Protein misfolding cyclic amplification (PMCA): Current status and future directions.

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Protein misfolding cyclic amplification (PMCA): Current status and future directions Paula Saá ∗ , Larisa Cervenakova Transmissible Diseases Department, American National Red Cross, Biomedical Services, Holland Laboratory, 15601 Crabbs Branch Way, Rockville, MD 20855, United States

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Article history: Available online xxx Keywords: Transmissible spongiform encephalopathies Protein conformational disorders Neurodegenerative diseases Protein misfolding cyclic amplification Prions In vitro

a b s t r a c t 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. © 2014 Elsevier B.V. All rights reserved.

Abbreviations: TSEs, transmissible spongiform encephalopathies; PrPTSE , disease associated misfolded PrP; PrPC , cellular prion protein; PrPres , proteinase K resistant misfolded PrP; PrP27–30, proteinase K resistant core of misfolded PrP; rPrP, recombinant PrP; PMCA, protein misfolding cyclic amplification; aPMCA, automated PMCA; saPMCA, serial automated PMCA; PK, proteinase K; Mo-vCJD, mouse-adapted variant Creutzfeldt-Jakob disease; 263K-BH, scrapie 263K-infected hamster brain homogenate; poly(A), polyadenylic acid; poly(rA), polyriboadenylic acid; poly(G), polyglutamic acid; POPG, 1-palmitoyl-2-oleoylphosphatidylglycerol; CWD, chronic wasting disease; BSE, bovine spongiform encephalopathy; CNS, central nervous system; EV, extracellular vesicles; EVP, extracellular vesicle proteins; SP-SC, murine spleen-derived stromal cell cultures; CPD, citrate-phosphate dextrose; BH, brain homogenate; wt-MoBH, wild type-mouse brain homogenate; D2 O, deuterium oxide; RT, room temperature; PBS, phosphate-buffered saline; FVB, FVB/NCr mouse; CSF, cerebral spinal fluid; PE, phosphatidylethanolamine; ␣-Syn, alpha-synuclein; AD, Alzheimer’s disease; PD, Parkinson’s disease; A␤, amyloid beta; GPI, glycosylphosphatidylinositol; sCJD, sporadic Creutzfeldt-Jakob disease; PrPCWD , CWD-associated misfolded PrP; rPrP-respoly(rA) , PK resistant rPrP generated by saPMCA in the presence of poly(rA); BC, buffy coat; WBC, white blood cells. ∗ Corresponding author. Tel.: +1 240 314 3529; fax: +1 301 610 4120. E-mail address: [email protected] (P. Saá). http://dx.doi.org/10.1016/j.virusres.2014.11.007 0168-1702/© 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: Saá, P., Cervenakova, L., Protein misfolding cyclic amplification (PMCA): Current status and future directions. Virus Res. (2014), http://dx.doi.org/10.1016/j.virusres.2014.11.007

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ARTICLE IN PRESS P. Saá, L. Cervenakova / Virus Research xxx (2014) xxx–xxx

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1. Introduction Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative diseases characterized by the accumulation in the brain and sometimes in the lymphoid tissues of conformationally misfolded prion protein (PrPTSE ) (Doi et al., 1988; Prusiner, 1982), which originates from a conformational transformation of a cellular membrane-bound prion protein (PrPC ) (Pan et al., 1993). PrPTSE is considered to be the only (Castilla et al., 2005a; Deleault et al., 2007; Legname et al., 2004; Wang et al., 2010) or the major (Couzin, 2004; Dickinson et al., 1989; Weissmann, 1991) constituent of the infectious agent, the prion, whose misfolding and aggregation follows a nucleation-dependent polymerization mechanism (Jarrett and Lansbury, 1993). TSEs are characterized by a long, clinically silent incubation period that may exceed 50 years in humans (Collinge et al., 2006), followed by a short and aggressive clinical course of several months (Collinge, 2001). Owing to unusual infectious properties, prions have been the cause of two major epidemics in animals: (i) bovine spongiform encephalopathy (BSE) that has mainly affected cattle in the UK and France but did not spare other European countries and countries around the world, and which has been causatively linked to variant Creutzfeldt-Jakob disease that resulted in the death of 227 individuals (http://www.cjd.ed.ac.uk/data.html); (ii) chronic wasting disease (CWD), which spreads with unprecedented speed through cervid populations in North America presenting a new threat to other animal species, including ungulates, cattle and felines, and to humans. Given their transmissible nature, these diseases can be modeled by inoculation of infectious prions into different laboratory animals through various routes (Prusiner et al., 2004). However, the disadvantages of these time demanding and costly experiments lead to the development of several cell-based (Clarke and Haig, 1970; Clarke and Millson, 1976; Giri et al., 2006; Markovits et al., 1983; Milhavet et al., 2006; Raymond et al., 2006; Rubenstein et al., 1984; Taraboulos et al., 1990; Vilette et al., 2001) as well as cell-free in vitro assays (Atarashi et al., 2008; Deleault et al., 2003; Kocisko et al., 1994; Saborio et al., 2001). More than a decade ago, protein misfolding cyclic amplification (PMCA) emerged as a technology designed to achieve sensitive levels of PrPTSE detection by unlimited continuing replication of misfolded aggregates using a mechanism similar to DNA amplification by PCR (Saborio et al., 2001), where a template of PrPTSE aggregates grows at the expense of a substrate (PrPC ) in a cyclic reaction (Fig. 1). Conceptually based on the nucleation-dependent polymerization model for prion replication, PMCA combines cycles of sonication that result in hydrodynamic shearing of PrPTSE aggregates into smaller nuclei, and incubation, during which PrPTSE molecules present in the nuclei imprint their abnormal conformation onto PrPC , which is subsequently incorporated into growing PrPTSE aggregates (Saborio et al., 2001). These cycles of sonication–incubation result in the exponential amplification of minute quantities of PrPTSE present in a sample (Saá et al., 2006b; Saborio et al., 2001; Soto et al., 2002). In this manuscript, we provide an overview of the PMCA technology from its origin and optimization to its widespread application to the study of TSEs and other neurodegenerative diseases caused by the accumulation of misfolded proteins.

2. Evolution of the PMCA technology: from early development to optimization 2.1. Protein misfolding cyclic amplification (manual PMCA) In vitro conversion of purified radiolabeled PrPC into PrPTSE was first achieved by Caughey and colleagues by means of the

cell-free conversion assay (Kocisko et al., 1994) which was later on used to investigate the molecular basis of TSE transmission between species and within species with different PrP genotypes (Bossers et al., 1997; Kocisko et al., 1995). However, the use of non-physiological conditions and the low yields of PrPTSE of this assay limited its further applications. Several years later, the original PMCA system was developed using a Bandelin Sonoplus HD2070 sonicator equipped with a Titanium microtip MS73 (Bandelin Electronic, Berlin, Germany). Proof-of-concept experiments were performed by serially diluting a scrapie 263K-infected hamster brain homogenate (263K-BH), used as source of PrPTSE , into a healthy hamster BH, source of PrPC and other co-factors important for prion replication. Serial dilutions of 263K-BH were performed to mimic samples from various tissues and/or distinct phases of TSE that contain different levels of PrPTSE . Aliquots of these samples were immediately deep-frozen at −80 ◦ C, while the residual samples were subjected to various cycles of incubation and sonication. Thereafter, frozen and amplified samples were digested with proteinase K (PK) and the success of conversion was evaluated by Western blotting. This procedure resulted in a dramatic increase in PrPres levels in amplified samples as compared to equivalent frozen controls where the PrPres signal disappeared after a 3000-fold dilution of the infected brain. In contrast, PrPres could still be detected in a 500,000-fold diluted 263K-BH after PMCA. Densitometric analysis estimated that >99% of PrPres present in amplified samples was PMCA-generated (Saborio et al., 2001). 2.2. Serial automated PMCA (saPMCA) In its original format, PMCA allowed an increase of sensitivity for PrPres detection between 10- and 60-fold; and represented a technical breakthrough in prion research by providing a new in vitro system to investigate important aspects of prion biology such, as the molecular requirements for prion replication (Deleault et al., 2003; Lucassen et al., 2003; Nishina et al., 2004), the strain and species specificity of prion conversion (Lucassen et al., 2003; Soto et al., 2005), and to evaluate replication inhibitory drugs with a potential application in disease therapy (Barret et al., 2003). However, it was inefficient to sustain continuous PrPres replication, thus preventing evaluation of the biological properties of newly generated molecules (Bieschke et al., 2004). Since the level of PMCA-induced amplification is directly proportional to the number of cycles applied to the sample, the overall efficiency of the assay was limited by the cycles performed during a working day. To overcome this limitation, the system was automated by using a programmable sonicator equipped with a microplate horn (Q700, Qsonica LLC, Newtown, CT) which allowed continuous operation (Castilla et al., 2005a,b; Saá et al., 2005, 2006b). PMCA automation resulted in routine performance of more cycles with lower processing times, higher consistency by eliminating operator-dependent variability, and prevented loss of material and cross-contamination by removing probe intrusion into the sample. Moreover, higher throughput screening of samples was achieved by processing up to 96 samples at a time, making this technology amenable for large-scale biochemical detection (Castilla et al., 2006, 2005b; Saá et al., 2005, 2006a,b). Amplification of 263K-BH PrPTSE in 140 cycles of the newly automated PMCA (aPMCA) enabled the detection of PrPres in a 6.6 million-fold dilution of the infected brain; which represented a sensitivity increase of 5 orders of magnitude relative to the manual PMCA (Castilla et al., 2005b). During the course of these experiments, it became evident that the conversion efficiency leveled off at approximately 140 cycles (70 h) and started to decrease after 150 cycles of PMCA. Because not all PrPC present in the substrate had been converted into PrPres , the reduced efficiency was likely related to decreasing levels of PrPC or other brain cofactors needed for




