Physiol Rev 89: 1105–1152, 2009;
doi:10.1152/physrev.00006.2009.
Prions: Protein Aggregation and Infectious Diseases
ADRIANO AGUZZI AND ANNA MARIA CALELLA
Institute of Neuropathology, University Hospital of Zurich, Zurich, Switzerland
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I. Properties of Prions
A. Introduction
B. Development of the prion concept
C. Discovery of the prion protein
D. PrP amyloid
E. Formation of PrPSc
F. Prion diversity
G. Prion-like properties of additional misfolded proteins
II. Function of the Cellular Prion Protein
A. Teachings from Prnp-ablated mice
B. PrPC and its partners
C. PrP’s double and its trouble
III. Mechanisms of Prion Toxicity
A. Alteration of PrPC mediated signaling
B. PrPC mislocalization
C. PrP-derived oligomeric species
IV. Clinical and Pathological Aspects of Prion Diseases
A. Scrapie
B. Bovine spongiform encephalopathy
C. Chronic wasting disease of deer and elk
D. Human prion diseases
V. Peripheral Routes of Prion Infectivity
A. Pathway of orally administered prions
B. Transepithelial enteric passage of prions
C. Uptake of prion through the skin
VI. Immunological Aspects of Prion Disease
A. The lymphoid system in prion disease
B. Tropism of prions for inflammatory foci
C. Neuroinflammation in prion pathogenesis
VII. Spread of Prions Along Peripheral and Central Nervous System Pathways
VIII. Towards Antiprion Therapeutics
A. Innate immunity and antiprion defense
B. Adaptive immunity and preexposure prophylaxis against prions
C. Soluble prion antagonists
IX. Progress in the Diagnostics of Prion Diseases
X. Concluding Remarks
Aguzzi A, Calella AM. Prions: Protein Aggregation and Infectious Diseases. Physiol Rev 89: 1105–1152, 2009;
doi:10.1152/physrev.00006.2009.—Transmissible spongiform encephalopathies (TSEs) are inevitably lethal neurodegenerative diseases that affect humans and a large variety of animals. The infectious agent responsible for TSEs is
the prion, an abnormally folded and aggregated protein that propagates itself by imposing its conformation onto the
cellular prion protein (PrPC) of the host. PrPC is necessary for prion replication and for prion-induced neurodegeneration, yet the proximal causes of neuronal injury and death are still poorly understood. Prion toxicity may arise
from the interference with the normal function of PrPC, and therefore, understanding the physiological role of PrPC
may help to clarify the mechanism underlying prion diseases. Here we discuss the evolution of the prion concept and
how prion-like mechanisms may apply to other protein aggregation diseases. We describe the clinical and the
pathological features of the prion diseases in human and animals, the events occurring during neuroinvasion, and the
possible scenarios underlying brain damage. Finally, we discuss potential antiprion therapies and current developments in the realm of prion diagnostics.
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0031-9333/09 $18.00 Copyright © 2009 the American Physiological Society
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I. PROPERTIES OF PRIONS
A. Introduction
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TABLE
1.
Human prion diseases
Disease
Kuru
Creutzfeldt-Jakob disease
Iatrogenic
Sporadic
Familial
Variant
Gerstmann-Sträussler-Scheinker disease
Fatal familial insomnia
Etiology
Infection
Infection
Unknown
PRNP mutation
Presumed BSE infection
PRNP mutation
PRNP mutation
BSE, bovine spongiform encephalopathy.
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Prion diseases, also termed transmissible spongiform
encephalopathies (TSEs), are inevitably fatal neurodegenerative conditions that affect humans and a wide variety
of animals. Prion diseases may present with certain morphological and pathophysiological features that parallel
other progressive encephalopathies, such as Alzheimer’s
and Parkinson’s disease (12). However, prion diseases
were considered unique in that they are transmissible. As
it happens, the latter assertion may not be entirely correct. It has been reported that aggregates of amyloid-␤
(〈␤) peptide associated with Alzheimer’s disease behave
like an infectious agent when injected into the brain of a
mouse model of Alzheimer’s disease, showing a pattern of
A␤ deposition that depends on both the host and the
agent (345). These findings suggest that what was considered to be a unique feature of prion disease may be a more
general property of amyloids.
The agent that causes TSEs was termed “prion” by
Stanley B. Prusiner and is defined as “a small proteinaceous infectious particle that is resistant to inactivation
by most procedures that modify nucleic acids” (403).
Prions certainly differ from all other known infectious
pathogens in several respects. First, prions do not appear
to contain an informational nucleic acid genome longer
than 50 bases that encodes for their progeny (423). Second, the only known component of the prion is a modified
form of the cellular prion protein, PrPC, which is encoded
by the gene Prnp. PrPC is a cell surface glycoprotein (481)
of unknown function that has been identified in all mammals and birds examined to date, as well as in Xenopus
laevis (482) and fish (425). Third, the central event in
prion pathogenesis is the conformational conversion of
PrPC into PrPSc, an insoluble and partially protease-resistant isoform that propagates itself by imposing its abnormal conformation onto PrPC molecules.
Numerous experiments have provided evidence that
PrPC is a key player in prion replication as well as in
prion-induced neurodegeneration. Mice lacking the prion
gene are resistant to the disease (84), and PrPC expression is required for neurodegeneration in host neurons,
because the presence of PrPSc alone does not cause disease. For example, when neurografts propagating PrPSc
were implanted into Prnp knockout mice, no pathological
changes were seen in PrP-deficient tissue, even in the
immediate vicinity of the grafts (64). Additionally, transgenic mice expressing only a secreted form of PrPC lacking a glycosylphosphatidylinositol (GPI) anchor do not
develop clinical signs of prion disease, although prion
inoculation induces PrPSc formation and aggregation of
amyloid plaques (108). These findings suggest that expres-
sion of membrane-anchored PrPC is necessary to initiate
disease. Finally, neuron-specific ablation of PrPC in transgenic mice (321) or RNAi knockdown of PrPC expression
in mice with established prion disease (519) rescues early
neuronal dysfunction and prolongs the survival of mice
despite the accumulation of extraneuronal PrPSc.
Attempts to identify posttranslational modifications
that distinguish PrPSc from PrPC have been mostly unsuccessful (480); however, increasing evidence indicates that
they may exist (89, 122). Almost 45% of the PrPC protein
is ␣-helical with two very short stretches of ␤-sheet (421).
Conversion to PrPSc results in a protein comprised of
⬃30% ␣-helix and 45% ␤-sheet. The mechanism by which
PrPC is converted into PrPSc is unknown, but PrPC appears to bind to PrPSc, perhaps in combination with ancillary proteins, to form an intermediate complex during
the formation of nascent PrPSc (343). Transgenic mouse
studies have provided genetic evidence that incoming
prions in the inoculum interact preferentially with homotypic PrPC during the propagation of prions (412, 447).
Prion disease is characterized by widespread neurodegeneration; therefore, affected individuals exhibit clinical symptoms of both cognitive and motor dysfunction.
In addition, the disease is characterized by the propagation of infectious prions and, in many instances, the formation of amyloid plaques. The human prion diseases
include Kuru, Creutzfeldt-Jakob disease (CJD), variant
CJD (vCJD), Gerstmann-Sträussler-Scheinker (GSS) disease and fatal familial insomnia (FFI) (Table 1). The most
common prion diseases of animals are scrapie, which
affects sheep and goats, bovine spongiform encephalopathy (BSE) or “mad cow” disease, and chronic wasting
disease (CWD), which affects deer and elk. Kuru was the
first of the human prion diseases to be transmitted to
experimental animals, and Gajdusek discovered that Kuru
spread among the Fore people of Papua New Guinea
through ritual cannibalism (172). The experimental and
presumed human-to-human transmission of Kuru led to
the belief that prion diseases are infectious disorders
caused by unusual viruses similar to those causing scrapie
in sheep and goats. Yet autosomal dominant inheritance
of CJD was first reported almost 90 years ago (260, 342),
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executed testing procedures. In any case, these findings
have exposed our deficit in knowledge about prion epidemiology and possible transmission routes of prion diseases in humans and animals.
B. Development of the Prion Concept
Scrapie was originally hypothesized to be a muscle
disease caused by parasites (310, 311). The transmissibility of scrapie was demonstrated in an early, seminal study
(128) and led to the hypothesis that scrapie is caused by
a “filterable” virus (527). The findings of Tikvah Alper and
colleagues (23, 24) that scrapie infectivity was resistant to
inactivation by ultraviolet (UV) and ionizing irradiation
resulted in a myriad of hypotheses on the chemical nature
of the scrapie agent, including the clairvoyant conjecture
by Griffith (197) that it may consist of a self-replicating
protein.
The experimental transmission of scrapie from sheep
to mice (105) resulted in a much more convenient laboratory model, which yielded considerable information on
the unusual infectious pathogen that causes scrapie (23–
25, 182, 350, 383). Yet progress was, and in many instances continues to be, slow because quantification of
infectivity in a single sample required holding many mice
for at least 1 yr before accurate scoring could be accomplished (105).
After scrapie incubation times were reported to be
⬃50% shorter in Syrian hamsters than in mice (329),
studies were undertaken to determine whether the incubation times in hamsters could be related to the titer of
the inoculated sample. There was an inverse correlation
between the length of the incubation time and the logarithm of the dose of inoculated prions, which allowed for
a more rapid and economical (albeit considerably less
precise) bioassay (406, 408).
The development of a quantitative, highly sensitive
cell-based bioassay by Klohn and Weissmann was crucial
for many practical purposes (270). In the scrapie cell
assay (SCA), susceptible neuroblastoma cells (N2a) are
exposed to prion-containing samples for 3 days, grown to
confluence, passaged several times, and then the proportion of PrPSc-containing cells is determined. In a log-log
plot, the dose response is linear over two logs of prion
concentrations. The SCA is about as sensitive as the
mouse bioassay, 10 times faster, ⬎2 orders of magnitude
less expensive, and suitable for automation. SCA performed in a more time-consuming end point titration format (SCEPA) extends the sensitivity and shows that infectivity titers measured in tissue culture and in the
mouse are similar.
Once an effective protocol was developed for the
preparation of partially purified fractions of scrapie agent
from hamster brain, it became possible to demonstrate
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suggesting that CJD was also a genetic disease. The significance of familial CJD (fCJD) remained largely unappreciated until mutations were discovered in the protein
coding region of the PRNP gene on the short arm of
chromosome 20 (478). The earlier finding that brain extracts from patients who had died of familial prion diseases inoculated into experimental animals often transmit
disease posed a conundrum that was resolved with the
genetic linkage of these diseases to mutations of the
PRNP gene that encodes PrPC (496). To date, all known
cases of familial prion disease cosegregate with PRNP
mutations.
The most common form of prion disease in humans is
“sporadic” CJD (sCJD) whose cause is unknown. Indeed,
many attempts to show that the sporadic prion diseases
are caused by infection have been unsuccessful (71, 125,
324). The discovery that inherited prion diseases are
caused by germ-line mutations in the PRNP gene raised
the possibility that sporadic forms of these diseases might
result from somatic mutations (402). Alternatively, since
PrPSc is derived from PrPC by a posttranslational process
(58), sporadic prion diseases may result from the spontaneous conversion of PrPC into PrPSc.
The incidence of sCJD is low in all ethnicities and has
been often said to affect one person in one million annually. However, in countries with active surveillance programs, the reported CJD incidence is often higher (13),
and in Switzerland it has reached 3.0/106 per year (187,
429), suggesting that many cases may go undetected. Less
than 1% of CJD cases arise from exposure to infected
material, and most of these seem to be iatrogenic. Between 5 and 15% of prion disease cases are inherited,
whereas the remaining cases are sporadic. Kuru was once
the most common cause of death among New Guinea
women in the Fore region of the Highlands (171, 173), but
the incidence has since dissipated with the cessation of
ritualistic cannibalism (341).
In the past 20 years, more than 280,000 cattle suffering from BSE has provoked a major worldwide food crisis
with thus far incomparable economic consequences for
the European Union and other countries. In addition,
transmission of BSE to humans is believed to have caused
ⱖ200 cases of variant CJD (vCJD). Direct experimental
proof that vCJD is caused by the transmission of BSE
prions to humans has not been demonstrated. However,
epidemiological, biochemical, and neuropathological evidence, as well as transmission studies, strongly suggest
that BSE has been transmitted to humans resulting in
vCJD (106, 231, 523).
In recent years, the United States has witnessed an
enigmatic rise of CWD cases affecting elk and deer, and
there has been a recrudescence of scrapie outbreaks
among European sheep flocks (e.g., Sweden, Austria, and
Sardinia). This resurgence of new cases might be linked
to an increased sensitivity and frequency of the currently
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protein (461). Moderate overexpression in transgenic
mice of a mouse PrP with two point mutations (170N,
174T) that subtly affect the structure of its globular domain causes a fully penetrant lethal spongiform encephalopathy with cerebral PrP plaques. This genetic disease
was reproduced with 100% attack rate by intracerebral
inoculation of brain homogenate to mice overexpressing
wild-type PrP (154), and from the latter to wild-type mice,
but not to PrP-deficient mice. This work provides an
important step forward in elucidating the role of a specific
domain of the prion protein in the generation of infectivity
(471).
C. Discovery of the Prion Protein
The dependence of prion infectivity on a protein
intensified the search for a scrapie-specific protein. Although the insolubility of scrapie infectivity made purification problematic, this property, along with its relative
resistance to degradation by proteases, was used by
Prusiner to facilitate purification (409, 411). Radioiodination of partially purified brain fractions revealed a protein
unique to preparations from scrapie-infected hamsters
(55, 404), and in subcellular fractions of hamster brain
enriched for scrapie infectivity, a protease-resistant
polypeptide of 27–30 kDa (designated PrP27–30) was identified (55, 337).
Purification of PrP27–30 to near-homogeneity allowed
determination of its NH2-terminal amino acid sequence,
which permitted the synthesis of an isocoding mixture of
oligonucleotides that was subsequently used to identify
incomplete PrP cDNA clones from hamster (370) and
mouse (107). cDNA clones encoding the entire open reading frames (ORFs) of Syrian hamster and mouse PrP were
subsequently isolated (39, 305). The above experiments
established that PrP is encoded by a chromosomal gene,
and not by a nucleic acid in the infectious scrapie prion
particle (39, 370). Levels of PrP mRNA remain unchanged
throughout the course of scrapie infection, an unexpected
observation that led to the identification of the normal
Prnp gene product, a protein of 33–35 kDa designated
PrPC (39, 370). PrPC is protease sensitive and soluble in
nondenaturing detergents, whereas PrP27–30 is the protease-resistant core of a 33- to 35-kDa disease-specific
protein, designated PrPSc, which is insoluble in detergents
(346).
The entire ORF of all known mammalian and avian
Prnp genes is contained within a single exon (39, 170, 230,
515). This feature of the Prnp gene eliminates the possibility that PrPSc arises from alternative RNA splicing (39,
516), although mechanisms such as RNA editing, protein
splicing, and alternative initiation of translation remain a
possibility (54, 241, 246). The PRNP gene is located in the
short arm of human chromosome 20 and in a homologous
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that the procedures that modify or hydrolyze proteins
produce a diminution in scrapie infectivity (405, 406). At
the same time, tests done in search of a scrapie-specific
nucleic acid were unable to demonstrate any dependence
of infectivity on a polynucleotide (403, 424), in agreement
with earlier studies reporting the extreme resistance of
infectivity to UV irradiation at 254 nm (23). On the basis of
these findings, it seemed unlikely that the infectious
pathogen capable of transmitting scrapie was a virus or a
viroid. For this reason, the term prion was introduced by
Prusiner to distinguish the proteinaceous infectious particles that cause scrapie, CJD, GSS, and Kuru from both
viroids and viruses (403).
A wealth of evidence has accumulated over the past
decade supporting the protein-only hypothesis, which
proposes that the main or perhaps only constituent of the
infectious agent is the mammalian prion PrPSc. Final
proof of this hypothesis could be achieved if pure PrPC or,
better yet, recombinant PrP produced in E. coli, could be
converted into a form that elicits prion disease. A first,
seminal step in this direction was the demonstration that
radioactive PrPC incubated with unlabeled PrPSc in a
cell-free system generated radioactive PrPSc, as characterized by its physical properties (97). However, an increase of infectivity was not demonstrated in these experiments (510). Another important advance was provided by the invention of protein misfolding cyclic
amplification (PMCA), a technique by which PrPSc is amplified by cycles of sonication followed by incubation
with brain homogenate (433, 434). Soto and co-workers
(94) showed that amplification of PrPSc was accompanied
by an amplification of infectivity. The use of purified PrPC
instead of brain homogenate as a substrate for the in vitro
PrPSc generation decreased the efficiency of amplification, suggesting that additional cofactors may facilitate
the conversion of PrPC to PrPSc (134). In addition, initial
attempts to use recombinant forms of PrP as a substrate
in PMCA have failed. Caughey and co-workers (30) have
since succeeded in carrying out PMCA using bacterially
expressed hamster PrP as a substrate. While this represents a major advance in many ways, the sensitivity is not
quite as high as with brain homogenate (5, 30).
In an intriguing study, Supattapone and co-workers
(135) identified the minimal components (PrPC, copurified lipids, and single-stranded polyanionic molecules)
required for the amplification of proteinase K-resistant
PrP, and they convincingly showed that prion infectivity can be generated de novo in brain homogenates
derived from healthy hamsters using PMCA. Inoculation of healthy hamsters with de novo formed prions
caused prion disease (135), providing strong support
for the prion hypothesis.
Additional support for the prion hypothesis comes
from the demonstration that infectious material can be
generated in mice by altering the sequence of the prion
AGGREGATION AND INFECTIOUS DISEASE
region in mouse chromosome 2 (479). Hybridization studies demonstrated ⬍0.002 Prnp gene sequences per ID50
unit in purified prion fractions, strongly suggesting that a
nucleic acid encoding PrPSc cannot constitute a component of the infectious prion particle (370). This is a major
feature that distinguishes prions from viruses, including
retroviruses which carry cellular oncogenes and “satellite
viruses” bearing coat proteins from viruses that had previously infected the host.
D. PrP Amyloid
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diseases characterized by protein aggregation, such as
Alzheimer’s disease and other amyloidoses, the formation
of large amyloid fibrils might be a protective process that
sequesters the more dangerous subfibrillar oligomers of
the amyloidogenic peptide or protein into relatively innocuous deposits.
Prion-like amyloids also exist in lower eukaryotes
such as yeast. Fungal prions are non-PrP related molecules that include HET-s, Ure2p, and Sup35 proteins,
which can adopt both nonamyloid and self-perpetuating
amyloid structures. In contrast to PrPSc, the conversion of
these proteins into their prion-like conformations has
been shown to have important physiological functions in
yeast. The conversion of Ure2p and Sup35 into their amyloid forms (URE3 and PSI⫹, respectively) regulates the
transcription and translation of specific yeast genes (237,
492). Aggregated HET-s regulates heterokaryon incompatibility, a fungal self/non-self recognition phenomenon that
prevents various forms of parasitism (509). So far there
have been only a few reported instances of mammalian
proteins that are functionally regulated in a nonpathological way by interconversion between nonamyloid and amyloid forms. A remarkable example is the synthesis of
melanin, which involves the formation of amyloid structures (158). In addition, it has been proposed that proteins
involved in establishing long-term memory might do so by
converting reversibly to and from an amlyoid-like state
(455, 457).
