- Email: [email protected]
Unraveling prion diseases through molecular genetics
21 Brown, D. A. and Adams, P. R. (1980) Nature 283,673-676 22 Pennefather, P., Lancaster, B., Adams, P. R. and Nicoll, R. A. (1985) Proc. Natl Acad. Sci. USA 82, 3040-3044 23 Madison, D. V. and Nicoll, R. A. (1984) J. Physiol. (London) 354, 319-331 24 Siggins, G. R. and Gruol, D. L. (1986) in Handbook of
Physiology (Sect. 1: The Nervous System; Pt IV Intrinsic Regulatory Systems of the Brain), pp, 1-114, Williams & Wilkins and the American Physiological Society 25 Armstrong-James, M. and Fox, K. (1983)J. PhysioL (London) 335, 427--447 26 Sillito, A. M. and Kemp, J. A. (1983) Brain Res. 289, 143-155 27 Lamour, Y., Dutar, P., Jobert, A. and Dykes, R. W. (1988) J. Neurophysiol. 60, 725-750 28 Waterhouse, B. D. et al. (1988) Brain Res. Bull. 21,425-432 29 De Lima, A. D. and Singer, W. (1987) J. Comp. Neurol. 259, 92-121 30 De Lima, A. D., Montero, V. M. and Singer, W. (1985) Exp. Brain Res. 59, 206-212 31 Steriade, M. and Llin,~s, R. R. (1988) Physiol. Rev. 68, 649-742 32 McCormick, D. A. and Prince, D. A. (1987) J. PhysioL (London) 392, 147-165 33 McCormick, D. A. and Prince, D. A. (1986) Nature 319, 402--405 34 McCormick, D. A. and Pape, H-C. (1988) Nature 334, 246-248
35 Kayama, Y. (1985) Vision Res. 25, 339-347 36 Rogawski, M. and Aghajanian, G. K. (1980) Nature 287, 731-734 37 Sillito, A. M., Kemp, J, A. and Berardi, N. (1983) Brain Res. 280, 299-307 38 Francesconi, W., Muller, C. M. and Singer, W. (1988) J. Neurophysiol. 59, 1690-1718 39 Jahnsen, H. and Llin&s, R. R. (1984)J. Physiol. (London)349, 205-247 40 Feeser, H. R. and McCormick, D. A. (1988) Soc. Neurosci. Abstr. 14, 277 41 Livingstone, M. S. and Hubel, D. H. (1981) Nature 291, 554-561 42 Sherman, S. M. and Koch, C. (1986) Exp. Brain Res. 63, 1-20 43 Singer, W. (1977) Physiol. Rev. 57, 386-420 44 Ahlsen, G., Lindstrom, S. and Lo, F. S. (1984) J. Physiol. (London) 347, 593-609 45 Eysel, U. T., Pape, H-C. and Van Schayck, R. (1986) J. Physiol. (London) 370, 233-254 46 Steriade, M., Domich, L., Oakson, G. and Deschenes, M. (1987) J. Neurophysiol. 57, 260-273 47 Steriade, M. and Deschenes, M. (1988) in Cellular Thalamic Mechanisms (Bentivoglio, M. and Spreafico, R., eds), pp. 37-62, Elsevier 48 Hirsch, J. C., Fourment, A. and Marc, M. E. (1983) Brain Res. 259, 308--312
Acknowledgements I thank Mircea Steriade, Martin Desch#nes,June Hirsch and their colleagues for permission to reprint portions of their data in this article. I am indebted to David Princeand HansChristian Papefor their collaboration on portions of these projects. Supported by JacobsJavits Centerin Neuroscience, NINCDS, and a fellowship from the Klingenstien Foundation.
Unravelingprion diseasesthrough moleculargenetics David Westaway,
G e o r g e A. C a r l s o n a n d S t a n l e y B. P r u s i n e r
Prions are transmissible pathogens that cause degenerative diseases in humans and animals. Unique attributes of prion diseases include infectious, sporadic and genetic manifestations, as well as progression to death, all in the absence of a detectable immune response. Prions are resistant to chemical procedures that modify or destroy nucleic acids and are composed largely of a protein, designated PrP so. Molecular cloning of a co~,nate cDNA established a cellular host origin for PrP °c protein and a convergence with the genetics of host susceptibility. The murine PrP gene is linked to the Prn-i gene which determines incubation times in experimental scrapie. Mice with long incubation times have unusual PrP alleles encoding phenylalanine and valine at codons 108 and 189. Moreover, the ataxic form of Gerstmann-Strdussler syndrome (a rare human neurodegenerative disorder) has been defined as an autosomal dominant disorder with a PrP mis-sense mutation at codon 102 linked to the predisposition locus. These studies argue that amino acid substitutions in 'PrP' genes may modulate initiation and development of prion diseases.
scrapie infectivity was filterable, the aetiological agent was highly resistant to a variety of procedures that destroy or modify nucleic acids 1. Instead, infectivity was associated with a proteinaceous component, subsequently identified as the scrapie prion protein, PrP so. Prion diseases are distinguished also from conventional infectious disease by their unusual biological features. Scrapie, CJD and GSS proceed in the apparent absence of a detectable immune response 2. When the familial versions of the human priori diseases are excluded, these diseases occur in isolation ('sporadically') or iatrogenically. Given the experimental transmissibility of the diseases, it was reasonable to attribute sporadic disease to cryptic vertical or lateral spread from an affected individual or from an animal reservoir. However, epidemiologists have failed to identify such vectorial 3 spread and studies of maternal transmission of kuru and CJD prions were similarly negative 4'5. Tripartite manifestation - infectious, familial and sporadic - may be an intrinsic feature of all prion diseases in nature. Molecular genetic studies summarized here reveal that PrP sc molecules are encoded by the host, and Seven prion diseases of animals and humans are now that this genetic origin may contribute to the unique recognized. All are degenerative neurological dis- biological attributes of prions. orders that can be transmitted by inoculation. The pathological features of prion diseases include neur- Prions contain PrP sc PrP27-30, the protease-resistant core of PrP sc, onal vacuolafion, astrocytic gliosis and deposition of amyloid plaques. In animals, prions are the cause of was discovered by enriching brain fractions for scrapie scrapie of sheep and goats, of transmissible mink infectivity6'7. Development of a more rapid and encephalopathy, of chronic wasting disease of mule economical bioassay8 greatly facilitated purification of deer and elk and of bovine spongiform encephal- the hamster scrapie agent 7. PrP27-30 migrates opathy. In humans, prions cause kuru, Creutzfeldt- during SDS-PAGE as a broad band with an apparent Jakob disease (CJD) and Gerstmann-Str~iussler syn- molecular weight of 27 000-30 000. drome (GSS). Correspondence should be addressed to David Westaway: The unusual physiochemical nature of the prions Department of Neurology, HSE-781, University of Cafifornia, San was apparent more than two decades ago. Although Francisco, CA 94143-0518, USA. TINS, Vol. 12, No. 6, 1989
© 1989.ElsevierSciencePublishersLtd,(UK) 0166-2236/89/$02.00
David Westawayand Stanley B. Prusinerare at the Departments of Neurology, Biochemistryand Biophysics, University of California, San Francisco, CA 94143, USA and GeorgeA. Carlsonis at the McLaughlin Research Institute, 6reat Falls, MT 5940f, USA.
