Note: Due to the interest in Dr. Prusiner's article, Science is offering its readers free access to the associated News item: Prusiner Recognized for Once-Heretical Prion Theory, by Gretchen Vogel.

Prion Diseases and the BSE Crisis

Stanley B. Prusiner

Bovine spongiform encephalopathy (BSE) and human Creutzfeldt-Jakob disease (CJD) are among the most notable central nervoussystem degenerative disorders caused by prions. CJD may presentas a sporadic, genetic, or infectious illness. Prions are transmissibleparticles that are devoid of nucleic acid and seem to be composedexclusively of a modified protein (PrPSc). The normal, cellular prion protein (PrPC) is converted into PrPSc through a posttranslational process during which it acquiresa high beta -sheet content. It is thought that BSE is a result ofcannibalism in which faulty industrial practices produced prion-contaminatedfeed for cattle. There is now considerable concern that bovineprions may have been passed to humans, resulting in a new formof CJD.

Department of Neurology (address for correspondence) and Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143, USA.

During the past two decades, a previously unknown mechanism of disease has been described in humans and animals. Several fatalillnesses, often referred to as the prion diseases and includingscrapie of sheep, BSE, and CJD of humans, are caused by the accumulationof a posttranslationally modified cellular protein. Indeed, thehallmark of all prion diseases--whether sporadic, dominantly inherited,or acquired by infection--is that they involve the aberrant metabolismand resulting accumulation of the prion protein (Table 1) (1,2). The conversion of PrPC (the normal cellular protein) into PrPSc (the abnormal disease-causing isoform) involves a conformationchange whereby the alpha -helical content diminishes and the amountof beta sheet increases (3). This structural transition is accompaniedby profound changes in the properties of the protein: PrPC is soluble in nondenaturing detergents, whereas PrPSc is not (4); and PrPC is readily digested by proteases, whereas PrPSc is partially resistant (5).

Table 1. The prion diseases.

Disease Mechanism of pathogenesis

Human diseases
Kuru (Fore people) Infection through ritualistic cannibalism
Iatrogenic Creutzfeldt-Jakob disease Infection from prion-contaminated HGH, dura mater grafts, and so forth
Variant Creutzfeldt-Jakob disease Infection from bovine prions?
Familial Creutzfeldt-Jakob disease Germline mutations in PrP gene
Gerstmann-Sträussler-Scheinker disease Germline mutations in PrP gene
Fatal familial insomnia Germline mutation in PrP gene (D178N and M129)
Sporadic Creutzfeldt-Jakob disease Somatic mutation or spontaneous conversion of PrPC into PrPSc?
Animal diseases
Scrapie (sheep) Infection in genetically susceptible sheep
Bovine spongiform encephalopathy (cattle) Infection with prion-contaminated MBM
Transmissible mink encephalopathy (mink) Infection with prions from sheep or cattle
Chronic wasting disease (mule deer, elk) Unknown
Feline spongiform encephalopathy (cats) Infection with prion-contaminated MBM
Exotic ungulate encephalopathy (greater kudu, nyala, oryx) Infection with prion-contaminated MBM

Investigations of the prion diseases have taken on new importance with the reports of 20 cases of an atypical, variant CJD(vCJD) in 3 teenagers and 17 adults (6, 7). All of thesecases have been reported from Great Britain and France to date.It now seems possible that bovine prions from "mad cows" werepassed to humans through the consumption of tainted beef products.In this article, I discuss the information on prions with respectto the origins of BSE and vCJD. I raise the possibility that aparticular conformation of bovine PrPSc was selected for heat resistance during the manufacture of meatand bone meal (MBM), thought to be the source of prions responsiblefor BSE. I also address the issue of preventing prion diseasesand developing therapeutic approaches.

Human Prion Diseases

Most humans afflicted with prion disease present with a rapidly progressive dementia but some manifest a cerebellar ataxia.Although the brains of patients appear grossly normal upon postmortemexamination, they usually show spongiform degeneration and astrocyticgliosis under the microscope. The human prion diseases can presentas sporadic, genetic, or infectious disorders (8) (Table 1).

