Co-existence of scrapie prion protein types 1 and 2 in sporadic Creutzfeldt-Jakob disease: its effect on the phenotype and prion-type characteristics.
ABSTRACT Five phenotypically distinct subtypes have been identified in sporadic Creutzfeldt-Jakob disease (sCJD), based on the methionine/valine polymorphic genotype of codon 129 of the prion protein (PrP) gene and the presence of either one of the two protease K-resistant scrapie prion protein (PrP(Sc)) types identified as 1 and 2. The infrequent co-existence of both PrP(Sc) types in the same case has been known for a long time. Recently, it has been reported, using type-specific antibodies, that the PrP(Sc) type 1 is present in all cases of sCJD carrying PrP(Sc) type 2. The consistent co-occurrence of both PrP(Sc) types complicates the diagnosis and the current classification of sCJD, and has implications for the pathogenesis of naturally occurring prion diseases. In the present study, we investigated the prevalence of PrP(Sc) types 1 and 2 co-occurrence, along with its effects on the disease phenotype and PrP(Sc) strain characteristics, comparatively analysing 34 cases of sCJD, all methionine homozygous at codon 129 of the PrP gene (sCJDMM). To minimize overestimating the prevalence of the sCJDMM cases carrying PrP(Sc) types 1 and 2 (sCJDMM1-2), we used proteinase K concentrations designed to hydrolyse all fragments resulting from an incomplete digestion, while preserving the protease-resistant PrP(Sc) core. Furthermore, we used several antibodies to maximize the detection of both PrP(Sc) types. Our data show that sCJDMM cases associated exclusively with either PrP(Sc) type 1 (sCJDMM1) or PrP(Sc) type 2 (sCJDMM2) do exist; we estimate that they account for approximately 56% and 5% of all the sCJDMM cases, respectively; while in 39% of the cases, both PrP(Sc) types 1 and 2 are present together (sCJDMM1-2) either mixed in the same anatomical region or separate in different regions. Clinically, sCJDMM1-2 had an average disease duration intermediate between the other two sCJDMM subtypes. The histopathology was also intermediate, except for the cerebellum where it resembled that of sCJDMM1. These features, along with the PrP immunostaining pattern, offer a diagnostic clue. We also observed a correlation between the disease duration and the prevalence of PrP(Sc) type 2 and sCJDMM2 phenotypes. The use of different antibodies and of the conformational stability immunoassay indicated that the co-existence of types 1 and 2 in the same anatomical region may confer special conformational characteristics to PrP(Sc) types 1 and 2. All of these findings indicate that sCJDMM1-2 should be considered as a separate entity at this time.
[show abstract] [hide abstract]
ABSTRACT: Antibody response in mice to scrapie-associated fibril proteins (protease-resistant proteins [PrPs]) was generated to different epitopes depending on the source of antigen. Mice responded differently to PrPs isolated from scrapie-infected animals of homologous (mouse) versus heterologous (hamster) species. An enzyme-linked immunosorbent assay established to monitor this antibody response in mice immunized with PrPs was unable to detect such a response in scrapie-infected mice. A monoclonal antibody (MAb), 263K 3F4, derived from a mouse immunized with hamster 263K PrPs reacted with hamster but not mouse PrPs. MAb 263K 3F4 also recognized normal host protein of 33 to 35 kilodaltons in brain tissue from hamsters and humans but not from bovine, mouse, rat, sheep, or rabbit brains. This is the first demonstration of epitope differences on this host protein in different species. The defining of various epitopes on PrP through the use of MAbs will lead to a better understanding of the relationship of PrPs to their host precursor protein and to the infectious scrapie agent.Journal of Virology 01/1988; 61(12):3688-93. · 5.40 Impact Factor
Article: A refined method for molecular typing reveals that co-occurrence of PrP(Sc) types in Creutzfeldt-Jakob disease is not the rule.[show abstract] [hide abstract]
ABSTRACT: Molecular typing in Creutzfeldt-Jakob disease (CJD) relies on the detection of distinct protease-resistant prion protein (PrP(Sc)) core fragments, which differ in molecular mass or glycoform ratio. However, the definition and correct identification of CJD cases with a co-occurrence of PrP(Sc) types remains a challenge. With antibodies recognizing a linear epitope in the octapeptide repeat PrP region, supposed to distinguish between the two major PrP(Sc) isoforms (ie, types 1 and 2), it was recently shown that all type 2 cases display an associated band with a type 1 migration pattern, which led to the conclusion that multiple PrP(Sc) types regularly coexist in CJD. We studied brain samples from 53 sporadic CJD and 4 variant CJD subjects using a high-resolution electrophoresis, a wide range of proteinase K (PK) activities, the 'type 1-selective' antibody 12B2, and several unselective antibodies. We found that the type 1-like band detected by 12B2 in all CJD subtypes associated with PrP(Sc) type 2 is not a PK-resistant PrP(Sc) core but rather matches the physicochemical properties of partially cleaved fragments, which result from the several PK cleavage sites included in the N-terminal portion of PrP(Sc). Furthermore, using gels with high resolution and a relatively high PK activity, we were able to increase the detection sensitivity of either type 1 or 2, when coexisting, to amount corresponding to 3-5% of the total PrP(Sc) signal (ie, weak band of one type/total PrP(Sc)). Our results show that there are many pitfalls associated with the use of 'type 1 selective' antibodies for CJD typing studies and that co-occurrence of PrP(Sc) types in CJD is not the rule. The present results further validate the rationale basis of current CJD classification and the qualitative nature of molecular typing in CJD.Laboratory Investigation 12/2007; 87(11):1103-12. · 3.64 Impact Factor
A JOURNAL OF NEUROLOGY
Co-existence of scrapie prion protein types 1 and 2
in sporadic Creutzfeldt–Jakob disease: its effect on
the phenotype and prion-type characteristics
Ignazio Cali,1Rudolph Castellani,2Amer Alshekhlee,3Yvonne Cohen,1Janis Blevins,1
Jue Yuan,1Jan P. M. Langeveld,4Piero Parchi,5Jiri G. Safar,1Wen-Quan Zou1and
1 Department of Pathology, Case Western Reserve University, Cleveland, OH 44106, USA
2 Division of Neuropathology, Department of Pathology, University of Maryland, Baltimore, MD 21201, USA
3 Department of Neurology, Case Western Reserve University, Cleveland, OH 44106, USA
4 Central Veterinary Institute of Wageningen UR, NL-8203 AA 2004, Lelystad, The Netherlands
5 Dipartimento di Scienze Neurologiche, Universita ` di Bologna, 40123 Bologna, Italy
Correspondence to: Dr Pierluigi Gambetti,
Department of Pathology,
Case Western Reserve University,
2085 Adelbert Road,
Cleveland, OH 44106,
Correspondence may also be addressed to: Dr Wen-Quan Zou,
Five phenotypically distinct subtypes have been identified in sporadic Creutzfeldt–Jakob disease (sCJD), based on the
methionine/valine polymorphic genotype of codon 129 of the prion protein (PrP) gene and the presence of either one of the
two protease K-resistant scrapie prion protein (PrPSc) types identified as 1 and 2. The infrequent co-existence of both PrPSctypes
in the same case has been known for a long time. Recently, it has been reported, using type-specific antibodies, that the PrPSc
type 1 is present in all cases of sCJD carrying PrPSctype 2. The consistent co-occurrence of both PrPSctypes complicates the
diagnosis and the current classification of sCJD, and has implications for the pathogenesis of naturally occurring prion diseases.
In the present study, we investigated the prevalence of PrPSctypes 1 and 2 co-occurrence, along with its effects on the disease
phenotype and PrPScstrain characteristics, comparatively analysing 34 cases of sCJD, all methionine homozygous at codon 129
of the PrP gene (sCJDMM). To minimize overestimating the prevalence of the sCJDMM cases carrying PrPSctypes 1 and 2
(sCJDMM1-2), we used proteinase K concentrations designed to hydrolyse all fragments resulting from an incomplete digestion,
while preserving the protease-resistant PrPSccore. Furthermore, we used several antibodies to maximize the detection of both
PrPSctypes. Our data show that sCJDMM cases associated exclusively with either PrPSctype 1 (sCJDMM1) or PrPSctype 2
(sCJDMM2) do exist; we estimate that they account for approximately 56% and 5% of all the sCJDMM cases, respectively;
while in 39% of the cases, both PrPSctypes 1 and 2 are present together (sCJDMM1-2) either mixed in the same anatomical
region or separate in different regions. Clinically, sCJDMM1-2 had an average disease duration intermediate between the other
two sCJDMM subtypes. The histopathology was also intermediate, except for the cerebellum where it resembled that of
sCJDMM1. These features, along with the PrP immunostaining pattern, offer a diagnostic clue. We also observed a correlation
doi:10.1093/brain/awp196Brain 2009: 132; 2643–2658 |
Received February 18, 2009. Revised May 16, 2009. Accepted June 8, 2009. Advance Access publication September 4, 2009
? The Author (2009). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.
For Permissions, please email: firstname.lastname@example.org
between the disease duration and the prevalence of PrPSctype 2 and sCJDMM2 phenotypes. The use of different antibodies and
of the conformational stability immunoassay indicated that the co-existence of types 1 and 2 in the same anatomical region may
confer special conformational characteristics to PrPSctypes 1 and 2. All of these findings indicate that sCJDMM1-2 should be
considered as a separate entity at this time.
