Introduction
Prion diseases are a group of neurodegenerative disorders of humans and other mammals characterized by misfolding of the cellular prion protein (PrP
C). In the disease, PrP
C is structurally converted into a pathogenic isoform, called scrapie prion protein (PrP
Sc), showing an increase in β-sheet content and a partial resistance to proteases in its C-terminal region [
27]. As a consequence of PrP
C conversion, oligomers and amyloid fibrils of aggregated PrP
Sc accumulate in the CNS, leading to neurodegeneration.
Sporadic Creutzfeldt-Jakob disease (sCJD), the most common prion disease in humans, can be classified into 6 major phenotypic variants, according to molecular, histopathological, and clinical features [
21,
24,
25]. These variants or histotypes largely correlate at molecular level with the genotype at the polymorphic
PRNP codon 129, encoding for methionine (M) or valine (V), and the relative molecular mass of PrP
Sc core fragment generated after proteolytic digestion, which can be 21 (type 1) or 19 kDa (type 2) [
22]. These are C-terminal fragments that differ from each other for an epitope spanning residues 82–96, which is present in type 1 and removed in type 2. Other physico-chemical properties distinguishing PrP
Sc aggregates among sCJD variants, associated with either type 1 or type 2, include the relative amount of the truncated C-terminal fragments, named CTF12–13 based on their molecular mass, and the so-called glycoform ratio, that is the ratio among the three differently glycosylated (e.g. di-, mono-, and unglycosylated) PrP
Sc forms [
20,
22,
32].
Five out of six of these major sCJD variants were shown to propagate in syngeneic hosts as distinct prion strains [
2,
17,
23]. These are defined as natural isolates of infectious prions characterized by distinctive clinical and neuropathological features, which are faithfully recapitulated upon serial passage within the same host genotype [
3,
4]. As the only exception, sCJDVV2 and MV2K converged to a single phenotype/strain after experimental transmission [
15,
23], suggesting a host-genotypic effect determined by codon 129. Interestingly, the strain isolated from sCJDMV2K and VV2, currently designated as V2, has also been associated with kuru as well many iatrogenic cases of CJD secondary to contaminated growth hormone or dura mater grafts (d-CJD) [
13,
23,
28]. Moreover, at variance with sCJD, iatrogenic CJD patients linked to the V2 strain include subjects carrying MM at codon 129 in addition to those carrying VV or MV [
13,
14,
28].
PrP-amyloid plaques represent a distinctive histopathological feature in CJD since they show a strong correlation with both prion strain and
PRNP genotype. The presence of florid plaques is a well-documented signature of vCJD (BSE strain) [
31], while kuru-type plaques are the hallmark of the CJD V2 strain, although only in subjects carrying MV or MM at
PRNP codon 129, since they are virtually lacking in those carrying VV despite the widespread focal PrP plaque-like deposits [
24,
28].
Experimental transmissions have linked sCJDMM1 to a distinctive prion strain, named M1 [
2,
23], which is typically associated with a diffuse, synaptic type of PrP deposition rather than with focal plaque-like protein aggregates. As a significant exception, however, Kobayashi et al. [
12] described 3 sCJD cases, all with a relatively long disease duration and quite severe pathology, resembling the MM1 subtype in most features but the presence of PrP-amyloid plaques in both subcortical and deep nuclei white matter. This observation raises questions about the origin of this phenotype, namely the role of disease duration, prion strain and host genetic background in the formation of white matter PrP plaques.
To contribute to answering these questions, in this study we report the clinical, histopathological and PrPSc biochemical characterization of five European MM1 cases with white matter plaques and the results of the experimental transmission to bank voles of one of these cases. Results are compared to those obtained in the typical MM1 subtype.
