Protein misfolding cyclic amplification (PMCA) is an in vitro assay for the study of prion conversion and strain and species barriers. It can also be used as a prion detection assay.
Prions are infectious agents that cause the inevitably fatal transmissible spongiform encephalopathy (TSE) in animals and humans9,18. The prion protein has two distinct isoforms, the non-infectious host-encoded protein (PrPC) and the infectious protein (PrPSc), an abnormally-folded isoform of PrPC 8.
One of the challenges of working with prion agents is the long incubation period prior to the development of clinical signs following host inoculation13. This traditionally mandated long and expensive animal bioassay studies. Furthermore, the biochemical and biophysical properties of PrPSc are poorly characterized due to their unusual conformation and aggregation states.
PrPSc can seed the conversion of PrPC to PrPSc in vitro14. PMCA is an in vitro technique that takes advantage of this ability using sonication and incubation cycles to produce large amounts of PrPSc, at an accelerated rate, from a system containing excess amounts of PrPC and minute amounts of the PrPSc seed19. This technique has proven to effectively recapitulate the species and strain specificity of PrPSc conversion from PrPC, to emulate prion strain interference, and to amplify very low levels of PrPSc from infected tissues, fluids, and environmental samples6,7,16,23 .
This paper details the PMCA protocol, including recommendations for minimizing contamination, generating consistent results, and quantifying those results. We also discuss several PMCA applications, including generation and characterization of infectious prion strains, prion strain interference, and the detection of prions in the environment.
1. Preparing the Equipment
2. Preparing the Samples
3. Preparing Conversion Buffer
4. Amplification of Prion Strains Using PMCA
5. Avoiding Cross-contamination
Critical steps have to be taken to minimize cross-contamination and the occurrence of false positives due to de novo formation of protease resistant products.
Protein misfolding cyclic amplification (PMCA) is used to amplify PrPSc in vitro7, 12, 14, 19, 24. A successful PrPSc amplification is shown by an increase in band intensity on Western blots of the PK-resistant prion protein (migrating between 19 and 30 kDa for hamster-derived prion strains) as shown in Figure 3. The increase in its band intensity after PMCA indicates amplification of the PK-resistant PrPSc material. Successful amplification of the hamster-derived prion protein, HaCWD and DY TME, is shown in the WB analysis of Figure 3 by comparing the band intensities before (lanes 5 and 7) and after (lanes 4 and 6) PMCA.
PK digestion of PrPSc followed by WB analysis shows an upper, middle and lower band corresponding to the di-, mono-, and unglycosylated prion protein, respectively. Some prion strains can be differentiated by their electrophoretic mobility. For instance, there is a two-kDa difference in migration between the HaCWD and DY TME prion strains. (Figure 3, lanes 1 and 2, respectively).
A high fidelity PrPSc amplification is achieved by the PMCA1, 7, 12, 19, 23, 24. This high fidelity in PMCA amplification is observed by a similar electrophoretic migration of the PMCA-amplified prion strains (HaCWD and DY TME) when compared to their corresponding seeds (Figure 3; lanes 1 & 4 and 2 & 6 for HaCWD and DY TME, respectively).
If no cross-contamination or de novo PrPSc formation occurs, WB analysis of mock control PMCA samples should remain clear after PK digestion (Figure 3, lanes 8 and 9).
Figure 1. To avoid water condensation, a regular aquarium heat-pad is placed above the lid of the microplate horn (panel A). A brand new sonicator should be allowed a break-in period of continuous sonication for about two months. Panel B shows the erosion on the titanium microplate horn after the break-in period. Panel C shows a possible arrangement of PCR tube strips that would allow an optimal sonication of the samples.
Figure 2. Flow chart for PMCA (panel A), sPMCA (panel B), and WB analysis (panel C). PrPSc is the PMCA seed and the uninfected brain homogenate (UN BH) is the PMCA substrate. The quantification of the amplified PrPSc for each PMCA round is obtained by dividing the band intensity after PMCA by its corresponding band intensity before PMCA.
