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Protein misfolding cyclic amplification (PMCA) is an amplification technique (conceptually like polymerase chain reaction (PCR) but not involving nucleotides) to multiply misfolded prions originally developed by Soto and colleagues.^[1] It is a test for spongiform encephalopathies like chronic wasting disease (CWD)^[2] or bovine spongiform encephalopathy (BSE).

Technique

The technique initially incubates a small amount of abnormal prion with an excess of normal protein, so that some conversion takes place. The growing chain of misfolded protein is then blasted with ultrasound, breaking it down into smaller chains and so rapidly increasing the amount of abnormal protein available to cause conversions.^[1]^[3] By repeating the cycle, the mass of normal protein is rapidly changed into the prion being tested for.^{[citation needed]}

Development

PMCA was originally developed to, in vitro, mimic prion replication with a similar efficiency to the in vivo process, but with accelerated kinetics.^[1] PMCA is conceptually analogous to the polymerase chain reaction - in both systems a template grows at the expense of a substrate in a cyclic reaction, combining growing and multiplication of the template units.^{[citation needed]}

Replication

PMCA has been applied to replicate the misfolded protein from diverse species.^[4]^[5]^[6] The newly generated protein exhibits the same biochemical, biological, and structural properties as brain-derived PrP^Sc and strikingly it is infectious to wild type animals, producing a disease with similar characteristics as the illness produced by brain-isolated prions.^[7]

Automation

The technology has been automated, leading to a dramatic increase in the efficiency of amplification. Now, a single cycle results in a 2500-fold increase in sensitivity of detection over western blotting,^[8] whereas 2 and 7 consecutive cycles result in 6 million and 3 billion-fold increases in sensitivity of detection over western blotting, a technique widely used in BSE surveillance in several countries.^[8]

Sensitivity

It has been shown that PMCA is capable of detecting as little as a single molecule of oligomeric infectious PrP^Sc.^[8] PMCA possesses the ability to generate millions infectious units, starting with the equivalent to one PrP^Sc oligomer; well below the infectivity threshold.^[8] This data demonstrates that PMCA has a similar power of amplification as PCR techniques used to amplify DNA. It opens a great promise for development of a highly sensitive detection of PrP^Sc, and for understanding the molecular basis of prion replication. Indeed, PMCA has been used by various groups to PrP^Sc in blood of animals experimentally infected with prions during both the symptomatic^[9] and pre-symptomatic phases^[10] as well as in urine.^[11]

Uses

The PMCA technology has been used by several groups to understand the molecular mechanism of prion replication, the nature of the infectious agent, the phenomenon of prion strains and species barrier, the effect of cellular components, to detect PrP^Sc in tissues and biological fluids and to screen for inhibitors against prion replication.^[12]^[13]^[14] Recent studies by the groups of Supattapone and Ma were able to produce prion replication in vitro by PMCA using purified PrP^C and recombinant PrP^C with the sole addition of synthetic polyanions and lipids.^[15]^[16] These studies have shown that infectious prions can be produced in the absence of any other cellular component and constitute some of the strongest evidence in favor of the prion hypothesis.

Research in 2020 concluded that protein misfolding cyclic amplification could be used to distinguish between two progressive neurodegenerative diseases, Parkinson's disease and multiple system atrophy, being the first process to give an objective diagnosis of Multiple System Atrophy instead of just a differential diagnosis.^[17]^[18]

