Pathological Prion Protein PrPsc Research Articles

Astrocyte in prion disease: a double-edged sword.

Prion diseases are infectious protein misfolding disorders of the central nervous system that result from misfolding of the cellular prion protein (PrPC) into the pathologic isoform PrPSc. Pathologic hallmarks of prion disease are depositions of pathological prion protein PrPSc, neuronal loss, spongiform degeneration and astrogliosis in the brain. Prion diseases affect human and animals, there is no effective therapy, and they invariably remain fatal. For a long time, neuronal loss was considered the sole reason for neurodegeneration in prion pathogenesis, and the contribution of non-neuronal cells like microglia and astrocytes was considered less important. Recent evidence suggests that neurodegeneration during prion pathogenesis is a consequence of a complex interplay between neuronal and non-neuronal cells in the brain, but the exact role of these non-neuronal cells during prion pathology is still elusive. Astrocytes are non-neuronal cells that regulate brain homeostasis under physiological conditions. However, astrocytes can deposit PrPSc aggregates and propagate prions in prion-infected brains. Additionally, sub-populations of reactive astrocytes that include neurotrophic and neurotoxic species have been identified, differentially expressed in the brain during prion infection. Revealing the exact role of astrocytes in prion disease is hampered by the lack of in vitro models of prion-infected astrocytes. Recently, we established a murine astrocyte cell line persistently infected with mouse-adapted prions, and showed how such astrocytes differentially process various prion strains. Considering the complexity of the role of astrocytes in prion pathogenesis, we need more in vitro and in vivo models for exploring the contribution of sub-populations of reactive astrocytes, their differential regulation of signaling cascades, and the interaction with neurons and microglia during prion pathogenesis. This will help to establish novel in vivo models and define new therapeutic targets against prion diseases. In this review, we will discuss the complex role of astrocytes in prion disease, the existing experimental resources, the challenges to analyze the contribution of astrocytes in prion disease pathogenesis, and future strategies to improve the understanding of their role in prion disease.

Disulfide-crosslink scanning reveals prion–induced conformational changes and prion strain–specific structures of the pathological prion protein PrPSc

Prions are composed solely of the pathological isoform (PrPSc) of the normal cellular prion protein (PrPC). Identification of different PrPSc structures is crucially important for understanding prion biology because the pathogenic properties of prions are hypothesized to be encoded in the structures of PrPSc. However, these structures remain yet to be identified, because of the incompatibility of PrPSc with conventional high-resolution structural analysis methods. Previously, we reported that the region between the first and the second α-helix (H1∼H2) of PrPC might cooperate with the more C-terminal side region for efficient interactions with PrPSc. From this starting point, we created a series of PrP variants with two cysteine substitutions (C;C-PrP) forming a disulfide-crosslink between H1∼H2 and the distal region of the third helix (Ctrm). We then assessed the conversion capabilities of the C;C-PrP variants in N2a cells infected with mouse-adapted scrapie prions (22L-ScN2a). Specifically, Cys substitutions at residues 165, 166, or 168 in H1∼H2 were combined with cysteine scanning along Ctrm residues 220–229. We found that C;C-PrPs are expressed normally with glycosylation patterns and subcellular localization similar to WT PrP, albeit differing in expression levels. Interestingly, some C;C-PrPs converted to protease-resistant isoforms in the 22L-ScN2a cells, but not in Fukuoka1 prion-infected cells. Crosslink patterns of convertible C;C-PrPs indicated a positional change of H1∼H2 toward Ctrm in PrPSc–induced conformational conversion. Given the properties of the C;C-PrPs reported here, we propose that these PrP variants may be useful tools for investigating prion strain–specific structures and structure–phenotype relationships of PrPSc.

Zinc significantly changes the aggregation pathway and the conformation of aggregates of human prion protein

Prion diseases are caused by the conformational change of cellular prion protein PrPC into pathological prion protein PrPSc. Here we study the effect of zinc on the aggregation and conformational change of human prion protein (PrP). As revealed by thioflavin T binding assays, Sarkosyl-soluble SDS-PAGE, and transmission electron microscopy, aggregation of wild-type PrP in the absence of Zn2+ undergoes four steps: amorphous aggregates, profibrils, mature fibrils, and fragmented fibrils. When the molar ratio of Zn2+ to PrP was 9:1, however, aggregation of wild-type PrP undergoes another pathway in which wild-type PrP forms oligomers quickly and then forms short-rod aggregates. Unlike wild-type PrP, the octarepeats deletion mutant PrPΔocta forms typical mature fibrils either with or without zinc. As evidenced by isothermal titration calorimetry, Fourier transform infrared spectroscopy, and proteinase K digestion assays, Zn2+ strongly binds to wild-type PrP monomers with the first binding constant exceeding 107M−1 under denaturing conditions, and changes the conformation of wild-type PrP aggregates remarkably, but weakly binds to PrPΔocta with binding affinity around 104M−1 and has no obvious effects on the conformation of PrPΔocta aggregates. Our data demonstrate that zinc significantly changes the aggregation pathway and the conformation of wild-type PrP aggregates mainly via interaction with its octarepeat region. Our findings could explain how zinc modifies pathological PrP conformation associated with prion diseases.

