Michael P. McKinley

A cellular gene encodes scrapie PrP 27-30 protein.

A clone encoding PrP 27-30, the major protein in purified preparations of scrapie agent, was selected from a scrapie-infected hamster brain cDNA library by oligonucleotide probes corresponding to the N terminus of the protein. Southern blotting with PrP cDNA revealed a single gene with the same restriction patterns in normal and scrapie-infected brain DNA. A single PrP-related gene was also detected in murine and human DNA. PrP-related mRNA was found at similar levels in normal and scrapie-infected hamster brain, as well as in many other normal tissues. Using antisera against PrP 27-30, a PrP-related protein was detected in crude extracts of infected brain and to a lesser extent in extracts of normal brain. Proteinase K digestion yielded PrP 27-30 in infected brain extract, but completely degraded the PrP-related protein in normal brain extract. No PrP-related nucleic acids were found in purified preparations of scrapie prions, indicating that PrP 27-30 is not encoded by a nucleic acid carried within the infectious particles.

Scrapie prions aggregate to form amyloid-like birefringent rods

A large scale purification protocol employing zonal rotor centrifugation has been developed for scrapie prions. The extensively purified fractions derived using this protocol contained only one major protein, designated PrP, and rod-shaped particles. The rods measured 10 to 20 nm in diameter and 100 to 200 nm in length by negative staining; no other particles were consistently observed. SDS denaturation caused the rods to disappear, prion infectivity to diminish, and PrP to become sensitive to protease digestion. Arrays of prion rods ultrastructurally resembled purified amyloid and showed green birefringence by polarization microscopy after staining with Congo red dye. The rods appear to represent a polymeric form of the scrapie prion; each rod may contain as many as 1,000 PrP molecules. Our findings raise the possibility that the amyloid plaques observed in transmissible, degenerative neurological diseases might consist of prions.

A protease-resistant protein is a structural component of the Scrapie prion

Fractions purified from scrapie-infected hamster brain contain a unique protein, designated PrP. It was labeled with N-succinimidyl 3-(4-hydroxy-5-[125I]-iodophenyl) propionate, which did not alter the titer of the scrapie prion. The concentration of PrP was found to be directly proportional to the titer of the infectious prion. Both PrP and prion infectivity were resistant for 2 hr at 37 degrees C to hydrolysis by proteinase K under nondenaturing conditions. Prolonging the digestion resulted in a concomitant decrease in both PrP and the scrapie prion. When the amino-acid-specific proteases trypsin or SV-8 protease were used instead of proteinase K, no change in either PrP or the prion was detected. The parallel changes between PrP and the prion provide evidence that PrP is a structural component of the infectious prion. Our findings also suggest that the prion contains only one major protein, namely PrP.

Distinct prion proteins in short and long scrapie incubation period mice

The Prn-i gene, controlling scrapie incubation period, is linked to or congruent with the murine prion protein (PrP) gene, Prn-p. In prototypic mouse strains with long (l/Ln) and short (NZW) incubation periods, Prn-p transcription is initiated at similar multiple sites. The predicted NZW and l/Ln PrP proteins differ at codons 108 and 189. Codon 189, highly conserved in mammals, lies within a polymorphic BstEll site that is retained in 17 mouse strains known to have short or intermediate incubation times, but is absent in l/Ln and two other inbred mice with long incubation times. Codon 108 in mice with short or intermediate incubation times encodes Leu; in mice with long incubation times it encodes Phe. The correlation of PrP sequence with length of scrapie incubation period suggests, but does not formally prove, congruency between Prn-p and Prn-i.

Identification of prion amyloid filaments in scrapie-infected brain

Extracellular collections of abnormal filaments composed of prion proteins have been identified in the brains of scrapie-infected hamsters using immunoelectron microscopy. Some of the filaments were 1500 nm in length; generally, they exhibited a uniform diameter of 16 nm. Rarely, the filaments had a twisted appearance, raising the possibility that they are flattened cylinders or are composed of helically wound protofilaments. The prion filaments possess the same diameter and limited twisting as the shorter rod-shaped particles observed in purified preparations of prions. Both the filaments and rods are composed of PrP 27-30 molecules, as determined by immunoelectron microscopy using affinity-purified antibodies. The ultrastructural features of the prion filaments are similar to those reported for amyloid in many tissues including brain. These results provide the first evidence that prion proteins assemble into filaments within the brain and that these filaments accumulate in extracellular spaces to form amyloid plaques.

