Prion review 2011

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doi: 10.1111/j.1365-2796.2011.02387.x
Prions and protein-folding diseases
E. Norrby
FromtheCenter for theHistoryofScience,RoyalSwedishAcademyofSciences,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 intodifferent 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-
degenerativediseases inhumansandanimals.These
diseasesaresometimes infectiousandhence referred
to as transmissible spongiform encephalopathies. In
the present review, the remarkable evolution of the
heterodoxprion 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,kuruandCreutzfeldt-Jakobdisease (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
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-
raryuseof aprion-contaminatedprotein-rich feed for
cows. The latter caused a major outbreak of bovine
spongiformencephalopathy,which spread tomanby
human consumption of contaminatedmeat, causing
approximately 200 cases of variant CJD. All these
epidemicsnowappeartobeoverbecauseofmeasures
takento curtail further spreadofprions.Recentstud-
ies have shown that themechanism of protein aggre-
gation may apply to a wider range of diseases in and
possiblyalsooutside thebrain, someofwhichare rel-
atively commonsuchasAlzheimer’s andParkinson’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 theestablishmentof long-termmemory.
Keywords: iatrogenicdisease,prions,protein folding.
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
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
Review |
In this review, our emerging understanding of the
mechanisms of prion diseaseswill first be discussed,
in particular their uniquemechanisms of spread will
be considered. The original belief in relatively firm
barriers preventing the spreadofprionsbetweenspe-
cieswasquestionedwhen it becameclear 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 fromman to
manbyblood transfusion.
Next, the possibility of a much wider application of
thepathogenicmechanismof cell destructionbypro-
tein aggregation, including degenerative processes
causing for exampleAlzheimer’sandParkinson’sdis-
eases,willbediscussed.The rapidlygrowingappreci-
ation of the significance of signalling between pro-
teinsoutside thecanonical stepsof thecentraldogma
ofmolecularbiologywill finallybeconsidered.
Funeral rites and infectious diseases
Unexpectedly, it was the fatal neurological disease
kuru, amongst the stone-age Fore people inNewGui-
nea, which provided the first insight into what came
to be called prion diseases. Studies of brain samples
collectedunderprimitive conditions from individuals
who had died of kuru provided a principal link be-
tween thepathogenesis of two, until then considered,
unrelated illnesses, scrapie in sheepandCJD inman
[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-
nizedanimals.
Creutzfeldt-Jakob disease was identified by twoGer-
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
formsof thedisease generallywithuniquepatternsof
symptoms.Approximately85%ofall casesofCJDare
sporadic, whereas about 10% are familial, leaving a
small minority of cases with an iatrogenic (environ-
mentallyacquired) 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
frompatientswithsporadicsCJD,but therewerealso
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 collaboratorsweresuccessful in transmittingfirst
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 thiskindof 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 deceasedrelative 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 ⁄CJDas slow
viruses, and this situation did not change until Prus-
inerbegan togain insights into thechemicalnatureof
theagentsusinghamster scrapieasamodel 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
2 ª 2011 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine 270; 1–14
nucleic acid in this preparation, he named it prion
(proteinaceous and infectious particle) [9]. The pro-
tein preparation – referred to as PrP 27–30 (prionpro-
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 informationmade it
possible to determine that the gene responsible for
the synthesis of the prionproteinwasnot foreign, but
a normal host cell gene [10]. Thus, it becamepossible
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 toourowntissues.
Following the identification of the PrP gene, it was
possible to produce mice deficient in PrP using
‘knockout’ technology [11]. At this time, itwasknown
that the PrP protein appears in the brain early during
embryonic development. It was found, unexpectedly,
thatdevelopmentand life spanappearedtobenormal
in animals without PrP protein. The PrP-knockout
mice could be used for two types of critical follow-up
experiments.
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 inpriondiseases.
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 deletionof 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-
genicprocess (asdiscussedbelow).The latterenabled
the evaluation of the species specificity of PrP pro-
teins. Mice are normally infected only by prions from
othermiceorrodents,suchashamsters,butnot from
more distant species. However, by generation of
transgenic mice carrying for example a human or a
bovinePrP gene, this species barrier can be overcome
providing important opportunities for studies of dif-
ferent kinds of pathogenic PrP proteins of relevance
forhumandisease.
Inappropriate folding and aggregation of proteins can cause disease
Diseases caused by protein aggregation have been
known fora long time.Theyarecollectively 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 theywere in fact composedof vari-
ous kinds of proteins. The amyloid deposits have
characteristic staining properties and show birefrin-
genceunderpolarized light.Proteinaggregatesshow-
ing these characteristics were demonstrated in the
brainsofpatientswithCJD.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
patientswithother formsof thedisease.
It then became possible to determine the structure of
purified PrP using nuclearmagnetic 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,
1000
800
600
400
200
N
um
be
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f d
ea
th
s Total
< 20 years
Males
5-years time periods
57– 62– 67– 72– 77– 82– 87– 92– 97– 02–
61 66 71 76 81 86 91 96 01 06
Fig. 1 The epidemiology of kuru amongst the Fore people,
which in 1960 had a total population of about 300 000 indi-
viduals.Thenumberof cases inyoung individuals rapidlyde-
clined after 1960, the year inwhich the practice of ritual can-
nibalism ceased. Themajority of victimswerewomen. Single
cases have appeared into the present century, implying an
incubation time exceeding 40 years. Figure kindly provided
byPaulBrown.
