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Vol. 95, Issue 23, 13363-13383, November 10, 1998
Nobel Lecture
Prions
Stanley B.
Prusiner
Departments of Neurology and of Biochemistry and Biophysics,
University of California, San Francisco, CA 94143
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ABSTRACT |
Prions are unprecedented infectious pathogens that cause a group of
invariably fatal neurodegenerative diseases by an entirely novel
mechanism. Prion diseases may present as genetic, infectious, or
sporadic disorders, all of which involve modification of the prion
protein (PrP). Bovine spongiform encephalopathy (BSE), scrapie of
sheep, and Creutzfeldt-Jakob disease (CJD) of humans are among the
most notable prion diseases. Prions are transmissible particles that
are devoid of nucleic acid and seem to be composed exclusively of a
modified protein (PrPSc). The normal, cellular PrP
(PrPC) is converted into PrPSc through a
posttranslational process during which it acquires a high 
-sheet
content. The species of a particular prion is encoded by the sequence
of the chromosomal PrP gene of the mammals in which it last replicated.
In contrast to pathogens carrying a nucleic acid genome, prions appear
to encipher strain-specific properties in the tertiary structure of
PrPSc. Transgenetic studies argue that PrPSc
acts as a template upon which PrPC is refolded into a
nascent PrPSc molecule through a process facilitated by
another protein. Miniprions generated in transgenic mice expressing
PrP, in which nearly half of the residues were deleted, exhibit unique
biological properties and should facilitate structural studies of
PrPSc. While knowledge about prions has profound
implications for studies of the structural plasticity of proteins,
investigations of prion diseases suggest that new strategies for the
prevention and treatment of these disorders may also find application
in the more common degenerative diseases.
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ARTICLE |
The torturous path of the scientific investigation that led to an
understanding of familial Creutzfeldt-Jakob disease (CJD) chronicles a
remarkable scientific odyssey. By 1930, the high incidence of familial
(f) CJD in some families was known (1, 2). Almost 60 years were to pass
before the significance of this finding could be appreciated (3-5).
CJD remained a curious, rare neurodegenerative disease of unknown
etiology throughout this period of three score years (6). Only with
transmission of disease to apes after inoculation of brain extracts
prepared from patients who died of CJD did the story begin to unravel
(7).
Once CJD was shown to be an infectious disease, relatively little
attention was paid to the familial form of the disease since most cases
were not found in families. It is interesting to speculate how the
course of scientific investigation might have proceeded had
transmission studies not been performed until after the molecular genetic lesion had been identified. Had that sequence of events transpired, then the prion concept, which readily explains how a single
disease can have a genetic or infectious etiology, might have been
greeted with much less skepticism (8).
Epidemiologic studies designed to identify the source of the CJD
infection were unable to identify any predisposing risk factors, although some geographic clusters were found (9-12). Libyan Jews living in Israel developed CJD about 30 times more frequently than
other Israelis (13). This finding prompted some investigators to
propose that the Libyan Jews had contracted CJD by eating lightly cooked brain from scrapie-infected sheep when they lived in Tripoli prior to emigration. Subsequently, the Libyan Jewish patients were all
found to carry a mutation at codon 200 in their prion protein (PrP)
gene (14-16).
My own interest in the subject began with a patient dying of CJD in the
fall of 1972. At that time, I was beginning a residency in neurology
and was most impressed by a disease process that could kill my patient
in 2 months by destroying her brain while her body remained unaffected
by this process. No febrile response, no leukocytosis or pleocytosis,
no humoral immune response, and yet I was told that she was infected
with a "slow virus."
Slow Viruses.
The term "slow virus" had been coined by
Bjorn Sigurdsson in 1954 while he was working in Iceland on scrapie and
visna of sheep (17). Five years later, William Hadlow had suggested
that kuru, a disease of New Guinea highlanders, was similar to scrapie and thus, it, too, was caused by a slow virus (18). Seven more years
were to pass before the transmissibility of kuru was established by
passaging the disease to chimpanzees inoculated intracerebrally (19).
Just as Hadlow had made the intellectual leap between scrapie and kuru,
Igor Klatzo made a similar connection between kuru and CJD (20). In
both instances, these neuropathologists were struck by the similarities
in light microscopic pathology of the central nervous system (CNS) that
kuru exhibited with scrapie or CJD. In 1968, the transmission of CJD to
chimpanzees after intracerebral inoculation was reported (7).
In scrapie, kuru, CJD, and all of the other disorders now referred to
as prion diseases (Table 1),
spongiform degeneration and astrocytic gliosis is found upon
microscopic examination of the CNS (Fig.
1) (21). The degree of
spongiform degeneration is quite variable, whereas the
extent of reactive gliosis correlates with the degree of neuron loss
(22).
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Fig. 1.
Neuropathologic changes in Swiss mice after
inoculation with RML scrapie prions. (a)
Hematoxylin and eosin stain of a serial section of the
hippocampus shows spongiform degeneration of the neuropil, with
vacuoles 10-30 µm in diameter. Brain tissue was immersion fixed in
10% buffered formalin solution after the animals had been sacrificed
and was then embedded in paraffin. Py, pyramidal cell layer; SR,
stratum radiatum. (b) Glial fibrillary acidic protein (GFAP)
immunohistochemistry of a serial section of the hippocampus shows
numerous reactive astrocytes. (Bar in b = 50 µm
and also applies to a.) Photomicrographs were prepared
by Stephen J. DeArmond.
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Prions: A Brief Overview.
Before proceeding with a detailed
discussion of our current understanding of prions causing scrapie and
CJD, I provide a brief overview of prion biology. Prions are
unprecedented infectious pathogens that cause a group of invariably
fatal neurodegenerative diseases mediated by an entirely novel
mechanism. Prion diseases may present as genetic, infectious, or
sporadic disorders, all of which involve modification of the prion
protein (PrP), a constituent of normal mammalian cells (23). CJD
generally presents as progressive dementia, whereas scrapie of sheep
and bovine spongiform encephalopathy (BSE) are generally manifest as
ataxic illnesses (Table 1) (24).
Prions are devoid of nucleic acid and seem to be composed exclusively
of a modified isoform of PrP designated
PrPSc.
The normal, cellular PrP, denoted
PrPC, is converted into
PrPSc through a process whereby a portion of its
-helical and coil structure is refolded into -sheet (25). This
structural transition is accompanied by profound changes in the
physicochemical properties of the PrP. The amino acid sequence of
PrPSc corresponds to that encoded by the PrP gene
of the mammalian host in which it last replicated. In contrast to
pathogens with a nucleic acid genome that encode strain-specific
properties in genes, prions encipher these properties in the tertiary
structure of PrPSc (26-28). Transgenetic studies
argue that PrPSc acts as a template upon which
PrPC is refolded into a nascent
PrPSc molecule through a process facilitated by
another protein.
More than 20 mutations of the PrP gene are now known to cause the
inherited human prion diseases, and significant genetic linkage has
been established for five of these mutations (4, 16, 29-31). The prion
concept readily explains how a disease can be manifest as a heritable
as well as an infectious illness.
Resistance of Scrapie Agent to Radiation.
My fascination with
CJD quickly shifted to scrapie once I learned of the remarkable
radiobiological data that Tikvah Alper and her colleagues had collected
on the scrapie agent (32-34). The scrapie agent had been found to be
extremely resistant to inactivation by UV and ionizing radiation, as
was later shown for the CJD agent (35). It seemed to me that the most
intriguing question was the chemical nature of the scrapie agent;
Alper's data had evoked a torrent of hypotheses concerning its
composition. Suggestions as to the nature of the scrapie agent ranged
from small DNA viruses to membrane fragments to polysaccharides to proteins, the last of which eventually proved to be correct (36-42).
