Introduction
The key pathogenic event of all prion diseases is the conversion of a normal or cellular prion protein (PrP
c) into a misfolded and disease-associated isoform commonly identified as scrapie prion protein (PrP
Sc) or prion. Newly converted PrP
Sc then propagates and accumulates preferentially in the central nervous system (CNS) typically accompanied by spongiform degeneration, gliosis and neuronal cell death. These pathogenetic features apply especially to Creutzfeldt-Jakob disease (CJD), by far the most common human prion disease. The pathogenic mechanism based on the conformational conversion of PrP
C into PrP
Sc, which then acts as a seed, can conceptually accommodate not only PrP
Sc accumulation and propagation in the affected subject, but also the potential transmission of the process from affected to non-affected subjects. Although the sporadic and inherited forms account for the majority of human prion diseases [
24], less than 1% of the prion diseases is acquired from animals or humans via an infectious mechanism [
7]. Within this group, iatrogenic CJD (iCJD) is of particular interest. More than 492 cases of iCJD have been reported worldwide [
4]. Most cases have been associated with the administration of growth hormone (GH) extracted from cadaveric pituitary glands and the application of cadaveric dura mater (DM) grafts [
7]. The existence of iCJD and experimental evidence have firmly established the infectious property of prion diseases [
18,
26,
27].
The central pathogenic event in Alzheimer’s disease (AD), the most common cause of dementia, is the deposition of amyloid β (Aβ), a truncated fragment of the amyloid precursor protein (
APP), which leads to the formation of extracellular Aβ plaques [
22,
30]. Deposition of highly phosphorylated, microtubule-associated tau protein (p-tau) also occurs [
57] and, is believed to be a downstream event [
33]. Most evidence indicates that deposition of Aβ and p-tau in the affected brain is stereotypical and hierarchical [
5,
63]. More recently, it has been pointed out that Aβ and p-tau mimic several major characteristics of PrP
Sc including mechanisms of accumulation and propagation and formation of distinct Aβ and p-tau species that fulfill some of the characteristics of strains [
16,
28,
56,
67,
68]. Like PrP
Sc, Aβ has been detected in tissues other than CNS parenchyma including dura mater and pituitary glands [
35,
43]. Furthermore, Aβ and p-tau pathologies (but not fully developed AD) have been replicated following Aβ or p-tau intracerebral or peripheral inoculations to transgenic mice that express human
APP harboring AD pathogenic mutations as well as wild type human
APP, indicating that Aβ pathology is transmissible [
11,
20,
47,
48,
70]. In contrast to prion diseases, there is no evidence that AD and/or tauopathy (e.g., frontotemporal lobar degeneration) transmit from human-to-human.
Five recent studies have independently reported the presence of significant Aβ pathology in 4 of 8, 18 of 33, and 1 of 24 cases of iCJD from the United Kingdom (UK) and France, linked to injections of cadaveric GH (GH-iCJD) [
19,
37,
53], and in 5 of 7 Swiss and Austrian iCJD and 13 of 16 Japanese iCJD cases who had received DM grafts (DM-iCJD) [
23,
31]. In the most severely affected cases, Aβ amorphous aggregates were also detected in the pituitary gland and DM [
37,
43]. The concomitant presence of Aβ and PrP
Sc deposits resulting in mixed AD and CJD phenotypes has been reported [
8,
25,
50,
65]. However, while the AD-CJD mixed phenotype has been observed in older CJD-affected individuals [
29,
64], most of the 28 iCJD cases harboring significant Aβ pathology were under 55 years of age [
19,
23,
37,
53]. A possible explanation for the higher prevalence of Aβ pathology at a relatively young age in iCJD-affected subjects is that both Aβ and PrP
Sc seeds are transmitted during the iatrogenic procedures and then accumulate and propagate concurrently. However, Ritchie and coworkers have also observed brain Aβ pathology in absence of PrP
Sc deposition in recipients of cadaveric GH [
53]. This important finding strongly argues for human-to-human transmission of an Aβ-related condition independently from the PrP
Sc seeding process. Nonetheless, whether the neuroendocrine deficiency may favor the Aβ pathology in some cases is still unknown [
21].