221
EXON I
INTRON
EXON II
(10 An amino acid substitution in the human PrP gene is genetically linked to the development of GSS. The last two lines of investigation listed above describe rather striking genetic findings which implicate PrP sc as an intrinsic component of the prion. The results of these molecular genetic studies are disG:C cussed in some detail in later sections. Many investigators have confirmed the presence of mRNA PrP 27-30 in brains infected with the scrapie or CJD agent21'27-29. Although the amino acid sequence of PrP has been confirmed and there is agreement that PrP is glycosylated29'3°, some investigators have suggested that PrP 27-30 may not be a component of the scrapie agent31'32. One argument revolves around . the inability of some investigators to detect either PrP mRNA or PrP sc in spleens of scrapie-infected rodents33'34; however, others have clearly shown that both PrP message and PrP sc are present in spleen Fig. 1. Organization and expression of the hamster PrP gene. The features tissue 13'35'36. Indeed, recent studies have shown an presented were deduced from the nucleotide sequences of PrP genomic and excellent correlation between the concentration of cDNA clones. Untranslated regions of the mRNA are indicated by hatched PrP mRNA in scrapie-infected rodent tissues and boxes. An open reading frame or protein coding region is indicated by the prion titer37. Another argument centers on a loss of open box. The diagonal fines show a spficing event that joins the 5' leader sequences to the remainder of the coding sequences. (Taken, with permission, infectivity of CJD agents bound to a lectin column. Neither denaturation of the PrP CJD nor neutralizfrom Ref. 43.) ation of CJD infectivity by immobilization on the lectin column matrix was considered as an explanation 29. Many lines of investigation have converged to Denaturation of PrP sc has been demonstrated to be establish that the protein PrP 27-30 is a component of accompanied by a loss of scrapie infectivity9'1°. To the infectious prion particle. date, no experimental studies have been reported (1) PrP27-30 and the scrapie agent co-purify 7'9'10 where fractions with high levels of scrapie infectivity using detergent extractions and limited proteolysis to are found to contain less than one PrP sc (or PrP promote aggregation of prions into amyloid rods which 27-30) molecule per infectious unit. are collected by centrifugation. PrP 27-30 is the most abundant macromolecule in purified preparations TM. Prion protein genes and isoforms The genetic provenance of PrP sc was resolved by (2) The PrP 27-30 concentration is proportional to the prion titer 11. PrP 27-30, or its precursor PrP sc, is retrieving a PrP clone from a scrapie-infected brain absent in normal, uninfected animals 12'13. cDNA library 13. The molecular clone failed to (3) Procedures that denature, hydrolyse, or selec- hybridize with proteinase-digested/phenol-extracted tively modify PrP 27-30 also diminish the prion titer 11. samples of scrapie prions but hybridized instead to The unusual kinetics of PrP 27-30 hydrolysis cata- genomic DNA and cellular RNA. These results lysed by proteases has been shown to correlate with indicated that a host mRNA, rather than a packaged nucleic acid, encode PrP sc. There was no significant the diminution of the scrapie prion titer. (4) PrP 27-30 and scrapie infectivity partition quantitative difference between a 2.1 kb PrP mRNA together into many different forms: membranes; detected at different times after infection, or between rods; spheres; detergent-lipid-protein complexes infected and control animals, consistent with the (DLPC); and liposomes. These dramatically different failure to observe PrP cDNAs in experiments using physical forms all contain PrP 27-30 and high prion 'subtractive' hybridization techniques3&39. Messenger titers 7,9.lo, 14-17. RNAs for PrP in uninfected animals suggested the (5) Scrapie and CJD prion proteins have been iden- presence of a corresponding translation product. This tiffed only in tissues of animals and humans with molecule has a Mr of 33 000-35 000 and is designated transmissible neurodegenerative diseases and not in PrP c (C for cellular). A similar analysis of infected those with other neurological disorders 7'lo, 18-23. brain proteins reveals the related, but more abundant, (6) Cultured murine neuroblastoma cells have been molecule PrP sc (Sc for scrapie), which, when treated infected with both scrapie and CJD prions 24. Clones of with proteinase K in vitro yields PrP 27-30 (Ref. 13). the scrapie-infected cells were found to produce Both PrP c and PrP sc are glycoproteins that possess PrP so, whereas clones showing no infectivity lacked asparagine (Asn)-linked oligosaccharides and glycosyl prp sc. phosphatidylinositol (GPI) anchors4°. Whether the (7) Rabbit antisera raised against PrP 27-30 purified features that distinguish PrP sc from PrP c arise from by SDS-PAGE were found to neutralize scrapie differences in their Asn-linked oligosaccharides 41 or infectivity in DLPC (Ref. 25). GPI anchors is unknown. (8) Co-purification of scrapie prion infectivity and The organization and structure of the hamster Prp PrP sc was found in fractions eluted from a column of gene have been elucidated; the entire open reading monoclonal antibody to PrP (Refs 25, 26). frame (ORF) or protein-coding region is contained (9) The PrP gene of mice (Prn-p) is linked to a gene within a single exon42 (Fig. 1). The 5' end of the PrP controlling scrapie incubation times (Prn-i). The PrP gene contains multiple transcription initiation sites gene of long incubation period mice encodes a variant which map within a G + C-rich promoter region. An prion protein. intron of - 1 0 kb separates exons I and II. Six lines of
repeats
I
@
222
1 kb
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evidence argue that the two PrP isoforms have the same amino acid sequence. (1) No evidence for rearrangement of the PrP gene in scrapie has been found 13,42. (2) The organization of the PrP gene provides no possibility for alternative splicing within the ORF 42. (3) Only one PrP mRNA of 2.1 kb has been detected
and its concentration does not change throughout the course of scrapie infection in hamster brain la. (4) 70% of the PrP sc and 87% of the PrP 27-30 have been sequenced by gas-phase protein sequencing (Teplow, D. et al., unpublished observations). These sequences correspond precisely with the translated genomic DNA sequence. However, currently, we do
Fig. 2. Comparison of ORFs. Comparison of predicted mammalian prion protein amino acid sequences 4 2 .4.4. .4 5 4 9 51 . Gaps are indicated by dashes. Variant amino acids are indicated above or below the NZW mouse sequence, arbitrarily chosen as a prototype. Sequences presumed missing from a partial rat PrP cDNA are shown by question marks. The ORF predicted from the human PrP cDNA described by Liao et al. 53 differs from a consensus established by 11 independent cDNA and genomic molecular clones, indicating that this clone may contain aberrant 5" sequences (an attendant error in the numbering of the codons in this done is perpetuated in the rat PrP sequence 51. Other human PrP sequences differ in the 5' untranslated region (Ref. 49; Hsiao, K., unpublished observations). Whether these different 5' sequences reflect polymorphisms, spficing variants, or features of the PrP mRNA that interfere with the fidelity of cDNA synthesis remains to be determined a2. Abbreviation: Syr, Syrian &olden hamster.
