Sheep Scrapie Agent Research Articles

Integrity of Helix 2-Helix 3 Domain of the PrP Protein Is Not Mandatory for Prion Replication

The process of prion conversion is not yet well understood at the molecular level. The regions critical for the conformational change of PrP remain mostly debated and the extent of sequence change acceptable for prion conversion is poorly documented. To achieve progress on these issues, we applied a reverse genetic approach using the Rov cell system. This allowed us to test the susceptibility of a number of insertion mutants to conversion into prion in the absence of wild-type PrP molecules. We were able to propagate several prions with 8 to 16 extra amino acids, including a polyglycine stretch and His or FLAG tags, inserted in the middle of the protease-resistant fragment. These results demonstrate the possibility to increase the length of the loop between helices H2 and H3 up to 4-fold, without preventing prion replication. They also indicate that this loop probably remains unstructured in PrP(Sc). We also showed that bona fide prions can be produced following insertion of octapeptides in the two C-terminal turns of H2. These insertions do not interfere with the overall fold of the H2-H3 domain indicating that the highly conserved sequence of the terminal part of H2 is not critical for the conversion. Altogether these data showed that the amplitude of modifications acceptable for prion conversion in the core of the globular domain of PrP is much greater than one might have assumed. These observations should help to refine structural models of PrP(Sc) and elucidate the conformational changes underlying prions generation.

Recovery of Small Infectious PrPres Aggregates from Prion-infected Cultured Cells

Prion diseases are characterized by deposits of abnormal conformers of the PrP protein. Although large aggregates of proteinase K-resistant PrP (PrP(res)) are infectious, the precise relationships between aggregation state and infectivity remain to be established. In this study, we have fractionated detergent lysates from prion-infected cultured cells by differential ultracentrifugation and ultrafiltration and have characterized a previously unnoticed PrP species. This abnormal form is resistant to proteinase K digestion but, in contrast to typical aggregated PrP(res), remains in the soluble fraction at intermediate centrifugal forces and is not retained by filters of 300-kDa cutoff. Cell-based assay and inoculation to animals demonstrate that these entities are infectious. The finding that cell-derived small infectious PrP(res) aggregates can be recovered in the absence of strong in vitro denaturating treatments now gives a biological basis for investigating the role of small PrP aggregates in the pathogenicity and/or the multiplication cycle of prions.

Prions and neuronal death

Dear Editor, It is widely accepted that a prominent neuron cell loss occurs in the brain in the course of prion diseases (PDs) or transmissible spongiform encephalopathies (TSEs),1 which are a group of slowly progressive and invariably fatal neurological disorders affecting man and animals.2 Despite this, the inner mechanisms underlying prion-induced neuronal death at the central nervous system (CNS) level are still largely unclear.3 A number of peripheral tissues of the host are colonized by the infectious agent during the preclinical phase of sheep scrapie, the TSE ‘prototype'. These include several lymphoreticular system districts, such as palatine tonsils, ileal Peyer's patches (PPs), and enteric nervous system (ENS) plexuses, from which ‘neuroinvasion' – the long journey of prions to the CNS – is believed to take place.1 In this respect, calbindin (CALB)-immunoreactive (IR), neuronal nitric oxide synthase (nNOS)-IR (Figure 1), and somatostatin-IR neurons have been recently identified as three distinct cell populations residing within ileal ENS plexuses that are selectively targeted during natural and oral experimental sheep scrapie infection.4, 5 However, differently from that reported at the CNS level,1 we did not observe significant differences in total (HuC/D-IR) as well as in CALB-IR and nNOS-IR neuron cell counts between the ileal ENS plexuses from scrapie-affected sheep and those from uninfected control sheep carrying an identical ‘susceptible' (ARQ/ARQ) PrP genotype.4 Furthermore, no evidence of gut dysfunction and/or functional loss was found in these scrapie-affected sheep, which is also in open contrast with that constantly observed in the brain of any PD-affected individual.1, 2, 3 Figure 1 Sheep. Tangential histological sections from an ileal myenteric plexus (MP) of a natural scrapie-affected Sarda breed sheep carrying the homozygous (scrapie-susceptible) ARQ/ARQ PrP genotype. Specific anti-nNOS (neuronal nitric oxide synthase) (a) and ... These striking results may argue in favour of an ‘interaction pattern' between the sheep scrapie agent (and, presumably, also between other TSE agents), on one side, and the host's neurons, on the other, which is different when dealing with either CNS or ENS neurons. Alternatively, a regenerative activity allowing damaged ENS neuronal cells to be numerically restored (at least in part) should not be ruled out. In conclusion, there is no doubt that this highly fascinating and still largely ‘mysterious' topic will continue to be a real challenge for neuropathologists and neuroscientists in the years to come.

