In vitro prion protein conversion suggests risk of bighorn sheep (Ovis canadensis) to transmissible spongiform encephalopathies

The cellular prion protein (PrPc) is a glycolipid-anchored cell surface protein that usually exhibits three glycosylation states. Its post-translationally modified isoform, PrPsc, is involved in the pathogenesis of various transmissible spongiform encephalopathies (TSEs). In bovine species, BSE infectivity appears to be restricted to the central nervous system; few or no detectable infectivity is found in lymphoid tissues in contrast to scrapie or variant CJD. Since expression of PrPc is a prerequisite for prion replication, we have investigated PrPc expression by bovine immune cells. Lymphocytes from blood and five different lymph organs were isolated from the same animal to assess intra- and interindividual variability of PrPc expression, considering six individuals. As shown by flow cytometry, this expression is absent or weak on granulocytes but is measurable on monocytes, B and T cells from blood and lymph organs. The activation of the bovine cells produces an upregulation of PrPc. The results of our in vitro study of PrPc biosynthesis are consistent with previous studies in other species. Interestingly, western blotting experiments showed only one form of the protein, the diglycosylated band. We propose that the glycosylation state could explain the lack of infectivity of the bovine immune cells.

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Sulphated glycosaminoglycans prevent the neurotoxicity of a human prion protein fragment

Biochemical Journal ◽

10.1042/bj3350369 ◽

1998 ◽

Vol 335 (2) ◽

pp. 369-374 ◽

Cited By ~ 44

Author(s):

Mar PÉREZ ◽

Francisco WANDOSELL ◽

Camilo COLAÇO ◽

Jesús AVILA

Keyword(s):

Prion Protein ◽

Amyloid Fibrils ◽

Transmissible Spongiform Encephalopathies ◽

Therapeutic Effects ◽

Protein Fragment ◽

Spongiform Encephalopathies ◽

Sulphated Polysaccharides ◽

Human Prion Protein ◽

Sulphated Glycosaminoglycans

Although a number of features distinguish the disease isoform of the prion protein (PrPSc) from its normal cellular counterpart (PrPC) in the transmissible spongiform encephalopathies (TSEs), the neuropathogenesis of these diseases remains an enigma. The amyloid fibrils formed by fragments of human PrP have, however, been shown to be directly neurotoxic in vitro. We show here that sulphated polysaccharides (heparin, keratan and chondroitin) inhibit the neurotoxicity of these amyloid fibrils and this appears to be mediated via inhibition of the polymerization of the PrP peptide into fibrils. This provides a rationale for the therapeutic effects of sulphated polysaccharides and suggests a rapid in vitro functional screen for TSE therapeutics.

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A Promising Antiprion Trimethoxychalcone Binds to the Globular Domain of the Cellular Prion Protein and Changes Its Cellular Location

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01441-17 ◽

2017 ◽

Vol 62 (2) ◽

Cited By ~ 6

Author(s):

N. C. Ferreira ◽

L. M. Ascari ◽

A. G. Hughson ◽

G. R. Cavalheiro ◽

C. F. Góes ◽

...

Keyword(s):