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223
PRION GENE COMPLEX (Prn)
A Pm-i
Prn-p
Chr 2
B Prnoi
Chr 2 Prn -p Fig. 3. Two models for the prion gene complex. Genes are indicated by open boxes. Abbreviation: Chr 2, chromosome 2.
not know whether all, or only a fraction of the PrP 27-30 molecules within our preparations are associated with infectivity. (5) Both scrapie hamster brain and mouse PrP cDNAs have translated ORF sequences that are identical with the corresponding translated genomic sequences 13,42,44,45. (6) Both PrP isoforms have the same amino-terminal amino acid sequences, as determined by N-terminal sequencing 46. Presumably, the difference in the properties of the two prion proteins is due to a posttranslational event 42. Nevertheless, we cannot exclude the findings that most PrP mRNA molecules encode PrP sc, while a few with a different ORF encode PrP sc. A hypothetical mis-sense codon could arise via two mechanisms, somatic mutation or RNA 'editing' (Ref. 47). The human and mouse PrP genes have been shown to be located on chromosomes 20 and 2, respectively, which are homologous48. This finding indicates that PrP genes existed prior to the speciafion of mammals. PrP genes from humans 49, Syrian hamsters 42, Chinese hamsters 5°, Armenian hamsters 5°, mice 44'45, rats 51 and sheep 52 have been sequenced (Fig. 2). Presumably, all mammals have PrP genes and all are capable of developing prion diseases 54. Whether eukaryotes other than mammals have authentic PrP genes remains to be established. Nucleotide sequences of all the PrP ORFs predict prion proteins of approximately 250 amino acids. All encode N-terminal signal peptides, consensus sites for Asn-linked glycosylafion, as well as two cysteines within the carboxy-terminal half of the molecule. All of the ORFs also possess a series of glycine and prolinerich repeats in the N-terminal portion of the PrP molecule. PrP sc and PrP c are modified by the addition of a phosphatidylinositol glycolipid anchor 4°. Divergence in the PrP carboxyl terminus can be reconciled with the notion that glycolipidation is not dependent on a primary structure motiP 5. A signal pepfide of 22 amino acids is cleaved during the biosynthesis of PrP c and PrP sc (Refs 29, 46, 56). A putative C-terminal cleavage site has been positioned 23 amino acids from the C-terminus of the full-length molecule57, in fair agreement with a C-terminal truncation of 19 residues deduced from amino acid analyses 13. When these cleaved N- and C-terminal peptide domains are excluded from sequence comparisons, PrP molecules are 96.7% identical in humans, hamsters, rats and 224
mice, implying that PrP c performs a crucial cellular function. Database searches have been unenlightening, with no similarities to proteins of known function detected to date. Location of glycolipidated PrP c on the external face of the cell membrane perhaps suggests a role in signal transduction or cellular communication. The Asn-linked oligosaccharides of PrP so, and presumably PrP c, contain the x-antigenic determinant [Gal[51-->4 (Fucod--->3) GIcNAc] associated with cell surface molecules involved in recognition (Endo, T., Groth, D. F., Prusiner, S. B. and Kobata, A., unpublished observations). The location of PrP c may form the molecular basis for the immunobiology (or lack thereof) of prion diseases. Expression of extracellularly oriented PrP c in several tissues would result in immune recognition as 'self and this tolerance could also encompass the diseaseassociated PrP sc isoform.
PrP mRNA Both in hamsters and in mice, the highest PrP mRNA levels are found in brain. In situ hybridization of normal and scrapie-infected hamster brains has shown that neurons contain the highest levels of PrP mRNA ( - 5 0 copies per cell); glial cells contain less than three mRNA copies per cell49. In hamsters, PrP mRNAs are detected in a number of peripheral organs, including spleen, heart and hmg 13. While PrP mRNA levels are constant in adult rodents 13, the expression of the PrP gene is regulated during development 58. During the first 20 days after birth, both PrP and [5-amyloid precursor protein ([5APP is the precursor molecule for the amyloid protein of Alzheimer's disease 59'6°) mRNAs increase in the neonatal hamster brain at different rates in various regions of the brain. In the septum, the kinetics of the increase in PrP and [5-APP mRNAs paralleled the kinetics of choline acetyltransferase. The steady-state level of both PrP and [5-APP mRNAs as well as the synthesis of choline acetyltransferase are stimulated by intraventricular injections of nerve growth factor 57. Prion gene complex The time that elapses from inoculation of an animal to death is a repeatable objective observation. The genetic control of time from inoculation to death is a crucial avenue for research, as manipulation of the host loci which control these times would provide an important approach for therapy; for example, if inoculation-to-death times can be altered to exceed the normal lifespan of the organism. Two genes, Pidl and Prn-i, that influence the time course of prion infections in mice have been assigned to chromosomes 17 and 2, respectively48. Pidl is located within the D subregion of the H-2 complex61. Of greater influence than Pidl in experimental scrapie is the Prn-i gene, which has been shown to be linked to the gene encoding the prion protein (Prn-p). The dominant allele of Prn-i increases the time from inoculation to death 62. Prn-i and Prn-p form the prion gene complex (Prn) (Fig. 3). Whether the Prn-i and Prn-p genes are separate, but linked genes or are identical remains to be determined. Dickinson and colleagues63'64, using inbred strains of mice and 'strains' of scrapie agent, defined an autosomal locus in mice that they labeled Sinc. Studies in congenic mice suggest that Sinc is linked to Prn-p, raising the TINS, Vol. 12, No. 6, 1989
possibility that Prn-i and Sinc are the same genetic locus 65-67. The most compelling genetic evidence for a central role of PrP in the pathogenesis of scrapie comes from molecular cloning showing that inbred mice in which scrapie infections have short (NZW strain) and long (I/Ln strain) time courses have distinct prion protein gene alleles 45. A comparison of the PrP sequences of NZW (Prn-p ~) and I/Ln (Prn-p b) mice shows that the amino acid at codon 108 is changed from leucine to phenylanaline and that the highly conserved codon at position 189 is changed from threonine to valine (Figs 2, 4). Mice in which scrapie infections have short and intermediate time courses possess a leucine at codon 108 and a threonine at codon 189; all three inbred strains of mice known to have long time courses of infection have variant amino acids at these two codons45'65. Although this argues for congruence of Prn-p and Prn-i, genetic subtleties of Prn-p b mice 65, including a possible 'founder' effect, demand functional studies to establish this point beyond dispute.
PrP alleles and genesis of prion isolates Dickinson, Kimberlin and co-workers have argued that their isolation of numerous isolates of scrapie agent is sufficient evidence for the existence of a hostindependent genome 7°. Many studies, including subtractive hybridization experiments 38'39, have failed to identify a discrete scrapie-specific nucleic acid molecule that is associated with infectivity; however, lack of evidence cannot be used to exclude the possibility that such a molecule exists. Irrespective of the exact relationship between Prn-p and Prn-i, Prn-p a and err/-p b mice encode PrP sc allotypes prpSC-A and prpSc-B, which differ at two amino acid residues. This is an important point as C57 and VM mice have been used to derive some distinguishable scrapie isolates. C57 mice are Prn-p a and VM mice are presumed to be Prn-p b [tie IM mice45. To test whether distinguishable isolates are a transient effect of Prn-p genotype, the Chandler isolate of scrapie prions was serially passaged in NZW, Swiss (also Prn-pa), I/LnJ, and NZW × I/LnJ F1 mice (Carlson, G. A., Westaway, D., DeArmond, S. J., Peterson-Torchia, M. and Prusiner, S. B., unpublished observations). A single passage through I/LnJ mice caused a dramatic shortening of incubation and survival times (and variance thereof) in I/LnJ mice with no further shortening in a second syngeneic passage. Conversely, the inoculum obtained after a single passage in I/LnJ mice delayed the onset of disease and death as well as increasing the variance of onset in NZW mice when compared with inocula passaged through NZW and Swiss mice. The decrease in incubation time in going from I/LnJ to NZW or Swiss mice happened in a single step, as it also appears to do in the case of scrapie isolate 22A (Re£ 71). Two observations implicate the Prn genotype in this phenomenon. Swiss and NZW mice behave similarly; although both are Prn-p ~, they differ at many other loci. Second, in a NZW × I/LnJ F2 cross inoculated with the Chandler scrapie isolate, and segregating for genes in addition to Prn-p, high variance was seen only in mice homozygous for Prn-p b after inoculation with prions containing prpSC-A. These results suggest that the different properties perceived for NZW- and I/LnJderived prion isolates reflect an inbred mouse strain TINS, VoL 12, No. 6, 1989
barrier, defined by the Prn genotype. The species barrier for transmission of scrapie may be, at least in part, analogous to the Prn-pa/Prn-p b allotype barrier. Certainly, all species susceptible to scrapie examined to date encode distinct prion proteins 42'44'5°-52. Prion isolates with true-breeding yet divergent incubation times in the same inbred host are more compelling as bona fide 'strains '64"72-74. Although there are no biochemical or fluctuation test 75 data to equate genesis of these isolates with nucleic acid mutation, this clearly warrants additional scrutiny.
