PMCA for Ultrasensitive Detection of Prions and To Study Disease Biology

The emergence of a novel class of infectious agent composed exclusively of a misfolded protein (termed prions) has been a challenge in modern biomedicine. Despite similarities on the behavior of prions with respect to conventional pathogens, the many uncertainties regarding the biology and virulence of prions make them a worrisome paradigm. Since prions do not contain nucleic acids and rely on a very different way of replication and spreading, it was necessary to invent a novel technology to study them. In this article, we provide an overview of such a technology, termed protein misfolding cyclic amplification (PMCA), and summarize its many applications to detect prions and understand prion biology

Seed Amplification Assay for the Detection of Pathologic Alpha-Synuclein Aggregates in Cerebrospinal Fluid

Misfolded alpha-synuclein (αSyn) aggregates are a hallmark event in Parkinson’s disease (PD) and other synucleinopathies. Recently, αSyn seed amplification assays (αSyn-SAAs) have shown promise as a test for biochemical diagnosis of synucleinopathies. αSyn-SAAs use the intrinsic self-replicative nature of misfolded αSyn aggregates (seeds) to multiply them in vitro. In these assays, αSyn seeds circulating in biological fluids are amplified by a cyclical process that includes aggregate fragmentation into smaller self-propagating seeds, followed by elongation at the expense of recombinant αSyn (rec-αSyn). Amplification of the seeds allows detection by fluorescent dyes specific for amyloids, such as thioflavin T. Several αSyn-SAA reports have been published in the past under the names ‘protein misfolding cyclic amplification’ (αSyn-PMCA) and ‘real-time quaking-induced conversion’. Here, we describe a protocol for αSyn-SAA, originally reported as αSyn-PMCA, which allows detection of αSyn aggregates in cerebrospinal fluid samples from patients affected by PD, dementia with Lewy bodies or multiple-system atrophy (MSA). Moreover, this αSyn-SAA can differentiate αSyn aggregates from patients with PD versus those from patients with MSA, even in retrospective samples from patients with pure autonomic failure who later developed PD or MSA. We also describe modifications to the original protocol introduced to develop an optimized version of the assay. The optimized version shortens the assay length, decreases the amount of rec-αSyn required and reduces the number of inconclusive results. The protocol has a hands-on time of ~2 h per 96-well plate and can be performed by personnel trained to perform basic experiments with specimens of human origin

Preventive and Therapeutic Reduction of Amyloid Deposition and Behavioral Impairments in a Model of Alzheimer’s Disease by Whole Blood Exchange

Alzheimer\u27s disease (AD) is the major form of dementia in the elderly population. The main neuropathological changes in AD patients are neuronal death, synaptic alterations, brain inflammation, and the presence of cerebral protein aggregates in the form of amyloid plaques and neurofibrillary tangles. Compelling evidence suggests that the misfolding, aggregation, and cerebral deposition of amyloid-beta (Aβ) plays a central role in the disease. Thus, prevention and removal of misfolded protein aggregates is considered a promising strategy to treat AD. In the present study, we describe that the development of cerebral amyloid plaques in a transgenic mice model of AD (Tg2576) was significantly reduced by 40-80% through exchanging whole blood with normal blood from wild type mice having the same genetic background. Importantly, such reduction resulted in improvement in spatial memory performance in aged Tg2576 mice. The exact mechanism by which blood exchange reduces amyloid pathology and improves memory is presently unknown, but measurements of Aβ in plasma soon after blood exchange suggest that mobilization of Aβ from the brain to blood may be implicated. Our results suggest that a target for AD therapy may exist in the peripheral circulation, which could open a novel disease-modifying intervention for AD

Detection of prions in blood from patients with variant Creutzfeldt-Jakob disease

Prions can be detected in blood from patients with variant Creutzfeldt-Jakob disease with high sensitivity and specificity.</jats:p

Development of a Methodology for Large-Scale Production of Prions for Biological and Structural Studies

