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https://doi.org/10.1038/s41467-019-10278-x

OPEN

Tau binding protein CAPON induces
tau aggregation and neurodegeneration

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Shoko Hashimoto 1, Yukio Matsuba1, Naoko Kamano1, Naomi Mihira1, Naruhiko Sahara2, Jiro Takano1,
Shin-ichi Muramatsu 3,4, Takaomi C. Saido 1 & Takashi Saito 1,5

To understand the molecular processes that link Aβ amyloidosis, tauopathy and neurodegeneration, we screened for tau-interacting proteins by immunoprecipitation/LC-MS. We
identiﬁed the carboxy-terminal PDZ ligand of nNOS (CAPON) as a novel tau-binding protein.
CAPON is an adaptor protein of neuronal nitric oxide synthase (nNOS), and activated by the
N-methyl-D-aspartate receptor. We observed accumulation of CAPON in the hippocampal
pyramidal cell layer in the AppNL-G-F -knock-in (KI) brain. To investigate the effect of CAPON
accumulation on Alzheimer’s disease (AD) pathogenesis, CAPON was overexpressed in
the brain of AppNL-G-F mice crossbred with MAPT (human tau)-KI mice. This produced
signiﬁcant hippocampal atrophy and caspase3-dependent neuronal cell death in the CAPONexpressing hippocampus, suggesting that CAPON accumulation increases neurodegeneration. CAPON expression also induced signiﬁcantly higher levels of phosphorylated, oligomerized and insoluble tau. In contrast, CAPON deﬁciency ameliorated the AD-related
pathological phenotypes in tauopathy model. These ﬁndings suggest that CAPON could
be a druggable AD target.

1 Laboratory for Proteolytic Neuroscience, RIKEN Center for Brain Science, 2-1 Hirosawa, Wako-City, Saitama 351-0198, Japan. 2 Department of Functional
Brain Imaging Research, National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology, 4-9-1
Anagawa, Inage-ku, Chiba-City, Chiba 263-8555, Japan. 3 Division of Neurology, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke-City, Tochigi
329-0498, Japan. 4 Center for Gene & Cell Therapy, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan. 5 Department of
Neuroscience and Pathobiology, Research Institute of Environmental Medicine, Nagoya University, Nagoya-City, Aichi 464-8601, Japan. Correspondence andrequests for materials should be addressed to S.H. (email: shoko.hashimoto@riken.jp) or to T.C.S. (email: saido@brain.riken.jp)
or to T.S. (email: takashi.saito.aa@riken.jp)

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lzheimer’s disease (AD) is the most common neurodegenerative disorder and the major cause of dementia1. It isalso a progressive disease, the symptoms of which worsen
over decades. The neuropathological hallmarks of AD include
extracellular deposits of amyloid-β (Aβ), the major component ofsenile plaques, and neuroﬁbrillary tangles (NFTs) composedof hyperphosphorylated tau protein. According to the amyloidcascade hypothesis, the initial elevation of Aβ levels is the unique
primary trigger of AD2, with a deterioration in Aβ metabolism
and the accumulation of Aβ plaques starting two decades prior
to the appearance of clinical symptoms3. In contrast, tauopathy,
which is observed in a variety of neurodegenerative disorders, issigniﬁcantly enhanced following Aβ amyloidosis3. However, themolecular mechanisms that link Aβ amyloidosis, tauopathy andneurodegeneration remain unresolved4.
We previously developed two lines of novel AD model micebased on an App (amyloid precursor protein) knock-in (KI)strategy5. The ﬁrst mouse model (AppNL-F/NL-F KI; AppNL-F)
carries two mutations (KM670/671NL: Swedish; I716F:
Iberian) identiﬁed in human familial AD. The second model(AppNL-G-F/NL-G-F; AppNL-G-F) carries an additional mutation(E639G: Arctic mutation), and exhibits aggressive pathology5,6.
Both lines, which produce humanized Aβ without overexpressing
APP, show pronounced Aβ amyloidosis, gliosis, and memory
deﬁcits5,7. Further, we established human MAPT (human tau)-KI
mice (hTau-KI), which express 6 isoforms of wild-type (WT)human tau instead of murine tau. Although we thought that the
double-KI mice generated by cross-breeding App KI and hTau-KI
mice might show greater AD pathology than the single App-KI
mice due to tau humanization, we noted no overt pathologicalchanges in the former mice. These results suggested the presenceof mechanisms/factors in addition to Aβ amyloidosis that induce
tauopathy and neurodegeneration.
In order to understand the molecular mechanism of tau
accumulation, we screened for tau-interacting proteins using
Wtau-transgenic (Tg) mice, which express WT human tau
tagged with a Flag epitope8. We isolated tau-binding proteinsby immunoprecipitation using a Flag-tag antibody and identiﬁedthem by LC-MS/MS analysis. The methods used to generate
the tau interactome were validated by identiﬁcation of the tubulin
beta-4A chain as one of the tau-binding proteins (Supplementary
Data 1), given that tau is a microtubule-binding protein.
Among the proteins identiﬁed in the tau interactome, we
focused on one protein, named carboxy-terminal PDZ ligand of
neuronal nitric oxide synthase (CAPON). CAPON is an adaptor
protein of neuronal nitric oxide synthase (nNOS), which acts as
an enzyme for the production of nitric oxide (NO), and is
involved in N-methyl-D-aspartate (NMDA) receptor-mediatedexcitotoxicity9. CAPON is generally assumed to recruit substrates
to nNOS, and facilitate their NO-mediated modiﬁcation
through protein-protein interactions9. Several studies havereported that CAPON polymorphisms are associated with schizophrenia, and other psychiatric disorders10,11. Moreover, Richieret al,12 demonstrated that CAPON positively regulates spine
density, while Li et al,13 reported that CAPON regulates neuronalcell death downstream of the NMDA receptor. It therefore
appears that CAPON contributes to neurotransmission andneuronal excitotoxicity. In addition, according to a report by
Hashimoto et al14, CAPON is upregulated in CA1 pyramidal cells
in the AD brain. These results imply that CAPON may play animportant role in the pathogenesis of AD, although the underlying mechanism(s) remain unknown.
To clarify the effect of CAPON on AD pathology, we used an
adeno-associated virus (AAV)-mediated approach to introduce
CAPON cDNA into the brain of AppNL-G-F and AppNL-G-F/
MAPT (hTau) double-KI mice which we newly developed. Given
2that the hTau-KI mouse expresses an endogenous level of WT
human tau, we were able to analyze the effects of the humantau protein. Our results revealed that CAPON expression facilitates hippocampal atrophy, with accompanying neuronal cell
death. We also veriﬁed that deﬁciency of CAPON in P301STau-Tg tauopathy mouse model suppressed tau pathology andneurodegeneration. In addition, we examined the molecularmechanisms that lead to CAPON-induced neuronal cell deathand AD pathology, i.e. tau phosphorylation and aggregation, Aβ
deposition, and gliosis, in CAPON-expressing mice.
Results
Generation and charactirization of human MAPT knockinmouse. In this study, to evaluate functions of a novel tau-binding
protein: CAPON on AD-related pathologies including tau
pathology, we used a new mouse model expressing tau proteinin the manner of human brain.
Normal adult human brains express six distinct isoforms which
are classiﬁed into 3-repeats (3R)-tau and 4-repeats (4R)-tau
depending on the number of repeated microtubule-binding domains.
On the other hand, adult mouse possesses 4R-tau only. Importantly,
NFTs in human AD comprise an equal mixture of all 3R and 4R tau
isoforms. Therefore, mouse model should express all tau isoforms
when we evaluate formation of tau pathology. In addition, ideally, themouse model of tau pathology should be based on the KI strategy
because overexpression of tau may disturb the normal physiological
functions of neurons, such as microtubule assembly and synaptic
functions. Accordingly, we generated human MAPT KI (hTau-KI)
mice, in which the entire human Mapt gene was inserted at themurine Mapt gene locus (Supplementary Fig. 1). Wild-type mice
predominantly expressed 4R tau, while MAPT KI mice expressed all
human tau isoforms as observed in human samples (Fig. 1a andSupplementary Fig. 2a, b). The relative ratio of mRNA for 4R-tau/3Rtau was 0.69 ± 0.07 (Fig. 1b, c), which is close to that of human brain.
MAPT KI mouse did not display acceralated neuroinﬂammation,neuronal cell death and brain atrophy (Supplementary Fig. 2c, d).
Moreover, cross-breeding of MAPT KI with AppNL-G-F KI did notalter amyloid pathology, neuroinﬂammation, and neuronal cell
death of AppNL-G-F KI (Fig. 1d, e). These ﬁndings indicate thathumanization of the Mapt gene does not affect neurodegenerative
processes.
AD pathology alters the expression pattern of CAPON. In
order to identify tau-binding proteins, we generated a tau interactome, based on mass spectrometry-based immunoprecipitation
proteomics. We performed immunoprecipitation with a Flag-tag
antibody using brain lysate from WT (negative control) and
Wtau-tg mice, which express WT human tau tagged with a Flag
epitope, to isolate tau and its binding proteins. Supplementary
Data 1 summarizes the proteins which were speciﬁcally identiﬁed
from the Wtau-tg mice. We subsequently focused on CAPON as
it is speciﬁcally expressed in the brain9, and polymorphisms havebeen identiﬁed in several psychiatric diseases10. Moreover,
CAPON is also upregulated in the hippocampal pyramidal cellsof AD patients14, and may therefore play a pivotal role in the
etiology of this disease.
According to Hashimoto et al14, although CAPON accumulates in hippocampal pyramidal cells in the AD brain, the overall
amount of the protein is signiﬁcantly lowered in AD patientscompared with healthy controls. We therefore ﬁrst examined
whether Aβ amyloidosis and tau pathology affect the level orpattern of CAPON expression in the brain. Western blot analysesof hippocampal and cortical tissues from 12–14-month-old WT,AppNL-G-F (with Aβ pathology), hTau single-KI (without Aβpathology) and AppNL-G-F / hTau double-KI (with Aβ pathology)

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a

b

Tau5

50

RD3
50

Tau
ladderWT

MAPT KI Tau(homo) ladder

c
50

RD4

β-tubulin
Mapt
KO

WT

MAPT KI (hetero)

MAPT KI (homo)

GAPDH
Tau WT
ladder

MAPT KI(homo)

