Lack of Prion Accumulation in Lymphoid Tissues of PRNP ARQ/ARR Sheep Intracranially Inoculated with the Agent of Scrapie

Sheep scrapie is a transmissible spongiform encephalopathy that can be transmitted horizontally. The prion protein gene (PRNP) profoundly influences the susceptibility of sheep to the scrapie agent and the tissue levels and distribution of PrP^Sc in affected sheep. The purpose of this study was to compare the survival time and PrP^Sc tissue distribution in sheep with highly resistant and highly susceptible PRNP genotypes after intracranial inoculation of the agent of scrapie. Five sheep each of genotype VRQ/VRQ, VRQ/ARR or ARQ/ARR were inoculated. Sheep were euthanized when clinical signs of scrapie became severe. Clinical signs, microscopic lesions, and western blot profiles were uniform across genotypes and consistent with manifestations of classical scrapie. Mean survival time differences were associated with the 171 polymorphic site with VRQ/VRQ sheep surviving 18 months, whereas VRQ/ARR and ARQ/ARR sheep survived 60 and 56 months, respectively. Labeling of PrP^Sc by immunohistochemistry revealed similar accumulations in central nervous system tissues regardless of host genotype. Immunoreactivity for PrP^Sc in lymphoid tissue was consistently abundant in VRQ/VRQ, present but confined to tonsil or retropharyngeal lymph node in 4/5 VRQ/ARR, and totally absent in ARQ/ARR sheep. The results of this study demonstrate the susceptibility of sheep with the ARQ/ARR genotype to scrapie by the intracranial inoculation route with PrP^Sc accumulation in CNS tissues, but prolonged incubation times and lack of PrP^Sc in lymphoid tissue.     

Nanopore analysis reveals differences in structural stability of ovine PrPC proteins corresponding to scrapie susceptible (VRQ) and resistance (ARR) genotypes

Species, as well as individuals within species, have unique susceptibilities to prion infection that are likely based on sequence differences in cellular prion protein (PrP^C). Species barriers to transmission also reflect PrP^C sequence differences. Defining the structure-activity relationship of PrP^C/PrP^Sc with respect to infectivity/susceptibility will benefit disease understanding and assessment of transmission risks. Here, nanopore analysis is employed to investigate genotypes of sheep PrP^C corresponding to differential susceptibilities to scrapie infection. Under non-denaturing conditions scrapie resistant (ARR) and susceptible (VRQ) genotypes display similar, type I (bumping) predominant event profiles, suggesting a conserved folding pattern. Under increasingly denaturing conditions both proteins shift to type II (intercalation/translocation) events but with different sensitivities to unfolding. Specifically, when pre-incubated in 2M Gdn-HCl, the VRQ variant had more of type II events as compared with the ARR protein, suggesting a more flexible unfolding pattern. Addition of PrP^Sc-specific polyclonal antibody (YML) to the ARR variant, pre-incubated in 2M Gdn-HCl, reduced the number of type II events with no clear intercalation/translocation peak, whereas for VRQ, type II events above blockades of 90 pA bound YML. A second PrP^Sc-specific antibody (SN6b) to a different cryptic epitope reduced type II events for VRQ but not the ARR variant. Collectively, the event patterns associated with sequential denaturation, as well as interactions with PrP^Sc-specific antibodies, support unique patterns and/or propensities of misfolding between the genotypes. Overall, nanopore analysis identifies intermediate conformations that occur during the unfolding pathways of ARR and VRQ genotypes and may help to understand the correlation of structural properties that induce protein misfolding.     

Ovine plasma prion protein levels show genotypic variation detected by C-terminal epitopes not exposed in cell-surface PrPC

