A trend of molecular genetics on prion diseases and prion protein

Infectious amyloid filaments designated as prion rods or scrapie associated fibrils (SAF) present in brain tissues affected by transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease (CJD), Gerstmann-Str?ussler-Scheinker disease (GSS) and kuru of humans, and scrapie of sheep. A hydrophobic glycoprotein, PrPSc is a major component of SAF, and is known to be associated with the infectivity of these diseases. Both PrPSc and the normal isoform of this glycoprotein, PrPC are encoded by a single host gene, PrP gene, and the conversion of PrPC to PrPSc is a posttranslational event. Several mutations on the PrP gene are associated with variations of the phenotype and the occurrence in familial CJD and GSS.     

The human 37-kDa laminin receptor precursor interacts with the prion protein in eukaryotic cells

Prions are thought to consist of infectious proteins that cause transmissible spongiform encephalopathies. According to overwhelming evidence, the pathogenic prion protein PrPSc converts its host encoded isoform PrPC into insoluble aggregates of PrPSc, concomitant with pathological modifications (for review, see refs. 1-3). Although the physiological role of PrPC is poorly understood, studies with PrP knockout mice demonstrated that PrPC is required for the development of prion diseases. Using the yeast two-hybrid technology in Saccharomyces cerevisiae, we identified the 37-kDa laminin receptor precursor (LRP) as interacting with the cellular prion protein PrPC. Mapping analysis of the LRP-PrP interaction site in S. cerevisiae revealed that PrP and laminin share the same binding domain (amino acids 161 to 180) on LRP. The LRP-PrP interaction was confirmed in vivo in insect (Sf9) and mammalian cells (COS-7). The LRP level was increased in scrapie-infected murine N2a cells and in brain and spleen of scrapie-infected mice. In contrast, the LRP concentration was not significantly altered in these organs from mice infected with the bovine spongiform encephalopathic agent (BSE), which have a lower PrPSc accumulation. LRP levels, however, were dramatically increased in brain and pancreas, slightly increased in the spleen and not altered in the liver of crapie-infected hamsters. These data show that enhanced LRP concentrations are correlated with PrPSc accumulation in organs from mice and hamsters. The laminin receptor precursor, which is highly conserved among mammals and is located on the cell surface, may act as a receptor or co-receptor for the prion protein on mammalian cells.     

The use of transgenic mice in the investigation of transmissible spongiform encephalopathies

The prion, the transmissible agent that causes spongiform encephalopathies such as scrapie, bovine spongiform encephalopathy and Creutzfeldt-Jakob disease, is believed to be devoid of nucleic acid and to be identical to PrPSc (prion protein: scrapie form), a modified form of the normal host protein PrPC (prion protein: cellular form) which is encoded by the single copy gene Prnp. The 'protein only' hypothesis proposes that PrPSc, when introduced into a normal host, causes the conversion of PrPC into PrPSc; it therefore predicts that an animal devoid of PrPC should be resistant to prion diseases. The authors generated homozygous Prnp(o/o) ('PrP knockout') mice and showed that, after inoculation with prions, these mice remained free from scrapie for at least two years while wild-type controls all died within six months. There was no propagation of prions in the Prnp(o/o) animals. Surprisingly, heterozygous Prnp(o/+) mice, which express PrPC at about half the normal level, also showed enhanced resistance to scrapie despite high levels of infectious agent and PrPSc in the brain at an early stage. After introduction of murine PrP transgenes, Prnp(o/o) mice became highly susceptible to mouse--but not to hamster--prions, while the insertion of Syrian hamster PrP transgenes rendered the mice susceptible to hamster prions but much less susceptible to mouse prions. These complementation experiments enabled the application of reverse genetics. The authors prepared animals transgenic for genes encoding PrP with amino terminal deletions of various lengths and found that PrP that lacks 48 amino proximal amino acids (which comprise four of the five octa repeats of PrP) is still biologically active.     

