REVIEW
published: 31 March 2022
doi: 10.3389/fnins.2022.807473

Recent Advances in the Modeling of
Alzheimer’s Disease
Hiroki Sasaguri 1 , Shoko Hashimoto 1 , Naoto Watamura 1 , Kaori Sato 1,2 , Risa Takamura 1,2 ,Kenichi Nagata 3 , Satoshi Tsubuki 1 , Toshio Ohshima 2 , Atsushi Yoshiki 4 , Kenya Sato 1,5 ,Wakako Kumita 1,5 , Erika Sasaki 5,6 , Shinobu Kitazume 7 , Per Nilsson 8 , Bengt Winblad 8 ,Takashi Saito 9,10* , Nobuhisa Iwata 11* and Takaomi C. Saido 1*
1

Edited by:
Brian Hansen,
Aarhus University, Denmark
Reviewed by:
Eugenio Gutiérrez Jiménez,Aarhus University, Denmark
Yingjun Zhao,
Xiamen University, China*Correspondence:
Takashi Saito
saito-t@med.nagoya-cu.ac.jp
Nobuhisa Iwata
iwata-n@nagasaki-u.ac.jp
Takaomi C. Saido
takaomi.saido@riken.jp
Specialty section:
This article was submitted to
Neurodegeneration,a section of the journal
Frontiers in Neuroscience
Received: 02 November 2021
Accepted: 22 February 2022
Published: 31 March 2022
Citation:
Sasaguri H, Hashimoto S,
Watamura N, Sato K, Takamura R,
Nagata K, Tsubuki S, Ohshima T,
Yoshiki A, Sato K, Kumita W,
Sasaki E, Kitazume S, Nilsson P,
Winblad B, Saito T, Iwata N and
Saido TC (2022) Recent Advancesin the Modeling of Alzheimer’s
Disease. Front. Neurosci. 16:807473.
doi: 10.3389/fnins.2022.807473

Laboratory for Proteolytic Neuroscience, RIKEN Center for Brain Science, Wako, Japan, 2 Laboratory for Molecular Brain
Science, Department of Life Science and Medical Bioscience, Waseda University, Shinjuku City, Japan, 3 Departmentof Functional Anatomy and Neuroscience, Nagoya University Graduate School of Medicine, Nagoya, Japan, 4 Experimental
Animal Division, RIKEN BioResource Research Center, Tsukuba, Japan, 5 Department of Marmoset Biology and Medicine,
Central Institute for Experimental Animals, Kawasaki, Japan, 6 Laboratory for Marmoset Neural Architecture, RIKEN Center
for Brain Science, Wako, Japan, 7 Department of Clinical Laboratory Sciences, School of Health Sciences, Fukushima
Medical University, Fukushima, Japan, 8 Division of Neurogeriatrics, Department of Neurobiology, Care Sciences and Society,
Bioclinicum, Karolinska Institutet, Stockholm, Sweden, 9 Department of Neurocognitive Science, Institute of Brain Science,
Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan, 10 Department of Neuroscienceand Pathobiology, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan, 11 Departmentof Genome-Based Drug Discovery and Leading Medical Research Core Unit, Graduate School of Biomedical Sciences,
Nagasaki University, Nagasaki, Japan

Since 1995, more than 100 transgenic (Tg) mouse models of Alzheimer’s disease (AD)have been generated in which mutant amyloid precursor protein (APP) or APP/presenilin
1 (PS1) cDNA is overexpressed (1st generation models). Although many of thesemodels successfully recapitulate major pathological hallmarks of the disease such as
amyloid β peptide (Aβ) deposition and neuroinﬂammation, they have suffered from
artiﬁcial phenotypes in the form of overproduced or mislocalized APP/PS1 and theirfunctional fragments, as well as calpastatin deﬁciency-induced early lethality, calpain
activation, neuronal cell death without tau pathology, endoplasmic reticulum stresses,
and inﬂammasome involvement. Such artifacts bring two important uncertainties into
play, these being (1) why the artifacts arise, and (2) how they affect the interpretationof experimental results. In addition, destruction of endogenous gene loci in someTg lines by transgenes has been reported. To overcome these concerns, single App
knock-in mouse models harboring the Swedish and Beyreuther/Iberian mutations withor without the Arctic mutation (AppNL−G−F and AppNL−F mice) were developed (2ndgeneration models). While these models are interesting given that they exhibit Aβpathology, neuroinﬂammation, and cognitive impairment in an age-dependent manner,
the model with the Artic mutation, which exhibits an extensive pathology as earlyas 6 months of age, is not suitable for investigating Aβ metabolism and clearance
because the Aβ in this model is resistant to proteolytic degradation and is therefore
prone to aggregation. Moreover, it cannot be used for preclinical immunotherapy
studies owing to the discrete afﬁnity it shows for anti-Aβ antibodies. The weaknessof the latter model (without the Arctic mutation) is that the pathology may require up
to 18 months before it becomes sufﬁciently apparent for experimental investigation.

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Nevertheless, this model was successfully applied to modulating Aβ pathology bygenome editing, to revealing the differential roles of neprilysin and insulin-degrading
enzyme in Aβ metabolism, and to identifying somatostatin receptor subtypes involved inAβ degradation by neprilysin. In addition to discussing these issues, we also provide
here a technical guide for the application of App knock-in mice to AD research.
Subsequently, a new double knock-in line carrying the AppNL−F and Psen1P117L/WT
mutations was generated, the pathogenic effect of which was found to be synergistic.
A characteristic of this 3rd generation model is that it exhibits more cored plaquepathology and neuroinﬂammation than the AppNL−G−F line, and thus is more suitablefor preclinical studies of disease-modifying medications targeting Aβ. Furthermore, aderivative AppG−F line devoid of Swedish mutations which can be utilized for preclinicalstudies of β-secretase modiﬁer(s) was recently created. In addition, we introduce a
new model of cerebral amyloid angiopathy that may be useful for analyzing amyloidrelated imaging abnormalities that can be caused by anti-Aβ immunotherapy. Use of the
App knock-in mice also led to identiﬁcation of the α-endosulﬁne-KATP channel pathway
as components of the somatostatin-evoked physiological mechanisms that reduce Aβ
deposition via the activation of neprilysin. Such advances have provided new insights
for the prevention and treatment of preclinical AD. Because tau pathology plays anessential role in AD pathogenesis, knock-in mice with human tau wherein the entire
murine Mapt gene has been humanized were generated. Using these mice, the carboxyterminal PDZ ligand of neuronal nitric oxide synthase (CAPON) was discovered as amediator linking tau pathology to neurodegeneration and showed that tau humanization
promoted pathological tau propagation. Finally, we describe and discuss the current
status of mutant human tau knock-in mice and a non-human primate model of AD that
we have successfully created.
Keywords: Alzheimer’s disease, amyloid – beta, amyloidosis, tau propagation, somatostatin, mouse model, nonhuman primate (NHP)

