Diagnosis in Scrapie: Conventional Methods and New Biomarkers.

Scrapie, a naturally occurring prion disease affecting goats and sheep, comprises classical and atypical forms, with classical scrapie being the archetype of transmissible spongiform encephalopathies. This review explores the challenges of scrapie diagnosis and the utility of various biomarkers and their potential implications for human prion diseases. Understanding these biomarkers in the context of scrapie may enable earlier prion disease diagnosis in humans, which is crucial for effective intervention. Research on scrapie biomarkers bridges the gap between veterinary and human medicine, offering hope for the early detection and improved management of prion diseases.

Detection of classical BSE prions in asymptomatic cows after inoculation with atypical/Nor98 scrapie.

The emergence of bovine spongiform encephalopathy (BSE) prions from atypical scrapie has been recently observed upon experimental transmission to rodent and swine models. This study aimed to assess whether the inoculation of atypical scrapie could induce BSE-like disease in cattle. Four calves were intracerebrally challenged with atypical scrapie. Animals were euthanized without clinical signs of prion disease and tested negative for PrPSc accumulation by immunohistochemistry and western blotting. However, an emergence of BSE-like prion seeding activity was detected during in vitro propagation of brain samples from the inoculated animals. These findings suggest that atypical scrapie may represent a potential source of BSE infection in cattle.

Susceptibility of Ovine Bone Marrow-Derived Mesenchymal Stem Cell Spheroids to Scrapie Prion Infection.

In neurodegenerative diseases, including prion diseases, cellular in vitro models appear as fundamental tools for the study of pathogenic mechanisms and potential therapeutic compounds. Two-dimensional (2D) monolayer cell culture systems are the most used cell-based assays, but these platforms are not able to reproduce the microenvironment of in vivo cells. This limitation can be surpassed using three-dimensional (3D) culture systems such as spheroids that more effectively mimic in vivo cell interactions. Herein, we evaluated the effect of scrapie prion infection in monolayer-cultured ovine bone marrow-derived mesenchymal stem cells (oBM-MSCs) and oBM-MSC-derived spheroids in growth and neurogenic conditions, analyzing their cell viability and their ability to maintain prion infection. An MTT assay was performed in oBM-MSCs and spheroids subjected to three conditions: inoculated with brain homogenate from scrapie-infected sheep, inoculated with brain homogenate from healthy sheep, and non-inoculated controls. The 3D conditions improved the cell viability in most cases, although in scrapie-infected spheroids in growth conditions, a decrease in cell viability was observed. The levels of pathological prion protein (PrPSc) in scrapie-infected oBM-MSCs and spheroids were measured by ELISA. In neurogenic conditions, monolayer cells and spheroids maintained the levels of PrPSc over time. In growth conditions, however, oBM-MSCs showed decreasing levels of PrPSc throughout time, whereas spheroids were able to maintain stable PrPSc levels. The presence of PrPSc in spheroids was also confirmed by immunocytochemistry. Altogether, these results show that a 3D culture microenvironment improves the permissiveness of oBM-MSCs to scrapie infection in growth conditions and maintains the infection ability in neurogenic conditions, making this model of potential use for prion studies.

Exploring differentially expressed genes in hypothalamic, pars tuberalis and pineal gland transcriptomes in different sexual behavior phenotypes in rams using RNA-Seq.

