From the Institut für Molekularbiologie der Universität Zürich, Abteilung I, Winterthurerstrasse 190, 8057 Zürich, Switzerland
Transmissible spongiform encephalopathies
are degenerative disorders of the central nervous system that occur
naturally in man and in a variety of other animals. They include
scrapie of sheep and goats, bovine spongiform encephalopathy
(BSE)1 in cattle, and several
human diseases such as Creutzfeldt-Jakob disease (CJD),
Gerstmann-Sträussler-Scheinker syndrome (GSS), and
fatal familial insomnia (FFI). Since its first recognition in 1986, BSE
reached epidemic proportions in the UK (1), and there is substantial
evidence that a new variant of CJD may be because of consumption of
BSE-contaminated products (2).
The unusual properties of the transmissible agent, or prion, early on
suggested that it might be devoid of nucleic acid (3). Currently, the
most widely accepted proposal is the "protein only" hypothesis, first outlined in general terms by Griffith (4) and
enunciated in its updated and detailed form by Prusiner (5, 6). It
proposes that the prion is identical with a conformational isoform of
PrPC (5), a normal host protein (7-9) found predominantly
on the outer surface of neurons. Introduction of the abnormal conformer into the organism would result in the conversion of PrPC
into a likeness of itself (Fig. 1).
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REFERENCES
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Fig. 1.
The "protein only" model.
A, in the normal cell PrPC is synthesized,
transported to the cell surface and recycled. B, the protein
only model assumes that the prion is identical with PrPSc
(or PrP*). Exogenous prions cause the conversion of the normal cellular
protein PrPC into PrPSc, either at the cell
surface or after internalization. C, PrPSc
accumulates intracellularly in late endosomes or lysosomes, and the
cell surface is depleted of PrPC. PrPSc is also
released into the extracellular space.
During the course of prion disease, a largely protease-resistant,
aggregated form of PrP, designated PrPSc, accumulates
mainly in brain (7, 10). Prusiner and co-workers (11) proposed that
PrPSc is the main or only constituent of the prion. Because
no chemical differences were found between PrPC and
PrPSc, the two species are believed to differ in their
conformation. The three-dimensional structure of PrPC has
been elucidated (12) but not that of PrPSc; however, the
-sheet content of PrPSc was found to be high whereas
that of PrPC is low (13). Because the ratio of infectious
units to PrPSc molecules is only about 1:100,000 (14), the
structure of the PrP molecule actually associated with infectivity
cannot be definitively inferred. For this reason the PrP species
responsible for infectivity is presently better designated as PrP*
(15). The conclusion that some form of PrP is the essential (perhaps
only) constituent of the infectious agent is based on compelling
biochemical and genetical evidence (5, 16).
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Familial Spongiform Encephalopathies Are Associated with Mutations in the PrP Gene |
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Although most cases of human prion disease are sporadic, about
10% are familial and linked to one of a number of mutations in the
PrP gene (Fig. 2) (for reviews
see Refs. 17 and 18). It is believed that these mutations allow
spontaneous conversion of PrPC into PrPSc with
a frequency sufficient to cause disease within the lifetime of the
individual (5, 19). Sporadic CJD could be attributed to rare instances
of spontaneous conversion of PrPC into PrPSc or
rare somatic mutations in the Prnp gene. In both cases the initial conversion would be followed by autocatalytic propagation. Mice
overexpressing a murine PrP transgene with a mutation
corresponding to the human GSS mutation Pro-102 Leu spontaneously
contract a lethal scrapie-like disease, and it has been reported that
this disease can be transmitted to mice expressing the same mutant transgene at lower levels, which do not lead to spontaneous disease (20, 21).
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Mice Devoid of PrPC Are Resistant to Scrapie |
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The "protein only" hypothesis predicts that in the absence of PrPC mice should be resistant to scrapie and fail to multiply the infectious agent. Mice devoid of PrP (Prnpo/o) were generated by homologous recombination and found to be essentially normal (22). When challenged with mouse prions, mice devoid of PrP were completely protected against scrapie disease, and prions failed to accumulate in spleen and brain, in contrast to wild-type mice (23, 24). Introduction of murine Prnp transgenes into Prnpo/o mice resulted in several lines with varying expression levels of PrPC, which were susceptible to mouse prions; the higher the PrPC content of the brain, the shorter the incubation times (25).
