BSE can propagate in sheep co-infected or pre-infected with scrapie.

To understand the possible role of mixed-prion infections in disease presentation, the current study reports the co-infection of sheep with bovine spongiform encephalopathy (BSE) and scrapie. The bovine BSE agent was inoculated subcutaneously into sheep with ARQ/ARQ or VRQ/ARQ PRNP genotypes either at the same time as subcutaneous challenge with scrapie, or three months later. In addition, VRQ/VRQ sheep naturally infected with scrapie after being born into a scrapie-affected flock were challenged subcutaneously with BSE at eight or twenty one months-of-age. Sheep were analysed by incubation period/attack rate, and western blot of brain tissue determined the presence of BSE or scrapie-like PrPSc. Serial protein misfolding cyclic amplification (sPMCA) that can detect very low levels of BSE in the presence of an excess of scrapie agent was also applied to brain and lymphoreticular tissue. For VRQ/ARQ sheep challenged with mixed infections, scrapie-like incubation periods were produced, and no BSE agent was detected. However, whilst ARQ/ARQ sheep developed disease with BSE-like incubation periods, some animals had a dominant scrapie western blot phenotype in brain, but BSE was detected in these sheep by sPMCA. In addition, VRQ/VRQ animals challenged with BSE after natural exposure to scrapie had scrapie-like incubation periods and dominant scrapie PrPSc in brain, but one sheep had BSE detectable by sPMCA in the brain. Overall, the study demonstrates for the first time that for scrapie/BSE mixed infections, VRQ/ARQ sheep with experimental scrapie did not propagate BSE but VRQ/VRQ sheep with natural scrapie could propagate low levels of BSE, and whilst BSE readily propagated in ARQ/ARQ sheep it was not always the dominant PrPSc strain in brain tissue. Indeed, for several animals, a dominant scrapie biochemical phenotype in brain did not preclude the presence of BSE prion.

Airway compliance measurements in mouse models of respiratory diseases.

The quantification of airway compliance (Caw) is essential to the study of airway alterations in disease models. However, the required measurements of airway pressure and volume are difficult to acquire in mice. We hypothesized that the inflation limb of full-range pressure-volume (PV) curves could be used to quantify Caw, as it contains a segment where only the airway tree is distended. The study objective was to assess the feasibility of the approach by analysis of full-range PV curves previously collected in three mouse models: an elastase model of emphysema, a genetic model spontaneously developing emphysema (leukotriene C4 synthase knockout; LTC4S-KO), and a bleomycin model of lung fibrosis. Attempts to validate results included Caw change relative to respiratory system compliance (ΔCaw/ΔC), the minute work of breathing (mWOB), and the elastance at 20.5 Hz (Ers_20.5) from prior respiratory mechanics measurements in the same subjects. Caw was estimated at 3% of total compliance in healthy mice or 2.3 ± 1 µL/cmH2O (n = 17). The technique detected changes in models of respiratory obstructive and restrictive diseases relative to control mice as well as differences in the two emphysema models studied. The changes in Caw were consistent with those seen in ΔCaw/ΔC, mWOB, or Ers_20.5, with some variations according to the model, as well as with results reported in the literature in humans and mice. Direct Caw measurements in subjects as small as mice could prove useful to further characterize other respiratory disease models associated with airway remodeling or to assess treatment effects.

A high-throughput method for unbiased quantitation and categorization of nuclear morphology†.

The physical arrangement of chromatin in the nucleus is cell type and species-specific, a fact particularly evident in sperm, in which most of the cytoplasm has been lost. Analysis of the characteristic falciform ("hook shaped") sperm in mice is important in studies of sperm development, hybrid sterility, infertility, and toxicology. However, quantification of sperm shape differences typically relies on subjective manual assessment, rendering comparisons within and between samples difficult. We have developed an analysis program for morphometric analysis of asymmetric nuclei and characterized the sperm of mice from a range of inbred, outbred, and wild-derived mouse strains. We find that laboratory strains have elevated sperm shape variability both within and between samples in comparison to wild-derived inbred strains, and that sperm shape in F1 offspring from a cross between CBA and C57Bl6J strains is subtly affected by the direction of the cross. We further show that hierarchical clustering can discriminate distinct sperm shapes with greater efficiency and reproducibility than even experienced manual assessors, and is useful both to distinguish between samples and also to identify different morphological classes within a single sample. Our approach allows for the analysis of nuclear shape with unprecedented precision and scale and will be widely applicable to different species and different areas of biology.