1st, 2nd, AND 3rd GENERATION MOUSE
MODELS OF ALZHEIMER’S DISEASE(Kwart et al., 2019) and transcriptional malfunctions (Nalivaeva
et al., 2014), respectively, may be induced. Other overexpression
artifacts include calpain activation (Saito et al., 2016), calpastatin
deﬁciency-induced early lethality (Higuchi et al., 2012), andendoplasmic reticulum stresses (Hashimoto et al., 2018).
Furthermore, it was demonstrated that the random insertion oftransgene(s) resulted in the destruction of unexpectedly large
regions of endogenous gene loci in the host animal (Gamache
et al., 2019). We suggest that all transgenic mouse models
being used in research in which APP or APP/PSEN1 areoverexpressed should undergo whole genome sequencing (WGS)
so that destroyed loci that possibly aﬀect their phenotypes can be
identiﬁed (Sasaguri et al., 2017).
To overcome these drawbacks, single App knock-in mice, i.e.,
AppNL−G−F/NLG−F knock-in (AppNL−G−F ) and AppNL−F/NL−F
knock-in (AppNL−F ) lines, were generated that harbor the
Swedish (KM670/671NL) (Citron et al., 1992; Mullan et al.,1992) and Beyreuther/Iberian (I716F) (Lichtenthaler et al., 1999)
mutations with or without the Arctic (E693G) (Nilsberth et al.,2001) mutation (2nd generation models) (Figure 2) (Saito et al.,2014; Sasaguri et al., 2017). These mice, which exhibit typicalAβ pathology, neuroinﬂammation and memory impairment

The deposition of amyloid β peptide (Aβ) in the brain is
the major pathological hallmark of Alzheimer’s disease (AD),which is considered the most common type of dementia in
the world (Karran and De Strooper, 2016; Selkoe and Hardy,2016). To date, disease-associated mutations in the presenilin
1 (PSEN1) and presenilin 2 (PSEN2) genes number more than
300, while more than 50 mutations have been reported in theamyloid precursor protein (APP) gene (Alzforum1 ). In responseto these ﬁndings, many transgenic mouse models overexpressing
mutant APP or APP/PSEN1 cDNAs have been developed(1st generation models) (Sasaguri et al., 2017), however theyoften suﬀer from experimental limitations resulting from themislocalization of APP (Figure 1) and by the overproductionof APP fragments such as the C-terminal fragment of APP
generated by β-secretase (CTF-β) and APP intracellular domain(AICD). Neither of these fragments appears to accumulate in
AD brains, meaning that artiﬁcial endosomal abnormalities1

http://www.alzforum.org

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genotype (Morishima-Kawashima et al., 2000), although there
is no experimental evidence for this. In contrast, the AppNL−F
line accumulates wild-type human Aβ, but it may take up to
18 months for the pathology to become suﬃciently evident forinvestigational studies to be carried out (Saito et al., 2014), whichis too long for researchers to wait in a practical sense. Therefore,a new mouse model that accumulates wild-type human Aβ as
quickly as the AppNL−G−F model, but did not depend on thepresence of the Arctic mutation was desired.
To achieve this, the heterozygous Psen1P117L/WT mutant
line (Psen1P117L ) which, of the several Psen1 mutants, exhibits
the largest increase in Aβ42 /Aβ40 ratio in the brain (Sasaguri
et al., 2018) was utilized. The Psen1P117L line was generated
by base editing technology (Komor et al., 2016). The AppNL−F
mice were crossed with Psen1P117L mice, despite it being
unclear whether their pathogenic eﬀects, both of which act
on the γ-cleavage of CTF-β, would be additive or not in vivo
(Figure 4). The pathological phenotypes of AppNL−F mice weremarkedly enhanced in a synergistic manner with the Psen1P117L
mutation (Sato et al., 2021), with AppNL−F X Psen1P117L/WT
mice showing a more aggressive cored plaque pathology andneuroinﬂammation than the AppNL−G−F mice (Figure 5). These
double mutant mice (3rd generation model) will likely become
highly relevant tools for examining the pathologic mechanisms
upstream of Aβ deposition. Moreover, these mice can be highly
useful for the preclinical screening of disease-modifying therapy
candidates promoting Aβ degradation or disaggregation, without
the added concern associated with artiﬁcial eﬀects caused by
the Arctic mutation. We expect the double homozygous line,
AppNL−F X Psen1P117L/P117L , to exhibit an even more-aggressive
pathology. In any case, the AppNL−F X Psen1P117L mice aresuperior to the AppNL−G−F mice or the AppNL−G−I mice
(Xia et al., 2021) for universal and unbiased drug screening
particularly because the Aβ-degrading enzyme, neprilysin (NEP:
Neutral endopeptidase), has become a therapeutic target. The
AppNL−G−I mice are similarly designed as the AppNL−G−F mice,in which the Beyreuther/Iberian mutation was replaced by theAustrian mutation (Kumar-Singh et al., 2000). The characteristicsof the App knock-in mouse lines are listed in Table 3.

FIGURE 1 | Mislocalization of APP in APP-overexpressing mice. App KO
mice, WT mice, APP23 (APP-overexpressing mice) and App KI mice
(AppNL−F/NL−F ) were subjected to immunohistochemistry using antibodies to
APP, 22C11 (upper panels) and synaptophysin, a synaptic vesicle marker(lower panels) as indicated. App KO mice were used as negative controls for
APP staining. While APP is selectively expressed in the axons of WT and KI
mice, APP23 expresses unphysiologically high levels of APP not only in theaxons but also in the soma and dendrites. The scale bar indicates 2 mm.

