Cell, Vol. 93, 337–348, May 1, 1998, Copyright 1998 by Cell Press

Prion Protein Biology Review

and seem to be composed exclusively of a modifiedStanley B. Prusiner,*† Michael R. Scott,*Stephen J. DeArmond,‡* and Fred E. Cohen†§‖ isoform of PrP, designated PrPSc. The normal cellular

PrP, denoted PrPC, is converted into PrPSc through a*Department of Neurology†Department of Biochemistry and Biophysics process whereby a portion of its a-helical and coil struc-

ture is refolded into b sheet (Pan et al., 1993). This struc-‡Department of Pathology§Department of Molecular and Cellular Pharmacology tural transition is accompanied by profound changes in

the physicochemical properties of the PrP. While PrPC‖Department of MedicineUniversity of California is soluble in nondenaturing detergents, PrPSc is not. PrPC

is readily digested by proteases, whereas PrPSc is par-San Francisco, California 94143tially resistant (Oesch et al., 1985). Because prions ap-pear to be composed entirely of a protein that adoptsIntroductionan abnormal conformation, it is not unreasonable tothink of prions as infectious proteins (Pan et al., 1993;It is interesting to contemplate how the course of scien-Telling et al., 1996). But we hasten to add that we stilltific investigation might have proceeded had studies oncannot eliminate the possibility of a small ligand boundthe transmissibility of inherited prion diseases not beento PrPSc as an essential component of the infectiousperformed until after the molecular genetic lesion hadprion particle.been identified (Meggendorfer, 1930; Roos et al., 1973;

In a more broad view, prions are elements that impartHsiao et al., 1989). Had the prion protein (PrP) gene beenand propagate variability through multiple conformersidentified in families with prion disease by positionalof a normal cellular protein. The species of a particularcloning or through the purification and sequencing ofprion is encoded by the sequence of the chromosomalPrP in amyloid plaques before brain extracts werePrP gene of the mammal in which it last replicated. Inshown to be transmissible, the prion concept might havecontrast to pathogens with a nucleic acid genome thatbeen more readily accepted (Prusiner, 1998).encode strain-specific properties in genes, prions seemBut that is not the course of events that led to ourto encipher these properties in the tertiary structurecurrent understanding of prions. Creutzfeldt-Jakob dis-of PrPSc (Bessen and Marsh, 1994; Telling et al., 1996;ease (CJD) remained a curious, rare neurodegenerativePrusiner, 1997).disease of unknown etiology for more than three score

The discovery that mutations of the PrP gene causedyears. Only the transmission of CJD to apes by inocula-dominantly inherited prion diseases in humans linkedtion of brain extracts from patients who had died of CJDthe genetic and infectious forms of prion diseases andinitiated a path of scientific investigation that was topresented another hurdle for investigators who contin-demystify that fascinating area of biomedicine (Gibbsued to argue that prion diseases are caused by viruses.et al., 1968). Not unexpectedly, once CJD was shownMore than 20 mutations of the PrP gene are now knownto be an infectious disease, relatively little attention wasto cause the inherited human prion diseases, and signifi-paid to the familial form of the disease since most casescant genetic linkage has been established for five ofare sporadic and familial clusters account for a minority.these mutations (Hsiao et al., 1989) (for review, see Prus-In this review, we focus on the prion particles thatiner, 1997). The prion concept readily explains how acause scrapie, bovine spongiform encephalopathy (BSE),disease can be manifest as a heritable as well as anand CJD. Some of the fascinating studies that led toinfectious illness. Moreover, the hallmark common toour knowledge of prions are not discussed here butall of the prion diseases whether sporadic, dominantlyhave recently been reviewed elsewhere (Prusiner, 1998).inherited, or acquired by infection is that they involvePrion diseases may present as genetic, infectious, orthe aberrant metabolism of the prion protein (Prusiner,sporadic disorders, all of which involve modification of1991).the prion protein (PrP), a constituent of normal mamma-

Although PrPSc is the only known component of thelian cells. CJD generallypresents as progressive demen-infectious prion particles, these unique pathogens sharetia while scrapie of sheep and BSE are generally mani-several phenotypic traits with other infectious entitiesfest as ataxic illnesses (Table 1) (Wells et al., 1987). Insuch as viruses. Because some features of the diseasesCJD, scrapie, and BSE, as well as all of the other disor-caused by prions and viruses are similar, some scien-ders frequently referred to as prion diseases (Table 1),tists have difficulty accepting the existence of prionsspongiform degeneration and astrocytic gliosis aredespite a wealth of scientific data supporting this con-found upon microscopic examination of the CNS. Thecept (Chesebro and Caughey, 1993; Manuelidis anddegree of spongiform degeneration is quite variableFritch, 1996; Lasmezas et al., 1997; Chesebro, 1998).while the extent of reactive gliosis correlates with the

degree of neuron loss (Masters and Richardson, 1978).

Molecular Genetics of Prion DiseasesOnce a PrP cDNA probe became available, molecularThe Prion Particle

Perhaps, the best current working definition of a prion genetic studies were undertaken to determine whetherthe PrP gene controls scrapie incubation times in mice.is a proteinaceous infectious particle that lacks nucleic

acid (Prusiner, 1997). A wealth of data supports the Independent of the enriching of brain fractions forscrapie infectivity that led to the discovery of PrPSc, thecontention that scrapie prions are devoid of nucleic acid

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Table 1. The Prion Diseases

Disease Host Mechanism of Pathogenesis

Kuru Fore people Infection through ritualistic cannibalismiCJD Humans Infection from prion-contaminated HGH, dura mater

grafts, etc.vCJD Humans Infection from bovine prions?fCJD Humans Germline mutations in PrP geneGSS Humans Germline mutations in PrP geneFFI Humans Germline mutation in PrP gene (D178N, M129)sCJD Humans Somatic mutation or spontaneous conversion of PrPC

into PrPSc?FSI Humans Somatic mutation or spontaneous conversion of PrPC

into PrPSc?