Scrapie of sheep and goats possesses a history no less fascinating than
that of CJD. The resistance of the scrapie agent to inactivation by
formalin and heat treatments (43), which were commonly used to produce
vaccines against viral illnesses, suggested that the scrapie agent
might be different from viruses, but it came at a time before the
structure of viruses was understood. Later, this resistance was
dismissed as an interesting observation but of little importance since
some viruses can survive such treatments; indeed, this was not an
unreasonable viewpoint. More than two decades were to pass before
reports of the extreme resistance of the scrapie agent to inactivation
by radiation again trumpeted the novelty of this infectious pathogen.
Interestingly, British scientists had argued for many years about
whether natural scrapie was a genetic or an infectious disease
(44-46). Scrapie, like kuru and CJD, produced death of the host
without any sign of an immune response to a "foreign infectious agent."
My initial studies focused on the sedimentation properties of scrapie
infectivity in mouse spleens and brains. From these studies, I
concluded that hydrophobic interactions were responsible for the
nonideal physical behavior of the scrapie particle (47, 48). Indeed,
the scrapie agent presented a biochemical nightmare: infectivity was
spread from one end to the other of a sucrose gradient and from the
void volume to fractions eluting at 5-10 times the included volume of
chromatographic columns. Such results demanded new approaches and
better assays (49).
Bioassays.
As the number of hypotheses about the molecular
nature of the scrapie agent began to exceed the number of laboratories
working on this problem, the need for new experimental approaches
became evident. Much of the available data on the properties of the
scrapie agent had been gathered on brain homogenates prepared from mice with clinical signs of scrapie. These mice had been inoculated 4-5
months earlier with scrapie agent that originated in sheep but had been
passaged multiple times in mice. Once an experiment was completed on
these homogenates, an additional 12 months was required to obtain the
results of an endpoint titration in mice (50). Typically, 60 mice were
required to determine the titer of a single sample. This slow, tedious,
and expensive system discouraged systematic investigation.
Although the transmission of scrapie to mice had ushered in a new era
of research, the 1.5- to 2-year intervals between designing experiments
and obtaining results discouraged sequential studies. Infrequently, the
results of one set of experiments were used as a foundation for the
next and so on. Moreover, the large number of mice needed to measure
the infectivity in a single sample prevented studies where many
experiments were performed in parallel. These problems encouraged
publication of inconclusive experimental results.
In 1972, when I became fascinated by the enigmatic nature of the
scrapie agent, I thought that the most direct path to determining the
molecular structure of the scrapie agent was purification. Fortunately,
I did not appreciate the magnitude of that task, although I had
considerable experience and training in the purification of enzymes
(51). Although many studies had been performed to probe the
physicochemical nature of the scrapie agent by using the mouse endpoint
titration system, few systematic investigations had been performed on
the fundamental characteristics of the infectious scrapie particle
(42). In fact, 12 years after introduction of the mouse bioassay, there
were few data on the sedimentation behavior of the scrapie particle.
Since differential centrifugation is frequently a useful initial step
in the purification of many macromolecules, some knowledge of the
sedimentation properties of the scrapie agent under defined conditions
seemed mandatory. To perform such studies, Swiss mice were inoculated
intracerebrally with the Chandler isolate of scrapie prions and the
mice were sacrificed about 30 and 150 days later, when the titers in
their spleens and brains, respectively, were expected to be at maximal levels. The two tissues were homogenized, extracted with detergent, and
centrifuged for increasing times and speeds (47, 52). The disappearance
of scrapie infectivity was measured in supernatant fractions by
endpoint titration, which required 1 year to score.
Incubation time assays in hamsters.
In view of these daunting
logistical problems, the identification of an inoculum that produced
scrapie in the golden Syrian hamster (SHa) in 
70 days after
intracerebral inoculation proved to be an important advance (53, 54)
once an incubation time assay was developed (55, 56). In earlier
studies, SHa had been inoculated with prions, but serial passage with
short incubation times was not reported (57). Development of the
incubation time bioassay reduced the time required to measure prions in
samples with high titers by a factor of 5: only 70 days were required instead of the 360 days previously needed. Equally important, 4 animals
could be used in place of the 60 that were required for endpoint
titrations, making possible a large number of parallel experiments.
With this bioassay, research on the nature of the scrapie agent was
accelerated nearly 100-fold and the hamster with high prion titers in
its brain became the experimental animal of choice for biochemical studies.
The incubation time assay enabled development of effective purification
schemes for enriching fractions for scrapie infectivity. It provided a
means to assess quantitatively those fractions that were enriched for
infectivity and those that were not. Such studies led rather rapidly to
the development of a protocol for separating scrapie infectivity from
most proteins and nucleic acids. With a 100-fold purification of
infectivity relative to protein, >98% of the proteins and
polynucleotides were eliminated, permitting more reliable probing of
the constituents of these enriched fractions.
The Prion Concept.
As reproducible data began to accumulate
indicating that scrapie infectivity could be reduced by procedures that
hydrolyze or modify proteins but was resistant to procedures that alter nucleic acids, a family of hypotheses about the molecular architecture of the scrapie agent began to emerge (58). These data established, for
the first time, that a particular macromolecule was required for
infectivity and that this macromolecule was a protein. The experimental
findings extended earlier observations on resistance of scrapie
infectivity to UV irradiation at 250 nm (33) in that the four different
procedures used to probe for a nucleic acid are based on physical
principles that are independent of UV radiation damage.
Once the requirement of protein for infectivity was established, I
thought that it was appropriate to give the infectious pathogen of
scrapie a provisional name that would distinguish it from both viruses
and viroids. After some contemplation, I suggested the term
"prion," derived from proteinaceous and
infectious (58). At that time, I defined prions as proteinaceous infectious particles that resist inactivation by procedures that modify nucleic acids. I never imagined the irate reaction of some scientists to the word "prion"
it was truly remarkable!
Current definitions.
Perhaps the best current working
definition of a prion is a proteinaceous infectious particle that lacks
nucleic acid (28). Because a wealth of data supports the contention
that scrapie prions are composed entirely of a protein that adopts an
abnormal conformation, it is not unreasonable to define prions as
infectious proteins (25, 27, 59, 60). But I hasten to add that we still
cannot eliminate a small ligand bound to PrPSc as
an essential component of the infectious prion particle. Learning how
to renature PrPSc accompanied by restoration of
prion infectivity or to generate prion infectivity de novo
by using a synthetic polypeptide should help address this
as-yet-unresolved issue (61). From a broader perspective, prions are
elements that impart and propagate conformational variability.
Although PrPSc is the only known
component of the infectious prion particles, these unique pathogens
share several phenotypic traits with other infectious entities such as
viruses. Because some features of the diseases caused by prions and
viruses are similar, some scientists have difficulty accepting the
existence of prions despite a wealth of scientific data supporting this concept (62-67).
Families of hypotheses.
Once the requirement for a protein was
established, it was possible to revisit the long list of hypothetical
structures that had been proposed for the scrapie agent and to
eliminate carbohydrates, lipids, and nucleic acids as the infective
elements within a scrapie agent devoid of protein (58). No longer could
structures such as a viroid-like nucleic acid, a replicating
polysaccharide, or a small polynucleotide surrounded by a carbohydrate
be entertained as reasonable candidates to explain the puzzling
properties of the scrapie agent (58, 68).
