Accumulation and dissemination of prion protein in experimental sheep scrapie in the natural host

ABSTRACT Sheep scrapie is the prototypical transmissible spongiform encephalopathy (prion disease), which has a fundamental pathogenesis involving conversion of normal cellular prion protein (PrPC [C superscript stands for cellular]) to disease-associated prion protein (PrPSc [Sc superscript stands for sheep scrapie]). Sheep microglial cell cultures, derived from a prnp 136VV/171QQ near-term fetal brain, were developed to study sheep scrapie in the natural host and to investigate potential cofactors in the prion conversion process. Two culture systems, a primary cell culture and a cell line transformed with the large T antigen of simian virus 40, were developed, and both were identified as microglial in origin as indicated by expression of several microglial phenotype markers. Following exposure to PrPSc, sheep microglial cells demonstrated relatively low levels (transformed cell line) to high levels (primary cell line) of PrPSc accumulation over time. The accumulated PrPSc demonstrated protease resistance, an inferred beta-sheet conformation (as determined by a commercial enzyme-linked immunosorbent assay), specific inhibition by anti-PrP antibodies, and was transmissible in a dose-dependent manner. Primary microglia coinfected with a small-ruminant lentivirus (caprine arthritis encephalitis virus-Cork strain) and PrPSc demonstrated an approximately twofold increase in PrPSc accumulation compared to that of primary microglia infected with PrPSc alone. The results demonstrate the in vitro utility of PrPSc-permissive sheep microglial cells in investigating the biology of natural prion diseases and show that small-ruminant lentiviruses enhance prion conversion in cultured sheep microglia.

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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

10.1101/800698 ◽

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.

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Sensitivity of the Western blot detection of prion protein PrPres in natural sheep scrapie

Journal of Virological Methods ◽

10.1016/s0166-0934(98)00113-x ◽

1998 ◽

Vol 75 (2) ◽

pp. 169-177 ◽

Cited By ~ 18

Author(s):

J.-Y. Madec ◽

M.H. Groschup ◽

A. Buschmann ◽

P. Belli ◽

D. Calavas ◽

...

Keyword(s):

Western Blot ◽

Prion Protein ◽

Sheep Scrapie

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Prion protein genetics: Host PrP control of TSE susceptibility

The Biochemist ◽

10.1042/bio02704020 ◽

2005 ◽

Vol 27 (4) ◽

pp. 20-23

Author(s):

Wilfred Goldmann

Keyword(s):

Prion Protein ◽

Transmissible Spongiform Encephalopathies ◽

Spongiform Encephalopathy ◽

Spongiform Encephalopathies ◽

Degenerative Disorders ◽

Jakob Disease ◽

The Central Nervous System ◽

Important Host ◽

Disease Family ◽

Sheep Scrapie

TSEs (transmissible spongiform encephalopathies) are fatal, degenerative disorders of the central nervous system. The best-known members of this disease family are sheep scrapie, cattle BSE (bovine spongiform encephalopathy) and human CJD (Creutzfeldt–Jakob disease). By far the most important host gene in TSEs is the PrP (prion protein) gene. It modulates TSE susceptibility at many levels and is the crucial element in the treatment and eradication of these diseases. This article will highlight the advances in our understanding of PrP genetics in animals and man.

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Glycan-Controlled Epitopes of Prion Protein Include a Major Determinant of Susceptibility to Sheep Scrapie

Journal of Virology ◽

10.1128/jvi.78.20.11449.2004 ◽

2004 ◽

Vol 78 (20) ◽

pp. 11449-11449 ◽

Cited By ~ 1

Author(s):

Mohammed Moudjou ◽

Eric Treguer ◽

Human Rezaei ◽

Elifsu Sabuncu ◽

Erdmute Neuendorf ◽

...

Keyword(s):

Prion Protein ◽

Major Determinant ◽

Sheep Scrapie

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Genetic and Pathological Follow-Up Study of Goats Experimentally and Naturally Exposed to a Sheep Scrapie Isolate

Journal of Virology ◽

10.1128/jvi.01262-15 ◽

2015 ◽

Vol 89 (19) ◽

pp. 10044-10052 ◽

Cited By ~ 10

Author(s):

Caterina Maestrale ◽

Maria G. Cancedda ◽

Davide Pintus ◽

Mariangela Masia ◽

Romolo Nonno ◽

...