Scrapie Sheep Infection in genetically susceptible sheepBSE Cattle Infection with prion-contaminated MBMTME Mink Infection with prions from sheep or cattleCWD Mule deer, elk UnknownFSE Cats Infection with prion-contaminated beefExotic ungulate encephalopathy Greater kudu, nyala, oryx Infection with prion-contaminated MBM

Abbreviations: BSE, bovine spongiform encephalopathy; CJD, Creutzfeldt-Jakob disease; sCJD, sporadic CJD; fCJD, familial CJD; iCJD,iatrogenic CJD; vCJD, (new) variant CJD; CWD, chronic wasting disease; FFI, fatal familial insomnia; FSE, feline spongiform encephalopathy;FSI, fatal sporadic insomnia; GSS, Gerstmann-Straussler-Scheinker disease; HGH, human growth hormone; MBM, meat and bone meal; TME,transmissible mink encephalopathy.

PrP gene was shown to be genetically linked to a locus process (Prusiner et al., 1990). The length of the incuba-tion time after inoculation with SHa prions was inverselycontrolling the incubation time (Carlson et al., 1986).proportional to the level of SHaPrPC in the brains ofSubsequently, mutation of the PrP gene was shown toTg(SHaPrP) mice (Prusiner et al., 1990). Bioassays ofbe genetically linked to the development of familial prionbrain extracts from clinically ill Tg(SHaPrP) mice inocu-disease (Hsiao et al., 1989). At the same time, expressionlated with mouse (Mo) prions revealed that only Moof a Syrian hamster (SHa) PrP transgene in mice wasprions but no SHa prions were produced. Conversely,shown to render the animals highly susceptible to SHainoculation of Tg(SHaPrP) mice with SHa prions led toprions, which demonstrated that expression of a foreignthe synthesis of only SHa prions. Thus, the rate of PrPScPrP gene could abrogate the species barrier (Scott etsynthesis appears to be a function of the level of PrPCal., 1989). Later, PrP-deficient (Prnp0/0) mice were foundexpression in Tg mice; however, the level to which PrPSc

to be resistant to prion infection and failed to replicateaccumulates appears to be independent of PrPC con-prions, as expected (Bueler et al., 1993; Prusiner et al.,centration (Prusiner et al., 1990).1993). The results of these studies indicated PrP mustPrP-Deficient Miceplay a central role in the transmission and pathogenesisThe development and lifespan of two lines of Prnp0/0

of prion disease, but equally important, they establishedmice were indistinguishable from controls (Bueler et al.,that the abnormal isoform is an essential component of1992; Manson et al., 1994) while another line exhibitedthe prion particle (Prusiner, 1991).ataxia and Purkinje cell degeneration at z70 weeks ofPrP Gene Dosage Controls Lengthage (Sakaguchi et al., 1996). In the former two lines withof Incubation Timenormal development, altered sleep–wake cycles (ToblerScrapie incubation times in mice were used to distin-et al., 1996) and synaptic behavior in brain slices haveguish prion strains and to identify a gene controlling itsbeen reported (Collinge et al., 1994), but the synaptic

length (Dickinson et al., 1968; Scott et al., 1997). Thischanges could not be confirmed by others (Lledo et al.,

gene was initially called Sinc based on genetic crosses1996).

between C57Bl and VM mice, which exhibited short and Prnp0/0 mice are resistant to prions (Bueler et al., 1993;long incubation times, respectively (Dickinson et al., Prusiner et al., 1993). Prnp0/0 mice were sacrificed 5, 60,1968). Because the distribution of VM mice was re- 120, and 315 days after inoculation with RML prions andstricted, we searched for another mouse with long incu- brain extracts bioassayed in CD-1 Swiss mice. Exceptbation times. I/Ln mice proved to be a suitable substitute for residual infectivity from the inoculum detected at 5for VM mice; eventually, I/Ln and VM mice were found days after inoculation, no infectivity was detected in theto be derived from a common ancestor. Subsequently, brains of Prnp0/0 mice (Prusiner et al., 1993). One groupthe PrP gene was shown to control the length of the of investigators found that Prnp0/0 mice inoculated withscrapie incubation time in mice (Carlson et al., 1994; RML prions and sacrificed 20 weeks later had 103.6 ID50

Moore et al., 1998). units/ml of homogenate by bioassay (Bueler et al., 1993).Overexpression of wtPrP Transgenes Others have used this report to argue that prion infectiv-Mice were constructed expressing different levels of the ity replicates in the absence of PrP (Chesebro andwild-type (wt) SHaPrP transgene (Scott et al., 1989). Caughey, 1993; Lasmezas et al., 1997). Neither we norInoculation of these Tg(SHaPrP) mice with SHa prions the authors of the initial report could confirm the findingdemonstrated abrogation of the species barrier resulting of prion replication in Prnp0/0 mice (Prusiner et al., 1993;

Sailer et al., 1994).in abbreviated incubation times due to a nonstochastic

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Prion Protein StructureOnce cDNA probes for PrP became available, the PrPgene was found to be constitutively expressed in adult,uninfected brain (Chesebro et al., 1985; Oesch et al.,1985). This finding eliminated the possibility that PrPSc

stimulated production of more of itself by initiating tran-scription of the PrP gene as proposed nearly two de-cades earlier (Griffith, 1967). Determination of the struc-ture of the PrP gene eliminated a second possiblemechanism that might explain the appearance of PrPSc

in brains already synthesizing PrPC. Since the entire pro-tein coding region was contained within a single exon,there was no possibility that the two PrP isoforms werethe products of alternatively spliced mRNAs (Basler etal., 1986). Next, a posttranslational chemical modifica-tion that distinguishes PrPSc from PrPC was consideredbut none was found in an exhaustive study (Stahl et al.,1993), and we considered it likely that PrPC and PrPSc

differed only in their conformations, a hypothesis alsoproposed earlier (Griffith, 1967).