The family of hypotheses that remained after identifying a protein
component was still large and required a continued consideration of all
possibilities in which a protein was a critical element (49). The prion
concept evolved from a family of hypotheses in which an infectious
protein was only one of several possibilities. With the accumulation of
experimental data on the molecular properties of the prion, it became
possible to discard an increasing number of hypothetical structures. In
prion research, as in many other areas of scientific investigation, a
single hypothesis is all too often championed at the expense of a
reasoned approach that requires entertaining a series of complex
arguments until one or more can be discarded on the basis of
experimental data (69).
Genes and DNA.
In some respects, the early development of the
prion concept mirrors the story of DNA (70-72). Prior to the
acceptance of DNA as the genetic material of life (73, 74), many
scientists asserted that the DNA preparations must be contaminated with
protein that is the true genetic material (75). The prejudices of these scientists were similar in some ways to those of investigators who have
disputed the prion concept. But the scientists who attacked the
hypothesis that genes are composed of DNA had no well proven alternative; they had only a set of feelings derived from poorly substantiated data sets that genes are made of protein. In contrast, those who attacked the hypothesis that the prion is composed only of
protein had more than 30 years of cumulative evidence showing that
genetic information in all organisms on our planet is encoded in DNA
and that biological diversity resides in DNA. Studies of viruses and
eventually viroids extended this concept to these small infectious
pathogens (76) and showed that genes could also be composed of RNA (77,
78).
Discovery of the Prion Protein.
The discovery of the prion
protein transformed research on scrapie and related diseases (79, 80).
It provided a molecular marker that was subsequently shown to be
specific for these illnesses as well as the major, and very likely the
only, constituent of the infectious prion.
PrP 27-30 was discovered by enriching fractions from SHa brain for
scrapie infectivity (79, 80). This protein is the protease-resistant core of PrPSc and has an apparent molecular mass
of 27-30 kDa (81, 82). Although resistance to limited proteolysis
proved to be a convenient tool for many but not all studies, use of
proteases to enrich fractions for scrapie infectivity created a problem
when the NH2-terminal sequence of PrP 27-30 was
determined (81). The ragged NH2 terminus of PrP
27-30 yielded three sets of signals in almost every cycle of the Edman
degradation. Only after these signals were properly interpreted and
placed in correct register could a unique sequence be assigned for the
NH2 terminus of PrP 27-30. The determination of
the amino acid sequence of the NH2 terminus of
PrP 27-30 made subsequent molecular cloning studies of the PrP gene
possible (83, 84).
The finding that PrP mRNA levels were similar in normal uninfected and
scrapie-infected tissues caused some investigators to argue that PrP
27-30 was not related to the infectious prion particle (83). An
alternate interpretation prompted a search for a prion protein in
uninfected animals that was found to be protease sensitive and soluble
in nondenaturing detergents, unlike PrP 27-30. This isoform was
designated PrPC (Fig.
2) (84, 85). Deduced amino acid
sequences from PrP cDNA as well as immunoblotting studies revealed that
PrP 27-30 was NH2-terminally truncated and was
derived from a larger molecule, designated PrPSc,
that was unique to infected animals (81, 82, 84-86).
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Fig. 2.
Prion protein isoforms. (A) Western
immunoblot of brain homogenates from uninfected (lanes 1 and 2) and
prion-infected (lanes 3 and 4) SHa. Samples in lanes 2 and 4 were
digested with 50 µg/ml proteinase K for 30 min at 37°C.
PrPC in lanes 2 and 4 was completely hydrolyzed under these
conditions, whereas approximately 67 amino acids were digested from the
NH2 terminus of PrPSc to generate PrP 27-30.
After polyacrylamide gel electrophoresis (PAGE) and electrotransfer,
the blot was developed with anti-PrP R073 polyclonal rabbit antiserum.
Molecular size markers are in kilodaltons (kDa). (B) Bar
diagram of SHaPrP, which consists of 254 amino acids. Attached
carbohydrate (CHO) and a glycosyl-phosphatidylinositol (GPI) anchor are
indicated. After processing of the NH2 and COOH termini,
both PrPC and PrPSc consist of 209 residues.
After limited proteolysis, the NH2 terminus of
PrPSc is truncated to form PrP 27-30, which is composed of
approximately 142 amino acids.
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With the discovery of PrP 27-30 and the production of antiserum (87),
brains from humans and animals with putative prion diseases were
examined for the presence of this protein. In each case, PrP 27-30 was
found, and it was absent in other neurodegenerative disorders such as
Alzheimer's disease, Parkinson's disease, and amyotrophic lateral
sclerosis (88-91). The extreme specificity of
PrPSc for prion disease is an important feature
of the protein and is consistent with the postulated role of
PrPSc in both the transmission and pathogenesis
of these illnesses (Table 2)
(92).
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Table 2.
Arguments for prions being composed largely, if not
entirely, of PrPSc molecules and devoid of
nucleic acid
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The accumulation of PrPSc contrasts markedly with
that of glial fibrillary acidic protein (GFAP) in prion disease. In
scrapie, GFAP mRNA and protein levels rise as the disease progresses
(93), but the accumulation of GFAP is neither specific nor necessary for either the transmission or the pathogenesis of disease. Mice deficient for GFAP show no alteration in their incubation times (94,
95).
Except for PrPSc, no macromolecule has been found
in tissues of patients dying of the prion diseases that is specific for
these encephalopathies. In searches for a scrapie-specific nucleic
acid, cDNAs have been identified that are complementary to mRNAs
encoding other proteins with increased expression in prion disease
(96-98). Yet none of the proteins has been found to be specific for
prion disease.
Attempts to Falsify the Prion Hypothesis.
Numerous attempts to
disprove the prion hypothesis over the past 15 years have failed. Such
studies have tried unsuccessfully to separate scrapie infectivity from
protein and more specifically from PrPSc. No
preparations of purified prions containing less than one PrPSc molecule per ID50
unit have been reported (99), and no replication of prions in
PrP-deficient (Prnp0/0) mice was found
(100-104).
Copurification of PrP 27-30 and scrapie infectivity demands that the
physicochemical properties as well as antigenicity of these two
entities be similar (105) (Table 2). The results of a wide array of
inactivation experiments demonstrated the similarities in the
properties of PrP 27-30 and scrapie infectivity (61, 106-109). To
explain these findings in terms of the virus hypothesis, it is
necessary to postulate either a virus that has a coat protein which is
highly homologous with PrP or a virus that binds tightly to
PrPSc. In either case, the PrP-like coat protein
or the PrPSc/virus complex must display
properties indistinguishable from PrPSc alone.
With each species that the putative virus invades, it must incorporate
a new PrP sequence during replication.
Search for a scrapie-specific nucleic acid.
The inability to
inactivate preparations highly enriched for scrapie infectivity by
procedures that modify nucleic acids militates against the existence of
a scrapie-specific nucleic acid (58, 110, 111). To explain the findings
in terms of a virus, one must argue that PrPSc or
an as-yet-undetected PrP-like protein of viral origin protects the
viral genome from inactivation. The notion that the putative scrapie
virus encodes a PrP-like protein was refuted by nucleic acid
hybridization studies using a PrP cDNA probe. Less than 0.002 nucleic
acid molecule encoding PrP per ID50 unit was
found in purified preparations of SHa prions (84). To circumvent this finding, it could be hypothesized that the genetic code used by the PrP
gene differs so greatly from that found in the cell that a PrP cDNA
probe failed to detect it in highly purified preparations.