(Saito et al., 2014; Masuda et al., 2016), are being used in morethan 500 research laboratories world-wide. At present, the
AppNL−G−F line is being used more frequently than the AppNL−F
line given that it develops Aβ pathology approximately threetimes faster (Saito et al., 2014) and can be used to analyze
downstream events such as neuroinﬂammation (Shirotani et al.,2019; Chen et al., 2020; Sobue et al., 2021), pericyte signaling(Nortley et al., 2019), oxidative stress (Hashimoto et al., 2019;
Hongo et al., 2020; Uruno et al., 2020), tau propagation (Saito
et al., 2019), and spatial memory impairment (Masuda et al.,2016; Jun et al., 2020; Sutoko et al., 2021; Table 1). Human
Arctic mutation carriers are indistinguishable from other familialand sporadic AD patients in pathological and neurological terms
except for low retention of 11 C-labeled Pittsburgh compound B(PiB) in PET study (Basun et al., 2008), indicating that the mutant
mice are relevant models for studying AD in general.
In addition to AppNL−F and AppNL−G−F models, App knock-in
mice devoid of the Swedish mutations (AppG−F mice) have been
recently developed, in which the Swedish mutations (NL) were
replaced by a wild-type sequence (KM) (Figure 3 and Table 2).
The AppG−F mice are more suitable for preclinical studies of βsecretase inhibition given that the Swedish mutation aﬀects thereactivity of APP to β-site amyloid precursor protein cleaving
enzyme 1 (BACE1) and most AD patients do not carry Swedish
mutations (Watamura et al., 2021b).
Despite the advantages mentioned above, the AppNL−G−F line
is not suitable for investigating the metabolism, clearance ordeposition of Aβ because the Arctic mutation present in the
middle of the Aβ sequence results in an Aβ that is resistant toproteolytic degradation (Tsubuki et al., 2003) and susceptible
to aggregation (Nilsberth et al., 2001). Moreover, this model
is not suitable for use in preclinical immunotherapy studies
due to its aﬃnity for anti-Aβ antibodies, even in the presenceof guanidine hydrochloride (GuHCl) (Saito et al., 2014). The
Arctic mutation may also directly or indirectly interfere withinteractions between Aβ deposition and the apolipoprotein E

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PRECAUTIONS REGARDING THE
UTILITY OF App KNOCK-IN MICE
There are several precautions to be aware of to make the best useof the App knock-in mice.

Nomenclature
A number of the App knock-in mouse users use incorrect
nomenclature such as APP-NLF, APPNLF and APPNL−F insteadof the AppNL−F mice, which accords with international rulesof standard genomic nomenclature. Genetic names always need
to be italicized.

Line-Ups and Biochemical Analyses
Approximately 20 lines of mutant mice, published or
unpublished, can currently or in the very near future be

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FIGURE 2 | Second generation mouse models of Alzheimer’s disease. See text for detailed explanation.

TABLE 1 | Successful application of the 2nd generation mouse models.
(1) Behavioral analysis using IntelliCage (Masuda et al., 2016; Sutoko et al., 2021).
(2) Three-dimensional visualization of amyloid pathology by transparency (Hama et al., 2015; Susaki et al., 2020).
(3) Impairment of gamma oscillations in medial entorhinal cortex (Nakazono et al., 2017).
(4) Additional genetic manipulation of the 2nd generation models through genome editing (Nagata et al., 2018; Watamura et al., 2021b).
(5) Generation of the double knock-in mouse models (Hashimoto et al., 2019; Saito et al., 2019; Sato et al., 2021).
(6) Assessment of vascular dysfunction in the 2nd generation models (Nortley et al., 2019; Tachida et al., 2020).
(7) Assessment of sleep dysfunction in the 2nd generation models (Maezono et al., 2020).
(8) Assessment of place cell dysfunction in the 2nd generation models (Jun et al., 2020; Takamura et al., 2021).
(9) Analyses of various aspects of neuroinﬂammation (Shirotani et al., 2019; Chiasseu et al., 2020; Salobrar-García et al., 2020; Barrett et al., 2021; Sobue et al., 2021).
(10) Application of spatial transcriptomics (Chen et al., 2020).

FIGURE 3 | AppG−F mice suitable for studies of BACE1 inhibitors. The AppG−F line is devoid of the Swedish mutation that inﬂuences the β-secretase activity andelevates the quantity of CTFβ. (The AppG−F line instead carries a wild-type sequence: KM.) The AppG−F model would be appropriate for use in preclinical studies of
β-secretase inhibitors without the interference of the Swedish mutation.

provided to academic and not-for-proﬁt researchers for
non-commercial research in a timely fashion with minimum
restrictions (Table 2; Saito et al., 2014; Sasaguri et al., 2018). It
is also recommended that scientists use optimized protocols for

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isolating Aβ from animal brain as previously described (Iwata
et al., 2005; Figure 6). The method allows the most sensitive
quantiﬁcation of both soluble and insoluble Aβ with the smallest
protocol deviations.

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TABLE 2 | List of mutant mice that are and will be made available to the research community.
Strains

Gene(s)Modiﬁcation Information

Availability*5

References*5

AppNL KI

App*1

KM670/671NL (Swedish)

RBRC*6 (RBRC06342)

Saito et al., 2014

AppNL−F KI

App*1

KM670/671NL (Swedish), I716F(Iberian/Beyreuther)

RBRC (RBRC06343)

Saito et al., 2014

AppNL−G−F KI

App*1

KM670/671NL (Swedish), E693G(Arctic), I716F (Iberian/Beyreuther)

RBRC (RBRC06344)

Saito et al., 2014

AppG−F KI

App*1

E693G (Arctic), I716F(Iberian/Beyreuther)Soon to be available from RBRC

Watamura et al., 2021b

ApphuAβ KI

App*1

No mutation (humanized Aβ
sequence)

Soon to be available from RBRC

Watamura et al., 2021b

Psen1P436S KI

Psen1*2

P436S

Available*7

Sasaguri et al., 2018

Psen1P117L/A KI

Psen1*3

P117L/A

Available*7

Sasaguri et al., 2018; Satoet al., 2021

AppNL−F KI/ Psen1P117L KI

App*1 , Psen1*3

App: KM670/671NL, I716F Psen1:
P117L

Soon to be available from RBRC

Sato et al., 2021

MAPT KI

MAPT ∗4

Humanization of the Mapt gene

RBRC (RBRC09995)

