Mouse-adapted ovine scrapie prion strains are characterized by different conformers of PrPSc

The agent responsible for prion disease may exist in different forms, commonly referred to as strains, with each carrying the specific information that determines its own distinct biological properties, such as incubation period and lesion profile. Biological strain typing of ovine scrapie isolates by serial passage in conventional mice has shown some diversity in ovine prion strains. However, this biological diversity remains poorly supported by biochemical prion strain typing. The protein-only hypothesis predicts that variation between different prion strains in the same host is manifest in different conformations adopted by PrPSc. Here we have investigated the molecular properties of PrPSc associated with two principal Prnp(a) mouse-adapted ovine scrapie strains, namely, RML and ME7, in order to establish biochemical prion strain typing strategies that may subsequently be used to discriminate field cases of mouse-passaged ovine scrapie isolates. We used a conformation-dependent immunoassay and a conformational stability assay, together with Western blot analysis, to demonstrate that RML and ME7 PrPSc proteins show distinct biochemical and physicochemical properties. Although RML and ME7 PrPSc proteins showed similar resistance to proteolytic digestion, they differed in their glycoform profiles and levels of proteinase K (PK)-sensitive and PK-resistant isoforms. In addition, the PK-resistant core (PrP27-30) of ME7 was conformationally more stable following exposure to guanidine hydrochloride or Sarkosyl than was RML PrP27-30. Our data show that mouse-adapted ovine scrapie strains can be discriminated by their distinct conformers of PrPSc, which provides a basis to investigate their diversity at the molecular level.     

Detection of prion epitopes on PrP and PrP of transmissible spongiform encephalopathies using specific monoclonal antibodies to PrP

Amino acid residues 90-120 of the prion protein (PrP) are likely to be critical for the conversion of PrP(c) to PrP(sc) in the transmissible spongiform encephalopathies. We raised 10 monoclonal antibodies against the 90-120 amino acid region, mapped the epitope specificity of these anti-PrP antibodies, and investigated the expression of epitopes recognized by the antibodies in both PrP(c) and PrP(sc). Four out of five of the anti-PrP antibodies raised in a prion knockout mouse immunized with the linear peptide of PrP90-120 could detect PrP(sc) in 'native' and denatured forms and PrP(c) in normal cells, as well as recognize epitopes within PrP93-112 residues. In contrast, the other six anti-PrP reagents, including five raised from the two knockout mice immunized with conformationally modified PrP90-120 peptide, could detect PrP(c) and recognize epitopes within PrP93-107 residues. Four of these reagents could also detect denatured PrP(sc) on western blots but not PrP(sc) plaques in brain tissue. The results indicate that residues PrP93-102 are exposed in PrP(c) but are buried upon conversion to the PrP(sc) isoform. Furthermore, PrP103-107 residues are partially buried in PrP(sc) while only the PrP107-112 epitope remains exposed, suggesting that the region PrP93-112 undergoes conformational changes during its conversion to PrP(sc).     

Molecular biology of prion diseases

What is the nature of the transmissible agent responsible for neurodegenerative diseases such as scrapie and mad-cow disease in animals and Creutzfeldt-Jakob disease in man? There is now weighty evidence that PrP(Sc), a modified version of the ubiquitously expressed host protein PrP(C), is responsible for pathogenesis of these diseases and that conversion of PrP(C) into PrP(Sc) under the influence of PrP(Sc) is the process leading to the propagation of PrP(Sc) and disease progression.     

