cro

ARTICLE

Humanization of the entire murine Mapt gene provides amurine model of pathological human tau propagation
Received for publication, May 22, 2019, and in revised form, June 27, 2019 Published, Papers in Press, July 4, 2019, DOI 10.1074/jbc.RA119.009487

Edited by Paul E. Fraser

In cortical regions of brains from individuals with preclinicalor clinical Alzheimer’s disease (AD), extracellular ␤-amyloid(A␤) deposition precedes the aggregation of pathological intracellular tau (the product of the gene microtubule-associatedprotein tau (MAPT)). To our knowledge, current mouse modelsof tauopathy reconstitute tau pathology by overexpressing
mutant human tau protein. Here, through a homologous recombination approach that replaced the entire murine Mapt genewith the human ortholog, we developed knock-in mice withhumanized Mapt to create an in vivo platform for studying
human tauopathy. Of note, the humanized Mapt expressed all
six tau isoforms present in humans. We next cross-bred the
MAPT knock-in mice with single amyloid precursor protein(App) knock-in mice to investigate the A␤–tau axis in AD etiology. The double-knock-in mice exhibited higher tau phosphorylation than did single MAPT knock-in mice but initially lacked
apparent tauopathy and neurodegeneration, as observed in the
single App knock-in mice. We further observed that tau humanization significantly accelerates cell-to-cell propagation of AD
brain-derived pathological tau both in the absence and presenceof A␤-amyloidosis. In the presence of A␤-amyloidosis, tau accumulation was intensified and closely associated with dystrophic
neurites, consistently showing that A␤-amyloidosis affects tau
This work was supported in part by research grants from the RIKEN Center for
Brain Science, research grants from the RIKEN Aging Project, a Grant-inAid-for Scientific Research (B) from MEXT (to T. S.), Precursory Research for
Embryonic Science and Technology from JST (to T. S.), the Cell Science
Research Foundation (to T. S.), the Brain Mapping by Integrated Neurotechnologies for Disease Studies (Brain/MINDS) from the Japan Agency for
Medical Research and Development (AMED) (Grant JP18dm027001) (toT. C. S.), Astellas Pharma, Inc. JSPS KAKENHI Grant JP16H06277 (to S. M.),and National Institutes of Health Grants AG10124 and AG17586 (toV. M. Y. L. and J. Q. T.). T. S., Y. M., S. H., and T. C. S. serve as advisor, director,
researcher, and CEO, respectively, for RIKEN BIO Co. Ltd., which sublicenses
animal models, App KI mice, to for-profit organizations, the proceeds fromwhich are used for the identification of disease biomarkers. The content issolely the responsibility of the authors and does not necessarily represent
the official views of the National Institutes of Health.
This article contains Fig. S1.
1
To whom correspondence may be addressed. E-mail: takashi.saito.
aa@riken.jp.
2
To whom correspondence may be addressed. E-mail: saido@brain.riken.jp.

pathology. Our results also indicated that the pathologicalhuman tau interacts better with human tau than with murinetau, suggesting species-specific differences between theseorthologous pathogenic proteins. We propose that the MAPT
knock-in mice will make it feasible to investigate the behaviors
and characteristics of human tau in an animal model.

Alzheimer’s disease (AD)3 is the primary cause of dementia
in the elderly and imposes a social and economic burden on
modern society. In cortical regions of the preclinical and clinical AD brains, extracellular A␤ deposition precedes the aggregation of pathological intracellular tau (MAPT gene product).
These aggregates, composed of hyperphosphorylated tau, formin the brain and are followed by irreversible progressive neurodegeneration. This pathological chronology agrees with thecause-and-effect relationship proposed by the amyloid cascade
hypothesis (1–3).
Despite current knowledge, the molecular mechanisms bywhich A␤ deposition induces the formation of neurofibrillarytangles (NFTs) remain elusive. We believe that this is in partcaused by a lack of relevant animal models. Accordingly, wedemonstrated nonphysiological aspects of the existing models
in our previous studies (4 –6), with the major problem beingthat these models overexpress mutant amyloid precursor protein (APP) and/or presenilin-1 (PS1) and tau. Of particularnote, MAPT gene mutations are known to cause frontotemporal dementia with Parkinsonism linked to chromosome 17(FTDP-17), but not AD, thus calling into question the relevanceof using mutant tau-overexpressing mice in AD research. Nevertheless, tau mutations can be considered as relevant tauopa3

The abbreviations used are: AD, Alzheimer’s disease; A␤, amyloid ␤; APP,
amyloid precursor protein; FTDP-17, frontotemporal dementia with Parkinsonism linked to chromosome 17; KI, knock-in; dKI, double knock-in;
LLPS, the liquid–liquid phase separation; PS1, presenilin-1; NFT, neurofibrillary tangle; 3R and 4R, three- and four-repeat, respectively; GuHCl, guanidine HCl; KO, knockout; ANOVA, analysis of variance; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

12754 J. Biol. Chem. (2019) 294(34) 12754 –12765© 2019 Saito et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.

Downloaded from http://www.jbc.org/ at Nagoya City University Library on August 27, 2019

X Takashi Saito‡§1, Naomi Mihira‡, Yukio Matsuba‡, Hiroki Sasaguri‡, X Shoko Hashimoto‡, Sneha Narasimhan¶,
Bin Zhang¶, Shigeo Murayama储, Makoto Higuchi**, Virginia M. Y. Lee¶, John Q. Trojanowski¶,and Takaomi C. Saido‡2
From the ‡Laboratory for Proteolytic Neuroscience, RIKEN Center for Brain Science, 2-1 Hirosawa, Wako-city, Saitama 351-0198,
Japan, the §Department of Neuroscience and Pathobiology, Research Institute of Environmental Medicine, Nagoya University,
Nagoya, Aichi 464-8601, Japan, the ¶Department of Pathology and Laboratory Medicine, Institute on Aging and Center for
Neurodegenerative Disease Research, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, the储Department of Neuropathology, Tokyo Metropolitan Geriatric Hospital, 35-2 Sakaecho, Itabashi, Tokyo 173-0015, Japan, and the**Department of Functional Brain Imaging Research, National Institute of Radiological Sciences, National Institutes for Quantum
and Radiological Science and Technology, Chiba 263-8555, Japan

Humanization of murine Mapt gene

Results
Characterization of human MAPT KI mice
Normal adult human brains express six distinct isoforms of
tau generated by alternative splicing (14). They are classified
into 3R-tau and 4R-tau, depending on the number of repeatedmicrotubule-binding domains (3 repeats in 3R and 4 in 4R).
Importantly, NFTs in human AD comprise an equal mixture of
all 3R and 4R tau isoforms (14). In contrast, 4R-tau aggregates
may cause corticobasal degeneration or progressive supranuclear palsy, whereas deposition of 3R-tau aggregates characterize Pick’s disease (14, 16).

