Ultra-efficient replication of infectious prions by automated protein misfolding cyclic amplification. J Biol Chem

Universidad Autónoma de Madrid, Madrid, Madrid, Spain
Journal of Biological Chemistry (Impact Factor: 4.57). 12/2006; 281(46):35245-52. DOI: 10.1074/jbc.M603964200
Source: PubMed

ABSTRACT Prions are the unconventional infectious agents responsible for transmissible spongiform encephalopathies, which appear to be composed mainly or exclusively of the misfolded prion protein (PrPSc). Prion replication involves the conversion of the normal prion protein (PrPC) into the misfolded isoform, catalyzed by tiny quantities of PrPSc present in the infectious material. We have recently developed the protein misfolding cyclic amplification (PMCA) technology to sustain the autocatalytic replication of infectious prions in vitro. Here we show that PMCA enables the specific and reproducible amplification of exceptionally minute quantities of PrPSc. Indeed, after seven rounds of PMCA, we were able to generate large amounts of PrPSc starting from a 1x10(-12) dilution of scrapie hamster brain, which contains the equivalent of approximately 26 molecules of protein monomers. According to recent data, this quantity is similar to the minimum number of molecules present in a single particle of infectious PrPSc, indicating that PMCA may enable detection of as little as one oligomeric PrPSc infectious particle. Interestingly, the in vitro generated PrPSc was infectious when injected in wild-type hamsters, producing a disease identical to the one generated by inoculation of the brain infectious material. The unprecedented amplification efficiency of PMCA leads to a several billion-fold increase of sensitivity for PrPSc detection as compared with standard tests used to screen prion-infected cattle and at least 4000 times more sensitivity than the animal bioassay. Therefore, PMCA offers great promise for the development of highly sensitive, specific, and early diagnosis of transmissible spongiform encephalopathy and to further understand the molecular basis of prion propagation.