Reproductive seasonality is a limiting factor in sheep production. Sexual behavior is a key element in reproductive efficiency, and this function is regulated by the hypothalamus-pituitary-gonadal (HPG) axis. To understand the mechanisms of sexual behavior, transcriptomic sequencing technology was used to identify differentially expressed genes (DEGs) in the hypothalamus (HT), pars tuberalis (PT) and pineal gland (PG) in Rasa Aragonesa rams with different sexual behavior. Bioinformatics analysis of the 16,401 identified genes by RNA-Seq revealed 103 and 12 DEGs in the HT and the PG, respectively, at a false discovery rate (FDR) of 5% with an absolute value of expression ≥ 1 (log2FC). However, no DEGs were found in the PT. Functional annotation and pathway enrichment analysis showed that DEGs of HT were enriched mainly in neuroactive ligand-receptor interactions and signaling pathways, including notable candidate genes such as MTNR1A, CHRNA2, FSHB, LHB, GNRHR, AVP, PRL, PDYN, CGA, GABRD, and TSHB, which play a crucial role in sexual behavior. The GnRH and cAMP signaling pathways were also highlighted. In addition, gene set enrichment analysis (GSEA) identified potential pathways, dominated mainly by biological process category, that could be responsible for the differences in sexual behavior observed in rams. The intracellular protein transport and pattern specification process were enriched within the PT and the transcription factor binding and protein ubiquitination pathways for the PG. Thus, these pathways together may play an important role in the regulation of the sexual behavior in Rasa Aragonesa rams through the hypothalamic-pituitary-gonadal axis. The validation of 5 DEGs using reverse transcription quantitative polymerase chain reaction (RT-qPCR) showed expression patterns like the found with RNA-Seq. Overall, these results contribute to understanding the genomic basis of sexual behavior in rams. Our study demonstrates that multiple networks and pathways orchestrate sexual behavior in sheep.

Male sexual behavior is a key factor in reproduction, especially in seasonal breeders such as sheep. The identification of differentially expressed genes (DEGs) in brain regions involved in male reproduction and sexual behavior between rams with different sexual activity by RNA high-throughput sequencing can provide useful information to the sheep meat industry. This work aimed to determine the possible molecular mechanisms underlying the sexual behavior of Rasa Aragonesa rams by investigating transcriptional changes in the hypothalamus (HT), pars tuberalis (PT) and pineal gland (PG) between active (A) and nonactive (NA) rams. Comparative analysis revealed 103 and 12 DEGs between the A vs. NA comparison in the HT and the PG, respectively, but no DEGs were found in the PT. Gene ontology (GO) enrichment analysis of DEGs in HT samples revealed significant pathways, associated mainly with neuroactive ligand-receptor interactions, and the GnRH and cAMP signaling pathways. Furthermore, gene set enrichment analysis (GSEA) detected many overrepresented pathways related to sexual behavior via an interaction network within the hypothalamic-pituitary-gonadal axis. These data will be helpful for further investigations to look for mutations or functional single nucleotide polymorphisms (SNPs) that may be used for genetic assisted selection to improve sexual behavior in sheep.

Peripheral neuropathy caused by fenugreek (Trigonella foenum-graecum) straw intoxication in cattle and experimental reproduction in sheep and goats.

Trigonella foenum-graecum (fenugreek) is a legume widely used as a food supplement in humans and less frequently in ruminants. Toxicity has been described sporadically in ruminants grazing mature fenugreek plants or stubble; however, the pathological features are unclear. This report describes a natural outbreak of intoxication in cattle fed fenugreek straw and the experimental reproduction using 8 sheep and 8 goats. Affected cattle presented clinical signs approximately 1 month after consuming the straw and 100 of 400 cattle (25%) were affected, of which 60 of 100 (60%) died or were euthanized. Clinical signs were characterized by proprioceptive positioning defects with abnormal postures and weakness of hindlimbs. Forelimbs were also affected in severely affected animals, and cattle became recumbent. Locomotion was characterized by trembling, and some cattle showed high-stepping movements of their forelimbs and knuckled over in their fetlocks. Experimental intoxication induced clinical signs only in sheep and were similar to cattle, although with signs starting in the forelegs. Gross and microscopic lesions were similar in spontaneous and experimental intoxications. Macroscopic changes corresponded with muscular hemorrhages and edema, mainly surrounding the peripheral nerves. Microscopic examination only demonstrated lesions in the distal peripheral nerves, which included edema, hemorrhages, and Wallerian degeneration. Neurofilament immunohistochemistry revealed altered axon labeling and S100 showed a decrease in myelin intensity and loss of its typical compact arrangement around axons. Biochemical and hematological abnormalities included elevated levels of muscle and liver enzymes and thrombocytopenia. These findings indicate that fenugreek straw induces peripheral neuropathy in cattle and sheep, but not in goats.