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Fig. 1. Protein misfolding cyclic amplification (PMCA). The figure shows a schematic representation of the PMCA methodology. A small amount of PrPTSE (reaction template) is mixed with a large amount of PrPC (reaction substrate) and sonicated to fractionate PrPTSE aggregates into smaller nuclei. Samples are thereafter incubated at 37 ◦ C to allow for PrPTSE molecules to imprint their aberrant conformation onto PrPC and further incorporation into growing PrPTSE aggregates (labeled as PrPPMCA ). Repetition of cycles of sonication-incubation results in the exponential amplification of minute quantities of PrPTSE present in the sample.

conversion due to degradation or proteolysis despite the presence of protease inhibitors, since the reaction conditions require constant sample incubation at 37 ◦ C for more than 3 days. Interestingly, this effect was overcome with the introduction of a second round of amplification, involving the dilution of PMCA-amplified material into fresh BH followed by additional 118 cycles of PMCA. By performing serial rounds of amplification/dilution a new form of PMCA was developed, the so-called serial automated PMCA (saPMCA), which allowed the amplification efficiency to increase by factor of 10 million-fold, achieving a sensitivity of detection of 20 fg/ml (4 × 105 equivalent molecules of PrPres per milliliter) (Castilla et al., 2005b). Since its original report, several protocols have been published describing the PMCA procedure in great detail for various applications (Barria et al., 2012; Boerner et al., 2013; Castilla et al., 2006; Morales et al., 2012; Saá et al., 2005, 2006b; Saunders et al., 2012). 3. The use of PMCA in basic prion research 3.1. The protein-only hypothesis and the generation of infectious prions in vitro The newly developed saPMCA technology was instrumental to demonstrate the autocatalytic nature of PrPTSE , but most importantly the infectious properties of saPMCA-generated PrPres . The prevalent hypothesis in the prion field, the protein-only hypothesis, states that the infectious agent is mainly or exclusively composed of PrPTSE (Prusiner, 1982). This theory is highly controversial in the essence that it does not follow the central dogma of molecular biology, namely DNA-RNA-Protein, and it is widely accepted that final proof would be achieved if pure or recombinant PrPC (rPrPC ) could be converted into PrPTSE and elicit disease in recipient animals (Weissmann, 2005). In a seminal study by Castilla et al. (2005a), a 263K-BH sample was subjected to 33 rounds of saPMCA separated by up to 1000-fold dilutions. These rounds of dilution-amplification resulted in a 10−55 dilution of the original BH, and an estimated amplification rate of at least 1000, given that the signal intensity recovered after dilution-amplification was unchanged. Since according to mathematical calculations, the very last molecule of brain-derived PrPTSE was presumably diluted out after a 10−14

dilution, continuous replication of PrPTSE by saPMCA thereafter, demonstrated the autocatalytic nature of newly generated PrPTSE . Additionally, the possibility of maintaining PrPTSE replicating indefinitely in an in vitro system, allowed the generation of a sample devoid of brain-derived infectious material and the evaluation of the infectious properties of in vitro generated PrPTSE . Intracerebral inoculation of hamsters with saPMCA-generated samples resulted in the development of TSE with a clinical disease indistinguishable from that caused by the inoculation of brain-derived preparations not subjected to saPMCA. A detailed analysis of saPMCA-generated PrPres confirmed identical biochemical and structural properties to that of brain-derived PrPres , demonstrating that saPMCA faithfully reproduces the PrPres replication process that takes place in vivo (Castilla et al., 2005a). Puzzling however, was the fact that primary inoculation of saPMCA-generated material resulted in a longer incubation phase, which shortened upon secondary transmission, becoming identical to that produced by the injection of similar levels of brain-derived PrPres , as determined by Western blotting after PK digestion (Castilla et al., 2005a). Because, no sample titration studies were performed at that time, it could not be assessed whether the extended incubation period resulted from lower specific infectivity of saPMCA-generated material or from inoculation of lower number of PrPTSE molecules. Later experiments addressing this discrepancy attributed the differences in infectivity to particle size variation and enhanced susceptibility of saPMCA material to clearance upon intracerebral injection (Weber et al., 2006, 2007). Recently, a comprehensive study by Klingeborn et al. (2011), comparing infectivity titers and PrPres levels, provided compelling evidence that saPMCAgenerated PrPTSE carries lower specific infectivity, probably due to the formation of non-infectious PrPres during saPMCA. This phenomenon on its own deserves further comprehensive investigation. Interestingly, an independent study performed by Deleault et al. showed that when hamster brain-purified PrPC was used as a substrate in saPMCA reactions seeded with Sc237 hamster scrapie, a strain equivalent to 263K, the prion infectivity levels were 4-fold lower as compared to brain-derived material (Bennett et al., 1992; Deleault et al., 2007). This result, together with subsequent studies performed with various strains of hamster scrapie (Castilla et al., 2008a) as well as with prion strains from different animal species,


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in which identical length of the incubation periods was observed upon injection of animals with brain- and saPMCA-derived samples (Castilla et al., 2008b; Di Bari et al., 2013; Green et al., 2008; Meyerett et al., 2008; Saá et al., 2012; Vidal et al., 2013), point out to a 263K strain-specific rather than to a generalized phenomenon associated with PMCA replication. 3.1.1. saPMCA reactions with simplified substrates Although the work by Castilla and colleagues convincingly demonstrated generation of PrPres and infectivity in vitro, and provided compelling evidence in favor of the prion-only hypothesis, it could not rule out the role of other molecules in amplification reactions as it was not carried out with purified components or rPrPC . Nonetheless, this study set the ground for later experiments addressing the capability of purified PrPC molecules to sustain PrPTSE replication by saPMCA (Deleault et al., 2007). Empirical analysis of various substrate mixtures showed that purified PrPC , equimolar quantities of 20-carbon fatty acids and synthetic polyadenylic acid [poly(A)] RNA molecules sufficed to initiate and sustain replication of purified Sc237 and 139H PrP27–30 scrapie molecules. In addition, intracerebral injection of saPMCA-generated material elicited disease in recipient hamsters, albeit produced preparations failed to maintain strain-specific differences triggering clinically similar diseases after identical incubation time and biochemically indistinguishable PrP profile (Deleault et al., 2007). Various reasons could explain the loss of strain identity following in vitro amplification with minimal components: the digestion of strain-specific, protease-sensitive PrPTSE conformers in the saPMCA seed, the removal of the N-terminal octarepeat region from protease-resistant PrPTSE (Deleault et al., 2007), or the lack of a molecular co-factor conferring strain-specific properties. On the other hand, the purification process may have altered the structural properties of PrPC leading to the acquisition of an alternative “default” infectious conformation independently of the strain used to seed conversion.

