Prions: Protein Aggregation and Infectious Diseases - Physiological ...

Prions: Protein Aggregation and Infectious Diseases 

ADRIANO AGUZZI AND ANNA MARIA CALELLA 

Institute of Neuropathology, University Hospital of Zurich, Zurich, Switzerland 

I. Properties of Prions 1106 

A. Introduction 1106 

B. Development of the prion concept 1107 

C. Discovery of the prion protein 1108 

D. PrP amyloid 1109 

E. Formation of PrP Sc 

Physiol Rev 89: 1105–1152, 2009; 

doi:10.1152/physrev.00006.2009. 

F. Prion diversity 1112 

G. Prion-like properties of additional misfolded proteins 1114 

II. Function of the Cellular Prion Protein 1116 

A. Teachings from Prnp-ablated mice 1116 

B. PrP C and its partners 1117 

C. PrP’s double and its trouble 1117 

III. Mechanisms of Prion Toxicity 1118 

A. Alteration of PrP C mediated signaling 1119 

B. PrP C mislocalization 1119 

C. PrP-derived oligomeric species 1121 

IV. Clinical and Pathological Aspects of Prion Diseases 1121 

A. Scrapie 1121 

B. Bovine spongiform encephalopathy 1122 

C. Chronic wasting disease of deer and elk 1122 

D. Human prion diseases 1123 

V. Peripheral Routes of Prion Infectivity 1126 

A. Pathway of orally administered prions 1126 

B. Transepithelial enteric passage of prions 1127 

C. Uptake of prion through the skin 1127 

VI. Immunological Aspects of Prion Disease 1127 

A. The lymphoid system in prion disease 1127 

B. Tropism of prions for inflammatory foci 1130 

C. Neuroinflammation in prion pathogenesis 1131 

VII. Spread of Prions Along Peripheral and Central Nervous System Pathways 1131 

VIII. Towards Antiprion Therapeutics 1134 

A. Innate immunity and antiprion defense 1135 

B. Adaptive immunity and preexposure prophylaxis against prions 1137 

C. Soluble prion antagonists 1138 

IX. Progress in the Diagnostics of Prion Diseases 1138 

X. Concluding Remarks 1139 

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 (PrP C ) of the host. PrP C 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 PrP C , and therefore, understanding the physiological role of PrP C 

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. 

www.prv.org 0031-9333/09 $18.00 Copyright © 2009 the American Physiological Society 

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1106 ADRIANO AGUZZI AND ANNA MARIA CALELLA 

I. PROPERTIES OF PRIONS 

A. Introduction 

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, PrP C , which is encoded 

by the gene Prnp. PrP C 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 

PrP C into PrP Sc , an insoluble and partially protease-resistant 

isoform that propagates itself by imposing its abnormal 

conformation onto PrP C molecules. 

Numerous experiments have provided evidence that 

PrP C 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 PrP C expression 

is required for neurodegeneration in host neurons, 

because the presence of PrP Sc alone does not cause disease. 

For example, when neurografts propagating PrP Sc 

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 PrP C lacking 

a glycosylphosphatidylinositol (GPI) anchor do not 

develop clinical signs of prion disease, although prion 

inoculation induces PrP Sc formation and aggregation of 

amyloid plaques (108). These findings suggest that expres- 

sion of membrane-anchored PrP C is necessary to initiate 

disease. Finally, neuron-specific ablation of PrP C in transgenic 

mice (321) or RNAi knockdown of PrP C expression 

in mice with established prion disease (519) rescues early 

neuronal dysfunction and prolongs the survival of mice 

despite the accumulation of extraneuronal PrP Sc . 

Attempts to identify posttranslational modifications 

that distinguish PrP Sc from PrP C have been mostly unsuccessful 

(480); however, increasing evidence indicates that 

they may exist (89, 122). Almost 45% of the PrP C protein 

is -helical with two very short stretches of -sheet (421). 

Conversion to PrP Sc results in a protein comprised of 

30% -helix and 45% -sheet. The mechanism by which 

PrP C is converted into PrP Sc is unknown, but PrP C appears 

to bind to PrP Sc , perhaps in combination with ancillary 

proteins, to form an intermediate complex during 

the formation of nascent PrP Sc (343). Transgenic mouse 

studies have provided genetic evidence that incoming 

prions in the inoculum interact preferentially with homotypic 

PrP C 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), 

TABLE 1. Human prion diseases 

Physiol Rev VOL 89 OCTOBER 2009 www.prv.org 

Disease Etiology 

Kuru Infection 

Creutzfeldt-Jakob disease 

Iatrogenic Infection 

Sporadic Unknown 

Familial PRNP mutation 

Variant Presumed BSE infection 

Gerstmann-Sträussler-Scheinker disease PRNP mutation 

Fatal familial insomnia PRNP mutation 

BSE, bovine spongiform encephalopathy.