To further investigate the iatrogenic seeding of Aβ pathology, we examined whether this condition occurs not only with UK and French GH as previously described but with receipt of GH produced before 1977 in the United States (US) as well. All US GH-iCJD patients to date received GH made before 1977, when a GH purification procedure was adopted that reduced or eliminated prion contamination. We also wanted to further examine the phenotypes of the DM-iCJD linked to the use of the Lyodura® brand. Out of 27 cases of definite iCJD, we selected 21 which were suitable for detailed histopathological examination and were provided by national prion surveillance centers of Australia, France, Italy, and the US. Cases of sporadic CJD (sCJD), non-neurodegenerative disorders (non-ND), and AD were used as comparison groups. Two autopsied cases who underwent GH treatment but did not develop iCJD were also examined. We report that (i) over 50% of iCJD cases harbored significant Aβ pathology, which included cerebral amyloid angiopathy (CAA) in all cases; (ii) the prevalence of the Aβ-positive iCJD subset was significantly higher than that of Aβ-positive sCJD cases, which were on average 17 years older; (iii) p-tau pathology was present but did not distinguish Aβ-positive iCJD cases from the sCJD controls, and seemed to be age-related; (iv) the phenotypic characteristics of the Aβ pathology in iCJD were distinct from those of typical AD.
Materials and methods
Reagents and antibodies
Dulbecco’s Phosphate Buffered Saline (DPBS), NaCl, Nonidet P-40, Sodium deoxycholate, Tris–HCl, Phenylmethanesulfonyl fluoride (PMSF), proteinase K (PK), Thioflavin S, and Kodak Biomax MR and XAR films were from Sigma-Aldrich (St. Louis, MO, USA). Tween 20, β–mercaptoethanol, Tris buffered saline (TBS), 2X Laemmli sample buffer, non-fat dry milk, 15% Criterion Tris–HCl polyacrylamide precast gels, 30% Acrylamide/Bis solution, tetramethylethylenediamine (TEMED), 10% sodium dodecyl sulfate (SDS) and ammonium persulphate (APS) were from Bio-Rad Laboratories (Hercules, CA, USA). Odyssey Blocking Buffer was from LI-COR Biosciences (Lincoln, NE, USA); polyvinylidene difluoride (PVDF) membrane (Immobilon-FL and Immobilon-P) was from EMD Millipore (Billerica, MA, USA). The primary antibodies (Abs) included the AT8 (to human phospho-tau residues Ser202 and Thr205) from Thermo Fisher Scientific Inc. (Waltham, MA, USA), 4G8 (to human Aβ residues 17–24), and 3F4 (to human PrP residues 106–110) [
38,
71] from Richard Kascsak at the N.Y.S. Institute for Basic Research; 12B2 (to human PrP residues 89–93) [
45] was from the Wageningen University & Research (Lelystad, Netherlands), whereas Tohoku-2 (to human PrP residues 97–103) [
42] was kindly provided by Dr. Tetsuyuki Kitamoto. Secondary Abs were the sheep anti-mouse IgG from Life Sciences (Piscataway, NJ, USA), IRDye 800CW goat anti-mouse IgG (1 mg/ml) and IRDye 680RD goat anti-rabbit IgG (1 mg/ml) from LI-COR Biosciences (Lincoln, NE, USA). Reagents ECL and ECL plus were from GE Healthcare, Life Sciences (Piscataway, NJ, USA); Envision Flex Peroxidase Blocking Reagent, Envision Flex/HRP and Envision Flex DAB were from Dako (Dako North America Inc., Carpinteria, USA); the Vectashield mounting medium for fluorescence was from Vector Laboratories Inc. (Burlingame, CA, USA).