Cell Surface ◽

Real Time ◽

Prion Protein ◽

Molecular Mechanisms ◽

Cellular Prion Protein ◽

Transmissible Spongiform Encephalopathies ◽

Globular Domain ◽

Mrna Levels ◽

Spongiform Encephalopathies

ABSTRACTThe search for antiprion compounds has been encouraged by the fact that transmissible spongiform encephalopathies (TSEs) share molecular mechanisms with more prevalent neurodegenerative pathologies, such as Parkinson's and Alzheimer's diseases. Cellular prion protein (PrPC) conversion into protease-resistant forms (protease-resistant PrP [PrPRes] or the scrapie form of PrP [PrPSc]) is a critical step in the development of TSEs and is thus one of the main targets in the screening for antiprion compounds. In this work, three trimethoxychalcones (compounds J1, J8, and J20) and one oxadiazole (compound Y17), previously identifiedin vitroto be potential antiprion compounds, were evaluated through different approaches in order to gain inferences about their mechanisms of action. None of them changed PrPCmRNA levels in N2a cells, as shown by reverse transcription-quantitative real-time PCR. Among them, J8 and Y17 were effective in real-time quaking-induced conversion reactions using rodent recombinant PrP (rPrP) from residues 23 to 231 (rPrP23–231) as the substrate and PrPScseeds from hamster and human brain. However, when rPrP from residues 90 to 231 (rPrP90–231), which lacks the N-terminal domain, was used as the substrate, only J8 remained effective, indicating that this region is important for Y17 activity, while J8 seems to interact with the PrPCglobular domain. J8 also reduced the fibrillation of mouse rPrP23–231seeded within vitro-produced fibrils. Furthermore, most of the compounds decreased the amount of PrPCon the N2a cell surface by trapping this protein in the endoplasmic reticulum. On the basis of these results, we hypothesize that J8, a nontoxic compound previously shown to be a promising antiprion agent, may act by different mechanisms, since its efficacy is attributable not only to PrP conversion inhibition but also to a reduction of the PrPCcontent on the cell surface.

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Prion protein interconversions†

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2000.0765 ◽

2001 ◽

Vol 356 (1406) ◽

pp. 197-202 ◽

Cited By ~ 17

Author(s):

Byron Caughey

Keyword(s):

Neurodegenerative Diseases ◽

Prion Protein ◽

Prion Diseases ◽

Transmissible Spongiform Encephalopathies ◽

Biological Parameters ◽

Spongiform Encephalopathies ◽

Lead Compounds ◽

Resistant Form

The transmissible spongiform encephalopathies (TSEs), or prion diseases, remain mysterious neurodegenerative diseases that involve perturbations in prion protein (PrP) structure. This article summarizes our use of in vitro models to describe how PrP is converted to the disease–associated, protease–resistant form. These models reflect many important biological parameters of TSE diseases and have been used to identify inhibitors of the PrP conversion as lead compounds in the development of anti–TSE drugs.

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Neuroprotective role of PrPC against kainate-induced epileptic seizures and cell death depends on the modulation of JNK3 activation by GluR6/7–PSD-95 binding

Molecular Biology of the Cell ◽

10.1091/mbc.e11-04-0321 ◽

2011 ◽

Vol 22 (17) ◽

pp. 3041-3054 ◽

Cited By ~ 43

Author(s):

Patricia Carulla ◽

Ana Bribián ◽

Alejandra Rangel ◽

Rosalina Gavín ◽

Isidro Ferrer ◽

...

Keyword(s):

Prion Protein ◽

Knockout Mice ◽

Neuronal Excitability ◽

Epileptic Seizures ◽

Cellular Prion Protein ◽

Transmissible Spongiform Encephalopathies ◽

Spongiform Encephalopathies ◽

Glycosyl Phosphatidylinositol

Cellular prion protein (PrPC) is a glycosyl-phosphatidylinositol–anchored glycoprotein. When mutated or misfolded, the pathogenic form (PrPSC) induces transmissible spongiform encephalopathies. In contrast, PrPC has a number of physiological functions in several neural processes. Several lines of evidence implicate PrPC in synaptic transmission and neuroprotection since its absence results in an increase in neuronal excitability and enhanced excitotoxicity in vitro and in vivo. Furthermore, PrPC has been implicated in the inhibition of N-methyl-d-aspartic acid (NMDA)–mediated neurotransmission, and prion protein gene (Prnp) knockout mice show enhanced neuronal death in response to NMDA and kainate (KA). In this study, we demonstrate that neurotoxicity induced by KA in Prnp knockout mice depends on the c-Jun N-terminal kinase 3 (JNK3) pathway since Prnpo/oJnk3o/o mice were not affected by KA. Pharmacological blockage of JNK3 activity impaired PrPC-dependent neurotoxicity. Furthermore, our results indicate that JNK3 activation depends on the interaction of PrPC with postsynaptic density 95 protein (PSD-95) and glutamate receptor 6/7 (GluR6/7). Indeed, GluR6–PSD-95 interaction after KA injections was favored by the absence of PrPC. Finally, neurotoxicity in Prnp knockout mice was reversed by an AMPA/KA inhibitor (6,7-dinitroquinoxaline-2,3-dione) and the GluR6 antagonist NS-102. We conclude that the protection afforded by PrPC against KA is due to its ability to modulate GluR6/7-mediated neurotransmission and hence JNK3 activation.

