Effect of Non-Concentrated and Concentrated Vaporized Hydrogen Peroxide on Scrapie Prions

To date, there have been no studies on the sterilization of prions by non-concentrated and concentrated vaporized hydrogen peroxide (VHP) applied by the same instrument. Here, the effect of the two types of VHP applied using an ES-700 sterilizer on prions was investigated. Brain homogenate from scrapie (Chandler) prion-infected mice was spotted on a cover glass and subjected to ES-700 treatment in soft (non-concentrated VHP from 59% hydrogen peroxide) or standard (concentrated VHP from 80% hydrogen peroxide) mode. Proteinase K-resistant prion protein (PrPres), an indicator of the abnormal isoform of prion protein (PrPSc), was reduced by ES-700 treatment under several conditions: SFT1/4 (soft mode, quarter cycle), SFT1/2 (soft mode, half cycle), SFT1 (soft mode, full cycle), and STD1/2 (standard mode, half cycle). PrPres was detected after the first and second rounds of protein misfolding cyclic amplification (PMCA) of untreated samples, but was undetectable in SFT1/4, SFT1/2, SFT1, and STD1/2 treated samples. In a mouse bioassay, SFT1/2 and STD1/2 treatment of prions significantly prolonged survival time, suggesting that prion infectivity is reduced after ES-700 treatment. In summary, both non-concentrated and concentrated VHP inactivate prions and may be useful for the low-temperature sterilization of prion-contaminated medical devices.

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Inactivation of Prions by Low-Temperature Sterilization Technology Using Vaporized Gas Derived from a Hydrogen Peroxide–Peracetic Acid Mixture

Pathogens ◽

10.3390/pathogens10010024 ◽

2020 ◽

Vol 10 (1) ◽

pp. 24

Author(s):

Akikazu Sakudo ◽

Daiki Anraku ◽

Tomomasa Itarashiki

Keyword(s):

Hydrogen Peroxide ◽

Medical Devices ◽

Neurodegenerative Disorders ◽

Peracetic Acid ◽

Protein Misfolding ◽

Prion Diseases ◽

Proteinase K ◽

Acid Mixture ◽

Cover Glass ◽

Standard Mode

Prion diseases are proteopathies that cause neurodegenerative disorders in humans and animals. Prion is highly resistant to both chemical and physical inactivation. Here, vaporized gas derived from a hydrogen peroxide–peracetic acid mixture (VHPPA) was evaluated for its ability to inactivate prion using a STERIACE 100 instrument (Saraya Co., Ltd.). Brain homogenates of scrapie (Chandler strain) prion-infected mice were placed on a cover glass, air-dried, sealed in a Tyvek package, and subjected to VHPPA treatment at 50–55 °C using 8% hydrogen peroxide and <10% peracetic acid for 47 min (standard mode, SD) or 30 min (quick mode, QC). Untreated control samples were prepared in the same way but without VHPPA. The resulting samples were treated with proteinase K (PK) to separate PK-resistant prion protein (PrPres), as a marker of the abnormal isoform (PrPSc). Immunoblotting showed that PrPres was reduced by both SD and QC VHPPA treatments. PrPres bands were detected after protein misfolding cyclic amplification of control but not VHPPA-treated samples. In mice injected with prion samples, VHPPA treatment of prion significantly prolonged survival relative to untreated samples, suggesting that it decreases prion infectivity. Taken together, the results show that VHPPA inactivates prions and might be applied to the sterilization of contaminated heat-sensitive medical devices.

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PMCA-generated prions from the olfactory mucosa of patients with Fatal Familial Insomnia cause prion disease in mice

eLife ◽

10.7554/elife.65311 ◽

2021 ◽

Vol 10 ◽

Author(s):

Edoardo Bistaffa ◽

Alba Marín-Moreno ◽

Juan Carlos Espinosa ◽

Chiara Maria Giulia De Luca ◽

Federico Angelo Cazzaniga ◽

...

