Review | doi: 10.1111/j.1365-2796.2011.02387.
Prions and protein-folding diseases
E. Norrby
From the Center for the History of Science, Royal Swedish Academy of Sciences, Stockholm, Sweden
Abstract. Norrby E (Royal Swedish Academy of Sci-
ences, Stockholm, Sweden). Prions and protein-
folding diseases (Review). J Intern Med 2011; 270:
1–14.
Prions represent a group of proteins with a unique
capacity to fold into different conformations. One iso-
form is rich in beta-pleated sheets and can aggregate
into amyloid that may be pathogenic. This abnormal
form propagates itself by imposing its confirmation
on the homologous normal host cell protein. Patho-
genic prions have been shown to cause lethal neuro-
degenerative diseases in humans and animals. These
diseases are sometimes infectious and hence referred
to as transmissible spongiform encephalopathies. In
the present review, the remarkable evolution of the
heterodox prion concept is summarized. The origin of
this phenomenon is based on information transfer
between homologous proteins, without the involve-
ment of nucleic acid-encoded mechanisms. Histori-
cally, kuru and Creutzfeldt-Jakob disease (CJD) were
the first infectious prion diseases to be identified in
man. It was their relationship to scrapie in sheep and
experimental rodents that allowed an unravelling of
the particular molecular mechanism that underlie
Introduction
The history of the identification of the infectious nat-
ure of prion diseases and the discovery of the chemi-
cal nature of this type of infectious agent is remark-
able. The great advances in this field of research
have been recognized by two Nobel Prizes in Physiol-
ogy or Medicine: one in 1976 to D. Carleton Gajd-
usek (a prize shared with Baruch S. Blumberg, for
the discovery of hepatitis B virus) and the other in
1997 to Stanley B. Prusiner. Infectious prion dis-
eases represent relatively rare phenomena mostly
observed in the context of the spread of the agent by
human intervention. However, the principal molecu-
lar mechanisms that lead to disease may have appli-
cations for a number of much more common non-
contagious diseases. Two fundamental observations
are relevant to the understanding of the molecular
mechanisms involved. The first is that the same
the disease process. Transmission between humans
has been documented to have occurred in particular
contexts, including ritual cannibalism, iatrogenic
transmission because of pituitary gland-derived
growth hormone or the use in neurosurgical proce-
dures of dura mater from cadavers, and the tempo-
rary use of a prion-contaminated protein-rich feed for
cows. The latter caused a major outbreak of bovine
spongiform encephalopathy, which spread to man by
human consumption of contaminated meat, causing
approximately 200 cases of variant CJD. All these
epidemics now appear to be over because of measures
taken to curtail further spread of prions. Recent stud-
ies have shown that the mechanism of protein aggre-
gation may apply to a wider range of diseases in and
possibly also outside the brain, some of which are rel-
atively common such as Alzheimer’s and Parkinson’s
diseases. Furthermore, it has become apparent that
the phenomenon of prion aggregation may have a
wider physiological importance, but a full under-
standing of this remains to be defined. It may involve
maintaining neuronal functions and possibly con-
tributing to the establishment of long-term memory.
Keywords: iatrogenic disease, prions, protein folding.
polypeptide chain, depending on the environmental
conditions, including the possible presence of
homologous proteins with a predetermined folding
pattern, may take on dramatically altered folding. In
certain cases, major aggregates of homologous pro-
teins may be formed and such aggregates in turn
may display cytopathogenic effects. A degenerative
disease may ensue. The second relevant observation
is that much still remains to be learnt about pro-
tein-folding phenomena and the role of information
transfer systems engaging only proteins. Many pro-
teins have sequences of amino acids that make
them potentially prionogenic. Such sequences that
under certain circumstances may be the cause of
disease in mammals can, in another context, play a
central role in physiological functions, for example,
as the source of epigenetic mechanisms of protein
signalling of importance for the survival of yeast
cells.
ª 2011 The Association for the Publication of the Journal of Internal Medicine 1
 E. Norrby
| Review: Prions and protein-folding diseases
In this review, our emerging understanding of the
mechanisms of prion diseases will first be discussed,
in particular their unique mechanisms of spread will
be considered. The original belief in relatively firm
barriers preventing the spread of prions between spe-
cies was questioned when it became clear that bovine
spongiform encephalopathy (BSE), also known as
‘mad cow disease’, could be transmitted to man. It
was then shown, surprisingly, that the disease con-
tracted from infected cattle could spread from man to
man by blood transfusion.
Next, the possibility of a much wider application of
the pathogenic mechanism of cell destruction by pro-
tein aggregation, including degenerative processes
causing for example Alzheimer’s and Parkinson’s dis-
eases, will be discussed. The rapidly growing appreci-
ation of the significance of signalling between pro-
teins outside the canonical steps of the central dogma
of molecular biology will finally be considered.
Funeral rites and infectious diseases
Unexpectedly, it was the fatal neurological disease
kuru, amongst the stone-age Fore people in New Gui-
nea, which provided the first insight into what came
to be called prion diseases. Studies of brain samples
collected under primitive conditions from individuals
who had died of kuru provided a principal link be-
tween the pathogenesis of two, until then considered,
unrelated illnesses, scrapie in sheep and CJD in man
[1, 2]. Scrapie had been known since the eighteenth
century, but it was in the 1930s that the disease was
shown to be infectious and caused by an agent the
size of viruses or smaller. The infectious agent was
found to be remarkably stable, as it survived the for-
malin treatment of an experimental vaccine against
louping ill disease in sheep and spread to the immu-
nized animals.
Creutzfeldt-Jakob disease was identified by two Ger-
man neuropathologists in the 1920s. The disease is
characterized by a progressive destruction of the
brain, referred to as spongiform encephalopathy. It
occurs sporadically, with a frequency of about one
case per million individuals per year worldwide. In
certain groups of people, the frequency is higher,
emphasizing the role of a genetic predisposition. One
example of such a difference has been identified in
studies of Sephardi Jews, who contract spontaneous
CJD (sCJD) at a frequency 30 times higher then Ash-
kenazi Jews. In addition, there are certain familial
forms of the disease generally with unique patterns of