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 PrP C (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 

PrP Sc is derived from PrP C by a posttranslational process 

(58), sporadic prion diseases may result from the spontaneous 

conversion of PrP C into PrP Sc . 

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 3.0/10 6 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 

200 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 

AGGREGATION AND INFECTIOUS DISEASE 1107 


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 PrP Sc -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


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 PrP Sc . Final 

proof of this hypothesis could be achieved if pure PrP C 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 PrP C incubated with unlabeled PrP Sc in a 

cell-free system generated radioactive PrP Sc , 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 PrP Sc is amplified 

by cycles of sonication followed by incubation 

with brain homogenate (433, 434). Soto and co-workers 

(94) showed that amplification of PrP Sc was accompanied 

by an amplification of infectivity. The use of purified PrP C 

instead of brain homogenate as a substrate for the in vitro 

PrP Sc generation decreased the efficiency of amplification, 

suggesting that additional cofactors may facilitate 

the conversion of PrP C to PrP Sc (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 (PrP C , 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 

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 PrP 27–30 ) was identified 

(55, 337). 

Purification of PrP 27–30 to near-homogeneity allowed 

determination of its NH 2-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 

PrP C (39, 370). PrP C is protease sensitive and soluble in 

nondenaturing detergents, whereas PrP 27–30 is the protease-resistant 

core of a 33- to 35-kDa disease-specific 

protein, designated PrP Sc , 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 PrP Sc 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

egion in mouse chromosome 2 (479). Hybridization studies 

demonstrated 0.002 Prnp gene sequences per ID 50 

unit in purified prion fractions, strongly suggesting that a 

nucleic acid encoding PrP Sc 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 

The discovery of PrP 27–30 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 PrP 27–30 into 

liposomes with retention of infectivity (169) suggests that 

large PrP polymers are not required for infectivity, although 

it is certainly possible that PrP Sc 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 PrP Sc -containing aggregates, Caughey and coworkers 

(464) partially disaggregated PrP Sc . 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 


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 PrP Sc , 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 PrP Sc 


It remains to be established whether any form of 

PrP C can act as a substrate for PrP Sc formation, or 

whether a restricted subset of PrP molecules are precursors 

for PrP Sc (511). It is also unknown whether reactive 

transition states between the two exist. Several experimental 

results argue that PrP molecules destined to 

become PrP Sc exit to the cell surface prior to their conversion 

into PrP Sc (59, 99, 494). Similar to other GPIanchored 

proteins, PrP C appears to localize in cholesterolrich, 

nonacidic, detergent-insoluble membranes known as 

rafts (26, 195, 253, 456, 495). Within the raft compartment, 

GPI-anchored PrP C is apparently either converted into 

PrP Sc or partially degraded (343, 495). Chemical and enzymatic 

treatment of purified PrP 27–30 leads to the release 

of glycolipid components, suggesting that PrP Sc could be 

tethered to the membrane by a GPI anchor (481). 

The role of the GPI membrane anchor in the formation 

of PrP Sc 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 PrP Sc , they developed significant amyloid 

plaque pathology in the brain, but clinical manifesta-


tions were minimal. In contrast, combined expression of 

anchorless and wild-type PrP resulted in accelerated clinical 

scrapie (108). 

1. Structures of purified PrP C and PrP Sc 

Early low-resolution structural studies indicated that 

PrP C 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 PrP C by nuclear 

magnetic resonance spectroscopy (Fig. 1A) (229, 421), 

and later also by crystallographic studies (271). The NH2 proximal half of the molecule is not structured at all, 

whereas the COOH-proximal half is arranged in three 

-helices corresponding, for the human PrP C , 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 PrP C acquires a defined 

structure when PrP C is present within membrane rafts 

(362). 

In contrast to PrP C , the -sheet content of PrP Sc 

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 PrP Sc , although 

interesting models have been conjectured on the basis of 

electron crystallography studies (524). Prion rods of the 

NH2-terminally truncated form of PrP Sc derived by limited 

proteolysis, PrP 27–30 , exhibit green-gold birefringence after 

staining with Congo red, indicating that this isoform 

has a high -sheet content (410). Indeed, PrP 27–30 poly- 

FIG. 1. Structural features of PrP C and PrP Sc . 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 

NH 2-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 PrP C (black line), PrP Sc (gray line), and PrP 27–30 (dotted line). 