incubation times and with different attack rates that were stabilized on secondary transmission. Neuropathological examination revealed similar lesion pattern and severity of spongiform degeneration in hamsters inoculated with rShaPrP-(23–231)PMCA and rShaPrP-(90–231)PMCA . Importantly, observed pathological features differed from those elicited by the inoculation of 263K, and strongly suggested the emergence of a new strain of scrapie (Kim et al., 2010). In the lack of specific knowledge regarding the molecular and/or physicochemical determinants of strain properties, the aspects that might influence the fidelity of strain propagation can only be discussed with reservation. rPrPC differs from brain-derived PrPC in a number of ways but predominantly in the absence of glycosylation and glycosylphosphatidylinositol (GPI) anchor. However, it has been shown that strain fidelity was maintained upon saPMCA replication even when PrPC was not glycosylated (Piro et al., 2009). On the other hand, propagation of brain-derived mouse scrapie and misfolded rPrPTSE in transgenic mice expressing anchorless PrPC led to the emergence of a new strain with novel cell tropism as determined by the extended cell panel assay (Mahal et al., 2012) and to the selection and propagation of new synthetic prion strains (Raymond et al., 2012). These limited data highlight the need to fully understand the role which GPI plays in strain fidelity. Once again, PrPTSE produced in PMCA (rShaPrP-(23–231)PMCA and rShaPrP-(90–231)PMCA ) had lower infectivity as compared to brain derived PrPTSE , phenomenon which in this case can be attributed to various factors: (1) the absence of brain cofactors that might promote infectivity or (2) the generation of noninfectious PrPres by PMCA, as previously suggested (Kim et al., 2010; Klingeborn et al., 2011). Interestingly and similarly to previous reports (Castilla et al., 2005a; Deleault et al., 2007), this material characterized by lower infectivity was originally seeded with 263K hamster scrapie, leaving open the question as to whether seeding with a different strain would have resulted in the same or higher infectivity levels comparable to brain-derived material.

3.1.2. Use of rPrPC as saPMCA substrate The generation of infectious prions by saPMCA led to the rapid incorporation of this technology in many laboratories and further development. One of the most relevant advances, from a fundamental standpoint, was the successful conversion of rPrPC into rPrPres in saPMCA. In vitro reactions were seeded with scrapie PrPres purified from hamster brain, in the absence of other brain cofactors (Atarashi et al., 2007). Strikingly, similar to previous findings obtained with brain-derived material, rPrPTSE replication was autocatalytic and could be maintained indefinitely in vitro; characteristics central to the protein-only hypothesis and the nucleation-dependent polymerization mechanism of conversion (Atarashi et al., 2007; Jarrett and Lansbury, 1993; Prusiner, 1982). Several advantages arise from the use of rPrPC as PMCA substrate that may facilitate fundamental studies of structure and formation of PrPTSE . Bacterially expressed rPrPC can be produced in large amounts and high purity, reducing the difficulties of purifying replication competent PrPC , eliminating ethical issues and reducing the labor intensity and costs associated with the use of BH. Moreover, rPrPC can be easily mutated or labeled to study conformational changes and molecular interactions, leading to a better understanding of the conversion mechanism. However, these studies strongly rely on the demonstration of the infectious properties of misfolded rPrPTSE . After nine rounds of saPMCA and a cumulative 10−14 dilution of the original 263K-BH seed, the infectious properties of full length (23–231) and truncated (90–231) misfolded Syrian hamster rPrPTSE [rShaPrP-(23–231)PMCA and rShaPrP-(90–231)PMCA ] were evaluated by intracerebral inoculation into hamsters (Kim et al., 2010). As a result, a number of inoculated animals in both groups developed clinical signs of TSE after variable, extended

3.1.3. De novo generation of prion infectivity Prion diseases can be subdivided into three etiological groups: sporadic, genetic and environmentally acquired (i.e. infectious). Sporadic Creutzfeldt-Jakob disease (sCJD) is the most common form, occurring without any obvious cause with a worldwide frequency of 1 case per million people per year. Interestingly, during the course of saPMCA optimization, the scattered appearance of PrPres in unseeded reactions after many rounds of amplification was observed; and in light of these findings, de novo generation of PrPres was considered together with the possibility of crosscontamination (Saá et al., 2006b). Since its original report, several studies have been published reporting de novo generation of PrPres from different substrates of various species (e.g. rPrPC , purified PrPC ), that can be subdivided in two groups: (i) originated from PMCA studies (Abdallah et al., 2012; Atarashi et al., 2007; Barria et al., 2009; Deleault et al., 2007, 2012a,b; Geoghegan et al., 2007; Wang et al., 2010, 2012) and (ii) created by in vitro aggregation experiments (Colby et al., 2009, 2010; Legname et al., 2004; Makarava et al., 2010). Building upon preliminary findings (Saá et al., 2006b) and by modifying the saPMCA conditions in favor of longer incubation periods between rounds, Barria et al. (2009) were able to generate PrPres in the absence of pre-existing seeds using hamster and mouse, but not human, BH as source of PrPC in saPMCA reactions. Intracerebral inoculation of hamsters with de novo generated PrPTSE resulted in a new disease phenotype with unique clinical, neuropathological and biochemical features (Barria et al., 2009). De novo generation of PrPres and infectivity was also achieved when hamster brain-purified PrPC , containing equimolar amounts of 20-carbon fatty acids, was incubated with poly(rA) in saPMCA




reactions, suggesting that lipids and RNA are the minimal components required to generate infectious PrPTSE in vitro (see further discussion in Section 3.3) (Deleault et al., 2007). The disease phenotype in hamsters injected with these preparations was different from that elicited by the inoculation of Sc237seeded- or Sc237 brain-derived material. Similar results were obtained when mixtures of mouse rPrP, synthetic 1-palmitoyl-2oleoylphosphatidylglycerol (POPG) and liver RNA were subjected to rounds of saPMCA, albeit producing a preparation with higher infectivity levels. Nevertheless, and similarly to other studies, de novo saPMCA-generated PrPTSE produced a disease phenotype different from that caused by the RML scrapie strain, the only brain-derived strain used in that lab (Wang et al., 2010). Given the ultrasensitive properties of the assay (Saá et al., 2006b), crosscontamination has always been a concern in saPMCA reactions, which has been described under certain experimental conditions (Cosseddu et al., 2011). However, the aforementioned studies have reported to entertain stringent precautionary measures to prevent cross-contamination. Moreover, the new disease phenotypes produced by de novo generated PrPTSE argues against this possibility. Collectively, the confirmation of de novo generation of PrPTSE and infectivity in vitro has important implication in prion research since it provides compelling evidence in favor of the protein-only hypothesis. In addition, these findings open a new ways to study not only sporadic, but also genetic forms of TSE, as de novo generation of PrPres has also been reported when rPrP harboring the Y145Stop mutation was subjected to saPMCA reactions in the absence of brain-derived PrPTSE (Abdallah et al., 2012). Interestingly, some studies on the spontaneous generation of PrPTSE have shown formation of non infectious, misfolded, PK-resistant and self-propagating forms of prion protein, together with infectious species (Timmes et al., 2013; Zhang et al., 2013). These findings together with the fact that de novo PrPTSE produced under various conditions can give rise to new disease phenotypes, indicate that the spectrum of possible different conformations that PrP can adopt is not restricted to those currently known, and that new infectious conformations may spontaneously arise under certain environmental conditions, resulting in new forms of TSE (Barria et al., 2009). Indeed, unseeded saPMCA experiments performed with BHs prepared from rabbits, a species regarded as TSE-resistant, resulted in spontaneous generation of rabbit prions causing the disease in wild-type rabbits, although with low attack rate and a long incubation period of 766 days upon primary passage, and to transgenic mice over-expressing rabbit prion protein (Chianini et al., 2012). 3.2. Prion strain and species barrier phenomena The facts that prions appear in the form of different strains, and that interspecies propagation of TSEs is defined by a transmission barrier, are perhaps the most puzzling and interesting phenomena in TSE research. These two aspects of prion diseases are highly interrelated as strains from one species have different capacity to cross-seed conversion of PrPC from a different species, as highlighted by the generation of different disease phenotypes and brain lesion profiles upon inoculation (Bruce et al., 1997); and cross-species transmission results in the emergence of new prion strains on some occasions (Kimberlin et al., 1987). Importantly, prion strains exhibit specific biological and biochemical characteristics (Bessen and Marsh, 1992; Bruce, 1993). To accommodate the existence of prion strains within the framework of the protein-only hypothesis, it has been posited that strains arise when the same amino acid sequence adopts different pathologic conformations (Telling et al., 1996). Experimental data have been collected supporting the notion that strain properties are encoded in the structure of PrPTSE (Caughey et al., 1998; Safar et al., 1998).

