Received: 18 May 2020 | Accepted: 4 January 2021

DOI: 10.1111/bpa.12941

R E SEA RCH A RTICLE

Accumulation of cellular prion protein within β-­amyloid oligomer

plaques in aged human brains

Reisuke H. Takahashi1,2 | Mayumi Yokotsuka1 | Minoru Tobiume2 | Yuko Sato2 |

Hideki Hasegawa2 | Toshitaka Nagao1 | Gunnar K. Gouras3

1
Department of Anatomic Pathology,
Tokyo Medical University, Tokyo, Japan Abstract
2
Department of Pathology, National Alzheimer’s disease (AD) is the main cause of dementia, and β-­amyloid (Aβ) is
Institute of Infectious Diseases, Tokyo,
Japan a central factor in the initiation and progression of the disease. Different forms
3
Experimental Dementia Research Unit, of Aβ have been identified as monomers, oligomers, and amyloid fibrils. Many
Department of Experimental Medical
Science, Lund University, Lund, Sweden proteins have been implicated as putative receptors of respective forms of Aβ.
Correspondence Distinct forms of Aβ oligomers are considered to be neurotoxic species that
Reisuke H. Takahashi, Department of trigger the pathophysiology of AD. It was reported that cellular prion protein
Anatomic Pathology, Tokyo Medical
University, Nishi-­Shinjuku 6-­7-­1, Shinjuku, (PrPC) is one of the most selective and high-­affinity binding partners of Aβ oli-
Tokyo, 160-­0 023, Japan.
Email: [email protected] gomers. The interaction of Aβ oligomers with PrPC is important to synaptic dys-
function and loss. The binding of Aβ oligomers to PrPC has mostly been studied
Funding information
Japan Agency for Medical Research and with synthetic peptides, cell culture, and murine models of AD by biochemi-
Development, Grant/Award Number:
19mk0101104j0702; JSPS Grant-­i n-­A id cal and biological methods. However, the molecular mechanisms underlying
for Scientific Research (C), Grant/Award the relationship between Aβ oligomers and PrPC remain unclear, especially in
Number: 17K08524 and 17K08705
the human brain. We immunohistochemically investigated the relationship be-
tween Aβ oligomers and PrPC in human brain tissue with and without amyloid
pathology. We histologically demonstrate that PrPC accumulates with aging
in human brain tissue even prior to AD mainly within diffuse-­type amyloid
plaques, which are composed of more soluble Aβ oligomers without stacked β-­
sheet fibril structures. Our results suggest that PrPC accumulating plaques are
associated with more soluble Aβ oligomers, and appear even prior to AD. The
investigation of PrPC accumulating plaques may provide new insights into AD.

K EY WOR DS
amyloid plaque, Aβ oligomer, human brain, neuropathology, PrPC

1 | I N T RODUC T ION γ-­secretases. Aβ can exist in multiple forms as full length

and various N-­and C-­truncated monomers, low and
A characteristic hallmark of Alzheimer’s disease (AD) high molecular weight soluble oligomers, protofibrils,
is the deposition of amyloid plaques, which consist of and fibrils, which form from the self-­associated assem-
β-­amyloid peptides (Aβ), in the brain. Aβ is produced bly. Soluble Aβ oligomers, precursors of amyloid fibrils,
from the amyloid precursor protein (APP) by β-­ and are proposed to be the main neurotoxic species in AD,

This is an open access article under the terms of the Creative Commons Attribution-­NonCommercial-­NoDerivs License, which permits use and distribution in any
medium, provided the original work is properly cited, the use is non-­c ommercial and no modifications or adaptations are made.
© 2021 The Authors. Brain Pathology published by John Wiley & Sons Ltd on behalf of International Society of Neuropathology

