Proteinase K-resistant PrP Sc Research Articles

Anle138b prevents PrP plaque accumulation in Tg(PrP-A116V) mice but does not mitigate clinical disease.

Anle138b is an anti-aggregating compound previously shown to delay the onset of scrapie, a transmissible prion disease, although its in vivo efficacy against other prion disease subtypes has not been fully assessed. TgGSS mice that model Gerstmann-Sträussler-Scheinker disease (GSS) via expression of mouse PrPA116V accumulate PrP amyloid plaques in their brains and develop progressive ataxia leading to death in ~160 days. When allowed to feed on food pellets containing anle138b from weaning until death, the brains of TgGSS mice displayed significant reductions in PrP plaque burden, insoluble PrP, and proteinase K-resistant PrPSc at end stage, compared with TgGSS mice allowed to feed on placebo food pellets. Despite these effects on biological markers of disease, there was no difference in the onset of symptoms or the age at death between the two treatment groups. In contrast, scrapie-inoculated wild-type mice treated with anle138b survived nearly twice as long (254 days) as scrapie-inoculated mice fed placebo (~136 days). They also displayed greater reductions in insoluble and PK-resistant PrPSc than TgGSS mice. Although these results support an anti-aggregating effect of anle138b, the discordance in clinical efficacy noted between the two prion disease models tested underscores the pathophysiological differences between them and highlights the need to consider differences in susceptibilities among prion subtypes when assessing potential therapies for prion diseases.

Iatrogenic Creutzfeldt-Jakob disease with Amyloid-\u03b2 pathology: an international study

The presence of pathology related to the deposition of amyloid-β (Aβ) has been recently reported in iatrogenic Creutzfeldt-Jakob disease (iCJD) acquired from inoculation of growth hormone (GH) extracted from human cadaveric pituitary gland or use of cadaveric dura mater (DM) grafts.To investigate this phenomenon further, a cohort of 27 iCJD cases – 21 with adequate number of histopathological sections – originating from Australia, France, Italy, and the Unites States, were examined by immunohistochemistry, amyloid staining, and Western blot analysis of the scrapie prion protein (PrPSc), and compared with age-group matched cases of sporadic CJD (sCJD), Alzheimer disease (AD) or free of neurodegenerative diseases (non-ND).Cases of iCJD and sCJD shared similar profiles of proteinase K-resistant PrPSc with the exception of iCJD harboring the “MMi” phenotype. Cerebral amyloid angiopathy (CAA), either associated with, or free of, Thioflavin S-positive amyloid core plaques (CP), was observed in 52% of 21 cases of iCJD, which comprised 37.5% and 61.5% of the cases of GH- and DM-iCJD, respectively. If only cases younger than 54 years were considered, Aβ pathology affected 41%, 2% and 0% of iCJD, sCJD and non-ND, respectively. Despite the patients’ younger age CAA was more severe in iCJD than sCJD, while Aβ diffuse plaques, in absence of Aβ CP, populated one third of sCJD. Aβ pathology was by far most severe in AD. Tau pathology was scanty in iCJD and sCJD.In conclusion, (i) despite the divergences in the use of cadaveric GH and DM products, our cases combined with previous studies showed remarkably similar iCJD and Aβ phenotypes indicating that the occurrence of Aβ pathology in iCJD is a widespread phenomenon, (ii) CAA emerges as the hallmark of the Aβ phenotype in iCJD since it is observed in nearly 90% of all iCJD with Aβ pathology reported to date including ours, and it is shared by GH- and DM-iCJD, (iii) although the contributions to Aβ pathology of other factors, including GH deficiency, cannot be discounted, our findings increase the mounting evidence that this pathology is acquired by a mechanism resembling that of prion diseases.

Transmissibility of H-Type Bovine Spongiform Encephalopathy to Hamster PrP Transgenic Mice.

