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MRC Prion Unit,
Scripps Research Institute
Transmissible spongiform encephalopathies (TSEs,or
prion diseases), such as Creutzfeldt–Jakob disease
(CJD) and kuru in man, bovine spongiform
encephalopathy (BSE) in cattle or scrapie in sheep,
still present major challenges to biomedical research.
The first phase of investigation of these diseases was
dominated by experiments at the classical biological
level and led to the discovery of transmissibility of
scrapie,kuru and CJD to appropriate recipients,the
existenceof different STRAINSand recognition of the pec-
uliar properties of the transmissible agent, such as
lack of immunogenicity and long incubation times.
The unusual resistance of these agents to radiation led
to the proposal that the agent, later named ‘PRION’,
might be devoid of nucleic acid (in this review I will
use the term prion to signify the infectious TSE agent,
regardless of its composition or structure).
The next phase of research involved the isolation
and biochemical characterization of PrPSc,a protein
that is found only in scrapie-infected animals1.This
was followed by the cloning of PrP cDNA2,3and Prnp
(the cognate gene)4, the recognition that this gene
encodes a normal host protein,PrPC,from which PrPSc
is derived by conformational rearrangement4–6,and
the establishment of a link between Prnpand familial
In the third phase of research,transgenic experi-
mentation strengthened the link between the PrP
gene and susceptibility to prion disease by showing
that the so-called species barrier could be overcome,at
least in some cases,by introducing the PrP gene of a
donor into a recipient8.The strongest evidence for the
essential role of PrP in prion disease was the demon-
stration that PrP-knockout mice9were resistant to
prion disease and were incapable of propagating the
infectious agent10.The discovery of‘yeast prions’has
provided profound insights into the mechanism of
self-propagating conformational changes11.
The precise nature of the transmissible agent has
been debated since the mid-1960s. As a virus is
understood to consist of a protein-encased nucleic
acid encoding some or all of its constituent proteins,
the concept of a ‘slow’or ‘unconventional’virus has
lost support because intense efforts in many laborato-
ries have failed to identify a TSE-specific nucleic acid,
or even a nucleic acid that is long enough to encode a
small protein12.Nonetheless,the idea that a virus —
perhaps an endogenous virus, the nucleic acid of
which would not score as extraneous — is responsible
for TSEs persists in some quarters13,14. The VIRINO
hypothesis, which asserts that the infectious agent
consists of an agent-specific nucleic acid enveloped in
a host-specified protein,was proposed to explain the
lack of an immune response by the host as well as
strain variation15(FIG.1c).However,the failure to iden-
tify a TSE-associated nucleic acid, the biochemical
linkage of the mouse scrapie prions and PrPSc(which
is a protease-resistant, aggregated conformational
isoform of the normal host protein PrPC), and the
link between the PrP gene and susceptibility to prions
and familial prion diseases have provided support for
an updated version of the ‘protein-only’hypothesis16,
namely that the infectious agent is PrPSc,which ‘multi-
plies’by catalysing the conversion of PrPCinto a like-
nessof itself17(FIG.1b).The finding that PrP knockout
mice were resistant to scrapie fulfilled a central predic-
tion of the protein-only hypothesis,and significantly
promoted its acceptance,without however proving it.
THE STATE OF THE PRION
Abstract | There is little doubt that the main component of the transmissible agent of spongiform
encephalopathies — the prion — is a conformational variant of the ubiquitous host protein PrPC,
and that the differing properties of various prion strains are associated with different abnormal
conformations of this protein. The precise structure of the prion is not yet known, nor are the
mechanisms of infection, conformational conversion and pathogenesis understood.
Types of prions differing in
regard to the clinical course of
the disease and the
neuropathology they elicit,
their transmissibility and the
physico-chemical properties of
the PrP isoforms that they are
agent causing transmissible
(TSE),unusually resistant to
agents known to inactivate
nucleic acids.As used in this
article the term does not imply
any specific components or
An infectious particle that is
conjectured to consist of a
TSE-specific nucleic acid
enveloped by PrPSc.
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A further proposal18attempted to mediate between the
two camps (protein only and protein plus nucleic acid)
by hypothesizing that the conversion of PrPCto an
abnormal conformer that can cause disease is indeed
the essential pathogenic event,but that a small host-
specified nucleic acid associated with PrPSc— the
‘co-prion’— is a crucial component that is required
to modulate strain specificity. These different ideas
are reviewed here in the light of recent developments.
Abnormal forms of PrP
PrPC, which is the normal form of the protein
encoded by Prnp,is attached to the outer surface of the
plasma membrane by a glycosylphosphatidyl inositol
(GPI) anchor (see BOX 1 for prion nomenclature).It is
readily released from the cell surface by cleavage with
phosphatidyl inositol-specific phospholipase C
(PIPLC)and is highly susceptible to proteinase K (PK)
digestion. PrPScwas originally defined as a form of
PrP that was largely resistant to PK digestion under
conditions in which PrPCand most other proteinswere
readily degraded19,but is somewhat confusingly now
also used as the designation for the infectious iso-
form of PrP, whatever its properties might be20,21.