These proteins were suspended in a buffer in D 2O 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 PrP C , PrP Sc , and Prion rods, composed of 

PrP 27–30 , that were negatively stained with uranyl acetate. Bar, 100 nm. 

[From Pan et al. (379).] 

Physiol Rev VOL 89 OCTOBER 2009 www.prv.org

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 

PrP 27–30 , neither purified PrP C nor PrP Sc forms aggregates 

detectable by electron microscopy (338, 379). 

2. Models describing the replication of prions 

Transgenic mouse studies have provided genetic 

(412) and biochemical (343) evidence that the conversion 

of PrP C to PrP Sc occurs through the formation of a PrP C / 

PrP Sc complex. However, such a complex has never been 

isolated to purity. Consequently, it is unclear whether 

PrP C binds to one or more additional macromolecules 

during the process of PrP C to PrP Sc conversion. The formation 

of a PrP C /PrP Sc 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 PrP Sc formation is more important for the 

conversion to protease resistance than for the initial binding 

steps, additional intermolecular interactions between 

PrP C and PrP Sc may be required to complete the process 

of conversion to the protease-resistant state (228). 

The level of PrP C expression appears to be directly 

proportional to the rate of PrP Sc formation and, thus, inversely 

related to the length of the incubation time. Prnp o/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 PrP C into PrP Sc . The “template-directed refolding” 

hypothesis predicates an instructionist role for 

PrP Sc on PrP C (Fig. 2A). Alternatively, the seeded nucleation 

hypothesis suggests that PrP Sc exists in equilibrium 

with PrP C . In a nondisease state, such an equilibrium 

would be heavily shifted toward the PrP C conformation, 

such that only minute amounts of PrP Sc 

would coexist with PrP C . If this were the case, PrP Sc 

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 PrP Sc molecules. 

The aggregated state would be an intrinsic property 

of infectivity. Monomeric PrP Sc would be harmless, 

but it might be prone to incorporation into nascent 

PrP Sc aggregates (Fig. 2B) (19). 

The recently developed PMCA (92, 93, 196, 432, 

434) is designed to mimic PrP Sc autocatalytic replication 

(Fig. 3). In the PMCA, PrP Sc is amplified in a cyclic 



FIG. 2. Models for the conformational conversion of PrP C into 

PrP Sc . A: the “refolding” or template assistance model postulates an 

interaction between exogenously introduced PrP Sc and endogenous 

PrP C , which is induced to transform itself into further PrP Sc . A highenergy 

barrier may prevent spontaneous conversion of PrP C into PrP Sc . 

B: the “seeding” or nucleation-polymerization model proposes that PrP C 

and PrP Sc are in a reversible thermodynamic equilibrium. Only if several 

monomeric PrP Sc molecules are mounted into a highly ordered seed, can 

further monomeric PrP Sc be recruited and eventually aggregate to amyloid. 

Fragmentation of PrP Sc aggregates increases the number of nuclei, 

which can recruit further PrP Sc and thus results in apparent replication 

of the agent. 

manner by incubating small amounts of PrP Sc - 

containing brain homogenate with uninfected, PrP C - 

containing brain homogenate, resulting in the conversion 

of PrP C to PrP Sc . The latter forms aggregates that 

are sonicated to generate multiple smaller units functioning 

as a seed for the continued formation of new 

PrP Sc aggregates.


FIG. 3. Schematic representation of the protein misfolding cyclic amplification (PMCA) reaction. PrP C is recruited into growing aggregates of 

PrP Sc ; hence, it undergoes conformational conversion and becomes PrP Sc . During PMCA, the growing PrP Sc 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 

PrP C . The whole procedure is repeated several times. PrP C is shown as light gray spheres. PrP Sc is shown as trapezium. The original seed is in dark 

gray, and the newly formed PrP Sc is in light gray. 

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 

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 PrP Sc 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

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. 

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. Synopsis of currently used prion strain differentiation assays 


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, PrP Sc 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 PrP Sc 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- 

Assay Principle Test Substrate Speed Cost Reference Nos. 