The major difference between tau isoforms present in adultmouse and human brains is that the former possesses 4R-tau
only, although they express 3R-tau in development likehumans, whereas normal adult human brains also possess
3R-tau (17). In contrast, almost all existing mouse models of
tauopathy overexpress one of the six possible tau isoforms (18).
These mice, with or without FTDP-17 mutations, do not fully
recapitulate the pathophysiology of tauopathy. In addition,
because the presence of murine tau may hamper human tau
aggregation, models in which endogenous murine tau is deleted
are preferred (19). Ideally, the mouse model of tauopathy
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 (20).
We therefore generated mutant mice, in which the entire
murine Mapt gene has been humanized (MAPT KI mice) byhomologous recombination as described (21). The tau protein
in MAPT KI mice localized to axons (e.g. mossy fibers) in amanner similar to endogenous tau in WT mice (10) (Fig. 1, A
and B). In clear contrast, P301S-tau transgenic mice, which
express human tau protein at 5-fold higher levels than endogenous mouse tau (22) (Fig. 1B), exhibit an abnormal distributionof tau, such as dendritic mislocalization (23) (Fig. 1C). Moreover, the humanization of murine tau did not affect A␤ levels inthe brain (Fig. 1D). These observations suggest that the humantau proteins in MAPT KI mice are functionally, physiologically,and structurally normal and that MAPT KI incorporates correctly into murine neuronal systems.
Humanization of Mapt gene in App KI mice
We next cross-bred MAPT KI mice with AppNL-F andAppNL-G-F mice (5). 6-Month-old AppNL-G-F/MAPT dKI mice
exhibited a significantly higher tau phosphorylation status thansingle MAPT KI mice at Ser-202/Thr-205, Ser-396/Ser-404,
and Ser-422, as recognized by AT8, PHF-1, and anti-pS422
antibodies, respectively (Fig. 2, A and B). Similarly, 6-monthold single AppNL-G-F KI mice also showed greater tau phosphorylation than WT mice in a similar manner (Fig. 2, C and D).
Immunohistochemistry allowed us to detect AT8 signals indystrophic neurites around A␤ plaques, but we observed no
NFTs in single AppNL-G-F KI mice or the dKI mice, where weused P301S-tau Tg and AD brains as positive controls (Fig. 2, E
and F). Humanization of the Mapt gene did not affect A␤-amyloid deposition, neuroinflammation (Fig. 3), or memory in
AppNL-G-F mice (Fig. S1).
At 24 months of age, AppNL-G-F/MAPT dKI mice exhibited
significantly higher tau phosphorylation than single MAPT KI
mice at Ser-202/Thr-205, Ser-396/Ser-404, and Ser-422 (Fig. 4,
A and B). A␤-amyloidosis accelerated phosphorylation ofmurine and human tau proteins to a similar extent in the App
KI and App/MAPT dKI mice, respectively (Fig. 4, C and D),
indicating that murine and human tau proteins behave comparably in terms of A␤-amyloidosis– evoked phosphorylation.
We also observed that A␤-amyloidosis induced more prominent neuroinflammation in these mutant mice (21). Consistently, the single AppNL-G-F KI and AppNL-G-F/MAPT dKI mice
exhibited more dystrophic neurites than the WT and single
MAPT KI mice without NFT formation (Fig. 4, E and F).
J. Biol. Chem. (2019) 294(34) 12754 –12765

12755

Downloaded from http://www.jbc.org/ at Nagoya City University Library on August 27, 2019thy-accelerating factors if they are used in a nonoverexpression
paradigm.
To overcome these drawbacks, we have created novelA␤-amyloidosis models, using a knock-in (KI) strategy andreported the advantages of KI models over the overexpression
models (5, 7). However, we did not observe NFTs in these mice
during their lifespan. One possible explanation for this is thatmurine tau might be incompatible for NFT formation, presumably due to differences in the isoform expression profile
between humans and mice (8, 9). In the present study, we havecreated mutant mice in which the murine Mapt gene, including
all exons and introns, are humanized (MAPT KI mice) to overcome the above concern. These mice express all six isoforms of
tau in a manner similar to that in humans; in contrast, normal
adult mouse brains express only the three four-repeat (4R) tau
isoforms (8). Tau proteins in the KI mice show normal axonal
localization, unlike that in tau-overexpressing mice (10). Whenwe cross-bred the MAPT KI mice with single App KI mice
(AppNL-G-F mice), we confirmed that the double knock-in (dKI)mice exhibited pathological and cognitive features similar to
those of single AppNL-G-F mice. These observations indicatethat human tau in the mouse brain plays physiological andpathological roles essentially identical to those of murine tau,suggesting that tau humanization does not cause any artificial
phenotypes in these mice.
One of the characteristic features of pathological tau is that it
propagates from neuron to neuron in the brain (11). This may
explain aging-associated expansion of tau pathology describedby Braak and Braak (12). Recently, He et al. (13) demonstrated
that presence of the A␤ plaque milieu was linked to accelerated
propagation of pathological tau particularly in association withdystrophic plaque-associated neurite formation. We thus analyzed the difference in tau propagation between WT andhuman MAPT KI mice. We were surprised to see that humanization of murine tau significantly accelerated propagation ofpathological tau in the presence and absence of A␤-amyloidosis. Apparently, the pathological human tau interacted betterwith normal human tau than with murine tau in the human
MAPT KI mice. These results suggest the presence of speciesdefined preference between human versus mouse tau proteins.
The divergence may be attributed to the differences in theamino acid sequences and in the alternative splicing-dependent
isoform expression (14). The MAPT KI mice will serve as avaluable platform for creating a relevant model(s) of FTDP-17
using genome-editing technology (15).

Humanization of murine Mapt gene

AppNL-F mice exhibited a much less aggressive pathology thanthe AppNL-G-F mice (5), but AppNL-F/MAPT dKI mice exhibited
higher tau phosphorylation than single MAPT KI mice (Fig. 5).
Thus, both in AppNL-F and AppNL-G-F mice, A␤-amyloidosis
augmented tau phosphorylation.
Several studies have shown an aberrant increase of exon
10 – containing 4R-tau isoforms in AD and tauopathy cases (24,25). Exon 10 encodes one of the microtubule-binding domains,and its inclusion gives rise to the production of 4R-tau. However, it has been difficult to analyze tau splicing using existingmouse models because of the lack of a suitable model express-

12756 J. Biol. Chem. (2019) 294(34) 12754 –12765

ing all human isoforms without overexpression of tau. To
examine the contribution of A␤ pathology to tau splicing, we
determined the level of each tau isoform in MAPT KI andAppNL-G-F/MAPT dKI mice. The relative expression ratio ofmRNA for 4R-tau/3R-tau was almost the same between aged
MAPT KI mice and AppNL-G-F/MAPT dKI mice, with values of
0.71 ⫾ 0.08 and 0.69 ⫾ 0.11 (n ⫽ 4 each), respectively, obtainedfor each strain (Fig. 4, G and H). The isoform profile of tau
protein remained unaltered in the aged dKI mice (Fig. 4, I and J),
suggesting that A␤-amyloidosis does not directly affect alternative splicing of the tau gene.

Downloaded from http://www.jbc.org/ at Nagoya City University Library on August 27, 2019

Figure 1. Biochemical and pathological properties of tau protein in MAPT KI mice. A, tau expression in MAPT KI mice was immunostained with theanti-tau antibodies Tau13, T49, and Tau5. B, axonal distribution of tau at mossy fiber in MAPT KI mouse brains. Brain sections from MAPT KI mice and
P301S-tau transgenic mice were immunostained with the anti-pan-tau antibody, Tau1. Arrows, mossy fibers. C, localization of tau in MAPT KI brains. Brain
sections from MAPT KI mice were doubly immunostained with Tau5 (red) and anti-MAP2 antibody as a dendritic marker (green). Scale bars, 500 ␮m (A),250 ␮m (B), and 50 ␮m (C). D, A␤ levels in the brains of MAPT KI mice. Biochemical quantification of A␤ in mouse brains. A␤40 and A␤42 in the TS and
GuHCl fractions of cortical tissue from 12-month-old mouse brains were quantified by sandwich ELISA. Data represent mean ⫾ S.E. (error bars) (n ⫽ 3(WT) and n ⫽ 4 (MAPT KI), Student’s t test (TS-A␤40: t(5) ⫽ 0.287, p ⫽ 0.392; TS-A␤42: t(5) ⫽ 0.460, p ⫽ 0.332; GuHCl-A␤40: t(5) ⫽ 0.679, p ⫽ 0.736;
GuHCl-A␤42: t(5) ⫽ 0.121, p ⫽ 0.546)).

