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

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 Variably Protease-Sensitive Prionopathy: An Update

pathogen

Transcriptomics Profiling of Acer pseudosieboldianum Molecular Mechanism against Freezing Stress

Low temperature is an important environmental factor that affects the growth and development of trees and leads to the introduction of failure in the genetic improvement of trees. Acer pseudosieboldianum is a tree species that is well-known for its bright red autumn leaf color. These trees are widely used in landscaping in northeast China. However, due to their poor cold resistance, introduced A. pseudosieboldianum trees suffer severe freezing injury in many introduced environments. To elucidate the physiological indicators and molecular mechanisms associated with freezing damage, we analyzed the physiological indicators and transcriptome of A. pseudosieboldianum, using kits and RNA-Seq technology. The mechanism of A. pseudosieboldianum in response to freezing stress is an important scientific question. In this study, we used the shoots of four-year-old A. pseudosieboldianum twig seedlings, and the physiological index and the transcriptome of A. pseudosieboldianum under low temperature stress were investigated. The results showed that more than 20,000 genes were detected in A. pseudosieboldianum under low temperature (4 °C) and freezing temperatures (−10 °C, −20 °C, −30 °C, and −40 °C). There were 2505, 6021, 5125, and 3191 differential genes (DEGs) between −10 °C, −20°C, −30°C, −40 °C, and CK (4 °C), respectively. Among these differential genes, 48 genes are involved in the MAPK pathway and 533 genes are involved in the glucose metabolism pathway. In addition, the important transcription factors (MYB, AP2/ERF, and WRKY) involved in freezing stress were activated under different degrees of freezing stress. A total of 10 sets of physiological indicators of A. pseudosieboldianum were examined, including the activities of five enzymes and the accumulation of five hormones. All of the physiological indicators except SOD and GSH-Px reached their maximum values at −30 °C. The enzyme activity of SOD was highest at −10 °C, and that of GSH-Px was highest at −20 °C. Our study is the first to provide a more comprehensive understanding of the differential genes (DEGs) involved in A. pseudosieboldianum under freezing stress at different temperatures at the transcriptome level. These results may help to clarify the molecular mechanism of cold tolerance of A. pseudosieboldianum and provide new insights and candidate genes for the genetic improvement of the freezing tolerance of A. pseudosieboldianum

Post-translational modifications in PrP expand the conformational diversity of prions in vivo

Misfolded prion protein aggregates (PrPSc) show remarkable structural diversity and are associated with highly variable disease phenotypes. Similarly, other proteins, including amyloid-beta, tau, alpha-synuclein, and serum amyloid A, misfold into distinct conformers linked to different clinical diseases through poorly understood mechanisms. Here we use mice expressing glycophosphatidylinositol (GPI)anchorless prion protein, PrPC, together with hydrogen-deuterium exchange coupled with mass spectrometry (HXMS) and a battery of biochemical and biophysical tools to investigate how posttranslational modifications impact the aggregated prion protein properties and disease phenotype. Four GPI-anchorless prion strains caused a nearly identical clinical and pathological disease phenotype, yet maintained their structural diversity in the anchorless state. HXMS studies revealed that GPIanchorless PrPSc is characterized by substantially higher protection against hydrogen/deuterium exchange in the C-terminal region near the N-glycan sites, suggesting this region had become more ordered in the anchorless state. For one strain, passage of GPI-anchorless prions into wild type mice led to the emergence of a novel strain with a unique biochemical and phenotypic signature. For the new strain, histidine hydrogen-deuterium mass spectrometry revealed altered packing arrangements of beta-sheets that encompass residues 139 and 186 of PrPSc. These findings show how variation in posttranslational modifications may explain the emergence of new protein conformations in vivo and also provide a basis for understanding how the misfolded protein structure impacts the disease.Funding Agencies|National Institutes of Health [NS069566, NS076896, AI106705, NS083687]; Swedish Foundation for Strategic Research (KPRN)</p

Further characterization of glycoform-selective prions of variably protease-sensitive prionopathy

International audiencePrion is an infectious protein (PrPSc ) that is derived from a cellular glycoprotein (PrPC ) through a conformational transition and associated with a group of prion diseases in animals and humans. Characterization of proteinase K (PK)-resistant PrPSc by western blotting has been critical to diagnosis and understanding of prion diseases including Creutzfeldt-Jakob disease (CJD) and Gerstmann-Sträussler-Scheinker (GSS) disease in humans. However, formation as well as biochemical and biological properties of the glycoform-selective PrPSc in variably protease-sensitive prionopathy (VPSPr) remain poorly understood. Here we reveal that formation of the ladder-like PrPSc in VPSPr is a PK-dependent two-step process, which is enhanced by basic pH. Two sets of PrPSc fragments can be identified with antibodies directed against an intermediate or a C-terminal domain of the protein. Moreover, antibodies directed against specific PrP glycoforms reveal faster electrophoretic migrations of PrP fragments mono-glycosylated at residue 181 and 197 in VPSPr than those in sporadic CJD (sCJD). Finally, RT-QuIC assay indicates that PrPSc-seeding activity is lower and its lag time is longer in VPSPr than in sCJD. Our results suggest that the glycoform-selective PrPSc in VPSPr is associated with altered glycosylation, resulting in different PK-truncation and aggregation seeding activity compared to PrPSc in sCJD

Artificial strain of human prions created in vitro

The molecular mechanism that determines under physiological conditions transmissibility of the most common human prion disease, sporadic Creutzfeldt-Jakob disease (sCJD) is unknown. We report the synthesis of new human prion from the recombinant human prion protein expressed in bacteria in reaction seeded with sCJD MM1 prions and cofactor, ganglioside GM1. These synthetic human prions were infectious to transgenic mice expressing non-glycosylated human prion protein, causing neurologic dysfunction after 459 and 224 days in the first and second passage, respectively. The neuropathology, replication potency, and biophysical profiling suggest that a novel, particularly neurotoxic human prion strain was created. Distinct biological and structural characteristics of our synthetic human prions suggest that subtle changes in the structural organization of critical domains, some linked to posttranslational modifications of the pathogenic prion protein (PrPSc), play a crucial role as a determinant of human prion infectivity, host range, and targetting of specific brain structures in mice models