Demyelinating polyneuropathy in goats lacking prion protein.

Studies in mice with ablation of Prnp, the gene that encodes the cellular prion protein (PrPC ), have led to the hypothesis that PrPC is important for peripheral nerve myelin maintenance. Here, we have used a nontransgenic animal model to put this idea to the test; namely, goats that, due to a naturally occurring nonsense mutation, lack PrPC . Teased nerve fiber preparation revealed a demyelinating pathology in goats without PrPC . Affected nerves were invaded by macrophages and T cells and displayed vacuolated fibers, shrunken axons, and onion bulbs. Peripheral nerve lipid composition was similar in young goats with or without PrPC , but markedly different between corresponding groups of adult goats, reflecting the progressive nature of the neuropathy. This is the first report of a subclinical demyelinating polyneuropathy caused by loss of PrPC function in a nontransgenic mammal.

Goats naturally devoid of PrPC are resistant to scrapie.

Prion diseases are progressive and fatal, neurodegenerative disorders described in humans and animals. According to the "protein-only" hypothesis, the normal host-encoded prion protein (PrPC) is converted into a pathological and infectious form (PrPSc) in these diseases. Transgenic knockout models have shown that PrPC is a prerequisite for the development of prion disease. In Norwegian dairy goats, a mutation (Ter) in the prion protein gene (PRNP) effectively blocks PrPC synthesis. We inoculated 12 goats (4 PRNP+/+, 4 PRNP+/Ter, and 4 PRNPTer/Ter) intracerebrally with goat scrapie prions. The mean incubation time until clinical signs of prion disease was 601 days post-inoculation (dpi) in PRNP+/+ goats and 773 dpi in PRNP+/Ter goats. PrPSc and vacuolation were similarly distributed in the central nervous system (CNS) of both groups and observed in all brain regions and segments of the spinal cord. Generally, accumulation of PrPSc was limited in peripheral organs, but all PRNP+/+ goats and 1 of 4 PRNP+/Ter goats were positive in head lymph nodes. The four PRNPTer/Ter goats remained healthy, without clinical signs of prion disease, and were euthanized 1260 dpi. As expected, no accumulation of PrPSc was observed in the CNS or peripheral tissues of this group, as assessed by immunohistochemistry, enzyme immunoassay, and real-time quaking-induced conversion. Our study shows for the first time that animals devoid of PrPC due to a natural mutation do not propagate prions and are resistant to scrapie. Clinical onset of disease is delayed in heterozygous goats expressing about 50% of PrPC levels.

Cell and context-dependent sorting of neuropathy-associated protein NDRG1 - insights from canine tissues and primary Schwann cell cultures.

BACKGROUND: Mutations in the N-myc downstream-regulated gene 1 (NDRG1) can cause degenerative polyneuropathy in humans, dogs, and rodents. In humans, this motor and sensory neuropathy is known as Charcot-Marie-Tooth disease type 4D, and it is assumed that analogous canine diseases can be used as models for this disease. NDRG1 is also regarded as a metastasis-suppressor in several malignancies. The tissue distribution of NDRG1 has been described in humans and rodents, but this has not been studied in the dog. RESULTS: By immunolabeling and Western blotting, we present a detailed mapping of NDRG1 in dog tissues and primary canine Schwann cell cultures, with particular emphasis on peripheral nerves. High levels of phosphorylated NDRG1 appear in distinct subcellular localizations of the Schwann cells, suggesting signaling-driven rerouting of the protein. In a nerve from an Alaskan malamute homozygous for the disease-causing Gly98Val mutation in NDRG1, this signal was absent. Furthermore, NDRG1 is present in canine epithelial cells, predominantly in the cytosolic compartment, often with basolateral localization. Constitutive expression also occurs in mesenchymal cells, including developing spermatids that are transiently positive for NDRG1. In some cells, NDRG1 localize to centrosomes. CONCLUSIONS: Overall, canine NDRG1 shows a cell and context-dependent localization. Our data from peripheral nerves and primary Schwann cell cultures suggest that the subcellular localization of NDRG1 in Schwann cells is dynamically influenced by signaling events leading to reversible phosphorylation of the protein. We propose that disease-causing mutations in NDRG1 can disrupt signaling in myelinating Schwann cells, causing disturbance in myelin homeostasis and axonal-glial cross talk, thereby precipitating polyneuropathy.