E. Norrby | Review: Prions and protein-folding diseases
ª 2011 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine 270; 1–14 3
whereas there is a predominance of beta-pleated
sheets in the pathological PrP-TSE protein (Fig. 2). It
is the PrP-TSEprotein thatmay formamyloid 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 theTSE formbyanas
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-generatingproteins.
Iatrogenic CJD
The awareness that CJD was a disease that could be
transmitted to experimental animals immediately
raisedthequestionof towhatextent itmightbe trans-
missible between humans. There was no epidemio-
logical evidence of connections between cases of
CJD, except for an increased frequencyof 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 casesmust
be because of inheritedmutations 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
emergenceofamisfoldedPrPprotein (thenucleation–
polymerizationmodel) during the lifetime of the indi-
vidual. As alreadymentioned, to date it has not been
possible to find evidence for transmissionof 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-
mentalblood 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, themajority of infectiousprionswere 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-
tionwith a corneal transplant [20], and later the simi-
lar spread to two relatively young individuals was
demonstratedasa result ofusing electrodes for intra-
cerebral recording [21].
Three epidemics of strikingly different origins have
beendocumented, andall 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-
dredsofpituitaryglands fromcadavers [see19for 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-
videdbyPaulBrown.
E. Norrby | Review: Prions and protein-folding diseases
4 ª 2011 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine 270; 1–14
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
and28 of 7700, in theUSA. In theUSA, an additional
step of purification was introduced in 1977, which
may have reduced the risk of transmission of infec-
tiousprions.
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 typeof graftwasbanned in theUK in1989and in
Japan in1997. In1987adisinfectionstepwithNaOH
was introduced, but eventually this was not consid-
eredsafe.
The alternatives used today are synthetic duramater
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 thepresentawareness of the situation, any
potential occupational risk of disease, for example,
for surgeons and nurses involved in brain surgical
procedures,can inpractical termsbeeliminated.
The main focus of interest during the last 15 years
has been the third epidemic, the unexpected spread
ofprions fromcattle toman.
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
casesofBSE (cowsdisplayingsymptomsofdiseaseor
with demonstrable PrP in their tissues) have so far
been identified.Themean incubation time isapproxi-
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
Fig. 3 Schematic model of conversion of PrP-C to PrP-TSE. In the nucleation-polymerizationmodel, there is a rapid conversion of
PrP proteinbetween thePrP-C (circles) andPrP-TSE forms (squares), but the former ismore stable. In the presenceof anaggregate
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-assistancemodel, theconversionofPrP-Coramodifiedconformation,PrP-INT (intermediate), toPrp-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 ispotentially responsible for thediseaseprocess.Modified fromref.17.
E. Norrby | Review: Prions and protein-folding diseases
ª 2011 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine 270; 1–14 5
chickens. Such amixed food had already been in use
forsome time,but in the late1970s,apreviouslyused
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
sourceofspreadofprionsofeitherbovineorovineori-
gin. As soon as this was recognized in 1988, its use
was forbidden. Soon after, a test to detect PrP in tis-
sues of animals was successfully developed, but
unfortunatelywasnotapplicable toblood. In farms in
which an infected animal was detected, the entire
herdwasculled.
In 1989 mandatory changes in slaughtering tech-
niqueswere introduced. These changes ensured that
thebrainandspinal cord, themainsourcesofprions,
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 fromcattle directly toman, as therehadnever
been any evidence that the scrapie agent could
spread fromsheep toman. Inprinciple, thesamespe-
cies barrier that had prevented such a spread for
hundreds of yearswas expected to exist also between
cowsandman.However, in1994 thefirst caseofCJD
ofbovineoriginwas identified inman [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 laterwas 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 probablynon-human, origin. The new form
ofCJDwas 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 fromabrain extract or bydirect con-
tact between brainmaterials in neurosurgical proce-
dures, the spread in the case of vCJDwas oral. It was
known from previous studies of prions from cases of
28
24
20
16
12
8
4
0
28
24
20
16
12
8
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0
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24
20
16
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1953 19631967198919911993 1995 19971999 2001 20032005 20072009
vCJD – prion contaminated meat
Injection of growth hormone from pituitary glands
Transplant of dura mater grafts
Fig. 4 Comparison of threeCreutzfeldt-Jakobdisease (CJD)
epidemics.Eachof theepidemics involvedabout200patientsandwascausedbydifferenthuman interventions: iatrogenic
CJD by intra-muscular injection of pituitary gland-derived
growth hormone; iatrogenic CJD as a result of dura mater
transplantation;andvariantCJDbecauseof ingestionof con-
taminatedmeat. Theaverage incubation time in the three epi-
demicswas15,11andabout11–12 years, respectively.