Automated Nuclear Cartography Reveals Conserved Sperm Chromosome Territory Localization across 2 Million Years of Mouse Evolution.

Measurements of nuclear organization in asymmetric nuclei in 2D images have traditionally been manual. This is exemplified by attempts to measure chromosome position in sperm samples, typically by dividing the nucleus into zones, and manually scoring which zone a fluorescence in-situ hybridisation (FISH) signal lies in. This is time consuming, limiting the number of nuclei that can be analyzed, and prone to subjectivity. We have developed a new approach for automated mapping of FISH signals in asymmetric nuclei, integrated into an existing image analysis tool for nuclear morphology. Automatic landmark detection defines equivalent structural regions in each nucleus, then dynamic warping of the FISH images to a common shape allows us to generate a composite of the signal within the entire cell population. Using this approach, we mapped the positions of the sex chromosomes and two autosomes in three mouse lineages (Mus musculus domesticus, Mus musculus musculus and Mus spretus). We found that in all three, chromosomes 11 and 19 tend to interact with each other, but are shielded from interactions with the sex chromosomes. This organization is conserved across 2 million years of mouse evolution.

Ultrastructure and pathology of prion protein amyloid accumulation and cellular damage in extraneural tissues of scrapie-infected transgenic mice expressing anchorless prion protein.

In most human and animal prion diseases the abnormal disease-associated prion protein (PrPSc) is deposited as non-amyloid aggregates in CNS, spleen and lymphoid organs. In contrast, in humans and transgenic mice with PrP mutations which cause expression of PrP lacking a glycosylphosphatidylinositol (GPI)-anchor, most PrPSc is in the amyloid form. In transgenic mice expressing only anchorless PrP (tg anchorless), PrPSc is deposited not only in CNS and lymphoid tissues, but also in extraneural tissues including heart, brown fat, white fat, and colon. In the present paper, we report ultrastructural studies of amyloid PrPSc deposition in extraneural tissues of scrapie-infected tg anchorless mice. Amyloid PrPSc fibrils identified by immunogold-labeling were visible at high magnification in interstitial regions and around blood vessels of heart, brown fat, white fat, colon, and lymphoid tissues. PrPSc amyloid was located on and outside the plasma membranes of adipocytes in brown fat and cardiomyocytes, and appeared to invaginate and disrupt the plasma membranes of these cell types, suggesting cellular damage. In contrast, no cellular damage was apparent near PrPSc associated with macrophages in lymphoid tissues and colon, with enteric neuronal ganglion cells in colon or with adipocytes in white fat. PrPSc localized in macrophage phagolysosomes lacked discernable fibrils and might be undergoing degradation. Furthermore, in contrast to wild-type mice expressing GPI-anchored PrP, in lymphoid tissues of tg anchorless mice, PrPSc was not associated with follicular dendritic cells (FDC), and FDC did not display typical prion-associated pathogenic changes.

PrP aggregation can be seeded by pre-formed recombinant PrP amyloid fibrils without the replication of infectious prions.

