Protein Misfolding Cyclic Amplification Reactions Research Articles

Alpha-Synuclein Oligomers and Neurofilament Light Chain Predict Phenoconversion of Pure Autonomic Failure.

To explore the role of alpha-synuclein (αSyn) oligomers and neurofilament light chain (NfL) in cerebrospinal fluid (CSF) of patients with pure autonomic failure (PAF) as markers of future phenoconversion to multiple system atrophy (MSA). Well-characterized patients with PAF (n=32) were enrolled between June 2016 and February 2019 at Mayo Clinic Rochester and followed prospectively with annual visits to determine future phenoconversion to MSA, Parkinson's disease (PD), or dementia with Lewy bodies (DLB). ELISA was utilized to measure NfL and protein misfolding cyclic amplification (PMCA) to detect αSyn oligomers in CSF collected at baseline. Patients were followed for a median of 3.9 years. Five patients converted to MSA, 2 to PD, and 2 to DLB. NfL at baseline was elevated only in patients who later developed MSA, perfectly separating those from future PD and DLB converters as well as non-converters. ASyn-PMCA was positive in all but two cases (94%). The PMCA reaction was markedly different in five samples with maximum fluorescence and reaction kinetics previously described in MSA patients; all of these patients later developed MSA. αSyn-PMCA is almost invariably positive in the CSF of patients with PAF establishing this condition as α-synucleinopathy. Both NfL and the magnitude and reaction kinetics of αSyn PMCA faithfully predict which PAF patients will eventually phenoconvert to MSA. This finding has important implications not only for prognostication, but also for future trials of disease modifying therapies, allowing for differentiation of MSA from Lewy body synucleinopathies before motor symptoms develop. ANN NEUROL 2021;89:1212-1220.

Sensitive protein misfolding cyclic amplification of sporadic Creutzfeldt\u2013Jakob disease prions is strongly seed and substrate dependent

Unlike variant Creutzfeldt–Jakob disease prions, sporadic Creutzfeldt–Jakob disease prions have been shown to be difficult to amplify in vitro by protein misfolding cyclic amplification (PMCA). We assessed PMCA of pathological prion protein (PrPTSE) from 14 human sCJD brain samples in 3 substrates: 2 from transgenic mice expressing human prion protein (PrP) with either methionine (M) or valine (V) at position 129, and 1 from bank voles. Brain extracts representing the 5 major clinicopathological sCJD subtypes (MM1/MV1, MM2, MV2, VV1, and VV2) all triggered seeded PrPTSE amplification during serial PMCA with strong seed- and substrate-dependence. Remarkably, bank vole PrP substrate allowed the propagation of all sCJD subtypes with preservation of the initial molecular PrPTSE type. In contrast, PMCA in human PrP substrates was accompanied by a PrPTSE molecular shift during heterologous (M/V129) PMCA reactions, with increased permissiveness of V129 PrP substrate to in vitro sCJD prion amplification compared to M129 PrP substrate. Combining PMCA amplification sensitivities with PrPTSE electrophoretic profiles obtained in the different substrates confirmed the classification of 4 distinct major sCJD prion strains (M1, M2, V1, and V2). Finally, the level of sensitivity required to detect VV2 sCJD prions in cerebrospinal fluid was achieved.

Detection of alpha-synuclein aggregates in gastrointestinal biopsies by protein misfolding cyclic amplification

