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Background Chronic wasting disease (CWD) is a prion disease of cervids first reported in North America in the 1960s. In Europe, CWD was first diagnosed in 2016 in a wild reindeer in Norway. Detection of two more cases in the same mountain area led to the complete culling of this partially confined reindeer population of about 2400 animals. A total of 19 CWD positive animals were identified. The affected area is extensively used for the grazing of sheep during summers. There are many mineral licks intended for sheep in the area, but these have also been used by reindeer. This overlap in area use raised concerns for cross-species prion transmission between reindeer and sheep. In this study, we have used global positioning system (GPS) data from sheep and reindeer, including tracking one of the CWD positive reindeer, to investigate spatial and time-relevant overlaps between these two species. Since prions can accumulate in lymphoid follicles following oral uptake, samples of gut-associated lymphoid tissue (GALT) from 425 lambs and 78 adult sheep, which had grazed in the region during the relevant timeframe, were analyzed for the presence of prions. The recto-anal mucosa associated lymphoid tissue (RAMALT) from all the animals were examined by histology, immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA), and the ileal Peyer's patch (IPP) from a subsample of 37 lambs were examined by histology and IHC, for the detection of prions. Results GPS data showed an overlap in area use between the infected reindeer herd and the sheep. In addition, the GPS positions of an infected reindeer and some of the sampled sheep showed temporospatial overlap. No prions were detected in the GALT of the investigated sheep even though the mean lymphoid follicle number in RAMALT and IPP samples were high. Conclusion The absence of prions in the GALT of sheep that have shared pasture with CWD-infected reindeer, may suggest that transmission of this novel CWD strain to sheep does not easily occur under the conditions found in these mountains. We document that the lymphoid follicle rich RAMALT could be a useful tool to screen for prions in sheep.

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-MarieTooth 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. (C) 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license.

Additional file 2. Neurological examination of goats.

Additional file 3. Primer sequences used for qPCR analysis.

Additional file 6. Distribution of PrP Sc in the CNS and peripheral tissues.

Additional file 5. Semi-quantitative scoring of GFAP.

Additional file 1. Kinship and degree of inbreeding.

Additional file 4. Sequence alignment of PRNP in 12 goats.

A homozygous mutation has been identified in the N-myc downstream-regulated gene 1 (NDRG1) in recent cases of polyneuropathy in Alaskan malamute dogs from the Nordic countries and USA. The objective of the present study was to determine if cases diagnosed 30–40 years ago with polyneuropathy in the Alaskan malamute breed in Norway had the same hereditary disease as the recent cases. Fourteen historical cases and 12 recently diagnosed Alaskan malamute dogs with hereditary polyneuropathy, and their parents and littermates (n = 88) were included in this study (total n = 114). After phenotyping of historical and recent cases, NDRG1 genotyping was performed using DNA extracted from archived material from five Norwegian dogs affected by the disease in the late 1970s and 1980s. In addition, pedigrees were analysed. Our study concluded that historical and recent phenotypic polyneuropathy cases were carrying the same NDRG1-mutation. The pedigree analysis showed that all affected Alaskan malamute cases with polyneuropathy could be traced back to one common ancestor of North American origin. By this study, a well-documented example of the silent transmission of recessive disease-causing alleles through many generations is provided, demonstrated by the re-emergence of a phenotypically and genetically uniform entity in the Scandinavian Alaskan malamute population.

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-1β, 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.

Gene name, gene abbreviations and primer sequences used in the real time qPCR analysis. For previously untested primer assays, two primer pairs were designed for each transcript. (DOCX 22Â kb)

International audience; Despite intensive studies since the 1990s, the physiological role of the cellular prion protein (PrPC) remains elusive. Here, we present a novel concept suggesting that PrPC contributes to immunological quiescence in addition to cell protection. PrPC is highly expressed in diverse organs that by multiple means are particularly protected from inflammation, such as the brain, eye, placenta, pregnant uterus, and testes, while at the same time it is expressed in most cells of the lymphoreticular system. In this paradigm, PrPC serves two principal roles: to modulate the inflammatory potential of immune cells and to protect vulnerable parenchymal cells against noxious insults generated through inflammation. Here, we review studies of PrPC physiology in view of this concept.

