In vitro conversion and seeded fibrillization of posttranslationally modified prion protein.

The conversion of the cellular isoform of the prion protein (PrP(C)) into the pathologic isoform (PrP(Sc)) is the key event in prion diseases. To study the conversion process, an in vitro system based on varying the concentration of low amounts of sodium dodecyl sulfate (SDS) has been employed. In the present study, the conversion of full-length PrP(C) isolated from Chinese hamster ovary cells (CHO-PrP(C)) was examined. CHO-PrP(C) harbors native, posttranslational modifications, including the GPI anchor and two N-linked glyco-sylation sites. The properties of CHO-PrP(C) were compared with those of full-length and N-terminally truncated recombinant PrP. As shown earlier with recombinant PrP (recPrP90-231), transition from a soluble α-helical state as known for native PrP(C) into an aggregated, ß-sheet-rich PrP(Sc)-like state could be induced by dilution of SDS. The aggregated state is partially proteinase K (PK)-resistant, exhibiting a cleavage site similar to that found with PrP(Sc). Compared to recPrP (90-231), fibril formation with CHO-PrP(C) requires lower SDS concentrations (0.0075%), and can be drastically accelerated by seeding with PrP(Sc) purified from brain homogenates of terminally sick hamsters. Our results show that recPrP 90-231 and CHO-PrPC behave qualitatively similar but quantitatively different. The in vivo situation can be simulated closer with CHO-PrP(C) because the specific PK cleave site could be shown and the seed-assisted fibrillization was much more efficient.

Human neurospheres as three-dimensional cellular systems for developmental neurotoxicity testing.

BACKGROUND: Developmental neurotoxicity (DNT) of environmental chemicals is a serious threat to human health. Current DNT testing guidelines propose investigations in rodents, which require large numbers of animals. With regard to the "3 Rs" (reduction, replacement, and refinement) of animal testing and the European regulation of chemicals [Registration, Evaluation, and Authorisation of Chemicals (REACH)], alternative testing strategies are needed in order to refine and reduce animal experiments and allow faster and less expensive screening. OBJECTIVES: The goal of this study was to establish a three-dimensional test system for DNT screening based on human fetal brain cells. METHODS: We established assays suitable for detecting disturbances in basic processes of brain development by employing human neural progenitor cells (hNPCs), which grow as neurospheres. Furthermore, we assessed effects of mercury and oxidative stress on these cells. RESULTS: We found that human neurospheres imitate proliferation, differentiation, and migration in vitro. Exposure to the proapoptotic agent staurosporine further suggests that human neurospheres possess functioning apoptosis machinery. The developmental neurotoxicants methylmercury chloride and mercury chloride decreased migration distance and number of neuronal-like cells in differentiated hNPCs. Furthermore, hNPCs undergo caspase-independent apoptosis when exposed toward high amounts of oxidative stress. CONCLUSIONS: Human neurospheres are likely to imitate basic processes of brain development, and these processes can be modulated by developmental neurotoxicants. Thus, this three-dimensional cell system is a promising approach for DNT testing.

Mechanisms of prion protein assembly into amyloid.

The conversion of the alpha-helical, cellular isoform of the prion protein (PrP(C)) to the insoluble, beta-sheet-rich, infectious, disease-causing isoform (PrP(Sc)) is the key event in prion diseases. In an earlier study, several forms of PrP were converted into a fibrillar state by using an in vitro conversion system consisting of low concentrations of SDS and 250 mM NaCl. Here, we characterize the structure of the fibril precursor state, that is, the soluble state under fibrillization conditions. CD spectroscopy, analytical ultracentrifugation, and chemical cross-linking indicate that the precursor state exists in a monomer-dimer equilibrium of partially denatured, alpha-helical PrP, with a well defined contact site of the subunits in the dimer. Using fluorescence with thioflavin T, we monitored and quantitatively described the kinetics of seeded fibril formation, including dependence of the reaction on substrate and seed concentrations. Exponential, seed-enhanced growth can be achieved in homogeneous solution, which can be enhanced by sonication. From these data, we propose a mechanistic model of fibrillization, including the presence of several intermediate structures. These studies also provide a simplified amplification system for prions.

Counting of single prion particles bound to a capture-antibody surface (surface-FIDA).

Hitherto accredited prion tests use the PK resistance of PrP(Sc), the pathogenic isoform of the prion protein, as a marker for the disease. Because of variations in the amount of disease-related aggregated PrP, which is not PK-resistant, these prion tests offer only limited sensitivity. Therefore, a prion detection method that does not rely on PK digestion would allow for the detection of both PK-resistant as well as PK-sensitive PrP(Sc). Furthermore, single particle counting is more sensitive than methods measuring an integrated signal. Our new test system is based on dual-colour fluorescence correlation spectroscopy (FCS). This method quantifies the number of protein aggregates that have been simultaneously labelled with two different antibodies using dual-colour fluorescence intensity distribution analysis (2D-FIDA). This only counts PrP aggregates, and not PrP monomers. To increase the sensitivity, PrP(Sc) was concentrated in a two-dimensional space by immobilizing it so that the antibodies could be captured on the surface of the slide (surface-FIDA). When the surface was systematically scanned, even single prion particles were detected. Using this new technique, the sensitivity to identify samples from scrapie-infected hamster as well as BSE-infected cattle can be dramatically increased in comparison with identification using FIDA in solution.

Detection of prion particles in samples of BSE and scrapie by fluorescence correlation spectroscopy without proteinase K digestion.

A characteristic feature of prion diseases such as bovine spongiform encephalopathy (BSE) is the accumulation of a pathological isoform of the host-encoded prion protein, PrP. In contrast to its cellular isoform PrP(C), the pathological isoform PrP(Sc) forms insoluble aggregates. All commercial BSE tests currently used for routine testing are based on the proteinase K (PK) resistance of PrP, but not all pathological PrP is PK-resistant. In the present study, single prion particles were counted by fluorescence correlation spectroscopy (FCS). The property of PK resistance is not required, i.e., both the PK-resistant and the PK-sensitive parts of the prion particles are detectable. PrP aggregates were prepared from the brains of BSE-infected cattle, as well as from scrapie-infected hamsters, by the NaPTA precipitation method without PK digestion. They were labeled using two different PrP-specific antibodies for FCS measurements in the dual-color mode (2D-FIDA). Within the limited number of samples tested, BSE-infected cattle and scrapie-infected hamsters in the clinical stage of the disease could be distinguished with 100% specificity from a control group. Thus, a diagnostic tool for BSE detection with complete avoidance of PK treatment is presented, which should have particular advantages for testing animals in the preclinical stage.

The polysaccharide scaffold of PrP 27-30 is a common compound of natural prions and consists of alpha-linked polyglucose.

An inert polysaccharide scaffold identified as a 5-15% component of prion rods (PrP 27-30) is unambiguously distinguishable from the N-glycosyl groups and the GPI anchor of PrP, and consists predominantly of 1,4-linked glucose with some branching via 1,4,6-linked glucose. We show that this polysaccharide scaffold is a common secondary component of prions found in hamster full-length PrP(Sc), prion rods and in mouse ScN2a prions from cell culture. The preparation from prion rods was improved, resulting in a polysaccharide scaffold free of remaining infectivity. Furthermore, we determined the stereochemistry of the glycoside linkages as pre-dominantly if not entirely alpha-glycosidic. The origin of the polysaccharide, its interaction with PrP and its potential relation to glycogen and corpora amylacea are discussed.