ABSTRACT
Prion diseases result from the misfolding of the physiological prion protein (PrPC) to a pathogenic conformation (PrPSc). Compelling evidence indicates that prevention and/or reduction of PrPSc replication are promising therapeutic strategies against prion diseases. However, the existence of different PrPSc conformations (or strains) associated with disease represents a major problem when identifying anti-prion compounds. Efforts to identify strain-specific anti-prion molecules are limited by the lack of biologically relevant high-throughput screening platforms to interrogate compound libraries. Here, we describe adaptations to the protein misfolding cyclic amplification (PMCA) technology (able to faithfully replicate PrPSc strains) that increase its throughput to facilitate the screening of anti-prion molecules. The optimized PMCA platform includes a reduction in sample and reagents, as well as incubation/sonication cycles required to efficiently replicate and detect rodent-adapted and cervid PrPSc strains. The visualization of PMCA products was performed via dot blots, a method that contributed to reduced processing times. These technical changes allowed us to evaluate small molecules with previously reported anti-prion activity. This proof-of-principle screening was evaluated for six rodent-adapted prion strains. Our data show that these compounds targeted either none, all or some PrPSc strains at variable concentrations, demonstrating that this PMCA system is suitable to test compound libraries for putative anti-prion molecules targeting specific PrPSc strains. Further analyses of a small compound library against deer prions demonstrate the potential of this new PMCA format to identify strain-specific anti-prion molecules. The data presented here demonstrate the use of the PMCA technique in the selection of prion strain-specific anti-prion compounds.
Subject(s)
PrPSc Proteins , Protein Folding , Animals , Protein Folding/drug effects , PrPSc Proteins/metabolism , PrPSc Proteins/chemistry , Mice , Prion Diseases/drug therapy , Prion Diseases/metabolism , Prions/metabolismABSTRACT
Chronic wasting disease (CWD) strains present a novel challenge to defining and mitigating this contagious prion disease of deer, elk, moose, and reindeer. Similar to strains of other prion diseases (bovine spongiform encephalopathy, sheep scrapie), CWD strains can affect biochemical and neuropathological properties of the infectious agent, and importantly interspecies transmission. To date, ten CWD strains have been characterized. The expanding range of CWD in North America and its presence in South Korea as well as Scandinavian countries will potentially result in millions of cervids infected with CWD; thus, novel strains will continue to emerge. In this review, we will summarize the characteristics of known CWD strains and describe the impact of prion protein gene polymorphisms on the generation of strains. We will also discuss the evidence that individual cervids can harbor more than one CWD strain, complicating strain analysis, and affecting selection and adaptation of strains in new hosts.
Subject(s)
Deer , Prions , Wasting Disease, Chronic , Cattle , Animals , Sheep , Wasting Disease, Chronic/genetics , Wasting Disease, Chronic/metabolism , Deer/metabolism , Prion Proteins/metabolism , Prions/geneticsABSTRACT
Mammalian prions are lethal transmissible pathogens that cause fatal neurodegenerative diseases in humans and animals. They consist of fibrils of misfolded, host-encoded prion protein (PrP) which propagate through templated protein polymerisation. Prion strains produce distinct clinicopathological phenotypes in the same host and appear to be encoded by distinct misfolded PrP conformations and assembly states. Despite fundamental advances in our understanding of prion biology, key knowledge gaps remain. These include precise delineation of prion replication mechanisms, detailed explanation of the molecular basis of prion strains and inter-species transmission barriers, and the structural definition of neurotoxic PrP species. Central to addressing these questions is the determination of prion structure. While high-resolution definition of ex vivo prion fibrils once seemed unlikely, recent advances in cryo-electron microscopy (cryo-EM) and computational methods for 3D reconstruction of amyloids have now made this possible. Recently, near-atomic resolution structures of highly infectious, ex vivo prion fibrils from hamster 263K and mouse RML prion strains were reported. The fibrils have a comparable parallel in-register intermolecular ß-sheet (PIRIBS) architecture that now provides a structural foundation for understanding prion strain diversity in mammals. Here, we review these new findings and discuss directions for future research.
