Abstract
Prions are proteinaceous infectious agents responsible for the transmission of prion diseases. The lack of a procedure for cultivating prions in the laboratory has been a major limitation to the study of the unorthodox nature of this infectious agent and the molecular mechanism by which the normal prion protein (PrPC) is converted into the abnormal isoform (PrPSc). Protein misfolding cyclic amplification (PMCA), described in detail in this protocol, is a simple, fast and efficient methodology to mimic prion replication in the test tube. PMCA involves incubating materials containing minute amounts of infectious prions with an excess of PrPC and boosting the conversion by cycles of sonication to fragment the converting units, thereby leading to accelerated prion replication. PMCA is able to detect the equivalent of a single molecule of infectious PrPSc and propagate prions that maintain high infectivity, strain properties and species specificity. A single PMCA assay takes little more than 3 d to replicate a large amount of prions, which could take years in an in vivo situation. Since its invention 10 years ago, PMCA has helped to answer fundamental questions about this intriguing infectious agent and has been broadly applied in research areas that include the food industry, blood bank safety and human and veterinary disease diagnosis.
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Acknowledgements
We thank the many former laboratory members whose input has been important for reaching the optimal PMCA procedure, especially G. Saborio, L. Anderes, C. Adessi, K. Maundrell, J. Castilla, P. Saa, K. Abid, M. Barria, D. Gonzalez-Romero and B. Chen. We also thank M.-J. Liberona for critical reading of the manuscript. This work was partially supported by US National Institutes of Health grants R01 NS049173, P01 AI077774 and P01 AG014359 to C.S.
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R.M. designed the experiments, carried out the work, analyzed the results and wrote part of the manuscript. C.D.-A. performed various PMCA assays, wrote part of the manuscript and drafted the figures. M.V.C. performed various PMCA optimizations and wrote part of the manuscript. R.D.-E. performed experiments with recombinant prion protein and wrote part of the manuscript, and C.S. is the principal investigator on the project and was responsible for coordinating research activity, funding and producing the final version of the article.
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Dr Soto is inventor on several patents related to the PMCA technology and is currently Founder, Chief Scientific Officer and Vice -President of Amprion Inc., a biotech company focusing on the commercial exploitation of PMCA for prion diagnosis.
Supplementary information
Supplementary Fig. 1
Schematic representation of a PMCA procedure using purified PrPC and PrPSc components. Purified PrPC and PrPSc submitted to PMCA in the sole presence of conversion buffer will not show any PrP27-30 signal as shown in the Western blot of the left panel. However, complementing the PMCA reaction with tissue homogenate from different sources or purified molecules will result in a typical PrPSc amplification (middle and right Western blots). This system could be applied to investigate in more detail the role of different molecules in the misfolding and aggregation processes of mammalian prions. Samples shown in the Western blots were all PK treated. (PDF 654 kb)
Supplementary Fig. 2
Perfused brains for PMCA substrate. A proper preparation of homogenates from non-infected animals is one of the most important steps in the PMCA procedure. We have observed that blood components can dramatically decrease the PrPSc amplification in a PMCA reaction. For that reason, brain perfusion is critical when preparing a good quality brain homogenate. Properly perfused brains (A) are easily differentiated from those that are not well perfused (B) by their appearance and color. (PDF 3423 kb)
Supplementary Fig. 3
Quality of the sonicator horn. Sonicator platforms from automated sonicators adapted for PMCA release white sediments that decrease the sonication efficiency inside the tube. The release of these sediments is especially abundant in new sonicators. We strongly recommend periodically cleaning the sonicator horn, especially when a new PMCA assay starts. A: Sonicator horn with typical white sediment. As observed, it is impossible to see the sonication platform. B: Sonicator horn filled with clean water. Note the rusted-like appearance in the sonicator platform typically observed when sonicator ages. (PDF 5005 kb)
Supplementary Fig. 4
Quantification of PrPSc by qPMCA. A: A PrPSc standard for qPMCA is prepared by partially purifying PrPSc from the brain of infected animals by a series of sarkosyl precipitation steps. The concentration of PrPSc in the standard inoculum is estimated by comparison with known amounts of recombinant PrP (rPrP), produced as described in Supplementary Method 1. For better comparison of the signal, partially purified PrPSc can be deglycosylated by PNGase as described in Box 1 and treated with PK. The asterisk indicates a band of PrPSc incompleted digested by PK. B: In order to assess the extent of amplification in the standard sample, aliquots of PrPSc ranging from 10-8 to 10-21 g of partially purified PrPSc are subjected to sPMCA using standard conditions. All samples shown in B were PK digested with the sole exception of NBH which corresponds to the PrPC signal from a normal (non-infected) brain homogenate which is used as a control of electrophoretical mobility. (PDF 2120 kb)
Supplementary Methods
PMCA using purified components. (PDF 232 kb)
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Morales, R., Duran-Aniotz, C., Diaz-Espinoza, R. et al. Protein misfolding cyclic amplification of infectious prions. Nat Protoc 7, 1397–1409 (2012). https://doi.org/10.1038/nprot.2012.067
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DOI: https://doi.org/10.1038/nprot.2012.067
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