Characterization of the pars tuberalis and hypothalamus transcriptome in female sheep under different reproductive stages.

For understanding the molecular events underlying the follicular (F) and luteal (L) phases of estrous cycle, and anestrous (A) phase, the pars tuberalis (PT), and hypothalamus (HT) transcriptomes of 21 ewes were studied. In HT, 72 and 3 differential expression genes (DEGs) were found when comparing F vs. A and L vs. A, respectively. In PT, 6 and 4 DEGs were found in F vs. A and L vs. A comparisons, respectively. Enrichment analysis for DEGs between the F and A phases in the HT revealed significant clusters, mainly associated with actin-binding, and cytoskeleton, that are related to neural plasticity modulated by gonadal steroid hormones, as well as with oxytocin signaling. We found that DEGs in PT had higher differences in expression levels than those found in HT. In this sense, the ITLN was highly upregulated in the F and L vs. A phases, being MRPL57 and IRX4 highly downregulated in L vs. A comparison. The DDC gene in PT, related to LH regulation, was upregulated in the F phase. The gene set enrichment analysis (GSEA) revealed multiple pathways related to neurotransmission and neuronal plasticity. Our study reveals new candidate genes involved in the reproductive stages' transitions in seasonal sheep.

Bona fide atypical scrapie faithfully reproduced for the first time in a rodent model.

Atypical Scrapie, which is not linked to epidemics, is assumed to be an idiopathic spontaneous prion disease in small ruminants. Therefore, its occurrence is unlikely to be controlled through selective breeding or other strategies as it is done for classical scrapie outbreaks. Its spontaneous nature and its sporadic incidence worldwide is reminiscent of the incidence of idiopathic spontaneous prion diseases in humans, which account for more than 85% of the cases in humans. Hence, developing animal models that consistently reproduce this phenomenon of spontaneous PrP misfolding, is of importance to study the pathobiology of idiopathic spontaneous prion disorders. Transgenic mice overexpressing sheep PrPC with I112 polymorphism (TgShI112, 1-2 × PrP levels compared to sheep brain) manifest clinical signs of a spongiform encephalopathy spontaneously as early as 380 days of age. The brains of these animals show the neuropathological hallmarks of prion disease and biochemical analyses of the misfolded prion protein show a ladder-like PrPres pattern with a predominant 7-10 kDa band. Brain homogenates from spontaneously diseased transgenic mice were inoculated in several models to assess their transmissibility and characterize the prion strain generated: TgShI112 (ovine I112 ARQ PrPC), Tg338 (ovine VRQ PrPC), Tg501 (ovine ARQ PrPC), Tg340 (human M129 PrPC), Tg361 (human V129 PrPC), TgVole (bank vole I109 PrPC), bank vole (I109I PrPC), and sheep (AHQ/ARR and AHQ/AHQ churra-tensina breeds). Our analysis of the results of these bioassays concludes that the strain generated in this model is indistinguishable to that causing atypical scrapie (Nor98). Thus, we present the first faithful model for a bona fide, transmissible, ovine, atypical scrapie prion disease.

Lesiones cutáneas por síndrome de disfunción multiorgánica: caso clínico / Skin injuries associated with multiple organ dysfunction syndrome: clinical case

Se presenta el caso de una mujer de 95 años, derivada a urgencias por presentar de forma súbita lesiones necróticas parcheadas en cuero cabelludo, que tras realización de analítica y pruebas complementarias se identifica como un cuadro de sepsis de origen multifactorial. Posteriormente, se realiza preparación del lecho de las heridas con desbridamiento cortante con bisturí, aplicando posteriormente colagenasa e hidrogel para continuar con desbridamiento enzimático y autolítico. La paciente fallece a las 4 h tras realizar el desbridamiento. Dado el cuadro de sepsis de origen multifactorial, muy probablemente se trate de lesiones cutáneas por compromiso vital grave, y dentro de ellas de lesiones cutáneas por síndrome de disfunción multiorgánica, que se puede confundir con lesiones por presión, pero dada la localización de las lesiones y el cuadro de sepsis de origen multifactorial, se descarta esta etiología. Los pacientes que sufren este tipo de lesiones tienen una condición clínica irreversible (AU)