symptoms. Approximately 85% of all cases of CJD are
2 ª 2011 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine 270; 1–14
sporadic, whereas about 10% are familial, leaving a
small minority of cases with an iatrogenic (environ-
mentally acquired) cause.
Unique samples of brain collected by Gajdusek al-
lowed identification of the histopathology of kuru. It
showed many similarities to the changes in brains
from patients with sporadic sCJD, but there were also
similarities to the changes in brains from sheep with
scrapie as noted by Hadlow [2]. His observation
encouraged Gajdusek to attempt to transmit these
noninflammatory diseases to chimpanzees. He and
his collaborators were successful in transmitting first
kuru [3] and then CJD [4] by intracerebral inocula-
tion of chimpanzees. The term transmissible spongi-
form encephalitis (TSE) was commonly used to refer
to this kind of infection.
Next, after determining that kuru was infectious, the
route of transmission was found to be the ritual can-
nibalism practised by the Fore people [5]. The body
of the deceased relative was prepared for the funeral
meal, and the central nervous system containing the
largest concentration of the infectious agent was
consumed mainly by the women and children; thus,
they were primarily affected by the disease (Fig. 1).
However, no child born after 1960, the year in which
the practice of ritual cannibalism ceased, has con-
tracted kuru. The incubation time of the disease can
be long, potentially longer than the life span of the
individual, and cases of kuru have occurred in the
present century [6]. The infectious nature of sCJD
had been demonstrated, but it was initially unclear
how it might spread. In fact, under normal condi-
tions, CJD is not infectious. To date, careful studies
of transfusion of blood from people incubating or
even displaying early signs of CJD have not revealed
any spread of the agent between individuals by this
route [7]. Only under conditions of medical interven-
tions involving brain material could an iatrogenic
spread of the spontaneous form of the disease be
demonstrated.
Prions
Throughout the 1970s, Gajdusek continued to refer
to the infectious agents that cause kuru ⁄ CJD as slow
viruses, and this situation did not change until Prus-
iner began to gain insights into the chemical nature of
the agents using hamster scrapie as a model to isolate
and characterize them. He continued to purify the
infectious material from hamster brains until he
produced a relatively pure protein preparation [8].
Because of the lack of any evidence of participating
 E. Norrby
| Review: Prions and protein-folding diseases
1000
800
Number of deaths
Total
600
400
< 20 years
200
Males
57– 62– 67– 72– 77– 82– 87– 92– 97– 02–
61 66 71 76 81 86 91 96 01 06
5-years time periods
Fig. 1 The epidemiology of kuru amongst the Fore people,
which in 1960 had a total population of about 300 000 indi-
viduals. The number of cases in young individuals rapidly de-
clined after 1960, the year in which the practice of ritual can-
nibalism ceased. The majority of victims were women. Single
cases have appeared into the present century, implying an
incubation time exceeding 40 years. Figure kindly provided
by Paul Brown.
nucleic acid in this preparation, he named it prion
(proteinaceous and infectious particle) [9]. The pro-
tein preparation – referred to as PrP 27–30 (prion pro-
tein with a molecular weight 27–30 kD) – was pure
enough to determine a short section of its amino ter-
minal amino acid sequence. This information made it
possible to determine that the gene responsible for
the synthesis of the prion protein was not foreign, but
a normal host cell gene [10]. Thus, it became possible
to explain the absence of any inflammatory response
in the tissues in the presence of this infectious dis-
ease; under healthy conditions, we do not develop
immunological reactions to our own tissues.
Following the identification of the PrP gene, it was
possible to produce mice deficient in PrP using
‘knockout’ technology [11]. At this time, it was known
that the PrP protein appears in the brain early during
embryonic development. It was found, unexpectedly,
that development and life span appeared to be normal
in animals without PrP protein. The PrP-knockout
mice could be used for two types of critical follow-up
experiments.
ª 2011 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine 270; 1–14
First, after infection with different doses of infectious
prion material, the knockout mice were found to be
completely refractory to development of disease [12,
13]. Furthermore, they could mount an immune re-
sponse to the PrP protein, as this no longer repre-
sented an endogenous product. For the first time,
antibodies could be generated against different parts
of the protein. The protein that Prusiner et al. had
identified was indeed critical to the pathogenic pro-
cess in prion diseases.
The second type of experiment was to investigate the
effects of reintroducing into the PrP-knockout mice
either a modified homologous PrP gene (carrying a
substitution or deletion of a single or a stretch of ami-
no acids) or a PrP gene from a different species. The
former experiment enabled attempts to dissect the
role of different parts of the PrP protein in the patho-
genic process (as discussed below). The latter enabled
the evaluation of the species specificity of PrP pro-
teins. Mice are normally infected only by prions from
other mice or rodents, such as hamsters, but not from
more distant species. However, by generation of
transgenic mice carrying for example a human or a
bovine PrP gene, this species barrier can be overcome
providing important opportunities for studies of dif-
ferent kinds of pathogenic PrP proteins of relevance
for human disease.
Inappropriate folding and aggregation of proteins can cause disease
Diseases caused by protein aggregation have been
known for a long time. They are collectively referred to
as amyloid diseases, a term reflecting the original
misconception that the observed stainable deposits
contained starch (amylum in Latin). Later, it was
demonstrated that they were in fact composed of vari-
ous kinds of proteins. The amyloid deposits have
characteristic staining properties and show birefrin-
gence under polarized light. Protein aggregates show-
ing these characteristics were demonstrated in the
brains of patients with CJD. However, amyloid forma-
tion is not always a feature of prion disease [14]. It
is in fact found in only about 10% of the brains of
patients with sCJD, but at a much higher rate in
patients with other forms of the disease.
It then became possible to determine the structure of
purified PrP using nuclear magnetic resonance anal-
ysis [15, 16]. It was shown that PrP can appear in two
completely different forms, albeit with an identical
amino acid sequence. The ‘healthy’ protein, referred
to as PrP-C (control), has a structure involving
predominantly two large alpha-helix structures,
3
 E. Norrby
| Review: Prions and protein-folding diseases