Humanization of murine Mapt gene
We previously observed no atrophy in these regions in aged
AppNL-G-F/MAPT dKI mice from 18 to 24 months and detected
only a low level of caspase-3 activation in AppNL-G-F/MAPT dKI

mice as well as in AppNL-G-F KI mice (21). These findings indicate thathumanization of the Mapt gene does not affect neurodegenerative
processes in these mouse models of extensive A␤-amyloidosis.

Downloaded from http://www.jbc.org/ at Nagoya City University Library on August 27, 2019

J. Biol. Chem. (2019) 294(34) 12754 –12765

12757

Humanization of murine Mapt gene

Propagation of AD brain-derived pathological tau proteins in
WT, App KI, MAPT KI, and AppNL-G-F/MAPT dKI mice
Propagation of pathological proteins associated with neurodegeneration, such as tau, A␤, ␣-synuclein, and TDP-43, hasbeen shown to represent the primary mechanism that accounts
for progressive spreading of pathogenic agents (26). Based on
the experimental paradigm described previously (13), we analyzed whether humanization of murine tau protein affects pathological tau propagation in WT and App KI mouse brains(Fig. 6).
The first notable observation is that humanization of murine
tau in WT mice boosted propagation of AD brain-derived pathological tau (Fig. 6A, left panels), indicating that humanizationof the host animal tau affects the transmission of the pathogenic
agents. In addition, the presence of A␤ plaques augmented tau
pathology seeded by the AD brain– derived pathological tau
proteins in single AppNL-G-F KI mice (Fig. 6, A and B) in agreement with the previous report (13). Of particular note,
AppNL-G-F/MAPT dKI mice exhibited greater pathologicalpropagation than AppNL-G-F KI mice (Fig. 6, A and B). In addition, we observed many more AT8-positive tau aggregates indystrophic neurites surrounding A␤ plaques in AppNL-G-F/
MAPT dKI than single AppNL-G-F KI mice (Fig. 6, C and D), but

we did not detect thioflavin S– or Gallyas silver–positive NFT
formation (data not shown). These differences between WT
and MAPT KI mice or App KI and App KI/MAPT dKI mice
were undetectable unless the AD brain– derived pathological
tau was introduced. Our findings probably imply that there
exists a species preference (i.e. mice versus humans) in the
dynamic interactions between the pathological and normal tau
proteins. This may possibly apply to some other pathogenic
proteins, such as ␣-synuclein, associated with neurodegenerative diseases.
The experimental inoculation of the pathological tau protein
derived from AD brain, however, did not evoke NFT formationor neurodegeneration in AppNL-G-F/MAPT dKI mice. Thesefindings suggest that tau propagation in dystrophic neurites islikely an initial stage of tau pathology formation and neurodegeneration, which proceeds over decades in human brains (12).
Because many of the pathogenic mutations in the MAPT genethat cause FTDP-17 are located in the genomic regions corresponding to microtubule-binding domains (27) and becausesome mutations in the exon 10 –intron 10 interface are also
pathogenic by affecting alternative splicing (27), introductionof these mutations into the human MAPT KI mice may lead toa generation of relevant tauopathy models by accelerating the

Figure 2. Pathological changes in 6-month-old of AppNL-G-F/MAPT dKI mice. A and B, status of tau phosphorylation in 6-month-old single MAPT KI andAppNL-G-F/MAPT dKI mice was immunoblotted with the anti-tau antibodies AT8, PHF-1, pS422, and Tau13. Data represent mean ⫾ S.E. (n ⫽ 8 (MAPT KI) and n ⫽8 (AppNL-G-F/MAPT dKI), Student’s t test (AT8: t(14) ⫽ 5.853 (**, p ⫽ 2.1 ⫻ 10⫺5); PHF-1: t(14) ⫽ 2.551 (*, p ⫽ 0.012); pS422: t(14) ⫽ 3.758 (**, p ⫽ 0.001); Tau13:t(14) ⫽ 1.165, p ⫽ 0.868). C and D, status of tau phosphorylation in 6-month-old single AppNL-G-F KI mice. Data represent mean ⫾ S.E. (n ⫽ 7 (WT) and n ⫽ 7(AppNL-G-F KI)); *, p ⬍ 0.05, Student’s t test (AT8: t(12) ⫽ 2.500 (*, p ⫽ 0.014); PHF-1: t(12) ⫽ 6.464 (**, p ⫽ 1.55 ⫻ 10⫺5); pS422: t(12) ⫽ 2.584 (*, p ⫽ 0.012); Tau5:t(12) ⫽ 2.656 (p ⫽ 0.07)). E and F, immunostaining of pathological tau in mouse brains. Brain sections were immunostained with the anti-A␤ antibody, A␤1-X(green), and AT8 (red). Scale bars, 50 ␮m. Data represent mean ⫾ S.E. (error bars) (n ⫽ 8 (AppNL-G-F KI) and n ⫽ 9 (AppNL-G-F/MAPT dKI), where each n reflects one
mouse (with four 4-␮m sections averaged to a single value per mouse)); Student’s t test (AT8: t(15) ⫽ 0.050, p ⫽ 0.481).

12758 J. Biol. Chem. (2019) 294(34) 12754 –12765

Downloaded from http://www.jbc.org/ at Nagoya City University Library on August 27, 2019

Figure 3. Amyloid deposition and neuroinflammation in 6-month-old AppNL-G-F/MAPT dKI mice. A, biochemical quantification of A␤ in mouse brains.
A␤40 and A␤42 in the TS and GuHCl fractions of cortical tissue from 6-month-old mouse brains were quantified by sandwich ELISA. Data represent mean ⫾ S.E.
(error bars) (n ⫽ 3 (AppNL-G-F KI) and n ⫽ 4 (AppNL-G-F/MAPT dKI), Student’s t test (TS-A␤40: t(5) ⫽ 0.168, p ⫽ 0.563; TS-A␤42: t(5) ⫽ 0.408, p ⫽ 0.650; GuHCl-A␤40:
t(5) ⫽ 0.377, p ⫽ 0.361; GuHCl-A␤42: t(5) ⫽ 0.419, p ⫽ 0.346)). B, amyloid depositions and neuroinflammation were detected by triple staining of brains from
6-month-old mice using anti-A␤1-x antibody (82E1) (blue), anti-GFAP antibody (green), and anti-Iba1 antibody (red) as markers of A␤ plaque, astrocytosis, andmicrogliosis, respectively. Scale bars, 100 ␮m.

Humanization of murine Mapt geneDownloaded from http://www.jbc.org/ at Nagoya City University Library on August 27, 2019

12759

J. Biol. Chem. (2019) 294(34) 12754 –12765

Humanization of murine Mapt genepathological processes without depending on the overexpression paradigm.

Discussion
Human tau and murine tau differ from each other not only interms of their primary structure but also in their isoform profile
created by alternative splicing (17). There are six isoforms of tau
in adult human brain and three isoforms in murine brain. This
probably explains why humanization of murine tau resulted inenhanced propagation of pathological human tau not only inthe absence but also in the presence of A␤-amyloidosis. Our
results indicate that humanization of the murine Mapt gene
exerts little effect, if any, on the physiological and pathologicalparameters of WT and single App KI mouse models of preclinical AD (7). This indicates that the humanization of murine tau
does not artificially perturb the structure and function of mousebrain and thus makes the MAPT KI mouse an ideal platformfrom which to study different forms of tauopathy, including
FTDP-17. In this way, the transgenic overexpression paradigm,
which may abolish endogenous gene loci and/or induce artificial phenotypes can be avoided (7, 28).
The MAPT KI mouse model can be applied to the study ofneurodegenerative diseases involving tauopathy in a number ofways. For instance, the mice would be a useful tool for investigating the molecular mechanisms underlying pathogenesis of
Pick’s disease, as the MAPT KI mice exhibited a relatively
higher 3R-tau expression (21). This is because the 3R-tau aggregates accumulate in the brain of Pick’s disease patients (14),whereas the adult mouse brain only expresses 4R-tau. Consistently, some mutations in the MAPT gene that affect the alternative splicing cause FTDP-17 (27), making the murine Maptgene a less attractive target for gene manipulation. Thus, introduction of mutations associated with FTDP-17 (27) into theexons and introns of the MAPT gene in the App/MAPT dKI