Patients
Brain tissue from 27 confirmed iCJD patients was collected in this study. Thirteen of these cases were associated with cadaveric GH extracted from pituitary glands, whereas the other 14 cases were associated with cadaveric DM graft (Table
1 and Additional file
1: Table S1). All GH-iCJD were from the US and were treated with GH under the US National Hormone and Pituitary Program and started their treatment between 1969 and 1974. Tissue from 12 of the 13 US GH-iCJD was obtained at autopsy and one at biopsy. Two DM-iCJD were US cases, but only one -a 26 years old man -had received the Lyodura® brand dura (B. Braun Melsungen AG, Melsungen, Germany) (DM
L-iCJD) whereas the other case -a 39 years old woman -had received the Tutoplast® brand dura (Pfrimmer-Viggo GmbH + Co, Erlangen, Germany) (DM
T-iCJD). An important difference between these brands of dural graft is that unlike Tutoplast, Lyodura brand dural grafts were intermingled with many other dural grafts during the manufacturing procedure, thereby increasing the risk of cross-contamination. Other DM-iCJD cases included 1) two cases from the Australian National CJD Registry (Melbourne, Australia), 2) six cases from the Réseau National de Référence de maladies de Creutzfeldt-Jakob and Centre National de Référence des agents transmissibles non conventionnels (Paris, France), and 3) four cases from the Istituto Superiore di Sanita’ (Rome, Italy). With the exception of one French DM-iCJD case, for which the medical product Lyodura® brand could not be confirmed, all cases were DM
L-iCJD. The histopathology and/or clinical and molecular features have been published for two US DM-iCJD (cases 1 and 2, Table
1) [
3,
10,
32], three US GH-iCJD (cases 6, 9 and 10, Table
1) [
10], and one Australian DM
L-iCJD (case 17, Table
1) [
58]. The five distinct comparison groups included 1) two US non-CJD patients who received the GH between years 1973–1977 (50 years) or between years 1977–1981 (46 years). The latter case may not have received any of the pre-1977 produced higher risk US GH material because 1977 was the transition year when a new purification procedure of GH extraction was started [
1]; 2) 67 confirmed sCJD cases from Australia (
N = 4), France (
N = 11), Italy (
N = 8) and US (
N = 44) (Additional file
1: Tables S1 and S2); 3) 11 US autopsied cases of non-ND with age at death of 40 ± 12 years (range, 25–59 years) following diagnosis of blood cancer (
N = 3; 25, 27 and 41 years), intracranial tumors (
N = 2; 35 and 44 years), hemoglobin sickle cell disease (28 years), scrotal abscess and pulmonary embolus (31 years), type II diabetes mellitus (47 years), gastrointestinal hemorrhage (50 years), systemic lupus erythematosus (56 years), end stage renal disease and cardiac arrest (59 years); 4) seven US cases of AD with age at death of 64 ± 8 years, and disease duration of 75 ± 39 months [
12]. Disease duration was not available in one AD case. All sporadic and iatrogenic CJD cases were classified according to Parchi and collaborators (1999). Since all Aβ-positive cases had multiple cortical sections not all of which were positive, we excluded from the study six Aβ-negative cases with only one section available.
Table 1
All examined cases of iCJD from countries of treatment with clinical, molecular and histopathological features
1
b,c
| DML | United States | M | 26 | 5 | 19 | na | na | MM(MV)1 | yes |
2d,e | DMT | | F | 39 | 4 | 6 | MM | 1 | MM(MV)1 | yes |
3 | GH | | M | 33 | 6 | 23 | VV | na | VV2 | yes |
4 | GH | | M | 39 | 17 | 24 | MV | na | MV2K | yes |