Keyword(s):

Prion Protein ◽

Gene Sequence ◽

Protein Gene ◽

Brain Homogenate ◽

Prion Protein Gene ◽

Wild Type ◽

Related Factors ◽

Sheep And Goats ◽

First Time ◽

Sheep Scrapie

ABSTRACTThirty-seven goats carrying different prion protein genotypes (PRNP) were orally infected with a classical scrapie brain homogenate from wild-type (ARQ/ARQ) sheep and then mated to obtain 2 additional generations of offspring, which were kept in the same environment and allowed to be naturally exposed to scrapie. Occurrence of clinical or subclinical scrapie was observed in the experimentally infected goats (F0) and in only one (F1b) of the naturally exposed offspring groups. In both groups (F0and F1b), goats carrying the R154H, H154H, R211Q, and P168Q-P240P dimorphisms died of scrapie after a longer incubation period than wild-type, G37V, Q168Q-P240P, and S240P goats. In contrast, D145D and Q222K goats were resistant to infection. The immunobiochemical signature of the scrapie isolate and its pathological aspects observed in the sheep donors were substantially maintained over 2 goat generations, i.e., after experimental and natural transmission. This demonstrates that the prion protein gene sequence, which is shared by sheep and goats, is more powerful than any possible but unknown species-related factors in determining scrapie phenotypes. With regard to genetics, our study confirms that the K222 mutation protects goats even against ovine scrapie isolates, and for the first time, a possible association of D145 mutation with scrapie resistance is shown. In addition, it is possible that the sole diverse frequencies of these genetic variants might, at least in part, shape the prevalence of scrapie among naturally exposed progenies in affected herds.IMPORTANCEThis study was aimed at investigating the genetic and pathological features characterizing sheep-to-goat transmission of scrapie. We show that in goats with different prion protein gene mutations, the K222 genetic variant is associated with scrapie resistance after natural and experimental exposure to ovine prion infectivity. In addition, we observed for the first time a protective effect of the D145 goat variant against scrapie. Importantly, our results demonstrate that the phenotypic characteristic of the wild-type sheep scrapie isolate is substantially preserved in goats carrying different susceptiblePRNPgene variants, thus indicating that the prion protein gene sequence, which is shared by sheep and goats, plays a fundamental role in determining scrapie phenotypes.

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Efficient Transmission of Two Different Sheep Scrapie Isolates in Transgenic Mice Expressing the Ovine PrP Gene

Journal of Virology ◽

10.1128/jvi.75.11.5328-5334.2001 ◽

2001 ◽

Vol 75 (11) ◽

pp. 5328-5334 ◽

Cited By ~ 57

Author(s):

Carole Crozet ◽

Frederic Flamant ◽

Anna Bencsik ◽

Denise Aubert ◽

Jacques Samarut ◽

...

Keyword(s):

Transgenic Mice ◽

Prion Protein ◽

Neuron Specific Enolase ◽

Open Reading Frame ◽

Protein Accumulation ◽

Spongiform Encephalopathies ◽

Reading Frame ◽

Neurological Signs ◽

Prp Gene ◽

Sheep Scrapie

ABSTRACT We produced transgenic mice expressing the sheep prion protein to obtain a sensitive model for sheep spongiform encephalopathies (scrapie). The complete open reading frame, with alanine, arginine, and glutamine at susceptibility codons 136, 154, and 171, respectively, was inserted downstream from the neuron-specific enolase promoter. A mouse line, Tg(OvPrP4), devoid of the murine PrP gene, was obtained by crossing with PrP knockout mice. Tg(OvPrP4) mice were shown to selectively express sheep PrP in their brains, as demonstrated in mRNA and protein analysis. We showed that these mice were susceptible to infection by sheep scrapie following intracerebral inoculation with two natural sheep scrapie isolates, as demonstrated not only by the occurrence of neurological signs but also by the presence of the spongiform changes and abnormal prion protein accumulation in their brains. Mean times to death of 238 and 290 days were observed with these isolates, but the clinical course of the disease was strikingly different in the two cases. One isolate led to a very early onset of neurological signs which could last for prolonged periods before death. Independently of the incubation periods, some of the mice inoculated with this isolate showed low or undetectable levels of PrPsc, as detected by both Western blotting and immunohistochemistry. The development of experimental scrapie in these mice following inoculation of the scrapie infectious agent further confirms that neuronal expression of the PrP open reading frame alone is sufficient to mediate susceptibility to spongiform encephalopathies. More importantly, these mice provide a new and promising tool for studying the infectious agents in sheep spongiform encephalopathies.

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Proteinase K-sensitive disease-associated ovine prion protein revealed by conformation-dependent immunoassay

Biochemical Journal ◽

10.1042/bj20061264 ◽

2006 ◽

Vol 401 (2) ◽

pp. 475-483 ◽

Cited By ~ 64

Author(s):

Alana M. Thackray ◽

Lee Hopkins ◽

Raymond Bujdoso

Keyword(s):

