Tubulofilaments in negatively stained scrapie-infected brains: relationship to scrapie-associated fibrils.

Abstract

A simple method was devised for negativestain transmission electron microscopy of brain infected with the agent of scrapie. Brains of infected' hamsters contained large masses of tubuloframentous structures with irregular fuzzy surfaces. Brains of mice infected with Creutzfeldt-Jakob disease agent contained similar tubulofilaments in smaller numbers. The abnormal tubulofilaments resembled but were distinguished from normal microtubules. On grids soaked in sodium dodecyl sulfate the abnormal tubuloframents were found in stages of fragmentation, an outer coat appearing to strip from the surface to reveal thinner fibrillary structures resembling scrapie-associated fibrils (SAF). The unmasked fibrils were identified as SAF by immunogold labeling, while the larger tubulofilaments were not labeled. The findings indicate that in infected brain tissue SAF may occur as an internal part of a larger structure that is disrupted by detergent and are not likely to be an artifact formed during extraction procedures.Unique "virus-like" tubulovesicular particles (Fig. 1) have been consistently observed by electron microscopy (EM) in thin sections of plastic-embedded brain tissues from several types of animals and from humans infected with the agents of scrapie and Creutzfeldt-Jakob disease (CJD) (1-6). However, progress in characterizing those particles or investigating their relationship to the etiological agents of the spongiform encephalopathies has been impeded by failure to identify them except in plastic sections, where they are relatively inaccessible to studies other than morphological analyses. We studied unembedded preparations of brain tissue by negative staining (7) with the goal of obtaining such particles in a state in which they could be concentrated and purified. Not only were abnormal tubulofilaments identified in brains of rodents with spongiform encephalopathies but also an unanticipated association was observed between those structures and filaments of the amyloid-like protein variously designated as scrapie-associated fibrils (SAF) (8) and prion protein 27-30 (PrP 27-30) (9, 10). MATERIALS AND METHODSScrapie and CJD Agents. The strains of agent used have been previously described. The 263K strain of hamsteradapted scrapie agent (11, 12) was used at the 4th passage level (13). The KFu strain of mouse-adapted CJD agent (14) was used at our 3rd passage level (15).Animals and Inoculations. Weanling female LGV/LAK golden Syrian hamsters were inoculated intracerebrally with 0.03-ml aliquots of 10% suspension of scrapie-infected brain as previously described. The inoculum of scrapie agent contained at least 107 LD50 units per 0.03 ml; animals became ill as early as 65 days later, and all had died by 90 days after inoculation. Weanling female National Institutes of Health white Swiss mice were inoculated intracerebrally with 0.03 ml of a 10-3 dilution of CJD-agent infected brain suspension; the inoculum contained at least 50 LD50 units per 0.03 ml.Mice became ill 141 days later and were killed 149 days after...

Evidence of ssDNA in Tubulofilamentous Particles: Their Relationship to Scrapie-Associated Fibrils

“…The grids prepared by the impression method were treated with several different proteases and nucleases. This study confirms the previous observations [9,15] that the tubulofilamen tous particle contains a central core with a single-stranded DN A (ssDNA) which is pro tected by a protein coat of unknown origin.…”

“…Recently, experi ments have shown that SAF/PrP can be de tected in brains of infected mice and ham sters, but not in their spleen samples with a similar titre of infectivity [10], Infectivity also has been demonstrated in the absence of prion rods [11] and can be separated from any identifiable structure [12], In addition to SAF, one other type of structure, tubulofilamentous particle, is seen in a thin section by electron microscopy in all spongiform encephalopathies -experimen tal and natural [13,14]. Using scrapie-infected hamster brains, negative staining of grids prepared by an 'impression' technique de monstrated that SAF form the core of tubulofilamentous particles [9,15]. The present study was extended to mice infected with four different strains of scrapie.…”

“…A number of studies show that scrapie infectivity is intimately associated with amy loid fibrils called scrapie-associated fibrils (SAF) [1,2] or prion rods [3,4], SAF and prion rods are composed of the same major protease-resistant sialoglycoprotein of 27-30 kD (PrP 27-30) [5][6][7]. Antibodies prepared against SAF/PrP prion rods cross-react with each other [8,9]; therefore, the terms most likely are synonymous. Recently, experi ments have shown that SAF/PrP can be de tected in brains of infected mice and ham sters, but not in their spleen samples with a similar titre of infectivity [10], Infectivity also has been demonstrated in the absence of prion rods [11] and can be separated from any identifiable structure [12], In addition to SAF, one other type of structure, tubulofilamentous particle, is seen in a thin section by electron microscopy in all spongiform encephalopathies -experimen tal and natural [13,14].…”

Evidence that DNA is present in abnormal tubulofilamentous structures found in scrapie.

“…The 263K strain of scrapie agent (9) was used at the fourth passage level as described (1 units/ml diluted in buffer recommended by the supplier; and (ix) diluent alone. Grids were immersed under 0.5 ml of solution and incubated in a moist chamber.…”

“…In addition, after a 30-min treatment with proteinase K, collagenase, or trypsin, grids were washed and then treated with 2% NaDod-S04 or stained with 0.02% aqueous ethidium bromide (Calbiochem) by floating on a drop for 5 min; grids were then washed five times with distilled water, fixed, and stained as above. After incubations of 30, 60, 90, or 120 min, 2 grids were removed from each group, washed three times with 0.1 M MgCl2 and three times with distilled water, and then fixed with ruthenium red, glutaraldehyde, and osmium tetroxide as described (1). Finally grids were stained with phosphotungstic acid (pH 6.6) for 30 sec, edge-drained on filter paper, and air-dried.…”