Activation of innate immune genes in caprine blood leukocytes after systemic endotoxin challenge.

BACKGROUND: Sepsis is a serious health problem associated with a range of infectious diseases in animals and humans. Early events of this syndrome can be mimicked by experimental administration of lipopolysaccharides (LPS). Compared with mice, small ruminants and humans are highly sensitive to LPS, making goats valuable in inflammatory models. We performed a longitudinal study in eight Norwegian dairy goats that received LPS (0.1 µg/kg, Escherichia coli O26:B6) intravenously. A control group of five goats received corresponding volumes of sterile saline. Clinical examinations were performed continuously, and blood samples were collected throughout the trial. RESULTS: Characteristic signs of acute sepsis, such as sickness behavior, fever, and leukopenia were observed within 1 h of LPS administration. A high-throughput longitudinal gene expression analysis of circulating leukocytes was performed, and genes associated with the acute phase response, type I interferon signaling, LPS cascade and apoptosis, in addition to cytokines and chemokines were targeted. Pro-inflammatory genes, such as IL1B, CCL3 and IL8, were significantly up-regulated. Interestingly, increased mRNA levels of seven interferon stimulated genes (ISGs) were observed peaking at 2 h, corroborating the increasing evidence that ISGs respond immediately to bacterial endotoxins. A slower response was manifested by four extrahepatic acute phase proteins (APP) (SAA3, HP, LF and LCN2) reaching maximum levels at 5 h. CONCLUSIONS: We report an immediate induction of ISGs in leukocytes in response to LPS supporting a link between the interferon system and defense against bacterial infections. The extrahepatic expression of APPs suggests that leukocytes contribute to synthesis of these proteins at the beginning of a systemic inflammation. Taken together, these findings provide insights into the dynamic regulation of innate immune genes, as well as raising new questions regarding the importance of ISGs and extrahepatic APPs in leukocytes after systemic endotoxin challenge.

The PrP(C) C1 fragment derived from the ovine A136R154R171PRNP allele is highly abundant in sheep brain and inhibits fibrillisation of full-length PrP(C) protein in vitro.

Expression of the cellular prion protein (PrP(C)) is crucial for the development of prion diseases. Resistance to prion diseases can result from reduced availability of the prion protein or from amino acid changes in the prion protein sequence. We propose here that increased production of a natural PrP α-cleavage fragment, C1, is also associated with resistance to disease. We show, in brain tissue, that ARR homozygous sheep, associated with resistance to disease, produced PrP(C) comprised of 25% more C1 fragment than PrP(C) from the disease-susceptible ARQ homozygous and highly susceptible VRQ homozygous animals. Only the C1 fragment derived from the ARR allele inhibits in-vitro fibrillisation of other allelic PrP(C) variants. We propose that the increased α-cleavage of ovine ARR PrP(C) contributes to a dominant negative effect of this polymorphism on disease susceptibility. Furthermore, the significant reduction in PrP(C) ß-cleavage product C2 in sheep of the ARR/ARR genotype compared to ARQ/ARQ and VRQ/VRQ genotypes, may add to the complexity of genetic determinants of prion disease susceptibility.

The Zoonotic Potential of Chronic Wasting Disease-A Review.

Prion diseases are transmissible neurodegenerative disorders that affect humans and ruminant species consumed by humans. Ruminant prion diseases include bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep and goats and chronic wasting disease (CWD) in cervids. In 1996, prions causing BSE were identified as the cause of a new prion disease in humans; variant Creutzfeldt-Jakob disease (vCJD). This sparked a food safety crisis and unprecedented protective measures to reduce human exposure to livestock prions. CWD continues to spread in North America, and now affects free-ranging and/or farmed cervids in 30 US states and four Canadian provinces. The recent discovery in Europe of previously unrecognized CWD strains has further heightened concerns about CWD as a food pathogen. The escalating CWD prevalence in enzootic areas and its appearance in a new species (reindeer) and new geographical locations, increase human exposure and the risk of CWD strain adaptation to humans. No cases of human prion disease caused by CWD have been recorded, and most experimental data suggest that the zoonotic risk of CWD is very low. However, the understanding of these diseases is still incomplete (e.g., origin, transmission properties and ecology), suggesting that precautionary measures should be implemented to minimize human exposure.