Importantly, digestion of PrPScby PK (but not by
other proteinases such as trypsin) causes cleavage at
residues 87 to 91 (the exact position depends on the
prion strain) of the mature PrP sequence,leading to a
characteristic electrophoretic mobility shift of the
three bands that correspond to di-, mono- and un-
glycosylated species (designated PrP27-30).It should
be noted that ‘protease resistance’is a relative concept;
the ‘resistant’moiety is also susceptible to degradation,
at a rate that depends critically not only on the concen-
tration of PK22,23and the PK to protein ratio,but also
on the prion strain with which the PrPScis associated24.
Some forms of PrP are more resistant to PK than PrPC
but are nonetheless non-infectious25,26.
Purified preparations of prions contain aggregates
of PrPSc(or PrP27-30 if PK-treated) as the main protein
component, but also contain several non-protein
components such as glycosaminoglycans and poly-
saccharides27. Strikingly, hamster prion infectivity
shows a similar resistance to PK as PrPScand,under
stringent digestion conditions,PrPScconcentration
and infectivity decrease at similar rates23.Complete
dissociation of the aggregates by alkali,strong acids,
detergents or chaotropic agents leads to a loss of
infectivity that so far has remained irreversible; so,
attribution of infectivity to a monomeric component
of the aggregates has not been achieved.
Usually,the number of PrP molecules in a scrapie-
infected brain homogenate or in purified preparations
is several orders of magnitude greater than the number
of infectious units28,which raises the question as to
whether the infectious process is very inefficient,the
infectious entity is an aggregate of a large number of
PrPScmolecules or whether it is a minority component
of the preparation,perhaps a different isoform of PrP,
generically designated PrP* (REF.29).Several studies
have reported very low30or undetectable levels of
a Normal cell
b Scrapie-infected cell: Protein-only model
c Scrapie-infected cell: Virino model
Figure 1 | Models for the propagation of the TSE agent (prion). a | In a normal cell, PrPC
(yellow square) is synthesized, transported to the cell surface and eventually internalized.
b | The protein-only model postulates that the infectious entity, the prion, is congruent with an
isoform of PrP, here designated as PrPSc(blue circle). Exogenous PrPSccauses catalytic
conversion of PrPCto PrPSc, either at the cell surface or after internalization. c | The virino model
postulates that the infectious agent consists of a TSE-specific nucleic acid associated with or
packaged in PrPSc. The hypothetical nucleic acid is replicated in the cell and associates with PrPC,
which is thereby converted to PrPSc. Reproduced with permission from REF.123 © (1994) Elsevier.
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R E V I E W S
authors use the term ‘PrP-res’for protease-resistant
PrP.However,as mentioned above,protease resistance
is a relative term,and the main issue is whether one is
dealing with the naturally occurring,well-characterized
α-helix-rich PrPC,or with one of the many isoforms
that are found in association with different host
species and/or prion strains,or generated in vitro,for
which one might propose the generic designation
Spontaneous forms of prion disease
The most common form of human prion disease is the
so-called sporadic Creutzfeldt–Jakob disease (sCJD),
which occurs with an incidence ofabout 1–2 per million
individuals per year.It is considered to be spontaneous
because no epidemiological evidence for association
with any exogenous factors, in particular animal or
PrPScin homogenates31,or partially purified prepara-
tions,of prion-infected hamster brains14and brains of
fatal familial insomnia patients32,which suggests that
PrPScis not required for infectivity33.However,failure to
detect PrPScin infectious brain homogenates,or frac-
tions thereof,does not disprove that PrPScis all or part
of the infectious agent unless it can be shown that the
detection limits for PrPScare commensurate with
those for infectivity,a hurdle that is difficult to over-
come. Within the framework of the protein-only
hypothesis it could be argued that at least some infec-
tivity might be associated with an abnormal isoform
of PrP that is sensitive to PK digestion (designated
sensitive PrPScor sPrPSc)20,34,35.
It would be useful to revise the PrP nomenclature
in the near future.As PrPScimplies ‘scrapie-specific
PrP’and therefore relates to the sheep disease,some
Box 1 | Prion nomenclature
The α-helix-rich form ofPrP,represented by PrPC.
The β-sheet-rich forms ofPrP can be generated from the oxidized and from the reduced form ofPrP by exposure to
various chemical treatments.They can form fibrillary structures,particularly when amino-terminally truncated.
The physiologically occurring,mainly GPI-linked form ofPrP,or prion protein,that can be glycosylated on one or both
oftwo asparagine residues with a variety ofglycans.As shown by NMR and X-ray crystallography,it is rich in α-helical
structure and contains only a little β-sheet structure.
A designation I propose,for any stable form of PrP that differs from PrPConly by virtue of its conformation but not
primary structure.Such differences may currently be detected by a variety of methods,such as reactivity to certain
monoclonal antibodies,conformation-dependent immunoassay,susceptibility to proteinases,including the location
of cleavage site(s),and optical measurements such as infra-red or circular dichroism.PrPisocomprises,among others,
PrP-res,PrPScor sPrPSc,as defined below.