Creutzfeldt-Jakob disease prion proteins in human brains

Creutzfeldt-Jakob disease is caused by a slow infectious pathogen, or prion. We found that purified fractions from the brains of two patients with Creutzfeldt-Jakob disease contained protease-resistant proteins ranging in apparent molecular weight from 10,000 to 50,000. These proteins reacted with antibodies raised against the scrapie prion protein PrP 27-30. Rod-shaped particles were found in the brain tissue of the patients that were similar to those isolated from rodents with either scrapie or experimental Creutzfeldt-Jakob disease. After being stained with Congo red dye, the protein polymers from patients with Creutzfeldt-Jakob disease exhibited green birefringence when examined under polarized light. Our findings suggest that the amyloid plaques found in the brains of patients with Creutzfeldt-Jakob disease may be composed of paracrystalline arrays of prions similar to those in prion diseases in laboratory animals.

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.

Purified scrapie prions resist inactivation by procedures that hydrolyze, modify, or shear nucleic acids

Prions were purified from scrapie-infected hamster brains and incubated for 24 hr at 65 degrees with 2 mM Zn2+ or 5 mM Mg2+; no loss of infectivity was observed. Bacteriophage M13, tobacco mosaic virus (TMV), potato virus X, and potato spindle tuber viroid were all inactivated by divalent metal ions under these conditions. Prions also resisted inactivation by prolonged digestions with DNase I, RNases A and T1, and micrococcal nuclease. Prions were resistant to psoralen photoadduct formation using high concentrations of psoralens; in contrast, M13 bacteriophage was inactivated by low concentrations of all these psoralens. Hydroxylamine failed to inactivate prions even after lengthy exposures to concentrations as high as 1 M, while TMV and M13 were both inactivated. Sonication of prions failed to decrease infectivity even though rod-shaped aggregates were disrupted while both M13 and TMV lost infectivity.

On the biology of prions

SummaryPrions cause scrapie and Creutzfeldt-Jakob disease (CJD); these infectious pathogens are composed largely, if not entirely, of protein molecules. No prion-specific polynucleotide has been identified. Purified preparations of scrapie prions contain high titers (≥109.5 ID50/ml), one protein (PrP 27-30) and amyloid rods (10–20 nm in diameter ×100–200 nm in length). Considerable evidence indicates that PrP 27-30 is required for and inseparable from scrapie infectivity. PrP 27-30 is encoded by a cellular gene and is derived from a larger protein, denoted PrPSc or PrP 33-35Sc, by protease digestion. A cellular isoform, designated PrPC or PrP 33-35C, is encoded by the same gene as PrPSc and both proteins appear to be translated from the same 2.1 kb mRNA. Monoclonal antibodies to PrP 27-30, as well as antisera to PrP synthetic peptides, specifically react with both PrPC and PrPSc, establishing their relatedness. PrPC is digested by proteinase K, while PrPSc is converted to PrP 27-30 under the same conditions. Prion proteins are synthesized with signal peptides and are integrated into membranes. Detergent extraction of microsomal membranes isolated from scrapie-infected hamster brains solubilizes PrPC but induces PrPSc to polymerize into amyloid rods. This procedure allows separation of the two prion protein isoforms and the demonstration that PrPSc accumulates during scrapie infection, while the level of PrPC does not change. The prion amyloid rods generated by detergent extraction are identical morphologically, except for length, to extracellular collections of prion amyloid filaments which form plaques in scrapie- and CJD-infected brains. The prion amyloid plaques stain with antibodies to PrP 27-30 and PrP peptides. PrP 33-35C does not accumulate in the extracellular space. Prion rods composed of PrP 27-30 can be dissociated into phospholipid vesicles with full retention of scrapie infectivity. The murine PrP gene (Prn-p) is linked to thePrn-i gene which controls the length of the scrapie incubation period. Prolonged incubation times are a cardinal feature of scrapie and CJD. While the central role of PrPSc in scrapie pathogenesis is well established, the chemical as well as conformational differences between PrPC and PrPSc are unknown but probably arise from post-translational modifications.

Developmental expression of prion protein gene in brain

Synthesis of the cellular isoform of the prion protein (PrPC) was found to be regulated during development of the hamster brain. PrP poly A(+) RNA was readily detectable 10 days postpartum; after 20 days of age, no change in its level could be detected through 13 months of age. Low levels of PrP poly A(+) RNA were detectable 1 day after birth. By contrast, myelin basic protein poly A(+) RNA was found at high levels in brain at 30 days of age and thereafter declined steadily. Using monospecific PrP antisera, immunoprecipitable cell-free translation products were detected at low levels 2 days after birth and increased progressively through 10 days of age. How the levels of PrP mRNA participate in brain development and function remains to be established.