Ovine PBMCs (peripheral blood mononuclear cells) express PrP(C) [cellular PrP (prion-related protein)] and have the potential to harbour and release disease-associated forms of PrP during scrapie in sheep. Cell-surface PrP(C) expression by PBMCs, together with plasma PrP(C) levels, may contribute to the regulatory mechanisms that determine susceptibility and resistance to natural scrapie in sheep. Here, we have correlated cell-surface PrP(C) expression on normal ovine PBMCs by FACS with the presence of PrP(C) in plasma measured by capture-detector immunoassay. FACS showed similar levels of cell-surface PrP(C) on homozygous ARR (Ala136-Arg154-Arg171), ARQ (Ala136-Arg154-Gln171) and VRQ (Val136-Arg154-Gln171) PBMCs. Cell-surface ovine PrP(C) showed modulation of N-terminal epitopes, which was more evident on homozygous ARR cells. Ovine plasma PrP(C) levels showed genotypic variation and the protein displayed C-terminal epitopes not available in cell-surface PrP(C). Homozygous VRQ sheep showed the highest plasma PrP(C) level and homozygous ARR animals the lowest. For comparison, similar analyses were performed on normal bovine PBMCs and plasma. PrP(C) levels in bovine plasma were approx. 4-fold higher than ovine homozygous ARQ plasma despite similar levels of PBMC cell-surface PrP(C) expression. Immunoassays using C-terminal-specific anti-PrP monoclonal antibodies as capture and detector reagents revealed the highest level of PrP(C) in both ovine and bovine plasma, whilst lower levels were detected using N-terminal-specific monoclonal antibody FH11 as the capture reagent. This suggested that a proportion of plasma PrP(C) was N-terminally truncated. Our results indicate that the increased susceptibility to natural scrapie displayed by homozygous VRQ sheep correlates with a higher level of plasma PrP(C).     

α-Cleavage of cellular prion protein

The cellular prion protein (PrP^C) is subjected to various processing under physiological and pathological conditions, of which the α-cleavage within the central hydrophobic domain not only disrupts a region critical for both PrP toxicity and PrP^C to PrP^Sc conversion but also produces the N1 fragment that is neuroprotective and the C1 fragment that enhances the pro-apoptotic effect of staurosporine in one report and inhibits prion in another. The proteases responsible for the α-cleavage of PrP^C are controversial. The effect of ADAM10, ADAM17, and ADAM9 on N1 secretion clearly indicates their involvement in the α-cleavage of PrP^C, but there has been no report of direct PrP^C α-cleavage activity with any of the three ADAMs in a purified protein form. We demonstrated that, in muscle cells, ADAM8 is the primary protease for the α-cleavage of PrP^C, but another unidentified protease(s) must also play a minor role. We also found that PrP^C regulates ADAM8 expression, suggesting that a close examination on the relationships between PrP^C and its processing enzymes may reveal novel roles and underlying mechanisms for PrP^C in non-prion diseases such as asthma and cancer.     

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.     

Amyloidogenic unfolding intermediates differentiate sheep prion protein variants

Sheep is a unique example among mammalian species to present a strong correlation between genotype and prion disease susceptibility phenotype. Indeed a well-defined set of PrP polymorphisms at positions 136, 154 and 171 (sheep numbering) govern scrapie susceptibility, ranging from very high susceptibility for V136-R154-Q171 variant (VRQ) to resistance for A136-R154-R171 variant (ARR).To get better insight into the molecular mechanisms of scrapie susceptibility/resistance, the unfolding pathways of the different full-length recombinant sheep prion protein variants were analysed by differential scanning calorimetry in a wide range of pH. In the pH range 4.5-6.0, thermal unfolding occurs through a reversible one-step process while at pH <4.5 and >6.0 unfolding intermediates are formed, which are stable in the temperature range 65-80 degrees C. While these general behaviours are shared by all variants, VRQ and ARQ (susceptibility variants) show higher thermal stability than AHQ and ARR (resistance variants) and the formation of their unfolding intermediates requires higher activation energy than in the case of AHQ and ARR. Furthermore, secondary structures of the unfolding intermediates differentiate variants: ARR unfolding intermediate exhibits random coil structure, contrasting with the beta-sheet structure of VRQ and ARQ unfolding intermediates. The rate of the unfolding intermediate formation allows us to classify genetic variants along increasing scrapie susceptibility at pH 4.0, VRQ and ARQ rates being the highest. Rather poor correlation is observed at pH 7.2. Upon cooling, these intermediates refold into stable species, which are rich in beta-type secondary structures and, as revealed by thioflavin T fluorescence and electron microscopy, share amyloid characteristics. These results highlight the prion protein plasticity genetically modulated in sheep, and might provide a molecular basis for sheep predisposition to scrapie taking into account both thermodynamic stability and transconformation rate of prion protein.     