BSE [0–40 000]
vCJD blood transmission [0–40]
vCJD undefined route [0–40]
40 000
35 000
30 000
25 000
20 000
15 000
10 000
5000
0
<19881989 1991 1993 1995 1997 1999 2001 2003 2005 2007
40
35
30
25
20
15
10
5
0
Fig. 5 The epizootic of bovine spongiform encephalopathy
(bluebars), theepidemicof variantCreutzfeldt-Jakobdisease
(vCJD) in humans (red bars) and three cases of transmission
of vCJDbetweenhumansbyblood transfusion (yellowbars).
E. Norrby | Review: Prions and protein-folding diseases
6 ª 2011 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine 270; 1–14
sCJDthat feedingof experimentalanimalsbytheoral
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, theepidemicpeaked in theyear2000andnow
appears tohavecometoanend (Figs4and5).Todate,
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 thepeaksof
the BSE epizootic and the vCJD epidemic is 9 years.
However, it should be noted that slaughtering tech-
niques toexcludethemostcontaminatedpartsofcat-
tle were introduced by 1989, suggesting that true
incubation times exceed an average of 11–12 years.
Noonebornafter1989hascontracted thedisease.
As alreadymentioned,mutational changes in thePrP
protein can influence its propensity to fold incor-
rectly. In the case of vCJD, it has been found that the
aminoacid inposition129 in theproteinhasacritical
role for the development of thedisease.Depending on
the nucleotides in this position, it can specify either
valine or methionine, but all cases of clinical vCJD
identified to date have hadmethionine 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
willprobablybe fewer than inthe recentepidemic.
High-tech ‘cannibalism’
Essentially cannibalism is not practised in modern
society; however, there are an increasing number of
situations inwhich body fluids, tissues or organs are
transferred between individuals. Blood transfusions
havebeenperformedsince a long time, but theproce-
duredidnotbecomesafeuntil thediscoveryof thehu-
man blood groups in the beginning of the 1900s.
Since the beginning of the 1960s, transplantation of
solidorgansandbonemarrowhasbecomeacommon
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
vCJDcouldbespreadbyblood transfusioncameasa
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
detail in Fig. 6. Case one was a 69-year-oldman who
became ill 7 years after receiving blood from a 24-
year-old donor who himself started to show symp-
toms of vCJD3 years after he had donated the blood.
Thisfirst identified caseprompteda reviewof individ-
ualswhohad received blood fromdonorswhohad la-
ter developed vCJD.The second case of proven trans-
missionwas a 77-year-old patient, who died because
ofa rupturedaorticaneurysm5 yearsafterhehadre-
ceivedblood fromayoungerdonorwho18 months la-
ter developed vCJD. The recipient of the bloodhadno
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 took7.5and8.5 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
alreadybeen 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 finda reliable test have failed to
date. Nevertheless, other precautions have been
taken. Primarily the selection of donors became
96 97 98 99 00 01 02 03 04 05 06
Case # 1
Case # 2
Case # 3
Case # 4
3 1/3 years
6 1/2 years
1 1/2 years
1 2/3 years
1 1/3 years
8 1/2 years
7 1/2 years
5 years
Fig. 6 Four cases of variant Creutzfeldt-Jakob disease
(vCJD) prion infection caused by blood transfusion. Cases 3
and4were infectedby transfusionofblood fromthesamedo-
nor. Ineachcase, theupperbarshowsthe timeuntil thedonor
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.
E. Norrby | Review: Prions and protein-folding diseases
ª 2011 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine 270; 1–14 7
stricter to try toexclude individualswhomightpoten-
tially have an increased risk of carrying vCJD prions.
Furthermore, it had been reported that mice and
hamsters infected with scrapie prions had low titres
outside the nervous system and that these prions
preferentially occurred in white blood cells [27]. It
was therefore decided, first in the UK in the late
1990s and then in other European countries, that
white blood cells should be removed from blood to be
used for transfusion. From animal experiments, it
was deduced that this would reduce, but not elimi-
nate, the amount of possibly contaminating prions
[28]. Since the introduction of this precautionary
measure, no further cases of vCJD caused by blood
transfusion have been seen. Additionally, because
the number of cases of vCJD has progressively de-
creasedsince2000, the risks for thespreadof thedis-
ease between humans by blood should by now have
beeneliminated.
The risk of spread of vCJD prions by blood also ap-
plies to products that are prepared using blood as a
source. The history of the spread of virus infections
by products containing components of blood is long.
HepatitisBviruswas transmitted to300 000soldiers
duringWorldWar II bya yellow fever vaccine contain-
ing live virus stabilized by human albumin. Much la-
ter, it was demonstrated that the albumin used had
been contaminated by hepatitis B virus. During the
mid 1980s, a large number of haemophilia patients
became infected with human immunodeficiency
virus (HIV) when treated with Factor VIII or Factor IX
preparations. Not until techniques to screen for the
presence of a virus, or antibodies against it, in blood
had been developed, could safe preparations be
developed. Increased awareness of the importance of
excluding contaminationbyviruses frombloodprod-
ucts led to the development of general methods of
elimination.Treatment with organic solvents was
introduced to eliminate enveloped viruses and other
procedures, including the more recently introduced
nanofiltration, have led to a high degree of efficacy in
preventing the spread of conventional viruses by
blood-derivedproducts forhumanuse.However, pri-
ons representedadifferent challenge because of their
unique stability against physical ⁄chemical treat-
ments. The particular resistance of this kind of
infectious agent was observed at an early stage and
stimulated speculation on the possible means of
replicationwithoutnucleicacid [29,30].