The physiological role of the cellular prion protein (PrP(C)) is incompletely understood. The expression of PrP(C) in hematopoietic stem cells and immune cells suggests a role in the development of these cells, and in PrP(C) knockout animals altered immune cell proliferation and phagocytic function have been observed. Recently, a spontaneous nonsense mutation at codon 32 in the PRNP gene in goats of the Norwegian Dairy breed was discovered, rendering homozygous animals devoid of PrP(C). Here we report hematological and immunological analyses of homozygous goat kids lacking PrP(C) (PRNP(Ter/Ter) ) compared to heterozygous (PRNP (+/Ter)) and normal (PRNP (+/+)) kids. Levels of cell surface PrP(C) and PRNP mRNA in peripheral blood mononuclear cells (PBMCs) correlated well and were very low in PRNP (Ter/Ter), intermediate in PRNP (+/Ter) and high in PRNP (+/+) kids. The PRNP (Ter/Ter) animals had a shift in blood cell composition with an elevated number of red blood cells (RBCs) and a tendency toward a smaller mean RBC volume (P = 0.08) and an increased number of neutrophils (P = 0.068), all values within the reference ranges. Morphological investigations of blood smears and bone marrow imprints did not reveal irregularities. Studies of relative composition of PBMCs, phagocytic ability of monocytes and T-cell proliferation revealed no significant differences between the genotypes. Our data suggest that PrP(C) has a role in bone marrow physiology and warrant further studies of PrP(C) in erythroid and immune cell progenitors as well as differentiated effector cells also under stressful conditions. Altogether, this genetically unmanipulated PrP(C)-free animal model represents a unique opportunity to unveil the enigmatic physiology and function of PrP(C).

The first cases of early-onset progressive polyneuropathy appeared in the Alaskan Malamute population in Norway in the late 1970s. Affected dogs were of both sexes and were ambulatory paraparetic, progressing to non-ambulatory tetraparesis. On neurologic examination, affected dogs displayed predominantly laryngeal paresis, decreased postural reactions, decreased spinal reflexes and muscle atrophy. The disease was considered eradicated through breeding programmes but recently new cases have occurred in the Nordic countries and the USA. The N-myc downstream-regulated gene (NDRG1) is implicated in neuropathies with comparable symptoms or clinical signs both in humans and in Greyhound dogs. This gene was therefore considered a candidate gene for the polyneuropathy in Alaskan Malamutes. The coding sequence of the NDRG1 gene derived from one healthy and one affected Alaskan Malamute revealed a non-synonymous G>T mutation in exon 4 in the affected dog that causes a Gly98Val amino acid substitution. This substitution was categorized to be "probably damaging" to the protein function by PolyPhen2 (score: 1.000). Subsequently, 102 Alaskan Malamutes from the Nordic countries and the USA known to be either affected (n = 22), obligate carriers (n = 7) or healthy (n = 73) were genotyped for the SNP using TaqMan. All affected dogs had the T/T genotype, the obligate carriers had the G/T genotype and the healthy dogs had the G/G genotype except for 13 who had the G/T genotype. A protein alignment showed that residue 98 is conserved in mammals and also that the entire NDRG1 protein is highly conserved (94.7%) in mammals. We conclude that the G>T substitution is most likely the mutation that causes polyneuropathy in Alaskan Malamutes. Our characterization of a novel candidate causative mutation for polyneuropathy offers a new canine model that can provide further insight into pathobiology and therapy of human polyneuropathy. Furthermore, selection against this mutation can now be used to eliminate the disease in Alaskan Malamutes.