Monitoring of chronic wasting disease (CWD) (IV).

The European Commission requested an analysis of the Chronic Wasting Disease (CWD) monitoring programme in Norway, Sweden, Finland, Iceland, Estonia, Latvia, Lithuania and Poland (9 January 2017-28 February 2022). Thirteen cases were detected in reindeer, 15 in moose and 3 in red deer. They showed two phenotypes, distinguished by the presence or absence of detectable disease-associated normal cellular prion protein (PrP) in lymphoreticular tissues. CWD was detected for the first time in Finland, Sweden and in other areas of Norway. In countries where the disease was not detected, the evidence was insufficient to rule out its presence altogether. Where cases were detected, the prevalence was below 1%. The data also suggest that the high-risk target groups for surveillance should be revised, and 'road kill' removed. Data show that, in addition to differences in age and sex, there are differences in the prion protein gene (PRNP) genotypes between positive and negative wild reindeer. A stepwise framework has been proposed with expanded minimum background surveillance to be implemented in European countries with relevant cervid species. Additional surveillance may include ad hoc surveys for four different objectives, specific to countries with/without cases, focusing on parallel testing of obex and lymph nodes from adult cervids in high-risk target groups, sustained over time, using sampling units and a data-driven design prevalence. Criteria for assessing the probability of CWD presence have been outlined, based on the definition of the geographical area, an annual assessment of risk of introduction, sustained minimum background surveillance, training and engagement of stakeholders and a surveillance programme based on data-driven parameters. All positive cases should be genotyped. Sample sizes for negative samples have been proposed to detect and estimate the frequency of PRNP polymorphisms. Double-strand sequencing of the entire PRNP open reading frame should be undertaken for all selected samples, with data collated in a centralised collection system at EU level.

Healthy goats naturally devoid of prion protein.

Prion diseases such as scrapie in small ruminants, bovine spongiform encephalopathy (BSE) in cattle and Creutzfeldt-Jakob disease (CJD) in man, are fatal neurodegenerative disorders. These diseases result from the accumulation of misfolded conformers of the host-encoded prion protein (PrP) in the central nervous system. To date naturally-occurring PrP free animals have not been reported. Here we describe healthy non-transgenic animals, Norwegian Dairy Goats, lacking prion protein due to a nonsense mutation early in the gene. These animals are predicted to be resistant to prion disease and will be valuable for research and for production of prion-free products.

A secretory Golgi bypass route to the apical surface domain of epithelial MDCK cells.

Proteins leave the endoplasmic reticulum (ER) for the plasma membrane via the classical secretory pathway, but routes bypassing the Golgi apparatus have also been observed. Apical and basolateral protein secretion in epithelial Madin-Darby canine kidney (MDCK) cells display differential sensitivity to Brefeldin A (BFA), where low concentrations retard apical transport, while basolateral transport still proceeds through intact Golgi cisternae. We now describe that BFA-mediated retardation of glycoprotein and proteoglycan transport through the Golgi apparatus induces surface transport of molecules lacking Golgi modifications, possessing those acquired in the ER. Low concentrations of BFA induces apical Golgi bypass, while higher concentrations were required to induce basolateral Golgi bypass. Addition of the KDEL ER-retrieval sequence to model protein cores allowed observation of apical Golgi bypass in untreated MDCK cells. Basolateral Golgi bypass was only observed after the addition of BFA or upon cholesterol depletion. Thus, in MDCK cells, an apical Golgi bypass route can transport cargo from pre-Golgi organelles in untreated cells, while the basolateral bypass route is inducible.

Atypical prion diseases in humans and animals.

