Gerstmann-Sträussler-Scheinker disease revisited: accumulation of covalently-linked multimers of internal prion protein fragments

Despite their phenotypic heterogeneity, most human prion diseases belong to two broadly defined groups: Creutzfeldt-Jakob disease (CJD) and Gerstmann-Sträussler-Scheinker disease (GSS). While the structural characteristics of the disease-related proteinase K-resistant prion protein (resPrPD) associated with the CJD group are fairly well established, many features of GSS-associated resPrPD are unclear. Electrophoretic profiles of resPrPD associated with GSS variants typically show 6-8 kDa bands corresponding to the internal PrP fragments as well as a variable number of higher molecular weight bands, the molecular nature of which has not been investigated. Here we have performed systematic studies of purified resPrPD species extracted from GSS cases with the A117V (GSSA117V) and F198S (GSSF198S) PrP gene mutations. The combined analysis based on epitope mapping, deglycosylation treatment and direct amino acid sequencing by mass spectrometry provided a conclusive evidence that high molecular weight resPrPD species seen in electrophoretic profiles represent covalently-linked multimers of the internal ~ 7 and ~ 8 kDa fragments. This finding reveals a mechanism of resPrPD aggregate formation that has not been previously established in prion diseases

Effects of different experimental conditions on the PrPSc core generated by protease digestion: implications for strain typing and molecular classification of CJD.

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

Clinicopathological Correlates in a PRNP P102L Mutation Carrier with Rapidly Progressing Parkinsonism-dystonia

Parkinsonism-dystonia is rare in carriers of PRNP P102L mutation. Severity and distribution of prion protein (PrP) deposition may influence the clinical presentation. We present such clinic-pathological correlation in a 56-year-old male with a PRNP P102L mutation associated with a phenotype characterized by rapidly progressing parkinsonism-dystonia. The patient was studied clinically (videotaped exams, brain MRIs); molecular genetically (gene sequence analysis); and neuropathologically (histology, immunohistochemistry) during his 7-month disease course. The patient had parkinsonism, apraxia, aphasia, and dystonia, which progressed rapidly. Molecular genetic analysis revealed PRNP P102L mutation carrier status. Brain MRIs revealed progressive global volume loss and T2/FLAIR hyperintensity in neocortex and basal ganglia. Postmortem examination showed neuronal loss, gliosis, spongiform changes, and PrP deposition in the striatum. PrP immunohistochemistry revealed widespread severe PrP deposition in the thalamus and cerebellar cortex. Based on the neuropathological and molecular-genetic analysis, the rapidly progressing parkinsonism-dystonia correlated with nigrostriatal, thalamic, and cerebellar pathology

A novel mechanism of phenotypic heterogeneity in Creutzfeldt-Jakob disease

One of remarkable features of sporadic Creutzfeldt-Jakob disease (sCJD) is the great phenotypic variability. Understanding the molecular basis of this variability has important implications for the development of therapeutic approaches. It is well established that, in many cases, phenotypic heterogeneity of sCJD is under control of two determinants: the genotype at the methionine (M)/valine (V) polymorphic codon 129 of the human prion protein gene and the type, 1 or 2, of the pathogenic and disease-related form of the prion protein, PrPD. However, this scenario fails to explain the existence of distinct heterozygous sCJDMV2 subtypes, where heterogeneity occurs without any variation of the 129 allotype and PrPD type. One of these subtypes, denoted sCJDMV2C, associated with PrPD type 2, is characterized by widespread spongiform degeneration of the cerebral cortex (C). The second variant, denoted sCJDMV2K, features prominent deposition of PrPD amyloid forming kuru type (K) plaques. Here we used a mass spectrometry based approach to test the hypothesis that phenotypic variability within the sCJDMV2 subtype is at least partly determined by the abundance of 129 M and 129 V polymorphic forms of proteinase K-resistant PrPD (resPrPD). Consistent with this hypothesis, our data demonstrated a strong correlation of the MV2C and MV2K phenotypes with the relative populations of protease-resistant forms of the pathogenic prion proteins, resPrPD-129 M and resPrPD-129 V, where resPrPD-129 M dominated in the sCJDMV2C variant and resPrPD-129 V in the sCJDMV2K variant. This finding suggests an important, previously unrecognized mechanism for phenotypic determination in human prion diseases