Microdeletions within the hydrophobic core region of cellular prion protein alter its topology and metabolism

The cellular prion protein (PrP^C) is a GPI-anchored cell-surface protein. A small subset of PrP^C molecules, however, can be integrated into the ER-membrane via a transmembrane domain (TM), which also harbors the most highly conserved regions of PrP^C, termed the hydrophobic core (HC). A mutation in HC is associated with prion disease resulting in an enhanced formation of a transmembrane form (^CtmPrP), which has thus been postulated to be a neurotoxic molecule besides PrP^Sc. To elucidate a possible physiological function of the transmembrane domain, we created a set of mutants carrying microdeletions of 2-8 aminoacids within HC between position 114 and 121. Here, we show that these mutations display reduced propensity for transmembrane topology. In addition, the mutants exhibited alterations in the formation of the C1 proteolytic fragment, which is generated by α-cleavage during normal PrP^C metabolism, indicating that HC might function as recognition site for the protease(s) responsible for PrP^C α-cleavage. Interestingly, the mutant G113V, corresponding to a hereditary form of prion disease in humans, displayed increased ^CtmPrP topology and decreased α-cleavage in our in vitro assay. In conclusion, HC represents an essential determinant for transmembrane PrP topology, whereas the high evolutionary conservation of this region is rather based upon preservation of PrP^C α-cleavage, thus highlighting the biological importance of this cleavage.     

The PrP genotype of sheep of the improved Valachian breed

In a worldwide majority of sheep breeds an excessive susceptibility to scrapie associated with the PrP gene alleles coding for valine (V; at the 136 codon) and glutamine (Q; at the 171 codon) (e.g., VRQ/VRQ, VRQ/ARQ, or ARQ/ARQ) was demonstrated. Particularly the PrPVRQ allele is closely associated with the high-risk development of the disease; the PrPARQ allele can also fulfill this function but under certain limited conditions. Polymorphism in the PrP gene sequences (conclusively related to the increased susceptibility of sheep to scrapie) of improved Valachian sheep from two Slovak regions, Orava and Spis, was determined. Examination of 735 sheep showed that ARR/ARQ was the most frequent genotype (45.2%). High-risk genotypes were determined in 32.4% of sheep (ARQ/ARQ 19.3, ARR/VRQ 9.0, ARR/VRQ 3.5, VRQ/VRQ 0.3, ARR/VRR 0.3). Low-risk genotypes were found in 67.7% of sheep (ARR/ARQ 45.2, ARR/ARR 10.9, ARR/AHQ 5.7, ARQ/ARQ 4.9, AHQ/AHQ 0.7, ARR/AHR 0.3). Despite the geographically distant flocks of improved Valachian sheep investigated no difference in the occurrence of individual PrP genotypes was observed.     

Prion protein gene polymorphisms in sheep in the state of Paraná, Brazil

To determine the polymorphisms of the prion protein gene in sheep from the state of Paraná, Brazil, 323 animals of meat breeds (Suffolk, Hampshire Down, Texel, Ile de France, Dorper, Dorset, Santa Inês and crossbreds) were genotyped by restriction fragment length polymorphism (RFLP) analysis. The most frequent allele was ARQ, with a frequency of 0.61, followed by ARR (0.30). VRQ and AHQ alleles were present at very low frequencies (0.13 and 0.05 respectively), and the ARH allele was not found. Seven genotypes were identified (ARR/ARR, ARR/ARQ, ARQ/ARQ, ARR/VRQ, ARR/AHQ, ARQ/VRQ and ARQ/AHQ), of which ARQ/ARQ was the most frequent (0.41). The Santa Inês breed and crossbred animals showed the highest genotypic variability.     