E. Formation of PrPSc
It remains to be established whether any form of
PrPC can act as a substrate for PrPSc formation, or
whether a restricted subset of PrP molecules are precursors for PrPSc (511). It is also unknown whether reactive
transition states between the two exist. Several experimental results argue that PrP molecules destined to
become PrPSc exit to the cell surface prior to their conversion into PrPSc (59, 99, 494). Similar to other GPIanchored proteins, PrPC appears to localize in cholesterolrich, nonacidic, detergent-insoluble membranes known as
rafts (26, 195, 253, 456, 495). Within the raft compartment,
GPI-anchored PrPC is apparently either converted into
PrPSc or partially degraded (343, 495). Chemical and enzymatic treatment of purified PrP27–30 leads to the release
of glycolipid components, suggesting that PrPSc could be
tethered to the membrane by a GPI anchor (481).
The role of the GPI membrane anchor in the formation of PrPSc in vivo has been addressed by Chesebro et
al. (108), who have established a transgenic mouse model
expressing anchorless, and hence secreted, PrP. When
these transgenic mice were subsequently infected with
protease-resistant PrPSc, they developed significant amyloid plaque pathology in the brain, but clinical manifesta-
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The discovery of PrP27–30 in fractions enriched for
scrapie infectivity was accompanied by the identification
of rod-shaped particles in the same fractions (404, 410).
The fine structure of these particles, which had been
originally described by Mertz as “scrapie-associated
fibrils” (344), failed to reveal any regular substructure
characteristic of most viruses (526). Conversely, prion
rods are indistinguishable from many purified amyloids
(114). This analogy was extended when the prion rods
were found to display the tinctorial properties of amyloids
(410). Small amyloidotropic dyes, such as derivatives of
Congo red and thioflavins, bind with various degrees of
selectivity to protein aggregates having an extensive cross
␤-pleated sheet conformation and sufficient structural
regularity and give rise to an enhanced fluorescence (thioflavins) or apple-green birefringence under cross-polarized light (Congo red) (47, 138). However, these dyes are
not suitable for recognizing prefibrillary species and amyloid deposits of diverse morphological origin. Advancement in this direction has been provided by luminescent
conjugated polymers (LCPs), a recently developed, novel
class of amyloidotropic dyes (216, 366, 367).
The amyloid plaques in the brains of humans and
other animals with prion disease contain PrP, as determined by immunoreactivity and amino acid sequencing
(45, 132, 265, 426, 488). Solubilization of PrP27–30 into
liposomes with retention of infectivity (169) suggests that
large PrP polymers are not required for infectivity, although it is certainly possible that PrPSc oligomers may
constitute nucleation centers pivotal to prion replication
(238, 288). To systematically evaluate the relationship
between infectivity, converting activity, and the size of
various PrPSc-containing aggregates, Caughey and coworkers (464) partially disaggregated PrPSc. The resulting
species were fractionated by size and analyzed by light
scattering and nondenaturing gel electrophoresis. Intracerebral inoculation of the different fractions into hamsters
revealed that with respect to PrP content, infectivity
peaked markedly with 17–27 nm (300 – 600 kDa) particles.
These results suggest that nonfibrillar particles, with
masses equivalent to 14 –28 PrP molecules, are the most
efficient initiators of TSE disease (464). As with other
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tions were minimal. In contrast, combined expression of
anchorless and wild-type PrP resulted in accelerated clinical scrapie (108).
1. Structures of purified PrPC and PrPSc
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Early low-resolution structural studies indicated that
PrPC had a high ␣-helix content (⬃40% of the protein) and
relatively little ␤-sheet (3% of the protein) (379). These
findings were further refined by Wüthrich and colleagues
who determined the fine structure of PrPC by nuclear
magnetic resonance spectroscopy (Fig. 1A) (229, 421),
and later also by crystallographic studies (271). The NH2proximal half of the molecule is not structured at all,
whereas the COOH-proximal half is arranged in three
␣-helices corresponding, for the human PrPC, to the residues 144 –154, 173–194, and 200 –228, interspersed with
an antiparallel ␤-pleated sheet formed by ␤-strands at
residues 128 –131 and 161–164. A single disulfide bond is
found between cysteine residues 179 and 214 (421, 422,
531). It is unlikely that the NH2 terminus is randomly
coiled in vivo, since functional studies in transgenic mice
imply that the domain comprising amino acids 32–121
carries out important physiological functions (453). It is
possible that the flexible tail of PrPC acquires a defined
structure when PrPC is present within membrane rafts
(362).
In contrast to PrPC, the ␤-sheet content of PrPSc
comprises ⬃40% of the protein, whereas ␣-helices comprise 30% of the protein as measured by Fourier-transform
infrared (379) and CD spectroscopy (Fig. 1B) (436). No
high-resolution structure is available for PrPSc, although
interesting models have been conjectured on the basis of
electron crystallography studies (524). Prion rods of the
NH2-terminally truncated form of PrPSc derived by limited
proteolysis, PrP27–30, exhibit green-gold birefringence after staining with Congo red, indicating that this isoform
has a high ␤-sheet content (410). Indeed, PrP27–30 poly-
C
Sc
FIG. 1. Structural features of PrP and PrP . A: NMR structure of
the mouse prion protein domain (121–231). The ribbon diagram indicates the positions of the three helices (yellow) and the antiparallel
two-stranded ␤-sheet (cyan). The connecting loops are displayed in
green if their structure is well defined and in magenta otherwise. The
disulfide bond between Cys-179 and Cys-214 is shown in white. The
NH2-terminal segment of residues 121–124 and the COOH-terminal segment 220 –231 are disordered and not displayed. (Figure kindly provided
by S. Hornemann.) B: Fourier transform infrared spectroscopy of prion
proteins. The amide I⬘ band (1,700 –1,600 cm⫺1) of transmission FTIR
spectra of PrPC (black line), PrPSc (gray line), and PrP27–30 (dotted line).
These proteins were suspended in a buffer in D2O containing 0.15 M
sodium chloride/10 mM sodium phosphate, pD 7.5 (uncorrected)/0.12%
ZW. The spectra are scaled independently to be full scale on the ordinate
axis (absorbance). [From Pan et al. (379).] C: electron micrographs of
negatively stained and immunogold-labeled prion proteins. Each panel
shows in following order PrPC, PrPSc, and Prion rods, composed of
PrP27–30, that were negatively stained with uranyl acetate. Bar, 100 nm.
[From Pan et al. (379).]
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merized into rod-shaped particles with an ultrastructural
appearance of amyloid (Fig. 1C) containing ⬃50% ␤-sheet
and 20% ␣-helix content (103, 176, 435). In contrast to
PrP27–30, neither purified PrPC nor PrPSc forms aggregates
detectable by electron microscopy (338, 379).
2. Models describing the replication of prions
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C
FIG. 2. Models for the conformational conversion of PrP into
PrPSc. A: the “refolding” or template assistance model postulates an
interaction between exogenously introduced PrPSc and endogenous
PrPC, which is induced to transform itself into further PrPSc. A highenergy barrier may prevent spontaneous conversion of PrPC into PrPSc.
B: the “seeding” or nucleation-polymerization model proposes that PrPC
and PrPSc are in a reversible thermodynamic equilibrium. Only if several
monomeric PrPSc molecules are mounted into a highly ordered seed, can
further monomeric PrPSc be recruited and eventually aggregate to amyloid. Fragmentation of PrPSc aggregates increases the number of nuclei,
which can recruit further PrPSc and thus results in apparent replication
of the agent.
manner by incubating small amounts of PrP S c containing brain homogenate with uninfected, PrPCcontaining brain homogenate, resulting in the conversion of PrPC to PrPSc. The latter forms aggregates that
are sonicated to generate multiple smaller units functioning as a seed for the continued formation of new
PrPSc aggregates.
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Transgenic mouse studies have provided genetic
(412) and biochemical (343) evidence that the conversion
of PrPC to PrPSc occurs through the formation of a PrPC/
PrPSc complex. However, such a complex has never been
isolated to purity. Consequently, it is unclear whether
PrPC binds to one or more additional macromolecules
during the process of PrPC to PrPSc conversion. The formation of a PrPC/PrPSc complex may be facilitated when
the amino acid sequences of the two PrP isoforms are
identical. For instance, hamster PrP differs from mouse
PrP at 16 of 254 positions, perhaps explaining the strong
species barrier between mice and hamsters. Differences
in amino acid sequence delay the onset of prion disease
by prolonging the incubation time (446). As the sequence
specificity of PrPSc formation is more important for the
conversion to protease resistance than for the initial binding steps, additional intermolecular interactions between
PrPC and PrPSc may be required to complete the process
of conversion to the protease-resistant state (228).
The level of PrPC expression appears to be directly
proportional to the rate of PrPSc formation and, thus, inversely related to the length of the incubation time. Prnpo/o
mice are resistant to prions and do not propagate scrapie
infectivity (84, 407, 439). Mice hemizygous (Prnp⫹/o) for
ablation of the Prnp gene have prolonged incubation times
when inoculated with mouse prions (83, 326).
According to the “protein only” hypothesis, there
are two models to explain the conformational conversion of PrPC into PrPSc. The “template-directed refolding” hypothesis predicates an instructionist role for
PrPSc on PrPC (Fig. 2A). Alternatively, the seeded nucleation hypothesis suggests that PrPSc exists in equilibrium with PrPC. In a nondisease state, such an equilibrium would be heavily shifted toward the PrPC conformation, such that only minute amounts of PrPSc
would coexist with PrPC. If this were the case, PrPSc
could not possibly represent the infectious agent, since
it would be ubiquitous. According to this “seeded nucleation” hypothesis (238), the infectious agent would
consist of a highly ordered aggregate of PrPSc molecules. The aggregated state would be an intrinsic property of infectivity. Monomeric PrPSc would be harmless,
but it might be prone to incorporation into nascent
PrPSc aggregates (Fig. 2B) (19).
The recently developed PMCA (92, 93, 196, 432,
434) is designed to mimic PrPSc autocatalytic replication (Fig. 3). In the PMCA, PrPSc is amplified in a cyclic
1112
ADRIANO AGUZZI AND ANNA MARIA CALELLA
An important part of the prion replication process is
the propagation of prions through the fragmentation of
existing fibrils. This mechanism has been experimentally
verified for yeast prions (62, 491). There are indications
that similar processes govern the growth of mammalian
prions (94, 295) as well as non-prion-related amyloid
fibrils (139).
Analysis of yeast prions has shown that the mechanical frangibility of formed prion fibrils plays a key role in
fibril growth, complexity, and diversity (492). The fragility
of yeast prions varies substantially between different
classes of fibrils, but is relatively homogeneous within a
single class (237, 492). Yeast prion classes with an aggressive tendency to multiply in vivo have been found to be
composed of amyloid fibrils with the highest propensity to
fragment. It is interesting to note that in terms of their
mechanical rigidity, amyloid fibrils can be very heterogeneous (272), implying a substantial potential for intrinsic
variability in their breakage rates. Although prion propagation in mammalian tissues is likely to be inherently
more convoluted, recent observations indicate that the
propagation of mammalian prion strains may be kinetically similar to fungal prions. Indeed, shorter fibrils,
which may result from breakage of frangible structures,
are more infectious than longer fibrils (464), an observation consistent with the fact that a larger number of free
Physiol Rev • VOL
ends in a short fibril population leads to more rapid
conversion of soluble cellular prion protein into misfolded fibrillar form and eventually overwhelming cellular
clearance mechanisms.
In agreement with this idea, an inverse correlation
was recently found between the stability of prion aggregates and incubation times in vivo (296), a finding that is
closely analogous to results obtained in yeast prions
(499). Furthermore, in in vitro growth assays of many
amyloid fibril systems, it has been noted that agitation
significantly enhances the overall conversion rate of proteins into fibrillar forms (255, 371, 467), indicating that
fibril breakage is an essential factor determining the rate
of amyloid formation. In living systems, the rate constants
for prion replication are clearly influenced not only by the
intrinsic strength of PrPSc aggregates, but also by other
cellular components. Molecular chaperones in yeast, for
instance, have been identified as important players in the
rate of prion replication (454).
F. Prion Diversity
One of the most puzzling phenomena in prion biology
is the existence of prion strains. Prion strains are defined
as infectious isolates that, when transmitted to identical
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C
FIG. 3. Schematic representation of the protein misfolding cyclic amplification (PMCA) reaction. PrP is recruited into growing aggregates of
PrPSc; hence, it undergoes conformational conversion and becomes PrPSc. During PMCA, the growing PrPSc species are disrupted by repeated
sonication in the presence of detergents. This treatment generates an expanded population of converting units for the continuous recruitment of
PrPC. The whole procedure is repeated several times. PrPC is shown as light gray spheres. PrPSc is shown as trapezium. The original seed is in dark
gray, and the newly formed PrPSc is in light gray.
1113
AGGREGATION AND INFECTIOUS DISEASE
1. Prion strains and variations in patterns of disease
Although defining of prion strains according to biochemical characteristics is useful in terms of nomenclature and classification of prion diseases, it certainly does
not explain why strains display different organ tropisms.
TABLE
2.
For example, some prion strains preferentially propagate
in the CNS and exhibit low abundance in secondary lymphoid organs. This neurotropic pattern of tissue distribution is observed in “classical” BSE in bovines. In other
strains, PrPSc and/or prion infectivity are detected not
only in the CNS but also, to a large extent, in secondary
lymphoid organs. These lymphotropic prion strains include vCJD, CWD (460), and many scrapie strains (20).
However, these differences are not absolute. For example, in patients with sCJD, it was originally believed that
prions were present in the CNS and other peripheral
tissues, but not in secondary lymphoid organs (74). However, it is now acknowledged that PrPSc can also be found
in the spleen and skeletal muscle of patients with sCJD
(183, 388).
What defines the organ tropism of a given prion strain
is still a completely open question. One might speculate
that tropism is defined by the tertiary or (more probably)
by the supramolecular structure of the prion agent, which
influences its ability to bind to or interact with specific
molecules (for example, receptors), and as a result affects
specific cell types (9). The phenomenon of cellular prion
tropism has been elegantly demonstrated by Charles
Weissmann’s group in an array of cell lines which replicate different prion strains to varying degrees (319). The
differential cell tropism of the various prion strains suggests the requirement for cell-specific cofactors, be they
chaperones, specific uptake receptors, RNA species, a
particular lipid environment, a specific posttranslationally
modified PrP molecule, or a particular prion replicating
subcellular environment. This hypothesis relies on the
assumption that cofactors supporting the conversion of
distinct prion strains exist in defined cell groups (16).
2. Molecular basis of prion strains
The mechanism by which isolate-specific information
is carried by prions remains enigmatic. A dwindling num-
Synopsis of currently used prion strain differentiation assays
Assay Principle
Test Substrate
Speed
Cost
Reference Nos.
Incubation period in indicator mice
Histological lesion profile
Histoblots
Conformation-dependent immunoassay
Conformational stability assay
Conformation assay
PK cleavage site
Detection with NH2-terminal antibodies
Glycosylation profile
Amyloid detection by thioflavin and Congo red stains
Luminescent-conjugated polymers
Cell panel assay
Mice
Mice
Immunohistology
ELISA
Western blot
Infrared spectroscopy
Western blot
Western blot
Western blot
Histochemistry
Histochemistry
Cell culture
Years
Years
Days
Days
Days
Days
Days
Days
Days
Hours
Hours
Weeks
⫹⫹⫹
⫹⫹⫹
⫹⫹
⫹
⫹⫹
⫹⫹
⫹
⫹
⫹
⫹
⫹
⫹⫹
164
79, 163
493
437
390
101
51
396
220
460
462
319
Most assays have high discriminatory power between few specific strains (e.g., glycosylation profiles for bovine spongiform encephalopathy and variant Creutzfeldt-Jakob disease) but may perform poorly with other strains. ⫹, Low; ⫹⫹, high; ⫹⫹⫹, extremely high. 关Adapted
from Aguzzi (9).兴
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hosts, exhibit distinct prion-disease phenotypes. The phenotypic traits may include distinct patterns of protein
aggregate deposition, incubation times, histopathological
lesion profiles, and specific neuronal target areas. Most of
these traits are relatively stable across serial passages.
Often new distinct strains can be observed upon transmission of prions across an interspecies barrier or into
animals of the same species expressing different polymorphisms of the prion gene, a phenomenon sometimes referred to as a “strain mutation” (76, 504).
The diversity of scrapie prions was initially noted in
goats inoculated with “hyper” and “drowsy” isolates (386).
Scrapie isolates or “strains” from goats with a drowsy
syndrome transmitted a similar syndrome to inoculated
recipients, whereas those from goats with a “hyper” or
ataxic syndrome transmitted an ataxic form of scrapie to
recipient goats. Subsequent studies in mice have also
demonstrated the existence of diverse scrapie strains that
produce characteristic phenotypes in inoculated recipients (78, 136, 137) with respect to incubation times, the
distribution of central nervous system (CNS), and the
formation of amyloid plaques, although the clinical signs
of scrapie tend to be similar.
The identification of strains through the inoculation
of mice is time-consuming, cumbersome, and prohibitively expensive. Moreover, the reliability of the resulting
classification is based on mere correlative evidence. However, over time, a number of biochemical correlates for
prion strains have been identified (Table 2) (9), and it is
expected that these criteria will facilitate the study and
the diagnosis of prion strains.
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ADRIANO AGUZZI AND ANNA MARIA CALELLA
Physiol Rev • VOL
Sc
FIG. 4. Representation of the three glycosylated PrP
moieties
(un-, mono-, and diglycosylated PrPSc) in immunoblots of brain extracts
after digestion with proteinase K. Different inocula result in specific
mobilities of the three PrP bands as well as different predominance of
certain bands (top panel). These characteristic patterns can be retained,
or changed to other predictable patterns after passage in wild-type mice
(bottom panel). On the basis of the fragment size and the relative
abundance of individual bands, three distinct patterns (PrPSc types 1–3)
were defined for sCJD and iCJD cases. In contrast, all cases of vCJD and
of BSE displayed a novel pattern, designated as type 4 pattern.
the disease remain unanswered. It has been proposed
that the relative prevalence of distinct glycoforms may
determine the structure of infectious PrP seeds and
thereby determine strain properties (116), although little
physical evidence for this assertion existed until recently.
This hypothesis is certainly compatible with the report
that strains differ in their ability to infect hosts with
restricted PrP glycosylation, and certain strains may only
produce disease in hosts whose PrPC harbors specific
carbohydrate side chains (501).
G. Prion-like Properties of Additional
Misfolded Proteins
The “seeded nucleation” theory (238) described for
PrP may also apply to amyloid formation in other protein
misfolding disorders (PMDs). In fact, protein conformational changes associated with the pathogenesis of most
PMDs result in the formation of abnormal proteins that
are rich in ␤-sheet structure, partially resistant to proteolysis, and have a high tendency to form larger-order
aggregates, similar to PrPSc. Amyloid formation depends
on the slow interaction of misfolded protein monomers to
form oligomeric nuclei, around which a faster phase of
elongation takes place. The ability of oligomeric species
to seed their own growth is analogous to the self-propagating activity of prions.