Sporadic forms of prion disease comprise most cases of CJD and possibly a few cases of Gerstmann-Sträussler-Scheinker disease(GSS) (9, 10). In these patients, mutations of the PrP geneare not found. It is not known how disease-causing prions arisein patients with sporadic forms; hypotheses include horizontaltransmission of prions from humans or animals (11), somaticmutation of the PrP gene, and spontaneous conversion of PrPC into PrPSc (8, 12). Numerous attempts to establish an infectious linkbetween sporadic CJD and a preexisting prion disease in animalsor humans have been unrewarding (13, 14).

To date, 20 different mutations in the human PrP gene, resulting in nonconservative substitutions, have been found that segregatewith the inherited prion diseases (Fig. 1). Familial CJD (fCJD)cases suggested that genetic factors might influence pathogenesis(15), but this was difficult to reconcile with the transmissibilityof fCJD and GSS (16). The discovery of genetic linkage betweenthe PrP gene and scrapie incubation times in mice (17) raisedthe possibility that mutation might be an aspect of the hereditaryhuman prion diseases. The Pro102rightarrow Leu (P102L) mutation was the first PrP mutation to be geneticallylinked to central nervous system (CNS) dysfunction in GSS (Fig.1B) (10) and has since been found in many GSS families throughoutthe world (18). Indeed, a mutation in the protein-coding regionof the PrP gene has been found in all reported kindreds with inheritedhuman prion disease; besides the P102L mutation, genetic linkagehas been established for four other mutations (19).

Fig. 1. Species variations and mutations of the gene encoding the prion protein. (A) Species variations. The x axis representsthe human PrP sequence, showing the five octarepeats and H1 throughH4 as well as the three alpha helices A, B, and C and the two beta strandsS1 and S2. Vertical bars above the axis indicate the number ofspecies that differ from the human sequence at each position.Below the axis, the length of the bars indicates the number ofalternative amino acids at each position in the alignment. (B)Mutations causing inherited human prion disease and polymorphismsin human, mouse, and sheep. Above the line of the human sequenceare mutations that cause prion disease. Below the lines are polymorphisms,some but not all of which are known to influence the phenotypeof disease. Parentheses indicate corresponding human codons. [Datacompiled by P. Bamborough and F. E. Cohen][View Larger Version of this Image (29K GIF file)]

Transgenic (Tg) studies confirmed that mutations of the PrP gene can cause neurodegeneration. The P102L mutation of GSS wasintroduced into the mouse PrP (MoPrP) gene, and five lines ofTg(MoPrP-P101L) mice expressing large amounts of mutant PrP developedCNS degeneration consisting of widespread vacuolation of the neuropil,astrocytic gliosis, and PrP amyloid plaques (20, 21). Brainextracts prepared from spontaneously ill Tg(MoPrP-P101L) micetransmitted CNS degeneration to Tg196 mice (21). Although theTg196 mice did not develop spontaneous disease, they expressedsmall amounts of the protein encoded by the mutant transgene MoPrP-P101Land were deficient for MoPrP (Prnp0/0) (22). Prions from patients who died of GSS could be transmittedto apes and monkeys (16) or to Tg(MHu2M-P101L) mice (MHu2M designatesa chimeric human-mouse PrP) (23, 24). Together, these resultsdemonstrate that prions are generated de novo by mutations inPrP. Additionally, an artificial set of mutations in a PrP transgene(consisting of Ala113rightarrow Val, Ala115rightarrow Val, and Ala118rightarrow Val) produced neurodegeneration in neonatal mice; brain extractsfrom these mice transmitted disease to hamsters and Tg mice expressinga chimeric Syrian hamster-mouse PrP (25).

The infectious prion diseases include kuru of the Fore people in New Guinea, where prions were transmitted by ritualisticcannibalism (11, 26, 27). With the cessation of cannibalismat the urging of missionaries, kuru began to decline long beforeit was known to be transmissible (Fig. 2). Sources of prions causinginfectious CJD include improperly sterilized depth electrodes,transplanted corneas, human growth hormone (HGH) and gonadotropinderived from cadaveric pituitaries, and dura mater grafts (28).More than 90 young adults have developed CJD after treatment withcadaveric HGH, with incubation periods ranging from 3 years tomore than 20 years (29). Dura mater grafts implanted duringneurosurgical procedures seem to have caused more than 60 casesof CJD, with incubation periods ranging from 1 year to more than14 years (30).