Peripheral nerves transmit signals between peripheral tissue and the central nervous system, essential for normal motor and sensory function. To enable this, the integrity of both the axon and Schwann cell is crucial. The Schwann cell forms a myelin sheath around large diameter axons, necessary for rapid nerve conduction and axonal survival. Neuropathies, diseases affecting peripheral nerves, have diverse etiologies and may cause severe clinical signs in both human and animals. Some neuropathies are caused by inherited deficiencies in proteins necessary for normal axonal function or formation/maintenance of the myelin sheath. In this work, we have investigated how deficiencies in NDRG1 or PrPC affect peripheral nerve myelin maintenance by using morphological studies in two unique, spontaneous animal models. The hereditary polyneuropathy of Alaskan Malamutes is associated with a mutation in N-myc downstream-regulated gene 1 (NDRG1). Other mutations in the same gene cause polyneuropathies in humans, rodent models and Greyhound show dogs. However, the function of NDRG1 in peripheral nerves is largely unknown and the pathomorphological changes described in NDRG1-associated neuropathies of different species diverge. Whereas the human disease, denominated Charcot-Marie-Tooth disease type 4D, is considered a primary demyelinating disease with secondary axonal loss, axonal degeneration dominated in the polyneuropathy of Greyhounds. A detailed mapping of NDRG1 in canine tissues and cells presented in this thesis revealed high NDRG1 expression in epithelia and myelinating Schwann cells. A phosphorylated isoform of the protein localized to the centrosomes and cleavage furrow in a primary canine Schwann cell culture. Phosphorylated NDRG1 was restricted to the abaxonal cytoplasm of myelinating Schwann cells in control dogs, but this signal was not observed in an affected Alaskan Malamute. Ultrastructural studies and teased fiber preparations of nerves from neuropathic Alaskan Malamutes uncovered a demyelinating neuropathy associated with accumulation of actin in the Schwann cell and prominent myelin infoldings. The latter ultimately segregated the axon into pockets with aggregates of organelles, suggestive of early axonal degeneration. We therefore suggest that impaired NDRG1 function in the myelinating Schwann cells of affected Alaskan Malamutes results in deranged myelin maintenance, demyelination and secondary axonal degeneration. The cellular prion protein (PrPC) has been studied extensively because of its role in the development of transmissible spongiform encephalopathies in several species, such as Creutzfeldt-Jakobs disease in humans, bovine spongiform encephalopathy (“mad cow disease”) in cattle and scrapie in small ruminants. In these diseases, a misfolded isoform of the protein, PrPSc, accumulates in the brain and ultimately results in neuronal death. The normal functions of PrPC are not fully understood. Some of the phenotypes reported from transgenic mice lacking PrPC have later been ascribed to other genes, but recent reports have suggested that PrPC is important in dampening pro-inflammatory signaling and in myelin maintenance. We have used a nontransgenic, spontaneous model to investigate the latter; a line of Norwegian dairy goats lacking PrPC. Morphological studies revealed a demyelinating neuropathy with infiltration of macrophages and lymphocytes, vacuolated fibers and paranodal myelin outfoldings. Peripheral nerve lipid composition of adult goats lacking PrPC was significantly different from controls, while no difference was observed in young goats, suggesting a progressive disease. In conclusion, this is the first report of a demyelinating polyneuropathy caused by loss of PrPC in a nontransgenic mammal, supporting that PrPC has a vital role in peripheral nerve myelin maintenance. Perifere nerver overfører nerveimpulser mellom perifert vev og sentralnervesystemet, og er derfor essensielle for normal motorisk og sensorisk funksjon. For