If prions contained a genome with a unique genetic code, then it is
likely that this genome would encode some specialized proteins required
for replication as well as some unique tRNAs. But both UV and ionizing
radiation inactivation studies as well as physical studies have
eliminated the possibility of a large nucleic acid hiding within
purified preparations of prions (110-112). Only oligonucleotides of
fewer than 50 bases were found at a concentration of one molecule per
ID50 unit in prion preparations highly enriched for scrapie infectivity (113, 114). These small nucleic acids were of
variable length and are thought to be degradation byproducts generated
during purification of prions. Failure to find a bona fide genome was
attributed to the unusual properties of the putative viral nucleic acid
or technical incompetence on the part of the investigators who were
unable to find it (63, 115).
PrP amyloid.
In preparations highly enriched for scrapie
infectivity and containing only PrP 27-30 by silver staining of gels
after SDS/PAGE, numerous rod-shaped particles were seen by electron
microscopy after negative staining (Fig.
3) (107). Each of the rods was slightly different, in contrast to viruses, which exhibit extremely uniform structures (116). These irregular rods, composed largely, if
not entirely, of PrP 27-30, were indistinguishable morphologically from many other purified amyloids (117). Studies of the prion rods with
Congo red dye demonstrated that the rods also fulfilled the tinctorial
criteria for amyloid (107), and immunostaining later showed that PrP is
a major component of amyloid plaques in some animals and humans with
prion disease (118-120). Subsequently, it was recognized that the
prion rods were not required for scrapie infectivity (121);
furthermore, the rods were shown to be an artifact of purification
during which limited proteolysis of PrPSc
generated PrP 27-30 that polymerized spontaneously in the presence of
detergent (Fig. 3) (122).
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Fig. 3.
Electron micrographs of negatively stained and
ImmunoGold-labeled prion proteins. (A) PrPC.
(B) PrPSc. Neither PrPC nor
PrPSc forms recognizable, ordered polymers. (C)
Prion rods composed of PrP 27-30 were negatively stained. The prion
rods are indistinguishable from many purified amyloids. (Bar = 100 nm.)
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The idea that scrapie prions were composed of an amyloidogenic protein
was truly heretical when it was introduced (107). Since the prevailing
view at the time was that scrapie is caused by an atypical virus, many
argued that amyloid proteins are mammalian polypeptides and not viral proteins!
Some investigators have argued that the prion rods are synonymous with
scrapie-associated fibrils (123-125) even though morphologic and
tinctorial features of these fibrils clearly differentiated them from
amyloid and as such from the prion rods (126, 127). The
scrapie-associated fibrils were identified by their unique ultrastructure in which two or four subfilaments were helically wound
around each other (126) and were proposed to represent the first
example of a filamentous animal virus (128). After the argument for a
filamentous animal virus causing scrapie faded, it was hypothesized
that a virus induces the formation of PrP amyloid to explain the
accumulation of PrPSc in prion diseases (129).
Besides the lack of evidence for a virus of any shape, no compelling
data have been offered in support of the idea that prion diseases are
caused by a filamentous bacterium called a spiroplasma (130).
Search for the ubiquitous "scrapie virus."
When PrP gene
mutations were discovered to cause familial prion diseases (4), it was
postulated that PrPC is a receptor for the
ubiquitous scrapie virus that binds more tightly to mutant than to wt
PrPC (131). A similar hypothesis was proposed to
explain why the length of the scrapie incubation time was found to be
inversely proportional to the level of PrP expression in transgenic
(Tg) mice and why Prnp0/0 mice are
resistant to scrapie (132). The higher the level of PrP expression, the
faster the spread of the putative virus, which results in shorter
incubation times; conversely, mice deficient for PrP lack the receptor
required for spread of the virus (63). The inability to find virus-like
particles in purified preparations of PrPSc was
attributed to these particles being hidden (115) even though tobacco
mosaic viruses could be detected when one virion was added per
ID50 unit of scrapie prions (121).
Recent studies on the transmission of mutant prions from FFI and
fCJD(E200K) to Tg(MHu2M) mice, which results in the formation of two
different PrPSc molecules (27), has forced a
corollary to the ubiquitous virus postulate. To accommodate this
result, at least two different viruses must reside worldwide, each of
which binds to a different mutant HuPrPC and each
of which induces a different MHu2M PrPSc
conformer when transferred to Tg mice. Even more difficult to imagine
is how one ubiquitous virus might acquire different mutant PrPSc molecules corresponding to FFI or
fCJD(E200K) and then induce different MHu2M PrPSc
conformers upon transmission to Tg mice.
Artificial prions.
To explain the production of artificial
prions from chimeric or mutant PrP transgenes in terms of a virus
(133-135), mutated PrPSc molecules must be
incorporated into the virus. In the case of mice expressing chimeric
PrP transgenes, artificial prions are produced with host ranges not
previously found in nature. Similarly, deleting specific regions of PrP
resulted in the formation of "miniprions" with a unique host range
and neuropathology as described below. The production of artificial
prions that were generated by modifying the PrP gene sequence and
exhibit unique biological properties is another compelling argument
against the proposition that scrapie and CJD are caused by viruses.
Skepticism once well justified.
While the skepticism about
prions was once well justified and formed the basis for a vigorous
scientific debate, the wealth of available data now renders such
arguments moot. In summary, no single hypothesis involving a virus can
explain the findings summarized above (Table 2); instead, a series of
ad hoc hypotheses, virtually all of which can be refuted by
experimental data, must be constructed to accommodate a steadily
enlarging body of data.
It is notable that the search for an infectious pathogen with a nucleic
acid genome as the cause of scrapie and CJD has done little to advance
our understanding of these diseases. Instead, studies of PrP have
created a wealth of data that now explain almost every aspect of these
fascinating disorders. While no single experiment can refute the
existence of the "scrapie virus," all of the data taken together
from numerous experimental studies present an impressive edifice which
argues that the 50-year quest for a virus has failed because it does
not exist!
Prions Defy Rules of Protein Structure.
Once cDNA probes for
PrP became available, the PrP gene was found to be constitutively
expressed in adult, uninfected brain (83, 84). This finding eliminated
the possibility that PrPSc stimulated production
of more of itself by initiating transcription of the PrP gene as
proposed nearly two decades earlier (37). Determination of the
structure of the PrP gene eliminated a second possible mechanism that
might explain the appearance of PrPSc in brains
already synthesizing PrPC. Since the entire
protein coding region was contained within a single exon, there was no
possibility for the two PrP isoforms to be the products of
alternatively spliced mRNAs (82). Next, a posttranslational chemical
modification that distinguishes PrPSc from
PrPC was considered, but none was found in an
exhaustive study (59) and we considered it likely that
PrPC and PrPSc differed
only in their conformation, a hypothesis also proposed earlier (37).
However, this idea was no less heretical than that of an infectious protein.
For more than 25 years, it had been widely accepted that the amino acid
sequence specifies one biologically active conformation of a protein
(136). Yet in scrapie we were faced with the possibility that one
primary structure for PrP might adopt at least two different conformations to explain the existence of both
PrPC and PrPSc. When the
secondary structures of the PrP isoforms were compared by optical
spectroscopy, they were found to be markedly different (25).
Fourier-transform infrared (FTIR) and circular dichroism (CD) studies
showed that PrPC contains about 40% -helix
and little -sheet, whereas PrPSc is composed
of about 30% -helix and 45% -sheet (25, 137). Nevertheless,
these two proteins have the same amino acid sequence!
Prior to comparative studies on the structures of
PrPC and PrPSc, we found by
metabolic labeling studies that the acquisition of
PrPSc protease resistance is a posttranslational
process (138). In our quest for a chemical difference that would
distinguish PrPSc from
PrPC, we found ethanolamine in hydrolysates of
PrP 27-30, which signaled the possibility that PrP might contain a GPI
anchor (139). Both PrP isoforms were found to carry GPI anchors, and
PrPC was found on the surface of cells where it
could be released by cleavage of the anchor. Subsequent studies showed
that PrPSc formation occurs after
PrPC reaches the cell surface (140, 141) and is
localized to caveolae-like domains (142-145).