Materials and methods
Patients and tissues
We studied 5 subjects affected by CJDMM1 associated with PrP
Sc plaque-type deposits in white matter (hereafter indicated as p-CJDMM1) and 8 cases affected by typical CJDMM1 (hereafter indicated as np-CJDMM1). All cases were referred for diagnosis to the Laboratory of Neuropathology, University of Bologna, Italy between 2005 and 2016 as part of the National Surveillance program on CJD and related disorders or (one p-CJDMM1) in the context of a collaborative effort with the Dutch Surveillance Centre for Prion Diseases on the molecular characterization of autopsy confirmed prion cases [
10]. The 8 selected np-CJDMM1 control cases were representative of the spectrum of clinical and histopathologic features of the sCJDMM1 subtype [
21,
25] including disease duration (range 1–14 months).
Brains were obtained at autopsy, one half, or tissue blocks from representative areas, were immediately frozen at −80 °C, whereas the rest was fixed in formalin.
Clinical and diagnostic evaluation
We collected and reviewed all available medical information from hospital reports, including results of neurologic examination(s), cerebral magnetic resonance imaging (MRI) studies and electroencephalographic (EEG) recordings. We defined the date of disease onset as the time when unexplained progressive neurological or psychiatric symptoms first occurred, and as ‘onset symptom(s)’ the first neurological disturbance(s) complained by the patient. We measured total tau (t-tau) protein levels in the cerebrospinal fluid (CSF) by quantitative ELISA (INNOTEST hTAU Ag, Innogenetics) according to the manufacturer’s instructions, considering as an optimal cut-off value 1250 pg/mL on the basis of receiver operating characteristic curve analysis, as previously described [
16]. Semi-quantitative detection of CSF 14–3-3 protein was performed by western blotting, as previously described [
16].
Genetic analysis
Genomic DNA was extracted from blood or frozen brain tissue. Genotyping of the
PRNP coding region was performed as described [
10]
.
Neuropathology
We semi-quantitatively evaluated gray matter spongiform change and astrogliosis in 10 brain regions on hematoxylin and eosin stained sections, as reported [
21].
For PrP immunohistochemistry, paraffin sections from formalin-fixed and formic acid treated blocks were processed using the monoclonal antibody (mAb) 3F4 (1:400, Signet Labs), according to published protocols [11, 22], with some modifications. Briefly, after de-waxing and re-hydration, sections were incubated for 15 min in 8% hydrogen peroxide solution in methanol to block endogenous peroxidase. Sections were then washed, immersed in 98% formic acid for 1 h, rewashed and microwaved in 1.5 mM HCl for 25 min, incubated with reagent A of Histostain-Plus IHC Kit (Thermo-Fisher Scientific) for 10 min and then probed overnight with mAb 3F4. After two sequential incubations with reagent B and C of Histostain-Plus IHC Kit interspersed with washing steps in TBS 1X, sections were treated with Romulin AEC Chromogen (Biocare Medical) for 5 min and Mayer’s hematoxylin for 15 s before being dehydrated, cleared and coverslipped.
We carried out a specific assessment of white matter changes of myelin, axons, astrocytes and microglia by means of Luxol Fast Blue (LFB) and several immunohistochemical stainings in section from the frontal, temporal and occipital cortices and the cerebellum. To this aim the following antibodies were used: 1) anti-myelin proteolipid protein (PLP) (1:3000, Biorad-Serotec MCA739G) for the assessment of demyelination (in combination with LFB staining), 2) anti-APP (1:10000, Merck-Millipore MAB348), anti-synaptophysin (1:100, Monosan Monx 10,779) and anti-neurofilament protein (1:100, Dako M0762) for axonal damage, 3) anti-GFAP (1:100 Dako M0761) for astrocytosis and 4) anti-HLA-DR (1:400, Dako M0775) for microgliosis. All antibodies but the anti-PLP required an antigen retrieval step (15 min microwave incubation after boiling) in Sodium Citrate buffer pH 6.0 (APP, GFAP, HLA-DR and neurofilament) or Tris/EDTA buffer pH 9.0 (synaptophysin).