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Prion protein as a target for therapeutic interventions

Pure and Applied Chemistry ◽

10.1351/pac200476050915 ◽

2004 ◽

Vol 76 (5) ◽

pp. 915-920 ◽

Cited By ~ 4

Author(s):

P. P. Liberski

Keyword(s):

Prion Protein ◽

Phenotypic Expression ◽

Therapeutic Interventions ◽

Transmissible Spongiform Encephalopathies ◽

Spongiform Encephalopathy ◽

Public Health Perspective ◽

Spongiform Encephalopathies ◽

Mad Cow Disease

Transmissible spongiform encephalopathies (TSEs), currently known as prion diseases, are neurodegenerative disorders of the central nervous system (CNS) caused by an elusive infectious agent called “prion” (proteinaceous infectious particle). These dis orders include: kuru, Creutzfeldt –Jakob disease (CJD) and its variant (vCJD), Gerstmann–Sträussler–Scheinker (GSS) disease and fatal familial insomnia (FFI) in humans, scrapie in sheep and goats, bovine spongiform encephalopathy (BSE) or mad cow disease, and chronic wasting disease (CWD) in cervids. According to the widely accepted “prion hypothesis”, prion is an aggregate of the abnormal isoform of prion protein (PrPSc). Prion protein is a cell-derived glycoprotein (this normal isoform is called PrPc) encoded by a gene on chromosome 20 in humans (PRNP). In familial forms of TSEs, mutations within the ORF of PRNP are linked to the phenotypic expression of the disease. TSEs are important from public health perspective, and “mad cow disease has created the greatest threat to the safety of human food supply in modern times. vCJD threatens the safety of the blood supply worldwide”. Thus, to search for effective therapy is more than an urgent task. In TSEs, aggregates of PrPSc accumulate in the brain in a form of plaques, or synaptic deposits. The conversion of PrPc into PrPSc and subsequent deposits of PrPSc are targets for therapeutic interventions. These include: tricyclic compounds—acridine and phenothiazine derivatives; quinacrine; anti-PrPSc antibodies; dendrimers; polyethylene antibiotics (amphotericin B, MS-8209); pentosan polysulfate; and dextran sulfate. All these compounds are active in many in vitro and in vivo assays, but not proved definitely active in humans. Thus, albeit interesting and promising, the chemotherapy of TSEs is still in the infant phase.

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Evaluation of new cell culture inhibitors of protease-resistant prion protein against scrapie infection in mice

Journal of General Virology ◽

10.1099/vir.0.80082-0 ◽

2004 ◽

Vol 85 (8) ◽

pp. 2479-2483 ◽

Cited By ~ 25

Author(s):

David A. Kocisko ◽

John D. Morrey ◽

Richard E. Race ◽

Jiancao Chen ◽

Byron Caughey

Keyword(s):

