-
Silvio Notari,
Liuting Qing,
Maurizio Pocchiari,
Ayuna Dagdanova,
Kristin Hatcher,
Arend Dogterom,
Jose F Groisman,
Ib Bo Lumholtz,
Maria Puopolo,
Corinne Lasmezas,
Shu G Chen,
Qingzhong Kong,
Pierluigi Gambetti
[show abstract]
[hide abstract]
ABSTRACT: Prion diseases are neurodegenerative conditions associated with a misfolded and infectious protein, scrapie prion protein (PrP(Sc)). PrP(Sc) propagate prion diseases within and between species and thus pose risks to public health. Prion infectivity or PrP(Sc) presence has been demonstrated in urine of experimentally infected animals, but there are no recent studies of urine from patients with Creutzfeldt-Jakob disease (CJD). We performed bioassays in transgenic mice expressing human PrP to assess prion infectivity in urine from patients affected by a common subtype of sporadic CJD, sCJDMM1. We tested raw urine and 100-fold concentrated and dialyzed urine and assessed the sensitivity of the bioassay along with the effect of concentration and dialysis on prion infectivity. Intracerebral inoculation of transgenic mice with urine from 3 sCJDMM1 patients failed to demonstrate prion disease transmission, indicating that prion infectivity in urine from sCJDMM1 patients is either not present or is <0.38 infectious units/mL.
Emerging Infectious Diseases 01/2012; 18(1):21-8. · 6.79 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Prion diseases are believed to propagate by the mechanism involving self-perpetuating conformational conversion of the normal form of the prion protein, PrP(C), to the misfolded, pathogenic state, PrP(Sc). One of the most intriguing aspects of these disorders is the phenomenon of prion strains. It is believed that strain properties are fully encoded in distinct conformations of PrP(Sc). Strains are of practical relevance to human prion diseases as their diversity may explain the unusual heterogeneity of these disorders. The first insight into the molecular mechanisms underlying heterogeneity of human prion diseases was provided by the observation that two distinct disease phenotypes and their associated PrP(Sc) conformers co-distribute with distinct PrP genotypes as determined by the methionine/valine polymorphism at codon 129 of the PrP gene. Subsequent studies identified six possible combinations of the three genotypes (determined by the polymorphic codon 129) and two common PrP(Sc) conformers (named types 1 and 2) as the major determinants of the phenotype in sporadic human prion diseases. This scenario implies that each 129 genotype-PrP(Sc) type combination would be associated with a distinct disease phenotype and prion strain. However, notable exceptions have been found. For example, two genotype-PrP(Sc) type combinations are linked to the same phenotype, and conversely, the same combination was found to be associated with two distinct phenotypes. Furthermore, in some cases, PrP(Sc) conformers naturally associated with distinct phenotypes appear, upon transmission, to lose their phenotype-determining strain characteristics. Currently it seems safe to assume that typical sporadic prion diseases are associated with at least six distinct prion strains. However, the intrinsic characteristics that distinguish at least four of these strains remain to be identified.
Acta Neuropathologica 11/2010; 121(1):79-90. · 9.32 Impact Factor
-
Ayuna Dagdanova,
Serguei Ilchenko, Silvio Notari,
Qiwei Yang,
Mark E Obrenovich,
Kristen Hatcher,
Peter McAnulty,
Lequn Huang,
Wenquan Zou,
Qingzhong Kong,
Pierluigi Gambetti,
Shu G Chen
[show abstract]
[hide abstract]
ABSTRACT: The presence of the prion protein (PrP) in normal human urine is controversial and currently inconclusive. This issue has taken a special relevance because prion infectivity has been demonstrated in urine of animals carrying experimental or naturally occurring prion diseases, but the actual presence and tissue origin of the infectious prion have not been determined. We used immunoprecipitation, one- and two-dimensional electrophoresis, and mass spectrometry to prove definitely the presence of PrP in human urine and its post-translational modifications. We show that urinary PrP (uPrP) is truncated mainly at residue 112 but also at other residues up to 122. This truncation makes uPrP undetectable with some commonly used antibodies to PrP. uPrP is glycosylated and carries an anchor which, at variance with that of cellular PrP, lacks the inositol-associated phospholipid moiety, indicating that uPrP is probably shed from the cell surface. The detailed characterization of uPrP reported here definitely proves the presence of PrP in human urine and will help determine the origin of prion infectivity in urine.
