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PrP(Sc) is an infectious protein. The only experimentally verified difference between PrP(Sc) and its normal cellular isoform (PrP(C)) is conformational. This work describes an approach to determining the presence of surface exposed or sequestered amino acids present in the PrP(Sc) isoform. The N-hydroxysuccinimide esters of acetic acid and 4-trimethylammoniumbutyric acid were synthesized and reacted with detergent-solubilized brain extracts from Me7-infected mice, uninfected mice, 263K-infected hamsters or uninfected hamsters. These reaction mixtures were analyzed by western blots probed with the antibodies 3F4, 6D11, 7D9, AG4, AH6, GE8 or MAB5424. The 3F4, 6D11, AH6, and GE8 antibodies recognize an epitope that is encrypted in the PrP(Sc) isoform, but exposed in the PrP(C) isoform. These reagents permit the detection of prion infected brain extracts without the need for proteinase K digestion. In addition they can be used, with an appropriate antibody, to determine which amino acids of PrP(Sc) are exposed on the surface and which are encrypted, thus providing useful structural information. This approach was used to distinguish between the 263K and drowsy strains of hamster-adapted scrapie without the use of proteinase K.

Prionen verursachen transmissible spongiforme Enzephalopathien wie Scrapie, die bovine spongiforme Enzephalopathie oder die Creutzfeldt-Jakob-Krankheit und bestehen aus fehlgefaltetem, aggregierten Prionprotein (PrP). Aufgrund ihrer hohen Toleranz gegenüber üblichen Desinfektionsverfahren stellen sie sowohl eine besondere Herausforderung als auch ein exzellentes Paradigma für die Aufbereitung von chirurgischen Instrumenten dar. Tatsächlich haben sich Prionen bereits als aussagekräftige Modellpathogene für die Entwicklung neuer Formulierungen zur hochwirksamen Desinfektion auch von Bakterien, Viren und Pilzen bewährt. Um Prionen zukünftig in größerem Umfang als Testerreger zur Entwicklung neuer Mittel und Verfahren für die Breitspektrumdesinfektion ohne Rückgriff auf Tierversuche nutzen zu können, wurden in dieser Arbeit in vitro- Ersatzmethoden für Prion-Bioassays in Nagetieren aufgebaut und validiert. Zur quantitativen biochemischen Bestimmung der PrP-konvertierenden Keimaktivität des Scrapie-assoziierten Prionproteins wurde ein spezifisch adaptierter Protein Misfolding Cyclic Amplification (PMCA)-Assay etabliert. Mit hamster- adaptierten Scrapie-Prionen (Stamm 263K) kontaminierte Stahlstifte wurden verschiedenen Desinfektionsbehandlungen und anschließend einer quantitativen PMCA unterzogen. Die Ergebnisse des PMCA-Assays wurden dann mit bereits früher erhobenen Bioassay-Daten zur Reduktion infektiöser Priontiter durch die angewandten Desinfektionsverfahren verglichen. Zusätzlich dazu wurde ein Zellkulturassay mit primären Gliazellen aus Hamstern zum biologischen in vitro-Nachweis der Keimaktivität des Prionproteins entwickelt. Der Nachweis von Scrapie-assoziierter Keimaktivität an Stahlstiftprüfkörpern in der quantitativen PMCA übertraf die Sensitivität des Nachweises von Prioninfektiosität im Tierversuch um mindestens eine Größenordnung. Dabei konnte die Reduktion der Prioninfektiosität an Stahlstiftprüfkörpern durch 16 verschiedene Desinfektions-verfahren mit Titerreduktionen von weniger als 101-fach bis zu größer oder gleich 105,5-fach durchgängig korrekt abgeschätzt werden. Mittels PMCA biochemisch nachgewiesene Keimaktivität ließ sich auch biologisch in der Zellkultur detektieren. Konzeptionell deuten die Befunde dieser Arbeit darauf hin, dass die proteinöse Keimaktivität und pathogene Infektiosität von 263K Scrapie-Prionen biochemische und biologische Manifestationen desselben molekularen Prinzips darstellen. In praktischer Hinsicht steht mit der Gliazellkultur-gekoppelten PMCA potentiell eine gleichwertige oder sogar überlegene Alternative zum Prion-Bioassay in Tieren zur Verfügung., Prions are misfolded, aggregated prion proteins (PrP) and the causative agents of transmissible spongiform encephalopathies such as scrapie, bovine spongiform encephalopathy (BSE) or Creutzfeldt-Jakob disease. Prions are pathogens with an unusually high tolerance to inactivation and constitute a complex challenge as well as an informative paradigm for the re-processing of surgical instruments. They have been exploited successfully as model pathogens for the development of novel disinfectants with simultaneous activity also against bacteria, viruses and fungi. In order to further facilitate the use of prions as model agents in the search for novel broad-range disinfectants without the use of animal experiments we established and validated an experimental platform for the sensitive quantitative measurement and biological detection of scrapie seeding activity in vitro. We used specifically adapted serial protein misfolding cyclic amplification (PMCA) for the quantitative biochemical detection of the seeding activity of scrapie associated prion protein. Steel wires contaminated with 263K scrapie prions were subjected to different decontamination procedures and subsequently analysed by quantitative PMCA. The results of the PMCA assay were validated by comparison to bioassay data previously established for similar treatments of steel wires. Furthermore a novel cell culture assay of cerebral glial cell cultures from hamsters was established for the biological detection of 263K scrapie seeding activity. The detection limit for scrapie seeding activity on steel wires in the PMCA assay exceeded that for prion infectivity detected by bioassay at least tenfold. The residual scrapie infectivity’s on test wires treated with 16 different disinfection procedures yielding scrapie titre reductions of ≤101- to ≥105.5-fold could be correctly deduced, for all examined procedures, from our quantitative seeding activity assay. In addition, we found that scrapie seeding activity biochemically detected by PMCA could be also demonstrated biologically in the cell culture assay. Conceptually, the findings from this work suggested the seeding activity and infectivity of 263K scrapie prions as corresponding biochemical and biological manifestations of the same replicative molecular principle. In practical terms, our biochemical and biological in vitro assays, when combined, potentially provide a coequal or even superior alternative to titrations of scrapie infectivity in animals. In vitro-detection of the seeding activity of scrapie associated prion protein: A superior alternative to titrations of scrapie infectivity in animals

