GPI anchor in prion propagation between the anchorless mutant PrP and wild-type PrP relies on the cellular distribution of the protein.

ObjectiveTo determine whether (1) immunofluorescence is a reproducible technique in detecting misfolded α-synuclein in skin nerves and subsequently whether (2) immunofluorescence and real-time quaking-induced conversion (RT-QuIC) (both in skin and CSF) show a comparable in vivo diagnostic accuracy in distinguishing synucleinopathies from non-synucleinopathies in a large cohort of patients.MethodsWe prospectively recruited 90 patients fulfilling clinical and instrumental diagnostic criteria for all synucleinopathies variants and non-synucleinopathies (mainly including Alzheimer disease, tauopathies, and vascular parkinsonism or dementia). Twenty-four patients with mainly peripheral neuropathies were used as controls. Patients underwent skin biopsy for immunofluorescence and RT-QuIC; CSF was examined in patients who underwent lumbar puncture for diagnostic purposes. Immunofluorescence and RT-QuIC analysis were made blinded to the clinical diagnosis.ResultsImmunofluorescence showed reproducible results between 2 pairs of neighboring skin samples. Both immunofluorescence and RT-QuIC showed high sensitivity and specificity in discriminating synucleinopathies from non-synucleinopathies and controls but immunofluorescence presented higher diagnostic accuracy. Immunofluorescence presented a good level of agreement with RT-QuIC in both skin and CSF in synucleinopathies.ConclusionsBoth immunofluorescence and RT-QuIC showed high diagnostic accuracy, although immunofluorescence displayed the better value as well as optimal reproducibility; they presented a good level of agreement in synucleinopathies, supporting the use of less invasive tests such as skin immunofluorescence or RT-QuIC instead of CSF RT-QuIC as a diagnostic tool for synucleinopathies.Classification of EvidenceThis study provides Class III evidence that immunofluorescence or RT-QuIC accurately distinguish synucleinopathies from non-synucleinopathies.

Tau aggregates are present in a large number of neurodegenerative diseases known as “tauopathies”, including Alzheimer’s disease (AD). As there are six human tau isoforms in brain tissues and both 3R and 4R isoforms have been observed in the neuronal inclusions, we tested whether tau isoforms behave differently in aggregation. We discovered that all six tau isoforms are capable of forming PHF-tau like filaments and the 3R tau isoforms aggregate significantly faster than their 4R counterparts. We further mapped key segments of tau isoforms that contribute to their aggregation kinetics, where it was determined that microtubule binding domains R2 and R3 were the major contributors to tau aggregation. To evaluate the feasibility of using the six recombinant tau isoforms as substrates to amplify misfolded tau, we demonstrated that full-length human tau isoforms can seed and detect misfolded tau from the post-mortem AD brain tissues with high specificity by an ultrasensitive technology termed real-time quaking-induced conversion (RT-QuIC). Mass spectrometric analysis of PHF-tau samples extracted from AD brains identified peptides corresponding to all major forms of human brain tau isoforms along with a consensus hyperphosphorylated peptide near the C-terminus. Together, our findings not only reveal new aggregation kinetic properties of human tau isoforms, support the development of methods to quantitatively measure misfolded human tau isoforms in AD brains, but also uncover the capability of full-length human tau isoforms as substrates for “prion-like” tau seeding by RT-QuIC assays that may be used for new biomarker development for AD and other tauopathy diagnosis.

Prion or PrPSc is the proteinaceous infectious agent causing prion diseases in various mammalian species. Despite decades of research, the structural basis for PrPSc formation and prion infectivity remains elusive. To understand the role of the hydrophobic region in forming infectious prion at the molecular level, we report X-ray crystal structures of mouse (Mo) prion protein (PrP) (residues 89–230) in complex with a nanobody (Nb484). Using the recombinant prion propagation system, we show that the binding of Nb484 to the hydrophobic region of MoPrP efficiently inhibits the propagation of proteinase K resistant PrPSc and prion infectivity. In addition, when added to cultured mouse brain slices in high concentrations, Nb484 exhibits no neurotoxicity, which is drastically different from other neurotoxic anti-PrP antibodies, suggesting that the Nb484 can be a potential therapeutic agent against prion disease. In summary, our data provides the first structure-function evidence supporting a crucial role of the hydrophobic region of PrP in forming an infectious prion., Author summary Prion disease is caused by the misfolding of normal prion protein (PrPC) to its pathogenic isoform, termed PrPSc. The underlying mechanism for such fatal misfolding is still unknown, which greatly impedes the development of efficacious therapeutic strategies against these currently incurable diseases. Previously, we reported the crystal structure of human PrP in complex with a nanobody (Nb484), which could inhibit mouse prion propagation in a cell culture model. To understand the molecular mechanism for the inhibitory action of Nb484, in this study, firstly we determine the crystal structures of mouse PrP-Nb484 complexes and show that Nb484 binds to and stabilizes the hydrophobic region of PrP. Then in our newly developed recombinant prion propagation system, we show that Nb484 inhibits prion propagation through the competitive inhibition mechanism. We further demonstrate that, contrary to certain neurotoxic anti-PrP antibodies, the inhibitory Nb484 exhibits no neurotoxicity on cultured organotypic brain slices, indicating the great therapeutic potential of Nb484. In summary, our current study provides the first structure-function evidence supporting a critical role of the hydrophobic region in forming the infectious prion and highlights the therapeutic potential of Nb484.

SUMMARY Since the term protein was first coined in 1838 and protein was discovered to be the essential component of fibrin and albumin, all cellular proteins were presumed to play beneficial roles in plants and mammals. However, in 1967, Griffith proposed that proteins could be infectious pathogens and postulated their involvement in scrapie, a universally fatal transmissible spongiform encephalopathy in goats and sheep. Nevertheless, this novel hypothesis had not been evidenced until 1982, when Prusiner and coworkers purified infectious particles from scrapie-infected hamster brains and demonstrated that they consisted of a specific protein that he called a “prion.” Unprecedentedly, the infectious prion pathogen is actually derived from its endogenous cellular form in the central nervous system. Unlike other infectious agents, such as bacteria, viruses, and fungi, prions do not contain genetic materials such as DNA or RNA. The unique traits and genetic information of prions are believed to be encoded within the conformational structure and posttranslational modifications of the proteins. Remarkably, prion-like behavior has been recently observed in other cellular proteins—not only in pathogenic roles but also serving physiological functions. The significance of these fascinating developments in prion biology is far beyond the scope of a single cellular protein and its related disease.

In Alzheimer disease (AD), amyloid-β (Aβ) oligomer is suggested to play a critical role in imitating neurodegeneration, although its pathogenic mechanism remains to be determined. Recently, the cellular prion protein (PrP(C)) has been reported to be an essential co-factor in mediating the neurotoxic effect of Aβ oligomer. However, these previous studies focused on the synaptic plasticity in either the presence or the absence of PrP(C) and no study to date has reported whether PrP(C) is required for the neuronal cell death, the most critical element of neurodegeneration in AD. Here, we show that Prnp(-/-) mice are resistant to the neurotoxic effect of Aβ oligomer in vivo and in vitro. Furthermore, application of an anti-PrP(C) antibody or PrP(C) peptide prevents Aβ oligomer-induced neurotoxicity. These findings are the first to demonstrate that PrP(C) is required for Aβ oligomer-induced neuronal cell death, the pathology essential to cognitive loss.