Prion Protein ◽

Phosphotungstic Acid ◽

Limited Proteolysis ◽

Proteinase K ◽

Genotypic Differences ◽

Time Resolved Fluorescence ◽

Time Resolved ◽

Natural Scrapie ◽

Proteinase K Treatment ◽

Sheep Scrapie

PrPSc [abnormal disease-specific conformation of PrP (prion-related protein)] accumulates in prion-affected individuals in the form of amorphous aggregates. Limited proteolysis of PrPSc results in a protease-resistant core of PrPSc of molecular mass of 27–30 kDa (PrP27–30). Aggregated forms of PrP co-purify with prion infectivity, although infectivity does not always correlate with the presence of PrP27–30. This suggests that discrimination between PrPC (normal cellular PrP) and PrPSc by proteolysis may underestimate the repertoire and quantity of PrPSc subtypes. We have developed a CDI (conformation-dependent immunoassay) utilizing time-resolved fluorescence to study the conformers of disease-associated PrP in natural cases of sheep scrapie, without using PK (proteinase K) treatment to discriminate between PrPC and PrPSc. The capture-detector CDI utilizes N-terminal- and C-terminal-specific anti-PrP monoclonal antibodies that recognize regions of the prion protein differentially buried or exposed depending on the extent of denaturation of the molecule. PrPSc was precipitated from scrapie-infected brain stem and cerebellum tissue following sarkosyl extraction, with or without the use of sodium phosphotungstic acid, and native and denatured PrPSc detected by CDI. PrPSc was detectable in brain tissue from homozygous VRQ (V136 R154 Q171) and ARQ (A136 R154 Q171) scrapie-infected sheep brains. The highest levels of PrPSc were found in homozygous VRQ scrapie-infected brains. The quantity of PrPSc was significantly reduced, up to 90% in some cases, when samples were treated with PK prior to the CDI. Collectively, our results show that the level of PrPSc in brain samples from cases of natural scrapie display genotypic differences and that a significant amount of this material is PK-sensitive.

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Immunological characterization of abnormal prion protein from atypical scrapie cases in sheep using a panel of monoclonal antibodies

Journal of General Virology ◽

10.1099/vir.0.81816-0 ◽

2006 ◽

Vol 87 (12) ◽

pp. 3715-3722 ◽

Cited By ~ 31

Author(s):

Anja Gretzschel ◽

Anne Buschmann ◽

Jan Langeveld ◽

Martin H. Groschup

Keyword(s):

Monoclonal Antibodies ◽

Prion Protein ◽

Proteinase K ◽

Atypical Scrapie ◽

Surveillance Programme ◽

Rapid Tests ◽

The Brain ◽

Sheep Scrapie ◽

Scrapie Case

After the implementation of an active surveillance programme for scrapie in sheep in the EU, the number of diagnosed classical scrapie cases rose sharply and a novel kind of so-called atypical scrapie case was discovered. These atypical scrapie cases display unusual features concerning the distribution of the abnormal prion protein (PrPSc) in the brain, a distinct electrophoretic profile of PrPSc and an inconsistent reaction pattern in the currently used rapid tests. In this report, PrPSc of two German atypical sheep scrapie cases was characterized by epitope mapping using a panel of 18 monoclonal antibodies that were directed against epitopes located throughout the prion protein. This analysis suggests that PrPSc derived from atypical scrapie cases and treated with proteinase K is largely composed of an 11 kDa fragment (previously referred to as the 12 kDa band) and of polymeric fragments thereof. The 11 kDa band corresponds to a prion protein fragment spanning approximately aa 90–153 and may therefore represent a novel PrPSc type.

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Prion protein degradation by lichens of the genusCladonia

The Lichenologist ◽

10.1017/s0024282912000102 ◽

2012 ◽

Vol 44 (4) ◽

pp. 523-531 ◽

Cited By ~ 2

Author(s):

James P. BENNETT ◽

Cynthia M. RODRIGUEZ ◽

Christopher J. JOHNSON

Keyword(s):

Protein Degradation ◽

Serine Protease ◽

Prion Protein ◽

Prion Disease ◽

Prion Diseases ◽

Usnic Acid ◽

Phylogenetic Position ◽

Wasting Disease ◽

Degradation Activity ◽

Sheep Scrapie

AbstractIt has recently been discovered that lichens contain a serine protease capable of degrading the pathogenic prion protein, the etiological agent of prion diseases such as sheep scrapie and cervid chronic wasting disease. Limited methods are available to degrade or inactivate prion disease agents, especially in the environment, and lichens or their serine protease could prove important for management of these diseases. Scant information is available regarding the presence or absence of the protease responsible for degrading prion protein (PrP) in lichen species and, in this study, we tested the hypothesis that PrP degradation activity in lichens is phylogenetically-based by testing 44 species ofCladonialichens, a genus for which a significant portion of the phylogeny is well established. We categorized PrP degradation activity among the 44 species (high, moderate, low or none) and found that activity inCladoniaspecies did not correspond with phylogenetic position of the species. Degradation of PrP did correspond, however, with three classical taxonomic characters within the genus: species with brown apothecia, no usnic acid, and the presence of a cortex. Of the 44 species studied, 18 (41%) had either high or moderate PrP degradation activity, suggesting the protease may be frequent in this genus of lichens.

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