Molecular and transmission characteristics of primary-passaged ovine scrapie isolates in conventional and ovine PrP transgenic mice

A more complete assessment of ovine prion strain diversity will be achieved by complementing biological strain typing in conventional and ovine PrP transgenic mice with a biochemical analysis of the resultant PrPSc. This will provide a correlation between ovine prion strain phenotype and the molecular nature of different PrP conformers associated with particular prion strains. Here, we have compared the molecular and transmission characteristics of ovine ARQ/ARQ and VRQ/VRQ scrapie isolates following primary passage in tg338 (VRQ) and tg59 (ARQ) ovine PrP transgenic mice and the conventional mouse lines C57BL/6 (Prnp^a), RIII (Prnp^a), and VM (Prnp^b). Our data show that these different genotypes of scrapie isolates display similar incubation periods of >350 days in conventional and tg59 mice. Facilitated transmission of sheep scrapie isolates occurred in tg338 mice, with incubation times reduced to 64 days for VRQ/VRQ inocula and to ≤210 days for ARQ/ARQ samples. Distinct genotype-specific lesion profiles were seen in the brains of conventional and tg59 mice with prion disease, which was accompanied by the accumulation of more conformationally stable PrPSc, following inoculation with ARQ/ARQ compared to VRQ/VRQ scrapie isolates. In contrast, the lesion profiles, quantities, and stability of PrPSc induced by the same inocula in tg338 mice were more similar than in the other mouse lines. Our data show that primary transmission of different genotypes of ovine prions is associated with the formation of different conformers of PrPSc with distinct molecular properties and provide the basis of a molecular approach to identify the true diversity of ovine prion strains.     

High prion and PrPSc levels but delayed onset of disease in scrapie-inoculated mice heterozygous for a disrupted PrP gene.

BACKGROUND: It has been proposed that the prion, the infectious agent of transmissible spongiform encephalopathies, is PrPSc, a post-translationally modified form of the normal host protein PrPC. We showed previously that mice devoid of PrPC (Prn-p0/0) are completely resistant to scrapie. We now report on the unexpected response of heterozygous (Prn-p0/+) mice to scrapie infection. MATERIALS AND METHODS: Prn-p0/+, Prn-p0/0 and Prn-p+/+ mice were obtained from crosses of Prn-p0/+ mice. Mice were inoculated intracerebrally with mouse-adapted scrapie agent and the clinical progression of the disease recorded. Mice were sacrificed at intervals, PrPSc was determined as protease-resistant PrP and the prion titer by the incubation time assay. RESULTS: Prn-p0/+ mice, which have about half the normal level of PrPC in their brains, show enhanced resistance to scrapie, as manifested by a significant delay in onset and progression of clinical disease. However, while in wild type animals an increase in prion titer and PrPSc levels is followed within weeks by scrapie symptoms and death, heterozygous Prn-p0/+ mice remain free of symptoms for many months despite similar levels of scrapie infectivity and PrPSc. CONCLUSIONS: Our findings extend previous reports showing an inverse relationship between PrP expression level and incubation time for scrapie. However, contrary to expectation, overall accumulation of PrPSc and prions to a high level do not necessarily lead to clinical disease. These findings raise the question whether high titers of prion infectivity could also persist for long periods under natural circumstances in the absence of clinical symptoms.     

Molecular biology and transgenetics of prion diseases.

Considerable progress has been made deciphering the role of an abnormal isoform of the prion protein (PrP) in scrapie of animals and Gerstmann-Str?ussler syndrome (GSS) of humans. Some transgenic (Tg) mouse (Mo) lines that carry and express a Syrian hamster (Ha) PrP gene developed scrapie 75 d after inoculation with Ha prions; non-Tg mice failed to show symptoms after greater than 500 d. Brains of these infected Tg(HaPrP) mice featured protease-resistant HaPrPSc, amyloid plaques characteristic for Ha scrapie, and 10(9) ID50 units of Ha-specific prions upon bioassay. Studies on Syrian, Armenian, and Chinese hamsters suggest that the domain of the PrP molecule between codons 100 and 120 controls both the length of the incubation time and the deposition of PrP in amyloid plaques. Ataxic GSS in families shows genetic linkage to a mutation in the PrP gene, leading to the substitution of Leu for Pro at codon 102. Discovery of a point mutation in the Prp gene from humans with GSS established that GSS is unique among human diseases--it is both genetic and infectious. These results have revised thinking about sporadic Creutzfeldt-Jakob disease, suggesting it may arise from a somatic mutation. These findings combined with those from many other studies assert that PrPSc is a component of the transmissible particle, and the PrP amino acid sequence controls the neuropathology and species specificity of prion infectivity. The precise mechanism of PrPSc formation remains to be established. Attempts to demonstrate a scrapie-specific nucleic acid within highly purified preparations of prions have been unrewarding to date. Whether transmissible prions are composed only of PrPSc molecules or do they also contain a second component such as small polynucleotide remains uncertain.     