whereas there is a predominance of beta-pleated
sheets in the pathological PrP-TSE protein (Fig. 2). It
is the PrP-TSE protein that may form amyloid protein
aggregates. The reason for the two completely differ-
ent configurations of the same protein is not known,
but a critical observation is that if a small amount of
PrP-TSE is added to a larger amount of PrP-C, the
‘healthy’ protein is converted to the TSE form by an as
yet undefined contagious ‘snowball’ effect. Two theo-
retical models, nucleation-polymerization and tem-
plate assistance, have been proposed to explain this
([17], Fig. 3). However, as discussed in the next sec-
tion, only certain kinds of proteins are capable of
forming amyloid and are truly infectious, and the
term prion is reserved for them; the term prionogenic
has been introduced to include noninfectious amy-
loid-generating proteins.
Iatrogenic CJD
The awareness that CJD was a disease that could be
transmitted to experimental animals immediately
raised the question of to what extent it might be trans-
missible between humans. There was no epidemio-
logical evidence of connections between cases of
CJD, except for an increased frequency of occurrence
in certain families and ethnic groups. In the light of
understanding the seminal importance of PrP in the
disease, it could be deduced that familial cases must
be because of inherited mutations in different sites of
the PrP gene, whereas sporadic cases were likely to
be caused by mutation(s) accumulated in somatic
(possibly brain) cells or alternatively a spontaneous
emergence of a misfolded PrP protein (the nucleation–
polymerization model) during the lifetime of the indi-
vidual. As already mentioned, to date it has not been
possible to find evidence for transmission of disease
from individuals with sCJD by blood transmission
[7], but relatively recent data provide evidence for a
possible transmission of scrapie in sheep by experi-
mental blood transfusion [18].
Gajdusek and collaborators at an early stage began
to search for evidence of the spread of CJD through
medical intervention. They found that in CJD, as in
scrapie, the majority of infectious prions were located
in the brain; indeed, brain tissue is about 100 000
times more infectious than peripheral tissues, such
as blood [19]. The first case of iatrogenic spread of
CJD between two individuals was found in connec-
tion with a corneal transplant [20], and later the simi-
lar spread to two relatively young individuals was
demonstrated as a result of using electrodes for intra-
cerebral recording [21].
Three epidemics of strikingly different origins have
been documented, and all comprise slightly in excess
of two hundred cases with the maximum number of
cases at the end of the 1990s (Fig. 4). The first of the
three epidemics was caused by the use of human
growth hormone prepared from pools of many hun-
dreds of pituitary glands from cadavers [see 19 for ref-
erences]. Because these preparations were used in
growing individuals, many of the victims of the dis-
ease were relatively young. When this iatrogenic
spread of CJD prions was discovered, the product
was rapidly withdrawn, and it was progressively re-
placed by growth hormone prepared by recombinant
DNA technology. The average incubation time of this
parenterally injected material was estimated to be
15 years (range 4–36 years). The total number of
cases to date is 206, and the epidemic seems to have