The unexpected threat, and later demonstration, of
transmission of vCJD prions by blood transfusions
caused a major review of the possible means of
excluding such infectious agents from blood and
blood products. First and foremost, it is important to
decide fromthehistoryof thedonors that the risk that
they carry vCJD prions is minimized. In the absence
of any effective technique to detect PrP-TSE in blood,
a number of experimental studies using materials
‘spiked’ with prions from rodents have been used to
developgeneralmethods toreduce thecontentofpos-
sibly contaminating prion proteins. The safety of the
products used has beenmarkedly improved [31–35],
but it is impossible to be absolutely certain that there
is no risk of transmission. Still it should be kept in
mind that the recent use of different coagulation fac-
tors in man has an impressive track record. A com-
prehensive review of approximately 20 000 patients
with haemophilia treated with concentrates of differ-
ent factorshasnot revealedasinglecaseofvCJD [36].
The only exception is aBritishmanwithhaemophilia
in whom a transfer of vCJD prions might have oc-
curred (personal information by Tor-Einar Svae, Oc-
tapharma, Vienna). This patient was treated during
the 1990s with a concentrate of Factor VIII produced
in the UK. It was found that material from a donor
who later developed vCJD was included in two
batches, and it was subsequently shown that the
technique of purification did not exclude vCJD pri-
ons. This purificationmethod has not been in use for
manyyears.
The physiological role of the prion protein
Once itbecameclear that thepresenceof thePrPgene
was absolutely essential to the development of PrP-
TSE-derived diseases and that animals without PrP,
unexpectedly, seemed to develop normally, it was
important to determine the physiological role of the
PrP protein. However, despite many studies, its fun-
damental function(s) still remains to be definitively
identified. Different studies have highlighted a wide
rangeofdifferent functions [37–42].
The chromosomal gene denoted Prnp encodes PrP. It
is a member of the Prn gene family, which also genes
encoding two other proteins. The PrP open reading
frame is encoded within a single exon directing the
synthesis of aproteinwith254aminoacids. Thispro-
tein is post-translationally modified by removal of a
22-amino acid, amino terminal signal peptide and a
23-amino acid carboxy terminal. The latter directs
the addition of a glycosylphosphatidyl inositol mem-
brane anchor. Under normal conditions, PrP is a
membrane-bound protein, but it can also show bio-
logical activity and cause infectious amyloid disease
in a nonmembrane-bound form [43]. In addition, the
E. Norrby | Review: Prions and protein-folding diseases
8 ª 2011 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine 270; 1–14
proteinhas twoglycosylation sites andan 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 67amino acids from theami-
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
throughevolution [42].However, there arealsodiffer-
ences, which explain the species barrier to disease
transmissionmentionedearlier.
A number of different functions have been proposed
for the normal protein: modulation of signal path-
waysof importance for thesurvival 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 isanysignificance to thispossible 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-termmaintenance of neuronal func-
tions. One study involving four independently tar-
getedmouse strains depleted of PrP-C demonstrated
a role of the gene product for peripheralmyelinmain-
tenance [45]. Ablationof theprotein triggeredchronic
demyelinating neuropathy. Other recent results sug-
gest that the seminal role ofnormal PrP is tomaintain
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 inanimals,whereas inmammals, theymaygive
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
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
prionstrainsand, in relation to this, howprionsrepli-
cate and whether this replication can be mimicked
in vitro are important issues that remain to be
addressed.
Prion strains: their diversity and origin
Theearly studiesof transmissibleprionbraindisease
inmiceandhamsters revealed thatagentsofdifferent
origin and ⁄or passage history could cause disease
after different incubation times and with different
histopathologies [51].Originally, thiswas interpreted
to mean that nucleic acids must play a role in prion
inheritance because it was believed at the time that
only this typeofmoleculecouldbethesourceofstably
inherited properties. However, the more the system
was analysed, themore 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
correlationswithmore than40differentmutations in
the PrP gene [see ref 42]. These genetic variants in-
clude single-nucleotide base changes, deletions and
occurrenceofavaryingnumberofsegmental repeats.
The effects of many of these types of genetic changes
have now been mimicked by the use of transgenic
mice. It has beenshown that prions exist as conform-ationally diversepopulations 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
fourdifferentapproaches.
E. Norrby | Review: Prions and protein-folding diseases
ª 2011 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine 270; 1–14 9
1. Synthetic peptides exploring amino acid-depen-
dent conformational differences that determine the
emergence of polymorphic amyloid fibrils structur-
allymimickingprionstrains.
2. Performance of bioinformatic proteome-wide sur-
veys forprionogenicproteins incertainspecies.
