doi:10.1093/brain/awl013
Brain (2006), 129, 676–685
Distinct glycoform ratios of protease resistant
prion protein associated with PRNP point
mutations
Andrew F. Hill,1 Susan Joiner, Jonathan A. Beck, Tracy A. Campbell, Andrew Dickinson,
Mark Poulter, Jonathan D. F. Wadsworth and John Collinge
MRC Prion Unit and Department of Neurodegenerative Disease, Institute of Neurology, University College
London, National Hospital for Neurology and Neurosurgery, Queen Square, London, UK
1
Present addresses: Department of Biochemistry & Molecular Biology, Bio21 Molecular Science and Biotechnology
Institute and Department of Pathology, University of Melbourne, Parkville, Victoria 3010, Australia
Correspondence to: Professor John Collinge, MRC Prion Unit and Department of Neurodegenerative Disease,
Institute of Neurology, University College London, National Hospital for Neurology and Neurosurgery,
Queen Square, London WC1N 3BG, UK
E-mail: [email protected]
Inherited prion diseases are neurodegenerative disorders caused by autosomal dominant mutations in the
human prion protein gene (PRNP). Kindred with inherited prion disease can show remarkable phenotypic
variability that has yet to be explained. Here we report analysis of protease resistant disease-related prion
protein (PrPSc) isoforms from a range of inherited prion disease cases (point mutations P102L, D178N, E200K
and 2-, 4- and 6-octapeptide repeat insertions) and show that the glycoform ratios of PrPSc associated with PRNP
point mutations are distinct from those observed in sporadic, iatrogenic and variant Creutzfeldt–Jakob disease.
Patients with the same PRNP mutation can also propagate PrPSc with distinct conformations. These data
extend the spectrum of recognized PrPSc types seen in human prion diseases and provide further insight
into the generation of diverse clinicopathological phenotypes associated with inherited prion disease.
Keywords: Creutzfeldt-Jakob disease; prion disease; prion protein; fatal familial insomnia
Abbreviations: CJD = Creutzfeldt–Jakob disease; FFI = fatal familial insomnia; GSS = Gerstmann–Straüssler–Scheinker
syndrome; MHs = methionine homozygotes; vCJD = variant CJD
Received September 5, 2005. Revised November 27, 2005. Accepted December 22, 2005. Advance Access publication January 16, 2006
Introduction
Human prion diseases are fatal, transmissible neurodegenerative disorders that include Creutzfeldt–Jakob disease (CJD),
variant CJD (vCJD), Gerstmann–Sträussler–Scheinker
syndrome (GSS), fatal familial insomnia (FFI) and kuru
(Collinge, 2001). The central feature of prion diseases is
the post-translational conversion of cellular prion protein
(PrPC) to an abnormal protease-resistant isoform designated
PrPSc (Prusiner, 1998; Collinge, 2001). Inherited prion
diseases account for 15% of human cases and are caused
by autosomal dominant mutations in the human prion protein gene (PRNP) (Collinge, 2001; Wadsworth et al., 2003).
Traditionally, inherited prion diseases have been classified by
the presenting clinical or pathological syndrome, falling into
three main sub-divisions of either GSS, CJD or FFI (Collinge
#
and Palmer, 1997; Collinge, 1998; Kovacs et al., 2002;
Gambetti et al., 2003; Harder et al., 2004). Remarkably,
however, some families show extensive phenotypic variability,
which can encompass both CJD- and GSS- like disease as well
as other cases that do not conform to either CJD or GSS
phenotypes (Collinge et al., 1992; Hainfellner et al., 1995;
Mallucci et al., 1999; Kovacs et al., 2002).
Within the framework of the protein-only hypothesis of
prion propagation, distinct clinical and neuropathological
phenotypes are thought to be determined by the propagation
of distinct disease-related PrPSc isoforms with divergent physicochemical properties (Bessen and Marsh, 1994; Collinge
et al., 1996; Parchi et al., 1996; Gambetti et al., 2003; Hill et al.,
2003; Zanusso et al., 2004). To date we have identified four
The Author (2006). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: [email protected]
PrPSc glycosylation in inherited prion diseases
major types of human PrPSc associated with sporadic and
acquired human prion diseases that can be differentiated
on immunoblots after limited proteinase K digestion of
brain homogenates (Collinge et al., 1996; Wadsworth et al.,
1999; Hill et al., 2003). PrPSc types 1–3 are seen in classical
(sporadic or iatrogenic) CJD brain, whereas type 4 PrPSc is
uniquely seen in vCJD brain (Collinge et al., 1996; Wadsworth
et al., 1999; Hill et al., 2003). An earlier classification of PrPSc
types seen in classical CJD described only two banding patterns (Parchi et al., 1996) with PrPSc types 1 and 2 that we
describe corresponding with the type 1 pattern of Gambetti
and co-workers (2003), and our type 3 fragment size corresponding to their type 2 pattern (Parchi et al., 1997, 1999).