Prion diseases are a group of infectious neurodegenerative diseases produced by the conversion of the normal prion protein (PrPC) into the disease-associated form (PrPSc). Extensive evidence indicate that the main or sole component of the infectious agent is PrPSc, which can replicate in affected individuals in the absence of nucleic acids. However, the mechanism of PrPC-to-PrPSc conversion remains elusive, which has been attributed to the lack of sufficient structural information of infectious PrPSc and a reliable system to study prion replication in vitro. In this article we adapted the Protein Misfolding Cyclic Amplification (PMCA) technology for rapid and efficient generation of highly infectious prions in large-scale. Murine prions of the RML strain were efficiently propagated in volumes up to 1,000-fold larger than conventional PMCA. The large-scale PMCA (LS-PMCA) procedure enabled to produce highly infectious prions, which maintain the strain properties of the seed used to begin the reaction. LS-PMCA was shown to work with various species and strains of prions, including mouse RML and 301C strains, hamster Hyper prion, cervid CWD prions, including a rare Norwegian CWD prion, and human CJD prions. We further improved the LS-PMCA into a bioreactor format that can operate under industry-mimicking conditions for continuous and unlimited production of PrPSc without the need to keep adding brain-derived prions. In our estimation, this bioreactor can produce in 1d an amount of prions equivalent to that present in 25 infected animals at the terminal stage of the disease. Our LS-PMCA technology may provide a valuable tool to produce large quantities of well-defined and homogeneous infectious prions for biological and structural studies

Development of a methodology for large-scale production of prions for biological and structural studies

Prion diseases are a group of infectious neurodegenerative diseases produced by the conversion of the normal prion protein (PrPC) into the disease-associated form (PrPSc). Extensive evidence indicate that the main or sole component of the infectious agent is PrPSc, which can replicate in affected individuals in the absence of nucleic acids. However, the mechanism of PrPC-to-PrPSc conversion remains elusive, which has been attributed to the lack of sufficient structural information of infectious PrPSc and a reliable system to study prion replication in vitro. In this article we adapted the Protein Misfolding Cyclic Amplification (PMCA) technology for rapid and efficient generation of highly infectious prions in large-scale. Murine prions of the RML strain were efficiently propagated in volumes up to 1,000-fold larger than conventional PMCA. The large-scale PMCA (LS-PMCA) procedure enabled to produce highly infectious prions, which maintain the strain properties of the seed used to begin the reaction. LS-PMCA was shown to work with various species and strains of prions, including mouse RML and 301C strains, hamster Hyper prion, cervid CWD prions, including a rare Norwegian CWD prion, and human CJD prions. We further improved the LS-PMCA into a bioreactor format that can operate under industry-mimicking conditions for continuous and unlimited production of PrPSc without the need to keep adding brain-derived prions. In our estimation, this bioreactor can produce in 1d an amount of prions equivalent to that present in 25 infected animals at the terminal stage of the disease. Our LS-PMCA technology may provide a valuable tool to produce large quantities of well-defined and homogeneous infectious prions for biological and structural studies

Central and Peripheral α-Synuclein in Parkinson Disease Detected by Seed Amplification Assay

Objectives Detection of α‐synuclein aggregates by seed amplification is a promising Parkinson disease biomarker assay. Understanding intraindividual relationships of α‐synuclein measures could inform optimal biomarker development. The objectives were to test accuracy of α‐synuclein seed amplification assay in central (cerebrospinal fluid) and peripheral (submandibular gland) sources, compare to total α‐synuclein measures, and investigate within‐subject relationships. Methods The Systemic Synuclein Sampling Study aimed to characterize α‐synuclein in multiple tissues and biofluids within Parkinson disease subjects (n = 59) and compared to healthy controls (n = 21). Motor and non‐motor measures and dopamine transporter scans were obtained. Four measures of α‐synuclein were compared: seed amplification assay in cerebrospinal fluid and formalin‐fixed paraffin‐embedded submandibular gland, total α‐synuclein quantified in biofluids using enzyme‐linked immunoassay, and aggregated α‐synuclein in submandibular gland detected by immunohistochemistry. Accuracy of seed amplification assay for Parkinson disease diagnosis was examined and within‐subject α‐synuclein measures were compared. Results Sensitivity and specificity of α‐synuclein seed amplification assay for Parkinson disease diagnosis was 92.6% and 90.5% in cerebrospinal fluid, and 73.2% and 78.6% in submandibular gland, respectively. 25/38 (65.8%) Parkinson disease participants were positive for both cerebrospinal fluid and submandibular gland seed amplification assay. Comparing accuracy for Parkinson disease diagnosis of different α‐synuclein measures, cerebrospinal fluid seed amplification assay was the highest (Youden Index = 83.1%). 98.3% of all Parkinson disease cases had ≥1 measure of α‐synuclein positive. Interpretation α‐synuclein seed amplification assay (cerebrospinal fluid\u3esubmandibular gland) had higher sensitivity and specificity compared to total α‐synuclein measures, and within‐subject relationships of central and peripheral α‐synuclein measures emerged