Modeling PrP structures.
Molecular modeling studies predicted
that PrPC is a four-helix bundle protein
containing four regions of secondary structure denoted H1, H2, H3, and
H4 (Fig. 4) (146, 147).
Subsequent NMR studies of a synthetic PrP peptide containing residues
90-145 provided good evidence for H1 (148). This peptide contains the residues 113-128, which are most highly conserved among all species studied (Fig. 4A) (147, 149, 150) and correspond to a
transmembrane region of PrP that was delineated in cell-free
translation studies (151, 152). Recent studies show that a
transmembrane form of PrP accumulates in GSS caused by the A117V
mutation and in Tg mice overexpressing either mutant or wild-type
(wt)PrP (153). The paradoxical lack of evidence for an -helix in
this region from NMR studies of recombinant PrP in aqueous buffers
(154-156) could be explained if the recombinant PrPs correspond to the
secreted form of PrP that was also identified in cell-free translation studies. This contention is supported by studies with recombinant antibody fragments (Fabs) showing the GPI-anchored
PrPC on the surface of cells exhibits an
immunoreactivity similar to that of recombinant PrP that was prepared
with an -helical conformation (157, 158). GPI-anchored
PrPC is synthesized within the secretory pathway
and transported to the surface of the cell (139, 159).
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Fig. 4.
Species variations and mutations of the prion
protein gene. (A) Species variations. The
x-axis represents the human PrP sequence, with the five
octarepeats and H1-H4 regions of putative secondary structure shown as
well as the three -helices A, B, and C and the two -strands S1
and S2 as determined by NMR. The precise residues corresponding to each
region of secondary structure are given in Fig. 5. Vertical bars above
the axis indicate the number of species that differ from the human
sequence at each position. Below the axis, the length of the bars
indicates the number of alternative amino acids at each position in the
alignment. (B) Mutations causing inherited human prion
disease and polymorphisms in human, mouse, and sheep. Above the line of
the human sequence are mutations that cause prion disease. Below the
lines are polymorphisms, some but not all of which are known to
influence the onset as well as the phenotype of disease. Data were
compiled by Paul Bamborough and Fred E. Cohen.
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Optical spectroscopic measurements of PrPC
provided the necessary background for more detailed structural studies
(25). Unable to produce crystals of PrP, we and others utilized NMR to
determine the structure of an -helical form of a recombinant PrP.
The NMR structure of a COOH-terminal fragment of MoPrP consisting of
111 residues showed three helices, two of which corresponded to H3 and
H4 in the PrPC model, and two small -strands
each consisting of three residues (154). How the structure of this
MoPrP(121-231) fragment differs from PrPC is of
interest because this fragment is lethal when expressed in Tg mice
(160). Subsequently, structural studies were performed on a longer
fragment of PrP containing residues 90-231 and corresponding to SHaPrP
27-30 (155, 161, 162). Expression of PrP(90-231) in Tg mice did not
produce spontaneous disease (163, 164). More recently, NMR structures
of recombinant full-length PrP have been reported (156, 165).
Models of PrPSc suggested that formation of the
disease-causing isoform involves refolding of residues within the
region between residues 90 and 140 into -sheets (166); the single
disulfide bond joining COOH-terminal helices would remain intact
because the disulfide is required for PrPSc
formation (Fig. 5E)
(167, 168). The high -sheet content of PrPSc
was predicted from the ability of PrP 27-30 to polymerize into amyloid
fibrils (107). Subsequent optical spectroscopy confirmed the presence
of -sheet in both PrPSc and PrP 27-30 (25,
169-171). Deletion of each of the regions of putative secondary
structure in PrP, except for the NH2-terminal 66 amino acids (residues 23-88) (163, 172) and a 36-amino acid region
(mouse residues 141-176) prevented formation of
PrPSc as measured in scrapie-infected cultured
neuroblastoma cells (168). With anti-PrP Fabs selected from phage
display libraries (157) and two monoclonal antibodies (mAbs) derived
from hybridomas (173-175), the major conformational change that occurs
during conversion of PrPC into
PrPSc has been localized largely, but not
entirely, to a region bounded by residues 90 and 112 (158). Similar
conclusions were drawn from studies with an anti-PrP IgM mAb (176).
While these results indicate that PrPSc formation
involves primarily a conformational change in the domain composed of
residues 90-112, mutations causing inherited prion diseases have been
found throughout the protein (Fig. 4B). Interestingly, all
of the known point mutations in PrP with biological significance occur
either within or adjacent to regions of putative secondary structure in
PrP and as such, appear to destabilize the structure of PrP (147, 148,
154).
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Fig. 5.
Structures of prion proteins. (A)
NMR structure of SHa recombinant (r) PrP(90-231).
Presumably, the structure of the -helical form of rPrP(90-231)
resembles that of PrPC. rPrP(90-231) is viewed from the
interface where PrPSc is thought to bind to
PrPC. The color scheme is as follows: -helices A
(residues 144-157), B (172-193), and C (200-227) in pink; disulfide
between Cys-179 and Cys-214 in yellow; conserved hydrophobic region
composed of residues 113-126 in red; loops in gray; residues 129-134
in green encompassing strand S1 and residues 159-165 in blue
encompassing strand S2; the arrows span residues 129-131 and 161-163,
as these show a closer resemblance to -sheet (155). (B)
NMR structure of rPrP(90-231) is viewed from the interface where
protein X is thought to bind to PrPC. Protein X appears to
bind to the side chains of residues that form a discontinuous epitope:
some amino acids are in the loop composed of residues 165-171 and at
the end of helix B (Gln-168 and Gln-172 with a low-density van der
Waals rendering), whereas others are on the surface of helix C (Thr-215
and Gln-219 with a high-density van der Waals rendering) (178).
(C) PrP residues governing the transmission of prions (180).
NMR structure of recombinant SHaPrP region 121-231 (155) shown with
the putative epitope formed by residues 184, 186, 203, and 205 highlighted in red. Residue numbers correspond to SHaPrP. Additional
residues (138, 139, 143, 145, 148, and 155) that might participate in
controlling the transmission of prions across species are depicted in
green. Residues 168, 172, 215, and 219 that form the epitope for the
binding of protein X are shown in blue. The three helices (A, B, and C)
are highlighted in pink. (D) Schematic diagram showing the
flexibility of the polypeptide chain for PrP(29-231) (156). The
structure of the portion of the protein representing residues 90-231
was taken from the coordinates of PrP(90-231) (155). The remainder of
the sequence was hand-built for illustration purposes only. The color
scale corresponds to the heteronuclear
{1H}-15N nuclear Overhauser enhancement
data: red for the lowest (most negative) values, where the polypeptide
is most flexible, to blue for the highest (most positive) values in the
most structured and rigid regions of the protein. (E)
Plausible model for the tertiary structure of HuPrPSc
(166). Color scheme is as follows: S1 -strands are 108-113 and
116-122 in red; S2 -strands are 128-135 and 138-144 in green;
-helices H3 (residues 178-191) and H4 (residues 202-218) in gray;
loop (residues 142-177) in yellow. Four residues implicated in the
species barrier are shown in ball-and-stick form (Asn-108, Met-112,
Met-129, Ala-133).
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NMR structure of recombinant PrP.