Whereas type 4 PrPSc is readily distinguished from the PrPSc
types seen in classical CJD by a predominance of the diglycosylated PrP glycoform, type 4 PrPSc also has a distinct
proteolytic fragment size (Hill et al., 2003), although this is
not recognized by the alternative classification, which designates type 4 PrPSc as type 2b (Parchi et al., 1997).
Efforts to produce a unified international classification and
nomenclature of human PrPSc types has been complicated by
the fact that the N-terminal conformation of some PrPSc
subtypes seen in sporadic CJD can be altered in vitro via
changes in metal-ion occupancy (Wadsworth et al., 1999;
Hill et al., 2003) or solvent pH (Zanusso et al., 2001;
Notari et al., 2004). Although it has recently been proposed
that pH alone determines PrPSc N-terminal structure (Notari
et al., 2004), this interpretation has not been supported by
other studies (Lewis et al., 2005; Polymenidou et al., 2005),
and strain-specific PrPSc conformations show critical dependence upon the presence of copper or zinc ions under conditions where pH 7.4 is strictly controlled (Wadsworth et al.,
1999). Although agreement has yet to be reached on methodological differences, nomenclature and the biological
importance of relatively subtle biochemical differences in
PrPSc, there is strong agreement between laboratories that
phenotypic diversity in human prion disease relates to the
propagation of disease-related PrP isoforms with distinct physicochemical properties (Collinge et al., 1996; Parchi et al.,
1996, 1999, 2000; Wadsworth et al., 1999; Gambetti et al.,
2003; Hill et al., 2003; Zanusso et al., 2004).
Polymorphism at residue 129 of human PrP [encoding
either methionine (M) or valine (V)] powerfully affects
genetic susceptibility to human prion diseases (Collinge
et al., 1991; Palmer et al., 1991; Lee et al., 2001; Mead et al.,
2003). Within a conformational selection model of prion
transmission barriers (Collinge, 1999, 2001; Hill and
Collinge, 2003) it is predicted that coding changes in PrP
act to specify thermodynamic preferences for diseaserelated PrP conformations. Although a wealth of data from
acquired or sporadic CJD indicates that residue 129 polymorphism critically dictates thermodynamic preferences
for disease-related PrP isoforms (Collinge, 2001; Hill and
Collinge, 2003; Hill et al., 2003; Hosszu et al., 2004;
Wadsworth et al., 2004), the full spectrum of effects that
different pathogenic PRNP mutations may have remains
Brain (2006), 129, 676–685
677
unclear. However, numerous studies of abnormal PrP
isoforms in inherited prion diseases have established that
the detection of PrPSc in the molecular mass range of
21–30 kDa is by no means a consistent feature. Instead,
some cases, in particular those in which amyloid plaques
are a prominent feature, show smaller protease resistant fragments of 7–15 kDa (Parchi et al., 1998; Piccardo et al., 1998,
2001; Tagliavini et al., 2001; Kovacs et al., 2002). PrPSc glycoform ratios in patients with FFI or GSS also show dramatic
differences from PrPSc seen in sporadic CJD (Furukawa et al.,
1998; Parchi et al., 1998; Cardone et al., 1999) and have been
reported to be more similar to that of type 4 PrPSc seen in
vCJD (Furukawa et al., 1998). In order to further define the
spectrum of abnormal PrP isoforms seen in human prion
disease we performed detailed analysis of protease-resistant
PrP species in patients with different PRNP mutations,
including those classified as CJD, GSS and FFI. Our findings
indicate that pathogenic PrP point mutations may have a
profound effect in specifying the physicochemical properties
of disease-related PrP isoforms and provide a critical insight
into the potential mechanisms underlying phenotypic variability in inherited prion disease.
Methods
Biosafety
All procedures were carried out in a microbiological containment
level 3 facility with strict adherence to safety protocols.
Selection of patients
This study was performed under approval from the Institute of
Neurology/National Hospital for Neurology and Neurosurgery
Local Research Ethics Committee. Tissues were derived from an
unselected series of patients with a neuropathologically proven diagnosis of inherited prion disease who had provided consent to use
autopsy material for research purposes. Ethical permission
for research on autopsy materials stored in the National CJD Surveillance Unit was obtained from Lothian Region Ethics Committee.
Genetic analysis of PRNP
DNA was extracted from frozen brain tissue or peripheral blood.
PCR of the PRNP open reading frame was performed using previously described oligonucleotide primers, which were selected so
that they did not overlay a polymorphism in the intron 50 to the open
reading frame, which prevents the amplification of some alleles
(Palmer et al., 1996). The PCR fragments were size fractionated
on 1.5% agarose gels to investigate insertions or deletions in the
octapeptide repeat region. The PCR products were then sequenced
using both strands on an Applied Biosystems 377 DNA sequencer.