Figure 4. Pathological changes at 24 months old of AppNL-G-F/MAPT dKI mice. A and B, status of tau phosphorylation in 24-month-old single MAPT KI andAppNL-G-F/MAPT dKI mice were immunoblotted with the anti-tau antibodies, AT8, PHF-1, pS422, and Tau13. Data represent mean ⫾ S.E. (error bars) (n ⫽ 8 (MAPT
KI) and n ⫽ 9 (AppNL-G-F/MAPT dKI), Student’s t test (AT8: t(15) ⫽ 5.126 (**, p ⫽ 6.21 ⫻ 10⫺5); PHF-1: t(15) ⫽ 2.999 (**, p ⫽ 0.0045); pS422: t(15) ⫽ 3.521 (**, p ⫽0.002); Tau13: t(15) ⫽ 0.637 (p ⫽ 0.733))). C and D, phosphorylation of murine tau in 24-month-old single AppNL-G-F KI mice. Data represent mean ⫾ S.E. (n ⫽ 3(WT) and n ⫽ 4 (AppNL-G-F KI) (**, p ⬍ 0.01), Student’s t test (AT8: t(5) ⫽ 4.239, p ⫽ 0.0041; PHF-1: t(5) ⫽ 3.396, p ⫽ 0.0097; pS422: t(5) ⫽ 1.572, p ⫽ 0.912; Tau5:t(5) ⫽ 1.220, p ⫽ 0.861)). E and F, immunostaining of pathological tau in mouse brains. Brain sections were immunostained with A␤1-X (green), and AT8 (red).
Scale bars, 50 ␮m. Data represent mean ⫾ S.E. (n ⫽ 8 (AppNL-G-F KI) and n ⫽ 8 (AppNL-G-F/MAPT dKI), where each n reflects one mouse (with four 4-␮m sections
averaged to a single value per mouse); Student’s t test (AT8: t(14) ⫽ 0.095, p ⫽ 0.537). G and H, the relative ratio of 4R-tau/3R-tau in aged mice was calculated
by semiquantitative RT-PCR. Data represent mean ⫾ S.E. (n ⫽ 4 each; Student’s t test: t(6) ⫽ 0.122, p ⫽ 0.547). I and J, Tau isoforms in aged AppNL-G-F/MAPT dKI
mouse brains. Dephosphorylated biochemical extracts of the brains were subjected to Western blot analysis using Tau13. The relative expression of tau
isoforms in AppNL-G-F/MAPT dKI mouse brains was calculated by densitometric analysis. Data represent mean ⫾ S.E. (n ⫽ 5 (MAPT KI) and n ⫽ 6 (AppNL-G-F/MAPTdKI); Student’s t test (0N3R: t(9) ⫽ 0.211, p ⫽ 0.581; 0N4R: t(9) ⫽ 1.157, p ⫽ 0.139; 1N3R: t(9) ⫽ 1.146, p ⫽ 0.141; 1N4R: t(9) ⫽ 0.522, p ⫽ 0.693; 2N3R: t(9) ⫽ 0.897,
p ⫽ 0.803; 2N4R: t(9) ⫽ 0.407, p ⫽ 0.347)).

12760 J. Biol. Chem. (2019) 294(34) 12754 –12765

Downloaded from http://www.jbc.org/ at Nagoya City University Library on August 27, 2019

Figure 5. Amyloid deposition and neuroinflammation in 24-month-old AppNL-F/MAPT dKI mice. A and B, status of tau phosphorylation in 24-month-old
single MAPT KI and AppNL-F/MAPT dKI mice was immunoblotted with the anti-tau antibodies AT8, PHF-1, pS422, and Tau13. Data represent mean ⫾ S.E. (errorbars) (n ⫽ 3 (MAPT KI) and n ⫽ 4 (AppNL-F/MAPT dKI), Student’s t test (AT8: t(5) ⫽ 5.108 (**, p ⫽ 0.0019); PHF-1: t(5) ⫽ 2.473 (*, p ⫽ 0.028); pS422: t(5) ⫽ 0.004, p ⫽0.501; Tau13: t(5) ⫽ 1.220, p ⫽ 0.861)). C, immunostaining of pathological tau in 24-month-old AppNL-F/MAPT dKI mouse brains. Brain sections were immunostained with A␤1-X (green) and AT8 (red). D, amyloid depositions and neuroinflammation were detected by triple staining of 24-month-old mice using 82E1
(blue), anti-GFAP antibody (green), and anti-Iba1 antibody (red) as markers of A␤ plaque, astrocytosis, and microgliosis, respectively. Scale bars, 50 ␮m (C) and100 ␮m (D).

Humanization of murine Mapt geneDownloaded from http://www.jbc.org/ at Nagoya City University Library on August 27, 2019

12761

J. Biol. Chem. (2019) 294(34) 12754 –12765

Humanization of murine Mapt gene
yloidosis, neuroinflammation, etc. could induce the kinetic
changes represented by the liquid–liquid phase separation(LLPS) of tau (35). Recently, Boyko et al. (36) demonstrated thattau LLPS was largely regulated by intermolecular electrostaticinteractions of tau between negatively charged N terminus andpositively charged middle/C terminus, and various posttranslational modification of tau could modulate tau LLPS. Although
the amino acid sequence of middle to C terminus of murine tau
is identical to human tau, the N terminus of murine tau is relatively different from human tau, suggesting that difference ofN-terminal motif of tau between murine and human mightcause tau LLPS-related species preference of tau (37).
Taken together, our results indicate that human tau protein
expressed in the mouse brain plays physiological and pathological roles that are essentially identical to those of murine tau, as
the tau humanization did not cause any artificial phenotypes inthese mice. At the same time, tau humanization led to significantly amplified interaction between the AD brain– derived
pathological tau and normal tau residing in the host brain, indicating that human and murine tau proteins are not 100% identical to each other in pathological terms, whereas their physiological properties are essentially indistinguishable. On the basisof these facts and assumptions, we propose that single MAPT
KI mice as well as App/MAPT dKI mice should serve as usefulmodels for further elucidating the molecular mechanisms
underlying the pathogenesis of tauopathies represented by AD
and for performing preclinical studies of these disorders
accompanying tau pathology.

Experimental procedures
Mutant mice
AppNL-G-F mice, which include the Swedish, Arctic, and Iberian mutations, and MAPT KI mice were maintained as
described previously (5, 21). P301S-tau transgenic mice (PS19)
were described previously (22) and then backcrossed to
C57BL/6J mice. Tau-deficient mice were obtained from theJackson Laboratory (stock no. 007251). All mice used in theexperiments were on the C57BL/6J background. We used
the male mice for the 6-month-old analysis and the female micefor 24-month-old analysis in the present study. All animal
experiments were carried out according to the RIKEN Brain
Science Institute’s guidelines for animal experimentation.
ELISA of A␤
Soluble materials from mouse cortical hemispheres were dissolved in Tris-HCl– buffered saline (TS fraction) and insoluble
materials in guanidine HCl solution (GuHCl fraction) as