Keywords: prion protein; prion disease; co-existence; conformation; sporadic Creutzfeldt–Jakob disease
Abbreviations: CSI=conformational stability immunoassay; mAb=monoclonal antibodies; MM=homozygous methionine;
MV=heterozygous methionine/valine; NPDPSC=National Prion Disease Pathology Surveillance Centre; PK=proteinase K;
PrP=prion protein; sCJD=sporadic Creutzfeldt–Jakob disease; VV=homozygous valine
One of the major characteristics of human prion diseases is
the heterogeneity of the clinical and pathological phenotype
(Monari et al., 1994; Parchi et al., 1996, 1999; Gambetti et al.,
2003; Kong et al., 2004). There is a general consensus that in
sporadic prion diseases the genotype at codon 129 of the prion
protein (PrP) gene and the presence of distinct types of the
(hereafter identified as PrPSc) are the major determinants of the
disease phenotype (Gambetti et al., 2003). Several years ago, we
proposed a classification of human sporadic prion diseases based
on the codon 129 genotype and the PrPSctype that distinguishes
five subtypes of sporadic Creutzfeldt–Jakob disease (sCJD) (Parchi
et al., 1996, 1999, 2000; Gambetti et al., 2003). The genotype of
codon 129 is determined by the methionine/valine polymorphism,
which results in three patient populations: homozygous methionine
(MM), homozygous valine (VV) and heterozygous methionine/
valine (MV). The PrPScpresent in the majority of the sCJD patients
can be distinguished in two groups according to the electrophoretic
mobility of the fragments resistant to proteinase K (PK) digestion. In
PrPSctype 1, the unglycosylated isoform migrates to the 21kDa
region of the gel, while the corresponding type 2 isoform migrates
to 19kDa (Parchi et al., 1996, 1997; Gambetti et al., 2003). The
combination of the 129 genotype and the PrPSctype has allowed for
the distinction of five subtypes of sCJD with different clinical and
histopathological features that are identified as: (i) sCJDMM1 and
sCJDMV1; (ii) sCJDVV1; (iii) sCJDMV2; (iv) sCJDVV2; and (v) the
sCJDMM2 (Parchi et al., 1999).
When we originally proposed the sCJD classification, we
observed that 14 out of the 300 cases examined (about 5%)
had both PrPSctypes 1 and 2 (Parchi et al., 1999). Since then,
the co-occurrence of PrPSctypes 1 and 2 in a relatively small
percentage of sCJD cases has been confirmed by a number of
subsequent studies (Puoti et al., 1999; Kova ´cs et al., 2002; Haı ¨k
et al., 2004; Head et al., 2004; Lewis et al., 2005; Schoch et al.,
2006; Uro-Coste et al., 2008). Recently, however, two studies
using antibodies to PrP that specifically recognize PrPSctype 1
have suggested that all cases of sCJD associated with PrPSc
type 2 also invariably contain PrPSctype 1 (Polymenidou et al.,
2005; Yull et al., 2006). This finding has raised a number of
questions: (i) Is the sCJD classification that we proposed, which
is largely based on the presence of either PrPSctype 1 or 2,
still valid or should it be changed? (ii) Does the co-occurrence of
both PrPSctypes generate a distinct phenotype? (iii) Are the
type occurring alone, if this condition exists? (iv) What are the
mechanisms of type co-occurrence?
We have decided to address these questions separately in three
groups of sCJD determined according to the MM, VV and MV
129 genotypes with the purpose of keeping the issue of PrPSctype
co-occurrence relatively simple by reducing heterogeneity. In this
initial study, we selected 34 cases of sCJD and followed a complex
strategy to analyse in detail the presence, distribution and
characteristics of PrPSctypes 1 and 2 occurring independently or
combined. We then correlated these findings with the clinical and
histopathological phenotypes of these cases.
Materials and Methods
Reagents and antibodies
PK, phenylmethylsulphonyl fluoride (PMSF), 10% sodium dodecyl
sulphate solution (10% SDS) and guanidine hydrochloride (GdnHCl)
were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tris–HCl
buffers, 30% Acrylamide/Bis, Tetramethylethylenediamine (TEMED)
Laboratories (Richmond, CA, USA). Reagents for enhanced chemilumi-
nescence (ECL plus) and the horseradish peroxidase-conjugated
antibodies were produced by Amersham Biosciences (Piscataway, NJ,
USA). The following anti-human PrP monoclonal antibodies (mAb)
were used: 3F4 to PrP residues 109–112 (Kascsak et al., 1987), 12B2
to PrP residues 89–93 (Langeveld et al., 2006), 1E4 to PrP residues
97–108 (Cell Sciences, Canton, MA, USA; Yuan et al., 2008), 9A2 to
PrP residues 99–101 (Langeveld et al., 2006), 6H4 to PrP residues
Switzerland) and 8H4 to PrP residues 175–185 (Zanusso et al., 1998).
The rabbit polyclonal antibody 2301 to PrP residues 220–231 was also
used (Chen et al., 1995; Zou et al., 2003).
We selected 34 subjects, all of whom were methionine homozygous at
codon 129 (MM) of the human PrP gene (PRNP), and had a definitive
diagnosis of sCJD (sCJDMM) at the National Prion Disease Pathology
Surveillance Centre (NPDPSC) in Cleveland, OH, USA. Based on
routine western blot (WB) examination of two or three brain regions
(including frontal, occipital and cerebellum cortices) with the mAb 3F4,
the scrapie PrP (PrPSc) type had been classified (i) PrPSctype 1 only
(sCJDMM1) (n=13); (ii) PrPSctype 2 only (sCJDMM2) (n=9); or
(iii) PrPSctypes 1 and 2 (sCJDMM1-2) (n=12). Patients lacked
Brain 2009: 132; 2643–2658 I. Cali et al.
pathogenic mutations in the PRNP and had no history of familial
diseases or known exposure to prion agents. These cases underwent
detailed analyses of the PrPScto ascertain the accuracy of their original
classification as sCJDMM1, sCJDMM2 or sCJDMM1-2.
Retrospective chart review was carried out for all subjects paying
particular attention to cardinal clinical signs of sCJD such as dementia,
ataxia and myoclonus. The findings were also recorded on electroen-
cephalography and brain magnetic resonance imaging when available.
Chi-square or Fisher exact tests were used to compare the proportions
and Wilcoxon rank sum test was used to compare median of age
and disease duration between the groups. We omitted the missing
observations during analysis.
Coronal sections of human brain tissues were obtained at autopsy and
stored at ?80?C. The entire cortical gyrus, large amounts of cerebellar
and sub-cortical tissues were taken from each brain and used for
molecular analyses. The other symmetric cerebral hemisphere was
fixed in formalin and used for histological and immunohistochemical
purposes. For all cases of sCJDMM1-2 (according to the revised-type
classification), six brain regions were sampled for western blotting:
frontal (middle gyrus) and occipital (visual) neocortices, subiculum
and entorhinal cortex, basal ganglia (putamen), thalamus and cerebel-
lum. Additional brain regions were investigated from the frontal
[superior (n=8), inferior (n=1) and more posterior middle (n=3)
gyrus] and occipital [superior (n=1) and inferior (n=8)] neocortices.
For sCJDMM1 and sCJDMM2 cases, three samples of frontal (superior
and more posterior middle gyri) and occipital cortex (inferior gyrus)
were obtained, in addition to the above six brain regions. Additional
samples including the gyrus rectus and the superior gyrus of the
occipital cortex were investigated in one sCJDMM1 and one
sCJDMM2 case, respectively. Finally, the tectum and periaqueductal
grey of the midbrain, along with the periventricular grey and inferior
olive were sampled in two sCJDMM2 cases in which the brainstem
DNA was extracted from frozen brain tissues in all the cases, and
genotypic analysis of PRNP coding region was performed as described
(Parchi et al., 1996, 2000).
Histopathology and PrP
The percent distribution of the large vacuole spongiosis throughout
the brain and fine spongiform degeneration in the molecular layer
of the cerebellum was determined by comparing haematoxylin
and eosin-stained sections in nine sCJDMM1, five sCJDMM2 and
19 sCJDMM1-2 cases (according to the revised classification). Ten
brain regions were examined: frontal, temporal, parietal, entorhinal
and visual cortices, hippocampus, basal ganglia (putamen), thalamus
(anterior and mediodorsal nuclei), substantia nigra and cerebellum. The
sCJDMM1-2 cases were divided into two groups according to the
or 58 months (8–13 months). Immunohistochemistry, according to
Parchi et al. (1996), was performed to determine: (i) the percent
areas of the cortical (frontal, temporal, parietal, entorhinal, visual
and hippocampus) and subcortical
thalamus) occupied by perivacuolar and coarse PrP immunostaining
patterns; (ii) the type of PrP immunostaining pattern in the cerebellum
(i.e. the diffuse PrP immunostaining pattern described for the
sCJDMM1 or the coarse PrP immunostaining pattern characteristic of
the sCJDMM2 subtype); and (iii) positive or negative PrP immuno-
staining of the hippocampus.
regions (basal gangliaand
Proteinase K digestions
Brain homogenates (10%wt/vol) were prepared in lysis buffer with
100mM Tris–HCl (LB100) (100mM NaCl, 10mM EDTA, 0.5% NP-40,
0.5% sodium deoxycholate, 100mM Tris–HCl, pH 8.0) (Notari et al.,
2004) and then centrifuged at 1000g for 10min to collect the
supernatant (S1). Homogenates were incubated with different PK
concentrations [48U/mg specific activity at 37?C, with 1U/ml
equal to 20.8mg/ml PK]. In an initial study, PK concentrations ranging
from 0 to 160U/ml were used to separate the bona fide PrPSc
fragments from those that were incompletely digested. As a result of
this preliminary study, a concentration of 5U/ml PK was adopted
to determine the ratio of PrPSctypes 1 and 2 when the two types
co-occurred in the same brain region, while resistance of PrPScto
digestion with 20U/ml PK was the requirement for identifying the
bona fide PrPSccore fragments (Fig. 1). When needed, P2 fractions
were prepared as previously described (Zou et al., 2003). All PK
digestions were carried out at 37?C for 1h, and then stopped by the
addition of 2mM PMSF. Samples were mixed in an equal volume of
2? sample buffer (6% SDS, 5% b-mercaptoethanol, 20% glycerol,
4mM EDTA, 125mM Tris–HCl, pH 6.8) and boiled for 10min.