3.Examination of the product(s) of replication of pri-
ons of different molecular characteristics in trans-
genicmicewith a PrP gene construct of a preselected,
potentially different species origin (possibly a chi-
mera), with different molecular characteristics and
displayingvarying levelsofexpression.
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 vivosystem.
Syntheticpeptidesof limited length (<10aminoacids)
havebeenused tomimicaggregationphenomenagiv-
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 transferofprotein-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. Thismay facilitate survival of
yeastpopulations indifferent environments.
Prion replication inmammalian systems requires the
presenceofbothPrP-CandPrP-TSE.The latterserves
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
proteins. Prusiner and collaborators referred to pro-
tein X as a cofactor. Many putative X proteins have
been identified, but transgenic knockouts for the
responsiblegeneshavegivenequivocal 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,
itwasdemonstrated that theconformational stability
of the prions (either native or synthetic) correlated
with the incubation period of disease [65–68]. Even
protease-sensitive formsof PrPhavebeen found tobe
capableof inducingdisease [69].
In vitro replicationof infectiousPrPusingamixture in
which all reagents are defined and employing a cell
culture read out system as not yet been demon-
strated.Nevertheless, there isgeneral 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 informationstored innucleicacids.
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
10 ª 2011 The Association for the Publication of the Journal of Internal Medicine Journal of InternalMedicine 270; 1–14
Reviewing theextensiveandgrowing literatureonpri-
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-
tionwas found tobe transcribedwith ahighdegree 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 formsof regulatoryRNA,dotheyuseadifferentpat-
tern of signals in their use of potentially loop-forming
single-stranded RNA? Can there be a secondary
information-carrying systemwith its own codebased
ondifferentqualitiesofRNA loopformation?
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 toinformation stored in
DNA;histoneproteinscontrol theavailabilityofgenes
for expression – there is even talk of a ‘histone code’ –
and this in turn is controlled viamethylation or other
chemicalmodifications brought about by other sepa-
rate proteins. This arrangement allows for several
intertwined feed-back loops.
However asmentionedearlier, it has become increas-
ingly realized that there is an extensive flow of
information, or cross-talk, between proteins. Many
proteinsdonothaveafirm 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 inmany 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 innucleic acid sequences, is a rapidly expand-
ingfieldandthere isroomforstillmoresurprises from
thestudyofprions.
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
manyother diseases, someofwhichare common [71,
72]. Several examples are given in Table 1.One inter-
esting case is thebeta-amyloid protein,whichplays a
central role inAlzheimer’s disease. It has been shown
in experiments with transgenic mice that injection of
Table 1 Prions and potential prio-
noids
Disease Protein
Molecular
transmissibility
Infectious
life cycle
Priondiseases PrP-TSE Yes Yes
Alzheimer’sdisease Amyloid-beta Yes Not shown
Tauopathies Tau Yes Not shown
Parkinson’sdisease Alpha-synuclein Host-to-graft Not shown
AAamyloidosis AmyloidA Yes Possible
Huntington’sdisease Polyglutamine Yes Not shown
Modified fromref [71]
E. Norrby | Review: Prions and protein-folding diseases
ª 2011 The Association for the Publication of the Journal of Internal Medicine Journal of InternalMedicine 270; 1–14 11
this typeofamyloidmaterial cancauseabraindegen-
erative disease with characteristics dependent on
both the inoculum and the host [73, 74]. Brain ex-
tracts from transgenicmice expressingmutant 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
theprotein leads to polymerizationandsubsequently
produces filamentous protein aggregates. There is
also evidence for prion-like transmission of polyglu-
tamineproteinaggregates, characteristic ofHunting-
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-
tionofprionoidproteins, it shouldbenoted that there
is no evidence that they may be transmitted by an
infectiousprocess.
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
proteinsmayalso,as inthecaseof fungi, carry impor-
tant physiological functions remains to be deter-
mined. It was recently demonstrated that the cyto-
plasmicpolyadenylationelementbindingprotein can
form prion-like multimers in sensory neurons in the
nervous systemof thegiantmarine snailAplysia [80].
Thismodification has been proposed to serve a func-
tion in long-termmemory.Thus, for readerswhohave
followed this review to theend, recollectionof thesali-
ent factsandspeculationspresented - if stored for the
future -maybedue toaggregationofprionogenicpro-
teins in the brain, provided of course that the funda-
mental long-termmemorymechanismsof thehuman
brainaresimilar to thoseofAplysia.
Acknowledgements
Bruce Chesebro, David Eisenberg, Susan Lindquist
and Stanley Prusiner provided valuable information
for this review.PaulBrowngenerouslyprovidedsome
of thefigures.
Conflict of interest statement
Noconflict of interestwasdeclared.
References
1 Gajdusek DC, Zigas V. Degenerative disease of the central
nervous system in New Guinea: the endemic occurrence
of ‘‘kuru’’ in thenativepopulation.NEnglJMed1957;257:974–
8.
2 HadlowWJ.Scrapieandkuru.Lancet1959;2:289–90.
3 Gajdusek DC, Gibbs CJ Jr, Alpers M. Experimental transmis-
sionofakuru-likesyndrometochimpanzees.Nature1966;209:
794–6.
