Prion diseases are transmissible neurological disorders associated with the presence of abnormal, disease-related prion protein (PrPD). The detection of PrPD in the brain is the only definitive ...diagnostic evidence of prion disease and its identification in body fluids and peripheral tissues are valuable for pre-mortem diagnosis. Protein misfolding cyclic amplification (PMCA) is a technique able to detect minute amount of PrPD and is based on the conversion of normal or cellular PrP (PrPC) to newly formed PrPD, sustained by a self-templating mechanism. Several animal prions have been efficiently amplified by PMCA, but limited results have been obtained with human prions with the exception of variant-Creutzfeldt–Jakob-disease (vCJD). Since the total or partial absence of glycans on PrPC has been shown to affect PMCA efficiency in animal prion studies, we attempted to enhance the amplification of four major sporadic-CJD (sCJD) subtypes (MM1, MM2, VV1, and VV2) and vCJD by single round PMCA using partially or totally unglycosylated PrPC as substrates. The amplification efficiency of all tested sCJD subtypes underwent a strong increase, inversely correlated to the degree of PrPC glycosylation and directly related to the matching of the PrP polymorphic 129 M/V genotype between seed and substrate. This effect was particularly significant in sCJDMM2 and sCJDVV2 allowing the detection of PK-resistant PrPD (resPrPD) in sCJDMM2 and sCJDVV2 brains at dilutions of 6 × 107 and 3 × 106. vCJD, at variance with the tested sCJD subtypes, showed the best amplification with partially deglycosylated PrPC substrate reaching a resPrPD detectability at up to 3 × 1016 brain dilution. The differential effect of substrate PrPC glycosylations suggests subtype-dependent PrPC–PrPD interactions, strongly affected by the PrPC glycans. The enhanced PMCA prion amplification efficiency achieved with unglycosylated PrPC substrates may allow for the developing of a sensitive, non-invasive, diagnostic test for the different CJD subtypes based on body fluids or easily-accessible-peripheral tissues.
Objective
Several prion amplification systems have been proposed for detection of prions in cerebrospinal fluid (CSF), most recently, the measurements of prion seeding activity with second‐generation ...real‐time quaking‐induced conversion (RT‐QuIC). The objective of this study was to investigate the diagnostic performance of the RT‐QuIC prion test in the broad phenotypic spectrum of prion diseases.
Methods
We performed CSF RT‐QuIC testing in 2,141 patients who had rapidly progressive neurological disorders, determined diagnostic sensitivity and specificity in 272 cases that were autopsied, and evaluated the impact of mutations and polymorphisms in the PRNP gene, and type 1 or type 2 human prions on diagnostic performance.
Results
The 98.5% diagnostic specificity and 92% sensitivity of CSF RT‐QuIC in a blinded retrospective analysis matched the 100% specificity and 95% sensitivity of a blind prospective study. The CSF RT‐QuIC differentiated 94% of cases of sporadic Creutzfeldt–Jakob disease (sCJD) MM1 from the sCJD MM2 phenotype, and 80% of sCJD VV2 from sCJD VV1. The mixed prion type 1‐2 and cases heterozygous for codon 129 generated intermediate CSF RT‐QuIC patterns, whereas genetic prion diseases revealed distinct profiles for each PRNP gene mutation.
Interpretation
The diagnostic performance of the improved CSF RT‐QuIC is superior to surrogate marker tests for prion diseases such as 14‐3‐3 and tau proteins, and together with PRNP gene sequencing the test allows the major prion subtypes to be differentiated in vivo. This differentiation facilitates prediction of the clinicopathological phenotype and duration of the disease—two important considerations for envisioned therapeutic interventions. ANN NEUROL 2017;81:79–92
Prions are self‐perpetuating proteins able to switch between a soluble state and an aggregated‐and‐transmissible conformation. These proteinaceous entities have been widely studied in yeast, where ...they are involved in hereditable phenotypic adaptations. The notion that such proteins could play functional roles and be positively selected by evolution has triggered the development of computational tools to identify prion‐like proteins in different kingdoms of life. These algorithms have succeeded in screening multiple proteomes, allowing the identification of prion‐like proteins in a diversity of unrelated organisms, evidencing that the prion phenomenon is well conserved among species. Interestingly enough, prion‐like proteins are not only connected with the formation of functional membraneless protein–nucleic acid coacervates, but are also linked to human diseases. This review addresses state‐of‐the‐art computational approaches to identify prion‐like proteins, describes proteome‐wide analysis efforts, discusses these unique proteins' functional role, and illustrates recently validated examples in different domains of life.
Prions are proteins able to populate two conformational states, one of which is transmissible. The discovery of functional prions in yeast triggered the development of distinct computational approaches to discover prion‐like proteins in other organisms. This review delves into the rationale behind these algorithms and discusses the new insights gathered from proteome‐wide data analysis in different kingdoms of life.
