Paradoxical shortening of scrapie incubation times by expression of prion protein transgenes derived from long incubation period mice.

Prolonged incubation times for experimental scrapie in I/LnJ mice are dictated by a dominant gene linked to the prion protein gene (Prn-p). Transgenic mice were analyzed to discriminate between an effect of the I/LnJ Prn-pb allele and a distinct incubation time locus designated Prn-i. Paradoxically, 4 independent Prn-pb transgenic mouse lines had scrapie incubation times shorter than nontransgenic controls, instead of the anticipated prolonged incubation periods. Aberrant or overexpression of the Prn-pb transgenes may dictate abbreviated incubation times, masking genuine Prn-p/Prn-i congruence; alternatively, a discrete Prn-i gene lies adjacent to Prn-p.     

Linkage of prion protein and scrapie incubation time genes

A single gene (Prn-i) that affects scrapie incubation period in mice has been identified. I/LnJ mice have a very long incubation period after inoculation of scrapie prions (200-385 days) and NZW/LacJ mice have a short one (113 +/- 2.8 days). (NZW X I/Ln)F1 hybrid mice had incubation times of 223 +/- 2.8 days indicating longer incubation times were dominant. Incubation periods in the backcross progeny of (NZW/LacJ X I/LnJ)F1 X NZW/LacJ segregated into two groups (64 mice, 130 +/- 1.1 d; 66 mice, 195 +/- 1.9 d) indicating single gene control. NZW/LacJ and 20 other inbred strains have the Prn-pa allele which is identified as a 3.8 kb Xbal fragment using a hamster PrP (prion protein) cDNA probe. I/LnJ and three other Prn-pb mouse strains have a 5.5 kb Xbal restriction fragment. Analysis of DNA from 66 backcross mice indicated Prn-i is tightly linked to Prn-p, the structural gene for PrP.     

Three hamster species with different scrapie incubation times and neuropathological features encode distinct prion proteins.

Given the critical role of the prion protein (PrP) in the transmission and pathogenesis of experimental scrapie, we investigated the PrP gene and its protein products in three hamster species, Chinese (CHa), Armenian (AHa), and Syrian (SHa), each of which were found to have distinctive scrapie incubation times. Passaging studies demonstrated that the host species, and not the source of scrapie prions, determined the incubation time for each species, and histochemical studies of hamsters with clinical signs of scrapie revealed characteristic patterns of neuropathology. Northern (RNA) analysis showed the size of PrP mRNA from CHa, AHa, and SHa hamsters to be 2.5, 2.4, and 2.1 kilobases, respectively. Immunoblotting demonstrated that the PrP isoforms were of similar size (33 to 35 kilodaltons); however, the monoclonal antibody 13A5 raised against SHa PrP did not react with the CHa or AHa PrP molecules. Comparison of the three predicted amino acid sequences revealed that each is distinct. Furthermore, differences within the PrP open reading frame that uniquely distinguish the three hamster species are within a hydrophilic segment of 11 amino acids that includes polymorphisms linked to scrapie incubation times in inbred mice and an inherited prion disease of humans. Single polymorphisms in this region correlate with the presence or absence of amyloid plaques for a given hamster species or mouse inbred strain. Our findings demonstrate distinctive molecular, pathological, and clinical characteristics of scrapie in three related species and are consistent with the hypothesis that molecular properties of the host PrP play a pivotal role in determining the incubation time and neuropathological features of scrapie.     

Identification of two prion protein regions that modify scrapie incubation time

A series of prion transmission experiments was performed in transgenic (Tg) mice expressing either wild-type, chimeric, or truncated prion protein (PrP) molecules. Following inoculation with Rocky Mountain Laboratory (RML) murine prions, scrapie incubation times for Tg(MoPrP)4053, Tg(MHM2)294/Prnp(0/0), and Tg(MoPrP, Delta23-88)9949/Prnp(0/0) mice were approximately 50, 120, and 160 days, respectively. Similar scrapie incubation times were obtained after inoculation of these lines of Tg mice with either MHM2(MHM2(RML)) or MoPrP(Delta23-88)(RML) prions, excluding the possibility that sequence-dependent transmission barriers could account for the observed differences. Tg(MHM2)294/Prnp(0/0) mice displayed prolonged scrapie incubation times with four different strains of murine prions. These data provide evidence that the N terminus of MoPrP and the chimeric region of MHM2 PrP (residues 108 through 111) both influence the inherent efficiency of prion propagation.     