Fig. 2 Fundamentally different

structures of normal and inap-
propriately folded PrP protein.
The latter has a predominance
of beta-pleated sheets, which
gives it a propensity to aggregate
with other homologous proteins
potentially causing destruction
of tissues. Figure kindly pro-
vided by Paul Brown.

4 ª 2011 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine 270; 1–14
 E. Norrby
| Review: Prions and protein-folding diseases

Fig. 3 Schematic model of conversion of PrP-C to PrP-TSE. In the nucleation-polymerization model, there is a rapid conversion of
PrP protein between the PrP-C (circles) and PrP-TSE forms (squares), but the former is more stable. In the presence of an aggregate
large enough to act as a stable nucleus, illustrated by the collection of PrP-TSE squares, a change from PrP-C to PrP-TSE is
favoured. In the template-assistance model, the conversion of PrP-C or a modified conformation, PrP-INT (intermediate), to Prp-TSE
is extremely slow in the absence of PrP-TSE, but the process of conversion is essentially irreversible. PrP-TSE is able to propagate
itself by catalysing the conversion of other PrP-INT molecules to the PrP-TSE confirmation. The final product of the two models is
amyloid, which is potentially responsible for the disease process. Modified from ref. 17.

just reached an end. Most cases occurred in France,
109 of 1700 treated individuals. The corresponding
figures are 56 of 1848 treated individuals in the UK
and 28 of 7700, in the USA. In the USA, an additional
step of purification was introduced in 1977, which
may have reduced the risk of transmission of infec-
tious prions.
The second epidemic has a distinctly different iatro-
genic background. It relates to the previous use of
heterologous cadaveric dura mater material to im-
prove the healing process after neurosurgical inter-
ventions [see 19 for references]. The total number of
cases registered to date is 196 (only 142 shown in
Fig. 4), the majority (63%) of which have been in Ja-
pan. The estimated average incubation time was
11 years (range 16 months – 23 years). The use of
this type of graft was banned in the UK in 1989 and in
Japan in 1997. In 1987 a disinfection step with NaOH
was introduced, but eventually this was not consid-
ered safe.
The alternatives used today are synthetic dura mater
material, connective tissue (fascia lata orfascia tem-
poralis) from the patient or material of animal origin
(bovine pericardium)’. Overall, it seems that the
threat of iatrogenic spread of CJD is now minimal
[19]. With the present awareness of the situation, any
potential occupational risk of disease, for example,
for surgeons and nurses involved in brain surgical
procedures, can in practical terms be eliminated.
The main focus of interest during the last 15 years
has been the third epidemic, the unexpected spread
of prions from cattle to man.
The spread of BSE to man
The BSE epizootic began in the mid 1980s (Fig. 5). It
developed rapidly and reached its peak in 1992, but
still to the present time, spurious cases of infected
animals are being identified. Extensive culling of
cows took place on farms in which BSE-infected ani-
mals were identified. In spite of this, about 180 000
cases of BSE (cows displaying symptoms of disease or
with demonstrable PrP in their tissues) have so far
been identified. The mean incubation time is approxi-
mately 5 years. The source of the epizootic was a no-
vel type of feed introduced to provide protein, in par-
ticular for diary cows. The natural cow feed of plant
proteins was supplemented with meat and bone
meal prepared from offal of cattle, sheep, pigs and

ª 2011 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine 270; 1–14 5
 E. Norrby
| Review: Prions and protein-folding diseases

BSE [0–40 000] vCJD undefined route [0–40]

Injection of growth hormone from pituitary glands vCJD blood transmission [0–40]
28 40 000 40

24 35 000 35

30 000 30
20
25 000 25
16
20 000 20
12
15 000 15
8
10 000 10
4
5000 5
0
0 0
<19881989 1991 1993 1995 1997 1999 2001 2003 2005 2007
Transplant of dura mater grafts
28

24
Fig. 5 The epizootic of bovine spongiform encephalopathy
20 (blue bars), the epidemic of variant Creutzfeldt-Jakob disease
16
(vCJD) in humans (red bars) and three cases of transmission
of vCJD between humans by blood transfusion (yellow bars).
12

4 sues of animals was successfully developed, but

0 unfortunately was not applicable to blood. In farms in
which an infected animal was detected, the entire
vCJD – prion contaminated meat
herd was culled.
28

24
20
16
12
8
4
0 been any evidence that the scrapie agent could
1953 1963 1967 1989 1991 1993 1995 19971999 2001 20032005 20072009
Fig. 4 Comparison of three Creutzfeldt-Jakob disease (CJD)
epidemics. Each of the epidemics involved about 200 patients
and was caused by different human interventions: iatrogenic
CJD by intra-muscular injection of pituitary gland-derived
growth hormone; iatrogenic CJD as a result of dura mater
transplantation; and variant CJD because of ingestion of con-
taminated meat. The average incubation time in the three epi-
demics was 15, 11 and about 11–12 years, respectively.
chickens. Such a mixed food had already been in use
for some time, but in the late 1970s, a previously used
hydrocarbon solvent-extraction method was aban-
doned resulting in a markedly increased fat content
of the product. It was this modified feed that was the
source of spread of prions of either bovine or ovine ori-
gin. As soon as this was recognized in 1988, its use
was forbidden. Soon after, a test to detect PrP in tis-
In 1989 mandatory changes in slaughtering tech-
niques were introduced. These changes ensured that
the brain and spinal cord, the main sources of prions,
were excluded from products used for human con-
sumption. The precaution was taken even though at
the time it was not anticipated that prions could
spread from cattle directly to man, as there had never
spread from sheep to man. In principle, the same spe-
cies barrier that had prevented such a spread for
hundreds of years was expected to exist also between
cows and man. However, in 1994 the first case of CJD
of bovine origin was identified in man [22]. For several
reasons, it was concluded that this case was caused
by transmission of prions from cows. First, the histo-
pathological changes in the brain showed unique
characteristics. Second, the patients were younger,
as it later was shown, by about 40 years compared to
those with sCJD. Finally, the molecular characteris-
tics of the agent were unique, demonstrating a differ-
ent, most probably non-human, origin. The new form
of CJD was referred to as variant CJD (vCJD). In con-
trast to the above-mentioned forms of iatrogenic
CJD, which were caused by a parenterally injected
drug originating from a brain extract or by direct con-
tact between brain materials in neurosurgical proce-
dures, the spread in the case of vCJD was oral. It was
known from previous studies of prions from cases of