Brain lysate(30 μg protein)d

NL-G-FGAPDHDNA Mapt
ladder KO

Brain lysate(5 μg protein)50

Tauladder

4R-Tau3R-Tau

200
400
200

GAPDH

2N4R
2N3R
1N4R
1N3R
0N4R
0N3R

600
400

Tau
ladder

MAPT KI*
Relative expression of tau

2N4R
2N3R
Tau13
(human tau) 1N4R
1N3R
0N4R
0N3R

1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
3RTau

4RTau

NL-G-F/MAPT dKI

WT

MAPT KI

App

KI

App

WT

MAPT KIAppNL-G-F KI

App

Aβ/Iba1/GFAPe

NL-G-F/MAPT dKI

caspase 3/Hoechst

Fig. 1 Characterization of human MAPT KI mice. a Tau isoforms in MAPT KI mouse brains were determined. Brain extracts derived from 3-month-old WT,
heterozygous (hetero) and homozygous (homo) mice were subjected to western blot analysis after dephosphorylation (n = 3 each). b, c The relative
expression of 3R and 4R tau were calculated by semi-quantiative RT-PCR. Data reperesent mean ± SEM (n = 6, Stutent t test (t(10) = 1.883, *p = 0.0446).
d Amyloid pathology and neuroinﬂammation were detected in 24-month-old WT, single MAPT KI, AppNL-G-F single-KI and AppNL-G-F /MAPT double-KI
mice. The brain sections were triple-immunostained using 82E1 (blue), anti-GFAP (green) and anti-Iba1 antibody (red). Scale bar: 100 μm. e The brain
sections of 24-month-old WT, MAPT single-KI, AppNL-G-F single-KI and AppNL-G-F /MAPT double-KI mice were immunostained by cleaved-caspase 3
antibody. Scale bar: 100 μm. Source data are provided as a Source Data ﬁlemice revealed that presence of with Aβ pathology resulted inhigher CAPON expression levels in these animals (Fig. 2a andSupplementary Fig. 4a, b). Intriguingly, immunohistochemical
analyses showed a signiﬁcantly stronger CAPON signal in the
pyramidal cell layer of AppNL-G-F mice than in the WT mice
(Fig. 2b). These results indicate that Aβ pathology elevates thelevel and localization of CAPON in hippocampal pyramidal cells.
On the other hand, P301S-Tau-Tg mice (tauopathy model)
exhibited an age-dependent decline in CAPON protein (Fig. 2c),suggesting that severe pathology such as that associated withfrontotemporal dementia with parkinsonism-17 (FTDP-17)
mutant tau-induced cytotoxicity decreases the level of CAPON,
presumably because the number of neuronal cells where CAPON
is mainly expressed is reduced (Supplementary Fig. 6). Indeed,
P301S-Tau-Tg mice display severe brain atrophy in association
with neuronal cell death, in an age-dependent manner15.
How does Aβ pathology alter the expression pattern of
CAPON? We selected two candidates as potential mediators of
this change: nNOS and neuroinﬂammation. Further analysis
revealed a dramatic reduction in the protein levels of CAPON,
without any change in its mRNA levels, in the nNOS-deﬁcientmouse brain, suggesting that stabilization of the CAPON protein
is highly dependent on its interaction with nNOS (Supplementary
Fig. 5a, b, c). Because pathogenic Aβ species interact with andactivate NMDA receptor16,17, nNOS is considered to be activatedunder amyloid pathology18. Therefore, we quantiﬁed the interactions between CAPON and nNOS in the WT and AppNL-G-F
mouse brain using the Duolink system19. In this system, if nNOS
is located in a neighboring distance from CAPON, optical signals
will be detected as dots. As shown in Fig. 3a, b, we observed
no difference in the levels of nNOS protein or in the CAPONnNOS interaction between WT and AppNL-G-F tissue, indicatingthat interaction with nNOS does not contribute to CAPON
accumulation in the AppNL-G-F mouse.
We next examined the involvement of neuroinﬂammation.
Shao et al20 reported that lipopolysaccharide (LPS) stimulationinduces CAPON expression, and Cheng et al21 proposed thatCAPON increases after spinal cord injury. Therefore, neuroinﬂammation could also be involved in the elevation of CAPON
expression under amyloid pathology. Indeed, AppNL-G-F micedisplay marked activation of microglia and astrocytes5. Similar to
Shao et al, we examined the expression pattern of CAPON 1 week

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Hippocampus

***

1.4
CAPON/β-actin protein(fold of WT)

1.6
CAPON/β-actin protein(fold of WT)

cCortex

*
1.4
1.2
1.0
0.8
0.6
0.4
0.2

2.0
CAPON/β-actin protein(fold of 7M WT)a

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1.2
1.0
0.8
0.6
0.4

*
1.5

n.s.

1.0
0.5
0.0

0.2

WT

0.0

0.0
WT

NL-G-FAppWT

NL-G-F

App

CAPON

64

CAPON

64

CAPON

β-actin50

β-actin

50

Tau

P301S
-Tau-Tg
7M

WT

P301S
-Tau-Tg
14M
64
64

NL-G-F

WT

App

WT

NL-G-FApp50
50

β-actinWT

Tau-Tg

WT

b

14 M
WT

Tau-Tg
14M

7M

24 M
App

NL-G-F

WTApp

NL-G-F

14 M

NL-G-F

WTApp

NL-G-F15
CAPON/Hoechst intensity(fold of WT)AppCAPON/Hoechst intensity(fold of WT)

WT

24 M

*

6.0

4.0

2.0

*
10

5

0

0.0
WT

AppNL-G-F

WT

AppNL-G-F

Fig. 2 Endogenous CAPON expression in AppNL-G-F mice. a The protein levels of CAPON in the hippocampi and cortices of 14-month-old (M) WT andmale AppNL-G-F mice were determined. Values shown in the graph represent the band intensity of CAPON divided by the intensity of β-actin, expressed as
the mean level ± SEM (n = 3, *p < 0.05, ***p < 0.001). b Representative images of the hippocampal CA1 region of 14- and 24-month-old WT and male
AppNL-G-F mice immunohistochemically stained for CAPON. Arrows indicate the hippocampal pyramidal cell layer. The ﬂuorescence intensity in the
pyramidal cell layer is also quantitatively represented as the mean intensity level ± SEM (n = 3, *p < 0.05). Scale bar: 100 μm. c The protein level of CAPON
in the hippocampi of male 7- and 14-month-old WT and P301S-Tau-Tg mice was determined. The values shown in the graph are the band intensity of
CAPON divided by the intensity of β-actin, with the results expressed as the mean relative expression level of CAPON ± SEM (n = 3, *p < 0.05). Source
data are provided as a Source Data ﬁleafter intraventricular injection of LPS. Immunohistochemical
analyses revealed signiﬁcant induction of CAPON in regions
where the microglia was highly activated (Fig. 3c-left). However,
the CAPON signals did not colocalize with Iba1 (Fig. 3c-left, andSupplementary Fig. 7) and non-neuronal cells markers (Supplementary Fig. 7), whereas the signals colocalized with the nNOS
immunoreactivity (Fig. 3c-right), and other neuronal cell markers(Supplementary Fig. 7). Therefore, CAPON appears partially
increased in neuronal rather than glial cells. These results indicatethat neuroinﬂammation associated with Aβ pathology may
possibly be involved in CAPON elevation.
Expression of CAPON cDNA induces neuronal cell deathmediated hippocampal atrophy. We then hypothesized that theincrease in CAPON protein in AppNL-G-F mice aggravates thepathological events that occur in response to Aβ pathology, i.e.
tauopathy and neurodegeneration. To investigate whether
CAPON inﬂicts damage on the brain, we evaluated the effect of
CAPON cDNA expression on AD pathology in AppNL-G-F / hTau
double-KI mice. Humanization of tau itself did not change
CAPON levels in hippocampus and cortex, but cross-breedingwith AppNL-G-F elevated CAPON levels (Supplementary Fig.4),
suggesting that amyloid pathology increased CAPON level also in
hTau-KI mice.
4

AAV was used to introduce the CAPON or GFP gene,
expressed under the control of the synapsin I promoter(Supplementary Fig. 8a), bilaterally into the ventricles of 12month-old double-KI mice (Supplementary Fig. 8b, c). This
produced strong gene expression, particularly in the hippocampus (Supplementary Fig. 8d, e). To assess brain atrophy,
magnetic resonance imaging (MRI) was performed 7 days and3 months after AAV injection (Supplementary Fig. 8c). Whereas
the AAV-GFP-injected mice did not show any signiﬁcantdifference in brain volume between the two time-points, the
AAV-CAPON-injected mice exhibited a large decrease inhippocampal volume at 3 months, falling to approximately 77%of the control (Fig. 4a, b). The rate of reduction in the
hippocampal volume correlated negatively with the expression
level of CAPON (Supplementary Fig. 13a).
We also examined hippocampal atrophy and neuronal cell
death using histochemical analyses. Signiﬁcant shrinkage of the
hippocampus was conﬁrmed by hematoxylin and eosin (H&E)
staining of brain sections (Fig. 4c). We also observed a signiﬁcantdecrease in NeuN (neuron marker) -positive neuronal cells andan increase in cleaved caspase 3 (cell death marker) signals in the
hippocampus of CAPON-expressing mice (Fig. 4d). The signalsof cleaved caspase 3 colocalized with neuronal cell markers(Fig. 4e). Moreover, TUNEL-positive cells were also detected in

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a Duolink (nNOS and CAPON)/Hoechst

b

WT

n.s.

AppNL-G-F

1.0
0.8
0.6
0.4

CAPON-KO

1.0
0.8
0.6
0.4
0.2
0.0

0.2
0.0

n.s.

1.2
nNOS/β-actin protein(fold of WT)

Duolink/Hoechst intensity(fold of WT)

1.2

AppNL-G-F

WT
WT AppNL-G-F

148

nNOS

50

β-actinAppNL-GF

WT

c

Iba1

CAPON

Iba1/CAPONnNOS/CAPON

PBS

5.0

**

Overlapping/CAPON (%)

LPSCAPON intensity (fold of PBS)