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Available from: Paula Saá, Dec 09, 2014
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    • "Many similarities can be found between DNA amplification by PCR and PrP TSE amplification by saPMCA, one of them being the possibility of cross-contamination, which is most likely to take place during sample manipulation for next round preparation in the latter . Due to the ultrasensitive nature of saPMCA, capable of detecting one single infectious unit of PrP TSE (Saá et al., 2006b), stringent precautionary measures need to be put in place to minimize such risk and appropriate negative controls need to be included in each experiment. Interestingly, real time quantitative PCR (RT-qPCR) is widely used in molecular disease diagnosis and it is the method of choice in some cases despite associated specificity issues being reported (Phillips et al., 2009a,b). "
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    ABSTRACT: Transmissible spongiform encephalopathies (TSEs) most commonly known as prion diseases are invariably fatal neurological disorders that affect humans and animals. These disorders differ from other neurodegenerative conformational diseases caused by the accumulation in the brain of misfolded proteins, sometimes with amyloid properties, in their ability to infect susceptible species by various routes. While the infectious properties of amyloidogenic proteins, other than misfolded prion protein (PrPTSE), are currently under scrutiny, their potential to transmit from cell to cell, one of the intrinsic properties of the prion, has been recently shown in vitro and in vivo. Over the decades, various cell culture and laboratory animal models have been developed to study TSEs. These assays have been widely used in a variety of applications but showed to be time consuming and entailed elevated costs. Novel economic and fast alternatives became available with the development of in vitro assays that are based on the property of conformationally abnormal PrPTSE to recruit normal cellular PrPC to misfold. These include the cell-free conversion assay, protein misfolding cyclic amplification (PMCA) and quaking induced conversion assay (QuIC), of which the PMCA has been the only technology shown to generate infectious prions. Moreover, it allows indefinite amplification of PrPTSE with strain-specific biochemical and biological properties of the original molecules and under certain conditions may give rise to new spontaneously generated prions. The method also allows addressing the species barrier phenomena and assessing possible risks of animal-to-animal and animal-to-human transmission. Additionally, its unprecedented sensitivity has made possible the detection of as little as one infectious dose of PrPTSE and the biochemical identification of this protein in different tissues and biological fluids, including blood, cerebral spinal fluid (CSF), semen, milk, urine and saliva during the pre-clinical and clinical phases of the disease. The mechanistic similarities between TSEs and other conformational disorders have resulted in the adaptation of the PMCA to the amplification and detection of various amyloidogenic proteins. Here we provide a compelling discussion of the different applications of this technology to the study of TSEs and other neurodegenerative diseases.
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    • "More recently, in vitro amplification methods [serial protein misfolding cyclic amplification (sPMCA) (Saborio et al., 2001), amyloid seeding assay (ASA) (Colby et al., 2007) and real-time quaking-induced conversion (RT-QuIC) (Atarashi et al., 2011a)], all of which employ conversion of substrate PrP C to the disease-specific conformation, have expanded the sensitivity and mechanistic possibilities of prion detection assays. For example, sPMCA, with its alternating cycles of elongation and sonication to facilitate fragmentation of nascent amyloid filaments, can be sufficiently sensitive to detect only a few molecules of PrP Res (Castilla et al., 2005a, 2008; Saá et al., 2006; Saborio et al., 2001). The ASA and RT-QuIC are generally analogous to sPMCA, but employ recombinant PrP C instead of brain homogenate as substrate for disease-specific, in vitro, amyloid formation (Atarashi et al., 2007, 2011a; Colby et al., 2007). "
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    ABSTRACT: Prions are amyloid-forming proteins that cause transmissible spongiform encephalopathies through a process involving the templated conversion of the normal cellular prion protein (PrPC) to a pathogenic misfolded conformation. Templated conversion has been modeled in several in vitro assays, including serial protein misfolding amplification (sPMCA), amyloid seeding, and real time quaking induced conversion (RT-QuIC). Because RT-QuIC measures formation of amyloid fibrils in real time, it can be used to estimate the rate of seeded conversion. Here we use samples from deer infected with chronic wasting disease (CWD) in RT-QuIC to show that serial dilution of prion seed is linearly related to the rate of amyloid formation over a range of 10-3 to 10-8 µg. We then used an amyloid formation rate standard curve derived from a bioassayed reference sample (CWD+ brain homogenate) to estimate the prion seed concentration and infectivity in tissues, body fluids and excreta. Using these methods we estimate that urine and saliva from CWD-infected deer contain between 1 and 5 LD50 per 10 ml, respectively. Thus, over the 1 to 2 year course of infection, a substantial environmental reservoir of CWD prion contamination accumulates.
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    • "So far, PMCA has been applied to detect minute quantities of oligomeric misfolded prion protein (PrP Sc ) implicated in prion diseases (Morales et al., 2012). Using PMCA, we were able to detect the equivalent of a single particle of misfolded PrP oligomer (Saá et al., 2006b) and strikingly to identify PrP Sc in the blood and urine of infected animals at symptomatic and presymptomatic stages of the disease (Castilla et al., 2005; Saá et al., 2006a; Gonzalez-Romero et al., 2008). The basis for the PMCA technology is the fact that the process of misfolding and aggregation of Ab, PrP, and the other proteins implicated in PMDs follow a seeding-nucleation mechanism (Soto et al., 2002, 2006). "
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    ABSTRACT: Alzheimer's disease (AD) diagnosis is hampered by the lack of early, sensitive, and objective laboratory tests. We describe a sensitive method for biochemical diagnosis of AD based on specific detection of misfolded Aβ oligomers, which play a central role in AD pathogenesis. The protein misfolding cyclic amplification assay (Aβ-PMCA), exploits the functional property of Aβ oligomers to seed the polymerization of monomeric Aβ. Aβ-PMCA allowed detection of as little as 3 fmol of Aβ oligomers. Most importantly, using cerebrospinal fluid, we were able to distinguish AD patients from control individuals affected by a variety of other neurodegenerative disorders or nondegenerative neurological diseases with overall sensitivity of 90% and specificity of 92%. These findings provide the proof-of-principle basis for developing a highly sensitive and specific biochemical test for AD diagnosis.
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