Fig. 2. Disappearance of kuru and the BSE epidemic. (A) Number of annual cases of BSE in cattle in Great Britain; (B)number of biannual cases of kuru in Papua New Guinea. Data compiledfor BSE by J. Wilesmith at the Central Veterinary Laboratory,Weybridge, United Kingdom, and for kuru by M. Alpers at the Institutefor Human Disease, Goroka, Papua New Guinea.[View Larger Version of this Image (16K GIF file)]

The transmission of prions from one species to another is generally accompanied by a prolongation of the incubation time relativeto transmissions where the host species is the same. This prolongationis often referred to as the "species barrier" (31). From studieswith Tg mice, three factors have been identified that contributeto the species barrier: (i) the difference in PrP sequences betweenthe prion donor and recipient, (ii) the strain of prion, and (iii)the species specificity of protein X, a factor defined by moleculargenetic studies that binds to PrPC and facilitates PrPSc formation. This factor is likely to be a protein, hence the provisionaldesignation protein X (23, 32). The prion donor is the lastmammal in which the prion was passaged, and its PrP sequence representsthe "species" of the prion. The strain of prion, which seems tobe enciphered in the conformation of PrPSc, conspires with the PrP sequence (which is specified by the recipient)to determine the tertiary structure of nascent PrPSc. These principles are demonstrated by studies on the transmissionof Syrian hamster prions to mice, which showed that expressionof a Syrian hamster PrP (SHaPrP) transgene in mice abrogated thespecies barrier (Table 2) (33). Besides the PrP sequence, thestrain of prion also modified the transmission of SHa prions tomice (Table 2) (34, 35).

Table 2. Influence of prion species and strains on transmission across a species barrier in Tg mice [inoculum, SHa; data from (35,104, 109)].

Host Prion strain and inoculation time
Days (± SEM) n/n0 Days (± SEM) n/n0

SHa 77 ± 1 48/48 167 ± 1 94/94
Non-Tg mice >700 0/9 499 ± 15 11/11
Tg(SHaPrP)81/ FVB mice 75 ± 2 22/22 110 ± 2 19/19
Tg(SHaPrP)81/ Prnp0/0 mice 54 ± 1 9/9 65 ± 1 15/15

Protein X was postulated to explain the results of studies on the transmission of prions to Tg mice expressing either humanPrP (HuPrP) or MHu2M. Transgenic mice expressing HuPrP and MoPrPwere resistant to prions, whereas mice expressing only HuPrP orchimeric MHu2M were susceptible (Table 3) (23, 36). We producedmice expressing only HuPrP by crossing the Tg(HuPrP) mice withPrnp0/0 mice. These studies showed that MoPrPC prevented the conversion of HuPrPC into PrPSc but had little effect on the conversion of MHu2M into PrPSc. We interpreted these results in terms of MoPrPC binding to another mouse protein with a higher affinity thanto a foreign protein such as HuPrPC. We postulated that we had not seen this effect in Tg(SHaPrP)mice (Table 2) because SHaPrP is more closely related to MoPrPthan is HuPrP. In addition, MoPrPC had little effect on the formation of PrPSc from MHu2M (Table 3) because the COOH-termini of MoPrP and MHu2Mare identical in amino acid sequence.

Table 3. Evidence for protein X from studies of human prion transmission in Tg mice [inoculum, sCJD; data with inoculum RG from (23)].