Western blot analyses
Western Blots were performed as previously described with minor
modifications (Cali et al., 2006). Briefly, proteins were separated by
non-commercial 15% Tris–HCl, 20-cm-long SDS–polyacrylamide gel
electrophoresis (SDS–PAGE) gels then transferred to Polyvinylidene
Fluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA,
USA) for 2h at 60V. Each antibody was incubated for 2h at room
temperature: 3F4 (0.1mg/ml), 12B2 (0.05mg/ml), 1E4 (1mg/ml), 9A2
(2mg/ml), 6H4 (1.1mg/ml), 8H4 (1mg/ml) and 2301 (1:2000).
The experiments of 3F4 or 1E4 immunoreactivity with PrPSctype 2
that was either artificially mixed or naturally co-existing with PrPSc
type 1 (see below and Fig. 2B), were performed at the optimum
concentration of 2mg/ml for the two antibodies. In the sCJDMM1-2
cases, the ratio of the two unglycosylated PrPSctypes (T1:T2)
co-existing in the same anatomical region was calculated according
to 3F4 and 1E4 immunoreactivity to PrPSctype 2. When 3F4
immunoreacted with PrPSctype 2, the T1:T2 ratio (which was identical
to the one obtained with 6H4, 8H4 and 2301) was calculated by using
the same antibody. When 3F4 did not immunoreact with PrPSctype 2,
1E4 was employed. 1E4 could either fail to detect PrPSctype 2
(therefore, the T1:T2 ratio was equal to 100:0) or recognize a
PK-resistant fragment of 19kDa matching the core of PrPSctype 2.
In this latter case, the T1:T2 ratio was calculated as follows: (i) since
1E4 had, on average, ?12 times higher immunoreactivity to PrPSc
type 2 than PrPSctype 1, the densitometric value of PrPSctype 2
was divided by 12 (3F4 shows similar immunoreactivity for both
PrPSctypes); and (ii) the obtained value was then multiplied by two
because 3F4 has ?2 times more immunoreactivity to PrPSctype 2 than
Co-existence of PrPSctypes in sCJD Brain 2009: 132; 2643–2658 |
1E4 (as determined after antigen/antibody saturation curve studies).
Immunoreactivity of 3F4 and 1E4 to PrPSctypes 1 and 2 was also
investigated by artificially mixing low speed brain homogenates (S1)
from the sCJDMM1 and sCJDMM2 subtypes. PrPSctypes 1 and 2
were mixed in ratios of 100:0, 95:5, 90:10, 60:40, 45:55, 30:70,
15:85 and 0:100. Immunoreactivity of 3F4 and 1E4 antibodies to
both PrPSctypes did not change when PrPSctypes 1 and 2 were
denatured in the same or in different experimental tubes.
To exclude the co-existence of PrPSctypes when PrPSctype 1
appeared to be present alone after probing with 3F4, tissue was
further probed with 1E4. Similarly, if only PrPSctype 2 was suspected,
the co-existence of PrPSctype 1 was excluded with 12B2 which selec-
tively binds to PrPSctype 1. To maximize PrPSc-type immunoreactivity,
gels were loaded with up to three times the amount of tissue
homogenate loaded with 3F4, and PrPSc-enriched P2 fractions were
used when the total amount of PK-resistant PrPScwas low.
Conformational stability immunoassay
The conformational stability immunoassay (CSI) was performed as
described, with minor modifications (Zou et al., 2004; Pastore et al.,
2005). Briefly, aliquots of 20ml S1 were mixed with 20ml of GdnHCl
stock solution to give a final concentration of GdnHCl ranging from
0 to 4.0M. After 1.5h of incubation at room temperature, samples
were precipitated with 5-fold volume excess of pre-chilled methanol
overnight at ?20?C. Samples were centrifuged at 16000g for 30min
at 4?C, pellets were re-suspended in 20ml of LB100 (pH 8.0) and
sonicated. Each aliquot was digested with 5U/ml PK for 1h at
37?C. The reaction was stopped with 2mM of PMSF, denatured
and loaded onto 15% Tris–HCl pre-cast gels (Bio-Rad) for WB ana-
lyses. A total of 31 brain regions were examined: frontal [sCJDMM1
(n=1), sCJDMM1-2 (n=4)], visual [sCJDMM1 (n=7), sCJDMM2
(n=4) and sCJDMM1-2 (n=5)], entorhinal [sCJDMM1-2 (n=2)]
0 0.15 0.3 0.6 1.2 2.5 5 10 20 40 80 160
Unglyc. 20 kDacore
0 0.15 0.3 0.6 1.2 2.5
0 0.15 0.3 0.6 1.2 2.5 5 10 20 40 80 160
19 kDa core
19 kDa core
Figure 1 Validation of PrPSctyping by protease digestion in sCJDMM2 and sCJDMM1. S1 fractions from the frontal cortex were
incubated with several amounts of PK and probed with 3F4 (A, C) or 12B2 (B). (A) The core of the unglycosylated PrPSctype 2 (large
black solid arrow) is detectable after treatment with up to 40U/ml PK (up to 160U/ml in long exposure films). The partially digested
PrP fragments (dashed arrows) are visible between 0.15 (denoted by asterisk) and 0.6U/ml of PK treatment but are completely
hydrolyzed at relatively low PK concentrations ranging from 2.5 to 5U/ml (denoted by double asterisk). The bands indicated by the
black solid arrows in A* presumably represent monoglycosylated PK-resistant PrPScfragments. (B) The mAb 12B2, which binds to
the PrPSctype 1 but not to type 2 PK-resistant fragments, immunodetects the four partially digested PrP fragments but not the
unglycosylated PrPSctype 2 core confirming that the fragments are incompletely digested, while the ‘core’ belongs to type 2.
(C) Four partially cleaved PrP fragments (dashed arrows) are detectable between 0.15 and 0.6U/ml PK in sCJDMM1 preparations.
The core of the unglycosylated PrPSctype 1 (solid grey arrow) is resistant up to 40 U/ml (or up to 80U/ml PK in long exposure films).
For the small black solid arrows see (A*).
Brain 2009: 132; 2643–2658I. Cali et al.
cortices, striatum [sCJDMM1-2 (n=3)] and thalamus [sCJDMM1-2
(n=5)]. Of the 19 sCJDMM1-2 samples (13 cases), six had PrPSc
type 1 [frontal cortex (n=2) and striatum (n=1)] and type 2 [frontal
(n=1) and visual (n=2) cortices] present separately. In the remaining
13 sCJDMM1-2 samples, both PrPSctypes co-occurred in the same
anatomical brain area, i.e. in the frontal (n=1), visual (n=3) and
entorhinal (n=2) cortices, striatum (n=2) and thalamus (n=5). CSI
study of the unglycosylated PrPSctypes 1 and 2 co-existing in the
same brain region was performed in 11 out of the 13 sCJDMM1-2
samples that had the best resolution of the two unglycosylated PrPSc
bands. In 4 out of 11 sCJDMM1-2 cases, PrPSctype 2 was detectable
only by 1E4 mAb. For CSI analysis of the in vitro mixed PrPSctypes 1
and 2, S1 homogenates from sCJDMM1 (n=3) and sCJDMM2 (n=3)
were mixed to generate T1:T2 in ratios of 50:50 and incubated with
GdnHCl in the same experimental tube. Since the [GdnHCl]1/2values
vary according to the intensity of the PrP bands, we used the exposure
of the films at which the starting amount of PrPSc(as determined
at GdnHCl=0M) was similar for each sCJDMM case. Densitometric
analysis was performed with UN-SCAN-IT gel 5.1 software. Statistical
analysis was assessed by analysis of variance (ANOVA).
We investigated the effect of the following demographic, clinical and
laboratory variables on survival: race, sex, age at onset, electrophoretic
type of PrPScand stability of PrPScin GdnHCl (Safar et al., 1998;
Peretz et al., 2002). Cumulative survival curves were constructed by
the Kaplan–Meier method, both overall, and by stratifying for each of
the above variables. The 20 sCJDMM1-2 were compared with the
9 sCJDMM1 and 5 sCJDMM2 cases of the present study or with a
larger population of 166 sCJDMM1 and 19 sCJDMM2 received at the
NPDPSC from 2005 to 2007. For each type of PrPSc, we report as
descriptive statistics the survival times overall and stratified for each
variable; the comparisons of survival curves between groups were
carried out by the log rank (Mantel–Cox) and generalized Wilcoxon
test. To obtain estimates of the dependency of duration of the disease
on stability of PrPSc, the [GdnHCl]1/2 values were analysed by
non-linear regression. Statistical analyses were performed using SPSS
16 software (SPSS Inc., Chicago, IL, USA).