The demonstration that disruption of the PrP gene confers
resistance to scrapie and that reintroduction of a PrP-encoding transgene restores susceptibility paved the way to reverse genetics of
PrP, that is the introduction of deletions or mutations into the
Prnp gene and determination of the capacity of the modified gene to confer susceptibility to scrapie to a PrP knockout mouse. Transgenes encoding PrP with deletions extending to codon 93, but not
to 106, restored susceptibility to scrapie
(25).2
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Transport of Prions from the Periphery to the CNS |
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Acquired forms of prion diseases are mostly transmitted through oral uptake of prions or peripheral administration, raising the question as to how the agent finds its way to the CNS.
Prnp+/+ neuroectodermal tissue introduced into
the brain of newly born Prnpo/o mice develops
into differentiated Prnp+/+ nervous tissue.
Intracerebral inoculation of such engrafted mice leads to typical
scrapie pathology in the graft but not in the surrounding
Prnpo/o tissue (26). However, because
intraperitoneal inoculation of these mice does not lead to pathology in
the Prnp+/+ graft, transport of prions from the
periphery to the CNS is seen to require interposed PrP-bearing tissue
(27). It has also been shown that Prnp+/+
immunodeficient mice lacking mature B cells fail to transport prions
from the periphery to the CNS, suggesting a role of the lymphoreticular
system in transport (28). It is believed that the follicular dendritic
cells, which depend on B cells for their maintenance in a mature state,
are the site of prion replication in the spleen (29, 30) and that
transport to the CNS is, in addition, dependent on the peripheral
nervous system (31).
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The Species Barrier Is Abolished by Introducing the PrP Transgene of the Prion Donor into the Recipient |
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Prions are transmitted from one species to another much less
efficiently, if at all, than within the same species and only after
prolonged incubation times. In the case of prion transmission from
hamsters to mice, this so-called species barrier was overcome by
introducing hamster Prnp transgenes into recipient wild-type mice (32, 33). Importantly, the properties of the prions produced in
these transgenic mice corresponded to the prion species used for
inoculation (32), that is infection with hamster prions led to
production of hamster prions, but infection with mouse prions gave rise
to mouse prions. Within the framework of the "protein only"
hypothesis this means that hamster PrPC but not murine
PrPC (which differs from the former by 10 amino acids) is a
suitable substrate for conversion to hamster PrPSc by
hamster prions and vice versa. Interestingly, susceptibility of the mouse to prions from other species, such as hamster, mouse, or
man, is increased when the PrP transgenes are introduced
into a PrP knockout mouse, suggesting that the resident murine gene inhibits the propagation of the alien prions (23).
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The Puzzle of Prion Strains |
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Dickinson and colleagues (34, 35) showed that many distinct strains of scrapie prions can be derived from sheep isolates. These strains differ by their incubation times in various inbred mouse lines and by the lesion patterns they occasion in the affected brains. Interestingly, different strains can be propagated in one inbred mouse strain (homozygous with regard to its PrP gene) (35). Within the framework of the protein only hypothesis this is at first blush puzzling, because it means that one and the same polypeptide chain is able to mediate different strain phenotypes. The conformational hypothesis proposes that each strain is associated with a different conformation of PrPSc (or PrP*) and that each of these can convert the PrPC of its host into a likeness of itself (19). Marsh and colleagues (36) showed that the PrPSc species associated with two hamster-adapted scrapie strains, HY and DY, are cleaved to products of different length by proteinase K; the different susceptibility to protease is readily explained by different conformations. Similar findings were made with other prion strains propagated in the mouse (37, 38). Moreover, PrPSc of certain strains differ in the ratio of the diglycosylated to the monoglycosylated form (39).
