Protein Misfolding Cyclic Amplification Assay Research Articles

Identification of chronic wasting disease prions in decaying tongue tissues from exhumed white-tailed deer.

Chronic wasting disease (CWD) prions cause fatal neuropathies in farmed and free-ranging cervids. The deposition of prions in natural and humanmade environmental components has been implicated as a major mechanism mediating CWD spread in wild and captive populations. Prions can be deposited in the environment through excreta, tissues, and carcasses from pre-clinical and clinical animals. Furthermore, burial of CWD-positive animals may reduce but not completely mitigate prion spread from carcasses into the surrounding environment. Here, we analyzed exhumed, decaying deer carcasses for the presence of CWD prions. By analyzing tongue tissues through the protein misfolding cyclic amplification (PMCA) technique, we were able to identify seven out of 95 exhumed white-tailed deer carcasses as CWD prions carriers. Confirmatory analyses were performed using the real-time quaking-induced conversion (RT-QuIC) technique. In addition, we evaluated the potential contamination of the pens that housed these animals by swabbing feeders and waterers. PMCA analyses of swabs confirmed CWD contamination on farming equipment. This work demonstrates the usefulness of PMCA to detect CWD prions in a variety of contexts, including exhumed/decaying tissues. In addition, this is the first report demonstrating swabbing coupled with PMCA as a method for the detection of prion seeding activity on naturally exposed surfaces. Considering that this study was focused on a single site, further studies should confirm whether prion amplification assays are useful to identify CWD prions not only in animals but also in the environment that contains them. IMPORTANCE Environmental contamination is thought to be a major player in the spread of chronic wasting disease (CWD), a fatal prion disease affecting a wide variety of cervid species. At present, there are no officially approved methods allowing for the detection of prion infectivity in environmental components. Importantly, animal as well as anthropogenic activities are thought to contribute to prion environmental contamination. Here, we detected CWD prions in exhumed white-tailed deer carcasses by using the protein misfolding cyclic amplification (PMCA) assay. In addition, we identified CWD prions in feeders used within the infected facility. These results highlight the potential role of PMCA in identifying prion infectivity in a variety of scenarios, ranging from decaying tissues to farming equipment.

Conservation of vCJD Strain Properties After Extraction and In Vitro Propagation of PrPSc from Archived Formalin-Fixed Brain and Appendix Tissues Using Highly Sensitive Protein Misfolding Cyclic Amplification.

Three retrospective lymphoreticular tissue studies (Appendix I, II, and III) aimed to estimate the UK prevalence of variant Creutzfeldt-Jakob disease (vCJD), following exposure of the population to the bovine spongiform encephalopathy (BSE) agent, in the late 1980s and 1990s. These studies evaluated the presence of abnormal prion protein aggregates, in archived formalin-fixed paraffin-embedded (FFPE) appendectomy samples, by immunohistochemical detection. Although there was concordance in the estimated prevalence of vCJD from these studies, the identification of positive specimens from pre- and post-BSE-exposure periods in Appendix III study has raised questions regarding the nature and origin of the detected abnormal prion protein. We applied a robust and novel approach in the extraction of disease-associated prion protein (PrPSc) present in frozen and FFPE samples of brain and appendix from a patient with pathologically confirmed vCJD. The extracted material was used to seed the highly sensitive protein misfolding cyclic amplification assay (hsPMCA) to investigate the in vitro and in vivo propagation properties of the extracted abnormal prion protein. We demonstrate that PrPSc can be successfully extracted from FFPE appendix tissue and propagated in vitro. Bioassay in wild-type and gene-targeted mouse models confirmed that the extracted and amplified product is infectious and retains strain properties consistent with vCJD. This provides a highly sensitive and reliable platform for subsequent analysis of the archived FFPE appendix tissue derived from the Appendix II and III surveys, to further evaluate the nature of the abnormal PrP detected in the positive samples.

Abnormal prion protein, infectivity and neurofilament light-chain in blood of macaques with experimental variant Creutzfeldt-Jakob disease.

Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative infections. Variant Creutzfeldt-Jakob disease (vCJD) and sporadic CJD (sCJD) are human TSEs that, in rare cases, have been transmitted by human-derived therapeutic products. There is a need for a blood test to detect infected donors, identify infected individuals in families with TSEs and monitor progression of disease in patients, especially during clinical trials. We prepared panels of blood from cynomolgus and rhesus macaques experimentally infected with vCJD, as a surrogate for human blood, to support assay development. We detected abnormal prion protein (PrPTSE) in those blood samples using the protein misfolding cyclic amplification (PMCA) assay. PrPTSE first appeared in the blood of pre-symptomatic cynomolgus macaques as early as 2 months post-inoculation (mpi). In contrast, PMCA detected PrPTSE much later in the blood of two pre-symptomatic rhesus macaques, starting at 19 and 20 mpi, and in one rhesus macaque only when symptomatic, at 38 mpi. Once blood of either species of macaque became PMCA-positive, PrPTSE persisted through terminal illness at relatively constant concentrations. Infectivity in buffy coat samples from terminally ill cynomolgus macaques as well as a sample collected 9 months before clinical onset of disease in one of the macaques was assayed in vCJD-susceptible transgenic mice. The infectivity titres varied from 2.7 to 4.3 infectious doses ml-1. We also screened macaque blood using a four-member panel of biomarkers for neurodegenerative diseases to identify potential non-PrPTSE pre-symptomatic diagnostic markers. Neurofilament light-chain protein (NfL) increased in blood before the onset of clinical vCJD. Cumulatively, these data confirmed that, while PrPTSE is the first marker to appear in blood of vCJD-infected cynomolgus and rhesus macaques, NfL might offer a useful, though less specific, marker for forthcoming neurodegeneration. These studies support the use of macaque blood panels to investigate PrPTSE and other biomarkers to predict onset of CJD in humans.

Subclinical infection occurs frequently following low dose exposure to prions by blood transfusion

Infectious prion diseases have very long incubation periods, and the role that subclinical infections play in transmission, persistence and re-emergence of these diseases is unclear. In this study, we used a well-established model of vCJD (sheep experimentally infected with bovine spongiform encephalopathy, BSE) to determine the prevalence of subclinical infection following exposure by blood transfusion from infected donors. Many recipient sheep survived for years post-transfusion with no clinical signs and no disease-associated PrP (PrPSc) found in post mortem tissue samples by conventional tests. Using a sensitive protein misfolding cyclic amplification assay (PMCA), we found that the majority of these sheep had detectable PrPSc in lymph node samples, at levels approximately 105–106 times lower than in equivalent samples from clinically positive sheep. Further testing revealed the presence of PrPSc in other tissues, including brain, but not in blood samples. The results demonstrate that subclinical infection is a frequent outcome of low dose prion infection by a clinically relevant route for humans (blood transfusion). The long term persistence of low levels of infection has important implications for prion disease control and the risks of re-emergent infections in both humans and animals.

The Future of Seed Amplification Assays and Clinical Trials.

Prion-like seeded misfolding of host proteins is the leading hypothesised cause of neurodegenerative diseases. The exploitation of the mechanism in the protein misfolding cyclic amplification (PMCA) and real-time quaking-induced conversion (RT-QuIC) assays have transformed prion disease research and diagnosis and have steadily become more widely used for research into other neurodegenerative disorders. Clinical trials in adult neurodegenerative diseases have been expensive, slow, and disappointing in terms of clinical benefits. There are various possible factors contributing to the failure to identify disease-modifying treatments for adult neurodegenerative diseases, some of which include: limited accuracy of antemortem clinical diagnosis resulting in the inclusion of patients with the “incorrect” pathology for the therapeutic; the role of co-pathologies in neurodegeneration rendering treatments targeting one pathology alone ineffective; treatment of the primary neurodegenerative process too late, after irreversible secondary processes of neurodegeneration have become established or neuronal loss is already extensive; and preclinical models used to develop treatments not accurately representing human disease. The use of seed amplification assays in clinical trials offers an opportunity to tackle these problems by sensitively detecting in vivo the proteopathic seeds thought to be central to the biology of neurodegenerative diseases, enabling improved diagnostic accuracy of the main pathology and co-pathologies, and very early intervention, particularly in patients at risk of monogenic forms of neurodegeneration. The possibility of quantifying proteopathic seed load, and its reduction by treatments, is an attractive pharmacodynamic biomarker in the preclinical and early clinical stages of drug development. Here we review some potential applications of seed amplification assays in clinical trials.