Serial Passage of Sheep Scrapie Inoculum in Suffolk Sheep

Scrapie is a naturally occurring fatal neurodegenerative disease of sheep and goats. Susceptibility to the disease is partly dependent upon the genetic makeup of the host. In a recent study, it was shown that sheep intracerebrally inoculated with a US scrapie agent (No. 13-7) developed scrapie and survived for an average of 19 months post inoculation. In the present study, when this scrapie inoculum was further passaged for 3 successive generations, the survival time was reduced by approximately 8 months in scrapie-susceptible (QQ on prion protein gene [PRNP] at codon 171) Suffolk sheep. It is concluded that inoculum No. 13-7 appears to have been stabilized in susceptible (171 QQ) Suffolk sheep and may be considered a specific isolate of sheep scrapie agent in the USA and therefore that it can be used to evaluate other isolates of sheep scrapie in this country.

Strain‐specific viral properties of variant Creutzfeldt–Jakob disease (vCJD) are encoded by the agent and not by host prion protein

Human CJD, endemic sheep scrapie, epidemic bovine spongiform encephalopathy (BSE), and other transmissible spongiform encephalopathies (TSEs), are caused by a group of related but molecularly uncharacterized infectious agents. The UK-BSE agent infected many species, including humans where it causes variant CJD (vCJD). As in most viral infections, different TSE disease phenotypes are determined by both the agent strain and the host species. TSE strains are most reliably classified by incubation time and regional neuropathology in mice expressing wild-type (wt) prion protein (PrP). We compared vCJD to other human and animal derived TSE strains in both mice and neuronal cultures expressing wt murine PrP. Primary and serial passages of the human vCJD agent, as well as the highly selected mutant 263K sheep scrapie agent, revealed profound strain-specific characteristics were encoded by the agent, not by host PrP. Prion theory posits that PrP converts itself into the infectious agent, and thus short incubations require identical PrP sequences in the donor and recipient host. However, wt PrP mice injected with human vCJD brain homogenates showed dramatically shorter primary incubation times than mice expressing only human PrP, a finding not in accord with a PrP species barrier. All mouse passage brains showed the vCJD agent derived from a stable BSE strain. Additionally, both vCJD brain and monotypic neuronal cultures produced a diagnostic 19 kDa PrP fragment previously observed only in BSE and vCJD primate brains. Monotypic cultures can be used to identify the intrinsic, strain-determining molecules of TSE infectious particles.

Experimental Transmission of US Scrapie Agent by Nasal, Peritoneal, and Conjunctival Routes to Genetically Susceptible Sheep

Scrapie is a naturally occurring fatal neurodegenerative disease of sheep and goats. This study documents incubation periods, pathologic findings, and distribution of abnormal prion proteins (PrP(Sc)) by immunohistochemistry in tissues of genetically susceptible sheep inoculated with US sheep scrapie agent. Four-month-old Suffolk lambs (QQ at codon 171) were inoculated by 1 of 3 different routes (nasal, peritoneal, and conjunctival) with an inoculum (No. 13-7) consisting of a pool of scrapie-affected sheep brains. Except for 3 sheep, all inoculated animals were euthanized when advanced clinical signs of scrapie were observed between 19 and 46 months postinoculation (MPI). Spongiform lesions in the brains and labeling of PrP(Sc) in central nervous system and lymphoid tissues were present in these sheep. One intranasally inoculated sheep euthanized at 12 MPI had presence of PrP(Sc) that was confined to the pharyngeal tonsil. These results indicate that the upper respiratory tract, specifically the pharyngeal tonsil, may serve as a portal of entry for prion protein in scrapie-infected environments.