Lewy bodies and neurites, the pathological signatures found in the central nervous system of Parkinson's disease (PD) patients, are primarily composed of aggregated alpha-synuclein (aSyn). The observation that aSyn aggregates are also found in the enteric nervous system has prompted several studies aimed at developing a diagnostic procedure based on the detection of pathological aSyn in gastrointestinal (GI) biopsies. The existing studies, which have all used immunohistochemistry for the detection of pathological aSyn, have had conflicting results. In the current survey, we analyzed the seeding propensity of aSyn aggregates from GI biopsies.A total of 29 subjects participated to this study, 18 PD patients and 11 controls. For each patient, 2 to 4 GI biopsies were taken from the same site (antrum, sigmoid colon or rectum) and used to seed the aggregation of recombinant aSyn in an assay inspired from the protein misfolding cyclic amplification (PMCA) method. In a subset of patients and controls (14 and 3, respectively), one or two additional biopsies were analyzed by immunohistochemistry for the presence of phosphorylated aSyn histopathology (PASH) using antibodies against phosphorylated aSyn and PGP 9.5.Except for one subject, none of the control samples seeded aSyn aggregation in PMCA reaction. GI biopsies from patients with PD seeded aSyn aggregation in 10 out of 18 cases (7 from the sigmoid colon, 2 from the antrum and one from the rectum). There was good agreement between PMCA and immunohistochemistry results as, except for two cases, all PMCA-positive PD patients were also PASH-positive.Our findings show that the PMCA method we implemented is capable of detecting aSyn aggregates in routine GI biopsies. They also suggest that rectum biopsies do not contain sufficient amounts of aggregated aSyn to detect seeded assembly by PMCA. While encouraging, our findings indicate that further studies are needed to establish the diagnostic potential of the PMCA method we implemented to detect aSyn aggregates in upper GI biopsies.

Cross-seeding of prions by aggregated α-synuclein leads to transmissible spongiform encephalopathy.

Aggregation of misfolded proteins or peptides is a common feature of neurodegenerative diseases including Alzheimer’s, Parkinson’s, Huntington’s, prion and other diseases. Recent years have witnessed a growing number of reports of overlap in neuropathological features that were once thought to be unique to only one neurodegenerative disorder. However, the origin for the overlap remains unclear. One possibility is that diseases with mixed brain pathologies might arise from cross-seeding of one amyloidogenic protein by aggregated states of unrelated proteins. In the current study we examined whether prion replication can be induced by cross-seeding by α-synuclein or Aβ peptide. We found that α-synuclein aggregates formed in cultured cells or in vitro display cross-seeding activity and trigger misfolding of the prion protein (PrPC) in serial Protein Misfolding Cyclic Amplification reactions, producing self-replicating PrP states characterized by a short C-terminal proteinase K (PK)-resistant region referred to as PrPres. Non-fibrillar α-synuclein or fibrillar Aβ failed to cross-seed misfolding of PrPC. Remarkably, PrPres triggered by aggregated α-synuclein in vitro propagated in animals and, upon serial transmission, produced PrPSc and clinical prion disease characterized by spongiosis and astrocytic gliosis. The current study demonstrates that aggregated α-synuclein is potent in cross-seeding of prion protein misfolding and aggregation in vitro, producing self-replicating states that can lead to transmissible prion diseases upon serial passaging in wild type animals. In summary, the current work documents direct cross-seeding between unrelated amyloidogenic proteins associated with different neurodegenerative diseases. This study suggests that early interaction between unrelated amyloidogenic proteins might underlie the etiology of mixed neurodegenerative proteinopathies.

Ovine recombinant PrP as an inhibitor of ruminant prion propagation in vitro

ABSTRACTPrion diseases are fatal and incurable neurodegenerative diseases of humans and animals. Despite years of research, no therapeutic agents have been developed that can effectively manage or reverse disease progression. Recently it has been identified that recombinant prion proteins (rPrP) expressed in bacteria can act as inhibitors of prion replication within the in vitro prion replication system protein misfolding cyclic amplification (PMCA). Here, within PMCA reactions amplifying a range of ruminant prions including distinct Prnp genotypes/host species and distinct prion strains, recombinant ovine VRQ PrP displayed consistent inhibition of prion replication and produced IC50 values of 122 and 171 nM for ovine scrapie and bovine BSE replication, respectively. These findings illustrate the therapeutic potential of rPrPs with distinct TSE diseases.

Quiescin-sulfhydryl oxidase inhibits prion formation in vitro.