These theoretical considerations are supported by
extensive experimental data (474). In addition, it has been
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ber of investigators still contend that scrapie is caused by
a viruslike particle containing a scrapie-specific nucleic
acid that encodes the information expressed by each
isolate (78). But to date, no such polynucleotide has been
identified, despite the use of a wide variety of techniques,
including the measurement of nucleic acids in purified
preparations. An alternative hypothesis is that PrPSc alone
is capable of transmitting the disease and that the characteristics of PrPSc are modified by a cellular nucleic acid
(512). This accessory cellular polynucleotide is postulated
to induce its own synthesis upon transmission from one
host to another, but there is currently no experimental
evidence to support its existence.
In striking contrast, mice expressing Prnp transgenes
have demonstrated that the level of PrP expression is
inversely related to the incubation time (412). Taken together, these observations indicate that PrPSc itself is the
molecule in which prion “strain”-specific information is
encrypted. Preliminary immunochemical evidence suggests that PrPSc of different strains expose different
epitopes (438) and display different degrees of stability to
chaotropic salts (390). Infrared spectroscopy measurements have shown that types of PrPSc associated with
distinct hamster TSE strains can possess different conformations, even though they are derived from PrP with
the same amino acid sequence (101). This could be interpreted as indirect evidence for conformational differences or could indicate that different PrPSc strains associate with different molecules of proteinaceous or other
nature.
Both PrPC and PrPSc exist in three main glycosylation
states: unglycosylated, monoglycosylated, and diglycosylated. The relative ratios of these three forms of PrPSc
differ in various prion strains. In some cases, such differences are remarkably robust and are widely used for
prion strain typing. On the basis of the fragment size and
the relative abundance of individual bands identified by
gel electrophoresis, three distinct patterns of PrPSc (types
1–3) have been identified for sporadic and iatrogenic CJD
cases (118, 381). In contrast, all cases of vCJD display a
novel pattern, designated type 4 by Collinge and type III
by Parchi and Gambetti (Fig. 4). Interestingly, brain extracts from BSE-infected cattle as well as BSE-inoculated
macaques exhibited a type 4 pattern. Even more intriguing, transmission of BSE or vCJD to mice produced
mouse PrPSc with a type 4 pattern indistinguishable from
the original inoculum (219). These findings strongly support the hypothesis that vCJD is the result of BSE transmission to humans.
It is clear that in some patients suffering from CJD,
multiple distinct types of CJD-associated PrPSc molecules coexist within the same patient (396). However,
the molecular basis for these differences in glycosylation is poorly understood, and the mechanisms by
which differences in PrPSc glycosylation states affect
AGGREGATION AND INFECTIOUS DISEASE
Physiol Rev • VOL
clearly the case with legitimate TSE prions, or merely
enhances an already ongoing pathogenic process (96).
Several attempts have been made to transmit AD to
experimental animals, which have yielded intriguing, yet
conflicting, results. An early report of buffy coat-borne AD
transmission to hamsters (327) initially caused great consternation, but was later found to be irreproducible. Marmosets injected with brain homogenates from individuals
with AD developed scattered deposits of the amyloid-␤
protein (A␤) in the brain parenchyma and cerebral vasculature 6 –7 yr after inoculation (35). However, extensive
studies by Gajdusek and colleagues (74) have failed to
transmit AD and other dementias to primates.
Jucker and co-workers (345) showed that the injection of the A␤ peptide derived from human AD brains to
transgenic mice overexpressing the A␤ precursor protein
APP induced cerebral ␤-amyloidosis. The pathology in the
APP transgenic mice was determined by both the host and
the inoculum (345). The authors postulated the existence
of A␤ strains that can initiate and accelerate aggregation
and A␤ pathology. It remains to be seen whether different
A␤ strains with distinct biochemical or neuropathological
characteristics occur in humans. Once again, as was the
case with the AA inoculation studies mentioned above, it
is unclear whether inoculation with A␤ amyloid causes
amyloidosis or simply accelerates an ongoing pathogenic
process, since transgenic recipients ultimately develop
amyloid pathology in the brain, even without inoculation
with A␤ amyloid extracts.
Another major unresolved issue is the extent to
which the induced amyloidotic lesions can spread from
localized sites of seeding by the inoculum to other sites
within the host. Surprisingly, a recent study reported that
healthy fetal tissue grafted into the brains of Parkinson’s
disease patients acquired cytoplasmic ␣-synuclein-rich
Lewy bodies, suggesting that misfolded ␣-synuclein
spreads to healthy cells and acts as a template for the
conversion of native ␣-helices to pathogenic ␤-sheets
(299).
Type 2 diabetes is yet another disease whose pathogenesis may involve ordered protein aggregation. Evidence for this was discovered early on (373) but was
largely forgotten for almost a century. It is now evident
that aggregation of islet amyloid polypeptide (IAPP) is an
exceedingly frequent feature of type 2 diabetes. IAPP
amyloid damages the insulin-producing ␤-cells within
pancreatic islets and may crucially contribute to the
pathogenesis of diabetes (233). It is unknown, however,
whether IAPP deposition simply accrues linearly with
IAPP production, or whether it spreads in a prion-like
fashion from one pancreatic islet to the next.
In summary, the role of protein misfolding and aggregation in various human diseases has been clearly
established in the past decade, yet an important challenge
for the coming years will be to determine whether the
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reported that polyglutamine aggregates (420) and tau aggregates (167) can be internalized by mammalian cells in
culture. These internalized fibrillar aggregates may then
selectively recruit soluble cytoplasmic proteins and stimulate further misfolding of otherwise stable intracellular
proteins. From a molecular standpoint, all PMDs can be
considered “infectious” in the sense that misfolded and
aggregated conformers act as a templates for the recruitment of native proteins. However, in no PMDs other than
PrPSc pathologies has bona fide “microbiological” infectivity been detected, and none of the non-PrP aggregation
diseases has ever given rise to macroepidemics such as
Kuru and BSE.
Some experimental amyloidoses in mammals have
shown evidence of being transmissible in the sense that
amyloid taken from an amyloidotic individual can sometimes greatly accelerate amyloidosis in another individual. However, it is important to note that such “transmissions” have only been shown under experimental conditions in which the recipient animal is highly primed for
susceptibility to amyloidosis and would be expected to
spontaneously develop amyloidosis over time. Typical examples are AA amyloidosis and Alzheimer’s disease (AD).
Most cases of AA amyloidosis in mammals appear to
occur spontaneously due to chronic inflammation or genetic peculiarities that elevate blood levels of serum amyloid A (SAA) and predispose the organism to AA amyloid
formation. AA amyloid is characterized by the accumulation of aggregated SAA NH2-terminal-derived peptides in
organs such as the kidney, liver, and spleen. Inoculation
of unaffected mice with tissue extracts from amyloidotic
mice can rapidly induce amyloidosis, as long as the recipients are “primed” for amyloidosis with proinflammatory
treatments such as silver nitrate injections (261, 308).
Given the “infectious” characteristics of AA amyloidosis, transmission between captive cheetahs may explain
the high incidence of AA amyloidosis observed in this
species. Acceleration of amyloidosis in mice upon oral
administration of AA amyloid suggests a plausible fecaloral route for the natural transmission of AA amyloidosis
in cheetahs. Indeed, amyloidotic cheetahs shed AA amyloid in the feces, and this fecal amyloid can accelerate AA
amyloidosis when injected intravenously into silver nitrate-primed mice (533).
However, unlike wild cheetahs, captive cheetahs exhibit chronic inflammation and high blood SAA levels
(359). These factors could make them susceptible to
spontaneous amyloidosis, as it is the case with silver
nitrate-treated mice. Therefore, the high incidence of
amyloidosis in captive cheetahs might also be due to
spontaneous disease, rather than infection with amyloid
from other animals. This raises the important question of
whether, in the case of non-PrPSc amyloids, inoculation
with the amyloid actually initiates the disease, as is
1115
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ADRIANO AGUZZI AND ANNA MARIA CALELLA
prion mechanism of disease transmission might be operating in other human diseases, some of which are highly
prevalent.
whether the physiological function of PrPC is actually
linked to toxicity associated with prion diseases.
A. Teachings From Prnp-Ablated Mice
II. FUNCTION OF THE CELLULAR PRION
PROTEIN
FIG. 5. Outline of the primary structure of the cellular prion protein including posttranslational modifications. A secretory signal peptide resides
at the extreme NH2 terminus. CC1 and CC2 define the charged clusters. OR indicates the octapeptide repeat, and four are present. HC defines the
hydrophobic core. MA denotes the membrane anchor region. S-S indicates the single disulfide bridge, and the glycosylation sites are designated as
CHO. The numbers describe the position of the respective amino acids.
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PrPC, following the cleavage of a 22-amino acid
(aa) signal peptide, is exported to the cell surface as an
N-glycosylated, GPI-anchored protein of 208 –209 aa.
PrPC contains a long NH2-proximal flexible random-coil
sequence, which is followed by a globular COOH-proximal domain. The structurally less-defined NH2 proximal region consists of residues 23–124 and contains a
stretch of several octapeptide repeats (OR), flanked by
two positively charged clusters, CC1 (aa 23–27) and
CC2 (aa 95–110). These domains are linked by a hydrophobic stretch of amino acids known as the HC region
(aa 111–134) (Fig. 5).
The structure of the globular half of human PrPC is
identical to many other mammals, as expected from the
high degree of sequence identity (309). Interestingly, despite the low sequence identity between PrPC in chicken,
turtle, or frog, and the mammalian isoforms, the major
structural features of PrPC are remarkably preserved in
nonmammalian species (88).
Full-length PrPC is found in non-, mono-, or diglycosylated forms, corresponding to the variable occupancy of
glycosyl groups on residues Asn-181 and Asn-197 in human and Asn-180 and Asn-196 in mice (201). A rather large
variety of N-glycans are found attached to both full-length
and truncated PrPC (380, 428), which may be differentially
distributed in various areas of the central nervous system
(49, 133). The physiological significance of PrPC glycosylation is unknown.
Despite advances in the characterization of PrPC
structure, the physiological function of PrPC is still unclear. An unrealistic plethora of possible functions have
been ascribed to PrP. Therefore, there is a lingering suspicion that each of these reports represents a specific
aspect of a more complex reality whose full picture is still
not entirely understood. Importantly, it is not yet clear
Obviously, the study of PrP knockout mice has thus
far failed to elucidate the molecular function of the protein. This is particularly sobering when one considers the
fact that independent lines of mice lacking PrPC have
been generated by homologous recombination in embryonic stem cells in many laboratories. Mice with disruptive
gene modifications restricted to the open reading frame
are known as Prnpo/o (Zurich I) (85) and Prnp⫺/⫺ (Edinburgh) (325). These mice were found to develop normally,
although a number of subtle abnormalities have been
described (120, 368, 498) whose molecular basis remains
undefined. Among other functions, it has been suggested
that PrPC may have a role in the synaptic machinery of the
olfactory bulb (293). However, the reported involvement
of PrPC in synaptic hippocampal activity has been the
subject of intense controversy (301).
The only molecularly well-defined phenotype of
Prnpo/o mice is their resistance to prion inoculation
(84). However, it seems rather unlikely that a protein as
highly conserved among species as PrPC has evolved
purely for the purpose of bestowing susceptibility to
prion diseases upon organisms. If the function of PrPC
were completely unrelated to prion disease pathogenesis, PrPC would be just one of many proteins whose
function awaits clarification. Otherwise, the function of
PrPC may reflect, in a subtle way, prion-induced damage. However, Prnp ablation does not elicit disease,
even when induced postnatally (322). Therefore, prion
pathology is unlikely to occur due to a simple loss of
PrPC function. If PrPC possessed enzymatic activity or
transduced a signal (similarly to many other GPI-linked
proteins), one could hypothesize that conversion to
PrPSc alters PrPC substrate specificity or signal transduction strength, conferring as a result a toxic dominant function. Therefore, understanding the function of
PrPC may be instrumental to deciphering prion pathol-
AGGREGATION AND INFECTIOUS DISEASE
B. PrPC and Its Partners
Perhaps PrPC and PrPSc do not possess any intrinsic biological activity of their own, but modify the
function of other proteins. GPI-anchored proteins must
interact with transmembrane adaptors on the same cell
or an adjacent cell to influence intracellular signal
transduction pathway. This supposition has led to the
search for PrP-interacting partners using methods such
as yeast-two hybrid, cross-linking experiments, and coimmunoprecipitations. All known PrPC interaction partners have been summarized in a recent review (10).
Putative PrPC interacting proteins include membrane
proteins (receptors, enzymes, caveolin-1, Na ⫹ -K ⫹ ATPase, and a potassium channel), cytoplasmic proteins (components of the cytoskeleton, heat-shock proteins, and adaptor proteins involved in signaling), and
even nuclear proteins. Recently, transgenic mice expressing functional tagged prion protein have been
used to characterize the PrPC interactome (431). However, many studies used to identify such interactors are
fraught with methodological issues. PrPC is exposed to
the extracellular space, and therefore, it is questionable
whether conventional yeast two-hybrid screens, which
artificially expose PrPC to the cytosolic compartment,
represent an appropriate method to investigate PrPC
interaction partners. The same concerns may apply to
chemical cross-linking experiments. Cross-linking
fuses together any proteins that reside in the same
microenvironment, even if they do not necessarily interact with each other. Furthermore, the choice of detergent conditions in coimmunoprecipitation experiments is also crucial, since it should allow for weak and
transient protein-protein interactions, but should destroy any potential artificial or postlysis nonspecific
interactions. Ultimately, it would be desirable to validate reported interactions by physiological read-outs,
yet this lofty goal has not been attained for most of the
candidates described thus far.
Physiol Rev • VOL
C. PrP’s Double and Its Trouble
After the original report by the Weissmann laboratory (85), several independent Prnp knockout mouse
lines were generated, including Prnp⫺/⫺ (Nagasaki),
Rcm0, and Prnp⫺/⫺ (Zurich II) (356, 427, 440). These
endeavors have led to very surprising insights. All of the
latter mice, but none of the Zurich I mice, developed
ataxia and Purkinje cell loss later in life. The discrepancy
among the different lines of PrP knockout mice was not
resolved until a novel gene (Prnd), encoding a protein
called Doppel (Dpl), was discovered. Prnd is localized 16
kb downstream of Prnp. In all three lines of PrPC-deficient mice developing ataxia and Purkinje cell loss, a
splice acceptor site to the third exon of Prnp was deleted.
This placed Prnd under transcriptional control of the
Prnp promoter, resulting in the formation of chimeric
transcripts and overexpression of Dpl in the brain (513).
The Prnd gene is evolutionarily conserved from humans to sheep and cattle and shares roughly 25% homology with the COOH-proximal two-thirds of PrPC,
but it lacks the PrPC octapeptide repeats and hydrophobic region. Structural studies indicate that Dpl contains three ␣-helices, similar to PrPC, and two disulfide
bridges between the second and third helix (307, 465). Dpl
mRNA is expressed at high levels in testis and heart,
moderately in spleen and some other organs and, notably,
at very low levels in brain of adult wild-type mice. However, elevated Prnd mRNA transcripts have been detected
during embryogenesis and in the brains of newborn mice,
thus arguing for a possible function of Dpl in brain development (298). To probe the function of Dpl, the Prnd gene
was inactivated in embryonic stem cells (43). Similarly to
mice lacking PrPC, mice devoid of Dpl survive to adulthood and do not show obvious phenotypical alterations,
suggesting that Dpl is dispensable for embryogenesis and
postnatal development (44). The similarities between
PrPC and Dpl in primary amino acid sequence, in structure, and in subcellular localization suggest related biological functions (42). Therefore, a possible role of PrPC
and Dpl during development may be masked by functional redundancy. To address this possibility, Prnp/Prnd
double knockouts were generated using transallelic targeted meiotic recombination (177). Offspring carrying the
double deletion exhibited no abnormalities, except for
male sterility, and they did not develop ataxia, implicating
a causal role for Dpl overexpression in the Zurich II
phenotype. Furthermore, Dpl did not appear to compensate for the loss of any unrecognized function of PrP.
Why the overexpression of Dpl is deleterious is still
unclear. Neurodegeneration is not uniquely associated
with overexpression of Dpl. Transgenic mice overexpressing partially deleted Prnp variants provoke spontaneous neurodegenerative disease, and some domains are
also essential for restoring prion susceptibility (Table 3).
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ogy, and perhaps even to devising therapeutic approaches.
Glockshuber et al. (190) noted that PrPC had similarities to membrane-anchored signal peptidases, but
their observation has not been substantiated by functional data. PrPC can bind copper (67, 505), but reports
of increased copper content in neurons lacking PrPC
have been called into question by contradictory findings (506). The idea that PrPC might be a superoxide
dismutase (68, 69) was perceived as particularly attractive in view of its multiple copper-binding sites. However, PrPC does not provide any measurable contribution to dismutase activity in vivo (234, 506), although it
has been suggested that NH2-proximally-truncated PrPC
may suppress endogenous dismutase activity (441).
1117
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TABLE
ADRIANO AGUZZI AND ANNA MARIA CALELLA
3.
Summary of transgenic mutant for PrP
PrP Deletion Mutants
⌬1-22 231#
⌬23-88
⌬23-88 C178A
⌬23-88 ⌬95-107
⌬23-88 ⌬108-121
⌬23-88 ⌬122-140
⌬23-88 ⌬141-176
⌬23-88 ⌬141-221
⌬23-88 ⌬177-200
⌬23-88 ⌬201-217
⌬32-80
⌬32-93
⌬32-106
⌬32-121
⌬32-134
⌬94-134
⌬104-114
⌬105-125
⌬114-121
231#
144#
PG 14
Rescue by PrP (if applicable)
Cerebellar disorder
No
No
No
No
Short-lived protein
No
Cerebellar disorder
Storage disease
Storage disease
No
No
No
Cerebellar disorder
Cerebellar disorder
Cerebellar disorder
No
Cerebellar disorder
No
No
Short-lived protein
Cerebellar disorder
No
Prion Propagation
No
Yes
No
No
No
Yes
No
No
No
Yes
Yes
No
No
No
Not done
Not done
Not done
No
Yes
No
No
No
Yes
Yes
Yes
Yes
No
Yes
Reference Nos.
314
360, 361
360, 361
360, 361
360, 361
360
360, 483
484
360
360
153
154, 453
453*
453
453*
40
204
297
40, 225
108
360
109
The left column denotes the individual mutants. The column named “phenotype” indicates presence or absence of phenotypic abnormalities
in transgenic mice when expressed on a PrP-deficient genetic background; when the phenotype is rescued by endogenous PrP is indicated in the
next column. The column named “prion propagation” describes the susceptibility of transgenic mice or transfected cell lines to prions. #Stop codon.
*E. Flechsig, I. Hegyi, A. Aguzzi, and C. Weissman, unpublished results.
Deletions of amino acids 32–121 or 32–134 (collectively
termed ⌬PrP) confer strong neurotoxicity to PrPC in vivo,
which indicates that ⌬PrP is a functional antagonist of
PrPC (414, 453). Furthermore, a small deletion within the
hydrophobic stretch (amino acids 94 –134) is sufficient to
produce a highly neurotoxic molecule (40). This pathology can be abrogated by the reintroduction of wild-type,
full-length PrPC. Because the lack of PrPC itself does not
induce an obvious phenotype, the latter pathologies imply
the existence of pathways in which PrPC is functionally
active. Hence, mice expressing PrP variants may allow for
the identification of functionally relevant domains within
PrPC (10).