RT-QuIC and Related Assays for Detecting and Quantifying Prion-like Pathological Seeds of α-Synuclein.

Various disease-associated forms or strains of α-synuclein (αSynD) can spread and accumulate in a prion-like fashion during synucleinopathies such as Parkinson’s disease (PD), Lewy body dementia (DLB), and multiple system atrophy (MSA). This capacity for self-propagation has enabled the development of seed amplification assays (SAAs) that can detect αSynD in clinical samples. Notably, α-synuclein real-time quaking-induced conversion (RT-QuIC) and protein misfolding cyclic amplification (PMCA) assays have evolved as ultrasensitive, specific, and relatively practical methods for detecting αSynD in a variety of biospecimens including brain tissue, CSF, skin, and olfactory mucosa from synucleinopathy patients. However, αSyn SAAs still lack concordance in detecting MSA and familial forms of PD/DLB, and the assay parameters show poor correlations with various clinical measures. End-point dilution analysis in αSyn RT-QuIC assays allows for the quantitation of relative amounts of αSynD seeding activity that may correlate moderately with clinical measures and levels of other biomarkers. Herein, we review recent advancements in α-synuclein SAAs for detecting αSynD and describe in detail the modified Spearman–Karber quantification algorithm used with end-point dilutions.

Pure autonomic failure and the differential diagnosis of autonomic peripheral neuropathies.

Pure autonomic failure (PAF) is a peripheral autonomic neurodegenerative disease caused by alpha-synuclein deposition that is predominantly confined to peripheral autonomic neurons. Patients present with insidious features of autonomic failure that have a chronic course.In this review, we highlight the features of PAF, the differentiating features from other autonomic neuropathies, the diagnostic tests, and the predictors for conversion to a central synucleinopathy. Natural history studies have defined the predictors for and rate of conversion to a central alpha-synucleinopathy. Skin immunohistochemistry techniques and demonstration of length-dependent neuronal loss of both somatic and autonomic small fiber nerves, and intraneural phosphorylated synuclein deposition provide diagnostic biomarkers. In the future, diagnosis maybe supported by measuring cerebrospinal fluid alpha-synuclein oligomers using techniques, such as protein misfolding cyclic amplification assay and real-time quaking-induced conversion. PAF is a sporadic peripheral autonomic neurodegenerative disease that belongs to the group of disorders known as alpha-synucleinopathies. Peripheral autonomic manifestations are similar to those seen in other autonomic neuropathies, particularly, diabetic autonomic neuropathy, amyloid polyneuropathy, and autoimmune autonomic neuropathies. Novel diagnostic procedures like skin immunohistochemistry for alpha-synuclein, and protein amplification techniques are being investigated to provide an earlier and more specific diagnosis. A substantial number of PAF patients' phenoconvert to a central alpha-synucleinopathy.

The Neural Gut-Brain Axis of Pathological Protein Aggregation in Parkinson's Disease and Its Counterpart in Peroral Prion Infections.

A neuropathological hallmark of Parkinson’s disease (PD) is the cerebral deposition of abnormally aggregated α-synuclein (αSyn). PD-associated αSyn (αSynPD) aggregates are assumed to act, in a prion-like manner, as proteinaceous nuclei (“seeds”) capable of self-templated propagation. Braak and colleagues put forward the idea of a neural gut-brain axis mediating the centripetal spread of αSynPD pathology from the enteric nervous system (ENS) to the brain in PD. This has sparked great interest and initiated passionate discussions both in support of and opposing the suggested hypothesis. A precedent for the spread of protein seeds or seeding from the gastro-intestinal (GI) tract to the central nervous system (CNS) had been previously revealed for pathological prion protein in peroral prion infections. This article scrutinizes the similarities and dissimilarities between the pathophysiological spread of disease-associated protein aggregation along the neural gut–brain axis in peroral prion infections and PD. On this basis, evidence supporting the proposed neural gut–brain axis in PD is concluded to be not as robust as that established for peroral prion infections. New tools for the ultrasensitive detection of αSynPD-associated seeding activity in archived or fresh human tissue samples such as real-time quaking induced conversion (RT-QuIC) or protein misfolding cyclic amplification (PMCA) assays can possibly help to address this deficit in the future.