Prions are infectious proteins that cause a group of fatal transmissible diseases in animals and humans. The scrapie isoform (PrPSc) of the cellular prion protein (PrPC) is the only known component of the prion. Several lines of evidence have suggested that the formation and molecular features of PrPSc are associated with an abnormal unfolding/refolding process. Quiescin-sulfhydryl oxidase (QSOX) plays a role in protein folding by introducing disulfides into unfolded reduced proteins. Here we report that QSOX inhibits human prion propagation in protein misfolding cyclic amplification reactions and murine prion propagation in scrapie-infected neuroblastoma cells. Moreover, QSOX preferentially binds PrPSc from prion-infected human or animal brains, but not PrPC from uninfected brains. Surface plasmon resonance of the recombinant mouse PrP (moPrP) demonstrates that the affinity of QSOX for monomer is significantly lower than that for octamer (312 nM vs 1.7 nM). QSOX exhibits much lower affinity for N-terminally truncated moPrP (PrP89-230) than for the full-length moPrP (PrP23-231) (312 nM vs 2 nM), suggesting that the N-terminal region of PrP is critical for the interaction of PrP with QSOX. Our study indicates that QSOX may play a role in prion formation, which may open new therapeutic avenues for treating prion diseases.

Application of protein misfolding cyclic amplification to detection of prions in anaerobic digestate

The exceptional physio-chemical resistance of prions to established decontamination procedures poses a challenge to assessing the suitability of applied inactivation methods. Prion detection is limited by the sensitivity level of Western blotting or by the cost and time factors of bioassays. In addition, prion detection assays can be limited by either the unique or complex nature of matrices associated with environmental samples. To investigate anaerobic digestion (AD) as a practical and economical approach for potential conversion of specified risk materials (SRM) into value added products (i.e., renewable energy), challenges associated with detection of prions in a complex matrix need to be overcome to determine potential inactivation. Protein misfolding cyclic amplification (PMCA) assay, with subsequent Western blot visualization, was used to detect prions within the AD matrix. Anaerobic digestate initially inhibited the PMCA reaction and/or Western blot detection. However, at concentrations of ≤1% of anaerobic digestate, 263K scrapie prions could be amplified and semi-quantitatively detected. Infectious 263K prions were also proven to be bioavailable in the presence of high concentrations of digestate (10–90%). Development of the PMCA application to digestate provides extremely valuable insight into the potential degradation and/or fate of prions in complex biological matrices without requiring expensive and time-consuming bioassays.

In vitro amplification of scrapie and chronic wasting disease PrPres using baculovirus-expressed recombinant PrP as substrate

Protein misfolding cyclic amplification (PMCA) is an in vitro simulation of prion replication, which relies on the use of normal brain homogenate derived from host species as substrate for the specific amplification of abnormal prion protein, PrPSc. Studies showed that recombinant cellular PrP, PrPC, expressed in Escherichia coli lacks N-glycosylation and an glycophosphatidyl inositol anchor (GPI) and therefore may not be the most suitable substrate in seeded PMCA reactions to recapitulate prion conversion in vitro. In this study, we expressed 2 PRNP genotypes of sheep, V136L141R154Q171 and A136F141R154Q171, and one genotype of white-tailed deer (Q95G96, X132,Y216) using the baculovirus expression system and evaluated their suitability as substrates in seeded-PMCA. It has been reported that host-encoded mammalian RNA molecules and divalent cations play a role in the pathogenesis of prion diseases, and RNA molecules have also been shown to improve the sensitivity of PMCA assays. Therefore, we also assessed the effect of co-factors, such as prion-specific mRNA molecules and a divalent cation, manganese, on protein conversion. Here, we report that baculovirus-expressed recombinant PrPC shows a glycoform and GPI-anchor profile similar to mammalian brain-derived PrPC and supports amplification of PrPSc and PrPCWD derived from prion-affected animals in a single round of seeded PMCA in the absence of exogenous co-factors. Addition of species-specific in vitro transcribed PrP mRNA molecules stimulated the conversion efficiency resulting in increased PrPSc or PrPCWD production. Addition of 2 to 20 μM of manganese chloride (MnCl2) to unseeded PMCA resulted in conversion of recombinant PrPC to protease-resistant PrP. Collectively, we demonstrate, for the first time, that baculovirus expressed sheep and deer PrP can serve as a substrate in protein misfolding cyclic amplification for sheep and deer prions in the absence of additional exogenous co-factors.