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rather than amyloid fibrils with stacked β-­sheet struc- In this study, we immunohistochemically demon-
tures stained by thioflavin S (1–­4). Aβ oligomers induce strate the co-­localization of endogenous Aβ and PrPC
and accelerate Aβ seeding and play an important role in amyloid plaques of human brain tissue and charac-
in the early initiation of Aβ aggregation (5). Aβ oligo- terize the amyloid plaques within which PrPC accumu-
mers are associated with early neuritic degeneration, lates. Accumulation of PrPC within primitive or cored
especially in synaptic compartments (6,7), leading to neuritic plaques in AD and AD mouse model brains
cognitive impairment (8). Experimentally, mouse brains, was previously reported by several groups (30,33–­35).
brain slices, and primary neurons have been treated with However, PrPC accumulating plaques are even seen in
synthetic Aβ oligomers or natural Aβ oligomer enriched the aged brain without cognitive impairment, while they
extracts isolated from AD brains (9–­11). Memory im- are rare in the advanced AD brain and not present in
pairment, inhibition of long-­ term potentiation (LTP), juvenile brains without amyloid plaques. Specifically,
synaptic dysfunction, and loss of dendritic spines were here we show that PrPC accumulates within a subset
observed in such experiments (2,11,12). of diffuse-­type plaques, composed of more soluble and
The cellular prion protein (PrPC) is a membrane-­ oligomeric Aβ in aging human brains. Since PrPC ac-
bound cell surface glycoprotein that is C-­ terminally cumulating plaques appear in aged, but not in juvenile,
bound to the plasma membrane by a glycosylphospha- human brains, and are more likely to present with de-
tidylinositol (GPI) anchor and positioned in lipid rafts mentia, these plaques could result in the deterioration of
(13,14). PrPC is highly expressed on neurons, and it was cognition in early AD. More precise characterization of
recently reported that PrPC is also highly expressed on these PrP accumulating diffuse-­type plaques might also
neuronal exosomes (15). PrPC can be the substrate for the facilitate early diagnosis of AD.
production of the pathological isoform of PrP (PrPSC),
which plays an important role in prion diseases such as
Creutzfeldt-­Jakob disease (16). PrPC and the prion para- 2 | M AT E R I A L S A N D M ET HOD S
digm also play key roles in other neurodegenerative dis-
eases, including AD (17–­19). It was identified that PrPC is 2.1 | Human brain tissue
a high-­affinity binding partner of Aβ oligomers (20–­22).
The N-­terminus of PrPC contains the binding site for Aβ We examined 30 cases (age, 29–­95 years), 19 with biopsy
oligomers (23), and this interaction is involved in Aβ neu- specimens and 11 with specimens obtained at autopsy,
rotoxicity. The binding of Aβ oligomers to PrPC with a co-­ for this study (Table 1). The information on plaque la-
receptor, metabotropic glutamate receptor 5 (mGluR5), beling in Table 1 reflects labeling with PrP antibody
in dendrites appears to cause neuronal degeneration via 3F4. Among the cases with biopsy specimens, two pa-
the activation of the Src kinase Fyn (24), leading to a loss tients were clinically diagnosed with AD (Patient nos.
of surface N-­methyl-­D-­aspartate receptors (NMDARs) 1, 2; Table 1). Four were diagnosed with or suspected of
(25–­27). Aβ oligomers affect the trafficking and endocy- having dementia without AD (Patient nos. 3–­6; Table 1),
tosis of PrPC (28). The interaction between PrPC and Aβ and eight were not diagnosed with either AD or demen-
oligomers has been reported to impair synaptic plasticity tia (Patient nos. 7–­14; Table 1). Five biopsy specimens
(29) and LTP (9). Furthermore, dendritic spine loss (27), were obtained from young patients with glioblastoma at
spatial memory deficits (29), and tau pathology spread- 29–­39 years of age, and regions of brain tissue without
ing (30) were observed in AD mouse models due to this tumor were used as control subjects (Patient nos. 15–­19;
interaction. In an AD model mouse, the deletion of PrPC Table 1). Among the autopsy specimens, one patient was
rescued mice from the loss of synaptic markers, neuritic both clinically and pathologically diagnosed with ad-
degeneration, early death, and AD pathology (29,31,32). vanced AD (Patient no. 25; Table 1). One was only clini-
The mechanism by which Aβ oligomers cause neuro- cally diagnosed with AD (Patient no. 20; Table 1), and
toxicity mediated by PrPC is not well understood. It is one was diagnosed with dementia without AD (Patient
presumed that the neurotoxic effects start with the bind- no. 21; Table 1). Five patients were not diagnosed with ei-
ing of Aβ oligomers to PrPC on the surface of neurons. ther AD or dementia (Patient nos. 22–­24, 26, 27; Table 1).
Thus, Aβ oligomers and PrPC might be expected to co-­ Three autopsy specimens were from young individuals
localize and accumulate together. The co-­localization at 28–­49 years of age (Patient nos. 28–­30; Table 1). The
of PrPC and Aβ42 oligomers was shown in hippocampal use of brain samples was approved by the ethics commit-
mouse primary neurons (21). It was also demonstrated tee of the Tokyo Medical University (approval number:
that PrPC was especially distributed to dendritic spines T2020-­0230).
in neuronal cells and co-­localized with Aβ in AD human
brain tissues (25). However, it is possible that only spe-
cific Aβ oligomeric species are involved in these protein-­ 2.2 | Antibodies
protein interactions, and the endogenous types of Aβ
molecules that are relevant to these interactions have not The following well-­characterized antibodies were used at
been precisely elucidated. the appropriate concentrations for immunohistochemical
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PRPC ACCUMULATION WITHIN Aβ OLIGOMER PLAQUES    | 3 of 14

TA BL E 1 Characteristics of patients and pathological data

Biopsy

No. Age Gender AD Dementia Amyloid plaque PrPC Plaque* Diagnosis BA

1 84 F + + + + CAA, AD, involuntary movement TPL
2 77 F + + + + CAA, AD FL
3 83 F − + + + CAA, subdural hematoma, dementia, HT OL
4 78 F − + + + Cerebral hemorrhage, dementia TL
5 86 M − + + + Cerebral hemorrhage, dementia PL
6 79 F − + + + Glioblastoma, disorientation, hemiplegia FL
7 74 F − − + + Glioblastoma, hemiplegia PL
8 78 M − − + + Subcortical hemorrhage, diabetes mellitus
type II
9 75 M − − + − CAA
10 81 F − − + − CAA OL
11 71 F − − − − Subcortical hemorrhage, hemiplegia FL
12 81 M − − − − Glioblastoma FL
13 69 M − − − − Metastatic carcinoma FPL
14 71 M − − − − Glioblastoma, aphasia, cerebral edema TL
15 39 M − − − − Oligoastrocytoma TL
16 39 M − − − − Anaplastic oligodendroglioma epilepsy FL
17 38 F − − − − Glioblastoma FL
18 32 M − − − − Metastatic sarcoma
19 29 M − − − − Germ cell tumor, headache, double vision CBR
Autopsy
No. Age Gender AD Dementia Amyloid plaque PrPC Plaque Diagnosis BA PMT
20 95 M + + + + Aspiration pneumonia, AD FTL 9:42
21 83 M − + + + Ischemic enteritis, cognitive impairment TL 2:08
22 78 M − − + + Malignant lymphoma, multiple Infarction TL 14:35
23 70 M − − + + Lung cancer, brain metastasis PL 3:58
24 71 M − − + + OMI, multiple infarction FOL 3:08
25 75 M + + + − Dissecting aneurysm, AD TL 2:40
26 85 M − − + − Intestinal necrosis, old cerebral infarction FL 13:42
27 85 M − − − − Bronchitis, congestive pulmonary edema TL 6:00
28 49 F − − − − Multiple organ failure TL 15:11
29 37 M − − − − Stomach cancer, carcinomatous meningitis FL 11:14
30 28 M − − − − CPE, cerebral edema FL 2:21
Abbreviations: AD, Alzheimer’s disease; BA, brain area; CAA, cerebral amyloid angiopathy; CBR, cerebral basal region; CPE, congestive pulmonary edema; F,
frontal; HT, hypertension; L, lobe; O, occipital; OMI, old myocardial infarction; P, parietal; PMT, post mortem time; T, temporal.
*
All results are evaluated by 3F4 antibody.

