Identification of distinct N-terminal truncated forms of prion protein in different Creutzfeldt-Jakob disease subtypes

In prion diseases, the cellular prion protein (PrP(C)) is converted to an insoluble and protease-resistant abnormal isoform termed PrP(Sc). In different prion strains, PrP(Sc) shows distinct sites of endogenous or exogenous proteolysis generating a core fragment named PrP27-30. Sporadic Creutzfeldt-Jakob disease (sCJD), the most frequent human prion disease, clinically presents with a variety of neurological signs. As yet, the clinical variability observed in sCJD has not been fully explained by molecular studies relating two major types of PrP27-30 with unglycosylated peptides of 21 (type 1) and 19 kDa (type 2) and the amino acid methionine or valine at position 129. Recently, smaller C-terminal fragments migrating at 12 and 13 kDa have been detected in different sCJD phenotypes, but their significance remains unclear. By using two-dimensional immunoblot with anti-PrP antibodies, we identified two novel groups of protease-resistant PrP fragments in sCJD brain tissues. All sCJD cases with type 1 PrP27-30, in addition to MM subjects with type 2 PrP27-30, were characterized by the presence of unglycosylated PrP fragments of 16-17 kDa. Conversely, brain homogenates from patients VV and MV with type 2 PrP27-30 contained fully glycosylated PrP fragments, which after deglycosylation migrated at 17.5-18 kDa. Interestingly, PrP species of 17.5-18 kDa matched deglycosylated forms of the C1 PrP(C) fragment and were associated with tissue PrP deposition as plaque-like aggregates or amyloid plaques. These data show the presence of multiple PrP(Sc) conformations in sCJD and, in addition, shed new light on the correlation between sCJD phenotypes and disease-associated PrP molecules.     

pH-dependent prion protein conformation in classical Creutzfeldt-Jakob disease.

In transmissible spongiform encephalopathies, the cellular prion protein (PrP(C)) undergoes a conformational change from a prevailing alpha-helical structure to a beta-sheet-rich, protease-resistant isoform, termed PrP(Sc). PrP(C) has two characteristics: a high affinity for Cu(2+) and a strong pH-dependent conformation. Lines of evidence indicate that PrP(Sc) conformation is dependent on copper and that acidic conditions facilitate the conversion of PrP(C) --> PrP(Sc). In each species, PrP(Sc) exists in multiple conformations, which are associated with different prion strains. In sporadic Creutzfeldt-Jakob disease (sCJD), different biochemical types of PrP(Sc) have been identified according to the size of the protease-resistant fragments, patterns of glycosylation, and the metal-ion occupancy. Based on the site of cleavage produced by proteinase K, we investigated the conformational stability of PrP(Sc) under acidic, neutral, and basic conditions in 42 sCJD subjects. Our study shows that only one type of sCJD PrP(Sc), associated with the classical form, shows a pH-dependent conformation, whereas two other biochemical PrP(Sc) types, detected in distinct sCJD phenotypes, are unaffected by pH variations. This novel approach demonstrates the presence of three types of PrP(Sc) in sCJD.     

Identification of glycoinositol phospholipid linked and truncated forms of the scrapie prion protein

Analysis of carboxy-terminal peptides derived from endoproteinase Lys-C digests of the scrapie isoform of the hamster prion protein revealed that the majority of the molecules are glycoinositol phospholipid linked through ethanolamine attached at serin-231. However, approximately 15% of PrPSc had a carboxy-terminal peptide that ends at glycine-228. It is intriguing that this glycine is part of the PrP sequence Gly-Arg-Arg, which is an established target sequence for the proteolysis and release of bioactive peptides from larger precursors. The mechanism of formation, as well as the role of the truncated carboxy terminus in the dissemination and neuropathology of scrapie, remains to be determined.     