Hashimoto et al., 2019;
Saito et al., 2019

AppNL KI/hMAPT KI

App*1 , MAPT*4

App: KM670/671NL MAPT: No
mutation

RBRC (RBRC10041)

Saito et al., 2019

AppNL−F KI/hMAPT KI

App*1 , MAPT*4

App: KM670/671NL, I716F, MAPT:No mutation

RBRC (RBRC10042)

Saito et al., 2019

AppNL−G−F KI/hMAPT KI

App*1 , MAPT*4

App: KM670/671NL E693G, I716F,
MAPT: No mutation

RBRC (RBRC10043)

Saito et al., 2019hMAPTP301L KI

MAPT*4

P301L

Available*7

Watamura et al., 2021b

hMAPTP301S KI

MAPT*4

P301S

Available*7

Watamura et al., 2021b

hMAPTP301V KI

MAPT*4

P301V

Available*7

Watamura et al., 2021b

hMAPTIntron10+3G>A KI

MAPT*4Intron10 + 3 G > A

Available*7

Watamura et al., 2021b

hMAPTP301L;Intron10+3G>A KI

MAPT*4

P301L; Intron10 + 3 G > A

Available*7

Watamura et al., 2021b

hMAPTP301S;Intron10+3G>A KI

MAPT*4

P301S; Intron10 + 3 G > A

Available*7

Watamura et al., 2021b

hMAPTS305N;Intron10+3G>A KI

MAPT*4

S305N; Intron10 + 3 G > A

Available*7

Watamura et al., 2021b*1 Knock-in of APP sequence (from intron 15 to intron 17) including a humanized Aβregion.
*2 The mutation was introduced into the murine Psen1 gene by using Base Editor (BE) or Target-AID.
*3 The mutation was introduced into the murine Psen1 gene by using VQR-BE.
*4 Replaced the entire genomic sequence of the murine Mapt gene (from exon 1 to exon 14) with the human MAPT gene from the ATG codon of exon 1 to the 3 -UTR.
*5 As of September 30, 2021.
*6 RIKEN BioResource Research Center (https://web.brc.riken.jp/en/).
*7 All strains are available through TCS (takaomi.saido@riken.jp).

the Arctic Aβ produced by AppNL−G−F mice (Saito et al.,2014; Figure 7). Note that the AppNL−G−F is the mostfrequently used model because it recapitulates Aβ pathology andneuroinﬂammation much faster than other lines. This applies
to all other transgenic and knock-in mice that carry intraAβ mutations, including the Arctic and Dutch mutations (Van
Broeckhoven et al., 1990; Li et al., 2014). In contrast, there is
no restriction regarding use of antibodies for the AppNL−F X
Psen1P117L mice.

Maintaining Mouse Lines on a Clean
C57BL6/J BackgroundIn most cases, the knock-in mice are used in a homozygous
state to accelerate the generation of pathological andbehavioral phenotypes. However, the number of recessive
mutations increases over time if the breeding of mice ismaintained in this way. It is necessary therefore to back-crossheterozygous mutant mice with the wild-type C57B6/J mice
to remove these extraneous mutations, preferentially for 5–10
generations at an interval of 5–10 generations. Those groups
dealing with poor reproductive output of mice due to theirextremely inbred nature can contact RIKEN BioResource
Research Center (email: animal.brc@riken.jp), a national
mouse repository of Japan (Mizuno-Iijima et al., 2021), to
renew their strains.

Preclinical Nature of Behaviors
In our experience, the most sensitive and reproducible test
involves contextual fear conditioning, although its irreversible
nature can be problematic. In a more complex sense, theapplication of a multi-task paradigm such as IntelliCage(NewBehavior AG, Zurich, Switzerland) would be more
informative (Codita et al., 2010; Masuda et al., 2016). We
recently showed that the mouse genotypes can be predictedfrom their behavioral parameters by machine learning (Sutoko
et al., 2021). It should be emphasized that the App knock-in

Choosing Appropriate Anti-Aβ Antibodies
It was previously indicated that some anti-Aβ antibodies areinappropriate for biochemically and pathologically detecting

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FIGURE 4 | Scheme of AppNL−F × Psen1P 117L double-mutant mice. For the generation of the double-mutant mice, the AppNL−F line was crossbred with thePsen1P 117L line whose mutation was introduced in the endogenous Psen1 gene utilizing base editing technology. The synergistic effects of the pathogenic
mutations in the App and Psen1 genes strongly accelerates the deposition of wild-type human Aβ in mouse brains.

A NEW MODEL OF CEREBRAL AMYLOIDANGIOPATHY
Most AD patients exhibit parenchymal and vascular Aβ
deposition in the brains, and both pathologies seem tobe driven by impaired Aβ clearance within the interstitial
ﬂuid and perivascular drainage pathways (Greenberg et al.,2020). Iliﬀ et al. (2012) injected ﬂuorescent tracers into Tie2GFP:NG2-DsRed double reporter mice, which express GFP
in all cerebral blood vessels and DsRed in perivascular cells,
and successfully observed glymphatic pathway; subarachnoid
CSF inﬂux into the brain parenchyma and bulk ISF solute
clearance from the parenchyma within the perivascular spaces.
Importantly, in AD model mice, glymphatic CSF inﬂux is
reduced and the clearance of Aβ is severely impaired (Penget al., 2016). Impaired glymphatic pathway may contributeto the deposition of Aβ in the blood vessels of the brain,
cerebral amyloid angiopathy (CAA). Although CAA is
profoundly observed in most AD patients (Brenowitz et al.,2015), limited model mice, such as those with Dutch/Iowa
mutation, exhibit apparent CAA, thus making it diﬃcult
to determine how CAA contributes to the pathogenesis ofsporadic AD. Notably, human vascular endothelial cells
express signiﬁcant level of APP770 and human plasma
contains ∼100 ng/ml of sAPP770 (Kitazume et al., 2010).
Since peripheral blood cells other than platelet do not express
APP, and platelets release sAPP770 upon their activation(Miura et al., 2020), it is considered that plasma sAPP770 ismostly derived from endothelial APP770. Because in rodents

FIGURE 5 | AD pathology in the hippocampus of a 3rd generation model
mouse. A 12-month-old AppNL−F X Psen1P 117L/WT mouse was analyzed byimmunohistochemistry. Blue: Aβ plaques; red: microglia; green: astrocytes.
The bar indicates 25 μm.

mice are models of preclinical AD because the App knockin mice, like all the APP and APP/PS1 transgenic mice, do
not recapitulate tau pathology alone (Sasaguri et al., 2017).
Consistently, we observe only mild cognitive decline inthese mice. In contrast, the App knock-in mice crossbred
with mutant MAPT knock-in mice exhibited accelerated tau
pathology (Table 2).