opprettholdelse av denne funksjonen, er både aksonet og den Schwannske cellen viktige. Aksonet er en utløper fra nervecellen og sørger for overføring av nerveimpulser. Den Schwannske cellen danner en myelinskjede rundt større aksoner. Dette er nødvendig for rask nerveledningshastighet og aksonal overlevelse. Nevropatier, sykdommer i perifere nerver, har ulik etiologi og kan forårsake alvorlige kliniske tegn både hos mennesker og dyr. Noen nevropatier er forårsaket av arvelige forstyrrelser i proteiner som er nødvendig for normal aksonal funksjon eller dannelse/vedlikehold av myelinskjeden. I dette arbeidet har vi undersøkt hvordan mangel på NDRG1 eller PrPC påvirker myelinvedlikehold i perifere nerver ved å bruke morfologiske studier i to unike, spontane dyremodeller. Arvelig polynevropati hos alaskan malamute er assosiert med en mutasjon i N-myc downstream-regulated gene 1 (NDRG1). Andre mutasjoner i samme genet forårsaker polynevropatier hos menneske, gnagermodeller og utstillingslinjer av greyhound. Funksjonen til NDRG1 i perifere nerver er imidlertid ukjent, og de patomorfologiske forandringene ved NDRG1-assosierte nevropatier hos ulike arter divergerer. Mens den humane nevropatien, Charcot-Marie-Tooths sykdom type 4D, er ansett for å være en primær demyeliniserende sykdom med sekundært aksonalt tap, dominerte aksonal degenerasjon ved polynevropati hos greyhound. En detaljert kartlegging av NDRG1 i celler og vev fra hund presentert i denne avhandlingen viser høye nivåer av NDRG1 i epitel og myeliniserende Schwannske celler. En fosforylert isoform av proteinet ble lokalisert i centrosomer og kløyvingsfuren hos Schwannske celler isolert fra hund. Fosforylert NDRG1 var begrenset til abaksonale cytoplasma i myeliniserende Schwannske celler i kontrollhunder, men dette signalet ble ikke observert i en syk alaskan malamute. Ultrastrukturelle studier og teased nerve fibers av nerver fra affiserte alaskan malamuter avslørte en demyeliniserende nevropati assosiert med aktinaggregater i de myeliniserende Schwannske cellene og uttalte innfoldinger av myelin. Innfoldingene delte stedvis aksonene inn i atskilte lommer med aggregater av organeller, forenelig med tidlig aksonal degenerasjon. Resultatene tyder på at svekket NDRG1-funksjon i myeliniserende Schwannske celler hos affiserte alaskan malamuter fører til forstyrret vedlikehold av myelin, demyelinisering og sekundær aksonal degenerasjon. Det cellulære prionproteinet (PrPC) har blitt grundig studert på grunn av sin rolle i utvikling av overførbare spongiforme encefalopatier, eller prionsykdommer, hos flere arter, for eksempel Creutzfeldt-Jakobs sykdom hos menneske, bovin spongiform encefalopati («kugalskap») hos storfe og skrapesjuke hos små drøvtyggere. Ved disse sykdommene vil en misfoldet isoform av proteinet, PrPSc, akkumulere i hjernen og tilslutt resultere i nevronal celledød. Normalfunksjonen til PrPC er ikke fullstendig forstått, og forståelsen av denne kan være viktig for å belyse den molekylære patogenesen ved prionsykdommer. Noen fenotyper rapportert fra transgene mus uten PrPC ble senere vist å skyldes andre gener, men nylige rapporter foreslår at PrPC er viktig for å dempe proinflammatorisk signalering og myelinvedlikehold. Vi har brukt en ny, ikke-transgen, spontan modell - en linje av norske melkegeiter som mangler PrPC - til å undersøke om denne modellen kan bekrefte tidligere studier fra transgene mus som har vist myelinskader. Morfologiske studier hos geiter uten PrPC avslørte en demyeliniserende nevropati med infiltrasjon av makrofager og lymfocytter, vakuoliserte nervefibre og paranodale myelinutfoldinger. Lipidsammensetningen i perifere nerver hos voksne geiter uten PrPC var signifikant forskjellig fra kontrollene, mens ingen forskjell ble observert hos unge dyr, noe som tyder på en progredierende sykdom. Dette er den første rapporten av en demyeliniserende polynevropati forårsaket av PrPC-mangel hos et ikke-transgent pattedyr, og støtter at PrPC har en viktig rolle i myelinvedlikehold.