Decrease in Skin Prion-Seeding Activity of Prion-Infected Mice Treated with a Compound Against Human and Animal Prions: a First Possible Biomarker for Prion Therapeutics

Previous studies have revealed that the infectious scrapie isoform of prion protein (PrPSc) harbored in the skin tissue of patients or animals with prion diseases can be amplified and detected through the serial protein misfolding cyclic amplification (sPMCA) or real-time quaking-induced conversion (RT-QuIC) assays. These findings suggest that skin PrPSc-seeding activity may serve as a biomarker for the diagnosis of prion diseases; however, its utility as a biomarker for prion therapeutics remains largely unknown. Cellulose ethers (CEs, such as TC-5RW), widely used as food and pharmaceutical additives, have recently been shown to prolong the lifespan of prion-infected mice and hamsters. Here we report that in transgenic (Tg) mice expressing hamster cellular prion protein (PrPC) infected with the 263K prion, the prion-seeding activity becomes undetectable in the skin tissues of TC-5RW-treated Tg mice by both sPMCA and RT-QuIC assays, whereas such prion-seeding activity is readily detectable in the skin of untreated mice. Notably, TC-5RW exhibits an inhibitory effect on the in vitro amplification of PrPSc in both skin and brain tissues by sPMCA and RT-QuIC. Moreover, we reveal that TC-5RW is able to directly decrease protease-resistant PrPSc and inhibit the seeding activity of PrPSc from chronic wasting disease and various human prion diseases. Our results suggest that the level of prion-seeding activity in the skin may serve as a useful biomarker for assessing the therapeutic efficacy of compounds in a clinical trial of prion diseases and that TC-5RW may have the potential for the prevention/treatment of human prion diseases.

Multiple system atrophy-associated oligodendroglial protein p25\u03b1 stimulates formation of novel \u03b1-synuclein strain with enhanced neurodegenerative potential

Pathology consisting of intracellular aggregates of alpha-Synuclein (α-Syn) spread through the nervous system in a variety of neurodegenerative disorders including Parkinson’s disease, dementia with Lewy bodies, and multiple system atrophy. The discovery of structurally distinct α-Syn polymorphs, so-called strains, supports a hypothesis where strain-specific structures are templated into aggregates formed by native α-Syn. These distinct strains are hypothesised to dictate the spreading of pathology in the tissue and the cellular impact of the aggregates, thereby contributing to the variety of clinical phenotypes. Here, we present evidence of a novel α-Syn strain induced by the multiple system atrophy-associated oligodendroglial protein p25α. Using an array of biophysical, biochemical, cellular, and in vivo analyses, we demonstrate that compared to α-Syn alone, a substoichiometric concentration of p25α redirects α-Syn aggregation into a unique α-Syn/p25α strain with a different structure and enhanced in vivo prodegenerative properties. The α-Syn/p25α strain induced larger inclusions in human dopaminergic neurons. In vivo, intramuscular injection of preformed fibrils (PFF) of the α-Syn/p25α strain compared to α-Syn PFF resulted in a shortened life span and a distinct anatomical distribution of inclusion pathology in the brain of a human A53T transgenic (line M83) mouse. Investigation of α-Syn aggregates in brain stem extracts of end-stage mice demonstrated that the more aggressive phenotype of the α-Syn/p25α strain was associated with an increased load of α-Syn aggregates based on a Förster resonance energy transfer immunoassay and a reduced α-Syn aggregate seeding activity based on a protein misfolding cyclic amplification assay. When injected unilaterally into the striata of wild-type mice, the α-Syn/p25α strain resulted in a more-pronounced motoric phenotype than α-Syn PFF and exhibited a “tropism” for nigro-striatal neurons compared to α-Syn PFF. Overall, our data support a hypothesis whereby oligodendroglial p25α is responsible for generating a highly prodegenerative α-Syn strain in multiple system atrophy.

Preclinical transmission of prions by blood transfusion is influenced by donor genotype and route of infection.