Scrapie-associated PrP accumulation and agent replication: effects of sulphated glycosaminoglycan analogues

An abnormally protease-resistant and apparently neuropathogenic form of PrP accumulates in the brains of hosts with scrapie and related transmissible spongiform encephalopathies. Studies with scrapie-infected neuroblastoma cells have highlighted dramatic differences in the metabolism of the normal (protease-sensitive) and scrapie-associated (protease-resistant) isoforms of PrP. Furthermore, this model has been useful in identifying inhibitors of protease-resistant PrP accumulation and scrapie agent replication which are valuable as potential therapeutic agents and as probes of the mechanism of protease-resistant PrP formation. These inhibitors include the amyloid stain Congo red and certain sulphated glycans which are glycosaminoglycans themselves or glycosaminoglycan analogues. The relative potencies of various sulphated glycans correlate with their previously determined anti-scrapie activities in vivo, suggesting that the prophylactic effects of sulphated polyanions is due to inhibition of protease-resistant PrP accumulation. These and other observations suggest that an interaction of PrP with endogenous sulphated glycosaminoglycans or proteoglycans is important in protease-resistant PrP accumulation, and raise the possibility that therapies for transmissible spongiform encephalopathies and other amyloidoses could be based on blocking (pre)amyloid-glycosaminoglycan interactions.     

MS-8209, a new amphotericin B derivative, provides enhanced efficacy in delaying hamster scrapie

To test the efficacy of a new amphotericin B derivative, MS-8209, in delaying scrapie, hamsters were infected intracerebrally with the 263K scrapie agent and treated with MS-8209 either early in the course of the disease or continuously. The results show that (i) all treatments lengthened the incubation period of hamster scrapie, (ii) continuous treatment with MS-8209 doubled the length of the incubation period compared with that observed in infected, untreated animals, and (iii) all treatments delayed the accumulation of a proteinase-resistant prion protein and glial fibrillary acidic protein in the brain. These findings suggest that MS-8209 is a powerful tool for investigating the pathogenesis of transmissible subacute spongiform encephalopathies.     

Inhibition of scrapie-associated PrP accumulation. Probing the role of glycosaminoglycans in amyloidogenesis

Accumulation of an abnormal, protease-resistant form of an endogenous protein, PrP, is a characteristic feature of scrapie and related transmissible spongiform encephalopathies. This abnormal isoform is also present in the amyloid plaques that are often observed in these diseases. In mouse neuroblastoma cells persistently infected with scrapie, the abnormal protease-resistant isoform of PrP is derived from an operationally normal protease-sensitive precursor. Conversion of PrP to the protease-resistant state occurs either on the plasma membrane or along an endocytic pathway by an unknown mechanism. Inhibitors of protease-resistant PrP accumulation have been identified, and these include the amyloid-binding dye Congo red and certain sulfated glycans. The similarity of these compounds to sulfated glycosaminoglycans, which are components of all natural amyloids, has led to the hypothesis that the inhibitors act by competitively blocking an interaction between endogenous glycosaminoglycan(s) and PrP that is critical for amyloidogenic PrP accumulation. The proven prophylactic effect of these sulfated glycans in animal models of scrapie suggests that they represent a group of compounds that might interfere with the pathogenic formation of amyloid in a variety of diseases, such as Alzheimer's disease.     

Monoclonal antibody F89/160.1.5 defines a conserved epitope on the ruminant prion protein