Mammalian prions are unusual infectious agents, as they are thought to consist solely of aggregates of misfolded prion protein (PrP). Generation of synthetic prions, composed of recombinant PrP (recPrP) refolded into fibrils, has been utilised to address whether PrP aggregates are, indeed, infectious prions. In several reports, neurological disease similar to transmissible spongiform encephalopathy (TSE) has been described following inoculation and passage of various forms of fibrils in transgenic mice and hamsters. However, in studies described here, we show that inoculation of recPrP fibrils does not cause TSE disease, but, instead, seeds the formation of PrP amyloid plaques in PrP-P101L knock-in transgenic mice (101LL). Importantly, both WT-recPrP fibrils and 101L-recPrP fibrils can seed plaque formation, indicating that the fibrillar conformation, and not the primary sequence of PrP in the inoculum, is important in initiating seeding. No replication of infectious prions or TSE disease was observed following both primary inoculation and subsequent subpassage. These data, therefore, argue against recPrP fibrils being infectious prions and, instead, indicate that these pre-formed seeds are acting to accelerate the formation of PrP amyloid plaques in 101LL Tg mice. In addition, these data reproduce a phenotype which was previously observed in 101LL mice following inoculation with brain extract containing in vivo-generated PrP amyloid fibrils, which has not been shown for other synthetic prion models. These data are reminiscent of the "prion-like" spread of aggregated forms of the beta-amyloid peptide (Aß), α-synuclein and tau observed following inoculation of transgenic mice with pre-formed seeds of each misfolded protein. Hence, even when the protein is PrP, misfolding and aggregation do not reproduce the full clinicopathological phenotype of disease. The initiation and spread of protein aggregation in transgenic mouse lines following inoculation with pre-formed fibrils may, therefore, more closely resemble a seeded proteinopathy than an infectious TSE disease.

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

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

Comparative Susceptibility of Sheep of Different Origins, Breeds and PRNP Genotypes to Challenge with Bovine Spongiform Encephalopathy and Scrapie.

Sheep are natural hosts of the prion disease, scrapie. They are also susceptible to experimental challenge with various scrapie strains and with bovine spongiform encephalopathy (BSE), which affects cattle and has been accidentally transmitted to a range of other species, including man. Incidence and incubation period of clinical disease in sheep following inoculation is controlled by the PRNP gene, which has different alleles defined on the basis of polymorphisms, particularly at codons 136, 154 and 171, although other codons are associated with survival time, and the exact responses of the sheep may be influenced by other breed-related differences. Here we report the results of a long term single study of experimental scrapie and BSE susceptibility of sheep of Cheviot, Poll Dorset and Suffolk breeds, originating from New Zealand and of a wide range of susceptible and resistant PRNP genotypes. Responses were compared with those of sheep from a closed Cheviot flock of UK origin (Roslin Cheviot flock). The unusually long observation period (6-8 years for most, but up to 12 years for others) allows us to draw robust conclusions about rates of survival of animals previously regarded as resistant to infection, particularly PRNP heterozygotes, and is the most comprehensive such study reported to date. BSE inoculation by an intracerebral route produced disease in all genotype groups with differing incubation periods, although M112T and L141F polymorphisms seemed to give some protection. Scrapie isolate SSBP/1, which has the shortest incubation period in sheep with at least one VRQ PRNP allele, also produced disease following sub-cutaneous inoculation in ARQ/ARQ animals of New Zealand origin, but ARQ/ARQ sheep from the Roslin flock survived the challenge. Our results demonstrate that the links between PRNP genotype and clinical prion disease in sheep are much less secure than previously thought, and may break down when, for example, a different breed of sheep is moved into a new flock.

Stability of murine scrapie strain 87V after passage in sheep and comparison with the CH1641 ovine strain.

Breed- and prion protein (PRNP) genotype-related disease phenotype variability has been observed in sheep infected with the 87V murine scrapie strain. Therefore, the stability of this strain was tested by inoculating sheep-derived 87V brain material back into VM mice. As some sheep-adapted 87V disease phenotypes were reminiscent of CH1641 scrapie, transgenic mice (Tg338) expressing ovine prion protein (PrP) were inoculated with the same sheep-derived 87V sources and with CH1641. Although at first passage in VM mice the sheep-derived 87V sources showed some divergence from the murine 87V control, all the characteristics of murine 87V infection were recovered at second passage from all sheep sources. These included 100 % attack rates and indistinguishable survival times, lesion profiles, immunohistochemical features of disease-associated PrP accumulation in the brain and PrP biochemical properties. All sheep-derived 87V sources, as well as CH1641, were transmitted to Tg338 mice with identical clinical, pathological, immunohistochemical and biochemical features. While this might potentially indicate that sheep-adapted 87V and CH1641 are the same strain, profound divergences were evident, as murine 87V was unable to infect Tg338 mice but was lethal for VM mice, while the reverse was true for CH1641. These combined data suggest that: (i) murine 87V is stable and retains its properties after passage in sheep; (ii) it can be isolated from sheep showing a CH1641-like or a more conventional scrapie phenotype; and (iii) sheep-adapted 87V scrapie, with conventional or CH1641-like phenotype, is biologically distinct from experimental CH1641 scrapie, despite the fact that they behave identically in a single transgenic mouse line.