and immunofluorescent staining. The widely used and Well-­characterized antibodies 3F4 (1:1000, BioLegend,
well-­
established antibody 6E10 is directed at amino previously COVANCE, 800301, CA, USA), 6H4 (1:1000,
acid residues 3–­8 within the N-­terminus of human Aβ Prionics, 01-­
010, Schlieren-­Zurich, Switzerland), and
and detects also Aβ-­ containing APP full-­length and 12F10 (1:1000, Cayman CHEMICAL 189170, MI, USA),
fragments (1:1000, BioLegend, previously COVANCE, recognize amino acids 109–­112, 144–­152, and 142–­160
SIG-­39320, NJ, USA). 11A1 antibody (1:1000, Immuno-­ of PrP, respectively. Anti-­
prion antibody 3H2 (1:500)
Biological Laboratories, 10379, Gunma, Japan) was (37) recognizes the N-­terminus of PrP, amino acids 35–­
generated against a toxic conformer with a turn at 53. The PrP antibody T4 (1:1000 or 1:250) (38) is raised
amino acid residues 22 and 23 in Aβ42, and recog- against the C-­ terminus of bovine-­ PrP-­
peptide corre-
nizes oligomers rather than the monomer of Aβ (36). sponding to amino acids 221–­239 equivalent to amino
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4 of 14 |    TAKAHASHI et al.

acids 210–­228 of human PrP. T4 is a rabbit polyclonal 3 | R E SU LT S

antibody, while all other antibodies used in this study are
mouse monoclonals. Epitopes and predictive epitopes 3.1 | Specific accumulation of PrPC within
detected by the respective prion antibodies are depicted diffuse-­type amyloid plaques in aged non-­AD
in Figure S1. Additionally, Aβ oligomer binding sites are brain tissue
also depicted (21,23).
Amyloid plaques detected after autoclave pretreat-
ment by the 6E10 antibody which detects Aβ and Aβ-­
2.3 | Immunohistochemistry containing APP products were observed in aged patients
without dementia as evident in a representative case
Brain sections were deparaffinized and autoclaved for (Figure 1A, left). An adjacent brain section with the
30 min at 121°C in antigen retrieval buffer (Nichirei same pretreatment was subjected to PrP staining with an
Biosciences Inc. #415211, Tokyo, Japan) or incubated anti-­PrP antibody, 3F4 and showed the accumulation of
with 90% formic acid for 15 min at room tempera- PrPC within diffuse-­type amyloid plaques that had been
ture. Endogenous peroxidase was blocked, and sec- labeled by 6E10 in the adjacent section (Figure 1A, right).
tions were treated with 10% normal goat serum, and Since all human brain samples employed in this analysis
then incubated with the above-­ d escribed primary were from patients without prion diseases, only PrPC,
antibodies. and not PrPSC, was detected in the brain sections by 3F4
For the preabsorption experiment, the 3F4 antibody staining. However, PrPC is not accumulated within all
(1 mg/ml) was incubated with glutathione S-­transferase of the 6E10-­positive plaques, and the number of PrPC
(GST) protein (0.5 mg/ml) or GST-­PrPC recombinant accumulating plaques is much lower in comparison to
protein (0.5 mg/ml) in 1% bovine serum albumin (BSA). antibody 6E10 labeling of plaques. Aβ-­labeled plaques
Each protein was adjusted in 1% BSA to a concentration are shown in Figure 1 from a representative case from
10 times higher than that of the antibody (3F4, 1 μg/ml; whom brain tissue specimens were obtained both by bi-
GST-­PrPC recombinant protein, 10 μg/ml; GST protein, opsy and at autopsy (see Table 1 for characteristics of
10 μg/ml). After the preincubation of the 3F4 antibody the cases). Moreover, PrP-­positive plaques were not al-
with each protein overnight at 4°C, the brain sections ways observed in brain tissue with 6E10-­positive amyloid
were treated with the mixture of the 3F4 antibody and plaques (as indicated in Table 1). Results on PrP labeling
proteins. The sections were then incubated with second- of plaques in Table 1 were obtained using PrP antibody
ary antibodies followed by Envision+ (DakoCytomation, 3F4, which is one of the most established antibodies
CA, USA) and labeled peroxidase was detected using against prion protein and recognizes the epitope over-
diaminobenzidine. lapping with the putative Aβ oligomer binding site. The
specificity of the PrPC accumulation within the plaques
detected by the PrP antibody 3F4 was confirmed by
2.4 | Immunofluorescence preabsorption experiments employing GST protein and
GST-­PrPC recombinant protein. The PrPC accumulating
For dual immunofluorescent labeling brain sections plaques were still observed with treatment with the 3F4
were incubated with the 6E10 and T4 antibodies followed antibody and just the GST protein, even though the con-
by fluorescence-­ labeled secondary antibodies (1:200; centration of the GST was 10 times higher than that of
Molecular Probes, OR, USA). For dual label Thioflavin-­S the 3F4 antibody (Figure 1B, left). However, none of the
(ThS) and 1-­f luoro-­2,5-­bis (3-­carboxy-­4 -­hydroxystyryl) plaques were detected when treated with a combination
benzene (FSB) staining, the sections were incubated with of the 3F4 antibody and GST-­PrPC recombinant protein
filtered 0.01% ThS or FSB in 70% ethanol (ETOH) for (Figure 1B, right), supporting the specificity of 3F4 la-
20 min, and then rinsed sequentially with 70% and 100% beling. For this, the concentration of the recombinant
ETOH twice and embedded after immunofluorescent la- protein was 10 times higher than that of the antibody.
beling by the 3F4 antibody. Furthermore, the immunoreactivity detected by the 3F4
antibody was also observed with the other anti-­PrP anti-
bodies, 3H2, 6H4, 12F10, and T4 (Figure S2A-­D) in the
2.5 | Congo red and Direct Fast Scarlet adjacent section confirmed by the 6E10 antibody (Figure
(DFS) staining S3). All the PrP antibodies revealed similar diffuse-­type
plaques and plaque distributions.
Briefly, after incubating the deparaffinized brain Accumulation of PrPC within diffuse-­type amyloid
sections in 0.5% Congo red solution diluted by 100% plaques was also confirmed in the same section by dou-
ETOH or DFS stain solution (MUTO Pure Chemicals ble immunofluorescent staining with Aβ antibody 6E10
Co., LTD, Tokyo, Japan) for 20 min, the sections were and PrP antibody T4 (Figure 2A, upper row). Diffuse
rinsed in 0.2% KOH/80% ETOH solution or distilled plaques stained by 6E10 antibody were co-­localized with
water. PrPC stained by T4 antibody in biopsy brain tissue not
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PRPC ACCUMULATION WITHIN Aβ OLIGOMER PLAQUES    | 5 of 14