For haplotype analysis, oligonucleotide primers were designed to
perform allele specific PCR (asPCR) between the M129V
(ATG–GTG) transition and a second primer targeted either 50
(A117V, P102L and OPRI haplotype analysis) or 30 (D178N and
E200K haplotype analysis). Haplotyping of N-terminal variations
used reverse asPCR primers 50 -CTCATGGCACTTCCCAGCAT-30
(methionine allele) and 50 -CTCATGGCACTTCCCAGCAC-30
678
Brain (2006), 129, 676–685
(valine allele), with forward primer 50 -ACCTGGGCCTCTGCAAGAAG-30 . Annealing temperatures for met and val alleles were 67
and 69 C, respectively. Haplotyping of C-terminal variations
used forward asPCR primers 50 -GGGCCTTGGCGGCTACA-30
(methionine allele) and 50 -GGGCCTTGGCGGCTACG-30 (valine
allele), with the reverse primer 50 -AAGAGAGAAGAAAGAGTGAGACAC-30 . Annealing temperatures in this case were 66 C for
both alleles. Thermal cycling was performed using 95 C denature
for 5 min, followed by 35 cycles of 95 C for 30 s denature, anneal for
30 s at the temperature described above and extension at 72 C for
1 min, and a final extension at 72 C for 5 min. Allele specific PCR
products were sequenced using asPCR primers on an ABI 377
sequencer.
A. F. Hill et al.
anti-PrP monoclonal antibody 3F4 and enhanced chemiluminescence was performed as described previously (Wadsworth et al.,
2001).
High sensitivity immunoblotting
Sodium phosphotungstic acid precipitation of PrPSc from 10% tissue
homogenate, proteinase K digestion and immunoblotting with antiPrP monoclonal antibody 3F4 using high sensitivity enhanced chemiluminescence was performed as described previously (Wadsworth
et al., 2001). This method facilitates highly efficient recovery and
detection of PrPSc from tissue homogenate when present at levels
104–105-fold lower than those found in vCJD brain (Wadsworth
et al., 2001; Joiner et al., 2002; Frosh et al., 2004).
Immunoblot analysis
10% (w/v) brain homogenates were prepared in lysis buffer (100 mM
NaCl, 10mM EDTA, 10 mM Tris–HCl, 0.5% sodium deoxycholate,
0.5% NP-40, pH 7.4) by passage through needles of decreasing diameter. The homogenates were cleared by centrifugation at 800 r.p.m.
(100 g) in a microcentrifuge for 1 min. Samples of supernatant were
treated with proteinase K (50 mg/ml final concentration unless stated
otherwise) for 60 min at 37 C. The reaction was terminated by the
addition of Pefabloc (Boehringer Mannheim) to 1 mM final concentration. An equal volume of SDS loading buffer (125 mM
Tris–HCl, 4% SDS, 20% glycerol, 0.02% bromophenol blue, 4%
b-mercaptoethanol) was added and the samples boiled for 5 min
before electrophoresis on 16% Tris–glycine gels (Invitrogen). The
gels were electroblotted onto PVDF membrane (Immobilon-P,
Millipore) and blocked for 1 h in phosphate-buffered saline
(PBS)/0.05% Tween-20 (PBST) containing 5% non-fat milk powder.
Membranes were incubated with anti-PrP monoclonal antibody 3F4
(Kascsak et al., 1987) (Signet Pathology Inc) at 0.2 mg/ml final concentration in PBST for 1 h or overnight. After washing in PBST
(45 min), membranes were incubated with an alkaline phosphatase
conjugated goat anti-mouse IgG antibody (Sigma) diluted 1 : 10 000
in PBST. Following washing in PBST (45 min), membranes were
developed in chemifluorescent substrate (Attophos, Promega) and
visualized on a Storm 840 phosphoimager (Molecular Dynamics).
The glycoform profiles were quantitated using Imagequant software
(Molecular Dynamics) to measure the relative intensity of each PrPSc
band, which is then expressed as a percentage of the total signal. Each
case of inherited prion disease suitable for analysis was analysed at
least twice. Statistical analysis was performed using the INSTAT
program (GraphPad Software, San Diego). Immunoblotting with
Results
PrP analysis of inherited prion disease
cases in the absence of proteinase K
digestion
Immunoblotting of brain homogenate in the absence of proteinase K digestion showed no discernible difference in the
size of full length PrP from cases with PRNP point mutations
(P102L, E200K and D178N) when compared with wild-type
PrP in sporadic or vCJD (Fig. 1A). As expected however,
similar analysis of inherited prion disease cases caused by
2-, 4- or 6-octapeptide repeat insertional mutations
(OPRI) showed PrP species with a greater molecular mass
than wild-type PrP, this being most apparent with the larger
insert cases (Fig. 1B). Although theoretically all of the samples
analysed should show closely similar levels of PrP, it should be
emphasized that we are analysing post-mortem samples having intrinsic differences in post-mortem delay that can lead to
degradation of PrPC. The presence of truncated PrP fragments
(particularly of 20 kDa) generated by endogenous proteolytic activity in brain homogenate is well recognized (JiménezHuete et al., 1998) and levels of these can vary quite markedly
between samples. Although an 8 kDa PrP species can sometimes be detected in non-proteinase K digested P102L brain
homogenate (Piccardo et al., 1998; Zou et al., 2003) this was
not a prominent characteristic of our series of patient samples.