LPS

4.0
3.0
2.0
1.0
0.0
PBS

100
80
60
40
20
0

LPS

Iba1

nNOS

Fig. 3 Neuroinﬂammation and interaction with nNOS could be involved in CAPON expression. a Duolink signals (green) for the nNOS-CAPON interaction
were detected in WT, AppNL-G-F (14-month-old, male), and CAPON-deﬁcient (3-month-old, male; negative control) mice. Values shown in the graph are
the ﬂuorescence intensity of the Duolink signal divided by the intensity of Hoechst staining, expressed as the mean ± SEM (n = 3). Scale bar: 500 μm (left)or 100 μm (right). b The protein level of nNOS in the hippocampi of AppNL-G-F mice (14-month-old male) was determined. Values shown in the graph are
the band intensity of nNOS divided by the intensity of β-actin, expressed as the mean relative expression level of nNOS ± SEM (n = 3). c Representative
image of the cortical region of LPS- or PBS-treated WT mice (3-month-old male) immunohistochemically stained for CAPON (green), Iba1 (red; left) or
nNOS (red; right). The expression level of CAPON correlates with the level of Iba1, which is activated in LPS-induced neuroinﬂammation. The values shownin the graph are the ﬂuorescence intensity of CAPON with the results expressed as the mean relative levels of CAPON ± SEM. (n = 3, **p < 0.01). Scale bar:
50 μm. Source data are provided as a Source Data ﬁlethe hippocampus (Fig. 4f). These results indicate that CAPON
expression induces caspase 3-mediated neuronal cell death andresults in neuronal loss and hippocampal atrophy. We also
detected Gasdermin D (GSDMD) and Gasdermin E (GSDME)
activation in CAPON-overexpressing mouse brains (Fig. 4g).
Gasdermin family proteins possess novel membrane poreforming activity22. Especially, GSDMD acts as an essential
effector of pyroptosis, an inﬂammatory form of cell death
induced by various infectious and immunological challenges22.
This indicates that the pyroptotic pathway was also activated by
CAPON overexpression. Consistently, we observed signiﬁcant
increases in Iba1 and GFAP signals associated with neuroinﬂammation in the mouse brains treated with AAV-CAPON(Fig. 4h, i). In contrast, GSDME is activated through cleavage
by caspase 3 in the apoptotic pathway and induces necrosis andpyroptosis secondarily when apoptotic cells are not scavenged23.
Taken together, our results suggest that CAPON overexpressionmediated cell death is not caused only by a single characteristic
pathway such as apoptosis, pyroptosis and necrosis but also by
more complicated mechanisms that involve multiple pathways.
Notably, we observed almost identical pathological changes(neuron loss and activation of caspase 3) in single AppNL-G-F KI
and WT mice treated with AAV-CAPON (Fig. 5a). We thenshort-term overexpression of CAPON in hippocampus by direct
injection of AAV-CAPON to single hTau-KI mice and double-KI
mice (Fig. 5b). Hippocampal overexpression of CAPON also
induced neuronal cell death and neuroinﬂammation in hTau
single-KI mice as well as double-KI mice (Fig. 5c, d). These results
suggest that CAPON-induced neuronal cell death does notdepend on the humanization of tau or on Aβ pathology. Together

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7 daysc HE staining

3 months

GFP

GFP

CAPON#1

CAPON#1e
CAPON#1

CAPON#2

CAPON#1

CAPON#2

MAP2/caspase 3

AAV-GFPGFPd NeuN/caspase 3
GFP

AAV-CAPON

NeuN/caspase 3

CAPON#2

CAPON#2

40
35

76.9%

30

p = 0.00047
GFP

GFP

g

GSDMD

1.5
1.0
0.5

CAPON

CAPON
CAPON

n.d.

GFP

2.0

0.0
N
O
AP
C

G

i Iba1

4.0

FP

0.0

FP

CAPON

**

***

G

50

6.0
GSDME/β-actin protein(fold of GFP)36

GSDMD/β-actin protein(fold of CAPON)

36

GFP

GFP

GSDME

2.0

β-actin

0.0

3 monthsN

7 daysGSDME

1.0

h GFAP

CAPON

GSDMD

2.0

CAPON

2.5

*

2.0
1.5
1.0
0.5
0.0
GFP

CAPON

**
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
GFP

CAPON

O

0

CAPON

3.0

AP

25

GFP

***

4.0

GFAP intensity (fold of GFP)

100.2%45

CAPON#2

Iba1 intensity (fold of GFP)

50

5.0

C

Bilateral hippocampal
volume (mm3)

b

*

1.2
1.0
0.8
0.6
0.4
0.2
0.0

Cleaved-caspase 3 intensity(fold of GFP)

5 mm

NeuN intensity (fold of GFP)

f TUNEL/Hoechst
caspase 3

NeuN

Fig. 4 Neurodegeneration in CAPON-overexpressing AppNL-G-F/hTau double-KI mice. a Representative MRI scans of GFP-expressing and CAPONoverexpressing mouse brains scanned 7 days and 3 months after AAV introduction to 12-month-old double-KI mice. Scale bar: 5 mm. b Values shownin the graph represent the bilateral hippocampal volume calculated from 8 scanned MR images. The mean ± SEM (n = 4, p = 0.00047) is provided.
c, d The hippocampal area stained by H&E (c) or immunostained for NeuN (green)/cleaved-caspase 3 (red) (d). The values shown in the graph are
the ﬂuorescence intensity of NeuN and caspase 3 with the results expressed as the mean relative levels ± SEM. (n = 3, *p < 0.05, ***p < 0.001). Scalebar: 500 μm (c, d above) or 250 μm (d below). e Representative image of hippocampal CA1 region of control and AAV-CAPON introduced miceco-immunostained with cleaved-caspase 3 and neuronal cell markers. Signals of cleaved-caspase 3 (red) overlap with MAP2 or NeuN signals (Green).
Scale bar: 100 μm. f The hippocampal CA1 area of CAPON-overexpressing mice stained by TUNEL (green). Scale bar: 100 μm. g The protein levels ofcleaved-GSDMD and cleaved-GSDME was determined. Values shown in the graph are the band intensity divided by the intensity of β-actin, expressedas the mean relative expression levels ± SEM (GFP: n = 3, CAPON: n = 6, **p < 0.01, ***p < 0.001). h, i Brain sections from GFP-expressing or CAPONoverexpressing mice immunostained with an astrocyte marker (GFAP: h) or a microglial marker (Iba1: i). The values shown in the graph are theﬂuorescence intensity of GFAP or Iba1 with the results expressed as the mean relative levels ± SEM. (n = 3, *p < 0.05, **p < 0.01). Scale bar: 500 μm.
Source data are provided as a Source Data ﬁlewith the results shown in Fig. 2, this leads us to speculate that thisaction of CAPON occurs downstream of Aβ pathology, a notionthat is supported by the fact that CAPON expression had noeffect on the Aβ pathology of double-KI and AppNL-G-F single-KI
mice (Supplementary Fig. 9a, b).
Mitochondrial damage is involved in CAPON-induced neuronal cell death. Several studies have demonstrated that CAPON
mediates signaling from the NMDA receptor in association withnNOS9. NMDA receptor-induced cytotoxicity is closely related
to mitochondrial dysfunction because mitochondrial functions
are disturbed by disruption of calcium homeostasis24,25. Mitochondrial dysfunction is also considered to be related to amyloid
and tau pathology-induced cytotoxicity, and is detected in postmortem AD brain and AD mouse models26. We therefore
examined mitochondrial dysfunction in CAPON-overexpressing
6

mice. Upon mitochondrial damage, cytochrome C (CytC) is
released into the cytosol from the mitochondria. Immunohistochemistry revealed that CAPON overexpression increases the
CytC levels in the hippocampal pyramidal cell layer (Fig. 6a, b).
Moreover, the protein level of Bax, which is induced by mitochondrial damage and induces neuronal cell death, was also
elevated in AAV-CAPON-injected mice (Fig. 6e). We also analyzed the mitochondrial membrane potential and oxygen consumption rate of CAPON-overexpressing primary neurons:
CAPAN overexpression resulted in a decrease these cellular
fundamental functions compared to those of the controls (Fig. 6c,d). These results demonstrate that mitochondrial dysfunction
contributes to CAPON-induced neuronal cell death.
Dexras1, a Ras family protein, is known to act as a CAPONbinding protein, and its activation inhibits Raf, MEK and ERK
signaling27. On the other hand, activation of MEK-ERK signaling

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a

WT

CAPON#1

CAPON#2

GFP

CAPON

GFP

CAPON#1

CAPON#2

GFP

CAPON

GFP

CAPON#1

CAPON#2

GFP

CAPON

caspase 3/Hoechst

NeuN

HE staining

AppNL-G-F
GFPbdouble-KI

hTau-single-KI
64

CAPON

64

β-actin
Untreated AAV-GFP

Untreated

AAVCAPONd

HE staining

UntreatedGFPCAPON#1

CAPON#2

NeuN/
caspase 3

UntreatedGFPCAPON#1

CAPON#2

UntreatedGFPCAPON#1

CAPON#2

hTau-single-KI
Untreated

CAPON#1

CAPON#2

Untreated

CAPON#1

CAPON#2

Untreated

CAPON#1

CAPON#2

Untreated

CAPON#1

CAPON#2CAPON

CAPON#2

HE staining

CAPON#1

NeuN/
caspase 3

GFP

CAPON

Untreated

Iba1/GFAP

double-KI

Iba1/GFAPc

AAV-CAPON

500 μm

250 μm

250 μm

250 μmFig. 5 CAPON overexpression in single AppNL-G-F KI, wild-type or hTau-single-KI mice induces neuronal cell death. a AAV-GFP or AAV-CAPON was
introduced bilaterally into the ventricles of 6-month-old AppNL-G-F (left) and WT (right) male mice. Three months later, the mice were sacriﬁced andparafﬁn sections of their brains were prepared. The brain sections were subjected to H&E staining (upper) and immunostained using NeuN (middle) or
cleaved-caspase 3 (lower) antibody, respectively. CAPON overexpression enhanced hippocampal atrophy, neuron loss, and caspase 3 activation in both
AppNL-G-F single-KI and WT mice, as well as in the double-KI mouse. Scale bar: 500 μm or 250 μm (NeuN below). b AAV-GFP or AAV-CAPON was
introduced bilateral hippocampi of 3–4-month-old double-KI (male) and hTau-single-KI (mixture of male and female). c, d Three weeks later, the mice
were sacriﬁced and parafﬁn sections of their brains were prepared. Overexpression of CAPON was determined by immunostaining (the ﬁrst row). The
brain sections were subjected to H&E staining (second row) and immunostained using NeuN (Green)/caspase 3 (Red) (third row) or Iba1 (Green)/GFAP(Red) (fourth row) antibody, respectively. CAPON overexpression enhanced neuron loss, and caspase 3 activation in hTau-single-KI, as well as in the
double-KI. Scale bar: 500 μm (CAPON) or 250 μm (HE, NeuN/caspase 3, and Iba1/GFAP). Source data are provided as a Source Data ﬁlehas been implicated to mitochondrial damage28,29. We
therefore examined the levels of Dexras1 and MEK-ERK
phosphorylation. Our results revealed a marked decline in
Dexras1 and signiﬁcant acceleration of MEK-ERK phosphorylation in CAPON-overexpressing mice, suggesting that the
decline in Dexras1 activates MEK-ERK signaling (Fig. 6e). Taken
together, these ﬁndings indicate that CAPON could induce
mitochondrial damage and neuronal cell death by regulating
Dextras1-MEK-ERK signaling (Fig. 6e). Zhu et al suggested thatoverexpression of CAPON activates Dexras1 activity by facilitating the s-nitrosylation of Dexras127. They detected Dexras1
activation after several days of lentivirus-mediated overexpressionof CAPON in the mouse brain. In contrast, we observed adecrease in Dexras1 after 3 months, suggesting that prolonged
overexpression of CAPON decreases the level of the Dexras1

protein via negative feedback, and results in hyperactivationof the MEK-ERK signal. Incidentally, the baseline expressionsof Dextras1 and phosphorylated (p)-MEK were essentially
identical between WT and double-KI mice, and we also observed
alterations of these proteins in CAPON-overexpressing WT mice
(Supplementary Fig. 10).
CAPON expression facilitates tau phosphorylation and insolubility but not amyloid pathology. As we originally identiﬁed
CAPON as a tau-binding protein, the interaction between
CAPON and tau could be considered a cause of tau pathology,
resulting in the induction of neuronal cell death. We therefore
tested whether CAPON overexpression aggravates tau pathology(Fig. 7). We ﬁrst validated the interaction between tau and
CAPON in the WT, hTau-KI, AppNL-G-F, and double-KI mouse

