Infrared microspectroscopy detects protein misfolding cyclic amplification (PMCA)-induced conformational alterations in hamster scrapie progeny seeds.

The self-replicative conformation of misfolded prion proteins (PrP) is considered a major determinant for the seeding activity, infectiousness, and strain characteristics of prions in different host species. Prion-associated seeding activity, which converts cellular prion protein (PrP(C)) into Proteinase K-resistant, infectious PrP particles (PrP(TSE)), can be monitored in vitro by protein misfolding cyclic amplification (PMCA). Thus, PMCA has been established as a valuable analytical tool in prion research. Currently, however, it is under discussion whether prion strain characteristics are preserved during PMCA when parent seeds are amplified in PrP(C) substrate from the identical host species. Here, we report on the comparative structural analysis of parent and progeny (PMCA-derived) PrP seeds by an improved approach of sensitive infrared microspectroscopy. Infrared microspectroscopy revealed that PMCA of native hamster 263K scrapie seeds in hamster PrP(C) substrate caused conformational alterations in progeny seeds that were accompanied by an altered resistance to Proteinase K, higher sedimentation velocities in gradient ultracentrifugations, and a longer incubation time in animal bioassays. When these progeny seeds were propagated in hamsters, misfolded PrP from brain extracts of these animals showed mixed spectroscopic and biochemical properties from both parental and progeny seeds. Thus, strain modifications of 263K prions induced by PMCA seem to have been partially reversed when PMCA products were reinoculated into the original host species.

Ultrastructures and strain comparison of under-glycosylated scrapie prion fibrils.

Prions, composed primarily of misfolded, often fibrillar, polymers of prion protein, have poorly understood structures. Heavy surface glycosylation may obscure visualization of their fibrillar cores, so we purified severely under-glycosylated prion protein fibrils from scrapie-infected transgenic mice expressing anchorless prion protein. Using electron and atomic force microscopy, we obtained dimensions and morphological information about prion protein core protofilaments which variably intertwined to form scrapie fibrils. Occasional isolated protofilaments were observed, suggesting that the lateral association of protofilaments is neither essential nor invariant in prion protein polymerization. Strain comparisons suggested basic structural differences; ME7 and 22L fibrils contained thinner protofilaments, 22L fibrils preferred left-handed twists, and 22L fibril periodicities averaged 106nm per half-turn, compared with 64 and 66nm for RML and ME7 fibrils, respectively. The strains displayed overlapping fibril morphologies, providing evidence that prion fibril morphology is influenced, but not dictated, by strain-dependent differences in protofilament structure. These measurements of the amyloid core of scrapie fibrils should aid development of models of prion structure and strain determination.

Failure to detect abnormal prion protein and scrapie-associated fibrils 6 wk after intracerebral inoculation of genetically susceptible sheep with scrapie agent.

Detection of the scrapie-associated protease-resistant prion protein (PrPres) in sheep brains in the early phase after intracerebral inoculation of the scrapie agent has not been documented. Fourteen 4-mo-old, genetically susceptible lambs (QQ homozygous at codon 171 of the PrP gene) were obtained for this study. Twelve lambs were inoculated intracerebrally with a brain suspension from sheep naturally affected with scrapie, and 2 served as uninoculated controls. Two inoculated animals were euthanized at each of 6 times postinoculation (1 h to 6 wk), and their brains were collected for histopathological study, for detection of PrPres by the Western blot technique and an immunohistochemical (IHC) method, and for the detection of scrapie-associated fibrils (SAF) by negatively stained electron microscopy (EM). Microscopic lesions associated with introduction of the inoculum were seen in the brains of inoculated animals at all 6 times. However, both the Western blot and IHC techniques did not detect PrPres after the initial 3 d postinoculation, nor did EM detect SAF in any of the samples. From these findings, it is presumed that until host amplification has occurred, the concentration of PrPres in inoculum is insufficient for detection by currently available techniques.

Structural studies of the scrapie prion protein by electron crystallography.