Recombinant Prion Protein Refolded with Lipid and RNA Has the Biochemical Hallmarks of a Prion but Lacks In Vivo Infectivity

During prion infection, the normal, protease-sensitive conformation of prion protein (PrPC) is converted via seeded polymerization to an abnormal, infectious conformation with greatly increased protease-resistance (PrPSc). In vitro, protein misfolding cyclic amplification (PMCA) uses PrPSc in prion-infected brain homogenates as an initiating seed to convert PrPC and trigger the self-propagation of PrPSc over many cycles of amplification. While PMCA reactions produce high levels of protease-resistant PrP, the infectious titer is often lower than that of brain-derived PrPSc. More recently, PMCA techniques using bacterially derived recombinant PrP (rPrP) in the presence of lipid and RNA but in the absence of any starting PrPSc seed have been used to generate infectious prions that cause disease in wild-type mice with relatively short incubation times. These data suggest that lipid and/or RNA act as cofactors to facilitate the de novo formation of high levels of prion infectivity. Using rPrP purified by two different techniques, we generated a self-propagating protease-resistant rPrP molecule that, regardless of the amount of RNA and lipid used, had a molecular mass, protease resistance and insolubility similar to that of PrPSc. However, we were unable to detect prion infectivity in any of our reactions using either cell-culture or animal bioassays. These results demonstrate that the ability to self-propagate into a protease-resistant insoluble conformer is not unique to infectious PrP molecules. They suggest that the presence of RNA and lipid cofactors may facilitate the spontaneous refolding of PrP into an infectious form while also allowing the de novo formation of self-propagating, but non-infectious, rPrP-res.

Highly Efficient Amplification of Chronic Wasting Disease Agent by Protein Misfolding Cyclic Amplification with Beads (PMCAb)

Protein misfolding cyclic amplification (PMCA) has emerged as an important technique for detecting low levels of pathogenic prion protein in biological samples. The method exploits the ability of the pathogenic prion protein to convert the normal prion protein to a proteinase K-resistant conformation. Inclusion of Teflon® beads in the PMCA reaction (PMCAb) has been previously shown to increase the sensitivity and robustness of detection for the 263 K and SSLOW strains of hamster-adapted prions. Here, we demonstrate that PMCAb with saponin dramatically increases the sensitivity of detection for chronic wasting disease (CWD) agent without compromising the specificity of the assay (i.e., no false positive results). Addition of Teflon® beads increased the robustness of the PMCA reaction, resulting in a decrease in the variability of PMCA results. Three rounds of serial PMCAb allowed detection of CWD agent from a 6.7×10−13 dilution of 10% brain homogenate (1.3 fg of source brain). Titration of the same brain homogenate in transgenic mice expressing cervid prion protein (Tg(CerPrP)1536+/− mice) allowed detection of CWD agent from the 10−6 dilution of 10% brain homogenate. PMCAb is, thus, more sensitive than bioassay in transgenic mice by a factor exceeding 105. Additionally, we are able to amplify CWD agent from brain tissue and lymph nodes of CWD-positive white-tailed deer having Prnp alleles associated with reduced disease susceptibility.

Reply to Karapetyan: Viral synthesis and assembly is unlikely to occur under cell-free PMCA conditions

The letter to the editor by Karapetyan (1) takes issue with our conclusion in PNAS (2) that the generation of transmissible spongiform encephalopathy (TSE) infectivity in cell-free conditions is against the concept that the TSE infectious agent is a virus. Karapetyan's main point is that poliovirus has been shown to “replicate in a cell-free system” by Wimmer and colleagues (3). We would disagree with the interpretation that these poliovirus experiments are analogous to our protein misfolding cyclic amplification (PMCA) reactions. The divalent cations (Mg2+) and energy sources (ATP and GTP) required for synthesis of viral proteins and nucleic acids, as well as virus assembly (3, 4), should not be present at appropriate concentrations in the crude 1% Triton X-100 brain homogenates made in PBS with EDTA that were used for PMCA reactions. Furthermore, in scrapie PMCA experiments, we and others were able to passage and amplify infectivity serially over multiple reactions, whereas in the poliovirus experiments, serial cell-free passage of the assembled infectious viruses was not demonstrated and would probably not be possible without a mechanism for gently uncoating the viral RNA packaged within the particles. For all these reasons, the generation of new scrapie infectivity in the amounts we detected in PMCA reactions (up to 3 × 105 LD50/0.050 mL reaction mix) is not likely to represent viral synthesis and assembly into infectious TSE virus particles.

