NR AXYN
AU Weber,P.; Giese,A.; Piening,N.; Mitteregger,G.; Reznicek,L.; Thomzig,A.; Beekes,M.; Kretzschmar,H.A.
TI Size Matters
QU International Conference - Prion 2007 (26.-28.9.2007) Edinburgh International Conference Centre, Edinburgh, Scotland, UK - Book of Abstracts: Protein Misfolding P01.49
IA http://www.prion2007.com/pdf/Prion Book of Abstracts.pdf
PT Konferenz-Poster
AB
Background: In serial transmission protein misfolding cyclic amplification (sPMCA) experiments, newly formed misfolded and proteinase K-resistant PrP (PrPres) catalysed the structural conversion of cellular prion protein (PrPc) as efficiently as PrPsc from brain of scrapie (263K)-infected hamsters confirming an autocatalytic misfolding cascade. However, the fact, that PrPres generated in vitro was associated with ten times less infectivity than an equivalent quantity of brain-derived PrPsc casts doubt on the "protein-only" hypothesis of prion propagation and has supported theories invoking additional molecular species of infectious PrP or further agent-associated factors.
Aims: We reasoned that apart from conformational differences and potential cofactors, the specific biological infectivity of PrPres preparations should also depend on the size distribution of PrPres aggregates which in turn should affect the number of infectious units per given amount of PrPres.
Results/Discussion: Using bioassays of infectivity, we could clearly demonstrate that ultrasonic treatment breaks up aggregates of misfolded PrP into smaller units and that sonication-induced fragmentation of prion aggregates is associated with a pronounced prolongation of incubation times by reducing aggregate stability and facilitating clearance from the brain. In contrast, when coupled to NC-particles, PrPres generated in vitro by sPMCA induced clinical disease in wild-type hamsters as efficient as PrPsc derived from brains of diseased animals. Notably, if biological clearance was evaded by using N2a cells infected with differentially sonicated scrapie brain homogenates, the propagation of misfolded prion protein followed a bell-shaped curve. Applying the serial PMCA approach to a Misonix automate we achieved a 100,000-fold total amplification of hamster PrPres concurrently to a 2.514 (373,000fold) dilution of the initial PrPsc. An efficient automated PMCA system is a prerequisite to establish a routine diagnostic tool for the early detection of human TSE agents.
Conclusion: By combining sPMCA with prion delivery on carrier particles we could resolve the apparent discrepancy between the amount of PrPres and infectivity which we could relate to differences in the size distribution of PrP aggregates and consecutive differences in regard to biological clearance.
AD P. Weber, A. Giese, N. Piening, G. Mitteregger, L. Reznicek, H. Kretzschmar, LMU Munich, Center of Neuropathology and Prion Research, Germany; A. Thomzig, M. Beekes, RobertKoch-Institut, Germany
SP englisch
PO Schottland
EA pdf-Datei und Poster (L. Reznicek von der Autorenliste gestrichen)