The gut-associated lymphoid tissue (GALT) of sheep has been investigated for decades, and several immune cells receding within these structures have been described. The GALT of sheep has specialized compartments described to contain myeloid and lymphoid cells. The newly developed antibody against ovine NCR1 has been shown to label lymphocytes with conventional attributes of NK cells. However, the precise localization, phenotype and activation of NCR1+ cells in the small and large intestinal lymphoid compartments remain unknown. Investigations presented in this thesis were done using different types of immuno-labelling on tissue sections and in flow cytometry. The NCR1+ cells were detected in the gut of foetuses from day 70 in the gestation, and these cells resided in compartments where T cells usually are seen; the inter-follicular area, dome and lamina propria. The NCR1+ cells of the ovine foetal gut showed an increasing fraction of c-kit expression in the last period before birth. The localization of NCR1+ cells described in the three papers is stable in foetus, normal unchallenged lambs and during cryptosporidiosis. The studies have revealed two separate phenotypic subtypes, both exhibiting characteristics of conventional NK cells. However, based on the new classification of innate lymphoid cells described in mouse and human, some of the NCR1+ cells could be grouped as a subtype featuring regulatory and less cytotoxic functions, designated ILC22. From the youngest group of foetuses investigated we found NCR1+ cells to proliferate, whereas this fraction of cells decreased to a minimum and were almost absent in the juvenile lambs. During an experimental infection with Cryptosporidium parvum we found the NCR1+ cells to be faithful to their compartmentalization and showed an elevation of activation status. The results presented in this thesis may be useful for comparative considerations and for general understanding of NCR1+ cells in the sheep. It may be a basis for further investigations of the GALT and immune responses in the gut of sheep. Tarmassosiert lymfatisk vev har vært undersøkt i flere tiår, og dette lymfatiske vevet har spesialiserte områder, inneholdende myeloide og lymfoide celler. Det nylige utviklede antistoffet mot sau NCR1 er blitt vist å merke lymfocytter med klassiske karakteristika kjent for dreper(NK) celler. Det er imidlertid ennå ikke blitt beskrevet lokalisasjon, fenotype eller aktiveringsstatus hos NCR1+ celler i tynn- og tykktarmens lymfoide områder hos drøvtyggere. Undersøkelser som er presentert i denne avhandlingen ble gjort ved hjelp av ulike typer immunmerking på snitt av vev og i væskestrømcytometri. NCR1+ celler ble funnet i tarm hos fostre fra 70 dager i drektigheten og ble funnet i områder hvor T celler vanligvis er lokalisert; interfollikulær områder, dome og lamina propria. NCR1+ celler som ble funnet i sauefosterets tarm viste å ha økende uttrykk av c-kit i den siste perioden før fødsel. Studiene har avslørt to separate fenotypiske subtyper, der begge innehar typiske karakteristiske trekk av klassiske NK celler. Det er imidlertid kommet en ny klassifisering av medfødte lymfoide celler, som kan føre til at noen av de NCR1+ cellene kan bli klassifisert som en subtype vist å være mer regulatorisk og mindre cytotoksisk, kalt ILC22. Vi fant prolifererende NCR1+ celler i de yngste fostrene, der andelen av disse sank til et minimum mot fødsel og var nesten fraværende i unge lam. Lokaliseringen av NCR1+ celler beskrevet i de tre artiklene er stabil i fostre, normale friske lam og i cryptosporidieinfiserte lam. I løpet av en eksperimentell infeksjon med Cryptosporidium parvum fant vi at de NCR1+ cellene viste uendret lokalisasjon i tarmen og en økning av aktiveringsstatus. Resultatene som er presentert i denne avhandlingen kan være nyttige for komparative betraktninger og for å få en generell forståelse av NCR1+ celler i sau. Det kan være et grunnlag for videre undersøkelser hos tarmassosiert vev og immunrespons hos sau.