Change in the characteristics of ferritin induces iron imbalance in prion disease affected brains

Prion disease associated neurotoxicity is mainly attributed to PrP-scrapie (PrPSc), the disease associated isoform of a normal protein, the prion protein (PrPC). Participation of other proteins and processes is suspected, but their identity and contribution to the pathogenic process is unclear. Emerging evidence implicates imbalance of brain iron homeostasis as a significant cause of prion disease-associated neurotoxicity. The underlying cause of this change, however, remains unclear. We demonstrate that iron is sequestered in heat and SDS-stable protein complexes in sporadic-Creutzfeldt–Jakob-disease (sCJD) brains, creating a phenotype of iron deficiency. The underlying cause is change in the characteristics of ferritin, an iron storage protein that becomes aggregated, detergent-insoluble, and partitions with denatured ferritin using conventional methods of ferritin purification. A similar phenotype of iron deficiency is noted in the lumbar spinal cord (SC) tissue of scrapie infected hamsters, a site unlikely to be affected by massive neuronal death and non-specific iron deposition. As a result, the iron uptake protein transferrin (Tf) is upregulated in scrapie infected SC tissue, and increases with disease progression. A direct correlation between Tf and PrPSc suggests sequestration of iron in dysfunctional ferritin that either co-aggregates with PrPSc or is rendered dysfunctional by PrPSc through an indirect process. Surprisingly, amplification of PrPScin vitro by the protein-misfolding-cyclic-amplification (PMCA) reaction using normal brain homogenate as substrate does not increase the heat and SDS-stable pool of iron even though both PrPSc and ferritin aggregate by this procedure. These observations highlight important differences between PrPSc-protein complexes generated in vivo during disease progression and in vitro by the PMCA reaction, and the significance of these complexes in PrPSc-associated neurotoxicity.

Ultra-Efficient PrPSc Amplification Highlights Potentialities and Pitfalls of PMCA Technology

In order to investigate the potential of voles to reproduce in vitro the efficiency of prion replication previously observed in vivo, we seeded protein misfolding cyclic amplification (PMCA) reactions with either rodent-adapted Transmissible Spongiform Encephalopathy (TSE) strains or natural TSE isolates. Vole brain homogenates were shown to be a powerful substrate for both homologous or heterologous PMCA, sustaining the efficient amplification of prions from all the prion sources tested. However, after a few serial automated PMCA (saPMCA) rounds, we also observed the appearance of PK-resistant PrPSc in samples containing exclusively unseeded substrate (negative controls), suggesting the possible spontaneous generation of infectious prions during PMCA reactions. As we could not definitively rule out cross-contamination through a posteriori biochemical and biological analyses of de novo generated prions, we decided to replicate the experiments in a different laboratory. Under rigorous prion-free conditions, we did not observe de novo appearance of PrPSc in unseeded samples of M109M and I109I vole substrates, even after many consecutive rounds of saPMCA and working in different PMCA settings. Furthermore, when positive and negative samples were processed together, the appearance of spurious PrPSc in unseeded negative controls suggested that the most likely explanation for the appearance of de novo PrPSc was the occurrence of cross-contamination during saPMCA. Careful analysis of the PMCA process allowed us to identify critical points which are potentially responsible for contamination events. Appropriate technical improvements made it possible to overcome PMCA pitfalls, allowing PrPSc to be reliably amplified up to extremely low dilutions of infected brain homogenate without any false positive results even after many consecutive rounds. Our findings underline the potential drawback of ultrasensitive in vitro prion replication and warn on cautious interpretation when assessing the spontaneous appearance of prions in vitro.