新疆地区绵羊品种朊蛋白基因136、154和171位点的多态性

绵羊痒病是一种渐进性和致死性中枢神经系统疾病,绵羊朊蛋白基因(Prion protein gene,PRNP)多态性与痒病的易感或抗性有关,其中PRNP136位(V/A)、154位(H/R)和171位(H/Q/R)的基因多态性与该病发生最相关。为评价新疆地区主要绵羊品种对痒病的易感性,文章对新疆地区10个绵羊品种(阿勒泰、巴士拜、巴音布鲁克、多浪、和田、策勒黑、中国美利奴、德国肉用美利奴、特克赛尔和萨福克羊)共746只个体PRNP基因的136位(V/A)、154位(H/R)和171位(H/Q/R)的遗传多态性进行分析,检测到了ARQ、ARR、ARH、ARK、VRQ、AHR、AHQ、AHH8种等位基因,其中ARQ和ARR等位基因存在于所有品种中,且ARQ在所有品种中的基因频率最高。ARH存在于除萨福克和德国肉用美利奴羊外的8个绵羊品种中。仅在新疆地方品种阿勒泰、巴音布鲁克、巴士拜和多浪羊中检测到ARK等位基因。而VRQ、AHR、AHQ和AHH4种等位基因只在中国美利奴羊上存在,且频率极低。在10个品种中共检测到了ARQ/ARQ、ARQ/ARK、ARR/ARR、ARH/ARH、ARQ/ARR、ARH/ARQ、ARH/ARR、ARK/ARK、ARH/ARK、ARQ/VRQ、ARQ/AHQ、ARQ/AHR和ARH/AHH13种基因型,其中中度易感的ARQ/ARQ基因型频率最高,而抗性最强的ARR/ARR基因型仅存在于巴音布鲁克、策勒黑、中国美利奴、特克赛尔和德国肉用美利奴羊,且频率较低。文章首次在中国美利奴羊上发现了易感性很强的VRQ/ARQ基因型。上述结果提示新疆的主要绵羊品种对痒病的抗性较弱。     

Interaction of Peptide Aptamers with Prion Protein Central Domain Promotes α-Cleavage of PrP<Superscript>C</Superscript>

Prion diseases are infectious and fatal neurodegenerative diseases affecting humans and animals. Transmission is possible within and between species with zoonotic potential. Currently, no prophylaxis or treatment exists. Prions are composed of the misfolded isoform PrP^Sc of the cellular prion protein PrP^C. Expression of PrP^C is a prerequisite for prion infection, and conformational conversion of PrP^C is induced upon its direct interaction with PrP^Sc. Inhibition of this interaction can abrogate prion propagation, and we have previously established peptide aptamers (PAs) binding to PrP^C as new anti-prion compounds. Here, we mapped the interaction site of PA8 in PrP and modeled the complex in silico to design targeted mutations in PA8 which presumably enhance binding properties. Using these PA8 variants, we could improve PA-mediated inhibition of PrP^Sc replication and de novo infection of neuronal cells. Furthermore, we demonstrate that binding of PA8 and its variants increases PrP^C α-cleavage and interferes with its internalization. This gives rise to high levels of the membrane-anchored PrP-C1 fragment, a transdominant negative inhibitor of prion replication. PA8 and its variants interact with PrP^C at its central and most highly conserved domain, a region which is crucial for prion conversion and facilitates toxic signaling of Aβ oligomers characteristic for Alzheimer’s disease. Our strategy allows for the first time to induce α-cleavage, which occurs within this central domain, independent of targeting the responsible protease. Therefore, interaction of PAs with PrP^C and enhancement of α-cleavage represent mechanisms that can be beneficial for the treatment of prion and other neurodegenerative diseases.     

Separate mechanisms act concurrently to shed and release the prion protein from the cell

The cellular prion protein (PrP^C) is attached to the cell membrane via its glycosylphosphatidylinositol (GPI)-anchor and is constitutively shed into the extracellular space. Here, three different mechanisms are presented that concurrently shed PrP^C from the cell. The fast α-cleavage released a N-terminal fragment (N1) into the medium and the extreme C-terminal cleavage shed soluble full-length (FL-S) PrP and C-terminally cleaved (C1-S) fragments outside the cell. Also, a slow exosomal release of full-length (FL) and C1-fragment (C1) was demonstrated. The three separate mechanisms acting simultaneously, but with different kinetics, have to be taken into consideration when elucidating functional roles of PrP^C and also when processing of PrP^C is considered as a target for intervention in prion diseases. Further, in this study it was shown that metalloprotease inhibitors affected the extreme C-terminal cleavage and shedding of PrP^C. The metalloprotease inhibitors did not influence the α-cleavage or the exosomal release. Taken together, these results are important for understanding the different mechanisms acting in parallel in the shedding and cleavage of PrP^C.     