4 Gibbs CJ Jr, Gajdusek DC, Asher DM et al. Creutzfeldt-Jakob
disease (spongiformencephalopathy): transmission to thechim-
panzee.Science1968;161:388–9.
5 Glasse RM. Cannibalism in the kuru region of New Guinea.
TransNYAcadSci1967;29:748–54.
6 Collinge J, Whitfield J, McKintosh E et al. Kuru in the 21st cen-
tury – an acquired human prion disease with very long incuba-
tionperiods.Lancet2006;367:2068–74.
7 Dorsey KA, Zou S, Schonberger LB et al. Lack of evidence of
transfusion transmission of Creutzfeldt-Jakob disease in a US
surveillancestudy.Transfusion2009;49:977–84.
8 Prusiner SB, Groth DF, Bolton DC, Kent SB, Hood LE. Purifica-
tion and structural studies of amajor scrapie prion protein.Cell
1984;38:127–34.
9 Prusiner SB. Prions. In: Fra¨ngsmyr T, ed. Nobel Lecture. Stock-
holm:Almqvist&Wiksell International,1997;268–323.
10 Oesch B, Westaway D, Wa¨lchli M et al. A cellular gene encodes
scrapiePrP27-30protein.Cell1986;46:417–28.
11 Bu¨eler H, Fischer M, Lang Y et al. Normal development and
behaviour of mice lacking the neuronal cell-surface PrP protein.
Nature1992;356:577–82.
12 Bu¨elerH,Aguzzi A, SailerA et al.Micedevoid of PrP are resistant
toscrapie.Cell1993;73:1339–47.
13 Prusiner SB, Groth D, Serban A et al. Ablation of the prion pro-
tein (PrP) gene in mice prevents scrapie and facilitates produc-
tion of anti-PrP antibodies. Proc Natl Acad Sci USA 1993; 90:
10608–12.
14 Wille H, Prusiner SB, Cohen FE. Scrapie infectivity is indepen-
dentofamyloidstainingpropertiesof theN-terminally truncated
prionprotein.JStructBiol2000;130:323–38.
15 Donne DG, Viles JH, Groth D et al. Structure of the
recombinant full-length hamster protein PrP (29-231): the N
terminus is highly flexible. Proc Natl Acad Sci USA 1997; 94:
7279–82.
16 Riek R, Hornemann S, Wider G, Glockshuber R, Wu¨thrich
K. NMR characterization of the full-length recombinant
murine prion protein, mPrP (23-231). FEBS Lett 1997; 413:
282–8.
17 HorwichAL,WeissmanJS.Deadly conformations – Proteinmis-
folding inpriondisease.Cell1997;89:499–510.
18 Houston F, McCutcheon S, Goldmnann W et al. Prion diseases
are effectively transmitted by blood transfusion in sheep. Blood
2008;112:4739–45.
19 BrownP.Creutzfeldt-Jakobdisease: reflections on the risk from
bloodproduct therapy.Haemophilia2007;13:33–40.
20 Duffy P,Wolf J,CollinsG,DeVoeAG,SteetenB,CowenD.Possi-ble person to person transmission of Creutzfeldt-Jakobdisease.
NEnglJMed1974;299:692–3.
E. Norrby | Review: Prions and protein-folding diseases
12 ª 2011 The Association for the Publication of the Journal of Internal Medicine Journal of InternalMedicine 270; 1–14
21 Will RG, Mathews WB. Evidence for case-to-case transmission
of Creutzfeldt-Jakob disease. J Neurol Neurosurg Psychiatry
1982;45:235–8.
22 Will RG, Ironside JW, Zeidler M et al. A new variant of Creutz-
feldt-Jakobdisease intheUK.Lancet1996;347:921–5.
23 The National Creutzfeldt-Jakob Disease Surveillance Unit
(NCJDSU), University of Edinburgh, UK, Available at: http://
www.cjd.ed.ac.uk,AccessedNovember30,2010.
24 KaskiD,MeadS,HyareHetal.VariantCJD inan individualhet-
erozygous forPRNPcodon129.Lancet2009;374:2128.
25 LlewelynCA,HewittRE,KnightRSGetal.Possible transmission
of variant Creutzfeldt-Jakob disease by blood transfusion. Lan-
cet2004;363:417–21.
26 Peden AH, HeadMW, Ritchie DL, Bell JE, Ironside JW. Preclini-
cal vCJDafter blood transfusion in a PRNP codon 129 heterozy-
gouspatient.Lancet2004;364:527–9.
27 Kuroda Y, Gibbs CJ Jr, Amyx HL, Gajdusek DC. Creutzfeldt-
Jakob disease inmice: persistent viremia and preferential repli-
cationof virus in low-density lymphocytes. Infect Immunol1983;
41:154–61.
28 Gregori L,McCombie DP, PalmerD, Sowemimo-Coker AO, Giu-
livi A, RohwerRG. Effectiveness of leucoreduction for removal of
infectivity of transmissible spongiform encephalopathies from
blood.Lancet2004;264:529–31.
29 Alper T,CrampWA,HaigDA,ClarkeMC.Does theagent of scra-
pie replicatewithoutnucleicacid?Nature1967;214:764–6.