The soluble cellular prion protein (PrP(C)) is best known for its association with prion disease (PrD) through its conversion to a pathogenic insoluble isoform (PrP(Sc)). However, its deleterious effects independent of PrP(Sc) have recently been observed not only in PrD but also in Alzheimer disease (AD), two diseases which mainly affect cognition. At the same time, PrP(C) itself seems to have broad physiologic functions including involvement in cognitive processes. The PrP(C) that is believed to be soluble and monomeric has so far been the only PrP conformer observed in the uninfected brain. In 2006, we identified an insoluble PrP(C) conformer (termed iPrP(C) ) in uninfected human and animal brains. Remarkably, the PrP(Sc) -like iPrPC shares the immunoreactivity behavior and fragmentation with a newly-identified PrP(Sc) species in a novel human PrD termed variably protease-sensitive prionopathy. Moreover, iPrP(C) has been observed as the major PrP species that interacts with amyloid β (Aβ) in AD. This article highlights evidence of PrP involvement in two putatively beneficial and deleterious PrP-implicated pathways in cognition, and hypothesizes first, that beneficial and deleterious effects of PrP(C) are attributable to the chameleon-like conformation of the protein and second, that the iPrP(C) conformer is associated with PrD and AD.

The Prion protein (PrP) is a membrane-tethered glycoprotein that plays a central role in a unique class of neurodegenerative diseases that affect humans and other mammals. Prion diseases have genetic and sporadic origins, but their infectious nature sets them apart from other neurodegenerative disorders. According to the “protein-only” hypothesis, misfolded PrP conformers (prions) are responsible for both spongiform degeneration of the brain and disease transmissibility. Thus, understanding PrP conformational dynamics is key to developing effective therapies. Classic studies showing the different susceptibility to prion disease in mammals have recently found support in structural and transgenic studies with PrP from susceptible (mouse, hamster) and resistant (rabbit, horse, dog) animals. These studies identify key residues in PrP that determine both PrP structure and its propensity to acquire a β-structure conformation proposed to be neurotoxic.

The prion protein (PrP) is best known for its association with prion diseases. However, a controversial new role for PrP in Alzheimer disease (AD) has recently emerged. In vitro studies and mouse models of AD suggest that PrP may be involved in AD pathogenesis through a highly specific interaction with amyloid-β (Aβ42) oligomers. Immobilized recombinant human PrP (huPrP) also exhibited high affinity and specificity for Aβ42 oligomers. Here we report the novel finding that aggregated forms of huPrP and Aβ42 are co-purified from AD brain extracts. Moreover, an anti-PrP antibody and an agent that specifically binds to insoluble PrP (iPrP) co-precipitate insoluble Aβ from human AD brain. Finally, using peptide membrane arrays of 99 13-mer peptides that span the entire sequence of mature huPrP, two distinct types of Aβ binding sites on huPrP are identified in vitro. One specifically binds to Aβ42 and the other binds to both Aβ42 and Aβ40. Notably, Aβ42-specific binding sites are localized predominantly in the octapeptide repeat region, whereas sites that bind both Aβ40 and Aβ42 are mainly in the extreme N-terminal or C-terminal domains of PrP. Our study suggests that iPrP is the major PrP species that interacts with insoluble Aβ42 in vivo. Although this work indicated the interaction of Aβ42 with huPrP in the AD brain, the pathophysiological relevance of the iPrP/Aβ42 interaction remains to be established.

Prion diseases are neurodegenerative disorders caused by misfolding of the normal prion protein (PrP) into a pathogenic “scrapie” conformation. To better understand the cellular and molecular mechanisms that govern the conformational changes (conversion) of PrP, we compared the dynamics of PrP from mammals susceptible (hamster and mouse) and resistant (rabbit) to prion diseases in transgenic flies. We recently showed that hamster PrP induces spongiform degeneration and accumulates into highly aggregated, scrapie-like conformers in transgenic flies. We show now that rabbit PrP does not induce spongiform degeneration and does not convert into scrapie-like conformers. Surprisingly, mouse PrP induces weak neurodegeneration and accumulates small amounts of scrapie-like conformers. Thus, the expression of three highly conserved mammalian prion proteins in transgenic flies uncovered prominent differences in their conformational dynamics. How these properties are encoded in the amino acid sequence remains to be elucidated.

The vertical transmission of a prion disease from infected mothers to their offspring is believed to be one of the routes for the natural spread of animal prion diseases. Supporting this notion is the observation that prion infectivity occurs in the placenta of infected ewes. Furthermore, the prion protein (PrP), both in its cellular form (PrP(C)) and its pathological isoform (PrP(Sc)), has been observed at the fetal-maternal interface of scrapie-infected sheep. However, whether these features of prion infectivity also hold true for human prion diseases is currently unknown. To begin to address such an important question, we examined PrP in the uterus as well as gestational tissues, including the placenta and amniotic fluid, in a pregnant woman with sporadic Creutzfeldt-Jakob disease (CJD). Although the proteinase K (PK)-resistant prion protein, PrP27-30, was present in the brain tissues of the mother, the PrP detected in the uterus, placenta, and amniotic fluid was sensitive to PK digestion. Unlike PrP(C) in the brain and adjacent cerebrospinal fluid, the predominant PrP species in the reproductive and gestational tissues were N-terminally truncated, similar to urine PrP. Our study did not detect abnormal PrP in the reproductive and gestational tissues in this case of CJD. Nevertheless, examination by a highly sensitive bioassay is ongoing to ascertain possible prion infectivity from CJD in the amniotic fluid.

Bovine spongiform encephalopathy (BSE), the prion disease in cattle, was widely believed to be caused by only one strain, BSE-C. BSE-C causes the fatal prion disease named new variant Creutzfeldt-Jacob disease in humans. Two atypical BSE strains, bovine amyloidotic spongiform encephalopathy (BASE, also named BSE-L) and BSE-H, have been discovered in several countries since 2004; their transmissibility and phenotypes in humans are unknown. We investigated the infectivity and human phenotype of BASE strains by inoculating transgenic (Tg) mice expressing the human prion protein with brain homogenates from two BASE strain-infected cattle. Sixty percent of the inoculated Tg mice became infected after 20 to 22 months of incubation, a transmission rate higher than those reported for BSE-C. A quarter of BASE strain-infected Tg mice, but none of the Tg mice infected with prions causing a sporadic human prion disease, showed the presence of pathogenic prion protein isoforms in the spleen, indicating that the BASE prion is intrinsically lymphotropic. The pathological prion protein isoforms in BASE strain-infected humanized Tg mouse brains are different from those from the original cattle BASE or sporadic human prion disease. Minimal brain spongiosis and long incubation times are observed for the BASE strain-infected Tg mice. These results suggest that in humans, the BASE strain is a more virulent BSE strain and likely lymphotropic.