The transmissible spongiform encephalopathies are a heterogeneous group of fatal neurodegenerative disorders occurring in humans, mink, cats, and ruminant herbivores. The occurrence of novel transmissible spongiform encephalopathies in cattle in the United Kingdom and Europe and in mule deer and elk in parts of the United States has emphasized the need for reliable diagnostic tests with standardized reagents. Postmortem diagnosis is performed by histologic examination of brain sections from affected animals. The histopathological criteria for transmissible spongiform encephalopathies include gliosis, astrocytosis, neuronal degeneration, and spongiform change. These lesions vary in intensity and anatomic location depending on the host species and genetics, stage of disease, and infectious agent source. Diagnosis by histopathology alone may be ambiguous in hosts with early cases of disease and impossible if the tissue is autolyzed. Deposition of the prion protein (an abnormal isoform of a native cellular sialoglycoprotein) in the central nervous system is a reliable marker for infection, and immunohistochemical detection of this marker is a useful adjunct to histopathology. In the present paper we describe monoclonal antibody (MAb) F89/160.1.5, which reacts with prion protein in tissues from sheep, cattle, mule deer, and elk with naturally occurring transmissible spongiform encephalopathies. This MAb recognizes a conserved epitope on the prion protein in formalin-fixed, paraffin-embedded sections after hydrated autoclaving. MAb F89/160.1.5 will be useful in diagnostic and pathogenesis studies of the transmissible spongiform encephalopathies in these ruminant species.     

Neuropathological diagnostic criteria for Creutzfeldt-Jakob disease (CJD) and other human spongiform encephalopathies (prion diseases)

Neuropathological diagnostic criteria for Creutzfeldt-Jakob disease (CJD) and other human transmissible spongiform encephalopathies (prion diseases) are proposed for the following disease entities: CJD--sporadic, iatrogenic (recognised risk) or familial (same disease in 1st degree relative): spongiform encephalopathy in cerebral and/or cerebellar cortex and/or subcortical grey matter; or encephalopathy with prion protein (PrP) immunoreactivity (plaque and/or diffuse synaptic and/or patchy/perivacuolar types). Gerstmann-Str?ussler-Scheinker disease (GSS) (in family with dominantly inherited progressive ataxia and/or dementia): encephalo(myelo)pathy with multicentric PrP plaques. Familial fatal insomnia (FFI) (in member of a family with PRNP178 mutation): thalamic degeneration, variable spongiform change in cerebrum. Kuru (in the Fore population). Without PrP data, the crucial feature is the spongiform change accompanied by neuronal loss and gliosis. This spongiform change is characterised by diffuse or focally clustered small round or oval vacuoles in the neuropil of the deep cortical layers, cerebellar cortex or subcortical grey matter, which might become confluent. Spongiform change should not be confused with non-specific spongiosis. This includes status spongiosus ("spongiform state"), comprising irregular cavities in gliotic neuropil following extensive neuronal loss (including also lesions of "burnt-out" CJD), "spongy" changes in brain oedema and metabolic encephalopathies, and artefacts such as superficial cortical, perineuronal, or perivascular vacuolation; focal changes indistinguishable from spongiform change may occur in some cases of Alzheimer's and diffuse Lewy body diseases. Very rare cases might not be diagnosed by these criteria. Then confirmation must be sought by additional techniques such as PrP immunoblotting, preparations for electron microscopic examination of scrapie associated fibrils (SAF), molecular biologic studies, or experimental transmission.     

Chaperone-supervised conversion of prion protein to its protease-resistant form

Transmissible spongiform encephalopathies (TSEs) are lethal, infectious disorders of the mammalian nervous system. A TSE hallmark is the conversion of the cellular protein PrPC to disease-associated PrPSc (named for scrapie, the first known TSE). PrPC is protease-sensitive, monomeric, detergent soluble, and primarily alpha-helical; PrPSc is protease-resistant, polymerized, detergent insoluble, and rich in beta-sheet. The "protein-only" hypothesis posits that PrPSc is the infectious TSE agent that directly converts host-encoded PrPC to fresh PrPSc, harming neurons and creating new agents of infection. To gain insight on the conformational transitions of PrP, we tested the ability of several protein chaperones, which supervise the conformational transitions of proteins in diverse ways, to affect conversion of PrPC to its protease-resistant state. None affected conversion in the absence of pre-existing PrPSc. In its presence, only two, GroEL and Hsp104 (heat shock protein 104), significantly affected conversion. Both promoted it, but the reaction characteristics of conversions with the two chaperones were distinct. In contrast, chemical chaperones inhibited conversion. Our findings provide new mechanistic insights into nature of PrP conversions, and provide a new set of tools for studying the process underlying TSE pathogenesis.     