An isoform ofPrPCthat is almost invariably detected in TSE-infected tissues and cells.It comprises a carboxy-proximal
segment ofabout 140 residues that is resistant to defined conditions ofPK treatment.The term PrPScis used by some
interchangeably with prion,a usage that should be avoided.PrPScdesignates a structure,prion is a functional concept.
The implication that a particular form ofPrP is the only essential constituent ofthe prion remains to be proven.
The PrP fragment remaining after controlled PK digestion ofPrPSc.
Alternative designation for PrPSc,that has been proposed to generalize the term for all types ofTSEs and not only scrapie.
The designation for PrPCand forms ofPrP that are equally susceptible to PK digestion.
A hypothetical isoform ofPrP that is the essential component ofthe TSE agent or prion.
The gene encoding PrP.
Genotype in which both copies ofthe PrP gene are inactivated or ablated.
Denotes recombinant PrP.When produced in Escherichia coliit lacks the GPI anchor and the glycan residues.
A term used by Prusiner to designate a protease-sensitive isoform ofPrP that is detected in prion-infected tissue.
This terminology is contradictory because PrPScwas originally defined as a protease-resistant entity.
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The proponents of the virus or virino hypothesis
predicate that these agents are ubiquitous and that,
in the case of sCJD,the outbreak of disease is promoted
by unknown exogenous factors, in analogy perhaps
to herpesvirus, which is almost ubiquitous in the
population but only rarely gives rise to disease
symptoms.However,there is no evidence to support
Replication of prions in organisms
The protein-only hypothesis proposes that prions
consist of an isoform of PrPC(generically designated
PrP* and commonly assumed to be PrPSc) and that
their replication comes about by a self-propagating
conversion of PrPCto the pathogenic isoform (FIG.2).
According to the refolding model, PrPCunfolds to
some extent and refolds under the influence of a PrPSc
molecule17.The nucleation or seeding model however,
proposes that PrPCis in equilibrium with PrPSc(or a
precursor thereof), with the equilibrium strongly
favouring PrPC,and that PrPScis only stabilized when it
adds to a crystal-like seed of PrPSc.Once a seed is pre-
sent,monomer addition ensues rapidly38.Occasional
cleavage of aggregates must be postulated to explain the
exponential increase of PrPScduring infection39.
The nucleation or seeding model has found con-
vincing experimental support in the case ofyeast prions,
as discussed below, and more recently also with
mammalian prions (REF.40),where in vitroincubation
of the normal form of a protein with a seed of its mis-
folded,fibrillary counterpart leads to rapid conversion,
whereas spontaneous misfolding is a slow process.In
yeast,propagation of yeast prions depends on a crucial
concentration of the chaperone Hsp104 (REFS 41,42);
there is currently no evidence for a similar requirement
In the case of mammalian prions,PrPCexpression
(or even overexpression) by a cell culture or tissue,is not
sufficient to allow prion replication43,44,which might be
due to the absence of a receptor or of an auxiliary host
protein (such as the conjectured protein X; REF 45),or to
a rate ofdegradation ofPrPSc(REFS 43,46)that exceeds the
rate ofits formation,as detailed below.
A high degree of identity between the sequence of
the incoming PrPScand the resident PrPCis often8,47,but
not always,crucial for efficient prion replication.A few,
or even a single,amino acid change in the PrP that is
expressed by a normally susceptible host might confer
protection against prion disease.For example,Cheviot
sheep with two 171Q alleles (171Q/Q) are susceptible
to scrapie, whereas the configurations 171Q/R and
171R/R confer protection48.Prnpo/otransgenic mice
that expressed murine PrPQ167Rat about the same level
as wild-type animals remained healthy and did not
accumulate PrPScafter inoculation with mouse scrapie
prions.Moreover,expression of PrPQ167Raffords partial
protection to mice containing wild-type PrP; it has
been suggested that this is due to sequestration of the
conjectural protein X (REF.49). This protective effect
should not be confused with the partial protection
against sCJD that is afforded by the M/V heterozygosity
human sources of infection,has been uncovered.The
protein-only hypothesis proposes that either normal PrPC
rarely converts spontaneously to PrPSc,or that a somatic
mutation in PrP renders it susceptible to conversion to
PrPSc.Once PrPSchas been formed it is hypothesized
to elicit an autocatalytic conversion process.
All familial forms of prion disease are associated
with one of 20 or more mutations of the PrP gene,of
which most are amino acid substitutions but some are
insertions of supernumerary repeats in the octarepeat
region36. It is not understood how PrP mutations
favour the generation of prions;within the framework
of the protein-only hypothesis it is assumed that the
pathogenic mutations increase the probability of
spontaneous conversion of the mutated PrPCinto the
cognate PrPScform.There is however,no evidence that
any of the mutations significantly destabilize the PrPC
conformation,at least in studies with the recombinant,
a Refolding model
b Seeding model
Figure 2 | Models for the conversion of PrPCto PrPSc.
a | The refolding model. The conformational change is
kinetically controlled, a high activation energy barrier
preventing spontaneous conversion at detectable rates.