Although prion diseases, such as Creutzfeldt-Jakob disease (CJD) in humans and scrapie in sheep, have long been recognized, our understanding of their epidemiology and pathogenesis is still in its early stages. Progress is hampered by the lengthy incubation periods and the lack of effective ways of monitoring and characterizing these agents. Protease-resistant conformers of the prion protein (PrP), known as the "scrapie form" (PrP(Sc)), are used as disease markers, and for taxonomic purposes, in correlation with clinical, pathological, and genetic data. In humans, prion diseases can arise sporadically (sCJD) or genetically (gCJD and others), caused by mutations in the PrP-gene (PRNP), or as a foodborne infection, with the agent of bovine spongiform encephalopathy (BSE) causing variant CJD (vCJD). Person-to-person spread of human prion disease has only been known to occur following cannibalism (kuru disease in Papua New Guinea) or through medical or surgical treatment (iatrogenic CJD, iCJD). In contrast, scrapie in small ruminants and chronic wasting disease (CWD) in cervids behave as infectious diseases within these species. Recently, however, so-called atypical forms of prion diseases have been discovered in sheep (atypical/Nor98 scrapie) and in cattle, BSE-H and BSE-L. These maladies resemble sporadic or genetic human prion diseases and might be their animal equivalents. This hypothesis also raises the significant public health question of possible epidemiological links between these diseases and their counterparts in humans.

Impaired NDRG1 functions in Schwann cells cause demyelinating neuropathy in a dog model of Charcot-Marie-Tooth type 4D.

Mutations in the N-myc downstream-regulated gene 1 (NDRG1) cause degenerative polyneuropathy in ways that are poorly understood. We have investigated Alaskan Malamute dogs with neuropathy caused by a missense mutation in NDRG1. In affected animals, nerve levels of NDRG1 protein were reduced by more than 70% (p< 0.03). Nerve fibers were thinly myelinated, loss of large myelinated fibers was pronounced and teased fiber preparations showed both demyelination and remyelination. Inclusions of filamentous material containing actin were present in adaxonal Schwann cell cytoplasm and Schmidt-Lanterman clefts. This condition strongly resembles the human Charcot-Marie-Tooth type 4D. However, the focally folded myelin with adaxonal infoldings segregating the axon found in this study are ultrastructural changes not described in the human disease. Furthermore, lipidomic analysis revealed a profound loss of peripheral nerve lipids. Our data suggest that the low levels of mutant NDRG1 is insufficient to support Schwann cells in maintaining myelin homeostasis.

Salmonid T cells assemble in the thymus, spleen and in novel interbranchial lymphoid tissue.

In modern bony fishes, or teleost fish, the general lack of leucocyte markers has greatly hampered investigations of the anatomy of the immune system and its reactions involved in inflammatory responses. We have previously reported the cloning and sequencing of the salmon CD3 complex, molecules that are specifically expressed in T cells. Here, we generate and validate sera recognizing a peptide sequence of the CD3ε chain. Flow cytometry analysis revealed high numbers of CD3ε(+) or T cells in the thymus, gill and intestine, whereas lower numbers were detected in the head kidney, spleen and peripheral blood leucocytes. Subsequent morphological analysis showed accumulations of T cells in the thymus and spleen and in the newly discovered gill-located interbranchial lymphoid tissue. In the latter, the T cells are embedded in a meshwork of epithelial cells and in the spleen, they cluster in the white pulp surrounding ellipsoids. The anatomical organization of the salmonid thymic cortex and medulla seems to be composed of three layers consisting of a sub-epithelial medulla-like zone, an intermediate cortex-like zone and finally another cortex-like basal zone. Our study in the salmonid thymus reports a previously non-described tissue organization. In the intestinal tract, abundant T cells were found embedded in the epithelium. In non-lymphoid organs, the presence of T cells was limited. The results show that the interbranchial lymphoid tissue is quantitatively a very important site of T cell aggregation, strategically located to facilitate antigen encounter. The interbranchial lymphoid tissue has no resemblance to previously described lymphoid tissues.

Antler cannibalism in reindeer.