6 ª 2011 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine 270; 1–14
 E. Norrby
| Review: Prions and protein-folding diseases
sCJD that feeding of experimental animals by the oral
route was a very inefficient way of initiating an infec-
tion with this kind of agent. Yet still the disease
spread, which initiated speculation about the possi-
ble number of case that might be expected. Fortu-
nately, the epidemic peaked in the year 2000 and now
appears to have come to an end (Figs 4 and 5). To date,
the total number of individuals infected by vCJD is
214, of which 167 cases have occurred in the UK, 25
in France and most of the rest in other European
countries [23]. The time interval between the peaks of
the BSE epizootic and the vCJD epidemic is 9 years.
However, it should be noted that slaughtering tech-
niques to exclude the most contaminated parts of cat-
tle were introduced by 1989, suggesting that true
incubation times exceed an average of 11–12 years.
No one born after 1989 has contracted the disease.
As already mentioned, mutational changes in the PrP
protein can influence its propensity to fold incor-
rectly. In the case of vCJD, it has been found that the
amino acid in position 129 in the protein has a critical
role for the development of the disease. Depending on
the nucleotides in this position, it can specify either
valine or methionine, but all cases of clinical vCJD
identified to date have had methionine in both allelic
positions. The recent vCJD epidemic peaked in
2000 ⁄ 2001 and apparently ended in 2008, but there
have been speculations of a possible additional wave
of the disease in individuals with methionine ⁄ valine
or valine ⁄ valine in position 129 [24]. However, it is
doubtful whether infected individuals with this ge-
netic background can develop clinical disease and in
the unlikely event that they do, the number of cases
will probably be fewer than in the recent epidemic.
High-tech ‘cannibalism’
Essentially cannibalism is not practised in modern
society; however, there are an increasing number of
situations in which body fluids, tissues or organs are
transferred between individuals. Blood transfusions
have been performed since a long time, but the proce-
dure did not become safe until the discovery of the hu-
man blood groups in the beginning of the 1900s.
Since the beginning of the 1960s, transplantation of
solid organs and bone marrow has become a common
practice in human healthcare. Because, as men-
tioned earlier, blood transfusion has not been known
to cause the spread of sCJD, the report in 2004 that
vCJD could be spread by blood transfusion came as a
great surprise [25]. Within a short period, four cases
of transmission of vCJD prions were observed ([26]
and see ref [19]). These four cases are described in
ª 2011 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine 270; 1–14
3 1/3 years
6 1/2 years
Case # 1
1 1/2 years
5 years
Case # 2
1 2/3 years
7 1/2 years
Case # 3
1 1/3 years
8 1/2 years
Case # 4
96 97 98 99 00 01 02 03 04 05 06
Fig. 6 Four cases of variant Creutzfeldt-Jakob disease
(vCJD) prion infection caused by blood transfusion. Cases 3
and 4 were infected by transfusion of blood from the same do-
nor. In each case, the upper bar shows the time until the donor
developed disease and the lower bar the time until disease
appeared in the recipient or, as in case 2, vCJD prions were
demonstrated in the tissues. Figure kindly provided by Paul
Brown.
detail in Fig. 6. Case one was a 69-year-old man who
became ill 7 years after receiving blood from a 24-
year-old donor who himself started to show symp-
toms of vCJD 3 years after he had donated the blood.
This first identified case prompted a review of individ-
uals who had received blood from donors who had la-
ter developed vCJD. The second case of proven trans-
mission was a 77-year-old patient, who died because
of a ruptured aortic aneurysm 5 years after he had re-
ceived blood from a younger donor who 18 months la-
ter developed vCJD. The recipient of the blood had no
neurological symptoms; however, PrP-TSE could be
demonstrated in the spleen and in one lymph node
(but not in the brain). It is possible that this individ-
ual, with time, might have developed a degenerative
disease. It should be noted in this context that this
patient was heterozygous for methionine ⁄ valine at
position 129 in the PrP protein. The third and fourth
cases received transfusions from the same donor,
who developed vCJD about 1.5 years after donating.
It took 7.5 and 8.5 years, respectively, until the recip-
ients of the transfusions developed the disease. Be-
fore these cases of iatrogenic transmittance of prions
causing vCJD had been documented, measures had
already been taken to reduce the risk of their possible
spread by blood. Attempts were made to develop a
blood test that could demonstrate, as in the case of
hepatitis B and human immunodeficiency virus
infections, whether a potential donor was infected.
However, attempts to find a reliable test have failed to
date. Nevertheless, other precautions have been
taken. Primarily the selection of donors became
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 E. Norrby
| Review: Prions and protein-folding diseases

stricter to try to exclude individuals who might poten- excluding such infectious agents from blood and
tially have an increased risk of carrying vCJD prions. blood products. First and foremost, it is important to
Furthermore, it had been reported that mice and decide from the history of the donors that the risk that
hamsters infected with scrapie prions had low titres they carry vCJD prions is minimized. In the absence
outside the nervous system and that these prions of any effective technique to detect PrP-TSE in blood,
preferentially occurred in white blood cells [27]. It a number of experimental studies using materials
was therefore decided, first in the UK in the late ‘spiked’ with prions from rodents have been used to
1990s and then in other European countries, that develop general methods to reduce the content of pos-
white blood cells should be removed from blood to be sibly contaminating prion proteins. The safety of the
used for transfusion. From animal experiments, it products used has been markedly improved [31–35],
was deduced that this would reduce, but not elimi- but it is impossible to be absolutely certain that there
nate, the amount of possibly contaminating prions is no risk of transmission. Still it should be kept in
[28]. Since the introduction of this precautionary mind that the recent use of different coagulation fac-
measure, no further cases of vCJD caused by blood tors in man has an impressive track record. A com-
transfusion have been seen. Additionally, because prehensive review of approximately 20 000 patients
the number of cases of vCJD has progressively de- with haemophilia treated with concentrates of differ-
creased since 2000, the risks for the spread of the dis- ent factors has not revealed a single case of vCJD [36].
ease between humans by blood should by now have The only exception is a British man with haemophilia
been eliminated. in whom a transfer of vCJD prions might have oc-
curred (personal information by Tor-Einar Svae, Oc-
The risk of spread of vCJD prions by blood also ap- tapharma, Vienna). This patient was treated during
plies to products that are prepared using blood as a the 1990s with a concentrate of Factor VIII produced
source. The history of the spread of virus infections in the UK. It was found that material from a donor
by products containing components of blood is long. who later developed vCJD was included in two
Hepatitis B virus was transmitted to 300 000 soldiers batches, and it was subsequently shown that the
during World War II by a yellow fever vaccine contain- technique of purification did not exclude vCJD pri-
ing live virus stabilized by human albumin. Much la- ons. This purification method has not been in use for
ter, it was demonstrated that the albumin used had many years.
been contaminated by hepatitis B virus. During the
mid 1980s, a large number of haemophilia patients
The physiological role of the prion protein
became infected with human immunodeficiency
virus (HIV) when treated with Factor VIII or Factor IX Once it became clear that the presence of the PrP gene
preparations. Not until techniques to screen for the was absolutely essential to the development of PrP-
presence of a virus, or antibodies against it, in blood TSE-derived diseases and that animals without PrP,
had been developed, could safe preparations be unexpectedly, seemed to develop normally, it was
developed. Increased awareness of the importance of important to determine the physiological role of the
excluding contamination by viruses from blood prod- PrP protein. However, despite many studies, its fun-
ucts led to the development of general methods of damental function(s) still remains to be definitively
elimination. Treatment with organic solvents was identified. Different studies have highlighted a wide
introduced to eliminate enveloped viruses and other range of different functions [37–42].
procedures, including the more recently introduced
nanofiltration, have led to a high degree of efficacy in The chromosomal gene denoted Prnp encodes PrP. It
preventing the spread of conventional viruses by is a member of the Prn gene family, which also genes
blood-derived products for human use. However, pri- encoding two other proteins. The PrP open reading
ons represented a different challenge because of their frame is encoded within a single exon directing the
unique stability against physical ⁄ chemical treat- synthesis of a protein with 254 amino acids. This pro-
ments. The particular resistance of this kind of tein is post-translationally modified by removal of a
infectious agent was observed at an early stage and 22-amino acid, amino terminal signal peptide and a
stimulated speculation on the possible means of 23-amino acid carboxy terminal. The latter directs
replication without nucleic acid [29, 30]. the addition of a glycosylphosphatidyl inositol mem-
brane anchor. Under normal conditions, PrP is a
The unexpected threat, and later demonstration, of membrane-bound protein, but it can also show bio-
transmission of vCJD prions by blood transfusions logical activity and cause infectious amyloid disease
caused a major review of the possible means of in a nonmembrane-bound form [43]. In addition, the