Dynamics of the natural transmission of bovine spongiform encephalopathy within an intensively managed sheep flock.

Sheep are susceptible to the bovine spongiform encephalopathy (BSE) agent and in the UK they may have been exposed to BSE via contaminated meat and bone meal. An experimental sheep flock was established to determine whether ovine BSE could be naturally transmitted under conditions of intensive husbandry. The flock consisted of 113 sheep of different breeds and susceptible PRNP genotypes orally dosed with BSE, 159 sheep subsequently born to them and 125 unchallenged sentinel controls. BSE was confirmed in 104 (92%) orally dosed sheep and natural transmission was recorded for 14 of 79 (18%) lambs born to BSE infected dams, with rates varying according to PRNP genotype. The likelihood of natural BSE transmission was linked to stage of incubation period of the dam: the attack rate for lambs born within 100 days of the death of BSE infected dams was significantly higher (9/22, 41%) than for the rest (5/57, 9%). Within the group of ewes lambing close to death, those rearing infected progeny (n = 8, for 9/12 infected lambs) showed a significantly greater involvement of lymphoid tissues than those rearing non-infected offspring (n = 8, for 0/10 infected lambs). Horizontal transmission to the progeny of non-infected mothers was recorded only once (1/205, 0.5%). This low rate of lateral transmission was attributed, at least partly, to an almost complete absence of infected placentas. We conclude that, although BSE can be naturally transmitted through dam-lamb close contact, the infection in this study flock would not have persisted due to low-efficiency maternal and lateral transmissions.

Archival search for historical atypical scrapie in sheep reveals evidence for mixed infections.

Natural scrapie in sheep occurs in classical and atypical forms, which may be distinguished on the basis of the associated neuropathology and properties of the disease-associated prion protein on Western blots. First detected in 1998, atypical scrapie is known to have occurred in UK sheep since the 1980s. However, its aetiology remains unclear and it is often considered as a sporadic, non-contagious disease unlike classical scrapie which is naturally transmissible. Although atypical scrapie tends to occur in sheep of prion protein (PRNP) genotypes that are different from those found predominantly in classical scrapie, there is some overlap so that there are genotypes in which both scrapie forms can occur. In this search for early atypical scrapie cases, we made use of an archive of fixed and frozen sheep samples, from both scrapie-affected and healthy animals (∼1850 individuals), dating back to the 1960s. Using a selection process based primarily on PRNP genotyping, but also on contemporaneous records of unusual clinical signs or pathology, candidate sheep samples were screened by Western blot, immunohistochemistry and strain-typing methods using tg338 mice. We identified, from early time points in the archive, three atypical scrapie cases, including one sheep which died in 1972 and two which showed evidence of mixed infection with classical scrapie. Cases with both forms of scrapie in the same animal as recognizable entities suggest that mixed infections have been around for a long time and may potentially contribute to the variety of scrapie strains.

Intraperitoneal Infection of Wild-Type Mice with Synthetically Generated Mammalian Prion.

The prion hypothesis postulates that the infectious agent in transmissible spongiform encephalopathies (TSEs) is an unorthodox protein conformation based agent. Recent successes in generating mammalian prions in vitro with bacterially expressed recombinant prion protein provide strong support for the hypothesis. However, whether the pathogenic properties of synthetically generated prion (rec-Prion) recapitulate those of naturally occurring prions remains unresolved. Using end-point titration assay, we showed that the in vitro prepared rec-Prions have infectious titers of around 104 LD50/µg. In addition, intraperitoneal (i.p.) inoculation of wild-type mice with rec-Prion caused prion disease with an average survival time of 210-220 days post inoculation. Detailed pathological analyses revealed that the nature of rec-Prion induced lesions, including spongiform change, disease specific prion protein accumulation (PrP-d) and the PrP-d dissemination amongst lymphoid and peripheral nervous system tissues, the route and mechanisms of neuroinvasion were all typical of classical rodent prions. Our results revealed that, similar to naturally occurring prions, the rec-Prion has a titratable infectivity and is capable of causing prion disease via routes other than direct intra-cerebral challenge. More importantly, our results established that the rec-Prion caused disease is pathogenically and pathologically identical to naturally occurring contagious TSEs, supporting the concept that a conformationally altered protein agent is responsible for the infectivity in TSEs.