A 95-year-old woman, brought to emergency department who suddenly present patchy necrotic skin injuries on the scalp, after performing laboratory tests and complementary tests, is associated with sepsis of multifactorial origin. Subsequently, the wound bed was prepared with sharp debridement with a scalpel, applying collagenase and hydrogel to continue with enzymatic and autolytic debridement. The patient died 4 h after debridement. Given the picture of sepsis of multifactorial origin, it is very likely to be skin injuries associated with serious life threatening situations, and within them skin injuries associated with multiple organ dysfunction syndrome, which could be confused with pressure skin injuries, but given the location of skin injuries and sepsis of multifactorial origin, this etiology is ruled out. Patients who suffer this type of skin injuries have an irreversible clinical condition (AU)

Glycans are not necessary to maintain the pathobiological features of bovine spongiform encephalopathy.

The role of the glycosylation status of PrPC in the conversion to its pathological counterpart and on cross-species transmission of prion strains has been widely discussed. Here, we assessed the effect on strain characteristics of bovine spongiform encephalopathy (BSE) isolates with different transmission histories upon propagation on a model expressing a non-glycosylated human PrPC. Bovine, ovine and porcine-passaged BSE, and variant Creutzfeldt-Jakob disease (vCJD) isolates were used as seeds/inocula in both in vitro and in vivo propagation assays using the non-glycosylated human PrPC-expressing mouse model (TgNN6h). After protein misfolding cyclic amplification (PMCA), all isolates maintained the biochemical characteristics of BSE. On bioassay, all PMCA-propagated BSE prions were readily transmitted to TgNN6h mice, in agreement with our previous in vitro results. TgNN6h mice reproduced the characteristic neuropathological and biochemical hallmarks of BSE, suggesting that the absence of glycans did not alter the pathobiological features of BSE prions. Moreover, back-passage of TgNN6h-adapted BSE prions to BoTg110 mice recovered the full BSE phenotype, confirming that the glycosylation of human PrPC is not essential for the preservation of the human transmission barrier for BSE prions or for the maintenance of BSE strain properties.

Neurogranin and Neurofilament Light Chain as Preclinical Biomarkers in Scrapie.

Prion diseases are diagnosed in the symptomatic stage, when the neuronal damage is spread throughout the central nervous system (CNS). The assessment of biological features that allow the detection of asymptomatic cases is needed, and, in this context, scrapie, where pre-symptomatic infected animals can be detected through rectal biopsy, becomes a good study model. Neurogranin (Ng) and neurofilament light chain (NfL) are proteins that reflect synaptic and axonal damage and have been studied as cerebrospinal fluid (CSF) biomarkers in different neurodegenerative disorders. In this study, we evaluated Ng and NfL both at the protein and transcript levels in the CNS of preclinical and clinical scrapie-affected sheep compared with healthy controls and assessed their levels in ovine CSF. The correlation between these proteins and the main neuropathological events in prion diseases, PrPSc deposition and spongiosis, was also assessed. The results show a decrease in Ng and NfL at the protein and gene expression levels as the disease progresses, and significant changes between the control and preclinical animals. On the contrary, the CSF levels of NfL increased throughout the progression of the disease. Negative correlations between neuropathological markers of prion disease and the concentration of the studied proteins were also found. Although further research is needed, these results suggest that Ng and NfL could act as biomarkers for neurodegeneration onset and intensity in preclinical cases of scrapie.

Classical BSE prions emerge from asymptomatic pigs challenged with atypical/Nor98 scrapie.