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a CytCbCAPON #1

CAPON #2

1.0
0.5

Cytochrome C

CA3CAPON #2

1.5
1.0
0.5
0.0
GFP CAPON

1.5

***
Bax

*

22

Dexras1

36

p-ERK1/2

50

ERK1/2

50

p-MEK

50

MEK

50

β-actin

50

2.0
1.5
1.0
0.5

0.2
0.0
GFP CAPONCAPON

Dexras1

*

1.0

0.5

GFP

*

**

p-MEK/MEK protein(fold of GFP)

4.0

2.0

1.0

CAPON

p-MEK

p-ERK1/2
p-ERK/ERK protein(fold of GFP)

16

1.5

CAPON

0.0
GAPDH

0.4

0.0
GFP

3.0

GFP CAPON

Untreated

0.6

GFP CAPON

*

0.0

1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
–0.2

0.8

GFP CAPON

2.5

1.0

0.5

1.0

β-actin

0.0

Dexras1/β-actin protein(fold of GFP)***

Untreated GAPDH CAPON Ionomycin

Oxygen consumption rate
(fold of untreated)

2.0

1.2

Bax

0.0

d

*

e

1.5
Unhealthy/healthy (ratio)

2.0

Bax/β-actin protein(fold of GFP)c*

Cytochrome C intensity (CA3)(fold of GFP)CAPON #1

CA3

GFP

Cytochrome C intensity (CA1)(fold of GFP)

CA1

CA1
2.5

*
1.4

Cytochrome C/β-actin protein(fold of GFP)

GFP

3.0
2.0
1.0
0.0

GFP

CAPON

GFP

CAPON

Fig. 6 CAPON induces mitochondrial damage via Dexras1/MEK/ERK signaling. a AAV was introduced into 12-month-old male double-KI, and brain
samples were prepared 3 months after injection. Hippocampal CA1 or CA3 region of GFP-expressing or CAPON-overexpressing double-KI mice
immunostained with CytC. The values shown in the graph are the ﬂuorescence intensity of CytC in CA1 and CA3 region with the results expressed as
the mean relative levels ± SEM. (n = 3, *p < 0.05). Scale bar: 100 μm. b The protein level of Cyrochrome C in the cytosolic fraction of hippocampi. Values
shown in the graph are the band intensity divided by the intensity of β-actin, expressed as the mean relative expression levels ± SEM (GFP: n = 3, CAPON:n = 6, *p < 0.05). c CAPON or GAPDH (Control) was overexpressed in primary cortical neurons from WT mice and mitochondrial membrane potential
was detected using the JC-1 Mitochondrial Membrane Potential Assay. Ionomycine was treated to obtain a membrane depolarization positive control.
Values show the ﬂuorescence ratio for healthy membrane potential to unhealthy membrane potential (n = 4, ***p < 0.001). d Oxygen consumption rate
was measured using CAPON or GAPDH (Control) was overexpressed in primary cortical neurons from WT by Oxygen Consumption Rate Assay Kit.
Values show the relative oxygen consumption rate which are calculated from change of ﬂuorescence intensity per 1 min while the ﬂuorescence rise linearly.
e Levels of Dexras1/MEK/ERK signaling-related proteins were detected using hippocampal samples from GFP-expressing or CAPON-overexpressing
double-KI mice. The values shown in the graph are the band intensity of proteins divided by the intensity of β-actin, with the results expressed as themean level ± SEM (n = 3, *p < 0.05, **p < 0.01). Source data are provided as a Source Data ﬁlebrain using the Duolink system (Fig. 7a and Supplementary
Fig. 11a). We then assessed pathological changes in tau in
CAPON-overexpressing double-KI mice (Fig. 7b–f). Intriguingly,
CAPON overexpression signiﬁcantly increased the phosphorylation level at all the phosphorylation sites which we tested, without
any change in the total tau level (Fig. 7b). To investigate theformation of insoluble inclusions such as NFTs in CAPONoverexpressing mice, we isolated a 1% sarkosyl-insoluble fractionof hippocampal protein and detected insoluble tau by immunoblotting using the Tau5 antibody. A greater amount of sarkosylinsoluble Tau was detected in CAPON-overexpressing micecompared to control mice (Fig. 7c). We also observed a higher
amount of soluble tau oligomer, which could be detected by nonreducing SDS-PAGE, in the CAPON-overexpressing mice
(Fig. 7d). An increase in AT8-positive tau was also seen byimmunohistochemistry not only around Aβ plaques but also overa wide area of the hippocampus (Fig. 7e). In addition, we foundthat the MC1 antibody, which recognizes the pathological conformation of tau protein, detected some neuronal cells in CA1
8pyramidal cell layer (Fig. 7f). Consistently, these cells were also
stained for phosphorylated-Ser404 (pS404) rabbit antibody(Fig. 7f). These results indicate that CAPON can drive the progression of tau pathology in the mouse brain. We also observed
the induction of tau pathology by AAV-CAPON not only indouble-KI mice but also in AppNL-G-Fsingle-KI and WT mice
(Supplementary Fig. 11b, c), demonstrating that amyloid
deposition or the humanization of tau did not affect the CAPONinduced tau pathology.
How does CAPON facilitate the phosphorylation and accumulation of tau? Many reports indicate that CAPON essentiallyfunctions as a scaffold protein for nNOS. We therefore speculated
that it induces the nitration of tau, which is associated with theprogression of tauopathy. To investigate this, we analyzed
nitration at the 18th tyrosine (Y18) and Y29 of tau using
nitrated-tyrosine antibodies, revealing signiﬁcant enhancement of
nitration at Y29 in CAPON-overexpressing mice (Fig. 7g). Thissuggests that nitration at Y29 is enhanced by CAPON, resulting
in pathological tau aggregation. Indeed, Reynolds et al, have

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Tau5

AT8
1.2

64

64

64

50

50

50

CAPON-KOGFP CAPONGFP CAPONGFP CAPON

pS396

pS404

pS422

64

64

Tau5/β-actin protein(fold of GFP)AppNL-GF X hTau-KI

Tau1

n.s.

1.0
0.8
0.6
0.4
0.2
0.0

64

GFP CAPON

β-actin50

GFP CAPON

0.8
0.6
0.4
0.2
0.0

1% sarkosyl insoluble
fraction(detected by Tau5)

d

0.0

pS422

*

**

2.0

2.0
1.5
1.0
0.5
0.0

1.5
1.0
0.5

Nitrated Y18

Nitrated Y18
1.6

AT8/N1D(Aβ)
CAPON

GFP
64

GFP CAPON

50
50

Nitrated Y29

36
GFP

CAPON

GFP

0.5

GFP CAPON

CAPON

50
98

1.0

g
GFP

64

1.5

0.0

250
148

98

1.2
1.0
0.8
0.6
0.4
0.2
0.0
GFP CAPONCAPON

Nitrated Y29

3.0
2.0
1.0

6.0

*

f
AT8

MC1

pS404

MC1/pS404

50

4.0
GFP

2.0

CAPON

β-actin
50

0.0

0.0
GFP CAPONGFP CAPON

GFP

CAPON

nY29-tau/β-actin protein(fold of GFP)

4.0

Tau oligomer (fold of GFP)Sarkosyl insoluble tau(fold of GFP)

***

n.s.

1.4

64
5.0

*

2.0

GFP CAPON

64

250
148

2.5

0.0

AT8/Hoechst
Well

1.0

GFP CAPONe

Non-reducing
SDS-PAGE(detected by Tau5)

2.0

pS404

GFP CAPON

c

*

3.0

GFP CAPON

pS404/β-actin protein(fold of GFP)

pS396/β-actin protein(fold of GFP)GFP CAPON

1.0

pS396
2.5

GFP CAPON

1.2

4.0

GFP CAPON

50

50

50

Tau-KO

AT8

n.s.

1.4

AT8/β-actin protein(fold of GFP)

Tau1

pS422/β-actin protein(fold of GFP)

Tau5

nY18-tau/β-actin protein(fold of GFP)b

Duolink (Tau and CAPON)/Hoechst

Tau5/β-actin protein(fold of GFP)

a

2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0

**

GFP CAPON

Fig. 7 Tau pathology in the CAPON-overexpressing AppNL-G-F/hTau double-KI mouse. a Duolink signals (green) generated by tau-CAPON interaction
were detected in AppNL-G-F/hTau double-KI, Tau-KO and CAPON-deﬁcient (negative control) mice. Scale bar: 500 μm (left) or 100 μm (right). b AAV
was introduced into 12-month-old male double-KI, and brain samples were prepared 3 months after injection. The total-tau and phosphorylated-tau levelsin the soluble (S1) fraction from GFP-expressing or CAPON-overexpressing double-KI mice were determined using several tau antibodies. The values
shown in the graphs are the band intensity of the proteins divided by the intensity of β-actin, with the results expressed as the mean ± SEM (n = 4,
*p < 0.05, **p < 0.01). c The sarkosyl-insoluble (P3) fraction was obtained from the hippocampus, and insoluble tau levels were determined using
Tau5 antibody (n = 4). d Tau oligomers in the S1 fraction were detected by non-reducing SDS-PAGE and immunoblotting with Tau5 antibody (n = 4,***p < 0.001). The red box shows bands which appear to be tau oligomer (n = 4, *p < 0.05). e AT8-positive tau was detected by immunohistochemistry.
The lower panels show the hippocampal CA1 region double-stained with AT8 and N1D (Aβ). Scale bar: 500 μm (above) or 100 μm (below). f Magniﬁed
image of CAPON-overexpressing double-KI mice immunostained with AT8, MC1 or pS404-tau antibody. The images show hippocampal CA1 region.
Scale bar: 25 μm. g Nitrated-tau levels in the S1 fraction were determined using anti-nitrated-Y18 (nY18) and nY29 antibodies. The values shown in
the graph are the band intensity of proteins divided by the intensity of β-actin, with the results expressed as the mean ± SEM (n = 4, **p < 0.01).
Source data are provided as a Source Data ﬁlereported that tau with nitrated Y29 is more abundant in NFTs
than soluble tau, with the authors suggesting that nitration at Y29
is a disease-related event that may alter the intrinsic ability of tau
polymerization30.
We next questioned which event is ﬁrst initiated in CAPONoverexpressing mice, tau pathology or neurodegeneration. To
answer this question, we analyzed the pathology of AAVCAPON-expressing WT mice in which overexpression of
CAPON occurs at a low level (Fig. 8a). Low level overexpressionof CAPON did not induce hippocampal atrophy, neuron loss, or
caspase 3 activation in double-KI mice (Fig. 8b–d). On the other
hand, these mice exhibited a signiﬁcant increase in phosphorylated tau (Fig. 8e). Moreover, we observed a signiﬁcant increase inAT8-positive tau without neuronal cell death 2 weeks after the
introduction of AAV-CAPON (Fig. 8f–h). These results suggestthat tau pathology is induced by a lower level of CAPON, whereas

neuronal cell death needs a higher level or more prolonged actionof the protein.
CAPON induces neuronal cell death in tau-KO mice. From theabove results, it appears that tau pathology precedes neuronal cell
death in CAPON-overexpressing mice. This conclusion led us toinvestigate whether CAPON-induced neuronal cell death ismediated by tau pathology. To this end, we assessed neuronal cell
death and brain atrophy in AAV-CAPON-expressing tau-KO
mice (Fig. 9). We injected AAV-CAPON into 6-month-old tauKO or WT mice (Supplementary Fig. 12), and assessed their brain
pathologies 3 months later. Interestingly, much higher levels of
CAPON were expressed in the tau-KO mice, although the reason
for this remains unknown (Fig. 9a). MRI and histochemical
analyses revealed that the degree of hippocampal atrophy