III. MECHANISMS OF PRION TOXICITY
Despite considerable knowledge about the characteristics of the TSE infectious agent, its mechanism of replication, and the association of PrPSc with neurodegeneration, the molecular pathways leading to cerebral
damage are, for the most part, unknown. Depletion of
PrPC is an unlikely cause, in view of the finding that
abrogation of PrP does not cause scrapielike neuropathological changes (85), even when elicited postnatally (322).
Moreover, it was observed that depletion of PrPC in mice
with established prion infection reversed early spongiform degeneration and prevented neuronal loss and progression to clinical disease (321, 323). Importantly, this
occurred despite the accumulation of extraneuronal
Physiol Rev • VOL
PrPSc to levels similar to terminally ill, wild-type infected
animals. These data suggest that PrPSc might not be directly responsible for neurodegeneration; it is more likely
that the toxicity of PrPSc depends on some PrPC-dependent process culminating in neuronal dysfunction and
death (63, 64).
The first experimental evidence in support of this
hypothesis was provided when neural tissue overexpressing PrPC was grafted into the brains of PrP-deficient mice
(153). Following scrapie inoculation, these mice remained
free of symptoms for at least 70 wk, exceeding the survival time of scrapie-infected donor mice by at least sevenfold (64). Therefore, the presence of a continuous
source of PrPSc does not exert any clinically detectable
adverse effects on a mouse devoid of PrPC. On the other
hand, the grafted tissue developed characteristic histopathological features of scrapie after inoculation. The
course of the disease in the graft was very similar to that
observed in the brain of scrapie-inoculated wild-type mice
(63). Since the grafts had extensive contact with the
recipient brain, prions could navigate between the two
compartments, as shown by the fact that inoculation of
wild-type animals engrafted with PrP-expressing neuroectodermal tissue resulted in scrapie pathology in both graft
and host tissue. Nonetheless, histopathological changes
never extended into host tissue, even at the latest stages
(⬎450 days), although PrPSc was detected in both grafts
and recipient brain, and immunohistochemistry revealed
PrP deposits in the hippocampus, and occasionally in the
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PrP
PrP
PrP
PrP
PrP
PrP
PrP
PrP
PrP
PrP
PrP
PrP
PrP
PrP
PrP
PrP
PrP
PrP
PrP
PrP
PrP
PrP
Phenotype
AGGREGATION AND INFECTIOUS DISEASE
A. Alteration of PrPC Mediated Signaling
Although it is unlikely that the toxic activity of PrPSc
results primarily from loss of PrPC function, neurons
derived from Prnp-deficient mice were originally reported to be more susceptible to the induction of apoptosis by serum deprivation. This effect could be rescued by
expression of either B-cell lymphoma protein 2 (BCL2) or
PrPC (286). Furthermore, it was shown that PrPC exerts
its cytoprotective function by decreasing the rate of apoptosis induced by particular apoptotic stimuli such as Bax
overexpression or tumor necrosis factor (TNF)-␣. Bax
overexpression normally induces apoptosis in human
neuronal cells, but coexpression of Bax with wild-type
PrPC, but not of PrP lacking the octarepeats, could reverse this effect (60). Therefore, PrPC can be neuroprotective against various insults, suggesting that its conversion to PrPSc could abrogate this function and induce
neurodegeneration.
A second possibility is that the binding of PrPSc to
C
PrP triggers a signal transduction pathway leading to
Physiol Rev • VOL
neuronal damage. This hypothesis is supported by a report that injection of antibodies that recognize and crosslink PrPC in the mouse hippocampus results in rapid and
extensive neuronal apoptosis (469), although in the intervening 5 years since this publication, no further insights
have come forth to explain this phenomenon.
B. PrPC Mislocalization
Abnormal topology or altered trafficking of PrPC has
also been proposed to underlie PrP-related neuronal toxicity. After processing in the endoplasmic reticulum (ER)
and Golgi apparatus, where it is glycosylated, mature
PrPC is attached to the cell surface by its GPI anchor at its
COOH terminus. PrPC can adopt at least two transmembrane topologies in the ER, CtmPrP and NtmPrP, which
have either their COOH or NH2 terminus in the ER
lumen, respectively. It has been suggested that CtmPrP
and NtmPrP normally comprise only a small portion of
cellular PrPC, and an excess of CtmPrP induces neurodegeneration (204, 205). Some PrP mutations favor
Ctm
PrP formation and are associated with a disease state
characterized by low levels of PrPSc accumulation. Thus
the propensity for the formation of the CtmPrP and NtmPrP
intermediates might lead to neurotoxicity in the absence
of PrPSc formation (Fig. 6).
Another suggested mechanism of PrP-mediated neurotoxicity is aberrant PrPC trafficking resulting in the
toxic accumulation of PrPC in the cytoplasm (Fig. 6).
Here, it is important to consider the mechanisms controlling protein synthesis and degradation. The fidelity of
protein synthesis is ensured by quality control within the
ER; misfolded proteins undergo retrograde transport to
the cytosol, become polyubiquitinylated, and are degraded by the proteasome through a process called ERassociated degradation (ERAD). Both wild-type and misfolded forms of PrPC undergo ERAD (313, 529). It has
been shown in vitro that the transfer of newly synthesized
PrPC to the cytoplasm by ERAD, and its cytosolic accumulation through the pharmacological inhibition of the
proteasome, leads to PrPSc-like formation and neurotoxicity (312). Furthermore, the artificial expression of PrP in
the cytosol in transgenic animals expressing a construct
lacking ER-targeting and GPI-anchoring signals causes
severe neurodegeneration (314).
Recently, Rane et al. (417) have described a possible
mechanism to explain the accumulation of cy(PrP) during
TSE neurodegeneration. Chronic ER stress, produced by
PrPSc accumulation, leads to persistent activation of a
“preemptive” quality control system (pQC) that aborts ER
translocation of PrP, thereby facilitating proteasome-mediated degradation of PrP in the cytosol. This pathway
constitutes a defense mechanism to prevent nascent protein entry into the ER lumen during conditions of com-
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parietal cortex, of all animals (64). The distribution of
PrPSc in the white matter tracts of the host brain suggests
diffusion within the extracellular space (239) rather than
axonal transport. Thus prions moved from the grafts to
some regions of the PrP-deficient host brain without causing pathological changes or clinical disease.
These findings suggest that the expression of PrPC by
an infected cell, rather than the extracellular deposition
of PrPSc, is one of the critical prerequisites for the development of scrapie pathology. Furthermore, what is important is not only the expression of PrP, but also its
location at the plasma membrane. Indeed, expression of a
Prnp variant that lacks the peptide responsible for anchoring membrane GPI molecules resulted in transgenic
mice that failed to develop disease after prion infection in
spite of PrPSc plaque formation in their brains (108). Also,
the blood and hearts of GPI-negative transgenic mice
contained both abnormal protease-resistant PrP as well
as infectious prions (500). Evidently, removal of the GPI
anchor abolishes the activation of downstream pathways
and susceptibility to clinical disease while preserving the
competence of soluble PrPC to support prion replication.
This observation is consistent with a growing body of
evidence that PrPC may function as a signaling molecule.
However, it is still possible that abnormal topology or
altered trafficking of PrPC might also underlie toxicity, or
perhaps the formation of a toxic form of PrP termed PrP*
by Charles Weissmann (511), other than PrPSc is responsible for spongiosis, gliosis, and neuronal death. This
theory could explain why in several instances (e.g., fatal
familial insomnia) localized spongiform pathology is detectable although very little PrPSc is present (21).
1119
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ADRIANO AGUZZI AND ANNA MARIA CALELLA
promised ER function. However, under chronic ER stress
conditions, the proteasome may become overwhelmed,
resulting in PrP accumulation in the cytosol. According to
the study of Rane et al. (417), even a modest increase in
PrP routing to pQC for prolonged periods of time causes
clinical and histological neurodegenerative changes reminiscent, in some aspects, of those observed in prion
diseases.
Although ER stress in prion diseases is well documented (217), the relevance or even the existence of cy(PrP)
continues to be rather controversial (142, 152). Furthermore, the transgenic mice with increased PrP translocation
to the pQC pathway reported by Rane et al. (417) showed a
relatively mild neurodegenerative phenotype that resembles
only a subset of TSE pathology, and other cellular pathways
Physiol Rev • VOL
may also be contributing to prion-induced neurodegeneration (472). A role for dysfunction of the ubiquitin-proteasome system (UPS) in the pathogenesis of prion disease was
also suggested: neuronal propagation of prions in the presence of mild proteasome impairment triggered a neurotoxic
mechanism involving the intracellular formation of cytosolic
PrPSc aggresomes that, in turn, activated caspase-dependent
neuronal apoptosis. A similar effect was also seen in vivo in
brains of prion-infected mice (285). In a follow-up study, the
same group reported that disease-associated prion protein
specifically inhibited the proteolytic ␤-subunits of the 26S
proteasome. Upon challenge with recombinant prion and
other amyloidogenic proteins, only the prion protein in a
nonnative ␤-sheet conformation inhibited the 26S proteasome at stoichiometric concentrations. Furthermore, there
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C
C
FIG. 6. Toxicity mediated by abnormal topology or altered trafficking of PrP . The normal cellular isoform of prion protein, PrP (green coils),
is synthesized, folded, and glycosylated in the endoplasmic reticulum (ER), where its glycosyl phosphatidylinositol (GPI) anchor is added, before
further modification in the Golgi complex. Mature PrPC translocates to the outer leaflet of the plasma membrane. Instead, CtmPrP and NtmPrP are
unusual transmembrane forms, generated in the ER, which have their COOH or NH2 terminus in the ER lumen, respectively. It has been suggested
that misfolded and aberrantly processed PrP (cyPrP and CtmPrP, respectively) (orange coils), which would normally be degraded by the proteasomes
through the ER-associated degradation (ERAD) pathway, aggregate in the cytoplasm and cause cell death. Putative proteasomal inhibition or
malfunction during prion disease would contribute to this route of toxicity. Induction of ER stress by PrPSc may lead to translocation of nascent
PrPC molecules to the cytosol for proteasomal degradation as a way to alleviate the overloaded ER (pQC pathway). However, this mechanism of
defense turns negative under chronic ER stress conditions, overwhelming the proteasome and leading to the cytosolic accumulation of potentially
toxic PrP molecules (dashed lines).
AGGREGATION AND INFECTIOUS DISEASE
appears to be a direct relationship between prion neuropathology and impairment of the UPS in prion-infected UPSreporter mice (284). Together, these data suggest a possible
mechanism of intracellular neurotoxicity mediated by misfolded prion protein.
C. PrP-Derived Oligomeric Species
Physiol Rev • VOL
amyloidosis (517) and familial amyloidotic polyneuropathy (124). The primary toxic species in TTR-associated
diseases appear to be low-molecular-weight oligomers
that do not stain with Congo red (470, 477).
The toxicity of aggregated “preamyloid” proteins appears to be a fairly general phenomenon. For example, the
prefibrillar forms of the non-disease-related HypF-N from
E. coli, the SH3 domain from bovine phosphatidylinositol
3-kinase, lysozyme from horse, and apomyoglobin from
sperm whale are all highly toxic to cultured fibroblasts
and neurons, whereas the monomeric native states and
the amyloid-like fibrils (formed in vitro) displayed very
little, if any, toxicity (81, 320, 466). The finding that polyclonal antisera can bind to protofibrillar species from
different sources, but not to their corresponding monomeric or fibrillar states, suggests that soluble amyloid
oligomers have some important common structural elements (252).
With the assumption that oligomeric intermediates of
a particular structure are common initiators of neurotoxicity observed in prion disease and in other protein misfolding disorders, the crucial question remains why these
protein species are toxic to cells. The conversion of a
protein from its native state into an oligomeric form invariably generates a wide distribution of misfolded species which exposes an array of groups that are normally
masked in globular proteins. The improper interactions
between oligomers and cellular components, such as
membranes, small metabolites, proteins, or other macromolecules, may conceivably lead to the malfunctioning of
crucial aspects of the cellular machinery. Depending on
the cell type involved, the result could be impairment of
axonal transport, oxidative stress, sequestration of essential proteins, or a combination of disparate factors, ultimately leading to apoptosis or other forms of cell death.
Small aggregates have a higher proportion of residues on
their surfaces than larger aggregates, including mature
amyloid fibrils, and therefore may be likely, in general, to
have a higher relative toxicity (110).
Elucidation of the mechanism of tissue damage by
amyloid fibril proteins is undoubtedly an important issue
in the development of therapeutic approaches, although
the optimum strategy may be to prevent aggregation or
even production of the amyloidogenic protein before it
can generate any potentially damaging intermediates.
IV. CLINICAL AND PATHOLOGICAL ASPECTS
OF PRION DISEASES
A. Scrapie
Even though scrapie was recognized as a distinct
disorder of sheep with respect to its clinical manifestations as early as 1738, the disease remained an enigma,
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The presence of highly organized and stable fibrillar
deposits in the brain of patients and animals suffering
from prion diseases initially led to the reasonable conjecture that this material is a causative agent of neurodegeneration. However, the most highly infective form of the
mammalian prion protein is an oligomeric species of ⬃20
molecules, and such small aggregates are the most effective initiators of transmissible spongiform encephalopathies (464).
More recent findings from other disorders associated
with protein misfolding and aggregation, such as AD and
Parkinson’s disease, have raised the possibility that precursors to amyloid fibrils, particularly including low-molecular-weight oligomers and/or structured protofibrils,
may represent the most toxic pathogenic species. In this
respect, it is certainly plausible to speculate that the
mechanism leading to neurotoxicity in prion disease
could be similar to that of other disorders associated with
protein misfolding.
In AD, the severity of cognitive impairment correlates with the levels of low-molecular-weight species of
A␤, rather than with the amyloid burden (202, 451). In
addition, transgenic mice (290, 352), as well as Drosophila expressing A␤1– 42 and A␤1– 40 (127, 236), show deficits in synaptic plasticity before significant accumulation
of amyloid. The existence of a toxic oligomeric intermediate is further supported by evidence that the aggressive
“arctic” (E693G) mutation of the amyloid ␤-precursor
protein, associated with heritable early-onset manifestation of AD, has been found in vitro to enhance protofibril,
but not fibril, formation (364).
The toxicity of early aggregates has also been shown
for Parkinson’s disease. In patients harboring mutations
associated with juvenile Parkinson’s disease or early-onset forms of Parkinsonism, the loss of nigral and locus
coeruleus neurons occurs in the absence of Lewy bodies
(intracellular fibrillar deposits) (262). In transgenic fly or
rat models of Parkinson’s disease, neuronal degeneration
is not associated with the formation of detectable intracellular deposits (32, 304). Furthermore, transgenic mice
exhibit substantial motor deficiencies and loss of dopaminergic neurons in the presence of nonfibrillar ␣-synuclein
deposits in various regions of the brain (330).
Toxicity of smaller aggregates appears not to be restricted to the brain. For example, transthyretin (TTR) is
an amyloidogenic protein implicated in systemic senile
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ADRIANO AGUZZI AND ANNA MARIA CALELLA
B. Bovine Spongiform Encephalopathy
In 1986, an epidemic of a previously unknown disease appeared in cattle in Great Britain (514): bovine
spongiform encephalopathy (BSE) or “mad cow” disease. BSE was found to be a prion disease upon the
discovery of protease-resistant PrP in the brains of ill
cattle (226). On the basis mainly of epidemiological
evidence, it has been proposed that BSE represents a
massive common source epidemic that has caused
more than 190,000 cases to date (http://www.oie.int/)
(Fig. 7A). In Britain, cattle, particularly dairy cows,
were routinely fed meat and bone meal as a nutritional
supplement (521). Since 1988, the practice of using
dietary protein supplements for domestic animals derived from rendered sheep or cattle offal has been
forbidden in the United Kingdom.
In 1992, the BSE epidemic reached a peak, with over
35,000 cattle afflicted. In 1993, fewer than 32,000 cattle
were diagnosed with BSE, and in 1994, the number was
⬃22,000. By 2003, BSE had become a rare disease in
British and European cattle, but regrettably (and quite
inexplicably) it still has not completely disappeared.
Brain extracts from BSE cattle have transmitted disease to mice, cattle, sheep, and pigs after intracerebral
inoculation (75, 130, 131, 165). Of particular importance to
Physiol Rev • VOL
the BSE epidemic is the transmission of BSE to the nonhuman primate marmoset after intracerebral inoculation
followed by a prolonged incubation period (34).
C. Chronic Wasting Disease of Deer and Elk
Chronic wasting disease of deer and elk (CWD) was
initially reported in 1980 as a TSE in captive research deer
in Colorado and Wyoming. The disease origin still remains
completely obscure (525). Since 1980, cases of CWD in
free-ranging mule deer (Odocoileus hemionus), whitetailed deer (O. virginanus), and Rocky Mountain elk
(Cervus elaphus nelsoni) have been detected in the same
region of Colorado and Wyoming. Recently, increased
surveillance efforts across the United States and Canada
have startlingly revealed CWD in adjacent states (Nebraska, New Mexico, and Utah) but also distant (Wisconsin, Illinois, Canada) from this original endemic region.
Thus far, CWD is only known to occur in North America
and in South Korea. That said, international testing for
CWD has been minimal with the exception of a CWD
surveillance program in Germany (458).
CWD is believed to be horizontally transmitted
among cervids with high efficiency (348). Exposure
through grazing in areas contaminated by prion-infected
secretions, excretions (saliva, urine, feces), tissues (placenta), or decomposed carcasses is most likely the major
source of spreading. Insightful experimental studies have
recently revealed two key findings: 1) saliva from CWDinfected deer can transmit disease (332), and 2) CWDinfected carcasses allowed to decay naturally in confined
pastures can lead to CWD infections in captive deer (349).
Additionally, abundant CWD prion accumulation within
lymphoid tissues may also lead to CWD prion buildup in
nonlymphoid organs with lymphoid follicles, as was recently demonstrated in kidney, potentially shifting shedding routes (200). It is unknown whether other types of
inflammation, such as the granulomatous inflammation in
the intestine seen in Johne’s disease (Mycobacterium
avium subsp. paratuberculosis; affects ruminants, including deer and elk) or parasitic inflammation could lead
to or perhaps increase prion excretion by fecal routes.
The potential for CWD transmission to other species
is clearly an area of great concern. Wild predators and
scavengers are presumably feeding on CWD-infected carcasses. Skeletal muscle has been shown to harbor CWD
prion infectivity (29), indicating that other species are
almost certainly being exposed to CWD through feeding.
However, CWD has not been successfully transmitted by
oral inoculation to species outside of the cervid family,
suggesting a strong species barrier for heterologous PrP
conversion (458). Human susceptibility to CWD is still
unclear, although we can be somewhat reassured in that
there have been no large-scale outbreaks of human TSE
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even with respect to its pathology, for more than two
centuries (382). Some veterinarians thought that scrapie
was a disease of muscle caused by parasites, whilst others
thought that it was a dystrophic process (311). An investigation into the etiology of scrapie followed the vaccination of sheep for louping-ill virus with formalin-treated
extracts of ovine lymphoid tissue unknowingly contaminated with scrapie prions (194). Two years later, more
than 1,500 sheep developed scrapie from this vaccine.