Percentage distribution of the sCJDMM
The original PrPSc-type distribution of the sCJDMM cases examined
by the NPDPSC was estimated based on the corrections of the type
classification made in this study, as the result of the detailed analyses
of the PrPSctype. The NPDPSC has received and classified 234
sCJDMM consecutive cases during 2005–2007, a three-year period.
Based only on routine western blotting examination, without any
PK-resistant Type 1
PK-resistant Type 2
Figure 2 Diagram of epitopes of various anti-PrP antibodies and western blotting of PrPScmixed naturally and artificially.
(A) Representation of PK cleavage sites, PK-sensitive and PK-resistant regions and epitope location in human PrPSc. Long and short
arrows identify the primary and secondary PK cleavage sites located along the variable region G74-S103. This region is depicted out of
proportion. The locations of the relevant residues for PrPSctypes 1 and 2 are indicated with white and black arrows, respectively.
Tryptophan 89 (W89*) represents the expected PK cleavage site (see PeptideCutter Program) generating the PrPSctype 1 fragment of
?20kDa when samples are homogenized at pH ?8.0. The curly brackets indicate the PrP epitopes recognized by the antibodies used in
this study. The basis for the reactivity of mAb 12B2 with PrPSctype 1 but not type 2 is illustrated with the dotted lines. (B) Both mAb
3F4 and 1E4 immunoreacted with PrPSctype 2 that had been artificially mixed with PrPSctype 1 (lanes 1 and 4). In contrast, PrPSc
type 2 from sCJDMM1-2, where types 1 and 2 co-occurred in the same brain region, was detected by 1E4 only (lanes 5 and 6). Lanes
1 and 4 were generated with tissue from the frontal cortex of sCJDMM1 and sCJDMM2; lanes 2 and 5 with thalamic tissue and lanes
3 and 6 with the cerebellum of two sCJDMM1-2 cases, respectively (T1=PrPSctype 1; T2=PrPSctype 2).
Co-existence of PrPSctypes in sCJDBrain 2009: 132; 2643–2658 |
special study, 188 (80%) of these cases were classified as sCJDMM1,
23 (10%) as sCJDMM2 and 23 (10%) as sCJDMM1-2. In the present
study, 4 of the 13 cases originally classified as sCJDMM1 and four of
the nine classified as sCJDMM2 were reclassified as sCJDMM1-2. The
original NPDPSC percent distribution of the 234 sCJDMM cases was
then corrected according to the type changes resulting from the
detailed analyses used in this study.
Validation of PrPSctyping
Optimal conditions of PK digestion
A major challenge in PrPSc-type determination is avoiding
incomplete protein digestion, most often caused by hydrolysis
with an insufficient amount of PK or performed at an unsuitable
pH (Notari et al., 2004; Cali et al., 2006). Inappropriate conditions
generate a number of smaller PrPScfragments representing the
product of the partial PrPScdigestion, which, especially in the
case of PrPSctype 2, may erroneously be construed as represent-
ing the co-occurrence of types 1 and 2 (Notari et al., 2007). Since
the major goal of this study is to explore the correlations between
the disease phenotype and the co-occurrence of both PrPSctypes,
it was imperative to distinguish the genuine cases of sCJDMM1-2
from those caused by artefacts. This was done largely according
to Notari et al. (2007). Incubation of low speed supernatant (S1)
(see Methods section) from sCJDMM1 and sCJDMM2 with PK
concentrations between 0 and 160U/ml (1U/ml equal to
20.8mg/ml PK) resulted in as many as seven higher molecular
weight (MW) PrPScfragments which could be detected in long
exposure films following treatment with low doses of PK (up to
0.6U/ml) (Fig. 1). In the sCJDMM2 preparations, of the seven
bands only the 19.0kDa band was still well detectable after diges-
tion with high doses of PK (up to 160 U/ml) in long exposure films
(Fig. 1A). The mAb 12B2, which binds to the PrPSctype 1 but not
to PrPSctype 2 PK-resistant fragment, immunoreacted with four
of the higher MW fragments but not, as expected, with the
19.0kDa fragment (Fig. 1B) (Yull et al., 2006; Notari et al.,
2007; Uro-Coste et al., 2008). This finding indicates that the
four higher MW fragments had cleavage sites closer to the
N-terminus than the19.0kDa
incomplete PK digestion, while the highly PK-resistant 19.0kDa
fragment represents the PrPSctype 2 original PK-resistant ‘core’,
thereafter referred to as type 2 core.
The same experiments carried out on sCJDMM1 preparations
revealed the same disparity in PK sensitivity (Fig. 1C) leading to
the conclusion that the four fragments migrating in sCJDMM1
above the 20.0kDa were also probably generated by incomplete
PK digestion, while the 20.0kDa band represents the unglycosy-
lated, highly PK-resistant core of PrPSctype 1 (Fig. 1C).
The two remaining higher MW bands that were still detectable
at PK concentrations of 20U/ml in both sCJDMM2 and sCJDMM1
likely carry the monoglycosylated PrPSccore of types 2 and 1,
respectively, since in the sCJDMM2 preparations they did not
react with 12B2 (Fig. 1A*–C).
fragment, probablydue to
Since 5U/ml was the PK concentration at which (i) the
bona fide PrPSccore was well represented in both sCJDMM1
and sCJDMM2; and (ii) all the PrPScfragments associated with
sCJDMM2 that resulted from PK incomplete digestion were
hydrolysed, we adopted this PK concentration for determining
the ratio of PrPSctypes 1 and 2 when they co-occurred, while
the resistance to at least 20U/ml PK was the requirement
for identifying PrPScas the bona fide PrPSccore fragment in
sCJDMM1 and sCJDMM2 (Fig. 1).
We primarily used 3F4 and 1E4 to maximize our ability to detect
both PrPSctypes 1 and 2, while 12B2 was used to identify
incompletely digested fragments (Figs 1A and 2A). The combined
use of mAb 1E4 and 3F4 was especially valuable in establishing
the ratios of types 1 and 2: (i) when types 1 and 2 co-existed
naturally in sCJDMM1-2; or (ii) in control experiments in which
PrPSctypes 1 and 2 preparations obtained from the sCJDMM1 and
sCJDMM2 were mixed in vitro. These two antibodies bound to
both PK-resistant PrPSctypes when they were mixed in vitro
(Fig. 2B). In contrast, when the PrPSctypes 1 and 2 co-existed
naturally, mAb 3F4, as well as 6H4, 8H4, 9A2 and 2301
sCJDMM1-2 cases (Fig. 2A and B and data not shown). In
contrast, 1E4 reaction was much more consistent; for example,
while 3F4 detected the co-occurrence of PrPSctypes 1 and 2 in
46 out of 114 sCJDMM1-2 brain regions, 1E4 increased this
number to 79, corresponding to 72% more brain regions with
co-existing PrPSc types. Surprisingly, the lack of 3F4 immuno-
reaction, when it occurred, was complete, i.e. no reaction could
be detected regardless of the length of exposure or amount
of loading (Fig. 2B). Therefore, in some sCJDMM1-2 cases, the
co-existence of PrPSctypes 1 and 2 in the same anatomical region
remained undetected unless 1E4 was used. Furthermore, the
complete lack of immunoreaction of PrPSctype 2 with 3F4, only
when types 1 and 2 co-existed in the same anatomical region,
indicates that in some cases the native PrPSc
sCJDMM1-2 is different from that of sCJDMM2 cases and that
the close co-existence may modify the structural characteristics of
PrPSctype 2. The detection of the PK-resistant PrPScin various
brain regions of sCJDMM1, sCJDMM2 and sCJDMM1-2 is
exemplified in Fig. 3.
2only in someofthe
type 2 in
Based on the aforementioned analyses, the original PrPSctype
classification of the 34 cases initially selected for this study had
to be changed. Four of the 13 cases classified as sCJDMM1
following routine examination and four of the nine classified as
sCJDMM2 were reclassified as sCJDMM1-2. Therefore, 8 of the
22 sCJDMM cases originally thought to be associated with only
one type of PrPScwere found to have both types. Age and gender
were similar in these three groups (P40.05). The newly formed
groups were then comparatively examined in detail with regard to
the PrPSccharacteristics, histopathological and clinical phenotype
and correlation between PrPSctype and phenotype.
Brain 2009: 132; 2643–2658I. Cali et al.
PrPSclevels and type distributions in
sCJDMM1, sCJDMM2 and
In the 20 sCJDMM1-2 subjects, both types were present in 69%
of all brain regions (79 out of 114 samples); PrPSctype 1 was
found in 21% and PrPSctype 2 in 10% (Table 1). All regions
could contain PrPSctypes 1 and 2, but the combined sub-cortical
regions, which accounted for 82%, and above all, the thalamus
accounting for 90% were best represented (Table 1). In contrast,
the cerebellum had the lowest occurrence of PrPSctypes 1 and 2
(56%); type 1 alone was observed in the remaining 44% of cases,
about three times those of the cortical and sub-cortical regions.
Type 2 alone was not observed in the cerebellum and was rare
(2%) in the sub-cortical regions, while it had the same prevalence
as type 1 alone in the cerebral cortex (Table 1).