8 ª 2011 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine 270; 1–14
 E. Norrby
| Review: Prions and protein-folding diseases
protein has two glycosylation sites and an internal di-
sulphide bond. All these properties are shared be-
tween molecules exerting their normal physiological
function(s) and proteins causing prion diseases. The
majority, but not all, PrP proteins are relatively resis-
tant to protease digestion. This property was used in
the early attempts to purify the protein. Proteinase
digestion cleaves about 67 amino acids from the ami-
no terminal of the 209-amino acid final protein prod-
uct. This produces PrP 27–30, a truncated protein,
which can still form amyloid. This was the protein
used by Prusiner et al. to identify the nature of PrP.
Alignment of PrP sequences of different mammalian
origin shows a striking degree of conservation, high-
lighting a crucial biological function preserved
through evolution [42]. However, there are also differ-
ences, which explain the species barrier to disease
transmission mentioned earlier.
A number of different functions have been proposed
for the normal protein: modulation of signal path-
ways of importance for the survival of cells, protection
against oxidative stress and binding of copper. It was
recently reported that membrane-bound PrP repre-
sents the major cellular receptor for the oligomeric
beta-amyloid involved in Alzheimer’s disease [44].
Whether there is any significance to this possible con-
nection between mechanisms of development of
these two neurodegenerative diseases that both de-
pend on transmission of inappropriately folded pro-
teins remains to be seen. A number of recent studies
points towards the particular importance of the PrP
protein for long-term maintenance of neuronal func-
tions. One study involving four independently tar-
geted mouse strains depleted of PrP-C demonstrated
a role of the gene product for peripheral myelin main-
tenance [45]. Ablation of the protein triggered chronic
demyelinating neuropathy. Other recent results sug-
gest that the seminal role of normal PrP is to maintain
brain cells in good condition and protect them from
overexcitement.
The role of prion proteins in fungi differs markedly
from their potential significance for disease develop-
ment in animals, whereas in mammals, they may give
rise to transmissible neurodegenerative disease,
their function in fungi appears to be to produce heri-
table and sometimes beneficial phenotypes [46, 47].
Lindquist and colleagues conducted pioneering
studies of the potential benefit of prion proteins in
yeast and have provided solid evidence that they are
crucial for non-Mendelian inheritance in this spe-
cies [48–50]. Comprehensive studies of yeast have
clarified the central role of prion proteins for survival
ª 2011 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine 270; 1–14
under different conditions of stress. The results
obtained emphasize that biological information can
be conveyed not only by nucleic acids but also, sepa-
rately, by certain proteins that can initiate a self-
perpetuating spread of modified folding of homolo-
gous proteins. A broadening of our understanding of
the significance of prion-based epigenetic phenom-
ena clearly will have an influence on our interpreta-
tion of diseases in man and animals. The origin of
prion strains and, in relation to this, how prions repli-
cate and whether this replication can be mimicked
in vitro are important issues that remain to be
addressed.
Prion strains: their diversity and origin
The early studies of transmissible prion brain disease
in mice and hamsters revealed that agents of different
origin and ⁄ or passage history could cause disease
after different incubation times and with different
histopathologies [51]. Originally, this was interpreted
to mean that nucleic acids must play a role in prion
inheritance because it was believed at the time that
only this type of molecule could be the source of stably
inherited properties. However, the more the system
was analysed, the more it became clear that proteins
alone could be a source of diversity, the expression of
which to a considerable extent was dependent on the
environmental conditions. To date, studies of here-
ditable human prion diseases have demonstrated
correlations with more than 40 different mutations in
the PrP gene [see ref 42]. These genetic variants in-
clude single-nucleotide base changes, deletions and
occurrence of a varying number of segmental repeats.
The effects of many of these types of genetic changes
have now been mimicked by the use of transgenic
mice. It has been shown that prions exist as conform-
ationally diverse populations and that amongst these
there are different strains that can replicate with
independent reproducibility. Prion transformation
may occur by competition and selection [52]. Other
studies have focused on the effect of deletion of the
part of the PrP-TSE protein that is responsible for
anchoring to the cytoplasmic membrane [43]. The
soluble form of PrP-TSE can still cause disease, but
there is a major change in the incubation time and
in histopathological changes in the infected brain
[53, 54].
Further studies of the nature of prions
The propensity of certain proteins to form potentially
pathogenic aggregates can be examined currently by
four different approaches.
9
 E. Norrby
| Review: Prions and protein-folding diseases
1. Synthetic peptides exploring amino acid-depen-
dent conformational differences that determine the
emergence of polymorphic amyloid fibrils structur-
ally mimicking prion strains.
2. Performance of bioinformatic proteome-wide sur-
veys for prionogenic proteins in certain species.
3. Examination of the product(s) of replication of pri-
ons of different molecular characteristics in trans-
genic mice with a PrP gene construct of a preselected,
potentially different species origin (possibly a chi-
mera), with different molecular characteristics and