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Nevertheless, definite proof depends on the in vitro generation of PrPTSE in reactions seeded with various strains, resulting in the maintenance of strain-specific properties. The development of the saPMCA made available a new technology to investigate the molecular aspects of the strain and species barrier phenomena. As a result, strain fidelity was first replicated in a cell-free environment by seeding mouse and human brain homogenates with five different strains of mouse scrapie and four human prion strains, respectively (Castilla et al., 2008b). Likewise, the same group was capable to overcome in vitro the species barrier hamster–mouse–hamster with the concomitant generation of novel prion strains with new biochemical and pathological features in both species (Castilla et al., 2008a). Furthermore, the latter studies demonstrated adaptation of the in vitro-produced PrPTSE to the new host in a way reminiscent of strain stabilization after serial transmission in vivo (Castilla et al., 2008a; Race et al., 2002, 2001). These observations were supported later on when the CWD-field isolate D10, propagated in saPMCA and injected into transgenic mice over-expressing cervid PrPC caused the disease after a shorter incubation period. The brain lesion profile, PrPres glycoform ratio and conformational stability were identical to those produced by in vivo sub-passage of D10 (Meyerett et al., 2008). These experiments were instrumental to demonstrate the applicability of the PMCA technology to study the certain underlying mechanisms of prion diseases and to evaluate the potential risks of prion strain transmission to human and animals by assessing the strength of the species barrier in a semi-quantitative fashion (Fernandez-Borges et al., 2009). One of the advantages that the PMCA technology offers relates to the possibility of using a variety of different prion strains to evaluate the zoonotic potential of animal forms of TSE in a fast paced, inexpensive approach that cannot be easily ascertained in vivo. In this way, Jones et al. (2009) reproduced and confirmed in vitro the ability of BSE prions to convert human prion protein, and the resistance of the latter to be converted with sheep scrapie prions. The existence of a causative link between BSE and vCJD created a new precedent and set the basis to investigate the transmissibility of other animal diseases, like CWD, to humans. CWD, originally described in farmed mule-deer in Wyoming four decades ago (Williams ES, 1980) has now been identified in free-ranging cervids in 22 US states and 2 Canadian provinces. This widespread epidemic is considered to be one of the largest zoonotic problems in wild animals in North America. The exact prevalence of the disease is unknown, as it is the risk of transmission of CWD to other species, including human. Histopathological and biochemical evaluations have found no association between CWD and the development of sCJD in unusually young patients who were game hunters, consumed venison or lived in a CWD endemic area (Belay et al., 2004). Likewise, several attempts have failed to transmit CWD to transgenic mice either over-expressing or expressing physiological levels of human PrPC (Kong et al., 2005; Sandberg et al., 2010; Tamguney et al., 2006; Wilson et al., 2012). CWD is highly contagious among cervids, and PrPTSE and/or infectivity have been found in blood (Rubenstein et al., 2010), saliva (Haley et al., 2009b; Mathiason et al., 2006), urine, skeletal muscle (Daus et al., 2011) and feces (Haley et al., 2009a,b; Pulford et al., 2012) of naturally and experimentally infected animals either by mouse bioassay and/or saPMCA. Moreover, CWD prions persist in the environment through the adherence to clay in soil (Johnson et al., 2006; Miller et al., 2004) as demonstrated by the successful transmission of soil-bound PrPCWD by intranasal and oral exposure (Miller et al., 2004; Nichols et al., 2013). Additionally, PrPCWD was detected in one of two environmental water samples from a CWD endemic area by saPMCA, although the amounts contained in these samples, prior amplification, were too low to cause the infection in experimental animals (Nichols et al., 2009). Extremely disturbing




is the notion of experimental CWD transmission to domestic cats (Mathiason et al., 2013), a species closely interacting with humans. Although it is not clear whether CWD can be naturally transmitted to cattle, experimental infection has been achieved through intracerebral but not oral inoculation (Hamir et al., 2011). These findings are of upmost importance given the possibility for wild cervids to be in contact with cattle at domestic-wildlife interfaces, giving rise to the hypothesis that transmission to humans may occur through the exposure to CWD-infected cattle. In the absence of epidemiological evidence of CWD transmission to humans these findings raise the concern that under certain conditions such transmission can occur directly or through intermediate hosts in the future. To this end, saPMCA could help predict whether conversion of human PrPC to PrPres can be induced by CWD. Importantly, conversion of human PrPC by PrPCWD -seeding has been achieved by means of this technology (Barria et al., 2011), however this study did not address whether in vitro generated PrPTSE is infectious to transgenic mice expressing human prion protein that represent a highly susceptible animal model for addressing transmissibility of some forms of human TSEs. It would be equally important to investigate if prions from CWD-infected cattle or cats, or any other species that share the environment with humans can induce conversion of human PrPC . 3.3. Understanding the molecular basis for prion replication Several groups have reported that RNA molecules facilitate in vitro amplification of infectious PrPTSE (Deleault et al., 2007; Gonzalez-Montalban et al., 2011; Wang et al., 2010). These studies were led by experiments demonstrating that the enzymatic removal of RNA but not DNA, dsRNA or RNA:DNA hybrids inhibited in vitro prion replication, which was specifically restored by the addition of total RNA from brain, but not by other polyanions such as heparan sulfate proteoglycan, ssDNA, poly(A), polyglutamic acid [poly(G)] or heparan sulfate, thus indicating the importance of RNA in in vitro PrPTSE replication (Deleault et al., 2003). Additional experiments performed by the same group showed that other endogenous polyanions such as DNA, heparan sulfates or lipids may come into play in conditions of RNA deficiency (Deleault et al., 2005). Unfortunately, the use of crude BH in these experiments complicated direct demonstration of RNA stimulatory effect on the conversion reaction because it could not be established whether RNA act alone or in combination with other cofactors. Later, saPMCA experiments performed with purified PrPC provided evidence that successful prion replication requires accessory polyanionic molecules (Deleault et al., 2007). Infectious preparations were generated using a mixture of hamster brain-purified PrPC and synthetic poly(A) RNA in Sc237- and 139H-seeded and unseeded saPMCA reactions. Because purification of PrPC from brain yielded a PrPC preparation containing equimolar quantities of 20-carbon fatty acids, the findings suggested that lipids and polyanionic molecules suffice to induce (unseeded reactions) and propagate (seeded reactions) the misfolding process (Deleault et al., 2007). The role of both components was supported further in experiments employing unseeded reactions when highly infectious rPrPTSE was formed in preparations containing rPrP plus the synthetic phospholipid POPG and liver RNA (Wang et al., 2010). Further experiments showed that generation of these infectious prion molecules did not rely on the presence of genetic informational RNA. However, these studies could not rule out whether these molecules are required for strain preservation (Wang et al., 2012). When a mixture containing synthetic poly(rA), POPG and rPrPC was seeded with rPrPres , generated de novo in the presence of liver RNA and POPG (Wang et al., 2010), the resulting rPrP-respoly(rA) induced a clinical phenotype similar to the seed and reminiscent of TSE, but after a significantly longer incubation time (220 versus