The physiological function of the cellular prion protein (PrPC) remains enigmatic. A misfolded, infectious conformer of the protein, known as the scrapie isoform PrPSc, is able to aggregate in brain tissue and cause a group of fatal transmissible neurodegenerative disorders in humans and animals. Amongst them are Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep and goats, bovine spongiform encephalopathy (BSE) in cattle and chronic wasting disease (CWD) in cervids. PrPC is evolutionary conserved and abundantly expressed in neurons, but also widely in other non-neuronal tissues, depicting a central role in cellular physiology. Several methods have been used for studies of PrPC function and the development of Prnp knockout mice during the 1990s, provided researchers with a new and valuable tool for studies of in vivo consequences of PrPC ablation. Interestingly, apart from being completely resistant to prion disease, Prnp knockout mice displayed no major aberrant phenotypes; rather they developed normally and exhibited normal lifespans. Further studies have however attributed several phenotypes to the loss of PrPC, including inhibition of apoptosis, protection against oxidative stress, a role in synapses, NMDA receptor signaling and modulation of behavior, amongst others. Recently, a line of Norwegian Dairy Goats was found to carry a nonsense mutation at codon 32 of the PRNP reading frame that completely blocks PrPC synthesis. A non-transgenic, non-rodent mammalian PrPC-null model represents a new and valuable resource for prion research that could, in combination with other model systems, shed light on PrPC physiology. The studies going into this thesis represent the first sets of analysis and experiments characterizing this unique line of goats. Firstly, we set out to characterize goats with (PRNP+/+) and without (PRNPTer/Ter) PrPC with regard to standard health parameters. Hematological analyses revealed that PRNPTer/Ter goats presented with an increased number of red blood cells (RBCs) with a smaller mean cell volume (MCV) as well as a tendency for increased levels of neutrophils, an alteration we collectively coined a “hematological shift”. This observation suggests that PrPC may serve a role in bone marrow hematopoiesis. The bone marrow morphology, however, did not deviate between the genotypes and further studies are needed to clarify PrPC’s role in RBC development and/or physiology. Based on the moderate, but dynamic expression of PrPC in mononuclear cells and previous research showing that PrPC might play a role in modulating basic immune cell functions and immune responses, we pursued this by investigating basal immune cell traits. Numbers of peripheral blood mononuclear cells (PBMCs) were similar between the PRNP genotypes. Moreover, basal immune cells functions such as monocyte phagocytosis and lymphocyte proliferation were also similar between the genotypes, suggesting that PrPC deficiency had no major effects on these important processes, which is contrast to some observations in mouse models. A deeper analysis of PrPC loss was undertaken by investigating the transcriptome of PBMCs from PRNP+/+ and PRNPTer/Ter animals. Analysis of differentially expressed genes revealed a significant upregulation of type I interferon (IFN)-responsive genes in PRNPTer/Ter cells, which could not be attributed to differences in cell populations or altered expression of genes encoding major components of the type I IFN signaling pathway, indicating that PrPC somehow downregulates tonic type I INF signaling. By using several clones of human neuroblastoma SH-SY5Y cells, stably expressing different levels of PrPC, we were able to demonstrate that mock-transfected cells with very low levels of PrPC responded with increased transcription of the IFN-responsive gene MX2 after treatment with IFN-α, compared with clones expressing moderate or high levels of PrPC. Although, providing support to the observations from the PBMCs, the data from the SH-SY5Y cells did not reveal a simple dose-response relationship between PrPC levels and apparent sensitivity to INF-α. However, an independent dataset from an in vivo lipopolysaccharide (LPS) challenge of goats with and without PrPC, showed a similar gene expression signature in circulating leukocytes both at basal level and after LPS exposure, demonstrating that the phenotype is also present and functional in vivo. Further studies are needed to reveal the molecular mechanisms behind these observations and to clarify at which level PrPC impacts type I IFN signaling. Goats naturally devoid of PrPC can have significant breeding value. Knowing that PrPC is normally present at high levels in the male genital tract, including ejaculated spermatozoa, we wanted to investigate if lack of PrPC could influence semen quality and spermatozoa stress resilience, as suggested by mouse studies. Our data confirmed the prominent presence of PrPC in testicle and epididymis as well as lower levels in spermatozoa in PRNP+/+ animals. However, analysis of 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 ROS after exposure to FeSO4 and H2O2 revealed no differences between the PRNP genotypes. Since cytoprotective roles have been assigned to PrPC, we wanted to broaden our analysis by inclusion of PBMCs with and without PrPC in our in vitro analysis of oxidative and genotoxic stress. Similar to the observations from spermatozoa, PrPC appeared dispensable for in vitro stress resilience of PBMCs. Expression levels of genes involved in DNA damage repair and ROS scavenging in PBMCs were also unaffected by PrPC loss. In conclusion, no PRNP genotype related differences in stress resilience were detected concerning viability and global accumulation of DNA damage in PBMCs after treatment with H2O2, doxorubicin or MMS. These observations were corroborated by data from SH-SY5Y cells expressing very low or moderate levels of PrPC, again revealing no direct cyto-protective function of PrPC, under these in vitro conditions. Altogether, the degree to which PrPC is able to confer cytoprotection during stressful situations in vitro is questioned, as we were unable to detect any differences between cells with and without PrPC in our studies. However, PrPC could still execute functions in neuro-immune crosstalk at rest and during inflammatory stress, possibly protecting immune-privileged tissues. Taken together, this thesis has provided unique new knowledge concerning PrPC’s normal physiological function by the use of a non-transgenic animal model. Animals without PrPC displayed a hematological shift, but no effects were observed in circulating leukocytes or basal immune cell functions. The finding of an immunological signature dominated by increased level of type I IFN-responsive genes in PBMCs is a previously unrecognized phenotype in cells without PrPC, which can function as a gateway for further studies needed to dissect the pathway in which PrPC might be involved. No stress-protective properties could be found in vitro in the cells with PrPC assessed in this work, however it remains to be investigated if these effects are better explored in vivo. Det cellulære prionproteinets (PrPC) fysiologiske funksjon er, til tross for årevis med forskning, fortsatt ukjent. En feilfoldet, infeksiøs isoform av prionproteinet, bedre kjent som scrapievarianten, PrPSc, har evne til å aggregere i hjernevev og forårsake en rekke fatale, overførbare nevrodegenerative sjukdommer hos mennesker og dyr. Blant disse er Creutzfeldt Jakobs sjukdom hos menneske, skrapesjuke hos sau og geit, bovin spongiform encefalopati eller kugalskap hos storfe, og skrantesjuke hos hjortedyr.