displaying varying levels of expression.
4. Treatment of prion inocula prior to inoculation by
different procedures to attempt to increase the infec-
tiousness of the preparation. This is referred to as in
vitro generation of prions, but it should be kept in
mind that the read-out of prion ‘replication’ is always
an in vivo system.
Synthetic peptides of limited length (<10 amino acids)
have been used to mimic aggregation phenomena giv-
ing rise to different forms of amyloid and prion strain
structures [55–57]. These short structures fibrillize
to form different kinds of tightly packed, highly com-
plementary beta-sheets. The term ‘steric zippers’ was
introduced to denote the critical parts of the model
molecules. The packing polymorphism observed can
provide an insight into the basis for prion strains and
for transfer of protein-encoded information.
To identify the diversity of prionogenic proteins in
yeast, a bioinformatic survey of the whole genome of
this organism was performed [48]. Some 100 candi-
date proteins were examined and 19 of these were
found to be capable of forming prions. The potential
physiological role of these different proteins remains
to be determined, but some have already been recog-
nized to have interesting functions. The self-perpetu-
ating characteristics of these proteins suggest that
they represent a vast source of heritable phenotypic
variation in yeast cells. This may facilitate survival of
yeast populations in different environments.
Prion replication in mammalian systems requires the
presence of both PrP-C and PrP-TSE. The latter serves
as a seeding nucleus or a template onto which the
physiological form of the protein is refolded into the
infectious conformation (see Fig. 3). To undergo con-
version, it is likely that PrP-C must develop an inter-
mediate state. In vivo, this is assumed to be achieved
by the assistance of additional as yet unidentified
10 ª 2011 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine 270; 1–14
proteins. Prusiner and collaborators referred to pro-
tein X as a cofactor. Many putative X proteins have
been identified, but transgenic knockouts for the
responsible genes have given equivocal results [58].
Experimentally induced increase in the infectious-
ness of a prion-containing material can be achieved
in vivo or in vitro. Many different experiments have
demonstrated that it is the characteristics of the
seeding nucleus or template that decides the nature
of the final product. The roles of the seeding nuclei
and templates have been examined in studies with
transgenic mice [42]. Certain kinds of seeding nuclei
or templates result in an increase in the titre of infec-
tious prions in the inoculum [59]. Dissociation an-
d ⁄ or denaturation treatments by sonication have
been used to increase the titre of infectious PrP in vi-
tro [60–64]. The infectiousness of the preparations,
which include both native and recombinant forms of
PrP-TSE, has been facilitated by addition of polya-
nions.
In further experiments, including denaturation by
guanidine hydrochloride at varying concentrations,
it was demonstrated that the conformational stability
of the prions (either native or synthetic) correlated
with the incubation period of disease [65–68]. Even
protease-sensitive forms of PrP have been found to be
capable of inducing disease [69].
In vitro replication of infectious PrP using a mixture in
which all reagents are defined and employing a cell
culture read out system as not yet been demon-
strated. Nevertheless, there is general agreement that
the successful generation of new infectious material
that has been achieved both in vivo and in vitro rules
out the possibility that prion replication is dependent
on information stored in nucleic acids.
A further complication to understanding the nature
of prions has been provided by the recent demon-
stration that infectious prions can arise spontane-
ously from normal brain [70]. The catalyst in this
study by Edgeworth et al. was steel wires, which had
been previously shown to effectively bind infectious
prions. Surprisingly, even noninfected mouse brain
homogenate attaching to the wires could induce a
prion infection in cell cultures, which was transmis-
sible to mice. This finding may emphasize that
the emergence of a single or a few misfolded PrP pro-
teins – not necessarily originating from mutational
changes in the PrP gene – might be enough to initiate
a cascade of misfolding events involving homologous
proteins.
 E. Norrby
| Review: Prions and protein-folding diseases
Reviewing the extensive and growing literature on pri-
ons and prionogenic proteins, one is struck by the
important and longstanding relative contributions of
Prusiner and his collaborators. It is often said that a
Nobel Prize may hamper later creative contributions
in science, but this does not appear to apply to
Prusiner.
Flow of information between proteins
The establishment around 1960 of the existence of
the ‘central dogma of genetics’ was an overwhelming
advance in our understanding of the process of infor-
mation storage and transfer in biology. Information
could be safely preserved in digital form and accu-
rately reproduced in the form of DNA. This informa-
tion was found to be transcribed with a high degree of
fidelity to RNA, which by interaction with the ribo-
somal machinery could transfer the linear message
into three-dimensional operative molecules, the pro-
teins. In other words, the hardware could build its
own software. Still, the more we learn the more we
understand that this system cannot answer all our
questions. The more refined sequencing of genomes,
not least our own, has revealed that there are more
nonprotein-coding units, possibly genes, than pro-
tein-coding genes. What is the functional diversity of
catalytic RNA and how has this evolved using a com-
bination of processes? And what about the plethora
of forms of regulatory RNA, do they use a different pat-