Prion diseases, or transmissible spongiform encephalopathies (TSEs), are a group of fatal and transmissible neurodegenerative disorders characterized by long incubation periods, misfolded prion ...protein (PrP) deposition, and usually spongiform vacuolation. These devastating diseases affect many mammals, with the best known examples being Creutzfeldt–Jakob disease (CJD), fatal familial insomnia (FFI), or Kuru in humans; and scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle, and chronic wasting disease (CWD) in cervids. Despite major achievements in research of TSEs, there are still many unresolved key issues that hamper the development of effective therapies. However, the last decade has been particularly prolific in advances in the prion field. Among others, prion propagation in vitro has been achieved, leading to new diagnostic methods; the basic architecture of infectious prions has been deciphered; new prion disease types have been described in humans and other animals; and prion disorders have emerged in places that had not previously reported the disorders, as is the case for CWD in Europe.This Special Issue will focus on the state of the art of our knowledge of PrPSc: on what we know about its structure and propagation, the basis of strains and transmission barriers, the mechanisms of PrPSc toxicity, the possible function of PrPSc’s properly folded precursor, PrPC and its evolutionary history, and recent technical breakthroughs in diagnostics and therapy development among other key aspects of PrPSc prion biology.
The bank vole (BV) prion protein (PrP) can function as a universal acceptor of prions. However, the molecular details of BVPrP's promiscuity for replicating a diverse range of prion strains remain ...obscure. To develop a cultured cell paradigm capable of interrogating the unique properties of BVPrP, we generated monoclonal lines of CAD5 cells lacking endogenous PrP but stably expressing either hamster (Ha), mouse (Mo), or BVPrP (M109 or I109 polymorphic variants) and then challenged them with various strains of mouse or hamster prions. Cells expressing BVPrP were susceptible to both mouse and hamster prions, whereas cells expressing MoPrP or HaPrP could only be infected with species‐matched prions. Propagation of mouse and hamster prions in cells expressing BVPrP resulted in strain adaptation in several instances, as evidenced by alterations in conformational stability, glycosylation, susceptibility to anti‐prion small molecules, and the inability of BVPrP‐adapted mouse prion strains to infect cells expressing MoPrP. Interestingly, cells expressing BVPrP containing the G127V prion gene variant, identified in individuals resistant to kuru, were unable to become infected with prions. Moreover, the G127V polymorphic variant impeded the spontaneous aggregation of recombinant BVPrP. These results demonstrate that BVPrP can facilitate cross‐species prion replication in cultured cells and that a single amino acid change can override the prion‐permissive nature of BVPrP. This cellular paradigm will be useful for dissecting the molecular features of BVPrP that allow it to function as a universal prion acceptor.
CAD5 cells lacking endogenous prion protein (PrP) expression (CAD5‐PrP−/−) were stably transfected with vectors encoding expression of wild‐type bank vole PrP or bank vole PrP containing the G127V substitution. Cells were challenged with either mouse or hamster prions and then passaged several times. Expression of bank vole PrP facilitated cross‐species prion infection, as revealed by the presence of proteinase K‐resistant PrP in cell lysates. In contrast, the addition of the protective G127V polymorphism to bank vole PrP completely blocked the ability of cells to become infected with mouse or hamster prions.
Prion diseases (PrD) or transmissible spongiform encephalopathies (TSE) are invariably fatal and pathogenic neurodegenerative disorders caused by the self-propagated misfolding of cellular prion ...protein (PrP
C
) to the neurotoxic pathogenic form (PrP
TSE
) via a yet undefined but profoundly complex mechanism. Despite several decades of research on PrD, the basic understanding of where and how PrP
C
is transformed to the misfolded, aggregation-prone and pathogenic PrP
TSE
remains elusive. The primary clinical hallmarks of PrD include vacuolation-associated spongiform changes and PrP
TSE
accumulation in neural tissue together with astrogliosis. The difficulty in unravelling the disease mechanisms has been related to the rare occurrence and long incubation period (over decades) followed by a very short clinical phase (few months). Additional challenge in unravelling the disease is implicated to the unique nature of the agent, its complexity and strain diversity, resulting in the heterogeneity of the clinical manifestations and potentially diverse disease mechanisms. Recent advances in tissue isolation and processing techniques have identified novel means of intercellular communication through extracellular vesicles (EVs) that contribute to PrP
TSE
transmission in PrD. This review will comprehensively discuss PrP
TSE
transmission and neurotoxicity, focusing on the role of EVs in disease progression, biomarker discovery and potential therapeutic agents for the treatment of PrD.