The NMR structure of
recombinant (r) SHaPrP(90-231) derived from Escherichia
coli was determined after the protein was purified and refolded
(Fig. 5A). Residues 90-112 are not shown because marked
conformational heterogeneity was found in this region, while residues
113-126 constitute the conserved hydrophobic region, which also
displays some structural plasticity (162). Although some features of
the structure of rPrP(90-231) are similar to those reported earlier
for the smaller recombinant MoPrP(121-231) fragment (154, 177),
substantial differences were found. For example, the loop at the
NH2 terminus of helix B is well defined in
rPrP(90-231) but is disordered in MoPrP(121-231); in addition, helix
C is composed of residues 200-227 in rPrP(90-231) but extends only
from 200-217 in MoPrP(121-231). The loop and the COOH-terminal portion of helix C are particularly important because they form the
site to which protein X binds as described below (Fig. 5B) (178). Whether the differences between the two recombinant PrP fragments are because of (i) their different lengths,
(ii) species-specific differences in sequences, or
(iii) the conditions used for solving the structures remains
to be determined.
Studies of chimeric SHa/Mo and Hu/Mo PrP transgenes identified a
domain composed of residues 95-170, where PrPC
binds to PrPSc (133, 179). When chimeric bovine
(Bo)/Mo PrP transgenes failed to render mice sensitive to BSE prions,
we examined the differences among the sequences in the chimeric and
parent PrP genes (180). The findings identified a second domain in PrP
composed of residues 180-205 that seems to modulate the interaction
between PrPC and PrPSc
(Fig. 5C).
Recent NMR studies of full-length MoPrP(23-231) and SHaPrP(29-231)
have shown that the NH2 termini are highly
flexible and lack identifiable secondary structure under the
experimental conditions employed (Fig. 5D) (156, 165).
Studies of SHaPrP(29-231) indicate transient interactions between the
COOH terminus of helix B and the highly flexible,
NH2-terminal random-coil containing the
octareapeats (residues 29-125) (156); such interactions were not
reported for MoPrP(23-231) (165).
PrP appears to bind copper.
The highly flexible
NH2 terminus of recombinant PrP may be more
structured in the presence of copper. Each SHaPrP(29-231) molecule was
found to bind two atoms of Cu2+; other divalent
cations did not bind to PrP (181). Earlier studies with synthetic
peptides corresponding to the octarepeat sequence demonstrated the
binding of Cu2+ ions (182, 183), and optical
spectroscopy showed that Cu2+ induced an
-helix formation in these peptides (184). More recently, PrP-deficient (Prnp0/0) mice were
found to have lower levels of Zn/Cu superoxide dismutase (SOD)
activity than do controls (185); SOD activity has been shown to mirror
the state of copper metabolism (186). Measurements of membrane extracts
from brains of Prnp0/0 mice showed low
levels of Cu, whereas Fe and Zn were unchanged suggesting
PrPC might function as a
Cu2+-binding protein (187).
Disturbances in Cu2+ homeostasis leading to
dysfunction of the CNS are well documented in humans and animals but
are not known to be due to abnormalities in PrP metabolism: Menkes
disease is manifest at birth and is due to a mutation of the
MNK gene on the X chromosome, whereas Wilson's disease
appears in childhood and is due to a mutation of the WD gene
on chromosome 13 (188-191). Both the MNK and WD
genes encode copper-transporting ATPases. While both Menkes and
Wilson's diseases are recessive disorders, only Wilson's disease can
be treated with copper-chelating reagents. Interestingly, cuprizone, a
Cu2+-chelating reagent, has been used in mice to
induce neuropathological changes similar to those found in the prion
diseases (192, 193).
PrP Gene Structure and Expression.
The entire open reading
frame (ORF) of all known mammalian and avian PrP genes resides within a
single exon (4, 82, 194, 195). The mouse, sheep, cattle, and rat PrP
genes contain three exons with the ORFs in exon 3 (196-200) which is
analogous to exon 2 of the SHa gene (82). The two exons of the SHaPrP
gene are separated by a 10-kb intron: exon 1 includes a portion of the 5' untranslated leader sequence, while exon 2 includes the ORF and 3'
untranslated region (82). Recently, a low abundance SHaPrP mRNA
containing an additional small exon in the 5' untranslated region was
discovered that is encoded by the SHaPrP gene (201). Comparative
sequencing of sheep and Hu cosmid clones containing PrP genes revealed
an additional putative, small untranslated 5' exon in the HuPrP gene
(202). The promoters of both the SHa- and MoPrP genes contain multiple
copies of G+C-rich repeats and are devoid of TATA boxes. These G+C
nonamers represent a motif that may function as a canonical binding
site for the transcription factor Sp1 (203). Mapping of PrP genes to
the short arm of Hu chromosome 20 and to the homologous region of Mo
chromosome 2 argues for the existence of PrP genes prior to the
speciation of mammals (204, 205).
Although PrP mRNA is constitutively expressed in the brains of adult
animals (83, 84), it is highly regulated during development. In the
septum, levels of PrP mRNA and choline acetyltransferase were found to
increase in parallel during development (206). In other brain regions,
PrP gene expression occurred at an earlier age. In situ
hybridization studies show that the highest levels of PrP mRNA are
found in neurons (207).
PrPC expression in brain was defined by standard
immunohistochemistry (208) and by histoblotting in the brains of
uninfected controls (209). Immunostaining of PrPC
in the SHa brain was most intense in the stratum radiatum and stratum
oriens of the CA1 region of the hippocampus and was virtually absent
from the granule cell layer of the dentate gyrus and the pyramidal cell
layer throughout Ammon's horn. PrPSc staining
was minimal in those regions that were intensely stained for
PrPC. A similar relationship between
PrPC and PrPSc was found in
the amygdala. In contrast, PrPSc accumulated in
the medial habenular nucleus, the medial septal nuclei, and the
diagonal band of Broca; in contrast, these areas were virtually devoid
of PrPC. In the white matter, bundles of
myelinated axons contained PrPSc but were devoid
of PrPC. These findings suggest that prions are
transported along axons and are in agreement with earlier findings in
which scrapie infectivity was found to migrate in a pattern consistent
with retrograde transport (210-212).
Molecular Genetics of Prion Diseases.
Independent of enriching
brain fractions for scrapie infectivity that led to the discovery of
PrPSc, the PrP gene was shown to be genetically
linked to a locus controlling scrapie incubation times (213).
Subsequently, mutation of the PrP gene was shown to be genetically
linked to the development of familial prion disease (4). At the same
time, expression of a SHaPrP transgene in mice was shown to render the
animals highly susceptible to SHa prions, demonstrating that expression of a foreign PrP gene could abrogate the species barrier (214). Later,
PrP-deficient (Prnp0/0) mice were
found to be resistant to prion infection and failed to replicate
prions, as expected (100, 101). The results of these studies indicated
PrP must play a central role in the transmission and pathogenesis of
prion disease, but equally important, they argued that the abnormal
isoform is an essential component of the prion particle (23).
PrP gene dosage controls length of incubation time.
Scrapie
incubation times in mice were used to distinguish prion strains and to
identify a gene controlling its length (135, 215). This gene was
initially called Sinc on the basis of genetic crosses
between C57BL and VM mice that exhibited short and long incubation
times, respectively (215). Because of the restricted distribution of VM
mice, we searched for another mouse with long incubation times. I/Ln
mice proved to be a suitable substitute for VM mice (216); eventually,
I/Ln and VM mice were found to be derived from a common ancestor
(217). With a PrP cDNA probe, we demonstrated genetic linkage between
the PrP gene and a locus controlling the incubation time in crosses
between NZW/LacJ and I/Ln mice (213). We provisionally designated
these genes as components of the Prn complex but eventually
found that the incubation time gene, Prn-i, is either
congruent with or closely linked to the PrP gene, Prnp
(195).