Asparagine-linked glycosylation of the scrapie and cellular prion proteins

Post-translational modification of the scrapie prion protein (PrP) is thought to account for the unusual features of this protein. Molecular cloning of a PrP cDNA identified two potential Asn-linked glycosylation sites. Both the scrapie (PrPSc) and cellular (PrPC) isoforms were susceptible to digestion by peptide N-glycosidase F (PNGase F) but resistant to endoglycosidase H as measured by migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PNGase F digestion of PrPC yielded two proteins of Mr26K and 28K; however, the 26-k species was only a minor component. In contrast, PNGase F digestion of PrPSc yielded equimolar amounts of two proteins of Mr26K and 28K. The significance of this altered stoichiometry between the 26- and 28-kDa deglycosylated forms of PrP during scrapie infection remains to be established. Both isoforms as well as PrP 27-30, which is produced by limited proteolysis of PrPSc, exhibited a reduced number of charge isomers after PNGase F digestion. The molecular weight of PrP 27-30 was reduced from 27K-30K by PNGase F digestion to 20K-22K while anhydrous hydrogen fluoride or trifluoromethanesulfonic acid treatment reduced the molecular weight to 19K-21K and 20K-22K, respectively. Denatured PrP 27-30 was radioiodinated and then assessed for its binding to lectin columns. PrP 27-30 was bound to wheat germ agglutinin (WGA) or lentil lectins and eluted with N-acetylglucosamine or alpha-methyl-mannoside, respectively. Digestion of PrP 27-30 with sialidase prevented its binding to WGA but enhanced its binding to Ricinus communis lectin. These findings argue that PrP 27-30 probably possesses Asn-linked, complex oligosaccharides with terminal sialic acids, penultimate galactoses, and fucose residues attached to the innermost N-acetyl-glucosamine. Whether differences in Asn-linked oligosaccharide structure between PrPC and PrPSc exist and are responsible for the distinct properties displayed by these two isoforms remain to be established.     

Correlation analysis for the incubation period of prion disease

Previous studies have shown that genetic quantitative trait loci (QTL), strain barriers, inoculation dose and inoculation method modulate the incubation period of prion diseases. We examined the relationship between a diverse set of physical, genetic and immunological characteristics and the incubation period of prion disease using correlation analyses. We found that incubation period was highly correlated with brain weight. In addition, mean corpuscular volume and cell size were strongly correlated with incubation period, indicating that the physical magnitude of prion-infected organs or individual cells may be important in determining the incubation period. Given the same prion inoculation dose, animals with a lower brain weight, mean corpuscular volume or cell size may experience more virulent disease, as the effective concentration of abnormal prion, which might regulate the attachment rate of prions to aggregates, is increased with smaller capacity of brains and cells. This is partly consistent with previous theoretical modeling. The strong correlations between incubation period and physical properties of the brain and cells in this study suggest that the mechanism underlying prion disease pathology may be physical, indicating that the incubation process is governed by simple chemical stoichiometry.     

Correlation analysis for the incubation period of prion disease

Previous studies have shown that genetic quantitative trait loci (QTL), strain barriers, inoculation dose and inoculation method modulate the incubation period of prion diseases. We examined the relationship between a diverse set of physical, genetic and immunological characteristics and the incubation period of prion disease using correlation analyses. We found that incubation period was highly correlated with brain weight. In addition, mean corpuscular volume and cell size were strongly correlated with incubation period, indicating that the physical magnitude of prion-infected organs or individual cells may be important in determining the incubation period. Given the same prion inoculation dose, animals with a lower brain weight, mean corpuscular volume or cell size may experience more virulent disease, as the effective concentration of abnormal prion, which might regulate the attachment rate of prions to aggregates, is increased with smaller capacity of brains and cells. This is partly consistent with previous theoretical modeling. The strong correlations between incubation period and physical properties of the brain and cells in this study suggest that the mechanism underlying prion disease pathology may be physical, indicating that the incubation process is governed by simple chemical stoichiometry.     

Effects of the antiserum against a fraction enriched in scrapie-associated fibrils on the scrapie incubation period in mice

An antiserum against a fraction enriched for scrapie-associated fibrils (SAF), was examined for its effects on scrapie incubation period by inoculating mice either intraperitoneally or intracerebrally with various dilutions of the serum mixed with scrapie-infected mouse brain homogenate. After intraperitoneal inoculation the mean time of the incubation period increased with increasing concentrations of the antiserum in a statistically significant fashion, when the serum dilutions were made with phosphate-buffered saline. After intracerebral inoculation, however, there were no statistically significant differences between the control group and any of the antiserum-groups. When the antiserum dilutions were made with pre-immune serum, the mice inoculated intraperitoneally also showed no significant differences between the two groups. These results indicate that the specific antibodies to SAF have no effect on the scrapie infectivity.     