When the secondary structures of the PrP isoformswere compared by optical spectroscopy, they werefound to be markedly different (Pan et al., 1993). Fouriertransform infrared (FTIR) and circular dichroism (CD)spectroscopy studies showed that PrPC contains about40% a helix and little b sheet while PrPSc is composedof about 30% a helix and 45% b sheet (Pan et al., 1993).That the two PrP isoforms have the same amino acidsequence runs counter to the widely accepted view that

Figure 1. Species Variations and Mutations of the Prion Proteinthe amino acid sequence specifies only one biologically Geneactive conformation of a protein (Anfinsen, 1973). (A) Species variations. The x-axis represents the human PrP se-

Prior to comparative studies on the structures of PrPCquence, with the five octarepeats and H1–H4 regions of putative

and PrPSc, metabolic labeling studies showed that the secondary structure shown as well as the three a helices A, B, andC and the two b strands S1 and S2. Vertical bars above the axisacquisition of PrPSc protease resistance is a posttransla-indicate the number of species that differ from the human sequencetional process (Borchelt et al., 1990). In a search forat each position. Below the axis, the length of the bars indicates thechemical differences that would distinguish PrPSc fromnumber of alternative amino acids at each position in the alignment.

PrPC, we identified ethanolamine in hydrolysates of PrP (B) Mutations causing inherited human prion disease and polymor-27–30, which signaled the possibility that PrP might con- phisms in human, mouse, and sheep. Above the line of the humantain a glycosylphosphatidyl inositol (GPI) anchor (Stahl sequence are mutations that cause prion disease. Below the lines

are polymorphisms, some but not all of which are known to influenceet al., 1987). Both PrP isoforms were found to carry GPIthe onset as well as the phenotype of disease. Data were compiledanchors and PrPC was found on the surface of cellsby Paul Bamborough and Fred E. Cohen. Reprinted with permissionwhere it could be released by cleavage of the anchor.from Science 278, pp. 245–251 (copyright 1997 American Associa-

Subsequent studies showed that PrPSc formation occurs tion for the Advancement of Science).after PrPC reaches the cell surface (Caughey and Ray-mond, 1991) and is localized to caveolae-like domains

these recombinant PrPs correspond to the secreted(Gorodinsky and Harris, 1995; Taraboulos et al., 1995).form of PrP that was also identified in the cell-free trans-Computational Models and Optical Spectroscopylation studies. This contention is supported by studiesModeling studiesand subsequent nuclear magnetic res-with recombinant antibody fragments (Fabs) showingonance (NMR) investigations of a synthetic PrP peptidethat GPI-anchored PrPC on the surface of cells exhibitscontaining residues 90–145 suggested that PrPC mightan immunoreactivity similar to that of recombinant PrPcontain an a helix within this region (Figure 1) (Huangprepared with an a-helical conformation (Peretz et al.,et al., 1994). This peptide contains the residues, 113–1997).128, that are most highly conserved among all species

Models of PrPSc suggest that formation of thedisease-studied (Figure 1A) and that correspond to a transmem-causing isoform involves refolding of a region corre-brane region of PrP which was delineated in cell-freesponding roughly to residues 108–144 into b sheets (Hu-translation studies. A transmembrane form of PrP wasang et al., 1996); the single disulfide bond joining thefound in brains of patients with Gerstmann-Straussler-COOH-terminal helices would remain intact since theScheinker disease (GSS) caused by the A117V mutationdisulfide is required for PrPSc formation (Figure 2D) (Mur-and in Tg mice overexpressing either mutant or wtPrPamoto et al., 1996). Deletion of each of several regions(Hegde et al., 1998). That no evidence for an a helix inof putative secondary structure in PrP, except for thethis region has been found in NMR studies of recombi-NH2-terminal 66 amino acids (residues 23–88) and a 36–nant PrP in an aqueous environment (Riek et al., 1996;

Donne et al., 1997; James et al., 1997) suggests that amino acid stretch (Mo residues 141–176), prevented

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Figure 2. Structures of Prion Proteins

(A) NMR structure of Syrian hamster (SHa) recombinant (r) PrP(90–231). Presumably, the structure of the a-helical form 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 scheme: a helices A (residues144–157), B (172–193), and C (200–227) in pink; disulfide between Cys-179 and Cys-214 in yellow; conserved hydrophobic region composedof residues 113–126 in red; loops in gray; residues 129–134 in green encompassing strand S1 and residues 159–165 in blue encompassingstrand S2; the arrows span residues 129–131 and 161–163, as these show a closer resemblance to b sheet (James et al., 1997).(B) NMR structure of rPrP(90–231) is viewed from the interface where protein X is thought to bind to PrPC. Protein X appears to bind to theside chains of residues that form a discontinuous epitope: some amino acids are in the loop composed of residues 165–171 and at the endof helix B (Gln-168 and Gln-172 with a low density van der Waals rendering) while others are on the surface of helix C (Thr-215 and Gln-219with a high density van der Waals rendering) (Kaneko et al., 1997b).(C) Schematic diagram showing the flexibility of the polypeptide chain for PrP(29–231) (Donne et al., 1997). The structure of the portion of theprotein representing residues 90–231 was taken from the coordinates of PrP(90–231) (James et al., 1997). The remainder of the sequencewas hand-built for illustration purposes only. The color scale corresponds to the heteronuclear {1H}-15N NOE data: red for the lowest (mostnegative) values, where the polypeptide is most flexible, to blue for the highest (most positive) values in the most structured and rigid regionsof the protein. Reprinted with permission from Proc. Natl. Acad. Sci. USA 94, pp. 13452–13457 (copyright 1997 National Academy of Sciences).(D) Plausible model for the tertiary structure of human PrPSc (Huang et al., 1996). Color scheme: S1 b strands are 108–113 and 116–122 inred; S2 b strands are 128–135 and 138–144 in green; a helices H3 (residues 178–191) and H4 (residues 202–218) in gray; loop (residues142–177) in yellow. Four residues implicated in the species barrier are shown in ball-and-stick form (Asn-108, Met-112, Met-129, Ala-133).Reprinted with permission from Science 278, pp. 245–251 (copyright 1997 American Association for the Advancement of Science).

