Properties of the scrapie agent-endomembrane complex from hamster brain.

Subcellular fractionation of scrapie-infected hamster brain indicated the association of the scrapie agent with a component of the endomembrane system. Characterization by equilibrium density gradient centrifugation, electron microscopy, and marker enzymes suggested a primary association with rough and smooth endoplasmic reticulum and a possible incorporation into the plasma membrane. DNA polymerase activity demonstrated a direct correlation with regions of scrapie activity from the gradient fractions. A scrapie-related product was detected after (3H)TMP incorporation and analysis on 2.2% polyacrylamide gels. Analysis of nucleic acid species extracted from subcellular fractions resulted in a greater quantity from healthy brain; however, no qualitative distinctions were detected.     

Alteration of free radical metabolism in the brain of mice infected with scrapie agent.

Alteration of free radical metabolism in the mouse brain by scrapie infection was evaluated. The infection of mice with scrapie agent, 87V strain, slightly increased the activities of catalase and glutathione-S-transferase, while it had no effect on glutathione peroxidase, glutathione reductase, and Cu, Zn-superoxide dismutase. Results show that the scrapie infection decreased the activity of mitochondrial Mn-superoxide dismutase by 50% but increased that of monoamine oxidase (p < 0.05). Scrapie infection also increased the rate of mitochondrial superoxide generation (p < 0.05). Following scrapie infection, the level of free-sulfhydryl compounds in brain homogenates slightly decreased, but the content of thiobarbituric-acid-reactive substances and malondialdehyde increased significantly. Electron microscopy indicated that the ultrastructure of mitochondria was destroyed in the brain of scrapie-infected mice. These results suggest that elevated oxygen free radical generation and lowered scavenging activity in mitochondria might cause the free radical damage to the brain. Such deleterious changes in mitochondria may contribute to the development of prion disease.     

Immune system-dependent and -independent replication of the scrapie agent.

Using the severe combined immunodeficiency (SCID) mouse model, we investigated the requirement of the immune system for the development of scrapie after peripheral inoculation. A total of 33% of SCID mice, all but one immunologically reconstituted SCID mice (93%), and all CB17 control mice developed the disease. PrPres was detectable in the brains of all diseased animals and in the spleens of reconstituted SCID and CB17 control mice but not of the diseased non-immunologically reconstituted SCID mice. The immune system appears to be a primary target in the pathogenesis of scrapie, but direct spread to the central nervous system from the peritoneum via visceral nerve fibers can probably also occur.     

Karyological characteristics of continuous mouse cell lines infected with the scrapie agent

A karyological study of murine cell line L, latently infected with different strains of the scrapie agent (Compton and C-506) showed that the both variants number of chromosomes and the number of Robertson's translocations of chromosomes decrease insignificantly with an increase in the time of subcultivation. The use of the C-method of differential chromosome staining revealed four new analogous chromosomes in both experimental cell lines. This phenomenon is probably specific for this infected cell model.     

Towards purification of the scrapie agent

A method for the partial purification of scrapie infectivity from hamster brain is described. About a 100-1000-fold, 20-fold, and 200-fold enrichment in scrapie infectivity with respect to protein, RNA, and DNA content has been achieved using differential centrifugation, enzyme and detergent treatment. The inbred CLAC strain of hamsters used in our experiments contained about 10 times less infectivity in brain than has been found in randomly bred animals or other inbred strains.     

Congo red inhibition of scrapie agent replication.

Congo red inhibits the accumulation of protease-resistant PrP in scrapie-infected mouse neuroblastoma cells. Here we show that Congo red also inhibits the replication of scrapie infectivity in these cells. This observation is consistent with the idea that protease-resistant PrP is a vital component of the scrapie agent or that agent replication depends on the presence of protease-resistant PrP in the cell.     

Role of the GALT in scrapie agent neuroinvasion from the intestine

Following oral exposure, some transmissible spongiform encephalopathy (TSE) agents accumulate first upon follicular dendritic cells (FDCs) in the GALT. Studies in mice have shown that this accumulation is obligatory for the efficient delivery of the TSE agent to the brain. However, which GALTs are crucial for disease pathogenesis is uncertain. Mice deficient in specific GALT components were used here to determine their separate involvement in scrapie agent neuroinvasion from the intestine. In the combined absence of the GALTs and FDCs (lymphotoxin (LT)alpha(-/-) mice and LTbeta(-/-) mice), scrapie agent transmission was blocked. When FDC maturation was induced in remaining lymphoid tissues, mice that lacked both Peyer's patches (PPs) and mesenteric lymph nodes (wild-type (WT)-->LTalpha(-/-) mice) or PPs alone (WT-->LTbeta(-/-) mice) remained refractory to disease, demonstrating an important role for the PPs. Although early scrapie agent accumulation also occurs within the mesenteric lymph nodes, their presence in WT-->LTbeta(-/-) mice did not restore disease susceptibility. We have also shown that isolated lymphoid follicles (ILFs) are important novel sites of TSE agent accumulation in the intestine. Mice that lacked PPs but contained numerous FDC-containing mature ILFs succumbed to scrapie at similar times to control mice. Because the formation and maturation status of ILFs is inducible and influenced by the gut flora, our data suggest that such factors could dramatically affect susceptibility to orally acquired TSE agents. In conclusion, these data demonstrate that following oral exposure TSE agent accumulation upon FDCs within lymphoid tissue within the intestine itself is critically required for efficient neuroinvasion.     