Infectious, sporadic and genetic prion diseases The human prion diseases kuru, GSS and CJD illustrate three manifestations of CNS degeneration disorder: slow infection, sporadic disease and genetic disorder 76'77. That all three diseases can be transmitted to laboratory animals by inoculation is well documented 78-a°. Kuru is thought to have been spread exclusively through a slow infectious mechanism by means of ritualistic cannibalism 4'81. Although a few cases of CJD can be traced to iatrogenic inoculation with CJD-contaminated material or instruments - i.e. injections of human growth hormone 22'82'8a, transplantation of neural tissue, and implantation of cerebral electrodes - the vast majority appear to be sporadic, despite considerable effort to implicate scrapie-infected sheep as an exogenous source a1'84. In hamsters, scrapie infection by intracerebral inoculation is 109 times more efficient than infection by the oral route 85. Epidemiological studies have failed to reveal a common denominator in the sporadic disease, which occurs in all parts of the world at an apparently constant frequency of about one per million. It is possible, although unlikely, that sporadic CJD results from prions that are endemic in humans but have a very low efficiency for establishing a fully fledged infection.
A
Murine Prn-p
a---q
108
189
I
I
Leu
Thr
Phe
Val
r O.F
I
b
BstE II B
Human PRN-P
Odel
102
a Pro
Leu Fig. 4. Genetic variations in the PrP gene linked to altered susceptibility phenotypes for prion diseases. (A) Alleles a and b of murine PrP gene (Prn-p) from NZW and I/Ln mice, respectively45. (B) Alleles a and A of human PrP gene; the A allele is found in patients with Gerstmann-Str~ussler syndrome 68. (Taken, with permission, from Ref. 69.) 225
Gerstmann-Strfiussler syndrome and familial CJD (F-CJD) are rare neurodegenerative diseases that exhibit PrP immunoreactive amyloid plaques and are transmissible to experimental animals°s's6. About 5-15% of cases of CJD are familial, whereas most cases of GSS are inherited68'76'79. Pedigree studies suggested that GSS and F-CJD are inherited as autosomal dominant disorders 68'85'87. Until recently the significance of this observation was moot, as familial occurrence could be rationalized in terms of an -50% efficient vertical infection. Familial manifestation of GSS has now been defined as a bona fide genetic trait and its development linked to a mis-sense variant of the prion protein86'88. The PrP genes from a US patient affected with the ataxic variant of GSS were analysed (Ref. 88). One of the patient's alleles contained a proline to leucine substitution at codon 102. This polymorphism was absent from the chromosomes of 100 normal patients but present in an apparently unrelated GSS pedigree from the UK. The log of the odds (lod score) against a spurious association of polymorphism and disease phenotype was 3.26, assuming an allele frequency of 0.01. These data establish linkage of an 'ataxic GSS' gene and the PrP gene on chromosome 20 (Ref. 88). As with Prn-i/Prn-p, the precise relationship between these genes is not yet established. Exclusion of the vertical infection mechanism for GSS is in accord with a failure to observe maternal transmission of kuru, an ataxic CJD variant, and experimental CJD4'5. The GSS data differ from those of experimental scrapie 6~ in one respect, in that the codon 102 polymorphisms in the two pedigrees may reflect independent mutational events. The C to T transition where the C lies within a CpG dinucleotide, a motif liable to methylation and to an enhanced forward mutation rate to thymine, is particularly provocative in this regard sg. Interestingly, a structural rearrangement of the amino terminus of a PrP allele has been reported in an F-CJD pedigree from the UK9°. Further study of GSS will be informative about the mechanisms of natural prion diseases. The 'GSS gene' may be a susceptibility locus; this poses an interesting conundrum about the ubiquity of prions in the human population (see below). A second view is that inheritance of the mutant 'GSS gene' is sufficient for development of the disease. The latter view in turn suggests a mechanism for sporadic CJD, if prions contain only PrP sc (prpCJD). A somatic PrP mutation in a single cell could generate PrP cJD in that cell; prions would then spread to neighboring cells upon exit of the PrP cJD molecules. The precedent set by GSS dictates an openmindedness to genetic and infectious manifestation in prion diseases outside the laboratory. Contaminated feedstuffs have been strongly implicated in a recent outbreak of bovine spongiform encephalopathy91. The situation with respect to natural scrapie is less clear. This disease has a strong hereditary component and was commonly regarded as a genetic disease92. Experimental transmission of scrapie was established in the 1930s (Ref. 93), suggesting spread by lateral or vertical infection as a plausible alternative. While scrapie can be transmitted orally in an experimental settings~'94'95, it is still unclear how the disease spreads naturally among sheep and goats. Some investigators favored infection while others were 226
proponents of genetic transmission92'96. In retrospect, both points of view are probably correct.
Unprecedented features of prions That PrP is encoded by a cellular gene and not by a putative nucleic acid carried within the prion is a major feature distinguishing prions from viruses. This discovery coupled with recent genetic studies showing linkage between a PrP mis-sense variant and GSS demand that scrapie and CJD no longer be considered virological disorders. Although prion diseases resemble viral illnesses in some respects, the structure, cell biology and genetics of prions clearly separate them from viruses. Indeed it now seems reasonable to equate the latent host-encoded pathogen postulated to underlie these types of disease 4 with the PrP gene and to seek an origin for sporadic CJD (and sporadic natural scrapie) in stochastic cellular events that prompt conversion of PrP c to the infection-associated PrP cJD and PrP sc isoforms. Whether prions are composed only of an abnormal isoform of the prion protein or whether they contain some additional molecule is still unresolved. Certain lines of evidence argue that PrP sc is the sole component of pfions, for example, the co-partitioning of PrP sc and infectivity in a wide variety of different forms 7'9'10'14-17. Similarly, attempts to inactivate scrapie prion infectivity by procedures that hydrolyse or modify nucleic acids have consistently been unsuccessful. Ultraviolet irradiation of membranes, rods and detergent-lipid-protein complexes suggests that if prions have an essential nucleic acid, then it will have less than five bases if single-stranded, or 30-45 bp if double-stranded. Ionizing radiation studies give a target size too small to protect a large nucleic acid but do not rule out some other macromolecule. Linkage of the PrP mis-sense allele with a GSS predisposition gene bears upon this question. If prions contain a second component, then such molecules must be dispersed throughout the world in order to explain sporadic CJD with an incidence of perhaps one per million and yet paradoxically close to 100% endemic in the rare families that are segregating F-CJD or GSS predisposition genes. Arguments in favor of a second prion component are: (1) prion infectivity has not been recovered from denatured samples after attempts at renaturation; and (2) many 'strains' of prions have been reported. The first argument raises the possibility of a second component, but it need not necessarily be a nucleic acid. The second argument focuses on prion diversity and offers a nucleic acid genome as the basis for this diversity. Just as GSS and familial CJD are unprecedented human illnesses since they are both genetic traits and infectious, the etiological prion particles seem equally novel. Learning the chemical mechanisms responsible for converting PrP c or a precursor into PrP sc will be extremely important. Whether proteins other than PrP are converted from benign, cellular isoforms into pathogenic molecules resulting in degenerative diseases remains to be established.