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., Author Summary Prion diseases are neurodegenerative disorders that propagate as multiple strains in a variety of mammalian species. The detection of all such prion types by a single ultrasensitive assay, such as the Real Time Quaking-induced Conversion (RT-QuIC) assay, would facilitate prion disease diagnosis, surveillance, and research. Here we show detection of minute amounts of 28 different prion types from humans, cattle, sheep, cervids and rodents, some of which were previously undetectable, using a single recombinant bank vole prion protein substrate. We also demonstrate the generation of prion type-dependent RT-QuIC conversion products which may help with prion strain discrimination and the characterization of distinct classes of prion templates. Finally, we describe a practical strategy for prion strain discrimination, e.g. classical and atypical L-type bovine spongiform encephalopathy; classical and atypical Nor98 sheep scrapie; and human sporadic and variant Creutzfeldt-Jakob disease. Thus, our study provides a basis for wide-ranging prion detection and strain discrimination.

Chronic wasting disease (CWD), a transmissible prion disease that affects elk and deer, poses new challenges to animal and human health. Although the transmission of CWD to humans has not been proven, it remains a possibility. If this were to occur, it is important to know whether the "acquired" human prion disease would show a phenotype including the scrapie prion protein (PrP(Sc)) features that differ from those associated with human sporadic prion disease. In this study, we have compared the pathological profiles and PrP(Sc) characteristics in brains of CWD-affected elk and deer with those in subjects with sporadic Creutzfeldt-Jakob disease (CJD), as well as CJD-affected subjects who might have been exposed to CWD, using histopathology, immunohistochemistry, immunoblotting, conformation stability assay, and N-terminal protein sequencing. Spongiform changes and intense PrP(Sc) staining were present in several brain regions of CWD-affected animals. Immunoblotting revealed three proteinase K (PK)-resistant bands in CWD, representing different glycoforms of PrP(Sc). The unglycosylated PK-resistant PrP(Sc) of CWD migrated at 21 kDa with an electrophoretic mobility similar to that of type 1 human PrP(Sc) present in sporadic CJD affecting subjects homozygous for methionine at codon 129 (sCJDMM1). N-terminal sequencing showed that the PK cleavage site of PrP(Sc) in CWD occurred at residues 82 and 78, similar to that of PrP(Sc) in sCJDMM1. Conformation stability assay also showed no significant difference between elk CWD PrP(Sc) and the PrP(Sc) species associated with sCJDMM1. However, there was a major difference in glycoform ratio of PrP(Sc) between CWD and sCJDMM1 affecting both subjects potentially exposed to CWD and non-exposed subjects. Moreover, PrP(Sc) of CWD exhibited a distinct constellation of glycoforms distinguishable from that of sCJDMM1 in two-dimensional immunoblots. These findings underline the importance of detailed PrP(Sc) characterization in trying to detect novel forms of acquired prion disease.

Foodborne transmission of bovine spongiform encephalopathy (BSE) to humans as variant Creutzfeldt-Jakob disease (CJD) has affected over 100 individuals, and probably millions of others have been exposed to BSE-contaminated food substances. Despite these obvious public health concerns, surprisingly little is known about the mechanism by which PrP-scrapie (PrPSc), the most reliable surrogate marker of infection in BSE-contaminated food, crosses the human intestinal epithelial cell barrier. Here we show that digestive enzyme (DE) treatment of sporadic CJD brain homogenate generates a C-terminal fragment similar to the proteinase K-resistant PrPSccore of 27-30 kDa implicated in prion disease transmission and pathogenesis. Notably, DE treatment results in a PrPSc-protein complex that is avidly transcytosed in vesicular structures across anin vitromodel of the human intestinal epithelial cell barrier, regardless of the amount of endogenous PrPCexpression. Unexpectedly, PrPScis cotransported with ferritin, a prominent component of the DE-treated PrPSc-protein complex. The transport of PrPSc-ferritin is sensitive to low temperature, brefeldin-A, and nocodazole treatment and is inhibited by excess free ferritin, implicating a receptor- or transporter-mediated pathway. Because ferritin shares considerable homology across species, these data suggest that PrPSc-associated proteins, in particular ferritin, may facilitate PrPScuptake in the intestine from distant species, leading to a carrier state in humans.

Although prion diseases are rare, affecting about one person per million per year worldwide, they have recently received remarkable attention because of the epidemic of the newly recognized bovine spongiform encephalopathy (BSE) and the causative association of BSE with human variant Creutzfeldt–Jakob disease (vCJD). At present, prion diseases are incurable. Here, we review current strategies in the modeling of human prion propagation and human prion diseases in vitro and in vivo. Modeling of prion diseases has contributed and will continue to contribute to the understanding of the pathogenesis as well as of the therapeutics of these diseases, but crucial experiments remain elusive.

In transmission studies with Alzheimer's disease (AD) animal models, the formation of Aβ plaques is proposed to be initiated by seeding the inoculated amyloid β (Aβ) peptides in the brain. Like the misfolded scrapie prion protein (PrPSc) in prion diseases, Aβ in AD shows a certain degree of resistance to protease digestion while the biochemical basis for protease resistance of Aβ remains poorly understood. Using in vitro assays, histoblotting, and electron microscopy, we characterize the biochemical and morphological features of synthetic Aβ peptides and Aβ isolated from AD brain tissues. Consistent with previous observations, monomeric and oligomeric Aβ species extracted from AD brains are insoluble in detergent buffers and resistant to digestions with proteinase K (PK). Histoblotting of AD brain tissue sections exhibits an increased Aβ immunoreactivity after digestion with PK. In contrast, synthetic Aβ40 and Aβ42 are soluble in detergent buffers and fully digested by PK. Electron microscopy of Aβ40 and Aβ42 synthetic peptides shows that both species of Aβ form mature fibrils. Those generated from Aβ40 are longer but less numerous than those made of Aβ42. When spiked into human brain homogenates, both Aβ40 and Aβ42 acquire insolubility in detergent and resistance to PK. Our study favors the hypothesis that the human brain may contain cofactor(s) that confers the synthetic Aβ peptides PrPSc-like physicochemical properties.