Fig. 1 Immunoblots of PrP in brain homogenate from classical CJD, vCJD and cases of inherited prion disease analysed without proteinase
K digestion. A and B, respectively, show cases of inherited prion diseases associated with PRNP point mutations or octapeptide repeat
insertions (OPRI). Immunoblots were developed with anti-PrP monoclonal antibody 3F4 using a chemiluminescent substrate.
PrPSc glycosylation in inherited prion diseases
Brain (2006), 129, 676–685
679
Table 1 Study cohort of patients with inherited prion disease, showing PRNP codon 129 genotype, PRNP mutation,
the codon 129 genotype of the mutated allele and PrPSc proteolytic fragment size using the MRC Prion Unit classification
of PrPSc types (Collinge et al., 1996; Hill et al., 2003)
Laboratory reference
number
PRNP mutation
PRNP codon
129 genotype
PRNP haplotype
(mutation – codon 129 genotype)
PrPSc type
1929
1911
1756
2179
3070
6271
6202
3185
2190
1394
2177
1261
2163
2169
2193
2191
3069
2165
2268
2507
362
6197
1477
6354
1276
P102L
P102L
P102L
P102L
P102L
P102L
P102L
A117V
D178N
D178N
D178N
E200K
E200K
E200K
E200K
E200K
E200K
2-OPRI
4-OPRI
4-OPRI
6-OPRI
6-OPRI
6-OPRI
6-OPRI
6-OPRI
MM
MM
MV
MM
MV
MM
MM
MV
MM
MM
MM
MM
MV
MM
MV
VV
MM
MM
MM
MM
MV
MM
MM
MM
MM
102L-M129
102L-M129
102L-M129
102L-M129
102L-M129
102L-M129
102L-M129
117V-V129
178N-M129
178N-M129
178N-M129
200K-M129
200K-M129
200K-M129
200K-M129
200K-V129
200K-M129
2-OPRI-M129
4-OPRI-M129
4-OPRI-M129
6-OPRI-M129
6-OPRI-M129
6-OPRI-M129
6-OPRI-M129
6-OPRI-M129
1
2
1
2
Negative,
Negative,
Negative,
Negative,
2
2
Negative,
1
2
1
2/3
3
1
2
1
2
2
3
Negative
Negative
Negative
8kDa
8kDa
8kDa
8kDa
8kDa
PRNP point mutations are designated by the wild-type amino acid preceding the codon number followed by the mutant residue using single
letter amino acid nomenclature. (OPRI, octapeptide repeat insertion mutations).
PrPsc analysis in inherited prion disease
cases
We analysed protease-resistant PrP species present in a series
of patients with inherited prion disease. A total of 25 cases
were studied of which 17 had PRNP point mutations and 8 had
insertions in the octapeptide repeat region (OPRI mutations)
(Table 1). Previous findings in sporadic CJD of detection of
two apparently different PrPSc types in different regions of the
same brain (Puoti et al., 1999; Head et al., 2004; Polymenidou
et al., 2005) indicate the need to compare the same brain
region from different patients for comparative PrPSc typing.
The samples used in our study were from frontal cortex and
each sample was routinely analysed at least twice using appropriate controls of known PrPSc type on every immunoblot.
Figure 2 illustrates the comparison in PrPSc fragment size and
degree of glycosylation between classical CJD (types 1–3),
vCJD (type 4) and inherited cases caused by octapeptide
repeat insertion mutations (OPRI) or point mutations in
the PRNP gene.
PrPsc analysis of cases with octapeptide
repeat insertion (OPRI) mutations
We examined frontal cortex from patients with three different
OPRI mutations (2-OPRI, 4-OPRI and 6-OPRI). All were
Fig. 2 Immunoblots of proteinase K digested brain homogenate
from cases of classical CJD and vCJD (A) showing PrPSc types 1–4,
and (B–E) inherited prion disease showing protease-resistant PrP
fragments of 21–30 kDa. The types of CJD or pathogenic PRNP
mutation are shown above the samples, and the PrPSc type [using
the Collinge classification of PrPSc types (Collinge et al., 1996; Hill
et al., 2003)] and codon 129 genotype of the sample are shown
below each lane. Immunoblots were developed with monoclonal
antibody 3F4 using a chemiluminescent substrate.