Keyword(s):

Prion Protein ◽

Prion Disease ◽

Protein Misfolding ◽

Brain Homogenate ◽

Olfactory Mucosa ◽

Fatal Familial Insomnia ◽

Prpsc Deposition ◽

Astroglial Activation ◽

The Brain ◽

D178n Mutation

Background:Fatal Familial Insomnia (FFI) is a genetic prion disease caused by the D178N mutation in the prion protein gene (PRNP) in coupling phase with methionine at PRNP 129. In 2017, we have shown that the olfactory mucosa (OM) collected from FFI patients contained traces of PrPSc detectable by Protein Misfolding Cyclic Amplification (PMCA).Methods:In this work, we have challenged PMCA-generated products obtained from OM and brain homogenate of FFI patients in BvPrP-Tg407 transgenic mice expressing the bank vole prion protein to test their ability to induce prion pathology.Results:All inoculated mice developed mild spongiform changes, astroglial activation, and PrPSc deposition mainly affecting the thalamus. However, their neuropathological alterations were different from those found in the brain of BvPrP-Tg407 mice injected with raw FFI brain homogenate.Conclusions:Although with some experimental constraints, we show that PrPSc present in OM of FFI patients is potentially infectious.Funding:This work was supported in part by the Italian Ministry of Health (GR-2013-02355724 and Ricerca Corrente), MJFF, ALZ, Alzheimer’s Research UK and the Weston Brain Institute (BAND2015), and Euronanomed III (SPEEDY) to FM; by the Spanish Ministerio de Economía y Competitividad (grant AGL2016-78054-R [AEI/FEDER, UE]) to JMT and JCE; AM-M was supported by a fellowship from the INIA (FPI-SGIT-2015-02).

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Alteration of Prion Strain Emergence by Nonhost Factors

mSphere ◽

10.1128/msphere.00630-19 ◽

2019 ◽

Vol 4 (5) ◽

Cited By ~ 1

Author(s):

Sara A. M. Holec ◽

Qi Yuan ◽

Jason C. Bartz

Keyword(s):

Protein Misfolding ◽

Cumulative Effect ◽

Brain Homogenate ◽

Proteinase K ◽

Silty Clay ◽

Surface Binding ◽

Prion Strain ◽

Weathering Processes ◽

Mechanism Of Inhibition ◽

Environmental Treatment

ABSTRACT Prions can persist in the environment for extended periods of time after adsorption to surfaces, including soils, feeding troughs, or fences. Prion strain- and soil-specific differences in prion adsorption, infectivity, and response to inactivation may be involved in strain maintenance or emergence of new strains in a population. Extensive proteinase K (PK) digestion of Hyper (HY) and Drowsy (DY) PrPSc resulted in a greater reduction in the level of DY PrPSc than of HY PrPSc. Use of the PK-digested material in protein misfolding cyclic amplification strain interference (PMCAsi) resulted in earlier emergence of HY PrPSc than of undigested controls. This result established that strain-specific alteration of the starting ratios of conversion-competent HY and DY PrPSc can alter strain emergence. We next investigated whether environmentally relevant factors such as surface binding and weathering could alter strain emergence. Adsorption of HY and DY PrPSc to silty clay loam (SCL), both separately and combined, resulted in DY interfering with the emergence of HY in PMCAsi in a manner similar to that seen with unbound controls. Similarly, repeated cycles of wetting and drying of SCL-bound HY and DY PrPSc did not alter the emergence of HY PrPSc compared to untreated controls. Importantly, these data indicate that prion strain interference can occur when prions are bound to surfaces. Interestingly, we found that drying of adsorbed brain homogenate on SCL could restore its ability to interfere with the emergence of HY, suggesting a novel strain interference mechanism. Overall, these data provide evidence that the emergence of a strain from a mixture can be influenced by nonhost factors. IMPORTANCE The prion strain, surface type, and matrix containing PrPSc can influence PrPSc surface adsorption. The cumulative effect of these factors can result in strain- and soil-specific differences in prion bioavailability. Environmental weathering processes can result in decreases in PrPSc conversion efficiency and infectivity. Little is known about how incomplete inactivation of surface-bound PrPSc affects transmission and prion strain emergence. Here, we show that strain interference occurs with soil-bound prions and that altering the ratios of prion strains by strain-specific inactivation can affect strain emergence. Additionally, we identify a novel mechanism of inhibition of prion conversion by environmental treatment-induced changes at the soil-protein interface altering strain emergence. These novel findings suggest that environmental factors can influence strain emergence of surface-bound prions.

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Inhibition of neuroinflammatory nitric oxide signalling supresses protein glycation and recovers neuronal dysfunction in prion disease

10.21203/rs.3.rs-20075/v1 ◽

2020 ◽

Author(s):

Julie-Myrtille Bourgognon ◽

Jereme G. Spiers ◽

Sue Robinson ◽

Hannah Scheiblich ◽

Catharine Ortori ◽

...