For LFB staining, slides were immersed overnight in LFB solution (final concentration, 0.1% solvent blue and 0.5% acetic acid in 95% alcohol) at 60 °C. After immersion in 95% alcohol and washing, sections were immersed 5 s in 0.05% lithium carbonate and rewashed. The latter steps were repeated until suitable gray matter discoloration. The obtained sections were then processed for PAS staining through immersion in periodic acid for 10 min and, after a washing step in deionized water, incubation in dark condition with Schiff’s reagent for 15 min. Subsequently, slides were washed, incubated with Mayer’s hematoxylin for 1 min, immersed in warm water and rewashed.
Transmission to bank voles
Brain tissue from the p-CJDMM1 index case (case #1 described below) and from 4 control cases without plaques (three sCJDMM1 and one sCJDMV1) were homogenized at 10% (
w/
v) concentration in phosphate buffered saline (PBS) and stored at −80 °C. Two genetic lines of bank voles, Bv109M and Bv109I carrying methionine or isoleucine homozygosity at
PRNP codon 109, were injected by the intracerebral route (20 μl) into the left cerebral hemisphere under ketamine anesthesia. Beginning one month after inoculation, voles were examined twice per week until the appearance of neurological signs, and evaluated daily thereafter. The animals were sacrificed with carbon dioxide when they reached the terminal stage of the disease. Survival time was calculated as the interval between inoculation and sacrifice, attack rate as the number of animals developing disease with respect to the total number of inoculated animals [
18].
The lesion profile was based on the severity of vacuolation, with a score from 0 to 5, in nine grey-matter brain areas on hematoxylin and eosin-stained sections, as previously described [
7]. Vacuolation scores derived from at least 6 individual voles per group and were reported as mean ± standard error (SEM).
Preparation of human and bank vole brain total homogenates (THs) and PK digestion
Tissues from occipital cortex grey matter (human) and vole brain (50 mg) were homogenized (10%
w/
v) in lysis buffer (100 mM NaCl, 10 mM EDTA, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 100 mM Tris) at pH 6.9 (as reported in [
19]) and digested with proteinase K (PK) (Roche Diagnostics) at a final concentration of 8 U/ml for 1 h at 37 °C. Digestion was blocked with phenylmethylsulfonyl fluoride (PMSF, final concentration 3.6 mM), then samples were boiled in sample buffer (final concentration: 3% SDS, 4% β-mercaptoethanol, 10% glycerol, 2 mM EDTA, 62.5 mM Tris) for 6 min at 100 °C.
Preparation of white matter total homogenates and PK digestion
Frontal and parietal cortical white matter was obtained from case #1 (p-CJDMM1), and one np-CJDMM1. PrP
Sc was purified from 350 mg of white matter, following a previously published protocol [
29] and re-suspended in 200 μl of lysis buffer at pH 6.9. PK digestion was carried out at a final concentration of 4 U/ml for 1 h at 37 °C.
PrP deglycosylation
N-Linked glycans were removed by using a peptide-N-glycosidase F kit (New England Biolabs) according to the manufacturer’s instructions.
PK titration curves
Grey matter tissues were homogenized (10% w/v) in lysis buffer at pH 8. Total protein concentration was measured by means of a standard colorimetric method based on bicinchoninic acid (Pierce) and then adjusted to a final value of 4200 μg/ml. Samples were digested using serial dilutions of PK activity ranging from 2 to 256 U/ml, for 1 h at 37 °C. Digested samples were treated as previously described.
Thermo-solubilization assay (TSA)
TSA was performed as described [
6]. Briefly, grey matter THs (10% w/v in lysis buffer at pH 6.9) were digested with 8 U/ml PK for 1 h at 37 °C with mild shaking (300 rpm). PK digestion was inactivated with PMSF (final concentration, 3.6 mM). Aliquots were mixed with an equal volume of loading buffer (final concentrations, 1.5% SDS, 2% β-mercaptoethanol, 5% glycerol, 1 mM EDTA, 31.2 mM Tris) and heated to temperatures ranging from 25 °C to 95 °C (ΔT = 10 °C) for 6 min with shaking in a thermomixer at 1000 rpm before loading.