Pigs are susceptible to infection with the classical bovine spongiform encephalopathy (C-BSE) agent following experimental inoculation, and PrPSc accumulation was detected in porcine tissues after the inoculation of certain scrapie and chronic wasting disease isolates. However, a robust transmission barrier has been described in this species and, although they were exposed to C-BSE agent in many European countries, no cases of natural transmissible spongiform encephalopathies (TSE) infections have been reported in pigs. Transmission of atypical scrapie to bovinized mice resulted in the emergence of C-BSE prions. Here, we conducted a study to determine if pigs are susceptible to atypical scrapie. To this end, 12, 8-9-month-old minipigs were intracerebrally inoculated with two atypical scrapie sources. Animals were euthanized between 22- and 72-months post inoculation without clinical signs of TSE. All pigs tested negative for PrPSc accumulation by enzyme immunoassay, immunohistochemistry, western blotting and bioassay in porcine PrP mice. Surprisingly, in vitro protein misfolding cyclic amplification demonstrated the presence of C-BSE prions in different brain areas from seven pigs inoculated with both atypical scrapie isolates. Our results suggest that pigs exposed to atypical scrapie prions could become a reservoir for C-BSE and corroborate that C-BSE prions emerge during interspecies passage of atypical scrapie.

Effect of Scrapie Prion Infection in Ovine Bone Marrow-Derived Mesenchymal Stem Cells and Ovine Mesenchymal Stem Cell-Derived Neurons.

Scrapie is a prion disease affecting sheep and goats and it is considered a prototype of transmissible spongiform encephalopathies (TSEs). Mesenchymal stem cells (MSCs) have been proposed as candidates for developing in vitro models of prion diseases. Murine MSCs are able to propagate prions after previous mouse-adaptation of prion strains and, although ovine MSCs express the cellular prion protein (PrPC), their susceptibility to prion infection has never been investigated. Here, we analyze the potential of ovine bone marrow-derived MSCs (oBM-MSCs), in growth and neurogenic conditions, to be infected by natural scrapie and propagate prion particles (PrPSc) in vitro, as well as the effect of this infection on cell viability and proliferation. Cultures were kept for 48-72 h in contact with homogenates of central nervous system (CNS) samples from scrapie or control sheep. In growth conditions, oBM-MSCs initially maintained detectable levels of PrPSc post-inoculation, as determined by Western blotting and ELISA. However, the PrPSc signal weakened and was lost over time. oBM-MSCs infected with scrapie displayed lower cell doubling and higher doubling times than those infected with control inocula. On the other hand, in neurogenic conditions, oBM-MSCs not only maintained detectable levels of PrPSc post-inoculation, as determined by ELISA, but this PrPSc signal also increased progressively over time. Finally, inoculation with CNS extracts seems to induce the proliferation of oBM-MSCs in both growth and neurogenic conditions. Our results suggest that oBM-MSCs respond to prion infection by decreasing their proliferation capacity and thus might not be permissive to prion replication, whereas ovine MSC-derived neuron-like cells seem to maintain and replicate PrPSc.

Classical and Atypical Scrapie in Sheep and Goats. Review on the Etiology, Genetic Factors, Pathogenesis, Diagnosis, and Control Measures of Both Diseases.

Prion diseases, such as scrapie, are neurodegenerative diseases with a fatal outcome, caused by a conformational change of the cellular prion protein (PrPC), originating with the pathogenic form (PrPSc). Classical scrapie in small ruminants is the paradigm of prion diseases, as it was the first transmissible spongiform encephalopathy (TSE) described and is the most studied. It is necessary to understand the etiological properties, the relevance of the transmission pathways, the infectivity of the tissues, and how we can improve the detection of the prion protein to encourage detection of the disease. The aim of this review is to perform an overview of classical and atypical scrapie disease in sheep and goats, detailing those special issues of the disease, such as genetic factors, diagnostic procedures, and surveillance approaches carried out in the European Union with the objective of controlling the dissemination of scrapie disease.

Neuroimmune Response Mediated by Cytokines in Natural Scrapie after Chronic Dexamethasone Treatment.