Cell Culture ◽

Prion Protein ◽

Tannic Acid ◽

Grape Seed ◽

Transmissible Spongiform Encephalopathies ◽

Spongiform Encephalopathies ◽

Scrapie Infection ◽

Approved Drugs

In vitro inhibitors of the accumulation of abnormal (protease-resistant) prion protein (PrP-res) can sometimes prolong the lives of scrapie-infected rodents. Here, transgenic mice were used to test the in vivo anti-scrapie activities of new PrP-res inhibitors, which, because they are approved drugs or edible natural products, might be considered for clinical trials in humans or livestock with transmissible spongiform encephalopathies (TSEs). These inhibitors were amodiaquine, thioridazine, thiothixene, trifluoperazine, tetrandrine, tannic acid and polyphenolic extracts of tea, grape seed and pine bark. Test compounds were administered for several weeks beginning 1–2 weeks prior to, or 2 weeks after, intracerebral or intraperitoneal 263K scrapie challenge. Tannic acid was also tested by direct preincubation with inoculum. None of the compounds significantly prolonged the scrapie incubation periods. These results highlight the need to assess TSE inhibitors active in cell culture against TSE infections in vivo prior to testing these compounds in humans and livestock.

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Cells Expressing Anchorless Prion Protein Are Resistant to Scrapie Infection

Journal of Virology ◽

10.1128/jvi.02412-08 ◽

2009 ◽

Vol 83 (9) ◽

pp. 4469-4475 ◽

Cited By ~ 40

Author(s):

Kristin L. McNally ◽

Anne E. Ward ◽

Suzette A. Priola

Keyword(s):

Transgenic Mice ◽

Prion Protein ◽

Prion Diseases ◽

Transmitted Disease ◽

Transmissible Spongiform Encephalopathies ◽

Gpi Anchor ◽

Spongiform Encephalopathies ◽

Infected Cells

ABSTRACT The hallmark of transmissible spongiform encephalopathies (TSEs or prion diseases) is the accumulation of an abnormally folded, partially protease-resistant form (PrP-res) of the normal protease-sensitive prion protein (PrP-sen). PrP-sen is attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. In vitro, the anchor and the local membrane environment are important for the conversion of PrP-sen to PrP-res. In vivo, however, the anchor is not necessary because transgenic mice expressing anchorless PrP-sen accumulate PrP-res and replicate infectivity. To clarify the role of the GPI anchor in TSE infection, cells expressing GPI-anchored PrP-sen, anchorless PrP-sen, or both forms of PrP-sen were exposed to the mouse scrapie strain 22L. Cells expressing anchored PrP-sen produced PrP-res after exposure to 22L. Surprisingly, while cells expressing anchorless PrP-sen made anchorless PrP-res in the first 96 h postinfection, no PrP-res was detected at later passes. In contrast, when cells expressing both forms of PrP-sen were exposed to 22L, both anchored and anchorless PrP-res were detected over multiple passes. Consistent with the in vitro data, scrapie-infected cells expressing anchored PrP-sen transmitted disease to mice whereas cells expressing anchorless PrP-sen alone did not. These results demonstrate that the GPI anchor on PrP-sen is important for the persistent infection of cells in vitro. Our data suggest that cells expressing anchorless PrP-sen are not directly infected with scrapie. Thus, PrP-res formation in transgenic mice expressing anchorless PrP-sen may be occurring extracellularly.

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New inhibitors of prion replication that target the amyloid precursor

Journal of General Virology ◽

10.1099/vir.0.009084-0 ◽

2009 ◽

Vol 90 (5) ◽

pp. 1294-1301 ◽

Cited By ~ 9

Author(s):

Mathieu Charvériat ◽

Marlène Reboul ◽

Qian Wang ◽

Christèle Picoli ◽

Natacha Lenuzza ◽

...