Selected references 1 Prusiner, S. B. (1982) Science 216, 136-144 2 Kasper, K. C., Bowman, H., Stites, D. P. and Prusiner, S. B. (1981) in Hamster Immune Responses in Infectious and Oncologic Disease (Streilein, J. W., Hart, D. A., Stein-Streilein,
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3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
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Acknowledgements We would like to thank D.Oppenheimer, T. Rademacher, M. Simon, T. Diener, J. Bockman, D. KinEsbury, D. Borchelt, B. Oesch, D. Bredesen,K. Hsiao, D. Lowenstein, and A. Taraboulosfor commentsand
discussionsduring the preparation of this manuscript. Thiswork wassupportedby grants from the NIH, the AmericanHealth Assistance Foundationand the USDepartmentof Agriculture, as wellas by gifts from Sherman FairchildFoundation, RJR/Nabiscoand NationalMedical Enterprises.
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Acknowledgements I thank Mircea Steriade, Martin Desch#nes,June Hirsch and their colleagues for permission to reprint portions of their data in this article. I am indebted to David Princeand HansChristian Papefor their collaboration on portions of these projects. Supported by JacobsJavits Centerin Neuroscience, NINCDS, and a fellowship from the Klingenstien Foundation.
Unravelingprion diseasesthrough moleculargenetics David Westaway,
G e o r g e A. C a r l s o n a n d S t a n l e y B. P r u s i n e r
Prions are transmissible pathogens that cause degenerative diseases in humans and animals. Unique attributes of prion diseases include infectious, sporadic and genetic manifestations, as well as progression to death, all in the absence of a detectable immune response. Prions are resistant to chemical procedures that modify or destroy nucleic acids and are composed largely of a protein, designated PrP so. Molecular cloning of a co~,nate cDNA established a cellular host origin for PrP °c protein and a convergence with the genetics of host susceptibility. The murine PrP gene is linked to the Prn-i gene which determines incubation times in experimental scrapie. Mice with long incubation times have unusual PrP alleles encoding phenylalanine and valine at codons 108 and 189. Moreover, the ataxic form of Gerstmann-Strdussler syndrome (a rare human neurodegenerative disorder) has been defined as an autosomal dominant disorder with a PrP mis-sense mutation at codon 102 linked to the predisposition locus. These studies argue that amino acid substitutions in 'PrP' genes may modulate initiation and development of prion diseases.
scrapie infectivity was filterable, the aetiological agent was highly resistant to a variety of procedures that destroy or modify nucleic acids 1. Instead, infectivity was associated with a proteinaceous component, subsequently identified as the scrapie prion protein, PrP so. Prion diseases are distinguished also from conventional infectious disease by their unusual biological features. Scrapie, CJD and GSS proceed in the apparent absence of a detectable immune response 2. When the familial versions of the human priori diseases are excluded, these diseases occur in isolation ('sporadically') or iatrogenically. Given the experimental transmissibility of the diseases, it was reasonable to attribute sporadic disease to cryptic vertical or lateral spread from an affected individual or from an animal reservoir. However, epidemiologists have failed to identify such vectorial 3 spread and studies of maternal transmission of kuru and CJD prions were similarly negative 4'5. Tripartite manifestation - infectious, familial and sporadic - may be an intrinsic feature of all prion diseases in nature. Molecular genetic studies summarized here reveal that PrP sc molecules are encoded by the host, and Seven prion diseases of animals and humans are now that this genetic origin may contribute to the unique recognized. All are degenerative neurological dis- biological attributes of prions. orders that can be transmitted by inoculation. The pathological features of prion diseases include neur- Prions contain PrP sc PrP27-30, the protease-resistant core of PrP sc, onal vacuolafion, astrocytic gliosis and deposition of amyloid plaques. In animals, prions are the cause of was discovered by enriching brain fractions for scrapie scrapie of sheep and goats, of transmissible mink infectivity6'7. Development of a more rapid and encephalopathy, of chronic wasting disease of mule economical bioassay8 greatly facilitated purification of deer and elk and of bovine spongiform encephal- the hamster scrapie agent 7. PrP27-30 migrates opathy. In humans, prions cause kuru, Creutzfeldt- during SDS-PAGE as a broad band with an apparent Jakob disease (CJD) and Gerstmann-Str~iussler syn- molecular weight of 27 000-30 000. drome (GSS). Correspondence should be addressed to David Westaway: The unusual physiochemical nature of the prions Department of Neurology, HSE-781, University of Cafifornia, San was apparent more than two decades ago. Although Francisco, CA 94143-0518, USA. TINS, Vol. 12, No. 6, 1989
© 1989.ElsevierSciencePublishersLtd,(UK) 0166-2236/89/$02.00
David Westawayand Stanley B. Prusinerare at the Departments of Neurology, Biochemistryand Biophysics, University of California, San Francisco, CA 94143, USA and GeorgeA. Carlsonis at the McLaughlin Research Institute, 6reat Falls, MT 5940f, USA.