The actual role of prion protein-induced glial activation and subsequent cytokine secretion during prion diseases is still incompletely understood. The overall aim of this study is to assess the effect of an anti-inflammatory treatment with dexamethasone on different cytokines released by neuroglial cells that are potentially related to neuroinflammation in natural scrapie. This study emphasizes the complex interactions existent among several pleiotropic neuromodulator peptides and provides a global approach to clarify neuroinflammatory processes in prion diseases. Additionally, an impairment of communication between microglial and astroglial populations mediated by cytokines, mainly IL-1, is suggested. The main novelty of this study is that it is the first one assessing in situ neuroinflammatory activity in relation to chronic anti-inflammatory therapy, gaining relevance because it is based on a natural model. The cytokine profile data would suggest the activation of some neurotoxicity-associated route. Consequently, targeting such a pathway might be a new approach to modify the damaging effects of neuroinflammation.

SARS-CoV-2 Outbreak on a Spanish Mink Farm: Epidemiological, Molecular, and Pathological Studies.

Farmed minks have been reported to be highly susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and may represent a risk to humans. In this study, we describe the first outbreak of SARS-CoV-2 occurred on a mink farm in Spain, between June and July 2020, involving 92,700 animals. The outbreak started shortly after some farm workers became seropositive for SARS-CoV-2. Minks showed no clinical signs compatible with SARS-CoV-2 infection throughout the outbreak. Samples from 98 minks were collected for histopathological, serological, and molecular studies. Twenty out of 98 (20.4%) minks were positive by RT-qPCR and 82 out 92 (89%) seroconverted. This finding may reflect a rapid spread of the virus at the farm with most of the animals overcoming the infection. Additionally, SARS-CoV-2 was detected by RT-qPCR in 30% of brain samples from positive minks. Sequencing analysis showed that the mink sequences were not closely related with the other mink SARS-CoV-2 sequences available, and that this mink outbreak has its probable origin in one of the genetic variants that were prevalent in Spain during the first COVID-19 epidemic wave. Histological studies revealed bronchointerstitial pneumonia in some animals. Immunostaining of viral nucleocapsid was also observed in nasal turbinate tissue. Farmed minks could therefore constitute an important SARS-CoV-2 reservoir, contributing to virus spread among minks and humans. Consequently, continuous surveillance of mink farms is needed.

Neuroimmune Response in Natural Preclinical Scrapie after Dexamethasone Treatment.

A recently published report on chronic dexamethasone treatment for natural scrapie supported the hypothesis of the potential failure of astroglia in the advanced stage of disease. Herein, we aimed to extend the aforementioned study on the effect of this anti-inflammatory therapy to the initial phase of scrapie, with the aim of elucidating the natural neuroinflammatory process occurring in this neurodegenerative disorder. The administration of this glucocorticoid resulted in an outstanding reduction in vacuolation and aberrant protein deposition (nearly null), and an increase in glial activation. Furthermore, evident suppression of IL-1R and IL-6 and the exacerbation of IL-1α, IL-2R, IL-10R and IFNγR were also demonstrated. Consequently, the early stage of the disease is characterized by an intact neuroglial response similar to that of healthy individuals attempting to re-establish homeostasis. A complex network of neuroinflammatory markers is involved from the very early stages of this prion disease, which probably becomes impaired in the more advanced stages. The in vivo animal model used herein provides essential observations on the pathogenesis of natural scrapie, as well as the possibility of establishing neuroglia as potential target cells for anti-inflammatory therapy.

Assessment of Glial Activation Response in the Progress of Natural Scrapie after Chronic Dexamethasone Treatment.

Neuroinflammation has been correlated with the progress of neurodegeneration in many neuropathologies. Although glial cells have traditionally been considered to be protective, the concept of them as neurotoxic cells has recently emerged. Thus, a major unsolved question is the exact role of astroglia and microglia in neurodegenerative disorders. On the other hand, it is well known that glucocorticoids are the first choice to regulate inflammation and, consequently, neuroglial inflammatory activity. The objective of this study was to determine how chronic dexamethasone treatment influences the host immune response and to characterize the beneficial or detrimental role of glial cells. To date, this has not been examined using a natural neurodegenerative model of scrapie. With this aim, immunohistochemical expression of glial markers, prion protein accumulation, histopathological lesions and clinical evolution were compared with those in a control group. The results demonstrated how the complex interaction between glial populations failed to compensate for brain damage in natural conditions, emphasizing the need for using natural models. Additionally, the data showed that modulation of neuroinflammation by anti-inflammatory drugs might become a research focus as a potential therapeutic target for prion diseases, similar to that considered previously for other neurodegenerative disorders classified as prion-like diseases.