Journal of Biological Chemistry 10/2010; 285(40):30489-95. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Six clinico-pathological phenotypes of sporadic Creutzfeldt-Jakob disease have been characterized which correlate at the molecular level with the type (1 or 2) of the abnormal prion protein, PrP(TSE), present in the brain and with the genotype of polymorphic (methionine or valine) codon 129 of the prion protein gene. However, to what extent these phenotypes with their corresponding molecular combinations (i.e. MM1, MM2, VV1 etc.) encipher distinct prion strains upon transmission remains uncertain. We studied the PrP(TSE) type and the prion protein gene in archival brain tissues from the National Institutes of Health series of transmitted Creutzfeldt-Jakob disease and kuru cases, and characterized the molecular and pathological phenotype in the affected non-human primates, including squirrel, spider, capuchin and African green monkeys. We found that the transmission properties of prions from the common sporadic Creutzfeldt-Jakob disease MM1 phenotype are homogeneous and significantly differ from those of sporadic Creutzfeldt-Jakob disease VV2 or MV2 prions. Animals injected with iatrogenic Creutzfeldt-Jakob disease MM1 and genetic Creutzfeldt-Jakob disease MM1 linked to the E200K mutation showed the same phenotypic features as those infected with sporadic Creutzfeldt-Jakob disease MM1 prions, whereas kuru most closely resembled the sporadic Creutzfeldt-Jakob disease VV2 or MV2 prion signature and neuropathology. The findings indicate that two distinct prion strains are linked to the three most common Creutzfeldt-Jakob disease clinico-pathological and molecular subtypes and kuru, and suggest that kuru may have originated from cannibalistic transmission of a sporadic Creutzfeldt-Jakob disease of the VV2 or MV2 subtype.
Brain 10/2010; 133(10):3030-42. · 9.46 Impact Factor
-
Wen-Quan Zou,
Gianfranco Puoti,
Xiangzhu Xiao,
Jue Yuan,
Liuting Qing,
Ignazio Cali,
Miyuki Shimoji,
Jan P M Langeveld,
Rudy Castellani, Silvio Notari, [......],
Sabina Capellari,
Giorgio Giaccone,
Ermias D Belay,
Lawrence B Schonberger,
Mark Cohen,
George Perry,
Qingzhong Kong,
Piero Parchi,
Fabrizio Tagliavini,
Pierluigi Gambetti
[show abstract]
[hide abstract]
ABSTRACT: The objective of the study is to report 2 new genotypic forms of protease-sensitive prionopathy (PSPr), a novel prion disease described in 2008, in 11 subjects all homozygous for valine at codon 129 of the prion protein (PrP) gene (129VV). The 2 new PSPr forms affect individuals who are either homozygous for methionine (129MM) or heterozygous for methionine/valine (129MV).
Fifteen affected subjects with 129MM, 129MV, and 129VV underwent comparative evaluation at the National Prion Disease Pathology Surveillance Center for clinical, histopathologic, immunohistochemical, genotypical, and PrP characteristics.
Disease duration (between 22 and 45 months) was significantly different in the 129VV and 129MV subjects. Most other phenotypic features along with the PrP electrophoretic profile were similar but distinguishable in the 3 129 genotypes. A major difference laid in the sensitivity to protease digestion of the disease-associated PrP, which was high in 129VV but much lower, or altogether lacking, in 129MV and 129MM. This difference prompted the substitution of the original designation with "variably protease-sensitive prionopathy" (VPSPr). None of the subjects had mutations in the PrP gene coding region.
Because all 3 129 genotypes are involved, and are associated with distinguishable phenotypes, VPSPr becomes the second sporadic prion protein disease with this feature after Creutzfeldt-Jakob disease, originally reported in 1920. However, the characteristics of the abnormal prion protein suggest that VPSPr is different from typical prion diseases, and perhaps more akin to subtypes of Gerstmann-Sträussler-Scheinker disease.