Quantitative Detection and Biological Propagation of Scrapie Seeding Activity In Vitro Facilitate Use of Prions as Model Pathogens for Disinfection

Prions are pathogens with an unusually high tolerance to inactivation and constitute a complex challenge to the re-processing of surgical instruments. On the other hand, however, they provide an informative paradigm which has been exploited successfully for the development of novel broad-range disinfectants simultaneously active also against bacteria, viruses and fungi. Here we report on the development of a methodological platform that further facilitates the use of scrapie prions as model pathogens for disinfection. We used specifically adapted serial protein misfolding cyclic amplification (PMCA) for the quantitative detection, on steel wires providing model carriers for decontamination, of 263K scrapie seeding activity converting normal protease-sensitive into abnormal protease-resistant prion protein. Reference steel wires carrying defined amounts of scrapie infectivity were used for assay calibration, while scrapie-contaminated test steel wires were subjected to fifteen different procedures for disinfection that yielded scrapie titre reductions of ≤101- to ≥105.5-fold. As confirmed by titration in hamsters the residual scrapie infectivity on test wires could be reliably deduced for all examined disinfection procedures, from our quantitative seeding activity assay. Furthermore, we found that scrapie seeding activity present in 263K hamster brain homogenate or multiplied by PMCA of scrapie-contaminated steel wires both triggered accumulation of protease-resistant prion protein and was further propagated in a novel cell assay for 263K scrapie prions, i.e., cerebral glial cell cultures from hamsters. The findings from our PMCA- and glial cell culture assays revealed scrapie seeding activity as a biochemically and biologically replicative principle in vitro, with the former being quantitatively linked to prion infectivity detected on steel wires in vivo. When combined, our in vitro assays provide an alternative to titrations of biological scrapie infectivity in animals that substantially facilitates the use of prions as potentially highly indicative test agents in the search for novel broad-range disinfectants

Transmission, Strain Diversity, and Zoonotic Potential of Chronic Wasting Disease

Chronic wasting disease (CWD) is a prion disease affecting several species of captive and free-ranging cervids. In the past few decades, CWD has been spreading uncontrollably, mostly in North America, resulting in a high increase of CWD incidence but also a substantially higher number of geographical regions affected. The massive increase in CWD poses risks at several levels, including contamination of the environment, transmission to animals cohabiting with cervids, and more importantly, a putative transmission to humans. In this review, I will describe the mechanisms and routes responsible for the efficient transmission of CWD, the strain diversity of natural CWD, its spillover and zoonotic potential and strategies to minimize the CWD threat

