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

Prion Protein Biology

Stanley B. Prusiner,*† Michael R. Scott,* Stephen J. DeArmond,‡* and Fred E. Cohen†§k * Department of Neurology † Department of Biochemistry and Biophysics ‡ Department of Pathology § Department of Molecular and Cellular Pharmacology k Department of Medicine University of California San Francisco, California 94143 Introduction It is interesting to contemplate how the course of scientific investigation might have proceeded had studies on the transmissibility of inherited prion diseases not been performed until after the molecular genetic lesion had been identified (Meggendorfer, 1930; Roos et al., 1973; Hsiao et al., 1989). Had the prion protein (PrP) gene been identified in families with prion disease by positional cloning or through the purification and sequencing of PrP in amyloid plaques before brain extracts were shown to be transmissible, the prion concept might have been more readily accepted (Prusiner, 1998). But that is not the course of events that led to our current understanding of prions. Creutzfeldt-Jakob disease (CJD) remained a curious, rare neurodegenerative disease of unknown etiology for more than three score years. Only the transmission of CJD to apes by inoculation of brain extracts from patients who had died of CJD initiated a path of scientific investigation that was to demystify that fascinating area of biomedicine (Gibbs et al., 1968). Not unexpectedly, once CJD was shown to be an infectious disease, relatively little attention was paid to the familial form of the disease since most cases are sporadic and familial clusters account for a minority. In this review, we focus on the prion particles that cause scrapie, bovine spongiform encephalopathy (BSE), and CJD. Some of the fascinating studies that led to our knowledge of prions are not discussed here but have recently been reviewed elsewhere (Prusiner, 1998). Prion diseases may present as genetic, infectious, or sporadic disorders, all of which involve modification of the prion protein (PrP), a constituent of normal mammalian cells. CJD generally presents as progressive dementia while scrapie of sheep and BSE are generally manifest as ataxic illnesses (Table 1) (Wells et al., 1987). In CJD, scrapie, and BSE, as well as all of the other disorders frequently referred to as prion diseases (Table 1), spongiform degeneration and astrocytic gliosis are found upon microscopic examination of the CNS. The degree of spongiform degeneration is quite variable while the extent of reactive gliosis correlates with the degree of neuron loss (Masters and Richardson, 1978). The Prion Particle Perhaps, the best current working definition of a prion is a proteinaceous infectious particle that lacks nucleic acid (Prusiner, 1997). A wealth of data supports the contention that scrapie prions are devoid of nucleic acid

Review

and seem to be composed exclusively of a modified isoform of PrP, designated PrPSc. The normal cellular PrP, denoted PrPC, is converted into PrPSc through a process whereby a portion of its a-helical and coil structure is refolded into b sheet (Pan et al., 1993). This structural transition is accompanied by profound changes in the physicochemical properties of the PrP. While PrPC is soluble in nondenaturing detergents, PrPSc is not. PrPC is readily digested by proteases, whereas PrPSc is partially resistant (Oesch et al., 1985). Because prions appear to be composed entirely of a protein that adopts an abnormal conformation, it is not unreasonable to think of prions as infectious proteins (Pan et al., 1993; Telling et al., 1996). But we hasten to add that we still cannot eliminate the possibility of a small ligand bound to PrPSc as an essential component of the infectious prion particle. In a more broad view, prions are elements that impart and propagate variability through multiple conformers of a normal cellular protein. The species of a particular prion is encoded by the sequence of the chromosomal PrP gene of the mammal in which it last replicated. In contrast to pathogens with a nucleic acid genome that encode strain-specific properties in genes, prions seem to encipher these properties in the tertiary structure of PrPSc (Bessen and Marsh, 1994; Telling et al., 1996; Prusiner, 1997). The discovery that mutations of the PrP gene caused dominantly inherited prion diseases in humans linked the genetic and infectious forms of prion diseases and presented another hurdle for investigators who continued to argue that prion diseases are caused by viruses. More than 20 mutations of the PrP gene are now known to cause the inherited human prion diseases, and significant genetic linkage has been established for five of these mutations (Hsiao et al., 1989) (for review, see Prusiner, 1997). The prion concept readily explains how a disease can be manifest as a heritable as well as an infectious illness. Moreover, the hallmark common to all of the prion diseases whether sporadic, dominantly inherited, or acquired by infection is that they involve the aberrant metabolism of the prion protein (Prusiner, 1991). Although PrPSc is the only known component of the infectious prion particles, these unique pathogens share several phenotypic traits with other infectious entities such as viruses. Because some features of the diseases caused by prions and viruses are similar, some scientists have difficulty accepting the existence of prions despite a wealth of scientific data supporting this concept (Chesebro and Caughey, 1993; Manuelidis and Fritch, 1996; Lasme´zas et al., 1997; Chesebro, 1998). Molecular Genetics of Prion Diseases Once a PrP cDNA probe became available, molecular genetic studies were undertaken to determine whether the PrP gene controls scrapie incubation times in mice. Independent of the enriching of brain fractions for scrapie infectivity that led to the discovery of PrPSc, the

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Table 1. The Prion Diseases Disease

Host

Mechanism of Pathogenesis

Kuru iCJD

Fore people Humans

vCJD fCJD GSS FFI sCJD

Humans Humans Humans Humans Humans

FSI

Humans

Infection through ritualistic cannibalism Infection from prion-contaminated HGH, dura mater grafts, etc. Infection from bovine prions? Germline mutations in PrP gene Germline mutations in PrP gene Germline mutation in PrP gene (D178N, M129) Somatic mutation or spontaneous conversion of PrP C into PrP Sc? Somatic mutation or spontaneous conversion of PrP C into PrP Sc?