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TABLE 3 | Characteristics of the App knock-in mouse lines.
Stain

Gene mutations

Genetic
backgroundAβ plaques(ﬁrst
appearance)

Tangles Neuronal
loss

Cognitive
impairment

Strengths

Weaknesses

App

Psen1

ApphuAβ

Humanized Aβ–

C57BL/6J

–

NR

NR

NR

A control forother models

–

AppNL

Humanized Aβ –KM670/671NL

C57BL/6J

–

–

–

–

A control forother models

No amyloid
pathology No
cognitive deﬁcits
Increased CTF-βAppNL−F

Humanized Aβ
KM670/671NL
I716F

–

C57BL/6J

6 months––

18 months

Deposition of wild Long time required
type human Aβ
for amyloid
pathology andcognitive deﬁcits
Increased CTF-βAppNL−G−F Humanized Aβ
KM670/671NL
E693G I716F–

C57BL/6J

2 months––

6 months

Early appearance The Arctic mutation
inside the Aβof amyloid
sequencepathology
Increased CTF-βAppG−F–

C57BL/6J

4 months

NR

NR

NR

The Arctic mutation
Absence of theinside the Aβ
Swedishsequencemutation No
increase of CTF-β

App and Psen1 AppNL−F
Humanized Aβ P117L C57BL/6J
double mutant Psen1P117L KM670/671NL
I716F

3 months

NR

NR

NR

Early appearance Mutations in both
App and Psen1of amyloid
genes
pathology
Deposition of wild
type human AβSingle App
knock-in

Humanized Aβ
E693G I716F

The Swedish mutations; KM670/671NL.
The Iberian/Beyreuther mutation; I716F.
The Arctic mutation; E693G.
NR denotes data not reported.

plasma sAPP is a markedly lower (∼100 pg/ml) than thatof humans (Kitazume et al., 2012), it’s possible that low
level of endothelial APP expression in mice could be oneof the reasons that App knock-in mice exhibit mild CAA
pathology. To overcome this, a mouse line that speciﬁcally
expresses human APP770 in endothelial cells has just been
generated (unpublished). In brief, ﬂoxed hAPP770NL mice
under the CMV early enhancer/chicken β-actin promoter wereﬁrst generated. These mice were then crossed with Tie2-Cre
mice, in which the Tie2 promoter directs the expression of
Cre recombinase in the endothelial cells to obtain doubletransgenic (Tg) mice.
App knock-in mouse models were previously produced by
Li et al. (2014) who used multiple pathogenic mutations. These
mice carry the Swedish (K670N/M671L), Dutch (E693Q), andLondon (V717I) mutations with the humanized Aβ sequence.
The Dutch mutation results in an intensive CAA pathology inhumans, thereby causing brain hemorrhage and early mortality(Levy et al., 1990; Van Broeckhoven et al., 1990). This mutationis therefore not speciﬁcally responsible for causing FAD. These
mice did not develop prominent Aβ deposits over their lifespan,
but when they were crossbred with Psen1M 146V knock-in mice, anage-dependent deposition of Aβ was seen in the resultant double

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knock-in mice. The deposition of Aβ was detected not only inthe parenchyma of the cerebral cortex but also in the cerebral
vasculature, similar to that seen in CAA in humans. Double
knock-in mice that did not have the Dutch mutation exhibited
virtually no vascular pathology. In this way, if the authors had
used the Beyreuther/Iberian or Austrian mutation instead of the
London mutation in the mouse App gene then they probably
would not have had to introduce the Psen1 knock-in mice.
Knock-in mice harboring the Dutch mutation can still serve as
relevant models for CAA; however they may not be appropriate
for examining the eﬀect of immunotherapy on CAA because theDutch mutation is present in the middle of the Aβ sequence.

NEPRILYSIN-SENSITIVEAMYLOIDOGENIC Aβ AS A PROBABLE
CAUSE FOR SPORADIC ALZHEIMER’SDISEASENEP and insulin-degrading enzyme (IDE) are considered thetwo major catabolic enzymes that degrade Aβ (Qiu et al., 1998;
Iwata et al., 2000, 2001; Farris et al., 2003; Leissring et al., 2003).
NEP is capable of degrading both soluble and insoluble Aβ

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FIGURE 6 | Outlined protocols for extraction and quantiﬁcation of Aβ from tissues. See text for detailed explanation.

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FIGURE 7 | Reactivity of different antibodies to Arctic Aβ in AppNL−G−F mice. (A) Epitope map of anti-Aβ antibodies. (B,C) Quantiﬁcation of Arctic Aβ species using
BNT77 as a capture antibody. BNT77 binds to the mid-portion of Aβ [see epitope map (A)]. A sandwich ELISA kit (Wako, Japan) was used to quantify Aβx-40 (C)and Aβx-42 (D), respectively. (D,E) Quantiﬁcation of Arctic Aβ species using BAN50 as a capture antibody. BAN50 binds to the N-terminal region of Aβ [see epitope
map (A)]. A sandwich ELISA kit (Wako, Japan) was used to quantify Aβx-40 (D) and Aβx-42 (E), respectively. BNT77 and BAN50 captured Arctic Aβ more weakly
than wild-type Aβ. (F) Immunohistochemistry using various anti-Aβ antibodies. Brain sections derived from AppNL−F mice (24 months old) were immunostainedusing antibodies with different epitopes after antigen retrieval as indicated (upper panels); those of AppNL−G−F mice (9 months old) were similarly immunostained(lower panels). Scale bars represent 500 μm.

