The conversion of the ␣-helical, cellular isoform of the prion protein (PrP C ) to the insoluble, ␤-sheet-rich, infectious, diseasecausing 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, ␣-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.dimer ͉ seeding ͉ fibril ͉ precursor state

The conversion of the alpha-helical, protease sensitive and noninfectious form of the prion protein (PrP(C)) into an insoluble, protease resistant, predominantly beta-sheeted and infectious form (PrP(Sc)) is the fundamental event in prion formation. In the present work, two soluble and stable intermediate structural states are newly identified for recombinant Syrian hamster PrP(90-231) (recPrP), a dimeric alpha-helical state and a tetra- or oligomeric, beta-sheet rich state. In 0.2% SDS at room temperature, recPrP is soluble and exhibits alpha-helical and random coil secondary structure as determined by circular dichroism. Reduction of the SDS concentration to 0.06% leads first to a small increase in alpha-helical content, whereas further dilution to 0.02% results in the aquisition of beta-sheet structure. The reversible transition curve is sigmoidal within a narrow range of SDS concentrations (0.04 to 0.02%). Size exclusion chromatography and chemical crosslinking revealed that the alpha-helical form is dimeric, while the beta-sheet rich form is tetra- or oligomeric. Both the alpha-helical and beta-sheet rich intermediates are soluble and stable. Thus, they should be accessible to further structural and mechanistic studies. At 0.01% SDS, the oligomeric intermediates aggregated into large, insoluble structures as observed by fluorescence correlation spectroscopy. Our results are discussed with respect to the mechanism of PrP(Sc) formation and the propagation of prions.

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Recent studies indicate that small amyloid-β peptide (Aβ) oligomers are the major toxic species responsible for development and progression of Alzheimer's disease (AD). Therefore, we suggest that the number of Aβ oligomers in body fluids is the most direct and relevant biomarker for AD. Determination of the Aβ oligomer content of cerebrospinal fluid (CSF) samples from 14 AD patients and 12 age-matched controls revealed a clear distinction between both groups. All samples of the control group showed homogenously low numbers of Aβ oligomers, while the samples of the AD group exhibited significantly higher levels of Aβ oligomers. The Aβ oligomer numbers correlated with the patients' Mini-Mental State Examination scores. This indicates that the quantity of Aβ oligomers in CSF reflects the severity of the disease and that Aβ oligomers play a crucial role in AD pathology and in turn can be used as a diagnostic biomarker.