150 days), and with milder degree of spongiosis in the brains of inoculated mice (Wang et al., 2012). Secondary transmission studies will be necessary to determine whether these differences can be further maintained, therefore confirming the generation of a strain different from the one used to seed the reaction. Despite the aforementioned studies support a role for RNA in in vitro prion replication, it is not fully understood whether RNA molecules act as mere catalysts of the misfolding process, or if they associate with the infectious particle conferring prion strain characteristics. Some experiments have shown colocalization of RNA with extracellular aggregates of PrPTSE present in scrapieaffected hamster brains (Geoghegan et al., 2007); and structural analysis of PrP misfolding demonstrated that cofactor molecules were responsible for the transformation undergone by PrPC during PMCA conversion (Miller et al., 2013). Recent studies suggest a species-dependent requirement for PrPTSE amplification in vitro, with hamster-derived strains being largely dependent on the presence of RNA while these molecules appear to be not necessary for mouse PrPTSE in vitro amplification (Deleault et al., 2010; GonzalezMontalban et al., 2011). However, the fact that photodegradation of polyanions did not affect prion infectivity led some authors to speculate that these molecules assist in the misfolding event but do not confer strain-specific properties (Piro et al., 2011). The latter observation was further supported by studies evaluating the conversion efficiency, of six in vivo-propagated plus three additional saPMCA-generated murine scrapie strains in RNA-depleted mouse BH. After RNase A treatment of mouse BH and RNA removal assessment by quantitative real time PCR, Saá and colleagues observed a significant reduction in PMCA conversion efficiency for some prion strains (Saá et al., 2012). Moreover, this effect was independent of the intrinsic conversion capacity of each individual strain under normal conditions and highlighted strain-specific differences in their requirements for the presence of RNA for efficient prion replication. Interestingly, continuous replication of RML strain under RNA-depleted conditions did not affect strain identity, as determined by mouse bioassay, despite the observed dramatic decrease in conversion efficacy (Saá et al., 2012). The latter findings together with the aforementioned photodegradation experiments (Piro et al., 2011) contributed to the suggested hypothesis that RNA molecules may act as strain-specific catalysts of prion replication, but are not required to confer strain-specific properties (Piro et al., 2011; Saá et al., 2012). Additional evidence suggesting a role for RNA in prion replication came from saPMCA experiments conducted with rPrP and phosphatidylethanolamine (PE) in the absence of nucleic acids. Under these experimental conditions, spontaneous conversion of rPrPC was achieved, producing a rPrPTSE conformation characterized by a protease-resistant core of ∼18 kDa which was infectious to wild-type mice upon intracerebral inoculation. Nevertheless, the long incubation phase of almost 400 days post inoculation manifested the important role that other brain-derived cofactors (i.e. RNA or other lipids) play in generating highly infectious prion preparations (Deleault et al., 2012a). Collectively, there is sufficient evidence supporting the need for brain cofactors in efficient prion replication in vitro since no preparations of PrPTSE harboring high levels of specific infectivity have been generated under conditions in which a single cofactor or no cofactor has been utilized (Deleault et al., 2012a; Kim et al., 2010). Moreover, two facts: in situ photodegradation of polyanionic molecules being incorporated into newly generated rPrPTSE aggregates during their assisted misfolding (Piro et al., 2011; Piro and Supattapone, 2011) and the enzymatic removal of RNA molecules during prion replication (Saá et al., 2012), resulting in the maintenance of infectious and strain-specific properties in both cases, suggest that these molecules are catalysts of prion conversion. Additional studies will be, however, required to




elucidate the molecular aspects of the synergy between brain cofactors during prion replication, in which the PMCA technology will again play a central role.

4. PMCA in TSE diagnosis At present, definite diagnosis of human and animal TSEs is established postmortem by neuropathological examination and detection of PrPTSE either by immuno-histochemistry, immunohistoblot or Western blot (Budka et al., 1995; Schiermeier, 2001; Weber et al., 1997; Windl and Dawson, 2012). Transfusion transmission of vCJD has been evidenced in four recipients of nonleukoreduced red blood cells and a number of healthy individuals have been found to harbor PrPTSE in lymphoreticular tissues (de Marco et al., 2010; Gill et al., 2013; Knight, 2010; Wadsworth et al., 2011). These findings, in a scenario where uncertainty exists in regard to the number of people incubating vCJD (Gill et al., 2013; Hilton et al., 2004), raise an enormous concern about the possible spread of TSEs via blood and blood-derived products; and highlight the need of developing reliable ante-mortem assays to diagnose affected people and animals. PrPTSE is the only validated surrogate marker of TSEs and the identification of this protein in human or animal tissues is key for diagnosis (Prusiner, 1991). However, the levels of PrPTSE in tissues other than brain are significantly lower, and a few experimental detection methods rely on concentration and/or amplification techniques. saPMCA offers the opportunity to amplify PrPTSE to levels detectable by standard biochemical methods and presents itself as an ideal system to achieve biochemical detection of PrPTSE in samples containing minute amounts of this protein. The ultrasensitive nature of this technology, capable of detecting as little as 26 molecules or ∼1 ag of PrPTSE (Saá et al., 2006b) made possible the identification of PrPTSE in buffy coats (BC) isolated during the preclinical, as early as 20 days post inoculation, and at clinical phases of the disease, from hamsters intracerebrally and intraperitoneally injected with 263K scrapie (Castilla et al., 2005b; Saá et al., 2006a), and at 75 days post inoculation, when hamsters were per-orally infected with Sc237 scrapie (Murayama et al., 2007). Subsequent studies involving the use of saPMCA facilitated the detection of PrPTSE in various blood components, including the following: in plasma from preclinical and clinically sick scrapie-affected sheep and hamsters and CWD-infected white-tailed deer (Chen et al., 2010; Murayama et al., 2007; Rubenstein et al., 2010), and from clinically ill mice infected with mouse-adapted BSE (Fujihara et al., 2009), in white blood cells (WBC) from sheep of the VRQ/VRQ genotype clinically affected with scrapie (Thorne and Terry, 2008), and in whole blood collected during the clinical phase from scrapieinfected mice (Tattum et al., 2010). More recently, Lacroux et al. (2014) applied the saPMCA technology to demonstrate the presence of vCJD prions in WBC obtained during the pre-symptomatic and symptomatic phases from sheep orally challenged with BSE. Additionally, PrPTSE was detected by means of this assay in WBC and BC collected from clinically ill cynomolgus macaques intravenously challenged with a brain homogenate or whole blood from a primate infected with vCJD; and in BC collected during the preclinical phase from the same animals. The originality of the approach presented by this group was the development of a heterologous system where the PrP sequence of the seed (experimental sample under evaluation) and the substrate, derived from different species. Empirical comparison of the saPMCA converting efficiency of brain homogenates prepared from wild-type mice or transgenic mice expressing bovine, 129M human and ARQ or VRQ sheep PrPC , unexpectedly showed that ARQ and VRQ ovine PrPC homogenates were the best substrate to amplify vCJD prions. Notably, these conditions allowed detection of PrPTSE in WBC from one patient and BC from

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two out of three patients afflicted with vCJD (Lacroux et al., 2014). Although the low number of samples analyzed limits the conclusions of this study, these findings confirm the applicability of the saPMCA to the clinical diagnosis of TSEs. Further confirmation of the saPMCA potential application in clinical diagnosis was recently provided in experiments conducted with urine samples. Presence of PrPTSE in urine was first demonstrated by saPMCA in samples collected from hamsters intracerebrally and per-orally challenged with Sc237 prions (Murayama et al., 2007), and later, in urine samples from hamsters intraperitoneally injected with 263K and HY scrapie strains (Chen et al., 2010; Gonzalez-Romero et al., 2008), from symptomatic scrapie-infected sheep, and from CWD-infected pre-clinical or clinical white-tailed deer (Rubenstein et al., 2011). While these findings represented an important advance toward a non-invasive test to diagnose TSEs in animals, the evidence of this protein in human urine remained elusive until recently, when the Soto group reported the specific detection by saPMCA of PrPTSE in urine from patients affected with vCJD, but not in urine of sCJD patients or individuals afflicted with other forms of TSE or other degenerative or non-degenerative neurological diseases (Moda et al., 2014).

4.1. PrPTSE co-localizes with plasma extracellular vesicles (EVs) with biochemical and morphological characteristics reminiscent of exosomes Despite numerous attempts in different laboratories, no study has fully addressed in what blood component(s) TSE infectivity and/or PrPTSE are present and whether or how blood contributes to the dissemination of the agent from the periphery to the central nervous system (CNS). PrPC has been identified in extracellular vesicles (EVs) of 30–120 nm in diameter, known as exosomes, released by platelets, and in annexin V-positive EVs released by apoptotic endothelial cells (Robertson et al., 2006; Simak et al., 2002). Moreover, exosomes purified from the conditioned media of TSEchronically infected cell cultures have been shown to contain PrPTSE and PrPC (Alais et al., 2008; Castro-Seoane et al., 2012; Coleman et al., 2012; Fevrier et al., 2004; Mattei et al., 2009; Veith et al., 2009; Vella et al., 2007; Wik et al., 2012), and intracerebral inoculation of vesicles from infected cell cultures has induced TSE in mice (Fevrier et al., 2004; Vella et al., 2007). Interestingly, it has been shown that some viruses take advantage of the mechanism for exosome biogenesis and release to disseminate within the body (Gould et al., 2003; Nguyen et al., 2003). Likewise, serum amyloid A, tau, ␣-synuclein (␣-Syn) and ␤-amyloid peptides have been detected in exosomes leading to the speculation that they may contribute to the prion-like spread of these proteins (Rajendran et al., 2006; Saman et al., 2012; Schneider and Simons, 2012; Surgucheva et al., 2012; Tasaki et al., 2010; Vingtdeux et al., 2012). While the distribution of PrPTSE in blood is still under investigation, PrPC has been identified on exosomes isolated from human plasma (Ritchie et al., 2013). It is well established that plasma of TSE-infected experimental rodents contains infectivity (Brown et al., 1999, 1998; Cervenakova et al., 2003). Altogether, these findings suggest with high probability that endothelial and blood cells may be capable of releasing PrPTSE in association with exosomes and other EVs, thus contributing to the transfusion transmission of TSEs and to the distribution of PrPTSE from the periphery to the CNS. By combining EVs isolation from plasma samples collected from clinically sick FVB/NCr (FVB) mice infected with mouse adapted vCJD (Mo-vCJD) with saPMCA, we attempted more precise identification of PrPTSE in this bodily fluid, and were capable of demonstrating the colocalization of PrPres with Hsp70-containing EVs of 114 nm in diameter, which are reminiscent of exosomes.