Amyotrophic lateral sclerosis (ALS) and prionopathies, such as Creutzfeldt-Jakob disease (CJD), are devastating neurodegenerative diseases with a relatively early age of onset and a rapid disease course. Prior to 1983, slowly progressing dementia with motor neuron signs was often considered part of the CJD spectrum, termed the “amyotrophic form” of CJD [4]. The landmark study by Salazar and colleagues demonstrated that “amyotrophic CJD” was not a transmissible form of CJD, but was more akin to classic ALS [7], with the spongiosis noted in such cases now recognized as a nonspecific feature of frontotemporal degenerations [1]. While 27 cases with amyotrophy and prion disease have been reported, only 4 have had immunohistochemical proof of prion disease, while 7 had anterior horn cell pathology without mention of evidence of upper motor neuron pathology [8]. Moreover, none had immunohistochemistry with the molecular pathologic marker of ALS, TAR DNA-binding protein of 43 kDa (TDP-43), which labels neuronal and glial inclusions in ALS [5]. In 2008, a novel human prion disease was discovered by Gambetti and colleagues characterized by variable sensitivity of prion protein to protease digestion [2]. Named “variably protease-sensitive prionopathy” (VPSPr), the clinical presentation, age of onset, and disease duration as well as the histopathology of this disorder differed between PRNP genetic variants (129V/V, 129M/V, 129M/M) and from CJD [3, 9]. We report a 74-year-old woman followed longitudinally as part of prospective studies of dementia with Lewy bodies at Mayo Clinic who had a one-year history of gradually progressive dementia, cognitive fluctuations, excessive daytime sleepiness, visual hallucinations, and probable rapid eye movement sleep behavior disorder. General motor exam revealed normal strength and muscle tone. Magnetic resonance imaging indicated mild generalized cortical atrophy. By age 75, she developed agitation, behavioral disturbances, incontinence, and struggles with activities-of-daily-living. A year later, she had dysarthria, and difficulty ambulating, with stooped posture, shuffling, and unsteady gait. By age 77, she had dysphagia and was wheelchair bound and unresponsive. She died at the age of 78. Macroscopic examination of the fixed left hemibrain revealed diffuse cortical atrophy, enlargement of lateral ventricles, and thinning of the cortical ribbon. The hippocampus and amygdala were atrophic, but the basal ganglia and thalamus were unremarkable. There was visible pigmentation in the substantia nigra, but pallor of the locus ceruleus. Microscopic examination showed spongiform change and gliosis throughout the neocortex with ballooned neurons (Fig. 1a). Spongiform change was also noted in the hippocampus, basal ganglia (Fig. 1b), and minimally in the thalamus. Purkinje cell loss and Bergmann gliosis was seen in the cerebellum, and was associated with dense-cored plaques with thioflavin S fluorescent microscopy. In addition, skein-like inclusions, hyaline inclusions, and Bunina bodies were observed (Fig. 1c). Prion protein (PrP) immunohistochemistry (3F4 antibody; 1:50,000) revealed many plaque-like lesions and coarse granular immunoreactivity in the neocortex (Fig. 1d), dentate fascia, amygdala, and basal ganglia, with small dense plaques in the cerebellum (Fig. 1e). PrP pathology and spongiform change was uniform in severity across the neocortex. Immunohistochemistry for TDP-43 (MC2085 antibody; 1:2,500), a neuropathologic hallmark of ALS, revealed neuronal cytoplasmic inclusions in residual Betz cells, as well as skein-like and Lewy-like hyaline inclusions in the hypoglossal nucleus (Fig. 1g). No additional white matter TDP-43 pathology or extramotor TDP-43 pathology was detected. Marked neuronal cell loss of anterior horn cells was observed in the cervical spinal cord in addition to the aforementioned TDP-43 immunoreactive lesions (Fig. 1h). There was mild demyelination and microglial activation in the pyramidal tract with Luxol fast blue for myelin and IBA-1 immunohistochemistry (1:3000) for microglia (data not shown). Together, these findings were consistent with lower motor neuron predominant ALS. No Lewy bodies or Lewy neurites were detected by α-synuclein immunohistochemistry. There were no neurofibrillary tangles or amyloid angiopathy on thioflavin-S fluorescent microscopy. Many diffuse, non-neuritic senile plaques were seen throughout the neocortex on thioflavin-S microscopy as well as in the hippocampus with Aβ immunohistochemistry (33.1.1 antibody; 1:1,000). C9RANT immunohistochemistry (Rb5823 antibody; 1:5,000), a specific marker of the C9ORF72 hexanucleotide repeat expansion, was negative. Ultrastructural analysis confirmed spongiform change and PrP-positive plaques in the hippocampus (Fig. 1f) as well as skein-like inclusions in motor neurons composed of TDP-43-positive ~10-nm diameter granule-coated filaments (Fig. 1i). Fig. 1 Neuropathologic profile of VPSPr-129M/V and ALS case. H&E shows balloon neurons in the superior temporal gyrus (a) and spongiform change in the caudate (b). H&E also revealed skein-like inclusions (black arrow) and Bunina bodies (green ... Brain homogenates were prepared in LB100 lysis buffer, treated with 2 U/ml of proteinase K for 1 hour at 37°C, and run on 15% Tris/glycine SDS-polyacrylamide gels [6]. Western blot examination (with 3F4 and 1E4 antibodies) demonstrated a PrP five band ladder profile consistent with VPSPr compared to the three band profile of sporadic CJD (Fig. 2) [2, 9]. Sequencing of PRNP failed to detect pathogenic mutations and demonstrated methionine/valine heterozygosity at codon 129 [9]. These results led to the final neuropathologic diagnosis of coincident VPSPr and ALS. Fig. 2 Detection and characterization of PrP. Immunoblot of total homogenates, treated with proteinase K, obtained from the frontal cortex of the present case and of the most common sporadic CJD subtypes, sCJD-MM1 and sCJD-VV2. Membranes were probed with the ... The comorbidity of prion disease and ALS is exceedingly rare, but may be more likely with VPSPr given the later disease-onset and longer disease duration compared to typical CJD. Initial clinical features consistent with dementia with Lewy bodies in this patient were likely related to VPSPr since there was no extramotor TDP-43 pathology. Later gait disturbances and dysphagia were probably due to ALS involvement of motor tracts and motor neurons in spinal cord and brainstem. In summary, this is the first report of a patient with both VPSPr and ALS, confirmed with PrP biochemistry, as well as light and electron microscopic TDP-43 immunohistochemistry.

Variably protease-sensitive prionopathy (VPSPr) is a recently described novel prion disease biochemically characterized by abnormal prion protein (PrP) which has various degrees of sensitivity to proteases and generates a distinct profile on Western blots 1. VPSPr is the second most common sporadic prion protein disease after CJD 2. All reported cases to date have exhibited cognitive, behavioral, and psychiatric deficits with an age at onset range of 48-81 years and disease duration range of 7-72 months 1-4. Initial clinical diagnoses included normal pressure hydrocephalus (NPH), dementia with Lewy bodies (DLB), or frontotemporal dementia (FTD) 1,5, but Creutzfeldt-Jakob disease (CJD) often was considered clinically after rapid decline became apparent 1. All VPSPr cases evaluated have lacked mutations in the prion protein gene (PRNP) 1,2,5. Initial reports indicated all cases were homozygous for valine at codon 129 (VV) in PRNP 1. However, more recent reports have found VPSPr in all three codon 129 genotypes 2,5-7. Here we report a case of VPSPr in which there were no known clinical manifestations of prion disease during life and yet pathognomonic findings were revealed at autopsy.