(Iwata et al., 2000, 2001; Huang et al., 2006), but it is not clearlyshown whether endogenous IDE could degrade insoluble Aβ in
the mouse brains (Farris et al., 2003), rather IDE appears to be
involved in metabolism of AICD. Unfortunately, their roles in Aβ
metabolism in vivo have never been compared in an impartial andside-by-side manner. Once double mutants crossbred single App
knock-in mice with NEP (Mme) KO mice and with IDE (Ide) KO
mice were analyzed in detail for their biochemical properties andAβ pathology properties, it would be clear their distinct roles in
APP metabolism and the AD pathogenesis.
Further to the above, a deﬁciency of NEP had no signiﬁcant
impact on the levels of various neuropeptides (Sasaguri et al.,2018) as well as enkephalins (Saria et al., 1997) that are wellknown to be in vitro substrates for NEP (Turner et al., 1996,
2000, 2001; Turner and Nalivaeva, 2006) in the cerebral cortex
and hippocampal formation of mice, presumably because NEP
is mainly expressed in secretory vesicles and on the presynaptic
membranes of excitatory neurons (Iwata et al., 2002, 2004,
2013), while most if not all neuropeptides are secreted from

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inhibitory neurons. This argues against the concern that NEP
up-regulation for the treatment of preclinical AD would reduce
the levels of these neuropeptides. These ﬁndings also indicatethat NEP relatively selectively degrades Aβ in the brain. Whereasfamilial AD (FAD) is unambiguously caused by an increased
anabolism of Aβ, and of Aβ42 and Aβ43 in particular (Selkoe andHardy, 2016), the anabolism of Aβ appears unaﬀected prior toits deposition in the brain that subsequently leads to the onsetof sporadic AD (SAD). These observations suggest that NEPsensitive amyloidogenic Aβ likely plays a primary pathogenic rolein the etiology of SAD. Our ﬁndings are consistent with the agingdependent decline of NEP expression in the human brain andwith recent genome-wide association studies (GWAS) indicatingthat variants of the gene encoding NEP (MME) are associatedwith the risk of SAD development (Bellenguez et al., 2020). Taken
together, our results imply that the aging-associated decrease in
NEP expression is a primary cause of SAD and could thus be atarget for the treatment of preclinical AD once other factors suchas apolipoprotein E genotypes have also been considered.

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REGULATION OF Aβ METABOLISM VIA
SOMATOSTATIN RECEPTOR SUBTYPES
THROUGH MODULATION OF NEP
ACTIVITY
Since NEP is a major Aβ-degrading enzyme and it isdownregulated upon aging, its decreased levels in the brain will
most likely lead to increased Aβ levels (Yasojima et al., 2001;
Carpentier et al., 2002; Iwata et al., 2002; Maruyama et al.,2005; Hellström-Lindahl et al., 2008). NEP is regulated by theneuropeptide somatostatin (Saito et al., 2005). Somatostatin,
which binds to somatostatin receptors, is also decreased upon
aging and in AD possibly due to loss of somatostatin-positive
interneurons (Davies et al., 1980; Beal et al., 1985; Bergström
et al., 1991; Hayashi et al., 1997; van de Nes et al., 2002; Lu et al.,
2004; Gahete et al., 2010). Somatostatin, which was ﬁrst identiﬁed
to regulate secretion of growth hormone from pituitary, hasbeen traditionally abbreviated as SRIF (somatotropin releaseinhibiting factor) (Møller et al., 2003), so we will keep tothis nomenclature in this review. SST1 , SST2 , SST3 , SST4 , andSST5 are used to express somatostatin receptor subtypes 1–5.
Interestingly, mutations in SRIF are linked to AD (Vepsäläinen
et al., 2007). By using a combination of in vitro and in vivo
approaches to identify the subtype speciﬁcity of the ﬁve SSTs
expressed in the brain and considered to be involved in theregulation of NEP. We would like to emphasize that it is necessary
to use a co-culture system of primary neurons from the cortex,
hippocampus, and striatum for in vitro experiments (Kakiya et al.,2012; Nilsson et al., 2020; Watamura et al., 2021a).
Using a battery of Sst double knockout (dKO) mice, we foundthat NEP is regulated by SST1 and SST4 in a redundant manner.
Sst1 and Sst4 dKO mice exhibit a speciﬁc decrease of presynaptic
NEP in the Lacunosum molecular layer. Moreover, a genetic
deﬁciency of Sst 1 and Sst4 in the App knock-in mice aggravated
the Aβ pathology in the hippocampus. As a ﬁrst proof of concept
towards an Aβ-lowering strategy involving NEP, a treatment withan agonist selective for SST1 and SST4 could ameliorates the Aβ
pathology and improves cognitive outcomes in the App knockin AD mouse model as schematized in Figure 8 (Nilsson et al.,2020). These results indicate that a combination of SST1 and SST4
homodimers or the SST1 and SST4 heterodimer may become atarget for pharmaceutical intervention to treat preclinical AD.
Of note, the expression of SRIF in human brain declines withaging and in AD (Davies et al., 1980; Lu et al., 2004) and may
causally contribute to AD pathogenesis via reduction of NEP
activity/expression.

FIGURE 8 | Somatostatin receptor subtypes 1 and 4 (SST1/4 ) regulate theAβ-degrading enzyme NEP. The neuropeptide somatostatin (SRIF) was
identiﬁed as a regulator of NEP activity through in vitro screening. Subsequent
analysis of the effect of genetic deletion of somatostatin receptor (SST)
subtypes in mice revealed that SST1 and SST4 regulate NEP in a redundantmanner. This was further conﬁrmed by concurrently deleting SST1 and SST4
in App KI mice, which aggravated the Aβ pathology. SST1/4 can be either acombination of SST1 and SST4 homodimers or an SST1 /SST4 heterodimer.

elucidated. Proteomic analyses enabled α-endosulﬁne (ENSA),an endogenous ligand of the ATP-sensitive potassium (KATP )channel, to be identiﬁed as a negative regulator of NEP
downstream of SRIF signaling (Watamura et al., 2021a). The
expression of ENSA is signiﬁcantly increased in AD mousemodels and in patients with AD. In addition, NEP directly
contributes to the degradation of ENSA, suggesting a substratedependent feedback loop regulating NEP activity.
It was also discovered the speciﬁc KATP channel subtype[sulfonylurea receptor subunit 1 (SUR1) and inwardly rectifying
K+ channel 6.2 (Kir6.2)] that modulates NEP activity, resulting
in altered Aβ levels in the brain. Pharmacological intervention
targeting this particular KATP channel by diazoxide attenuatedAβ deposition, with impaired memory function rescued via
the NEP activation in our AD mouse model. These ﬁndings
provide a mechanism explaining the molecular link between
KATP channels and NEP activation. They also provide new
insights into how ENSA and the KATP channel could proﬁleas a new therapeutic target for lowering Aβ and thus provide
an alternative strategy to prevent AD. Figure 9 summarizes the
NEP activation mechanism that involves SRIF receptor subtypes,
ENSA and KATP channel involvement.