4.1.1. Isolation and characterization of plasma EVs. EVs preparations are positive for the exosomal marker Hsp70 Experimental studies in mice were reviewed and approved by the Institutional Animal Care and Use Committee of the American Red Cross. To characterize EV preparations, we analyzed the presence of the exosomal marker Hsp70 and the absence of cellular markers such as the Golgi marker GM130. EVs were isolated from plasma of a healthy FVB mouse (Charles River Laboratories, Andover, MA). Mouse blood was collected into citrate-phosphate dextrose (CPD) by cardiac puncture under isoflurane anesthesia (Patterson Veterinary, Devens, MA). Samples were spun at 2300 × g for 10 min, at room temperature (RT). Upper phase, containing plasma was collected, aliquoted and stored at −80 ◦ C. Plasma aliquots were thawed at 37 ◦ C and spun for 15 min at 3000 × g and 4 ◦ C. The resulting supernatant was mixed with ExoQuickTM (System Biosciences, Mountain View, CA) and incubated overnight at 4 ◦ C. Samples were thereafter centrifuged for 30 min at 1500 × g and 4 ◦ C. EV pellets were collected and further purified through a 30% sucrose cushion following a protocol adapted from Thery et al. (2006). Briefly, the ExoQuickTM pellet was thawed at RT, resuspended in PBS and layered on top of 30% sucrose prepared in a Tris/deuterium oxide (D2 O) solution (30 g sucrose, 2.4 g Tris base, D2 O up to 100 ml). After ultracentrifugation at 110,000 × g and 4 ◦ C for 75 min, three fractions were collected; “infected top”, “infected middle” containing the sucrose and the PBS/sucrose interface, and the pellet (“infected bottom”). Samples were subjected to a second ultracentrifugation at 110,000 × g for 70 min. Purification through a 30% sucrose cushion eliminated non-specifically associated proteins, which are sedimented by centrifugation but do not float on a sucrose gradient, yielding a highly purified exosomal preparation (Thery et al., 2006). The top, middle and bottom fractions were resuspended in EV protein (EVP) lysis buffer (100 mM NaCl, 10 mM EDTA, 0.5% Triton X-100, 0.5% sodium deoxycholate, and 10 mM Tris pH 7.4) to solubilize membrane proteins which were thereafter concentrated by methanol precipitation and resuspended in 1% Sarkosyl in PBS. EVs were also isolated from the conditioned media of murine spleenderived stromal cell cultures (SP-SC) with ExoQuickTM -TC. SP-SC cells were developed in our laboratory and have been previously described (Akimov et al., 2009, 2008). Cells were plated in 25 cm2 flasks with OPTI-MEM media (Life TechnologiesTM , Carlsbad, CA) that was not supplemented with fetal bovine serum to avoid contamination with exogenous EVs. Cells were allowed to grow at 37 ◦ C and 5% CO2 for two to three days. The cell culture media was collected when cells reached confluence and centrifuged at 3000 × g and 4 ◦ C for 15 min to remove cells and cell debris. Supernatants were mixed with ExoQuickTM -TC (System Biosciences) and incubated at 4 ◦ C overnight. The mixture was centrifuged for 30 min at 1500 × g and 4 ◦ C. EV pellets were collected, resuspended in EVP lysis buffer, methanol precipitated and resuspended in 1% Sarkosyl in PBS (“cell EVs”). This preparation was used as positive control for Hsp70. Additionally, SP-SC cells were lysed in EVP lysis buffer (“SP-SC cell lysate”) and used as control for GM130. Proteins were separated by SDS-PAGE and electro-blotted onto a nitrocellulose membrane (Life TechnologiesTM ). Western blots were probed with the primary rabbit monoclonal antibodies anti-Hsp70 (Epitomics, Burlingame, CA) and anti-GM130 (Abcam, Cambridge, MA) and the corresponding HRP-conjugated mouse anti-rabbit IgG Rabbit Trueblot® secondary antibody (Rockland, Gilbertsville, PA). Immunoreactive bands were visualized using West Pico (Pierce, Rockford, IL). Analysis of samples obtained from plasma, conditioned media and cell lysates revealed the presence of Hsp70 in the conditioned media sample (“cell EVs”) and sucrose middle plasma fraction but not in sucrose top and bottom plasma fractions obtained after ultracentrifugation (data not shown). These findings confirmed the presence of exosomes in samples prepared

Fig. 2. Determination of plasma-derived EVs particle size. EVs were purified from uninfected mouse plasma. The particle size distribution represented in the figure relative to the particle concentration was determined in a NanoSight LM-10 instrument. A main population of 114 nm-particles was identified, which is consistent with the exosome size range of 30–120 nm.

from plasma using ExoQuickTM . As expected, the Golgi marker GM130 was exclusively detected in cell lysates used as positive controls. 4.1.2. EVs preparations contain particles of exosomal size To identify the size of EVs, and further confirm the presence of exosomes in our preparations, samples purified with ExoQuickTM from plasma of healthy FVB mice were resuspended in PBS and the suspension further diluted 1000-fold prior to being analyzed. The mean hydrodynamic diameters of the EVs were measured for 90 s using a NanoSight LM10 system (NanoSight Ltd., Amesbury, UK). The Nanoparticle Tracking Analysis 2.3 analytical software was used for capturing and analyzing the data. The vesicle size ranged from 50 nm to 300 nm, with an average hydrodynamic size of 114 nm (Fig. 2) which was consistent with the size range reported for plasma EVs (Dragovic et al., 2011). 4.1.3. PrPres co-localizes with plasma EVs The presence of PrPres was investigated in EVs isolated from plasma samples collected from clinically sick Mo-vCJD-infected and uninfected control FVB mice. Mice were intracerebrally injected with 30 ␮l of a 10−2 diluted Mo-vCJD-infected BH. Control mice received a similar injection of physiological saline. Mice in the experimental group were euthanized when they developed clinical signs of TSE. Negative controls were euthanized at the end of the experiment. TSE in mice was confirmed by detecting PrPres in brain extracts by Western blot and/or immunohistochemistry as described elsewhere (Cervenakova et al., 2011). EVs were isolated from plasma samples as described above (Section 4.1.1), stored at −80 ◦ C and labeled as “non-sucrose”. EVs were likewise isolated from a second aliquot of pooled plasma and further purified through a 30% sucrose cushion, after which three fractions were collected as described. Samples were resuspended in 10% wt-MoBH (source of PrPC ) which was prepared in conversion buffer [1% Triton X-100 in PBS with 1× complete protease inhibitor cocktail (Roche, Mannheim, Germany)]. The exosomal suspension was thereafter subjected to four rounds of saPMCA. Briefly, samples were aliquoted into PCR tubes containing zirconia/silica beads (Biospec Products Inc., Bartlesville, OK) and amplified by 48 cycles (one round) of incubation at 37 ◦ C, followed by a 20 s pulse of sonication at power 4 in a Q700MPX microplate horn sonicator (QSonica, Newtown, CT) (Castilla et al., 2005a; Saá et al., 2006b). After each round, sample aliquots were mixed 1:1 with 10% wt-MoBH to perform the next round of




saPMCA. Additionally, aliquots of 9 ␮l of samples subjected to saPMCA were treated with 20 ␮g/ml of PK (Novagen® , Darmstadt, Germany) for 1 h at 37 ◦ C with agitation at 450 rpm. PK treatment was stopped by sample denaturation in NuPAGE LDS sample buffer (Life TechnologiesTM , Carlsbad, CA) at 100 ◦ C for 10 min. Under these conditions, PrPres was specifically detected after saPMCA in “non-sucrose” plasma exosomal preparations from infected mice, as determined by Western blotting with the anti-PrP primary antibody 6D11(Covance® , Berkeley, CA) (Fig. 3A, “infected non-sucrose”). Presence of PrPres could be confirmed in “nonsucrose” plasma exosomal preparations from infected mice after just one round of saPMCA when the Western blotting was probed with anti-PrP primary antibody 6D11 and HRP-conjugated rat antimouse IgG Mouse Trueblot® secondary antibody (Rockland) (data not shown). Likewise, saPMCA revealed the presence of PrPres in the pellet fraction obtained after purification through a sucrose cushion (Fig. 3B, lanes 3 and 4). In addition, PrPres was detected in the “top” layer (Fig. 3B, lanes 1 and 2). The floatation density and lipidic appearance of this sample suggests that it corresponds to a fraction of co-purified PrPres that is likely to be associated with lipids, and lipoproteins (Safar et al., 2006). Notably, PrPres was also detected in the “middle” fraction (Fig. 3B, lanes 5 and 8), containing plasma exosomes as suggested by the presence of the exosomal marker Hsp70 (data not shown). No PrPres was detected in corresponding fractions from plasma EV preparations from uninfected mice (Fig. 3A, lanes 5–8, and Fig. 3B, lanes 6, 7 and 9–12). Conclusively, by combining EV isolation with ExoQuickTM and PrPTSE amplification by saPMCA, we were able to demonstrate the presence of PrPres in plasma samples collected from clinically sick mice infected with Mo-vCJD, and showed for the first time its association with blood-circulating EVs.