Enzymatic Degradation of PrPSc by a Protease Secreted from Aeropyrum pernix K1

BackgroundAn R30 fraction from the growth medium of Aeropyrum pernix was analyzed for the protease that can digest the pathological prion protein isoform (PrPSc) from different species (human, bovine, deer and mouse).Methodology/Principal FindingsDegradation of the PrPSc isoform by the R30 fraction and the purified protease was evaluated using the 6H4 anti-PrP monoclonal antibody. Fragments from the N-terminal and C-terminal of PrPSc were also monitored by Western blotting using the EB8 anti-PrP monoclonal antibody, and by dot blotting using the C7/5 anti-PrP monoclonal antibody, respectively. For detection of smaller peptides from incomplete digestion of PrPSc, the EB8 monoclonal antibody was used after precipitation with sodium phosphotungstate. Characterization of the purified active protease from the R30 fraction was achieved, through purification by fast protein liquid chromatography, and identification by tandem mass spectrometry the serine metalloprotease pernisine. SDS-PAGE and zymography show the purified pernisine plus its proregion with a molecular weight of ca. 45 kDa, and the mature purified pernisine as ca. 23 kDa. The purified pernisine was active between 58°C and 99°C, and between pH 3.5 and 8.0. The temperature and pH optima of the enzymatic activity of the purified pernisine in the presence of 1 mM CaCl2 were 105°C ±0.5°C and pH 6.5±0.2, respectively.Conclusions/SignificanceOur study has identified and characterized pernisine as a thermostable serine metalloprotease that is secreted from A. pernix and that can digest the pathological prion protein PrPSc.

Accumulation of Pathological Prion Protein PrPSc in the Skin of Animals with Experimental and Natural Scrapie

Prion infectivity and its molecular marker, the pathological prion protein PrPSc, accumulate in the central nervous system and often also in lymphoid tissue of animals or humans affected by transmissible spongiform encephalopathies. Recently, PrPSc was found in tissues previously considered not to be invaded by prions (e.g., skeletal muscles). Here, we address the question of whether prions target the skin and show widespread PrPSc deposition in this organ in hamsters perorally or parenterally challenged with scrapie. In hamsters fed with scrapie, PrPSc was detected before the onset of symptoms, but the bulk of skin-associated PrPSc accumulated in the clinical phase. PrPSc was localized in nerve fibres within the skin but not in keratinocytes, and the deposition of PrPSc in skin showed no dependence from the route of infection and lymphotropic dissemination. The data indicated a neurally mediated centrifugal spread of prions to the skin. Furthermore, in a follow-up study, we examined sheep naturally infected with scrapie and detected PrPSc by Western blotting in skin samples from two out of five animals. Our findings point to the skin as a potential reservoir of prions, which should be further investigated in relation to disease transmission.

Preclinical deposition of pathological prion protein PrPSc in muscles of hamsters orally exposed to scrapie.

Recently, pathological prion protein PrP(Sc), the putative key constituent of infectious agents causing transmissible spongiform encephalopathies (TSEs), was found in muscles of rodents experimentally infected with scrapie and in patients with Creutzfeldt-Jakob disease (CJD). For the assessment of risk scenarios originating from these findings (e.g., alimentary transmission of pathogens associated with bovine spongiform encephalopathy [BSE] and chronic wasting disease [CWD] via tainted beef and game or iatrogenic dissemination of CJD agent through contaminated surgical instruments) more detailed information about the time course of PrP(Sc) accumulation in muscles at preclinical and clinical stages of incubation is needed. Here we show that PrP(Sc) in muscles of hamsters fed with scrapie can be detected prior to the onset of clinical symptoms, but that the bulk of PrP(Sc) was deposited late in clinical disease. Additionally, regarding the question of how muscles become invaded, we report on the intramuscular location of PrP(Sc) and substantial indications for centrifugal spread of infection from spinal motor neurons to myofibers. Our findings in a well-established animal model for TSEs contribute to a better assessment of the risks for public health emanating from "Prions in skeletal muscle" and provide new insights into the pathophysiological spread of TSE agents through the body.

Preclinical deposition of pathological prion protein PrPSc in muscles of hamsters orally exposed to scrapie

Preclinical deposition of pathological prion protein PrPSc in muscles of hamsters orally exposed to scrapie

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.

Cortical neurons of Creutzfeldt–Jakob disease patients express the urokinase-type plasminogen activator receptor

Plasminogen belongs to the plasminogen activator system of cell signaling proteins and has recently been identified to bind to pathological prion protein PrP SC, but not to its normal conformer, PrP C. Plasminogen binds specifically to the urokinase-type plasminogen activator receptor (uPAR) to promote pericellular proteolysis, regulate integrin function, and mediate cell signaling. By using immunohistochemistry, we observed that significantly more cortical neurons in eight postmortem brains of patients who died with sporadic Creutzfeldt–Jakob disease (CJD) are immunoreactive for uPAR compared with controls. These data provide the pathophysiological basis for detailed analyses of the role of the plasminogen activator system in CJD and related diseases.