Host MoPrP gene Incubation time
Days (± SEM) n/n0

Tg(HuPrP) Prnp+/+ 721 1/10
Tg(HuPrP)Prnp0/0 Prnp0/0 263 ± 2 6/6
Tg(MHu2M) Prnp+/+ 238 ± 3 8/8
Tg(MHu2M)Prnp0/0 Prnp0/0 191 ± 3 10/10

Characteristics of Prions

PrPSc is the major, and very probably the only, component of the infectious prion particle. PrPSc formation is a posttranslational process involving only a conformationalchange in PrPC (3, 37). Molecular modeling studies predicted that PrPC is a four-helix bundle protein containing four regions of secondarystructure, denoted H1 through H4 (Fig. 1) (38, 39). Fouriertransform infrared (FTIR) and circular dichroism (CD) studiesshowed that PrPC contains about 40% alpha helix and little beta sheet, consistent withthe structural predictions (3, 40). Subsequent nuclear magneticresonance (NMR) studies of a synthetic PrP peptide containingresidues 90 to 145 provided good evidence for H1 (41). Thispeptide contains residues 113 to 128, the most highly conservedresidues among all species studied (Fig. 1A) (39, 42). Whenthe peptide is extended to include alpha helix A (Fig. 3A), this formsthe central domain of PrPC (approximately residues 95 to 170) that binds to PrPSc during the formation of nascent PrPSc (43). This domain shows higher homology between cattle andhumans than between sheep and humans, which raises the possibilitythat prion transmission from cattle to humans may occur more readilythan from sheep to humans (44).
Fig. 3. Structures of prion proteins. (A) NMR structure of Syrian hamster rPrP(90-231). Presumably, the structure of the alpha -helicalform of rPrP(90-231) resembles that of PrPC. rPrP(90-231) is viewed from the interface where PrPSc is thought to bind to PrPC. Color code: pink, alpha helices A (residues 144 to 157), B (172to 193), and C (200 to 227); yellow, disulfide between Cys179 and Cys214; red, conserved hydrophobic region (composed of residues 113to 126); gray, loops; green, residues 129 to 134 encompassingstrand S1; and blue, residues 159 to 165 encompassing strand S2.The arrows span residues 129 to 131 and 161 to 163, which showa closer resemblance to beta sheet (47). (B) NMR structureof rPrP(90-231), viewed from the interface where protein X isthought to bind to PrPC. Protein X appears to bind to the side chains of residues thatform a discontinuous epitope: some amino acids are in the loopcomposed of residues 165 to 171 and at the end of helix B (Gln168 and Gln172 with a low-density van der Waals rendering), while others areon the surface of helix C (Thr215 and Gln219 with a high-density van der Waals rendering) (32). Images in(A) and (B) were generated with Midasplus. (C) Plausiblemodel for the tertiary structure of human PrPSc (52). Color code: red, S1 beta strands (residues 108 to 113 and116 to 122); green, S2 beta strands (residues 128 to 135 and 138to 144); gray, alpha helices H3 (residues 178 to 191) and H4 (residues202 to 218); and yellow, loop (residues 142 to 177). Four residuesimplicated in the species barrier (Asn108, Met112, Met129, and Ala133) are shown in ball-and-stick form (color code: dark gray, carbon;light gray, hydrogen; blue, nitrogen; red, oxygen; and yellow,sulfur). [View Larger Version of this Image (27K GIF file)]

The NMR structure of an alpha -helical form of a recombinant PrP (rPrP), containing residues 90 to 231 and corresponding to SHaPrP27-30 (1), presumably resembles that of PrPC (45-47). Residues 90 to 112 are not shown because marked conformationalheterogeneity was found in this region, whereas residues 113 to126 constitute the conserved hydrophobic region that also displayssome structural plasticity (46) (Fig. 3A). The NH2-terminaldomain of PrPC is thought to form the interface where PrPSc binds, whereas the COOH-terminal region appears to contain thesite for protein X binding (Fig. 3B). Although some features ofthe structure of rPrP(90-231) are similar to those reported earlierfor a smaller recombinant MoPrP fragment containing residues 121to 231 (48, 49), substantial differences were found. For example,the loop at the NH2-terminus of helix B is well defined in rPrP(90-231)but is disordered in MoPrP(121-231); in addition, helix C is composedof residues 200 to 227 in rPrP(90-231) but encompasses only residues200 to 217 in MoPrP(121-231). The loop and the COOH-terminal portionof helix C are particularly important because they form the siteto which protein X binds (Fig. 3B) (32). It is not yet knownwhether the differences between the two recombinant PrP fragmentsare attributable to their different lengths, to species-specificdifferences in sequences, or to the conditions used for solvingthe structures.