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7 days3 months

Bilateral hippocampal
3
volume (mm )

b

a
CAPON

β-actinDegeneration

Non-degeneratione

HE staining

97.1%

30
20
10
0

7 days 3 months

Tau5

0.5

O
AP

at
re
nt

U

N

ed

0.0
C

N

1.0

U

C

PHF-1/β-actin protein (fold of control)

4.0

O

ed
at
re
nt

CAPON

U

Untreated

0.0

*

1.5

AP

1.4

0.0

1.0

ed

50

β-actin

0.2

2.0

2.0

C

50

3.0

at

PHF-1

0.4

4.0

AP

CAPON

0.6

5.0

re

64

NeuN/caspase 3

PHF-1

*

nt

d

0.8

AT8/β-actin protein (fold of control)

50

1.0

6.0

AT8/β-actin protein (fold of control)

AT8

AT8

n.s.

N

64

Tau5
1.2

O

CAPON

50

Untreated

40

64

Tau5/β-actin protein (fold of control)

Untreated

5 mm

Tau5/β-actin protein (fold of control)

c

CAPON

Untreated

n.s.

50

h

HE staining

64

U

Untreated

2.0

1.0

O

N

0.0
C
AP

0.0

n.s.

nt
re
at
ed

1.0

3.0

U

0.0
nt
re

β-actin

0.2

PHF-1/β-actin protein (fold of control)

50
50

0.4

N

PHF-1

0.6

O

64

2.0

C
AP

50

0.8

3.0

nt
re
at
ed

CAPON

1.0

PHF-1

*

U

AT8

NeuN/caspase 3
Untreated

1.2

N

64

AT8

n.s.

O

50

g

Tau5

64

Tau5

AP

CAPON

C

CAPON

ed

Untreated

at

f

CAPON

Fig. 8 A low level of CAPON overexpression induces tau phosphorylation without neuronal cell death. a–e AAV-CAPON was introduced into 6-month-old
WT male mice. Data were obtained 3 months after AAV injection. a Western blots show the overexpression levels of CAPON in the hippocampus of micewith and without neurodegeneration. b Representative MRI scan of the low-level-CAPON-overexpressing mouse brain, which does not show neuronal cell
death. Scale bar: 5 mm. c, d Hippocampal area of low-level-CAPON-overexpressing mice stained by H&E (c) or immunostained for NeuN (green)/cleavedcaspase 3 (red) (d). Scale bar: 500 μm. e The levels of total tau and phosphorylated tau in low-level-CAPON-overexpressing mice were determined using
several tau antibodies. Values shown in the graph represent the band intensity of proteins divided by the intensity of β-actin, with the results expressed as
the mean ± SEM (n = 3, *p < 0.05). f–h AAV-CAPON was introduced into 3-month-old WT male mice. Data were obtained 2 weeks after AAV-injection.
f, g Hippocampal area of CAPON-overexpressing mice stained by H&E (f) or immunostained for NeuN (green)/cleaved-caspase 3 (red) (g). Scale bar:
500 μm. h The levels of CAPON, total tau and phosphorylated tau were determined in CAPON-overexpressing mice. The values shown in the graphrepresent the band intensity of proteins divided by the intensity of β-actin, with the results expressed as the mean ± SEM (n = 3, *p < 0.05). Source data areprovided as a Source Data ﬁlecorresponded with the expression level of CAPON (Fig. 9a–d andSupplementary Fig.13). Moreover, we observed signiﬁcant activation of caspase 3 and neuronal loss in the AAV-CAPONexpressing tau-KO mice (Fig. 9e). These results indicate thatCAPON induces neuronal cell death via a tau-independent
mechanism or through a combination of both tau-dependent andtau-independent mechanisms. We would also like to point out
that tau is not the only microtubule-binding protein (MAP) andthat the other MAPs such as MAP2 might be involved although
we do not have any experimental evidence31,32.
Deﬁciency of CAPON restores AD-related pathological phenotypes in P301S-Tau-Tg mice. We thus far analyzed the effectof CAPON overexpression on pathological characteristics indouble-KI mice. As a proof of evidence, we next examined theeffect of CAPON deﬁciency on tau pathology and neurodegeneration in tauopathy mouse model. The P301S-Tau-Tg mice on
the CAPON KO background33 exhibited signiﬁcantly reduced
levels of total and phosphorylated tau (Fig. 10a). CAPON deﬁciency also resulted in reduction of Sarkosyl-insoluble tau in
10

P301S-Tau-Tg mice (Fig. 10b), suggesting that CAPON contributes to progression of tau pathology in P301S-Tau-Tg mice.
Moreover, CAPON deﬁciency ameliorated brain atrophy andneuron loss in P301S-Tau-Tg mice (Fig. 10c, d). These results
suggest that the CAPON-induced neuronal cell death is closely
associated with the pathological tau protein although there
appears to be a tau-independent mechanism as well (Fig. 9a).
Discussion
In this study, we have demonstrated that Aβ pathology leads tothe accumulation of CAPON protein, and that the increase in
CAPON induces tau pathology and neuronal cell death. Ourﬁndings suggest that CAPON is one of the novel mediators thatlink Aβ, tau and neurodegeneration. CAPON was originallyidentiﬁed as a nNOS-binding protein and is considered to be
involved in NMDA receptor-mediated excitotoxicity9. Although
one report proposed that CAPON prevents excitotoxicity bycompetitively inhibiting nNOS-PSD95 binding9, recent studies
have indicated that it mediates NMDA receptor-induced neuronal toxicity13,34,35. Our study supports the latter proposal, given

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a

b
64

CAPON

7 days3 months

Tau-KO#1

Tau-KO#1

Tau-KO#2

Tau-KO#2

Tau-KO#3

Tau-KO#3

Tau-KO#4

Tau-KO#450

β-actinc#1 #2 #3 #4
AAV-CAPON
injected WT#1 #2 #3 #4
AAV-CAPON
injected Tau-KO

AAV-CAPON

Untreated

Bilateral hippocampal
volume (mm3)

50
40
74.6%

30

p = 0.01371
20
10

#1
#2
#3
#4

0
7 days3 monthsd
HE staining
Untreated

AAV-CAPON
Tau-KO#2

Tau-KO#3

Tau-KO#2

Tau-KO#3

Tau-KO#4e

Tau-KO#4

NeuN intensity (fold of GFP)AAV-CAPON

*

*

1.5

Cleaved-caspase 3 intensity(fold of GFP)

NeuN/caspase 3
Untreated

1.0

0.5

0.0
GFP

CAPON

400
300
200
100
0
GFP

CAPON

Fig. 9 CAPON overexpression in tau-KO mice induces neurodegeneration. a AAV-CAPON was introduced into 6-month-old WT (male) or tau-KO
mice (#1, #2 and #4 are male, and #3 is female), and expression levels of CAPON in the hippocampus were determined 3 months after injection.
b Representative MRI scan of the CAPON-overexpressing tau-KO mouse brain scanned 7 days and 3 months after the introduction of AAV. Scale bar:
5 mm. c The values shown in the graph are the bilateral hippocampal volume calculated from 8 scanned MR images (n = 4, p = 0.01371). d, e The
hippocampal area of CAPON-overexpressing tau-KO mice stained by H&E (d) or immunostained for NeuN (green)/cleaved-caspase 3 (red) (e). Scale bar:
500 μm (d, e above) or 100 μm (e below). Source data are provided as a Source Data ﬁleour observation that overexpression of CAPON leads to neuronalcell death and hippocampal atrophy. We also demonstrated thecontribution of mitochondrial dysfunction to this pathology. Aβoligomers have been reported to upregulate reactive oxygen/nitrogen species (ROS/RNS) and induce mitochondrial dysfunction via their interaction with the NMDA receptor36. Abnormal
activation of nNOS signaling together with mitochondrial Ca2+
overload produces excess ROS and RNS37. This in turn leads toabnormal s-nitrosylation, sulfonation and the accumulation ofperoxides, resulting in protein misfolding and dysfunction37–39.
For example, s-nitrosylation of dynamin-related protein 1 (Drp1)
is linked to mitochondrial dysfunction and neuronal injury, andprotein disulﬁde isomerase is associated with protein misfoldingin neurodegenerative disease40,41. Dysfunction of CAPON maycontribute to disruption of these proteins, thereby producing
pathological changes. Mitochondrial dysfunction also induces
caspase activation and cell death42.
In CAPON-overexpressing mice, we detected not only apoptosis markers such as cleaved-caspase 3 but also non-apoptotic
cell death markers, i.e. cleaved-GSDMD and GSDME. GSDMD isa physiological substrate for inﬂammatory caspases, and its
activation is required for initiation of pyroptosis. GSDME activation by caspase 3 induces pyroptosis and necrosis secondarily if

apoptotic cells are not scavenged23. Our observation suggests thatCAPON-induced neuronal cell death is not composed of a single
simple pathway but also of complicated mechanisms, which
involve multiple pathways. Indeed, activation of several cell deathpathways were also detected in AD samples and AD mousemodels. For instance, NLRP1-dependent neuronal pyroptosis isdetected in APPswe/PS1dE943. Also, activation of necroptosis
pathway, a programmed form of necrosis, is observed in AD
samples44. Taken together, the mechanisms of neurodegeneration
observed in the present study appear quite complex, and we thus
need further investigation to understand the entire landscapewhile taking contribution of neuroinﬂammation into consideration. It is possible that neuroinﬂammation may be attributed as acause of cell death induced by CAPON overexpression.
We observed a signiﬁcant decrease in Dexras1 and enhanced
MEK-ERK phosphorylation in CAPON-overexpressing mice.
Activation of MEK-ERK signaling is related to the induction of
mitochondrial damage28,29. We therefore concluded that
Dexras1-MEK-ERK signaling is one of the mediators of CAPONinduced neuronal cell death. In contrast, Chen et al45 demonstrated that a deﬁciency in Dexras1 inhibits NMDA-induced
neuronal cell death. Moreover, Zhu et al27 reported that CAPON
overexpression facilitates Dexras1 activity via enhancement of its