Prion diseases are characterized by the presence of the abnormal prion protein PrP Sc , which is believed to be generated by the conversion of the α-helical structure that predominates in the normal PrP isoform into a β-sheet structure resistant to proteinase K (PK). In human prion diseases, two major types of PrP Sc , type 1 and 2, can be distinguished based on the difference in electrophoretic migration of the PK-resistant core fragment. In this study, protein sequencing was used to identify the PK cleavage sites of PrP Sc in 36 cases of prion diseases. We demonstrated two primary cleavage sites at residue 82 and residue 97 for type 1 and type 2 PrP Sc , respectively, and numerous secondary cleavages distributed along the region spanning residues 74–102. Accordingly, we identify three regions in PrP Sc : one N-terminal (residues 23–73) that is invariably PK-sensitive, one C-terminal (residues 103–231) that is invariably PK-resistant, and a third variable region (residues 74–102) where the site of the PK cleavage, likely reflecting the extent of the β-sheet structure, varies mostly as a function of the PrP genotype at codon 129.

The history of the transmissible spongiform encephalopathies or prion diseases is teeming with dramatic findings. Originally described as neurodegenerative conditions, these diseases were shown to be experimentally transmissible to animals; subsequently, human-to-human transmissibility through the tribal custom of cannibalism or through medical procedures was reported.1-4 Then in 1996, with the discovery of variant Creutzfeldt-Jakob disease (vCJD), prion diseases assumed renewed and dangerous relevance.5 The appearance of vCJD followed a prion disease epidemic named bovine spongiform encephalopathy (BSE) in cattle, and it was clear that vCJD was caused by the consumption of prion-contaminated meat from BSE-affected cattle.6-8 Prion diseases thereby proved themselves capable of jumping the species barrier and of being naturally transmissible from animals to humans. Originally discovered in the United Kingdom, vCJD has subsequently been reported in other countries but especially in France. In this issue of the Annals of Neurology, Brandel and colleagues9 present a detailed comparison between the clinical, histopathological, and prion-protein (PrP) characteristics of the vCJD identified in France and in the United Kingdom. Human prion diseases include three major forms. One form encompasses prion diseases that are acquired through infection including vCJD, kuru of New Guinea, and iatrogenic CJD. The other two forms comprise sporadic CJD, the most common form of human prion diseases, and the genetically determined or familial CJD. However, regardless of their form, all prion diseases share a fundamental and identical pathogenetic mechanism: the conversion of the normal or cellular PrP into an isoform, identified as scrapie PrP (PrPSc), which is insoluble in detergent, often resistant to protease, and pathogenic.10 Another typical characteristic of prion diseases is that they are recognizable and can be compared with each other based on two major molecular features: the physicochemical, especially electrophoretic, characteristics of PrPSc, which largely define the so-called prion strain, and the genotype of the patient based on the methionine/valine polymorphism at codon 129 of the PrP gene.6,11,12 These two features are crucial in distinguishing between the types of prion diseases and their respective origins.6,8,12 For instance, through these features, it has been shown that the electrophoretic characteristics of PrPSc associated with vCJD observed in the British patients are similar to those of PrPSc associated with the British BSE, from which it can be inferred that the former originated in the latter.6,8 This conclusion has been supported by the transmissibility experiments, which provided overwhelming evidence that vCJD is an acquired, not a spontaneous, human disease.6-8 The comparative analyses of the British vCJD with other prion diseases have also been facilitated by the clinical and histopathological characteristics of vCJD, which are homogeneous yet highly differentiated from those of other known human prion diseases.13 Brandel and colleagues’9 study uses all these points of reference to show that the British and French vCJD cases manifest similar clinical, histopathological, and molecular features, from which it can be confidently concluded that the two vCJD sets of patients share the same PrPSc isoform or strain.9 The clinical disease duration is also similar. The only major divergence emerging from this study appears in the age at onset, which is more than 8 years older in the French than in the British cases (36.4 vs 28.3 years). This difference may stem from an average exposure to lower amounts of BSE PrPSc in the French patient population (because of different dietary habits), as the authors suggest,9 or from greater dilution of PrPSc in the meat consumed by the French patient population. Reduced exposure would explain the longer incubation time.14 However, other possibilities, such as a slight change in the French prion strain, cannot be ruled out. The finding that the duration of the symptomatic disease is similar agrees with the experimental transmission data, which likewise demonstrated that the clinical disease duration is not related to incubation time.15 Brandel and colleagues’9 findings bring back to the fore at least two issues. First, it raises the question of the origin of the infectious prion in the French vCJD: whether it originates from British BSE or from indigenous French BSE; second, whether vCJD worldwide is caused by one prion strain or whether all vCJD cases carry the same British prion strain. It is critical that these two issues be clarified so that the spread of the BSE epidemics through different continents can be understood and arrested. Concerning the origin of the prion strain associated with the French cases of vCJD, the broad similarity of the phenotypic and PrPSc characteristics between French and British vCJD cases is consistent with the notion that the French vCJD prion strain derives from the consumption of prion-contaminated beef imported from the United Kingdom.9,16 This notion is also supported by the absence of correlation between the prevalence of vCJD and the incidence of BSE observed in several countries such as Switzerland and Germany, where there is a greater incidence of BSE but no cases of vCJD.17 Alternatively, French vCJD might have resulted from indigenous BSE, whereas the PrPSc had strain characteristics similar to the British BSE possibly because the prion-infected high-protein diet, the so-called meat and bone meal that triggered BSE in the two countries, had the same characteristics. Either origin is consistent with the 5-year delay in the peak incidence of French vCJD compared with that of British vCJD, as well as the 9-year delay between the peak incidence of the British and French BSE (Table 1).18 A ban on British beef import was introduced in France and the whole European Union in March 1996.19 The origin of the French vCJD from indigenous French BSE would become highly compelling if cases of vCJD were discovered among people born in France after the ban went into effect. Table 1 Number of Reported Variant Creutzfeldt-Jakob Disease and Bovine Spongiform Encephalopathy Cases in France and the United Kingdom To investigate further the origin of French vCJD, it would be necessary for investigators to conduct a detailed comparative study of the strain characteristics of PrPSc associated with British and French BSE and vCJD using direct analyses and transmissibility studies in transgenic mice expressing bovine PrP, and monitoring incubation time, attack rate, and phenotypic characteristics of the transmitted diseases. Similar studies extended to the BSE and vCJD observed in other countries around the world where these two prion diseases have been reported (Table 2) may answer the question whether vCJD identified in these countries shares the same prion strain with British vCJD as the French cases likely do.9,20 The high homogeneity of the phenotypic and PrPSc characteristics indeed suggests that all cases of vCJD around the world have been caused by a prion strain indistinguishable from that of the British cases. Table 2 Number of Reported Cases of Bovine Spongiform Encephalopathy and Variant Creutzfeldt-Jakob Disease in the Countries with the two prion diseases Although the incidence of vCJD and BSE has been decreasing after the epidemic, Brandel and colleagues study,9 together with other studies mentioned earlier, as well as the continuing reporting of new cases, all clearly indicate that the battle against vCJD and BSE is far from over.