Abrogation of Complex Glycosylation by Swainsonine Results in Strain- and Cell-specific Inhibition of Prion Replication

Neuroblastoma-derived N2a-PK1 cells, fibroblastic LD9 cells, and CNS-derived CAD5 cells can be infected efficiently and persistently by various prion strains, as measured by the standard scrapie cell assay. Swainsonine, an inhibitor of Golgi α-mannosidase II that causes abnormal N-glycosylation, strongly inhibits infection of PK1 cells by RML, 79A and 22F, less so by 139A, and not at all by 22L prions, and it does not diminish propagation of any of these strains in LD9 or CAD5 cells. Misglycosylated PrP(C) formed in the presence of swainsonine is a good substrate for conversion to PrP(Sc), and misglycosylated PrP(Sc) is fully able to trigger infection and seed the protein misfolding cyclic amplification reaction. Distinct subclones of PK1 cells mediate swainsonine inhibition to very different degrees, implicating misglycosylation of one or more host proteins in the inhibitory process. The use of swainsonine and other glycosylation inhibitors described herein enhances the ability of the cell panel assay to differentiate between prion strains. Moreover, as shown elsewhere, the susceptibility of prions to inhibition by swainsonine in PK1 cells is a mutable trait.

Assessment of the Genetic Susceptibility of Sheep to Scrapie by Protein Misfolding Cyclic Amplification and Comparison with Experimental Scrapie Transmission Studies

The susceptibility of sheep to scrapie is influenced mainly by the prion protein polymorphisms A136V, R154H, and Q171R/H. Here we analyzed the ability of protein misfolding cyclic amplification (PMCA) to model the genetic susceptibility of sheep to scrapie. For this purpose, we studied the efficiency of brain homogenates from sheep with different PrP genotypes to support PrP(Sc) amplification by PMCA using an ARQ/ARQ scrapie inoculum. The results were then compared with those obtained in vivo using the same sheep breed, genotypes, and scrapie inoculum. Genotypes associated with susceptibility (ARQ/ARQ, ARQ/AHQ, and AHQ/ARH) were able to sustain PrP(Sc) amplification in PMCA reactions, while genotypes associated with resistance to scrapie (ARQ/ARR and ARR/ARR) were unable to support the in vitro conversion. The incubation times of the experimental infection were then compared with the in vitro amplification factors. Linear regression analysis showed that the efficiency of in vitro PrP(Sc) amplification of the different genotypes was indeed inversely proportional to their incubation times. Finally, the rare ARQK₁₇₆/ARQK₁₇₆ genotype, for which no in vivo data are available, was studied by PMCA. No amplification was obtained, suggesting ARQK₁₇₆/ARQK₁₇₆ as an additional genotype associated with resistance, at least to the isolate tested. Our results indicate a direct correlation between the ability of different PrP genotypes to undergo PrP(C)-to-PrP(Sc) conversion by PMCA and their in vivo susceptibility and point to PMCA as an alternative to transmission studies and a potential tool to test the susceptibility of numerous sheep PrP genotypes to a variety of prion sources.

Highly Efficient Protein Misfolding Cyclic Amplification

Protein misfolding cyclic amplification (PMCA) provides faithful replication of mammalian prions in vitro and has numerous applications in prion research. However, the low efficiency of conversion of PrPC into PrPSc in PMCA limits the applicability of PMCA for many uses including structural studies of infectious prions. It also implies that only a small sub-fraction of PrPC may be available for conversion. Here we show that the yield, rate, and robustness of prion conversion and the sensitivity of prion detection are significantly improved by a simple modification of the PMCA format. Conducting PMCA reactions in the presence of Teflon beads (PMCAb) increased the conversion of PrPC into PrPSc from ∼10% to up to 100%. In PMCAb, a single 24-hour round consistently amplified PrPSc by 600-700-fold. Furthermore, the sensitivity of prion detection in one round (24 hours) increased by 2-3 orders of magnitude. Using serial PMCAb, a 1012-fold dilution of scrapie brain material could be amplified to the level detectible by Western blotting in 3 rounds (72 hours). The improvements in amplification efficiency were observed for the commonly used hamster 263K strain and for the synthetic strain SSLOW that otherwise amplifies poorly in PMCA. The increase in the amplification efficiency did not come at the expense of prion replication specificity. The current study demonstrates that poor conversion efficiencies observed previously have not been due to the scarcity of a sub-fraction of PrPC susceptible to conversion nor due to limited concentrations of essential cellular cofactors required for conversion. The new PMCAb format offers immediate practical benefits and opens new avenues for developing fast ultrasensitive assays and for producing abundant quantities of PrPSc in vitro.