Keyword(s):

Nitric Oxide ◽

Prion Protein ◽

Prion Disease ◽

Protein Misfolding ◽

Brain Homogenate ◽

Protein Glycation ◽

Therapeutic Effects ◽

Normal Brain ◽

Gene And Protein Expression ◽

Nos Inhibitor

Abstract Background: Several neurodegenerative diseases associated with protein misfolding (Alzheimer’s, Parkinson’s disease) exhibit oxidative and nitrergic stress following initiation of neuroinflammatory pathways. Associated nitric oxide (NO)-mediated post-translational modifications impact upon protein functions that can exacerbate pathology. Non-enzymatic and irreversible glycation signalling has been implicated as an underlying pathway that promotes protein misfolding, but the direct interactions between both pathways are poorly understood. Methods: Here we investigated the potential therapeutic effects of supressing neurotoxic NO signalling during early progression of prion disease. Tg37 mice aged 3-4 weeks were inoculated by intracerebral injection with either 1% brain homogenate of Rocky Mountain Laboratory (RML) scrapie prion protein or control normal brain homogenate (NBH). Hippocampal gene and protein expression levels of oxidative and nitrergic stress markers were analysed and electrophysiological characterisations of pyramidal neurons were performed in 6-10 weeks old RML and NBH mice. Mice were injected with a NO synthase (NOS) inhibitor and the time course of disease markers was compared to controls. Electrophysiology, immunoblotting and immunocytochemistry studies were performed to identify the effects of NOS inhibition on neurophysiology, glycation, prion protein misfolding and cell death. Statistical analyses employed two-tailed unpaired Student’s t-test, one-way or two-way ANOVA as required and data were considered significant with P<0.05.Results: Increased neuroinflammatory signalling was observed in mice between 6 and 10 weeks post inoculation (w.p.i.) with scrapie prion protein which was characterised by enhanced nitrergic stress and associated with a decline in hippocampal neuronal function by 9 w.p.i.. Daily in vivo administration of the NOS inhibitor L-NAME between 6 and 9 w.p.i. at 20 mg/kg abolished the functional degeneration of hippocampal neurons in prion mice. We further found that this intervention in diseased mice ameliorated 3-nitrotyrosination of triose-phosphate isomerase, an enzyme involved in the formation of disease-associated glycation signalling. Furthermore, L-NAME application reduced the expression of the receptor for advanced glycation end products and the accumulation of hippocampal prion misfolding. Conclusions: Our data suggest that alleviating nitrergic stress during early phases of neurodegeneration reduces neurotoxic post-translational NO signalling and glycation-assisted prion misfolding in the hippocampus, a mechanism which might be applicable to other protein misfolding neurodegenerative conditions.

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Significant differences in incubation times in sheep infected with bovine spongiform encephalopathy result from variation at codon 141 in the PRNP gene

Journal of General Virology ◽

10.1099/vir.0.039008-0 ◽

2012 ◽

Vol 93 (12) ◽

pp. 2749-2756 ◽

Cited By ~ 10

Author(s):

Boon Chin Tan ◽

Anthony R. Alejo Blanco ◽

E. Fiona Houston ◽

Paula Stewart ◽

Wilfred Goldmann ◽

...

Keyword(s):

Amino Acid ◽

Bovine Spongiform Encephalopathy ◽

Prion Protein ◽

Protein Misfolding ◽

Prnp Gene ◽

Brain Homogenate ◽

Spongiform Encephalopathy ◽

Direct Effects ◽

Prion Infection ◽

Incubation Periods

The susceptibility of sheep to prion infection is linked to variation in the PRNP gene, which encodes the prion protein. Common polymorphisms occur at codons 136, 154 and 171. Sheep which are homozygous for the A136R154Q171 allele are the most susceptible to bovine spongiform encephalopathy (BSE). The effect of other polymorphisms on BSE susceptibility is unknown. We orally infected ARQ/ARQ Cheviot sheep with equal amounts of BSE brain homogenate and a range of incubation periods was observed. When we segregated sheep according to the amino acid (L or F) encoded at codon 141 of the PRNP gene, the shortest incubation period was observed in LL141 sheep, whilst incubation periods in FF141 and LF141 sheep were significantly longer. No statistically significant differences existed in the expression of total prion protein or the disease-associated isoform in BSE-infected sheep within each genotype subgroup. This suggested that the amino acid encoded at codon 141 probably affects incubation times through direct effects on protein misfolding rates.

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Association of Bcl-2 with Misfolded Prion Protein Is Linked to the Toxic Potential of Cytosolic PrP

Molecular Biology of the Cell ◽

10.1091/mbc.e06-01-0083 ◽

2006 ◽

Vol 17 (8) ◽

pp. 3356-3368 ◽

Cited By ~ 67

Author(s):

Angelika S. Rambold ◽

Margit Miesbauer ◽

Doron Rapaport ◽

Till Bartke ◽

Michael Baier ◽

...