5 | GH | | M | 39 | 7 | 27 | MM | 2 | Atypical | yes |
6e | GH | | F | 41 | 2 | 26 | MM | 1 | MM(MV)1 | yes |
7 | GH | | M | 43 | 26 | 23 | MV | i+2 | MV2K | yes |
8
| GH | | M | 44 | 18 | 32.5 | MV | i+2 | MV2K | yes |
9
e
| GH | | M | 51 | 14 | 38 | MM | i | MMi | yes |
10
e
| GH | | M | 54 | 2 | 41.5 | MM | 1 | MM(MV)1 | yes |
11 | GH | | M | 23 | 19 | 10 | na | na | MV2K or MMi | no |
12 | GH | | M | 34 | 18 | 23 | na | na | MV2K or MMi | no |
13 | GH | | M | 37 | 3 | 21 | na | na | MM(MV)1 | no |
14 | GH | | M | 42 | 5 | >28f | MM | na | Undeterminedg | no |
15h | GH | | M | 52 | 4 | 43 | na | na | MM(MV)1 | no |
mean±SDi | | | | 41±8.5 | 11±8 | 28±9.5j | | | | |
16
| DML | Australia | M | 32 | 3.5 | 16.5 | MV | na | MM(MV)1 | yes |
17
k
| DML | | M | 62 | 2 | 5 | na | na | MM(MV)1 | yes |
mean±SD | | | | 47±21 | 3±1 | 11±8 | | | | |
18 | DML | France | M | 25 | 8 | 8 | MV | na | MM(MV)1 | yes |
19
| DMUnk | | M | 29 | 6 | 25 | MV | 1 | MM(MV)1 | yes |
20 | DML | | M | 42 | 5 | 6 | VV | na | VV2 | yes |
21
| DML | | F | 50 | 14 | 11 | MM | na | MMi | yes |
22
| DML | | F | 62 | 4 | 4 | VV | na | VV2 | yes |
23
| DML | | F | 71 | 4 | 4 | MV | na | MV2K | yes |
mean±SD | | | | 46.5±18 | 7±4 | 10±8 | | | | |
24 | DML | Italy | F | 23 | 27 | 21 | VV | 2 | VV2 | yes |
25 | DML | | F | 26 | 6 | 12 | MM | na | MM(MV)1 | no |
26 | DML | | M | 42 | 3.5 | 18 | MM | 1+2 | MM1+2 | yes |
27
| DML | | M | 75 | 3 | 18 | VV | 2 | VV2 | yes |
mean±SD | | | | 41.5±24 | 10±11.5 | 17±4 | | | | |
All of the US iCJD and the two CJD-free recipients of GH were collected at the National Prion Disease Pathology Surveillance Center (NPDPSC) in Cleveland (OH) in collaboration with the National Institute of Diabetes, Digestive and Kidney Diseases, NIH, (Bethesda, MD) and Division of High Consequence Pathogens and Pathology, CDC, (Atlanta, GA) and Westat Agency (Rockville, MD). The AD and US sCJD controls were from the NPDPSC whereas the 11 non-ND cases were collected in the repository of the Department of Pathology at Case Western Reserve University. The neuropathological study was carried out either in our laboratory or in the laboratories of the participating countries. Neuropathology was reviewed by IC and MC. Western blot (WB) examination of iCJD cases was performed in our laboratory in Cleveland (cases 2, 6–10, 24, 26 and 27, Table
1), at University of California, San Francisco (UCSF) (case 5) as well as in France (case 19, Table
1) and Italy (cases 24, 26 and 27, Table
1).
Histology and immunohistochemistry
Formalin-fixed brain tissue was treated as previously described [
9]. Briefly, sections were deparaffinized and rehydrated, immersed in 1X Tris buffered saline-Tween 20 (TBS-T), and endogenous peroxidase blocked after incubation with the Envision Flex Peroxidase Blocking Reagent for 10 minutes (min). Sections were washed, immersed in 1.5 mmol/L hydrochloric acid, microwaved for 15 min and probed with Abs 3F4 (1:1000), 4G8 (1:3000), and AT8 (1:200) for 1 hour (h). After washing and incubation with Envision Flex/HRP polymer for 30 min, sections were treated with Envision Flex DAB to show the immunostaining.
Thioflavin S staining
After deparaffinization, formalin-fixed sections were stained in Thioflavin S for 7 min, washed three times in 80% alcohol, dehydrated in ethanol, cleared in xylene, and cover slipped with Vectashield mounting medium for fluorescence. Sections were kept in the dark at 4 °C for 30 min before being viewed under the fluorescence microscope (Olympus IX71).
