scholarly journals Increased Infectivity of Anchorless Mouse Scrapie Prions in Transgenic Mice Overexpressing Human Prion Protein

2015 ◽  
Vol 89 (11) ◽  
pp. 6022-6032 ◽  
Author(s):  
Brent Race ◽  
Katie Phillips ◽  
Kimberly Meade-White ◽  
James Striebel ◽  
Bruce Chesebro
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Transgenic Mice ◽  
Prion Protein ◽  
Prion Diseases ◽  
Human Species ◽  
Species Barrier ◽  
Gpi Anchor ◽  
Prion Infection ◽  
Barrier Model

ABSTRACTPrion protein (PrP) is found in all mammals, mostly as a glycoprotein anchored to the plasma membrane by a C-terminal glycosylphosphatidylinositol (GPI) linkage. Following prion infection, host protease-sensitive prion protein (PrPsen or PrPC) is converted into an abnormal, disease-associated, protease-resistant form (PrPres). Biochemical characteristics, such as the PrP amino acid sequence, and posttranslational modifications, such as glycosylation and GPI anchoring, can affect the transmissibility of prions as well as the biochemical properties of the PrPres generated. Previousin vivostudies on the effects of GPI anchoring on prion infectivity have not examined cross-species transmission. In this study, we tested the effect of lack of GPI anchoring on a species barrier model using mice expressing human PrP. In this model, anchorless 22L prions derived from tg44 mice were more infectious than 22L prions derived from C57BL/10 mice when tested in tg66 transgenic mice, which expressed wild-type anchored human PrP at 8- to 16-fold above normal. Thus, the lack of the GPI anchor on the PrPres from tg44 mice appeared to reduce the effect of the mouse-human PrP species barrier. In contrast, neither source of prions induced disease in tgRM transgenic mice, which expressed human PrP at 2- to 4-fold above normal.IMPORTANCEPrion protein (PrP) is found in all mammals, usually attached to cells by an anchor molecule called GPI. Following prion infection, PrP is converted into a disease-associated form (PrPres). While most prion diseases are species specific, this finding is not consistent, and species barriers differ in strength. The amino acid sequence of PrP varies among species, and this variability affects prion species barriers. However, other PrP modifications, including glycosylation and GPI anchoring, may also influence cross-species infectivity. We studied the effect of PrP GPI anchoring using a mouse-to-human species barrier model. Experiments showed that prions produced by mice expressing only anchorless PrP were more infectious than prions produced in mice expressing anchored PrP. Thus, the lack of the GPI anchor on prions reduced the effect of the mouse-human species barrier. Our results suggest that prion diseases that produce higher levels of anchorless PrP may pose an increased risk for cross-species infection.

scholarly journals The amino acid residue in position 163 of canine PrPC is critical to the exceptional resistance of dogs to prion infections: evidence from transgenic mouse models

2019 ◽  
Author(s):  
Enric Vidal ◽  
Natalia Fernández-Borges ◽  
Hasier Eraña ◽  
Beatriz Parra ◽  
Belén Pintado ◽  
...  
Keyword(s):  
Amino Acid ◽  
Mouse Model ◽  
Experimental Evidence ◽  
Transgenic Mouse ◽  
Prion Protein ◽  
Amino Acid Residue ◽  
Prion Diseases ◽  
Prion Infection ◽  
The One ◽  
Sheep Scrapie

ABSTRACTUnlike other species, such as cattle, cats or humans, prion disease has never been described in dogs, even though they were similarly exposed to the bovine spongiform encephalopathy (BSE) agent. This resistance prompted a thorough analysis of the canine PRNP gene and the presence of a negatively charged amino acid residue in position 163 was readily identified as potentially fundamental as it differed from all known susceptible species. Furthermore, recent results from our group demonstrated that mouse PRNP with the dog substitution N158D (mouse equivalent to position 163) rendered mice resistant to prion infection. In the present study, a transgenic mouse model was generated expressing dog prion protein (with glutamic acid at position 163) and challenged intracerebrally with a panel of prion isolates (including cattle BSE, sheep scrapie, atypical sheep scrapie, atypical BSE-L, sheep-BSE and chronic wasting disease, among others) none of which could infect them. The brains of these mice were subjected to in vitro prion amplification and failed to find even minimal amounts of misfolded prions providing definitive experimental evidence that dogs are resistant to prion disease. Subsequently, a second transgenic model was generated in which aspartic acid in position 163 was substituted for asparagine (the most common amino acid in this position in prion susceptible species) and this mutation resulted in susceptibility to BSE-derived isolates.These findings strongly support the hypothesis that the amino acid residue at position 163 of canine PrPC is a major determinant of the exceptional resistance of the canidae family to prion infection and establish this as a promising therapeutic target for prion diseases.AUTHOR SUMMARYCats, cattle, people and dogs were all exposed to mad cow disease but, unlike the other three, dogs never succumbed to the disease. We generated a mouse model expressing canine prion protein (instead of mouse prion protein) to provide experimental evidence that dogs are resistant to prion infection by challenging the mice with a panel of prion isolates. None of the prions could infect our transgenic mice that expressed dog prion protein. When the prion protein amino acid sequence of dogs was compared to that of other susceptible species, one amino acid in a specific position was found to be different to all the prion-susceptible animals. To determine if this amino acid was the one responsible for dogs’ resistance to prions, a second mouse model was generated with the canine prion protein but the critical amino acid was substituted for the one susceptible species have. When this model was challenged with the same panel of prions it could be infected with at least one of them. These results demonstrate the relevance of this amino acid position in determining susceptibility or resistance to prions, and this information can be used to design preventative treatments for prion diseases.