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a

b

Tau5

***

2.0

250

*

150
100

400

75

200

0.0

50

uTa

37

P3

C
01 AP
S- O
Ta N –/–
u- X
Tg

T
W
S01

P3

P3

01

50

P3

S-

Ta

u-

W

AT8

Tg

0
T

75

AT8/β-actin protein(fold of WT)

50

4.0

Tg

Tau5

6.0

Tau5

***

800

C
01 AP
S- O
Ta N –/–
u- X
Tg

75

Tau5/β-actin protein(fold of WT)

8.0

CAPON

AT8

**

P301S-Tau-Tg

WT

75

PHF1

P301S-Tau-Tg–/–

CAPON XP301S-Tau-Tg

4.0
2.0

0.5

n.d.
T

0.0

Ta
01

P3

S-

uTa
S01
P3

1.0

W

Tg

T

–/–

CAPON

W

WT

C
01 AP
S- O
Ta N –/–
u- X
Tg

0.0

*

***

Tg

37
WT

Tau5 (insoluble)
1.5

6.0

u-

PHF-1/β-actin protein(fold of WT)

β-actin

***

***

P3

P3

d

C
01 AP
S- O
Ta N –/–
u- X
Tg

8.0
50

Tau5/β-actin protein(fold of P301S-Tau-Tg)

PHF-1

c

–/–

CAPON XP301S-Tau-Tg

NeuN
CAPON-/-

–/–

CAPON

–/–

CAPON XP301S-Tau-Tg

P301S-Tau-Tg–/–

CAPON XP301S-Tau-Tg

***

1.0
0.8

*

0.6
0.4
0.2
0.0

S01

P3

P3

C

AP

O

W

N –/–

T

P301S-Tau-Tg

NeuN intensity (fold of WT)

1.2

Ta
uTg
C
01 AP
S- O
Ta N –/–
u- X
Tg

WT

Fig. 10 CAPON deﬁciency in P301S-Tau-Tg mice attenuates tau pathology and neurodegeneration. a The total-tau and AT8/PHF1 posotive-tau levels in thesoluble (S1) fraction from 12-month-old WT, P301S-Tau-Tg or CAPON−/−/P301S-Tau-Tg mice (mixed-sex) were determined. Values are the band
intensity of the Tau5/AT8/PHF1 divided by the intensity of β-actin, with the results expressed as the mean ± SEM (WT, P301S-Tau-Tg: n = 4,
CAPON−/−/P301S-Tau-Tg:n = 3, *p < 0.05, **p < 0.01, ***p < 0.001). b The total-tau sarkosyl insoluble fraction were determined. Values are the band
intensity of the Tau5, with the results expressed as the mean ± SEM (WT, P301S-Tau-Tg: n = 4, CAPON−/−/P301S-Tau-Tg:n = 3, *p < 0.05, ***p < 0.001).
c, d The sections of WT, CAPON−/−, P301S-Tau-Tg or CAPON−/−/P301S-Tau-Tg mice stained by H&E (c) or immunostained for NeuN (d). The values
shown in the graph are ﬂuoresence intensity of NeuN, with the results expressed as the mean level ± SEM (WT, P301S-Tau-Tg: n = 4, CAPON−/−/P301STau-Tg:n = 3, *p < 0.05, ***p < 0.001). Scale bar: 1 mm (c) or 500 μm (d). Source data are provided as a Source Data ﬁles-nitrosylation, and that this inactivates MEK-ERK signaling. We
assumed that this inconsistency is due to the difference inexperimental conditions between studies, as Zhu et al,27 used alenti-virus to overexpress CAPON, observing activation of Dexras1 and inactivation of MEK-ERK signaling 4 days after introduction of the virus. We also assumed that a reduction in
activated Dexras1 rather than a deﬁcit in Dexras1 per se facilitated
MEK-ERK signaling, and disrupted mitochondrial function. Thiseffect could be mediated by prolonged overexpression of CAPON.
Although we could detect a signiﬁcant increase in CAPON
protein in the hippocampus and cortex of the AppNL-G-F mouse,
these mice do not exhibit severe neuronal cell death or tau
pathology5. In sporadic AD patients, Aβ accumulation starts
about 20 years prior to the clinical onset of the disease. We
therefore assume that the prolonged effect of Aβ leads to theaccumulation of CAPON and results in tau pathology and neuronal cell death.
We found that the nitration of tyrosine at Y29 is one of themechanisms by which tau pathology is induced. Nitrated-Y29positive tau is predominately detected in tau inclusions such as
NFTs, suggesting that nitration at Y29 facilitates the aggregationof tau30. We attempted to validate the contribution of nNOS
to tau nitration using nNOS-deﬁcient mice that overexpress
12

CAPON. However, we were unable to successfully overexpress
CAPON by AAV delivery in nNOS-KO mice, probably
because the protein is not stable in these animals. CAPON
overexpression-induced neuroinﬂammation may also contributeto acceleration of tau pathology through other possible
mechanisms. For instance, genome-wide and rare variant association studies have shown linkage between single-nucleotide
polymorphisms in genes related to immune system and etiologyof AD. In particular, Triggering receptor expressed on myeloid
cells 2 (TREM2), the mutation in which increases the risk of AD,
is associated with tau pathology and synaptic loss in post mortem
human cortical samples from AD patients46. Also, Asai et al47
demonstrated that depletion of microglia and inhibition of exosome synthesis interrupted tau propagation, suggesting thatmicroglia and exosomes contribute to the progression of tauopathy. Thus, although there is still room for argument about
relationship between neuroinﬂammation and tau pathology,
CAPON may be a candidate facilitater of tau pathology in suchprocesses.
Given that tau pathology occurred earlier than neurodegeneration in CAPON-overexpressing mice, we predicted that thispathology was driving neurodegeneration. However, we observed
CAPON-induced hippocampal atrophy and neuronal cell death

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not only in double-KI mice but also in tau-KO mice. This result
indicates that CAPON-induced neuronal cell death occurs via atau pathology-independent manner or through a combination ofboth tau-dependent and tau-independent mechanisms. Since
CAPON deﬁciency in tauopathy mouse model attenuated tau
accumulation and neuron loss, the CAPON-induced cell deathcan be attributed at least in part to a tau-dependent mechanism.
A positional correlation between tau pathology and neuron loss
is observed in the human AD brain, while it remains unclear
whether tau is the actual killer of neuronal cells. Althoughoverexpression of tauopathy-associated mutant tau induces neuronal cell death, overexpression of WT tau or artiﬁcial facilitationof tau propagation by aggregate seeding almost never inducessuch death. Hence, the relationship between tau pathology andneurodegeneration still requires further investigation.
Numerous studies have reported that CAPON is a risk gene forschizophrenia and other psychiatric disorders, including autism
spectrum disorder, obsessive-compulsive disorder, post-traumaticstress disorder and depression10,48–50. Psychiatric symptoms are amajor feature of AD, and sometimes appear from the early stagesof the disease51,52. The disruption of CAPON expression induced
by Aβ may therefore link these clinical conditions.
A recent study used the APP/PS1 mouse and an in vitro system
to demonstrate that the interaction between CAPON with nNOS
is enhanced under amyloid pathologies, and that this facilitatesneuronal toxicity53. In contrast, we did not detect an enhanced
interaction between CAPON and nNOS in App KI mice. However, because these mice, unlike APP/PS1 animals, do not present
signiﬁcant neuron loss or neuronal cell death, it is possible thatsuch an effect might not be detected. We assume that the gradualaccumulation of CAPON over long periods could enhance the
CAPON-nNOS interaction and induce neuronal toxicity, particularly as Aβ accumulation starts more than two decades before
the onset of the clinical phenotype. Another reason for the difference between App KI and APP/PS1 mice could lie in theoverexpression of APP and PS1 in the latter case. We have previously suggested that APP and/or presenilin overexpression itself
alters calcium homeostasis without regard for amyloid pathology54–57. For example, unlike App KI mice, APP-overexpressing
mice display calpain-dependent conversion of p35 to p25, which
upregulates the kinase activity of CDK556. Moreover, APP/PS1
mice exhibit enhanced endoplasmic reticulum stress, whereas
App KI and APP-single transgenic mice do not55,57. CAPON andnNOS function could therefore be subjected to the action of
calcium signaling, and care is required when interpreting results
obtained using the APP/PS1 model.
It is desirable to prevent the progression of AD before the onsetof neurodegeneration. Many groups have been developing novel
therapeutic agents to target Aβ and tau accumulation58. However,few studies are targeting the connection between amyloid
pathology and tau pathology or neuronal cell death. We believethat CAPON is a promising drug target to break the connection
between Aβ and tau or cell death. Inhibition of the interaction
between CAPON-tau or CAPON-nNOS could therefore be anovel approach for the treatment of AD and related diseases.
Methods
Animals. All animal experiments were conducted in accordance with theguidelines of the RIKEN Center for Brain Science. We previously produced the
AppNL-G-F/NL-G-F KI (AppNL-G-F) mouse using genomic DNA of introns 15 to 17
of mouse App, which humanized the Aβ sequence and introduced KM670/671NL(Swedish), I716F (Iberian), and E693G (Arctic) mutations5. Human MAPT KI(hTau-KI) mice, in which the entire human Mapt gene was inserted at the murine
Mapt gene locus, were generated. The strategy for generating this mouse isdescribed in the Supplementary information (Fig. S1). The Wtau-Tg mouse
expresses wild-type (WT) human 4 repeat Tau tagged with myc and Flag epitopes
which are regulated by the CAM kinase II promoter8. The P301S-Tau-Tg