Variant Creutzfeldt-Jakob disease (vCJD) is a human prion disease resulting from zoonotic transmission of bovine spongiform encephalopathy (BSE). Documented cases of vCJD transmission by blood transfusion necessitate on-going risk reduction measures to protect blood supplies, such as leucodepletion (removal of white blood cells, WBCs). This study set out to determine the risks of prion transmission by transfusion of labile blood components (red blood cells, platelets, plasma) commonly used in human medicine, and the effectiveness of leucodepletion in preventing infection, using BSE-infected sheep as a model. All components were capable of transmitting prion disease when donors were in the preclinical phase of infection, with the highest rates of infection in recipients of whole blood and buffy coat, and the lowest in recipients of plasma. Leucodepletion of components (<106 WBCs/unit) resulted in significantly lower transmission rates, but did not completely prevent transmission by any component. Donor PRNP genotype at codon 141, which is associated with variation in incubation period, also had a significant effect on transfusion transmission rates. A sensitive protein misfolding cyclic amplification (PMCA) assay, applied to longitudinal series of blood samples, identified infected sheep from 4 months post infection. However, in donor sheep (orally infected), the onset of detection of PrPSc in blood was much more variable, and generally later, compared to recipients (intravenous infection). This shows that the route and method of infection may profoundly affect the period during which an individual is infectious, and the test sensitivity required for reliable preclinical diagnosis, both of which have important implications for disease control. Our results emphasize that blood transfusion can be a highly efficient route of transmission for prion diseases. Given current uncertainties over the prevalence of asymptomatic vCJD carriers, this argues for the maintenance and improvement of current measures to reduce the risk of transmission by blood products.

Seeding Propensity and Characteristics of Pathogenic αSyn Assemblies in Formalin-Fixed Human Tissue from the Enteric Nervous System, Olfactory Bulb, and Brainstem in Cases Staged for Parkinson's Disease.

We investigated α-synuclein’s (αSyn) seeding activity in tissue from the brain and enteric nervous system. Specifically, we assessed the seeding propensity of pathogenic αSyn in formalin-fixed tissue from the gastric cardia and five brain regions of 29 individuals (12 Parkinson’s disease, 8 incidental Lewy body disease, 9 controls) using a protein misfolding cyclic amplification assay. The structural characteristics of the resultant αSyn assemblies were determined by limited proteolysis and transmission electron microscopy. We show that fixed tissue from Parkinson’s disease (PD) and incidental Lewy body disease (ILBD) seeds the aggregation of monomeric αSyn into fibrillar assemblies. Significant variations in the characteristics of fibrillar assemblies derived from different regions even within the same individual were observed. This finding suggests that fixation stabilizes seeds with an otherwise limited seeding propensity, that yield assemblies with different intrinsic structures (i.e., strains). The lag phase preceding fibril assembly for patients ≥80 was significantly shorter than in other age groups, suggesting the existence of increased numbers of seeds or a higher seeding potential of pathogenic αSyn with time. Seeding activity did not diminish in late-stage disease. No statistically significant difference in the seeding efficiency of specific regions was found, nor was there a relationship between seeding efficiency and the load of pathogenic αSyn in a particular region at a given neuropathological stage.

An intact membrane is essential for small extracellular vesicle‐induced modulation of α‐synuclein fibrillization

The misfolding and fibrillization of the protein, α‐synuclein (αsyn), is associated with neurodegenerative disorders referred to as the synucleinopathies. Understanding the mechanisms of αsyn misfolding is an important area of interest given that αsyn misfolding contributes to disease pathogenesis. While many studies report the ability of synthetic lipid membranes to modulate αsyn folding, there is little data pertaining to the mechanism(s) of this interaction. αSyn has previously been shown to associate with small lipid vesicles released by cells called extracellular vesicles (EVs) and it is postulated these interactions may assist in the spreading of pathological forms of this protein. Together, this presents the need for robust characterisation studies on αsyn fibrillization using biologically‐derived vesicles. In this study, we comprehensively characterised the ability of lipid‐rich small extracellular vesicles (sEVs) to alter the misfolding of αsyn induced using the Protein Misfolding Cyclic Amplification (PMCA) assay. The biochemical and biophysical properties of misfolded αsyn were examined using a range of techniques including: Thioflavin T fluorescence, transmission electron microscopy, analytical centrifugation and western immunoblot coupled with protease resistance assays and soluble/insoluble fractionation. We show that sEVs cause an acceleration in αsyn fibrillization and provide comprehensive evidence that this results in an increase in the abundance of mature insoluble fibrillar species. In order to elucidate the relevance of the lipid membrane to this interaction, sEV lipid membranes were modified by treatment with methanol, or a combination of methanol and sarkosyl. These treatments altered the ultrastructure of the sEVs without changing the protein cargo. Critically, these modified sEVs had a reduced ability to influence αsyn fibrillization compared to untreated counterparts. This study reports the first comprehensive examination of αsyn:EV interactions and demonstrates that sEVs are powerful modulators of αsyn fibrillization, which is mediated by the sEV membrane. In doing so, this work provides strong evidence for a role of sEVs in contributing directly to αsyn misfolding in the synucleinopathy disorders.