The immunoreactivity characteristics of PrPSctypes 1 and 2
differed also in gyri of the same lobe, i.e. the superior and
middle frontal gyri or visual and non-visual occipital cortices. For
example, 50% of the pairs of samples from two adjacent gyri
differed on whether the PrPSctype 2 component was detected
when only 3F4 was used but this discrepancy was reduced to
510% after also probing with 1E4. It indicates that adjacent
brain regions could be populated by distinct type 2 species: one
recognized by both 3F4 and 1E4 and the other by only 1E4.
In addition, a difference in the ratio (but not in the number) of
the two PrPSctypes was often seen in sample pairs obtained from
the same gyrus at different anterior–posterior locations.
The study of the quantitative distribution after probing with
both 3F4 and 1E4 showed that the mean ratios of PrPSctypes 1
and 2, regardless of being found in the same or different brain
locations and expressed as percentage of the total PrPSctypes 1
and 2 (1:2), was 54%:46% in the cortical regions combined,
67%:33% in the thalamus and striatum and 84%:16% in the
cerebellum, indicating that PrPSctype 1 dominated in all the
regions examined, but especially in the sub-cortical regions and
in the cerebellum.
Characterization of PrPSctypes 1 and 2
by conformational stability
The CSI is based on the concept that slight differences in protein
structure can be determined by the measurement of the confor-
mational stability when the protein is exposed to a denaturant
such as GdnHCl in an appropriate range of concentrations
(Shirley, 1995). In the case of PrPSc, with increasing concentrations
Gr FCMPVC OCI
EC ST TH CE T2
T1 FCSFCMFCMPVC OCIEC ST TH PaqG Tct PvG Olv CE T1
10 20510 20
5 10 20
Figure 3 Examples of PK-resistant PrPScdistribution in various
brain regions in sCJDMM1, sCJDMM2 and sCJDMM1-2
according to the criteria applied in this study. (A–C) Only the
unglycosylated PrPScisoform is shown in each box. (A) In
sCJDMM1, PrPSctype 1 (20kDa, arrow) but not PrPSctype 2 is
detected in several brain regions after probing with mAb 1E4;
PrPSctype 2 (T2) is shown at each edge of the gel as a control.
(B) The PrPScassociated with sCJDMM2 (19kDa, arrow)
immunoreact with mAb 3F4. A PK-resistant PrPScfragment of
18kDa is often associated with PrPSctype 2 (18kDa, arrow)
(Pan et al., 2005); PrPSctype 1 (T1) is shown at each edge of
the gel as control. (C) A case of sCJDMM1-2 with PrPSctypes
1 and 2 co-occurring (20 and 19kDa, arrows) in four brain
regions (FC, VC, EC and CE) after incubation with 3F4.
However, with 1E4a 19kDa band matching PrPSctype 2 was
detected in the striatum (denoted by asterisk) and thalamus
(denoted by double asterisk) even after loading one-half of the
original amount used for PrPScincubation with 3F4.
FCS=frontal cortex (cx), superior gyrus; FCM=frontal cx,
middle gyrus; FCMP=frontal cx, more posterior middle gyrus;
VC=visual cx; OCI=occipital cx, inferior gyrus; EC=entorhinal
cx; ST=striatum; TH=thalamus; PaqG=periaqueductal gray;
Tct=tectum; PvG=periventricular grey; Olv=inferior olive;
CE=cerebellum; T2=PrPSctype 2; T1=PrPSctype 1.
Table 1 Topographic representation of PrPSctypes 1
and 2, type 1, and type 2 in 20 sCJDMM1-2 expressed
as percentage of the regions containing each of the three
Brain regionPrPSctypes 1
and 2 (%)
17 (n=10)17 (n=10)
82 (n=31)16 (n=6)2 (n=1)
56 (n=10) 44 (n=8)0
a Mean of frontal, occipital and entorhinal cortex.
b Total samples examined.
c Detected PrPSctype.
d Mean of striatum and thalamus.
Co-existence of PrPSctypes in sCJDBrain 2009: 132; 2643–2658 |
of GdnHCl, PrPScdissociates and unfolds from native b-sheet-
structured aggregates, becoming protease sensitive.
In the present study, the conformational characteristics of PrPSc
associated with the sCJDMM1-2 were analysed with the aim of
determining whether they (i) were similar to those of type 1 PrPSc;
(ii) were similar to those of type 2 PrPSc; (iii) matched those of
PrPSctypes 1 and 2 mixture; or (iv) were different from those
previously reported. As expected, the PrPSc
sCJDMM1 and sCJDMM2 had significantly different stability
characteristics as revealed by CSI (Fig. 4A and B). The guanidine
concentrations needed to render half of the PrPScsensitive to PK,
the so-called [GdnHCl]1/2, were 2.76M for sCJDMM1 and 1.42M
in sCJDMM2 indicating that the PrPScassociated with sCJDMM1
is ?1.9-fold more stable than that of sCJDMM2 (P50.0001)
(Fig. 4A and B). The average [GdnHCl]1/2 in the sCJDMM1-2
examined was 2.05M, i.e. roughly in between the [GdnHCl]1/2
of sCJDMM2 and sCJDMM1 (sCJDMM1-2 versus sCJDMM1,
P=0.00086; sCJDMM1-2 versus sCJDMM2, P=0.008) (Fig. 4A
and B). PrPScfrom the sCJDMM1-2 cases could be divided into
three groups according to whether (i) PrPSctypes 1 and 2 were
present separately; (ii) both PrPSctypes co-occurred in the same
brain region after probing with 3F4; and (iii) both PrPSctypes were
detected together in the same brain region after incubation
with only 1E4. To determine the CSI characteristics of the PrPSc
types 1 and 2 present together in the same brain region, it
was necessary to analyse the unglycosylated forms, which can
be monitored individually with ease. This approach allowed for
the CSI monitoring of the PrPSctypes 1 and 2, individually but
in the same preparation (Fig. 4A). Preliminary analyses with PrPSc
types 1 and 2 present separately confirmed that the [GdnHCl]1/2
data generated by CSI using the unglycosylated forms were
representative of those generated from the analyses of all the
bands (Supplementary Fig. S1) (P40.05). As expected, the
[GdnHCl]1/2of PrPScfrom sCJDMM1-2 cases with PrPSctypes 1
and 2 present separately were similar to those of the sCJDMM1
and sCJDMM2 subtypes, respectively (Fig. 4C). Surprisingly, when
the two PrPSctypes were present together in the same anatomical
region, PrPSctype 1 showed a mean [GdnHCl]1/2value similar to
that of PrPSctype 2 (2.01 and 1.75M, respectively) (Fig. 4C)
suggesting that when they occur jointly, PrPSctype 2 maintains
its original stability characteristics while PrPSctype 1 adopts those
of PrPSctype 2. To assess the specificity of this finding, we
examined the [GdnHCl]1/2of PrPSctypes 1 and 2 obtained from
sCJDMM1 and sCJDMM2, respectively, after mixing these two
species in vitro (Fig. 4C). We observed a similar result: the
[GdnHCl]1/2of the unglycosylated PrPSctype 1 from the in vitro
mixture shifted similarly towards that of the type 2 present in
the same mixture (Fig. 4C). This finding makes it impossible to
determine whether the stability shift of PrPSctype 1 towards PrPSc
type 2 takes place only in vitro or also in intact tissue when the
two types occur together. Finally, a difference in [GdnHCl]1/2
immunoreacting only with the mAb 1E4 and the PrPSctype 2
recognized by both 1E4 and 3F4 mAb (2.25 and 1.60M, respec-
tively), but the difference did not reach statistical significance
immunohistochemistry in sCJDMM1,
sCJDMM2 and sCJDMM1-2
Type and topography of the lesions in sCJDMM1 and sCJDMM2
were similar to those previously described (Parchi et al., 1996,
1999). Briefly, in sCJDMM1, the spongiform degeneration was
characterized by fine vacuoles homogeneously distributed within
the affected brain regions (Fig. 5A and B). None of the nine
sCJDMM1 cases, including three cases having disease duration
of 8, 11 and 24 months, respectively, showed the large vacuoles
typical of sCJDMM2. The cortical and sub-cortical distributions of
the spongiform degeneration were similar to those previously
reported (Fig.5A) (Parchi
showed moderate vacuolization, often focal, except for one case
of 24-month duration, where the spongiosis was ubiquitous
In sCJDMM2, the spongiform degeneration consisted mostly of
large vacuoles, the hallmark of this subtype, often mixed with fine
spongiform degeneration indistinguishable from that of sCJDMM1
(Fig. 5C) (Parchi et al., 1996). Overall, spongiosis and astrogliosis
were more pervasive than in sCJDMM1. The lesion distribution
was as previously reported (Fig. 5A) (Parchi et al., 1999). As in
sCJDMM1, no spongiform degeneration was associated with the
hippocampal gyrus, but contrary to sCJDMM1, the cerebellum
was virtually spared (Fig. 5A).