Scrapie BSE TME CWD FSE Exotic ungulate encephalopathy

Sheep Cattle Mink Mule deer, elk Cats Greater kudu, nyala, oryx

Infection in genetically susceptible sheep Infection with prion-contaminated MBM Infection with prions from sheep or cattle Unknown Infection with prion-contaminated beef 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-Stra¨ussler-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 controlling the incubation time (Carlson et al., 1986). Subsequently, mutation of the PrP gene was shown to be genetically linked to the development of familial prion disease (Hsiao et al., 1989). At the same time, expression of a Syrian hamster (SHa) PrP transgene in mice was shown to render the animals highly susceptible to SHa prions, which demonstrated that expression of a foreign PrP gene could abrogate the species barrier (Scott et al., 1989). Later, PrP-deficient (Prnp 0/0) mice were found to be resistant to prion infection and failed to replicate prions, as expected (Bu¨eler et al., 1993; Prusiner et al., 1993). The results of these studies indicated PrP must play a central role in the transmission and pathogenesis of prion disease, but equally important, they established that the abnormal isoform is an essential component of the prion particle (Prusiner, 1991). PrP Gene Dosage Controls Length of Incubation Time Scrapie incubation times in mice were used to distinguish prion strains and to identify a gene controlling its length (Dickinson et al., 1968; Scott et al., 1997). This gene was initially called Sinc based on genetic crosses between C57Bl and VM mice, which exhibited short and long incubation times, respectively (Dickinson et al., 1968). Because the distribution of VM mice was restricted, we searched for another mouse with long incubation times. I/Ln mice proved to be a suitable substitute for VM mice; eventually, I/Ln and VM mice were found to be derived from a common ancestor. Subsequently, the PrP gene was shown to control the length of the scrapie incubation time in mice (Carlson et al., 1994; Moore et al., 1998). Overexpression of wtPrP Transgenes Mice were constructed expressing different levels of the wild-type (wt) SHaPrP transgene (Scott et al., 1989). Inoculation of these Tg(SHaPrP) mice with SHa prions demonstrated abrogation of the species barrier resulting in abbreviated incubation times due to a nonstochastic

process (Prusiner et al., 1990). The length of the incubation time after inoculation with SHa prions was inversely proportional to the level of SHaPrPC in the brains of Tg(SHaPrP) mice (Prusiner et al., 1990). Bioassays of brain extracts from clinically ill Tg(SHaPrP) mice inoculated with mouse (Mo) prions revealed that only Mo prions but no SHa prions were produced. Conversely, inoculation of Tg(SHaPrP) mice with SHa prions led to the synthesis of only SHa prions. Thus, the rate of PrPSc synthesis appears to be a function of the level of PrPC expression in Tg mice; however, the level to which PrPSc accumulates appears to be independent of PrPC concentration (Prusiner et al., 1990). PrP-Deficient Mice The development and lifespan of two lines of Prnp0/0 mice were indistinguishable from controls (Bu¨eler et al., 1992; Manson et al., 1994) while another line exhibited ataxia and Purkinje cell degeneration at z70 weeks of age (Sakaguchi et al., 1996). In the former two lines with normal development, altered sleep–wake cycles (Tobler et al., 1996) and synaptic behavior in brain slices have been reported (Collinge et al., 1994), but the synaptic changes could not be confirmed by others (Lledo et al., 1996). Prnp0/0 mice are resistant to prions (Bu¨eler et al., 1993; Prusiner et al., 1993). Prnp0/0 mice were sacrificed 5, 60, 120, and 315 days after inoculation with RML prions and brain extracts bioassayed in CD-1 Swiss mice. Except for residual infectivity from the inoculum detected at 5 days after inoculation, no infectivity was detected in the brains of Prnp0/0 mice (Prusiner et al., 1993). One group of investigators found that Prnp0/0 mice inoculated with RML prions and sacrificed 20 weeks later had 103.6 ID50 units/ml of homogenate by bioassay (Bu¨eler et al., 1993). Others have used this report to argue that prion infectivity replicates in the absence of PrP (Chesebro and Caughey, 1993; Lasme´zas et al., 1997). Neither we nor the authors of the initial report could confirm the finding of prion replication in Prnp0/0 mice (Prusiner et al., 1993; Sailer et al., 1994).

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Prion Protein Structure Once cDNA probes for PrP became available, the PrP gene 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 transcription of the PrP gene as proposed nearly two decades earlier (Griffith, 1967). Determination of the structure of the PrP gene eliminated a second possible mechanism that might explain the appearance of PrPSc in brains already synthesizing PrPC. Since the entire protein coding region was contained within a single exon, there was no possibility that the two PrP isoforms were the products of alternatively spliced mRNAs (Basler et al., 1986). Next, a posttranslational chemical modification that distinguishes PrPSc from PrPC was considered but 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 also proposed earlier (Griffith, 1967). When the secondary structures of the PrP isoforms were compared by optical spectroscopy, they were found to be markedly different (Pan et al., 1993). Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopy studies showed that PrPC contains about 40% a helix and little b sheet while PrPSc is composed of about 30% a helix and 45% b sheet (Pan et al., 1993). That the two PrP isoforms have the same amino acid sequence runs counter to the widely accepted view that the amino acid sequence specifies only one biologically active conformation of a protein (Anfinsen, 1973). Prior to comparative studies on the structures of PrPC and PrPSc, metabolic labeling studies showed that the acquisition of PrPSc protease resistance is a posttranslational process (Borchelt et al., 1990). In a search for chemical differences that would distinguish PrPSc from PrPC, we identified ethanolamine in hydrolysates of PrP 27–30, which signaled the possibility that PrP might contain a glycosylphosphatidyl inositol (GPI) anchor (Stahl et al., 1987). Both PrP isoforms were found to carry GPI anchors and PrPC was found on the surface of cells where it could be released by cleavage of the anchor. Subsequent studies showed that PrPSc formation occurs after PrPC reaches the cell surface (Caughey and Raymond, 1991) and is localized to caveolae-like domains (Gorodinsky and Harris, 1995; Taraboulos et al., 1995). Computational Models and Optical Spectroscopy Modeling studies and subsequent nuclear magnetic resonance (NMR) investigations of a synthetic PrP peptide containing residues 90–145 suggested that PrPC might contain an a helix within this region (Figure 1) (Huang et al., 1994). This peptide contains the residues, 113– 128, that are most highly conserved among all species studied (Figure 1A) and that correspond to a transmembrane region of PrP which was delineated in cell-free translation studies. A transmembrane form of PrP was found in brains of patients with Gerstmann-Stra¨usslerScheinker disease (GSS) caused by the A117V mutation and in Tg mice overexpressing either mutant or wtPrP (Hegde et al., 1998). That no evidence for an a helix in this region has been found in NMR studies of recombinant PrP in an aqueous environment (Riek et al., 1996; Donne et al., 1997; James et al., 1997) suggests that