Improving the Predictive Value of Prion Inactivation Validation Methods to Minimize the Risks of Iatrogenic Transmission With Medical Instruments.

Prions are pathogenic infectious agents responsible for fatal, incurable neurodegenerative diseases in animals and humans. Prions are composed exclusively of an aggregated and misfolded form (PrPSc) of the cellular prion protein (PrPC). During the propagation of the disease, PrPSc recruits and misfolds PrPC into further PrPSc. In human, iatrogenic prion transmission has occurred with incompletely sterilized medical material because of the unusual resistance of prions to inactivation. Most commercial prion disinfectants validated against the historical, well-characterized laboratory strain of 263K hamster prions were recently shown to be ineffective against variant Creutzfeldt-Jakob disease human prions. These observations and previous reports support the view that any inactivation method must be validated against the prions for which they are intended to be used. Strain-specific variations in PrPSc physico-chemical properties and conformation are likely to explain the strain-specific efficacy of inactivation methods. Animal bioassays have long been used as gold standards to validate prion inactivation methods, by measuring reduction of prion infectivity. Cell-free assays such as the real-time quaking-induced conversion (RT-QuIC) assay and the protein misfolding cyclic amplification (PMCA) assay have emerged as attractive alternatives. They exploit the seeding capacities of PrPSc to exponentially amplify minute amounts of prions in biospecimens. European and certain national medicine agencies recently implemented their guidelines for prion inactivation of non-disposable medical material; they encourage or request the use of human prions and cell-free assays to improve the predictive value of the validation methods. In this review, we discuss the methodological and technical issues regarding the choice of (i) the cell-free assay, (ii) the human prion strain type, (iii) the prion-containing biological material. We also introduce a new optimized substrate for high-throughput PMCA amplification of human prions bound on steel wires, as translational model for prion-contaminated instruments.

Chronic wasting disease (CWD) prion detection in blood from pre-symptomatic white-tailed deer harboring PRNP polymorphic variants

Chronic wasting disease (CWD) is a prionopathy affecting wild and farmed cervids. This disease is endemic in North America and has been recently identified in Europe. Ante-mortem CWD tests of pre-clinical cervids may be an important tool in helping control the spread of this disease. Unfortunately, current CWD diagnostic methods are not suitable for non-tissue type samples. We reported that CWD prions can be detected in blood of pre-clinical CWD-infected white-tailed deer (WTD) with high sensitivity and specificity using the Protein Misfolding Cyclic Amplification (PMCA) assay. However, that report only included animals homozygous for codon 96G, the most common polymorphic version of the prion protein within this animal species. Here, we report CWD prion detection using blood of naturally infected WTD coding one or two copies of the PrP-96S polymorphic variant. Our results, from a blinded screening, show 100% specificity and ~ 58% sensitivity for animals harboring one 96S codon, regardless of their stage within the pre-clinical phase. Detection efficiency for PrP-96S homozygous animals was substantially lower, suggesting that this allele affect peripheral prion replication/tropism. These results provide additional information on the influence of codon 96 polymorphisms and the ability of PMCA to detect CWD in the blood of pre-clinical WTD.

Inhibition of α-Synuclein Aggregation and Mature Fibril Disassembling With a Minimalistic Compound, ZPDm.