Expression of mu-opiate receptor in human epidermis and keratinocytes

Chronic wasting disease (CWD), a member of the transmissible spongiform encephalopathies (TSEs), was first identified in captive mule and black-tail deer in 1967. Due to the failure to transmit CWD to rodents, we investigated the use of ferrets (Mustela putorius furo) as a small animal model of CWD. The inoculation of CWD into ferrets resulted in an incubation period of 17-21 months on primary passage that shortened to 5 months by the third ferret passage. The brain tissue of animals inoculated with ferret-passaged CWD exhibited spongiform degeneration and reactive astrocytosis. Western blot analysis of ferret-passaged CWD demonstrated the presence of PrP-res. Unlike mule deer CWD, ferret-passaged CWD was transmissible to Syrian golden hamsters (Mesocricetus auratus). Increasing the passage number of CWD in ferrets increased the pathogenicity of the agent for hamsters. This increase in host range of a field isolate on interspecies transmission emphasizes the need for caution when assessing the potential risk of transmission of TSEs, such as bovine spongiform encephalopathy, to new host species.     

Prions

Prions are unprecedented infectious pathogens that cause a group of invariably fatal neurodegenerative diseases by an entirely novel mechanism. Prion diseases may present as genetic, infectious, or sporadic disorders, all of which involve modification of the prion protein (PrP). Bovine spongiform encephalopathy (BSE), scrapie of sheep, and Creutzfeldt-Jakob disease (CJD) of humans are among the most notable prion diseases. Prions are transmissible particles that are devoid of nucleic acid and seem to be composed exclusively of a modified protein (PrPSc). The normal, cellular PrP (PrPC) is converted into PrPSc through a posttranslational process during which it acquires a high beta-sheet content. The species of a particular prion is encoded by the sequence of the chromosomal PrP gene of the mammals in which it last replicated. In contrast to pathogens carrying a nucleic acid genome, prions appear to encipher strain-specific properties in the tertiary structure of PrPSc. Transgenetic studies argue that PrPSc acts as a template upon which PrPC is refolded into a nascent PrPSc molecule through a process facilitated by another protein. Miniprions generated in transgenic mice expressing PrP, in which nearly half of the residues were deleted, exhibit unique biological properties and should facilitate structural studies of PrPSc. While knowledge about prions has profound implications for studies of the structural plasticity of proteins, investigations of prion diseases suggest that new strategies for the prevention and treatment of these disorders may also find application in the more common degenerative diseases.     

The spatial dynamics of prion disease

An important component of the latency period of the transmissible spongiform encephalopathies (prion diseases) can be attributed to delays during the propagation of the infectious prion isoform, PrPSc, through peripheral nervous tissues. A growing body of data report that the host prion protein, PrPC, is required in both peripheral and central nervous tissues for susceptibility to infection. We introduce a mathematical model, which treats the PrPSc as a mobile infectious pathogen, and show how peripheral delays can be understood in terms of the intercellular dispersal properties of the PrPSc strain, its decay rate, and its efficiency at transforming the PrPC. It has been observed that when two pathogenic strains co-infect a host, the presence of the first inoculated strain can slow down, or stop completely, the spread of the second strain. This is thought to result from a reduced concentration of host protein available for conversion by the second strain. Our model can explain the mechanisms of such interstrain competition and the time-course of the increased delay. The model provides a link between those data suggesting a role for a continuous chain of PrP-expressing tissue linking peripheral sites to the brain, and data on prion strain competition.     