Prion diseases constitute a class of invariably fatal and degenerative encephalopathies. Chronic Wasting Disease (CWD) is a contagious prion disease among cervids, which is spreading and causing marked population declines in USA and Canada. The first outbreak of CWD in Europe was discovered in a reindeer population in Norway in 2016. In the worst-case scenario with continental-wide spreading of CWD in Eurasia, an annual harvest of around 4 million cervids is at stake only in Europe, with huge economic and cultural significance. An in situ origin of CWD was suspected, and it appear urgent to identify the likely cause to prevent future emergences. Here, we document the novel phenomenon of extensive antler cannibalism prior to shedding among reindeer in the CWD-infected population. The extent of antler cannibalism increased over the last decades when CWD emerged, and included ingestion of vascularized antlers. Ingestion of tissues from conspecifics is a risk factor for the emergence of prion diseases, where the presence of extensive antler cannibalism opens the intriguing possibility of a 'Kuru-analogue' origin of CWD among the reindeer in Europe. Based on general insight on pathology of prion diseases and strain selection processes, we propose an hypothesis for how contagious CWD may emerge from sporadic CWD under the unique epidemiological conditions we document here. More research is required to document the presence of prions in reindeer antlers, and whether antler cannibalism actually led to a strain selection process and the emergence of a contagious form of CWD from a sporadic form of CWD.

DNA glycosylase Neil2 contributes to genomic responses in the spleen during clinical prion disease.

The DNA glycosylase Neil2 is a member of the base excision repair (BER) family of enzymes, which are important for repair of oxidative DNA damage. Specifically, Neil2 participates in repair of oxidized bases in single-stranded DNA of transcriptionally active genes. Mice with genetic ablation of Neil2 (Neil2-/-) display no overt phenotypes, but an age-dependent accumulation of oxidative DNA damage and increased inflammatory responsiveness. In young mice intra-cerebrally inoculated with prions, vigorous prion propagation starts rapidly in the germinal follicles of the spleen due to inoculum spillover. Here, we compare experimental prion disease in Neil2-/- mice with that in wild-type mice at disease onset and end-stage. Specifically, we investigated disease progression, accumulation of DNA damage, and mitochondrial respiratory complex activity in brain and spleen. We used genome-wide RNA sequencing of the spleen to compare the immune responses to prion propagation between the two groups of mice, at both onset and end-stage prion disease. The Neil2-/- mice deteriorated more rapidly than wild-type mice after onset of clinical signs. Levels of DNA damage in brain increased in both mouse groups, slightly more in the Neil2-/- mice. Transcriptome data from spleen at disease onset were similar between the mouse groups with moderate genomic responses. However, at end-stage a substantial response was evident in the wild-type mice but not in Neil2-/- mice. Our data show that Neil2 counteracts toxic signaling in clinical prion disease, and this is separate from gross pathological manifestations and PrPSc accumulation.

Chronic wasting disease associated with prion protein gene (PRNP) variation in Norwegian wild reindeer (Rangifer tarandus).

The emergence of CWD in Europe in 2016 and the first natural infection in wild reindeer warranted disease management. This led to the testing of 2424 hunted or culled reindeer during 2016-2018, from the infected subpopulation in the Nordfjella mountain range in Southern Norway. To identify any association between PRNP variation and CWD susceptibility, we characterized the open reading frame of the PRNP gene in 19 CWD positive reindeer and in 101 age category- and sex-matched CWD negative controls. Seven variant positions were identified: 6 single nucleotide variants (SNVs) and a 24 base pair (bp) deletion located between nucleotide position 238 and 272, encoding four instead of five octapeptide repeats. With a single exception, all variant positions but one were predicted to be non-synonymous. The synonymous SNV and the deletion are novel in reindeer. Various combinations of the non-synonymous variant positions resulted in the identification of five PRNP alleles (A-E) that structured into 14 genotypes. We identified an increased CWD risk in reindeer carrying two copies of the most common allele, A, coding for serine in position 225 (Ser225) and in those carrying allele A together with the 24 bp deletion.

The prion protein in neuroimmune crosstalk.