Polymorphisms of the prion protein gene in Italian patients with Creutzfeldt-Jakob disease

Creutzfeldt-Jakob disease (CJD) is a transmissible neurodegenerative disorder characterized by the accumulation of the amyloid protein PrP in the CNS. Two coding polymorphisms of the PrP gene (PRNP) are a methionine (Met) to valine (Val) change at codon 129, and a deletion in the octapeptide coding region. In the United Kingdom, homozygosity at codon 129 appears to be associated with a predisposition to develop CJD. However, in Japan, where allelic frequencies and genotype distribution are significantly different, such an association has not been demonstrated. To determine whether such deletion(s) or codon 129 polymorphisms of PRNP predispose to the development of CJD in Italian patients, 31 sporadic CJD patients with no known PRNP mutations, and 186 unrelated control subjects were studied. Genotypic frequencies at codon 129 in these Italian CJD patients revealed a significant excess of methionine alleles, and a different genotype distribution in comparison with the normal Italian population. Deletions of a 24-bp segment located in the PrP octapeptide coding region were found in two control subjects, but in none of the sporadic CJD patients. These data suggest that Met homozygosity at codon 129 may contribute, with other enviromental or endogenous factors, to CJD development.     

Heterogeneity of normal prion protein in two- dimensional immunoblot: presence of various glycosylated and truncated forms

The common use of one-dimensional (1-D) immunoblot with a single monoclonal antibody (Mab) engenders the notion that the normal or cellular prion protein (PrP(C) ) comprises few and simple forms. In this study we used two-dimensional (2-D) immunoblot with a panel Mabs to various regions of the prion protein to demonstrate the complexity of the PrP(C) present in human brain. We distinguished over 50 immunoblot spots, each representing a distinct PrP(C) species based on combinations of different molecular weights and isoelectric points (pIs). The PrP(C) heterogeneity is due to the presence of a full-length and two major truncated forms as well as to the diversity of the glycans linked to most of these forms. The two major truncated forms result from distinct cleavage sites located at the N-terminus. In addition, enzymatic removal of sialic acid and lectin binding studies indicate that the glycans linked to the full-length and truncated PrP(C) forms differ in their structure and ratios of the glycoforms. The truncation of PrP(C) and the heterogeneity of the linked glycans may play a role in regulating PrP(C) function. Furthermore, the presence of relatively large quantities of different PrP(C) species may provide additional mechanisms by which the diversity of prion strains could be generated.     

Iatrogenic and sporadic Creutzfeldt-Jakob disease in 2 sisters without mutation in the prion protein gene

ABSTRACT

Human genetic prion diseases have invariably been linked to alterations of the prion protein (PrP) gene PRNP. Two sisters died from probable Creutzfeldt-Jakob disease (CJD) in Switzerland within 14 y. At autopsy, both patients had typical spongiform change in their brains accompanied by punctuate deposits of PrP. Biochemical analyses demonstrated proteinase K-resistant PrP. Sequencing of PRNP showed 2 wild-type alleles in both siblings. Retrospectively, clinical data revealed a history of dural transplantation in the initially deceased sister, compatible with a diagnosis of iatrogenic CJD. Clinical and familial histories provided no evidence for potential horizontal transmission. This observation of 2 siblings suffering from CJD without mutations in the PRNP gene suggests potential involvement of non-PRNP genes in prion disease etiology.     

Amyloid- and FDG-PET in sporadic Creutzfeldt-Jakob disease: Correlation with pathological prion protein in neuropathology

Introduction. The role of positron emission tomography (PET) in Creutzfeldt-Jakob disease is less defined than in other neurodegenerative diseases. We studied the correlation between the uptake of ¹⁸F-florbetaben and ¹⁸F-fluorodeoxyglucose with pathological prion protein deposition in histopathology in a case.Methods. A patient with 80 y old with a rapid neurological deterioration with a confirmed diagnosis of CJD was studied. PET and MRI studies were performed between 13–20 d before the death. A region of interest analysis was performed using Statistical Parametric Mapping.Results. MRI showed atrophy with no other alterations. FDG-PET showed extensive areas of hypometabolism including left frontoparietal lobes as well as bilateral thalamus. Correlation between uptake of ¹⁸F-florbetaben and pathological prion protein deposition was r = 0.786 (p < 0.05). Otherwise, correlation between uptake of ¹⁸F-FDG and pathological prion protein was r = 0.357 (p = 0.385). Immunohistochemistry with β-amyloid did not show amyloid deposition or neuritic plaques.Conclusions. Our study supports the use of FDG-PET in the assessment of CJD. FDG-PET may be especially useful in cases of suspected CJD and negative MRI. Furthermore, this case report provides more evidence about the behavioral of amyloid tracers, and the possibility of a low-affinity binding to other non-amyloid proteins, such as the pathological prion protein, is discussed.     

