Cecilie Ersdal

Sub-cellular pathology of scrapie: coated pits are increased in PrP codon 136 alanine homozygous scrapie-affected sheep

Sub-cellular studies of transmissible spongiform encephalopathies (TSEs) have been carried out on several animal species and human beings. However, studies of optimal perfusion-fixed tissues have largely been confined to examination of rodents. Using a recently developed technique, heads of scrapie-affected sheep and controls were perfusion fixed with mixed aldehydes. The obexes were immunohistochemically labelled with PrP antibodies, and the dorsal motor nucleus of the vagal nerve was examined by electron microscopy. Irregular neuritic profiles with highly invaginated membranes, associated with coated pits were found in all scrapie-affected sheep, but not in controls. Interestingly, they were consistently more frequent in the homozygous A136 sheep. This is the first report describing sub-cellular differences in pathology associated with different PrP genotypes. Rarely, amorphous material, or sparse fibrillar structures, were present in the extracellular space. The changes were often associated with irregular plasmalemma and frequent coated pits. Vacuolation typical of TSEs, dystrophic neurites and variable gliosis were present. Herniation of membranes and organelles from apparently healthy processes into adjacent vacuoles and dendrites was also observed. We suggest that the increase in coated pits and plasmalemma invagination is related to an attempted internalisation of aggregated disease-specific PrP, or protofilaments, from the extracellular space.

Relationships between ultrastructural scrapie pathology and patterns of abnormal prion protein accumulation

On immunohistochemical examination several morphological types of disease-specific prion protein (PrPd) accumulation are recognised in the brain of sheep suffering from scrapie. The present study examined the relationship between the type of PrPd deposits seen by light microscopy and ultrastructural changes in the olivary nuclei and the dorsal motor nucleus of the vagus (DMNV) in naturally infected sheep with clinical scrapie. The nature and magnitude of sub-cellular morphological changes found in the olivary nuclei differed from the patterns of degeneration previously described in the DMNV. In the olivary nuclei, lamellar bodies in the neuronal perikaryon were found to correlate with marked intraneuronal PrPd accumulation. Bizarre, coated, spiral invaginations of the plasmalemma were only found in A136 homozygous sheep in this nucleus, where few coated pits were usually observed. Neuropil vacuolation in the olivary nuclei was mild and correlated with sparse extracellular PrPd deposition. In the DMNV, the magnitude of extracellular immunolabelling in the neuropil was prominent. These extracellular PrPd aggregates coincided with intense neuropil vacuolation, increased numbers of coated pits, and with the presence of pre-amyloid changes and infrequent short fibrils in the extracellular space. Scrapie-infected neurons in the two neuroanatomic sites examined, therefore, appear to process and respond to the presence of PrPd differently. We hypothesise that vacuolation, coated pits and spiral invaginations of the plasmalemma may be responses to extracellular PrPd molecules, and that lamellar bodies are changes associated with the high levels of intraneuronal PrPd.

LPS-induced systemic inflammation reveals an immunomodulatory role for the prion protein at the blood-brain interface

BackgroundThe cellular prion protein (PrPC) is an evolutionary conserved protein abundantly expressed not only in the central nervous system but also peripherally including the immune system. A line of Norwegian dairy goats naturally devoid of PrPC (PRNPTer/Ter) provides a novel model for studying PrPC physiology.MethodsIn order to explore putative roles for PrPC in acute inflammatory responses, we performed a lipopolysaccharide (LPS, Escherichia coli O26:B6) challenge of 16 goats (8 PRNP+/+ and 8 PRNPTer/Ter) and included 10 saline-treated controls (5 of each PRNP genotype). Clinical examinations were performed continuously, and blood samples were collected throughout the trial. Genome-wide transcription profiles of the choroid plexus, which is at the blood-brain interface, and the hippocampus were analyzed by RNA sequencing, and the same tissues were histologically evaluated.ResultsAll LPS-treated goats displayed clinical signs of sickness behavior, which were of significantly (p < 0.01) longer duration in animals without PrPC. In the choroid plexus, a substantial alteration of the transcriptome and activation of Iba1-positive cells were observed. This response included genotype-dependent differential expression of several genes associated with the immune response, such as ISG15, CXCL12, CXCL14, and acute phase proteins, among others. Activation of cytokine-responsive genes was skewed towards a more profound type I interferon response, and a less obvious type II response, in PrPC-deficient goats. The magnitude of gene expression in response to LPS was smaller in the hippocampus than in the choroid plexus. Resting state expression profiles revealed a few differences between the PRNP genotypes.ConclusionsOur data suggest that PrPC acts as a modulator of certain pathways of innate immunity signaling, particularly downstream of interferons, and probably contributes to protection of vulnerable tissues against inflammatory damage.