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EXON I
INTRON
EXON II
(10 An amino acid substitution in the human PrP gene is genetically linked to the development of GSS. The last two lines of investigation listed above describe rather striking genetic findings which implicate PrP sc as an intrinsic component of the prion. The results of these molecular genetic studies are disG:C cussed in some detail in later sections. Many investigators have confirmed the presence of mRNA PrP 27-30 in brains infected with the scrapie or CJD agent21'27-29. Although the amino acid sequence of PrP has been confirmed and there is agreement that PrP is glycosylated29'3°, some investigators have suggested that PrP 27-30 may not be a component of the scrapie agent31'32. One argument revolves around . the inability of some investigators to detect either PrP mRNA or PrP sc in spleens of scrapie-infected rodents33'34; however, others have clearly shown that both PrP message and PrP sc are present in spleen Fig. 1. Organization and expression of the hamster PrP gene. The features tissue 13'35'36. Indeed, recent studies have shown an presented were deduced from the nucleotide sequences of PrP genomic and excellent correlation between the concentration of cDNA clones. Untranslated regions of the mRNA are indicated by hatched PrP mRNA in scrapie-infected rodent tissues and boxes. An open reading frame or protein coding region is indicated by the prion titer37. Another argument centers on a loss of open box. The diagonal fines show a spficing event that joins the 5' leader sequences to the remainder of the coding sequences. (Taken, with permission, infectivity of CJD agents bound to a lectin column. Neither denaturation of the PrP CJD nor neutralizfrom Ref. 43.) ation of CJD infectivity by immobilization on the lectin column matrix was considered as an explanation 29. Many lines of investigation have converged to Denaturation of PrP sc has been demonstrated to be establish that the protein PrP 27-30 is a component of accompanied by a loss of scrapie infectivity9'1°. To the infectious prion particle. date, no experimental studies have been reported (1) PrP27-30 and the scrapie agent co-purify 7'9'10 where fractions with high levels of scrapie infectivity using detergent extractions and limited proteolysis to are found to contain less than one PrP sc (or PrP promote aggregation of prions into amyloid rods which 27-30) molecule per infectious unit. are collected by centrifugation. PrP 27-30 is the most abundant macromolecule in purified preparations TM. Prion protein genes and isoforms The genetic provenance of PrP sc was resolved by (2) The PrP 27-30 concentration is proportional to the prion titer 11. PrP 27-30, or its precursor PrP sc, is retrieving a PrP clone from a scrapie-infected brain absent in normal, uninfected animals 12'13. cDNA library 13. The molecular clone failed to (3) Procedures that denature, hydrolyse, or selec- hybridize with proteinase-digested/phenol-extracted tively modify PrP 27-30 also diminish the prion titer 11. samples of scrapie prions but hybridized instead to The unusual kinetics of PrP 27-30 hydrolysis cata- genomic DNA and cellular RNA. These results lysed by proteases has been shown to correlate with indicated that a host mRNA, rather than a packaged nucleic acid, encode PrP sc. There was no significant the diminution of the scrapie prion titer. (4) PrP 27-30 and scrapie infectivity partition quantitative difference between a 2.1 kb PrP mRNA together into many different forms: membranes; detected at different times after infection, or between rods; spheres; detergent-lipid-protein complexes infected and control animals, consistent with the (DLPC); and liposomes. These dramatically different failure to observe PrP cDNAs in experiments using physical forms all contain PrP 27-30 and high prion 'subtractive' hybridization techniques3&39. Messenger titers 7,9.lo, 14-17. RNAs for PrP in uninfected animals suggested the (5) Scrapie and CJD prion proteins have been iden- presence of a corresponding translation product. This tiffed only in tissues of animals and humans with molecule has a Mr of 33 000-35 000 and is designated transmissible neurodegenerative diseases and not in PrP c (C for cellular). A similar analysis of infected those with other neurological disorders 7'lo, 18-23. brain proteins reveals the related, but more abundant, (6) Cultured murine neuroblastoma cells have been molecule PrP sc (Sc for scrapie), which, when treated infected with both scrapie and CJD prions 24. Clones of with proteinase K in vitro yields PrP 27-30 (Ref. 13). the scrapie-infected cells were found to produce Both PrP c and PrP sc are glycoproteins that possess PrP so, whereas clones showing no infectivity lacked asparagine (Asn)-linked oligosaccharides and glycosyl prp sc. phosphatidylinositol (GPI) anchors4°. Whether the (7) Rabbit antisera raised against PrP 27-30 purified features that distinguish PrP sc from PrP c arise from by SDS-PAGE were found to neutralize scrapie differences in their Asn-linked oligosaccharides 41 or infectivity in DLPC (Ref. 25). GPI anchors is unknown. (8) Co-purification of scrapie prion infectivity and The organization and structure of the hamster Prp PrP sc was found in fractions eluted from a column of gene have been elucidated; the entire open reading monoclonal antibody to PrP (Refs 25, 26). frame (ORF) or protein-coding region is contained (9) The PrP gene of mice (Prn-p) is linked to a gene within a single exon42 (Fig. 1). The 5' end of the PrP controlling scrapie incubation times (Prn-i). The PrP gene contains multiple transcription initiation sites gene of long incubation period mice encodes a variant which map within a G + C-rich promoter region. An prion protein. intron of - 1 0 kb separates exons I and II. Six lines of
repeats
I
@
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1 kb
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evidence argue that the two PrP isoforms have the same amino acid sequence. (1) No evidence for rearrangement of the PrP gene in scrapie has been found 13,42. (2) The organization of the PrP gene provides no possibility for alternative splicing within the ORF 42. (3) Only one PrP mRNA of 2.1 kb has been detected
and its concentration does not change throughout the course of scrapie infection in hamster brain la. (4) 70% of the PrP sc and 87% of the PrP 27-30 have been sequenced by gas-phase protein sequencing (Teplow, D. et al., unpublished observations). These sequences correspond precisely with the translated genomic DNA sequence. However, currently, we do
Fig. 2. Comparison of ORFs. Comparison of predicted mammalian prion protein amino acid sequences 4 2 .4.4. .4 5 4 9 51 . Gaps are indicated by dashes. Variant amino acids are indicated above or below the NZW mouse sequence, arbitrarily chosen as a prototype. Sequences presumed missing from a partial rat PrP cDNA are shown by question marks. The ORF predicted from the human PrP cDNA described by Liao et al. 53 differs from a consensus established by 11 independent cDNA and genomic molecular clones, indicating that this clone may contain aberrant 5" sequences (an attendant error in the numbering of the codons in this done is perpetuated in the rat PrP sequence 51. Other human PrP sequences differ in the 5' untranslated region (Ref. 49; Hsiao, K., unpublished observations). Whether these different 5' sequences reflect polymorphisms, spficing variants, or features of the PrP mRNA that interfere with the fidelity of cDNA synthesis remains to be determined a2. Abbreviation: Syr, Syrian &olden hamster.
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223
PRION GENE COMPLEX (Prn)
A Pm-i
Prn-p
Chr 2
B Prnoi
Chr 2 Prn -p Fig. 3. Two models for the prion gene complex. Genes are indicated by open boxes. Abbreviation: Chr 2, chromosome 2.
not know whether all, or only a fraction of the PrP 27-30 molecules within our preparations are associated with infectivity. (5) Both scrapie hamster brain and mouse PrP cDNAs have translated ORF sequences that are identical with the corresponding translated genomic sequences 13,42,44,45. (6) Both PrP isoforms have the same amino-terminal amino acid sequences, as determined by N-terminal sequencing 46. Presumably, the difference in the properties of the two prion proteins is due to a posttranslational event 42. Nevertheless, we cannot exclude the findings that most PrP mRNA molecules encode PrP sc, while a few with a different ORF encode PrP sc. A hypothetical mis-sense codon could arise via two mechanisms, somatic mutation or RNA 'editing' (Ref. 47). The human and mouse PrP genes have been shown to be located on chromosomes 20 and 2, respectively, which are homologous48. This finding indicates that PrP genes existed prior to the speciafion of mammals. PrP genes from humans 49, Syrian hamsters 42, Chinese hamsters 5°, Armenian hamsters 5°, mice 44'45, rats 51 and sheep 52 have been sequenced (Fig. 2). Presumably, all mammals have PrP genes and all are capable of developing prion diseases 54. Whether eukaryotes other than mammals have authentic PrP genes remains to be established. Nucleotide sequences of all the PrP ORFs predict prion proteins of approximately 250 amino acids. All encode N-terminal signal peptides, consensus sites for Asn-linked glycosylafion, as well as two cysteines within the carboxy-terminal half of the molecule. All of the ORFs also possess a series of glycine and prolinerich repeats in the N-terminal portion of the PrP molecule. PrP sc and PrP c are modified by the addition of a phosphatidylinositol glycolipid anchor 4°. Divergence in the PrP carboxyl terminus can be reconciled with the notion that glycolipidation is not dependent on a primary structure motiP 5. A signal pepfide of 22 amino acids is cleaved during the biosynthesis of PrP c and PrP sc (Refs 29, 46, 56). A putative C-terminal cleavage site has been positioned 23 amino acids from the C-terminus of the full-length molecule57, in fair agreement with a C-terminal truncation of 19 residues deduced from amino acid analyses 13. When these cleaved N- and C-terminal peptide domains are excluded from sequence comparisons, PrP molecules are 96.7% identical in humans, hamsters, rats and 224
mice, implying that PrP c performs a crucial cellular function. Database searches have been unenlightening, with no similarities to proteins of known function detected to date. Location of glycolipidated PrP c on the external face of the cell membrane perhaps suggests a role in signal transduction or cellular communication. The Asn-linked oligosaccharides of PrP so, and presumably PrP c, contain the x-antigenic determinant [Gal[51-->4 (Fucod--->3) GIcNAc] associated with cell surface molecules involved in recognition (Endo, T., Groth, D. F., Prusiner, S. B. and Kobata, A., unpublished observations). The location of PrP c may form the molecular basis for the immunobiology (or lack thereof) of prion diseases. Expression of extracellularly oriented PrP c in several tissues would result in immune recognition as 'self and this tolerance could also encompass the diseaseassociated PrP sc isoform.