Two distinct forms of atypical bovine spongiform encephalopathies (H-BSE and L-BSE) can be distinguished from classical (C-) BSE found in cattle based on biochemical signatures of disease-associated prion protein (PrPSc). H-BSE is transmissible to wild-type mice—with infected mice showing a long survival period that is close to their normal lifespan—but not to hamsters. Therefore, rodent-adapted H-BSE with a short survival period would be useful for analyzing H-BSE characteristics. In this study, we investigated the transmissibility of H-BSE to hamster prion protein transgenic (TgHaNSE) mice with long survival periods. Although none of the TgHaNSE mice manifested the disease during their lifespan, PrPSc accumulation was observed in some areas of the brain after the first passage. With subsequent passages, TgHaNSE mice developed the disease with a mean survival period of 220 days. The molecular characteristics of proteinase K-resistant PrPSc (PrPres) in the brain were identical to those observed in first-passage mice. The distribution of immunolabeled PrPSc in the brains of TgHaNSE mice differed between those infected with H-BSE as compared to C-BSE or L-BSE, and the molecular properties of PrPres in TgHaNSE mice infected with H-BSE differed from those of the original isolate. The strain-specific electromobility, glycoform profiles, and proteolytic cleavage sites of H-BSE in TgHaNSE mice were indistinguishable from those of C-BSE, in which the diglycosylated form was predominant. These findings indicate that strain-specific pathogenic characteristics and molecular features of PrPres in the brain are altered during cross-species transmission. Typical H-BSE features were restored after back passage from TgHaNSE to bovinized transgenic mice, indicating that the H-BSE strain was propagated in TgHaNSE mice. This could result from the overexpression of the hamster prion protein.

Prion-induced and spontaneous formation of transmissible toxicity in PrP transgenic Drosophila.

Prion diseases are fatal transmissible neurodegenerative diseases of various mammalian species. Central to these conditions is the conversion of the normal host prion protein PrP(C) into the abnormal prion conformer PrP(Sc). Mature PrP(C) is attached to the plasma membrane by a glycosylphosphatidylinositol anchor, whereas during biosynthesis and metabolism cytosolic and secreted forms of the protein may arise. The role of topological PrP(C) variants in the mechanism of prion formation and prion-induced neurotoxicity during prion disease remains undefined. In the present study we investigated whether Drosophila transgenic for ovine PrP targeted to the plasma membrane, to the cytosol or for secretion, could produce transmissible toxicity following exposure to exogenous ovine prions. Although all three topological variants of PrP were efficiently expressed in Drosophila, cytosolic PrP was conformationally distinct and required denaturation before recognition by immunobiochemical methods. Adult Drosophila transgenic for pan neuronally expressed ovine PrP targeted to the plasma membrane, to the cytosol or for secretion exhibited a decreased locomotor activity after exposure at the larval stage to ovine prions. Proteinase K-resistant PrP(Sc) was detected by protein misfolding cyclic amplification in prion-exposed Drosophila transgenic for membrane-targeted PrP. Significantly, head homogenate from all three variants of prion-exposed PrP transgenic Drosophila induced a decreased locomotor activity when transmitted to PrP recipient flies. Drosophila transgenic for PrP targeted for secretion exhibited a spontaneous locomotor defect in the absence of prion exposure that was transmissible in PrP transgenic flies. Our data are consistent with the formation of transmissible prions in PrP transgenic Drosophila.