Abnormal isoform of prion protein accumulates in follicular dendritic cells in mice with Creutzfeldt-Jakob disease.

We established that follicular dendritic cells (FDCs) are the site of abnormal prion protein (PrPCJD) accumulations in lymphoid tissues from mice infected with Creutzfeldt-Jakob disease. Evidence of positive FDC staining was observed in Creutzfeldt-Jakob disease-infected mice irrespective of the inoculation route, while no such staining was seen in the control mice. We also found that the severe combined immunodeficiency mouse trait is transmittable via the intracranial route but not via the intraperitoneal route. Mice with severe combined immunodeficiency did not have PrPCJD accumulation in FDCs.     

Creutzfeldt-Jakob disease associated with a V203I homozygous mutation in the prion protein gene

We report a Japanese patient with Creutzfeldt-Jakob disease (CJD) with a V203I homozygous mutation of the prion protein gene (PRNP). A 73-year-old woman developed rapidly progressive gait disturbance and cognitive dysfunction. Four months after the onset, she entered a state of an akinetic mutism. Gene analysis revealed a homozygous V203I mutation in the PRNP. Familial CJD with a V203I mutation is rare, and all previously reported cases had a heterozygous mutation showing manifestations similar to those of typical sporadic CJD. Although genetic prion diseases with homozygous PRNP mutations often present with an earlier onset and more rapid clinical course than those with heterozygous mutations, no difference was found in clinical phenotype between our homozygous case and reported heterozygous cases.     

Cell surface accumulation of a truncated transmembrane prion protein in Gerstmann-Straussler-Scheinker disease P102L

A familial prion disorder with a proline to leucine substitution at residue 102 of the prion protein (PrP(102L)) is typically associated with protease-resistant PrP fragments (PrP(Sc)) in the brain parenchyma that are infectious to recipient animals. When modeled in transgenic mice, a fatal neurodegenerative disease develops, but, unlike the human counterpart, PrP(Sc) is lacking and transmission to recipient animals is questionable. Alternate mice expressing a single copy of PrP(102L) (mouse PrP(101L)) do not develop spontaneous disease, but show dramatic susceptibility to PrP(Sc) isolates from different species. To understand these discrepant results, we studied the biogenesis of human PrP(102L) in a cell model. Here, we report that cells expressing PrP(102L) show decreased expression of the normal 18-kDa fragment on the plasma membrane. Instead, a 20-kDa fragment, probably derived from transmembrane PrP ((Ctm)PrP), accumulates on the cell surface. Because the 20-kDa fragment includes an amyloidogenic region of PrP that is disrupted in the 18-kDa form, increased surface expression of 20-kDa fragment may enhance the susceptibility of these cells to PrP(Sc) infection by providing an optimal substrate, or by amplifying the neurotoxic signal of PrP(Sc). Thus, altered susceptibility of PrP(101L) mice to exogenous PrP(Sc) may be mediated by the 20-kDa (Ctm)PrP fragment, rather than PrP(102L) per se.     

The effects of lysosomal and proteasomal inhibitors on abnormal forms of prion protein degradation in murine macrophages

It has been reported that macrophages degrade infectious forms of prion protein (PrP(Sc) ). In order to investigate the mechanisms underlying PrP(Sc) degradation in macrophages, the effects of lysosomal and proteasomal inhibitors on macrophage cell lines which were incubated with scrapie-affected brain homogenate were studied. PrP(Sc) degradation was inhibited in the presence of both proteasomal and lysosomal inhibitors. Indirect fluorescence assays to determine the cellular localization of PrP(Sc) were undertaken. PrP(Sc) colocalized with the lysosomal membrane protein Lamp-1 and ubiquitin, a protein that is related to the proteasome. The present data indicate that macrophages might degrade PrP(Sc) via the lysosomal and proteasomal pathways.     

