j . Comp, Path. 1991 Vol. 105

The Ultrastructural Diversity of Scrapie-associated Fibrils Isolated from Experimental Scrapie and

Creutzfeldt-Jakob Disease

P. P. Liberski*~, P. Brown*, Shu-Yan Xiao* and D. C. Gajdusek*

* Laborato,y of Cenlral Nervous System Studies, NINDS, National Institutes of Health, Bethesda, MD 20892, U.S.A. and "IElectron Microscopy Laboratory, Dept. Oncology,

Medical Academy, ~&l.~, Poland

Summary Several different sa,nples of scrapie-associated fibriIs (SAF) were extracted in identical fashion from the brains of golden Syri;m hamsters infected with the 263K strain of scrapie agent and NIH Swiss mice intiected with the Fujisaki strain o[" Creutzfeldt-Jakob disease (CJD) agent. Based on a total &over 500 measurements in individual fibrils in different extracts, hamster fibrils were more abundant, thicker and had better defined substructure than mouse fibrils. Hamster protofibrils were usually either twisted helically or in parallel arrays, whereas mouse protofibrils were often twisted, occasionally parallel, or could not be morphologically defined. Thus, SAF preparations from scrapie-affected hamsters can be uhrastructurally distinguished from those of CJD-affected mice, an observation that presumably reflects differences in their respective host-encoded amyloid protein subunits.

Introduction

Scrapie-associated fibrils (SAF) have been consistently observed in all disorders caused by unconventional viruses (Merz, Somerville, Wisniewski and Iqbal, 1981; Merz, Somerville, Wisniewski, Manuelidis and Manuelidis, 1983a; Merz, Somerville and Wisniewski, 1983b; Merz, Rohwer, Kascak, Wisniewski, Somerville, Gibbs and Gajdusek, 1984; Diringer, Gerdelblom, Hilmert, Ozel, Edelbluth and Kimberlin, 1983; Hilmert and Diringer, 1984; Prusiner, McKinley, Bowman, Bohon, Bendheim, Groth and Glenner, 1983; Kascak, Rubenstein, Merz, Carp, Wisniewski and Diringer, 1985; Cho, 1986; Gibson, Somerville, Fraser, Foster and Kimberlin, 1987; Muhhaup, Diringer, Hilmert, Prinz, Heukeshoven and Beyreuther, 1985; Scott, Done, Venables and Daw- son, 1987; Rubenstein, Merz, Kascak, Carp, Scalici, Farna and Wisniewski, 1987; Hope, Multhaup, Reekie, Kimberlin and Beyreuther, 1988a; Hope, Reekie, Hunter, Muhhaup, Beyreuther, White, Scott, Stack, Dawson and

Corresponding authm': Dr P. P, Liberski, Chie[; Fteetron Microscopy l,al)oratory, Dept Oneology, Medical Academy,~6d~., Poland.

0021-9975/91/080377+ I0 $03.00/0 (C) 1991 Academic Press Limited

378 P.P. Liberski e t al .

Wells, 1988b; Liberski, Plucienniczak, Hrabec and Bogucki, 1989). The ultrastructural morphology of SAF varies slightly according to the procedure used for their purification, but generally SAF appear as rod-like fibrils 10 to 25 nm in diameter composed of two (SAF type I) to four (SAF type II) helically twisted or parallel 4 to 6 nm filaments (Merz et al., 1981, 1983b). They are similar to but easily differentiated from other types ofamyloid fibrils (Merz et al., 1981, 1983a, b; Merz, Wisniewski, Somerville, Bobin, Masters and Iqbal, 1983c).

SAF are composed of protease-resistant sialoglycoprotein (prion protein) PrP 27-30 (Prusiner et al., 1983; Diringer et al., 1983). PrP 27-30 is the cleavage product by proteinase K (PK) of a larger precursor protein PrP 33- 35 Sc from scrapie-affected brains (Oesch, Westaway, Walchii, McKinley, Kent, Aebersold, Barry, Tempst, Teplow, Hood, Prusiner and Weissmann, 1985; Barry, McKinley, Bendheim, Lewis, DeArmond and Prusiner, 1985; Multhaup el al., 1985). Under the same conditions the analogous protein from normal brains (PrP 33-35 So) is completely degraded by PK. The PrP proteins (PrPs) are host encoded and the sequences of their cDNA and homologous genes are very similar but not identical in all animal species (Oesch et al., 1985; Chesebro, Race, Wehrly, Nishio, Bloom, Lechner, Bergstrom, Robbins, Mayer, Keith, Garon and Haase, 1985; Basler, Oesch, Scott, Westaway, Walchii, Groth, McKinley, Prusiner and Weissmann, 1986).