Evaluation of the histopathological changes associated with Alzheimer’s disease and Prion disease
Histopathological evaluation was performed in 10 or more anatomical regions in most cases. Standard brain locations included the frontal, temporal, parietal, occipital and entorhinal cortices, hippocampus, striatum, thalamus, midbrain and cerebellar hemispheres and/or vermis. Histopathological evaluation included 1) Hematoxylin-eosin (HE) staining, to assess the presence of spongiform degeneration, gliosis, and amyloid Aβ cores; 2) Immunostaining with Abs 4G8, AT8 and 3F4 to Aβ, p-tau and PrP, respectively; 3) Staging of Aβ plaques using monoclonal Ab 4G8 and Thioflavin S, according to Thal et al. [
63]. This method identifies five major stages or phases of Aβ plaques deposition affecting the neocortex, including frontal, temporal, parietal and occipital cortices (Phase 1), hippocampus and entorhinal cortex (Phase 2), striatum thalamus and midbrain (Phase 3–4), and cerebellum (Phase 5); 4) Description of the morphology of the Aβ plaques, including a) diffuse plaques, b) core plaques (CP) (i.e., a plaque with a dense core surrounded by a halo and a corona of lightly stained Aβ), c) neuritc plaques (i.e., a core plaque surrounded by p-tau dystrophic neurites); 5) Staging of Aβ CAA, according to previous procedures [
61] with recognition of three major phases: CAA affecting the neocortex (Phase 1), hippocampus, entorhinal cortex, cerebellum and midbrain (Phase 2), striatum and thalamus (Phase 3); 6) Typing of CAA, identified as CAA type 1 or type 2 depending on the presence or absence of Aβ deposits in the cortical capillaries [
60]. The criteria for the identification of CAA type 1 were i) diameter of the vessels ≤ 10 μm, and ii) deposition of Aβ in the outer basement membrane; 7) Severity of CAA, based on a modified protocol by Vonsattel and coworkers that uses 4G8-stained sections (with the addition of Thioflavin S in some cases) instead of Congo red [
66]; 8) Brain distribution of neurofibrillary tangles (NFT) and neocortical distribution of dystrophic neurites (DN); 9) Severity of NFT and DN expressed as NFT or DN density in one microscopic field (area: 1.3 × 1.0 mm
2, using a 10X objective) harboring the highest density of NFT or DNs in one or more brain regions; severity was scored as mild (≤ 10 NFT or DN), moderate (> 10 to < 30 NFT or DN) and severe (≥ 30 NFT or DN); 10) Automated image acquisition and morphometric analysis to assess density (expressed as the percentage of the area of the cerebral cortex occupied by plaques) and size (diameter) of Thioflavin S-positive Aβ core plaques as well as size (perimeter) of the blood vessel; 11) Semiquantitative analysis of the percentage of 4G8-positive Aβ deposits along the wall of blood vessels; and 12) Double immunostaining with 4G8 and 3F4 to rule out the co-localization of PrP and Aβ [
25].
Image acquisition and statistical analysis
Image acquisition was carried out with a Leica DFC 425 digital camera mounted on a Leica DM 2000 microscope. Images were analyzed by the software Image-Pro Plus 7.0 (Media Cybernetics, Inc.). Cumulative survival curves were generated by the Kaplan–Meier analysis. Statistical significance between the survival curves of the individual groups were determined by the log rank (Mantel-Cox) test. When comparing different patient groups, P-values were calculated with Chi-square test, Fisher’s exact test, Student’s t-test (two-tailed). All the statistical analyses were performed using GraphPad Prism 6.0.