PRNP codon 129 methionine homozygotes (MHs) except
for one 6-OPRI case, which was a methionine/valine heterozygote (Table 1). PrPSc with a fragment size corresponding to
type 2 PrPSc seen in sporadic CJD (Fig. 2A) was detected in the
single 2-OPRI case (Fig. 2B) and PrPSc with fragment sizes
680
Brain (2006), 129, 676–685
Fig. 3 (A–D) Immunoblots of proteinase K digested brain
homogenate from cases of inherited prion disease showing
protease-resistant PrP fragments of 21–30 kDa. The PRNP
mutation is designated above each immunoblot with PrPSc
proteolytic fragment size [using the Collinge classification of PrPSc
types (Collinge et al., 1996; Hill et al., 2003)] and PRNP codon
129 genotype designated below. Immunoblots were developed
with anti-PrP monoclonal antibody 3F4 using a chemiluminescent
substrate.
corresponding to types 1 and 2 PrPSc seen in sporadic CJD
were detected in the two 4-OPRI cases (Table 1). Only two of
five 6-OPRI cases showed detectable levels of PrPSc, even after
reducing the concentration of proteinase K (to 12.5 mg/ml for
1 h at 37 C). The 6-OPRI codon 129 heterozygote case
showed PrPSc with a fragment size corresponding to type 2
PrPSc seen in sporadic CJD and one 6-OPRI codon 129
methionine homozygous case showed PrPSc with a fragment
size corresponding to type 3 PrPSc seen in sporadic CJD
(Fig. 3A). Analysis of the PrPSc glycoform ratio in these
OPRI cases by densitometry (high molecular mass glycoform,
20.59 6 2.53%; low molecular mass glycoform, 44.2 6 2.07%;
unglycosylated, 35.22 6 1.36%) revealed no statistically significant difference from PrPSc of the same type and codon 129
genotype seen in sporadic CJD (Fig. 5).
PrPsc analysis in cases with PRNP point
mutations
P102L
We examined seven P102L cases of which two were PRNP
codon 129 heterozygotes and the other five were codon 129
MHs (Table 1). Analysis of multiple homogenates prepared
from each specimen identified in four cases varying concentrations of proteinase K resistant PrP fragments of 21–30
kDa (Fig. 3B) together with variable levels of an 8 kDa protease resistant PrP fragment (data not shown), whereas only
the 8 kDa PrP fragment was detected in the other three cases
(Fig. 4). These data are consistent with those of other
researchers (Parchi et al., 1998; Piccardo et al., 1998) and
typify both the spectrum and spatial variability of proteinase
K-resistant PrP species seen in P102L inherited prion disease.
All four cases that propagated PrPSc proteolytic fragments of
21–30 kDa showed fragment sizes corresponding to either
PrPSc type 1 or type 2 seen in sporadic CJD (Table 1 and
Fig. 3B). In agreement with previous studies (Cardone et al.,
1999; Furukawa et al., 1998; Parchi et al., 1998) analysis of the
glycoform ratio of the PrPSc fragments of 21–30 kDa from
A. F. Hill et al.
Fig. 4 Immunoblots of proteinase K digested brain homogenate
from cases of inherited prion disease with PRNP mutations
showing protease-resistant PrP fragments of 7–12 kDa. The
PRNP point mutation is designated above each immunoblot with
codon 129 genotype designated below. Immunoblots were
developed with anti-PrP monoclonal antibody 3F4 using
a chemiluminescent substrate.
these patients revealed an abundance of diglycosylated PrP
(Fig. 2C). However, densitometry showed that this glycoform
ratio (high molecular mass glycoform, 40.46 6 2.60%; low
molecular mass glycoform, 39.18 6 2.06%; unglycosylated,
20.36 6 0.81%) differs significantly from PrPSc types 1–3 seen
in classical CJD and from type 4 PrPSc seen in vCJD (Fig. 5).
A117V
A single PRNP codon 129 heterozygote patient with the
A117V mutation was examined (Table 1). Following proteinase K digestion, PrPSc fragments of 21–30 kDa were not
detected but instead an 8 kDa protease-resistant PrP species
was observed (Fig. 4). This finding correlates well with the
findings of other researchers that only detected similar low
molecular mass protease-resistant PrP species in A117V cases
(Piccardo et al., 1998, 2001; Tagliavini et al., 2001).
D178N
We examined three patients with the D178N mutation all of
which were of PRNP 129MM genotype (Table 1). Two of
these cases propagated PrPSc with fragment sizes corresponding to type 2 PrPSc seen in sporadic CJD (Fig. 2D). The glycoform ratio of these PrPSc fragments showed an abundance
of the di-glycosylated PrP glycoform (high molecular mass
glycoform, 53.72 6 4.88%; low molecular mass glycoform,
38.84 6 6.81%; unglycosylated, 7.45 6 1.93%), similar to
those observed in the P102L cases, that was significantly different from PrPSc types 1–3 seen in sporadic CJD and from
type 4 PrPSc seen in vCJD (Fig. 5). The other D178N case had
no detectable PrPSc proteolytic fragments of 21–30 kDa,
which is consistent with previous reports of FFI (Budka
et al., 1997; Parchi et al., 1995), but instead showed an
8 kDa protease-resistant PrP species similar to that observed
in P102L or A117V samples (Fig. 4).
PrPSc glycosylation in inherited prion diseases
Fig. 5 Ratio of three principal protease-resistant PrP glycoforms
of 21–30 kDa seen in classical CJD, vCJD and cases of inherited
prion disease. Data points represent the mean relative proportions of di-, mono- and unglycosylated PrP as percentage 6 SEM.