The cellular prion protein (PrPC) is a medium-sized glycoprotein, attached to the cell surface by a glycosylphosphatidylinositol anchor. PrPC is encoded by a single-copy gene, PRNP, which is abundantly expressed in the central nervous system and at lower levels in non-neuronal cells, including those of the immune system. Evidence from experimental knockout of PRNP in rodents, goats, and cattle and the occurrence of a nonsense mutation in goat that prevents synthesis of PrPC, have shown that the molecule is non-essential for life. Indeed, no easily recognizable phenotypes are associate with a lack of PrPC, except the potentially advantageous trait that animals without PrPC cannot develop prion disease. This is because, in prion diseases, PrPC converts to a pathogenic "scrapie" conformer, PrPSc, which aggregates and eventually induces neurodegeneration. In addition, endogenous neuronal PrPC serves as a toxic receptor to mediate prion-induced neurotoxicity. Thus, PrPC is an interesting target for treatment of prion diseases. Although loss of PrPC has no discernable effect, alteration of its normal physiological function can have very harmful consequences. It is therefore important to understand cellular processes involving PrPC, and research of this topic has advanced considerably in the past decade. Here, we summarize data that indicate the role of PrPC in modulating immune signaling, with emphasis on neuroimmune crosstalk both under basal conditions and during inflammatory stress.

Stress Resilience of Spermatozoa and Blood Mononuclear Cells without Prion Protein.

The cellular prion protein PrPC is highly expressed in neurons, but also present in non-neuronal tissues, including the testicles and spermatozoa. Most immune cells and their bone marrow precursors also express PrPC. Clearly, this protein operates in highly diverse cellular contexts. Investigations into putative stress-protective roles for PrPC have resulted in an array of functions, such as inhibition of apoptosis, stimulation of anti-oxidant enzymes, scavenging roles, and a role in nuclear DNA repair. We have studied stress resilience of spermatozoa and peripheral blood mononuclear cells (PBMCs) derived from non-transgenic goats that lack PrPC (PRNPTer/Ter) compared with cells from normal (PRNP+/+) goats. Spermatozoa were analyzed for freeze tolerance, DNA integrity, viability, motility, ATP levels, and acrosome intactness at rest and after acute stress, induced by Cu2+ ions, as well as levels of reactive oxygen species (ROS) after exposure to FeSO4 and H2O2. Surprisingly, PrPC-negative spermatozoa reacted similarly to normal spermatozoa in all read-outs. Moreover, in vitro exposure of PBMCs to Doxorubicin, H2O2 and methyl methanesulfonate (MMS), revealed no effect of PrPC on cellular survival or global accumulation of DNA damage. Similar results were obtained with human neuroblastoma (SH-SY5Y) cell lines stably expressing varying levels of PrPC. RNA sequencing of PBMCs (n = 8 of PRNP+/+ and PRNPTer/Ter) showed that basal level expression of genes encoding DNA repair enzymes, ROS scavenging, and antioxidant enzymes were unaffected by the absence of PrPC. Data presented here questions the in vitro cytoprotective roles previously attributed to PrPC, although not excluding such functions in other cell types or tissues during inflammatory stress.

Characterization of the prion protein 3F4 epitope and its use as a molecular tag.

The monoclonal antibody (MAb) 3F4 has for nearly two decades been one of the most commonly used tools in prion research. This MAb has contributed significantly to our understanding of the normal cell biology of the prion protein (PrP(C)), as well as the disease related abnormalities occurring in prion diseases. The 3F4 antibody binds strongly to human and hamster PrP, with a specific requirement of two Met residues at positions 109 and 112 in the human PrP. Other species in which PrP lack one of the Met residues, like cattle and sheep, or both, like rat and mouse, do not react with the 3F4 antibody. These and other observations have led to the commonly accepted notion that the 3F4 epitope consists of the tetra-peptide Met-Lys-His-Met. In this study, we have identified the minimal epitope for 3F4 by studying its binding to synthetic peptides and by analysis of mutated ovine PrP::GFP constructs expressed in cell culture. We have found that the 3F4 epitope consists of a hepta-peptide (Lys-Thr-Asn-Met-Lys-His-Met), which in sheep encompass residues 109-115. We found that Lys 109 is critically important for 3F4 binding, as omission, or substitution of this residue to Ala resulted in no binding. We also demonstrate that the hepta-peptide constituting the minimal 3F4 epitope, can be used as a discrete, moveable high-affinity molecular tag. Thus, the 3F4 antibody can find its use beyond prion research.