Evidence That Bank Vole PrP Is a Universal Acceptor for Prions

Bank voles are uniquely susceptible to a wide range of prion strains isolated from many different species. To determine if this enhanced susceptibility to interspecies prion transmission is encoded within the sequence of the bank vole prion protein (BVPrP), we inoculated Tg(M109) and Tg(I109) mice, which express BVPrP containing either methionine or isoleucine at polymorphic codon 109, with 16 prion isolates from 8 different species: humans, cattle, elk, sheep, guinea pigs, hamsters, mice, and meadow voles. Efficient disease transmission was observed in both Tg(M109) and Tg(I109) mice. For instance, inoculation of the most common human prion strain, sporadic Creutzfeldt-Jakob disease (sCJD) subtype MM1, into Tg(M109) mice gave incubation periods of ∼200 days that were shortened slightly on second passage. Chronic wasting disease prions exhibited an incubation time of ∼250 days, which shortened to ∼150 days upon second passage in Tg(M109) mice. Unexpectedly, bovine spongiform encephalopathy and variant CJD prions caused rapid neurological dysfunction in Tg(M109) mice upon second passage, with incubation periods of 64 and 40 days, respectively. Despite the rapid incubation periods, other strain-specified properties of many prion isolates—including the size of proteinase K–resistant PrPSc, the pattern of cerebral PrPSc deposition, and the conformational stability—were remarkably conserved upon serial passage in Tg(M109) mice. Our results demonstrate that expression of BVPrP is sufficient to engender enhanced susceptibility to a diverse range of prion isolates, suggesting that BVPrP may be a universal acceptor for prions.

Protease-sensitive prions with 144-bp insertion mutations

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.

Propagation of RML prions in mice expressing PrP devoid of GPI anchor leads to formation of a novel, stable prion strain.

PrPC, a host protein which in prion-infected animals is converted to PrPSc, is linked to the cell membrane by a GPI anchor. Mice expressing PrPC without GPI anchor (tgGPI- mice), are susceptible to prion infection but accumulate anchorless PrPSc extra-, rather than intracellularly. We investigated whether tgGPI− mice could faithfully propagate prion strains despite the deviant structure and location of anchorless PrPSc. We found that RML and ME7, but not 22L prions propagated in tgGPI− brain developed novel cell tropisms, as determined by the Cell Panel Assay (CPA). Surprisingly, the levels of proteinase K-resistant PrPSc (PrPres) in RML- or ME7-infected tgGPI− brain were 25–50 times higher than in wild-type brain. When returned to wild-type brain, ME7 prions recovered their original properties, however RML prions had given rise to a novel prion strain, designated SFL, which remained unchanged even after three passages in wild-type mice. Because both RML PrPSc and SFL PrPSc are stably propagated in wild-type mice we propose that the two conformations are separated by a high activation energy barrier which is abrogated in tgGPI− mice.

Proteasome inhibitors promote the sequestration of PrPSc into aggresomes within the cytosol of prion-infected CAD neuronal cells

Dysfunction of the endoplasmic reticulum associated protein degradation/proteasome system is believed to contribute to the initiation or aggravation of neurodegenerative disorders associated with protein misfolding, and there is some evidence to suggest that proteasome dysfunctions might be implicated in prion disease. This study investigated the effect of proteasome inhibitors on the biogenesis of both the cellular (PrP(C)) and abnormal (PrP(Sc)) forms of prion protein in CAD neuronal cells, a newly introduced prion cell system. In uninfected cells, proteasome impairment altered the intracellular distribution of PrP(C), leading to a strong accumulation in the Golgi apparatus. Moreover, a detergent-insoluble and weakly protease-resistant PrP species of 26 kDa, termed PrP(26K), accumulated in the cells, whether they were prion-infected or not. However, no evidence was found that, in infected cells, this PrP(26K) species converts into the highly proteinase K-resistant PrP(Sc). In the infected cultures, proteasome inhibition caused an increased intracellular aggregation of PrP(Sc) that was deposited into large aggresomes. These findings strengthen the view that, in neuronal cells expressing wild-type PrP(C) from the natural promoter, proteasomal impairment may affect both the process of PrP(C) biosynthesis and the subcellular sites of PrP(Sc) accumulation, despite the fact that these two effects could essentially be disconnected.

Current status of scrapie.