In some cases the error bars were smaller than the symbols used.
The number of cases analysed were: sCJD type 1PrPSc 129MM
(n = 17), sCJD type 2 PrPSc 129, MM,MV,VV (n = 57), sCJD type 3
PrPSc 129MM (n = 1), sCJD type 3 PrPSc 129MV (n = 8), vCJD type
4 129MM (n = 30), P102L 129MM (n = 4), D178N 129MM (n = 2),
E200K 129, MM,MV,VV (n = 5), OPRI mutations 129 MM, MV
(n = 5). For PRNP point mutations P102L, E200K and D178N,
there is a statistically significant difference in the proportions of
di- and un-glycosylated PrP glycoforms when compared with
PrPSc types 1–3 in classical CJD (P < 0.0001 for the di- and
un-glycosylated bands; unpaired t-test) and in the proportions of
mono- and un-glycosylated PrP glycoforms compared with type
4 PrPSc seen in vCJD (P < 0.004 for either glycoform; unpaired
t-test). The PrP glycoform ratio in OPRI cases shows no significant
difference from PrPSc seen in sporadic CJD cases of the
same codon 129 genotype (P > 0.1).
E200K
Six cases of inherited prion disease with the E200K mutation
were examined. Three cases were PRNP codon 129 MHs, two
were methionine/valine heterozygotes and one was a valine
homozygote (Table 1). All three codon 129 MHs propagated
PrPSc with a fragment size corresponding to type 1 PrPSc seen
in sporadic CJD (Fig. 2E), accompanied by weak variable
labelling of a 12 kDa protease resistant fragment (Fig. 4).
One PRNP 129MV heterozygote propagated PrPSc with a
fragment size corresponding to type 2 PrPSc seen in sporadic
CJD (Fig. 3C) and the single PRNP 129VV homozygote propagated PrPSc with a fragment size corresponding to type 3
PrPSc seen in sporadic CJD (Fig. 3C and D). The other PRNP
129MV heterozygote patient showed PrPSc with fragment
sizes corresponding to both type 2 and type 3 PrPSc seen
in sporadic CJD (Fig. 3D). The presence of PrPSc with two
clearly defined proteolytic fragment sizes is also occasionally
observed in brain homogenates from sporadic CJD cases
(Parchi et al., 1999; Hill et al., 2003) and may represent
the propagation of two different PrPSc types in the same
region of the brain (Puoti et al., 1999; Head et al., 2004;
Brain (2006), 129, 676–685
681
Fig. 6 Immunoblots of proteinase-K digested brain homogenates
from human sporadic CJD and E200K inherited prion disease
cases were analysed by enhanced chemiluminescence using
anti-PrP monoclonal antibody 3F4. Lanes 1 and 2, sporadic CJD
type 1 PrPSc (PRNP genotype 129MM); lanes 3 and 4, E200K
case (PDG 3069) with type 1 PrPSc fragment size (PRNP genotype
129MM); lanes 5 and 6, E200K case (PDG 2163) with type 2 PrPSc
fragment size (PRNP genotype 129MV); lanes 7 and 8, E200K case
(PDG 2191) with type 3 PrPSc fragment size (PRNP genotype
129VV). Samples were proteinase K digested in the absence ()
or presence (+) of 25 mM EDTA.
Polymenidou et al., 2005). The glycoform ratio of PrPSc fragments seen in these E200K cases was similar to the P102L and
D178N cases in this study (high molecular mass glycoform,
42.76 6 1.59%; low molecular mass glycoform, 41.53 6
2.17%; unglycosylated, 15.71 6 1.52%), and again was significantly different to PrPSc types 1–3 seen in sporadic CJD
and type 4 PrPSc seen in vCJD (Fig. 5). Thus in the six cases
studied with the E200K mutation, we were able to observe all
of the common PrPSc fragment sizes previously reported in
sporadic and iatrogenic prion disease (Collinge et al., 1996;
Wadsworth et al., 1999; Hill et al., 2003) (Fig. 3C), but with
the additional superimposition of a distinct glycoform ratio
(Fig. 5). We also investigated the behaviour of E200K samples
following proteinase K digestion in the presence of 25 mM
EDTA (Wadsworth et al., 1999; Hill et al., 2003). All three
PRNP 129MM homozygous cases with type 1 PrPSc fragment
size showed altered proteinase K digestion products after
inclusion of EDTA during proteolysis, generating a fragment
size corresponding to type 2- generated from EDTA-treated
type 1 PrPSc in sporadic CJD (Fig. 6). In comparison, PrPSc
with type 2 fragment size from the codon 129 heterozygote
case and PrPSc with type 3 fragment size from the codon 129
valine homozygous case showed no alteration in the electrophoretic mobility of proteolytic fragments after digestion in
the presence of EDTA (Fig. 6).