Subject(s)
Neurodegenerative Diseases , Prion Diseases , Prions , Cricetinae , Humans , Mice , Animals , Prions/metabolism , Cryoelectron Microscopy , Prion Proteins , Mammals/metabolism , Prion Diseases/metabolismABSTRACT
Mammalian prion or PrPSc is a proteinaceous infectious agent that consists of a misfolded, self-replicating state of the prion protein or PrPC. PrPC and PrPSc are posttranslationally modified with N-linked glycans, which are sialylated at the terminal positions. More than 30 years have passed since the first characterization of the composition and structural diversity of N-linked glycans associated with the prion protein, yet the role of carbohydrate groups that constitute N-glycans and, in particular, their terminal sialic acid residues in prion disease pathogenesis remains poorly understood. A number of recent studies shed a light on the role of sialylation in the biology of prion diseases. This review article discusses several mechanisms by which terminal sialylation dictates the spread of PrPSc across brain regions and the outcomes of prion infection in an organism. In particular, relationships between the sialylation status of PrPSc and important strain-specific features including lymphotropism, neurotropism, and neuroinflammation are discussed. Moreover, emerging evidence pointing out the roles of sialic acid residues in prion replication, cross-species transmission, strain competition, and strain adaptation are reviewed. A hypothesis according to which selective, strain-specified recruitment of PrPC sialoglycoforms dictates unique strain-specific disease phenotypes is examined. Finally, the current article proposes that prion strains evolve as a result of a delicate balance between recruiting highly sialylated glycoforms to avoid an "eat-me" response by glia and limiting heavily sialylated glycoforms for enabling rapid prion replication.
Subject(s)
Prion Diseases , Prions , Animals , Prions/metabolism , Prion Proteins/metabolism , N-Acetylneuraminic Acid/chemistry , N-Acetylneuraminic Acid/metabolism , PrPSc Proteins/chemistry , PrPSc Proteins/genetics , PrPSc Proteins/metabolism , Prion Diseases/metabolism , Prion Diseases/pathology , Polysaccharides/metabolism , Mammals/metabolismABSTRACT
Chronic wasting disease (CWD) is a relentless epidemic disorder caused by infectious prions that threatens the survival of cervid populations and raises increasing public health concerns in North America. In Europe, CWD was detected for the first time in wild Norwegian reindeer (Rangifer tarandus) and moose (Alces alces) in 2016. In this study, we aimed at comparing the strain properties of CWD prions derived from different cervid species in Norway and North America. Using a classical strain typing approach involving transmission and adaptation to bank voles (Myodes glareolus), we found that prions causing CWD in Norway induced incubation times, neuropathology, regional deposition of misfolded prion protein aggregates in the brain, and size of their protease-resistant core, different from those that characterize North American CWD. These findings show that CWD prion strains affecting Norwegian cervids are distinct from those found in North America, implying that the highly contagious North American CWD prions are not the proximate cause of the newly discovered Norwegian CWD cases. In addition, Norwegian CWD isolates showed an unexpected strain variability, with reindeer and moose being caused by different CWD strains. Our findings shed light on the origin of emergent European CWD, have significant implications for understanding the nature and the ecology of CWD in Europe, and highlight the need to assess the zoonotic potential of the new CWD strains detected in Europe.
Subject(s)
Arvicolinae/physiology , Prions/metabolism , Wasting Disease, Chronic/epidemiology , Adaptation, Physiological , Animals , Brain/pathology , Nerve Degeneration/complications , Nerve Degeneration/pathology , North America/epidemiology , Norway/epidemiology , Phenotype , Species Specificity , Wasting Disease, Chronic/complications , Wasting Disease, Chronic/transmissionABSTRACT
Tau can adopt distinct fibril conformations in different human neurodegenerative diseases, which may invoke distinct pathological mechanisms. In a recent issue, Weismiller et al. showed that intramolecular disulfide links between cys291 and cys322 for a specific tau isoform containing four microtubule-binding repeats direct the formation of a structurally distinct amyloid polymorph. These findings have implications in how oxidative stress can flip switches of tau polymorphism in these diseases.