Goats without Prion Protein Display Enhanced Proinflammatory Pulmonary Signaling and Extracellular Matrix Remodeling upon Systemic Lipopolysaccharide Challenge.

A naturally occurring mutation in the PRNP gene of Norwegian dairy goats terminates synthesis of the cellular prion protein (PrPC), rendering homozygous goats (PRNPTer/Ter) devoid of the protein. Although PrPC has been extensively studied, particularly in the central nervous system, the biological role of PrPC remains incompletely understood. Here, we examined whether loss of PrPC affects the initial stage of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Acute pulmonary inflammation was induced by intravenous injection of LPS (Escherichia coli O26:B6) in 16 goats (8 PRNPTer/Ter and 8 PRNP+/+). A control group of 10 goats (5 PRNPTer/Ter and 5 PRNP+/+) received sterile saline. Systemic LPS challenge induced sepsis-like clinical signs including tachypnea and respiratory distress. Microscopic examination of lungs revealed multifocal areas with alveolar hemorrhages, edema, neutrophil infiltration, and higher numbers of alveolar macrophages, with no significant differences between PRNP genotypes. A total of 432 (PRNP+/+) and 596 (PRNPTer/Ter) genes were differentially expressed compared with the saline control of the matching genotype. When assigned to gene ontology categories, biological processes involved in remodeling of the extracellular matrix (ECM), were exclusively enriched in PrPC-deficient goats. These genes included a range of collagen-encoding genes, and proteases such as matrix metalloproteinases (MMP1, MMP2, MMP14, ADAM15) and cathepsins. Several proinflammatory upstream regulators (TNF-α, interleukin-1ß, IFN-γ) showed increased activation scores in goats devoid of PrPC. In conclusion, LPS challenge induced marked alterations in the lung tissue transcriptome that corresponded with histopathological and clinical findings in both genotypes. The increased activation of upstream inflammatory regulators and enrichment of ECM components could reflect increased inflammation in the absence of PrPC. Further studies are required to elucidate whether these alterations may affect the later reparative phase of ALI.

Loss of prion protein induces a primed state of type I interferon-responsive genes.

The cellular prion protein (PrPC) has been extensively studied because of its pivotal role in prion diseases; however, its functions remain incompletely understood. A unique line of goats has been identified that carries a nonsense mutation that abolishes synthesis of PrPC. In these animals, the PrP-encoding mRNA is rapidly degraded. Goats without PrPC are valuable in re-addressing loss-of-function phenotypes observed in Prnp knockout mice. As PrPC has been ascribed various roles in immune cells, we analyzed transcriptomic responses to loss of PrPC in peripheral blood mononuclear cells (PBMCs) from normal goat kids (n = 8, PRNP+/+) and goat kids without PrPC (n = 8, PRNPTer/Ter) by mRNA sequencing. PBMCs normally express moderate levels of PrPC. The vast majority of genes were similarly expressed in the two groups. However, a curated list of 86 differentially expressed genes delineated the two genotypes. About 70% of these were classified as interferon-responsive genes. In goats without PrPC, the majority of type I interferon-responsive genes were in a primed, modestly upregulated state, with fold changes ranging from 1.4 to 3.7. Among these were ISG15, DDX58 (RIG-1), MX1, MX2, OAS1, OAS2 and DRAM1, all of which have important roles in pathogen defense, cell proliferation, apoptosis, immunomodulation and DNA damage response. Our data suggest that PrPC contributes to the fine-tuning of resting state PBMCs expression level of type I interferon-responsive genes. The molecular mechanism by which this is achieved will be an important topic for further research into PrPC physiology.