PrP mRNA Both in hamsters and in mice, the highest PrP mRNA levels are found in brain. In situ hybridization of normal and scrapie-infected hamster brains has shown that neurons contain the highest levels of PrP mRNA ( - 5 0 copies per cell); glial cells contain less than three mRNA copies per cell49. In hamsters, PrP mRNAs are detected in a number of peripheral organs, including spleen, heart and hmg 13. While PrP mRNA levels are constant in adult rodents 13, the expression of the PrP gene is regulated during development 58. During the first 20 days after birth, both PrP and [5-amyloid precursor protein ([5APP is the precursor molecule for the amyloid protein of Alzheimer's disease 59'6°) mRNAs increase in the neonatal hamster brain at different rates in various regions of the brain. In the septum, the kinetics of the increase in PrP and [5-APP mRNAs paralleled the kinetics of choline acetyltransferase. The steady-state level of both PrP and [5-APP mRNAs as well as the synthesis of choline acetyltransferase are stimulated by intraventricular injections of nerve growth factor 57. Prion gene complex The time that elapses from inoculation of an animal to death is a repeatable objective observation. The genetic control of time from inoculation to death is a crucial avenue for research, as manipulation of the host loci which control these times would provide an important approach for therapy; for example, if inoculation-to-death times can be altered to exceed the normal lifespan of the organism. Two genes, Pidl and Prn-i, that influence the time course of prion infections in mice have been assigned to chromosomes 17 and 2, respectively48. Pidl is located within the D subregion of the H-2 complex61. Of greater influence than Pidl in experimental scrapie is the Prn-i gene, which has been shown to be linked to the gene encoding the prion protein (Prn-p). The dominant allele of Prn-i increases the time from inoculation to death 62. Prn-i and Prn-p form the prion gene complex (Prn) (Fig. 3). Whether the Prn-i and Prn-p genes are separate, but linked genes or are identical remains to be determined. Dickinson and colleagues63'64, using inbred strains of mice and 'strains' of scrapie agent, defined an autosomal locus in mice that they labeled Sinc. Studies in congenic mice suggest that Sinc is linked to Prn-p, raising the TINS, Vol. 12, No. 6, 1989
possibility that Prn-i and Sinc are the same genetic locus 65-67. The most compelling genetic evidence for a central role of PrP in the pathogenesis of scrapie comes from molecular cloning showing that inbred mice in which scrapie infections have short (NZW strain) and long (I/Ln strain) time courses have distinct prion protein gene alleles 45. A comparison of the PrP sequences of NZW (Prn-p ~) and I/Ln (Prn-p b) mice shows that the amino acid at codon 108 is changed from leucine to phenylanaline and that the highly conserved codon at position 189 is changed from threonine to valine (Figs 2, 4). Mice in which scrapie infections have short and intermediate time courses possess a leucine at codon 108 and a threonine at codon 189; all three inbred strains of mice known to have long time courses of infection have variant amino acids at these two codons45'65. Although this argues for congruence of Prn-p and Prn-i, genetic subtleties of Prn-p b mice 65, including a possible 'founder' effect, demand functional studies to establish this point beyond dispute.
PrP alleles and genesis of prion isolates Dickinson, Kimberlin and co-workers have argued that their isolation of numerous isolates of scrapie agent is sufficient evidence for the existence of a hostindependent genome 7°. Many studies, including subtractive hybridization experiments 38'39, have failed to identify a discrete scrapie-specific nucleic acid molecule that is associated with infectivity; however, lack of evidence cannot be used to exclude the possibility that such a molecule exists. Irrespective of the exact relationship between Prn-p and Prn-i, Prn-p a and err/-p b mice encode PrP sc allotypes prpSC-A and prpSc-B, which differ at two amino acid residues. This is an important point as C57 and VM mice have been used to derive some distinguishable scrapie isolates. C57 mice are Prn-p a and VM mice are presumed to be Prn-p b [tie IM mice45. To test whether distinguishable isolates are a transient effect of Prn-p genotype, the Chandler isolate of scrapie prions was serially passaged in NZW, Swiss (also Prn-pa), I/LnJ, and NZW × I/LnJ F1 mice (Carlson, G. A., Westaway, D., DeArmond, S. J., Peterson-Torchia, M. and Prusiner, S. B., unpublished observations). A single passage through I/LnJ mice caused a dramatic shortening of incubation and survival times (and variance thereof) in I/LnJ mice with no further shortening in a second syngeneic passage. Conversely, the inoculum obtained after a single passage in I/LnJ mice delayed the onset of disease and death as well as increasing the variance of onset in NZW mice when compared with inocula passaged through NZW and Swiss mice. The decrease in incubation time in going from I/LnJ to NZW or Swiss mice happened in a single step, as it also appears to do in the case of scrapie isolate 22A (Re£ 71). Two observations implicate the Prn genotype in this phenomenon. Swiss and NZW mice behave similarly; although both are Prn-p ~, they differ at many other loci. Second, in a NZW × I/LnJ F2 cross inoculated with the Chandler scrapie isolate, and segregating for genes in addition to Prn-p, high variance was seen only in mice homozygous for Prn-p b after inoculation with prions containing prpSC-A. These results suggest that the different properties perceived for NZW- and I/LnJderived prion isolates reflect an inbred mouse strain TINS, VoL 12, No. 6, 1989
barrier, defined by the Prn genotype. The species barrier for transmission of scrapie may be, at least in part, analogous to the Prn-pa/Prn-p b allotype barrier. Certainly, all species susceptible to scrapie examined to date encode distinct prion proteins 42'44'5°-52. Prion isolates with true-breeding yet divergent incubation times in the same inbred host are more compelling as bona fide 'strains '64"72-74. Although there are no biochemical or fluctuation test 75 data to equate genesis of these isolates with nucleic acid mutation, this clearly warrants additional scrutiny.