Recent NMR studies of full-length MoPrP(23-231) and SHaPrP(29-231) have shown that the NH2-termini are highly flexible andlack identifiable secondary structure under the experimental conditionsused (50, 51). Studies of SHaPrP(29-231) indicate transientinteractions between the COOH-terminal end of helix B and thehighly flexible NH2-terminal random coil containing the octarepeats(residues 29 to 125) (51); such interactions were not reportedfor MoPrP- (23-231) (50). The tertiary structure of the NH2-terminusis of considerable interest because it is within this region ofPrP that a profound conformational change occurs during the formationof PrPSc, as described below (59).

Models of PrPSc suggested that formation of the disease-causing isoform involves refolding of the NH2-terminal helices (H1 andH2) into beta sheets (52); the single disulfide bond joining COOH-terminalhelices would remain intact because the disulfide is requiredfor PrPSc formation (Fig. 3C) (53, 54). The high beta -sheet content ofPrPSc was predicted from the ability of PrP 27-30 to polymerize intoamyloid fibrils (55). Subsequent optical spectroscopy confirmedthe presence of beta sheet in both PrPSc and PrP 27-30 (3, 56). Deletion of each of the regions ofputative secondary structure in PrP, except for the NH2-terminal66 amino acids (residues 23 to 88) (57, 58) and the 36-aminoacid loop (mouse residues 141 to 176) between H2 and H3, preventedformation of PrPSc as measured in scrapie-infected cultured neuroblastoma cells(54). With the use of alpha -PrP Fabs selected from phage displaylibraries and two monoclonal antibodies derived from hybridomas,the major conformational change that occurs during conversionof PrPC into PrPSc has been localized to residues 90 to 112 (59). Although theseresults indicate that PrPSc formation primarily involves a conformational change at the NH2-terminus,mutations causing inherited prion diseases have been found throughoutthe protein (Fig. 1B). Interestingly, all of the known point mutationsin PrP occur either within or adjacent to regions of putativesecondary structure in PrP and, as such, appear to destabilizethe structure of PrP (39, 41, 48).

PrPSc Conformation Enciphers Prion Diversity

The existence of prion strains has posed a conundrum as to how biological information can be enciphered in any molecule otherthan nucleic acid (60, 61). Prions from cattle, nyala, kudu,and domestic cats were inoculated into C57BL, VM, and F1(C57BL× VM) mice for "strain typing" (60, 62); all of these prionsgave the same distribution of incubation times, which suggeststhat they all originated in cattle (63). Whether prions fromhumans with vCJD will give similar incubation times is unknown.

The typing of prion strains in C57BL, VM, and F1(C57BL × VM) mice began with isolates from sheep with scrapie. The prototypicstrains Me7 and 22A gave incubation times of ~150 and ~400 days,respectively, in C57BL mice (60, 62). The PrPs of C57BL andIlnJ mice (later shown to be genetically identical to VM mice)differ at two residues and control incubation times (Fig. 1B)(64). Besides incubation times, profiles of spongiform changehave been used to characterize prion strains (65), but recentstudies with PrP transgenes imply that such profiles are not anintrinsic feature of strains (66).

Until recently, support for the hypothesis that the tertiary structure of PrPSc enciphers strain-specific information (2) was minimal, exceptfor the DY strain isolated from mink with transmissible encephalopathy(67). PrPSc in DY prions showed diminished resistance to proteinase K digestionand greater truncation of the NH2-terminus. The DY strain presenteda puzzling anomaly because other prion strains exhibiting similarincubation times did not show this aberrant behavior of PrPSc (68). Also notable was the generation of new strains duringpassage of prions through animals with different PrP genes (34,68).