In sCJDMM1-2, most of the cortical sections had both large and
fine vacuoles as sCJDMM2, but in sCJDMM1-2 the spongiform
degeneration was more circumscribed. Overall, the sCJDMM1-2
spongiform profile (as determined by the relative regional area
occupied by the ‘grape-like’ type of spongiform degeneration)
ran approximately in between that of sCJDMM1 where the
large vacuoles were absent and that of sCJDMM2 where ?40%
of the cortex was occupied by large vacuoles (Fig. 5A). In
contrast, the cerebellum showed a degree of fine spongiform
degeneration in the molecular layer, comparable with that of
sCJDMM1and only inthe
significantly more severe (P50.002) than those of sCJDMM2,
matching in severity with those of sCJDMM1 (Fig. 5A).
The lesion profiles determined separately in sCJDMM1-2 cases
with 58-month and 58-month durations revealed no significant
difference between the two groups although lesions were consis-
tently more severe in the 58-month group; both groups closely
paralleled the lesion profile of sCJDMM2 with the exception of
the cerebellum (P50.03) (Fig. 5A).
The immunohistochemistry of the sCJDMM1 subtype demon-
strated the classical punctate or ‘synaptic’ pattern of staining with
the intensity distribution previously reported (Parchi et al., 1996).
In sCJDMM2, both the perivacuolar and coarse patterns of PrP
deposition were clearly distinguishable from the PrP staining
pattern of sCJDMM1. Semi-quantitative analysis revealed that
the temporal neocortex, with on average 95% of the surface
immunostained, was the most affected cortical region followed
by the parietal and occipital (80%), entorhinal (74%) and frontal
(70%) cortices (Table 3). The coarse immunostaining pattern was
Brain 2009: 132; 2643–2658 I. Cali et al.
invariably present in the molecular layer of the cerebellum. The
coarse pattern of PrP immunostaining in the five cases examined
occupied 70% of the averaged surfaces of the cortical (frontal,
occipital and entorhinal) and sub-cortical (basal ganglia and
thalamus) regions (Table 3). In the cerebellum, the coarse immu-
nostaining pattern occupied ?10% of the molecular layer.
In sCJDMM1-2, the brain surface showing the coarse and
perivacuolar immunostaining pattern was, on average, smaller in
0.51.0 1.52.0 2.53.0 3.5 4.0
Figure 4 CSIs of PrPScfrom each sCJDMM group of cases. (A) Representative WB of PK-resistant PrPScfrom sCJDMM1, sCJDMM2
and sCJDMM1-2 probed with 3F4at increasing molar (M) concentrations of GdnHCl and used for the CSI shown in (B). The case of
sCJDMM1-2 illustrated here is case 6 of Table 3, and is identified with an asterisk in (B). The two arrows indicate the unglycosylated
bands belonging to PrPSctype 1 (T1) and type 2 (T2) analysed for the sCJDMM1-2 of C. (B) GdnHCl molar amounts needed to render
PK-sensitive one-half of the PrPSc, [GdnHCl]1/2, as determined with CSI performed with the whole PrPScfrom sCJDMM1, sCJDMM2
and sCJDMM1-2 subtypes. PrPScfrom sCJDMM1 (blue circles), sCJDMM1-2 (black diamonds) and sCJDMM2 (red triangles) show
distinct [GdnHCl]1/2values. The sCJDMM1-2 group appears to include two populations with a mean [GdnHCl]1/2of 2.4?0.2 and
1.7?0.1, respectively. The [GdnHCl]1/2correlated with the disease duration, which was 4.4?2.8 months in the cases of the upper
cluster and 7.8?3.2 months in the lower. The lower cluster includes cases 4, 12, 13, 14 and 15 of Table 3. In 1 of the 7 sCJDMM1
cases and in patients 3 and 1 of the 13 cases of sCJDMM1-2, [GdnHCl]1/2represents the mean of 2 or 3 brain regions. The disease
duration of the seven sCJDMM1 cases spans from 1 month [(GdnHCl)1/2=2.54M] to 24 months [(GdnHCl)1/2=2.51M]. (C)
[GdnHCl]1/2of the unglycosylated PrPScisoform from sCJDMM1 (blue circles), sCJDMM1-2 (black diamonds), sCJDMM2 (red
triangles) and PrPSctypes 1 and 2 from sCJDMM1 and sCJDMM2 mixed in vitro (black squares). The 17 tests from the sCJDMM1-2
cases are grouped as follows: tests of PrPSctypes 1 or 2 present separately (alone), and of PrPSctypes 1 and 2 co-existing in the same
preparation but quantified separately by densitometric analysis of the corresponding unglycosylated band [see arrows in (A)] (co-exist).
(D) CSI curves of the unglycosylated PrPScisoform from the sCJDMM1-2 regions in which PrPSctype 2, co-existing with PrPSctype 1 in
the same brain region, was recognized either by both mAb 3F4 and 1E4 (green line) (n=7 regions) or exclusively by 1E4 (orange line)
(n=4 regions). The values of [GdnHCl]1/2for each curve are indicated on the X-axis; (M)=molarity.
Co-existence of PrPSctypes in sCJDBrain 2009: 132; 2643–2658 |
all of the combined cases than that of sCJDMM2 cases; the high-
est percentage was detected in frontal, occipital and entorhinal
cortices combined (23%), thalamus (22%) and striatum (8%)
(Table 3). Only 2% of the area of the molecular layer of the
cerebellum was occupied by the coarse PrP immunostaining
characteristic of sCJDMM2, while ?18% was associated with
the punctuate pattern of the sCJDMM1 (Fig. 5D).
Correlations of disease duration with
clinical features, PrPSctype,
immunostaining pattern and PrPSc
representation in sCJDMM1-2
There was no prevalence of any of the clinical signs and tests
examined among the three groups which would significantly
probably because the number of cases examined was too small
(Table 2). The mean ages at onset were also similar: 61 years in
sCJDMM1-2, 68 years in sCJDMM1 and 64 years in sCJDMM2.
However, sCJDMM1-2 subjects presented with progressive cogni-
tive decline in almost all the cases (95%) and had a relatively high
prevalence of MRI abnormalities (58%), like the subjects with
sCJDMM2. Other signs such as ataxia and myoclonus, along
with typical EEG changes, had a more similar prevalence to
those of sCJDMM1.
A major and readily measurable clinical feature that clearly
distinguishes sCJDMM1 and sCJDMM2 is the disease duration
(Parchi et al., 1999). The mean disease durations in sCJDMM1,
6.3?3.6 months, respectively. Therefore, the disease duration
sCJDMM1 case with the atypical 24-month duration is excluded,
the Kaplan–Meier analyses showed that patients with PrPSctype
1 had a significantly shorter survival (P=0.029) than patients
with PrPSctype 2, despite the same codon 129MM polymorph-
ism, age and sex distribution (data not shown; Table 2). There is
an apparent trend for longer survival in sCJDMM1-2 when com-
pared with those with pure type 1. However, this trend was not
sCJDMM1-2 is significantly shorter than that of sCJDMM2
(P=0.034). When the Kaplan–Meier analyses were performed
on 166 sCJDMM1 and 19 sCJDMM2 patients, consecutively
(P40.05). However,if the
Figure 5 Spongiform profile of the sCJDMM patients. (A) Percent distribution of large vacuole spongiform degeneration in sCJDMM1,
sCJDMM2 and sCJDMM1-2. Individual data points are the mean percentage of the surfaces occupied by large vacuole spongiform
degeneration characteristic of sCJDMM2. Standard deviations are omitted for clarity. In the cerebellum, the bars refer to the percentage
of the surface of the molecular layer occupied by the ‘fine’ type of spongiform degeneration characteristic of sCJDMM1. On average of
all regions, sCJDMM1-2 had 3.2 times and statistically significant less large vacuole spongiform degeneration than sCJDMM2
(P50.05). No large vacuole spongiform degeneration was seen in sCJDMM1. The profiles of sCJDMM1-2 of long (58 months) and
short (58 months) durations were not significantly different (P40.05). The fine spongiform degeneration in the cerebellum of
sCJDMM2 cases was significantly less (P50.05) than in the other subtypes. Statistical analyses were performed by ANOVA followed by
the Dunn’s multiple comparison post-test. (B) sCJDMM1 fine spongiform degeneration. (C) sCJDMM2 large vacuole spongiform
degeneration. (D) Fine, punctuate (circle) and plaque-like (squares) patterns of PrP immunoreactivity in the cerebellum of the
sCJDMM1-2. FC=frontal cortex; TC=temporal cortex; PC=parietal cortex; OC=occipital (visual) cortex; HI=hippocampus;
EC=entorhinal cortex; BG=basal ganglia (putamen); TH=thalamus (anterior and mediodorsal nuclei); SN=substantia nigra.
Brain 2009: 132; 2643–2658I. Cali et al.
received by the NPDPSC from 2005 to 2007, and compared with
our group of sCJDMM1-2 cases (Supplementary Fig. S2), the
sCJDMM1cases hada shorter
(P50.0001), whereas the disease duration of the sCJDMM1-2
was intermediate but significantly different from both sCJDMM1
(P=0.004) and sCJDMM2 (P=0.001). These findings support
the hypothesis that in sCJDMM, the type of PrPScdeposited in
the brain influences the disease duration. Therefore, we searched
for the correlation of the sCJDMM1-2 disease duration (which
ranges between 1 and 13 months) with (i) the extent of the
immunochemical features mimicking those of sCJDMM2; and
(ii) the relative amount of PrPSctype 2 after separating the
sCJDMM1-2 cases into two arbitrary groups according to
whether the disease duration was 58 months or 58 months
(Table 3). The first group of 12 cases had a mean disease dura-
tion of 3.7?1.8 months (range: 1–7 months); in the second
group of eight cases the mean disease duration was 10.1?1.7
months (range: 8–13 months). On average, the extent of the
sCJDMM2-like PrP immunostaining pattern and the amount of
PrPSctype 2 in cases with disease duration 58 months were
?2.0 and 1.8 times higher than that associated with 58-month
duration cases (P50.02–0.05). Furthermore, the analysis of the
relationship between disease duration and the extent of PrPSctype 2
immunostaining pattern along with percent representation of the
PrPSctype 2 revealed good correlations among these variables
(r=0.83 and 0.81, respectively), further strengthening the conclu-
sion that the disease duration is directly related to both PrPSctype 2
representation and the extent of sCJDMM2-like immunostaining
pattern (Supplementary Fig. S3).