PrP overdrive

Knockout of the cellular prion protein (PrP^C) in mice is tolerated, as is complete elimination of the protein’s N-terminal domain. However, deletion of select short segments between the N- and C-terminal domains is lethal. How can one reconcile this apparent paradox? Research over the last few years demonstrates that PrP^C undergoes α-cleavage in the vicinity of residue 109 (mouse sequence) to release the bioactive N1 and C1 fragments. In biophysical studies, we recently characterized the action of relevant members of the ADAM (A Disintegrin And Metalloproteinase) enzyme family (ADAM8, 10, and 17) and found that they all produce α-cleavage, but at 3 distinct cleavage sites, with proteolytic efficiency modulated by the physiologic metals copper and zinc. Remarkably, the shortest lethal deletion segment in PrP^C fully encompasses the three α-cleavage sites. Analysis of all reported PrP^C deletion mutants suggests that elimination of α-cleavage, coupled with retention of the protein’s N-terminal residues, segments 23–31 and longer, confers the lethal phenotype. Interestingly, these N-terminal residues are implicated in the activation of several membrane proteins, including synaptic glutamate receptors. We propose that α-cleavage is a general mechanism essential for downregulating PrP^C’s intrinsic activity, and that blockage of proteolysis leads to constitutively active PrP^C and consequent dyshomeostasis.     

Minimum Effective Dose of Cattle and Sheep BSE for Oral Sheep Infection

The minimum dose required to cause infection of Romney and Suffolk sheep of the ARQ/ARQ or ARQ/ARR prion protein gene genotypes following oral inoculation with Romney or Suffolk a sheep Bovine spongiform encephalopathy (BSE)-derived or cattle BSE-derived agent was investigated using doses ranging from 0.0005g to 5g. ARQ/ARQ sheep which were methionine (M) / threonine (T) heterozygous or T/T homozygous at codon 112 of the Prnp gene, dosed ARQ/ARR sheep and undosed controls did not show any evidence of infection. Within groups of susceptible sheep, the minimum effective oral dose of BSE was found to be 0.05g, with higher attack rates following inoculation with the 5g dose. Surprisingly, this study found no effect of dose on survival time suggesting a possible lack of homogeneity within the inoculum. All clinical BSE cases showed PrP^d accumulation in brain; however, following cattle BSE inoculation, LRS involvement within Romney recipients was found to be significantly lower than within the Suffolk sheep inoculated group which is in agreement with previous reports.     

��-Cleavage of cellular prion protein

The cellular prion protein (PrP^C) is subjected to various processing under physiological and pathological conditions, of which the α-cleavage within the central hydrophobic domain not only disrupts a region critical for both PrP toxicity and PrP^C to PrP^Sc conversion but also produces the N1 fragment that is neuroprotective and the C1 fragment that enhances the pro-apoptotic effect of staurosporine in one report and inhibits prion in another. The proteases responsible for the α-cleavage of PrP^C are controversial. The effect of ADAM10, ADAM17, and ADAM9 on N1 secretion clearly indicates their involvement in the α-cleavage of PrP^C, but there has been no report of direct PrP^C α-cleavage activity with any of the three ADAMs in a purified protein form. We demonstrated that, in muscle cells, ADAM8 is the primary protease for the α-cleavage of PrP^C, but another unidentified protease(s) must also play a minor role. We also found that PrP^C regulates ADAM8 expression, suggesting that a close examination on the relationships between PrP^C and its processing enzymes may reveal novel roles and underlying mechanisms for PrP^C in non-prion diseases such as asthma and cancer.     

Homodimerization as a molecular switch between low and high efficiency PrPC cell surface delivery and neuroprotective activity

PrP^C is associated with a variety of functions, and its ability to interact with a multitude of partners, including itself, may largely explain PrP multifunctionality and the lack of consensus on the genuine physiological function of the protein in vivo. In contrast, there is a consensus in the literature that alterations in PrP^C trafficking and intracellular retention result in neuronal degeneration. In addition, a proteolytic modification in the late secretory pathway termed the α-cleavage induces the secretion of PrPN1, a PrP^C-derived metabolite with fascinating neuroprotective activity against toxic oligomeric Aβ molecules implicated in Alzheimer disease. Thus, studies focusing on understanding the regulation of PrP^C trafficking to the cell surface and the modulation of α-cleavage are essential. The objective of this commentary is to highlight recent evidences that PrP^C homodimerization stimulates trafficking of the protein to the cell surface and results in high levels of PrPN1 secretion. We also discuss a hypothetical model for these results and comment on future challenges and opportunities.     