A Naturally Occurring Bovine Tauopathy Is Geographically Widespread in the UK.

Many human neurodegenerative diseases are associated with hyperphosphorylation and widespread intra-neuronal and glial associated aggregation of the microtubule associated protein tau. In contrast, animal tauopathies are not reported with only senescent animals showing inconspicuous tau labelling of fine processes albeit significant tau aggregation may occur in some experimental animal disease. Since 1986, an idiopathic neurological condition of adult cattle has been recognised in the UK as a sub-set of cattle slaughtered as suspect bovine spongiform encephalopathy cases. This disorder is characterised by brainstem neuronal chromatolysis and degeneration with variable hippocampal sclerosis and spongiform change. Selected cases of idiopathic brainstem neuronal chromatolysis (IBNC) were identified from archive material and characterised using antibodies specific to several tau hyperphosphorylation sites or different isoforms of the tau microtubule binding region. Labelling was also carried out for alpha synuclein, ubiquitin, TDP43, Aß 1-42, Aß 1-40. Widespread tau labelling was identified in all IBNC brains examined and with each of seven tau antibodies recognising different hyperphosphorylated sites. Labelling with each antibody was associated with dendrites, neuronal perikarya and glia. Thus IBNC is a sporadic, progressive neurological disease predominantly affecting aged cattle that occurs throughout the UK and is associated with hyperphosphorylation of tau, a rare example of a naturally-occurring tauopathy in a non-primate species. Secondary accumulation of alpha synuclein and ubiquitin was also present. The neuropathology does not precisely correspond with any human tauopathy. The cause of IBNC remains undetermined but environmental factors and exposure to agrochemicals needs to be considered in future aetiological investigations.

Susceptibility of European red deer (Cervus elaphus elaphus) to alimentary challenge with bovine spongiform encephalopathy.

European red deer (Cervus elaphus elaphus) are susceptible to the agent of bovine spongiform encephalopathy, one of the transmissible spongiform encephalopathies, when challenged intracerebrally but their susceptibility to alimentary challenge, the presumed natural route of transmission, is unknown. To determine this, eighteen deer were challenged via stomach tube with a large dose of the bovine spongiform encephalopathy agent and clinical signs, gross and histological lesions, presence and distribution of abnormal prion protein and the attack rate recorded. Only a single animal developed clinical disease, and this was acute with both neurological and respiratory signs, at 1726 days post challenge although there was significant (27.6%) weight loss in the preceding 141 days. The clinically affected animal had histological lesions of vacuolation in the neuronal perikaryon and neuropil, typical of transmissible spongiform encephalopathies. Abnormal prion protein, the diagnostic marker of transmissible encephalopathies, was primarily restricted to the central and peripheral nervous systems although a very small amount was present in tingible body macrophages in the lymphoid patches of the caecum and colon. Serial protein misfolding cyclical amplification, an in vitro ultra-sensitive diagnostic technique, was positive for neurological tissue from the single clinically diseased deer. All other alimentary challenged deer failed to develop clinical disease and were negative for all other investigations. These findings show that transmission of bovine spongiform encephalopathy to European red deer via the alimentary route is possible but the transmission rate is low. Additionally, when deer carcases are subjected to the same regulations that ruminants in Europe with respect to the removal of specified offal from the human food chain, the zoonotic risk of bovine spongiform encephalopathy, the cause of variant Creutzfeldt-Jakob disease, from consumption of venison is probably very low.

Membrane pathology and microglial activation of mice expressing membrane anchored or membrane released forms of Aß and mutated human Alzheimer's precursor protein (APP).