Figure 1. Species Variations and Mutations of the Prion Protein Gene (A) Species variations. The x-axis represents the human PrP sequence, with the five octarepeats and H1–H4 regions of putative secondary structure shown as well as the three a helices A, B, and C and the two b strands S1 and S2. Vertical bars above the axis indicate the number of species that differ from the human sequence at each position. Below the axis, the length of the bars indicates the number of alternative amino acids at each position in the alignment. (B) Mutations causing inherited human prion disease and polymorphisms in human, mouse, and sheep. Above the line of the human sequence are mutations that cause prion disease. Below the lines are polymorphisms, some but not all of which are known to influence the onset as well as the phenotype of disease. Data were compiled by Paul Bamborough and Fred E. Cohen. Reprinted with permission from Science 278, pp. 245–251 (copyright 1997 American Association for the Advancement of Science).

these recombinant PrPs correspond to the secreted form of PrP that was also identified in the cell-free translation studies. This contention is supported by studies with recombinant antibody fragments (Fabs) showing that GPI-anchored PrPC on the surface of cells exhibits an immunoreactivity similar to that of recombinant PrP prepared with an a-helical conformation (Peretz et al., 1997). Models of PrPSc suggest that formation of the diseasecausing isoform involves refolding of a region corresponding roughly to residues 108–144 into b sheets (Huang et al., 1996); the single disulfide bond joining the COOH-terminal helices would remain intact since the disulfide is required for PrPSc formation (Figure 2D) (Muramoto et al., 1996). Deletion of each of several regions of putative secondary structure in PrP, except for the NH 2-terminal 66 amino acids (residues 23–88) and a 36– 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 PrP C. rPrP(90–231) is viewed from the interface where PrPSc is thought to bind to PrPC. Color scheme: a helices A (residues 144–157), B (172–193), and C (200–227) in pink; disulfide between Cys-179 and Cys-214 in yellow; conserved hydrophobic region composed of residues 113–126 in red; loops in gray; residues 129–134 in green encompassing strand S1 and residues 159–165 in blue encompassing strand 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 the side chains of residues that form a discontinuous epitope: some amino acids are in the loop composed of residues 165–171 and at the end of 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-219 with 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 the protein representing residues 90–231 was taken from the coordinates of PrP(90–231) (James et al., 1997). The remainder of the sequence was hand-built for illustration purposes only. The color scale corresponds to the heteronuclear { 1H}-15N NOE data: red for the lowest (most negative) values, where the polypeptide is most flexible, to blue for the highest (most positive) values in the most structured and rigid regions of 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 in red; 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 (residues 142–177) 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 cultured neuroblastoma cells (Muramoto et al., 1996). With a-PrP Fabs selected from phage display libraries and two monoclonal antibodies (MAbs) derived from hybridomas, a major conformational change that occurs during conversion of PrPC into PrPSc has been localized to residues 90–112 (Peretz et al., 1997). Studies with an a-PrP IgM MAb, which was reported to immunoprecipitate PrP Sc selectively (Korth et al., 1997), support this conclusion. While these results indicate that PrPSc formation involves a conformational change at the NH 2 terminus, mutations causing inherited prion diseases have been found throughout the protein (Figure 1B). Interestingly, all of the known point mutations in PrP with biological significance occur either within or adjacent to regions

of putative secondary structure in PrP and, as such, appear to destabilize the structure of PrPC (Huang et al., 1994; Riek et al., 1996). NMR Structure of Recombinant PrP The NMR structure of recombinant SHaPrP(90–231) was determined after the protein was purified and refolded (Figure 2A). Residues 90–112 are not shown since marked conformational heterogeneity was found in this region while residues 113–126 constitute the conserved hydrophobic region that also displays some structural plasticity (James et al., 1997). Although some features of the structure of rPrP(90–231) are similar to those reported earlier for the smaller recombinant MoPrP(121– 231) fragment (Riek et al., 1996), substantial differences were found. For example, the loop at the NH2 terminus