Western blot
Samples were run in a 7 or 15 cm long separating gel and transferred to Immobilon-P membranes (Millipore). After blocking in 10% non-fat milk in Tween-Tris-buffered saline, membranes were probed overnight with the monoclonal antibody 3F4 with epitope at PrP residues 108–111 at 1:30000 working dilution (human samples). Immunoblots from bank voles samples were incubated overnight at 4 °C with the monoclonal antibody 9A2 (1:8000, PrP residues 99–101) [
26] instead of 3F4. In addition, all immunoblots were probed with the C-terminal antibody SAF60 (1:2000, PrP residues 157–161) [
20] in order to detect the CTF13. After four washings in Tween-Tris-buffered saline, membranes were incubated for 1 h at room temperature with an anti-mouse secondary antibody conjugated to horseradish peroxidase (GE Healthcare; working dilution, 1:4000) and washed again four times in Tween-Tris-buffered saline. The immunoreactive signal was visualized by enhanced chemiluminescence (Immobilon Western, Millipore) on an LAS 3000 camera (Fujifilm).
Quantitative analysis of protein signal
Densitometric analysis was performed using the software AIDA (Image Data Analyzer v.4.15, Raytest GmbH). For PK titration, a semi-logarithmic curve was obtained by plotting the percentage of protein remaining after digestion (with respect to the sample digested with 2 U/ml) against the corresponding PK concentration. The ED50 (i.e. the PK concentration needed to digest 50% of PrPSc) for each sample was calculated by means of the equation of the straight line that best fitted the linear portion of the curve (r2 ≥ 0.95). For TSA, the percentage of protein solubilized after heating treatment (with respect to the sample treated at 95 °C) was plotted against the corresponding heating temperature. The T50 (i.e. the temperature needed to solubilize 50% of PrPSc) for each sample was calculated from the equation describing the sigmoidal curve that best fitted the data (r2 ≥ 0.95).
Statistical analyses
All statistical analyses were performed with SigmaPlot 12.5 (Systat Software Inc.). Depending on the data distribution, Student’s t test or Mann-Whitney test were used to detect differences between two groups, while one-way analysis of variance (ANOVA), followed by Dunn’s or Holm-Sidak post hoc tests, was applied for three or more groups comparisons. P value <0.05 was considered statistically significant.
Discussion
The present data (i) add to previous studies reporting the rare occurrence of white matter PrP amyloid plaques in patients with an otherwise classic sCJDMM1 phenotype; (ii) originally report the occurrence of white matter PrP plaque-like deposits in genetic CJDMM1 and the results of the experimental transmission of p-sCJDMM1 to bank voles, and (iii) further address the issue of the molecular basis of amyloid plaque formation in CJD by providing an extensive characterization of the physico-chemical properties of PrPSc aggregates in p-CJDMM1.
No data are available on the relative frequency of this peculiar phenotype in the CJD population. Since the 5 cases described here were observed over a 15–20 year-period of diagnostic activity involving approximately 1000 CJD-affected brains from Italy and the Netherlands, we can estimate an incidence of p-CJDMM1 in western Europe around 0.5%.
A critical unsolved issue concerning the occurrence of white matter kuru-type amyloid plaques in sCJD carrying MM at codon 129 is whether or not this peculiar phenotype is linked to a specific prion strain. Our systematic analyses of PrPSc properties combined with the results of the experimental transmissions strongly argue for both the classic np-CJDMM1 and the atypical p-CJDMM1 phenotypes being linked to the same (M1) prion strain. Accordingly, amyloid plaque formation in such cases represents a host-derived, likely genetic, effect. To consider an alternative possibility, one would postulate the unlikely scenario of the co-occurrence in p-CJDMM1 of a distinct prion strain besides M1, not inducing a distinctive cerebral grey matter pathology, not affecting PrPSc properties, and not transmissible to bank voles.