Incubation period in indicator mice Mice Years 164 

Histological lesion profile Mice Years 79, 163 

Histoblots Immunohistology Days 493 

Conformation-dependent immunoassay ELISA Days 437 

Conformational stability assay Western blot Days 390 

Conformation assay Infrared spectroscopy Days 101 

PK cleavage site Western blot Days 51 

Detection with NH 2-terminal antibodies Western blot Days 396 

Glycosylation profile Western blot Days 220 

Amyloid detection by thioflavin and Congo red stains Histochemistry Hours 460 

Luminescent-conjugated polymers Histochemistry Hours 462 

Cell panel assay Cell culture Weeks 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. Adapted 

from Aguzzi (9). 



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 PrP Sc alone 

is capable of transmitting the disease and that the characteristics 

of PrP Sc 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 PrP Sc itself is the 

molecule in which prion “strain”-specific information is 

encrypted. Preliminary immunochemical evidence suggests 

that PrP Sc 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 PrP Sc 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 PrP Sc strains associate 

with different molecules of proteinaceous or other 

nature. 

Both PrP C and PrP Sc exist in three main glycosylation 

states: unglycosylated, monoglycosylated, and diglycosylated. 

The relative ratios of these three forms of PrP Sc 

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 PrP Sc (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 PrP Sc 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 PrP Sc 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 PrP Sc glycosylation states affect 


FIG. 4. Representation of the three glycosylated PrP Sc moieties 

(un-, mono-, and diglycosylated PrP Sc ) 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 (PrP Sc 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 PrP C 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 PrP Sc . 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

eported 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 

PrP Sc 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 NH 2-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-PrP Sc amyloids, inoculation 

with the amyloid actually initiates the disease, as is 



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


prion mechanism of disease transmission might be operating 

in other human diseases, some of which are highly 

prevalent. 

II. FUNCTION OF THE CELLULAR PRION 

PROTEIN 

PrP C , 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. 

PrP C contains a long NH 2-proximal flexible random-coil 

sequence, which is followed by a globular COOH-proximal 

domain. The structurally less-defined NH 2 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 PrP C is 

identical to many other mammals, as expected from the 

high degree of sequence identity (309). Interestingly, despite 

the low sequence identity between PrP C in chicken, 

turtle, or frog, and the mammalian isoforms, the major 

structural features of PrP C are remarkably preserved in 

nonmammalian species (88). 

Full-length PrP C 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 PrP C (380, 428), which may be differentially 

distributed in various areas of the central nervous system 

(49, 133). The physiological significance of PrP C glycosylation 

is unknown. 

Despite advances in the characterization of PrP C 

structure, the physiological function of PrP C 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 

whether the physiological function of PrP C is actually 

linked to toxicity associated with prion diseases. 

A. Teachings From Prnp-Ablated Mice 

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 PrP C 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 Prnp o/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 PrP C may have a role in the synaptic machinery of the 

olfactory bulb (293). However, the reported involvement 

of PrP C in synaptic hippocampal activity has been the 

subject of intense controversy (301). 

The only molecularly well-defined phenotype of 

Prnp o/o mice is their resistance to prion inoculation 

(84). However, it seems rather unlikely that a protein as 

highly conserved among species as PrP C has evolved 

purely for the purpose of bestowing susceptibility to 

prion diseases upon organisms. If the function of PrP C 

were completely unrelated to prion disease pathogenesis, 

PrP C would be just one of many proteins whose 

function awaits clarification. Otherwise, the function of 

PrP C 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 

PrP C function. If PrP C possessed enzymatic activity or 

transduced a signal (similarly to many other GPI-linked 

proteins), one could hypothesize that conversion to 

PrP Sc alters PrP C substrate specificity or signal transduction 

strength, conferring as a result a toxic dominant 

function. Therefore, understanding the function of 

PrP C may be instrumental to deciphering prion pathol- 

FIG. 5. Outline of the primary structure of the cellular prion protein including posttranslational modifications. A secretory signal peptide resides 

at the extreme NH 2 terminus. CC 1 and CC 2 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. 


ogy, and perhaps even to devising therapeutic approaches. 

Glockshuber et al. (190) noted that PrP C had similarities 

to membrane-anchored signal peptidases, but 

their observation has not been substantiated by functional 

data. PrP C can bind copper (67, 505), but reports 

of increased copper content in neurons lacking PrP C 

have been called into question by contradictory findings 

(506). The idea that PrP C might be a superoxide 

dismutase (68, 69) was perceived as particularly attractive 

in view of its multiple copper-binding sites. However, 

PrP C does not provide any measurable contribution 

to dismutase activity in vivo (234, 506), although it 

has been suggested that NH 2-proximally-truncated PrP C 

may suppress endogenous dismutase activity (441). 