Review 341

Figure 3. Schematic Diagram Showing Template-Assisted PrPSc Formation In the initial step, PrPC binds to protein X to form the PrP */protein X complex. Next, PrP Sc binds to PrP* that has already formed a complex with protein X. When PrP* is transformed into a nascent molecule of PrP Sc, protein X is released and a dimer of PrPSc remains. The inactivation target size of an infectious prion suggests that it is composed of a dimer of PrP Sc (Bellinger-Kawahara et al., 1988). In the model depicted here, a fraction of infectious PrP Sc dimers dissociate into uninfectious monomers as the replication cycle proceeds while most of the dimers accumulate in accord with the increase in prion 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 in MoPrP(121–231); in addition, helix C is composed of residues 200–227 in rPrP(90–231) but extends only from 200–217 in MoPrP(121–231). The loop and the COOHterminal portion of helix C are particularly important as described below (Figure 2B). Whether the differences between the two recombinant PrP fragments are due to (i) their different lengths, (ii) species-specific differences in sequences, or (iii) the conditions used for solving the structures remains to be determined. Recent NMR studies of full-length MoPrP(23–231) and SHaPrP(29–231) have shown that the NH2 termini are highly flexible and lack identifiable secondary structure under the experimental conditions employed (Figure 2C) (Donne et al., 1997). Studies of SHaPrP(29–231) indicate transient interactions between the COOH-terminal end of helix B and the highly flexible, NH2-terminal random-coil containing the octareapeats (residues 29–125) (Donne et al., 1997). Prion Replication In an uninfected cell, PrP C with the wild-type sequence exists in equilibrium in its monomeric a-helical, protease-sensitive state or bound to protein X (Figure 3). We denote the conformation of PrPC that is bound to protein X as PrP * (Cohen et al., 1994); this conformation is likely to be different from that determined under aqueous conditions for monomeric recombinant PrP. The PrP* /protein X complex will bind PrPSc, thereby creating a replication-competent assembly. Order of addition experiments demonstrate that for PrPC, protein X binding precedes productive PrPSc interactions (Kaneko et al., 1997b). A conformational change takes place wherein PrP, in a shape competent for binding to protein X and PrPSc , represents the initial phase in the formation of infectious PrPSc. Several lines of evidence argue that the smallest infectious prion particle is an oligomer of PrPSc , perhaps as small as a dimer (Bellinger-Kawahara et al., 1988). Upon purification, PrPSc tends to aggregate into insoluble multimers that can be dispersed into liposomes (Gabizon et al., 1988). Insolubility does not seem to be a prerequisite for PrPSc formation or prion infectivity, as suggested by some investigators (Gajdusek, 1988; Caughey et al., 1995); a protease-resistant PrPSc that is soluble in 1% Sarkosyl was generated in ScN2a cells by expression of a PrP deletion mutant consisting of 106 amino acid residues (Muramoto et al., 1996). In attempts to form PrPSc in vitro, PrPC has been exposed to 3 M guanidinium HCl and then diluted 10-fold prior to binding to PrPSc (Kocisko et al., 1994; Kaneko et al., 1997a). Based on these results, we presume that

exposure of PrPC to GdnHCl converts it into a PrP*-like molecule. Whether this PrP* -like protein is converted into PrPSc is unclear. Although the PrP*-like protein bound to PrPSc is protease-resistant and insoluble, this protease-resistant PrP has not been reisolated in order to assess whether or not it was converted into PrPSc. It is noteworthy that recombinant PrP can be refolded into either a-helical or b-sheet forms but none have been found to possess prion infectivity as judged by bioassay. Inherited and Sporadic Prion Diseases For inherited and sporadic prion diseases, the major question is how the first PrPSc molecules are formed. Once these are formed, replication presumably follows the mechanism outlined for infectious disease. Several lines of evidence suggest that PrPSc is more stable than PrPC and that a kinetic barrier precludes the formation of PrPSc under normal conditions. In the case of the initiation of inherited prion diseases, the barrier to PrPSc formation must be lower for the mutant (DPrPC) than the wild-type and thus DPrP* can spontaneously rearrange to form DPrPSc. While the known mutations would appear to be destabilizing to the structure of PrPC, we lack useful information about the structure of the transition state for either the mutant or wild-type sequences. Studies of PrP in the brains of patients who were heterozygous for the E200K mutation revealed DPrPSc(E200K) molecules that were both detergent-insoluble and resistant to limited proteolysis while most wtPrP was detergent-insoluble but protease-sensitive (Gabizon et al., 1996). These results suggest that in familial (f) CJD(E200K), insoluble wtPrP might represent a form of PrP* (Gabizon et al., 1996). In studies with CHO cells, expression of DPrP(E200K) was found to be accompanied with the posttranslational acquisition of resistance to limited proteolysis (Lehmann and Harris, 1996), but whether such cell lines expressing DPrP(E200K) produce infectious prions is unknown. It is noteworthy that levels of proteinase K used in the studies where DPrP(E200K) was expressed in CHO cells were lower by a factor of 10–100 compared to digestions of PrPSc derived from brain or ScN2a cells. Whether these alterations in the properties of DPrP(E200K) in CHO cells provide evidence for DPrP* or such changes lie outside the pathway of DPrPSc(E200K) formation remains to be determined. Initiation of sporadic disease may follow from a somatic mutation and thus follow a path similar to that for germline mutations in inherited disease. In this situation, the mutant PrPSc must be capable of co-opting wtPrPC, a process known to be possible for some mutations (e.g., E200K, D178N) but less likely for others (e.g., P102L) (Telling et al., 1995, 1996). Alternatively, the activation barrier separating wtPrP C from PrPSc could be