Mixtures of prion substrains in natural scrapie cases revealed by ovinised murine models.

Phenotypic variability in prion diseases, such as scrapie, is associated to the existence of prion strains, which are different pathogenic prion protein (PrPSc) conformations with distinct pathobiological properties. To faithfully study scrapie strain variability in natural sheep isolates, transgenic mice expressing sheep cellular prion protein (PrPC) are used. In this study, we used two of such models to bioassay 20 scrapie isolates from the Spain-France-Andorra transboundary territory. Animals were intracerebrally inoculated and survival periods, proteinase K-resistant PrP (PrPres) banding patterns, lesion profiles and PrPSc distribution were studied. Inocula showed a remarkable homogeneity on banding patterns, all of them but one showing 19-kDa PrPres. However, a number of isolates caused accumulation of 21-kDa PrPres in TgShp XI. A different subgroup of isolates caused long survival periods and presence of 21-kDa PrPres in Tg338 mice. It seemed that one major 19-kDa prion isoform and two distinct 21-kDa variants coexisted in source inocula, and that they could be separated by bioassay in each transgenic model. The reason why each model favours a specific component of the mixture is unknown, although PrPC expression level may play a role. Our results indicate that coinfection with more than one substrain is more frequent than infection with a single component.

Autophagy impairment in highly prion-affected brain areas of sheep experimentally infected with atypical scrapie.

Autophagy is a critical physiologic process contributing to the maintenance of cell homeostasis. Autophagy dysfunction has been directly linked to a growing number of neurodegenerative disorders, including prion diseases. However, little is known about the molecular mechanisms underlying autophagic failure and its connection with prion neuropathology. In a previous work we described alterations of this process in the central nervous system (CNS) of sheep naturally infected with classical scrapie, although specific neuronal populations such as Purkinje cells seemed to display an autophagy-related neuroprotective effect against prion toxicity. As atypical scrapie displays a lesion pattern different to the one observed in the classical form, using immunohistochemical analyses, we further investigated herein the role of autophagy in the CNS of sheep experimentally infected with atypical scrapie prions. While ATG5 protein showed a similar distribution in atypical scrapie to that observed in the classical form, expression of LC3-B and LC3-A did not change in any brain region. However, p62, a marker of impaired autophagy, was overexpressed in the most prion-affected areas, including Purkinje cells, which suggests that autophagic activity is deteriorated in the CNS of atypical scrapie and these cells are also susceptible to neurotoxicity and do not exhibit a general defensive mechanism based on autophagy. By comparing data from both clinical scrapie forms, we have demonstrated that autophagy impairment is highly dependent on the neuropathological lesion levels of the brain area analysed and may be implicated in prion neuropathology.

A Single Amino Acid Substitution, Found in Mammals with Low Susceptibility to Prion Diseases, Delays Propagation of Two Prion Strains in Highly Susceptible Transgenic Mouse Models.

Specific variations in the amino acid sequence of prion protein (PrP) are key determinants of susceptibility to prion diseases. We previously showed that an amino acid substitution specific to canids confers resistance to prion diseases when expressed in mice and demonstrated its dominant-negative protective effect against a variety of infectious prion strains of different origins and characteristics. Here, we show that expression of this single amino acid change significantly increases survival time in transgenic mice expressing bank vole cellular prion protein (PrPC), which is inherently prone to misfolding, following inoculation with two distinct prion strains (the CWD-vole strain and an atypical strain of spontaneous origin). This amino acid substitution hinders the propagation of both prion strains, even when expressed in the context of a PrPC uniquely susceptible to a wide range of prion isolates. Non-inoculated mice expressing this substitution experience spontaneous prion formation, but showing an increase in survival time comparable to that observed in mutant mice inoculated with the atypical strain. Our results underscore the importance of this PrP variant in the search for molecules with therapeutic potential against prion diseases.