In their ultrastructural morphology SAF are virtually indistit.aguishable from prion rods (Prusiner et al., 1983; McKinley, Barry, Braunfeld, Prusiner and DeArmond, 1986a; McKinley, Braunfeld, Bellinger and Prusiner, 1986b; McKinley, Braunfeld and Prusiner, 1987) and indeed most investigators regard SAF and prion rods as the same structures (Rohwer, 1984; Gajdusek, 1986; Hope and Kimberlin, I987; Kimberlin and Hope, 1987; Liberski et al., 1989). Furthermore, SAF are identified by antibodies against PrP 27-30 (Merz, Kascak, Rubenstein, Carp and Wisniewski, 1987). Despite the fact that subtle ultrastructural differences between SAF and prion rods probably reflect different procedures of purification and staining for electron microscopy, a few investigators continue to suggest that SAF and prion rods are different structures (McKinley el al., 1986a,b; 1987; Diener, 1987; Prusiner, 1988).

We report here the diversity of ultrastructural morphology of SAF isolated from hamster brains infected with the 263K strain ofscrapie agent and mouse brains infected with the Fujisaki strain of Creutzfeldt-Jakob disease (CJD) agent.

M a t e r i a l s a n d M e t h o d s

Agent Strains, Animals and Inoculation Procedures

The Fujisaki strain of CJD agent, isolated from a 56-year old man with progressive dementia (Tateishi, Ohta, Koga, Sato and Kuroiwa, 1978), was passaged three times in this laboratory (Kingsbury, Smeltzer, Amyx, Gibbs and Gajdusek, 1982). The 263K strain ofscrapie agertt was isolated fi-om hamsters (Kimberlin and Walker, 1977, 1978) infected originally with the "Chandler" isolate of a scrapie agent (Chandler and Turfey, 1972) and used at the fourth National Institutes of Health (NIH) passage. The Chandler strain had been isolated from "drowsy goats".

Diversity of Serapie-associated Fibrils 379

Weanling NIH Swiss mice (Animal Production Area, Frederick Cancer Research Facility, Frederick, MD, U.S.A.) were injected intracerebrally with 0"03 ml of a 10 per cent clarified suspension of brain prepared t?om mice terminally ill with the Fujisaki strain of CJD agent (infectivity titre, 3 ' lX104 LDs0 per 0.03ml (weight-volume- w/v), by the intracerebral route). Weanling golden Syrian hamsters (Charles River Lakeview Hamstery, Lakeview, NJ, U.S.A.) were inoculated with 0'03 ml of a 10 per cent (w/v) clarified suspension of the 263K strain ofscrapie agent (infectivity titre, 107.4 LDs0 per 0.03 ml, by the intracerebral route). The incubation period ranged from 16 to 18 weeks for mice infected with the Fujisaki strain of CJD agent, and from 8 to 10 weeks for hamsters infected with the 263K strain ofscrapie agent.

Purification of SAF The following protocol was uniformly employed in SAF purification (Hilmert and Diringer, 1984; Brown, Coker-Vann, Pomeroy, Franko, Asher, Gibbs and Gajdusek, 1986). Approximately 50 g of frozen brain was homogenized by sonication in Tris buffered saline (TBS) containing 10 gl per ml ofproteinase K (PK). The homogenate was centrifuged at 600 x g for 10 min and the pellet was resuspended by sonication in 50 ml of TBS containing 10 gl of PK. Following recentrifugation both supernates were combined and centrifuged at 22 000 x g for 40 min. The supernate was discarded and the pellet, resuspended by sonication in TBS containing 1 per cent sarkosyl, was centrifuged at 22 000 x g for 20 min. The supernate was recentrifuged at 200 000 x g for 120 min and the pellet suspended by sonication in TBS containing 1 per cent sarkosyl and 10 per cent NaC1. After centrifugation at 200000 Xg for 120min, the pellet was suspended in TBS containing 1 per cent sarkosyl and 10 per cent NaC1 and stirred overnight at 37°C. The next day PK was added at a final concentration of 5 gg per ml and the suspension was stirred at 370C for 2 h, then centrifuged at 7000 x g for 15 min. The pellet was resuspended in distilled water containing 1 per cent sarkosyl, stirred at 37°C for 1 h, recentrifuged at 7000 x g tbr 15 min, and finally resuspended in 2 ml of distilled water containing 0.1 per cent sarkosyl and stored at 4°C until examined by means of negative staining electron microscopy.