Keyword(s):

Prion Protein ◽

Protein Misfolding ◽

Apoptotic Cell ◽

Prion Diseases ◽

Protective Role ◽

Proteinase K ◽

Apoptotic Protein ◽

Induced Apoptosis ◽

Toxic Potential ◽

Cellular Compartments

Protein misfolding is linked to different neurodegenerative disorders like Alzheimer’s disease, polyglutamine, and prion diseases. We investigated the cytotoxic effects of aberrant conformers of the prion protein (PrP) and show that toxicity is specifically linked to misfolding of PrP in the cytosolic compartment and involves binding of PrP to the anti-apoptotic protein Bcl-2. PrP targeted to different cellular compartments, including the cytosol, nucleus, and mitochondria, adopted a misfolded and partially proteinase K–resistant conformation. However, only in the cytosol did the accumulation of misfolded PrP induce apoptosis. Apoptotic cell death was also induced by two pathogenic mutants of PrP, which are partially localized in the cytosol. A mechanistic analysis revealed that the toxic potential is linked to an internal domain of PrP (amino acids 115–156) and involves coaggregation of cytosolic PrP with Bcl-2. Increased expression of the chaperones Hsp70 and Hsp40 prevented the formation of PrP/Bcl-2 coaggregates and interfered with PrP-induced apoptosis. Our study reveals a compartment-specific toxicity of PrP misfolding that involves coaggregation of Bcl-2 and indicates a protective role of molecular chaperones.

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Hydrogen Peroxide Gas Plasma Sterilizer Combined with Dielectric Barrier Discharge and Corona Discharge Inactivates Prions

Applied Sciences ◽

10.3390/app11209777 ◽

2021 ◽

Vol 11 (20) ◽

pp. 9777

Author(s):

Akikazu Sakudo ◽

Yosuke Tsuji

Keyword(s):

Hydrogen Peroxide ◽

Corona Discharge ◽

Dielectric Barrier Discharge ◽

Prion Protein ◽

Discharge Plasma ◽

Barrier Discharge ◽

Cover Glass ◽

Dielectric Barrier ◽

Gas Plasma ◽

Corona Discharge Plasma

Prions are highly resistant to physical or chemical damage, although previous studies have shown that STERRAD®, a hydrogen gas plasma sterilizer using radiofrequency (RF) discharge, has an inactivation effect. Here, the effect of hydrogen peroxide gas combined with dielectric barrier discharge (DBD) plasma and corona discharge plasma using a RENO-S130 sterilizer on scrapie prions was examined. Scrapie prion-infected mouse brain homogenate was air-dried on a cover glass, sealed in a Tyvek pouch, and subjected to RENO-S130 treatment using either non-lumen mode (28 min) or Eco mode (45 min) with hydrogen peroxide gas derived from 50% hydrogen peroxide. Control (untreated) samples were prepared on a cover glass using the same procedure but without exposure to RENO-S130. PrPres (proteinase K (PK)-resistant prion protein), an index of the conformational variant of prion protein (PrPSc), was decreased by treatment with RENO-S130 under both modes of operation. Specifically, PrPres was identified after the 1st and 2nd cycles of protein misfolding cyclic amplification (PMCA) in control samples but was below the detection limit in RENO-S130-treated samples. A bioassay showed that treatment of prions with RENO-S130 (non-lumen or Eco mode) significantly prolonged mouse survival time. Taken together, these findings show hydrogen peroxide gas combined with DBD/corona discharge plasma can inactivate prions by reducing prion propagation and prion infectivity. This treatment is potentially applicable to the sterilization of prion-contaminated heat-sensitive medical devices.

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Propagation of CJD Prions in Primary Murine Glia Cells Expressing Human PrPc

Pathogens ◽

10.3390/pathogens10081060 ◽

2021 ◽

Vol 10 (8) ◽

pp. 1060

Author(s):

Joo-Hee Wälzlein ◽

Karla A. Schwenke ◽

Michael Beekes

Keyword(s):