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crossed on rare occasions when viewed in the context of a population. Most individuals would be spared while presentations in the elderly with an incidence of z1 per million would be seen. Mechanism of Prion Propagation? From the foregoing formalism, we can ask “What is the rate-limiting step in prion formation?” First, we must consider the impact of the concentration of PrPSc in the inoculum, which is inversely proportional to the length of the incubation time. Second, we must consider the sequence of PrPSc that forms an interface with PrPC. When the sequences of the two isoforms are identical, the shortest incubation times are observed. Third, we must consider the strain-specific conformation of PrPSc. Some prion strains exhibit longer incubation times than others; the mechanism underlying this phenomenon is not understood. From these considerations, there exists a set of conditions under which initial PrPSc concentrations can be rate-limiting. These effects presumably relate to the stability of the PrPSc, its targeting to the correct cells and subcellular compartments, and its ability to be cleared. Once infection in a cell is initiated and endogenous PrPSc production is operative, then the following discussion of PrPSc formation seems most applicable. If the assembly of PrPSc into a specific dimeric or multimeric arrangement were difficult, then a nucleation–polymerization (NP) formalism would be relevant. In NP processes, nucleation is the rate-limiting step and elongation or polymerization is facile. These conditions are frequently observed in peptide models of aggregation phenomena (Caughey et al., 1995); however, studies with Tg mice expressing foreign PrP genes suggest that a different process is occurring. From investigations with mice expressing both the SHaPrP transgene and the endogenous MoPrP gene, it is clear that PrPSc provides a template for directing prion replication where we define a template as a catalyst that leaves its imprint on the product of the reaction (Prusiner et al., 1990). Inoculation of these mice with SHaPrPSc leads to the production of nascent SHaPrPSc and not MoPrPSc. Conversely, inoculation of the Tg(SHaPrP) mice with MoPrPSc results in MoPrPSc formation and not SHaPrPSc. Even stronger evidence for templating has emerged from studies of prion strains passaged in Tg(MHu2M)Prnp0/0 mice expressing a chimeric Hu/MoPrP gene as described in more detail below (Telling et al., 1996; Prusiner, 1997). Even though the conformational templates were initially generated with PrPSc molecules having different sequences in patients with inherited prion diseases, these templates are sufficient to direct replication of distinct PrPSc molecules when the amino acid sequences of the substrate PrPs are identical. If the formation of this template were rate-limiting, then an NP model could apply. However, studies of PrPSc formation in ScN2a cells point to a distinct rate-limiting step. Cell biologic and transgenetic investigations argue for the existence of a chaperone-like molecule, referred to as protein X, that is required for PrPSc formation (Telling et al., 1995). As described below, mutagenesis experiments have created dominant negative forms of DPrPC that inhibit the formation of wtPrPSc by binding protein X (Kaneko et al., 1997b). This implies that the rate-limiting step in vivo in prion replication under conditions where

Table 2. Evidence for Protein X from Transmission Studies of Human Prionsa Inoculum

Host

MoPrP Gene

Incubation Time [days 6 SEM] (n/no)

sCJD sCJD sCJD sCJD

Tg(HuPrP) Tg(HuPrP)Prnp0/0 Tg(MHu2M) Tg(MHu2M)Prnp0/0

Prnp1/1 Prnp0/0 Prnp1/1 Prnp0/0

721 (1/10) 263 6 2 (6/6) 238 6 3 (8/8) 191 6 3 (10/10)

a

Data with inoculum RG from Telling et al., 1995.

PrPSc is sufficient must be the conversion of PrPC to PrP* since a dominant negative derived from a single point mutation could gate only a kinetically critical step in a cellular process. In the template-directed model, the conversion of PrPC to PrP* is a first order process. By contrast, NP processes follow higher order kinetics ([monomer]m, where m is the number of monomers in the nucleus). The experimental implications of these rate relationships are apparent in transgenic studies; if first order kinetics operate, halving the gene dose (hemizygotes) should double the incubation time while doubling the dose of a transgene array should halve the time to disease. This quantitative behavior has been observed in several studies in mice with altered levels of PrP expression (Prusiner et al., 1990, 1993; Bu¨eler et al., 1994; Carlson et al., 1994). The existence of prion strains that are conformational isoforms of PrPSc with distinct structures, incubation times, and neurohistopathology must also be considered in an analysis of the kinetics of PrPSc accumulation. Since the rate-limiting step in PrPSc formation cannot involve the unique template provided by a strain, differential rates of intercellular spread, cellular uptake, and clearance seem most likely to account for the variation in incubation times. This is consistent with the different patterns of protease sensitivity and glycosylation for distinct prion strains (Bessen and Marsh, 1994; Collinge et al., 1996; Telling et al., 1996; Somerville et al., 1997). However, we hasten to add that NP models can provide a useful description of other biologic phenomena. Under conditions when the monomer is relatively rare and/or the conformational change is facile (e.g., short peptides), the NP model will dominate. However, when the monomer is sufficiently abundant and/or the conformational conversion is difficult to accomplish, the template assistance formalism provides a more likely description of the process. Evidence for Protein X Protein X was postulated to explain the results on the transmission of human (Hu) prions to Tg mice (Table 2) (Telling et al., 1994, 1995). Mice expressing both Mo and HuPrP were resistant to Hu prions while those expressing only HuPrP were susceptible. These results argue that MoPrPC inhibited transmission of Hu prions, i.e., the formation of nascent HuPrP Sc. In contrast to the foregoing studies, mice expressing both MoPrP and chimeric MHu2M PrP were susceptible to Hu prions and mice expressing MHu2MPrP alone were only slightly more susceptible. These findings contend that MoPrPC has only a minimal effect on the formation of chimeric MHu2MPrPSc.