Besides the presence of amyloid plaques, another interesting feature, distinguishing the p-CJDMM1 reported by us and Kobayashi et al. [
12] from the np-CJDMM1 cases, is their significantly longer mean disease duration (22 months) in comparison to typical np-CJDMM1 cases (4 months). However, disease duration and the associated advanced pathology, although notoriously favoring the extent of plaque formation, cannot be the only causal factors since it is well established that most CJDMM1 patients with prolonged disease duration do not develop plaque-type depositions in the white matter. Moreover, the observations by Gelpi et al. and Berghoff et al. [
1,
9] in cases characterized by a short disease course, combined with our findings in case #5 and in a similar p-CJDMM1 case we recently obtained, also characterized by mild white matter changes (P. Parchi personal communication), clearly indicate that white matter amyloid plaques may develop early in the disease course and independently from a severe white matter damage. Interestingly, in our p-CJDMM1 cases, the onset and progression of clinical symptoms, including akinetic mutism, seem to be significantly delayed compared to np-CJDMM1 patients with similar disease duration. Taken together, these data support the hypothesis of a protective role of PrP amyloid, possibly by sequestering PrP
Sc into large fibrils and partially preventing the molecular interaction between monomeric PrP
C and PrP
Sc, that is essential for conversion and prion propagation. Since the mechanism of amyloid deposition seems to include the incorporation of lipid molecules into the aggregates [
30], white matter appears even more suitable for PrP amyloid plaque formation than the grey matter. In this regard, it is noteworthy that plaque-like PrP deposition in sCJDVV2 and MV2K is often best observed at the boundaries between gray and white matter.
Despite the intensive search, we failed to demonstrate a difference in the physico-chemical PrP
Sc properties between p-CJDMM1 and np-CJDMM1 that would correlate with plaque formation. Similarly, PrP
Sc properties did not differ between bank voles injected with the two CJD inocula. These data combined with the lack of PrP amyloid plaques or plaquelike deposits in the bank voles inoculated with p-CJDMM1 further point to a non-PrP factor of the host affecting PrP aggregation and fibrillation. It is well established that PrP
Sc spread within the peripheral and central nervous systems by axonal transport although the cellular mechanism of prion transport in axons and into peripheral tissue is largely unresolved. Thus, one possibility would be a modified molecular interactome for PrP
Sc during axonal transport favoring PrP
Sc aggregation and amyloid plaque formation. Since PrP-amyloid plaques in p-CJDMM1 cases sometimes co-localize with APP, a well-established marker of axonal damage, PrP
Sc deposition in white matter eventually disrupts axon integrity. The opposite scenario, namely axonal damage favoring PrP amyloid plaque formation, previously suggested by Kobayashi et al. [
12] seems unlikely given the observation of plaque formation in cases with short disease duration and/or lack of significant white matter damage [
1,
9] (and present cases #5).
Conclusions
The present study further establishes the existence of a rare CJD subtype, occurring in approximately 0.5% of CJD cases, designated as p-CJDMM1. The novel histotype largely overlaps with sCJDMM1 but shows, as a very distinctive feature, the presence of PrP-amyloid plaques of kuru-type in both subcortical and deep nuclei white matter. Likewise typical CJDMM1, p-CJDMM1 can also be observed in sCJD cases showing the co-occurrence of PrPSc types 1 and 2. Moreover, plaque-like PrP deposits in the white matter can be a feature of genetic CJD. Most significantly, p-CJDMM1 share both PrPSc and transmission properties with classic CJDMM1, strongly pointing to an host-dependent causal factor for amyloid plaque formation in this phenotype.
Acknowledgements
We wish to thank Barbara Polischi M.Sc., Silvia Piras, B.Sc., Stefano Marcon and Geraldina Riccardi for their valuable technical assistance; Claudia D’Agostino, DVM, Paolo Frassanito, Shimon Simson and the technical staff of the animal facility at ISS for their excellent animal care and work.