Figure 6. Propagation of AD brain– derived pathological tau in mouse brains. A and B, immunostaining of pathological tau in mouse brains. Brain sections
were immunostained with AT8 (red). Scale bars, 1 mm. The area of AT8 signal in the mice was quantified by image analysis. Data represent mean ⫾ S.E. (errorbars) (n ⫽ 6 (WT), n ⫽ 7 (AppNL-G-F KI), n ⫽ 5 (MAPT KI), and n ⫽ 5 (AppNL-G-F/MAPT dKI), where each n reflects one mouse (with four 4-␮m sections averaged toa single value per mouse); two-way ANOVA with post hoc Scheffe’s F test (ipsilateral of cortex: F(1,22) ⫽ 5.94 (*, p ⫽ 0.025); ipsilateral of hippocampus: F(1,22) ⫽18.42 (**, p ⫽ 0.0004); ipsilateral of subcortical region: F(1,22) ⫽ 0.474 (p ⫽ 0.499); contralateral of cortex: F(1,22) ⫽ 5.82 (*, p ⫽ 0.026); contralateral ofhippocampus: F(1,22) ⫽ 30.93 (**, p ⫽ 2.3 ⫻ 10⫺5); contralateral of subcortical region: F(1,22) ⫽ 0.164 (p ⫽ 0.690)). C and D, immunostaining of pathological tau
in mouse brains. Brain sections were immunostained with A␤1-X (green) and AT8 (red). Contralateral cortex images were shown in C. Scale bars, 50 ␮m. The ratioof AT8 signal/A␤1-X signal was calculated by image analysis. Data represent mean ⫾ S.E. (n ⫽ 7 (AppNL-G-F KI) and n ⫽ 5 (AppNL-G-F/MAPT dKI), where each n
reflects one mouse (with four 4-␮m sections averaged to a single value per mouse); Student’s t test (ipsilateral of cortex: t(10) ⫽ 2.558 (*, p ⫽ 0.014); ipsilateralof hippocampus: t(10) ⫽ 3.866 (**, p ⫽ 0.002); ipsilateral of subcortical region: t(10) ⫽ 0.192 (p ⫽ 0.574); contralateral of cortex: t(10) ⫽ 1.971 (*, p ⫽ 0.039);
contralateral of hippocampus: t(10) ⫽ 5.722 (**, p ⫽ 9.6 ⫻ 10⫺5); contralateral of subcortical region: t(10) ⫽ 0.009 (p ⫽ 0.497)). After injection of humanbrain– derived pathological tau, some of the mice died as follows: 2 of 8 WT mice; 3 of 8 MAPT KI mice; 1 of 8 AppNL-G-F KI mice; 2 of 7 AppNL-G-F/MAPT dKI mice.

12762 J. Biol. Chem. (2019) 294(34) 12754 –12765

Downloaded from http://www.jbc.org/ at Nagoya City University Library on August 27, 2019

mice by genome editing (15) may lead to generation of a pertinent model of AD if the mutations in the coding regions of the
MAPT gene mimic the pathogenic effect of tau hyperphosphorylation, which may contribute to detachment of the microtubule-bound tau. On the other hand, the molecular mechanisms
underlying pathogenesis of four-repeat tauopathy (e.g. progressive supranuclear palsy or corticobasal degeneration) are still
elusive (14). Despite such patients also expressing three-repeattau, only four-repeat tau forms aggregate at a specific region in
the brains of patients with the diseases. For further investigation, the MAPT KI mice would be needed.
Many of the tau propagation studies to date have used transgenic mice overexpressing mutant human tau protein (29). We
have shown here that the pathological tau protein from AD
brain interacts better with human tau than with murine tau invivo. This may signify that the presence of murine tau slows
human tau-seeded tau propagation in the brain. Thus, humanized tau models with/without A␤-amyloidosis are advantageous resources for the study of pathological tau propagation.
Tau protein resembles prion protein in its ability to propagate(30). The common underlying mechanisms likely involve conformational changes caused by the pathogenic forms as templates (31). Propagation of prion, however, is generally much
faster than that of tau (31), presumably because prion protein isa glycosylphosphatidylinositol-anchored protein with ␤-sheet
structure unlike tau protein (32).
The App/MAPT dKI mice showed a higher tau phosphorylation status than the single MAPT KI mice but lacked apparent
tauopathy and neurodegeneration, in a manner essentially
identical to that of single App KI mice, despite massive A␤
pathology and neuroinflammation. These observations, consistent with the chronology of clinical AD, in which A␤-amyloidosis precedes tauopathy and neurodegeneration by more thana decade, imply the presence of mechanism(s) that protect thebrain from tauopathy and neurodegeneration. Aggravating A␤
plaque-associated factors may also exist that promote tauopathy and neurodegeneration. The balance between aggravating
and protective factors may determine the timing of subsequent
pathological events, meaning that identifying such factors may
open new approaches for the development of preventive medications for neurodegenerative diseases.
Whereas the disease specificity of tauopathy is relatively
broad, that of A␤-amyloidosis is narrow (33), implying that thecausal and mechanistic specificity of the former is broader thanthat of the latter. Bearing in mind that essentially all familial AD
mutations cause both A␤-amyloidosis and tauopathy (34), we
propose that long-term pathological stresses caused by A␤-am-Humanization of murine Mapt genedescribed previously (38). A␤X-40 and A␤X-42 levels in brain
extracts were quantified using an A␤ ELISA kit (Wako, Tokyo,
Japan) according to the manufacturer’s instructions. A␤X-40
and A␤X-42 carrying the Arctic mutation were quantified basedon standard curves using synthetic human Arctic A␤ peptides(Peptide Institute, Osaka, Japan) as described previously (5).
Western blot analysis of tau

Immunohistochemical studies
Paraffin-embedded sections were immunostained using
antibodies specific to the N termini of A␤ (A␤1-X) (40) and 82E1
(catalog no. 10323, IBL, Gunma, Japan), to Iba1 (Wako), to
GFAP (catalog no. MAB3402, Merck Millipore, Darmstadt,
Germany), to tau (pan-tau–specific antibody, Tau1 (catalog no.
MAB3420, Merck Millipore, Darmstadt, Germany); murine
tau-specific antibody, T49 (catalog no. MABN827, Merck Millipore), Tau5, Tau13), to phosphorylated tau (AT8), and to
MAP2 as a dendritic marker (catalog no. M121, Leinco Technologies, St. Louis, MO). To show the specificity of antibodies,brain sections derived from Tau-KO mice were used as a negative control. When necessary, tyramide signal amplification(PerkinElmer Life Sciences) was used as described previously

RNA extraction and semiquantitative RT-PCR
Total RNA was extracted from brain samples using RNAiso
Plus (Takara, Shiga, Japan) according to the manufacturer’s
instructions. We performed semiquantitative RT-PCR for
detection of both 3R- and 4R-tau mRNA as described previously (42) with minor modifications. The primer pair used for
tau was 5⬘-AAGTCGCCGTCTTCCGCCAAG-3⬘ and 5⬘-GTCCAGGGACCCAATCTTCGA-3⬘, and that for GAPDH was
5⬘-CCATGGCACCGTCAAGGCTGA-3⬘ and 5⬘-GCCAGTAGAGGCAGGGATGAT-3⬘. PCR products of 288 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 tomortgage the linearity of the products, and the result was
decided by 26 cycles. Band intensity was calibrated using
ImageJ software.
Tau propagation analysis
AD-tau extracted from an AD patient’s brain, as described
previously (43), was prepared in the laboratory of V. M. Y. L.
and J. Q. T. In this study, AD-tau was provided from the samebatch of extracts (43). We injected AD brain– derived tau into
the mouse brain by stereotaxic injection as described previously(13, 44). The mice (19-month-old males) were deeply anesthetized and immobilized in a stereotaxic frame (Muromachi). AD
brain– derived tau was injected in dorsal hippocampus andoverlying cortex in the right hemisphere (bregma: ⫺2.5 mm;lateral ⫺2 mm; depth ⫺1.7 mm and ⫺0.5 mm from brain surface) with a total amount of 2 ␮g of AD-tau using a Hamilton
syringe as described (44). After 3 months, we collected thebrains, which were fixed in 4% paraformaldehyde, embedded inparaffin, and sectioned into 4-␮m thickness, as described previously (13), and performed immunohistochemical analysis.
Protocols involving the use of human tissue were approved by
the Institutional Review Board of the RIKEN Brain Science
Institute and University of Pennsylvania, respectively. All of thehuman materials were taken in accordance with the Declaration of Helsinki.
AD brain sections
AD brain sections were used as a positive control of tauopathy in all studies. Paraffin-embedded brain (neocortical andhippocampal region) sections from normal humans and AD
patients were purchased from Wako. Protocols involving theuse of human tissue were approved by the institutional review
board of the RIKEN Brain Science Institute.
Statistics
All data are shown as the mean ⫾ S.E. For comparison
between two groups, statistical analysis was performed byStudent’s t test. For comparisons among three or more groups,
J. Biol. Chem. (2019) 294(34) 12754 –12765