4.2. Potential pitfalls to the application of PMCA in prion diagnosis 4.2.1. Amplification of PrPTSE in apparently normal individuals Small amounts of detergent-insoluble, PrPres aggregates have been demonstrated in the brain of uninfected patients, cattle and hamsters (Yuan et al., 2006), and a number of healthy individuals have been found to harbor PrPres in lymphoreticular tissues (de Marco et al., 2010; Gill et al., 2013; Knight, 2010; Wadsworth et al., 2011). Consequently, the presence of these aggregates in blood or other biological fluids relevant for disease diagnosis, such as urine or CSF, from uninfected patients cannot be ruled out at the present time. Although a priori these findings may pose a potential pitfall for the application of techniques like saPMCA, which PrPTSE -amplification properties might turn positive clinically negative samples, it is not clear whether small quantities of PrP aggregates in otherwise healthy patients represent dormant prions awaiting activation by environmental changes or are suggestive of subclinical or carrier states awaiting transmission to more susceptible hosts (de Marco et al., 2010; Gill et al., 2013; Yuan et al., 2006). In such case, detection of PrPres aggregates by saPMCA would be extremely valuable to avoid iatrogenic spread of TSEs via blood transfusions or surgical procedures. Nevertheless, it is important to note that detection of PrPres in biological fluids has been achieved only in confirmed clinical cases or in samples from preclinical animals experimentally inoculated with TSE agents and not in negative controls, and therefore the reported specificity of saPMCA remains to be 100% (Castilla et al., 2005b; Chen et al., 2010; Fujihara et al., 2009; Gonzalez-Romero et al., 2008; Lacroux et al., 2014; Moda et al., 2014; Murayama et al., 2007; Rubenstein et al., 2010, 2011; Saá et al., 2006a; Tattum et al., 2010; Thorne and Terry, 2008).

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4.2.2. Spontaneous generation of misfolded PrPTSE While the description of spontaneous generation of PrPTSE by saPMCA has an important value from a biological stand point as it enables the study of sporadic forms of TSE, it is of concern when developing a diagnostic assay. Spontaneous generation of PrPTSE by saPMCA has resulted in new TSE strains and can therefore be distinguished from cross-contamination on the basis of their in vivo properties (Barria et al., 2009). However, the biological characterization of the resulting saPMCA amplified material is not feasible in routine disease diagnosis, and therefore very well defined saPMCA conditions, including a maximum number of amplification rounds, should be established for each TSE strain. De novo generation of PrPTSE takes place at a low and variable rate, under specific conditions and high saPMCA round numbers and it can therefore be avoided by, for example, limiting the number of rounds (Barria et al., 2009; Morales et al., 2012). Moreover, spontaneous PrPTSE generation has never been observed when human or transgenic mice over-expressing human PrPC were used as source of PrPC in saPMCA (Barria et al., 2009). 4.2.3. Cross-contamination Many similarities can be found between DNA amplification by PCR and PrPTSE 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 PrPTSE (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). In this line, it has been shown that establishing optimal Ct (cycle threshold, i.e. the cycle number at which the reporter dye emission intensities rises above background noise) cut-off values increases the specificity of the assay (Phillips et al., 2009a,b). If we again establish an analogy between PCR cycles and PMCA rounds, a round threshold (Rt) cut-off should be determined for each TSE strain, after which the appearance of positive PrPTSE results should be regarded as unspecific, but scientific consensus on this issue needs to be reached before the method will be adapted for diagnostic purposes. 4.2.4. Amplification by saPMCA of non-infectious PrPTSE Another potential drawback to the application of PMCA to TSE diagnosis is the potential amplification of certain PrPres species that may generate false positive results. This possibility has been suggested in light of PMCA experiments conducted with rPrPC and minimal components (Timmes et al., 2013). No such observation has ever been described in PMCA reactions prepared with animal or human-derived material and therefore it remains hypothetical at this moment. 5. PMCA beyond prion diseases TSEs belong to a larger group of protein misfolding diseases so called “conformational disorders” that includes among others a number of well known neurodegenerative diseases such as Alzheimer’s (AD), Parkinson’s (PD) and Huntington’s disease, fronto-temporal dementia and amyotrophic lateral sclerosis for which a conformational change in protein structure has been proposed as a triggering event leading to the deposition of aggregated proteins with amyloid properties and corresponding pathological changes, predominantly in the brain (Prusiner, 2012). The available data suggest that in all these disorders, the misfolded protein




Fig. 3. PrPTSE co-localizes with extracellular vesicles (EVs) isolated from plasma samples of Mo-vCJD-infected mice. EV samples were prepared using ExoQuickTM (System Biosciences) from the plasma of clinically sick or uninfected control mice. (A) EV pellets were mixed into 10% wild-type mouse brain homogenate (wt-MoBH), split into 4 aliquots and subjected to four rounds of saPMCA (non-sucrose). (B) EV pellets were used to purify exosomes by ultracentrifugation on a 30% sucrose cushion. Three fractions were collected [top, middle (containing exosomes) and bottom] and subjected to a second round of ultracentrifugation. Pellets were mixed into 10% wt-MoBH, split into 2 aliquots and subjected to four rounds of saPMCA. Aliquots of 9 ␮l were collected from each sample, treated with 20 ␮g/ml of proteinase K (PK), resolved by SDS-PAGE and subjected to Western blot using the PrP-specific antibody 6D11 (Covance) and HRP-conjugated secondary antibody (KPL). PK: wt-MoBH not treated with PK. Molecular mass is shown on the right. Samples were diluted 1:1 between rounds.

accumulation follows the same nucleation-dependent polymerization mechanism, which involves the formation of intermediate species such as oligomers and fibrils (Jarrett and Lansbury, 1993). Given the mechanistic similarities of protein misfolding between disorders, and the principles of PrPres replication by PMCA, this technology can be applied to study a broader range of diseases than TSEs. As occurs with other neurodegenerative diseases, including TSEs, identification of AD affected individuals before overt clinical symptoms develop is critical to the success of potential intervention approaches. Intermediary forms of amyloid formation, such as amyloid beta (A␤) oligomers, have been identified as potential disease biomarkers (Gao et al., 2010). Moreover, these molecules have been found circulating in cerebral spinal fluid (CSF) from clinically affected patients (Gao et al., 2010; Georganopoulou et al., 2005; Klyubin et al., 2008). Because amyloid formation is a slow process, which probably begins decades before disease onset (Braak, 1998; Collinge et al., 2006), detection of amyloid seeding activity in biological fluids during the preclinical phase of the disease might represent a good approach for early, minimally invasive diagnosis. In this line, the PMCA technology has been adapted to accelerate the conversion of monomeric synthetic A␤1–42 into A␤ oligomers in A␤-oligomer-seeded reactions, and has been successfully applied