F I G U R E 1 PrPC is present in diffuse amyloid plaques without AD. Immunoreactivity of Aβ and PrPC in brain tissue obtained at autopsy
from a representative 71-­year-­old patient without dementia, and absorption of the 3F4 antibody by GST-­PrPC recombinant protein. PrPC is
depicted as PrP in the figure. (A) Aβ accumulation was detected by antibody 6E10 after autoclave pretreatment as diffuse-­type amyloid plaques.
Neuritic-­type amyloid plaques were not seen by antibody 6E10 in this particular brain section (left). The accumulation of PrPC, reminiscent
of findings of diffuse-­type amyloid plaque staining, was also detected in adjacent brain sections by PrP antibody 3F4 staining. A subset of the
PrPC accumulations was clearly co-­localized within the plaques detected by the 6E10 antibody (arrowheads) (right). Focal accumulation of PrPC
in the same diffuse-­type plaques stained by the 6E10 antibody (asterisks) was observed with higher magnification (insets). (B) Immunoreactivity
of the 3F4 antibody preincubated with GST protein was preserved, and accumulation of PrPC within the plaques was observed in a section from
the same brain (left). 3F4 immunoreactivity was completely abolished in serial brain sections by preincubation with GST-­PrPC recombinant
protein (right). Bar: 250 μm

diagnosed with dementia. To confirm the specificity of now appeared following retrieval treatment after formic
the staining an adjacent section was stained without pri- acid (Figure 3B and inset). The same large plaque in an
mary 6E10 and T4 antibodies, respectively. The plaques adjacent section without formic acid did; however, show
were not observed at all even with longer exposure 3F4 antibody staining (Figure 3C and inset), and this
(Figure 2A, lower row). A representative diffuse-­type 3F4 immunoreactivity was completely abolished by the
plaque can be seen stained by both 6E10 and T4 anti- pretreatment with formic acid (Figure 3D). These results
bodies, while a plaque with an amyloid core was only suggest that PrPC accumulating plaques are composed
stained by the 6E10 and not with the T4 antibody in the of more soluble Aβ, and/or Aβ-­containing APP and/or
non-­AD case with dementia (Figure 2B). Thus, PrPC ac- APP products. Next, we tried to stain the same plaque in
cumulation can be observed within diffuse-­type amyloid an adjacent section with antibody 11A1 (36) specific for
plaques in non-­AD aged brain tissue. neurotoxic Aβ42 oligomers. 11A1 immunoreactivity was
observed in the same plaque (Figure 3E and inset) that
was mostly removed by formic acid treatment (Figure 3F
3.2 | PrPC accumulating plaques composed of and inset). At higher magnification, some amyloid depo-
more soluble Aβ oligomers without stacked β-­ sition seemed to be present after the treatment (Figure 3F
sheet structures inset), which might represent some labelings of insoluble
Aβ or alternatively may represent soluble Aβ oligomers
To better characterize the PrPC accumulating plaques in generated by the formic acid treatment. Intraneuronal
non-­AD cases, we performed pretreatment with formic Aβ42 detected by antibody 11A1 was also removed by
acid in addition to autoclave retrieval of brain tissue. We formic acid (Figure 3E inset, asterisk, and 3F inset).
focused on one independent plaque to facilitate precise These results suggest that most of the PrP antibody 3F4-­
observation of the effect of formic acid pretreatment positive plaques are composed of more soluble Aβ42
with serial sections. We saw that the 6E10 staining of this oligomers.
plaque was extremely diminished by formic acid treat- We next immunofluorescently investigated whether
ment (Figure 3A and inset), while several small plaques 3F4-­positive plaques in the non-­ AD with dementia
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6 of 14 |    TAKAHASHI et al.

F I G U R E 2 Co-­localization of Aβ and PrP in diffuse-­type amyloid plaques but not in cored plaques. (A) Diffuse plaques (arrowheads,
upper row) stained by Aβ antibody 6E10 (green) revealed co-­localization with PrP stained by antibody T4 (red) in biopsy brain tissue from
a 74-­year-­old female case not diagnosed with dementia. To show that the marked red dots are autofluorescence rather than from primary
antibody labeling, an adjacent section of the same area presented in the upper panel was stained without primary antibodies (lower row).
To confirm the close vicinity of the sections, the same blood vessel is evident in the upper right corner (arrows). Even with longer exposure,
the plaques were not observed without primary antibodies. In contrast, the green and red dot-­l ike labeling showing marked intensities were
completely co-­localized and thus represent autofluorescence. (B) A diffuse-­type plaque was stained by both 6E10 (green) and T4 antibodies
(red) in a section from a biopsy brain tissue of an 86-­year-­old male case diagnosed with dementia (arrowhead), while a plaque with an amyloid
core was only stained by the Aβ antibody 6E10 (arrow). A higher magnification view of the diffuse plaque (at arrowhead in the lower power
image) is shown in the inset. Bar: 100 μm

cases ever labeled with thioflavin S (ThS), which de- very small amyloid core was labeled by both ThS and
tects stacked β-­pleated amyloid fibrils. In a represen- 3F4 antibodies, obvious labeling of ThS was also not ob-
tative section, an amyloid core of a neuritic amyloid served in PrP accumulating diffuse-­type plaques in an-
plaque was stained by ThS, but not by 3F4 antibody other non-­AD case with dementia (Figure S5).
(Figure 4A and arrowheads). There was no evidence
of the co-­localization of ThS and antibody 3F4 in the
merged image, even at higher magnification (Figure 4A 3.3 | PrPC accumulation within various
inset). In contrast, there was no labeling of ThS within types of plaques in brain tissue clinically
3F4-­labeled plaques (Figure 4B and arrowheads) that diagnosed with AD
would, therefore, be classified as diffuse amyloid plaques.
Co-­localization of ThS and 3F4 labeling was also not In addition to the diffuse plaques, antibody 6E10
observed with higher magnification (Figure 4B arrow Aβ staining typically also reveals neuritic or classi-
and inset). In addition, we used the dye 1-­f luoro-­2,5-­bis cal plaques with amyloid cores in brain tissue, such
(3-­carboxy-­4 -­hydroxystyryl) benzene (FSB), which also as is seen in a case obtained by biopsy from a patient
detects β-­pleated amyloid fibrils, instead of ThS (Figure clinically diagnosed with AD (Patient no. 1; Table 1)
S4). Similarly, in another case, a diffuse plaque was not (Figure 5A). Similar to Figure 1A of aged brain tissue
labeled by ThS and labeled by 3F4 antibody (Figure S4 without AD, only a subset of the 6E10-­positive plaques
arrows), while a neuritic plaque with a very small am- in this AD brain was stained by the PrP antibody 3F4
yloid core was labeled by both ThS and 3F4 antibody (Figure 5B). However, in the neuritic plaques detected
(Figure S4 arrowheads). While a neuritic plaque with a by antibody 3F4 staining, most of the amyloid cores
 17503639, 2021, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bpa.12941 by Cochrane Mexico, Wiley Online Library on [12/06/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
PRPC ACCUMULATION WITHIN Aβ OLIGOMER PLAQUES    | 7 of 14