Characterization of scrapie agent isolated from sheep in Japan

A pathogenic agent isolated in mice from the brain of a sheep affected by scrapie-like disease was characterized. The incubation period of the disease in the primary transmission from the sheep to mice was longer than in the secondary and the tertiary transmission in the same strain of mice. Progressive dilution of the inoculum caused prolongation of the incubation period. The infectivity of the agent in a 10% brain homogenate persisted, but decreased about 10(3) to 10(4) times after heating at 100 C for 30 min. Histological changes in the diseased mouse brains consisted of vacuolation of the nerve cells and spongiform degeneration in the gray matter of the central nervous system. Fine rod-shaped granulae with a length of 3 to 5 nm were observed within the swollen neuropil, axon, and perivascular astrocytic process. No serum antibodies against available mouse viruses, parainfluenza type 1 virus, lymphocytic choriomeningitis virus, and mouse reovirus type 3, were detected in any mice used in the experiments. These findings demonstrate that the disease of the sheep was the first case of scrapie in Japan.     

The search for scrapie agent nucleic acid.

Despite decades of research, the identity of the scrapie agent has remained elusive. Recent studies have discovered much about the influence of the host genome upon scrapie infection, yet relatively little is known about the causative agent itself. The predominant hypothesis in the scrapie field (the prion hypothesis) argues that the disease is the result of an infectious protein and that nucleic acid is not required for infection. Biological studies of the scrapie agent, however, suggest that a nucleic acid may be involved in the disease. Sensitive molecular biology techniques have yet to identify this putative nucleic acid.     

Reflections on the nature of scrapie agent

The biochemical structure of scrapie and related agents is one of the fascinating questions of microbiology. The last 20 years of resesrch have been a story of exuberant speculation and experimental difficulties.     

Partial purification and evidence for multiple molecular forms of the scrapie agent.

A procedure for the partial purification of the scrapie agent from mouse spleen was developed based on its sedimentation profile. Differential centrifugation and detergent treatment with sodium deoxycholate yielded a fraction designated "P5" which was enriched for scrapie infectivity approximately 20-fold with respect to cellular protein. The P5 fraction was devoid of cellular membranes but heavily contaminated with ribosomes as judged by electron microscopy. On centrifugation of the fraction P5 to near equilibrium in a sucrose gradient scrapie infectivity was distributed over a range of densities from 1.08 to 1.30 g/cm3. Parallel rate-zonal analysis showed that the infectivity was distributed over a range of particle sizes with s20.w values from approximately 40 S to greater than 500 S. Incubation of P5 at 37 or 80 degrees C, under conditions that disrupt ribosomes, dramatically altered the rate-zonal gradient profile of the agent. Under these conditions, the agent sedimented as particles with s20.w greater than 500 S. The apparent heterogeneity of the scrapie agent with respect to both size and density and its ability to shift from one form to another suggest that the agent may contain hydrophobic domains on its surface.     

Isolation and structural studies of the intact scrapie agent protein

Purification of the scrapie agent by methods using digestion with proteinase K yields a protein product, PrP-27-30, with an apparent mass of 27-30 kDa (D. C. Bolton et al. (1982) Science 218, 1309-1311; S. B. Prusiner et al. (1982) Biochemistry 21, 6942-6950). In contrast, a 33-37 kDa glycoprotein, HaSp33-37, was the major protein component isolated from scrapie-affected hamster brain by a procedure that did not use protease digestion. The purified fractions containing HaSp33-37 had greater than 10(11) LD50 units of the scrapie agent per milligram of protein. Proteinase K digestion of HaSp33-37 gave a product indistinguishable from PrP-27-30 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The amino acid sequence of the first 22 residues of HaSp33-37 was determined. The sequence coincided with that predicted for the N-terminus of the precursor to PrP-27-30 (K. Basler et al. (1986) Cell 46, 417-428; N. K. Robakis et al. (1986) Proc. Natl. Acad. Sci. USA 83, 6377-6381) after processing by signal protease. HaSp33-37 was digested with N alpha-tosyl-L-phenylalanine chloromethyl ketone-trypsin to produce a 29-32 kDa protein fragment; following digestion this fraction retained complete biological activity. The amino terminal sequence of the 29-32 kDa protein corresponded to a position intermediate between the amino termini of HaSp33-37 and PrP-27-30. We conclude that HaSp33-37 is the intact form of the scrapie agent protein and that PrP-27-30 is produced by proteinase K degradation when this enzyme is introduced during isolation of the scrapie agent.