Annals of Neurology 08/2010; 68(2):162-72. · 11.09 Impact Factor
-
Silvio Notari,
Francisco J Moleres,
Stephen B Hunter,
Ermias D Belay,
Lawrence B Schonberger,
Ignazio Cali,
Piero Parchi,
Wun-Ju Shieh,
Paul Brown,
Sherif Zaki,
Wen-Quan Zou,
Pierluigi Gambetti
[show abstract]
[hide abstract]
ABSTRACT: Variant Creutzfeldt-Jakob disease (vCJD) is a prion disease thought to be acquired by the consumption of prion-contaminated beef products. To date, over 200 cases have been identified around the world, but mainly in the United Kingdom. Three cases have been identified in the United States; however, these subjects were likely exposed to prion infection elsewhere. Here we report on the first of these subjects.
Neuropathological and genetic examinations were carried out using standard procedures. We assessed the presence and characteristics of protease-resistant prion protein (PrP(res)) in brain and 23 other organs and tissues using immunoblots performed directly on total homogenate or following sodium phosphotungstate precipitation to increase PrP(res) detectability. The brain showed a lack of typical spongiform degeneration and had large plaques, likely stemming from the extensive neuronal loss caused by the long duration (32 months) of the disease. The PrP(res) found in the brain had the typical characteristics of the PrP(res) present in vCJD. In addition to the brain and other organs known to be prion positive in vCJD, such as the lymphoreticular system, pituitary and adrenal glands, and gastrointestinal tract, PrP(res) was also detected for the first time in the dura mater, liver, pancreas, kidney, ovary, uterus, and skin.
Our results indicate that the number of organs affected in vCJD is greater than previously realized and further underscore the risk of iatrogenic transmission in vCJD.
PLoS ONE 01/2010; 5(1):e8765. · 4.09 Impact Factor
-
Piero Parchi,
Rosaria Strammiello, Silvio Notari,
Armin Giese,
Jan P M Langeveld,
Anna Ladogana,
Inga Zerr,
Federico Roncaroli,
Patrich Cras,
Bernardino Ghetti,
Maurizio Pocchiari,
Hans Kretzschmar,
Sabina Capellari
[show abstract]
[hide abstract]
ABSTRACT: Six subtypes of sporadic Creutzfeldt-Jakob disease with distinctive clinico-pathological features have been identified largely based on two types of the abnormal prion protein, PrP(Sc), and the methionine (M)/valine (V) polymorphic codon 129 of the prion protein. The existence of affected subjects showing mixed phenotypic features and concurrent PrP(Sc) types has been reported but with inconsistencies among studies in both results and their interpretation. The issue currently complicates diagnosis and classification of cases and also has implications for disease pathogenesis. To explore the issue in depth, we carried out a systematic regional study in a large series of 225 cases. PrP(Sc) types 1 and 2 concurrence was detected in 35% of cases and was higher in MM than in MV or VV subjects. The deposition of either type 1 or 2, when concurrent, was not random and always characterized by the coexistence of phenotypic features previously described in the pure subtypes. PrP(Sc) type 1 accumulation and related pathology predominated in MM and MV cases, while the type 2 phenotype prevailed in VVs. Neuropathological examination best identified the mixed types 1 and 2 features in MMs and most MVs, and also uniquely revealed the co-occurrence of pathological variants sharing PrP(Sc) type 2. In contrast, molecular typing best detected the concurrent PrP(Sc) types in VV subjects and MV cases with kuru plaques. The present data provide an updated disease classification and are of importance for future epidemiologic and transmission studies aimed to identify etiology and extent of strain variation in sporadic Creutzfeldt-Jakob disease.