Because the insolubility of the scrapie prion protein (PrP(Sc)) has frustrated structural studies by x-ray crystallography or NMR spectroscopy, we used electron crystallography to characterize the structure of two infectious variants of the prion protein. Isomorphous two-dimensional crystals of the N-terminally truncated PrP(Sc) (PrP 27-30) and a miniprion (PrP(Sc)106) were identified by negative stain electron microscopy. Image processing allowed the extraction of limited structural information to 7 A resolution. By comparing projection maps of PrP 27-30 and PrP(Sc)106, we visualized the 36-residue internal deletion of the miniprion and localized the N-linked sugars. The dimensions of the monomer and the locations of the deleted segment and sugars were used as constraints in the construction of models for PrP(Sc). Only models featuring parallel beta-helices as the key element could satisfy the constraints. These low-resolution projection maps and models have implications for understanding prion propagation and the pathogenesis of neurodegeneration.

Evaluation of a rapid western immunoblotting procedure for the diagnosis of bovine spongiform encephalopathy (BSE) in the UK.

Bovine brain tissue samples from 625 UK cattle, clinically suspected as bovine spongiform encephalopathy (BSE) cases, were used in a blind analysis to assess a rapid Western immunoblotting technique (Prionics Check; Prionics AG, Zurich), which detects bovine disease-specific protease-resistant prion protein (PrP(Sc)). By means of statutory histopathological examination, 599 of the 625 cattle were confirmed as BSE cases by the demonstration of spongiform encephalopathy, the remaining 26 being classified as negative. Duplicate samples from the same animals were also examined by electron microscopy for the presence of abnormal brain fibrils (scrapie-associated fibrils; SAFs). The Prionics technique showed a high sensitivity, particularly when compared with the fibril detection test; the detection rates were 99.3% and 92.0% respectively, with histopathology being used as the "gold standard". The false negative results by the Prionics test were possibly related to the sampling procedure. Analysis of 50 BSE-positive samples revealed similar glycoprofiles, the majority of PrP(Sc)isoforms being di-glycosylated protein. The Prionics test also detected PrP(Sc)in the four brain samples from the 26 histopathologically negative animals, apparently reducing the specificity of the test to 84.6%; however, confirmatory positive results in these samples were obtained by demonstrating SAF or by immunohistochemical examination, or both. It was concluded that the Prionics test detected PrP(Sc)in a small percentage (0.64%) of clinically suspected BSE cases showing no spongiform change. Since January 2000, the Prionics Western blot test has been introduced as one of the statutory tests for the diagnosis of clinically suspected BSE and scrapie cases in the UK.

The reproducibility of scrapie-associated fibril and PrPSc detection methods after long-term cold storage of natural ovine scrapie-affected brain tissue.

A pool of grey matter (medulla/brain stem, cerebellum and frontal cerebral cortex) was prepared from the brains of 16 sheep with scrapie, diagnosed clinically and by the demonstration of spongiform encephalopathy. Aliquots from the pool of tissue were finely chopped or homogenized and stored at +4 degrees C or -70 degrees C, after undergoing one of several specific pre-treatments (storage with or without protease inhibitors or, alternatively, with or without the cryoprotectant, dimethyl sulphoxide). At intervals over a period of 2 years, the stored extracts were examined by electron microscopy for the presence of scrapie-associated fibrils (SAFs) and by Western immunoblotting for the disease-specific abnormal protein PrPSc. Throughout the 2-year period, SAFs and PrPSc were detected in the majority of all stored tissue extracts under all combinations of tissue preparation and pre-treatment. The combined detection rates for SAFs and PrPSc were 91% at +4 degrees C and 94% at -70 degrees C. There was no significant difference between the results obtained by the two detection methods and no specific combination of preparation method and pre-treatment was superior to any other. Storage of the samples at -70 degrees C appeared to give better results than storage at +4 degrees C, particularly with regard to fibril detection. For logistical reasons and ease of processing, and to avoid the effects of autolysis on recognizable brain regions, long-term storage at -70 degrees C, without any pre-treatment, would appear to be the method of choice.

Ultrastructural studies on scrapie prion protein crystals obtained from reverse micellar solutions.