30 Griffith JS. Self-replication and scrapie. Nature 1967; 215:
1043–4.
31 Poelser G, Berting A, Kindermann J et al. A new liquid intrave-
nous immunoglobulin with three dedicated virus reduction
steps: virus and prion reduction capacity. Vox Sang 2008; 94:
184–92.
32 Svae T-E, Neisser-Svae A, Bailey A et al. Prion safety of transfu-
sion plasma and plasma-derivatives typically used for prophy-
lactic treatment.TransfApheresSci2008;39:59–67.
33 Gregori L, Lambert B, Gurgel P et al.Reduction of transmissible
spongiform encephalopathy infectivity from human red blood
cells with prion protein affinity ligands. Transfusion 2009; 46:
1152–61.
34 Neisser-SvaeA,Bailey A,Gregori L et al.Prion removal effect of a
specific affinity ligand introduced into the manufacturing pro-
cess of thepharmaceutical quality solvent ⁄ detergent(S ⁄D)-trea-
tedplasmaOctaplasLG.VoxSang2009;97:226–33.
35 Heger A, Svae T-E, Neisser-Svae A, Jordan S, Behizad M, Ro¨-
misch J. Biochemical quality of the pharmaceutically licensed
plasma OctaplasLG after implementation of a novel prion pro-
tein (PrPsc) removal technology and reduction of the sol-
vent ⁄ detergent (S ⁄D)process time.VoxSang2009;97:219–25.
36 Health Protection Agency. Variant CJD and plasma products,
2009.
37 Aguzzi A, Polymenidou M. Mammalian prion biology: one cen-
turyof evolvingconcepts.Cell2004;116:313–27.
38 Aguzzi A, Calella AM. Prions: Protein aggregation and infectious
disease.PhysiolRev2009;89:1105–52.
39 CaugheyB,BaronGS.Prionsandtheirpartners incrime.Nature
2006;443:803–10.
40 Ramijak S, Asif AR, ArmstrongVW et al. Physiological role of the
cellular prion protein (PrP-C): protein profiling study in two cell
culturesystems.JProteomeRes2008;7:2681–95.
41 Linden R, Martins VR, Prado MA, Cammarota M, Izquierdo I,
Brentani RR. Physiology of the prion protein. Physiol Rev 2008;
88:673–728.
42 Colby DW, Prusiner SB. Prions. In: Morimoto R, Kelly J, Selkoe
D, ed. Additional Perspectives on Protein Homeostasis. Cold
SpringHarbPerspectBioldoi: 10.1101/cshperspect.a006833.
43 Chesebro B, Trifilo M, Race R et al. Anchorless prion protein
results in infectious amyloid disease without clinical scrapie.
Science2005;104:1435–9.
44 Laure´n J, Gimbel DA, NygaardHB, Gilbert JW, Strittmatter Jw.
Cellular prion protein mediates impairment of synaptic plastic-
itybyamyloid-betaoligomers.Nature2009;457:1128–33.
45 Bremer J, Baumann F, Tiberi C et al. Axonal prion protein is
required for peripheralmyelinmaintenance.NatNeurosci2010;
13:310–18.doi:10.1038/nn.2483.
46 Chien P,WeissmanJS,DePaceAH.Emerging principles of con-
formation-based prion inheritance. Annu Rev Biochem 2004;
73:617–56.
47 Ross ED, Minton A, Wickner RB. Prion domains: sequences,
structuresand interactions.NatCellBiol2005;7:1039–44.
48 Alberti S, Halfmann R, King O, Kapila A, Lindquist S. A system-
atic survey identifies prions and illuminates sequence features
ofprionogenicproteins.Cell2009;137:146–58.
49 HalfmannR,AlbertiS,LindquistS.Prions,proteinhomeostasis,
andphenotypicdiversity.TrendsCellBiol2010;20:125–34.
50 HalfmannR,LindquistS.Epigenetics in theextreme:prionsand
the inheritance of environmentally acquired traits. Science
2010;330:629–32.
51 Fraser H, Dickinson AG. Scrapie in mice. Agent-strain differ-
ences in the distribution and intensity of grey matter vacuola-
tion.JCompPathol1973;83:29–40.
52 Li J, Browning S, Mahal SP, Oelschlegel AM,Weissman C. Dar-
winian evolution of prions in cell culture. Science 2010; 327:
869–72.
53 Chesebro B, Race B, Meade-White K et al. Fatal transmissible
amyloid encephalopathy: a new type of prion disease associated
with lack of prion membrane anchoring. PLoS Pathog 2010; 6:
e1000800.
54 Klingeborn M, Race B, Meade-White KD, Rosenke R, Strie-
bel JF, Chesebro B Crucial role for prion protein membrane
anchoring in the neuroinvasion and neural spread of
prion infection. J Virol 2011; 85: 1484–94. PubMed PMID:
21123371.
55 Nelson R, Sawaya MR, Balbirnie M et al. Structure of the cross-
betaspineofamyloid-likefibrils.Nature2005;435:773–8.