12763

Downloaded from http://www.jbc.org/ at Nagoya City University Library on August 27, 2019

The cortical brain tissue samples were homogenized in lysis
buffer (50 mM Tris-HCl (pH 8.0) containing 150 mM NaCl, 50
mM EDTA, 1% Triton X-100, and protease/phosphatase inhibitor mixture) and centrifuged at 20,000 ⫻ g for 20 min at 4 °C, as
described previously (39). Brain extracts as the total fraction (15␮g of protein/lane) were subjected to Western blotting using
anti-Tau antibodies (both mouse and human tau-specific antibody, Tau5 (catalog no. AHB0042, Thermo Fisher Scientific;
epitope: amino acid residues 210 –214 on the longest isoform of
human tau); human tau-specific antibody, Tau13 (catalog no.
sc-21796, Santa Cruz Biotechnology, Inc.; epitope: amino acidresidues 2–18 on the longest isoform of human tau); 3R-tau–
specific antibody, RD3 (catalog no. 05-803, Merck Millipore;
epitope: a junction-flanking sequence coded by adjacent exons
9 and 11 in the absence of exon 10); 4R-tau–specific antibody,
RD4 (catalog no. MBS604301, MyBiosource, San Diego, CA;epitope: a junction-flanking sequence coded by adjacent exons
9 and 10 of Tau)), anti-phospho-Tau antibodies (AT8 (catalog
no. 90206, Innogenetics, Ghent, Belgium; epitope: phosphorylated serine 202 and threonine 205); PHF-1 (kindly provided
from Peter Davies, Albert Einstein College of Medicine;
epitope: phosphorylated serine 396 and 404); pS422 (catalog no.
ab62639, Abcam, Cambridge, UK; epitope: phosphorylated serine 422)), and internal controls (anti-␤-tubulin antibody (catalog no. ab11312, Abcam) and anti-GAPDH antibody (catalog
no. 2275-PC-100, TREVIGEN, Gaithersburg, MD)). To show
the specificity of antibodies, brain extract derived from
Tau-KO mice was used as a negative control. For analysis of theisoform pattern of tau, brain extracts were dephosphorylatedwith ␭-phosphatase (catalog no. sc-200312A, Santa Cruz Biotechnology) according to the manufacturer’s instructions. All
six isoforms of human Tau ladder were purchased from rPeptide. Each set of experiments was repeated at least three times
to confirm the results. Band intensity was determined with a
LAS4000 densitometer (Fujifilm, Tokyo, Japan).

(41). We performed antigen retrieval by autoclave (121 °C for
5 min).
We quantified the immunoreactive areas using MetaMorphimaging software (Universal Imaging Corp., West Chester, PA)
as described previously (41). To reduce the variation of results
across tissue sections, we used the average of data from at least
four sections per mouse.

Humanization of murine Mapt gene
two-way analysis of variance (ANOVA) or repeated measures
ANOVA followed by a post hoc test (Scheffe’s F test) were used.
Normality was tested using Statcel 3 (add-in software forMicrosoft Excel). Data were collected and processed in a randomized and blinded manner. No statistical methods were used
to predetermine sample size, but our sample sizes are similar to
those generally employed in the field.
Author contributions—T. S., T. C. S., V. M. Y. L., and J. Q. T. designed
this study and wrote the manuscript. T. S., Y. M., and N. M. performed animal experiments. S. N., B. Z., V. M. Y. L., and J. Q. T. provided AD brain-derived pathological tau and jointly discussed theresults. S. M. provided human brain tissues. T. S., H. S., S. H., M. H.,
and T. C. S. jointly analyzed the data.

References
1. Bateman, R. J., Xiong, C., Benzinger, T. L., Fagan, A. M., Goate, A., Fox,
N. C., Marcus, D. S., Cairns, N. J., Xie, X., Blazey, T. M., Holtzman, D. M.,
Santacruz, A., Buckles, V., Oliver, A., Moulder, K., et al. (2012) Clinical andbiomarker changes in dominantly inherited Alzheimer’s disease. N. Engl.
J. Med. 367, 795– 804 CrossRef Medline
2. Selkoe, D. J., and Hardy, J. (2016) The amyloid hypothesis of Alzheimer’s
disease at 25 years. EMBO Mol. Med. 8, 595– 608 CrossRef Medline
3. Robinson, J. L., Lee, E. B., Xie, S. X., Rennert, L., Suh, E., Bredenberg, C.,
Caswell, C., Van Deerlin, V. M., Yan, N., Yousef, A., Hurtig, H. I., Siderowf,A., Grossman, M., McMillan, C. T., Miller, B., et al. (2018) Neurodegenerative disease concomitant proteinopathies are prevalent, age-relatedand APOE4-associated. Brain 141, 2181–2193 CrossRef Medline
4. Higuchi, M., Iwata, N., Matsuba, Y., Takano, J., Suemoto, T., Maeda, J., Ji,
B., Ono, M., Staufenbiel, M., Suhara, T., and Saido, T. C. (2012) Mechanistic involvement of the calpain-calpastatin system in Alzheimer neuropathology. FASEB J. 26, 1204 –1217 CrossRef Medline
5. Saito, T., Matsuba, Y., Mihira, N., Takano, J., Nilsson, P., Itohara, S., Iwata,N., and Saido, T. C. (2014) Single App knock-in mouse models of Alzheimer’s disease. Nat. Neurosci. 17, 661– 663 CrossRef Medline
6. Saito, T., Matsuba, Y., Yamazaki, N., Hashimoto, S., and Saido, T. C. (2016)
Calpain activation in Alzheimer’s model mice is an artifact of APP andpresenilin overexpression. J. Neurosci. 36, 9933–9936 CrossRef Medline
7. Sasaguri, H., Nilsson, P., Hashimoto, S., Nagata, K., Saito, T., De Strooper,
B., Hardy, J., Vassar, R., Winblad, B., and Saido, T. C. (2017) APP mousemodels for Alzheimer’s disease preclinical studies. EMBO J. 36,
2473–2487 CrossRef Medline
8. Goedert, M., Spillantini, M. G., Jakes, R., Rutherford, D., and Crowther,R. A. (1989) Multiple isoforms of human microtubule-associated proteintau: sequences and localization in neurofibrillary tangles of Alzheimer’s
disease. Neuron 3, 519 –526 CrossRef Medline
9. Götz, J., Probst, A., Spillantini, M. G., Schäfer, T., Jakes, R., Bürki, K., andGoedert, M. (1995) Somatodendritic localization and hyperphosphorylation of tau protein in transgenic mice expressing the longest human brain
tau isoform. EMBO J. 14, 1304 –1313 CrossRef Medline

12764 J. Biol. Chem. (2019) 294(34) 12754 –12765

Downloaded from http://www.jbc.org/ at Nagoya City University Library on August 27, 2019

Acknowledgments—We thank Yoshitsugu Shitaka, Mitsuyuki Matsumoto, Yasuyuki Mitani, Hiroshi Yamada, Shinichi Miyake Mayuko
Okabe, Mayako Yamazaki, and Ni Keni (Astellas Pharma, Inc.,Tokyo, Japan) for valuable discussion. We also thank Naoko Kamanoand Mika Takahashi (RIKEN Center for Brain Science) for technicalassistance. We are grateful to the RIKEN Center for Brain Science
Research Resource Division for technical support in breeding of themice. We also thank Per Nilsson (Karolinska Institute) and Kaori
Tsukakoshi (Tokyo University of Agriculture and Technology) for valuable discussion. We also thank Maho Morishima (Tokyo Metropolitan Geriatric Hospital) for coordinating brain materials. Naruhiko
Sahara (National Institutes for Quantum and Radiological Scienceand Technology, Chiba, Japan) provided helpful suggestions.