AIMS: Alzheimer's disease and the transmissible spongiform encephalopathies or prion diseases accumulate misfolded and aggregated forms of neuronal cell membrane proteins. Distinctive membrane lesions caused by the accumulation of disease-associated prion protein (PrP(d)) are found in prion disease but morphological changes of membranes are not associated with Aß in Alzheimer's disease. Membrane changes occur in all prion diseases where PrP(d) is attached to cell membranes by a glycosyl-phosphoinositol (GPI) anchor but are absent from transgenic mice expressing anchorless PrP(d). Here we investigate whether GPI membrane attached Aß may also cause prion-like membrane lesions. METHODS: We used immunogold electron microscopy to determine the localization and pathology of Aß accumulation in groups of transgenic mice expressing anchored or unanchored forms of Aß or mutated human Alzheimer's precursor protein. RESULTS: GPI attached Aß did not replicate the membrane lesions of PrP(d). However, as with PrP(d) in prion disease, Aß peptides derived from each transgenic mouse line initially accumulated on morphologically normal neurite membranes, elicited rapid glial recognition and neurite Aß was transferred to attenuated microglial and astrocytic processes. CONCLUSIONS: GPI attachment of misfolded membrane proteins is insufficient to cause prion-like membrane lesions. Prion disease and murine Aß amyloidosis both accumulate misfolded monomeric or oligomeric membrane proteins that are recognized by glial processes and acquire such misfolded proteins prior to their accumulation in the extracellular space. In contrast to prion disease where glial cells efficiently endocytose PrP(d) to endolysosomes, activated microglial cells in murine Aß amyloidosis are not as efficient phagocytes.

Genotype-dependent molecular evolution of sheep bovine spongiform encephalopathy (BSE) prions in vitro affects their zoonotic potential.

Prion diseases are rare fatal neurological conditions of humans and animals, one of which (variant Creutzfeldt-Jakob disease) is known to be a zoonotic form of the cattle disease bovine spongiform encephalopathy (BSE). What makes one animal prion disease zoonotic and others not is poorly understood, but it appears to involve compatibility between the prion strain and the host prion protein sequence. Concerns have been raised that the United Kingdom sheep flock may have been exposed to BSE early in the cattle BSE epidemic and that serial BSE transmission in sheep might have resulted in adaptation of the agent, which may have come to phenotypically resemble scrapie while maintaining its pathogenicity for humans. We have modeled this scenario in vitro. Extrapolation from our results suggests that if BSE were to infect sheep in the field it may, with time and in some sheep genotypes, become scrapie-like at the molecular level. However, the results also suggest that if BSE in sheep were to come to resemble scrapie it would lose its ability to affect humans.

The protease inhibitor HAI-2, but not HAI-1, regulates matriptase activation and shedding through prostasin.

The membrane-anchored serine proteases, matriptase and prostasin, and the membrane-anchored serine protease inhibitors, hepatocyte growth factor activator inhibitor (HAI)-1 and HAI-2, are critical effectors of epithelial development and postnatal epithelial homeostasis. Matriptase and prostasin form a reciprocal zymogen activation complex that results in the formation of active matriptase and prostasin that are targets for inhibition by HAI-1 and HAI-2. Conflicting data, however, have accumulated as to the existence of auxiliary functions for both HAI-1 and HAI-2 in regulating the intracellular trafficking and activation of matriptase. In this study, we, therefore, used genetically engineered mice to determine the effect of ablation of endogenous HAI-1 and endogenous HAI-2 on endogenous matriptase expression, subcellular localization, and activation in polarized intestinal epithelial cells. Whereas ablation of HAI-1 did not affect matriptase in epithelial cells of the small or large intestine, ablation of HAI-2 resulted in the loss of matriptase from both tissues. Gene silencing studies in intestinal Caco-2 cell monolayers revealed that this loss of cell-associated matriptase was mechanistically linked to accelerated activation and shedding of the protease caused by loss of prostasin regulation by HAI-2. Taken together, these data indicate that HAI-1 regulates the activity of activated matriptase, whereas HAI-2 has an essential role in regulating prostasin-dependent matriptase zymogen activation.