F I G U R E 3 Abolition of PrP immunoreactivity following pretreatment with formic acid and labeling with Aβ oligomer antibody in
a diffuse-­type plaque. (A) Here we focused on one amyloid plaque to observe the alteration of the 6E10-­i mmunoreactivity by formic acid
pretreatment (arrowhead) and higher magnification of the markedly diminished plaque labeling is also depicted (inset). The brain tissue was
obtained at autopsy from a 71-­year-­old patient without dementia. (B) The Aβ immunoreactivity was markedly diminished by formic acid
(inset). (C) The same plaque was also immunolabeled by the PrP antibody 3F4 in serial brain sections (arrowhead) and was observed in a higher
magnification view (inset). (D) PrP immunoreactivity was completely abolished by formic acid pretreatment. (E) Moreover, this plaque was also
immunolabeled with the Aβ oligomer antibody 11A1 (arrowhead) and is also shown in a higher magnification view (inset). Intraneuronal Aβ
was detected by the 11A1 Aβ oligomer antibody (asterisk). (F) Similarly to 6E10-­i mmunoreactivity, the immunoreactivity of 11A1 was markedly
diminished by formic acid pretreatment (arrowhead) and amyloid deposition newly appeared after the treatment (inset). Intraneuronal Aβ was
also abolished by formic acid treatment (inset). Bar: 250μm

were not stained by antibody 3F4 in this case of AD, 3.4 | Much less PrP immunoreactivity in
while the surrounding dystrophic neurites were stained amyloid plaques in brain tissue clinically and
(Figure 5B, left inset). The amyloid core that was not pathologically diagnosed with advanced AD
stained by the 3F4 antibody was instead stained by
the Congo red dye (Figure 5B, right inset), denoting fi- While numerous and remarkable 6E10-­positive plaques
brillar amyloid. The 3F4-­positive plaques in AD were, were observed, faint staining by the 3F4 antibody was ob-
therefore, mainly of the diffuse type (Figure 5C), as served in plaques in a brain tissue specimen from a patient
detected in the other aged brain tissues. Moreover, in who was both clinically and pathologically diagnosed
brain tissues from cases with clinically diagnosed de- with advanced AD (Patient no. 25; Table 1) (Figure 6A,B).
mentia but not diagnosed AD (Patient no. 3; Table 1), In addition, 3F4-­positive diffuse-­type plaques were not
the amyloid cores were stained by the 3F4 antibody, in observed. At higher magnification, faint granular stain-
addition to the dystrophic neurites (Figure 5D). Thus, ing was only detected by the 3F4 antibody, especially in
some plaque cores have PrP labeling, although overall dystrophic neurites around amyloid cores in contrast to
there is more labeling of diffuse plaques and of dystro- amyloid plaques markedly stained by the 6E10 antibody
phic neurites. in the adjacent brain section (Figure 6B,C, asterisks).
 17503639, 2021, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bpa.12941 by Cochrane Mexico, Wiley Online Library on [12/06/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
8 of 14 |    TAKAHASHI et al.

F I G U R E 4 Lack of thioflavin S labeling of PrP-­positive plaques in non-­AD brains. (A) Thioflavin S (ThS) staining (green) for β-­pleated Aβ
fibrils revealed co-­localization with neuritic plaques in a brain tissue obtained by biopsy from an 83-­year-­old female case clinically diagnosed
with dementia (arrowheads). PrP-­i mmunoreactivity (red) was absent in this neuritic plaque. A higher magnification view of the neuritic plaque
is shown in the inset. (B) In contrast to the neuritic plaque, ThS staining was not evident in PrPC-­positive plaques (arrowheads). In a higher
magnification view of one PrP-­positive plaque (arrow), there was no overlap with ThS (inset). Bar: 50 μm

F I G U R E 5 PrPC is present in both diffuse and neuritic plaques of biopsy AD brain tissues. PrP immunoreactivity in plaques reminiscent
of both diffuse-­type and neuritic-­type is evident in aged brain tissue from a representative patient clinically diagnosed with AD. (A) Numerous
amyloid deposits are seen in diffuse and neuritic plaques, and in blood vessels, by the Aβ antibody 6E10 in brain tissue from an 84-­year-­old
patient. (B) Only parts of the plaques were detected by PrP antibody 3F4 staining. PrP immunoreactivity is not detectable in blood vessels.
Coarse and dense PrP accumulation, similar to dystrophic neurites of neuritic plaques, is evident (arrowhead); a higher magnification is also
shown (inset, left). The amyloid core was stained by Congo red in the same plaque (inset, right). (C) In a higher magnification view of the square
area of (B) the PrP-­positive plaques were mostly diffuse-­type plaques, while no remarkable PrP-­i mmunoreactivity was detected in the amyloid
cores of this brain section. (D) In another brain section from a biopsy specimen of an 83-­year-­old patient clinically diagnosed with dementia,
dense core PrP-­i mmunoreactivity was detected by 3F4 staining. Bars: 250 μm (A, B), 50 μm (C), 25 μm (D)

However, fewer and fainter immunolabeling of plaques that different labelings with PrP antibodies relates either
was observed by PrP antibodies 3H2 and T4 and none by to epitope blocking, antibody affinity, conformational
antibodies 6H4 and 12F10 (Figure S6). We hypothesize changes, and/or cleavage of PrP.
 17503639, 2021, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bpa.12941 by Cochrane Mexico, Wiley Online Library on [12/06/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
PRPC ACCUMULATION WITHIN Aβ OLIGOMER PLAQUES    | 9 of 14