The structural transition from the cellular prion protein (PrPC) that is rich in alpha-helices to the pathological form (PrPSc) that has a high beta-sheet content seems to be the fundamental event underlying the prion diseases. Determination of the structure of PrPSc and the N-terminally truncated PrP 27-30 has been complicated by their insolubility. Here we report the solubilization of PrP 27-30 through a system of reverse micelles that yields monomeric and dimeric PrP. Although solubilization of PrP 27-30 was not accompanied by any recognizable change in secondary structure as measured by FTIR spectroscopy, it did result in a loss of prion infectivity. The formation of small two- and three-dimensional crystals upon exposure to uranyl salts argues that soluble PrP 27-30 possesses considerable tertiary structure. The crystals of PrP 27-30 grown from reverse micellar solutions suggest a novel crystallization mechanism that might be applicable for other membrane proteins. A variety of different crystal lattices diffracted up to 1.85 nm by electron microscopy. Despite the lack of measurable biological activity, the structure of PrP 27-30 in these crystals may provide insight into the structural transition that occurs during PrPSc formation.

Comparison of scrapie-associated fibril detection and Western immunoblotting for the diagnosis of natural ovine scrapie.

Detergent- and proteinase K-treated extracts of grey matter were prepared from four regions of the brains of 106 sheep with scrapie, diagnosed clinically and by the demonstration of spongiform encephalopathy. The extracts were examined by electron microscopy for the presence of scrapie-associated fibrils and by Western immunoblotting for the disease-specific abnormal prion protein (PrPSc). As a diagnostic method, Western immunoblotting proved to be more sensitive than electron microscopy, the detection rates in the 106 sheep being 97 and 91% respectively (medulla), 99 and 76% (cerebellum), 95 and 88% (frontal cerebral cortex) and 93 and 61% (occipital cerebral cortex). Neither fibrils nor PrPSc could be detected in comparable brain extracts from 25 control sheep which had shown no clinical or histopathological evidence of scrapie.

Separation of scrapie prion infectivity from PrP amyloid polymers.

The prion protein (PrP) undergoes a profound conformational change when the cellular isoform (PrPC) is converted into the scrapie form (PrPSc). Limited proteolysis of PrPsc produces PrP 27-30 which readily polymerizes into amyloid. To study the structure of PrP amyloid, we employed organic solvents that perturb protein conformation. Hexafluoro-2-propanol (HFIP), which promotes alpha-helix formation, modified the ultrastructure of rod-shaped PrP amyloids; flattened ribbons with a more regular substructure were found. As the concentration of HFIP was increased, the beta-sheet content and proteinase K resistance of PrP 27-30 as well as prion infectivity diminished. HFIP reversibly decreased the binding of Congo red dye to the rods while inactivation of prion infectivity was irreversible. In contrast to 10% HFIP, 1,1,1-trifluoro-2-propanol (TFIP) did not inactivate prion infectivity but like HFIP, TFIP did alter the morphology of the rods and abolish Congo red binding. This study separates prion infectivity from the amyloid properties of PrP 27-30 and underscores the dependence of prion infectivity on PrPSc conformation. The results also demonstrate that the specific beta-sheet-rich structures required for prion infectivity can be differentiated from those needed for amyloid formation as determined by Congo red binding.

Comparative study of electron microscopical techniques for the detection of scrapie-associated fibrils.

Samples of cervical spinal cord and four anatomical regions of the brains of 12 sheep with natural scrapie and six control sheep were examined by electron microscopy, after the tissues had been stored at 4 degrees C and -20 degrees C. The tissues were tested for the presence of scrapie-associated fibrils by a centrifugal extraction technique and by a touch-grid technique. The touch-grid technique was no better than the centrifugal extraction technique for the detection of fibrils. Structures which could have been classified as tubulofilaments were detected in touch-grid preparations without detergent treatment. With the centrifugal extraction technique there was a significant reduction of the fibril scores in some of the tissue extracts stored at -20 degrees C, but not in any of the extracts stored at 4 degrees C. There was, however, a reduction in the fibril scores when the final extracted pellets were stored at 4 degrees C. The stability of the fibrils on the test grids was unaffected by six months storage at room temperature but the clarity of their ultrastructure did deteriorate. Poor hydrophilic spread of the sample on the test grids did not have a significant effect on the fibril scores.

X-ray diffraction of scrapie prion rods and PrP peptides.