PrP c Does Not Mediate Internalization of PrP Sc but Is Required at an Early Stage for De Novo Prion Infection of Rov Cells

We have studied the interactions of exogenous prions with an epithelial cell line inducibly expressing PrPc protein and permissive to infection by a sheep scrapie agent. We demonstrate that abnormal PrP (PrPSc) and prion infectivity are efficiently internalized in Rov cells, whether or not PrPc is expressed. At odds with earlier studies implicating cellular heparan sulfates in PrPSc internalization, we failed to find any involvement of such molecules in Rov cells, indicating that prions can enter target cells by several routes. We further show that PrPSc taken up in the absence of PrPc was unable to promote efficient prion multiplication once PrPc expression was restored in the cells. This observation argues that interaction of PrPSc with PrPc has to occur early, in a specific subcellular compartment(s), and is consistent with the view that the first prion multiplication events may occur at the cell surface.

Activation of the JNK–c‐Jun pathway during the early phase of neuronal apoptosis induced by PrP106–126 and prion infection

Prion diseases are neurodegenerative pathologies characterized by apoptotic neuronal death. Although the late execution phase of neuronal apoptosis is beginning to be characterized, the sequence of events occurring during the early decision phase is not yet well known. In murine cortical neurons in primary culture, apoptosis was first induced by exposure to a synthetic peptide homologous to residues 106-126 of the human prion protein (PrP), PrP106-126. Exposure to its aggregated form induced a massive neuronal death within 24 h. Apoptosis was characterized by nuclear fragmentation, neuritic retraction and fragmentation and activation of caspase-3. During the early decision phase, reactive oxygen species were detected after 3 h. Using immunocytochemistry, we showed a peak of phosphorylated c-Jun-N-terminal kinase (JNK) translocation into the nucleus after 8 h, along with the activation of the nuclear c-Jun transcription factor. Both pharmacological inhibition of JNK by SP600125 and overexpression of a dominant negative form of c-Jun significantly reduced neuronal death, while the MAPK p38 inhibitor SB203580 had no effect. Apoptosis was also studied after exposure of tg338 cortical neurons in primary culture to sheep scrapie agent. In this model, prion-induced neuronal apoptosis gradually increased with time and induced a 40% cell death after 2 weeks exposure. Immunocytochemical analysis showed early c-Jun activation after 7 days. In summary, the JNK-c-Jun pathway plays an important role in neuronal apoptosis induced by PrP106-126. This pathway is also activated during scrapie infection and may be involved in prion-induced neuronal death. Pharmacological blockade of early pathways opens new therapeutic prospects for scrapie PrP-based pathologies.

Prions can infect primary cultured neurons and astrocytes and promote neuronal cell death.

Transmissible spongiform encephalopathies arise as a consequence of infection of the central nervous system by prions, where neurons and glial cells are regarded as primary targets. Neuronal loss and gliosis, associated with the accumulation of misfolded prion protein (PrP), are hallmarks of prion diseases; yet the mechanisms underlying such disorders remain unclear. Here we introduced a cell system based on primary cerebellar cultures established from transgenic mice expressing ovine PrP and then exposed to sheep scrapie agent. Upon exposure to low doses of infectious agent, such cultures, unlike cultures originating from PrP null mice, were found to accumulate de novo abnormal PrP and infectivity, as assessed by mouse bioassay. Importantly, using astrocyte and neuron/astrocyte cocultures, both cell types were found capable of sustaining efficient prion propagation independently, leading to the production of proteinase K-resistant PrP of the same electrophoretic profile as in diseased brain. Moreover, contrasting with data obtained in chronically infected cell lines, late-occurring apoptosis was consistently demonstrated in the infected neuronal cultures. Our results provide evidence that primary cultured neural cells, including postmitotic neurons, are permissive to prion replication, thus establishing an approach to study the mechanisms involved in prion-triggered neurodegeneration at a cellular level.

Transmission of sheep scrapie to elk (Cervus elaphus nelsoni) by intracerebral inoculation: final outcome of the experiment.