The clinicopathological phenotype of the Gerstmann–Sträussler–Scheinker disease (GSS) variant linked to the codon 102 mutation in the prion protein (PrP) gene (GSS P102L) shows a high heterogeneity. This variability also is observed in subjects with the same prion protein gene PRNP haplotype and is independent from the duration of the disease. Immunoblot analysis of brain homogenates from GSS P102L patients showed two major protease-resistant PrP fragments (PrP-res) with molecular masses of ≈21 and 8 kDa, respectively. The 21-kDa fragment, similar to the PrP-res type 1 described in Creutzfeldt–Jakob disease, was found in five of the seven subjects and correlated with the presence of spongiform degeneration and “synaptic” pattern of PrP deposition whereas the 8-kDa fragment, similar to those described in other variants of GSS, was found in all subjects in brain regions showing PrP-positive multicentric amyloid deposits. These data further indicate that the neuropathology of prion diseases largely depends on the type of PrP-res fragment that forms in vivo . Because the formation of PrP-res fragments of 7–8 kDa with ragged N and C termini is not a feature of Creutzfeldt–Jakob disease or fatal familial insomnia but appears to be shared by most GSS subtypes, it may represent a molecular marker for this disorder.

Insertion of 144-base pair (bp) containing six extra octapeptide repeats between residues 51 and 91 of prion protein (PrP) gene is associated with inherited prion diseases. Most cases linked to this insertion examined by Western blotting showed detectable proteinase K-resistant PrPSc (rPrPSc) resembling PrPSc type 1 and type 2 in sporadic Creutzfeldt-Jakob disease (sCJD), or PrP7-8 in Gerstmann-Sträussler-Scheinker disease. However, cases lacking detectable rPrPSc also have been reported. Which PrP conformer is associated with neuropathological changes in the cases without detectable rPrPSc remains to be determined. Here we report that while all six but one subjects with the 144-bp insertion mutations examined display the pathognomonic PrP patches in the cerebellum, one of them exhibits no detectable typical rPrPSc even in PrPSc-enriched preparations. Instead, a large amount of abnormal PrP is captured from this case by gene 5 protein and sodium phosphotungstate, reagents that have been proved to specifically capture abnormal PrP. All captured abnormal PrP from the cerebellum and other brain regions is virtually sensitive to PK-digestion (termed sPrPSc). The presence of the predominant sPrPSc but absence of rPrPSc in this 144-bp insertion-linked inherited CJD case suggests that mutant sPrPSc is the main component of the PrP deposit patches and sPrPSc is sufficient to cause neurotoxicity and prion disease.

The detergent-soluble cellular prion protein (PrPC) and its detergent-insoluble infectious isoform (PrPSc) are two major conformers of the prion protein. Soluble PrPC has been the only isoform detected in the normal mammalian brain. In 2006, however, we identified an insoluble PrPC conformer (termed iPrPC) in uninfected human and animal brains. This article highlights the physiochemical properties of iPrPC, a conformer distinct from PrPC or PrPSc, and discusses its formation and probable pathophysiology.

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrP(C) into its pathogenic isoform PrP(Sc 1). PrP(C) is detergent-soluble and sensitive to proteinase K (PK)-digestion, whereas PrP(Sc) forms detergent-insoluble aggregates and is partially resistant to PK(2-6). The conversion of PrP(C) to PrP(Sc) is known to involve a conformational transition of α-helical to β-sheet structures of the protein. However, the in vivo pathway is still poorly understood. A tentative endogenous PrP(Sc), intermediate PrP* or "silent prion", has yet to be identified in the uninfected brain(7). Using a combination of biophysical and biochemical approaches, we identified insoluble PrP(C) aggregates (designated iPrP(C)) from uninfected mammalian brains and cultured neuronal cells(8, 9). Here, we describe detailed procedures of these methods, including ultracentrifugation in detergent buffer, sucrose step gradient sedimentation, size exclusion chromatography, iPrP enrichment by gene 5 protein (g5p) that specifically bind to structurally altered PrP forms(10), and PK-treatment. The combination of these approaches isolates not only insoluble PrP(Sc) and PrP(C) aggregates but also soluble PrP(C) oligomers from the normal human brain. Since the protocols described here have been used to isolate both PrP(Sc) from infected brains and iPrP(C) from uninfected brains, they provide us with an opportunity to compare differences in physicochemical features, neurotoxicity, and infectivity between the two isoforms. Such a study will greatly improve our understanding of the infectious proteinaceous pathogens. The physiology and pathophysiology of iPrP(C) are unclear at present. Notably, in a newly-identified human prion disease termed variably protease-sensitive prionopathy, we found a new PrP(Sc) that shares the immunoreactive behavior and fragmentation with iPrP(C 11, 12). Moreover, we recently demonstrated that iPrP(C) is the main species that interacts with amyloid-β protein in Alzheimer disease(13). In the same study, these methods were used to isolate Abeta aggregates and oligomers in Alzheimer's disease(13), suggesting their application to non-prion protein aggregates involved in other neurodegenerative disorders.

A 75-year-old man presented with a three-year history of progressively worsening insomnia and dementia. His mother and older sister had similar disorders. On initial examination, he was awake, apathetic, and disoriented but had no focal neurological deficits. Electroencephalography showed diffuse background slowing with neither periodic discharge nor sleeping activity. A single-photon emission CT scan showed significantly reduced cerebral perfusion in bilateral thalami, basal ganglia, and limbic cortices. In the late stage of his illness, he developed sphincter dysfunction. Laboratory studies showed increased T-lymphocytes and B-lymphocytes and reduced cortisol level. Cerebrospinal fluid 14-3-3 protein was absent. Genetic evaluations failed to show the aspartate to asparagine point mutation at codon 178 but disclosed an asparagine to serine substitution at codon 171 in one allele and a deletion of 24 base pairs in the other allele in the human prion protein gene. These findings led to a diagnosis of sporadic fatal insomnia, which is a recently described prion disease.

Background Sporadic fatal insomnia (sFI) and fatal familial insomnia (FFI) are rare human prion diseases. Case Presentation We report a case of a 33-year-old female who died of a prion disease for whom the diagnosis of sFI or FFI was not considered clinically. Following death of this patient, an interview with a close family member indicated the patient's illness included a major change in her sleep pattern, corroborating the reported autopsy diagnosis of sFI. Genetic tests identified no prion protein (PrP) gene mutation, but neuropathological examination and molecular study showed protease-resistant PrP (PrPres) in several brain regions and severe atrophy of the anterior-ventral and medial-dorsal thalamic nuclei similar to that described in FFI. Conclusions In patients with suspected prion disease, a characteristic change in sleep pattern can be an important clinical clue for identifying sFI or FFI; polysomnography (PSG), genetic analysis, and nuclear imaging may aid in diagnosis.