Purification and properties of the cellular and scrapie hamster prion proteins

During scrapie infection an abnormal isoform of the prion protein (PrP), designated PrPSc, accumulates and is found to copurify with infectivity; to date, no nucleic acid has been found which is scrapie-specific. Both uninfected and scrapie-infected cells synthesize a PrP isoform, denoted PrPC, which exhibits physical properties that differentiate it from PrPSc. PrPC was purified by immunoaffinity chromatography using a PrP-specific monoclonal antibody cross-linked to protein-A--Avidgel. PrPSc was purified by detergent extraction, poly(ethylene glycol) precipitation and repeated differential centrifugation of PrPSc polymers. Both PrP isoforms were found to have the same N-terminal amino acid sequence which begins at a predicted signal peptide cleavage site. The first 8 residues of PrPC were found to be KKXPKPGG and the first 29 residues of PrPSc were found to be KKXPKPGGWNTGGSXYPGQGSPGGNRYPP. Arg residues 3 and 15 in PrPSc and 3 in PrPC appear to be modified since no detectable signals (denoted X) were found at these positions during gas-phase sequencing. Both PrP isoforms were found to contain an intramolecular disulfide bond, linking Cys 179 and 214, which creates a loop of 36 amino acids containing the two N-linked glycosylation sites. Development of a purification protocol for PrPC should facilitate comparisons of the two PrP isoforms and lead to an understanding of how PrPSc is synthesized either from PrPC or a precursor.     

Quantitative trait loci affecting prion incubation time in mice

Although the gene encoding prion protein (PrP) is the major determinant of susceptibility to prion disease, other genes also affect prion incubation time in mice and may be involved in prion replication. Scrapie incubation time was analyzed as a quantitative trait using crosses between SJL/J and CAST/Ei mice; these mouse strains encode identical PrP molecules but have different incubation periods. Our analysis revealed loci on Chromosomes 9 and 11 that affect prion susceptibility.     

Identification of cellular proteins binding to the scrapie prion protein

The scrapie prion protein (PrPSc) is an abnormal isoform of the cellular protein PrPc. PrPSc is found only in animals with scrapie or other prion diseases. The invariable association of PrPSc with infectivity suggests that PrPSc is a component of the infectious particle. In this study, we report the identification of two proteins from hamster brain of 45 and 110 kDa (denoted PrP ligands Pli 45 and Pli 110) which were able to bind to PrP 27-30, the protease-resistant core of PrPSc on ligand blots. Pli 45 and Pli 110 also bound PrPC. Both Pli's had isoelectric points of approximately 5. The dissociation rate constant of the Pli 45/PrP 27-30 complex was 3 x 10(-6) s-1. Amino acid and protein sequence analyses were performed on purified Pli 45. Both the composition and the sequence were almost identical with those predicted for mouse glial fibrillary acidic protein (GFAP). Furthermore, antibodies to Pli 45 reacted with recombinant GFAP. The identification of proteins which interact with the PrP isoforms in normal and diseased brain may provide new insights into the function of PrPC and into the molecular mechanisms underlying prion diseases.     

Evidence for synthesis of scrapie prion proteins in the endocytic pathway.

Infectious scrapie prions are composed largely, if not entirely, of an abnormal isoform of the prion protein (PrP) which is designated PrPSc. A chromosomal gene encodes both the cellular prion protein (PrPC) as well as PrPSc. Pulse-chase experiments with scrapie-infected cultured cells indicate that PrPSc is formed by a post-translational process. PrP is translated in the endoplasmic reticulum, modified as it passes through the Golgi, and is transported to the cell surface. Release of nascent PrP from the cell surface by phosphatidylinositol-specific phospholipase C or hydrolysis with dispase prevented PrPSc synthesis. At 18 degrees C, the synthesis of PrPSc was inhibited under conditions that other investigators report a blockage of endosomal fusion with lysosomes. Our results suggest that PrPSc synthesis occurs after PrP transits from the cell surface. Whether all of the PrP molecules have an equal likelihood to be converted into PrPSc or only a distinct subset is eligible for conversion remains to be established. Identifying the subcellular compartment(s) of PrPSc synthesis should be of considerable importance in defining the molecular changes that distinguish PrPSc from PrPC.     