In 1998, aberrant cases of sheep scrapie were described in Norway, and the strain was newly classified as
Nor98 (46). Active European Union surveillance later revealed additional cases of atypical scrapie in several other
countries (87, 147); in fact, atypical scrapie appears to be
the rule rather than the exception in some geographical
areas.
Scrapie of sheep and goats seems to be readily communicable within flocks, but the mechanism of the natural
spread of scrapie among sheep is puzzling. The finding
that chronic inflammation can alter the tropism of prion
infectivity to organs hitherto believed prion free, like
mammary glands, raised concerns about scrapie transmission through milk (210, 300).
Polymorphisms at codons 136 and 171 of the Prnp
gene in sheep that produce amino acid substitutions have
been studied with respect to the occurrence of scrapie in
sheep (113).
AGGREGATION AND INFECTIOUS DISEASE
1123
cases in Colorado and Wyoming, where CWD has existed
for decades (334), and there is no doubt that people have
been exposed to CWD through venison consumption, particularly in light of recent data showing CWD prion accumulation in muscle.
Other indirect studies of human susceptibility to
CWD also suggest that the risk is low. In biochemical
conversion studies, the efficiency of CWD in converting
recombinant human PrP into amyloid fibrils was low, yet
similar to that of both BSE and scrapie (419). Accordingly,
it has also been reported that human PrP-overexpressing
mice were not susceptible to nine CWD isolates from
mule deer, white-tailed deer, and elk (490), whereas the
elk PrP expressing mice developed disease after only
118 –142 days postinoculation (275). However, mice have
Physiol Rev • VOL
a limited life span, and further passages may be necessary
to detect low levels of prion infectivity that may be
present subclinically.
D. Human Prion Diseases
Rapidly progressive dementia, myoclonus, visual or
cerebellar impairment, pyramidal/extrapyramidal signs,
and akinetic mutism clinically characterize fatal neurological diseases caused by prions. The human prion diseases
are classified as infectious, inherited or sporadic disorders, depending on the clinical, genetic, and neuropathological findings.
Prion disease, not only in inherited forms but also in
iatrogenic and sporadic forms, is influenced by an amino
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FIG. 7. Bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease (vCJD) cases reported worldwide. A: reported cases
of BSE in the United Kingdom (UK) (black) and in countries excluding the UK (gray). Non-UK BSE cases include cases from countries both within
and outside of the European Union. Data are as of December 2008 (http://www.oie.int). B: reported cases of vCJD in the UK (black) and in France
(gray). In the table are reported vCJD cases worldwide. Data are as of January 2009. °Three cases are reported as secondary cases due to blood
transfusion; #the third United States (US) patient was born and raised in Saudi Arabia and has lived permanently in the US since late 2005 and,
according to the US case report, the patient was most likely infected as a child when living in Saudi Arabia; *the case from Japan had resided in
the UK for 24 days in the period 1980 –1996 [see the National Creutzfeldt-Jakob Disease Surveillance Unit Web site for vCJD data to December 2008
(http://www.cjd.ed.ac. uk)].
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ADRIANO AGUZZI AND ANNA MARIA CALELLA
1. Iatrogenic prion diseases
Iatrogenic CJD is accidentally transmitted during the
course of medical or surgical procedures. The first documented case of iatrogenic prion transmission occurred in
1974 and was caused by corneal transplantation of a graft
derived from a patient suffering from sCJD (143). Iatrogenic CJD has also been transmitted by neurosurgery
using contaminated EEG electrode implantations (50)
and surgical operations using contaminated instruments
or apparatuses (129, 331). Moreover, the transmission of
CJD from contaminated human growth hormone (HGH)
preparations derived from human pituitaries was discovered when fatal cerebellar disorders with dementia occurred in ⬎80 patients ranging in age from 10 to over 60
yr (73, 82, 160). Five cases of CJD have occurred in
women receiving human pituitary gonadotropin (203). Although one case of spontaneous CJD in a 20-yr-old
woman has been reported (377), CJD in people under 40
yr of age is extremely rare.
Molecular genetic studies have revealed that most
patients developing iatrogenic CJD after receiving pituitary-derived HGH are homozygous for either Met or Val
at codon 129 of the PRNP gene (72, 117).
2. Inherited prion diseases
Familial TSEs are associated with an autosomal dominant PRNP gene alteration. There is variability in the
clinical and pathological findings, the age of onset, and
the duration depending on the particular PRNP mutation
involved (Table 4). Familial TSEs account for 10 –20% of
all TSE cases in humans and include fCJD, GSS, and FFI.
Clinically, fCJD is characterized by rapidly progressive
dementia, with myoclonus and pseudoperiodic discharges
on electroencephalogram; GSS by slow progression of
ataxia followed by later onset dementia, and FFI from
refractory insomnia, hallucinations, dysautonomia, and
motor signs. The clinical categories may be seen as phenotypic extremes of a continuum, because in reality the
syndromes overlap considerably (339).
fCJD have been associated with point mutations affecting the region between the second and the third helix
of the COOH terminus, insertions in the octarepeat region
in the NH2 terminus, and even a premature termination
codon at position 145. The inheritance is, in all cases,
autosomal dominant, often with very high penetrance. In
TABLE 4. Synopsis of all PRNP mutations and polymorphisms and their association with inherited
prion diseases
Presentation
Mutation in PRNP
Creutzfeldt-Jakob phenotype
Gerstmann-Sträussler-Scheinker phenotype
Fatal familial insomnia phenotype
Vascular amyloid depositions
Proven but unclassified prion disease
Neuropsychiatric disorder, no proven prion disease
Polymorphisms, established influence on phenotype
Polymorphisms, suggested influence on phenotype
Silent polymorphisms
D178N-129V, V180I, V180I-M232R, T183A, T188A, E196K, E200K, V203I, R208H,
V210I, E211Q, M232R, 96 bpi, 120 bpi, 144 bpi, 168 bpi, 48 bpd
P102L, P105L, A117V, G131V, F198S, D202N, Q212P, Q217R, M232T, 192 bpi
D178N-129M
Y145STOP
H187R, 216 bpi
I138M, G142S, Q160S, T188K, T188R, M232R, P238S, 24 bpi, 48 bpi
M129V
N171S, E219K
P68P, A117A, G124G, V161V, N173N, H177H, T188T, D202D, Q212Q, R228R, S230S
The wild-type human PRNP gene contains 5 octarepeats 关P(Q/H)GGG(G/–) WGQ兴 from codons 51–91. Deletion of a single octarepeat at codon
81 or 82 is not associated with prion disease (289, 413, 503); whether this deletion alters the phenotypic characteristics of a prion disease is unknown.
There are common polymorphisms at codons 117 (Ala3 Ala) and 129 (Met3 Val); homozygosity for Met or Val at codon 129 seems to increase
susceptibility to sporadic CJD (378). Octarepeat inserts of 16, 32, 40, 48, 56, 64, and 72 amino acids at codons 67, 75, or 83 are designated as bpi
(base pair insertion); bpd indicates base pair deletion. Point mutations are designated by the wild-type amino acid preceding the codon number and
the mutant residue follows, e.g., P102L. These point mutations segregate with the inherited prion diseases, and significant genetic linkage has been
demonstrated where sufficient specimens from family members are available. The single letter code for amino acids is as follows: A, Ala; D, Asp;
E, Glu; F, Phe; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; and Y, Tyr.
Physiol Rev • VOL
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acid polymorphism resulting in methionine (M)3valine
(V) substitution at PrP codon 129 (375). The finding that
homozygosity at codon 129 predisposes individuals to
CJD supports the theory that protective polymorphisms in
the human prion gene have been heavily selected for,
possibly due to evolutionary pressure from cannibalismpropagated prions (341). However, several publications
have pointed out that these conclusions may be incorrect
(449, 468). It was recently described that incubation periods of Kuru could be as long as 56 years. PRNP analysis
showed that most of those patients with Kuru were heterozygous at polymorphic codon 129, a genotype associated with extended incubation periods and resistance to
prion disease (119).
A second polymorphism resulting in glutamate
(E)3lysine (K) substitution at codon 219 has been identified in the Japanese population, and it has also been
reported to have an effect on the susceptibility of individuals to prion disease (264). Recently, additional candidate
loci have been identified by a genome-wide association
study of the risk of vCJD (340) which justifies functional
analyses of these biological pathways in prion disease.
AGGREGATION AND INFECTIOUS DISEASE
3. Sporadic Creutzfeldt-Jakob disease
Approximately 85% of all human prion diseases are
sporadic forms of CJD. For sCJD, there is no association
with a mutant PRNP allele, nor is there any epidemiological evidence for exposure to a TSE agent through contact
with people or animals infected with TSEs.
sCJD cases are currently subclassified according to
the methionine/valine polymorphism at codon 129 of the
PRNP gene and the size and glycoform ratio of proteaseresistant prion protein identified on western blot (type 1
or type 2) (174). Heterozygosity (Met/Val) at PrP codon
129 appears to be associated with a lower risk (378)
and/or prolonged incubation time (119, 387).
The lack of routine laboratory testing for preclinical
diagnosis makes the search for agent sources and other
risk factors extremely difficult. At present, the means of
acquisition of a TSE agent in these patients remains a
mystery. So far, there is no evidence for spontaneous
PrPSc formation in any animal or human TSE. In humans,
the peak age incidence of sporadic CJD is 55– 60 years.
However, if spontaneous misfolding were the primary
event, one might expect a continuously increasing incidence with age because more time would allow more
opportunity for rare misfolding events.
4. Variant Creutzfeldt-Jakob disease
The most recently identified form of CJD in humans,
new variant CJD (vCJD), was first described in 1996 and
has been linked to BSE (522).
At the time of this writing, vCJD has killed ⬃200
individual victims worldwide (http://www.cjd.ed.
ac.uk/). Most of the affected individuals lived in the
United Kingdom and France. Fortunately, in the United
Kingdom, the incidence appears to be decreasing since
2001 to five diagnosed cases yearly in 2006 and 2007
Physiol Rev • VOL
and one case in 2008. In contrast, in France, the number
of probable and definite cases of vCJD increased from
zero to three diagnosed cases per year in 1996 –2004 to
six per year in 2005 and 2006. In 2007, the number of
cases was back to three again, and no cases were
reported in 2008 (http://www.invs.sante.fr/publications/
mcj/donnees_mcj.html) (Fig. 7B).
vCJD represents a distinct clinicopathological entity
that is characterized at onset by psychiatric abnormalities,
sensory symptoms, and ataxia and eventually leads to dementia along with other features usually observed in sporadic CJD. What distinguishes vCJD from sporadic cases is
that the age of patients is abnormally low (vCJD: 19 –39 yr;
sporadic CJD: 55–70 yr) and duration of the illness is rather
long (vCJD: 7.5–22 mo; sporadic CJD: 2.5– 6.5 mo). Moreover, vCJD displays a distinct pathology within the brain
characterized by abundant “florid plaques,” decorated by a
daisylike pattern of vacuolation. Florid plaques are spherical
conglomerates of fibrillary birefringent material that stains
positive with “amyloidotropic” dyes such as Congo red.
What is the evidence that the agent causing vCJD may
be identical to that of BSE when transmitted to humans?
None of the arguments to date are conclusive. Considerable
effort has been devoted to characterizing the “strain properties” of the agent that affects cows and humans. Since the
molecular substrate underlying the phenomenon of prion
strains is not known, strain-typing of prions must rely on
surrogate markers.
Two such markers have been particularly useful. One is
the distribution of neuronal vacuoles in the brains of affected animals. BSE prions (extracted from the brains of
affected cows) and vCJD prions derived from the brains of
British patients produce the same lesion profile when transmitted to panels of susceptible mice (3, 22, 80). The second
marker for strain typing of prions comes from analyzing the
biochemical properties of disease-associated PrP recovered
from the brains of cattle and humans. These traits were
again found to be indistinguishable between BSE and human vCJD prions (118, 219). A third line of evidence linking
BSE to human vCJD comes from the epidemiology of the
disease. To date, the majority of vCJD cases have arisen in
the United Kingdom, with relatively few cases in other countries including Ireland, France (106), and Italy. With the
assumption that the quality of the epidemiological surveillance is similar in these countries and in the rest of Europe
(which has not reported cases of vCJD), the unavoidable
conclusion is that the incidence of vCJD correlates with the
prevalence of BSE.
Convincing as all of these arguments may seem, each
is phenomenological rather than causal. Given that a large
fraction of the European population may have been exposed to BSE prions, yet only a minute fraction actually
developed vCJD, there can be hardly any doubt that additional genetic modifiers exist, other than PRNP polymorphisms. So what will the numbers of vCJD victims be
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all fCJD, the clinicopathological disease phenotype varies
depending on the actual mutation, as well as on polymorphisms at codon 129, and most likely on a plethora of yet
unidentified modifiers and cofactors (280).
The first reports of GSS originate from 1928 and 1936
in an Austrian family (179, 199). GSS syndrome is a rare,
inherited autosomal dominant disease that is associated
with mutations in the PRNP gene (the most common are
at codons 102 and 198). GSS syndrome is characterized by
chronic progressive ataxia, terminal dementia, a long clinical duration (2–10 yr), and multicentric amyloid plaques
that can be visualized by antibodies directed against the
prion protein.
FFI is characterized by sleep disturbances as well as
vegetative and focal neurological signs as a result of
thalamic lesions. The clinical phenotype depends on the
D178N point mutation of the PRNP gene coupled with a
methionine at codon 129 (175).
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V. PERIPHERAL ROUTES OF PRION
INFECTIVITY
The fastest and most efficient method of inducing
spongiform encephalopathy in the laboratory is intracerebral inoculation with brain homogenate. Inoculation of
106 ID50 infectious units (defined as the amount of infectivity that will induce TSE with 50% likelihood in a given
host) will yield disease in approximately half a year; a
remarkably strict inverse relationship can be observed
between the logarithm of the inoculated dose and the
incubation time (406).
However, the above situation does not correspond to
what typically happens in the field. There, acquisition of
prion infectivity through any of several peripheral routes
is the rule. Prion infections can be induced by oral challenge (159) and occur naturally as a result of food-borne
contamination, as has been shown for Kuru, transmissible
mink encephalopathy, BSE, and vCJD (27, 119, 222, 328).
Physiol Rev • VOL
However, prion diseases can also be initiated by
intravenous and intraperitoneal injection (258) as well as
from the eye by conjunctival instillation (445), corneal
grafts (143), and intraocular injection (161). Two routes of
infection have suggested for a long time that immune cells
might be of importance for this phase of prion pathogenesis: oral challenge and administration by scarification
(7). Therefore, these routes will be discussed in some
detail in the following paragraphs.
A. Pathway of Orally Administered Prions
After oral infection, an early increase in prion infectivity is observed in the distal ileum. Within 2 wk of prion
ingestion, prions appear to enter peripheral nerves and
proceed by invasion of the dorsal motor nucleus of the
vagus in the brain, as has been shown in mouse and
hamster scrapie studies (336). Gastrointestinal infections
caused by viruses, bacteria, and parasites, as well as
idiopathic inflammatory diseases, could alter the dynamics of prion entry and systemic spread. It has been recently reported that moderate colitis, caused by an attenuated Salmonella strain, more than doubles the susceptibility of mice to oral prion infection and modestly
accelerates the development of disease after prion challenge (459).
How do prions reach the peripheral nerves after having entered the gastrointestinal tract? Following exposure, many acquired TSE agents accumulate in lymphoid
tissue, in the case of oral exposure, the gut-associated
lymphatic tissue (GALT), which includes Peyer’s patches
(PPs) and membranous epithelial cells (M cells), and
mesenteric lymph nodes. Following experimental intragastric or oral exposure of rodents to scrapie, or nonhuman primates and sheep to BSE, protease-resistant
prion protein accumulates rapidly in the GALT, PPs, and
ganglia of the enteric nervous system (41, 57, 156, 259),
long before they are detected in the CNS.
Recruitment of activated B lymphocytes to PPs requires ␣4␤7 integrin as an essential homing receptor; PPs
of mice that lack ␤7 integrin are normal in number, but
are atrophic and almost entirely devoid of B cells (507).
Therefore, it seemed interesting to investigate the susceptibility to orally administered prions of ␤7-deficient mice.
Surprisingly, minimal infectious dose and disease incubation after oral exposure to logarithmic dilutions of prion
inoculum were similar in ␤7-deficient and wild-type mice
(399). Despite their atrophy, PPs of both ␤7-knockout and
wild-type mice contained 3– 4 log LD50/g prion infectivity
at 125 or more days after challenge.
Why does reduced mucosal lymphocyte trafficking
not impair, as expected, the susceptibility to orally initiated prion disease? One possible reason may relate to the
fact that, despite marked reduction of B cells, M cells
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in the future? Although many mathematical models have
been generated (180, 181), the number of cases is still too
small to predict future developments with any certainty. A
30-yr mean incubation time of BSE/vCJD in humans is not
entirely implausible, and therefore, some authors have
predicted a multiphasic BSE endemic with a second increase in the incidence of vCJD affecting people heterozygous at codon 129 (119). Others, these authors included,
regard the incidence of vCJD as subsiding (28). It is
important to note, however, that the above considerations
apply primarily to the epidemiology of primary transmission from cows to humans. Although by now a pool of
preclinically infected humans may have been built, human-to-human transmission may present with characteristics very different from those of primary cow-to-human
transmission, including enhanced virulence, shortened incubation times, disregard of allelic PRNP polymorphisms
(129MM, MV or VV), and heterodox modes of infection
including blood-borne transmission. If we account for the
time it will take to eradicate these secondary transmissions in the population, vCJD is not likely to disappear
entirely in the coming four decades. Four cases (three
definite and one presumed) of vCJD transmission by
blood transfusions have been reported in the United Kingdom (303, 387, 528) (see also http://www.cjd.ed.ac.uk/
TMER/TMER.htm). Most worryingly, the Health Protection Agency of the United Kingdom has reported a case of
a hemophilic patient who has most likely acquired vCJD
prions through factor VIII preparation derived from
plasma donated by a “preclinical” vCJD patient (http://
www.hpa.org.uk).
The fact that preclinical infected individuals can
transmit vCJD underscores the important medical need
for sensitive diagnostic tools, which could be used for
screening blood units prior to transfusion.
AGGREGATION AND INFECTIOUS DISEASE
were still present in ␤7⫺/⫺ mice. In contrast, mice deficient in both TNF and lymphotoxin (LT)-␣ (TNF-␣⫺/⫺ ⫻
LT␣⫺/⫺) or in lymphocytes (RAG-1⫺/⫺, ␮MT), in which
Peyer’s patches are reduced in number, were highly resistant to oral challenge, and their intestines were virtually devoid of prion infectivity at all times after challenge.
Therefore, lymphoreticular requirements for enteric and
for intraperitoneal uptake of prions differ from each other
in that susceptibility to prion infection following oral
challenge correlates with the number of Peyer’s patches,
but is independent of the number of intestinal mucosaassociated lymphocytes (189, 399).