B. PrP C and Its Partners 

Perhaps PrP C and PrP Sc 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 PrP C interaction partners 

have been summarized in a recent review (10). 

Putative PrP C 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 PrP C interactome (431). However, 

many studies used to identify such interactors are 

fraught with methodological issues. PrP C is exposed to 

the extracellular space, and therefore, it is questionable 

whether conventional yeast two-hybrid screens, which 

artificially expose PrP C to the cytosolic compartment, 

represent an appropriate method to investigate PrP C 

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. 


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 PrP C -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 PrP C , 

but it lacks the PrP C octapeptide repeats and hydrophobic 

region. Structural studies indicate that Dpl contains 

three -helices, similar to PrP C , 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 PrP C , 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 

PrP C and Dpl in primary amino acid sequence, in structure, 

and in subcellular localization suggest related biological 

functions (42). Therefore, a possible role of PrP C 

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).


TABLE 3. Summary of transgenic mutant for PrP 

PrP Deletion Mutants Phenotype Rescue by PrP (if applicable) Prion Propagation Reference Nos. 

PrP 1-22 231# Cerebellar disorder No No 314 

PrP 23-88 No Yes 360, 361 

PrP 23-88 C178A No No 360, 361 

PrP 23-88 95-107 No No 360, 361 

PrP 23-88 108-121 No No 360, 361 

PrP 23-88 122-140 Short-lived protein 360 

PrP 23-88 141-176 No Yes 360, 483 

PrP 23-88 141-221 Cerebellar disorder No No 484 

PrP 23-88 177-200 Storage disease No No 360 

PrP 23-88 201-217 Storage disease No No 360 

PrP 32-80 No Yes 153 

PrP 32-93 No Yes 154, 453 

PrP 32-106 No No 453* 

PrP 32-121 Cerebellar disorder Yes No 453 

PrP 32-134 Cerebellar disorder Yes No 453* 

PrP 94-134 Cerebellar disorder Yes Not done 40 

PrP 104-114 No Not done 204 

PrP 105-125 Cerebellar disorder Yes Not done 297 

PrP 114-121 No No 40, 225 

PrP 231# No Yes 108 

PrP 144# Short-lived protein 360 

PrP PG 14 Cerebellar disorder No Yes 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 PrP C in vivo, 

which indicates that PrP is a functional antagonist of 

PrP C (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 PrP C . Because the lack of PrP C itself does not 

induce an obvious phenotype, the latter pathologies imply 

the existence of pathways in which PrP C is functionally 

active. Hence, mice expressing PrP variants may allow for 

the identification of functionally relevant domains within 

PrP C (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 PrP Sc with neurodegeneration, 

the molecular pathways leading to cerebral 

damage are, for the most part, unknown. Depletion of 

PrP C 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 PrP C 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 


PrP Sc to levels similar to terminally ill, wild-type infected 

animals. These data suggest that PrP Sc might not be directly 

responsible for neurodegeneration; it is more likely 

that the toxicity of PrP Sc depends on some PrP C -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 

PrP C 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 PrP Sc does not exert any clinically detectable 

adverse effects on a mouse devoid of PrP C . 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 PrP Sc was detected in both grafts 

and recipient brain, and immunohistochemistry revealed 

PrP deposits in the hippocampus, and occasionally in the

parietal cortex, of all animals (64). The distribution of 

PrP Sc 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 PrP C by 

an infected cell, rather than the extracellular deposition 

of PrP Sc , 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 PrP Sc 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 PrP C to support prion replication. 

This observation is consistent with a growing body of 

evidence that PrP C may function as a signaling molecule. 

However, it is still possible that abnormal topology or 

altered trafficking of PrP C might also underlie toxicity, or 

perhaps the formation of a toxic form of PrP termed PrP* 

by Charles Weissmann (511), other than PrP Sc 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 PrP Sc is present (21). 

A. Alteration of PrP C Mediated Signaling 

Although it is unlikely that the toxic activity of PrP Sc 

results primarily from loss of PrP C 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 

PrP C (286). Furthermore, it was shown that PrP C 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 

PrP C , but not of PrP lacking the octarepeats, could reverse 

this effect (60). Therefore, PrP C can be neuroprotective 

against various insults, suggesting that its conversion 

to PrP Sc could abrogate this function and induce 

neurodegeneration. 