HUMANIZATION OF THE ENTIRE
MURINE Mapt GENE TO GENERATEhMAPT KNOCK-IN MICE

SRIF-EVOKED Aβ CATABOLISM IN THE
BRAIN: MECHANISTIC INVOLVEMENT
OF THE α-ENDOSULFINE-KATP
CHANNEL PATHWAY

To date, most if not all, mouse models of tauopathy have been
unable to recapitulate the tau pathology without overexpressing
mutant human tau protein. As a novel in vivo platform forstudying human tauopathy, human MAPT knock-in mice havebeen developed in which the entire Mapt gene including all exons

Although SRIF is known to regulate Aβ catabolism by enhancing
NEP-catalyzed proteolytic degradation, the mechanism bywhich SRIF actually regulates NEP activity is yet to be fully

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NEP activation mechanism
Inactive NEP
SRIF

SST1/4ENSA

KATP channel
Active NEP
SUR1/Kir6.2

Aȕ degradation

FIGURE 9 | Role of ENSA in the regulation of NEP activity. Schematic
illustration of the mechanism describing NEP activity in the brain. ENSA, adownstream protein of SST-SST1/4 signaling, plays a role as a ligand of the
KATP channel composed of sulfonylurea receptor subunit 1 (SUR1) andinwardly rectifying K+ channel 6.2 (Kir6.2), resulting in the activation of NEP.
SST1/4 can be either a combination of SST1 and SST4 homodimers or an
SST1 /SST4 heterodimer.

FIGURE 10 | Propagation of AD-tau in mouse brains. Propagation of tau ineach mouse line was observed 3 months after AD-tau injection. Brain sections
were immunostained with AT8 (red). Humanization of the host animal tau
affects the transmission of the pathogenic agents. AppNL−G−F /MAPT dKI
mice exhibited greater pathological propagation than AppNL−G−F KI mice.

and introns are humanized (Hashimoto et al., 2019). In eachstrain, the MAPT and Mapt genes encoded human and murine
tau proteins, respectively. This was done by crossing MAPT
knock-in mice with single App knock-in mice in order to study
the role of the Aβ-tau axis in the etiology of AD. The double
knock-in mice exhibited a more pronounced tau phosphorylation
status than single MAPT knock-in mice but lacked evidence oftau pathology and neurodegeneration (in a manner similar to thatof single App knock-in mice) even after waiting until the mice
were 24 months old.
In both the absence and presence of Aβ amyloidosis, thetau humanization has been found to signiﬁcantly accelerate thepropagation of AD brain-derived pathological tau (Figure 10;
Saito et al., 2019). Tau accumulation was intensiﬁed in the lattercase and closely associated with dystrophic neurites, consistently
showing that Aβ amyloidosis aﬀects tau pathology. These results
indicated that pathological human tau interacted better withhuman tau than with murine tau, and suggest the presence ofa species-deﬁned preference between the pathogenic proteins.
The MAPT knock-in mice also facilitate the investigationof behavioral properties and of human tau characteristics
in living animal models. In addition, mutant MAPT knockin mice carrying various pathogenic mutations have been
generated (Table 2). These mice exhibit aging-dependent tau
aggregation and cognitive impairment in a manner accelerated
by Aβ pathology and are being provided to the researchcommunity upon request.

(Roberson et al., 2007) (Ittner et al., 2010). We also previously
identiﬁed a NMDAR-related molecule as a tau binding protein
which is involved in tau pathology- induced neurodegeneration.
To elucidate key molecules underlying tau accumulationinduced neurodegeneration, a comprehensive screening of tauinteracting proteins (tau interactome) was constructed. Taubinding proteins were isolated by immunoprecipitation-LCMS/MS (IP-MS) using a Flag-tag antibody and wild-type tau
Tg (wtau-Tg) mice, which is expressing human tau tagged witha ﬂag epitope (Kimura et al., 2007). Considering that tau is a
microtubule-binding protein, we validated the methods used togenerate the tau interactome by identifying the tubulin beta-4A
chain as one of the tau-binding proteins.
Of the many proteins identiﬁed in the tau interactome, we
focused on carboxy-terminal PDZ ligand of neuronal nitricoxide synthase (CAPON) (Hashimoto et al., 2019), which isan adaptor protein of neuronal nitric oxide synthase (nNOS).
CAPON acts as an enzyme for the production of nitricoxide (NO) and is involved in NMDAR-mediated excitotoxicity(Jaﬀrey et al., 1998). It is thought to recruit substrates tonNOS and facilitate their NO-mediated modiﬁcation throughprotein-protein interactions (Jaﬀrey et al., 1998). The presenceof CAPON polymorphisms associated with schizophrenia andother psychiatric disorders has been reported in several studies(Brzustowicz, 2008; Freudenberg et al., 2015). Moreover, CAPON
was shown to positively regulate spine density (Richier et al.,2010) and to regulate neuronal cell death downstream of the
NMDAR (Li et al., 2013). These ﬁndings suggest that CAPON
contributes to neurotransmission and neuronal excitotoxicity.
In addition, one report showed that CAPON is upregulated
in CA1 pyramidal cells in the AD brain (Hashimoto et al.,2012), implying that CAPON may play an important role in thepathogenesis of AD. The mechanism(s) underlying these eﬀects
nevertheless remain(s) unknown.
To further elucidate the eﬀects of CAPON on AD pathology,
we introduced CAPON cDNA into the brains of AppNL−G−F
and AppNL−G−F X MAPT (hTau) double-KI mice using a

THE ROLE OF CAPON IN TAUPATHOLOGY-MEDIATED TOXICITY
Pathological tau causes synaptic dysfunction and loss of synapses.
One of promising molecules that mediates tau pathology-induced
neurotoxicity is N-methyl-D-aspartate receptor (NMDAR). For
example, tau accumulation disturbs synaptic plasticity through
JAK2/STAT1-mediated suppression of NMDAR (Li et al., 2019).
Phosphorylation of tau at Tyr18, which is mediated by thetyrosine kinase Fyn, enhances NMDAR-dependent excitotoxicity(Guo et al., 2020). Several reports indicated that glutamateinduced excitotoxicity was prevented by downmodulation of tau

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FIGURE 12 | Photograph of common marmosets (Callithrix jacchus). The
photo shows members of captive common marmoset family. Their small bodysize, fecundity, and high cognitive functions are a suitable model forneuroscience. The photograph of marmosets was taken by WK at CIEA.