10. Kubo, A., Misonou, H., Matsuyama, M., Nomori, A., Wada-Kakuda, S.,
Takashima, A., Kawata, M., Murayama, S., Ihara, Y., and Miyasaka, T.
(2019) Distribution of endogenous normal tau in the mouse brain.
J. Comp. Neurol. 527, 985–998 CrossRef Medline
11. Spillantini, M. G., and Goedert, M. (2013) Tau pathology and neurodegeneration. Lancet Neurol. 12, 609 – 622 CrossRef Medline
12. Braak, H., and Braak, E. (1991) Neuropathological stageing of Alzheimerrelated changes. Acta Neuropathol. 82, 239 –259 CrossRef Medline
13. He, Z., Guo, J. L., McBride, J. D., Narasimhan, S., Kim, H., Changolkar, L.,
Zhang, B., Gathagan, R. J., Yue, C., Dengler, C., Stieber, A., Nitla, M.,
Coulter, D. A., Abel, T., Brunden, K. R., et al. (2018) Amyloid-␤ plaquesenhance Alzheimer’s brain tau-seeded pathologies by facilitating neuritic
plaque tau aggregation. Nat. Med. 24, 29 –38 CrossRef Medline
14. Buée, L., Bussière, T., Buée-Scherrer, V., Delacourte, A., and Hof, P. R.
(2000) Tau protein isoforms, phosphorylation and role in neurodegenerative disorders. Brain Res. Brain Res. Rev. 33, 95–130 CrossRef Medline
15. Sasaguri, H., Nagata, K., Sekiguchi, M., Fujioka, R., Matsuba, Y.,
Hashimoto, S., Sato, K., Kurup, D., Yokota, T., and Saido, T. C. (2018)
Introduction of pathogenic mutations into the mouse Psen1 gene by Base
Editor and Target-AID. Nat. Commun. 9, 2892 CrossRef Medline
16. Hong, M., Zhukareva, V., Vogelsberg-Ragaglia, V., Wszolek, Z., Reed, L.,
Miller, B. I., Geschwind, D. H., Bird, T. D., McKeel, D., Goate, A., Morris,
J. C., Wilhelmsen, K. C., Schellenberg, G. D., Trojanowski, J. Q., and Lee,V. M. (1998) Mutation-specific functional impairments in distinct tau
isoforms of hereditary FTDP-17. Science 282, 1914 –1917 CrossRef
Medline
17. Andreadis, A. (2005) Tau gene alternative splicing: expression patterns,
regulation and modulation of function in normal brain and neurodegenerative diseases. Biochim. Biophys. Acta 1739, 91–103 CrossRef Medline
18. Jankowsky, J. L., and Zheng, H. (2017) Practical considerations for choosing a mouse model of Alzheimer’s disease. Mol. Neurodegener. 12, 89
CrossRef Medline
19. Ando, K., Leroy, K., Heraud, C., Kabova, A., Yilmaz, Z., Authelet, M.,
Suain, V., De Decker, R., and Brion, J. P. (2010) Deletion of murine tau geneincreases tau aggregation in a human mutant tau transgenic mouse model.
Biochem. Soc. Trans. 38, 1001–1005 CrossRef Medline
20. Chen, Y., Liu, L., Li, M., Yao, E., Hao, J., Dong, Y., Zheng, X., and Liu, X.
(2018) Expression of human Tau40 in the medial entorhinal cortex impairs synaptic plasticity and associated cognitive functions in mice.
Biochem. Biophys. Res. Commun. 496, 1006 –1012 CrossRef Medline
21. Hashimoto, S., Matsuba, Y., Kamano, N., Mihira, N., Sahara, N., Takano,J., Muramatsu, S. I., Saido, T. C., and Saito, T. (2019) Tau binding protein
CAPON induces tau aggregation and neurodegeneration. Nat. Commun.
10, 2394 CrossRef Medline
22. Yoshiyama, Y., Higuchi, M., Zhang, B., Huang, S. M., Iwata, N., Saido,
T. C., Maeda, J., Suhara, T., Trojanowski, J. Q., and Lee, V. M. (2007)
Synapse loss and microglial activation precede tangles in a P301S tauopathy mouse model. Neuron 53, 337–351 CrossRef Medline
23. Hoover, B. R., Reed, M. N., Su, J., Penrod, R. D., Kotilinek, L. A., Grant,
M. K., Pitstick, R., Carlson, G. A., Lanier, L. M., Yuan, L. L., Ashe, K. H., andLiao, D. (2010) Tau mislocalization to dendritic spines mediates synaptic
dysfunction independently of neurodegeneration. Neuron 68, 1067–1081
CrossRef Medline
24. Glatz, D. C., Rujescu, D., Tang, Y., Berendt, F. J., Hartmann, A. M., Faltraco, F., Rosenberg, C., Hulette, C., Jellinger, K., Hampel, H., Riederer, P.,Möller, H. J., Andreadis, A., Henkel, K., and Stamm, S. (2006) The alternative splicing of tau exon 10 and its regulatory proteins CLK2 and TRA2BETA1 changes in sporadic Alzheimer’s disease. J. Neurochem. 96,
635– 644 CrossRef Medline
25. Schoch, K. M., DeVos, S. L., Miller, R. L., Chun, S. J., Norrbom, M.,
Wozniak, D. F., Dawson, H. N., Bennett, C. F., Rigo, F., and Miller, T. M.
(2016) Increased 4R-Tau induces pathological changes in a human-Tau
mouse model. Neuron 90, 941–947 CrossRef Medline
26. Luk, K. C., Covell, D. J., Kehm, V. M., Zhang, B., Song, I. Y., Byrne, M. D.,
Pitkin, R. M., Decker, S. C., Trojanowski, J. Q., and Lee, V. M. (2016)
Molecular and biological compatibility with host ␣-synuclein influences
fibril pathogenicity. Cell Rep. 16, 3373–3387 CrossRef MedlineHumanization of murine Mapt gene
37. Stefanoska, K., Volkerling, A., Bertz, J., Poljak, A., Ke, Y. D., Ittner, L. M.,
and Ittner, A. (2018) An N-terminal motif unique to primate tau enables
differential protein–protein interactions. J. Biol. Chem. 293, 3710 –3719
CrossRef Medline
38. Iwata, N., Mizukami, H., Shirotani, K., Takaki, Y., Muramatsu, S., Lu,
B., Gerard, N. P., Gerard, C., Ozawa, K., and Saido, T. C. (2004) Presynaptic localization of neprilysin contributes to efficient clearance ofamyloid-␤ peptide in mouse brain. J. Neurosci. 24, 991–998 CrossRef
Medline39. Higuchi, M., Tomioka, M., Takano, J., Shirotani, K., Iwata, N., Masumoto,H., Maki, M., Itohara, S., and Saido, T. C. (2005) Distinct mechanistic rolesof calpain and caspase activation in neurodegeneration as revealed in miceoverexpressing their specific inhibitors. J. Biol. Chem. 280, 15229 –15237
CrossRef Medline
40. Saido, T. C., Iwatsubo, T., Mann, D. M., Shimada, H., Ihara, Y., and Kawashima, S. (1995) Dominant and differential deposition of distinct ␤-amyloid peptide species, A␤ N3(pE), in senile plaques. Neuron 14, 457– 466
CrossRef Medline
41. Enya, M., Morishima-Kawashima, M., Yoshimura, M., Shinkai, Y., Kusui,K., Khan, K., Games, D., Schenk, D., Sugihara, S., Yamaguchi, H., andIhara, Y. (1999) Appearance of sodium dodecyl sulfate-stable amyloid
␤-protein (A␤) dimer in the cortex during aging. Am. J. Pathol. 154,
271–279 CrossRef Medline
42. Iovino, M., Patani, R., Watts, C., Chandran, S., and Spillantini, M. G.
(2010) Human stem cell-derived neurons: a system to study human taufunction and dysfunction. PLoS One 5, e13947 CrossRef Medline
43. Guo, J. L., Narasimhan, S., Changolkar, L., He, Z., Stieber, A., Zhang, B.,
Gathagan, R. J., Iba, M., McBride, J. D., Trojanowski, J. Q., and Lee, V. M.
(2016) Unique pathological tau conformers from Alzheimer’s brainstransmit tau pathology in nontransgenic mice. J. Exp. Med. 213,
2635–2654 CrossRef Medline
44. Narasimhan, S., and Lee, V. M. Y. (2017) The use of mouse models to study
cell-to-cell transmission of pathological tau. Methods Cell Biol. 141,
287–305 CrossRef Medline