References

^ ^a ^b ^c Saborio, G.P., Permanne, B. and Soto, C. (2001) Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding. Nature, 411, 810-813.
^ Patrice N Klein, CWD Program Manager USDA/APHIS. "Chronic Wasting Disease - APHIS Proposed Rule to Align BSE Import Regulations to OIE" (PDF). WHHCC Meeting – 5–6 February 2013. Archived from the original (PDF) on 26 September 2014.{{cite web}}: CS1 maint: location (link)
^ Soto, C., Saborio, G.P. and Anderes, L. (2002) Cyclic amplification of protein misfolding: application to prion- related disorders and beyond. Trends Neurosci., 25, 390-394.
^ Soto, C., Anderes, L., Suardi, S., Cardone, F., Castilla, J., Frossard, M.J., Peano, S., Saá, P., Limido, L., Carbonatto, M., Ironside, J., Torres, J.M., Pocchiari, M. and Tagliavini, F. (2005) Pre-symptomatic detection of prions by cyclic amplification of protein misfolding. FEBS Lett., 579, 638-642.
^ Jones, M., Peden, A.H., Prowse, C.V., Groner, A., Manson, J.C., Turner, M.L., Ironside, J.W., MacGregor, I.R. and Head, M.W. (2007) In vitro amplification and detection of variant Creutzfeldt–Jakob disease PrPSc. J.Pathol., 213, 21-26.
^ Kurt, T.D., Perrott, M.R., Wilusz, C.J., Wilusz, J., Supattapone, S., Telling, G.C., Zabel, M.D. and Hoover, E.A. (2007) Efficient in vitro amplification of chronic wasting disease PrPRES. J.Virol., 81, 9605-9608.
^ Castilla, J., Saá, P., Hetz, C. and Soto, C. (2005) In vitro generation of infectious scrapie prions. Cell, 121, 195-206.
^ ^a ^b ^c ^d Saa, P., Castilla, J. and Soto, C. (2006) Ultra-efficient replication of infectious prions by automated protein misfolding cyclic amplification. J.Biol.Chem., 281, 35245-35252.
^ Castilla, J., Saa, P. and Soto, C. (2005) Detection of prions in blood. Nat.Med., 11, 982-985.
^ Saa, P., Castilla, J. and Soto, C. (2006) Presymptomatic detection of prions in blood. Science, 313, 92-94.
^ Gonzalez-Romero, D., Barria, M.A., Leon, P., Morales, R. and Soto, C. (2008) Detection of infectious prions in urine. FEBS Lett., 582, 3161-3166.
^ Castilla, J., Gonzalez-Romero, D., Saa, P., Morales, R., De, C.J. and Soto, C. (2008) Crossing the species barrier by PrP(Sc) replication in vitro generates unique infectious prions. Cell, 134, 757-768.
^ Barria, M.A., Mukherjee, A., Gonzalez-Romero, D., Morales, R. and Soto, C. (2009) De novo generation of infectious prions in vitro produces a new disease phenotype. PLoS.Pathog., 5, e1000421.
^ Deleault, N.R., Lucassen, R.W. and Supattapone, S. (2003) RNA molecules stimulate prion protein conversion. Nature, 425, 717-720.
^ Deleault, N.R., Harris, B.T., Rees, J.R. and Supattapone, S. (2007) Formation of native prions from minimal components in vitro. Proc Natl Acad Sci U S A 104, 9741-9746.
^ Wang, F., Wang, X., Yuan, C.-G. and Ma, J. (2010) Generating a Prion with Bacterially Expressed Recombinant Prion Protein. Science 327, 1132-1135.
^ "Method Can Distinguish Parkinson's Disease From multiple system atrophy". Diagnostics from Technology Networks. Retrieved 23 February 2020.
^ Shahnawaz, Mohammad; Mukherjee, Abhisek; Pritzkow, Sandra; Mendez, Nicolas; Rabadia, Prakruti; Liu, Xiangan; Hu, Bo; Schmeichel, Ann; Singer, Wolfgang; Wu, Gang; Tsai, Ah-Lim; Shirani, Hamid; Nilsson, K. Peter R.; Low, Phillip A.; Soto, Claudio (5 February 2020). "Discriminating α-synuclein strains in Parkinson's disease and multiple system atrophy". Nature. 578 (7794): 273–277. Bibcode:2020Natur.578..273S. doi:10.1038/s41586-020-1984-7. PMC 7066875. PMID 32025029.