F I G U R E 6 PrP antibody 3F4 immunoreactivity was much lower in amyloid plaques of advanced AD brain. (A) Strong immunoreactivity of
the Aβ antibody 6E10 was observed in amyloid plaques from brain tissue of a 75-­year-­old patient clinically and pathologically diagnosed with
advanced AD. (B) In contrast, much less 3F4-­i mmunoreactivity was observed in an adjacent section. (C) The squared area in (B) was magnified
and the same plaques are visible, respectively (asterisks). Bars: 100 μm (A, B), 25 μm (C)

3.5 | No PrP immunoreactivity as plaques expression cloning, the PrPC accumulation in neu-
in brain tissue from young and aged patients ritic amyloid plaques in AD brain tissue was demon-
without amyloid plaques strated by immunohistochemical staining (33,40). We
previously reported that the accumulation of PrPC
While intraneuronal Aβ and/or Aβ domain-­containing was mainly detected within dystrophic neurites of AD
APP were immunohistochemically stained as granular brain tissue by immunohistochemical methods and
dots by the 6E10 antibody in brain tissues from eight that at times it was also detected in the amyloid cores
young patients (five biopsies, Patient nos. 15–­19; three au- of some neuritic plaques (35). Since PrPC is transported
topsies, Patient nos. 28–­30; Table 1), no obvious plaques to the distal regions of neurites in an anterograde man-
were detected in these young cases by either PrP or Aβ ner (41), we suggested that in AD brain where Aβ and
antibodies (Figure S7A,B). The intraneuronal accumu- other abnormal proteins aggregate in dystrophic neu-
lation of Aβ/APP was also revealed as granular dots by rites, PrPC seems to redistribute more in the proximal
6E10 staining in brain tissue specimens from 10 aged pa- part of such dystrophic neurites (35).
tients without dementia (eight biopsies, Patient nos. 7–­14; In this study, our immunohistochemical analyses now
two autopsies, Patient nos. 26, 27; Table 1) (Figure S7C demonstrated specific co-­localization of Aβ and PrPC
and inset), where amyloid plaques were not seen in five within diffuse-­type amyloid plaques in human brain tis-
cases (4 biopsies, Patient nos. 11–­14; one autopsy, Patient sue from aged patients. In addition to previous studies
nos. 27; Table 1) (Figure S7C). In these aged brain tis- that reported the accumulation of PrPC within amyloid
sue specimens without amyloid plaques from patients plaques in the brain tissue of patients with AD, we have
without dementia, no plaque was detected by the 3F4 now detected the accumulation of PrPC mainly within
antibody either (Figure S7D). In contrast, endogenous diffuse-­type amyloid plaques in human brain tissue from
PrPC was observed in neurites and neuronal cell bod- patients who were not diagnosed with AD. While the ac-
ies by 3F4 antibody at higher magnification (Figure S8). cumulation of PrPC in diffuse plaques was observed in
In one case involving an autopsied patient without de- specimens from several patients without dementia, it was
mentia who had an old cerebral infarction (Patient no. more evident in patients with dementia.
26; Table 1), numerous amyloid plaques were detected In a few brain tissues from patients only clinically, not
by 6E10 staining (Figure S7E and inset); however, none pathologically, diagnosed with AD we also demonstrated
of these plaques were detected by 3F4 staining (Figure marked immunoreactivity of PrP antibodies in dystro-
S7F). phic neurites and some amyloid cores. Interestingly, in
contrast to the brain tissue specimens from the patients
who were only clinically diagnosed with AD, in the au-
4 | DI SC US SION topsy brain tissue clinically and pathologically diagnosed
with advanced AD, far fewer PrP-­positive plaques were
The finding that PrPC binds to Aβ oligomers with detected and the plaques were more faintly stained with
high affinity has attracted considerable interest (21). PrP antibody in comparison to 6E10-­positive amyloid
Several lines of evidence have demonstrated that the plaques. Our results suggest that PrPC might preferen-
binding of Aβ oligomers to PrPC is relevant to the acti- tially accumulate in plaques of aged brains and in brain
vation of downstream proteins, leading to neurotoxic- tissue of patients with early-­stage dementia compared
ity (22,27,39). Notably, before PrPC was identified as to advanced AD. Alternatively, it is possible that the
a receptor for Aβ oligomers by genome-­w ide unbiased PrP epitope is more blocked or that PrPC is cleaved with
 17503639, 2021, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bpa.12941 by Cochrane Mexico, Wiley Online Library on [12/06/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
10 of 14 |    TAKAHASHI et al.

F I G U R E 7 Schema for PrPC labeling with amyloid plaques. (A) Aβ characteristics of PrP (+) plaques (PrP-­positive plaques). The PrP (+)
plaques were abolished by pretreatment with formic acid and were not stained by ThS, FSB, or Congo Red dyes. In addition, the plaques were
immunolabeled by the 11A1 antibody, which detects Aβ oligomers. The PrP (+) plaque thus should be composed of more soluble Aβ oligomers
and protofibrils. (B) Relationship between Aβ and PrPC. The binding affinity of PrPC might be much higher to Aβ oligomers and protofibrils,
and lower to fibrils and more insoluble-­types of Aβ. PrP showed markedly lower immunoreactivity in neuritic plaques in brain tissue with
advanced AD

increasing Aβ aggregation. We also noted that despite We histochemically characterized the PrPC accu-
low PrP labeling in advanced AD brain sections, more mulating plaques. The immunoreactivity of PrPC in
plaque labeling was detected with PrP antibodies 3H2 plaques was abolished by pretreatment with formic
and T4, which detect N-­terminal and C-­terminal region acid, while that of 6E10 was also significantly decreased.
of PrPC, compared to little to no labeling with antibod- Furthermore, PrP-­positive plaques were immunolabeled
ies 3F4, 6H4, and 12F10, which detect relatively central by an antibody that detects Aβ42 oligomers but was
regions of PrPC. In contrast, in non-­AD aged tissues, all not labeled by ThS, which detects stacked β-­sheet fibril
of the PrP antibodies labeled the diffuse plaques (Figure structures. Our results support the conclusion that initial
S2). It is possible that the Aβ oligomer binding site in PrPC PrPC accumulating diffuse plaques, PrP (+) plaques, are
is blocked, PrPC is cleaved or the PrP conformation is al- exclusively composed of more soluble Aβ oligomers and/
tered within amyloid plaques in advanced AD brain. Our or protofibrils rather than stacked β-­pleated fibrils (see
observations are in line with the findings of others that schema in Figure 7A). This conclusion is supported by
PrPC plaque load and the amount of PrPC accumulation PrP (+) plaques being abolished by pretreatment with for-
is lower in AD than non-­AD cases (30,42–­44). mic acid and that they are not stained by amyloid dyes.
 17503639, 2021, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bpa.12941 by Cochrane Mexico, Wiley Online Library on [12/06/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
PRPC ACCUMULATION WITHIN Aβ OLIGOMER PLAQUES    | 11 of 14