Shadoo (Sprn) and prion disease incubation time in mice

Prion diseases are transmissible neurodegenerative disorders of mammalian species and include scrapie, bovine spongiform encephalopathy (BSE), and variant Creutzfeldt-Jakob disease (vCJD). The prion protein (PrP) plays a key role in the disease, with coding polymorphism in both human and mouse influencing disease susceptibility and incubation time, respectively. Other genes are also thought to be important and a plausible candidate is Sprn, which encodes the PrP-like protein Shadoo (Sho). Sho is expressed in the adult central nervous system and exhibits neuroprotective activity reminiscent of PrP in an in vitro assay. To investigate the role of Sprn in prion disease incubation time we sequenced the open reading frame (ORF) in a diverse panel of mice and saw little variation except in strains derived from wild-trapped mice. Sequencing the untranslated regions revealed polymorphisms that allowed us to carry out an association study of incubation period in the Northport heterogeneous stock of mice inoculated with Chandler/RML prions. We also examined the expression level of Sprn mRNA in the brains of normal and prion-infected mice and saw no correlation with either genotype or incubation time. We therefore conclude that Sprn does not play a major role in prion disease incubation time in these strains of mice.     

Genetics and polymorphism of the mouse prion gene complex: control of scrapie incubation time.

The mouse prion protein (PrP) gene (Prn-p), which encodes the only macromolecule that has been identified in scrapie prions, is tightly linked or identical to a gene (Prn-i) that controls the duration of the scrapie incubation period in mice. Constellations of restriction fragment length polymorphisms distinguish haplotypes a to f of Prn-p. The Prn-pb allele encodes a PrP that differs in sequence from those encoded by the other haplotypes and, in inbred mouse strains, correlates with long scrapie incubation time (Westaway et al., Cell 51: 651-662, 1987). In segregating crosses of mice, we identified rare individuals with a divergent scrapie incubation time phenotype and Prn-p genotype, but progeny testing to demonstrate meiotic recombination was not possible because scrapie is a lethal disease. Crosses involving the a, d, and e haplotypes demonstrated that genes unlinked to Prn-p could modulate scrapie incubation time and that there were only two alleles of Prn-i among the mouse strains tested. All inbred strains of mice that had the Prnb haplotype were probably direct descendants of the I/LnJ progenitors. We established the linkage relationship between the prion gene complex (Prn) and other chromosome 2 genes; the gene order, proximal to distal, is B2m-II-1a-Prn-Itp-A. Recombination suppression in the B2m-Prn-p interval occurred during the crosses involved in transferring the I/LnJ Prnb complex into a C57BL/6J background. Transmission ratio distortion by Prna/Prnb heterozygous males was also observed in the same crosses. These phenomena, together with the founder effect, would favor apparent linkage disequilibrium between Prn-p and Prn-i. Therefore, transmission genetics may underestimate the number of genes in Prn.     

Transgenic mice expressing hamster prion protein produce species-specific scrapie infectivity and amyloid plaques

Three transgenic mouse lines designated Tg 69, 71, and 81 were produced harboring a Syrian hamster (Ha) prion protein (PrP) gene; all expressed the cellular HaPrP isoform in their brains. Inoculation of Tg 81 mice or hamsters with Ha prions caused scrapie in integral of 75 days; nontransgenic control mice failed to develop scrapie after greater than 500 days. Tg 71 mice inoculated with Ha prions developed scrapie in integral of 170 days. Both Tg 71 and Tg 81 mice exhibited spongiform degeneration and reactive astrocytic gliosis, and they produced the scrapie HaPrP isoform in their brains. Tg 81 brains also showed HaPrP amyloid plaques characteristic of Ha scrapie and contained integral of 10(9) ID50 units of Ha prions based on Ha bioassays. Our findings argue that the PrP gene modulates scrapie susceptibility, incubation times, and neuropathology; furthermore, they demonstrate synthesis of infectious scrapie prions programmed by a recombinant DNA molecule.     

Glycosylinositol phospholipid anchors of the scrapie and cellular prion proteins contain sialic acid.