Chronic wasting disease of elk and deer and Creutzfeldt-Jakob disease: comparative analysis of the scrapie prion protein

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.     

Mutation and polymorphism of the prion protein gene in Libyan Jews with Creutzfeldt-Jakob disease (CJD).

The inherited prion diseases are neurodegenerative disorders which are not only genetic but also transmissible. More than a dozen mutations in the prion protein gene that result in nonconservative amino acid substitutions segregate with the inherited prion diseases including familial Creutzfeldt-Jakob disease (CJD). In Israel, the incidence of CJD is about 1 case/10(4) Libyan Jews. A Lys200 substitution segregates with CJD and is reported here to be genetically linked to CJD with a lod score of > 4.8. Some healthy elderly Lys200 carriers > age 65 years were identified, suggesting the possibility of incomplete penetrance. In contrast, no linkage was found between the development of familial CJD and a polymorphism encoding either Met129 or Val129. All Libyan Jewish CJD patients with the Lys200 mutation encode a Met129 on the mutant allele. Homozygosity for Met129 did not correlate with age at disease onset or the duration of illness. The frequency of the Met129 allele was higher in the affected pedigrees than in a control population of Libyan Jews. The frequency of the Met129 and Val129 alleles in the control Libyan population was similar to that found in the general Caucasian population. The identification of three Libyan Jews homozygous for the Lys200 mutation suggests frequent intrafamilial marriages, a custom documented by genealogical investigations.     

Fatal prion disease in a mouse model of genetic E200K Creutzfeldt-Jakob disease

Genetic prion diseases are late onset fatal neurodegenerative disorders linked to pathogenic mutations in the prion protein-encoding gene, PRNP. The most prevalent of these is the substitution of Glutamate for Lysine at codon 200 (E200K), causing genetic Creutzfeldt-Jakob disease (gCJD) in several clusters, including Jews of Libyan origin. Investigating the pathogenesis of genetic CJD, as well as developing prophylactic treatments for young asymptomatic carriers of this and other PrP mutations, may well depend upon the availability of appropriate animal models in which long term treatments can be evaluated for efficacy and toxicity. Here we present the first effective mouse model for E200KCJD, which expresses chimeric mouse/human (TgMHu2M) E199KPrP on both a null and a wt PrP background, as is the case for heterozygous patients and carriers. Mice from both lines suffered from distinct neurological symptoms as early as 5-6 month of age and deteriorated to death several months thereafter. Histopathological examination of the brain and spinal cord revealed early gliosis and age-related intraneuronal deposition of disease-associated PrP similarly to human E200K gCJD. Concomitantly we detected aggregated, proteinase K resistant, truncated and oxidized PrP forms on immunoblots. Inoculation of brain extracts from TgMHu2ME199K mice readily induced, the first time for any mutant prion transgenic model, a distinct fatal prion disease in wt mice. We believe that these mice may serve as an ideal platform for the investigation of the pathogenesis of genetic prion disease and thus for the monitoring of anti-prion treatments.     

Cross-sequence transmission of sporadic Creutzfeldt-Jakob disease creates a new prion strain

The genotype (methionine or valine) at polymorphic codon 129 of the human prion protein (PrP) gene and the type (type 1 or type 2) of abnormal isoform of PrP (PrP(Sc)) are major determinants of the clinicopathological phenotypes of sporadic Creutzfeldt-Jakob disease (sCJD). Here we found that the transmission of sCJD prions from a patient with valine homozygosity (129V/V) and type 2 PrP(Sc) (sCJD-VV2 prions) to mice expressing human PrP with methionine homozygosity (129M/M) generated unusual PrP(Sc) intermediate in size between type 1 and type 2. The intermediate type PrP(Sc) was seen in all examined dura mater graft-associated CJD cases with 129M/M and plaque-type PrP deposits (p-dCJD). p-dCJD prions and sCJD-VV2 prions exhibited similar transmissibility and neuropathology, and the identical type of PrP(Sc) when inoculated into PrP-humanized mice with 129M/M or 129V/V. These findings suggest that p-dCJD could be caused by cross-sequence transmission of sCJD-VV2 prions.     