Subject(s)
Amyloidosis , Cysteine , Amyloid/genetics , Amyloidogenic Proteins , Humans , tau Proteins/geneticsABSTRACT
Prions cause infectious and fatal neurodegenerative diseases in mammals. Chronic wasting disease (CWD), a prion disease of cervids, spreads efficiently among wild and farmed animals. Potential transmission to humans of CWD is a growing concern due to its increasing prevalence. Here, we provide evidence for a zoonotic potential of CWD prions, and its probable signature using mice expressing human prion protein (PrP) as an infection model. Inoculation of these mice with deer CWD isolates resulted in atypical clinical manifestation with prion seeding activity and efficient transmissible infectivity in the brain and, remarkably, in feces, but without classical neuropathological or Western blot appearances of prion diseases. Intriguingly, the protease-resistant PrP in the brain resembled that found in a familial human prion disease and was transmissible upon second passage. Our results suggest that CWD might infect humans, although the transmission barrier is likely higher compared to zoonotic transmission of cattle prions. Notably, our data suggest a different clinical presentation, prion signature, and tissue tropism, which causes challenges for detection by current diagnostic assays. Furthermore, the presence of infectious prions in feces is concerning because if this occurs in humans, it is a source for human-to-human transmission. These findings have strong implications for public health and CWD management.
Subject(s)
Deer , Prions , Wasting Disease, Chronic , Animals , Blotting, Western , Cattle , Deer/metabolism , Humans , Mice , Prion Proteins/metabolism , Prions/metabolism , Wasting Disease, Chronic/metabolism , Wasting Disease, Chronic/pathologyABSTRACT
Although experimental transmission of bovine spongiform encephalopathy (BSE) to pigs and transgenic mice expressing pig cellular prion protein (PrPC) (porcine PrP [PoPrP]-Tg001) has been described, no natural cases of prion diseases in pig were reported. This study analyzed pig-PrPC susceptibility to different prion strains using PoPrP-Tg001 mice either as animal bioassay or as substrate for protein misfolding cyclic amplification (PMCA). A panel of isolates representatives of different prion strains was selected, including classic and atypical/Nor98 scrapie, atypical-BSE, rodent scrapie, human Creutzfeldt-Jakob-disease and classic BSE from different species. Bioassay proved that PoPrP-Tg001-mice were susceptible only to the classic BSE agent, and PMCA results indicate that only classic BSE can convert pig-PrPC into scrapie-type PrP (PrPSc), independently of the species origin. Therefore, conformational flexibility constraints associated with pig-PrP would limit the number of permissible PrPSc conformations compatible with pig-PrPC, thus suggesting that pig-PrPC may constitute a paradigm of low conformational flexibility that could confer high resistance to the diversity of prion strains.
Subject(s)
Encephalopathy, Bovine Spongiform , Prions , Scrapie , Animals , Brain/metabolism , Cattle , Encephalopathy, Bovine Spongiform/transmission , Mice , Mice, Transgenic , PrPSc Proteins , Prion Proteins , Prions/metabolism , SwineABSTRACT
The causative factors underlying conformational conversion of cellular prion protein (PrPC) into its infectious counterpart (PrPSc) during prion infection remain undetermined, in part because of a lack of monoclonal antibodies (mAbs) that can distinguish these conformational isoforms. Here we show that the anti-PrP mAb PRC7 recognizes an epitope that is shielded from detection when glycans are attached to Asn-196. We observed that whereas PrPC is predisposed to full glycosylation and is therefore refractory to PRC7 detection, prion infection leads to diminished PrPSc glycosylation at Asn-196, resulting in an unshielded PRC7 epitope that is amenable to mAb recognition upon renaturation. Detection of PRC7-reactive PrPSc in experimental and natural infections with various mouse-adapted scrapie strains and with prions causing deer and elk chronic wasting disease and transmissible mink encephalopathy uncovered that incomplete PrPSc glycosylation is a consistent feature of prion pathogenesis. We also show that interrogating the conformational properties of the PRC7 epitope affords a direct means of distinguishing different prion strains. Because the specificity of our approach for prion detection and strain discrimination relies on the extent to which N-linked glycosylation shields or unshields PrP epitopes from antibody recognition, it dispenses with the requirement for additional standard manipulations to distinguish PrPSc from PrPC, including evaluation of protease resistance. Our findings not only highlight an innovative and facile strategy for prion detection and strain differentiation, but are also consistent with a mechanism of prion replication in which structural instability of incompletely glycosylated PrP contributes to the conformational conversion of PrPC to PrPSc.