Analysis of lymphoreticular tissues for
the presence of PrPsc
Samples of lymphoreticular tissue were available for analysis
from six patients, four with PRNP point mutations and two
with a 6-OPRI insert mutation (Table 2). Using high sensitivity immunoblotting (Wadsworth et al., 2001) we were not
able to detect PrPSc in any of these tissues (Table 2). The
682
Brain (2006), 129, 676–685
A. F. Hill et al.
Table 2 High sensitivity immunoblot analysis of
lymphoreticular tissues from patients with inherited prion
disease
PRNP mutation
Laboratory
reference number
Tonsil PrPSc
Spleen PrPSc
P102L
2179
6202
6271
–
Negative
Negative
Negative*
Negative
Negative
Negative
Negative
Negative
–
–
Negative
E200K
6 repeat OPRI
6197
6354
The sensitivity of this method would detect PrPSc when present at
levels >0.1% of the maximal level of PrPSc observed in necropsy
vCJD tonsil (Wadsworth et al., 2001). *Tonsil biopsy specimen;
no brain tissue available for analysis.
detection sensitivity of this method indicates that if PrPSc is
present in lymphoreticular tissues from these cases of inherited prion disease then this is at a concentration <0.1% of the
maximal level seen in necropsy vCJD tonsil (Wadsworth et al.,
2001). To date, tonsil biopsy has shown 100% sensitivity and
specificity for diagnosis of vCJD (Hill et al., 1999; Wadsworth
et al., 2001; Frosh et al., 2004) and may allow diagnosis at an
early clinical stage (Wadsworth et al., 2003). Our present
findings suggest that the peripheral pathogenesis in inherited
prion disease more closely resembles that of classical CJD,
rather than vCJD, and therefore cases of inherited prion disease are unlikely to be detected by analysis of tonsil biopsy
specimens.
Discussion
In this study we have shown that cases of inherited prion
disease caused by the PRNP point mutations P102L,
D178N and E200K have a unique PrPSc glycoform ratio
that differs significantly from PrPSc glycoform ratios we
have observed in sporadic, iatrogenic and vCJD (Collinge
et al., 1996; Hill et al., 2003). Whereas the glycoform profile
of PrPSc in P102L cases has been reported by others to be
similar to that of type 4 PrPSc seen in vCJD (Furukawa et al.,
1998), our findings of a highly statistically significant difference (P < 0.004 for either mono, or un-glycosylated PrPSc
fragments) strongly support the hypothesis that vCJD is a
distinct human prion strain. Propagation of distinct PrPSc
isoforms appears to underlie phenotypic variability in
human prion diseases (Collinge et al., 1996; Parchi et al.,
1996; Puoti et al., 1999; Gambetti et al., 2003; Hill et al.,
2003; Head et al., 2004; Zanusso et al., 2004; Polymenidou
et al., 2005). Characteristic human PrPSc fragment sizes and
glycoform ratios can be maintained on passage in transgenic
mice expressing only human PrP and the propagation of these
distinct PrPSc types can be correlated with distinct neuropathological phenotypes (Collinge et al., 1996; Hill et al.,
1997; Asante et al., 2002; Wadsworth et al., 2004). These
data strongly support the ‘protein-only’ hypothesis of
prion infectivity and suggest that prion strain variation is
encoded through a combination of PrPSc conformation
and glycosylation.
The data presented here suggest that point mutations in
PRNP either destabilize non-glycosylated PrP, in turn reducing its relative abundance (Petersen et al., 1996), or directly
dictate the stoichiometry and packing order of the three PrP
glycoforms into disease-related fibrils or other aggregates. The
latter explanation is consistent with a conformational
selection model of prion transmission barriers (Collinge,
1999; Collinge, 2001; Hill and Collinge, 2003) that predicts
that coding changes in PrP act to specify structural preferences for disease-related PrP isoforms. Immunoprecipitation
studies using monoclonal antibodies, which distinguish
between PrP glycoforms, are also consistent with incorporation of glycoforms into PrPSc aggregates in strain-specific
ratios (Khalili-Shirazi et al., 2005). In contrast to the possible
effects of PRNP point mutations, OPRI mutations appear to
have little influence in determining the structure of the Cterminal protease-resistant core of PrPSc. Both the glycoform
ratio and proteolytic fragment sizes of PrP associated with
OPRI mutations are indistinguishable from those of PrPSc
seen in classical CJD.
The identification of distinct PrPSc glycoform ratios associated with PRNP point mutations extends the spectrum of
PrPSc isoforms seen in human prion disease and reinforces the
hypothesis that glycosylation may encode phenotypic information that defines distinct prion strains (Collinge et al.,
1996; Lawson et al., 2005; Collinge, 2001). Our findings
also support earlier studies (Piccardo et al., 1998, 2001) showing that individual PRNP mutations do not restrict abnormal
PrP isoforms to adopting a single pathogenic conformation.
Indeed, all of the PrPSc fragment sizes seen in sporadic CJD
(Hill et al., 2003) can be observed in PRNP E200K cases.