The cellular prion protein (PrPC) is known for its pivotal role in the development of prion diseases, such as Creutzfeldt-Jakob disease in humans and scrapie in small ruminants. In these diseases, PrPC is converted into a pathological form, PrPSc. The accumulation of this misfolded isoform in the brain results in fatal neurodegeneration. Several decades of research have revealed clues to the normal biological function of PrPC, yet still without a definite conclusion. Unravelling normal PrPC biology is important not only to understand the pathogenesis of prion disorders, but also to identify potential side effects of future treatments involving blocking of PrPC or removal of the PrPC-encoding gene (PRNP). We utilized a unique line of Norwegian dairy goats that carry a stop mutation early in the PRNP to study PrPC physiology. These goats are the only known mammals that are naturally devoid of PrPC (PRNPTer/Ter). By using next generation RNA sequencing, we identified a primed state of interferon-stimulated genes in circulating blood cells and tissues of PRNPTer/Ter goats (Papers II, III, and IV). When exposed to systemic lipopolysaccharide (LPS), PrPC-deficient goats suffered a prolonged sickness behavior and displayed an expression profile skewed towards a type I interferon signaling response in the brain (Paper III). In the lungs, a tissue with a more severe response to LPS, loss of PrPC resulted in increased activation of pro-inflammatory pathways and remodeling of the extracellular matrix (Paper IV), which indicates augmented damage to the lung parenchyma. Taken together, we propose that PrPC is a modulator of inflammatory pathways, particularly downstream of type I interferons, and protects vulnerable tissues against inflammatory stress. Being located at the cell surface, our findings corroborate the theory of PrPC as a scaffolding protein, which can interact with various forms of multiprotein complexes and regulate downstream signaling. In addition to exploring the function of PrPC, the current thesis extends the existing knowledge of the inflammatory response in small ruminants. Systemic administration of LPS mimics the initial events of sepsis and is a well-established method of studying the innate immune system. We provide evidence that both leukocytes and tissues increase transcription of acute-phase protein genes at the beginning of an inflammation, and we identify SAA3 as a particularly useful biomarker (Papers I, III, and IV). In the choroid plexus, a profound activation of the transcriptome and increased number of Iba1-positive cells were observed. Being located at the blood-brain interface, we confirm that this tissue plays a crucial role in the interaction between systemic circulation and the brain. Alterations in the hippocampus transcriptome were mild, which suggests that this brain region is relatively protected from circulating endotoxins. Yet, the clinical response and activation of astrocytes confirms that inflammation was present (Paper III). As described in paper IV, the lungs were the most affected organ, characterized by substantial activation of the transcriptome and morphological changes compatible with acute lung injury. The high pulmonary sensitivity of goats towards systemic endotoxins implies that limiting pulmonary inflammation is of special importance for treating septic patients. In light of the increasing problem of antimicrobial resistance against antibiotics, understanding the molecular mechanisms of inflammation is a prerequisite for designing new therapeutic strategies. Det normale prionproteinet (PrPC) finnes hos alle pattedyr og er kjent for sin rolle i utviklingen av prionsykdommer. Disse inkluderer blant annet Creutzfeldt-Jakobs sykdom hos mennesker, skrantesjuke (CWD) hos hjortedyr, og skrapesjuke (scrapie) hos småfe. Sykdommene skyldes at en unormal, feilfoldet variant av prionproteinet (PrPSc) akkumulerer i sentralnervesystemet og fører til degenerasjon av nerveceller. Prionsykdommer kan ikke behandles og er alltid dødelige. Flere tiår med forskning har indikert at PrPC er involvert i en rekke normale biologiske funksjoner, men noen endelig konklusjon har uteblitt. Kartlegging av PrPC sin fysiologiske funksjon er viktig for å forstå utviklingen av prionsykdommer, samt å identifisere potensielle bivirkninger av framtidige behandlingsformer som kan involvere blokkering av PrPC eller fjerning av genet som koder for prionproteinet (PRNP).