Previous studies have shown that the co-occurrence of PrPSctypes
1 and 2 in sCJDMM subjects may be over-diagnosed unless
stringent experimental conditions are followed, allowing the dis-
tinction of the bona fide PrPSctypes 1 and 2 fragments, referred
to as core fragments, from the partially digested fragments (Notari
et al., 2004, 2007). On the other hand, the co-occurrence of PrPSc
types 1 and 2 may be underestimated if appropriate antibodies are
not used or a sufficient number of brain regions are not
examined. To navigate these Scylla and Charybdis of PrPSc
typing, we used differential digestion to separate the PrPSccore
fragments that were highly resistant to PK hydrolysis from the
fragments generated during the stepwise digestion of PrPScas
previously shown by Notari et al. (2007). Since the partially
digested fragments are present in all cases regardless of the
PrPSctype and whether PrPSctypes occur independently or
combined, we inferred that they do not act as component of
the prion strain and, therefore, do not play a role in phenotype
The previous reports on high prevalence of co-occurrence of
both PrPSctypes in sCJDMM cases are based on the use of anti-
bodies that specifically recognize PK-resistant fragments of PrPSc
type 1, increasing the chances of detecting type 1 in sCJDMM2
cases. We have also used one of these antibodies, the mAb 12B2
(Langeveld et al., 2006) (Fig. 2A). To minimize the chances of
missing PrPSctype 2, we used mAb 1E4 which has been demon-
strated to have higher immunoreactivity to PrPSctype 2 and lower
to type 1, than 3F4 (Yuan et al., 2005, 2006, 2008; Gambetti
et al., 2008).
The identification of the types 1 and 2 core fragments, based on
the degree of protease resistance and the careful typing of the
core fragments with auxiliary antibodies, have in our opinion
maximized the detectability of ‘true’ PrPSctypes 1 and 2 in the
present study. The detailed search and analyses of PrPSctypes 1
and 2 in 277 brain regions from 34 sCJDMM cases, the correlation
between the PrPSctype with the disease phenotype and the PrPSc
characterization have lead to several conclusions that deserve
Table 2 Clinical features of sCJDMM1-2, sCJDMM1 and sCJDMM2
PresentationMM1-2 (n=20)MM1 (n=9) Statistical testa
MM2 (n=5)Statistical testa
Age at onset (mean?SD) (years)
Duration (mean?SD) (months)
PSWC on EEG
All values are given as n (%) unless otherwise mentioned.
a Chi-squared or Fisher exact tests were used to compare the proportions and Wilcoxon rank sum test used to compare median of age and disease duration between
b Cases with the feature listed/total cases examined.
c As determined with diffusion-weighted MR images.
Co-existence of PrPSctypes in sCJDBrain 2009: 132; 2643–2658 |
Existence of sCJDMM cases carrying
solely PrPScType 1 or 2, along with
cases carrying both PrPSc
types 1 and 2
Contrary to the notion put forward previously that all cases asso-
ciated with PrPSctype 2 invariably also carried type 1, our analysis
shows that along with sCJDMM cases carrying both PrPSctypes
(sCJDMM1-2) there are cases in which only either type 1
(sCJDMM1) or type 2 (sCJDMM2) can be detected (Parchi
et al., 1999; Puoti et al., 1999; Head et al., 2004; Schoch
et al., 2006; Uro-Coste et al., 2008). The precise prevalence of
the type ‘pure’ and ‘mixed’ cases cannot be established in the
present sCJDMM cases because they were not collected randomly
but based on the criteria of adequate representation of the three
sCJDMM case groups. Therefore, to obtain an estimate of
the sCJDMM-type distribution, we applied the same PrPSctype
corrections, which were prompted by the extensive analyses
carried out in this study, to the percent distributions of the
sCJDMM1, sCJDMM2 and sCJDMM1-2 cases received and
examined at the NPDPSC from 2005 to 2007 (see Methods
section for details). Using this approach, we estimate that of the
234 consecutive sCJDMM cases, 39% were sCJDMM1-2 while
the sCJDMM1 and sCJDMM2 cases accounted for 56% and
5%, respectively. Although showing conspicuous percentage of
sCJDMM1-2 cases, our data—along with those by Puoti et al.
et al. (2006),
Uro-Coste et al. (2008)—support the existence of sCJDMM
cases solely associated with PrPScof one type. The consideration
that more extensive sampling would have eventually uncovered
small amounts of the other PrPSctype making all cases types 1
and 2 cannot be dismissed in this and previous studies (Head
et al., 2004). Such consideration seems to be of theoretical
importance, but likely to be less relevant to the purpose of assessing
the effect that the co-occurrence of both PrPSctypes has on the
sCJDMM phenotype. Therefore, according to our findings, the cur-
rent classification of sCJDMM into the sCJDMM1 and sCJDMM2
subtypes should be maintained. Whether sCJDMM1-2 deserves to
be considered as a distinct subtype of sCJD will be considered below.
Phenotypically, sCJDMM1-2 Falls in
between sCJDMM1 and sCJDMM2
Although some differences in clinical features emerge between
sCJDMM1-2 and the other two subtypes, they are not sufficient
Table 3 Percentage of surface occupied by the PrP sCJDMM2 immunostaining pattern (IP), representation of type 1 or 2
immunostaining pattern in the hippocampus and relative amount of PrPSctype 2 in sCJDMM1-2 cases grouped according
to disease duration
CaseDurationsCJDMM2 PrP IP (%)a
PrP IHC patternPrPSctype 2 (%)b
ST TH CE
Cases with single PrPSctype 1 or 2
MM1 (n=9)6.1?7 (mean?SD)
MM2 (n=5)11.8?5 (mean?SD)
T2/T1 ratio 2/9
36?38 T2/T1 ratio 4/3
a Data obtained with 3F4 and expressed as percentage of the total surface examined.
b Data obtained from PrPScimmunoblots probed with 3F4 and 1E4 and expressed as percentage of total PrPScpresent.
c Average of frontal, occipital and entorhinal cortices.
d Data expressed as the percentage of the total immunostained area with 3F4 occupied by aggregates of coarse PrP immunostaining pattern (IP).
Brain 2009: 132; 2643–2658I. Cali et al.
to identify sCJDMM1-2 as a clinically identifiable entity. However,
it has been reported that patients with co-occurrence of PrPSc
types 1 and 2 presented with a clinical phenotype that was
different from those of the sCJDMM1 and sCJDMM2 subtypes
etal., 2006).The histopathological and immuno-
histochemical characteristics were also intermediate between
those of sCJDMM2 and sCJDMM1, except for the cerebellum
where the spongiform degeneration matched in type and severity
the sCJDMM1-2 PrPimmunostaining
sCJDMM1 pattern in 19 of 20 sCJDMM1-2 cases (95%), which
was absent in the cerebellum of the sCJDMM2 cases. Therefore, if
there isa defining histopathological
sCJDMM1-2, it is the association of abundant large vacuoles
and spongiform degeneration, characteristic of sCJDMM2, in
the cerebral cortex and sub-cortical regions along with the fine
spongiform degeneration and PrP immunostaining pattern of
sCJDMM1 in the molecular layer of the cerebellum.
in the cerebellum,
Characteristics of PrPScin sCJDMM1-2
The brain distribution of PrPSctypes 1 and 2, present either
together or separately, appeared to be non-random. Both types
combined were found in the thalamus of 90%, and in the
cerebellum of 56% of the cases. This diverse distribution suggests
that the surrounding tissues in different brain regions have a
distinct effect on the formation of types 1 and 2 in sCJDMM1-2
with the thalamus especially favouring the co-occurrence of the
two types. However, when occurring in isolation, the cerebral
cortex was the most permissive of the formation of type 2,
while the cerebellum, where PrPSctype 2 alone was never
observed, was the least permissive. Overall PrPSctype 1 was
better represented than type 2 when they were present separately
or together. When occurring separately, type 1 was observed in
69% of the brain regions examined from the 20 sCJDMM1-2
cases; type 2 in the remaining 31%. When present together,
the average ratio of PrPSctypes 1 and 2 expressed as a percentage
of the total PrPSctypes 1 and 2 found in the cortical, sub-cortical
regions and cerebellum, was 61% for type 1 and 39% for type 2.
Both sets of data argue that in sCJDMM1-2 the PrPCto PrPSc
conversion process is skewed in favour of type 1. This finding
is not surprising since methionine homozygosity at codon 129,
as in sCJDMM, strongly favours the formation of PrPSctype 1
(see below) (Gambetti et al., 2003). We observed a good
correlation between the representation of the PrPSctype 2 and
the extent of the corresponding histopathological lesions, as well
as PrP immunostaining pattern in agreement with the conclusions
by Puoti et al. (1999) that the PrPSc
histopathology in sCJDMM cases. Since the frontal cortex is the
anatomical region having the highest amount of PrPSctype 2 and
the thalamus having the highest co-occurrence of PrPSctype 1 and
2, these two brain regions are the most suitable for the tissue
diagnosis of sCJDMM1-2.