A second possibility is that the binding of PrP Sc to 

PrP C triggers a signal transduction pathway leading to 


neuronal damage. This hypothesis is supported by a report 

that injection of antibodies that recognize and crosslink 

PrP C 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. PrP C Mislocalization 


Abnormal topology or altered trafficking of PrP C 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 

PrP C is attached to the cell surface by its GPI anchor at its 

COOH terminus. PrP C can adopt at least two transmembrane 

topologies in the ER, Ctm PrP and Ntm PrP, which 

have either their COOH or NH 2 terminus in the ER 

lumen, respectively. It has been suggested that Ctm PrP 

and Ntm PrP normally comprise only a small portion of 

cellular PrP C , and an excess of Ctm PrP induces neurodegeneration 

(204, 205). Some PrP mutations favor 

Ctm PrP formation and are associated with a disease state 

characterized by low levels of PrP Sc accumulation. Thus 

the propensity for the formation of the Ctm PrP and Ntm PrP 

intermediates might lead to neurotoxicity in the absence 

of PrP Sc formation (Fig. 6). 

Another suggested mechanism of PrP-mediated neurotoxicity 

is aberrant PrP C trafficking resulting in the 

toxic accumulation of PrP C 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 PrP C undergo ERAD (313, 529). It has 

been shown in vitro that the transfer of newly synthesized 

PrP C to the cytoplasm by ERAD, and its cytosolic accumulation 

through the pharmacological inhibition of the 

proteasome, leads to PrP Sc -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 

PrP Sc 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-


FIG. 6. Toxicity mediated by abnormal topology or altered trafficking of PrP C . The normal cellular isoform of prion protein, PrP C (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 PrP C translocates to the outer leaflet of the plasma membrane. Instead, Ctm PrP and Ntm PrP are 

unusual transmembrane forms, generated in the ER, which have their COOH or NH 2 terminus in the ER lumen, respectively. It has been suggested 

that misfolded and aberrantly processed PrP (cyPrP and Ctm PrP, 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 PrP Sc may lead to translocation of nascent 

PrP C 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). 

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 


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 

PrP Sc 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

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 

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 A1–42 and A1–40 (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 


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,


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). 

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 


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

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 


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)]. 

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


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. 

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 D178N-129V, V180I, V180I-M232R, T183A, T188A, E196K, E200K, V203I, R208H, 

V210I, E211Q, M232R, 96 bpi, 120 bpi, 144 bpi, 168 bpi, 48 bpd 

Gerstmann-Sträussler-Scheinker phenotype P102L, P105L, A117V, G131V, F198S, D202N, Q212P, Q217R, M232T, 192 bpi 

Fatal familial insomnia phenotype D178N-129M 

Vascular amyloid depositions Y145STOP 

Proven but unclassified prion disease H187R, 216 bpi 

Neuropsychiatric disorder, no proven prion disease I138M, G142S, Q160S, T188K, T188R, M232R, P238S, 24 bpi, 48 bpi 

Polymorphisms, established influence on phenotype M129V 

Polymorphisms, suggested influence on phenotype N171S, E219K 

Silent polymorphisms P68P, A117A, G124G, V161V, N173N, H177H, T188T, D202D, Q212Q, R228R, S230S 

The wild-type human PRNP gene contains 5 octarepeats P(Q/H)GGG(G/–) WGQ 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 (Ala3Ala) and 129 (Met3Val); 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. 


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). 

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 

PrP Sc 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 



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


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. 

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 

10 6 ID 50 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). 


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 LD 50/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

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). 

B. Transepithelial Enteric Passage of Prions 

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. 



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-


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 PrP C -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 Prnp o/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 Prnp o/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 


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 

PrP C 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 PrP C 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

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 PrP Sc following scrapie infection 

(263), and prion replication in the spleen was reported to 

depend on PrP C -expressing FDCs, at least in the ME7 

prion strain (70). 

Inhibiting the LTR pathway in mice and nonhuman 

primates, by treatment with LTR-Ig, results in the disappearance 

or dedifferentiation of mature, functional FDCs 

(191, 192). In addition, treatment with LTR-Ig was found 

to impair peripheral prion pathogenesis (316, 355). Treatment 

of mice with LTR-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 PrP C -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 / , LTR / , or LT / TNF- / ) 

proved virtually noninfectible with 5 log LD 50 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 



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 PrP C and the LTR, even though they 

lacked FDCs and did not display lymphoneogenesis. After 

intraperitoneal prion inoculation, granulomas accumulated 

prion infectivity. Bone marrow transfers between 

Prnp / and Prnp o/o mice and administration of lymphotoxin 

signaling antagonists indicated that prion replication 

required radioresistant PrP C -expressing cells and 

LTR 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, 

PrP Sc retention, and disease kinetics and severity similar 

to those of wild-type mice. CD21/35 expression on hemopoietic 

cells also significantly affected these parameters,


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 

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 PrP Sc 

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 PrP C -expressing 

FDCs. Inflamed organs of mice lacking LT or LTR did 

not accumulate either PrP Sc 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 PrP Sc 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, 