Subject(s)
Antibodies, Monoclonal, Murine-Derived/chemistry , Epitopes/chemistry , PrPC Proteins/chemistry , Animals , Cell Line , Epitopes/metabolism , Glycosylation , PrPC Proteins/metabolism , RabbitsABSTRACT
The current classification of sporadic Creutzfeldt-Jakob disease (sCJD) includes six major clinicopathological subtypes defined by the physicochemical properties of the protease-resistant core of the pathologic prion protein (PrPSc), defining two major PrPSc types (i.e., 1 and 2), and the methionine (M)/valine (V) polymorphic codon 129 of the prion protein gene (PRNP). How these sCJD subtypes relate to the well-documented phenotypic heterogeneity of genetic CJD (gCJD) is not fully understood. We analyzed molecular and phenotypic features in 208 individuals affected by gCJD, carrying 17 different mutations, and compared them with those of a large series of sCJD cases. We identified six major groups of gCJD based on the combination PrPSc type and codon 129 genotype on PRNP mutated allele, each showing distinctive histopathological characteristics, irrespectively of the PRNP associated mutation. Five gCJD groups, named M1, M2C, M2T, V1, and V2, largely reproduced those previously described in sCJD subtypes. The sixth group shared phenotypic traits with the V2 group and was only detected in patients carrying the E200K-129M haplotype in association with a PrPSc type of intermediate size ("i") between type 1 and type 2. Additional mutation-specific effects involved the pattern of PrP deposition (e.g., a "thickened" synaptic pattern in E200K carriers, cerebellar "stripe-like linear granular deposits" in those with insertion mutations, and intraneuronal globular dots in E200K-V2 or -M"i"). A few isolated cases linked to rare PRNP haplotypes (e.g., T183A-129M), showed atypical phenotypic features, which prevented their classification into the six major groups. The phenotypic variability of gCJD is mostly consistent with that previously found in sCJD. As in sCJD, the codon 129 genotype and physicochemical properties of PrPSc significantly correlated with the phenotypic variability of gCJD. The most common mutations linked to CJD appear to have a variable and overall less significant effect on the disease phenotype, but they significantly influence disease susceptibility often in a strain-specific manner. The criteria currently used for sCJD subtypes can be expanded and adapted to gCJD to provide an updated classification of the disease with a molecular basis.
Subject(s)
Creutzfeldt-Jakob Syndrome/genetics , Creutzfeldt-Jakob Syndrome/pathology , Insomnia, Fatal Familial/genetics , Mutation/genetics , PrPSc Proteins/genetics , Prion Proteins/genetics , Adult , Aged , Codon , Cohort Studies , Female , Genotype , Humans , Insomnia, Fatal Familial/pathology , Male , Middle Aged , PhenotypeABSTRACT
Genetic Creutzfeldt-Jakob disease (gCJD) with a methionine to arginine substitution at codon 232 of the prion protein gene (gCJD-M232R) is rare and has only been reported in Japan. We report an autopsy case of gCJD-M232R showing alleles of codon 129 that were homozygous for methionine and the presence of multiple strains of the protease-resistant, abnormal isoform of prion protein (PrPSc ), M1 + M2C + M2T. The patient, a 54-year-old Japanese man, died after a clinical course of 21 months characterized by slowly progressive dementia and sleep disturbance. At autopsy, the neuropil of the cerebral neocortex showed a widespread and severe spongiform change. Grape-like clusters of large confluent vacuoles were admixed with fine vacuoles. Neuronal loss was moderate, but reactive astrocytosis was mild. The dorsomedial nucleus of the thalamus and the inferior olivary nucleus showed moderate and severe neuronal loss, respectively. Many amyloid plaques were present in the cerebellar molecular layer. PrPSc deposition pattern was predominantly the synaptic type in the cerebrum and corresponded to the plaques in the cerebellum. Perivacuolar deposition was also seen. Western blot analysis of PrPSc revealed the predominance of type 2. Moreover, by employing Western blot analysis in combination with the protein misfolding cyclic amplification (PMCA) method, which selectively amplifies the minor M2T prion strain, we demonstrated the presence of M2T, in addition to M1 and M2C strains, in the brain of the patient. PMCA was a powerful method for demonstrating the presence of the M2T strain, although the amount is often small and the transmission is difficult.