Interaction with exogenously introduced PrPSc(blue circle)
causes PrPC(yellow square) to undergo an induced
conformational change to yield PrPSc. This reaction could be
facilitated by an enzyme or chaperone. In the case of certain
mutations in PrPC, spontaneous conversion to PrPSccan
occur as a rare event, explaining why familial Creutzfeldt–
Jacob disease (CJD) or Gerstmann–Sträussler–Sheinker
syndrome (GSS) arise spontaneously, albeit late in life.
Sporadic CJD (sCJD) might arise when an extremely rare
event (occurring in about one in a million individuals per
year) leads to spontaneous conversion of PrPCto PrPSc.
b | The seeding model. PrPC(yellow square) and PrPSc(or a
PrPSc-like molecule; shown as a blue circle) are in equilibrium,
with PrPCstrongly favoured. PrPScis only stabilized when it
adds onto a crystal-like seed or aggregate of PrPSc. Seed
formation is rare; however, once a seed is present, monomer
addition ensues rapidly. To explain exponential conversion
rates, aggregates must be continuously fragmented,
generating increasing surfaces for accretion. Reproduced with
permission from REF.124 © (2002) National Academies of
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Replication of prions in cell lines
Only a few cell lines,including the murine neuroblastoma
line N2a (REF.53),the rat adrenal phaeochromocytoma
line PC12 (REF.54)and the hypothalamic neuronal line
GT1 (REF.55),can propagate scrapie prions — they accu-
mulate and maintain PrPScand infectivity,as shown by
the mouse bioassay — over many passages.Although
most cell lines that are used for prion propagation are of
at position 129 of human PrP because neither M/M nor
V/V is protective50and therefore this is not due to
dominance of an allele.As all clinical cases of variant
CJD (vCJD) have occurred in humans with the
129M/M polymorphism51it is possible that 129V exerts
a dominant-negative effect.This effect is however not
absolute because a subclinical case of vCJD was
detected post-mortem in a 129V/M heterozygote52.
PrPsc per cell (arbitrary units)
k = ln3/tD
k = ln2/tD
k = ln1.4/tD
Figure 3 | The dynamic susceptibility model for prion propagation. Even if the genetic and biochemical prerequisites for prion
replication in a cell population are given, propagation of prions seems to be subject to epigenetic determinants. The dynamic
susceptibility model proposes that the determinants are the relative rates of synthesis (ks) and degradation (kd) of prions in
individual cells, and that stable propagation of prions is only possible within narrow limits of k = ks/kd. The amount of infectivity per
cell can be calculated by P = P0× ektD, where P0is the infectivity content of the cell at the beginning of the cell cycle and tD is the
doubling timeof the host cell. After each cell division, infectivity per cell is halved. To successfully initiate infection, k must be equal
to, or larger than, ln2/tD, otherwise the amount of infectivity per cell would not increase, or continuously diminish, respectively, as
the cell divides. However, if k > tD, there will be a steady accumulation of infectious agent, which, if not arrested by an eventual
decrease in k, must lead to the death of the cell. In the figure, we assume that at the time of infection k = ln3/tD, resulting in an
overall logarithmic increase in the cellular content of infectivity. Starting after the third division, we plot the outcome if k continues at
ln3/tD(blue line), resulting in continuous accumulation of infectivity until the lethal limit is reached, if k diminishes to ln2/tD(red line),
leading to a stabilization of the infectivity level averaged over time, and if k decreases to ln1.4/tD(green line), leading to a decrease
of infectivity below the limits of detection (dashed line). An infected cell population is a mixture of cells with a wide variety of k
values. Infected cells secrete infectivity into the medium, infecting as-yet-uninfected cells59. Infected cells may die or lose their
infectivity, but the infection is maintained by cells with appropriate k values. The population is thus in a dynamic equilibrium.
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Conversion of PrPCto PrPScin cell-free systems
In vitro conversion of PrPCinto a PrPSc-like confor-
mation by incubation of PrPCor PrPC-containing
brain homogenates with PrPSchas been achieved.
Using 35S-labelled PrPC, Caughey and colleagues
showed that the conversion product resembles the
PrPSctemplate in regard to its electrophoretic mobility
pattern after PK digestion. As the amount of newly
formed PrPScremained sub-stoichiometric relative to the
amount of template64it is not surprising that a possible
increase in infectivity has not been reported.Modified
protocols have also been described for PrPSc-elicited
in vitroconversion ofPrPC,such as the cyclic amplifica-
tion procedure,in which samples are repeatedly sonicated
and incubated to give an overall amplification of30-fold
or sometimes more65.Supattapone and co-workers used
a similar protocol,but without sonication,to amplify
PrPSc,and reported that conversion was stimulated by
an RNA fraction from mammalian tissue and abolished
by RNases66. This indicates that other components
might be required,either in a structural or catalytic role,
to enable or promote in vitroconversion.So far it has
not been reported that an increase in the concentration
ofPrPScresults in an increase in infectivity.The discovery
of siRNAs and microRNAs,which would have escaped
notice in earlier analyses ofprion preparations,owing to
both their size and their host origin,provides candidates
for the hypothetical co-prion that has been proposed by
the UNIFIED THEORY18.