Although the amino acid substitutions in PrP that distinguish NZW
(Prnpa ) from I/Ln
(Prnpb) mice argued for
congruency of Prnp and Prn-i, experiments with Prnpa mice expressing
Prnpb transgenes demonstrated a
"paradoxical" shortening of incubation times (196). We had
predicted that these Tg mice would exhibit a prolongation of the
incubation time after inoculation with RML prions on the basis of
(Prnpa × Prnpb)F1 mice,
which do exhibit long incubation times. We described those findings as
"paradoxical shortening" because we and others had believed for
many years that long incubation times are dominant traits (213, 215).
From studies of congenic and transgenic mice expressing different
numbers of the a and b alleles of
Prnp, we learned that these findings were not paradoxical;
indeed, they resulted from increased PrP gene dosage (218). When the
RML isolate was inoculated into congenic and transgenic mice,
increasing the number of copies of the a allele was found to
be the major determinant in reducing the incubation time; however,
increasing the number of copies of the b allele also reduced
the incubation time, but not to the same extent as that seen with the
a allele. From the foregoing investigations, we concluded
that both Sinc and Prn-i are congruent with PrP
(218), and recent gene targeting studies have confirmed this view
(219).
Overexpression of wtPrP transgenes.
Mice were constructed
expressing different levels of the wt SHaPrP transgene (214).
Inoculation of these Tg(SHaPrP) mice with SHa prions demonstrated
abrogation of the species barrier, resulting in abbreviated incubation
times (220). The length of the incubation time after inoculation with
SHa prions was inversely proportional to the level of
SHaPrPC in the brains of Tg(SHaPrP) mice (220).
Bioassays of brain extracts from clinically ill Tg(SHaPrP) mice
inoculated with Mo prions revealed that only Mo prions but no SHa
prions were produced. Conversely, inoculation of Tg(SHaPrP) mice with
SHa prions led only to the synthesis of SHa prions. Although the rate
of PrPSc synthesis appears to be a function of
the level of PrPC expression in Tg mice, the
level to which PrPSc finally accumulates seems to
be independent of PrPC concentration (220).
During transgenetic studies, we discovered that uninoculated older mice
harboring numerous copies of wtPrP transgenes derived from Syrian
hamsters, sheep, and Prnpb mice
spontaneously developed truncal ataxia, hind-limb paralysis, and
tremors (198). These Tg mice exhibited a profound necrotizing myopathy
involving skeletal muscle, a demyelinating polyneuropathy, and focal
vacuolation of the CNS. Development of disease was dependent on
transgene dosage. For example, Tg(SHaPrP+/+)7
mice homozygous for the SHaPrP transgene array regularly developed disease between 400 and 600 days of age, whereas hemizygous
Tg(SHaPrP+/0)7 mice also developed disease, but
only after >650 days.
PrP-deficient mice.
The development and lifespan of two lines
of PrP-deficient (Prnp0/0) mice were
indistinguishable from those of controls (221, 222), whereas two other
lines exhibited ataxia and Purkinje cell degeneration at 70 weeks of
age (223) (R. Moore and D. Melton, personal communication). In the
former two lines with normal development, altered sleep-wake cycles
have been reported (224), and altered synaptic behavior in brain slices
was reported (225, 226) but could not be confirmed by others (227,
228).
Prnp0/0 mice are resistant to prions
(100, 101). Prnp0/0 mice were
sacrificed 5, 60, 120, and 315 days after inoculation with RML prions,
and brain extracts were bioassayed in CD-1 Swiss mice. Except for
residual infectivity from the inoculum detected at 5 days after
inoculation, no infectivity was detected in the brains of
Prnp0/0 mice (101). One group of
investigators found that Prnp0/0 mice
inoculated with RML prions and sacrificed 20 weeks later had
103.6 ID50 units/ml of
homogenate by bioassay (100). Others have used this report to argue
that prion infectivity replicates in the absence of PrP (67, 132).
Neither we nor the authors of the initial report could confirm the
finding of prion replication in
Prnp0/0 mice (101, 103).
Prion Species Barrier and Protein X.
The passage of prions
between species is often a stochastic process, almost always
characterized by prolonged incubation times during the first passage in
the new host (36). This prolongation is often referred to as the
"species barrier" (36, 229). Prions synthesized de novo
reflect the sequence of the host PrP gene and not that of the
PrPSc molecules in the inoculum derived from the
donor (90). On subsequent passage in a homologous host, the incubation
time shortens to that recorded for all subsequent passages. From
studies with Tg mice, three factors have been identified that
contribute to the species barrier: (i) the difference in PrP
sequences between the prion donor and recipient, (ii) the
strain of prion, and (iii) the species specificity of
protein X, a factor defined by molecular genetic studies that binds to
PrPC and facilitates PrPSc
formation. This factor is likely to be a protein, hence the provisional designation protein X (134, 178). The prion donor is the last mammal in
which the prion was passaged and its PrP sequence represents the
"species" of the prion. The strain of prion, which seems to be
enciphered in the conformation of PrPSc,
conspires with the PrP sequence, which is specified by the recipient, to determine the tertiary structure of nascent
PrPSc. These principles are demonstrated by
studies on the transmission of SHa prions to mice showing that
expression of a SHaPrP transgene in mice abrogated the species barrier
(Table 3) (214). Besides the PrP
sequence, the strain of prion modified transmission of SHa prions to
mice (Table 3) (135, 230, 231).
Transmission of Hu prions.
Protein X was postulated to explain
the results on the transmission of Hu prions to Tg mice (Table
4) (134, 179). Mice expressing
both Mo and HuPrP were resistant to Hu prions, whereas those expressing
only HuPrP were susceptible. These results argue that
MoPrPC inhibited transmission of Hu prionsi.e.,
the formation of nascent HuPrPSc. In contrast to
the foregoing studies, mice expressing both MoPrP and chimeric MHu2MPrP
were susceptible to Hu prions and mice expressing MHu2MPrP alone were
only slightly more susceptible. These findings contend that
MoPrPC has only a minimal effect on the formation
of chimeric MHu2MPrPSc.
Genetic evidence for protein X.
When the data on Hu prion
transmission to Tg mice were considered together, they suggested that
MoPrPC prevented the conversion of
HuPrPC into PrPSc but had
little effect on the conversion of MHu2M into
PrPSc by binding to another Mo protein. We
interpreted these results in terms of MoPrPC
binding to this Mo protein with a higher affinity than does
HuPrPC. We postulated that
MoPrPC had little effect on the formation of
PrPSc from MHu2M (Table 4) because MoPrP and
MHu2M share the same amino acid sequence at the COOH terminus. We
hypothesized that MoPrPC only weakly inhibited
transmission of SHa prions to Tg(SHaPrP) mice (Table 3) because SHaPrP
is more closely related to MoPrP than is HuPrP.
Using scrapie-infected Mo neuroblastoma cells transfected with chimeric
Hu/Mo PrP genes, we extended our studies of protein X. Substitution
of a Hu residue at position 214 or 218 prevented PrPSc formation (Fig. 5B) (178). The side chains
of these residues protrude from the same surface of the COOH-terminal
-helix, forming a discontinuous epitope with residues 167 and 171 in
an adjacent loop. Substitution of a basic residue at position 167, 171, or 218 prevented PrPSc formation; these mutant
PrPs appear to act as "dominant negatives" by binding protein X and
rendering it unavailable for prion propagation. Our findings within the
context of protein X explain the protective effects of basic
polymorphic residues in PrP of humans and sheep (199, 232, 233).