Figure 3. Western blot of PK-digested HaCWD and DY TME hamster-derived prion strains. PrPSc is detected using 3F4 anti-prion antibody. Analysis of the WB shows the abundance (blot intensity) and electrophoretic mobility of PrPSc. PMCA reactions were seeded with brain homogenate from hamsters infected with either the HaCWD (lanes 4-5) or DY TME (lanes 6-7) agents. Samples that underwent PMCA (PMCA +) show an increase in the PrPSc abundance when compared to their unamplified (PMCA -) controls (compare lanes 5 to 4 and 7 to 6). There is a two-kDa difference in the electrophoretic migration between the prion proteins of HaCWD and DY TME (lanes 1-2). This difference in migration is maintained in the PMCA-generated samples using HaCWD and DY TME homogenates (lanes 4 and 6, respectively). PMCA reactions seeded with uninfected brain (mock) homogenate fail to amplify PrPSc (lane 8). The locations for the 19 and 21 kDa molecular markers are indicated to the left of the panel.
Challenges of amplifying infectious prion proteins are the long incubation periods and the expenses of in vivo experiments. The PMCA technique is a cost effective means to amplify infectious prion agents. Several laboratories have confirmed the ability of PMCA to accurately amplify prion strains in vitro 7, 9, 12, 14, 19,24 .
Prion diseases can be transmitted between species. Bessen and Marsh have effectively inoculated hamsters with transmissible mink encephalopathy, which produced two distinct hamster-derived prion strains5 . In an elegant study, Telling and co-workers used sPMCA to amplify the mouse-derived prion strain, RML, using transgenic mice expressing cervid PrPC (Tg(CerPrP)1536+/-) as PMCA substrate. A rapid onset of disease was observed after inoculation of Tg(CerPrP)1536+/- with the PMCA-adapted material12 . Similarly, Kurt et al. were able to amplify chronic wasting disease (CWD), a prion disease in cervids, using non-cervid species PMCA substrate15. These data suggest that species barrier in prion diseases can be bypassed in vitro via PMCA.
Bessen and Marsh have shown that the hamster’s short incubation period strain, HY TME, has a quicker PrPSc accumulation when compared to its long incubation period counterpart, DY TME3, 5 . Similarly, this correlation between the incubation period and the accumulation rate is observed after PMCA in several hamster-derived prion strains1, 23, 24.
The amplification rate is a property intrinsic to each strain. PMCA has been used to quantify this amplification rate. Ayers et al. were able to calculate a unitless number, termed amplification coefficient, that represents the rate of amplification for a given hamster-derived prion strain1 .
Prion strains can interfere with each other when present in the same host. The presence of a long incubation period strain can extend the incubation period or even block the ability of a short incubation period strain to cause disease. This extension in incubation period is termed strain interference. Strain interference has been shown to occur in mice10 and hamsters22. Bartz and co-workers have used PMCA to study prion strain interference in hamsters. Their results show that the PMCA experiments correlate with the findings of a similar experiment in vivo22, 23.
Since there is a relatively low level of PrPSc outside the central nervous system, prions can only be accurately diagnosed post-mortem via dissection of the brain followed by immunohistochemistry. PMCA can be used as diagnostic tool, since it has shown a great capability of amplifying minute amounts of PrPSc 7 , even from hamster’s urine samples11.
The prion agent is able to withstand harsh environmental conditions and remain infectious16,17 . However, the amount of PrPSc in the environment is too small to be detected using conventional techniques such WB. PMCA has been used to amplify and calculate the approximate amount of PrPSc the environment, such as soil and water16, 17, 20, 21 .
The authors have nothing to disclose.
We would like to thank Dr. Vesper Fe Marie Ramos for critical reading of the manuscript. This work was supported by the National Center for Research Resources (P20 RR0115635-6, C06 RR17417-01 and G20RR024001) and the National Institute for Neurological Disorders and Stroke (2R01 NS052609).
Reagent / Equipment | Manufacturer | Cat. Number |
Misonix 3000 | Misonix | S-3000 |
Misonix 4000 | Misonix | S-4000 |
Tenbroeck Tissue Grinder | Kontes | 885000-0007 |
Neslab EX-7 Water Bath | Thermo Electron | Neslab EX-7 |
0.2 ml PCR Tube Strips | Thermo Scientific | AB-0451 |
Triton X-100 | Sigma Aldrich | T9284-100ML |
Complete Protease Inhibitor | Roche | 11 697 498 001 |
EDTA | J.T. Baker | 4040-00 |
DPBS | Mallinckrodt Baker Mediatech | 21-031-CV |
Versi-Dry Lab Soakers | Fisher Scientific | 14 206 28 |
Repti Therm Heater | Zoo Med Laboratories, Inc. | RH-4 |