formation of PrPSc as measured in scrapie-infected cul- of putative secondary structure in PrP and, as such,appear to destabilize the structure of PrPC (Huang et al.,tured neuroblastoma cells (Muramoto et al., 1996). With

a-PrP Fabs selected from phage display libraries and 1994; Riek et al., 1996).NMR Structure of Recombinant PrPtwo monoclonal antibodies (MAbs) derived from hybrid-

omas, a major conformational change that occurs during The NMR structure of recombinant SHaPrP(90–231) wasdetermined after the protein was purified and refoldedconversion of PrPC into PrPSc has been localized to resi-

dues 90–112 (Peretz et al., 1997). Studies with an a-PrP (Figure 2A). Residues 90–112 are not shown sincemarked conformational heterogeneity was found in thisIgM MAb, which was reported to immunoprecipitate

PrPSc selectively (Korth et al., 1997), support this conclu- region while residues 113–126 constitute the conservedhydrophobic region that also displays some structuralsion. While these results indicate that PrPSc formation

involves a conformational change at the NH2 terminus, plasticity (James et al., 1997). Although some featuresof the structure of rPrP(90–231) are similar to those re-mutations causing inherited prion diseases have been

found throughout the protein (Figure 1B). Interestingly, ported earlier for the smaller recombinant MoPrP(121–231) fragment (Riek et al., 1996), substantial differencesall of the known point mutations in PrP with biological

significance occur either within or adjacent to regions were found. For example, the loop at the NH2 terminus

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Figure 3. Schematic Diagram Showing Tem-plate-Assisted PrPSc Formation

In the initial step, PrPC binds to protein X toform the PrP*/protein X complex. Next, PrPSc

binds to PrP* that has already formed a com-plex with protein X. When PrP* is transformed

into a nascent molecule of PrPSc, protein X is released and a dimer of PrPSc remains. The inactivation target size of an infectious prion suggeststhat it is composed of a dimer of PrPSc (Bellinger-Kawahara et al., 1988). In the model depicted here, a fraction of infectious PrPSc dimersdissociate into uninfectious monomers as the replication cycle proceeds while most of the dimers accumulate in accord with the increase inprion titer that occurs during the incubation period. The precise stoichiometry of the replication process remains uncertain.

of helix B is defined in rPrP(90–231) but is disordered exposure of PrPC to GdnHCl converts it into a PrP*-likemolecule. Whether this PrP*-like protein is convertedin MoPrP(121–231); in addition, helix C is composed ofinto PrPSc is unclear. Although the PrP*-like proteinresidues 200–227 in rPrP(90–231) but extends only frombound to PrPSc is protease-resistant and insoluble, this200–217 in MoPrP(121–231). The loop and the COOH-protease-resistant PrP has not been reisolated in orderterminal portion of helix C are particularly important asto assess whether or not it was converted into PrPSc. Itdescribed below (Figure 2B). Whether the differencesis noteworthy that recombinant PrP can be refolded intobetween the two recombinant PrP fragments are dueeither a-helical or b-sheet forms but none have beento (i) their different lengths, (ii) species-specific differ-found to possess prion infectivity as judged by bioassay.ences in sequences, or (iii) the conditions used for solv-Inherited and Sporadic Prion Diseasesing the structures remains to be determined.For inherited and sporadic prion diseases, the majorRecent NMR studies of full-length MoPrP(23–231) andquestion is how the first PrPSc molecules are formed.SHaPrP(29–231) have shown that the NH2 termini areOnce these are formed, replication presumably followshighly flexible and lack identifiable secondary structurethe mechanism outlined for infectious disease. Severalunder the experimental conditions employed (Figure 2C)lines of evidence suggest that PrPSc is more stable than(Donne et al., 1997). Studies of SHaPrP(29–231) indicatePrPC and that a kinetic barrier precludes the formationtransient interactions between the COOH-terminal endof PrPSc under normal conditions. In the case of theof helix B and the highly flexible, NH2-terminal ran-initiation of inherited prion diseases, the barrier to PrPSc

dom-coil containing the octareapeats (residues 29–125)formation must be lower for the mutant (DPrPC) than the(Donne et al., 1997).wild-type and thus DPrP* can spontaneously rearrangeto form DPrPSc.While theknown mutationswould appearPrion Replicationto be destabilizing to the structure of PrPC, we lackIn an uninfected cell, PrPC with the wild-type sequenceuseful information about the structure of the transitionexists in equilibrium in its monomeric a-helical, prote-state for either the mutant or wild-type sequences. Stud-ase-sensitive state or bound to protein X (Figure 3). Weies of PrP in the brains of patients who were heterozy-denote the conformation of PrPC that is bound to pro-gous for the E200K mutation revealed DPrPSc(E200K)

tein X as PrP* (Cohen et al., 1994); this conformation ismolecules that were both detergent-insoluble and re-

likely to be different from that determined under aque-sistant to limited proteolysis while most wtPrP was

ous conditions for monomeric recombinant PrP. Thedetergent-insoluble but protease-sensitive (Gabizon

PrP*/protein X complex will bind PrPSc, thereby creating et al., 1996). These results suggest that in familial (f)a replication-competent assembly. Order of addition ex- CJD(E200K), insoluble wtPrP might represent a form ofperiments demonstrate that for PrPC, protein X binding PrP* (Gabizon et al., 1996). In studies with CHO cells,precedes productive PrPSc interactions (Kaneko et al., expression of DPrP(E200K) was found to be accompa-1997b). A conformational change takes place wherein nied with the posttranslational acquisition of resistancePrP, in a shape competent for binding to protein X and to limited proteolysis (Lehmann and Harris, 1996), butPrPSc, represents the initial phase in the formation of whether such cell lines expressing DPrP(E200K) pro-infectious PrPSc. duce infectious prions is unknown. It is noteworthy that