There was also a definite correlation between disease duration
and representation of PrPSctype 2 over that of PrPSctype 1 as well
as ofsCJDMM2 histological
brain region examined. This is of interest since perhaps the least
type influences the
phenotypic features in every
ambiguous clinical difference between sCJDMM1 and sCJDMM2
is the disease duration. The finding that in sCJDMM1-2, PrPSc
type 2 is under-represented in the cases of short duration but
increases over the type 1 (and the phenotype becomes progres-
sively more similar to that of sCJDMM2) with longer disease dura-
tion indicates that the rate of PrPSctype 2 formation exceeds that
of PrPSctype 1 and raises the question of whether the mere pres-
ence of PrPSctype 2 is the determinant of the disease duration or
the longer duration determined by other factors that allows for
accruing larger amounts of PrPSctype 2. This chicken-and-egg
question is difficult to answer. However, two considerations
seem to argue that the presence of PrPSctype 2 plays a role in
the determination of the disease duration. The first is that the
sCJDMM1 and of sCJDMM2, especially when the current, exten-
sively scrutinized sCJDMM1-2 cases are compared with large
series of cases of sCJDMM1 and sCJDMM2; the second is the
aforementioned strong correlation between PrPSctype 2 represen-
tation and duration of the disease. On the other hand, duration
alone is not sufficient to permit the formation of PrPSctype 2 in all
cases since three sCJDMM1 cases with disease duration of 8, 11
and 24 months carried PrPSctype 1 only and, as previously shown
(Cali et al., 2006), had the same CSI characteristics as the PrPScof
the short duration cases.
An unexpected finding in sCJDMM1-2 is the physical–chemical
features of the PrPSc. Using the two mAb, 3F4 and 1E4, which
recognize overlapping PrP epitopes but seemingly have distinct
affinities for different PrPScconformations (Yuan et al., 2006,
2008; Gambetti et al., 2008), we observed that, in sCJDMM1-2,
type 2 immunoreacted only with 1E4 and not with 3F4, regardless
of the immunoblot loading and film exposure. This 3F4 versus 1E4
immunoreactivity difference was observed only when PrPSctypes
1 and 2 shared the same anatomical region and not when they
were from different regions or cases or were mixed in vitro. CSI
that gauges the PrPScconformation by determining the resistance
to denaturation, showed a difference in stability of ?0.65M
between the PrPSctype 2, exclusively recognized by 1E4, and
that recognized by both but it was not statistically different, prob-
ably due to the limited number of cases tested. However, the
differential mAb recognition indicates that the co-occurrence is
likely to affect the conformation of PrPSctype 2 in sCJDMM1-2.
The 1E4 properties that determine this selectivity and differentiate
this mAb from other antibodies to PrP are unclear. In a recent
study (Yuan et al., 2008) we observed that, although 1E4 and
3F4 have adjacent epitopes along human PrP residues 97–112,
their accessibility to these epitopes is different because of different
neighbouring N-terminal residues. It is possible that the 1E4 selec-
tively detected PK-resistant PrP fragments have N-terminal starting
sites that are different from those of the well-characterized PrPr
types 1 and 2, even if the difference is based on only a few amino
acids. An alternative or complementary explanation is that 1E4 is a
partially conformation-sensitive antibody, which is affected by the
residual conformation of PrP maintained in polyacrylamide gels.
Additional analyses of 1E4, along the lines of the characterization
we carried out for a conformational mAb to PrPSc, are needed to
resolve this issue (Zou et al., 2004).
is differentfromthose of
Co-existence of PrPSctypes in sCJDBrain 2009: 132; 2643–2658 |
Extending the CSI analyses to all sCJDMM subtypes, we
observed (as expected) that when PrPSctypes 1 and 2 occur
separately, either in different cases (as in sCJDMM1 and
sCJDMM2) or in different anatomical regions of the same case
(as in some of the sCJDMM1-2 brain regions), the two types
have distinct CSI characteristics consistent with different con-
However, when PrPSctypes 1 and 2 occurred together in the
same anatomical region, PrPSctype 1 took on CSI characteristics
similar to those of the PrPSctype 2, although it apparently
maintained the original PK cleavage site. However, this shift in
PrPScfeatures did not require in vivo conditions, such as the
presence of intact tissue or long co-existence time of the two
types, since it was also observed in vitro after artificially mixing
the two PrPSctypes. Therefore, even in sCJDMM1-2 this CSI shift
might take place in vitro at the time of homogenization and not
bepresent in theintact tissue.
represents, to our knowledge, the first demonstration of a drastic
conformational effect of a naturally occurring PrPScstrain on
another when they are mixed. This finding raises challenging ques-
tions: does the new PrPSctype 2-like conformation confer pheno-
type-determining characteristics of the PrPSctype 2 to the re-
conformed PrPSctype 1? What is the mechanism by which the
protease cleavage site of PrPSctype 1 is maintained in the pres-
ence of drastic CSI conformational changes? What is the role of
each of the two conformations, one characterized by the protease
cleavage site and the other by the CSI features, in determining the
disease phenotype? How common is the shift of the PrPScCSI
characteristics in prion diseases? These questions must wait for
experiments involving transmission to suitable animal models and
protein misfolding cyclical amplification (Bartz et al., 2000; Jones
and Surewicz, 2005; Meyerett et al., 2008; Jones et al., 2008).
Does sCJDMM1-2 deserve to be
considered as a distinct subtype of
sCJDMM1-2 is different from those reported in large sCJDMM1
and sCJDMM2 patient populations (Parchi et al., 1999; Pocchiari
et al., 2004; Krasnianski et al., 2006; Collins et al., 2006;
Heinemann et al., 2007; this study). Furthermore, the contrasting
histopathological features (significant large vacuole spongiform
degeneration in the cerebral cortex and fine spongiform degener-
ation in the cerebellum) as well as PrP immunostaining pattern
(mixed sCJDMM1 and sCJDMM2 patterns) allow sCJDMM1-2
to be distinguished from sCJDMM1 and sCJDMM2. Finally, in
addition to being associated with both PrPSctypes, sCJDMM1-2
also seems to often be characterized by the presence of a unique
PrPSctype 2 species that immunoreacts with mAb 1E4, but not
with 3F4, and other PrP mAb and might have distinct CSI char-
acteristics. This argues that the PrPScassociated with sCJDMM1-2
is not simply a mixture of the two PrPSctypes but it has unique
features. Therefore, it seems justified to keep sCJDMM1-2 as a
separate subtype of sCJDMM at this time.
Speculations on the mechanisms of
PrPSctype co-occurrence in
Considerable evidence indicates that PrPSc-type formation is
influenced by the genotype at codon 129 of the PrP gene.
However, a considerable number of cases appear to escape this
rule. In a series of 172 cases of sCJD carrying either PrPSctype 2
or 1 examined in a previous study, PrPSctype 1 was associated
with methionine homozygosity at codon 129 (129MM) in 95% of
the cases. In contrast, when one or both 129 valine codons
(129MV or 129VV) were present, 86% of the cases had PrPSc
type 2 (Parchi et al., 1999; Gambetti et al., 2003; Gambetti
unpublished data). In the series of 234 sCJDMM cases corrected
for PrPSctype distribution reported in this study (see above), 56%
were associated with type 1 only and 5% with type 2 only, while
in 39%, both types were detectable. These data argue that
although the 129MM genotype favours the deposition of type 1
and the presence of one or two 129 valine codons favours PrPSc
type 2 deposition, at least 5–14% of the sCJD cases, including the
sCJDMM cases, are insensitive to the influence of codon 129.
We propose that these considerations equally apply to individual
brain regions. This suggestion carries several implications, which
are in agreement with the present findings. First, it is obvious that
the chances to detect cases of sCJDMM1-2 are directly related to
the number of areas examined. Second, different brain regions
may have different degrees of permissiveness for the formation
of the ‘escaped’ PrPSctype. We observed that the thalamus was
the most permissive location for the deposition of both PrPSctypes
and the cerebellum the least permissive, apparently not supporting
the formation of type 2 alone. Third, the examination of
the relative representation of the two PrPSctypes related to
disease duration suggests that PrPScdeposition in the cases of
sCJDMM1-2 starts with predominantly or exclusively type 1.
Then, after it appears, type 2 increases at a faster rate since,
relative to the type 1, its amount increases progressively with
the disease duration. The possibility that PrPScaugments because
of types 1 to 2 conversion cannot be excluded. However, this
possibility seems unlikely because of the strong correlation
between type and histopathological features. If the conversion
of types 1 and 2 occurred, the histopathological features asso-
ciated with PrPSctype 1 should be over-represented compared
to the amount of PrPSctype 1. This is because it is unlikely that
these features would disappear or convert into the features asso-
ciated with the type 2.
The authors are deeply grateful to Ms Diane Kofskey, Ms Phyllis
Scalzo and Ms Kay Edmonds for their invaluable technical help.
NIH Award AG-14359; CDC Award UR8/CCU515004; the
Charles S. Britton Fund; the CJD Foundation.
Brain 2009: 132; 2643–2658I. Cali et al.
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