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When the data on Hu prion transmission to Tg mice were considered together, they suggested that MoPrPC prevented the conversion of HuPrPC into PrPSc by binding to another Mo protein but had little effect on the conversion of MHu2M into PrPSc. We interpreted these results in terms of MoPrPC binding to this Mo protein with a higher affinity than does HuPrP C. We postulated that MoPrPC had little effect on the formation of PrPSc from MHu2M (Table 2) because MoPrP and MHu2M share the same amino acid sequence at the COOH terminus. This also suggested that MoPrPC only weakly inhibited transmission of SHa prions to Tg(SHaPrP) mice because SHaPrP is more closely related to MoPrP than is HuPrP. Using scrapie-infected mouse neuroblastoma cells transfected with chimeric Hu/Mo PrP genes, we extended our studies of protein X. Substitution of a Hu residue at position 214 or 218 prevented PrPSc formation (Figure 2B) (Kaneko et al., 1997b). The side chains of these residues protrude from the same surface of the COOH-terminal a helix forming a discontinuous epitope with residues 167 and 171 in an adjacent loop. Substitution of a basic residue at positions 167, 171, or 218 prevented PrPSc formation; these mutant PrPs appear to act as “dominant negatives” by binding protein X and rendering it unavailable for prion propagation. Our findings seem to explain the protective effects of basic polymorphic residues in PrP of humans and sheep (Hunter et al., 1993; Westaway et al., 1994; Shibuya et al., 1998). Is Protein X a Molecular Chaperone? Since PrP undergoes a profound structural transition during prion propagation, it seems likely that other proteins such as chaperones participate in this process. Whether protein X functions as a classical molecular chaperone or participates in PrP binding as part of its normal function but can also facilitate pathogenic aspects of PrP biology is unknown. Interestingly, scrapieinfected cells in culture display marked differences in the induction of heat-shock proteins (Tatzelt et al., 1995), and Hsp70 mRNA has been reported to increase in scrapie of mice (Kenward et al., 1994). While attempts to isolate specific proteins that bind to PrP have been disappointing (Oesch et al., 1990), PrP has been shown to interact with Bcl-2, Hsp60, and the laminin receptor protein by two-hybrid analysis in yeast (Kurschner and Morgan, 1996; Rieger et al., 1997). Although these studies are suggestive, no molecular chaperone involved in prion formation in mammalian cells has been identified.

Strains of Prions The existence of prion strains raises the question of how heritable biological information can be enciphered in any molecule other than nucleic acid (Dickinson et al., 1968). Strains or varieties of prions have been defined by incubation times and the distribution of neuronal vacuolation (Dickinson et al., 1968; Fraser and Dickinson, 1968). Subsequently, the patterns of PrPSc deposition were found to correlate with vacuolation profiles, and these patterns were also used to characterize strains of prions (DeArmond et al., 1987, 1997; Bruce et al., 1989). The typing of prion strains in C57Bl, VM, and

F1(C57Bl 3 VM) inbred mice began with isolates from sheep with scrapie. The prototypic strains called Me7 and 22A gave incubation times of z150 and z400 days in C57Bl mice, respectively (Dickinson et al., 1968). The PrPs of C57Bl and I/Ln (and later VM) mice differ at two residues and control incubation times (Carlson et al., 1994; Moore et al., 1998). Until recently, support for the hypothesis that the tertiary structure of PrPSc enciphers strain-specific information (Prusiner, 1991) was minimal except for the DY strain isolated from mink with transmissible encephalopathy (Bessen and Marsh, 1994). PrPSc in DY prions showed diminished resistance to proteinase K digestion as well as an anomalous site of cleavage. The DY strain presented a puzzling anomaly since other prion strains exhibiting similar incubation times did not show this altered susceptibility to proteinase K digestion of PrP Sc (Scott et al., 1997). Also notable was the generation of new strains during passage of prions through animals with different PrP genes (Scott et al., 1997). PrP Sc Conformation Enciphers Variation in Prions Persuasive evidence that strain-specific information is enciphered in the tertiary structure of PrPSc comes from transmission of two different inherited human prion diseases to mice expressing a chimeric MHu2M PrP transgene (Telling et al., 1996). In fatal familial insomnia (FFI), the protease-resistant fragment of PrPSc after deglycosylation has an Mr of 19 kDa; whereas in fCJD(E200K) and most sporadic prion diseases, it is 21 kDa (Monari et al., 1994). This difference in molecular size was shown to be due to different sites of proteolytic cleavage at the NH 2 termini of the two human PrPSc molecules reflecting different tertiary structures (Monari et al., 1994). These distinct conformations were not unexpected since the amino acid sequences of the PrPs differ. Extracts from the brains of FFI patients transmitted disease into mice expressing a chimeric MHu2M PrP gene about 200 days after inoculation and induced formation of the 19 kDa PrPSc ; whereas fCJD(E200K) and sCJD produced the 21 kDa PrPSc in mice expressing the same transgene (Telling et al., 1996). On second passage, Tg(MHu2M) mice inoculated with FFI prions showed an incubation time of z130 days and a 19 kDa PrP Sc while those inoculated with fCJD(E200K) prions exhibited an incubation time of z170 days and a 21 kDa PrPSc (Prusiner, 1997). The experimental data demonstrate that MHu2MPrPSc can exist in two different conformations based on the sizes of the protease-resistant fragments; yet, the amino acid sequence of MHu2MPrPSc is invariant. The results of our studies argue 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 both the generation and propagation of prion strains. Interestingly, the protease-resistant fragment of PrPSc after deglycosylation with an Mr of 19 kDa has been found in a patient who died after developing a clinical disease similar to FFI. Since both PrP alleles encoded the wild-type sequence and a Met at position 129, we