Abnormalities in stress proteins in prion diseases

1. Prion diseases include kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Str?ussler-Scheinker disease (GSS), and fatal familia insomnia (FFI) of humans, as well as scrapie and bovine spongiform encephalopathy (BSE) of animals. 2. All these disorders involve conversion of the normal, cellular prion protein (PrPC) into the corresponding scrapie isoform (PrPSc). PrPC adopts a structure rich in alpha-helices and devoid of beta-sheet, in contrast to PrPSc, which has a high beta-sheet content and is resistant to limited digestion by proteases. That a conformational transition features in the conversion of PrPC into PrPSc implies that prion diseases are disorders of protein conformation. 3. This concept has been extended by our studies with heat shock proteins (Hsp), many of which are thought to function as molecular chaperones. We found that the induction of some Hsps but not others was profoundly altered in scrapie-infected cells and that the distribution of Hsp73 is unusual in these cells. 4. Whether the conversion of PrPC into PrPSc is assisted by molecular chaperones, or if the accumulation of the abnormally folded PrPSc is complexed with Hsps remains to be established.     

Experimental evidence for the origin of ductal-type adenocarcinoma from the islets of Langerhans

To investigate the role of the islets of Langerhans in pancreatic carcinogenesis, freshly isolated islets from male Syrian hamsters were transplanted into the right submandibular glands of 50 female hamsters that were or were not pre-treated with streptozotocin. Thyroid gland fragments, cellulose powder, and immortal hamster pancreatic ductal cells were injected into the left submandibular gland of the same hamsters. All recipient hamsters were then treated with the potent pancreatic carcinogen N-nitrosobis(2-oxopropyl)amine weekly at a dose of 40 mg/kg of body weight for 3 weeks. Between 3 and 8 weeks later, 18 of 75 (24%) hamsters developed large ductal-type adenocarcinomas in the submandibular gland region, where islets were transplanted, but none developed tumors in the left submandibular gland. In 9 of 18 hamsters, tumors were multiple so that a total of 31 cancers were found. Eleven of these carcinomas were in the vicinity of transplanted islets, eight of which showed intra-insular ductular or cyst formation as seen in the pancreas of hamsters during pancreatic carcinogenesis. The formation of ductular structures within islets was also demonstrated in vitro. Some tumor cells in the vicinity of these islets were reactive with anti-insulin. Y chromosome message was found by polymerase chain reaction analysis in one of the three tumors examined. Also, like the induced pancreatic tumors, all three submandibular gland tumors that were examined had the mutation of the c-Ki-ras oncogene at codon 12 and all tumors expressed blood group A antigen. These and other findings strongly suggest that some components of islets, most probably stem cells, are the origin of ductal-type adenocarcinomas in this model.     

Vascular variant of prion protein cerebral amyloidosis with tau-positive neurofibrillary tangles: the phenotype of the stop codon 145 mutation in PRNP

Deposition of PrP amyloid in cerebral vessels in conjunction with neurofibrillary lesions is the neuropathologic hallmark of the dementia associated with a stop mutation at codon 145 of PRNP, the gene encoding the prion protein (PrP). In this disorder, the vascular amyloid in tissue sections and the approximately 7.5-kDa fragment extracted from amyloid are labeled by antibodies to epitopes located in the PrP sequence including amino acids 90-147. Amyloid-laden vessels are also labeled by antibodies against the C terminus, suggesting that PrP from the normal allele is involved in the pathologic process. Abundant neurofibrillary lesions are present in the cerebral gray matter. They are composed of paired helical filaments, are labeled with antibodies that recognize multiple phosphorylation sites in tau protein, and are similar to those observed in Alzheimer disease. A PrP cerebral amyloid angiopathy has not been reported in diseases caused by PRNP mutations or in human transmissible spongiform encephalopathies; we propose to name this phenotype PrP cerebral amyloid angiopathy (PrP-CAA).     