scholarly journals Cells Expressing Anchorless Prion Protein Are Resistant to Scrapie Infection

2009 ◽  
Vol 83 (9) ◽  
pp. 4469-4475 ◽  
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.

scholarly journals Analysis of Protein Levels of 24 Cytokines in Scrapie Agent-Infected Brain and Glial Cell Cultures from Mice Differing in Prion Protein Expression Levels

2009 ◽  
Vol 83 (21) ◽  
pp. 11244-11253 ◽  
Author(s):  
Déborah Tribouillard-Tanvier ◽  
James F. Striebel ◽  
Karin E. Peterson ◽  
Bruce Chesebro
Keyword(s):  
Transgenic Mice ◽  
Brain Damage ◽  
Prion Protein ◽  
Prion Diseases ◽  
Wild Type ◽  
Prion Infection ◽  
Protein Levels ◽  
Cytokine Responses ◽  
Scrapie Agent ◽  
High Level

ABSTRACT Activation of microglia and astroglia is seen in many neurodegenerative diseases including prion diseases. Activated glial cells produce cytokines as a protective response against certain pathogens and as part of the host inflammatory response to brain damage. In addition, cytokines might also exacerbate tissue damage initiated by other processes. In the present work using multiplex assays to analyze protein levels of 24 cytokines in scrapie agent-infected C57BL/10 mouse brains, we observed elevation of CCL2, CCL5, CXCL1, CXCL10, granulocyte-macrophage colony-stimulating factor (GM-CSF), gamma interferon (IFN-γ), interleukin 1α (IL-1α), IL-1β, IL-6, and IL-12p40. Scrapie agent-infected wild-type mice and transgenic mice expressing anchorless prion protein (PrP) had similar cytokine responses in spite of extensive differences in neuropathology. Therefore, these responses may be primarily a reaction to brain damage induced by prion infection rather than specific inducers of a particular type of pathology. To study the roles of astroglia and microglia in these cytokine responses, primary glial cultures were exposed to scrapie agent-infected brain homogenates. Microglia produced only IL-12p40 and CXCL10, whereas astroglia produced these cytokines plus CCL2, CCL3, CCL5, CXCL1, G-CSF, IL-1β, IL-6, IL-12p70, and IL-13. Glial cytokine responses from wild-type mice and transgenic mice expressing anchorless PrP differed only slightly, but glia from PrP-null mice produced only IL-12p40, indicating that PrP expression was required for scrapie agent induction of other cytokines detected. The difference in cytokine response between microglia and astroglia correlated with 20-fold-higher levels of PrP expression in astroglia versus microglia, suggesting that high-level PrP expression on astroglia might be important for induction of certain cytokines.

scholarly journals Characterization of four new monoclonal antibodies against the distal N-terminal region of PrPc

2014 ◽  
Author(s):  
Alessandro Didonna ◽  
Anja Colja Venturini ◽  
Katrina Hartman ◽  
Tanja Vranac ◽  
Vladka Curin Serbec ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Prion Protein ◽  
Biochemical Characterization ◽  
Prion Diseases ◽  
Physiological Role ◽  
Prion Infection ◽  
Promising Tool ◽  
C Terminus ◽  
N Terminus

Prion diseases are a group of fatal neurodegenerative disorders that affect humans and animals. They are characterized by the accumulation in the central nervous system of a pathological form of the host-encoded prion protein (PrPC). The prion protein is a membrane glycoprotein that consists of two domains: a globular, structured C-terminus and an unstructured N-terminus. The N-terminal part of the protein is involved in different functions in both health and disease. In the present work we discuss the production and biochemical characterization of a panel of four monoclonal antibodies (mAbs) against the distal N-terminus of PrPC using a well-established methodology based on the immunization of Prnp0/0 mice. Additionally, we show their ability to block prion (PrPSc) replication at nanomolar concentrations in a cell culture model of prion infection. These mAbs represent a promising tool for prion diagnostics and for studying the physiological role of the N-terminal domain of PrPC.