Several lines of evidence argue that thesmallest infec- levels of proteinase K used in the studies wheretious prion particle is an oligomer of PrPSc, perhaps as DPrP(E200K) was expressed in CHO cells were lowersmall as a dimer (Bellinger-Kawahara et al., 1988). Upon by a factor of 10–100 compared to digestions of PrPSc

purification, PrPSc tends to aggregate into insoluble derived from brain or ScN2a cells. Whether these alter-multimers that can be dispersed into liposomes (Gabi- ations in the properties of DPrP(E200K) in CHO cellszon et al., 1988). Insolubility does not seem to be a provide evidence for DPrP* or such changes lie outsideprerequisite for PrPSc formation or prion infectivity, the pathway of DPrPSc(E200K) formation remains to beas suggested by some investigators (Gajdusek, 1988; determined.Caughey et al., 1995); a protease-resistant PrPSc that is Initiation of sporadic disease may follow from a so-soluble in 1% Sarkosyl was generated in ScN2a cells matic mutation and thus follow a path similar to that forby expression of a PrP deletion mutant consisting of germline mutations in inherited disease. In this situation,106 amino acid residues (Muramoto et al., 1996). the mutant PrPSc must be capable of co-opting wtPrPC,

In attempts to form PrPSc in vitro, PrPC has been ex- a process known to be possible for some mutationsposed to 3 M guanidinium HCl and then diluted 10-fold (e.g., E200K, D178N) but less likely for others (e.g.,prior to binding to PrPSc (Kocisko et al., 1994; Kaneko P102L) (Telling et al., 1995, 1996). Alternatively, the acti-

vation barrier separating wtPrPC from PrPSc could beet al., 1997a). Based on these results, we presume that

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crossed on rare occasions when viewed in the context Table 2. Evidence for Protein X from Transmission Studies ofof a population. Most individuals would be spared while Human Prionsa

presentations in the elderly with an incidence of z1 perInoculum Host MoPrP Incubation Time

million would be seen. Gene [days 6 SEM] (n/no)Mechanism of Prion Propagation?sCJD Tg(HuPrP) Prnp1/1 721 (1/10)From the foregoing formalism, we can ask “What is thesCJD Tg(HuPrP)Prnp0/0 Prnp0/0 263 6 2 (6/6)rate-limiting step in prion formation?” First, we mustsCJD Tg(MHu2M) Prnp1/1 238 6 3 (8/8)

consider the impact of the concentration of PrPSc in the sCJD Tg(MHu2M)Prnp0/0 Prnp0/0 191 6 3 (10/10)inoculum, which is inversely proportional to the length

a Data with inoculum RG from Telling et al., 1995.of the incubation time. Second, we must consider thesequence of PrPSc that forms an interface with PrPC.When the sequences of the two isoforms are identical,

PrPSc is sufficient must be the conversion of PrPC to PrP*the shortest incubation times are observed. Third, wesince a dominant negative derived from a single pointmust consider the strain-specific conformation of PrPSc.mutation could gate only a kinetically critical step in aSome prion strains exhibit longer incubation times thancellular process. In the template-directed model, theothers; the mechanism underlying this phenomenon isconversion of PrPC to PrP* is a first order process. Bynot understood. From these considerations, there existscontrast, NP processes follow higher order kineticsa set of conditions under which initial PrPSc concentra-([monomer]m, where m is the number of monomers intions can be rate-limiting. These effects presumably re-the nucleus). The experimental implications of these ratelate to the stabilityof the PrPSc, its targetingto thecorrectrelationships are apparent in transgenic studies; if firstcells and subcellular compartments, and its ability to beorder kinetics operate, halving the gene dose (hemizy-cleared. Once infection in a cell is initiated and endoge-gotes) should double the incubation time while doublingnous PrPSc production is operative, then the followingthe dose of a transgene array should halve the time todiscussion of PrPSc formation seems most applicable.disease. This quantitative behavior has been observedIf the assembly of PrPSc into a specific dimeric orin several studies in mice with altered levels of PrPmultimeric arrangement were difficult, then a nucle-expression (Prusiner et al., 1990, 1993; Bueler et al.,ation–polymerization (NP) formalism would be relevant.1994; Carlson et al., 1994). The existence of prion strainsIn NP processes, nucleation is the rate-limiting step andthat are conformational isoforms of PrPSc with distinctelongation or polymerization is facile. These conditionsstructures, incubation times, and neurohistopathologyare frequently observed in peptide models of aggrega-must also be considered in an analysis of the kineticstion phenomena (Caughey et al., 1995); however, studiesof PrPSc accumulation. Since the rate-limiting step inwith Tg mice expressing foreign PrP genes suggest thatPrPSc formation cannot involve the unique template pro-a different process is occurring. From investigationsvided by a strain, differential rates of intercellular spread,with mice expressing both the SHaPrP transgene andcellular uptake, and clearance seem most likely to ac-the endogenous MoPrP gene, it is clear that PrPSc pro-count for thevariation in incubation times. This isconsis-vides a template for directing prion replication wheretent with the different patterns of protease sensitivitywe define a template as a catalyst that leaves its im-and glycosylation for distinct prion strains (Bessen andprint on the product of the reaction (Prusiner et al.,Marsh, 1994; Collinge et al., 1996; Telling et al., 1996;1990). Inoculation of these mice with SHaPrPSc leads toSomerville et al., 1997).the production of nascent SHaPrPSc and not MoPrPSc.

However, we hasten to add that NP models can pro-Conversely, inoculation of the Tg(SHaPrP) mice withvide a useful description of other biologic phenomena.MoPrPSc results in MoPrPSc formation and not SHaPrPSc.Under conditions when the monomer is relatively rareEven stronger evidence for templating has emerged fromand/or the conformational change is facile (e.g., shortstudies of prion strains passaged in Tg(MHu2M)Prnp0/0