N-terminal truncation of prion protein affects both formation and conformation of abnormal protease-resistant prion protein generated in vitro

Transmissible spongiform encephalopathy diseases are characterized by conversion of the normal protease-sensitive host prion protein, PrP-sen, to an abnormal protease-resistant form, PrP-res. In the current study, deletions were introduced into the flexible tail of PrP-sen (23) to determine if this region was required for formation of PrP-res in a cell-free assay. PrP-res formation was significantly reduced by deletion of residues 34-94 relative to full-length hamster PrP. Deletion of another nineteen amino acids to residue 113 further reduced the amount of PrP-res formed. Furthermore, the presence of additional proteinase K cleavage sites indicated that deletion to residue 113 generated a protease-resistant product with an altered conformation. Conversion of PrP deletion mutants was also affected by post-translational modifications to PrP-sen. Conversion of unglycosylated PrP-sen appeared to alter both the amount and the conformation of protease-resistant PrP-res produced from N-terminally truncated PrP-sen. The N-terminal region also affected the ability of hamster PrP to block mouse PrP-res formation in scrapie-infected mouse neuroblastoma cells. Thus, regions within the flexible N-terminal tail of PrP influenced interactions required for both generating and disrupting PrP-res formation.     

Cytosolic prion protein is the predominant anti-Bax prion protein form: exclusion of transmembrane and secreted prion protein forms in the anti-Bax function

Prion protein (PrP) prevents Bax-mediated cell death by inhibiting the initial Bax conformational change that converts cytosolic Bax into a pro-apoptotic protein. PrP is mostly a glycophosphatidylinositol-anchored cell surface protein but it is also retrotranslocated into cytosolic PrP (CyPrP) or can become a type 1 or type 2 transmembrane protein. To determine the form and subcellular location of the PrP that has anti-Bax function, we co-expressed various Syrian hamster PrP (SHaPrP) mutants that favour specific PrP topologies and subcellular localization with N-terminally green fluorescent protein tagged pro-apoptotic Bax (EGFP-Bax) in MCF-7 cells and primary human neurons. Mutants that generate both CyPrP and secreted PrP ((Sec)PrP) or only CyPrP have anti-Bax activity. Mutants that produce (Ctm)PrP or (Ntm)PrP lose the anti-Bax activity, despite their ability to also make (Sec)PrP. Transmembrane-generating mutants do not produce CyPrP and both normal and cognate mutant forms of CyPrP rescue against the loss of anti-Bax activity. (Sec)PrP-generating constructs also produce non-membrane attached (Sec)PrP. However, this form of PrP has minimal anti-Bax activity. We conclude that CyPrP is the predominant form of PrP with anti-Bax function. These results imply that the retrotranslocation of PrP encompasses a survival function and is not merely a pathway for the proteasomal degradation of misfolded protein.     

Diversity in neuroanatomical distribution of abnormal prion protein in atypical scrapie

Scrapie is a transmissible spongiform encephalopathy (TSE) in sheep and goats. In recent years, atypical scrapie cases were identified that differed from classical scrapie in the molecular characteristics of the disease-associated pathological prion protein (PrP(sc)). In this study, we analyze the molecular and neuropathological phenotype of nine Swiss TSE cases in sheep and goats. One sheep was identified as classical scrapie, whereas six sheep, as well as two goats, were classified as atypical scrapie. The latter revealed a uniform electrophoretic mobility pattern of the proteinase K-resistant core fragment of PrP(sc) distinct from classical scrapie regardless of the genotype, the species, and the neuroanatomical structure. Remarkably different types of neuroanatomical PrP(sc) distribution were observed in atypical scrapie cases by both western immunoblotting and immunohistochemistry. Our findings indicate that the biodiversity in atypical scrapie is larger than expected and thus impacts on current sampling and testing strategies in small ruminant TSE surveillance.