It was recently reported that recombinant truncated
PrPC(residues 89 to 230) could be converted in vitrointo
a β-sheet-rich fibrillary form.Injection of this material
into wild-type mice did not cause disease; however,
inoculation into transgenic mice overexpressing the PrP
89–230 fragment at a level 15-fold greater than that of
PrPcin wild-type mice elicited scrapie-like disease after
380–660 days.Brain homogenate from these sick trans-
genic mice caused scrapie in wild-type mice after 150
days,indicating that a more efficient form of infectious
agent was generated in the transgenic mice67.Although
non-injected transgenic controls failed to exhibit clinical
signs up to ~500 days,it was not reported whether these
controls succumbed to spontaneous disease at a later
time,whether they had histopathological changes in
their brains or whether homogenates from their brains
could elicit disease when injected into transgenic or
wild-type mice.The possibility that a spontaneous,
perhaps subclinical, form of scrapie can develop in
transgenic mice overexpressing PrP, and that this
process can be accelerated by the administration of
PrP isoforms,has still to be considered.
One of the many remarkable features of prion diseases
is the existence of distinct prion strains, which were
originally characterized by incubation time and the
neuropathology that they elicit in a particular host68.
The finding that several different strains can be propa-
gated indefinitely in hosts that are homozygous for the
PrP gene (Prnp) has been used to support the existence
of a nucleic-acid-containing agent;in the protein-only
neuronal origin,it is remarkable that the mouse fibroblast
lines NIH/3T3 and L929 support prion propagation56
and that a rabbit kidney epithelial line expressing ovine
PrP transgenes can propagate sheep scrapie prions57.
Interestingly,N2a lines and sublines are susceptible
to certain murine prion strains,such as RML,but not
to other strains,such as Me7 (REFS 58,59),87V or 22A
(REF.60),whereas L929 cells propagate RML,ME7 and
22L but not 87V prions56.A sensitive and rapid quan-
titative assay for murine RML scrapie prions that is
based on highly susceptible N2a cells has recently been
described and effectively replaces the slow and expensive
Overexpression of PrP is not a required parameter
for rendering cells highly susceptible to prions43,56.
Interestingly, overexpression of PrP in transgenic
mice accelerates the course of scrapie disease but
does not increase susceptibility to prions or the levels
of infectivity or concentrations of PrPScin the
affected brain61. So which property of a cell line
enables it to propagate prions and discriminate
between strains? Similarity between the sequence of
the PrPCthat is expressed in the cell and that of the
prion ‘donor’is crucial — as small differences not only
prevent conversion of the mismatched PrPC(REF. 47)
but also have a dominant-negative effect on the con-
version of matching PrPCin the same cell62. N2a cell
populations are heterogeneous in regard to their
susceptibility to infection43,58,59— cloned sublines can
exhibit a thousand-fold difference in susceptibility —
however,this property is not stable and changes during
prolonged propagation59,which might therefore reflect
an epigenetic mechanism.
The dynamic susceptibility model,which I propose
herewith,suggests that the capacity ofa cell to propagate
prions,all genetic features being equal,depends on the
ratio of prion synthesis to degradation (FIG.3).Contrary
to the earlier belief that PrPScis inherently very stable,
it has become clear that,at least in N2a cells,it has a
half-life ofless than 24 hours43,46.Ifthis property is shared
by prions,then clearly,ifthe rate ofprion formation does
not equal or exceed twice that ofits degradation(assum-
ing both rates to be constant),the infected state will be
eliminated at some time after exposure of cells to
exogenous prions.Conversely,if the rate of formation is
more than twice that of degradation,continuous accu-
mulation of PrPScmay eventually lead to the death of
the cell.So,persistent infection ofa cell might depend on
the maintenance ofa delicate balance ofsynthesis versus
degradation that is subject to both internal and external
factors. The unexpected finding that many murine
prion strains that are readily propagated in mice do not
infect N2a cells that are susceptible to the RML
strain58–60could be explained if,on average,the degrada-
tion of these strains is more rapid, or the synthesis
slower,than that of RML prions.As mentioned above,
PrPScspecies that are associated with different prion
strains have very different susceptibility to PK digestion63.
In the brain, different cells may have subtly different
prion synthesis and degradation rates for the various
This theory proposes that a PrP
isoform,PrP*,is indeed the
essential infectious component,
but that its properties can be
modified by a physically
associated small RNA,the
co-prion,such as a siRNA.
The co-prion would have to be
amplified in the host cell and
remain bound to the newly
formed PrP* to explain the
stability of strains,and different
siRNAs would be responsible for
the phenotypic differences
between prion strains.