The transmission of two different inherited human prion diseases to mice expressing a chimeric MHu2M PrP transgene (24)has provided persuasive evidence for the enciphering of strain-specificinformation in the tertiary structure of PrPSc. In fatal familial insomnia (FFI), the protease-resistant fragmentof PrPSc after deglycosylation has a relative molecular mass of 19 kD,whereas in other inherited and most sporadic prion diseases itis 21 kD (Table 4) (69, 70). This difference in molecularsize was shown to be attributable to different sites of proteolyticcleavage at the NH2-termini of the two human PrPSc molecules, reflecting different tertiary structures (69). Extractsfrom the brains of FFI patients transmitted disease to mice expressinga chimeric MHu2M PrP gene ~200 days after inoculation and inducedformation of the 19-kD PrPSc, whereas fCJD(E200K) and sporadic CJD produced the 21-kD PrPSc in mice expressing the same transgene (24). On second passage,Tg(MHu2M) mice inoculated with FFI prions showed an incubationtime of ~130 days and a 19-kD PrPSc, whereas those inoculated with fCJD(E200K) prions exhibited anincubation time of ~170 days and a 21-kD PrPSc. These findings imply that PrPSc acts as a template for the conversion of PrPC into nascent PrPSc. Imparting the size of the protease-resistant fragment of PrPSc through conformational templating provides a mechanism for boththe generation and propagation of prion strains.

Table 4. Distinct prion strains generated in humans with inherited prion diseases and transmitted to Tg mice [data from (24, 110)].

Inoculum Host species Host PrP genotype Incubation time
PrPSc (kD)
Days (± SEM) n/n0

None Human FFI(D178N, M129) 19
FFI Mouse Tg(MHu2M) 206 ± 7 7/7 19
FFI rightarrow Tg(MHu2M) Mouse Tg(MHu2M) 136 ± 1 6/6 19
None Human fCJD(E200K) 21
fCJD Mouse Tg(MHu2M) 170 ± 2 10/10 21
fCJD rightarrow Tg(MHu2M) Mouse Tg(MHu2M) 167 ± 3 15/15 21

Bovine Spongiform Encephalopathy

Understanding prion strains and the species barrier is paramount with respect to the BSE epidemic in Great Britain, wherealmost 1 million cattle are estimated to have been infected withprions (71). The mean incubation time for BSE is about 5 years.Most cattle were slaughtered between 2 and 3 years of age andtherefore did not manifest disease (72). Nevertheless, morethan 160,000 cattle, primarily dairy cows, have died of BSE overthe past decade (Fig. 2A) (71). BSE is a massive common-sourceepidemic that may be caused by MBM fed primarily to dairy cows(73). The MBM was prepared from the offal of sheep, cattle,pigs, and chickens as a high-protein nutritional supplement. Inthe late 1970s, the hydrocarbon-solvent extraction method usedin the rendering of offal began to be abandoned, resulting inMBM with a much higher fat content (73). It is now thought thatthis change in the rendering process allowed scrapie prions fromsheep to survive rendering and to be passed into cattle. Alternatively,some bovine prions may have been present before modification ofthe rendering process, and, with the processing change, survivedin sufficient numbers to initiate the BSE epidemic when inoculatedback into cattle orally through MBM. The latter hypothesis isinconsistent with the widespread geographical distribution throughoutEngland of the initial 17 cases of BSE, which occurred almostsimultaneously (74).

The origin of the bovine prions causing BSE cannot be determined by examining the amino acid sequence of PrPSc in cattle with BSE, because the PrPSc in these animals has the bovine sequence whether the initialprions in MBM came from cattle or sheep. The bovine PrP sequencediffers from that of sheep at seven or eight positions (75,76). In contrast to the many PrP polymorphisms found in sheep,only one PrP polymorphism has been found in cattle. Although mostbovine PrP alleles encode five octarepeats, some encode six. PrPalleles encoding six octarepeats do not seem to be overrepresentedin BSE (Fig. 1B) (77).