Variably protease-sensitive prionopathy (VPSPr) is a novel disease involving the prion protein (PrP) that has clinical similarities with non-Alzheimer’s dementias especially frontotemporal dementia, diffuse Lewis body disease, and normal pressure hydrocephalus. VPSPr can be distinguished from sporadic Creutzfeldt–Jakob disease (sCJD) especially for the characteristics of the abnormal PrP. Furthermore, although VPSPr like sCJD affects patients with the three PrP genotypes as determined by the common methionine/valine polymorphism, the allelic prevalence is very different in the two diseases. These findings suggest that VPSPr is basically different from classical prion diseases such as sCJD being perhaps more akin to other neurodegenerative dementias.

Six clinico-pathological phenotypes of sporadic Creutzfeldt-Jakob disease have been characterized which correlate at the molecular level with the type (1 or 2) of the abnormal prion protein, PrP(TSE), present in the brain and with the genotype of polymorphic (methionine or valine) codon 129 of the prion protein gene. However, to what extent these phenotypes with their corresponding molecular combinations (i.e. MM1, MM2, VV1 etc.) encipher distinct prion strains upon transmission remains uncertain. We studied the PrP(TSE) type and the prion protein gene in archival brain tissues from the National Institutes of Health series of transmitted Creutzfeldt-Jakob disease and kuru cases, and characterized the molecular and pathological phenotype in the affected non-human primates, including squirrel, spider, capuchin and African green monkeys. We found that the transmission properties of prions from the common sporadic Creutzfeldt-Jakob disease MM1 phenotype are homogeneous and significantly differ from those of sporadic Creutzfeldt-Jakob disease VV2 or MV2 prions. Animals injected with iatrogenic Creutzfeldt-Jakob disease MM1 and genetic Creutzfeldt-Jakob disease MM1 linked to the E200K mutation showed the same phenotypic features as those infected with sporadic Creutzfeldt-Jakob disease MM1 prions, whereas kuru most closely resembled the sporadic Creutzfeldt-Jakob disease VV2 or MV2 prion signature and neuropathology. The findings indicate that two distinct prion strains are linked to the three most common Creutzfeldt-Jakob disease clinico-pathological and molecular subtypes and kuru, and suggest that kuru may have originated from cannibalistic transmission of a sporadic Creutzfeldt-Jakob disease of the VV2 or MV2 subtype.

Human prion diseases are prominently heterogeneous. In sporadic Creutzfeldt-Jakob disease (sCJD), the most prevalent prion disease, heterogeneity is largely predicated on the common methionine (M)/valine (V) polymorphism at codon 129 of the prion protein (PrP) gene and the disease-associated PrP (PrPDis) that are distinguished in types 1 and 2 based on the electrophoretic mobility of their protease-resistant regions.1 However, despite this remarkable heterogeneity, all well-established sporadic prion diseases (here operationally defined as nonacquired prion diseases free of mutations in the PrP gene coding region) have been shown to share the same basic pathogenetic mechanism; PrPDis interacts with the normal or cellular PrP and converts it into PrPDis, triggering an autocatalytic process that leads to the accumulation of PrPDis and ultimately to the clinical disease.2 In 2008, we described 11 cases affected by a new disease involving PrP; we named this disease protease-sensitive prionopathy (PSPr).3 Subsequently, 2 additional cases of PSPr have been independently reported.4,5 PSPr differed from known sporadic prion diseases in the clinical presentation, in the histopathologic and immunohistochemical features, and in the basic characteristics of the PrPDis. Furthermore, all 11 cases had the 129VV genotype and no mutation in the PrP gene open reading frame (ORF). We now report 15 additional cases, all of which bear features of the PSPr as originally reported. However, the new cases also include, in addition to new 129VV subjects, individuals who are 129MV heterozygous and 129MM homozygous. Although the affected subjects belonging to the 3 genotypes share several important characteristics, they also display basic variations that allow the 3 corresponding phenotypes to be distinguished. Therefore, the new cases show that the disease originally described as PSPr, like sCJD, affects all 3 129 genotypes and to some extent mimics the 129-related phenotypic heterogeneity of sCJD, although the PSPr characteristics underline basic differences from sCJD and similarities with Gerstmann-Straussler-Scheinker disease (GSS), a rare phenotype, which to date has been reported as exclusively associated with PrP gene mutations. In view of the increased protease-resistance of the PrPDis associated with the new 129 genotypes compared to that of the 129VV cases, we propose to revise the original PSPr label to VPSPr or “variably protease-sensitive prionopathy.” Parts of these findings have been presented previously.6–9

Variant Creutzfeldt-Jakob disease, the human form of mad cow disease, triggered a health panic in the late twentieth century but it also greatly enhanced our understanding of prion diseases, a group of fatal transmissible spongiform encephalopathies that could be sporadic, familial or acquired by infection.

Sporadic Creutzfeldt–Jakob disease (sCJD) is the most common type of a group of fatal illnesses named transmissible spongiform encephalopathies or prion diseases, which have the unique features of being sporadic, genetically determined or acquired by infection. The three etiologies are associated with the same central pathogenic mechanism: a normal cellular protein, named prion protein, becomes insoluble, pathogenic and infectious by undergoing a change in conformation. sCJD is very heterogeneous due to the unique effect on the disease phenotype of the patient's prion protein genotype and characteristics of the abnormal prion protein. Based on these molecular features, five subtypes of sCJD with distinctive clinical and histopathological features have been identified. Increase in amount of 14–3–3 and/or tau in the cerebrospinal fluid and especially MRI are helpful in the diagnosis. Despite broad effort, no effective treatment is available.

Background Sporadic fatal insomnia is a rare prion disease that has recently been recognized. Objective To report a unique case of sporadic fatal insomnia in a woman with progressive cerebellar deterioration who was originally thought to have a paraneoplastic cerebellar syndrome. Design Case report describing a patient with autopsy-proven sporadic fatal insomnia. Patient A 56-year-old woman with progressive cerebellar ataxia who was found to have a retroperitoneal non-Hodgkin lymphoma. Results Autopsy demonstrated marked degenerative changes in the thalamus, cerebellum, and inferior olivary nucleus. A mild spongiform change was present in the thalamus and cortical gray matter. Western blot analysis confirmed the presence of abnormal, protease-resistant prion protein (PrP Sc ), characteristic of sporadic fatal insomnia. Conclusions Clinicians should be aware of this rare prion disease and should strongly consider the importance of autopsy toward the investigation of unusual neurological diseases.

From Creutzfeldt-Jakob disease (CJD) to variant CJD through Gerstmann-Straussler-Scheinker syndrome, kuru and fatal familial insomnia, the journey leading to current understanding of the basic aspects of human prion diseases has been full of unexpected, but often dramatic and always fascinating twists. Recent progress in modeling prion diseases and characterization of the various prion protein forms reveal that such a wide spectrum of the diseases is associated with the chameleon-like conformational features of prions.