Preparation of brain homogenates, proteinase K digestion and Western blot analysis
10% (wt/vol) brain homogenates (BH) prepared in 1X LB100 buffer (100 mM NaCl, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 10 mM EDTA, 100 mM Tris–HCl, pH 6.9 at 37 °C), were centrifuged at 1000 x g for 5 min at 4 °C, pellets discarded and supernatants (S1) collected. S1 aliquots were incubated with 100 U/ml PK at 37 °C for 1 h [PK specific activity was 48 U/mg at 37 °C, with 1 U/ml equal to 20.8 μg/ml PK]. The enzymatic digestion was stopped with PMSF (3 mM final concentration). Each sample was diluted with an equal volume of 2X Laemmli sample buffer (6% SDS, 20% glycerol, 4 mM EDTA, 5% β –mercaptoethanol, 125 mM Tris–HCl, pH 6.8) and denatured at 100 °C for 10 min. Proteins were separated on 15% Criterion™ Tris–HCl Precast Gels (W x L: 13.3 cm × 8.7 cm) at 120 Volts (V) for 20 min followed by 150 V for 1 h 45 min, or using 15% Tris–HCl SDS–polyacrylamide gels (W x L: 20 cm × 20 cm) at 25 mA/gel for 1 h 45 min followed by 35 mA/gel for 6 h 30 min (Bio-Rad PROTEAN® II xi cell system). For near-infrared WB analysis, proteins were blotted onto the Immobilon-FL PVDF membrane for 2 h, blocked with the Blocking Buffer Odyssey for 45 min and incubated with Abs 3F4 (1:20,000), 12B2 (200 ng/ml) or Tohoku-2 (1:10,000) for 2 h. Membranes were then washed with 1X DPBS containing 0.1% of Tween 20 (1X DPBS-T) and incubated with Abs IRDye 800CW goat anti-mouse IgG (1:15,000) or IRDye 680RD goat anti-rabbit IgG (1:15,000) for 1 h. After washing in 1X DPBS-T, membranes were developed with the Odyssey infrared imaging system (LI-COR Biosciences) as described by the manufacturer. For chemiluminescence, proteins were blotted onto the Immobilon-P PVDF membrane, blocked with 5% non-fat dry milk in 0.1% Tween 20-1X TBS (blocking buffer), and incubated with primary Abs and horseradish peroxidase-conjugated goat anti-mouse secondary Ab (1:3000). Membranes were developed by the enhanced chemiluminesce reaction using ECL and ECL plus reagents, and signal captured on MR and XAR films.
Clinical evaluation
Medical records were reviewed by a clinician (BSA) and data were collected on demographics (age at death, gender, and race/ethnicity). Disease onset was defined as the time at which the first persistent and consistent symptom of prion disease was observed. Data on family history of dementia as well as past medical and surgical history were also collected. The mean incubation period in iCJD was measured from the mid-point of GH therapy or date of receipt of the DM graft to the clinical onset of the disease.
Genetic analysis
DNA was extracted from frozen brain tissues and
APP, presenilin 1 (
PSEN1), presenilin 2 (
PSEN2), and
PRNP gene analysis was performed as previously described using Illumina and Sanger Sequencing for exons 4 and 5 in
PSEN1 [
12]. Sequencing analysis was performed using Mutation Surveyor Version 4.0.7 (Softgenetics, State College, PA). Genotyping of Apolipoprotein E (
ApoE) single-nucleotide polymorphisms was performed by Sanger sequencing (Center for Human Genetics, Cleveland, OH).
Discussion
We report on 21 cases of iCJD including GH-iCJD and DM-iCJD subsets collected in Australia, France, Italy and the US, 11 (52%) of which harbored significant Aβ pathology. Despite the diversity of their geographic origin, our iCJD cases were remarkably similar concerning the 129 genotype and CJD histopathological phenotype distributions, and the features of the associated Aβ pathology.
For DM-iCJD, the similarities may reflect the use of the same Lyodura product in all these countries. In contrast, the cases of GH-iCJD were all from the US and can only be compared with the similar cohorts studied in the UK and France, where methods of GH production differed from those in the US (SH and JPB, personal communication).
Genotype and phenotype distribution in our iCJD cohort showed a relatively high prevalence of MV and VV over MM codon 129 genotypes when compared to sCJD as also observed in previous studies [
54,
55]. Furthermore, we observed two iCJD with the MMi phenotype that has been shown to originate from adaptation of sCJDVV2 or sCJDMV2K to the 129MM background of the recipient [
39,
42,
54,
59]. The finding that this event, initially described in Japanese DM-iCJD, is also observed in our international iCJD cohort as well as the UK cohort, and thus is widespread, suggests that 129VV and 129MV individuals may have been preferentially selected as donors in several countries. Alternatively, subjects with 129MV and 129VV genotypes might be more susceptible to acquire CJD than the carriers of the 129MM genotype, in whom the sCJDMM1 phenotype might be difficult to reproduce requiring strain adaptation [
13,
46,
69]. The ~2 times longer incubation period in GH-iCJD than DM-iCJD cases resembles the longer incubation period associated with the peripheral (e.g., in the peritoneal cavity) vs. central (e.g., intracerebral) inoculations of prions or Aβ seeds in Tg mouse models for these proteinopathies [
20,
36]. Concerning the Aβ pathology, iCJD-affected subjects had CAA accompanied by parenchymal Aβ CP in five cases and subpial deposits in three. The Aβ-positive iCJD subset also had a relatively young age (mean 50.5 years, range 26–75 years) along with the relative severity and widespread distribution of CAA [
66]. Furthermore, with only one exception, CAA was type 2, i.e. did not affect cerebral capillaries unlike type 1 [
66]. Since CAA type 1 has been directly associated with AD severity, including tau pathology, age, and with
ApoE-ε4 allele frequency, the predominance of type 2 CAA in iCJD is not surprising given the relatively younger age and short duration of this condition [
52,
62].