Brain extracts from BSE cattle cause disease in cattle, sheep, mice, pigs, and mink after intracerebral inoculation (78),but prions in brain extracts from sheep with scrapie fed to cattleproduced illness substantially different from BSE (79). Theannual incidence of sheep with scrapie in Great Britain over thepast two decades has remained relatively low (80). In July 1988,the practice of feeding MBM to sheep and cattle was banned. Recentstatistics argue that the epidemic is now disappearing as a resultof this ruminant feed ban (Fig. 2A) (71), reminiscent of thedisappearance of kuru in the Fore people of New Guinea (11,27) (Fig. 2B).

Although many plans have been offered for the culling of older cattle to minimize the spread of BSE (71), it seems moreimportant to monitor the frequency of prion disease in cattleas they are slaughtered for human consumption. No reliable, specifictest for prion disease in live animals is available (81), butimmunoblotting of the brainstems of cattle for PrPSc might provide a reasonable approach to establishing the incidenceof subclinical BSE in cattle entering the human food chain (76,82).

Determining how early in the incubation period PrPSc can be detected by immunological methods is complicated by the lack ofa reliable, sensitive, and relatively rapid bioassay. Mice inoculatedintracerebrally with BSE brain extracts require more than a yearto develop disease (83-85). The number of inoculated animals developingdisease can vary over a wide range, depending on the titer ofthe inoculum, the structures of PrPC and PrPSc, and the structure of protein X (Table 2). Some investigatorshave stated that transmission of BSE to mice is quite variable,with incubation periods exceeding 1 year (85), while othersreport a low prion titer of 102.7 ID50 units per milliliter of 10% BSE brain homogenate (83)compared with 107 to 109 ID50 units per milliliter in rodent brain (86). Such problemswith the measurement of bovine prions demonstrate the urgent needfor Tg mice that are highly susceptible to bovine prions.

Have Bovine Prions Been Transmitted to Humans?

Cases of vCJD in Great Britain and France raise the possibility that BSE has been transmitted to humans (6, 7). Allbut one of the 20 vCJD patients are 40 years of age or younger;the only other group of young CJD patients are those who receivedpituitary HGH during childhood. The neuropathology of vCJD patientsis unusual, with numerous PrP amyloid plaques surrounded by intensespongiform degeneration (Fig. 4). These atypical neuropathologicchanges have not been seen in CJD cases in the United States,Australia, and Japan (87). Macaque monkeys and marmosets bothdeveloped neurologic disease several years after inoculation withbovine prions (88), but only the macaques exhibited numerousPrP plaques similar to those found in vCJD (89).
Fig. 4. Histopathology of vCJD in Great Britain. (A) Section from frontal cortex stained by the periodic acid-Schiff (PAS)method, showing a field with aggregates of plaques surroundedby spongiform degeneration. (B) Multiple plaques and amorphousdeposits are PrP-immunopositive. Scale bar, 50 µm. Photomicrographsprepared by S. J. DeArmond. [View Larger Version of this Image (118K GIF file)]

If the current cases of vCJD are caused by bovine prions, then the exposure must have occurred before the specified bovineoffals ban of November 1989 that prohibited human consumptionof CNS and lymphoid tissues from cattle older than 6 months ofage. This legislation was based on studies showing that the highesttiters of scrapie prions are found in these tissues in sheep (90).Because the bioassay for bovine prions in mice is so insensitive(83), the abundance of prions in bovine muscle remains unknown.If the distribution of bovine prions proves to be different fromthat presumed for sheep, then assumptions about the efficacy ofthe offal ban will need to be reassessed.

Attempts to predict the future number of cases of vCJD assuming exposure to bovine prions before the 1989 offal ban have beenuninformative, because so few cases of vCJD have occurred (7).The finding of only 9 new cases in the past 15 months since thefirst 11 cases were announced raises questions as to the originof vCJD. Epidemiological studies over the past three decades havefailed to find evidence for transmission of sheep prions to humans(14). Are we at the beginning of a human prion disease epidemicin Great Britain like those seen for BSE and kuru (Fig. 2), orwill the number of vCJD cases remain small, as seen with iatrogenicCJD caused by cadaveric HGH (29)? Until more time passes, assessingthe magnitude of vCJD will not be possible (7, 91, 92).