The most prominent hallmark of prion diseases is prion protein conversion and the subsequent deposition of the altered prions, PrPSc, at the pathological sites of affected individuals, particularly in the brain. A previous study has demonstrated that the N-terminus of the pathogenic prion isoform (PrPSc) is modified with advanced glycation end products (AGEs), most likely at one or more of the three Lys residues (positions 23, 24, and 27) in the N-terminus (23KKRPKP28). The current study investigated whether Nε-(carboxymethyl)lysine (CML), a major AGE form specific to Lys residues produced by nonenzymatic glycation, is an AGE adduct of the N-terminus of PrPSc. We show that CML is linked to at least one Lys residue at the N-terminus of PrPSc in 263K prion-infected hamster brains and at least one of the eight Lys residues (positions 101, 104, 106, 110, 185, 194, 204, and 220) in the proteinase K (PK)-resistant core region of PrPSc. The nonenzymatic glycation of the Lys residue(s) of PrPSc with CML likely occurs in the widespread prion-deposit areas within infected brains, particularly in some of the numerous tyrosine hydroxylase-positive thalamic and hypothalamic nuclei. CML glycation does not occur in PrPC but is seen in the pathologic PrPSc isoform. Furthermore, the modification of PrPSc with CML may be closely involved in prion propagation and deposition in pathological brain areas. Electronic supplementary material The online version of this article (doi:10.1007/s12035-015-9200-8) contains supplementary material, which is available to authorized users.

The mechanisms implicated in prion infection and tissue distribution are not completely understood. In this study we investigated the levels of 263K prions in brain and spleen of Syrian hamsters few days after intra-peritoneal challenge. For this purpose we utilized the PMCA technology which permits to detect as little as few PrPSc molecules. Our results show that peripherally administered prions directly reach the brain, although at levels below the minimum necessary to produce disease. PrPSc remains in the brain several days after administration suggesting inefficient clearance or early replication. Understanding the fate of the infectious agent after administration and its uptake in different organs and fluids may provide useful information to develop strategies to minimize further spreading of prion diseases.