The requirements for transepithelial passage of prions are of obvious interest to prion pathogenesis. M cells
are key sites of antigen sampling for the mucosal-associated lymphoid system (MALT) and have been recognized
as major ports of entry for enteric pathogens in the gut via
transepithelial transport (363).
Interestingly, maturation of M cells is dependent on
signals transmitted by intraepithelial B lymphocytes.
Efficient in vitro systems have been developed, in
which epithelial cells can be instructed to undergo differentiation to cells that resemble M cells, as judged by
morphological and functional-physiological criteria (254).
This led to the proposal that M cells could be a site of
prion entry, a hypothesis that has been substantiated in
coculture models (212). Although in vivo confirmation is
required, M cells are plausible sites for the transepithelial
transport of scrapie across the intestinal epithelium. However, the transport across the intestinal epithelium might
not rely entirely on M-cell-mediated transcytosis: several
reports have also indicated that prion transport may occur through enterocytes (372) and could be mediated by a
ferritin-dependent mechanism (351) or via laminin-receptor binding and endocytosis (357).
Dendritic cells (DCs), localized within the PPs beneath the M-cell intraepithelial pocket, are ideally situated
to acquire antigens that have been transcytosed from the
gut lumen and deliver them to peripheral nerves in lymphoid organs, thereby facilitating the process of neuroinvasion (232, 317). The contribution of CD11c⫹ DCs has
been investigated in vivo using CD11c-diphtheria toxin
receptor-transgenic mice in which CD11c⫹ DCs can be
specifically and transiently depleted (418). With the use of
two distinct scrapie agent strains (ME7 and 139A), depletion of CD11c⫹ DCs in the GALT and spleen before oral
exposure blocked early prion accumulation in these tissues. These data suggest that migratory CD11c⫹ DCs play
a role in the translocation of the scrapie agent from the
gut lumen to the GALT, from which neuroinvasion proper
may then ensue.
Physiol Rev • VOL
C. Uptake of Prion Through the Skin
Even less well understood, yet possibly much more
efficient than oral administration of prions, is challenge
by scarification. Removal of the most superficial layers of
the skin, and subsequent administration of prions, has
been known for long time to be a highly efficacious
method of inducing prion disease (90, 497). It is conceivable that dendritic cells in the skin may become loaded
with the infectious agent by this method, and in fact,
dendritic cells have been implicated as potential vectors
of prions in oral (232) and in hematogeneous spread (31)
of the agent.
Following inoculation with scrapie by skin scarification, replication in the spleen and subsequent neuroinvasion is critically dependent on mature follicular dendritic
cells (FDCs) (353). However, which lymphoid tissues are
crucial for TSE pathogenesis following inoculation via the
skin was not known until mice were created that lacked
the draining inguinal lymph node (ILN), but had functional FDCs in remaining lymphoid tissues such as the
spleen. In these mice, inoculated with the scrapie agent by
skin scarification, the disease susceptibility was dramatically reduced demonstrating that, following inoculation
by skin scarification, scrapie agent accumulation in FDCs
in the draining lymph node is critical for the efficient
transmission of disease to the brain (188).
It is equally possible (and maybe more probable),
however, that scarification induces direct neural entry of
prions into skin nerve terminals. This latter hypothesis
has not yet been experimentally tested, but it would help
explain the remarkable speed with which CNS pathogenesis ensues following inoculation by this route: dermal
inoculation of killed mice yields typical latency periods of
the disease that are similar to those obtained by intracerebral inoculation.
Rapid neuroinvasion was reported following intralingual inoculation and may also exploit a direct intraneural
pathway (38).
VI. IMMUNOLOGICAL ASPECTS OF PRION
DISEASE
In a discussion of the immunological aspects of prion
diseases, it is important to distinguish between an early
extraneural phase and a late CNS phase of pathogenesis.
There is no doubt that components of the immune system
participate in pathogenesis in both compartments, but
their respective functional implications are strikingly different.
A. The Lymphoid System in Prion Disease
More than 40 years ago it was discovered using bioassays that lymphoreticular organs contain prion infectiv-
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B. Transepithelial Enteric Passage of Prions
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primary splenic repository of prions: in fact, other experiments suggest that this is quite unlikely. Instead, bone
marrow transplantation may 1) transfer an ill-defined
population with the capability to replenish splenic stroma
and to replicate prions, or less probably, 2) donor-derived
PrPC expressing hematopoietic cells may confer prion
replication capability to recipient stroma by virtue of “GPI
painting,” i.e., the posttranslational cell-to-cell transfer of
glycophosphoinositol linked extracellular membrane proteins (277). Some evidence might be accrued for either
possibility: stromal splenic follicular dendritic cells have
been described by some authors to possibly derive from
hematopoietic precursors, particularly when donors and
recipients were young (250, 486). Conversely, instances
have been described in which transfer of GPI-linked proteins occurs in vivo with surprisingly high efficiency
(278). GPI painting has been described specifically for the
cellular prion protein (302).
While it has been known for a long time that specific
strains of prions may preferentially affect specific subsets
of neurons, the Blättler/Brown paradox may uncover an
analogous phenomenon in peripheral prion pathogenesis.
The latter question may be much more important than it
appears at face value, since the molecular and cellular
basis of peripheral tropism of prion strains is likely to be
directly linked to the potential danger of BSE in sheep
(77, 185, 249), as well the potential presence of vCJD
prions in human blood (6).
1. Follicular dendritic cells and prion replication
As mentioned above, prion infectivity rises very rapidly (in a matter of days) in the spleens of intraperitoneally infected mice. Although B lymphocytes are crucial for
neuroinvasion, all evidence indicates that most splenic
prion infectivity resides in a “stromal” fraction. Instead,
the apparent requirement for B lymphocytes in peripheral
pathogenesis is more likely to be derived, at least in part,
from indirect effects, including the provision of chemokines or cytokines to cells that efficiently replicate prions
in peripheral regions of the host body (14).
Any potential profiteer from these B-lymphocyte-derived signals would be localized in close proximity to the
B lymphocytes, be of stromal origin, and should also
display PrPC on its cell surface. FDCs fulfill each of these
criteria. FDCs support the formation and maintenance of
the lymphoid microarchitecture by expressing homeostatic chemokines and have a role in antigen trapping and
capturing of immune complexes by Fc␥ receptors. Identification of FDC-specific genes is extremely important
and useful, to assess the contribution of FDCs to prion
pathogenesis. Recently, the antigen FDC-M1 has been
identified as Milk fat globule EGF factor 8 (Mfge8) (282).
Gene deletion experiments in mice have shown that
signaling by both TNF and lymphotoxins is required for
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ity (162, 385). For scrapie in sheep and goats, infectivity
was assessed in a broad range of tissues using experimentally inoculated goats as donors and uninfected goats as
recipients (384), and both spleen and lymph nodes were
shown to transmit scrapie.
In the decades following these discoveries, our understanding of the extraneural immune phase advanced
considerably, and an impressive collection of data has
accrued that provides clues about peripheral prion pathogenesis. The normal prion protein was found to be consistently expressed, albeit at moderate levels, in circulating lymphocytes (91). Subsequently it was clearly shown
in a wealth of experimental paradigms that innate or
acquired deficiency of lymphocytes would impair peripheral prion pathogenesis, whereas no aspects of pathogenesis were affected by the presence or absence of lymphocytes upon direct transmission of prions to the CNS (263,
291). Klein and colleagues (4, 267) pinpointed the lymphocyte requirement to B cells. Interestingly, it was shown
that only B cells within the secondary lymphoid organs
accumulated prions in a PrPC-dependent fashion, while
circulating lymphocytes contained no detectable infectivity (415). A series of bone marrow transfer experiments
demonstrated that peripheral prion pathogenesis required
the physical presence of B cells, but the expression of the
cellular prion protein by these cells is dispensable for
pathogenesis upon intraperitoneal infection in the mouse
scrapie model (11). Adoptive transfer of Prnp⫹/⫹ bone
marrow to Prnpo/o-recipient mice did not suffice to restore infectibility of Prnp-expressing brain grafts, indicating that neuroinvasion was still defective (53), yet intraperitoneal infection occurred efficiently even in B-celldeficient hosts that had been engrafted with B cells from
Prnp knockout mice (268, 354).
Although the requirement for B cells appears to be
very stringent in most instances investigated, it has
emerged that not all strains of prions induce identical
patterns of peripheral pathogenesis, even when propagated in the same, isogenic strain of host organism (452).
Another interesting discrepancy that remains to be
addressed concerns the actual nature of the cells that
replicate and accumulate prions in lymphoid organs. In
the RML paradigm, four series of rigorously controlled
experiments over 5 years (18, 243, 269, 400) unambiguously reproduced the original observation by Blättler et
al. (53) that transfer of Prnp⫹/⫹ bone marrow cells (or
fetal liver cells) to Prnpo/o mice restored accumulation
and replication of prions in spleen. In contrast, Brown et
al. (70) reported a diametrically opposite outcome of
similar experiments when mice were inoculated with prions of the ME7 strain. This discrepancy may identify yet
another significant difference in the cellular tropism of
different prion strains.
It is important to note that the results of the Blättler
study do not necessarily indicate that lymphocytes are the
AGGREGATION AND INFECTIOUS DISEASE
Physiol Rev • VOL
nodes of TNFR1⫺/⫺ and TNF-␣⫺/⫺ mice had consistently
high infectivity titers (400). Even inguinal lymph nodes,
which are distant from the injection site and do not drain
the peritoneum, contained infectivity titers equal to all
other lymph nodes. Therefore, TNF deficiency prevents
lymphoreticular prion accumulation in spleen but not in
lymph nodes.
Why is susceptibility to peripheral prion challenge
preserved in the absence of TNFR1 or TNF-␣⫺/⫺, while
deletion of LT signaling components confers high resistance to peripheral prion infection? After all, each of
these defects (except TNFR2⫺/⫺) abolishes FDCs. For
one thing, prion pathogenesis in the lymphoreticular system appears to be compartmentalized, with lymph nodes
(rather than spleen) being important reservoirs of prion
infectivity during disease. Second, prion replication appears to take place in lymph nodes even in the absence of
mature FDCs; therefore, poorly defined immune cells or
possibly stromal precursor cells (for example, for FDCs)
might be capable of replicating the infectious agent.
In an unexpected turn of events, it was found that
lymphotoxin-dependent prion replication can occur in
inflammatory stromal cells that are distinct from FDCs
(208). Granulomas, a frequent feature of chronic inflammation, express PrPC and the LT␤R, even though they
lacked FDCs and did not display lymphoneogenesis. After
intraperitoneal prion inoculation, granulomas accumulated prion infectivity. Bone marrow transfers between
Prnp⫹/⫹ and Prnpo/o mice and administration of lymphotoxin signaling antagonists indicated that prion replication required radioresistant PrPC-expressing cells and
LT␤R signaling. The finding that a cell type other than
mature FDCs is involved in prion replication and accumulation within lymph nodes may be relevant to the development of postexposure prophylaxis strategies.
2. A role for the complement cascade in prion
accumulation
As FDCs interact with opsonized antigens through
the CD21/CD35 complement receptors, a pertinent question is whether there is a role for the complement cascade
in the pathology of prion diseases. Indeed, depletion of
either one of the early complement factors (C1q, Bf/C2
and C3) or the complement receptor (CD21/CD35) significantly delays the onset of disease symptoms in mice
injected with limiting doses of the scrapie strains RML
and ME7 (269, 315). The relative importance of CD21/35
on hemopoietic and stromal cell types has been assessed
through reciprocal reconstitution experiments by bone
marrow transplantation. CD21/35 expression exclusively
on FDCs in white pulp follicles resulted in prion titers,
PrPSc retention, and disease kinetics and severity similar
to those of wild-type mice. CD21/35 expression on hemopoietic cells also significantly affected these parameters,
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generation and maintenance of FDCs (145, 276). LTs belong to a family of structurally related cytokines, the TNF
superfamily (306), and are expressed primarily by B lymphocytes and activated T lymphocytes. LT␣ and LT␤ are
proinflammatory cytokines; ectopic expression of LTs in
the liver, kidney, or pancreas leads to the generation of
follicular structures resembling tertiary follicles (210,
283), which are very similar to activated lymphoid follicles found in the spleen or lymph nodes.
Membrane-bound LT-␣/␤ (LT-␣1/␤2 or LT-␣2/␤1) heterotrimers signal through the LT-␤ receptor (LT-␤R)
(508), thereby activating a signaling pathway required for
the development and maintenance of secondary lymphoid
organs (318, 333).
A series of studies has been performed to define the
effect of selective ablation of functional FDCs on the
pathogenesis of scrapie in mice.
FDCs accumulate PrPSc following scrapie infection
(263), and prion replication in the spleen was reported to
depend on PrPC-expressing FDCs, at least in the ME7
prion strain (70).
Inhibiting the LT␤R pathway in mice and nonhuman
primates, by treatment with LT␤R-Ig, results in the disappearance or dedifferentiation of mature, functional FDCs
(191, 192). In addition, treatment with LT␤R-Ig was found
to impair peripheral prion pathogenesis (316, 355). Treatment of mice with LT␤R-Ig for 1 wk before exposure to a
relatively low dose (intraperitoneally) of prions, followed
by further treatment for 2 wk postinoculation, resulted in
the complete protection of the mice from disease (15).
This suggests that B cell-deficient ␮MT mice (266) may be
resistant to intraperitoneal prion inoculation (267) because of impaired FDC maturation (268, 355).
However, additional experimentation appears to indicate that PrPC-expressing hematopoietic cells are required in addition to FDCs for efficient lymphoreticular
prion propagation (53, 243). This apparent discrepancy
called for additional studies of the molecular requirements for prion replication competence in lymphoid
stroma. Therefore, peripheral prion pathogenesis was
studied in mice lacking TNF-␣, LTa/␤, or their receptors.
After intracerebral inoculation, all treated mice developed
clinical symptoms of scrapie with incubation times, attack rates, and histopathological characteristics similar to
those of wild-type mice, indicating that TNF/LT signaling
is not relevant to cerebral prion pathogenesis. Upon intraperitoneal prion challenge, mice defective in LT signaling (LT␣⫺/⫺, LT␤⫺/⫺, LT␤R⫺/⫺, or LT␣⫺/⫺TNF-␣⫺/⫺)
proved virtually noninfectible with ⱕ5 log LD50 scrapie
infectivity, and establishment of subclinical disease (166)
was prevented. In contrast, TNFR1⫺/⫺ mice were almost
fully susceptible to all inoculum sizes, and TNF-␣⫺/⫺ mice
showed dose-dependent susceptibility. TNFR2⫺/⫺ mice
had intact FDCs and germinal centers and were fully
susceptible to scrapie. Unexpectedly, all examined lymph
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but far less than FDC expression of CD21/35. Therefore,
complement-mediated antigen trapping on FDCs is an
important mechanism for lymphoid prion accumulation
(530). The role of the classical complement cascade during peripheral prion infection certainly warrants further
investigations, since complement is clearly an important
determinant of pathogenesis yet its mechanics are not yet
fully understood (14).
B. Tropism of Prions for Inflammatory Foci
gous phenomena might occur in farm animals, since these
are commonly in contact with inflammogenic pathogens.
Indeed, PrPSc has been observed in the inflamed mammary glands of sheep with mastitis and which are also
infected with scrapie (300). These observations indicate
that inflammatory conditions induce accumulation and
replication of prions in organs previously considered to
be free from prion infection.
In addition, inflammatory conditions could result in
the shedding of the prion agent by excretory organs,
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Since proinflammatory cytokines and immune cells
are involved in lymphoid prion replication, it is likely that
chronic inflammatory conditions in nonlymphoid organs
could affect the dynamics of prion distribution. Indeed,
inclusion body myositis, an inflammatory disease of muscle, was reported to lead to the presence of large PrPSc
deposits in muscle (281). Many chronic inflammatory conditions, some of which are very common and include
rheumatoid arthritis, type 1 diabetes, Crohn’s disease,
Hashimoto’s disease and chronic obstructive pulmonary
disease, result in organized inflammatory foci of B lymphocytes, FDCs, DCs, macrophages, and other immune
cells associated with germinal centers (141, 224, 244, 489).
In addition, tertiary follicles can be induced, and are
surprisingly prevalent, in nonlymphoid organs by naturally occurring infections in ruminants (141, 502).
Lymphoid follicles with prominent FDC networks
can replicate prions in mice (210, 448), sheep (300), and
deer (200) (Fig. 8). In fact, in mice suffering from nephritis, hepatitis, or pancreatitis, prion accumulation occurs
in otherwise prion-free organs (210). The presence of
inflammatory foci consistently correlated with an upregulation of LTs and the ectopic induction of PrPC-expressing
FDCs. Inflamed organs of mice lacking LT␣ or LT␤R did
not accumulate either PrPSc or infectivity following prion
inoculation. These data have raised concerns that analo-
FIG. 8. Lymphoid follicles with prominent follicular dendritic cell
(FDC) networks can replicate prions in mice, sheep, and deer. A: in
prion-infected RIPLT␣-mouse, inflammatory foci in renal tissue overlap
with PrPSc deposition as shown by histoblot analysis of a renal cryosection blotted on a nitrocellulose membrane followed by proteinase K
(PK) treatment and HE staininig. (Figure kindly provided by M. Heikenwalder.) B: in sheep infected with scrapie and presenting with mastitis,
PrPSc deposition in a mammary gland coincides with tertiary follicles, as
confirmed by petblot analysis of a paraffin section blotted on a nitrocellulose membrane, followed by PK treatment and HE staining. [Adapted
from Ligios et al. (300).] C: in white-tailed deer experimentally inoculated with the agent of chronic wasting disease (CWD), PrPSc is present
in ectopic lymphoid tissue in the kidney. Note the presence of two
lymphoid follicles subjacent to the uroepithelial layer and PrPSc immunolabeling within the lymphoid follicles. (Figure kindly provided by
A. N. Hamir.) HE, hematoxylin and eosin; IHC, immunohistochemistry;
KM, medulla; RIPLT␣, transgenic mice expressing LT (lymphotoxin)
under the control of the rat insulin promoter (RIP) in pancreatic ␤-islet
cells and renal proximal convoluted tubules.
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C. Neuroinflammation in Prion Pathogenesis
In the CNS, prion pathology involves large-scale activation and proliferation of microglia (215), the CNS
equivalent of macrophages. The use of a prion organotypic slice culture assay (POSCA) (148) allowed pharmacogenetic ablation of microglia (Fig. 9A). In microgliadepleted slices, a 15-fold increase in prion titers and PrPSc
concentrations was observed. This suggests that the extensive microglia activation accompanying prion diseases
represents an efficacious defensive reaction (Fig. 9, B–E)
(149).
In addition, it has been reported that T cells also
infiltrate the CNS to some extent (52), which raises the
possibility of atypical CNS inflammation, since the expression of both proinflammatory and effector cytokines
are upregulated in prion infection (392). More importantly, intracerebral administration of prions has been
shown to induce TSE in mice that have various immune
deficiencies, including lack of B cells, T cells, interferon
Physiol Rev • VOL
receptors, TNF, LTs or their receptors (267), receptors for
IgG (Fc␥Rs), complement factors or their receptors (269),
Toll-like receptors (398), or chemokine receptors (397).