Accelerated clinical course of prion disease in mice compromised in repair of oxidative DNA damage

The detailed mechanisms of prion-induced neurotoxicity are largely unknown. Here, we have studied the role of DNA damage caused by reactive oxygen species in a mouse scrapie model by characterizing prion disease in the ogg1(-/-)mutyh(-/-) double knockout, which is compromised in oxidative DNA base excision repair. Ogg1 initiates removal of the major oxidation product 8-oxoguanine (8-oxoG) in DNA, and Mutyh initiates removal of adenine that has been misincorporated opposite 8-oxoG. Our data show that the onset of clinical signs appeared unaffected by Mutyh and Ogg1 expression. However, the ogg1(-/-)mutyh(-/-) mice displayed a significantly shorter clinical phase of the disease. Thus, accumulation of oxidative DNA damage might be of particular importance in the terminal clinical phase of prion disease. The prion-induced pathology and lesion profile were similar between knockout mice and controls. The fragmentation pattern of protease-resistant PrP as revealed in Western blots was also identical between the groups. Our data show that the fundamentals of prion propagation and pathological manifestation are not influenced by the oxidative DNA damage repair mechanisms studied here, but that progressive accumulation of oxidative lesions may accelerate the final toxic phase of prion disease.

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.

Neil3 induced neurogenesis protects against prion disease during the clinical phase

Base excision repair (BER) is the major pathway for repair of oxidative DNA damage. Mice with genetic knockout of the BER enzyme Neil3 display compromised neurogenesis in the sub-ventricular zone of the lateral ventricle and sub-granular layer of the dentate gyrus of the hippocampus. To elucidate the impact of oxidative DNA damage-induced neurogenesis on prion disease we applied the experimental prion disease model on Neil3-deficient mice. The incubation period for the disease was similar in both wild type and Neil3−/− mice and the overall neuropathology appeared unaffected by Neil3 function. However, disease in the Neil3−/− mice was of shorter clinical duration. We observed a mildly reduced astrogliosis in the hippocampus and striatum in the Neil3-deficient mice. Brain expression levels of neuronal progenitor markers, nestin (Nestin), sex determining region Box 2 (Sox2), Class III beta-tubulin (Tuj1) decreased towards end-stage prion disease whereas doublecortin (Dcx) levels were less affected. Neuronal nuclei (NeuN), a marker for mature neurons declined during prion disease and more pronounced in the Neil3−/− group. Microglial activation was prominent and appeared unaffected by loss of Neil3. Our data suggest that neurogenesis induced by Neil3 repair of oxidative DNA damage protects against prion disease during the clinical phase.

Controlled efficacy trial confirming toltrazuril resistance in a field isolate of ovine Eimeria spp.

BackgroundCoccidiosis due to Eimeria spp. infections in lambs causes increased mortality and substantial production losses, and anticoccidials are important for control of the infection. Anticoccidial resistance has been reported in poultry and swine, and we recently described reduced toltrazuril efficacy in ovine Eimeria spp. in some Norwegian sheep farms using a newly developed faecal oocyst count reduction test (FOCRT). The aim of the present study was to use a controlled efficacy trial to assess the efficacy of toltrazuril against a field isolate suspected of being resistant.MethodsTwenty lambs, 17–22 days old and raised protected against exposure to coccidia, were infected with a field isolate of 100,000 Eimeria spp. oocysts. This isolate was obtained from a farm with a previously calculated drug efficacy of 56% (95% confidence interval: -433.9 to 96.6%). At day 7 post-infection, 10 of the lambs were orally treated with 20 mg/kg toltrazuril (Baycox Sheep vet., Bayer Animal Health), while the other 10 lambs (controls) were given physiological saline. Clinical examinations were conducted, and weight gains recorded. Daily faecal samples were scored for diarrhoea on a scale from 1 to 5, and oocyst excretion was determined using a modified McMaster technique. Oocysts were morphologically identified to species level. At 17–24 days post-infection, the lambs were euthanized and necropsied.ResultsThe tested Eimeria isolate was resistant against toltrazuril, and resistance was seen in both pathogenic and non-pathogenic species. In addition, no significant differences in faecal score, growth, gross pathology or histological changes were identified between the two groups. The pathogenic E. ovinoidalis was the dominant species, and no significant difference in the individual prevalence of E. ovinoidalis post-treatment was found between treated (66.9%) and control lambs (61.9%). Other species identified included E. crandallis/weybridgensis, E. parva, E. marsica, E. faurei, E. pallida, E. ahsata and E. bakuensis.ConclusionsThis study confirms toltrazuril resistance in ovine Eimeria spp.; in addition, the data support the use of FOCRT as an appropriate tool for field evaluation of anticoccidial efficacy. Due to limited anticoccidial treatment alternatives, these findings may have important implications for the sheep industry, particularly in northern Europe.

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-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.