Aβ pathology was examined in age-group matched cohorts of sCJD and of non-ND used as controls to rule out the possibility that Aβ deposition in iCJD was either related to aging or resulted from the concomitant aggregation of prion or prion-like proteins by a cross-seeding mechanism. Furthermore, type and severity of the iCJD Aβ pathology was compared with that of AD. The 13% prevalence of Aβ pathology in the sCJD cohort (mean age 50 years, range 24–79 years) was 4 times lower than that in iCJD (52%) even though the Aβ-positive sCJD subset was 17 years older. In the non-ND cohort (age 40 ± 12 years, range 25–59 years), the 2 (18%) Aβ-positive cases were 56 and 59 years of age and the oldest of this cohort. When all Aβ-positive subsets were considered in the 20–54 years range, the prevalence of Aβ pathology in iCJD, sCJD and non-ND populations was 41%, 2% and 0%, respectively. Furthermore, the Aβ pathology in iCJD cases was more severe than sCJD, and showed a significantly more widespread CP and CAA distribution and a trend toward a significantly higher CAA severity score. On the contrary, Aβ pathology in AD was dramatically more severe than in Aβ-positive iCJD with regard to number, size and distribution of CP as well as of CAA in neocortical regions, even though the AD cohort used as control had a relatively young age (64 ± 8 years). These features clearly distinguish the Aβ pathology of iCJD from typical AD. Although we cannot rule out that the Aβ pathology might have evolved toward an AD phenotype had the Aβ positive iCJD patients lived longer and not died of CJD this possibility is unlikely since no significant differences were found between the UK GH-iCJD and hGH recipients free of CJD. Even though we used a different criterion for Aβ plaque selection, our findings on Aβ pathology do not significantly differ from those reported in five previous studies [
19,
23,
31,
37,
53], which collectively examined 95 cases of iCJD, including 67 GH-iCJD and 28 DM-iCJD. However, the 54% prevalence of the Aβ pathology in UK GH-iCJD is higher than the 37.5% we observed. Similarly, the 18 year mean incubation period of UK GH-iCJD is significantly shorter than that of US cases (28 years) (
P < 0.004). These differences may relate to the relatively high prevalence of UK GH-iCJD cases (~4.2%) compared to the 1.2% prevalence among US recipients of pre-1977 produced human GH (hGH) as well as to variations in selecting pituitary donors and in protocols of GH purification [
2,
4,
7,
54]. Regardless of the causes, the higher prevalence of the Aβ pathology, the significantly lower mean incubation period and the more than a decade younger age differential of UK Aβ-positive GH-iCJD cases, suggest that GH used in UK had a higher infectious dose not only of PrP
Sc but also of Aβ seeds. Remarkably, despite these differences, the Aβ phenotype in US and UK was similarly characterized by CAA alone or co-existing with CP. The small subset of Aβ-positive GH-iCJD cases harboring parenchymal Aβ deposits reported by Ritchie and co-workers (2017a) presumably included diffuse plaques, which may also account for the higher prevalence of positive Aβ pathology in UK GH-iCJD. Furthermore, CAA reached similar severity scores in US (1.6) and UK (2) and type 2 CAA markedly predominated in both countries [
37,
53]. The very low prevalence of the Aβ pathology (4%) in French GH-iCJD cases compared to the US and UK GH-iCJD cases has been explained possibly by the few years shorter incubation period in French GH-iCJD and to differences in GH preparations [
19]. The prevalence of the Aβ pathology in DM-iCJD reported here (61.5%) is similar to that of the previous studies combined (69%) but differs from the 81% prevalence reported by Hamaguchi et al. (2016). This difference may depend on the older age of the Japanese cohort compared to those examined by us and Frontzek et al. (2016) (10 and 16 years, respectively). The co-occurrence of CAA and Aβ parenchymal deposits was observed in all the cases of Frontzek and co-workers (2016) but in only half of ours (CAA occurred alone in the others). This discrepancy along with the aforementioned discrepancy of UK GH-iCJD cases are likely due to our different criteria of Aβ plaque validation. Unlike the five previous studies that also accepted diffuse plaques, we validated only CP (i.e. Aβ plaques that contained amyloid) to better distinguish Aβ pathology associated with iCJD from that related to aging [