Mutant PrPSc conformers induced by a synthetic peptide and several prion strains

Gerstmann-Sträussler-Scheinker (GSS) disease is a dominantly inherited, human prion disease caused by a mutation in the prion protein (PrP) gene. One mutation causing GSS is P102L, denoted P101L in mouse PrP (MoPrP). In a line of transgenic mice denoted Tg2866, the P101L mutation in MoPrP produced neurodegeneration when expressed at high levels. MoPrP^Sc(P101L) was detected both by the conformation-dependent immunoassay and after protease digestion at 4°C. Transmission of prions from the brains of Tg2866 mice to those of Tg196 mice expressing low levels of MoPrP(P101L) was accompanied by accumulation of protease-resistant MoPrP^Sc(P101L) that had previously escaped detection due to its low concentration. This conformer exhibited characteristics similar to those found in brain tissue from GSS patients. Earlier, we demonstrated that a synthetic peptide harboring the P101L mutation and folded into a β-rich conformation initiates GSS in Tg196 mice (29). Here we report that this peptide-induced disease can be serially passaged in Tg196 mice and that the PrP conformers accompanying disease progression are conformationally indistinguishable from MoPrP^Sc(P101L) found in Tg2866 mice developing spontaneous prion disease. In contrast to GSS prions, the 301V, RML, and 139A prion strains produced large amounts of protease-resistant PrP^Sc in the brains of Tg196 mice. Our results argue that MoPrP^Sc(P101L) may exist in at least several different conformations, each of which is biologically active. Such conformations occurred spontaneously in Tg2866 mice expressing high levels of MoPrP^C(P101L) as well as in Tg196 mice expressing low levels of MoPrP^C(P101L) that were inoculated with brain extracts from ill Tg2866 mice, with a synthetic peptide with the P101L mutation and folded into a β-rich structure, or with prions recovered from sheep with scrapie or cattle with bovine spongiform encephalopathy.The discovery that brain fractions enriched for prion infectivity contain a protein (rPrP^Sc) that is resistant to limited proteolytic digestion advanced prion research (8, 37). N-terminal truncation of rPrP^Sc produced a protease-resistant fragment, denoted PrP 27-30, that is readily measured by Western blotting, enzyme-linked immunosorbent assay, or immunohistochemistry. The measurement of PrP^Sc was dramatically changed with the development of the conformation-dependent immunoassay (CDI), which permitted detection of full-length rPrP^Sc as well as previously unrecognized protease-sensitive forms of PrP^Sc (39).The CDI depends on using anti-PrP antibodies that react with an epitope exposed in native PrP^C but that do not bind to native PrP^Sc. Upon denaturation, the buried epitope in PrP^Sc becomes exposed and readily reacts with anti-PrP antibodies. Using the CDI, we discovered that most PrP^Sc is protease sensitive, which we designate sPrP^Sc. Whether sPrP^Sc is an intermediate in the formation of rPrP^Sc remains to be determined. In Syrian hamsters inoculated with eight different strains of prions, the ratio of rPrP^Sc to sPrP^Sc was different for each strain and the concentration of sPrP^Sc was proportional to the length of the incubation time (39).In earlier studies, transgenic (Tg) mice, denoted Tg2866, expressing high levels of PrP(P101L) were used to model Gerstmann-Sträussler-Scheinker (GSS) disease caused by the P102L point mutation. In the brains of several lines of mice expressing high levels of PrP(P101L), no rPrP^Sc(P101L) was detectable (26, 27, 47). This was particularly perplexing since these Tg mice expressing high levels of PrP(P101L) developed all facets of prion-induced neurodegeneration, including multicentric PrP amyloid plaques. Moreover, brain extracts from ill Tg2866 mice transmitted disease to Tg196 mice expressing low levels of PrP(P101L) that infrequently developed spontaneous neurodegeneration (29).In humans with GSS, several different mutations of the PrP gene (PRNP) resulting in nonconservative amino acid substitutions have been identified (23). In these patients, the clinical presentation, disease course, and amounts of rPrP^Sc in the brain are variable. Brain extracts from humans who died of GSS were inoculated into apes and monkeys, but the transmission rates were not correlated with the levels of PrP^Sc in the inoculum (1, 2, 9, 32). In a limited study, GSS(P102L) was transmitted to Tg mice expressing a chimeric mouse-human (MHu2 M) PrP transgene carrying the P102L mutation but not to Tg mice expressing MHu2M PrP without the mutation (47). In another study, GSS(P102L) human prions were transmitted to Tg mice expressing MoPrP(P101L) in which the transgene was incorporated through gene replacement (31). The use of gene replacement permits all of the regulatory elements that control the wild-type (wt) MoPrP gene to modulate the expression of MoPrP(P101L). In these mice, the expression level of MoPrP(P101L) in brain is likely to be similar to that in Tg196 mice.When we synthesized a 55-mer MoPrP peptide composed of residues 89 to 143 containing the P101L mutation and folded it under conditions favoring a β-structure, it induced neurodegeneration in Tg196 mice (29). When the peptide was not folded into a β-structure, it did not produce disease in Tg196 mice. We report here that the peptide-initiated disease in Tg196 mice could be serially transmitted to other Tg196 mice using brain extracts from the peptide-inoculated Tg196 mice. Using procedures derived from the CDI, brain extracts from inoculated Tg196 mice were found to contain sPrP^Sc(P101L), from which a 22- to 24-kDa PrP fragment was generated by limited digestion with proteinase K (PK) at 4°C and selective precipitation with phosphotungstate (PTA) (25, 39). In the interest of clarity, we have designated digestion at 4°C as “cold PK” and simply refer to standard digestion at 37°C as “PK.” To aid in distinguishing rPrP^Sc(P101L) from sPrP^Sc(P101L), their properties based on the work reported here and in other previously published papers are listed in Table ​Table11 (39, 40).