scholarly journals Dimerization of the cellular prion protein inhibits propagation of scrapie prions

2018 ◽  
Vol 293 (21) ◽  
pp. 8020-8031 ◽  
Author(s):  
Anna D. Engelke ◽  
Anika Gonsberg ◽  
Simrika Thapa ◽  
Sebastian Jung ◽  
Sarah Ulbrich ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Prion Protein ◽  
Conformational Transition ◽  
Prion Diseases ◽  
Neuroblastoma Cells ◽  
Direct Interaction ◽  
Cellular Prion Protein ◽  
Dominant Negative ◽  
Dimerization Domain ◽  
Hydrophobic Domain

A central step in the pathogenesis of prion diseases is the conformational transition of the cellular prion protein (PrPC) into the scrapie isoform, denoted PrPSc. Studies in transgenic mice have indicated that this conversion requires a direct interaction between PrPC and PrPSc; however, insights into the underlying mechanisms are still missing. Interestingly, only a subfraction of PrPC is converted in scrapie-infected cells, suggesting that not all PrPC species are suitable substrates for the conversion. On the basis of the observation that PrPC can form homodimers under physiological conditions with the internal hydrophobic domain (HD) serving as a putative dimerization domain, we wondered whether PrP dimerization is involved in the formation of neurotoxic and/or infectious PrP conformers. Here, we analyzed the possible impact on dimerization of pathogenic mutations in the HD that induce a spontaneous neurodegenerative disease in transgenic mice. Similarly to wildtype (WT) PrPC, the neurotoxic variant PrP(AV3) formed homodimers as well as heterodimers with WTPrPC. Notably, forced PrP dimerization via an intermolecular disulfide bond did not interfere with its maturation and intracellular trafficking. Covalently linked PrP dimers were complex glycosylated, GPI-anchored, and sorted to the outer leaflet of the plasma membrane. However, forced PrPC dimerization completely blocked its conversion into PrPSc in chronically scrapie-infected mouse neuroblastoma cells. Moreover, PrPC dimers had a dominant-negative inhibition effect on the conversion of monomeric PrPC. Our findings suggest that PrPC monomers are the major substrates for PrPSc propagation and that it may be possible to halt prion formation by stabilizing PrPC dimers.

scholarly journals Prion Protein Expression Differences in Microglia and Astroglia Influence Scrapie-Induced Neurodegeneration in the Retina and Brain of Transgenic Mice

2007 ◽  
Vol 81 (19) ◽  
pp. 10340-10351 ◽  
Author(s):  
Lisa Kercher ◽  
Cynthia Favara ◽  
James F. Striebel ◽  
Rachel LaCasse ◽  
Bruce Chesebro
Keyword(s):  
Transgenic Mice ◽  
Prion Protein ◽  
Microglial Activation ◽  
Prion Diseases ◽  
Cell Types ◽  
Mouse Strains ◽  
Activation Markers ◽  
Activated Microglia ◽  
Morphological Criteria ◽  
Scrapie Infection

ABSTRACT Activated microglia and astroglia are known to be involved in a variety of neurodegenerative diseases, including prion diseases. In the present experiments, we studied activation of astroglia and microglia after intraocular scrapie infection in transgenic mice expressing prion protein (PrP) in multiple cell types (tg7 mice) or in neurons only (tgNSE mice). In this model, scrapie infection and protease-resistant PrP deposition occurs in the retinas of both strains of mice, but retinal degeneration is observed only in tg7 mice. Our results showed that the retinas of tg7 and tgNSE mice both had astroglial activation with increased chemokine expression during the course of infection. However, only tg7 retinas exhibited strong microglial activation compared to tgNSE retinas, which showed little microglial activation by biochemical or morphological criteria. Therefore, microglial PrP expression might be required for scrapie-induced retinal microglial activation and damage. Furthermore, microglial activation preceded retinal neurodegeneration in tg7 mice, suggesting that activated microglia might contribute to the degenerative process, rather than being a response to the damage. Surprisingly, brain differed from retina in that an altered profile of microglial activation markers was upregulated, and the profiles in the two mouse strains were indistinguishable. Microglial activation in the brain was associated with severe brain vacuolation and neurodegeneration, leading to death. Thus, retinal and brain microglia appeared to differ in their requirements for activation, suggesting that different activation pathways occur in the two tissues.

scholarly journals Prion Type-Dependent Deposition ofPRNPAllelic Products in Heterozygous Sheep