Prion diseases are fatal neurodegenerative conditions for which there is no effective treatment. Prion propagation involves the conversion of cellular prion protein, PrPC, to its conformational ...isomer, PrPSc, which accumulates in disease. Here, we show effective therapeutic knockdown of PrPC expression using RNAi in mice with established prion disease. A single administration of lentivirus expressing a shRNA targeting PrP into each hippocampus of mice with established prion disease significantly prolonged survival time. Treated animals lived 19% and 24% longer than mice given an "empty" lentivirus, or not treated, respectively. Lentivirally mediated RNAi of PrP also prevented the onset of behavioral deficits associated with early prion disease, reduced spongiform degeneration, and protected against neuronal loss. In contrast, mice receiving empty virus or no treatment developed early cognitive impairment and showed severe spongiosis and neuronal loss. The focal use of RNAi therapeutically in prion disease further supports strategies depleting PrPC, which we previously established to be a valid target for prion-based treatments. This approach can now be used to define the temporal, quantitative, and regional requirements for PrP knockdown for effective treatment of prion disease and to explore mechanisms involved in predegenerative neuronal dysfunction and its rescue.
Transmissible spongiform encephalopathies (TSEs) or prion diseases of animals notably include scrapie in small ruminants, chronic wasting disease (CWD) in cervids and classical bovine spongiform ...encephalopathy (C‐BSE). As the transmission barrier phenomenon naturally limits the propagation of prions from one species to another, and the lack of epidemiological evidence for an association with human prion diseases, the zoonotic potential of these diseases was for a long time considered negligible. However, in 1996, C‐BSE was recognized as the cause of a new human prion disease, variant Creutzfeldt‐Jakob disease (vCJD), which triggered an unprecedented public health crisis in Europe. Large‐scale epidemio‐surveillance programs for scrapie and C‐BSE that were implemented in the EU after the BSE crisis revealed that the distribution and prevalence of prion diseases in the ruminant population had previously been underestimated. They also led to the recognition of new forms of TSEs (named atypical) in cattle and small ruminants and to the recent identification of CWD in Europe. At this stage, the characterization of the strain diversity and zoonotic abilities associated with animal prion diseases remains largely incomplete. However, transmission experiments in nonhuman primates and transgenic mice expressing human PrP clearly indicate that classical scrapie, and certain forms of atypical BSE (L‐BSE) or CWD may have the potential to infect humans. The remaining uncertainties about the origins and relationships between animal prion diseases emphasize the importance of the measures implemented to limit human exposure to these potentially zoonotic agents, and of continued surveillance for both animal and human prion diseases.
Mammalian prion diseases are a group of neurodegenerative conditions caused by infection of the central nervous system with proteinaceous agents called prions, including sporadic, variant, and ...iatrogenic Creutzfeldt-Jakob disease; kuru; inherited prion disease; sheep scrapie; bovine spongiform encephalopathy; and chronic wasting disease. Prions are composed of misfolded and multimeric forms of the normal cellular prion protein (PrP). Prion diseases require host expression of the prion protein gene (
PRNP
) and a range of other cellular functions to support their propagation and toxicity. Inherited forms of prion disease are caused by mutation of
PRNP
, whereas acquired and sporadically occurring mammalian prion diseases are controlled by powerful genetic risk and modifying factors. Whereas some PrP amino acid variants cause the disease, others confer protection, dramatically altered incubation times, or changes in the clinical phenotype. Multiple mechanisms, including interference with homotypic protein interactions and the selection of the permissible prion strains in a host, play a role. Several non-
PRNP
factors have now been uncovered that provide insights into pathways of disease susceptibility or neurotoxicity.
Prion diseases are a unique group of rare neurodegenerative disorders characterized by tissue deposition of heterogeneous aggregates of abnormally folded protease-resistant prion protein (PrP
), a ...broad spectrum of disease phenotypes and a variable efficiency of disease propagation in vivo. The dominant clinicopathological phenotypes of human prion disease include Creutzfeldt⁻Jakob disease, fatal insomnia, variably protease-sensitive prionopathy, and Gerstmann⁻Sträussler⁻Scheinker disease. Prion disease propagation into susceptible hosts led to the isolation and characterization of prion strains, initially operatively defined as "isolates" causing diseases with distinctive characteristics, such as the incubation period, the pattern of PrP
distribution, and the regional severity of neuropathological changes after injection into syngeneic hosts. More recently, the structural basis of prion strains has been linked to amyloid polymorphs (i.e., variant amyloid protein conformations) and the concept extended to all protein amyloids showing polymorphic structures and some evidence of in vivo or in vitro propagation by seeding. Despite the significant advances, however, the link between amyloid structure and disease is not understood in many instances. Here we reviewed the most significant contributions of human prion disease studies to current knowledge of the molecular basis of phenotypic variability and the prion strain phenomenon and underlined the unsolved issues from the human disease perspective.
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