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infectious agent that is required to elicit clinical disease
and by the shorter incubation time.By the same criteria,
transmission of a TSE from one species to another is far
less efficient than transmission within the same species
and is sometimes impossible,giving rise to the concept
of a species barrier80.However,the more recent finding
that trans-species inoculation can lead to neuropatho-
logical changes,deposition of PrPScand/or propaga-
tion of infectivity in the absence of clinical disease,
albeit after long incubation times, undermines the
concept of a species barrier, at least of an absolute
one81,82.As it is not possible to determine whether clin-
ical symptoms would or would not appear if the life of
the affected animal could be prolonged sufficiently,the
use of the neutral term ‘asymptomatic disease’is better
than subclinical or preclinical disease.The concept of
species barrier is further complicated by the fact that
some prion strains but not others can cause clinical or
asymptomatic disease even though they are derived
from the same donor species.A striking example is the
greater susceptibility of wild-type mice to human
vCJD than to sCJD prions83.
Prusiner and colleagues showed that the barrier to
transmission of Syrian hamster prions to mice, as
assessed by the development of clinical disease,could
be abrogated by introducing Syrian hamster PrP genes
into wild-type mice;the incubation time was reduced
with increasing concentrations of the hamster PrPC
(REF.8) and even further reduced in the absence of the
resident murine PrP genes10. The interpretation of
these results was that lack of similarity between Syrian
hamster PrP and recipient murine PrP amino acid
sequences hindered the postulated conversion process
and that even if the recipient mouse was transgenic
for Syrian hamster PrPC,the conversion was still inef-
ficient owing to some form of competition by the
mouse PrPC.Such a competitive effect was even more
marked when human CJD prions were inoculated
into transgenic mice expressing human (Tg[HuPrP])
and mouse–human chimeric PrP genes (Tg[MHu2M]),
respectively.Although Tg[HuPrP] mice expressed high
levels of human PrPC(HuPrPC),they were resistant to
human prions but became susceptible after loss of
the mouse PrP gene.By contrast,mice expressing low
levels of the chimeric transgene mouse–human prion
protein (MHu2M) (which contained the carboxy-
terminal portion of the mouse gene) were susceptible
to human prions and disruption of the mouse PrP
gene led to only a small reduction in incubation
times.These findings were explained by postulating
the existence of a species-specific protein X that binds
near the C-terminus and is essential for the conver-
sion process45.However,in the case of the BSE prions,
mice expressing the chimeric PrP construct
mouse–bovine prion protein (MBo2M) were resis-
tant to infection,whereas mice carrying the complete
bovine PrP transgene were susceptible84.Equally dif-
ficult to explain is the finding mentioned above,that
wild-type mice are more susceptible to vCJD than to
sCJD prions,even though the donors have the same
hypothesis,the strain-specific properties would have to
be encoded in a feature of the pathogenic PrP other
than its amino acid sequence,such as its glycosylation
pattern or its conformation.In fact,in many instances
different strains are associated with PrPScspecies that
differ in physicochemical properties such as suscepti-
bility to PK digestion24,electrophoretic mobility after
PK treatment (which indicates different cleavage sites
in the amino-proximal region69–71),stability towards
denaturation agents21or the ratio of di-,mono- and
unglycosylated forms71.The conformation-dependent
immunoassay (CDI) provides a sensitive tool for differ-
entiating between different conformations of PrP that
are associated with distinct prion strains20,72,73.
Within the framework ofthe protein-only hypothesis
each strain is assumed to be associated with a different
isoform of PrP that can convert PrPCto a likeness of
itself.This assumption predicates that there are as many
stable conformations of PrP as there are stable prion
strains that can be propagated in mice of a particular
genotype, perhaps a dozen or more. The notion of
dozens of stable conformations of a protein seems
bizarre to classical protein biochemists thinking in terms
of functional enzymes,but is less odd if one considers
stable non-functional multimers or polymers. The
concept of ‘conformation templating’at the protein level
is supported by the cell-free conversion experiments of
Caughey and colleagues74,the seeding experiments with
PrPC(REF. 40) and particularly by recent experiments
with yeast prions (see below; REFS 75,76).Nonetheless,the
finding that a particular prion strain can be transmitted
between two species without changing strain-specific
properties,even though the PrP sequences of the two
species are very different77, is unexpected because it
implies that the same conformation can be imposed on
PrP molecules with different amino acid sequences
(primacy ofstrain).A further question that is not readily
explained by the protein-only hypothesis in its simplest
form is why different prion strains cause lesions and
PrPScdeposition at different locations in the brain;a role
for the nature of the glycosylation of the misfolded PrP
has been proposed to explain this78.The unanswered
questions in connection with the strain issue have been
used to support the proposal that the infectious agent
has a nucleic-acid component in addition to misfolded
PrP,as,for example,in the unified theory,or the pro-
posal that a virus-like agent is involved.The finding
that selected murine neuroblastoma (N2a) cell lines are
susceptible to some murine prion strains (such as RML)
but not to others (such as Me7)58,59,even though mice
are susceptible to both,might provide an approach to
analysing the basis ofsusceptibility to prions.