Is it possible that a dozen different strains of prions can differ in
regard to the conformation of the PrPSc they are associated
with and that all these conformations can "breed true"? It has
recently been claimed that PrPSc molecules of as many as
eight different strains could be differentiated by virtue of their
relative affinity for a monoclonal antibody directed against an epitope
that is fully available in PrPC but partially occluded in
PrPSc. In addition, some strains differed in their
susceptibility to denaturation by guanidinium chloride, adding further
credibility to the conformational hypothesis of strain specificity
(40).
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The Conversion Reaction |
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Conversion of PrPC to PrPSc in scrapie-infected cells is a late post-translational process, occurring after PrPC has reached its normal extracellular location or thereafter (41). Why is spontaneous formation of PrPSc an extremely rare event and how does scrapie infection promote conversion? The "refolding model" (Fig. 3A) proposes that conversion requires that PrPC be unfolded to some extent and refolded under the influence of a PrPSc molecule (19), a process that would have to overcome a high activation energy barrier and might require a chaperone and an energy source. The "nucleation model" (Fig. 3B) proposes that PrPC is in equilibrium with PrPSc (or a precursor thereof) and that PrPSc is only stabilized when it adds onto a crystal-like seed or aggregate of PrPSc. If a stable aggregate needs to consist minimally of a substantial number of PrPSc molecules, then its spontaneous formation would be a very rare event. However, once a seed is present, monomer addition could ensue at a rapid rate (42-44). Trapping of PrP by essentially irreversible aggregation would drive the bulk conversion process. The proposed process is akin to the assembly of (protease-sensitive) flagellin to (protease-resistant) flagellar filaments (45). Interestingly, the same flagellin molecule can assemble into two types of flagella, depending on the provenance of the seed (46), thereby providing an analogy for conformationally determined prion strain specificity.
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Caughey and colleagues (47) have shown that incubation of
35S-labeled PrPC with PrPSc yields
a radioactive product that resembled PrPSc with regard to
its protease resistance. The conversion reaction exhibited the
"species specificity" observed in vivo; thus
PrPC from mouse was readily converted to protease
resistance by murine PrPSc but poorly by bovine
PrPSc and vice versa (48). Moreover, even strain
specificity has been demonstrated in this type of reaction, in that
labeled PrPC incubated with PrPSc from the DY
or the HY hamster transmissible mink encephalopathy strains described
above yielded labeled PrPSc with the properties typical for
the input PrPSc (49). Despite efforts by several groups, it
has not been possible to demonstrate net synthesis of infectivity over
the background given by the PrPSc preparation.
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Implications and Outlook |
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Although each individual piece of evidence described above could be explained in several ways, the conjunction of data strongly supports the proposal that the prion is composed partly or entirely of a PrP-derived molecule (PrP* or PrPSc) and that protein-encoding nucleic acid is not an essential component. Probably the closest one could come to irrefutable proof for the protein only hypothesis would be the demonstration that biosynthetic, pure PrPC can be converted not only into a protease-resistant form but to infectious scrapie agent under defined conditions in vitro.
Many questions still need to be elucidated, in particular the mechanism and the requirements for the conversion reaction, the transport of the prion from periphery to CNS, and the mechanism of pathogenesis, to name but a few. From a practical side, early diagnosis of prion disease and treatments aimed at arresting or reversing the disease in humans are important goals.
Finally one may raise the question whether prion-like agents cause
other diseases or appear in non-vertebrate organisms. Although several
human diseases accompanied by amyloid formation are known, none of them
have been reproducibly transmitted. Interestingly, two yeast phenotypes
have been ascribed to "heritable protein conversion," namely the
[URE3] and the [PSI] systems (50), and have opened new perspectives
for the elucidation of this phenomenon.
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FOOTNOTES |
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* This minireview will be reprinted in the 1999 Minireview Compendium, which will be available in December, 1999. This is the first article of three in the "Prions of Mammals and Fungi Minireview Series."
To whom correspondence should be addressed. Tel.: 41-1-6353110;
Fax: 41-1-6356864; E-mail: weissma{at}molbio.unizh.ch.
2 D. Shmerling and E. Flechsig, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: BSE, bovine spongiform encephalopathy; CJD, Creutzfeldt-Jakob disease; GSS, Gerstmann-Sträussler-Scheinker syndrome; FFI, fatal familial insomnia; CNS, central nervous system..
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