TABLE 1.

Characteristics of PrP(P101L) isoforms

A naturally occurring C-terminal fragment of the prion protein (PrP) delays disease and acts as a dominant-negative inhibitor of PrPSc formation

The cellular prion protein (PrPC) undergoes constitutive proteolytic cleavage between residues 111/112 to yield a soluble N-terminal fragment (N1) and a membrane-anchored C-terminal fragment (C1). The C1 fragment represents the major proteolytic fragment of PrPC in brain and several cell types. To explore the role of C1 in prion disease, we generated Tg(C1) transgenic mice expressing this fragment (PrP(Δ23-111)) in the presence and absence of endogenous PrP. In contrast to several other N-terminally deleted forms of PrP, the C1 fragment does not cause a spontaneous neurological disease in the absence of endogenous PrP. Tg(C1) mice inoculated with scrapie prions remain healthy and do not accumulate protease-resistant PrP, demonstrating that C1 is not a substrate for conversion to PrPSc (the disease-associated isoform). Interestingly, Tg(C1) mice co-expressing C1 along with wild-type PrP (either endogenous or encoded by a second transgene) become ill after scrapie inoculation, but with a dramatically delayed time course compared with mice lacking C1. In addition, accumulation of PrPSc was markedly slowed in these animals. Similar effects were produced by a shorter C-terminal fragment of PrP(Δ23-134). These results demonstrate that C1 acts as dominant-negative inhibitor of PrPSc formation and accumulation of neurotoxic forms of PrP. Thus, C1, a naturally occurring fragment of PrPC, might play a modulatory role during the course of prion diseases. In addition, enhancing production of C1, or exogenously administering this fragment, represents a potential therapeutic strategy for the treatment of prion diseases.     