ARTICLE(Line PS19) mice were created on a B6C3H/F1 background15. For our experiments,
PS19 mice were back-crossed onto a C57BL/6 background. The details of the
CAPON (Nos1ap)-KO mouse have been described in Sugiyama et al33. Tau-KO(knockout) (B6.129 × 1-Mapttm1Hnd/J) and WT C57BL/6 mice were purchasedfrom Jackson Laboratory (Bar Harbor, ME). All strains were maintained on a
C57BL/6 background.
Materials. The cDNA encoding mouse CAPON (NCBI Reference Sequence:
NM_001109985) was obtained from Origene plasmid (CAT#: MC217011)(Origene, Rockville, MD). The AAV9 vectors were produced as previously
described59. The AAV vector plasmids contained an expression cassette, consistingof a synapsin (Syn) I promoter (GenBank, M55300.1), followed by cDNA encoding
CAPON or green ﬂuorescent protein (GFP), and a simian virus (SV) 40 polyadenylation signal sequence; and consisting of a Cytomegalovirus (CMV) promoter
followed by cDNA encoding GAPDH and a SV40 polyadenylation signal sequence.
The antibodies used for immunohistochemistry or western blotting are summarized in Supplementary Table 1. We conﬁrmed the speciﬁcity of the CAPON
antibodies using the CAPON-KO mouse33 (Supplementary Fig. 3).
Generation of human MAPT KI (hTau-KI) mice. As described in Supplementary
Figure 1a, we isolated genomic DNA of the human MAPT gene (NCBI Reference
Sequence: NG_007398) with H2 haplotype, from the ATG codon of exon 1 tothe 3’-untranslated region (67440 bp), from the human bacterial artiﬁcial chromosome (BAC) library. We inserted a neo gene cassette with a lox/FRT sequenceat the 3’-end of the isolated human MAPT gene which was subsequently inserted
into a mouse BAC vector, as a humanized BAC targeting vector.
Gene-replacement of the mouse Mapt gene with its human MAPT genecounterpart, which includes a region starting from the ATG codon of exon 1 to the
3’-untranslated region (58019 bp), was carried out with the homologous-based
recombination technique using BA1 C57BL/6 × 129SvEv hybrid embryonic stem(ES) cells. Targeted ES cells were microinjected into C57BL/6 blastocysts. Resulting
chimeras with a high percentage agouti coat color were mated to wild-type C57BL/
6 mice to generate F1 heterozygous offspring. We extracted DNA from the biopsied
tails of mouse pups and identiﬁed the F1 generation by PCR.
We then crossbred heterozygous mutant mice with EIIa-Cre Tg mice to remove
the neo gene and removed the EIIa-Cre transgene by crossing the mice with wildtype C57BL/6 mice. The generated MAPT KI mice were then back-crossed to the
C57BL/6 J strain. We genotyped the mice by PCR using the following primers:
Fwt5: 5′-GTCAGATCACTAGACTCAGC-3′, Rwt5: 5′CTGTGCTCCACTGTGACTGG-3′ and Rhm5: 5′CTGCTTGAGTTATCTTGGCC-3′.
Tau interactome. Tau was immunoprecipitated from brain extracts of 9-monthold WT or Wtau-Tg mice with Flag-tag antibody. The whole brains except theolfactory bulb and cerebellum were extracted in extraction buffer (phosphatebuffered saline (PBS) containing, protease inhibitor cocktail, phosphatase inhibitor), and insoluble material was removed by centrifugation at 1000 × g for
10 min. The resulting supernatant was adjusted to 1 mgmL−1 by adding extraction
buffer. Next, 1 mL of supernatant was incubated with anti-FLAG M2 magnetic
beads (Sigma-Aldrich, St. Louis, MO) for 2 h at 4 °C. After incubation, the magnetic beads were collected by placing the reaction tubes in a magnet stand, thenwashed 5 times with PBS. The precipitated proteins were extracted with 0.1%
triﬂuoroacetic acid (pH 2.5), and dried using a speed-vacuum system. Afterremoval of the solvent, the samples were dissolved in 100 mM ammonium bicarbonate, 0.2% RapiGest (Waters Corporation, Milford, MA), 2 mM CaCl2, and
100 ng μL−1 trypsin, then digested by incubation at 37 °C for 48 h. The determination of peptides was carried out by the Support Unit for Bio-Material Analysis,
Research Resource Division of the Center for Brain Science, using an Q Exactive
Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientiﬁc, Waltham, MA). The peptide identiﬁcation was performed using Proteome Discover(Thermo Fisher Scientiﬁc) and the Swiss-prot database. Co-immunoprecipitatedproteins which were speciﬁcally identiﬁed in Wtau-Tg mice are listed in Supplementary Data 1.
AAV injection. WT, AppNL-G-F/NL-G-F KI (AppNL-G-F), AppNL-G-F/NL-G-F KI X hTauKI (double-KI), hTau-KI and tau-KO mice (3–12 months old) were used for theexperiments. AAV vectors were diluted in PBS containing 2% PEG400 to 1 × 1010
genome copies per 5 μL or 1 μL. Mice were anesthetized with pentobarbital (50 mg
kg−1, i.p.) and placed in a stereotaxic apparatus before administration of the viral
vector solution. AAV solution (5 μL for icv injection per 1 μL for hippocampal
injection) was injected bilaterally into the cerebral ventricles (stereotaxic coordinates:
anteroposterior, −0.45 mm; mediolateral, ± 1 mm; dorsoventral, −2.4 mm) or hippocampi (stereotaxic coordinates: anteroposterior, −2.7 mm; mediolateral, ± 3.1 mm;
dorsoventral, −2.4 mm) using a needle equipped with a 50 mL NanoSyringe (Altair
Corporation, Yokohama, Japan), at a constant ﬂow rate of 1 μL min−1 (icv) or
0.2 μL min−1 (hippocampal) using a Legato 130 syringe pump (KD Scientiﬁc,Holliston, MA). After AAV injection, mice were bred in plastic cages with food(CE2, Clea Japan, Tokyo, Japan) and water, and were maintained on a 12/12 hlight–dark cycle for 3 months (icv) or 3 weeks (hippocampal).

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Magnetic resonance imaging. We conducted magnetic resonance imaging (MRI)of 21-month-old MAPT KI (Supplementary Fig. 2c), and AAV-CAPON/GFPexpressing mice 7 days and 3 months after AAV injection (Figs. 4a and 9b). The
mice were anchored in the apparatus under anesthesia with 1.5% (v/v) isoﬂurane.
During the scanning, the depth of anesthesia was monitored with a breathing
sensor. Coronal/horizontal T2-weighted (T2W) MRI scans (2D TurboRAGE) of
the whole brain were performed with a vertical-bore 9.4 T Bruker AVANCE
400WB imaging spectrometer with a 250 mTm−1 actively shielded imaging gradient insert (Bruker BioSpin, Billerica, MA) controlled by Paravision software.
T2W scans were performed with the following parameter settings: TR (repetitiontime) = 4342.2 ms, TE (echo time) = 53.8 ms, matrix dimensions = 256 × 256, ﬂip
angle = 180 degrees, ﬁeld of view = 1.8 cm × 1.8 cm. We used a slice thickness of
0.5 mm and 29 slices with a scan time of 22 min 47 s to image the whole brain.
Within the 29 scanned images, 8 images containing the hippocampal area wereselected for further analysis. The hippocampal volume of each mouse was calculated using ImageJ software.
Tissue ﬁxation and preparation of parafﬁn sections. Brain hemispheres were
ﬁxed by immersion in 4% paraformaldehyde in phosphate buffer solution (Nacalaitesque, Kyoto, Japan). Ethanol-ﬁxed brains were embedded in parafﬁn, and 4 μm
thick sections were mounted onto MAS-GP-coated glass slides. Sections were
stained with hematoxylin and eosin (H&E) or immunostained using primary andsecondary antibodies, the details of which are provided in Supplementary Table 1.
Histochemistry. H&E staining was carried out according to the following method.
After deparafﬁnization, sections were stained with Mayer’s hematoxylin solution(Wako, Tokyo, Japan) for 10 min, and then stained with eosin alcohol solution(Wako) for 4 min. Finally, the sections were dehydrated and coverslipped.
Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling(TUNEL) staining was carried out using the DeadEnd Fluorometric TUNEL
System (Promega Madison, WI), according to the manufacturer’s instructions.
Immunohistochemistry. In experiments in which we used anti-Aβ (N1D)60, a
direct immunoﬂuorescence method was applied. When we stained using antiCAPON, anti-GFP, anti-phospho-tau (AT8), anti-NeuN, anti-cleaved caspase 3,
anti-cytochrome C (CytC) or anti-nNOS, we applied a ﬂuorescence-indirect tyramide signal ampliﬁcation (TSA) technique (TSA System; NEN, Boston, MA).
When we stained using anti-GFAP, anti-Iba1, anti-Olig-2, anti-CD31 or antiMAP2, DAKO EnVision + System (Agilent Technologies, Santa Clara, CA) wasused as the second antibody and the signal was detected using tyramide-enhanced
ﬂuorescein isothiocyanate (FITC), as for the TSA method. When we stained using
anti-Synaptophysin, we used FITC-labeled antibody.
After deparafﬁnization, sections were heated in an autoclave at 121 °C for 5 minin 10 mM sodium citrate buffer (pH 6.0) for epitope retrieval, after which
endogenous peroxidase was inactivated by 0.3% hydrogen peroxide in methanol.
To block nonspeciﬁc immunoreactivity, sections were treated with the blocking
solutions (TSA Biotin System kit). First antibodies, diluted in TNT buffer (0.1 M
Tris-HCl, 0.15 M NaCl, 0.05% Tween20, pH 7.5), were reacted overnight at 4 °C.
The sections were then washed three times in TNT buffer for 5 min, and treatedwith secondary antibodies. In the case of the TSA method, the sections were treatedwith biotinylated goat anti-mouse/rabbit IgG (1:1000 dilution, Vector Laboratories,
Burlingame, CA) for 1 h, and then incubated with HRP-conjugated-avidin for
30 min (1:100 dilution in TNT, TSA System) at room temperature. Visualized wasachieved with tyramide-enhanced FITC (1:50 dilution in ampliﬁcation solution;
supplied in the TSA System) for 10 min. When the direct immunoﬂuorescencemethod was being used, the sections were treated with Alexa 488- or Alexa
546-conjugated anti-mouse/rabbit IgG (1:1000 dilution, Molecular Probes, Eugene,
OR). Finally, the sections were coverslipped using ProLong Gold Antifade
Mountant (Thermo Fisher Scientiﬁc), and the slides were scanned on a
NanoZoomer NDP system (Hamamatsu Photonics, Iwata, Japan) with ×20
resolution, an FSX100 microscope (Olympus, Tokyo, Japan), or All-in-one
Fluorescence Microscope BZ9000 (Keyence, Osaka, Japan). The signals were
quantiﬁed using Metamorph Imaging Software (Molecular Devices, San Jose, CA)
or Deﬁniens Tissue Studio (Deﬁniens, Parsippany, NJ). Fluorescence intensity ofeach protein is calculated as the product of average of ﬂuorescence intensity andﬂuorescence area.
Duolink. Duolink PLA probes and PLA detection reagents were purchased from
Sigma-Aldrich. Deparafﬁnization, epitope retrieval, inactivation of endogenous
peroxidase, and blocking were performed in the same manner as that used for
immunohistochemistry. Antibodies (mixture of CAPON/nNOS antibody or
CAPON/Tau5 antibody), diluted in TNT buffer, were initially reacted overnight at
4 °C. The sections were then washed three times in TNT buffer for 5 min, andreacted with PLA probes (a mixture of the Anti-Mouse Plus and Anti-Rabbit
Minus probes) in a humidiﬁed chamber at 37 °C. The ligation, ampliﬁcation, and
HRP labeling reactions were performed according to the manufacturer’s instructions. Interaction signals were visualized with tyramide-enhanced FITC (1:50
dilution in ampliﬁcation solution; supplied in the TSA Biotin System kit) for
10 min. The sections were then reacted with Hoechst solution, and coverslipped.
14