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labeled this case fatal sporadic insomnia (FSI). At autopsy, the spongiform degeneration, reactive astrogliosis, and PrPSc deposition were confined to the thalamus (Mastrianni et al., 1997). These findings argue that the clinicopathologic phenotype is determined by the conformation of PrPSc in accord with the results of the transmission of human prions from patients with FFI to Tg mice (Telling et al., 1996). Mechanism of Selective Neuronal Targeting? In addition to incubation times, neuropathologic profiles of spongiform change have been used to characterize prion strains (Fraser and Dickinson, 1968). However, recent studies with PrP transgenes argue that such profiles are not an intrinsic feature of strains (Carp et al., 1997; DeArmond et al., 1997). The mechanism by which prion strains modify the pattern of spongiform degeneration was perplexing since earlier investigations had shown that PrPSc deposition precedes neuronal vacuolation and reactive gliosis (DeArmond et al., 1987). When FFI prions were inoculated into Tg(MHu2M) mice, PrPSc was confined largely to the thalamus (Figure 4A) as is the case for FFI in humans (Telling et al., 1996). In contrast, fCJD(E200K) prions inoculated into Tg(MHu2M) mice produced widespread deposition of PrPSc throughout the cortical mantel and many of the deep structures of the CNS (Figure 4B) as is seen in fCJD(E200K) of humans. To examine whether the diverse patterns of PrPSc deposition are influenced by Asn-linked glycosylation of PrPC, we constructed Tg mice expressing PrPs mutated at one or both of the Asn-linked glycosylation consensus sites (DeArmond et al., 1997). These mutations resulted in aberrant neuroanatomic topologies of PrPC within the CNS, whereas pathologic point mutations adjacent to the consensus sites did not alter the distribution of PrPC. Tg mice with mutation of the second PrP glycosylation site exhibited prion incubation times of .500 days and unusual patterns of PrPSc deposition. These findings raise the possibility that glycosylation can modify the conformation of PrP and affect either the turnover of PrPC or the clearance of PrPSc. Regional differences in the rate of deposition or clearance would result in specific patterns of PrPSc accumulation. Yeast and Other Prions Although prions were originally defined in the context of an infectious mammalian pathogen (Prusiner, 1982), it is now becoming widely accepted that prions are elements that impart and propagate variability through multiple conformers of a normal cellular protein. Such a mechanism must surely not be restricted to a single class of transmissible pathogens. Indeed, it is likely that the original definition will need to be extended to encompass other situations where a similar mechanism of information transfer occurs. Two notable prion-like determinants, [URE3] and [PSI], have already been described in yeast and one in another fungus denoted [Het-s* ] (Wickner, 1994; Chernoff et al., 1995; Coustou et al., 1997). Studies of candidate prion proteins in yeast may prove particularly helpful in the dissection of some of the events that feature in PrPSc formation. Interestingly, different strains of yeast prions have been identified (Derkatch et al., 1996). Conversion to the prion-like [PSI] state in yeast requires the

Figure 4. Regional Distribution of PrP Sc Deposition in Tg(MHu2M)Prnp0/0 Mice Inoculated with Prions from Humans Who Died of Inherited Prion Diseases Histoblot of PrP Sc deposition in a coronal section of a Tg(MHu2M)Prnp0/0 mouse through the hippocampus and thalamus (Telling et al., 1996). (A) The Tg mouse was inoculated with brain extract prepared from a patient who died of FFI. (B) The Tg mouse was inoculated with extract from a patient with fCJD(E200K). Cryostat sections were mounted on nitrocellulose and treated with proteinase K to eliminate PrP C (Taraboulos et al., 1992). To enhance the antigenicity of PrPSc , the histoblots were exposed to 3 M guanidinium isothiocyanate before immunostaining using a-PrP 3F4 MAb (Kascsak et al., 1987). (C) Labeled diagram of a coronal section of the hippocampus/thalamus region. NC, neocortex; Hp, hippocampus; Hb, habenula; Th, thalamus; vpl, ventral posterior lateral thalamic nucleus; Hy, hypothalamus; Am, amygdala.

molecular chaperone Hsp104; however, no homolog of Hsp104 has been found in mammals (Chernoff et al., 1995). The NH2-terminal prion domains of Ure2p and Sup35 that are responsible for the [URE3] and [PSI] phenotypes in yeast have been identified. In contrast to PrP, which is a GPI-anchored membrane protein, both

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Ure2p and Sup35 are cytosolic proteins (Wickner, 1997). When the prion domains of these yeast proteins were expressed in E. coli, the proteins were found to polymerize into fibrils with properties similar to those of proteolytically trimmed PrP and other amyloids (Paushkin et al., 1997). Whether prions explain some other examples of acquired inheritance in lower organisms is unclear (Landman, 1991). For example, studies on the inheritance of positional order and cellular handedness on the surface of small organisms have demonstrated the epigenetic nature of these phenomena but the mechanism remains unclear (Frankel, 1990).

Therapeutic Approaches to Prion Diseases It seems likely that it will be possible to design effective therapeutics for prion diseases as our understanding of prion propagation increases. Because people at risk for inherited prion diseases can now be identified decades before neurologic dysfunction is evident, the development of an effective therapy for these fully penetrant disorders is imperative. Although we have no way of predicting the number of individuals who may develop neurologic dysfunction from bovine prions in the future, seeking an effective therapy now seems most prudent (Prusiner, 1997). Interfering with the conversion of PrPC into PrPSc seems to be the most attractive therapeutic target. Reasonable strategies are either stabilizing the structure of PrPC via the formation of a PrPC-drug complex or modifying the action of protein X, which may function as a molecular chaperone (Figure 2B). Whether it is more efficacious to design a drug that binds to PrPC at the protein X–binding site or one that mimics the structure of PrPC with basic polymorphic residues that seem to prevent scrapie and CJD remains to be determined (Kaneko et al., 1997b; Shibuya et al., 1998). Since PrPSc formation seems limited to caveolae-like domains (Gorodinsky and Harris, 1995; Taraboulos et al., 1995), drugs designed to inhibit this process need not penetrate the cytosol of cells but they do need to 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 R/R polymorphism at position 171 seem to be resistant to scrapie (Hunter et al., 1993; Westaway et al., 1994); presumably, this was the genetic basis of James Parry’s scrapie eradication program in Great Britain 30 years ago (Parry, 1962). A more effective approach using dominant negatives for producing prion-resistant domestic animals, including sheep and cattle, is probably the expression of PrP transgenes encoding R171 as well as additional basic residues at the protein X–binding site (Figure 2B) (Kaneko et al., 1997b). Such an approach can be readily evaluated in Tg mice, and once shown to be effective, it could be instituted by artificial insemination of sperm from males homozygous for the transgene. Less practical is the production of PrP-deficient cattle and sheep. Although such animals would not be susceptible to prion disease (Bu¨eler et al., 1993; Prusiner et al., 1993), they might suffer some deleterious effects from ablation of