Molecular genetics of human prion diseases in Germany

Human prion diseases may be acquired as infectious diseases, they may be inherited in an autosomal dominant fashion or occur sporadically. Mutations and polymorphisms in the sequence of the coding region of the prion protein gene (PRNP) have been established as an important factor in all of these three types of prion diseases. Therefore, a total of 578 patients with suspect prion diseases referred to the German Creutzfeldt-Jakob disease (CJD) surveillance unit over a period of 4.5 years have been examined for mutations and polymorphisms in the coding region of PRNP. We found 40 cases with a missense mutation previously reported as pathogenic. Amongst these, the aspartate to asparagine change at codon 178 (D178N) was the most common mutation. All of these cases carried the D178N mutation in coupling with methionine at codon 129, resulting in the typical fatal familial insomnia (FFI) genotype. Most cases with pathogenic mutations were not found in the group of clinically "probable" cases according to established clinical criteria, supporting the notion that inherited prion diseases often exhibit atypical features. Two novel missense mutations (T188R and P238S) and several silent polymorphisms were found, demonstrating the quality of our screening procedure based on a modified version of the single-stranded conformational polymorphism technique. In "definite" CJD cases with no pathogenic mutation, the patients clinically classified as "probable" were mostly homozygous for methionine at the common polymorphism at codon 129, whereas there was a marked over-representation of patients homozygous for valine amongst those clinically classified as "possible". This large study on suspect cases of human prion diseases in Germany clearly shows that PRNP genetics is essential for a comprehensive analysis of prion diseases.     

Molecular pathogenesis of sporadic prion diseases in man

The yeast, fungal and mammalian prions determine heritable and infectious traits that are encoded in alternative conformations of proteins. They cause lethal sporadic, familial and infectious neurodegenerative conditions in man, including Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS), kuru, sporadic fatal insomnia (SFI) and likely variable protease-sensitive prionopathy (VPSPr). The most prevalent of human prion diseases is sporadic (s)CJD. Recent advances in amplification and detection of prions led to considerable optimism that early and possibly preclinical diagnosis and therapy might become a reality. Although several drugs have already been tested in small numbers of sCJD patients, there is no clear evidence of any agent’s efficacy. Therefore, it remains crucial to determine the full spectrum of sCJD prion strains and the conformational features in the pathogenic human prion protein governing replication of sCJD prions. Research in this direction is essential for the rational development of diagnostic as well as therapeutic strategies. Moreover, there is growing recognition that fundamental processes involved in human prion propagation – intercellular induction of protein misfolding and seeded aggregation of misfolded host proteins – are of far wider significance. This insight leads to new avenues of research in the ever-widening spectrum of age-related human neurodegenerative diseases that are caused by protein misfolding and that pose a major challenge for healthcare.     

Molecular pathogenesis of sporadic prion diseases in man

The yeast, fungal and mammalian prions determine heritable and infectious traits that are encoded in alternative conformations of proteins. They cause lethal sporadic, familial and infectious neurodegenerative conditions in man, including Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS), kuru, sporadic fatal insomnia (SFI) and likely variable protease-sensitive prionopathy (VPSPr). The most prevalent of human prion diseases is sporadic (s)CJD. Recent advances in amplification and detection of prions led to considerable optimism that early and possibly preclinical diagnosis and therapy might become a reality. Although several drugs have already been tested in small numbers of sCJD patients, there is no clear evidence of any agent’s efficacy. Therefore, it remains crucial to determine the full spectrum of sCJD prion strains and the conformational features in the pathogenic human prion protein governing replication of sCJD prions. Research in this direction is essential for the rational development of diagnostic as well as therapeutic strategies. Moreover, there is growing recognition that fundamental processes involved in human prion propagation – intercellular induction of protein misfolding and seeded aggregation of misfolded host proteins – are of far wider significance. This insight leads to new avenues of research in the ever-widening spectrum of age-related human neurodegenerative diseases that are caused by protein misfolding and that pose a major challenge for healthcare.     