Is protein X a molecular chaperone?
Since PrP undergoes a
profound structural transition during prion propagation, it seems
likely that other proteins such as chaperones participate in this
process. Whether protein X functions as a molecular chaperone is
unknown. Interestingly, scrapie-infected cells in culture display
marked differences in the induction of heat-shock proteins (234, 235),
and Hsp70 mRNA has been reported to increase in scrapie of mice (236).
While attempts to isolate specific proteins that bind to PrP have been
disappointing (237), PrP has been shown to interact with Bcl-2 and
Hsp60 by two-hybrid analysis in yeast (238, 239). Although these
studies are suggestive, no molecular chaperone involved in prion
formation in mammalian cells has been identified.
Miniprions.
By using the four-helix bundle model of
PrPC (Fig. 4A) (147), each
region of proposed secondary structure was systematically deleted and
the mutant constructs were expressed in scrapie-infected neuroblastoma
(ScN2a) cells and Tg mice (164, 168). Deletion of any of the four
putative helical regions prevented PrPSc
formation, whereas deletion of the NH2-terminal
region containing residues 23-89 did not affect the yield of
PrPSc. In addition to the 67 residues at the
NH2 terminus, 36 residues from positions 141-176
could be deleted without altering PrPSc formation
(Figs. 6 and
7). The resulting PrP molecule of
106 amino acids was designated PrP106. In this mutant PrP, helix A as
well as the S2 -strand were removed. When PrP106 was expressed in
ScN2a cells, PrPSc106 was soluble in 1%
Sarkosyl. Whether the structure of PrPSc106 can
be more readily determined than that of full-length
PrPSc remains uncertain.
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Fig. 6.
Miniprions produced by deleting PrP residues
23-89 and 141-176. The deletion of residues 141-176 (green)
containing helix A and the S2 -strand is shown. Side chains of
residues 168, 172, 215, and 219, which are thought to bind protein X,
are shown in cyan.
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Fig. 7.
Tg(PrP106)Prnp0/0 mice
were inoculated with RML106 prions containing PrPSc106.
Sections of the hippocampus were stained with hematoxylin and eosin
(A and C) and immunostained for GFAP
(B and D). (A and
B) Control Tg(PrP106)Prnp0/0
mouse uninoculated and without neurologic deficits. (C and
D) Tg(PrP106)Prnp0/0 mouse
inoculated with RML106 prions and sacrificed after signs of neurologic
dysfunction were observed. (Bar = 50 µm.) Photomicrographs
prepared by Stephen J. DeArmond.
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Transgene-specified susceptibility.
Tg(PrP106)Prnp0/0 mice that expressed
PrP106 developed neurological dysfunction 300 days after inoculation
with RML prions previously passaged in CD-1 Swiss mice (S. Supattapone,
T. Muramoto, D. Peretz, S. J. DeArmond, A. Wallace, F. E. Cohen, S.B.P., and M. R. Scott, unpublished results). The
resulting prions containing PrPSc106 produced CNS
disease in 66 days upon subsequent passage in Tg(PrP106)Prnp0/0 mice (Table
5). Besides widespread spongiform
degeneration and PrP deposits, the pyramidal cells of the hippocampus
constituting the CA-1, CA-2, and CA-3 fields disappeared in
Tg(PrP106)Prnp0/0 mice inoculated with
prions containing PrPSc106 (Fig. 7). In no
previous study of Tg mice have we seen similar neuropathological
lesions. The Tg(MoPrP-A) mice overexpressing MoPrP are resistant to
RML106 miniprions but are highly susceptible to RML prions. These mice
require more than 250 days to produce illness after inoculation with
miniprions but develop disease in 50 days when inoculated with RML
prions containing full-length MoPrPSc.
Smaller prions and mythical viruses.
The unique incubation
times and neuropathology in Tg mice caused by miniprions are difficult
to reconcile with the notion that scrapie is caused by an
as-yet-unidentified virus. When the mutant or wt
PrPC of the host matched
PrPSc in the inoculum, the mice were highly
susceptible (Table 5). However, when there was a mismatch between
PrPC and PrPSc, the mice
were resistant to the prions. This principle of homologous PrP
interactions, which underlies the species barrier (Table 3), is
recapitulated in studies of PrP106 where the amino acid sequence has
been drastically changed by deleting nearly 50% of the residues. Indeed, the unique properties of the miniprions provide another persuasive argument supporting the contention that prions are infectious proteins.
Human Prion Diseases.
Most humans afflicted with prion disease
present with rapidly progressive dementia, but some manifest cerebellar
ataxia. Although the brains of patients appear grossly normal upon
postmortem examination, they usually show spongiform degeneration and
astrocytic gliosis under the microscope. The human prion diseases can
present as sporadic, genetic, or infectious disorders (5) (Table 1).
Sporadic CJD.
Sporadic forms of prion disease constitute most
cases of CJD and possibly a few cases of
Gerstmann-Sträussler-Scheinker disease (GSS) (Table 1) (4, 240,
241). In these patients, mutations of the PrP gene are not found. How
prions causing disease arise in patients with sporadic forms is
unknown; hypotheses include horizontal transmission of prions from
humans or animals (242), somatic mutation of the PrP gene, and
spontaneous conversion of PrPC into
PrPSc (5, 15). Because numerous attempts to
establish an infectious link between sporadic CJD and a preexisting
prion disease in animals or humans have been unrewarding, it seems
unlikely that transmission features in the pathogenesis of sporadic
prion disease (9-12, 243).
Inherited prion diseases.
To date, 20 different mutations in
the human PrP gene resulting in nonconservative substitutions have been
found that segregate with the inherited prion diseases (Fig.
4B). Familial CJD cases suggested that genetic factors might
influence pathogenesis (1, 2, 244), but this was difficult to reconcile
with the transmissibility of fCJD and GSS (3). The discovery of genetic
linkage between the PrP gene and scrapie incubation times in mice (213)
raised the possibility that mutation might feature in the hereditary human prion diseases. The P102L mutation was the first PrP mutation to
be genetically linked to CNS dysfunction in GSS (Fig. 4B)
(4) and has since been found in many GSS families throughout the world (245-247). Indeed, a mutation in the protein coding region of the PrP
gene has been found in all reported kindred with familial human prion
disease; besides the P102L mutation, genetic linkage has been
established for four other mutations (16, 29-31).
Tg mouse studies confirmed that mutations of the PrP gene can cause
neurodegeneration. The P102L mutation of GSS was introduced into the
MoPrP transgene, and five lines of Tg(MoPrP-P101L) mice expressing high
levels of mutant PrP developed spontaneous CNS degeneration consisting
of widespread vacuolation of the neuropil, astrocytic gliosis, and
numerous PrP amyloid plaques similar to those seen in the brains of
humans who die from GSS(P102L) (248-250). Brain extracts prepared from
spontaneously ill Tg(MoPrP-P101L) mice transmitted CNS degeneration to
Tg196 mice but contained no protease-resistant PrP (249, 250). The
Tg196 mice do not develop spontaneous disease but express low levels of
the mutant transgene MoPrP-P101L and are deficient for mouse PrP
(Prnp0/0) (221). These studies,
combined with the transmission of prions from patients who died of GSS
to apes and monkeys (3) or to Tg(MHu2M-P101L) mice (134), demonstrate
that prions are generated de novo by mutations in PrP.
Additionally, brain extracts from patients with some other inherited
prion diseases, fCJD(E200K) or FFI, transmit disease to Tg(MHu2M) mice
(27). An artific
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