In the present pathological study, we did not provide of circulating exosome-­bound Aβ in blood samples from
data demonstrating the direct binding of Aβ oligomers AD patients are correlated with amyloid plaque load on
and PrPC. However, since a number of laboratories have positron emission tomography (PET) (57). Investigation
reported evidence that the neurotoxicity of Aβ oligo- of the relationship between PrP (+) plaques and exosomes
mers emerges after binding with PrPC leading to AD by exosomal markers might serve to further characterize
pathology (17,21,30,45,46), this supports that PrPC and plaques.
soluble Aβ oligomers bind together in PrP (+) plaques. Different types of amyloid plaques have been reported
We propose a model of amyloid plaque formation and as neuritic plaques, primitive plaques, burnt-­out plaques,
the involvement of PrPC (see schema in Figure 7B). The cotton-­wool plaques, and diffuse plaques. And recently
binding of PrPC to Aβ may occur at an early phase in coarse-­g rained plaques in early onset AD were reported
the progression of AD or with aging in certain individ- (60). We demonstrated the specific co-­localization of Aβ
uals, while PrPC is not bound to or sequestered by amy- and PrPC within amyloid plaques in human brain tissue
loid plaques with the β-­sheet structure that is typically from aged patients who were not diagnosed with AD
observed in the advanced phase of AD. Alternatively, and demonstrated that such PrPC accumulating plaques
conformational alterations that block the PrP epitope are made up of more soluble Aβ oligomers. Not all the
as Aβ aggregates further might block PrP antibody diffuse-­type plaques, but rather a subset or even just
labeling. focal parts of the plaques, may be composed of Aβ oligo-
Whereas many previous studies have focused on mers with PrP (see Figure 1). We suggest that the PrP
downstream signaling pathways that affect the func- (+) plaque is related to aging and/or dementia and that
tion and activity of neurons, subsequent to the bind- it is another category of amyloid plaque. We, therefore,
ing of Aβ oligomers to PrPC at the neuronal surface propose the PrP (+) plaque as a new subtype of amyloid
(21,27,39,47,48), only a few groups have explored how Aβ plaque, and more specifically a subset of diffuse plaques.
oligomers bound to PrPC at the neuronal surface might While some neuritic plaques were immunolabeled by
act. It was suggested that the internalization of PrPC may anti-­PrP antibodies, the mechanisms of Aβ and PrPC
also allow Aβ oligomers to accumulate intracellularly in accumulation within diffuse plaques might be differ-
the cytoplasm of neurons, where they might affect cellu- ent from their mechanisms of accumulation in neuritic
lar functions, such as protein degradation by the prote- plaques. However, clear involvement of PrP (+) plaques
asome complex (49). Apolipoprotein E (APOE) has been in the pathophysiology of AD remains to be elucidated.
thought to be involved in the uptake of Aβ by neurons, A better understanding of the role of PrPC in AD could
and the blocking of the interaction between APOE and provide new insights to establish new therapies for AD.
Aβ by a non-­toxic synthetic peptide reduced intracellular
Aβ accumulation and Aβ oligomer levels and protected AC K NOW L E D G M E N T S
against synaptic protein loss (50,51). The blocking of Aβ The authors thank Drs. Hiroyuki Shimada and Kuniaki
internalization through PrPC might be a therapeutic tar- Tsuchiya for the precise diagnosis of AD in autopsied
get for AD therapy. brains. This study was supported by the JSPS Grant-­
Recent studies have supported that exosomes play im- in-­A id for Scientific Research (C) (No. 17K08524, No.
portant roles in the pathogenesis of neurodegenerative 17K08705). Additionally, it was supported by grants from
diseases, especially in AD and prion disease (52–­55). the Ministry of Health, Labor and Welfare, Japan (H29-­
Exosomes are small membrane vesicles (30–­150 nm in Shokuhin-­Ippan-­004) and partly by a grant for Research
diameter) that are released into the extracellular space on Regulatory Science of Pharmaceuticals and Medical
and body fluids (20,56). Membranes of multivesicular Devices from Japan Agency for Medical Research and
bodies (MVBs) are invaginated to form intraluminal Development (AMED) (19mk0101104j0702).
vesicles (ILVs). Following the fusion of MVBs with the
plasma membrane ILVs released into the extracellular C ON F L IC T OF I N T E R E ST
space are then called exosomes (55). It was reported that The authors declare no conflicts of interest in associa-
neuronal exosomes are highly enriched in PrPC, which tion with the present study.
binds to Aβ oligomers with high affinity (15), and also
that prefibrillar Aβ aggregates favorably bind to exo- AU T HOR C ON T R I BU T ION S
somes (57). Exosomes have been suggested to bind toxic Reisuke H. Takahashi made substantial contributions to
oligomers and convert them to non-­toxic fibril types the conceptual idea of this project, collection, analysis,
in amyloid plaques to protect from toxicity (58). PrP and interpretation of data. Reisuke H. Takahashi also
(+) plaques might be transiently and reversibly formed designed the experiments and drafted the manuscript.
prior to such non-­toxic amyloid plaques. Alternatively, Mayumi Yokotsuka and Yuko Sato contributed to ex-
since exosomal proteins are also enriched in plaques in perimental works. Minoru Tobiume, Hideki Hasegawa,
AD brains (55), PrPC could be transferred to amyloid and Toshitaka Nagao helped in funding this study.
plaques composed of soluble Aβ oligomers via exosomes Minoru Tobiume also provided technical advice for ex-
or ILVs (6,59). Recently, it was reported that the levels periments. Gunnar K. Gouras edited the manuscript.
 17503639, 2021, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bpa.12941 by Cochrane Mexico, Wiley Online Library on [12/06/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
12 of 14 |    TAKAHASHI et al.