56 Sawaya MR, Sambashivan S, Nelson R et al. Atomic structures
of amyloid cross-beta spines reveal varied steric zippers.Nature
2007;447:453–7.
57 Wiltzius JJ, Landau M, Nelson R et al. Molecular mechanisms
of protein-encoded inheritance. Nat Struct Mol Biol 2009; 16:
973–8.
58 Tamgu¨ney G, Giles K, Bouzamondo-Bernstein E et al. Genes
contributing to prion pathogenesis. J Gen Virol 2008; 89: 1777–
88.
59 Sigurdson CJ, Nilsson KP, Hornemann S et al.De novo genera-
tion of a transmissible spongiform encephalopathy by mouse
transgenesis.ProcNatlAcadSci2009;106:304–9.
60 Saborio GP, Permanne B, Soto C. Sensitive detection of patho-
logicalprionproteinbycyclicamplificationofproteinmisfolding.
Nature2001;411:810–3.
61 Castilla J, Saa´ P,HetzC, Soto C. In vitro generation of infectious
scrapieprions.Cell2005;121:136–44.
62 Deleault NR, Harris BT, Rees JR, Supattapone S. Formation of
native prions fromminimal components in vitro. Proc Natl Acad
SciUSA2007;104:9741–6.
E. Norrby | Review: Prions and protein-folding diseases
ª 2011 The Association for the Publication of the Journal of Internal Medicine Journal of InternalMedicine 270; 1–14 13
63 Barria MA, Mukherjee A, Gonzales-Romero D et al. De novo
generation of infectious prions in vitro produces a new disease
phenotype.PLoSPathog2009;5:e1000421.
64 WangF,WangX, YuanCG,MaJ.Generating a prionwith a bac-
terially expressed recombinant prion protein. Science 2010;
327:1132–5.
65 KanekoK,WilleH,Mehlhorn Ietal.Molecularproperties of com-
plexes formedbetween theprionproteinand synthetic peptides.
JMolBiol1997;270:574–86.
66 LegnameG, Baskakkov IV, Nguyen H-OB et al. Synthetic mam-
malianprions.Science2004;305:673–6.
67 Legname G, Nguyen H-OB, Peretz D et al. Continuum of prion
protein structures enciphers a multitude of prion isolate-spe-
cificphenotypes.ProcNatlAcadSci2006;103:19105–10.
68 ColbyDW, Giles K, LegnameG et al.Design and construction of
diversemammalian prion strains.ProcNatl AcadSci2009;106:
20417–22.
69 Colby DW, Wain R, Baskakov IV et al. Protease-sensitive syn-
theticprions.PLoSPathog2010;6:e1000736.
70 Edgeworth JA,Gros N, Alden J et al. Spontaneous generation of
mammalian prions. Proc Natl Acad Sci 2010; 107: 14402–6.
DOI:10.1073/pnas.1004036107.
71 Aguzzi A, Baumann F, Bremer J. The prion’s elusive reason for
being.AnnuRevNeurosci2008;31:439–77.
72 AguzziA.Beyondtheprionprinciple.Nature2009;459:924–5.
73 Meyer-LuehmannM,CoomaraswamyJ,BolmontT et al.Exoge-
nous induction of cerebral beta-amyloidogenesis is governed by
agentandhost.Science2006;313:1781–4.
74 Eisele YS,BolmontT,HeikenwalderM et al. Indictionof cerebral
beta-amyloidosis: intracerebral versussystemicA-beta inocula-
tion.ProcNatlAcadSci2009;106:12926–31.
75 Clavaguera F, Bolmont T, Crowther RA et al. Transmission and
spreading of tauopathy in transgenic mouse brain.Nat Cell Biol
2009;11:909–13.
76 RenPH, Lauckner JE, Kachirskaia I,Heuser JE,MelkiR,Kopito
RR. Cytoplasmic penetration and persistent infection of mam-
malian cells by polyglutamine aggregates. Nat Cell Biol 2009;
11:209–25.
77 Lundmark K, Westermark GT, Olsen A, Westermark P. Protein
fibrils in nature can enhance amyloid protein a amyloidosis in
mice: cross-seeding as a diseasemechanism. Proc Natl Acad Sci
USA2005;102:6098–102.
78 Olanow CW, Prusiner SB. Is Parkinson’s disease a prion disor-
der?ProcNatlAcadSci2009;106:12571–2.
79 Ho¨ppener JW, AhrenB, LipsCJ. Islet amyloid and type 2 diabe-
tesmellitus.NEnglJMed2000;343:411–9.
80 Si K, Choi YB, White-Grindley E, Majumdar A, Kandel ER.
Aplysia CFEB can form prion-like multimers in sensory
neurons that contribute to long-tem facilitation.Cell2010;140:
421–35.
Correspondence: Erling Norrby, Center for the History of Science,
The Royal Swedish Academy of Sciences, POBox 50005, Stockholm
10405,Sweden.
(fax:+4686739598;e-mail: erling.norrby@kva.se).
E. Norrby | Review: Prions and protein-folding diseases
14 ª 2011 The Association for the Publication of the Journal of Internal Medicine Journal of InternalMedicine 270; 1–14