Transmissibility of prions
Under ‘natural’circumstances — for example,in sheep
scrapie,chronic wasting disease of mule deer or BSE —
the transmissible agent is likely to be transmitted by
ingestion of contaminated foodstuff79.Experimental
transmission is usually several orders of magnitude
more efficient when administered intracerebrally
than peripherally or orally,as judged by the amount of
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R E V I E W S
compartment, presumably the peripheral nervous
system, is required. Transport of prions from the
peripheral entry site,particularly the digestive tract,to
the lymphoreticular system is attributed to myeloid
dendritic cells94.In the lymphoreticular system,prions
are replicated and accumulated.
B lymphocytes (not necessarily expressing PrPC)
are crucial for efficient peripheral prion spread and
neuroinvasion95. The dependence on lymphotoxin
(LT)-mediated signalling by B cells might explain the
requirement for B cells in peripheral pathogenesis:it is
the follicular dendritic cells (FDCs) that accumulate
PrPScafter scrapie infection96,and maturation of FDCs
requires signalling by B cells expressing LTα/LTβtrimers
on their surface97.Indeed,block of LTβ signalling by
administration ofsoluble LTβR-immunoglobulin causes
depletion of mature FDCs and markedly impairs
neuroinvasion and accumulation of peripheral PrPSc
and infectivity98,99.FDCs are crucial for efficient disease
progression after oral scrapie challenge,but only within
a short period oftime100.
After amplification in the spleen,prions are trans-
ferred to the CNS, presumably via the sympathetic
nervous system,which provides the main innervation
of lymphoid organs.Chemical SYMPATHECTOMYdelays the
onset of scrapie,whereas sympathetic hyperinnervation
enhances splenic prion replication and neuroinvasion101.
Massive peripheral inoculation of prions can however,
bypass the lymphoreticular system altogether and col-
onize the CNS,as shown by the susceptibility of mice
expressing PrP only in neuronal tissue102.
The major pathological changes resulting from prion
disease are found in the CNS,with vacuolation,neuronal
cell death and astrocytosis being the most common.As
abrogation ofPrP expression is not deleterious,whether
it is inborn9or elicited post-natally103,there is no reason
to believe that depletion of PrPCowing to its conversion
to PrPSc,if indeed such depletion occurs,is pathogenic.
This raises the obvious question of whether PrPScis
toxic.Several findings cast doubt on this possibility.A
first observation was that,although wild-type animals
succumbed to scrapie about 22 weeks after inoculation,
mice that were heterozygous for the PrP-knockout allele
accumulated levels of PrPScand infectivity as high as
wild-type mice by 20 weeks after inoculation and yet
survived without symptoms for more than 41 weeks104.
When inoculated with prions,PrP-knockout mice with
a PrP-expressing graft in their brains showed scrapie
pathology,PrPScand infectivity in the graft,whereas the
surrounding tissue that did not contain PrP showed no
changes,even when close to deposits of PrPScthat had
been exported from the graft105.Even more impressive is
the finding that mice in which neuronal expression of
PrP was prevented by conditional cre-lox knockout
about 7–8 weeks after intracerebral inoculation accumu-
lated vast amounts of infectivity and PrPScin astrocytes
without exhibiting clinical symptoms or evidence of
neuronal damage106.All these findings indicate that
neurons that are devoid ofPrPCare not affected by PrPSc.
Although PrP is essential for the propagation of
prions and progression ofdisease,other genes in various
loci of the mouse genome influence features such as
the incubation time and, perhaps, susceptibility to
infection;none ofthese genes has yet been identified85–88.
Ten years ago Reed Wickner proposed that unexplained
instances of ‘cytoplasmic inheritance’in yeast and other
fungi were due to the same phenomenon that had been
proposed to underlie prion diseases — a self-perpetuating
conformational change of a protein89,90.Although a
rare event,a particular protein in a yeast cell can flip
into a conformationally altered state,causing conver-
sion of its counterparts to the same state and giving rise
to functionally inactive aggregates.The resulting pheno-
type and the conformationally altered protein are
transmitted to the offspring of the ‘mutated’cell.In the
case of the translation termination factor Sup35,for
example, the conformational conversion results in
fibril formation and can be reproduced in a cell-free
system91.Interestingly,depending on the conditions,
different forms of fibrils can be generated in vitro,and
these forms can be propagated indefinitely by seeding
solutions of the recombinant protein with the fibrils.
When introduced into normal yeast cells the polymer-
ized forms of Sup35 cause them to adopt the ‘mutant’
or [PSI+] phenotype.It is of particular interest that the
different forms of aggregates generated in vitrounder
specific conditions give rise to different phenotypes in
the transformed yeast75,76, demonstrating the link
between protein conformation and phenotype.
It should be noted that yeast proteins showing the
‘prion phenomenon’are not homologous to vertebrate
PrPs at the level of amino acid sequence identity.