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44. Whitehouse IJ, Miners JS, Glennon EB, Kehoe PG, Love S, ognizes the C-­terminal region of PrP more than other
Kellett KA, et al. Prion protein is decreased in Alzheimer's brain PrP antibodies. One of the binding sites of Aβ oligomers
and inversely correlates with BACE1 activity, amyloid-­β levels
overlaps with the epitope on PrP recognized by antibody
and Braak stage. PLoS One. 2013;8:e59554.
45. Freir DB, Nicoll AJ, Klyubin I, Panico S, Mc Donald JM, 3F4 (21). GPI: glycosylphosphatidylinositol anchor. *The
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yloid β assemblies can be therapeutically targeted at multiple Chen et al (23) and Lauren et al (21), residues 23–­27 and
sites. Nat Commun. 2011;2:336. 95–­110, respectively
46. Younan ND, Chen KF, Rose RS, Crowther DC, Viles JH. Prion
FIGURE S2 PrPC accumulation in amyloid plaques is
protein stabilizes amyloid-­β (Aβ) oligomers and enhances Aβ
neurotoxicity in a Drosophila model of Alzheimer disease. J Biol seen with a panel of PrP antibodies. PrPC accumulation
Chem. 2018;293:13090–­9. consistent with diffuse-­type amyloid plaques with anti-­
47. Jarosz-­Griffiths HH, Noble E, Rushworth JV, Hooper NM. PrP antibodies in brain tissue from an aged patient with-
Amyloid-­β receptors: the good, the bad, and the prion protein. J out dementia. PrPC immunoreactivity was also detected
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in sections of aged brain tissue with PrP antibodies 3H2
48. Viola KL, Klein WL. Amyloid β oligomers in Alzheimer’s dis-
ease pathogenesis, treatment, and diagnosis. Acta Neuropathol. (A), 6H4 (B), 12F10 (C), and T4 (D). Bar: 250 μm
2015;129:183–­206. FIGURE S3 PrPC accumulating plaques labeled by PrP
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tion. Nature. 2009;457:1090–­1. 6E10. PrPC accumulations are observed in the same area
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of a serial brain section where amyloid plaques stained
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E and Aβ reduces intraneuronal accumulation of Aβ and inhibits by Aβ antibody 6E10 are seen
synaptic degeneration. Am J Pathol. 2013;182:1750–­68. FIGURE S4 Lack of FSB labeling of PrP-­positive plaques
51. Nomura S, Umeda T, Tomiyama T, Mori H. The E693Δ (Osaka) in non-­AD brains. No co-­localization of FSB with PrP-­
mutation in amyloid precursor protein potentiates cholesterol-­ positive plaques was observed, similarly to the ThS stain-
mediated intracellular amyloid β toxicity via its impaired choles-
ing pattern. (A) FSB staining (blue) was co-­localized
terol efflux. J Neurosci Res. 2013;91:1541–­50.
52. Asai H, Ikezu S, Tsunoda S, Medalla M, Luebke J, Haydar T, with neuritic plaques (arrowheads). (B) Conversely, FSB
et al. Depletion of microglia and inhibition of exosome synthesis staining revealed no PrP-­positive plaques (arrowheads).
halt tau propagation. Nat Neurosci. 2015;18:1584. Bar: 50 μm
53. Hartmann A, Muth C, Dabrowski O, Krasemann S, Glatzel M. FIGURE S5 Negative ThS labeling of PrP accumulat-
Exosomes and the prion protein: more than one truth. Front
ing diffuse plaque while positive ThS of neuritic plaque
Neurosci. 2017;11:194.
54. Jeon I, Cicchetti F, Cisbani G, Lee S, Li E, Bae J, et al. Human-­ with a very small amyloid core. A diffuse plaque was not
to-­mouse prion-­like propagation of mutant huntingtin protein. labeled by ThS and labeled by 3F4 antibody (arrows),
Acta Neuropathol. 2016;132:577–­92. while a neuritic plaque with a very small amyloid core
55. Rajendran L, Honsho M, Zahn TR, Keller P, Geiger KD, was labeled by both ThS and 3F4 antibodies (arrow-
Verkade P, et al. Alzheimer's disease β-­a myloid peptides are re-
heads) from a representative 74-­year-­old patient without
leased in association with exosomes. Proc Natl Acad Sci U S A.
2006;103:11172–­7. dementia. Bar: 100μm
 17503639, 2021, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bpa.12941 by Cochrane Mexico, Wiley Online Library on [12/06/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
14 of 14 |    TAKAHASHI et al.

FIGURE S6 . Low immunoreactivity of PrP antibodies spite of the remarkable amyloid deposition and large
in amyloid plaques of advanced AD brain tissue. (A, numbers of amyloid deposits (E, inset) in an 85-­year-­old
D) Compared to the immunoreactivity of Aβ antibody patient with an old infarction but without dementia (E),
6E10 in Figure 6A, fainter labeling of PrP antibodies no PrP-­immunoreactivity was detected. Bars: 250 μm,
3H2 (A) and T4 (D) are evident in serial sections of (inset, E 25 μm)
advanced AD brain tissue (arrows). (B, C) Almost no FIGURE S8 Localization of PrP in neurites and cell
immunoreactivity is evident in plaques in advanced bodies of neurons. With higher magnification of Figure
AD with PrP antibodies, 6H4 (B) and 12F10 (C). Bar: S7D, the ubiquitous localization of PrP immunoreactiv-
250 μm ity by antibody 3F4 is evident along neurites, which ap-
FIGURE S7 No PrP-­plaque was detected from young pear like lines in parallel (arrows). Additionally, PrPC is
and aged brain tissues without dementia. (A, B) The seen in cell bodies of neurons evident by stronger brown-
deposition of amyloid plaques was not detected by Aβ ish labeling (asterisks). Bar: 200 μm
antibody 6E10 in these cases (A), and (B) no PrP accu-
mulating plaques were observed in the brain tissue of
a 49-­year-­old patient. (C, D) Even in the brain tissue How to cite this article: Takahashi RH,
from an 85-­year-­old patient without dementia or amy- Yokotsuka M, Tobiume M, et al. Accumulation of
loid plaque deposition (C), no PrP-­positive plaque was cellular prion protein within β-­amyloid oligomer
detected (D). Intraneuronal Aβ/APP immunoreactivity plaques in aged human brains. Brain Pathology.
was observed as granular dots by the 6E10 antibody in 2021;31:e12941. https://doi.org/10.1111/bpa.12941
(A, C) and at higher magnification (C, inset). (E, F) In