In all of these models, pathogenesis after intracerebral
prion delivery proceeded with the same kinetics as in
wild-type immunocompetent mice, and in terminally sick
mice, intracerebral prion concentrations were unchanged
(20). This evidence indicates that, after prions are present
in the CNS, the disease course cannot be modified by
manipulating immunological parameters.
Furthermore, several studies have addressed the impact of the nuclear factor-␬B (NF␬B) pathway in prion
pathogenesis. NF␬B is involved in a variety of physiological and pathological responses and plays an essential role
in immune responses involving induction of inflammation,
proliferation, and the regulation of apoptosis (56, 251). In
acute and chronic neurodegenerative conditions such as
AD, NF␬B is activated in neurons and glia, and it has been
proposed that NF␬B might promote survival of neurons
(245). Alternatively, NF␬B may contribute to neuronal
degeneration through the production and release of inflammatory cytokines or of reactive oxygen molecules by
microglia and astrocytes. In prion diseases, NF␬B activity
was shown to be enhanced in parallel with the first neuronal pathological changes (256). Furthermore, the cytotoxic synthetic peptide PrP106 –126 activates NF␬B in microglia in vitro (33). Notably, it has been reported that
NF␬B activity leads to mitochondrial apoptosis after
prion infection (61). Despite these findings, it remains
unclear whether NF␬B activation is protective or pathogenic in prion disease. However, when the impact of
NF␬B signaling on prion disease was investigated in
mouse models with a CNS-restricted elimination of IKK␤
and in mice containing a nonphosphorylatable IKK subunit, no impairment of prion pathogenesis in the brain
was observed (242).
VII. SPREAD OF PRIONS ALONG PERIPHERAL
AND CENTRAL NERVOUS SYSTEM
PATHWAYS
The process of prion infectivity transfer from the
spleen to the CNS is termed “neuroinvasion.” In the last
several years, a biphasic model has emerged to explain
the process of prion. The first phase is characterized by
widespread colonization of lymphoreticular organs, the
details of which have been described in the previous
section. The second phase has long been suspected to
involve peripheral nerves. The innervation pattern of lymphoid organs is primarily sympathetic (150), and many
studies suggest that is the way how the prion agent travels
from lymphoid organs to the CNS (111, 115, 335). Not
surprisingly, the sympathetic nervous system is affected
in vCJD patients (198). The detection of PrPSc in the
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including the kidney. In fact, in various transgenic and
spontaneous mouse models of nephritis, prion infectivity
was observed in the urine with both subclinical and terminal scrapie (448). Recently, consistent levels of infectivity have been detected in colostrum and milk from
sheep incubating natural scrapie, several months prior to
clinical onset. The presence of ectopic lymphoid follicles
in ewes with lymphoproliferative mastitis could increase
prion shedding in milk, although the prion infectivity was
revealed in sheep with healthy mammary glands as well as
those with chronic lymphoproliferative mastitis (287). If
confirmed, these findings could have an impact on the risk
assessment of dairy foodstuffs (for example, milk) and
could lead to a readjustment of current rules. It is possible
that the horizontal spread of prions between hosts is
mediated by secreted body fluids (for example, urine and
milk) that are derived from potentially infectious secretory organs (for example, kidney and mammary glands).
However, chronic inflammation more commonly
gives rise to granulomas whose cellular composition is
different from that of lymphoid follicles. Granulomas occur, for example, in Crohn’s disease, sarcoidosis (235),
and most mycobacterial (66, 146, 430), fungal, and parasitic infections, as well as in foreign body reactions. A
recent study demonstrates that experimentally induced
granulomas in mice enable prion replication after intraperitoneal inoculation (208). Because granulomas can occur at any site of the body, these results suggest that
prions may colonize a broader spectrum of organs than
previously anticipated. With respect to the relevance of
the above findings for public health, it will be interesting
to test whether prion replication in granulomas occurs in
farm animals and in humans.
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FIG. 9. Role of microglia in prion disease. A: schematic representation for the conditional ablation of microglia in vitro and in vivo by using
the cytotoxic prodrug ganciclovir (GCV) in CD11b-HSVTK mice (213). B: histoblot analysis of PrPSc deposition in POSCA slices after 35 days in
culture shows that PrPSc accumulates predominantly in the molecular layer. Organotypic brain slices were prepared from 10-day-old tga20TK (mice
overexpressing PrP and carrying the CD11b-HSVTK transgene) and were inoculated with 100 ␮g RML (R) or uninfected homogenate (Ø). PrPSc was
detected with anti-PrP antibody (POM1). C: accumulation of PrPSc in slices was also shown by Western blot; tga20TK and prnpo/o slices were
cultured for 35 days, optionally digested with PK (␮g/ml), and probed with antibody POM1. D: microglia depletion in organotypic slices increases
the amount of PK-resistant PrP. Organotypic slices from mice overexpressing PrP with the CD11b-HSVTK transgene (tga20TK⫹) or without
(tga20TK⫺) were treated with GCV and inoculated with RML. Samples were PK-digested and PrP detected with POM1. E: microglia depletion in
organotypic slices increases the infectivity as evaluated by SCEPA of homogenates of tga20TK⫹ or prnpo/o/TK⫹ slices treated with GCV. Three
independent biological replicas of tga20TK⫹ and single replicas of prnpo/o/TK⫹ slices or RML were analyzed in 10-fold dilution steps using 6 –12
replica N2a-containing wells per dilution. Data are indicated as the number of infectious tissue culture units per gram of slice culture protein and
are the averages of biological replicas ⫾ SD. Slices from the same animal (pairs of ⫺GCV and ⫹GCV samples) are represented by the same color.
Sc, positive-control homogenate from brain of a mouse with scrapie; Inoc, inoculum. Left lane on all blots, molecular weight marker spiked with
recombinant PrPC, yielding a PrP signal at 23 kDa with a cleavage product at 15 kDa. [B–E are adapted from Falsig et al. (149).]
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Physiol Rev • VOL
short contact period of 30 min. Remarkably, this occurs
without any reduction in prion infectivity. These data
strongly suggest that contact-mediated transmission
rather than an exchange of the infectious agent likely
takes place. Consistent with these findings, cell-mediated
infection was shown in a tissue-culture model (247). This
study took advantage of genetically marked target cells in
combination with scrapie mouse brain (SMB) cells chronically propagating PrPSc (112). Several lines of evidence in
this study, in particular efficient transmission after aldehyde fixation of the SMB cells, suggest that direct contact
is responsible for the cell-to-cell transmission.
A second possibility is neuronal prion transfer by
vesicle-associated infectivity. Exosome-bound prion infectivity has been reported in two cell lines: Rov, a rabbit
epithelial cell line, and Mov, a neuroglial cell line, which
both express ovine PrP (151). This report of exosomes
containing prions in vitro is in agreement with a study that
suggests that retroviral infection robustly enhances the
release of prion infectivity in cell culture (294). For example, prion infectivity within exosomes, which in turn
may be released by prion-infected FDCs, may encounter
peripheral nerves and contribute to neuroinvasion (15,
397).
The third potential source of prion infectivity may be
cell-free, free-floating oligomeric or protofibrillar infectious particles (207). Several cell lines can be infected
with brain homogenates or brain fractions treated in a
way that renders the existence of intact cells or exosomes
highly unlikely. However, this most likely does not exclude the possibility of prion infectivity encapsulated in
micelles. One recent study that favors the latter hypothesis investigated the relationship between infectivity, converting activity, and the size of various PrPSc-containing
aggregates (464) and concluded that nonfibrillar particles,
with masses equivalent to 14 –28 PrP molecules, are the
most efficient initiators of prion disease.
All of these mechanisms shown in vitro potentially
play a role in vivo. However, there is currently no convincing evidence that implies an exclusive role of any of
the three possibilities in prion uptake by peripheral
nerves in vivo. It remains plausible, therefore, that more
than one of these pathways contributes to efficient prion
uptake by peripheral nerves simultaneously.
Although PrPC expression is known to modulate intranerval transport, it is unclear how prions are actually
transported within peripheral nerves (184). Axonal and
nonaxonal transport mechanisms may be involved, and
nonneuronal cells (such as Schwann cells) may also play
a role. Within the framework of the protein-only hypothesis, one may hypothesize a “domino” mechanism, by
which infiltrating PrPSc converts resident PrPC on the
axolemmal surface, sequentially propagating the infection. While speculative, this model is attractive, since it
may accommodate the finding that the velocity of neural
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spleens of sCJD patients (183) indicates that the interface
between cells of the immune and peripheral nervous systems might also be of relevance in sporadic prion diseases.
Sympathectomy appears to delay the transport of
prions from lymphatic organs to the thoracic spinal cord,
which is the entry site of sympathetic nerves to the CNS.
Denervation by injection of the drug 6-hydroxydopamine,
as well as “immunosympathectomy” by injection of antibodies against nerve growth factor (NGF) leads to a
rather dramatic decrease in the density of sympathetic
innervation of lymphoid organs, and significantly delayed
the development of scrapie (186). No alteration in lymphocyte subpopulations was detected in spleens at any
time point investigated. In particular, no significant differences in the content of FDCs were detected between
treated and untreated mice, which negate the possibility
that the observed protection may be due to modulation of
FDC microanatomy. Transgenic mice overexpressing
NGF under control of the K14 promoter, whose spleens
are hyperinnervated, developed scrapie significantly earlier than nontransgenic control mice.
The distance between FDCs and splenic nerves influences the rate of neuroinvasion (397). FDC positioning
was manipulated by ablation of the CXCR5 chemokine
receptor, directing lymphocytes towards specific microcompartments (155). As such, the distance between germinal center-associated FDCs and nerve endings was reduced. This process resulted in an increased rate of prion
entry into the CNS in CXCR5-deficient mice, probably
owing to the repositioning of FDCs in juxtaposition with
highly innervated, splenic arterioles.
The cellular and molecular prerequisites for prion
trafficking within the lymphoreticular system are not fully
understood. Genetic or pharmacological ablation of germinal center B cells, located in close vicinity to FDCs, has
shown no significant effects on splenic prion replication
efficacy, PrPSc distribution pattern, or latency to terminal
disease (206), although in a previous report, the ablation
of CD40L accelerated prion disease (86).
The mechanism by which prions reach neurons and
take advantage of neuronal tracks to propagate from the
periphery toward the CNS remains to be determined.
Three different hypothesis could explain how prions enter neurons: 1) cell-cell contact, 2) vesicle transport, or
3) “free-floating” prion infectivity.
Convincing evidence for contact-mediated transmission of prion infectivity was presented in a study by
Flechsig et al. (153a), where the inherent capacity of
prions to strongly adhere to steel wires was used to study
contact-mediated prion infection. Neuroblastoma cells
adhering to such prion-coated steel wires become infected in tissue culture. Additionally, subsequent use of
the identical steel wire mediates prion transmission efficiently to several PrPC-overexpressing indicator mice, in a
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cytes but are resistant to scrapie and do not replicate
prions in brain, spleen, and thymus after intraperitoneal
inoculation with scrapie prions (416). Engraftment of
these mice with PrP-overexpressing neuroectoderm did
not lead to the development of antibodies to PrP after
intracerebral or intraocular inoculation, presumably due
to clonal deletion of PrP-immunoreactive T-lymphocytes.
As before, intraocular inoculation with prions did not
provoke scrapie in the graft, supporting the conclusion
that lack of PrPC, rather than immune response to PrP,
prevented prion spread (65). Therefore, PrPC appears to
be necessary for the spread of prions along the retinal
projections and within the CNS.
These results indicate that intracerebral spread of
prions is based on a PrPC-paved chain of cells, perhaps
because they are capable of supporting prion replication.
When such a chain is interrupted by interposed cells that
lack PrPC, as in the case described here, no propagation
of prions to the target tissue can occur. Perhaps prions
require PrPC for propagation across synapses. PrPC is
present in the synaptic region (157) and certain synaptic
properties are altered in Prnpo/o mice (120, 520).
Although a wealth of data has been acquired about
the transport of PrPSc from the periphery into the brain in
recent years, the exact molecular mechanisms of neuronal prion transport are still unclear. It also is becoming
clear that investigation of the precise molecular transport
mechanisms within neurons is much more difficult than
previously thought. It is not trivial to test the involvement
of retrograde neuronal transport in prion pathogenesis
because a complete block of this transport system is incompatible with long-term survival. Furthermore, mouse models
with partially impaired transport mechanisms do not display
altered prion pathogenesis, yet such negative results by no
means disprove a role for axonal transport.
One possible method of circumventing problems associated with in vivo studies may be to establish cell
culture model systems of transport in vitro and ex vivo.
Such systems may pave the way to a more precise molecular understanding of prion transport. Another advantage of in vitro systems may be the ability to pharmacologically manipulate specific neuronal transport pathways, which may be difficult to perform in vivo (207).
VIII. TOWARDS ANTIPRION THERAPEUTICS
The current therapeutic landscape for prion diseases
gives little reason to rejoice. An impressive wealth of
molecules has been considered as potential antiprion lead
compounds. However, none of these therapeutic leads
has yet proven their usefulness in clinical settings, and
some have conspicuously failed. A startling variety of
substances appears to possess prion-curing properties. A
nonexhaustive list includes compounds as diverse as
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prion spread is extremely slow (257) and may not follow
the canonical mechanisms of fast axonal transport. The
second hypothesis is that prion transport occurs within
the axons of peripheral nerves similar to normal cargo
proteins. This is known as the “streetcar” hypothesis (17).
In the streetcar hypothesis, PrPSc is taken up at nerve
endings and then transported retrogradely, presumably by
a microtubule-based mechanism, to the CNS.
Despite evidence that neurons are the key mediators
of prion propagation toward the CNS, low-efficiency alternatives such as transport via blood cannot be excluded
completely: prion infectivity could directly invade the
brain via the blood-brain barrier (BBB) (36). The cellular
and molecular preconditions for such a process remain
elusive. However, invasion of prion infectivity through the
BBB could depend on the prion strain and the dose of
administered prions (207).
The existence of multiple prion strains is likely to
make neuronal transport even more complex. It is conceivable that distinct strains use different mechanisms for
uptake and transport (37). This may affect not only the
mechanisms of prion uptake by different neuronal cell
populations, and cell-to-cell transmissions, but may also
alter the route of neuroinvasion, type of transport, lesion
profile, or the incubation time of an infected host (8).
The spread of prions within the central nervous system has been studied by ocular administration of prions.
The retina is a part of the CNS, and intraocular injection
does not induce direct physical trauma to the brain, which
may disrupt the BBB and impair other aspects of brain
physiology.
Evidence that spread of prions occurs axonally relies
mainly on the demonstration of diachronic spongiform
changes along the retinal pathway after intraocular infection (161). Reports of iatrogenic prion transmissions in
humans via cornea transplants are consistent with the
idea that prions spread axonally, implying that prions
propagate retrogradely toward the cornea and anterograde from the cornea to the brain (143).
To investigate whether the spread of prions within
the CNS is dependent on PrPC expression in the visual
pathway, PrP-producing neural grafts have been used as
sensitive indicators of the presence of prion infectivity in
the brain of Prnpo/o hosts. After prion inoculation in the
eyes of grafted Prnpo/o mice, none of the grafts showed
signs of scrapie. It was concluded, therefore, that infectivity administered to the eye of PrP-deficient hosts cannot induce scrapie in a PrP-expressing brain graft (65).
Engraftment of Prnpo/o mice with PrPC-producing
tissue might lead to an immune response to PrP which
neutralizes PrPSc infectivity (214). To definitively rule out
the possibility that prion transport was disabled by a
neutralizing immune response, Prnpo/o mice were rendered tolerant by expressing PrPC under the control of the
lck promoter. These mice overexpress PrP on T-lympho-
AGGREGATION AND INFECTIOUS DISEASE
A. Innate Immunity and Antiprion Defense
Antiprion treatment strategies focusing on immunosuppression consist of conditional dedifferentiation of
Physiol Rev • VOL
FDCs with LT␤R-Ig, suppression of germinal centers, and
disruption of lymphoid microarchitecture. LT␤R-Ig treatment causes germinal center disruption due to the breakdown of FDC networks (Fig. 10A). The data from studies
using LT␤R-Ig indicate that postexposure treatment of
humans with LT␤R-Ig could only be considered for prophylaxis at well-defined, early time points in cases of
known prion exposure. These cases might include recipients of blood transfusions from patients with CJD and
researchers, pathologists, neurologists, neurosurgeons,
and technicians after accidental CJD injection. In these
situations, we think that a case could be made for experimental use of postexposure prophylaxis with LT␤R-Ig,
although it is not known how soon after exposure
LT␤R-Ig would need to be administered. It should be
considered that LT␤R-Ig will not offer any relief to patients with overt CJD, for whom neural entry has already
taken place.
Theoretically, LT␤R-Ig treatment might have unwanted effects on immune function. However, manipulation of the LT pathway has been carried out in
macaques without apparent side effects. Primary antibody responses to keyhole limpet hemocyanin were
found to be unaltered during a 20-day period (192).
Moreover, LT-fusion proteins are already entering clinical trials as a treatment for rheumatoid arthritis. Hopefully, the results of such studies will become available
in the foreseeable future (20).
The ordered aggregate state of PrPSc might render it
recognizable as a pathogen-associated molecular pattern;
therefore, the members of the Toll-like receptor (TLR)
family might be involved in the recognition and subsequent degradation of prions.
However, the kinetics of prion pathogenesis in
MyD88 (myeloid differentiation primary-response protein 88)-deficient mice inoculated with prions (by the
intraperitoneal route) are identical to the kinetics observed in the wild-type control mice (398), suggesting
that signaling by TLR1, -2, -6, and -9 (mediated by the
adapter protein MyD88; Ref. 1) are probably not involved in prion recognition and signaling. Nevertheless,
targeting TLR9 therapeutically might have other beneficial effects. TLR9 recognizes DNA sequences that are
overrepresented in bacterial DNA. For example, unmethylated cytosine phosphate guanosine (CpG) motifs
present in bacterial DNA can stimulate mouse and human immune responses through TLR9 (211) (Fig. 10B).
Repetitive CpG motifs in synthetic oligodeoxynucleotides (CpG-ODN) simulate bacterial unmethylated nucleic acid sequences, and thereby stimulate the innate
immune system through TLR9 expressed on various
immune cells, including monocytes, macrophages, and
dendritic cells.
CpG-ODN treatment has been discussed as a possible
therapy to delay prion disease, primarily based on prom-
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Congo red (98), amphotericin B, anthracyclins (487), sulfated polyanions (100), porphyrins (104, 401), branched
polyamines (485), “␤ sheet breakers” (475), the spice
curcumin (102), and the single-stranded phosphorothioated analogs of natural nucleic acids (273).
In drug discovery generally, and in TSE therapeutics
specifically, promising compounds are often initially
screened using relatively high-throughput in vitro tests,
prior to testing of the best candidates in vivo. However,
relatively few of the antiprion compounds that are active
in vitro have shown any efficacy in vivo. Additional hurdles may lie in the identification of drugs that have sufficient in vivo efficacies, bioavailabilities, pharmacokinetics, and safety profiles to allow for applicability in the
clinical phase of disease. Alternatively, one might conclude (and we lean towards this conclusion) that the
initial cell culture screens are not very good at predicting
in vivo antiprion activity.