Certain neurodegenerative diseases in humans and animals are caused by small proteinaceous infectious particles called prions. Limited proteolysis and detergent extraction of the prions containing PrPSc generate prion rods that are composed of a polypeptide having an apparent molecular mass of 27 to 30 kDa. This polypeptide, termed prion protein PrP 27-30, has a ragged N terminus that begins at about residue 90, but retains scrapie infectivity. Moreover, the findings in a patient having an inherited prion disease of a truncated PrP with its C terminus at residue 145 suggest that the residues 90 to 145 may be of particular importance in the pathogenesis of prion diseases. To determine the three-dimensional organization of prion rods and to identify the core region involved in amyloid formation, we recorded X-ray diffraction patterns from rods purified from scrapie-infected Syrian hamster (SHa) brains which contain PrP 27-30, and from synthetic SHaPrP peptides. Three peptides were studied corresponding to residues 113 to 120 (peptide A8A, an octamer composed of glycines and alanines), 109 to 122 (H1, the first predicted alpha-helical region of PrPC), and 90 to 145 (a 56 residue peptide containing both H1 and the second predicted alpha-helical region of PrPC, H2). Electron microscopy, carried out in parallel with the X-ray measurements, revealed that all the samples formed linear polymers which were approximately 60 to approximately 200 A wide, with fibrillar or ribbon-like morphology. Gels and dried preparations of prion rods gave X-ray patterns that indicated a beta-sheet conformation, in which the hydrogen bond distance was 4.72 A and the intersheet distance was 8.82 A. For the three PrP peptides, the intersheet spacings varied widely, owing to the side-chains of the residues involved in the formation of the beta-sheet interactions, i.e., 5.13 A for A8A, 5.91 A for lyophilized H1, 7.99 A from solubilized and dried H1 and 9.15 A for the peptide SHa 90-145. The intersheet distance of PrP 27-30 was thus within the observed range for the peptides, and suggests that the amyloidogenic core of PrP is closely modeled by the peptide SHa 90-145.

In situ hybridization and immunohistochemistry for prion protein (PrP) in bovine spongiform encephalopathy (BSE).

In about 5% of the cows showing clinical signs of bovine spongiform encephalopathy (BSE) the histopathological examination is not conclusive. In order to rule out BSE in these cases, additional methods are necessary. For that reason, non-radioactive in situ hybridization (ISH) was performed using a riboprobe against the messenger RNA coding for the prion protein (PrP). In addition, a polyclonal antibody for immunohistochemistry (IHC) was generated against a synthetic peptide derived from bovine PrP. ISH and IHC were used to analyse brain sections of cattle suffering from BSE and various neurological diseases including four cows with clinically suspect but histologically unconfirmed BSE. ISH revealed no differences in localization, distribution and neuronal levels of PrP mRNA between BSE positive and negative cattle. However, there was a BSE-specific staining pattern in IHC allowing to exclude BSE the four suspected cases. Additionally, IHC for PrP is an elegant alternative to search for scrapie associated fibrils by electron microscopy.

Pathology of the transmissible spongiform encephalopathies with special emphasis on ultrastructure.

The transmissible spongiform encephalopathies are a group of genetic and infectious disorders which are exemplified by scrapie in animals and Creutzfeldt-Jakob disease in humans. The spongiform encephalopathies are characterized by symmetrical vacuolation of neurons and neuropil. Amyloid plaque formation similar to that found in Alzheimer's disease is conspicuous in many, but not all, of these diseases. The sub-cellular pathology features of the spongiform encephalopathies have been studied by conventional transmission electron microscopy, scanning electron microscopy, freeze fracture, negative staining and most recently by application of immunogold labelling methods. Although these studies have revealed many unusual structures, convincing virus-like particles have not been demonstrated. Considerable data, including important transgenic mouse studies, now suggest that a single cellular protein, designated prion protein, is necessary for infection. Ultrastructural immunogold studies have shown that prion protein is released from the surface of neurons and neurites, diffuses through the extracellular space around infected cells where it accumulates and finally becomes aggregated as amyloid fibrils. It is likely that the accumulation of prion protein within the extracellular space is instrumental in causing nerve cell dysfunction and, ultimately, neurological disease.