Additionally, the conformational plasticity seen in the Nterminus of type 1 PrPSc in sporadic CJD (Wadsworth
et al., 1999; Hill et al., 2003) is similarly observed in PrPSc
with the same fragment size in PRNP E200K 129MM cases.
Collectively, these data provide an insight into how diverse
clinical phenotypes may arise in patients with the same PRNP
mutation.
Despite these advances, PrPSc type, as currently defined,
cannot satisfactorily explain all of the phenotypic differences
seen in inherited prion disease. For example, our data show
that cases of D178N FFI and cases of E200K CJD can share the
same PrPSc glycoform ratio and propagate the same type 2
PrPSc fragment size. Clearly, further studies are required to
investigate other differences in these PrPSc species. In this
context it is important to note that we probe PrPSc conformation by looking at accessibility to scissile bonds cleaved by
proteinase K at the N-terminus of PrPSc only. Given the site of
the PRNP point mutations it seems likely that conformational
heterogeneity may exist in the C-terminal structured domain
of PrPSc isoforms that are not revealed with proteinase K.
Neuropathology associated with specific PRNP mutations
PrPSc glycosylation in inherited prion diseases
may also be influenced by propagation of abnormal isoforms
of wild-type PrP (Gabizon et al., 1996; Chen et al., 1997;
Silvestrini et al., 1997), and it is also clear that propagation
of abnormal PrP isoforms and associated neuropathology
may be determined by genetic background (Lloyd et al.,
2001, 2004; Asante et al., 2002).
The absence of detectable PrPSc is a consistent feature in
inherited prion disease associated with certain PRNP mutations and a more variable feature in others (Collinge, 2001;
Kovacs et al., 2002). A possible explanation for some of these
findings may relate to sampling variation and in particular the
region of brain studied. We examined frontal cortex and
therefore with negative samples we cannot exclude the
propagation of PrPSc in other brain regions. Nevertheless,
sampling variation is unlikely to account for the emerging
consensus of similar findings from different laboratories.
These collective data indicate that PrPSc isoforms may be
generated in inherited prion disease with unique physicochemical properties, reflected by sensitivity to proteinase K
digestion or PrPSc/prion infectivity ratios that are very different from the PrPSc types propagated in sporadic and acquired
forms of human prion disease. For example in P102L inherited prion disease, it is now quite clear that patients propagate
at least two distinct abnormal PrP conformers. Abnormal PrP
conformers generating high molecular mass protease resistant
PrP fragments of 21–30 kDa are not uniformly detected
throughout the brain in P102L inherited prion disease and
appear to be restricted to areas of the brain showing synaptic
PrP deposition and spongiform vacuolation (Parchi et al.,
1998; Piccardo et al., 1998). In brain regions in which amyloid
plaques are a prominent feature an alternate abnormal PrP
isoform appears to predominate that generates smaller
protease-resistant PrP fragments of 8 kDa derived from
the central portion of PrP (Parchi et al., 1998; Piccardo
et al., 1998) indicating that both the N- and C-terminal
residues of this abnormal PrP isoform are digested by
proteinase K. Clearly, new experimental methods may be
required in order to fully document the spectrum of abnormal
PrP isoforms seen in inherited prion disease. In this context it
is important to note that not all inherited prion disease may
invoke disease through the same mechanism. For example the
PRNP A117V mutation gives rise to transmembrane forms of
PrP (PrPctm), which have been shown to invoke a neurological
disease without generation of PrPSc (Hegde et al., 1998). It is
also not presently known whether all inherited prion diseases
are transmissible by inoculation.
In summary, we have found that the glycoform ratios of
PrPSc in inherited prion disease associated with PRNP point
mutations P102L, D178N and E200K are similar to each
other, yet significantly different to glycoform ratios of
PrPSc observed in sporadic, iatrogenic and vCJD. Since the
glycoform ratio of PrPSc associated with PRNP point mutations is significantly different to those arising from PRNP
OPRI mutations this suggests that the point mutations
may act pathogenically by dictating preferred PrPSc assembly
states. Our findings have identified key isolates of inherited
Brain (2006), 129, 676–685
683
prion disease whose transmission properties are now being
investigated in appropriate lines of transgenic mice expressing
either wild-type or mutated forms of human PrP. These studies will be critical for further understanding the divergent
pathogenic mechanisms associated with distinct PRNP
mutations.
Acknowledgements
We specially thank all patients and their families for generously consenting to use of human tissues in this research and
the neuropathologists who have kindly helped in providing
these tissues, in particular James Ironside and Herbert Budka.
We thank Ray Young for preparation of figures. This research
was supported by the Medical Research Council (UK), the
Wellcome Trust and the European Commission. A.F.H. is an
Australian NHMRC RD Wright Fellow.
Conflict of interest statement
J.C. is a Director and J.C., A.F.H. and J.D.F.W. are shareholders and consultants of D-Gen Limited, an academic spinout company working in the field of prion disease diagnosis,
decontamination and therapeutics.
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