Mammalian Prions Generated from Bacterially Expressed Prion Protein in the Absence of Any Mammalian Cofactors

Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases that are associated with the conformational conversion of a normal prion protein, PrP(C), to a misfolded aggregated form, PrP(Sc). The protein-only hypothesis asserts that PrP(Sc) itself represents the infectious TSE agent. Although this model is supported by rapidly growing experimental data, unequivocal proof has been elusive. The protein misfolding cyclic amplification reactions have been recently shown to propagate prions using brain-derived or recombinant prion protein, but only in the presence of additional cofactors such as nucleic acids and lipids. Here, using a protein misfolding cyclic amplification variation, we show that prions causing transmissible spongiform encephalopathy in wild-type hamsters can be generated solely from highly purified, bacterially expressed recombinant hamster prion protein without any mammalian or synthetic cofactors (other than buffer salts and detergent). These findings provide strong support for the protein-only hypothesis of TSE diseases, as well as argue that cofactors such as nucleic acids, other polyanions, or lipids are non-obligatory for prion protein conversion to the infectious form.

Is RNA critical for the production of TSE infection?

Is RNA critical for the production of TSE infection?

Immunopurification of Pathological Prion Protein Aggregates

BackgroundPrion diseases are fatal neurodegenerative disorders that can arise sporadically, be genetically inherited or acquired through infection. The key event in these diseases is misfolding of the cellular prion protein (PrPC) into a pathogenic isoform that is rich in β-sheet structure. This conformational change may result in the formation of PrPSc, the prion isoform of PrP, which propagates itself by imprinting its aberrant conformation onto PrPC molecules. A great deal of effort has been devoted to developing protocols for purifying PrPSc for structural studies, and testing its biological properties. Most procedures rely on protease digestion, allowing efficient purification of PrP27-30, the protease-resistant core of PrPSc. However, protease treatment cannot be used to isolate abnormal forms of PrP lacking conventional protease resistance, such as those found in several genetic and atypical sporadic cases.Principal FindingsWe developed a method for purifying pathological PrP molecules based on sequential centrifugation and immunoprecipitation with a monoclonal antibody selective for aggregated PrP. With this procedure we purified full-length PrPSc and mutant PrP aggregates at electrophoretic homogeneity. PrPSc purified from prion-infected mice was able to seed misfolding of PrPC in a protein misfolding cyclic amplification reaction, and mutant PrP aggregates from transgenic mice were toxic to cultured neurons.SignificanceThe immunopurification protocol described here isolates biologically active forms of aggregated PrP. These preparations may be useful for investigating the structural and chemico-physical properties of infectious and neurotoxic PrP aggregates.

The role of glycophosphatidylinositol anchor in the amplification of the scrapie isoform of prion protein in vitro

Transmissible spongiform encephalopathies are associated with an autocatalytic conversion of normal prion protein, PrPC, to a protease-resistant form, PrPres. This autocatalytic reaction can be reproduced in vitro using a procedure called protein misfolding cyclic amplification (PMCA). Here we show that, unlike brain-derived PrPC, bacterially-expressed recombinant prion protein (rPrP) is a poor substrate for PrPres amplification in a standard PMCA reaction. The differences between PrPC and rPrP appear to be due to the lack of the glycophosphatidylinositol anchor in the recombinant protein. These findings shed a new light on prion protein conversion process and have important implications for the efforts to generate synthetic prions for structural and biophysical studies.