Incidence of infection in Prnp ARR/ARR sheep following experimental inoculation with or natural exposure to classical scrapie.

The prion protein gene (Prnp) is highly influential in determining risk and susceptibility of sheep exposed to classical scrapie. Sheep homozygous for alanine at codon 136 and arginine at codons 154 and 171 (ARR/ARR) of the Prnp gene are historically considered to be highly resistant to classical scrapie, although they form a significant fraction of cases of atypical scrapie. To date, experimental transmission of prions to ARR/ARR sheep has only been achieved with the BSE agent and mostly by the intracerebral route. We summarise here the results of six separate studies, in which 95 sheep of the ARR/ARR genotype were naturally exposed to (n = 18) or experimentally challenged with (n = 77) natural or experimental sources of classical scrapie by the oral, intra-intestinal, subcutaneous or intracerebral routes and allowed to survive for periods of up to 94 months post-infection. Only the intracerebral route resulted in disease and/or amplification of disease associated PrP (PrPd), and only in two of 19 sheep that survived for longer than 36 months. Discriminatory immunohistochemistry and Western blot confirmed the scrapie, non-BSE signature of PrPd in those two sheep. However, the neuropathological phenotype was different from any other scrapie (classical or atypical) or BSE source previously reported in sheep of any Prnp genotype. These studies confirm the widely held view that ARR/ARR sheep are highly resistant to classical scrapie infection, at least within their commercial lifespan. Moreover, within the constraints of the present studies (only two infected sheep), these results do not support the suggestion that atypical scrapie or BSE are generated by adaptation or mutation of classical scrapie in sheep of resistant ARR/ARR genotype.

Membrane-anchored Aß accelerates amyloid formation and exacerbates amyloid-associated toxicity in mice.

Pathological, genetic, and biochemical hallmarks of Alzheimer's disease (AD) are linked to amyloid-ß (Aß) peptide aggregation. Especially misfolded Aß42 peptide is sufficient to promote amyloid plaque formation. However, the cellular compartment facilitating the conversion of monomeric Aß to aggregated toxic Aß species remains unknown. In vitro models suggest lipid membranes to be the driving force of Aß conversion. To this end, we generated two novel mouse models, expressing either membrane-anchored or nonanchored versions of the human Aß42 peptide. Strikingly, membrane-anchored Aß42 robustly accelerated Aß deposition and exacerbated amyloid-associated toxicity upon crossing with Aß precursor protein transgenic mice. These in vivo findings support the hypothesis that Aß-membrane interactions play a pivotal role in early-onset AD as well as neuronal damage and provide evidence to study Aß-membrane interactions as therapeutic targets.

Membrane toxicity of abnormal prion protein in adrenal chromaffin cells of scrapie infected sheep.

Transmissible spongiform encephalopathies (TSEs) or prion diseases are associated with accumulations of disease specific PrP (PrP(d)) in the central nervous system (CNS) and often the lymphoreticular system (LRS). Accumulations have additionally been recorded in other tissues including the peripheral nervous system and adrenal gland. Here we investigate the effect of sheep scrapie on the morphology and the accumulation of PrP(d) in the adrenal medulla of scrapie affected sheep using light and electron microscopy. Using immunogold electron microscopy, non-fibrillar forms of PrP(d) were shown to accumulate mainly in association with chromaffin cells, occasional nerve endings and macrophages. PrP(d) accumulation was associated with distinctive membrane changes of chromaffin cells including increased electron density, abnormal linearity and invaginations. Internalisation of PrP(d) from the chromaffin cell plasma membrane occurred in association with granule recycling following hormone exocytosis. PrP(d) accumulation and internalisation from membranes is similarly associated with perturbations of membrane structure and trafficking in CNS neurons and tingible body macrophages of the LRS. These data suggest that a major toxic effect of PrP(d) is at the level of plasma membranes. However, the precise nature of PrP(d)-membrane toxicity is tissue and cell specific suggesting that the normal protein may act as a multi-functional scaffolding molecule. We further suggest that the co-localisation of PrP(d) with exocytic granules of the hormone trafficking system may provide an additional source of infectivity in blood.