Infectious, sporadic and genetic prion diseases The human prion diseases kuru, GSS and CJD illustrate three manifestations of CNS degeneration disorder: slow infection, sporadic disease and genetic disorder 76'77. That all three diseases can be transmitted to laboratory animals by inoculation is well documented 78-a°. Kuru is thought to have been spread exclusively through a slow infectious mechanism by means of ritualistic cannibalism 4'81. Although a few cases of CJD can be traced to iatrogenic inoculation with CJD-contaminated material or instruments - i.e. injections of human growth hormone 22'82'8a, transplantation of neural tissue, and implantation of cerebral electrodes - the vast majority appear to be sporadic, despite considerable effort to implicate scrapie-infected sheep as an exogenous source a1'84. In hamsters, scrapie infection by intracerebral inoculation is 109 times more efficient than infection by the oral route 85. Epidemiological studies have failed to reveal a common denominator in the sporadic disease, which occurs in all parts of the world at an apparently constant frequency of about one per million. It is possible, although unlikely, that sporadic CJD results from prions that are endemic in humans but have a very low efficiency for establishing a fully fledged infection.
A
Murine Prn-p
a---q
108
189
I
I
Leu
Thr
Phe
Val
r O.F
I
b
BstE II B
Human PRN-P
Odel
102
a Pro
Leu Fig. 4. Genetic variations in the PrP gene linked to altered susceptibility phenotypes for prion diseases. (A) Alleles a and b of murine PrP gene (Prn-p) from NZW and I/Ln mice, respectively45. (B) Alleles a and A of human PrP gene; the A allele is found in patients with Gerstmann-Str~ussler syndrome 68. (Taken, with permission, from Ref. 69.) 225
Gerstmann-Strfiussler syndrome and familial CJD (F-CJD) are rare neurodegenerative diseases that exhibit PrP immunoreactive amyloid plaques and are transmissible to experimental animals°s's6. About 5-15% of cases of CJD are familial, whereas most cases of GSS are inherited68'76'79. Pedigree studies suggested that GSS and F-CJD are inherited as autosomal dominant disorders 68'85'87. Until recently the significance of this observation was moot, as familial occurrence could be rationalized in terms of an -50% efficient vertical infection. Familial manifestation of GSS has now been defined as a bona fide genetic trait and its development linked to a mis-sense variant of the prion protein86'88. The PrP genes from a US patient affected with the ataxic variant of GSS were analysed (Ref. 88). One of the patient's alleles contained a proline to leucine substitution at codon 102. This polymorphism was absent from the chromosomes of 100 normal patients but present in an apparently unrelated GSS pedigree from the UK. The log of the odds (lod score) against a spurious association of polymorphism and disease phenotype was 3.26, assuming an allele frequency of 0.01. These data establish linkage of an 'ataxic GSS' gene and the PrP gene on chromosome 20 (Ref. 88). As with Prn-i/Prn-p, the precise relationship between these genes is not yet established. Exclusion of the vertical infection mechanism for GSS is in accord with a failure to observe maternal transmission of kuru, an ataxic CJD variant, and experimental CJD4'5. The GSS data differ from those of experimental scrapie 6~ in one respect, in that the codon 102 polymorphisms in the two pedigrees may reflect independent mutational events. The C to T transition where the C lies within a CpG dinucleotide, a motif liable to methylation and to an enhanced forward mutation rate to thymine, is particularly provocative in this regard sg. Interestingly, a structural rearrangement of the amino terminus of a PrP allele has been reported in an F-CJD pedigree from the UK9°. Further study of GSS will be informative about the mechanisms of natural prion diseases. The 'GSS gene' may be a susceptibility locus; this poses an interesting conundrum about the ubiquity of prions in the human population (see below). A second view is that inheritance of the mutant 'GSS gene' is sufficient for development of the disease. The latter view in turn suggests a mechanism for sporadic CJD, if prions contain only PrP sc (prpCJD). A somatic PrP mutation in a single cell could generate PrP cJD in that cell; prions would then spread to neighboring cells upon exit of the PrP cJD molecules. The precedent set by GSS dictates an openmindedness to genetic and infectious manifestation in prion diseases outside the laboratory. Contaminated feedstuffs have been strongly implicated in a recent outbreak of bovine spongiform encephalopathy91. The situation with respect to natural scrapie is less clear. This disease has a strong hereditary component and was commonly regarded as a genetic disease92. Experimental transmission of scrapie was established in the 1930s (Ref. 93), suggesting spread by lateral or vertical infection as a plausible alternative. While scrapie can be transmitted orally in an experimental settings~'94'95, it is still unclear how the disease spreads naturally among sheep and goats. Some investigators favored infection while others were 226
proponents of genetic transmission92'96. In retrospect, both points of view are probably correct.
Unprecedented features of prions That PrP is encoded by a cellular gene and not by a putative nucleic acid carried within the prion is a major feature distinguishing prions from viruses. This discovery coupled with recent genetic studies showing linkage between a PrP mis-sense variant and GSS demand that scrapie and CJD no longer be considered virological disorders. Although prion diseases resemble viral illnesses in some respects, the structure, cell biology and genetics of prions clearly separate them from viruses. Indeed it now seems reasonable to equate the latent host-encoded pathogen postulated to underlie these types of disease 4 with the PrP gene and to seek an origin for sporadic CJD (and sporadic natural scrapie) in stochastic cellular events that prompt conversion of PrP c to the infection-associated PrP cJD and PrP sc isoforms. Whether prions are composed only of an abnormal isoform of the prion protein or whether they contain some additional molecule is still unresolved. Certain lines of evidence argue that PrP sc is the sole component of pfions, for example, the co-partitioning of PrP sc and infectivity in a wide variety of different forms 7'9'10'14-17. Similarly, attempts to inactivate scrapie prion infectivity by procedures that hydrolyse or modify nucleic acids have consistently been unsuccessful. Ultraviolet irradiation of membranes, rods and detergent-lipid-protein complexes suggests that if prions have an essential nucleic acid, then it will have less than five bases if single-stranded, or 30-45 bp if double-stranded. Ionizing radiation studies give a target size too small to protect a large nucleic acid but do not rule out some other macromolecule. Linkage of the PrP mis-sense allele with a GSS predisposition gene bears upon this question. If prions contain a second component, then such molecules must be dispersed throughout the world in order to explain sporadic CJD with an incidence of perhaps one per million and yet paradoxically close to 100% endemic in the rare families that are segregating F-CJD or GSS predisposition genes. Arguments in favor of a second prion component are: (1) prion infectivity has not been recovered from denatured samples after attempts at renaturation; and (2) many 'strains' of prions have been reported. The first argument raises the possibility of a second component, but it need not necessarily be a nucleic acid. The second argument focuses on prion diversity and offers a nucleic acid genome as the basis for this diversity. Just as GSS and familial CJD are unprecedented human illnesses since they are both genetic traits and infectious, the etiological prion particles seem equally novel. Learning the chemical mechanisms responsible for converting PrP c or a precursor into PrP sc will be extremely important. Whether proteins other than PrP are converted from benign, cellular isoforms into pathogenic molecules resulting in degenerative diseases remains to be established.
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Acknowledgements We would like to thank D.Oppenheimer, T. Rademacher, M. Simon, T. Diener, J. Bockman, D. KinEsbury, D. Borchelt, B. Oesch, D. Bredesen,K. Hsiao, D. Lowenstein, and A. Taraboulosfor commentsand
discussionsduring the preparation of this manuscript. Thiswork wassupportedby grants from the NIH, the AmericanHealth Assistance Foundationand the USDepartmentof Agriculture, as wellas by gifts from Sherman FairchildFoundation, RJR/Nabiscoand NationalMedical Enterprises.
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