19,
23,
31,
37,
53]. The exclusion of the plaque amyloid requirement would have increased the the Aβ-positive iCJD cases from 11 to 13 and increased by 9 the number of Aβ-positive sCJD cases (Additional file
2: Table S5 and data not shown). The CP requirement in the Aβ pathology of iCJD might establish a qualitative difference in the Aβ phenotype between iCJD and sCJD in younger populations. Nonetheless, this and previous studies show that CAA, with or without CP, is the distinctive histopathological phenotype of Aβ deposition in iCJD [
19,
23,
31,
37,
53]. Our study taking advantage of the direct comparison, also shows that the Aβ phenotype is similar in GH- and DM-iCJD.
Because tau pathology is considered a consistent but secondary feature of AD, we also searched for the presence of the two most common tau related lesions, NFT and DN [
33,
34,
57]. NFT were present in Aβ-negative and Aβ-positive iCJD, as well as in sCJD controls with similar prevalences (45–53%), and they were age-related. Occurrence of NFT in absence of Aβ plaques was previously shown in sCJD and was considered as “primary age-related tauopathy” in the elderly [
17,
44,
51]. Overall, the NFT prevalence in cases with less than 52 years were similar in iCJD (44%) and sCJD (40%) cases, and did not differ from that reported in a large population of unselected individuals of similar age (41%) [
6]. Neocortical DN were observed in only three iCJD cases and one case of sCJD where they were occasionally associated with CP, as previously reported [
53]. In contrast, NFT and DN were consistently present in the AD cohort. These findings indicate that tau pathology is (i) a non-obligatory component of iCJD Aβ phenotype, (ii) likely develops independently from the Aβ pathology in iCJD, and (iii) further distinguishes iCJD Aβ pathology from AD.
Further important questions raised by our and previous studies are the origin of Aβ seeding, how Aβ seed reaches the brain, and whether Aβ-seeded diseases are contagious.
In hGH recipients, Ritchie and colleagues [
53] convincingly showed that Aβ deposition occurs in the absence of prion pathology and the phenotype associated with the Aβ deposition remains similar to that of Aβ-positive iCJD cases, suggesting that Aβ deposition is a primary co-pathology in GH-iCJD and that Aβ and PrP
Sc seeding processes occur independently.
Although we occasionally have observed Aβ-PrP mixed plaques supporting the possibility of co-seeding, the brunt of the two pathologies were anatomically segregated: Aβ deposition affected mostly vessel walls while PrP
Sc affected exclusively the brain parenchyma. Moreover, the fact that Aβ-positive iCJD is associated with different CJD subtypes argues against cross-seeding of Aβ by a specific prion strain. Additional support to the independent seeding of Aβ in DM-iCJD comes from two other observations. First, Aβ deposits occurred in the dura graft but not in the patient’s original dura [
43], secondly, the distribution of Aβ deposits is consistent with the propagation through the brain of Aβ pathology originating from the dura graft while the distribution of PrP
Sc pathology is uniform [
31,
43]. These findings point to the dura graft as the source of the Aβ seed and to a different tempo of Aβ and PrP
Sc propagation further strengthening the notion that in iCJD PrP
Sc and Aβ are independent pathologies. In GH-iCJD, the Aβ seed is likely to propagate from the site of cutaneous injection to the brain. Experimental data have definitely provided the proof of principle that human Aβ seed may reach the brain causing Aβ amyloidosis following systemic inoculation [
20]. Remarkably, a predominantly vascular distribution of the Aβ deposits consistent with Aβ-CAA was noted in these experiments [
20].