Prions in the Urine of Patients with Variant Creutzfeldt–Jakob Disease

BACKGROUND: Prions, the infectious agents responsible for transmissible spongiform encephalopathies, consist mainly of the misfolded prion protein (PrP(Sc)). The unique mechanism of transmission and the appearance of a variant form of Creutzfeldt–Jakob disease, which has been linked to consumption of prion-contaminated cattle meat, have raised concerns about public health. Evidence suggests that variant Creutzfeldt–Jakob disease prions circulate in body fluids from people in whom the disease is silently incubating. METHODS: To investigate whether PrP(Sc) can be detected in the urine of patients with variant Creutzfeldt–Jakob disease, we used the protein misfolding cyclic amplification (PMCA) technique to amplify minute quantities of PrP(Sc), enabling highly sensitive detection of the protein. We analyzed urine samples from several patients with various transmissible spongiform encephalopathies (variant and sporadic Creutzfeldt–Jakob disease and genetic forms of prion disease), patients with other degenerative or nondegenerative neurologic disorders, and healthy persons. RESULTS: PrP(Sc) was detectable only in the urine of patients with variant Creutzfeldt–Jakob disease and had the typical electrophoretic profile associated with this disease. PrP(Sc) was detected in 13 of 14 urine samples obtained from patients with variant Creutzfeldt–Jakob disease and in none of the 224 urine samples obtained from patients with other neurologic diseases and from healthy controls, resulting in an estimated sensitivity of 92.9% (95% confidence interval [CI], 66.1 to 99.8) and a specificity of 100.0% (95% CI, 98.4 to 100.0). The PrP(Sc) concentration in urine calculated by means of quantitative PMCA was estimated at 1×10(−16) g per milliliter, or 3×10(−21) mol per milliliter, which extrapolates to approximately 40 to 100 oligomeric particles of PrP(Sc) per milliliter of urine. CONCLUSIONS: Urine samples obtained from patients with variant Creutzfeldt–Jakob disease contained minute quantities of PrP(Sc). (Funded by the National Institutes of Health and others.

Photosynthetic trichomes contain a specific Rubisco with a modified pH9 dependent activity

Six subtypes of sporadic Creutzfeldt\u2013Jakob disease with distinctive clinico-pathological features have been identified largely based on two types of the abnormal prion protein, PrPSc, and the methionine (M)/valine (V) polymorphic codon 129 of the prion protein. The existence of affected subjects showing mixed phenotypic features and concurrent PrPSc 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. PrPSc 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. PrPSc 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 PrPSc type 2. In contrast, molecular typing best detected the concurrent PrPSc 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\u2013Jakob disease

Variable Protease-Sensitive Prionopathy Transmission to Bank Voles

Variably protease-sensitive prionopathy (VPSPr), a recently described human sporadic prion disease, features a protease-resistant, disease-related prion protein (resPrPD) displaying 5 fragments reminiscent of Gerstmann-Sträussler-Scheinker disease. Experimental VPSPr transmission to human PrP–expressing transgenic mice, although replication of the VPSPr resPrPD profile succeeded, has been incomplete because of second passage failure. We bioassayed VPSPr in bank voles, which are susceptible to human prion strains. Transmission was complete; first-passage attack rates were 5%–35%, and second-passage rates reached 100% and survival times were 50% shorter. We observed 3 distinct phenotypes and resPrPD profiles; 2 imitated sporadic Creutzfeldt-Jakob disease resPrPD, and 1 resembled Gerstmann-Sträussler-Scheinker disease resPrPD. The first 2 phenotypes may be related to the presence of minor PrPD components in VPSPr. Full VPSPr transmission confirms permissiveness of bank voles to human prions and suggests that bank vole PrP may efficiently reveal an underrepresented native strain but does not replicate the complex VPSPr PrPD profile

Transmission Characteristics of Variably Protease-Sensitive Prionopathy

Variably protease-sensitive prionopathy (VPSPr), a recently identified and seemingly sporadic human prion disease, is distinct from Creutzfeldt-Jakob disease (CJD) but shares features of Gerstmann-Sträussler-Scheinker disease (GSS). However, contrary to exclusively inherited GSS, no prion protein (PrP) gene variations have been detected in VPSPr, suggesting that VPSPr might be the long-sought sporadic form of GSS. The VPSPr atypical features raised the issue of transmissibility, a prototypical property of prion diseases. We inoculated VPSPr brain homogenate into transgenic mice expressing various levels of human PrP (PrPC). On first passage, 54% of challenged mice showed histopathologic lesions, and 34% harbored abnormal PrP similar to that of VPSPr. Surprisingly, no prion disease was detected on second passage. We concluded that VPSPr is transmissible; thus, it is an authentic prion disease. However, we speculate that normal human PrPC is not an efficient conversion substrate (or mouse brain not a favorable environment) and therefore cannot sustain replication beyond the first passage