Stimulated gracilis neosphincter: a new procedure for anal incontinence

Creutzfeldt-Jakob disease (CJD) is a transmissible neurodegenerative disorder characterized by the accumulation of proteinase-resistant prion protein (PrP) in the brain. Pathological changes in the cerebellum are common and include atrophy of the granular layer, spongiform change in the molecular layer, and astrocytic gliosis of the cerebellar cortex and white matter. In most cases of sporadic CJD immunohistochemistry for PrP shows widespread granular deposits of the scrapie isoform of the prion protein (PrPSc) in the cerebellar cortex. In a minority of cases plaque-like deposits of PrPSc are detectable. The genetic background of this phenomenon was investigated in 47 cases of sporadic CJD. Immunohistochemistry using antibodies against PrP was performed in brain autopsy specimens. A genetic analysis of the prion protein gene (PRNP) showed overrepresentation of homozygosity for either methionine (M/M) or valine (V/V) at the polymorphic codon 129 in CJD patients as compared to 74 controls. No significant difference in allele frequency between the 2 groups was found. Plaques or plaque-like PrPSc deposits were found in 9 cases of CJD and were associated with the presence of valine at codon 129 on at least 1 allele of PRNP. CJD patients homozygous for valine (V/V) were on an average more than 5 years younger than patients with M/M or M/V at codon 129.     

Synchrotron X-ray studies suggest that the core of the transthyretin amyloid fibril is a continuous beta-sheet helix

BACKGROUND: Amyloid diseases, which include Alzheimer's disease and the transmissible spongiform encephalopathies, are characterized by the extracellular deposition of abnormal protein fibrils derived from soluble precursor proteins. Although different precursors seem to generate similar fibrils, no adequate molecular structure of amyloid fibrils has been produced using modern techniques. Knowledge of the fibril structure is essential to understanding the molecular mechanism of amyloid formation and could lead to the development of agents to inhibit or reverse the process. RESULTS: The structure of amyloid fibrils from patients with familial amyloidotic polyneuropathy (FAP), which are derived from transthyretin (TTR) variants, has been investigated by fibre diffraction methods using synchrotron radiation. For the first time a significant high-angle diffraction pattern has been observed showing meridional reflections out to 2 A resolution. This pattern was fully consistent with the previously reported cross-beta structure for the fibril, but also reveals a new large scale fibre repeat of 115 A. We interpret this pattern as that of a repeating unit of 24 beta strands, which form a complete helical turn of beta sheet about an axis parallel to the fibre axis. This structure has not been observed previously. We have built a model of the protofilament of the FAP amyloid fibril based on this interpretation, composed of four beta sheets related by a single helix axis coincident with the fibre axis, and shown that it is consistent with the observed X-ray data. CONCLUSIONS: This work suggests that amyloid fibrils have a novel molecular structure consisting of beta sheets extended in regular helical twists along the length of the fibre. This implies that the polypeptide chains in the fibres are hydrogen-bonded together along the entire length of the fibres, thereby accounting for their great stability. The proposed structure of the FAP fibril requires a TTR building block that is structurally different from the native tetramer. This is likely to be either a monomer or dimer with reorganized or truncated beta sheets, suggesting that amyloid formation may require significant structural change in precursor proteins.     

Valproic acid overdose and L-carnitine therapy

We examined the primary ischaemic changes in the pancreas in 35 patients with disseminated intravascular coagulation. In 7 (20%), multiple patchy lesions composed of degenerative acinar cells indicating coagulation necrosis were noted. None of these lesions was accompanied by fat necrosis. The patchy lesions involved the islets of Langerhans in only 1 case. The interlobular arteries of the pancreas near these lesions contained fibrin thrombi in all 7 cases. We suggest that these lesions, without fat necrosis, are the distinctive ischaemic change associated with disseminated intravascular coagulation.     

Synergistic sterilization

Ten racing pigeons were infected experimentally with the paramyxovirus (PMV) type 1 of the pigeon. Within twelve weeks of observation, they were euthanized at different times. Their brains were examined for proteinase K resistant fibrils and histopathologically for spongiform lesions. No proteinase K resistant fibrils and no spongiform lesions could be detected in any case. Therefore, it is estimated that PMV type 1 of the pigeon is not likely to induce pathogenic mechanisms assumed for transmissible spongiform encephalopathies.