Negative Staining Electron Microscopy SAF samples from infected and sham-inoculated animals were mounted on freshly glow-discharged 300 mesh Formvar-coated carbon grids, and allowed to remain undisturbed for 1 rain. Sample volumes used to load the grids (10 to 20 ~1) were calculated to maintain a constant amount (5 gg) of added PrP 27-30, as determined by preceding Western immunoblot (Brown, Liberski, Wolff and Gajdusek, 1990a,b). The excess of fluid was drained with filter paper, and the grids were washed five times with distilled water, stained with 2 per cent phosphotungstic acid for 20 s and examined by means of a Philips 300 transmission electron microscope at 60 kV.

Measurements of SAF were performed directly on electron micrograph negatives of fields magnified x 42 000, by means of a 7 x magnifier supplied with metric graticule (1 division = 0' 1 ram). At least two grids were examined for each sample, and at least 10 to 20 SAF were measured on each negative. Since most SAF appeared as aggregated clusters, only SAF surrounding the margins of such clusters were measured.

R e s u l t s

Scrapie-inf ected Hamsler Brains

Most SAF ~brmed large aggregates, easily detectable even at low magnif icat ion (Fig. 1). A t endency of SAF to cluster a round other par t icula te structures,

3 8 0 P . P . L i b e r s k i et al.

mostly collagen fibres was also noted (Figs 1 and 2). At higher magnification SAF appeared as rod-like fibrils, 33 to 207 nm in length (average 111 nm; S.D., 13 nm) and 2'4 to 36 nm in width (average 13 nm; S.D., 8 nm). Each fibril was composed of two to four protofilaments, of which 56 per cent were twisted helically around each other, and 33 per cent formed parallel arrays; the substructure of 11 per cent of SAF was undefined (Fig. 3). A few fibrils were unwrapped at one end, forming flare-like structures (Fig. 3). Occasionally, tiny "spaghetti-like" filaments of wavy appearance and < 2 nm in diameter were observed in close proximity to SAF (Fig. 4) or as aggregates observed against the background of the grid.

CJD agent-infected Mouse Brains

SAF were also mostly clustered (Fig. 5) and appeared somewhat longer (31 to 333 nm; ave/~age = 115; S.D.=46) and thinner (2"4 to 23 nm; average= 10; S.D.=2'4) than those isolated from scrapie-infected hamsters. When these measurements were statistically compared with those of hamster fibrils (Cochran-Cox test), the difference in width was found to be statistically signi- ficant (P<0"01), but the difference in length was not (P>0"I) . In addition, the substructure of mouse fibrils was often difficult to classify: 60 per cent of protofibrils were twisted helically, 11 per cent formed parallel arrays and 28 per cent were undefined.

Fig. 1. SAF purified from hamster brain infected with the 263K strain ofscrapie agent. Note clustering ~1" fibrils along collagen fibre (arrowheads). Bar= i00 ran.

D i v e r s i t y o f S c r a p i e - a s s o c i a t e d F i b r i l s 3 8 ].

Fig, 2. A cluster of SAF purified fi'om hamster brain infected with the 263K strain of scrapie agent. Bar = 100 nm.

Fig. 3. SAF purified from hamster brain inlbcled with the 263K strain o[" scrapie agent. Note twisted (arrowheads), parallel (arrows) and "flare-like" arrays of filaments (open arrows). Bar= 100 nm.

382 P. P . L i b e r s k l et al .

Fig. 4. SAF purified from hamster brain infected with the 263K strain ofscrapie agent. Note "spaghet t i - like" filaments adjacent to SAF (arrowheads). Bar = I00 nm.