Was a particular conformation of bovine PrPSc selected for heat resistance during the rendering process and then reselectedmultiple times as cattle infected by ingesting prion-contaminatedMBM were slaughtered and their offal rendered into more MBM? Recentstudies of PrPSc from the brains of patients who died of vCJD show a pattern ofPrP glycoforms different from those found for sporadic or iatrogenicCJD (93). However, the utility of measuring PrP glycoforms isquestionable in trying to relate BSE to vCJD (94) because PrPSc is formed after the protein is glycosylated (37) and enzymaticdeglycosylation of PrPSc requires denaturation (95). Alternatively, it may be possibleto establish a relation between the conformations of PrPSc from cattle with BSE and those from humans with vCJD by usingTg mice, as was done for strains generated in the brains of patientswith FFI or fCJD (24).

It is also of interest to ask whether a particular strain of human prions was selected during ritualistic cannibalism amongthe Fore peoples of New Guinea when they cooked the brains oftheir dead relatives before eating them, or whether a strain wasselected during the purification of cadaveric HGH. The uniformconstellation of clinical signs of kuru and iatrogenic CJD causedby contaminated HGH contrasts with those found in other formsof prion disease (28, 96). Because the methods of preparationand the precise handling of brain tissue among the Fore are notwell documented (11, 26, 97), such speculation may provedifficult to substantiate.

Prevention and Therapeutics for Prion Diseases

As our understanding of prion propagation increases, it should be possible to design effective therapeutics. Because peopleat risk for inherited prion diseases can now be identified decadesbefore neurologic dysfunction is evident, the development of aneffective therapy for these fully penetrant disorders is imperative(98). Although we have no way of predicting the number of individualswho may develop neurologic dysfunction from bovine prions in thefuture (7), it seems prudent to seek an effective therapy now.

Interfering with the conversion of PrPC into PrPSc would seem to be the most attractive therapeutic target (99). One reasonabletherapeutic strategy would be to stabilize the structure of PrPC by binding a drug; another would be to modify the action of proteinX, which might function as a molecular chaperone (Fig. 3). Itremains to be determined whether a drug that binds to PrPC at the protein X binding site would be more efficacious thana drug that mimics the structure of PrPC with basic polymorphic residues that seem to prevent scrapieand CJD. Because PrPSc formation seems limited to caveolae-like domains (100), drugsdesigned to inhibit this process need not penetrate the cytosolof cells, but they must be able to enter the CNS. Alternatively,drugs that destabilize the structure of PrPSc might also prove useful.

The production of domestic animals that do not replicate prions may also be important with respect to preventing prion disease.Sheep encoding the Arg/Arg polymorphism at position 171 seem resistantto scrapie (Fig. 1B) (101); presumably, this was the geneticbasis of Parry's scrapie eradication program in Great Britain30 years ago (102). The use of dominant negatives to produceprion-resistant domestic animals, including sheep and cattle,through the expression of PrP transgenes encoding Arg171 as well as additional basic residues at the protein X bindingsite (Fig. 3B) (32) is likely a more effective approach. Suchan approach can be readily evaluated in Tg mice, and, if shownto be effective, it could be instituted by artificial inseminationof sperm from males homozygous for the transgene. Less practicalis the production of PrP-deficient cattle and sheep. Althoughsuch animals would not be susceptible to prion disease (103,104), they might suffer some deleterious effects from ablationof the PrP gene (105).

Understanding how PrPC unfolds and refolds into PrPSc not only has implications for interfering with the pathogenesis of priondiseases, but may open new approaches to deciphering the causesof and developing effective therapies for the more common neurodegenerativediseases, including Alzheimer's disease, Parkinson's disease,and amyotrophic lateral sclerosis (ALS). In addition, two differentstable metabolic states in yeast and one in a fungus have beenascribed to prion-like changes in protein conformation (106-108).Indeed, the expanding list of prion diseases and their novel modesof pathogenesis (Table 1), as well as the unprecedented mechanismsof prion propagation and information transfer (Table 4), indicatethat much more attention to these fatal disorders of protein conformationis urgently needed.


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