This is a final report of an experimental transmission of sheep scrapie agent by intracerebral inoculation to Rocky Mountain elk (Cervus elaphus nelsoni). It documents results obtained in experimental (n = 6) and control (n = 2) elk. During the first 2 years postinoculation (PI), 3 animals died or were euthanized because of infection or injuries other than spongiform encephalopathy (SE). In years 3 and 4 PI, 3 other inoculated elk died after brief terminal neurological episodes. Necropsy of these animals revealed moderate weight loss but no other gross lesions. Microscopically, characteristic lesions of SE were seen throughout the brain and spinal cord, and the tissue was positive for proteinase K-resistant prion protein (PrPres) by immunohistochemistry (IHC) and by Western blot. Scrapie-associated fibrils (SAF) were observed by negative-stain electron microscopy in the brain of elk with neurologic signs. PrPres and SAF were not detected in the 3 inoculated elk necropsied during the first 2 years or in the 2 control animals. Retrospective analysis of the gene-encoding cervid PrP revealed a polymorphism at codon 132. The elk with SE were either homozygous (MM) or heterozygous (LM). These findings confirm that intracerebral inoculation of sheep scrapie agent results in SE with accumulations of PrPres in the central nervous system of elk. Based on morphologic and IHC findings, the experimentally induced SE cannot be distinguished from chronic wasting disease of elk with currently available diagnostic techniques.

Cultured Peripheral Neuroglial Cells Are Highly Permissive to Sheep Prion Infection

Transmissible spongiform encephalopathies arise as a consequence of infection of the central nervous system (CNS) by prions. Spreading of the infectious agent through the peripheral nervous system (PNS) may represent a crucial step toward CNS neuroinvasion, but the modalities of this process have yet to be clarified. Here we provide further evidence that PNS glial cells are likely targets for infection by prions. Glial cell clones originating from dorsal root ganglia of transgenic mice expressing ovine PrP (tgOv) and simian virus 40 T antigen were found to be readily infectible by sheep scrapie agent. This led us to establish two stable cell lines that exhibited features of Schwann cells. These cells were shown to sustain an efficient and stable replication of sheep prion based on the high level of accumulation of abnormal PrP and infectivity in exposed cultures. We also provide evidence for abnormal PrP deposition in peripheral neuroglial cells from scrapie-infected tgOv mice and sheep. These findings have potential implications in terms of designing new cell systems permissive to prions and of peripheral pathobiology of prion infections.

Preliminary Observations on the Experimental Transmission of Scrapie to Elk (Cervus elaphus nelsoni) by Intracerebral Inoculation

To determine the transmissibility of scrapie to Rocky Mountain elk (Cervus elaphus nelsoni), six elk calves were inoculated intracerebrally with brain suspension from sheep naturally affected with scrapie. One elk developed a brain abscess and was euthanatized at 7 weeks postinoculation (PI), and two others died at 6 and 15 months PI because of physical injuries. At 25 and 35 months PI, two other elk died after brief terminal neurologic episodes. Necropsy of these revealed moderate weight loss but no other gross lesions. Microscopically, characteristic lesions of spongiform encephalopathy were seen throughout the brains and the spinal cords, and in both cases these tissues were positive for PrPres by immunohistochemistry. Brains of both animals were positive for PrPres by western blot and for scrapie-associated fibrils (SAFs) by negative stain electron microscopy. PrPres and SAFs were not detected in the three elk that died or were euthanatized because of coincidental causes. Over 3.5 years after initiation of this experiment, the one remaining inoculated elk and two uninoculated (control) elk are alive and apparently healthy. These preliminary findings demonstrate that 1) sheep scrapie agent can be transmitted to elk by intracerebral inoculation; 2) the infection can result in severe, widely distributed spongiform change and accumulations of PrPres in the central nervous system (CNS); and 3) based on the examination of a limited number of CNS sections from two cases, this condition cannot be distinguished from chronic wasting disease with currently available diagnostic techniques.

Resistance of domestic cats to a US sheep scrapie agent by intracerebral route.