Synucleinopathies are a group of disorders characterized by the accumulation of α-Synuclein amyloid inclusions in the brain. Preventing α-Synuclein aggregation is challenging because of the disordered nature of the protein and the stochastic nature of fibrillogenesis, but, at the same time, it is a promising approach for therapeutic intervention in these pathologies. A high-throughput screening initiative allowed us to discover ZPDm, the smallest active molecule in a library of more than 14.000 compounds. Although the ZPDm structure is highly related to that of the previously described ZPD-2 aggregation inhibitor, we show here that their mechanisms of action are entirely different. ZPDm inhibits the aggregation of wild-type, A30P, and H50Q α-Synuclein variants in vitro and interferes with α-Synuclein seeded aggregation in protein misfolding cyclic amplification assays. However, ZPDm distinctive feature is its strong potency to dismantle preformed α-Synuclein amyloid fibrils. Studies in a Caenorhabditis elegans model of Parkinson’s Disease, prove that these in vitro properties are translated into a significant reduction in the accumulation of α-Synuclein inclusions in ZPDm treated animals. Together with previous data, the present work illustrates how different chemical groups on top of a common molecular scaffold can result in divergent but complementary anti-amyloid activities.

Strain-Dependent Prion Infection in Mice Expressing Prion Protein with Deletion of Central Residues 91-106.

Conformational conversion of the cellular prion protein, PrPC, into the abnormally folded isoform, PrPSc, is a key pathogenic event in prion diseases. However, the exact conversion mechanism remains largely unknown. Transgenic mice expressing PrP with a deletion of the central residues 91–106 were generated in the absence of endogenous PrPC, designated Tg(PrP∆91–106)/Prnp0/0 mice and intracerebrally inoculated with various prions. Tg(PrP∆91–106)/Prnp0/0 mice were resistant to RML, 22L and FK-1 prions, neither producing PrPSc∆91–106 or prions in the brain nor developing disease after inoculation. However, they remained marginally susceptible to bovine spongiform encephalopathy (BSE) prions, developing disease after elongated incubation times and accumulating PrPSc∆91–106 and prions in the brain after inoculation with BSE prions. Recombinant PrP∆91-104 converted into PrPSc∆91–104 after incubation with BSE-PrPSc-prions but not with RML- and 22L–PrPSc-prions, in a protein misfolding cyclic amplification assay. However, digitonin and heparin stimulated the conversion of PrP∆91–104 into PrPSc∆91–104 even after incubation with RML- and 22L-PrPSc-prions. These results suggest that residues 91–106 or 91–104 of PrPC are crucially involved in prion pathogenesis in a strain-dependent manner and may play a similar role to digitonin and heparin in the conversion of PrPC into PrPSc.

Skin α-Synuclein Aggregation Seeding Activity as a Novel Biomarker for Parkinson Disease

Deposition of the pathological α-synuclein (αSynP) in the brain is the hallmark of synucleinopathies, including Parkinson disease (PD), Lewy body dementia (LBD), and multiple system atrophy (MSA). Whether real-time quaking-induced conversion (RT-QuIC) and protein misfolding cyclic amplification (PMCA) assays can sensitively detect skin biomarkers for PD and non-PD synucleinopathies remains unknown. To develop sensitive and specific skin biomarkers for antemortem diagnosis of PD and other synucleinopathies. This retrospective and prospective diagnostic study evaluated autopsy and biopsy skin samples from neuropathologically and clinically diagnosed patients with PD and controls without PD. Autopsy skin samples were obtained at 3 medical centers from August 2016 to September 2019, and biopsy samples were collected from 3 institutions from August 2018 to November 2019. Based on neuropathological and clinical diagnoses, 57 cadavers with synucleinopathies and 73 cadavers with nonsynucleinopathies as well as 20 living patients with PD and 21 living controls without PD were included. Specifically, cadavers and participants had PD, LBD, MSA, Alzheimer disease, progressive supranuclear palsy, or corticobasal degeneration or were nonneurodegenerative controls (NNCs). A total of 8 approached biopsy participants either refused to participate in or were excluded from this study due to uncertain clinical diagnosis. Data were analyzed from September 2019 to April 2020. Skin αSynP seeding activity was analyzed by RT-QuIC and PMCA assays. A total of 160 autopsied skin specimens from 140 cadavers (85 male cadavers [60.7%]; mean [SD] age at death, 76.8 [10.1] years) and 41 antemortem skin biopsies (27 male participants [66%]; mean [SD] age at time of biopsy, 65.3 [9.2] years) were analyzed. RT-QuIC analysis of αSynP seeding activity in autopsy abdominal skin samples from 47 PD cadavers and 43 NNCs revealed 94% sensitivity (95% CI, 85-99) and 98% specificity (95% CI, 89-100). As groups, RT-QuIC also yielded 93% sensitivity (95% CI, 85-97) and 93% specificity (95% CI, 83-97) among 57 cadavers with synucleinopathies (PD, LBD, and MSA) and 73 cadavers without synucleinopathies (Alzheimer disease, progressive supranuclear palsy, corticobasal degeneration, and NNCs). PMCA showed 82% sensitivity (95% CI, 76-88) and 96% specificity (95% CI, 85-100) with autopsy abdominal skin samples from PD cadavers. From posterior cervical and leg skin biopsy tissues from patients with PD and controls without PD, the sensitivity and specificity were 95% (95% CI, 77-100) and 100% (95% CI, 84-100), respectively, for RT-QuIC and 80% (95% CI, 49-96) and 90% (95% CI, 60-100) for PMCA. This study provides proof-of-concept that skin αSynP seeding activity may serve as a novel biomarker for antemortem diagnoses of PD and other synucleinopathies.