Subject(s)
Creutzfeldt-Jakob Syndrome/genetics , Creutzfeldt-Jakob Syndrome/pathology , Methionine/genetics , PrPSc Proteins/genetics , Atrophy/genetics , Atrophy/pathology , Autopsy , Blotting, Western , Cerebellum/pathology , Cerebrum/pathology , Humans , Japan , Magnetic Resonance Imaging , Male , Middle Aged , Mutation , Myocardium/pathology , Thalamus/pathologyABSTRACT
Many aggregation-prone proteins linked to neurodegenerative disease are post-translationally modified during their biogenesis. In vivo pathogenesis studies have suggested that the presence of post-translational modifications can shift the aggregate assembly pathway and profoundly alter the disease phenotype. In prion disease, the N-linked glycans and GPI-anchor on the prion protein (PrP) impair fibril assembly. However, the relevance of the two glycans to aggregate structure and disease progression remains unclear. Here we show that prion-infected knockin mice expressing an additional PrP glycan (tri-glycosylated PrP) develop new plaque-like deposits on neuronal cell membranes, along the subarachnoid space, and periventricularly, suggestive of high prion mobility and transit through the interstitial fluid. These plaque-like deposits were largely non-congophilic and composed of full length, uncleaved PrP, indicating retention of the glycophosphatidylinositol (GPI) anchor. Prion aggregates sedimented in low density fractions following ultracentrifugation, consistent with oligomers, and bound low levels of heparan sulfate (HS) similar to other predominantly GPI-anchored prions. Collectively, these results suggest that highly glycosylated PrP primarily converts as a GPI-anchored glycoform, with low involvement of HS co-factors, limiting PrP assembly mainly to oligomers. Since PrPC is highly glycosylated, these findings may explain the high frequency of diffuse, synaptic, and plaque-like deposits in the brain as well as the rapid conversion commonly observed in human and animal prion disease.
Subject(s)
Heparitin Sulfate/metabolism , Prion Diseases/metabolism , Prion Proteins/metabolism , Protein Aggregates/genetics , Protein Processing, Post-Translational/genetics , Animals , Brain/metabolism , Cell Membrane/metabolism , Female , Male , Mice , Mice, Transgenic , Prion Diseases/genetics , Prion Proteins/genetics , Protein Binding/geneticsABSTRACT
Chronic neuroinflammation is recognized as a major neuropathological hallmark in a broad spectrum of neurodegenerative diseases including Alzheimer's, Parkinson's, Frontal Temporal Dementia, Amyotrophic Lateral Sclerosis, and prion diseases. Both microglia and astrocytes exhibit region-specific homeostatic transcriptional identities, which under chronic neurodegeneration, transform into reactive phenotypes in a region- and disease-specific manner. Little is known about region-specific identity of glia in prion diseases. The current study was designed to determine whether the region-specific homeostatic signature of glia changes with the progression of prion diseases, and whether these changes occur in a region-dependent or universal manner. Also of interest was whether different prion strains give rise to different reactive phenotypes. To answer these questions, we analyzed gene expression in the thalamus, cortex, hypothalamus and hippocampus of mice infected with 22L and ME7 prion strains using a Nanostring Neuroinflammation panel at the subclinical, early clinical and advanced stages of the disease. We found that at the preclinical stage of the disease, the region-specific homeostatic identities were preserved. However, with the appearance of clinical signs, the region-specific signatures were partially lost and replaced with a neuroinflammation signature. While the same sets of genes were activated by both prion strains, the timing of neuroinflammation and the degree of activation in different brain regions was strain-specific. Changes in astrocyte function scored at the top of the activated pathways. Moreover, clustering analysis suggested that the astrocyte function pathway responded to prion infection prior to the Activated Microglia or Neuron and Neurotransmission pathways. The current work established neuroinflammation gene expression signature associated with prion diseases. Our results illustrate that with the disease progression, the region-specific homeostatic transcriptome signatures are replaced by the region-independent neuroinflammation signature, which is common for prion strains with different cell tropism. The prion-associated neuroinflammation signature identified in the current study overlapped only partially with the microglia degenerative phenotype and the disease-associated microglia phenotype reported for animal models of other neurodegenerative diseases.