Taking advantage of physiological proteolytic processing of the prion protein for a therapeutic perspective in prion and Alzheimer diseases

Prion and Alzheimer diseases are fatal neurodegenerative diseases caused by misfolding and aggregation of the cellular prion protein (PrP^C) and the β-amyloid peptide, respectively. Soluble oligomeric species rather than large aggregates are now believed to be neurotoxic. PrP^C undergoes three proteolytic cleavages as part of its natural life cycle, α-cleavage, β-cleavage, and ectodomain shedding. Recent evidences demonstrate that the resulting secreted PrP^C molecules might represent natural inhibitors against soluble toxic species. In this mini-review, we summarize recent observations suggesting the potential benefit of using PrP^C-derived molecules as therapeutic agents in prion and Alzheimer diseases.     

Association between the prion protein genotype and animal performance traits in a large multibreed sheep population

Genetic susceptibility to scrapie, a fatal disease of sheep and goats, is modulated by polymorphisms in the prion protein (PrP). Neither the frequency of the PrP genotypes nor their association with animal performance has been investigated in a large multibreed Irish sheep population. Scrapie genotypes were available on 16 416 animals; the breeds represented included purebred Belclare (733), Charollais (333), Suffolk (739), Texel (1 857), Vendeen (191), and crossbreds (12 563). Performance data on lambing, lamb and ewe performance as well as health traits were available. The association between alternative approaches of describing the PrP genotype (i.e. 15 individually called PrP genotypes, five genotype classes representing susceptibility to scrapie, or number of ARR haplotypes) and animal performance were quantified using animal linear mixed models. All 15 of the possible scrapie genotypes were detected, although the frequency differed by breed. The frequency of the five PrP haplotypes in the entire population were 0.70 (ARR), 0.15 (ARQ), 0.11 (ARH), 0.02 (AHQ) and 0.01 (VRQ); the most susceptible haplotype (VRQ) was only detected in purebred Texels and crossbreds. No association was detected between the PrP genotype of either the animal or dam and any of the lambing traits (i.e. lambing difficulty score, perinatal mortality and birth weight). With the exception of ultrasound muscle depth, no association between the PrP genotype and any of the lamb performance traits (i.e. lamb BW and carcass) was observed. Lambs carrying the category four PrP genotype (i.e. ARR/VRQ) had 1.20 (SE = 0.45) mm, 1.38 (SE = 0.12) mm, 1.47 (S = 0.25) mm shallower ultrasound muscle depth relative to lambs of the less susceptible scrapie categories of 1, 2, 3, respectively (P < 0.05). Nonetheless, no association between PrP genotype and lamb carcass conformation, the ultimate end goal of producers, was detected. Ewe litter size, body condition score or lameness did not differ by PrP genotype of the ewe (P > 0.05). For ewe mature BW, ARH/VRQ ewes differed from most other ewe PrP genotypes and were, on average, 3.79 (SE = 1.66) kg heavier than ARR/ARR genotype ewes. Lamb dag score differed by dam PrP genotype (P < 0.05), although the differences were small. Results from this study show that scrapie is segregating within the Irish sheep population, but the PrP genotype was not associated with most traits investigated and, where associations were detected, the biological significance was minimal. This suggests minimal impact of selection on PrP genotype on performance, at least for the traits investigated in the present study.     

Separate mechanisms act concurrently to shed and release the prion protein from the cell

The cellular prion protein (PrP^C) is attached to the cell membrane via its glycosylphosphatidylinositol (GPI)-anchor and is constitutively shed into the extracellular space. Here, three different mechanisms are presented that concurrently shed PrP^C from the cell. The fast α-cleavage released a N-terminal fragment (N1) into the medium and the extreme C-terminal cleavage shed soluble full-length (FL-S) PrP and C-terminally cleaved (C1-S) fragments outside the cell. Also, a slow exosomal release of full-length (FL) and C1-fragment (C1) was demonstrated. The three separate mechanisms acting simultaneously, but with different kinetics, have to be taken into consideration when elucidating functional roles of PrP^C and also when processing of PrP^C is considered as a target for intervention in prion diseases. Further, in this study it was shown that metalloprotease inhibitors affected the extreme C-terminal cleavage and shedding of PrP^C. The metalloprotease inhibitors did not influence the α-cleavage or the exosomal release. Taken together, these results are important for understanding the different mechanisms acting in parallel in the shedding and cleavage of PrP^C.