Molecular basis of cerebral neurodegeneration in prion diseases

The biochemical nature and the replication of infectious prions have been intensively studied in recent years. Much less is known about the cellular events underlying neuronal dysfunction and cell death. As the cellular function of the normal cellular isoform of prion protein is not exactly known, the impact of gain of toxic function or loss of function, or a combination of both, in prion pathology is still controversial. There is increasing evidence that the normal cellular isoform of the prion protein is a key mediator in prion pathology. Transgenic models were instrumental in dissecting propagation of prions, disease-associated isoforms of prion protein and amyloid production, and induction of neurodegeneration. Four experimental avenues will be discussed here which address scenarios of inappropriate trafficking, folding, or targeting of the prion protein.     

Mutational analysis of topological determinants in prion protein (PrP) and measurement of transmembrane and cytosolic PrP during prion infection

The prion protein (PrP) can adopt multiple membrane topologies, including a fully translocated form (SecPrP), two transmembrane forms (NtmPrP and CtmPrP), and a cytosolic form. It is important to understand the factors that influence production of these species, because two of them, CtmPrP and cytosolic PrP, have been proposed to be key neurotoxic intermediates in certain prion diseases. In this paper, we perform a mutational analysis of PrP synthesized using an in vitro translation system in order to further define sequence elements that influence the formation of CtmPrP. We find that substitution of charged residues in the hydrophobic core of the signal peptide increases synthesis of CtmPrP and also reduces the efficiency of translocation into microsomes. Combining these mutations with substitutions in the transmembrane domain causes the protein to be synthesized exclusively with the CtmPrP topology. Reducing the spacing between the signal peptide and the transmembrane domain also increases CtmPrP. In contrast, topology is not altered by mutations that prevent signal peptide cleavage or by deletion of the C-terminal signal for glycosylphosphatidylinositol anchor addition. Removal of the signal peptide completely blocks translocation. Taken together, our results are consistent with a model in which the signal peptide and transmembrane domain function in distinct ways as determinants of PrP topology. We also present characterization of an antibody that selectively recognizes CtmPrP and cytosolic PrP by virtue of their uncleaved signal peptides. By using this antibody, as well as the distinctive gel mobility of CtmPrP and cytosolic PrP, we show that the amounts of these two forms in cultured cells and rodent brain are not altered by infection with scrapie prions. We conclude that CtmPrP and cytosolic PrP are unlikely to be obligate neurotoxic intermediates in familial or infectiously acquired prion diseases.     

Rdj2, a J protein family member, interacts with cellular prion PrP(C)

PrP(C) is a glycosylphosphatidylinositol (GPI) anchored glycoprotein of unknown function. Misfolding of normal cellular PrP(C) to the pathogenic PrP(Sc) is the hallmark of prion diseases (transmissible spongiform encephalopathies). Prion diseases are characterized by extensive neurodegeneration and early death. Understanding how PrP(C) maintains its correct conformation is a major endeavor of current inquiry. Here we demonstrate a novel interaction between PrP(C) and the J protein family member, Rdj2 (DjA2; Dj3, Dnj3, Cpr3, and Hirip4). The importance of the J protein family in the cellular folding machinery has been recognized for many years. The PrP(C)/Rdj2 association was direct and concentration-dependent. Other J proteins such as CSPalpha and auxilin did not associate with PrP(C) in the absence of ATP, demonstrating the specificity of the PrP(C)/J protein interaction. These findings suggest that the J protein family serves as a 'folding catalyst' for PrP(C) and implicates Rdj2 as a factor in the protection against prion diseases.     