Keyword(s):

Protein Stability ◽

Prion Protein ◽

Prion Diseases ◽

Direct Interaction ◽

Cellular Prion Protein ◽

Transmissible Spongiform Encephalopathies ◽

Spongiform Encephalopathies ◽

Regulatory Pathways ◽

Erythromycin A

At present, there is no effective therapy for any of the neurodegenerative amyloidoses, despite renewed efforts to identify compounds active against the various implicated pathogenetic molecules. We have screened a library of 2960 natural and synthetic compounds in two cell lines chronically infected with mouse prions, and have identified eight new inhibitors of prion replication in vitro. They belong to two distinct chemical families that have not previously been recognised as effective in the field of transmissible spongiform encephalopathies: seven are 3-aminosteroids and one is a derivative of erythromycin A with an oxime functionality. Our results suggest that these aminosteroids inhibit prion replication by triggering a common target, possibly implicated in the regulatory pathways of cellular prion protein metabolism. Furthermore, using a quantitative approach for the study of protein stability, it was shown that the erythromycin A derivative altered prion protein stability by direct interaction. Such direct targeting of this amyloid precursor might provide new clues for the understanding of prion diseases and, more importantly, help to define new molecules that are active against prion diseases.

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In Vitro Approach To Identify Key Amino Acids in Low Susceptibility of Rabbit Prion Protein to Misfolding

Journal of Virology ◽

10.1128/jvi.01543-17 ◽

2017 ◽

Vol 91 (24) ◽

Cited By ~ 7

Author(s):

Hasier Eraña ◽

Natalia Fernández-Borges ◽

Saioa R. Elezgarai ◽

Chafik Harrathi ◽

Jorge M. Charco ◽

...

Keyword(s):

Amino Acid ◽

Prion Protein ◽

Amino Acid Residue ◽

Cellular Prion Protein ◽

Single Amino Acid ◽

Transmissible Spongiform Encephalopathies ◽

Spongiform Encephalopathy ◽

Experimental Conditions ◽

Spongiform Encephalopathies

ABSTRACT Prion diseases, or transmissible spongiform encephalopathies (TSEs), are a group of rare progressive neurodegenerative disorders caused by an abnormally folded prion protein (PrPSc). This is capable of transforming the normal cellular prion protein (PrPC) into new infectious PrPSc. Interspecies prion transmissibility studies performed by experimental challenge and the outbreak of bovine spongiform encephalopathy that occurred in the late 1980s and 1990s showed that while some species (sheep, mice, and cats) are readily susceptible to TSEs, others are apparently resistant (rabbits, dogs, and horses) to the same agent. To study the mechanisms of low susceptibility to TSEs of certain species, the mouse-rabbit transmission barrier was used as a model. To identify which specific amino acid residues determine high or low susceptibility to PrPSc propagation, protein misfolding cyclic amplification (PMCA), which mimics PrPC-to-PrPSc conversion with accelerated kinetics, was used. This allowed amino acid substitutions in rabbit PrP and accurate analysis of misfolding propensities. Wild-type rabbit recombinant PrP could not be misfolded into a protease-resistant self-propagating isoform in vitro despite seeding with at least 12 different infectious prions from diverse origins. Therefore, rabbit recombinant PrP mutants were designed to contain every single amino acid substitution that distinguishes rabbit recombinant PrP from mouse recombinant PrP. Key amino acid residue substitutions were identified that make rabbit recombinant PrP susceptible to misfolding, and using these, protease-resistant misfolded recombinant rabbit PrP was generated. Additional studies characterized the mechanisms by which these critical amino acid residue substitutions increased the misfolding susceptibility of rabbit PrP. IMPORTANCE Prion disorders are invariably fatal, untreatable diseases typically associated with long incubation periods and characteristic spongiform changes associated with neuronal loss in the brain. Development of any treatment or preventative measure is dependent upon a detailed understanding of the pathogenesis of these diseases, and understanding the mechanism by which certain species appear to be resistant to TSEs is critical. Rabbits are highly resistant to naturally acquired TSEs, and even under experimental conditions, induction of clinical disease is not easy. Using recombinant rabbit PrP as a model, this study describes critical molecular determinants that confer this high resistance to transmissible spongiform encephalopathies.

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