The structural differences between patient-derived \u03b1-synuclein strains dictate characteristics of Parkinson\u2019s disease, multiple system atrophy and dementia with Lewy bodies

Synucleinopathies, such as Parkinson’s disease (PD), multiple system atrophy (MSA), and dementia with Lewy bodies (DLB), are defined by the presence of α-synuclein (αSYN) aggregates throughout the nervous system but diverge from one another with regard to their clinical and pathological phenotype. The recent generation of pure fibrillar αSYN polymorphs with noticeable differences in structural and phenotypic traits has led to the hypothesis that different αSYN strains may be in part responsible for the heterogeneous nature of synucleinopathies. To further characterize distinct αSYN strains in the human brain, and establish a structure-pathology relationship, we pursued a detailed comparison of αSYN assemblies derived from well-stratified patients with distinct synucleinopathies. We exploited the capacity of αSYN aggregates found in the brain of patients suffering from PD, MSA or DLB to seed and template monomeric human αSYN in vitro via a protein misfolding cyclic amplification assay. A careful comparison of the properties of total brain homogenates and pure in vitro amplified αSYN fibrillar assemblies upon inoculation in cells and in the rat brain demonstrates that the intrinsic structure of αSYN fibrils dictates synucleinopathies characteristics. We report that MSA strains show several similarities with PD strains, but are significantly more potent in inducing motor deficits, nigrostriatal neurodegeneration, αSYN pathology, spreading, and inflammation, reflecting the aggressive nature of this disease. In contrast, DLB strains display no or only very modest neuropathological features under our experimental conditions. Collectively, our data demonstrate a specific signature for PD, MSA, and DLB-derived strains that differs from previously described recombinant strains, with MSA strains provoking the most aggressive phenotype and more similarities with PD compared to DLB strains.

Amplification Techniques for the Detection of Misfolded Prion Proteins in Experimental and Clinical Samples.

This article describes two methods for amplifying prions present in experimental and clinical samples: the protein misfolding cyclic amplification (PMCA) assay and the real-time quaking-induced conversion (RT-QuIC) assay. Protocols for preparation of amplification substrate and analysis of results are included in addition to those for the individual assays. For each assay, control and suspect samples are mixed with appropriate amplification substrate, which is whole brains from mice in the case of PMCA and recombinant prion protein produced in bacteria for RT-QuIC, followed by cyclic amplification over a number of cycles of sonication (PMCA) or shaking (RT-QuIC) at a consistent incubation temperature. The resultant amplification products are then assessed either by western blotting (PMCA) or based on fluorescent emissions (RT-QuIC). The equipment and expertise necessary for successfully performing either assay vary and will be important factors for individual laboratories to consider when identifying which assay is more appropriate for their experimental design. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Prion amplification via protein misfolding cyclic amplification Support Protocol 1: Collection of whole brains from mice and preparation of normal brain homogenate Basic Protocol 2: Prion amplification via real-time quaking-induced conversion Support Protocol 2: Preparation of recombinant truncated white-tailed-deer prion protein.