peptides), the NP model will dominate. However, whenmice expressing a chimeric Hu/MoPrP gene as de-the monomer is sufficiently abundant and/or the confor-scribed in more detail below (Telling et al., 1996; Prusi-mational conversion is difficult to accomplish, the tem-ner, 1997). Even though the conformational templatesplate assistance formalism provides a more likely de-were initially generated with PrPSc molecules having dif-scription of the process.ferent sequences in patients with inherited prion dis-Evidence for Protein Xeases, thesetemplates are sufficient todirect replicationProtein X was postulated to explain the results on theof distinct PrPSc molecules when the amino acid se-transmission of human (Hu) prions to Tg mice (Table 2)quences of the substrate PrPs are identical. If the forma-(Telling et al., 1994, 1995). Mice expressing both Mo andtion of this template were rate-limiting, then an NP modelHuPrP were resistant to Hu prions while those express-could apply. However, studies of PrPSc formation ining only HuPrP were susceptible. These results argueScN2a cells point to a distinct rate-limiting step.that MoPrPC inhibited transmission of Hu prions, i.e.,Cell biologic and transgenetic investigations argue forthe formation of nascent HuPrPSc. In contrast to thethe existence of a chaperone-like molecule, referred toforegoing studies, mice expressing both MoPrP and chi-as protein X, that is required for PrPSc formation (Tellingmeric MHu2M PrP were susceptible to Hu prions andet al., 1995). As described below, mutagenesis experi-mice expressing MHu2MPrP alone were only slightlyments have created dominant negative forms of DPrPC

more susceptible. These findings contend that MoPrPCthat inhibit the formation of wtPrPSc by binding protein Xhas only a minimal effect on the formation of chimeric(Kaneko et al., 1997b). This implies that the rate-limiting

step in vivo in prion replication under conditions where MHu2MPrPSc.

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When the data on Hu prion transmission to Tg mice F1(C57Bl 3 VM) inbred mice began with isolates fromwere considered together, they suggested that MoPrPC sheep with scrapie. The prototypic strains called Me7prevented the conversion of HuPrPC into PrPSc by bind- and 22A gave incubation times of z150 and z400 daysing to another Mo protein but had little effect on the in C57Bl mice, respectively (Dickinson et al., 1968). Theconversion of MHu2M into PrPSc. We interpreted these PrPs of C57Bl and I/Ln (and later VM) mice differ at tworesults in terms of MoPrPC binding to this Mo protein residues and control incubation times (Carlson et al.,with a higher affinity than does HuPrPC. We postulated 1994; Moore et al., 1998).that MoPrPC had little effect on the formation of PrPSc Until recently, support for the hypothesis that the ter-from MHu2M (Table 2) because MoPrP and MHu2M tiary structure of PrPSc enciphers strain-specific informa-share the same amino acid sequence at theCOOH termi- tion (Prusiner, 1991) was minimal except for the DY strainnus. This also suggested that MoPrPC only weakly inhib- isolated from mink with transmissible encephalopathyited transmission of SHa prions to Tg(SHaPrP) mice be- (Bessen and Marsh, 1994). PrPSc in DY prions showedcause SHaPrP is more closely related to MoPrP than is diminished resistance to proteinase K digestion as wellHuPrP. as an anomalous site of cleavage. The DY strain pre-

Using scrapie-infected mouse neuroblastoma cells sented a puzzling anomaly since other prion strains ex-transfected with chimeric Hu/Mo PrP genes, we ex- hibiting similar incubation times did not show this al-tended our studies of protein X. Substitution of a Hu tered susceptibility to proteinase K digestion of PrPSc

residue at position 214 or 218 prevented PrPSc formation (Scott et al., 1997). Also notable was the generation of(Figure 2B) (Kaneko et al., 1997b). The side chains of new strains during passage of prions through animalsthese residues protrude from the same surface of the with different PrP genes (Scott et al., 1997).COOH-terminal a helix forming a discontinuous epitope PrPSc Conformation Enciphers Variation in Prionswith residues 167 and 171 in an adjacent loop. Substitu- Persuasive evidence that strain-specific information istion of a basic residue at positions 167, 171, or 218 enciphered in the tertiary structure of PrPSc comes fromprevented PrPSc formation; these mutant PrPs appear transmission of two different inherited human prionto act as “dominant negatives” by binding protein X diseases to mice expressing a chimeric MHu2M PrPand rendering it unavailable for prion propagation. Our transgene (Telling et al., 1996). In fatal familial insomniafindings seem to explain the protective effects of basic (FFI), the protease-resistant fragment of PrPSc afterpolymorphic residues in PrP of humans and sheep deglycosylation has an Mr of 19 kDa; whereas in(Hunter et al., 1993; Westaway et al., 1994; Shibuya et fCJD(E200K) and most sporadic prion diseases, it is 21al., 1998). kDa (Monari et al., 1994). This difference in molecularIs Protein X a Molecular Chaperone? size was shown to be due to different sites of proteolyticSince PrP undergoes a profound structural transition cleavage at the NH2 termini of the two human PrPSc

during prion propagation, it seems likely that other pro- molecules reflecting different tertiary structures (Monariteins such as chaperones participate in this process. et al., 1994). These distinct conformations were not un-Whether protein X functions as a classical molecular expected since the amino acid sequences of the PrPschaperone or participates in PrP binding as part of its differ.normal function but can also facilitate pathogenic as- Extracts from the brains of FFI patients transmittedpects of PrP biology is unknown. Interestingly, scrapie- disease into mice expressing a chimeric MHu2M PrPinfected cells in culture display marked differences in gene about 200 days after inoculation and induced for-the induction of heat-shock proteins (Tatzelt et al., 1995), mation of the 19 kDa PrPSc; whereas fCJD(E200K) andand Hsp70 mRNA has been reported to increase in sCJD produced the 21 kDa PrPSc in mice expressingscrapie of mice (Kenward et al., 1994). While attempts

the same transgene (Telling et al., 1996). On secondto isolate specific proteins that bind to PrP have been

passage, Tg(MHu2M) mice inoculated with FFI prionsdisappointing (Oesch et al., 1990), PrP has been shown

showed an incubation time of z130 days and a 19 kDato interact with Bcl-2, Hsp60, and the laminin receptor