The absence of specific immune response is a hallmark of prion diseases. However, in vitro and in vivo experiments have provided evidence that an anti-PrP humoral response could have beneficial effects. Prophylactic passive immunization performed at the time of infection delayed or prevented disease. Nonetheless, the potential therapeutic effect of PrP antibodies administered shortly before the clinical signs has never been tested in vivo. Moreover, a recent study showed the potential toxicity of PrP antibodies administered intracerebrally. We aimed at evaluating the effect of a prolonged intracerebral anti-PrP antibody administration at the time of neuroinvasion in BSE infected Tg20 mice. Unexpectedly, despite a good penetration of the antibodies in the brain parenchyma, the treatment was not protective against the development of BSE. Instead, it led to an extensive neuronal loss, strong astrogliosis and microglial activation. Since this effect was observed after injection of anti-PrP antibodies as whole IgGs, F(ab')(2) or Fab fragments, the toxicity was directly related to the ability of the antibodies to recognize native PrP and to the intracerebral concentration achieved, and not to the Fc portion or the divalence of the antibodies. This experiment shows that a prolonged treatment with anti-PrP antibodies by the intracerebral route can induce severe side-effects and calls for caution with regard to the use of similar approaches for late therapeutic interventions in humans.

The cellular prion protein (PrPC), a protein most noted for its link to prion diseases, has been found to play a protective role in ischemic brain injury. To investigate the role of PrPC in the kidney, an organ highly prone to ischemia/reperfusion (IR) injury, we examined wild-type (WT) and PrPC knockout (KO) mice that were subjected to 30-min of renal ischemia followed by 1, 2, or 3 days of reperfusion. Renal dysfunction and structural damage was more severe in KO than in WT mice. While PrP was undetectable in KO kidneys, Western blotting revealed an increase in PrP in IR-injured WT kidneys compared to sham-treated kidneys. Compared to WT, KO kidneys exhibited increases in oxidative stress markers heme oxygenase-1, nitrotyrosine, and Nε-(carboxymethyl)lysine, and decreases in mitochondrial complexes I and III. Notably, phosphorylated extracellular signal-regulated kinase (pERK) staining was predominantly observed in tubular cells from KO mice following 2 days of reperfusion, a time at which significant differences in renal dysfunction, histological changes, oxidative stress, and mitochondrial complexes between WT and KO mice were observed. Our study provides the first evidence that PrPC may play a protective role in renal IR injury, likely through its effects on mitochondria and ERK signaling pathways.

Chronic wasting disease (CWD), a prion disease affecting free-ranging and captive cervids (deer and elk), is widespread in the United States and parts of Canada. The large cervid population, the popularity of venison consumption, and the apparent spread of the CWD epidemic are likely resulting in increased human exposure to CWD in the United States. Whether CWD is transmissible to humans, as has been shown for bovine spongiform encephalopathy (the prion disease of cattle), is unknown. We generated transgenic mice expressing the elk or human prion protein (PrP) in a PrP-null background. After intracerebral inoculation with elk CWD prion, two lines of “humanized” transgenic mice that are susceptible to human prions failed to develop the hallmarks of prion diseases after >657 and >756 d, respectively, whereas the “cervidized” transgenic mice became infected after 118–142 d. These data indicate that there is a substantial species barrier for transmission of elk CWD to humans.

Prion diseases are unique transmissible neurodegenerative diseases that have diverse phenotypes and can be familial, sporadic, or acquired by infection. Recent findings indicate that the PrP genotype and the PrP(Sc) type have a major influence on the disease phenotype in both sporadic and familial human prion diseases. This review attempts to classify and characterise sporadic and familial Creutzfeldt-Jakob disease (CJD) as a function of these two disease determinants. Based on the genotype at codon 129 on both PRNP alleles, the size of protease resistant PrP(Sc) fragments and disease phenotype, we divide sporadic CJD into six subtypes: sCJDMM1/sCJDMV1, sCJDVV2, sCJDMV2, sCJDMM2, sCJDVV1, and sporadic fatal insomnia (sFI). Familial CJD is classified into many haplotypes based on the PRNP mutation and codon 129 (and other polymorphic codons) on the mutant allele. The clinical and pathological features are summarised for each sporadic CJD subtype and familial CJD haplotype.

The heterogeneity of the clinicopathological phenotype in human prion diseases is associated with the presence of the different forms of the abnormal prion protein, PrPSc. We have previously shown that PrPSc in FFI and a subtype of familial CJD linked to the D178N mutation can be distinguished by their difference in gel mobility following proteinase K (PK) treatment. To further characterize the structural difference of PrPSc in familial prion diseases, N-terminal sequencing and mass spectrometry were used to identify the protease cleavage sites in PrPSc extracted from affected brains. We found that the main PK cleavage sites of PrPSc are located at residue 97 in FFI, and residue 82 in both CJD178 and a GSS subtype linked to the P102L mutation. The differential accessibility to protease in the native PrPSc suggests that PrPSc exist as distinct conformers in different disease states.

The hallmark of prion diseases is the presence of an aberrant isoform of the prion protein (PrP(res)) that is insoluble in nondenaturing detergents and resistant to proteases. We investigated the allelic origin of PrP(res) in brains of subjects heterozygous for the D178N mutation linked to fatal familial insomnia (FFI) and a subtype of Creutzfeldt-Jakob disease (CJD178), as well as for insertional mutations associated with another CJD subtype. We found that in FFI and CJD178 subjects, only mutant PrP was detergent-insoluble and protease-resistant. Therefore, PrP(res) derives exclusively from the mutant allele carrying the D178N mutation. In contrast, in the CJD subtype harboring insertional mutations, wild-type PrP was also detergent-insoluble and likely to be protease-resistant. Our findings indicate that the participation of the wild-type PrP in the formation of PrP(res) depends on the type of mutations, providing an insight into the molecular mechanisms underlying the phenotypic heterogeneity in familial prion diseases.

BACKGROUND: The increase of the 14-3-3 protein in CSF is used as a diagnostic test in Creutzfeldt-Jakob disease (CJD), but the sensitivity and specificity of the 14-3-3 test are disputed. One reason for the dispute may be the recently established heterogeneity of sporadic CJD. The relationship between CSF 14-3-3 protein and sporadic CJD subtypes, distinguished by electrophoretic mobility of proteinase K-resistant prion protein (PrP(Sc)) and genotype at codon 129 of the prion protein gene, has not been elucidated. METHODS: The authors examined the 14-3-3 protein test in 90 patients with sporadic CJD. PrP(Sc) type (type 1 or type 2) and the genotype at polymorphic codon 129 were determined in each patient. Mutations were excluded by prion gene sequencing. RESULTS: The authors' findings indicate that the sensitivity of the 14-3-3 test is higher in patients with molecular features of the classic sporadic CJD than in patients with the nonclassic CJD subtypes. The difference appears to be related to the PrP(Sc) type and not to the codon 129 genotype. Disease duration before 14-3-3 testing might also have an influence because it was shorter in classic sporadic CJD. CONCLUSION: The Creutzfeldt-Jakob disease clinical subtype should be considered when interpreting results of the 14-3-3 test.