A superior alternative to titrations of scrapie infectivity in animals

Prionen verursachen transmissible spongiforme Enzephalopathien wie Scrapie, die bovine spongiforme Enzephalopathie oder die Creutzfeldt-Jakob-Krankheit und bestehen aus fehlgefaltetem, aggregierten Prionprotein (PrP). Aufgrund ihrer hohen Toleranz gegenüber üblichen Desinfektionsverfahren stellen sie sowohl eine besondere Herausforderung als auch ein exzellentes Paradigma für die Aufbereitung von chirurgischen Instrumenten dar. Tatsächlich haben sich Prionen bereits als aussagekräftige Modellpathogene für die Entwicklung neuer Formulierungen zur hochwirksamen Desinfektion auch von Bakterien, Viren und Pilzen bewährt. Um Prionen zukünftig in größerem Umfang als Testerreger zur Entwicklung neuer Mittel und Verfahren für die Breitspektrumdesinfektion ohne Rückgriff auf Tierversuche nutzen zu können, wurden in dieser Arbeit in vitro- Ersatzmethoden für Prion-Bioassays in Nagetieren aufgebaut und validiert. Zur quantitativen biochemischen Bestimmung der PrP-konvertierenden Keimaktivität des Scrapie-assoziierten Prionproteins wurde ein spezifisch adaptierter Protein Misfolding Cyclic Amplification (PMCA)-Assay etabliert. Mit hamster- adaptierten Scrapie-Prionen (Stamm 263K) kontaminierte Stahlstifte wurden verschiedenen Desinfektionsbehandlungen und anschließend einer quantitativen PMCA unterzogen. Die Ergebnisse des PMCA-Assays wurden dann mit bereits früher erhobenen Bioassay-Daten zur Reduktion infektiöser Priontiter durch die angewandten Desinfektionsverfahren verglichen. Zusätzlich dazu wurde ein Zellkulturassay mit primären Gliazellen aus Hamstern zum biologischen in vitro-Nachweis der Keimaktivität des Prionproteins entwickelt. Der Nachweis von Scrapie-assoziierter Keimaktivität an Stahlstiftprüfkörpern in der quantitativen PMCA übertraf die Sensitivität des Nachweises von Prioninfektiosität im Tierversuch um mindestens eine Größenordnung. Dabei konnte die Reduktion der Prioninfektiosität an Stahlstiftprüfkörpern durch 16 verschiedene Desinfektions-verfahren mit Titerreduktionen von weniger als 101-fach bis zu größer oder gleich 105,5-fach durchgängig korrekt abgeschätzt werden. Mittels PMCA biochemisch nachgewiesene Keimaktivität ließ sich auch biologisch in der Zellkultur detektieren. Konzeptionell deuten die Befunde dieser Arbeit darauf hin, dass die proteinöse Keimaktivität und pathogene Infektiosität von 263K Scrapie-Prionen biochemische und biologische Manifestationen desselben molekularen Prinzips darstellen. In praktischer Hinsicht steht mit der Gliazellkultur-gekoppelten PMCA potentiell eine gleichwertige oder sogar überlegene Alternative zum Prion-Bioassay in Tieren zur Verfügung.Prions are misfolded, aggregated prion proteins (PrP) and the causative agents of transmissible spongiform encephalopathies such as scrapie, bovine spongiform encephalopathy (BSE) or Creutzfeldt-Jakob disease. Prions are pathogens with an unusually high tolerance to inactivation and constitute a complex challenge as well as an informative paradigm for the re-processing of surgical instruments. They have been exploited successfully as model pathogens for the development of novel disinfectants with simultaneous activity also against bacteria, viruses and fungi. In order to further facilitate the use of prions as model agents in the search for novel broad-range disinfectants without the use of animal experiments we established and validated an experimental platform for the sensitive quantitative measurement and biological detection of scrapie seeding activity in vitro. We used specifically adapted serial protein misfolding cyclic amplification (PMCA) for the quantitative biochemical detection of the seeding activity of scrapie associated prion protein. Steel wires contaminated with 263K scrapie prions were subjected to different decontamination procedures and subsequently analysed by quantitative PMCA. The results of the PMCA assay were validated by comparison to bioassay data previously established for similar treatments of steel wires. Furthermore a novel cell culture assay of cerebral glial cell cultures from hamsters was established for the biological detection of 263K scrapie seeding activity. The detection limit for scrapie seeding activity on steel wires in the PMCA assay exceeded that for prion infectivity detected by bioassay at least tenfold. The residual scrapie infectivity’s on test wires treated with 16 different disinfection procedures yielding scrapie titre reductions of ≤101- to ≥105.5-fold could be correctly deduced, for all examined procedures, from our quantitative seeding activity assay. In addition, we found that scrapie seeding activity biochemically detected by PMCA could be also demonstrated biologically in the cell culture assay. Conceptually, the findings from this work suggested the seeding activity and infectivity of 263K scrapie prions as corresponding biochemical and biological manifestations of the same replicative molecular principle. In practical terms, our biochemical and biological in vitro assays, when combined, potentially provide a coequal or even superior alternative to titrations of scrapie infectivity in animals. In vitro-detection of the seeding activity of scrapie associated prion protein: A superior alternative to titrations of scrapie infectivity in animal