The slides were scanned on a NanoZoomer NDP system (Hamamatsu Photonics,
Hamamatsu, Japan) with ×40 resolution, and signals were quantiﬁed using
Deﬁniens Tissue Studio.
Isolation of sarkosyl-insoluble fraction. Dissected brains were immediately
frozen by liquid nitrogen, and stored at −80 °C. The sarkosyl-insoluble fraction
was isolated according to the method described in Sahara et al61. Brieﬂy, thehippocampus was homogenized in 10× volumes of Hsiao TBS (50 mM Tris-HCl
pH 8.0, 274 mM NaCl, and 5 mM KCl) containing protease inhibitor cocktail andphosphatase inhibitor cocktail. The homogenates were centrifuged at 26,300 × g for
20 min at 4 °C. The resulting supernatants were stored at −80 °C as the S1 fraction.
The pellets (P1 fraction) were re-homogenized in 5× volumes of high salt andsucrose buffer (10 mM Tris-HCl, pH 7.4, 0.8 M NaCl, 10% sucrose, 1 mM EGTA)
containing protease inhibitor cocktail and phosphatase inhibitor cocktail, andcentrifuged at 26,300 × g for 20 min at 4 °C. The resulting supernatants (S2 fraction) were incubated with 1% sarkosyl (Sigma) for 1 h at 37 °C, and then, centrifuged at 150,000 × g at 1 h at 4 °C. The pellets (P3 fraction) were suspended in TE
buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA), and used as the sarkosyl-insoluble
fraction.
Western blotting. The S1 fraction or P3 fraction (sarkosyl-insoluble fraction) of
the hippocampus was used for analysis. Protein concentrations were determinedusing a BCA protein assay kit (Pierce, Rockford, IL). An equivalent amount ofprotein from each animal was mixed with a 4 x sample buffer with or without
2-mercaptoethanol, then separated by SDS-polyacrylamide gel electrophoresis, andtransferred electrophoretically to a PVDF membrane (Merck Millipore, Burlington,MA). The membrane was treated with the ECL prime blocking solutions (GEHealthcare, Little Chalfont, UK), and reacted with each antibody diluted in Trisbuffered saline containing 0.05% Tween20 (TNB), pH 7.5, overnight at 4 °C. The
membrane was washed three times in TNB for 5 min, and treated with HRPconjugated anti-rabbit or anti-mouse IgG (GE Healthcare) for 1 h. Immunoreactive
bands on the membrane were visualized with ECL select (GE Healthcare) andscanned with a LAS-3000mini LuminoImage analyzer (Fuji Film, Tokyo, Japan).
RNA extraction and semi-quantitative RT-PCR. Total RNA was extracted frombrain samples using RNAiso Plus (Takara, Shiga, Japan) according to the manufacturer’s instructions. We performed semi-quantitative RT-PCR for detection ofboth 3R- and 4R-tau mRNA as previously described19, with minor modiﬁcations.
The primer pairs used for tau were: 5′-AAGTCGCCGTCTTCCGCCAAG-3′ and5′-GTCCAGGGACCCAATCTTCGA-3′, and for glyceraldehyde-3-phosphate
dehydrogenase (GAPDH): 5′- CCATGGCACCGTCAAGGCTGA-3′ and 5′GCCAGTAGAGGCAGGGATGAT-3′. PCR products of 288 bp and 381 bp correspond to 3R-tau and 4R-tau, respectively. RT-PCR products were detected by 24,
26, 28, 30, 32, and 34 PCR cycles to mortgage the linearity of the products, andthe result was decided by 26 cycles. Band intensity was calibrated using Image J
software.
Separation of cortical neurons and cell-based assays. Cortices and hippocampi
were separated from E16–18 embryos of WT mice and placed in Neurobasal
medium (Thermo Fisher Scientiﬁc). The tissues were chopped with scalpels andtreated with 5 mL of 0.25% trypsin at 37 °C for 15 min with rotation. Then, 0.125
mL of 1% DNaseI was added and the solution was mixed by pipetting. Aftercentrifugation of the tissues at 1500 rpm for 3 min, 5 mL of Hank’s buffered salt
solution containing 0.125 mL of 1% DNaseI was added to the pellet, and incubatedat 37 °C for 5 min, with gentle movement, in a water bath. Following this, thetissues were again centrifuged at 1500 rpm for 3 min, and the resulting pellet weresuspended in 15 mL of Neurobasal medium containing B27 plus supplement(Thermo Fisher Scientiﬁc) and 25 μM glutamate. The cells were ﬁltered using a
Falcon 2360 Cell Strainer with 100 μm Nylon, and seeded in cell culture plates with
Neurobasal medium containing B27 and glutamate. Before use, the culture medium
was ﬁrst changed to Neurobasal medium without glutamate, and after 24 h, waschanged to Neurobasal medium containing AAV vector.
Mitochondrial membrane potential assay. The assay was performed using theJC-1 Mitochondrial Membrane Potential Assay Kit (Cayman Chemical Company,
Ann Arbor, MI). After 14 days in vitro, cortical neurons were treated with AAV,
and 48 h later, the assay was performed according to the manufacturer’s instructions. 5 × 104 cells per 1 well of a 96-well plate were used as one sample. The
ﬂuorescence intensity at emission 550 nm /excitation 590 nm was detected fromhealthy cells, and the intensity at emission 485 nm / excitation 535 nm was detectedfrom apoptotic (unhealthy) cells using a Inﬁnite Pro200 plate reader (Tecan,
Männedorf, Switzerland). The ratio of the ﬂuorescence intensity between healthy
and unhealthy cells was calculated.
Oxygen consumption rate assay. The assay was performed using the Oxygen
Consumption Rate Assay Kit based on the manufacture’s instruction (Cayman
Chemical Company, Ann Arbor, MI). After 15 days in vitro, cortical neurons weretreated with AAV, and 48 h later the assay was performed according to the

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NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-10278-x

manufacturer’s instructions. 5 × 104 cells per 1 well of a 96-well plate were used as
one sample. The ﬂuorescence intensity at emission 380 nm /excitation 650 nm
was recorded at 37 °C every 2 min for 140 min using an Inﬁnite Pro200 plate reader(Tecan). Change of ﬂuorescence intensity per 1 min while the ﬂuorescence rise
linearly was calculated as relative value of oxygen consumption rate.
Statistical analyses. All analyses were completed with Graphpad Prism7 Software(San Diego, CA, USA). Differences between groups were examined for statisticalsigniﬁcance with Student’s t test.
Reporting summary. Further information on research design is available in
the Nature Research Reporting Summary linked to this article.

Data availability
All relevant data are available from corresponding authors upon reasonable request. The
source data underlying Figs. 1a–c, 2a–c, 3a–c, 4b, 4d, 4g–i, 5b, 6a–e, 7b–d, 7g, 8a, 8b, 8e,
8f, 9a, 9c, 9e, 10a, 10b, 10d, Supplementary ﬁgure 2b, 2d, 2e, 4, 8, 9, and 10 were provided
in Supplementary information ﬁles (Supplementary ﬁgures 14–32) and Source data ﬁle.
Tau-interacting proteins identiﬁed by tau interactome were summarized in
Supplementary Data 1. Antibodies used for western blot (WB) andimmunohistochemical (IHC) analyses were shown in Supplementary Table 1.

Received: 1 October 2018 Accepted: 24 April 2019

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This work was supported by a Grant-in-Aid for Young Scientists (B) (a Ministry of
Education, Culture, Sports, Science and Technology (MEXT) grant) (15K19036) (SH),and research grants from the RIKEN Special Postdoctoral Research program (SH).
Support was also received via a Grant-in-Aid for Scientiﬁc Research (B) (a MEXT grant)(TS), and by the Brain Mapping by Integrated Neurotechnologies for Disease Studies(Brain/MINDS) from the Japan Agency for Medical Research and Development (AMED)(TCS) (JP18dm0207001), as well as from a research grant from the RIKEN Center for
Brain Science.

Author contributions
S.H., Y.M., N.K., N.M., N.S., and J.T. performed experiments. S.M. produced andprovided AAV vectors for the project. S.H., T.S. and T.C.S. supervised the research. S.H.
and T.C.S. wrote and edited the manuscript.

Additional information
Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467019-10278-x.
Competing interests: S.H., Y.M., T.S. and T.C.S. serve as a member, a director, anadvisor and a CEO, respectively, for RIKEN BIO Co. Ltd. S.M. servers as a director andowns equity in a gene therapy company (Gene Therapy Research Institution Co., Ltd.).
The remaining authors declare no competing interests.
Reprints and permission information is available online at http://npg.nature.com/
reprintsandpermissions/
Journal peer review information: Nature Communications thanks the anonymous
reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports
are available.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional afﬁliations.

Acknowledgements
We thank Hiroki Sasaguri (RIKEN Center for Brain Science) for beneﬁcial discussion;
Yukiko Nagai (RIKEN Center for Brain Science) for secretarial work; Ryo Fujioka, andResearch Resource Division (RIKEN Center for Brain Science) for technical assistance;and members of the Laboratory for Proteolytic Neuroscience (RIKEN Center for Brain
Science) for beneﬁcial suggestions. The authors also thank Mika Ito and Naomi Takino(Jichi Medical University) for their help with the production of the AAV vectors. We are
grateful to Dr. Akihiko Takashima (Gakushuin University) for providing the Wtau-Tg
mouse, to Virginia M.-Y. Lee (University of Pennsylvania) for providing P301S-Tau-Tg
mice, to Dr. Norihiro Kato and Tadashi Okamura (National Center for Global Healthand Medicine) for providing CAPON-deﬁcient mice, and to Peter Davis (Litwin Zucker
Center for Alzheimer’s Research, Long Island) for providing PHF-1 and MC1 antibody.
A part of this work was achieved by collaboration with Astellas Pharma Inc, and we
thank Yoshitsugu Shitaka, Mitsuyuki Matsumoto, Yasuyuki Mitani, Hiroshi Yamada,Shinich Miyake, Mayuko Okabe, Mayako Yamazaki, and Ni Keni for helpful suggestions.

16

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