Prion Protein ◽

In Vitro Propagation ◽

Cell Model ◽

Animal Experiments ◽

Cationic Lipid ◽

Brain Homogenate ◽

Proteinase K ◽

Lipid Mixture

There are various existing cell models for the propagation of animal prions. However, in vitro propagation of human prions has been a long-standing challenge. This study presents the establishment of a long-term primary murine glia culture expressing the human prion protein homozygous for methionine at codon 129, which allows in vitro propagation of Creutzfeldt–Jakob disease (CJD) prions (variant CJD (vCJD) and sporadic CJD (sCJD) type MM2). Prion propagation could be detected by Western blotting of pathological proteinase K-resistant prion protein (PrPSc) from 120 days post exposure. The accumulation of PrPSc could be intensified by adding a cationic lipid mixture to the infectious brain homogenate at the time of infection. Stable propagation of human prions in a long-term murine glia cell culture represents a new tool for future drug development and for mechanistic studies in the field of human prion biology. In addition, our cell model can reduce the need for bioassays with human prions and thereby contributes to further implementation of the 3R principles aiming at replacement, reduction and refinement of animal experiments.

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Inactivation of Scrapie Prions by the Electrically Charged Disinfectant CAC-717

Pathogens ◽

10.3390/pathogens9070536 ◽

2020 ◽

Vol 9 (7) ◽

pp. 536 ◽

Cited By ~ 5

Author(s):

Akikazu Sakudo ◽

Yoshifumi Iwamaru ◽

Koichi Furusaki ◽

Makoto Haritani ◽

Rumiko Onishi ◽

...

Keyword(s):

Infected Cell ◽

Prion Protein ◽

Protein Misfolding ◽

Prion Diseases ◽

Mouse Bioassay ◽

Median Dose ◽

Cell Lysates ◽

Log Reduction ◽

Bactericidal Effects ◽

Electrically Charged

Previous studies have revealed that the electrically charged disinfectant CAC-717 has strong virucidal and bactericidal effects but is safe for humans and animals. In this study, CAC-717 was further evaluated for its potential effects as a disinfectant against scrapie prions. Western blotting showed that CAC-717 reduced the amount of the abnormal isoform of prion protein (PrPSc) in prion-infected cell (ScN2a) lysates. Furthermore, the reduction of prion transmissibility was confirmed by a mouse bioassay, in which mice injected with scrapie prions pre-treated with CAC-717 survived longer than those injected with untreated scrapie prions. Lastly, to evaluate the seeding activity of ScN2a cell lysates treated with CAC-717, quantitative protein misfolding cyclic amplification (PMCA) was performed directly on ScN2a cell lysates treated with CAC-717, which showed that the median dose of PMCA (PMCA50) dropped from log9.95 to log5.20 after CAC-717 treatment, indicating more than a 4 log reduction. This suggests that the seeding activity of PrPSc is decreased by CAC-717. Collectively, these results suggest that CAC-717 has anti-prion activity, reducing both PrPSc conversion activity and prion transmissibility; thus, CAC-717 will be useful as a novel disinfectant in prion diseases.

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Detection of RNA in the Plasma of Patients with Sporadic Creutzfeldt–Jakob Disease, Gerstmann–Straüssler Syndrome and Other Non-Transmissible Spongiform Encephalopathy Brain Disorders

Microbiology Insights ◽

10.4137/mbi.s4043 ◽

2010 ◽

Vol 3 ◽

pp. MBI.S4043

Author(s):

Kazuo Tsukui ◽

Yasushi Iwasaki ◽

Masamitsu Nagaoka ◽

Kenji Tadokoro

Keyword(s):

Prion Protein ◽

Prion Diseases ◽

Transmissible Spongiform Encephalopathy ◽

Infectious Agent ◽

Brain Homogenate ◽

Proteinase K ◽

Spongiform Encephalopathy ◽

Brain Disorders ◽

Rna Molecules ◽

Jakob Disease

The infectious agent of transmissible spongiform encephalopathy (TSE) was assumed to be the aggregate of abnormal prion protein isoform (PrPsc). We observed that lowering the pH of 3% SDS-inoculated plasma or brain homogenate after PK digestion to 4.5 (acidic SDS condition) enabled to precipitate proteinase K-resistant prion protein (PrPres) in plasma as well as PrPres in the brain with synthetic poly-A RNA as affinity aggregate. Therefore, we determined if RNA molecules could be used for discriminating TSE patients from healthy individuals. We also examined the plasma of patients with classical Creutzfeldt–Jakob disease (CJD) and other brain disorders who were not diagnosed with TSE. The results indicated that RNA approximately 1.5–2.0 kb in length was commonly observed in the plasma of patients with brain disorders but was not detected in the plasma of healthy volunteers. Enhanced expression of RNA and its protection from endogenous nucleases might occur in the former group of patients. Moreover, we speculate that the non-transmissible neuronal disorders overlap with prion diseases.

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