Subject(s)
Astrocytes/metabolism , Inflammation/metabolism , Neuroglia/metabolism , Prion Diseases/metabolism , Alzheimer Disease/metabolism , Animals , Disease Models, Animal , Mice, Inbred C57BL , Microglia/metabolism , Neurons/metabolismABSTRACT
Adaptation of prions to new species is thought to reflect the capacity of the host-encoded cellular form of the prion protein (PrPC) to selectively propagate optimized prion conformations from larger ensembles generated in the species of origin. Here we describe an alternate replicative process, termed nonadaptive prion amplification (NAPA), in which dominant conformers bypass this requirement during particular interspecies transmissions. To model susceptibility of horses to prions, we produced transgenic (Tg) mice expressing cognate PrPC Although disease transmission to only a subset of infected TgEq indicated a significant barrier to EqPrPC conversion, the resulting horse prions unexpectedly failed to cause disease upon further passage to TgEq. TgD expressing deer PrPC was similarly refractory to deer prions from diseased TgD infected with mink prions. In both cases, the resulting prions transmitted to mice expressing PrPC from the species of prion origin, demonstrating that transmission barrier eradication of the originating prions was ephemeral and adaptation superficial in TgEq and TgD. Horse prions produced in vitro by protein misfolding cyclic amplification of mouse prions using horse PrPC also failed to infect TgEq but retained tropism for wild-type mice. Concordant patterns of neuropathology and prion deposition in susceptible mice infected with NAPA prions and the corresponding prion of origin confirmed preservation of strain properties. The comparable responses of both prion types to guanidine hydrochloride denaturation indicated this occurs because NAPA precludes selection of novel prion conformations. Our findings provide insights into mechanisms regulating interspecies prion transmission and a framework to reconcile puzzling epidemiological features of certain prion disorders.
Subject(s)
Host Specificity/physiology , PrPC Proteins/physiology , Prion Diseases/transmission , Prion Diseases/veterinary , Prions/physiology , Animals , Deer , Guanidine/pharmacology , Horses , Mice , Mice, Inbred C57BL , PrPC Proteins/chemistry , PrPC Proteins/genetics , Prions/chemistry , Protein Conformation , Protein Denaturation , Rabbits , Sheep , Species Specificity , Structure-Activity RelationshipABSTRACT
Mammalian prions are unconventional infectious agents that invade and replicate in an organism by recruiting a normal form of a prion protein (PrPC) and converting it into misfolded, disease-associated state referred to as PrPSc. PrPC is posttranslationally modified with two N-linked glycans. Prion strains replicate by selecting substrates from a large pool of PrPC sialoglycoforms expressed by a host. Brain regions have different vulnerability to prion infection, however, molecular mechanisms underlying selective vulnerability is not well understood. Toward addressing this question, the current study looked into a possibility that sialylation of PrPSc might be involved in defining selective vulnerability of brain regions. The current work found that in 22L -infected animals, PrPSc is indeed sialylated in a region dependent manner. PrPSc in hippocampus and cortex was more sialylated than PrPSc from thalamus and stem. Similar trends were also observed in brain materials from RML- and ME7-infected animals. The current study established that PrPSc sialylation status is indeed region-specific. Together with previous studies demonstrating that low sialylation status accelerates prion replication, this work suggests that high vulnerability of certain brain region to prion infection could be attributed to their low sialylation status.