Although mammalian prions have the ability to spread
laterally under natural conditions, as is the case in
scrapie and chronic wasting disease,penetrating their
host and using remarkable mechanisms to propagate
within it (see below), there seems to be no natural
mechanism for prion spread in yeast,and introduction
of yeast prions requires sophisticated experimental
intervention.Nonetheless,the findings with yeast prions
support the proposal that mammalian prion strains are
encoded in protein conformation.
Spread of prions in the organism
Under natural conditions, prion disease is mainly
acquired through oral infection. Prions then spread
from the periphery to the central nervous system (CNS;
neuroinvasion) and once there cause neurodegeneration.
PrPCexpression is required not only for prion
replication and pathogenesis but also for transporting
the infectious agent both from the peripheral sites to
the CNS (as shown by PrPC-expressing neurografts in
PrP-knockout mice)92and within the CNS93.However,
reconstitution ofPrnpo/omice with wild-type bone mar-
rowis insufficient to restore neuroinvasion in engrafted
Prnpo/omice, although the capacity of the spleen to
accumulate prions of the RML strain is reconstituted92.
This indicates that PrPCexpression in an additional
A chemical or surgical procedure
that destroys innervation by the
sympathetic nervous system.
deposits of protein.PrP in
amyloid form is found in some
but not all forms of prion disease
in humans and animals.
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VOLUME 2 | NOVEMBER 2004 | 869
R E V I E W S
Prion diseases differ from other proteinopathies in that
they are transmissible,not only under experimental
conditions but also naturally,predominantly by in-
gestion.Although in certain cases the inception of an
experimental amyloidosis can be accelerated by the
injection of amyloid into a predisposed host102,mam-
malian prions are exceptional in that they are able to
enter their hosts by natural portals and make their way
from the gut to the brain,using intermediate tissues for
amplification.In the case of microorganisms,including
viruses, acquisition of such sophisticated entry and
transport mechanisms is attributed to evolutionary
processes — genomic mutations and selection of
mutants that most readily enter their host and find a
suitable niche in which to efficiently replicate and/or
perpetuate.However,if the prion consists solely of a
protein encoded by the genome of its host,what drives
the prion to become efficient in replication and invasion
of its parent? We can only speculate.For example,the
‘misfolded’form ofPrP might have originated as a ‘mes-
senger’protein that on the one hand has or had a physio-
logical function,but on the other hand has a malignant
potential that is rarely realized and was not selected
against because evolutionary pressure does not operate
efficiently at post-reproductive age.It has been proposed
that,in yeast,a prion-like phenomenon involving Sup35
might confer a selective advantage on yeast growing
under fluctuating environmental conditions121.Another
possibility is that PrP/PrPScis derived from an ancient
pathogen,the genetic material of which was integrated
into the genome of its host and harnessed to fulfil a
useful function while its pathogenic potential was
minimized. More trivially, but equally improbably,
mammalian prion disease could result from the combi-
nation of the natural propensity of proteins to assume
β-sheet-rich conformations122,a failure of the organism
to prevent their formation and accumulation in some
cases, and the coincidental ability of the isoform to
penetrate organisms and cells through natural portals.
Toxicity due to a PrP conformer other than PrPSchas
been considered.Targeting ofPrP to the cytosol resulted
in rapidly lethal neurodegeneration (albeit without
accumulation of PrPSc), and proteasome inhibition
induced a slightly protease-resistant cytoplasmic PrP
species in cultured cells107,108.Therefore,prion toxicity
was proposed to start with retrotranslocation of PrPC
from the endoplasmic reticulum to the cytosol,in con-
junction with impaired proteasomal function.However,
other studies have found that cytosolic PrP retains its
secretory leader peptide and does not contain a GPI
anchor,suggesting that it never enters the endoplasmic
reticulum109.Moreover,the toxicity of cytosolic PrP has
been contested110,111.Lingappa and co-workers found
that PrPCassumes a transmembrane topology (CtmPrP),
the concentration of which correlates with neuro-
toxicity112,113.These data have been taken to indicate
that CtmPrP represents an important toxic moiety.
At least twenty human diseases are associated with
the deposition of β-sheet-rich protein aggregates,or
AMYLOID114,115.They are frequently designated ‘confor-
mational diseases’or ‘proteinopathies’,although it is
not always clear whether,or to what extent,the mis-
folded proteins are the cause of the disease rather than
TSEs or prion diseases should be distinguished from
PrP proteinopathies14,116,117.To qualify as a TSE or prion
disease,transmissibility within the same or a different
species must be demonstrated, a hurdle that cannot
always be overcome, particularly in human disease.
Although all human familial CJD cases co-segregate
with PRNP mutations,it is possible that some PRNP
mutations cause neurodegenerative diseases that are not
transmissible and are therefore proteinopathies rather
than prion diseases; many such examples have been
described in the mouse116and are exemplified by the
octapeptide repeat expansion mutants of both mouse118
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The author declares no competing financial interests.
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