Protein misfolding cyclic amplification of infectious prions

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 (PrP(C)) is converted into the abnormal isoform (PrP(Sc)). 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 PrP(C) 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 PrP(Sc) 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.

Figure 1

Schematic diagram for protein misfolding cyclic amplification. PMCA is based on the assumption that prion replication occurs by a seeding/nucleation model, in which PrP^Sc seeds bind and convert PrP^C by incorporating the protein into the polymer. In PMCA, PrP^Sc and PrP^C are mixed and incubated, allowing the misfolding of PrP^C, which permits the incorporation and growth of PrP^Sc aggregates. After incubation, samples are submitted to sonication in order to fragment PrP^Sc polymers, thereby generating new free ends suitable for continued prion replication. This process is cyclically repeated in order to produce an exponential amplification of PrP^C conversion.

Figure 2

PMCA flowchart. A conventional PMCA assay is usually composed of four main stages: substrate preparation, inoculum preparation, PMCA procedure and visualization of the extent of prion formation. After mixing PMCA substrate and inoculum, samples are submitted to incubation/sonication cycles that will allow an exponential generation of infectious prions. After the PMCA reaction is completed, samples are digested with PK and visualized by western blotting. Modifications to this technique have been used to replicate prions from different strains/species. PMCA has been adapted for many different applications, including PrP^Sc detection in body fluids and tissues and interspecies prion replication. PROCEDURE Steps are indicated in purple boxes with the step number(s) in parentheses. Critical steps are labeled in blue and Troubleshooting steps in green.

Figure 3

Schematic representation of the serial PMCA (sPMCA) assay. In a typical sPMCA assay, the resulting product of a PMCA round is used as an inoculum in a new PMCA reaction (usually by diluting the PMCA product tenfold in fresh brain homogenate substrate). (a,b) By using this procedure, it is possible to increase the detection levels in order to detect up to a single infectious prion. In the left side of the figure, schematic representations of the dilution setup is shown both for the experimental samples (a) and negative controls (b). (a) Western blots show how the detection of PrP27–30 increases among PMCA rounds (right), detecting the equivalent of a 10⁻¹⁰ 263K brain dilution in a second PMCA round. Numbers on top of each lane correspond to the dilution of the brain homogenate used to trigger amplification. Gaps in PrP27–30 detection as shown for the second PMCA round (10⁻⁹ dilution) are infrequently seen and are usually normalized in the following PMCA rounds. (b) Negative controls consisting only of NBH and submitted to the same procedures do not show any PrP^Sc signal if all proper precautions are followed (for details, please refer to the TROUBLESHOOTING section and references). In this panel, the numbers on top of the gel refer to multiple replicates. Results showed in this figure are obtained from PMCA assays performed as explained in the protocol using four silica beads per tube. MW, molecular weight ladder.

Figure 4

Distinguishing complete and incomplete proteolytic digestion of PrP^Sc. A frequent problem associated with the detection of PrP^Sc after PMCA amplification is ensuring that the product being detected is bona fide PrP^Sc and not some kind of amorphous aggregate that prevents access to the protease used to eliminate unconverted PrP^C. Fortunately, gel electrophoresis and western blotting enable these possibilities to be discriminated. Incomplete digestion (lane 1) leads to a signal with the molecular weight similar to PrP^C not treated with PK (lane 4), whereas the typical signal for PrP27–30 has a clearly lower molecular weight (around 29 kDa; lane 3). Lanes 1–3 show the results of experiments containing a similar amount of hamster PrP^Sc, in which PK digestion did not digest (lane 1), digested partially (lane 2) and digested completely (lane 3) the N-terminal fragment of the protein. To depict the migration of full-length PrP^C, lane 4 corresponds to a hamster NBH not treated with PK.