Subject(s)
Brain/metabolism , N-Acetylneuraminic Acid/metabolism , PrPC Proteins/metabolism , PrPSc Proteins/metabolism , Prion Diseases/metabolism , Protein Processing, Post-Translational , Animals , Brain/pathology , Female , Male , Mice , Prion Diseases/pathologyABSTRACT
The search for antiprion compounds has been encouraged by the fact that transmissible spongiform encephalopathies (TSEs) share molecular mechanisms with more prevalent neurodegenerative pathologies, such as Parkinson's and Alzheimer's diseases. Cellular prion protein (PrPC) conversion into protease-resistant forms (protease-resistant PrP [PrPRes] or the scrapie form of PrP [PrPSc]) is a critical step in the development of TSEs and is thus one of the main targets in the screening for antiprion compounds. In this work, three trimethoxychalcones (compounds J1, J8, and J20) and one oxadiazole (compound Y17), previously identified in vitro to be potential antiprion compounds, were evaluated through different approaches in order to gain inferences about their mechanisms of action. None of them changed PrPC mRNA levels in N2a cells, as shown by reverse transcription-quantitative real-time PCR. Among them, J8 and Y17 were effective in real-time quaking-induced conversion reactions using rodent recombinant PrP (rPrP) from residues 23 to 231 (rPrP23-231) as the substrate and PrPSc seeds from hamster and human brain. However, when rPrP from residues 90 to 231 (rPrP90-231), which lacks the N-terminal domain, was used as the substrate, only J8 remained effective, indicating that this region is important for Y17 activity, while J8 seems to interact with the PrPC globular domain. J8 also reduced the fibrillation of mouse rPrP23-231 seeded with in vitro-produced fibrils. Furthermore, most of the compounds decreased the amount of PrPC on the N2a cell surface by trapping this protein in the endoplasmic reticulum. On the basis of these results, we hypothesize that J8, a nontoxic compound previously shown to be a promising antiprion agent, may act by different mechanisms, since its efficacy is attributable not only to PrP conversion inhibition but also to a reduction of the PrPC content on the cell surface.
Subject(s)
Chalcones/pharmacology , Drugs, Investigational/pharmacology , Neurons/drug effects , Oxadiazoles/pharmacology , Prion Proteins/antagonists & inhibitors , Animals , Binding Sites , Cell Line, Tumor , Chalcones/chemical synthesis , Cloning, Molecular , Drugs, Investigational/chemical synthesis , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/ultrastructure , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Kinetics , Mice , Molecular Docking Simulation , Neurons/metabolism , Neurons/pathology , Oxadiazoles/chemical synthesis , Prion Proteins/chemistry , Prion Proteins/genetics , Prion Proteins/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Structure-Activity Relationship , ThermodynamicsABSTRACT
AIMS: The aim of this study was to describe the regional profiles of microglial activation in sporadic Creutzfeldt-Jakob disease (sCJD) subtypes and analyse the influence of prion strain, disease duration and codon 129 genotype. METHODS: We studied the amount/severity and distribution of activated microglia, protease-resistant prion protein (PrPSc ) spongiform change, and astrogliosis in eight regions of 57 brains, representative of the entire spectrum of sCJD subtypes. RESULTS: In each individual subtype, the regional extent and distribution of microgliosis significantly correlated with PrPSc deposition and spongiform change, leading to subtype-specific 'lesion profiles'. However, large differences in the ratio between PrPSc load or the score of spongiform change and microglial activation were seen among disease subtypes. Most significantly, atypical sCJD subtypes such as VV1 and MM2T showed a degree of microglial activation comparable to other disease variants despite the relatively low PrPSc deposition and the less severe spongiform change. Moreover, the mean microglial total load was significantly higher in subtype MM1 than in MM2C, whereas the opposite was true for the PrPSc and spongiform change total loads. Finally, some sCJD subtypes showed distinctive regional cerebellar profiles of microgliosis characterized by a high granular/molecular layer ratio (MV2K) and/or a predominant involvement of white matter (MVK and MM2T). CONCLUSIONS: Microglial activation is an early event in sCJD pathogenesis and is strongly influenced by prion strain, PRNP codon 129 genotype and disease duration. Microglial lesion profiling, by highlighting strain-specific properties of prions, contributes to prion strain characterization and classification of human prion diseases, and represents a valid support to molecular and histopathologic typing.
Subject(s)
Brain/pathology , Creutzfeldt-Jakob Syndrome/pathology , Gliosis/pathology , Microglia/pathology , Disease Progression , Humans , PhenotypeABSTRACT
Prion diseases are caused by a misfolding of the cellular prion protein (PrP) to a pathogenic isoform named PrPSc. Prions exist as strains, which are characterized by specific pathological and biochemical properties likely encoded in the three-dimensional structure of PrPSc. However, whether cofactors determine these different PrPSc conformations and how this relates to their specific biological properties is largely unknown. To understand how different cofactors modulate prion strain generation and selection, Protein Misfolding Cyclic Amplification was used to create a diversity of infectious recombinant prion strains by propagation in the presence of brain homogenate. Brain homogenate is known to contain these mentioned cofactors, whose identity is only partially known, and which facilitate conversion of PrPC to PrPSc. We thus obtained a mix of distinguishable infectious prion strains. Subsequently, we replaced brain homogenate, by different polyanionic cofactors that were able to drive the evolution of mixed prion populations toward specific strains. Thus, our results show that a variety of infectious recombinant prions can be generated in vitro and that their specific type of conformation, i.e., the strain, is dependent on the cofactors available during the propagation process. These observations have significant implications for understanding the pathogenesis of prion diseases and their ability to replicate in different tissues and hosts. Importantly, these considerations might apply to other neurodegenerative diseases for which different conformations of misfolded proteins have been described.