to specifically detect A␤ seeding activity in CSF collected form AD patients, further confirming the presence of A␤ oligomers in these samples (Salvadores et al., 2014). Because CSF collection is an invasive procedure, the challenge will be to demonstrate the presence of these aggregates in other biological samples like plasma or serum, which hitherto has been elusive probably due to the low concentration of A␤ oligomers in these fluids and the low detection sensitivity of current biochemical assays. Nevertheless, the increased sensitivity of the so called A␤-PMCA, capable of detecting 3 fmol of A␤ oligomers, may facilitate these studies (Salvadores et al., 2014). Another challenge will be to detect A␤ seeding activity in people at risk of developing AD to demonstrate the applicability of A␤-PMCA in preclinical diagnosis. A great deal of evidence has accumulated over the years suggesting that cellular toxicity and neurodegeneration are not produced by large amyloid deposits but non-fibrillar dimers and oligomeric species (Haass and Selkoe, 2007; Klein et al., 2004; Silveira et al., 2005; Simoneau et al., 2007; Volles and Lansbury, 2003). Nevertheless, the molecular mechanism(s) underlying transformation from soluble monomeric forms to disease-associated oligomeric aggregates are still under intense research. While studying mechanisms of ␣-Syn formation in PD, Kim et al. (2007) showed acceleration of fibril formation following periodic ultrasonication of ␣-Syn




aggregates into smaller nuclei, which further seeded the conversion of monomeric ␣-Syn during a quiescent incubation period. These findings greatly resembled the prion replication and PMCA mechanisms and provided compelling evidence that PD propagates via seed-dependent amyloidogenesis. A prion-like mechanism for PD propagation was further supported by in vivo and in vitro studies showing the capacity of ␣-Syn amyloids to transfer between cells (Desplats et al., 2009). PMCA studies have demonstrated a central role for ␣-Syn dimerization in the aggregation and fibrillogenesis processes observed in PD. Soluble monomeric ␣-Syn and genetically engineered ␣SynFv were incubated with pre-aggregated ␣-Syn and ␣-SynFv respectively, for 12 h with constant agitation. Analogous to PrPres replication by PMCA, this incubation phase allowed amyloid formation. Incubation was followed by a brief sonication of 30 s that resulted in fibril fractionation and seed multiplication for subsequent multiplication (Roostaee et al., 2013). Additionally, these studies proved the self-replicating properties of ␣-Syn as conversion could be maintained in serial reactions. It would be of importance to assess whether the saPMCA-generated material induce neurotoxicity or elicit PD upon injection into animal models. In addition to facilitating studies on the molecular mechanisms of replication, PMCA can also be applied to screen and assess the effectiveness of anti-amyloid compounds at reducing amyloid formation, and to detect low concentrations of intermediate species of amyloid fibers in biological samples collected from people at risk of developing protein conformational disorders. Contrary to the findings described by Roostaee et al. (2013), Herva and colleagues observed ␣-Syn fibril formation in unseeded saPMCA reactions set up with wild-type recombinant ␣-Syn. saPMCA-generated aggregates presented the biochemical hallmarks of misfolded ␣-Syn, like increased beta-sheet content and partial resistance to PK. Additionally, electron microscopy analysis showed the presence of fibrils of heterogeneous sizes in these preparations. This group used saPMCA-induced amyloid formation as the readout to evaluate amyloid-inhibitory drugs (Herva et al., 2014). Although these experiments showed the potential application of PMCA to anti-amyloid drug screening, an important caveat of the study was the absence of comparison of saPMCA- and brainderived ␣-Syn aggregates. As previously shown for prion diseases, and discussed in Sections 3.1.1–3.1.3, saPMCA reactions set up with different substrates and co-factors, result in preparations with very different biochemical and biological properties, some of them not being infectious to wild type animals and therefore, highly unlikely to be relevant to prion pathogenesis. The fact that some features of the saPMCA-produced ␣-Syn aggregates significantly differed from those generated by means of other in vitro systems, which have been demonstrated to resemble aggregates found in human brain (Freundt et al., 2012; Luk et al., 2012, 2009; Volpicelli-Daley et al., 2011), raise concerns about the biological relevance of the saPMCA generated material under the reported conditions and findings hereby obtained.

6. Concluding remarks After some controversial first years, PMCA has made its way into the prion field as a technological breakthrough essential for investigating biological and molecular aspects of TSE pathogenesis and PrPTSE amplification in various biological fluids and tissues to detectable levels. This technology has been instrumental for the in vitro generation of infectious material for the first time, demonstrating the autocatalytic nature of PrPTSE replication and allowing the dissection of its molecular basis. Moreover, and contrary to other in vitro assays, the PMCA system uncovers certain aspects of TSE etiology by mimicking the species barrier and strain

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phenomena characteristic of environmentally acquired TSEs, or by facilitating the spontaneous generation of infectious prions in a way resembling sporadic forms of human TSEs. This quality has allowed PMCA to be applied to the study of the zoonotic potential of animal prion diseases by determining the strength of the transmission barrier between different prion strains and different species. Another advantage of having a technology capable of generating genuine infection is that it provides an invaluable matrix for testing replication inhibitory molecules with potential therapeutic application. Likewise, the ultrasensitive properties of PMCA also facilitate risk assessment studies to identify potential infectionbearing biological fluids and to minimize the risk of iatrogenic transmission; and ultimately, to diagnose TSE affected people during the clinically silent phase. This achievement will make possible the administration of future therapeutic treatments before irreversible neurological damage takes place. Importantly, this system has not only been adopted by leading laboratories in the field of TSEs, but it is now being applied to investigate other protein conformational disorders such as AD and PD where the misfolding and aggregation of endogenous cellular proteins result in cellular dysfunction and tissue damage, ultimately leading to neurodegeneration. Acknowledgements We thank Oksana Yakovleva, Jorge de Castro and Irina Vasilyeva for their assistance with extracellular vesicle isolation, animal inoculation and surveillance, and cell culture maintenance; Donna Sobieski for editorial assistance and Anton Cervenak for technical support. We also thank the American National Red Cross vivarium staff for providing excellent animal care, and Drs. Jan Simak and Silvia Lacerda for their intellectual and technical assistance with nanoparticle tracking analysis. We are grateful to Dr. Moira Bruce for providing a Mo-vCJD-infected mouse brain for the infectivity studies. This study was partially funded by the American National Red Cross and the Fondation Alliance BioSecure, France. References Abdallah, A., Wang, P., Richt, J.A., Sreevatsan, S., 2012. Y145Stop is sufficient to induce de novo generation prions using protein misfolding cyclic amplification. Prion 6 (1). Akimov, S., Vasilyeva, I., Yakovleva, O., McKenzie, C., Cervenakova, L., 2009. Murine bone marrow stromal cell culture with features of mesenchymal stem cells susceptible to mouse-adapted human TSE agent, Fukuoka-1. Folia neuropathologica/Association of Polish Neuropathologists and Medical Research Centre. Pol. Acad. Sci. 47 (2), 205–214. Akimov, S., Yakovleva, O., Vasilyeva, I., McKenzie, C., Cervenakova, L., 2008. Persistent propagation of variant Creutzfeldt-Jakob disease agent in murine spleen stromal cell culture with features of mesenchymal stem cells. J. Virol. 82 (21), 10959–10962. Alais, S., Simoes, S., Baas, D., Lehmann, S., Raposo, G., Darlix, J.L., Leblanc, P., 2008. Mouse neuroblastoma cells release prion infectivity associated with exosomal vesicles. Biol. Cell/under the auspices of the Eur. Cell Biol. Organ. 100 (10), 603–615. Atarashi, R., Moore, R.A., Sim, V.L., Hughson, A.G., Dorward, D.W., Onwubiko, H.A., Priola, S.A., Caughey, B., 2007. Ultrasensitive detection of scrapie prion protein using seeded conversion of recombinant prion protein. Nat. Methods 4 (8), 645–650. Atarashi, R., Wilham, J.M., Christensen, L., Hughson, A.G., Moore, R.A., Johnson, L.M., Onwubiko, H.A., Priola, S.A., Caughey, B., 2008. Simplified ultrasensitive prion detection by recombinant PrP conversion with shaking. Nat. Methods 5 (3), 211–212. Barret, A., Tagliavini, F., Forloni, G., Bate, C., Salmona, M., Colombo, L., De Luigi, A., Limido, L., Suardi, S., Rossi, G., Auvre, F., Adjou, K.T., Sales, N., Williams, A., Lasmezas, C., Deslys, J.P., 2003. Evaluation of quinacrine treatment for prion diseases. J. Virol. 77 (15), 8462–8469. Barria, M.A., Gonzalez-Romero, D., Soto, C., 2012. Cyclic amplification of prion protein misfolding. Methods Mol. Biol. 849, 199–212. Barria, M.A., Mukherjee, A., Gonzalez-Romero, D., Morales, R., Soto, C., 2009. De novo generation of infectious prions in vitro produces a new disease phenotype. PLoS Pathog. 5 (5), e1000421.




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