PrPSc while those inoculated with fCJD(E200K) prionsprotein by two-hybrid analysis in yeast (Kurschner andexhibited an incubation time of z170 days and a 21 kDaMorgan, 1996; Rieger et al., 1997). Although these stud-PrPSc (Prusiner, 1997). The experimental data demon-ies are suggestive, no molecular chaperone involved instrate that MHu2MPrPSc can exist in twodifferent confor-prion formation in mammalian cells has been identified.mations based on the sizes of the protease-resistantfragments; yet, the amino acid sequence of MHu2MPrPSc

is invariant.Strains of PrionsThe results of our studies argue that PrPSc acts as aThe existence of prion strains raises the question of how

template for the conversion of PrPC into nascent PrPSc.heritable biological information can be enciphered inImparting the size of the protease-resistant fragmentany molecule other than nucleic acid (Dickinson et al.,of PrPSc through conformational templating provides a1968). Strains or varieties of prions have been definedmechanism for both the generation and propagation ofby incubation times and the distribution of neuronal vac-prion strains.uolation (Dickinson et al., 1968; Fraser and Dickinson,

Interestingly, the protease-resistant fragment of PrPSc1968). Subsequently, the patterns of PrPSc depositionafter deglycosylation with an Mr of 19 kDa has beenwere found to correlate with vacuolation profiles, andfound in a patient who died after developing a clinicalthese patterns were also used to characterize strains ofdisease similar to FFI. Since both PrP alleles encodedprions (DeArmond et al., 1987, 1997; Bruce et al., 1989).

The typing of prion strains in C57Bl, VM, and the wild-type sequence and a Met at position 129, we

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labeled this case fatal sporadic insomnia (FSI). At au-topsy, the spongiform degeneration, reactive astroglio-sis, and PrPSc deposition were confined to the thalamus(Mastrianni et al., 1997). These findings argue that theclinicopathologic phenotype is determined by the con-formation of PrPSc in accord with the results of the trans-mission of human prions from patients with FFI to Tgmice (Telling et al., 1996).Mechanism of Selective Neuronal Targeting?In addition to incubation times, neuropathologic profilesof spongiform change have been used to characterizeprion strains (Fraser and Dickinson, 1968). However,recent studies with PrP transgenes argue that such pro-files are not an intrinsic feature of strains (Carp et al.,1997; DeArmond et al., 1997). The mechanism by whichprion strains modify the pattern of spongiform degener-ation was perplexing since earlier investigations hadshown that PrPSc deposition precedes neuronal vacuola-tion and reactive gliosis (DeArmond et al., 1987). WhenFFI prions were inoculated into Tg(MHu2M) mice, PrPSc

was confined largely to the thalamus (Figure 4A) as is thecase for FFI in humans (Telling et al., 1996). In contrast,fCJD(E200K) prions inoculated into Tg(MHu2M) miceproduced widespread deposition of PrPSc throughoutthe cortical mantel and many of the deep structures ofthe CNS (Figure 4B) as is seen in fCJD(E200K) of hu-mans. To examine whether the diverse patterns of PrPSc

deposition are influenced by Asn-linked glycosylationof PrPC, we constructed Tg mice expressing PrPs mu-tated at one or both of the Asn-linked glycosylationconsensus sites (DeArmond et al., 1997). These muta-tions resulted in aberrant neuroanatomic topologies ofPrPC within the CNS, whereas pathologic point muta-tions adjacent to the consensus sites did not alter thedistribution of PrPC. Tg mice with mutation of thesecondPrP glycosylation site exhibited prion incubation timesof .500 days and unusual patterns of PrPSc deposition.These findings raise the possibility that glycosylationcan modify the conformation of PrP and affect either

Figure 4. Regional Distribution of PrPSc Deposition inthe turnover of PrPC or the clearance of PrPSc. RegionalTg(MHu2M)Prnp0/0 Mice Inoculated with Prions from Humans Whodifferences in the rate of deposition or clearance wouldDied of Inherited Prion Diseasesresult in specific patterns of PrPSc accumulation.Histoblot of PrPSc deposition in a coronal section of aTg(MHu2M)Prnp0/0 mouse through the hippocampus and thalamusYeast and Other Prions (Telling et al., 1996).

Although prions were originally defined in the context (A) The Tg mouse was inoculated with brain extract prepared fromof an infectious mammalian pathogen (Prusiner, 1982), a patient who died of FFI.

(B) The Tg mouse was inoculated with extract from a patient withit is now becoming widely accepted that prions are ele-fCJD(E200K). Cryostat sections were mounted on nitrocellulose andments that impart and propagate variability through mul-treated with proteinase K to eliminate PrPC (Taraboulos et al., 1992).tiple conformers of a normal cellular protein. Such aTo enhance the antigenicity of PrPSc, the histoblots were exposed

mechanism must surely not be restricted to a single to 3 M guanidinium isothiocyanate before immunostaining usingclass of transmissible pathogens. Indeed, it is likely that a-PrP 3F4 MAb (Kascsak et al., 1987).the original definition will need to beextended toencom- (C) Labeled diagram of a coronal section of the hippocampus/thala-

mus region. NC, neocortex; Hp, hippocampus; Hb, habenula; Th,pass other situations where a similar mechanism of in-thalamus; vpl, ventral posterior lateral thalamic nucleus; Hy, hypo-formation transfer occurs.thalamus; Am, amygdala.Two notable prion-like determinants, [URE3] and

[PSI], have already been described in yeast and one inanother fungus denoted [Het-s*] (Wickner, 1994; Cher-

molecular chaperone Hsp104; however, no homolog ofnoff et al., 1995; Coustou et al., 1997). Studies of candi-Hsp104 has been found in mammals (Chernoff et al.,date prion proteins in yeast may prove particularly help-1995). The NH2-terminal prion domains of Ure2p andful in the dissection of some of the events that featureSup35 that are responsible for the [URE3] and [PSI]in PrPSc formation. Interestingly, different strains of yeastphenotypes in yeast have been identified. In contrast toprions have been identified (Derkatch et al., 1996). Con-

version to the prion-like [PSI] state in yeast requires the PrP, which is a GPI-anchored membrane protein, both

Review345

Ure2p and Sup35 are cytosolic proteins (Wickner, 1997). the PrP gene (Collinge et al., 1994; Lledo et al., 1996;Sakaguchi et al., 1996; Tobler et al., 1996).When the prion domains of these yeast proteins were

expressed in E. coli, the proteins were found to polymer- Whether gene therapy for the human prion diseaseswill prove feasible using the dominant negative ap-ize into fibrils with properties similar to those of proteo-

lytically trimmed PrP and other amyloids (Paushkin et proach described above for prion-resistant animals de-pends on the availability of efficient vectors for deliveryal., 1997).