In our view, it is premature to treat patients with
alleged antiprion drugs on the sole basis of antiprion
efficacy in neuroblastoma cells. This shortcut was taken
in the case of quinacrine, which cures scrapie-infected
cultured cells with impressive efficacy (140), yet appears
to be utterly ineffective in scrapie-infected mice (121) and
in CJD patients (123), besides being severely hepatotoxic
(444). On the other hand, it is only fair to acknowledge
that these are daunting tasks for any drug discovery
project, even with great lead compounds in hand, and the
lack of success thus far in identifying drugs that impact
the course of prion disease in the clinical phase does not
diminish the importance of prion drug discovery efforts
that have been undertaken to date.
For therapeutic or prophylactic purposes, it is possible to exploit the roles of the lymphoid system and immune cells in prion pathogenesis. At least four alternatives can be envisaged on the basis of the information that
we have discussed: removal of functional FDCs and therefore ablation of lymphoid prion-replication sites; stimulation of the innate immune system; enhancement of elimination of PrPSc using PrP-specific antibodies; or binding
of available PrPC or PrPSc so that they are unavailable for
conversion (Fig. 10). All of these approaches, which include both suppression and stimulation of the immune
system, are now being tested in suitable in vivo systems
using mice experimentally infected with mouse-adapted
scrapie. However, because the lymphoid system has been
found to be involved in almost all forms of TSE, it is
reasonable to presume that mouse-adapted scrapie provides a realistic generic model for TSE therapy.
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ising results in mouse scrapie (450). The delay in the
development of prion disease in this model could be due
to the destruction of the primary site of peripheral prion
amplification, the lymphoid follicles (209). Alternatively,
the massive expansion of macrophage and dendritic cells
that is evident following repetitive CpG-ODN treatment
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might lead to enhanced PrPSc degradation or prion sequestration.
Macrophages might conceivably function as prion
transporters when exposed to high prion titers; on the
other hand, they might also inhibit prion infectivity when
confronted with manageable prion titers (48). However,
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FIG. 10. Immunotherapeutic strategies for prion disease. A: treatment with the lymphotoxin-␤ receptor fusion protein (LT␤R)-Ig breaks up the FDC
networks and disrupts lymphoid follicles. The best protection is achieved when the fusion protein is administered immediately after exposure to prions.
B: ablation of mature follicular dendritic cells (FDCs) delays the development of prion disease in mice. However, treatment with multiple doses of
CpG-containing oligodeoxynucleotides (CpG ODNs) produces severe unwanted side effects, including immunosuppression, liver necrosis, and thrombocytopenia. C: vaccination against a self-protein is difficult because of immune tolerance, and it has the potential to induce autoimmune disease. Transgenic
expression of an immunoglobulin ␮ chain containing the epitope-interacting region of 6H4, a high-affinity anti-PrP monoclonal antibody, associated with
endogenous ␬ and ␭ chains, leads to high anti-PrPC titers in Prnpo/o and Prnp⫹/⫹ mice. It suffices to block prion pathogenesis upon intraperitoneal prion
inoculation. After active immunization with full-length PrP in attenuated Salmonella vector, the mice develop high PrP specific antibody titers. D: treatment
with dimeric full-length PrP fused to the Fc portion of human IgG1 (PrP-Fc2) delays the development of prion disease in mice, most probably owing to
its interaction with the disease-associated PrP (PrPSc). LT-␣1␤2, LT heterotrimer; TLR, Toll-like receptor.
AGGREGATION AND INFECTIOUS DISEASE
despite these promising results, CpG-ODNs are not likely
to be a viable antiprion therapy owing to the severe toxic
side effects associated with repeated administration. One
might argue that severe side effects could be tolerable in
the case of a disease that is invariably fatal. However, if
the antiprion action of CpG ODNs is mediated by indiscriminate immune suppression, then there are alternative
ways to achieve this goal that would at least not lead to
hepatotoxicity.
An interesting alternative approach might include
phagocyte activation and targeted prion degradation without the reported adverse side effects associated with
CpG-ODNs (20).
For many conventional viral agents, vaccination is
the most effective method of infection control. But is it at
all possible to induce protective immunity in vivo against
prions? Prions are extremely sturdy, and their resistance
to sterilization is proverbial. Preincubation of PrPSc inoculum with anti-PrP antisera was reported to reduce the
prion titer of infectious hamster brain homogenates by up
to 2 log units (168), and an anti-PrP antibody was found to
inhibit formation of PrPSc in a cell-free system (227). Also,
antibodies (269) and F(ab) fragments raised against certain domains of PrP (144, 389) can suppress prion replication in cultured cells. However, it is difficult to induce
humoral immune responses against PrPC and PrPSc. This
is most likely due to tolerance of the mammalian immune
system to PrPC, which is an endogenous protein expressed rather ubiquitously. Ablation of the Prnp gene
(85) renders mice highly susceptible to immunization
with prions (65). Many of the best available monoclonal
antibodies to the prion protein have been generated in
Prnpo/o mice.
However, Prnpo/o mice are unsuitable for testing vaccination regimens, since they do not support prion pathogenesis (84). Second, it was generally thought (with good
reason) that antibodies specific for PrPC, if they could be
elicited at all, might lead to severe systemic immunemediated diseases, because PrP is expressed by many cell
types. Third, PrP-specific antibodies would be unlikely to
cross the BBB in therapeutic concentrations.
To test these various possibilities and circumvent the
problem of PrP tolerance, genes encoding high-affinity
anti-PrP antibodies (originally generated in Prnpo/o mice)
were used to reprogram B-cell responses of prion-susceptible mice that express PrPC. In 2001, it was reported that
mice transgenic for the ␮-chain of a PrP-specific antibody
were protected against prion disease after exposure by
intraperitoneal inoculation (214) (Fig. 10C). A blatant autoimmune disease as a consequence of antiprion immuPhysiol Rev • VOL
nization was not observed, unless transgenic PrPC was
artificially expressed at nonphysiological, extremely high
levels.
The strategy outlined above delivers proof-of-principle that a protective humoral response against prions can
be mounted by the mammalian immune system and suggests that B cells are not intrinsically tolerant to PrPC. The
challenges to practical antiprion immunization, however,
are enormous. While providing an encouraging proof of
principle, transgenic immunization cannot easily be reduced to practice. Furthermore, no protection was observed if treatment was started after the onset of clinical
symptoms, suggesting that passive immunization might be
a good candidate for prophylaxis rather than therapy of
TSEs. It was then found, in a follow-up study, that passive
transfer of anti-PrP monoclonal antibodies can delay the
onset of scrapie in prion-infected mice (518). Moreover,
intracranial delivery of PrPC-specific antibodies (those
having the epitope between aa 95–105 of the PrP sequence) has been shown to result in neuronal apoptosis in
the cerebellum and hippocampus, most likely through
clustering of PrPC, which is thought to trigger an abnormal signaling pathway (469). These results are alarming
and certainly reinforce the concept that adequate in vivo
safety studies must be carried out before prion immunoprophylaxis trials take place in humans. Active immunization, like in most antiviral vaccines, may be more effective, but is rendered exceedingly difficult by the stringent
tolerance to PrPC (476). The laboratory of Thomas
Wisnieswki was able to induce active immunization with
recombinant prion protein and a delay in the onset of
prion disease in wild-type mice was observed, although
the therapeutic effect was relatively modest and, eventually, all the mice succumbed to the disease (463). This
limited therapeutic effect may be explained by the observation that antibodies generated against prokaryotic PrP
often do not have a high affinity towards PrPC (394).
The possibility of producing protective immunity
against prions has captured the imagination of a considerable number of scientists, and additional reports have
since surfaced describing original methods of circumventing PrP tolerance and inducing an immune response in
animals that express the normal prion protein, including
mixing of the immunogenic moiety with bacterial chaperons (274). In a different study, tolerance was overcome by
oral inoculation of the PrP protein expressed in an attenuated Salmonella vector. This mucosal vaccine induced
gut anti-PrP immunoglobulins IgA and systemic anti-PrP
IgG in 100% of mice with a high mucosal anti-PrP IgA titer
and a high systemic IgG titer. Upon oral challenge with
PrPSc scrapie strain 139A, mice remained without symptoms of PrP infection at 400 days (193) (Fig. 10C). At
present, it is unclear whether vaccination approaches will
ultimately be translated to clinical practice. The predic-
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B. Adaptive Immunity and Preexposure
Prophylaxis Against Prions
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ADRIANO AGUZZI AND ANNA MARIA CALELLA
tive value of the different in vitro and in vivo experiments
is limited, and further investigations will be required.
C. Soluble Prion Antagonists
IX. PROGRESS IN THE DIAGNOSTICS OF
PRION DISEASES
As with any other disease, early diagnosis would
significantly advance the chances of success of any possible intervention approaches. Unfortunately, prion diagnostics continue to be rather primitive. Presymptomatic
diagnosis is virtually impossible, and the earliest possible
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In several paradigms, expression of two PrPC moieties with subtle differences antagonizes prion replication. The molecular basis for these effects is unknown.
Perhaps the subtly modified PrPC acts as a decoy by
binding incoming PrPSc (or protein X) and sequestering it
into a complex incapable of further replication.
To test the latter hypothesis, a transgenic mouse
was developed that expresses soluble full-length mouse
PrP rendered dimeric by fusion with the Fc portion of
human IgG1 (known as PrP-Fc2) (Fig. 10D). After prion
inoculation, these mice were surprisingly resistant to
prion disease (343). The PrP-Fc2 was not converted to
a prion-disease-causing isoform. Moreover, when the
transgenic mice expressing PrP-Fc2 were back-crossed
with wild-type mice and then inoculated with prions,
they showed marked retardation in the development of
prion disease, which was equivalent to a 105-fold reduction in titer of the prion-infected inoculum. This antiprion effect occurred in two different lines of PrP-Fc2expressing transgenic mice after either intracerebral or
intraperitoneal infection with scrapie. Therefore, it
seems that PrP-Fc2 effectively antagonizes prion accumulation in the spleen and brain. Because PrP-Fc2 cannot be converted to the protease-resistant, diseasecausing isoform, it might be effectively functioning as a
“sink” for PrPSc, by binding PrPSc and preventing the
binding and conversion of PrPC (20).
Delivery of the soluble prion antagonist PrP-Fc2 to
the brains of mice by lentiviral gene transfer impaired
replication of disease-associated PrPSc and delayed disease progression (178). These results suggest that somatic
gene transfer of prion antagonists may be effective for
postexposure prophylaxis of prion diseases. In addition, it
remains to be established whether the current form of
PrP-Fc2 has the strongest antiprion properties. For instance, the introduction of dominant-negative mutations
analogous to those described for Prnp (374, 391) might
considerably augment its efficacy. However, further research is needed to establish whether it may be effective
as a biopharmaceutical.
diagnosis is based on clinical signs and symptoms. Hence,
prion infection is typically diagnosed after the disease has
already progressed considerably.
A significant advance in prion diagnostics was accomplished in 1997 by the discovery that proteaseresistant PrPSc can be detected in tonsillar tissue of
vCJD patients (221). It was hence proposed that tonsil
biopsy may be the method of choice for diagnosis of
vCJD (218). Furthermore, there have been reports of
individual cases showing detectable amounts of PrPSc
at preclinical stages of the disease in the tonsil (443) as
well as in the appendix (223), indicating that lymphoid
tissue biopsy may represent a potential test for asymptomatic individuals. These observations triggered large
screenings of human populations for subclinical vCJD
prevalence, using appendectomy and tonsillectomy
specimens (187). PrPSc-positive lymphoid tissues were
long considered to be a vCJD-specific feature that
would not apply to any other forms of human prion
diseases (218). However, a successive survey of peripheral tissues of patients with sporadic CJD has led to the
identification of PrPSc in as many as one-third of skeletal muscle and spleen samples (183). In addition, PrPSc
was found in the olfactory epithelium of patients suffering from sCJD (532). These unexpected findings
raise the hope that minimally invasive diagnostic procedures may take the place of brain biopsy in intravital
CJD diagnostics.
For almost three decades, all gold standard methods
for the molecular diagnosis of prion diseases have relied
on the use of proteinase K (PK) to differentiate PrPC and
PrPSc. Recently, the complementary use of the protease
thermolysin has been introduced. This protease digests
PrPC but, unlike PK, leaves PrPSc intact without truncation of the NH2 terminus (126, 376).
The development of highly sensitive assays for biochemical detection of PrPSc in tissues and body fluids is a
top priority. One way to achieve this goal is to develop
high-affinity immunoreagents that recognize PrPSc. Examples include the “POM” series of antibodies that recognize
various well-defined conformational epitopes in the structured COOH-terminal region of PrPC, and linear epitopes
in the unstructured NH2-terminal region (395). Because of
the particular nature of the epitopes to which they are
directed, some of these antibodies have affinities for the
prion protein in the femtomolar range. Antibodies that
specifically bind PrPSc without binding PrPC have also
been reported (279, 358), yet their affinity seems to be
limited and their diagnostic value has awaited confirmation for more than one decade to no avail.
Searching for PrPSc binding reagents, Lau et al. (292)
discovered PrP-derived peptides that bound PrPSc. When
coupled with a sandwich ELISA for detection, these peptide
binding reagents create a sensitive assay that can detect
PrPSc nanoliter amounts of 10% (wt/vol) vCJD brain homog-
AGGREGATION AND INFECTIOUS DISEASE
Physiol Rev • VOL
also reported to be elevated in CSF of sCJD patients
(393). Recently, urinary ␣1-antichymotrypsin has also
been identified as a novel biomarker of prion infection
(347).
The gold standard of prion diagnostics is the ability
to detect prion infectivity itself. Until recently, the
animal bioassay was the only method available to
screen for prion infectivity, for example, by using transgenic mice overexpressing PrPC (tga20) (154). Recently, the bank vole (Clethrionomys glareolus) has
been suggested as a new and efficient model for bioassay of TSEs from different species, including human,
sheep, goat, mouse, and hamster (2, 369). Additionally,
the establishment of a human immune system in the mouse
could be an efficient tool to test the potential human pathogenicity of various prion strains in vivo (14).
However, these bioassays take 6 –7 mo to complete
and are very costly. More recently, the development of
highly susceptible, cloned neural cell lines (PK1 N2a)
have provided an assay that improves the precision,
cost, and time required to do prion detection bioassays,
and might lend itself to high-throughput automation
(270). Although such assays have the potential to advance methodologies aimed at the diagnostic assessment of whether the prion agent is present, it should be
noted that these cell lines are currently reported to be
permissive only to murine prions. Future advances will
hopefully provide a means by which the full range of
prion strains and species can be assayed.
X. CONCLUDING REMARKS
The study of prions has taken several unexpected
turns over the years. The development and appropriate
use of tools and technologies has enabled us to answer
some long-standing key questions; however, many questions are still left unanswered. The physiological function of PrPC remains unknown. Although the Prnp gene
encoding PrPC was identified in 1985 (39, 370) and the
Prnp knockout mice were described in 1992 (85), the
function of the protein is shrouded in mystery. Elucidation of the physiological function of PrPC has the
potential to help researchers understand the mechanisms involved in prion-induced pathogenesis.
The molecular mechanisms of prion replication are
not completely defined, and the possibility that other
proteins assist the process cannot be excluded. We still
do not understand how strain information is maintained
and transmitted, and we have no notion of the mechanisms that define the tropisms of prion strains. Finally, we
do not understand how neurotoxicity is induced by the
prion agent, and why it is less toxic to cells of the immune
system, where it also undergoes live replication. The ability to answer these questions in the future will rely mainly
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enate diluted in plasma, thereby yielding a powerful and
scalable assay that is very well suitable to blood testing.
PMCA is also a promising method for the sensitive
detection of the pathological prion protein (434, 473). It
was recently shown to increase sensitivity 6,600-fold over
standard detection methods (95). Amplifiable PrPSc was
detected in the blood of scrapie-infected hamsters by
PMCA during most of the presymptomatic phase of disease (432).
LCPs were recently developed as a novel class of
amyloidotropic dyes (216, 366). These dyes contain a
swiveling thiophene backbone, and the optical processes, e.g., the fluorescence from the dye, are highly
sensitive to the geometry of the thiophene backbone.
Hence, upon interaction with protein aggregates with a
distinct morphology, the rotational freedom of the LCP
backbone is restricted in a specific way and a unique
optical fingerprint is obtained for a given protein conformation. Instead of measuring simply the total
amount of aggregated protein, heterogeneous populations of specific protein aggregates can be differentiated by LCP staining. In a transgenic mouse model of
AD pathology, LCPs identified a striking heterogeneity
in amyloid plaques (365). LCPs also specifically bind
prion deposits, even those which were negative for
binding to other amyloidotropic dyes (Congo red and
ThT), and different prion strains could be discriminated
via individual staining patterns of LCPs with distinct
ionic side chains (462).
Another exciting prospect may be the combination
of PMCA with LCPs for PrPSc detection. The unique
photophysical properties of polythiophenes may pave
the way for a “real-time PMCA,” where the buildup,
lifetime, and conformational properties of prion aggregates may be visualized kinetically (9).
The use of surrogate biomarkers represents a diagnostic strategy fundamentally different from those
delineated above. Since they typically identify secondary host reactions, surrogate biomarkers of prion infection cannot aspire to match the specificity of PrPSc
detection. On the other hand, surrogate biomarkers
may be useful for identifying subjects at risk and specifying acceptance or deferral of blood donations. In
such cases, high sensitivity (i.e., the identification of all
suspect individuals) is more important than absolute
diagnostic specificity, as the latter can be supplied by
confirmatory assays. Ideally, these surrogate markers
should be detectable at preclinical stages of disease
and differentially expressed in easily accessible body
fluids such as blood or urine.
S-100, neuron-specific enolase, and 14-3-3 protein
have been reported to be elevated in cerebrospinal fluid
(CSF) of sCJD patients (240, 442). These proteins may
represent consequences of CNS damage and neuronal
death. The cysteine proteinase inhibitor cystatin C was
1139
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ADRIANO AGUZZI AND ANNA MARIA CALELLA
on the quality of the tools and technologies available to
the prion field. These unresolved questions provide an
opportunity for additional research which may have a
significant impact on further protein misfolding disorders.
12.
13.
ACKNOWLEDGMENTS
GRANTS
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
A. Aguzzi is supported by grants from the University of Zurich;
the Swiss Federal Offices of Education, Science, and Health; the
Swiss National Foundation; the National Center for Competence in
Research on neural plasticity and repair; the United Kingdom Department for Environment, Food, and Rural Affairs; and the
Stammbach Foundation. A. M. Calella was supported by the Roche
Research Foundation and an EMBO fellowship.
25.
26.
27.
28.
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This article honors the present and past members of the
Aguzzi laboratory, whose enthusiastic commitment led to much
of the work described here. Special thanks to Petra Schwarz for
maintaining our prion-infected mouse colony in impeccable
shape and to Rita Moos for excellent technical assistance.
Mathias Heikenwälder, Christina Sigurdson, Jeppe Falsig, and
Mario Nuvolone provided crucial help with the section on “Immunological aspects of Prion disease” and Simone Hornemann
with the section on “Structures of purified PrPC and PrPSc.”
Certain parts of the history of prion research were adapted from
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whose previous edition had been published by Dr. Stanley B.
Prusiner. We are grateful to Dr. Prusiner for giving us permission to utilize some of his material.
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