PrP Sc 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), PrP Sc is present 

in ectopic lymphoid tissue in the kidney. Note the presence of two 

lymphoid follicles subjacent to the uroepithelial layer and PrP Sc 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. 


gous phenomena might occur in farm animals, since these 

are commonly in contact with inflammogenic pathogens. 

Indeed, PrP Sc 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,

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. 

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 PrP Sc 

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 



receptors, TNF, LTs or their receptors (267), receptors for 

IgG (FcRs), 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 (NFB) pathway in prion 

pathogenesis. NFB 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, NFB is activated in neurons and glia, and it has been 

proposed that NFB might promote survival of neurons 

(245). Alternatively, NFB 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, NFB activity 

was shown to be enhanced in parallel with the first neuronal 

pathological changes (256). Furthermore, the cytotoxic 

synthetic peptide PrP 106–126 activates NFB inmicroglia 

in vitro (33). Notably, it has been reported that 

NFB activity leads to mitochondrial apoptosis after 

prion infection (61). Despite these findings, it remains 

unclear whether NFB activation is protective or pathogenic 

in prion disease. However, when the impact of 

NFB 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 PrP Sc in the


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 PrP Sc deposition in POSCA slices after 35 days in 

culture shows that PrP Sc accumulates predominantly in the molecular layer. Organotypic brain slices were prepared from 10-day-old tga20 TK (mice 

overexpressing PrP and carrying the CD11b-HSVTK transgene) and were inoculated with 100 g RML (R) or uninfected homogenate (Ø). PrP Sc was 

detected with anti-PrP antibody (POM1). C: accumulation of PrP Sc in slices was also shown by Western blot; tga20 TK and prnp o/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 (tga20 TK ) or without 

(tga20 TK ) 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 tga20 TK or prnp o/o/TK slices treated with GCV. Three 

independent biological replicas of tga20 TK and single replicas of prnp o/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 PrP C , yielding a PrP signal at 23 kDa with a cleavage product at 15 kDa. [B–E are adapted from Falsig et al. (149).] 


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, PrP Sc 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 PrP C -overexpressing indicator mice, in a 



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 PrP Sc (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 PrP Sc -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 PrP C 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 PrP Sc converts resident PrP C 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


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, PrP Sc 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 PrP C 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 Prnp o/o hosts. After prion inoculation in the 

eyes of grafted Prnp o/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 Prnp o/o mice with PrP C -producing 

tissue might lead to an immune response to PrP which 

neutralizes PrP Sc infectivity (214). To definitively rule out 

the possibility that prion transport was disabled by a 

neutralizing immune response, Prnp o/o mice were rendered 

tolerant by expressing PrP C under the control of the 

lck promoter. These mice overexpress PrP on T-lympho- 


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 PrP C , rather than immune response to PrP, 

prevented prion spread (65). Therefore, PrP C 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 PrP C -paved chain of cells, perhaps 

because they are capable of supporting prion replication. 

When such a chain is interrupted by interposed cells that 

lack PrP C , as in the case described here, no propagation 

of prions to the target tissue can occur. Perhaps prions 

require PrP C for propagation across synapses. PrP C is 

present in the synaptic region (157) and certain synaptic 

properties are altered in Prnp o/o mice (120, 520). 

Although a wealth of data has been acquired about 

the transport of PrP Sc 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

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 PrP Sc using PrP-specific antibodies; or binding 

of available PrP C or PrP Sc 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. 

A. Innate Immunity and Antiprion Defense 

Antiprion treatment strategies focusing on immunosuppression 

consist of conditional dedifferentiation of 



FDCs with LTR-Ig, suppression of germinal centers, and 

disruption of lymphoid microarchitecture. LTR-Ig treatment 

causes germinal center disruption due to the breakdown 

of FDC networks (Fig. 10A). The data from studies 

using LTR-Ig indicate that postexposure treatment of 

humans with LTR-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 LTR-Ig, 

although it is not known how soon after exposure 

LTR-Ig would need to be administered. It should be 

considered that LTR-Ig will not offer any relief to patients 

with overt CJD, for whom neural entry has already 

taken place. 