tern of signals in their use of potentially loop-forming
single-stranded RNA? Can there be a secondary
information-carrying system with its own code based
on different qualities of RNA loop formation?
In the next step of translation of RNA to protein, it is
readily accepted that this is a one-way process, but
what about the possibilities of flow of information be-
tween homologous or heterologous proteins? First, it
should be noted that many protein products have a
decisive influence on access to information stored in
Table 1 Prions and potential prio-
noids
Disease
Prion diseases
Alzheimer’s disease
Tauopathies
Parkinson’s disease
AA amyloidosis
Huntington’s disease
Modified from ref [71]
ª 2011 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine 270; 1–14
DNA; histone proteins control the availability of genes
for expression – there is even talk of a ‘histone code’ –
and this in turn is controlled via methylation or other
chemical modifications brought about by other sepa-
rate proteins. This arrangement allows for several
intertwined feed-back loops.
However as mentioned earlier, it has become increas-
ingly realized that there is an extensive flow of
information, or cross-talk, between proteins. Many
proteins do not have a firm three-dimensional form in
their native state, but represent a random coil; on
coming into contact with a specific part of another
protein or another chemical structure that they take
on a fixed three-dimensional structure. Others can,
under certain conditions, spontaneously move from
secondary to tertiary and even quaternary struc-
tures. Still, protein folding as a general phenomenon
has only been incompletely explained, and it is
known that in many cases assisting proteins, like the
chaperones, need to be present. It has been clearly
demonstrated that the same polypeptide chain may
take on very different conformations and that this
occurs under various environmental conditions, in
particular in the presence of homologous proteins
already folded into one form or another. Epigenetics,
i.e. the transfer of resilient genetic information not
stored in nucleic acid sequences, is a rapidly expand-
ing field and there is room for still more surprises from
the study of prions.
Inappropriate protein folding in human disease
Infectious prion diseases are rare, but the mecha-
nism of tissue destruction by aggregation of proteins
via their beta-pleated sheets seems to also apply to
many other diseases, some of which are common [71,
72]. Several examples are given in Table 1. One inter-
esting case is the beta-amyloid protein, which plays a
central role in Alzheimer’s disease. It has been shown
in experiments with transgenic mice that injection of
Molecular Infectious
Protein transmissibility life cycle
PrP-TSE Yes Yes
Amyloid-beta Yes Not shown
Tau Yes Not shown
Alpha-synuclein Host-to-graft Not shown
Amyloid A Yes Possible
Polyglutamine Yes Not shown
11
 E. Norrby
| Review: Prions and protein-folding diseases
this type of amyloid material can cause a brain degen-
erative disease with characteristics dependent on
both the inoculum and the host [73, 74]. Brain ex-
tracts from transgenic mice expressing mutant forms
of tau protein have been injected into brains of other
transgenic mice expressing human wild-type tau,
leading to development of aggregates of the human
tau [75]. Thus ‘tauopathies’ may be the result of a
prion-like process in which hyperphosphorylation of
the protein leads to polymerization and subsequently
produces filamentous protein aggregates. There is
also evidence for prion-like transmission of polyglu-
tamine protein aggregates, characteristic of Hunting-
ton’s disease [76]. Studies have shown that amyloid
protein A, the critical protein in secondary amyloido-
sis, injected into mouse brain can lead to degenera-
tive disease [77]. Additional studies of material from
patients with Parkinson’s disease have revealed that
the occurrence of inappropriate protein folding
can be transmitted from the cells of the host to
transplanted cells (see [78]). Also, diseases outside
the central nervous system can involve cells sub-
jected to degenerative processes induced by inappro-
priately folded proteins; one example of this is diabe-
tes type 2 [79]. Although these different diseases
appear to have their origin in self-sustained aggrega-
tion of prionoid proteins, it should be noted that there
is no evidence that they may be transmitted by an
infectious process.
To date, the focus in studies of mammalian prions
and prionogenic proteins has been on their potential
for development of disease. Whether this category of
proteins may also, as in the case of fungi, carry impor-
tant physiological functions remains to be deter-
mined. It was recently demonstrated that the cyto-
plasmic polyadenylation element binding protein can
form prion-like multimers in sensory neurons in the
nervous system of the giant marine snail Aplysia [80].
This modification has been proposed to serve a func-
tion in long-term memory. Thus, for readers who have
followed this review to the end, recollection of the sali-
ent facts and speculations presented - if stored for the
future - may be due to aggregation of prionogenic pro-
teins in the brain, provided of course that the funda-
mental long-term memory mechanisms of the human
brain are similar to those of Aplysia.
Acknowledgements
Bruce Chesebro, David Eisenberg, Susan Lindquist
and Stanley Prusiner provided valuable information
for this review. Paul Brown generously provided some
of the figures.
12 ª 2011 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine 270; 1–14
Conflict of interest statement
No conflict of interest was declared.
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Correspondence: Erling Norrby, Center for the History of Science,
The Royal Swedish Academy of Sciences, PO Box 50005, Stockholm
10405, Sweden.
(fax: +46 8 6739598; e-mail: erling.norrby@kva.se).