2015 ◽  
Vol 90 (2) ◽  
pp. 805-812 ◽  
Author(s):  
J. P. M. Langeveld ◽  
J. G. Jacobs ◽  
N. Hunter ◽  
L. J. M. van Keulen ◽  
F. Lantier ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Bovine Spongiform Encephalopathy ◽  
Prion Protein ◽  
Transmissible Spongiform Encephalopathy ◽  
Infected Sheep ◽  
Transmissible Spongiform Encephalopathies ◽  
Spongiform Encephalopathy ◽  
Sequence Variants ◽  
Spongiform Encephalopathies

ABSTRACTSusceptibility or resistance to prion infection in humans and animals depends on single prion protein (PrP) amino acid substitutions in the host, but the agent's modulating role has not been well investigated. Compared to disease incubation times in wild-type homozygous ARQ/ARQ (where each triplet represents the amino acids at codons 136, 154, and 171, respectively) sheep, scrapie susceptibility is reduced to near resistance in ARR/ARR animals while it is strongly enhanced in VRQ/VRQ carriers. Heterozygous ARR/VRQ animals exhibit delayed incubation periods. In bovine spongiform encephalopathy (BSE) infection, the polymorphism effect is quite different although the ARR allotype remains the least susceptible. In this study, PrP allotype composition in protease-resistant prion protein (PrPres) from brain of heterozygous ARR/VRQ scrapie-infected sheep was compared with that of BSE-infected sheep with a similar genotype. A triplex Western blotting technique was used to estimate the two allotype PrP fractions in PrPresmaterial from BSE-infected ARR/VRQ sheep. PrPresin BSE contained equimolar amounts of VRQ- and ARR-PrP, which contrasts with the excess (>95%) VRQ-PrP fraction found in PrP in scrapie. This is evidence that transmissible spongiform encephalopathy (TSE) agent properties alone, perhaps structural aspects of prions (such as PrP amino acid sequence variants and PrP conformational state), determine the polymorphic dependence of the PrPresaccumulation process in prion formation as well as the disease-associated phenotypic expressions in the host.IMPORTANCETransmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative and transmissible diseases caused by prions. Amino acid sequence variants of the prion protein (PrP) determine transmissibility in the hosts, as has been shown for classical scrapie in sheep. Each individual produces a separate PrP molecule from its two PrP gene copies. Heterozygous scrapie-infected sheep that produce two PrP variants associated with opposite scrapie susceptibilities (136V-PrP variant, high; 171R-PrP variant, very low) contain in their prion material over 95% of the 136V PrP variant. However, when these sheep are infected with prions from cattle (bovine spongiform encephalopathy [BSE]), both PrP variants occur in equal ratios. This shows that the infecting prion type determines the accumulating PrP variant ratio in the heterozygous host. While the host's PrP is considered a determining factor, these results emphasize that prion structure plays a role during host infection and that PrP variant involvement in prions of heterozygous carriers is a critical field for understanding prion formation.

scholarly journals Pathogenic Prion Protein Isoforms Are Not Present in Cerebral Organoids Generated from Asymptomatic Donors Carrying the E200K Mutation Associated with Familial Prion Disease

Pathogens ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 482
Author(s):  
Simote Foliaki ◽  
Bradley Groveman ◽  
Jue Yuan ◽  
Ryan Walters ◽  
Shulin Zhang ◽  
...  
Keyword(s):  
Prion Protein ◽  
Prion Disease ◽  
Neurological Disorders ◽  
Prion Diseases ◽  
Three Dimensional ◽  
Protein Isoforms ◽  
Genetic Modifiers ◽  
Neuronal Tissue ◽  
Prion Infection ◽  
E200k Mutation

Cerebral organoids (COs) are a self-organizing three-dimensional brain tissue mimicking the human cerebral cortex. COs are a promising new system for modelling pathological features of neurological disorders, including prion diseases. COs expressing normal prion protein (PrPC) are susceptible to prion infection when exposed to the disease isoforms of PrP (PrPD). This causes the COs to develop aspects of prion disease pathology considered hallmarks of disease, including the production of detergent-insoluble, protease-resistant misfolded PrPD species capable of seeding the production of more misfolded species. To determine whether COs can model aspects of familial prion diseases, we produced COs from donor fibroblasts carrying the E200K mutation, the most common cause of human familial prion disease. The mature E200K COs were assessed for the hallmarks of prion disease. We found that up to 12 months post-differentiation, E200K COs harbored no PrPD as confirmed by the absence of detergent-insoluble, protease-resistant, and seeding-active PrP species. Our results suggest that the presence of the E200K mutation within the prion gene is insufficient to cause disease in neuronal tissue. Therefore, other factors, such as further genetic modifiers or aging processes, may influence the onset of misfolding.

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