Fig. 5. SAF purified fi'om mouse brain infected with the Fujisaki strain of CJD agent. Note tile diversity of fibrils and abundance of protelnaceous material. Bar= 100 nm.

Diversity of Scrapie-associated Fibrils 383

D i s c u s s i o n

The ultrastructural criteria established for SAF by negative staining electron microscopy (Merz et al., 1981, 1983a,b, 1984) originally revealed two types of SAF. Type I was composed of two helically twisted 4 to 6 nm protofilaments yielding fibrils measuring 12 to 16 nm in diameter, while type II SAF was composed of four parallel 4 to 6 nm protofilaments in fibrils measuring 27 to 34 nm in diameter. The length of both types of SAF was 100 to 500 nm. In subsequent experiments it became apparent that the ultrastructural morpho- logy of SAF was more diverse than was at first supposed and that types I and II of" SAF merged with any intermediate fbrms. SAF purified from scrapie- infected hamsters and reported by Diringer et al. (1983), Cho (1986), and Liberski et al. (1989) were shorter and thicker than those purified by Merz and her colleagues (1981, 1983a,b, 1984). Prusiner et al. (1983) described "priori rods" that measured 25 nm in diameter and 100 to 200 nm in length by rotatory shadowing but 10 to 20 nm in diameter by negative staining electron microscopy. Prion rods and SAF are regarded by most (Rohwer, 1984; Gajdusek, 1986; Hope and Kimberlin, 1987; Kimberlin and Hope, 1987; Liberski et al., 1989) but not all investigators (McKinley et al., 1986a,b, 1987; Diener, 1987; Prusiner, 1988) to be the same structures.

Our morphometric data for SAF are consistent with those previously published by other investigators (Merz et al., 1981, 1983a,b,c, 1984; Cho, 1985; Diringer et al., 1983; Kascak et al., 1985; Bockman, Kingsbury, McKinley, Bendheim and Prusiner, 1985; Prusiner et al., 1983; Liberski el al., 1989).. SAF purified from hamster brains infected with the 263K strain of scrapie agent were significantly thicker than those purified from mice infected with the Fujisaki strain of CJD agent, and had better defined protofibril substructure. Because we used the same procedure to purify SAF from both hamsters and mice, these differences cannot be ascribed to technical artefact, as previously suggested (Diringer et al., 1983; Liberski et al., 1989). Instead, they presumably reflect differences in the respective host amyloid protein subunits of the fibrils, since a given host species responds to infection by either agent with produc- tion of its own protein (Bockman, Prusiner, Tateishi and Kingsbury, 1987; Scott, Foster, Mirenda, Serban, Coufal, Walchii, Torchia, Growth, Carlson, DeArmond, Westaway and Prusiner, 1989; Lowenstein, Butler, Westaway, McKinley, DeArmond and Prusiner, 1990).

The higher yield and clearer structure of SAF from scrapie than in CJD specimens has also been noted by other investigators (Bockman et al., 1985; Manuelidis, Valley and Manuelidis, 1985). Since the yield of SAF correlates with the infectivity titre (Prusiner et al., 1983; Somerville, Merz and Carp, 1986), different yields can be ascribed to higher titres of infectivity in scrapie- infected hamsters than in CJD-infected mice.

The nature and biological significance of wavy filamentous structures occasionally observed in specimens of SAF isolated fi'om hamsters infected with the 263K strain of scrapie agent but not from mice infected with the Fujisaki strain of CJD agent is unknown. Similar structures have been previously observed in SAF prepared according to a protocol similar to ours (Merz, 1988) or that exposed to 10 per cent sodium dodecyl sulfate (McKinley

384 P.P. Liberski et al.

el al., 1986b, 1987). We have observed a dramatic increase in these structures in SAF preparations treated with formaldehyde and autoclaved (Brown el al., 1990b) and it is therefore likely that they represent the morphological consequence of partial degradation.

Acknowledgments Dr Pawel P. Liberski was a recipient of a fellowship from Fogarty International Center while in NIH, Bethesda, U.S.A. We wish to thank Mr Kunio Nagashima for skilful technical assistance.

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I Received, January 17th, 1991 Accepted, May 24th, 1991 ]