Subject(s)
Brain/metabolism , Prion Diseases/metabolism , Prion Proteins/metabolism , Animals , Arvicolinae , Brain/pathology , Escherichia coli , Mice, Transgenic , Polymorphism, Genetic , Prion Proteins/genetics , Protein Folding , Recombinant Proteins/metabolismABSTRACT
The self-replicative conformation of misfolded prion proteins (PrP) is considered a major determinant for the seeding activity, infectiousness, and strain characteristics of prions in different host species. Prion-associated seeding activity, which converts cellular prion protein (PrP(C)) into Proteinase K-resistant, infectious PrP particles (PrP(TSE)), can be monitored in vitro by protein misfolding cyclic amplification (PMCA). Thus, PMCA has been established as a valuable analytical tool in prion research. Currently, however, it is under discussion whether prion strain characteristics are preserved during PMCA when parent seeds are amplified in PrP(C) substrate from the identical host species. Here, we report on the comparative structural analysis of parent and progeny (PMCA-derived) PrP seeds by an improved approach of sensitive infrared microspectroscopy. Infrared microspectroscopy revealed that PMCA of native hamster 263K scrapie seeds in hamster PrP(C) substrate caused conformational alterations in progeny seeds that were accompanied by an altered resistance to Proteinase K, higher sedimentation velocities in gradient ultracentrifugations, and a longer incubation time in animal bioassays. When these progeny seeds were propagated in hamsters, misfolded PrP from brain extracts of these animals showed mixed spectroscopic and biochemical properties from both parental and progeny seeds. Thus, strain modifications of 263K prions induced by PMCA seem to have been partially reversed when PMCA products were reinoculated into the original host species.
Subject(s)
PrPSc Proteins/chemistry , Animals , Brain Chemistry , Cricetinae , Endopeptidase K , Mesocricetus , Microscopy, Atomic Force , PrP 27-30 Protein/chemistry , PrP 27-30 Protein/metabolism , PrP 27-30 Protein/ultrastructure , PrPSc Proteins/metabolism , PrPSc Proteins/ultrastructure , Protein Conformation , Protein Folding , Protein Stability , Scrapie/metabolism , Scrapie/transmission , Spectroscopy, Fourier Transform Infrared/methodsABSTRACT
The transmission of prions across species is a critical aspect of their dissemination among mammalian hosts, including humans. This process often necessitates strain adaptation. In this study, we sought to investigate the mechanisms underlying prion adaptation while mitigating biases associated with the history of cross-species transmission of natural prion strains. To achieve this, we utilized the synthetic hamster prion strain S05. Propagation of S05 using mouse PrPC in Protein Misfolding Cyclic Amplification did not immediately overcome the species barrier. This finding underscores the involvement of factors beyond disparities in primary protein structures. Subsequently, we performed five serial passages to stabilize the incubation time to disease in mice. The levels of PrPSc increased with each passage, reaching a maximum at the third passage, and declining thereafter. This suggests that only the initial stage of adaptation is primarily driven by an acceleration in PrPSc replication. During the protracted adaptation to a new host, we observed significant alterations in the glycoform ratio and sialylation status of PrPSc N-glycans. These changes support the notion that qualitative modifications in PrPSc contribute to a more rapid disease progression. Furthermore, consistent with the decline in sialylation, a cue for "eat me" signaling, the newly adapted strain exhibited preferential colocalization with microglia. In contrast to PrPSc dynamics, the intensity of microglia activation continued to increase after the third passage in the new host. In summary, our study elucidates that the adaptation of a prion strain to a new host is a multi-step process driven by several factors.