Efficient inhibition of prion replication by PrP-Fc(2) suggests that the prion is a PrP(Sc) oligomer

Soluble dimeric prion protein (PrP-Fc(2)) binds to the disease-associated prion protein PrP(Sc), and inhibits prion replication when expressed in transgenic mice. Prion inhibition is effective even if PrP-Fc(2) is expressed at low levels, suggesting that its affinity for PrP(Sc) is higher than that of monomeric PrP(C). Here, we model prion accumulation as an exponential replication cycle of prion elongation and breakage. The exponential growth rate corresponding to this cycle is reflected in the incubation period of the disease. We use a mathematical model to calculate the exponential growth rate, and fit the model to in vivo data on prion incubation times corresponding to different levels of PrP(C) and PrP-Fc(2). We find an excellent fit of the model to the data. Surprisingly, targeting of PrP(Sc) can be effective at concentrations of PrP-Fc(2) lower than that of PrP(C), even if PrP-Fc(2) and PrP(C) have the same affinity for PrP(Sc). The best fit of our model to data predicts that the replicative prion consists of PrP(Sc) oligomers with a mean size of four to 15 units.     

PrP expression and replication by Schwann cells: implications in prion spreading

Prion infection relies on a continuous chain of PrP(c)-expressing tissues to spread from peripheral sites to the central nervous system (CNS). Direct neuroinvasion via peripheral nerves has long been considered likely. However, the speed of axonal flow is incompatible with the lengthy delay prior to the detection of PrP(Sc) in the brain. We hypothesized that Schwann cells could be the candidate implicated in this mechanism; for that, it has to express PrP(c) and to allow PrP(Sc) conversion. We investigated in vivo localization of PrP(c) in sciatic nerve samples from different strains of mice. We demonstrated that PrP(c) is mainly localized at the cell membrane of the Schwann cell. We also studied in vitro expression of PrP(c) in the Schwann cell line MSC-80 and demonstrated that it expresses PrP(c) at the same location. More specifically, we demonstrated that this glial cell line, when infected in vitro with the mouse Chandler prion strain, both produces the PrP(Sc) till after 18 passages and is able to transmit disease to mice, which then develop the typical signs of prion diseases. It is the first time that infection and replication of PrP(Sc) are shown in a peripheral glial cell line.     

Reconstitution of prion infectivity from solubilized protease-resistant PrP and nonprotein components of prion rods

The scrapie isoform of the prion protein, PrP(Sc), is the only identified component of the infectious prion, an agent causing neurodegenerative diseases such as Creutzfeldt-Jakob disease and bovine spongiform encephalopathy. Following proteolysis, PrP(Sc) is trimmed to a fragment designated PrP 27-30. Both PrP(Sc) and PrP 27-30 molecules tend to aggregate and precipitate as amyloid rods when membranes from prion-infected brain are extracted with detergents. Although prion rods were also shown to contain lipids and sugar polymers, no physiological role has yet been attributed to these molecules. In this work, we show that prion infectivity can be reconstituted by combining Me(2)SO-solubilized PrP 27-30, which at best contained low prion infectivity, with nonprotein components of prion rods (heavy fraction after deproteination, originating from a scrapie-infected hamster brain), which did not present any infectivity. Whereas heparanase digestion of the heavy fraction after deproteination (originating from a scrapie-infected hamster brain), before its combination with solubilized PrP 27-30, considerably reduced the reconstitution of infectivity, preliminary results suggest that infectivity can be greatly increased by combining nonaggregated protease-resistant PrP with heparan sulfate, a known component of amyloid plaques in the brain. We submit that whereas PrP 27-30 is probably the obligatory template for the conversion of PrP(C) to PrP(Sc), sulfated sugar polymers may play an important role in the pathogenesis of prion diseases.