J. Biol. Chem. (2019) 294(34) 12754 –12765

12765

Downloaded from http://www.jbc.org/ at Nagoya City University Library on August 27, 2019

27. Ghetti, B., Oblak, A. L., Boeve, B. F., Johnson, K. A., Dickerson, B. C., andGoedert, M. (2015) Invited review: frontotemporal dementia caused by
microtubule-associated protein tau gene (MAPT) mutations: a chameleon for neuropathology and neuroimaging. Neuropathol. Appl. Neurobiol. 41, 24 – 46 CrossRef Medline
28. Goodwin, L. O., Splinter, E., Davis, T. L., Urban, R., He, H., Braun, R. E.,
Chesler, E. J., Kumar, V., van Min, M., Ndukum, J., Philip, V. M., Reinholdt,
L. G., Svenson, K., White, J. K., Sasner, M., Lutz, C., and Murray, S. A.
(2019) Large-scale discovery of mouse transgenic integration sites reveals
frequent structural variation and insertional mutagenesis. Genome Res.
29, 494 –505 CrossRef Medline
29. Goedert, M., Eisenberg, D. S., and Crowther, R. A. (2017) Propagation of
tau aggregates and neurodegeneration. Annu. Rev. Neurosci. 40, 189 –210
CrossRef Medline
30. Kumar, H., and Udgaonkar, J. B. (2019) Mechanistic approaches to understand the prion-like propagation of aggregates of the human tau protein.
Biochim. Biophys. Acta Proteins Proteom. CrossRef Medline
31. Vasili, E., Dominguez-Meijide, A., and Outeiro, T. F. (2019) Spreading of
␣-synuclein and Tau: a systematic comparison of the mechanisms involved. Front. Mol. Neurosci. 12, 107 CrossRef Medline
32. Puig, B., Altmeppen, H., and Glatzel, M. (2014) The GPI-anchoring of PrP:
implications in sorting and pathogenesis. Prion 8, 11–18 CrossRef Medline
33. Wolfe, M. S. (2012) The role of tau in neurodegenerative diseases and its
potential as a therapeutic target. Scientifica (Cairo) 2012, 796024
CrossRef Medline
34. Lee, V. M., and Trojanowski, J. Q. (1999) Neurodegenerative tauopathies:human disease and transgenic mouse models. Neuron 24, 507–510
CrossRef Medline
35. Ambadipudi, S., Biernat, J., Riedel, D., Mandelkow, E., and Zweckstetter,
M. (2017) Liquid-liquid phase separation of the microtubule-binding repeats of the Alzheimer-related protein Tau. Nat. Commun. 8, 275
CrossRef Medline
36. Boyko, S., Qi, X., Chen, T.-H., Surewicz, K., and Surewicz, W. K. (2019)
Liquid–liquid phase separation of tau protein: the crucial role of electrostatic interactions. J. Biol. Chem. 274, 11054 –11059 CrossRef MedlineHumanization of the entire murine Mapt gene provides a murine model ofpathological human tau propagation
Takashi Saito, Naomi Mihira, Yukio Matsuba, Hiroki Sasaguri, Shoko Hashimoto,
Sneha Narasimhan, Bin Zhang, Shigeo Murayama, Makoto Higuchi, Virginia M. Y.
Lee, John Q. Trojanowski and Takaomi C. Saido
J. Biol. Chem. 2019, 294:12754-12765.
doi: 10.1074/jbc.RA119.009487 originally published online July 4, 2019

Access the most updated version of this article at doi: 10.1074/jbc.RA119.009487

Click here to choose from all of JBC's e-mail alerts
This article cites 44 references, 10 of which can be accessed free at
http://www.jbc.org/content/294/34/12754.full.html#ref-list-1

Downloaded from http://www.jbc.org/ at Nagoya City University Library on August 27, 2019

Alerts:• When this article is cited• When a correction for this article is posted

Supporting Information

Humanization of the entire murine Mapt gene provides a murine modelof pathological human tau propagation

Takashi Saito1,2*, Naomi Mihira1, Yukio Matsuba1, Hiroki Sasaguri1, Shoko Hashimoto1,
Sneha Narasimhan3, Bin Zhang3, Shigeo Murayama4, Makoto Higuchi5, Virginia M.Y.
Lee3, John Q. Trojanowski3, and Takaomi C. Saido1*
1

Laboratory for Proteolytic Neuroscience, RIKEN Center for Brain Science, 2-1
Hirosawa, Wako-city, Saitama 351-0198, Japan2

Department of Neuroscience and Pathobiology, Research Institute of Environmental
Medicine, Nagoya University, Nagoya, Aichi 464-8601, Japan3
Department of Pathology and Laboratory Medicine, Institute on Aging and Center for
Neurodegenerative Disease Research, University of Pennsylvania School of Medicine,Philadelphia, PA 19104, USA
4
Department of Neuropathology, Tokyo Metropolitan Geriatric Hospital, 35-2 Sakaecho,
Itabashi, Tokyo 173-0015, Japan
5

Department of Functional Brain Imaging Research, National Institute of Radiological

Sciences, National Institutes for Quantum and Radiological Science and Technology,
Chiba 263-8555, Japan.

Content
Supplemental Experimental procedure
Supplementary Figure: Figure S1

1

Supplemental Experimental procedureY-maze Test
The Y-maze test was performed as previously described (5). In this test, each mouse wasplaced in the center of the maze facing towards one of the arms and then allowed to
explore freely for 8 min. The light intensity of the platform was kept constant at 60 lux.
We recorded and analyzed the activity and spontaneous behavioral alternations of themice using Time YM2 for Y-maze software (Two Maze System, O’Hara & Co., Ltd,
Japan).

Figure S1. Memory tests on AppNL-G-F and AppNL-G-F/MAPT KI mice. The Y-maze
test was performed on 12-month-old of male mice. A and B, Total entries to theY-maze arm and the percent alternation were calculated using Time YM2 for Y-mazesoftware (Two Maze System, O’Hara & Co., Ltd, Japan). Data represent mean ± SEM(n = 12 each, *p < 0.05, **p < 0.01; two-way ANOVA with post-hoc Sheffe’s F test
(number of enrty: F(1,47) = 11.143, p = 0.0017; alteration: F(1,47) = 6.188, p = 0.017)).

2

3