the PrP gene (Collinge et al., 1994; Lledo et al., 1996; Sakaguchi et al., 1996; Tobler et al., 1996). Whether gene therapy for the human prion diseases will prove feasible using the dominant negative approach described above for prion-resistant animals depends on the availability of efficient vectors for delivery of the transgene to the CNS. Concluding Remarks Although the study of prions has taken several unexpected directions over the past three decades, a novel and fascinating story of prion biology is emerging. Investigations of prions have elucidated a previously unknown mechanism of disease in humans and animals. While learning the details of the structures of PrPs and deciphering the mechanism of PrPC transformation into PrPSc will be important, the fundamental principles of prion biology have become reasonably clear. Though some investigators prefer to view the composition of the infectious prion particle as unresolved (Manuelidis and Fritch, 1996; Lasme´zas et al., 1997; Chesebro, 1998), such a perspective denies an enlarging body of data, none of which refutes the prion concept. Moreover, the discovery of prion-like phenomena mediated by proteins unrelated to PrP in yeast and other fungi serves not only to strengthen the prion concept but also to widen it. The discovery that prion diseases in humans are uniquely both genetic and infectious greatly strengthened and extended the prion concept. To date, 20 different mutations in the human PrP gene, all resulting in nonconservative substitutions, have been found either to be linked genetically to or to segregate with the inherited prion diseases (Figure 1B). Yet, the transmissible prion particle is composed largely, if not exclusively, of an abnormal isoform of the prion protein designated PrPSc (Prusiner, 1991). Aberrant PrP Metabolism The hallmark of all prion diseases—whether sporadic, dominantly inherited, or acquired by infection—is that they involve the aberrant metabolism and resulting accumulation of the prion protein (Table 1) (Prusiner, 1991). The conversion of PrPC into PrPSc involves a conformation change whereby the a-helical content diminishes and the amount of b sheet increases (Pan et al., 1993). These findings provide a reasonable mechanism to explain the conundrum presented by the three different manifestations of prion disease. Understanding how PrPC unfolds and refolds into PrPSc will be of paramount importance in transferring advances in the prion diseases to studies of other degenerative illnesses. The mechanism by which PrPSc is formed must involve a templating process whereby existing PrPSc directs the refolding of PrPC into a nascent PrPSc with the same conformation. Not only will a knowledge of PrPSc formation help in the rational design of drugs that interrupt the pathogenesis of prion diseases, but it may also open new approaches to deciphering the causes of and to developing effective therapies for the more common neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis (ALS). Indeed, the expanding list of prion diseases and their novel modes of

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transmission and pathogenesis (Table 1), as well as the unprecedented mechanisms of prion propagation and information transfer, indicate that much more attention to these fatal disorders of protein conformation is urgently needed. But prions may have even wider implications than those noted for the common neurodegenerative diseases. If we think of prion diseases as disorders of protein conformation and do not require the diseases to be transmissible, then what we have learned from the study of prions may reach far beyond these common illnesses. Conformational Diversity The discovery that proteins may have multiple biologically active conformations may prove no less important than the implications of prions for diseases. How many different tertiary structures can PrPSc adopt? This query not only addresses the issue of the limits of prion diversity but also applies to proteins as they normally function within the cell or act to affect homeostasis in multicellular organisms. The expanding list of chaperones that assist in the folding and unfolding of proteins promises much new knowledge about this process. For example, it is now clear that proproteases can carry their own chaperone activity where the pro portion of the protein functions as a chaperone in cis to guide the folding of the proteolytically active portion before it is cleaved (Shinde et al., 1997). Such a mechanism might well feature in the maturation of polypeptide hormones. Interestingly, mutation of the chaperone portion of prosubtilisin resulted in the folding of a subtilisin protease with different properties than the one folded by the wild-type chaperone. Such chaperones have also been shown to work in trans (Shinde et al., 1997). Besides transient metabolic regulation within the cell and hormonal regulation of multicellular organisms, it is not unreasonable to suggest that assembly of proteins into multimeric structures such as intermediate filaments might be controlled at least in part by alternative conformations of proteins. Such regulation of multimeric protein assemblies might occur in either the proteins that form the multimers or the proteins that function to facilitate the assembly process. Additionally, apoptosis during development and throughout adult life might also be regulated at least in part by alternative tertiary structures of proteins. Future Studies The wealth of data establishing the essential role of PrP in the transmission of prions and the pathogenesis of prion diseases has provoked consideration of how many biological processes are controlled by changes in protein conformation. The extreme radiation-resistance of the scrapie infectivity suggested that the pathogen causing this disease and related illnesses would be different from viruses, viroids, and bacteria (Alper et al., 1967). Indeed, an unprecedented mechanism of disease has been revealed where an aberrant conformational change in a protein is propagated. The future of this emerging area of biology should prove even more interesting and productive as many new discoveries emerge.

by grants from the National Institute of Aging and the National Institute of Neurologic Diseases and Stroke of the National Institutes of Health, International Human Frontiers of Science Program, and American Health Assistance Foundation, as well as by gifts from the Sherman Fairchild Foundation, Keck Foundation, G. Harold and Leila Y. Mathers Foundation, Bernard Osher Foundation, John D. French Foundation, and Centeon.

Acknowledgments

Chesebro, B. (1998). Prion diseases: BSE and prions: uncertainties about the agent. Science 279, 42–43.

We thank G. Carlson, N. Nathanson, and J. Safar for carefully reviewing sections of this manuscript. This research was supported

Chesebro, B., and Caughey, B. (1993). Scrapie agent replication without the prion protein? Curr. Biol. 3, 696–698.

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