Theoretically, LTR-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 PrP Sc 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-


FIG. 10. Immunotherapeutic strategies for prion disease. A: treatment with the lymphotoxin- receptor fusion protein (LTR)-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-PrP C titers in Prnp o/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-Fc 2) delays the development of prion disease in mice, most probably owing to 

its interaction with the disease-associated PrP (PrP Sc ). LT- 1 2, LT heterotrimer; TLR, Toll-like receptor. 

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 


might lead to enhanced PrP Sc 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,

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). 

B. Adaptive Immunity and Preexposure 

Prophylaxis Against Prions 

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 PrP Sc 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 PrP Sc 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 PrP C and PrP Sc . This 

is most likely due to tolerance of the mammalian immune 

system to PrP C , 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 

Prnp o/o mice. 

However, Prnp o/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 PrP C , 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 Prnp o/o mice) 

were used to reprogram B-cell responses of prion-susceptible 

mice that express PrP C . 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 immu- 



nization was not observed, unless transgenic PrP C 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 PrP C . 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 PrP C -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 PrP C , 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 PrP C (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 PrP C (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 

PrP Sc 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-


tive value of the different in vitro and in vivo experiments 

is limited, and further investigations will be required. 

C. Soluble Prion Antagonists 

In several paradigms, expression of two PrP C moieties 

with subtle differences antagonizes prion replication. 

The molecular basis for these effects is unknown. 

Perhaps the subtly modified PrP C acts as a decoy by 

binding incoming PrP Sc (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-Fc 2) (Fig. 10D). After prion 

inoculation, these mice were surprisingly resistant to 

prion disease (343). The PrP-Fc 2 was not converted to 

a prion-disease-causing isoform. Moreover, when the 

transgenic mice expressing PrP-Fc 2 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 10 5 -fold reduction 

in titer of the prion-infected inoculum. This antiprion 

effect occurred in two different lines of PrP-Fc 2expressing 

transgenic mice after either intracerebral or 

intraperitoneal infection with scrapie. Therefore, it 

seems that PrP-Fc 2 effectively antagonizes prion accumulation 

in the spleen and brain. Because PrP-Fc 2 cannot 

be converted to the protease-resistant, diseasecausing 

isoform, it might be effectively functioning as a 

“sink” for PrP Sc , by binding PrP Sc and preventing the 

binding and conversion of PrP C (20). 

Delivery of the soluble prion antagonist PrP-Fc 2 to 

the brains of mice by lentiviral gene transfer impaired 

replication of disease-associated PrP Sc 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-Fc 2 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. 

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 


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 

PrP Sc 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 PrP Sc 

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). PrP Sc -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 PrP Sc in as many as one-third of skeletal 

muscle and spleen samples (183). In addition, PrP Sc 

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 PrP C and 

PrP Sc . Recently, the complementary use of the protease 

thermolysin has been introduced. This protease digests 

PrP C but, unlike PK, leaves PrP Sc intact without truncation 

of the NH 2 terminus (126, 376). 

The development of highly sensitive assays for biochemical 

detection of PrP Sc in tissues and body fluids is a 

top priority. One way to achieve this goal is to develop 

high-affinity immunoreagents that recognize PrP Sc . Examples 

include the “POM” series of antibodies that recognize 

various well-defined conformational epitopes in the structured 

COOH-terminal region of PrP C , and linear epitopes 

in the unstructured NH 2-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 PrP Sc without binding PrP C 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 PrP Sc binding reagents, Lau et al. (292) 

discovered PrP-derived peptides that bound PrP Sc . When 

coupled with a sandwich ELISA for detection, these peptide 

binding reagents create a sensitive assay that can detect 

PrP Sc nanoliter amounts of 10% (wt/vol) vCJD brain homog-

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 PrP Sc 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 PrP Sc 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 PrP Sc 

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 


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 PrP C (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 PrP C remains unknown. Although the Prnp gene 

encoding PrP C 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 PrP C 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


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. 

ACKNOWLEDGMENTS 

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 PrP C and PrP Sc .” 

Certain parts of the history of prion research were adapted from 

a previously published book chapter [A. Aguzzi. In: Topley & 

Wilson’s Microbiology and Microbial Infections (10 th ed.), 2005] 

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. 

Address for reprint requests and other correspondence: A. 

Aguzzi, Institute of Neuropathology, University Hospital Zürich, 

Schmelzbergstrasse 12, CH-8091 Zürich, Switzerland (e-mail: 

adriano.aguzzi@usz.ch). 

GRANTS 

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. 

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