Acta Neuropathologica 09/2009; 118(5):659-71. · 9.32 Impact Factor
-
Silvio Notari,
Rosaria Strammiello,
Sabina Capellari,
Armin Giese,
Maura Cescatti,
Jacques Grassi,
Bernardino Ghetti,
Jan P M Langeveld,
Wen-Quan Zou,
Pierluigi Gambetti,
Hans A Kretzschmar,
Piero Parchi
[show abstract]
[hide abstract]
ABSTRACT: In prion disease, the abnormal conformer of the cellular prion protein, PrP(Sc), deposits in fibrillar protein aggregates in brain and other organs. Limited exposure of PrP(Sc) to proteolytic digestion in vitro generates a core fragment of 19-21 kDa, named PrP27-30, which is also found in vivo. Recent evidence indicates that abnormal truncated fragments other than PrP27-30 may form in prion disease either in vivo or in vitro. We characterized a novel protease-resistant PrP fragment migrating 2-3 kDa faster than PrP27-30 in Creutzfeldt-Jakob disease (CJD) brains. The fragment has a size of about 18.5 kDa when associated with PrP27-30 type 1 (21 kDa) and of 17 kDa when associated with type 2 (19 kDa). Molecular mass and epitope mapping showed that the two fragments share the primary N-terminal sequence with PrP27-30 types 1 and 2, respectively, but lack a few amino acids at the very end of C terminus together with the glycosylphosphatidylinositol anchor. The amounts of the 18.5- or 17-kDa fragments and the previously described 13-kDa PrP(Sc) C-terminal fragment relatively to the PrP27-30 signal significantly differed among CJD subtypes. Furthermore, protease digestion of PrP(Sc) or PrP27-30 in partially denaturing conditions generated an additional truncated fragment of about 16 kDa only in typical sporadic CJD (i.e. MM1). These results show that the physicochemical heterogeneity of PrP(Sc) in CJD extends to abnormal truncated forms of the protein. The findings support the notion of distinct structural "conformers" of PrP(Sc) and indicate that the characterization of truncated PrP(Sc) forms may further improve molecular typing in CJD.
Journal of Biological Chemistry 09/2008; 283(45):30557-65. · 4.77 Impact Factor
-
Piero Parchi, Silvio Notari,
Petra Weber,
Heinz Schimmel,
Herbert Budka,
Isidre Ferrer,
Stéphane Haik,
Jean-Jacques Hauw,
Mark W Head,
James W Ironside,
Lucia Limido,
Agustin Rodriguez,
Thomas Ströbel,
Fabrizio Tagliavini,
Hans A Kretzschmar
[show abstract]
[hide abstract]
ABSTRACT: Molecular typing is of considerable importance for the surveillance and epidemiology of human transmissible spongiform encephalopathies (TSEs). It relies on the detection of distinct protease-resistant prion protein (PrP(Sc)) core fragments that differ in molecular mass and/or glycoform ratio. In this collaborative study, we tested the inter-laboratory agreement in TSE molecular typing. Sixteen characterized brain specimens from sporadic TSEs and variant Creutzfeldt-Jakob disease (vCJD) cases were distributed blindly to seven laboratories for molecular characterization by a defined protocol and classification. Agreement between laboratories in the classification of samples was excellent. In particular, there were no differences in the distinction between PrP(Sc) type 1, type 2A, and type 2B with one exception, which eventually was identified as a case with types 1 and 2 co-occurrence. This shows that the general technique and particular classification system used here are robust and represent a reliable basis for diagnostic and epidemiologic purposes. The subtle further distinction of subtypes among type 1 and type 2 groups requires high-sensitivity gel electrophoresis protocols that are unsuitable for routine diagnostic needs and must be reserved for research investigations. Further research is necessary on the identification and significance of co-occurrence of PrP(Sc) types 1 and 2 within one brain.
Brain Pathology 08/2008; 19(3):384-91. · 3.99 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Molecular typing in Creutzfeldt-Jakob disease (CJD) relies on the detection of distinct protease-resistant prion protein (PrP(Sc)) core fragments, which differ in molecular mass or glycoform ratio. However, the definition and correct identification of CJD cases with a co-occurrence of PrP(Sc) types remains a challenge. With antibodies recognizing a linear epitope in the octapeptide repeat PrP region, supposed to distinguish between the two major PrP(Sc) isoforms (ie, types 1 and 2), it was recently shown that all type 2 cases display an associated band with a type 1 migration pattern, which led to the conclusion that multiple PrP(Sc) types regularly coexist in CJD. We studied brain samples from 53 sporadic CJD and 4 variant CJD subjects using a high-resolution electrophoresis, a wide range of proteinase K (PK) activities, the 'type 1-selective' antibody 12B2, and several unselective antibodies. We found that the type 1-like band detected by 12B2 in all CJD subtypes associated with PrP(Sc) type 2 is not a PK-resistant PrP(Sc) core but rather matches the physicochemical properties of partially cleaved fragments, which result from the several PK cleavage sites included in the N-terminal portion of PrP(Sc). Furthermore, using gels with high resolution and a relatively high PK activity, we were able to increase the detection sensitivity of either type 1 or 2, when coexisting, to amount corresponding to 3-5% of the total PrP(Sc) signal (ie, weak band of one type/total PrP(Sc)). Our results show that there are many pitfalls associated with the use of 'type 1 selective' antibodies for CJD typing studies and that co-occurrence of PrP(Sc) types in CJD is not the rule. The present results further validate the rationale basis of current CJD classification and the qualitative nature of molecular typing in CJD.