Feline spongiform encephalopathy (FSE) is thought to have resulted from consumption of food contaminated with bovine spongiform encephalopathy and the latter is believed to result from the consumption of food contaminated with scrapie. However, no direct experimental documentation exists to indicate that the scrapie agent is capable of amplifying in cats, and, therefore, crossing the species barrier. During 1979, 6 cats ranging in age from 3.5 to 18 months were intracerebrally inoculated with sheep scrapie (inoculum G-639-PP) and were observed for an extended period. Inoculated cats did not develop neurologic disease, and microscopic lesions of spongiform encephalopathy were not evident. Immunohistochemistry and Western blot techniques failed to detect the abnormal form of prion protein (PrP(res)). These results indicate that the sheep scrapie agent (G-639-PP) used in this study was not capable of amplifying in cats and therefore was unable to cross the species barrier to produce FSE.

Molecular analysis of the abnormal prion protein during coinfection of mice by bovine spongiform encephalopathy and a scrapie agent.

Molecular features of the proteinase K-resistant prion protein (PrP res) may discriminate among prion strains, and a specific signature could be found during infection by the infectious agent causing bovine spongiform encephalopathy (BSE). To investigate the molecular basis of BSE adaptation and selection, we established a model of coinfection of mice by both BSE and a sheep scrapie strain (C506M3). We now show that the PrP res features in these mice, characterized by glycoform ratios and electrophoretic mobilities, may be undistinguishable from those found in mice infected with scrapie only, including when mice were inoculated by both strains at the same time and by the same intracerebral inoculation route. Western blot analysis using different antibodies against sequences near the putative N-terminal end of PrP res also demonstrated differences in the main proteinase K cleavage sites between mice showing either the BSE or scrapie PrP res profile. These results, which may be linked to higher levels of PrP res associated with infection by scrapie, were similar following a challenge by a higher dose of the BSE agent during coinfection by both strains intracerebrally. Whereas PrP res extraction methods used allowed us to distinguish type 1 and type 2 PrP res, differing, like BSE and scrapie, by their electrophoretic mobilities, in the same brain region of some patients with Creutzfeldt-Jakob disease, analysis of in vitro mixtures of BSE and scrapie brain homogenates did not allow us to distinguish BSE and scrapie PrP res. These results suggest that the BSE agent, the origin of which remains unknown so far but which may have arisen from a sheep scrapie agent, may be hidden by a scrapie strain during attempts to identify it by molecular studies and following transmission of the disease in mice.

Prions

proteinaceous infectious particles and slow, or unconventional, viruses. Prions are the causative agents of the transmissible spongiform encephalopathies (TSEs). We owe mad cow disease (bovine spongiform encephalopathy; BSE) and the human equivalent, new variant Creutzfeldt–Jakob disease, to one strain of these infectious little molecules but the most common TSE by far is scrapie, a neurodegenerative affliction of sheep and goats. As far as we know, only mammals seem to be susceptible to these diseases. So, no mad chickens yet. Antarctic, fairy, thin-billed or broad-billed prions (South Atlantic seabirds) or the wreckfish (Polyprion americanus), sole member of the Polyprionidae. Yes, Reed Wickner, a yeast geneticist, does. His definition of the prion encompasses not only mammalian disease agents but also proteins that allow non-Mendalian inheritance of certain traits in yeast and fungi. But the term really belongs to Stan Prusiner of UCSF, winner of the 1998 Nobel Prize for Physiology or Medicine, who resurrected J.S. Griffiths' 1967 idea of an infectious protein. After years in the intellectual wilderness, he gave this idea the West Coast treatment, repackaged it as the ‘prion hypothesis’, and never looked back. Crucially, he also discovered a candidate protein, PrP, and a great deal else. In its most common form, PrPC, this 208-amino-acid glycoprotein is tethered to nerve cell membranes by a single glycolipid anchor. But it is also found in lymphoid tissue and, at relatively high concentrations, as a free-floating monomeric protein in blood. This form is not infectious, nor does it seem to cause any harm. It's PrPSc, the abnormal isoform of PrP, that does the damage. This is the form found associated with the disease, and the infectious agent, which is relatively insoluble and resistant to proteolysis. Not very. Perhaps they differ by only a minor twist or turn of their polypeptide chains but dogma has it the interchange of PrPC to PrPSc is self-perpetuating, with twisted PrPSc serving as a template for the demonic conversion of PrPC. The conversion can be produced in the test tube but it is not autocatalytic in vitro, and the ‘new PrPSc’ has not yet been shown to be infectious in vivo. NMR spectroscopy has given us a picture of PrPC but the structure of PrPSc and the mechanism of conversion remain unknown. Last year it was simpler. Now, the convenient criteria used to distinguish ‘non-infectious’ from ‘infectious’ — relative protease-resistance and insolubility — have been demolished. In one recent study, a ‘soluble’ PrPSc of 106 amino-acids was genetically engineered in transgenic mice. In another study, a highly infectious prion strain obtained from a patient with a rare human TSE (Gerstmann–Staussler–Scheinker syndrome) transmitted a TSE-like disease to mice without noticeably changing their normal prion protein. Confused? Join the growing club of people who now doubt the classical description of the infectious agent as ‘PrPSc, a proteinase-resistant protein’ and are looking for other factors. Who knows? The protein binds copper and might serve as a copper transport protein in the cellular response to oxidative stress. But it's one of the many proteins that can be ‘knocked-out’ in transgenic mice without gross adverse effects. The mice eat, drink and mate to their heart's content, although some have trouble sleeping and remembering things … typical research students, actually. But most critically, the mice can't develop prion diseases and they can't replicate the infectious agent. And, yes, someone has patented a PrP knockout cow, with a view to curbing BSE. Stan Prusiner (of course), Charles Weissmann and Adriano Aguzzi, but the word prion has been shunned by Alan Dickinson, discoverer of the murine prion protein gene, Sinc. With Hugh Fraser, he was the first to produce rigorous criteria for typing different strains of sheep scrapie agent. Their criteria are based on the brain pathology and incubation times generated by these scrapie strains in mice of differing Sinc genotypes. Their methods were also critical in showing that BSE and new variant CJD are caused by a similar strain of agent. The lack of a molecular explanation for the variety of TSE strains continues to be a thorn in the side of those who believe a single protein could be the infectious agent. Sadly, no. Pentosan sulphate seems to be the most promising prophylactic agent (in mice, that is) but no compound has yet been effective in humans. J Hope, Institute for Animal Health, Compton, Berkshire, RG20 7NN, UK.