FIGURE 11 | Functions of CAPON in neurodegeneration. (A) Brain sections
from WT, P301S-Tau-Tg or nos1ap-/-/P301S-Tau-Tg mice stained by theconventional method using hematoxylin and eosin (H&E). A CAPON (Nos1ap)
deﬁciency restores AD-related pathological phenotypes in P301S-Tau-Tg
mice. (B) Scheme of CAPON action. Aβ pathology elevates the level andlocalization of CAPON in hippocampal pyramidal cells. CAPON-induced
neuronal cell death is closely associated with the pathological tau protein,although there appears to be a tau-independent mechanism in play as well.

adolescent marmosets exhibit tau hyperphosphorylation, but
not neuroﬁbrillary tangle formation, in the brain that increases
with aging (Rodriguez-Callejas et al., 2016). Their life spansin captivity are as long as 10–15 years, making them suitable
for age-related research (Tardif et al., 2011). Their immune
systems and metabolic functions resemble those of humans (tHart and Massacesi, 2009; Tardif et al., 2011) and thus may
aﬀect the pathogenic processes related to AD (Ennerfelt andLukens, 2020; Kellar and Craft, 2020; Rosario et al., 2020).
Because sleep disorder is an early clinical symptom of AD (Pyunet al., 2019), it is noteworthy that marmosets share with humans
the sleep phases composed of rapid eye movement (REM) andnon-REM cycles (Crofts et al., 2001). Among various non-humanprimate species, the marmoset seems most applicable to genetic
manipulation, i.e., generation of designed mutants, for which
their high reproductive eﬃcacy is advantageous (Sasaki et al.,2009; Sato et al., 2016; Park and Sasaki, 2021). Furthermore,
fecundity characteristics of marmosets, such as a short periodof sexual maturity, multiple births, and short gestation interval,
are suitable for producing genetically modiﬁed disease models(Tardif et al., 2003).
Majority of FAD-causing mutations reside in the PSEN1 gene
(Scearce-Levie et al., 2020). Typically, deletion mutations inexon 9 (Crook et al., 1998; Prihar et al., 1999; Smith et al.,
2001; Dumanchin et al., 2006; Le Guennec et al., 2017) orpoint mutations at the 3 splice site (acceptor site) of exon 9in the PSEN1 gene cause dominantly inherited FAD. The point
mutations instigate exon 9 elimination and S290C modiﬁcationin the corresponding mRNA at the junction sites of exons 8
and 10 via the conversion of alternative splicing (Hutton et al.,
1996; Kwok et al., 1997; Steiner et al., 1999; Brooks et al., 2003;
Blauwendraat et al., 2016). Thus, generation of a marmoset modelof AD is set out in which exon 9 of the PSEN1 gene product isdeleted using gene-editing technologies to produce AD marmoset

newly developed adeno-associated virus (AAV)-mediatedapproach. We analyzed the eﬀects of human tau protein as it
is known that the hTau-KI mouse expresses an endogenouslevel of WT human tau. These experiments revealed thatCAPON expression facilitates hippocampal atrophy inconjunction with neuronal cell death, and that a deﬁciency
of CAPON in the P301S-Tau-Tg tauopathy mouse model
suppressed tau pathology and neurodegeneration (Figure 11).
From our results, an intervention in the interaction between
CAPON-tau or CAPON-nNOS could be a new approach for thetreatment of AD.

GENERATION OF NON-HUMAN
PRIMATE MODELS OF FAMILIALALZHEIMER’S DISEASE
Common marmosets (marmosets, Callithrix jacchus) are small
non-human primates that belong to the New World Primates(Figure 12; Mansﬁeld, 2003). They have been increasingly
utilized in neuroscience because of advantages that wereobserved over other research primates (Okano, 2021; Parkand Sasaki, 2021). Marmosets possess physiological functions,brain structures and complex cognitive/social behaviors similar
to those of humans; they communicate mainly via visualand auditory measures. In association with AD research, theamino acid sequence of Aβ in marmosets is identical tothat of humans, with aged wild-type marmosets starting toaccumulate Aβ from 7 years of age or even earlier (Geulaet al., 2002; Rodriguez-Callejas et al., 2016). In addition,

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Alzheimer’s Models(HS); JSPS KAKENHI Grant Numbers JP15K19036 and
JP19K16271 and RIKEN Special Postdoctoral Research program(SH); JSPS KAKENHI Grant Number JP21K15378 (NW);
AMED Grant Number JP19dm0207065 (ES), AMED Grant
Number JP18am0101036 (SK); The Swedish Research Council
2018-02843 (BW); Hållsten Research Foundation (PN); JSPS
KAKENHI Grant Number JP20H03564/AMED Grant Numbers
JP21gm1210010s0102 and JP21dk0207050h001/JST [Moonshot
R&D] Grant Number JPMJMS2024/Grant-in-aid for Researchin Nagoya City University Grant Number 2021101/the Hori
Sciences and Arts Foundation, and Toyoaki ScholarshipFoundation (TS); AMED Grant Numbers JP16dm0107068h,
JP17dm0107068h, JP18dm0107068h (NI and TS); AMED Grant
Number JP15dm0207001 and RIKEN Aging Project (TCS).

models. Since TALEN exhibited high genome-editing eﬃcacy,
generates few oﬀ-target eﬀects, and produces little mosaicism, theTALEN would be a suitable tool for producing exon 9 deletion in
the PSEN1 gene (Sato et al., 2016; Zhang et al., 2019). Although it
is a non-peer review data, the exon 9 deletion in the PSEN1 genethat is an AD causing mutation has been successfully introduced
into non-human primates by TALEN (Sato et al., 2020).

AUTHOR CONTRIBUTIONS
All authors listed have made a substantial, direct, and intellectual
contribution to the work, and approved it for publication.

FUNDINGACKNOWLEDGMENTS
Part of the work described was supported by the following
research grants: JSPS KAKENHI Grant Number JP18K07402

We thank Yukiko Nagai-Watanabe for secretarial assistance.

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March 2022 | Volume 16 | Article 807473