Laboratory Investigation 12/2007; 87(11):1103-12. · 3.64 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Much progress has been made in understanding the molecular basis of phenotypic variability in Creutzfeldt-Jakob disease (CJD)
in the last ten years. The most significant advance was the discovery that the genotype at polymorphic codon 129 of PRNP and the “type” of the protease-resistant prion protein fragment, PrP-res, have a major influence on the disease phenotype
in all forms of CJD, irrespective of their etiology. The most widely accepted CJD classification includes six clinico-pathological
phenotypes and two major types of PrP-res, types 1 and 2, which can be distinguished on the basis of a ∼2 kDa difference in
relative molecular mass of the protein fragment. However, alternative classifications of human PrP-res types distinguished
three patterns of PrP-res molecular mass instead of two, thereby creating significant confusion in the field. Fortunately,
progress has been recently made in clarifying these disparities. Most significant in this regard, has been the finding that
pH variation among CJD brain homogenates in standard buffers influences the size of PrP-res. Thus, some of the PrP-res heterogeneity
used to identify putative strain-specific PrP-res types simply represents a technical “artefact” related to the experimental
conditions. On the other hand, recent data have also shown that PrP-res types 1 and 2 are heterogeneous biological species,
which can be further distinguished into molecular subtypes that fit the current histopathological classification of sporadic
CJD in 6 subtypes. Finally, novel truncated PrP-res fragments of smaller size than PrP-res types 1 and 2 have recently been
identified in CJD. Although more studies are needed to fully characterize the presence, characteristics and biological significance
of these peptides, preliminary results indicate that their search and characterization may be useful in the molecular diagnostics
of CJD subtypes.
06/2006: pages 77-95;
-
[show abstract]
[hide abstract]
ABSTRACT: The discovery of molecular subtypes of the pathological prion protein PrPSc has provided the basis for a novel classification of human transmissible spongiform encephalopathies (TSEs) and a potentially powerful method for strain typing. However, there is still a significant disparity regarding the understanding and nomenclature of PrPSc types. In addition, it is still unknown whether a specific PrPSc type is associated with each TSE phenotypic variant. In sporadic Creutzfeldt-Jakob disease (sCJD), five disease phenotypes are known, but only two major types of PrPSc, types 1 and 2, have been consistently reproduced. We further analyzed PrPSc properties in sCJD and variant CJD using a high resolution gel electrophoresis system and varying experimental conditions. We found that pH varies among CJD brain homogenates in standard buffers, thereby influencing the characteristics of protease-treated PrPSc. We also show that PrPSc type 1 and type 2 are heterogeneous species which can be further distinguished into five molecular subtypes that fit the current histopathological classification of sCJD variants. Our results shed light on previous disparities in PrPSc typing, provide a refined classification of human PrPSc types, and support the notion that the pathological TSE phenotype is related to PrPSc structure.
Journal of Biological Chemistry 05/2004; 279(16):16797-804. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The discovery of molecular subtypes of the pathological prion protein PrPSc has provided the basis for a novel classification of human transmissible spongiform encephalopathies (TSEs) and a potentially
powerful method for strain typing. However, there is still a significant disparity regarding the understanding and nomenclature
of PrPSc types. In addition, it is still unknown whether a specific PrPSc type is associated with each TSE phenotypic variant. In sporadic Creutzfeldt-Jakob disease (sCJD), five disease phenotypes
are known, but only two major types of PrPSc, types 1 and 2, have been consistently reproduced. We further analyzed PrPSc properties in sCJD and variant CJD using a high resolution gel electrophoresis system and varying experimental conditions.
We found that pH varies among CJD brain homogenates in standard buffers, thereby influencing the characteristics of protease-treated
PrPSc. We also show that PrPSc type 1 and type 2 are heterogeneous species which can be further distinguished into five molecular subtypes that fit the
current histopathological classification of sCJD variants. Our results shed light on previous disparities in PrPSc typing, provide a refined classification of human PrPSc types, and support the notion that the pathological TSE phenotype is related to PrPSc structure.
Journal of Biological Chemistry 04/2004; 279(16):16797-16804. · 4.77 Impact Factor