Second passage of a US scrapie agent in cattle

Scrapie and bovine spongiform encephalopathy are similar chronic neurodegenerative diseases of sheep and cattle. An earlier study showed that, on first passage in cattle, a US scrapie agent caused an encephalopathy that was distinct from bovine spongiform encephalopathy (BSE). The present report describes a second passage in cattle, carried out because diseases caused by the spongiform encephalopathy agents often change in character with additional passages in abnormal hosts. For this work, young calves were inoculated intracerebrally with a pooled suspension of brain from cattle that had died of encephalopathy after experimental inoculation with brain from scrapie-affected sheep. The second passage disease was essentially identical with the first passage disease, as judged by clinical signs, histopathological findings and distribution of "prion protein scrapie" (PrPsc). This represents additional evidence to suggest that the US sheep scrapie agent tested is incapable of causing BSE in cattle.

Intracerebral Transmission of Scrapie to Cattle

To determine if sheep scrapie agent(s) in the United States would induce a disease in cattle resembling bovine spongiform encephalopathy, 18 newborn calves were inoculated intracerebrally with a pooled suspension of brain from 9 sheep with scrapie. Half of the calves were euthanatized 1 year after inoculation. All calves kept longer than 1 year became severely lethargic and demonstrated clinical signs of motor neuron dysfunction that were manifest as progressive stiffness, posterior paresis, general weakness, and permanent recumbency. The incubation period was 14-18 months, and the clinical course was 1-5 months. The brain from each calf was examined for lesions and for protease-resistant prion protein. Lesions were subtle, but a disease-specific isoform of the prion protein was present in the brain of all calves. Neither signs nor lesions were characteristic of those for bovine spongiform encephalopathy.

Physicochemical and biological characterizations of distinct strains of the transmissible mink encephalopathy agent

Inoculation of the Stetsonville, Wisconsin source of transmissible mink encephalopathy (TME) into Syrian hamsters has identified two strains of the TME agent having distinct biological properties and producing disease-specific prion proteins (PrPTME) having different physicochemical properties. Although several strains of the sheep scrapie agent have been identified in Great Britain, this is the first indication that agents producing transmissible spongiform encephalopathies in the United States also are capable of producing distinct strains.