The only identified component of the scrapie prion is PrPSc, a glycosylinositol phospholipid (GPI)-linked protein that is derived from the cellular isoform (PrPC) by an as yet unknown posttranslational event. Analysis of the PrPSc GPI has revealed six different glycoforms, three of which are unprecedented. Two of the glycoforms contain N-acetylneuraminic acid, which has not been previously reported as a component of any GPI. The largest form of the GPI is proposed to have a glycan core consisting of Man alpha-Man alpha-Man-(NeuAc-Gal-GalNAc-)Man-GlcN-Ino. Identical PrPSc GPI structures were found for two distinct isolates or "strains" of prions which specify different incubation times, neuropathology, and PrPSc distribution in brains of Syrian hamsters. Limited analysis of the PrPC GPI reveals that it also has sialylated glycoforms, arguing that the presence of this monosaccharide does not distinguish PrPC from PrPSc.     

Immunological analysis of host and agent effects on Creutzfeldt-Jakob disease and scrapie prion proteins.

Creutzfeldt-Jakob disease (CJD) and scrapie are degenerative neurological diseases caused by unusual infectious pathogens. The term prion has been introduced to underscore the apparent distinctness of these agents from viruses and viroids. The only macromolecule shown to be associated with the infectious agent, the CJD or scrapie prion protein (PrPCJD or PrPSc, respectively), is encoded by the same gene as a normal cellular protein. In several studies biochemical differences have been reported in PrPScs derived from a common host species infected with different putative strains of the scrapie agent, suggesting agent-specific characteristics independent of the host. We analyzed various agent-host combinations by Western blotting of PrPs that were separated by size or charge. The profile of immunoreactive proteins for CJD prions isolated from mice, guinea pigs, and humans appeared distinct. Importantly, PrPCJDS purified from a human brain and from the corresponding first-passage mouse brains were clearly distinguishable. PrPCJDs isolated from CJD prions propagated in NAMRU or B10.Q mice, which are homozygous for a short-incubation-time gene; from the short-incubation-time backcross progeny of (B10.Q x I/LnJ)F1 x B10.Q; or from NAMRU mice inoculated with I/LnJ prions were identical to each other but distinguishable from those of I/LnJ mice, which are homozygous for the long-incubation-time gene. The PrPs from human CJD and ovine scrapie propagated in the same mouse strain appeared the same, but they were distinct from the same isolate of scrapie passaged in hamsters. Lastly, PrPScs purified from five different strains of scrapie propagated in C57BL mice were identical, including strains, ME7 and 139A, which were previously reported to be distinct. This evidence does not support, although it does not exclude, agent-mediated characteristics independent of host-mediated ones for scrapie and CJD.     

Different antigenicities of the N‐terminal region of cellular and scrapie prion proteins

Limited information is available about conformational differences between the abnormal isoform of prion protein (PrP^Sc) and cellular prion protein (PrP^C) under native conditions. To clarify conformational differences between these two isoforms, PrP‐deficient mice were immunized with brain homogenates of normal and scrapie‐infected animals. All mice generated anti‐PrP antibodies. Peptide array analysis of these serum samples revealed a distinctive epitope of PrP^Sc consisting of QGSPGGN (PrP41–47) at the N‐terminus. This study demonstrated a conformational dissimilarity at the N‐terminus between PrP^Sc and PrP^C, a finding that may provide novel information about conformational features of PrP^Sc.     

Differential release of cellular and scrapie prion proteins from cellular membranes by phosphatidylinositol-specific phospholipase C

The abnormal isoform of the scrapie prion protein PrPSc is both a host-derived protein and a component of the infectious agent causing scrapie. PrPSc and the normal cellular isoform PrPC have different physical properties that apparently arise from a posttranslational event. Both PrP isoforms are covalently modified at the carboxy terminus by a glycoinositol phospholipid. Using preparations of dissociated cells derived from normal and scrapie-infected hamster brain tissue, we find that the majority of PrPC is released from membranes by phosphatidylinositol-specific phospholipase C (PIPLC), while PrPSc is resistant to release. In contrast, purified denatured PrP 27-30 (which is formed from PrPSc during purification by proteolysis of the amino terminus) is completely cleaved by PIPLC. Incubation of the cell preparations with proteinase K cleaves PrPSc to form PrP 27-30, demonstrating that PrPSc is accessible to added enzymes. We have also developed a protocol involving biotinylation that gives a quantitative estimate of the fraction of a protein exposed to the cell exterior. Using this strategy, we find that a large portion of PrPSc in the cell preparations reacts with a membrane-impermeant biotinylation reagent. Whether alternative membrane anchoring of PrPSc, inaccessibility of the glycoinositol phospholipid anchor to PIPLC, or binding to another cellular component is responsible for the differential release of prion proteins from cells remains to be determined.