Whether prions explain some other examples of ac- of the transgene to the CNS.quired inheritance in lower organisms is unclear (Land-man, 1991). For example, studies on the inheritance of Concluding Remarks

Although the study of prions has taken several unex-positional order and cellular handedness on the surfaceof small organisms have demonstrated the epigenetic pected directions over the past three decades, a novel

and fascinating story of prion biology is emerging. In-nature of these phenomena but the mechanism remainsunclear (Frankel, 1990). vestigations of prions have elucidated a previously un-

known mechanism of disease in humans and animals.While learning the details of the structures of PrPs and

Therapeutic Approaches to Prion Diseases deciphering the mechanism of PrPC transformation intoIt seems likely that it will be possible to design effective PrPSc will be important, the fundamental principles oftherapeutics for prion diseases as our understanding of prion biology have become reasonably clear. Thoughprion propagation increases. Because people at risk for some investigators prefer to view the composition ofinherited prion diseases can now be identified decades the infectious prion particle as unresolved (Manuelidisbefore neurologic dysfunction is evident, the develop- and Fritch, 1996; Lasmezas et al., 1997; Chesebro,ment of an effective therapy for these fully penetrant 1998), such a perspective denies an enlarging body ofdisorders is imperative. Although we have no way of data, noneof which refutes the prionconcept. Moreover,predicting the number of individuals who may develop the discovery of prion-like phenomena mediated by pro-neurologic dysfunction from bovine prions in the future, teins unrelated to PrP in yeast and other fungi servesseeking an effective therapy now seems most prudent not only to strengthen the prion concept but also to(Prusiner, 1997). Interfering with the conversion of PrPC widen it.into PrPSc seems to be the most attractive therapeutic The discovery that prion diseases in humans aretarget. Reasonable strategies are either stabilizing the uniquely both genetic and infectious greatly strength-structure of PrPC via the formation of a PrPC-drug com- ened and extended the prion concept. To date, 20 differ-plex or modifying the action of protein X, which may ent mutations in the human PrP gene, all resulting infunction as a molecular chaperone (Figure 2B). Whether nonconservative substitutions, have been found eitherit is more efficacious to design a drug that binds to PrPC to be linked genetically to or to segregate with the inher-at the protein X–binding site or one that mimics the ited prion diseases (Figure 1B). Yet, the transmissiblestructure of PrPC with basic polymorphic residues that prion particle is composed largely, if not exclusively, ofseem to prevent scrapie and CJD remains to be deter- an abnormal isoform of the prion protein designatedmined (Kaneko et al., 1997b; Shibuya et al., 1998). Since PrPSc (Prusiner, 1991).PrPSc formation seems limited to caveolae-like domains Aberrant PrP Metabolism(Gorodinsky and Harris, 1995; Taraboulos et al., 1995), The hallmark of all prion diseases—whether sporadic,drugs designed to inhibit this process need not pene- dominantly inherited, or acquired by infection—is thattrate the cytosol of cells but they do need to be able to they involve the aberrant metabolism and resulting ac-enter the CNS. Alternatively, drugs that destabilize the cumulation of the prion protein (Table 1) (Prusiner, 1991).structure of PrPSc might also prove useful. The conversion of PrPC into PrPSc involves a conforma-

The production of domestic animals that do not repli- tion change whereby the a-helical content diminishescate prions may also be important with respect to pre- and the amount of b sheet increases (Pan et al., 1993).venting prion disease. Sheep encoding the R/Rpolymor- These findings provide a reasonable mechanism to ex-phism at position 171 seem to be resistant to scrapie plain the conundrum presented by the three different(Hunter et al., 1993; Westaway et al., 1994); presumably, manifestations of prion disease.this was the genetic basis of James Parry’s scrapie Understanding how PrPC unfolds and refolds intoeradication program in Great Britain 30 years ago (Parry, PrPSc will be of paramount importance in transferring1962). A more effective approach using dominant nega- advances in the prion diseases to studies of other de-tives for producing prion-resistant domestic animals, generative illnesses. The mechanism by which PrPSc isincluding sheep and cattle, is probably the expression formed must involve a templating process whereby ex-of PrP transgenes encoding R171 as well as additional isting PrPSc directs the refolding of PrPC into a nascentbasic residues at the protein X–binding site (Figure 2B) PrPSc with the same conformation. Not only will a knowl-(Kaneko et al., 1997b). Such an approach can be readily edge of PrPSc formation help in the rational design ofevaluated in Tg mice, and once shown to be effective, drugs that interrupt the pathogenesis of prion diseases,it could be instituted by artificial insemination of sperm but it may also open new approaches to decipheringfrom males homozygous for the transgene. Less practi- the causes of and to developing effective therapiescal is the production of PrP-deficient cattle and sheep. for the more common neurodegenerative diseases in-Although such animals would not be susceptible toprion cluding Alzheimer’s disease, Parkinson’s disease, anddisease (Bueler et al., 1993; Prusiner et al., 1993), they amyotrophic lateral sclerosis (ALS). Indeed, the ex-

panding list of prion diseases and their novel modes ofmight suffer some deleterious effects from ablation of

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by grants from the National Institute of Aging and the National Insti-transmission and pathogenesis (Table 1), as well as thetute of Neurologic Diseases and Stroke of the National Institutesunprecedented mechanisms of prion propagation andof Health, International Human Frontiers of Science Program, andinformation transfer, indicate that much more attentionAmerican Health Assistance Foundation, as well as by gifts from

to these fatal disorders of protein conformation is ur- the Sherman Fairchild Foundation, Keck Foundation, G. Harold andgently needed. Leila Y. Mathers Foundation, Bernard Osher Foundation, John D.

But prions may have even wider implications than French Foundation, and Centeon.those noted for the common neurodegenerative dis-

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