Abstract Despite being controlled in many developed countries, scrapie remains endemic in many parts of the world. Economic losses result from mortality and culling of small ruminants and from market restrictions. Moreover, it is difficult to develop all-inclusive guidelines that could establish a scrapie-free status for a country. Unfortunately, the global picture remains incomplete because in many countries confusion still remains regarding the clinical picture of scrapie and information is not available owing to the absence of adequate epidemiosurveillance networks. Currently, the predominant theory is that PrPSc is the infectious agent where host genetic factors play a central role. The precise transmission routes of scrapie and their relative contributions to the overall transmission intensity remain poorly documented and the physiopathology is not fully understood. However, it is evident that the purchase of female sheep from scrapie flocks, sharing pastures with scrapie flocks, sharing breeding rams and genetic host susceptibility are the main risk factors for the spread of the disease. A better understanding of the epidemiology of scrapie would greatly aid the development and evaluation of control and eradication strategies that were mainly based on selective depopulation of infected animals and genetically susceptible and/or related animals and also on the biosecurity and the use of selective genetic breeding programmes in healthy flocks. Some numbers of a new transmissible spongiform encephalopathy (TSE) form in small ruminants (atypical scrapie) have meanwhile been identified by TSE rapid testing using an assay, which also recognizes comparatively less proteinase K-resistant PrPSc. Uncertainties remain regarding the pathogenesis of this new TSE form, as well as regarding its potential transmissibility within the affected species and to other species. Thus far, no bovine spongiform encephalopathy (BSE) cases have been confirmed in sheep under natural conditions (a report of vertical transmission after experimental infection merits attention), but two historical cases of BSE in goats born in the 1990s have been identified. Currently BSE must also be considered in the differential diagnosis of scrapie. The development of prevention and control programmes should be assisted by new scientific findings.

Biochemical fingerprints of prion diseases: scrapie prion protein in human prion diseases that share prion genotype and type

The phenotype of human prion diseases is influenced by the prion protein (PrP) genotype as determined by the methionine (M)/valine (V) polymorphism at codon 129, the scrapie PrP (PrPSc) type and the etiology. To gain further insight into the mechanisms of phenotype determination, we compared two-dimensional immunoblot profiles of detergent insoluble and proteinase K-resistant PrP species in a type of sporadic Creutzfeldt-Jakob disease (sCJDMM2), variant CJD (vCJD) and sporadic fatal insomnia (sFI). Full-length and truncated PrP forms present in the insoluble fractions were also separately analyzed. These three diseases were selected because they have the same M/M PrP genotype at codon 129 and the same type 2 PrPSc, but different etiologies, also sCJDMM2 and sFI are sporadic, whereas vCJD is acquired by infection. We observed minor differences in the PrP detergent-insoluble fractions between sCJDMM2 and vCJD, although both differ in the corresponding fractions from sFI. We detected more substantial heterogeneity between sCJDMM2 and vCJD in the two-dimensional blots of the proteinase K-resistant PrP fraction suggesting that different PrP species are selected for conversion to proteinase K-resistant PrP in sCJDMM2 and vCJD. These differences are mostly, but not exclusively, due to variations in the type of the N-linked glycans. We also show that the over-representation of the highly glycosylated forms distinctive of the proteinase K-resistant PrPSc of vCJD in one-dimensional blots is due to differences in both the amount and the natures of the glycans. Overall, these findings underline the complexity of phenotypic determination in human prion diseases.

The abnormal isoform of the prion protein accumulates in late‐endosome‐like organelles in scrapie‐infected mouse brain

The prion encephalopathies are characterized by accumulation in the brain of the abnormal form PrPsc of a normal host gene product PrPc. The mechanism and site of formation of PrPsc from PrPc are currently unknown. In this study, ME7 scrapie-infected mouse brain was used to show, both biochemically and by double-labelled immunogold electron microscopy, that proteinase K-resistant PrPsc is enriched in subcellular structures which contain the cation-independent mannose 6-phosphate receptor, ubiquitin-protein conjugates, beta-glucuronidase, and cathepsin B, termed late endosome-like organelles. The glycosylinositol phospholipid membrane-anchored PrPc will enter such compartment for normal degradation and the organelles may therefore act as chambers for the conversion of PrPc into infectious PrPsc in this murine model of scrapie.