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Single App knock-in mousemodels of Alzheimer’s diseasenpg© 201 Nature America, Inc. All rights reserved.

Takashi Saito1,2, Yukio Matsuba1, Naomi Mihira1, Jiro Takano1,
Per Nilsson1, Shigeyoshi Itohara3, Nobuhisa Iwata1,4 &Takaomi C Saido1
Experimental studies of Alzheimer’s disease have largely
depended on transgenic mice overexpressing amyloid precursor
protein (APP). These mice, however, suffer from artificial
phenotypes because, in addition to amyloid b peptide (Ab),they overproduce other APP fragments. We generated knock-in
mice that harbor Swedish and Beyreuther/Iberian mutations
with and without the Arctic mutation in the APP gene.
The mice showed typical Ab pathology, neuroinflammationand memory impairment in an age-dependent manner.
Identification of a protective mutation in the APP gene against
sporadic Alzheimer’s disease (AD)1 has substantiated the AB hypothesis that was originally based on the discovery of gene mutations thatcause familial AD2. This hypothesis has led to the generation of anumber of transgenic (Tg) mouse models that overexpress APP3,4,
but these mice contain intrinsic problems that may induce artificial
phenotypes (Supplementary Table 1 and Supplementary Fig. 1a).
We overcame these problems by creating simple models that overproduce AB42 without overexpressing APP.
We manipulated the mouse App gene using a knock-in strategy(Supplementary Figs. 1b and 2) using Swedish (KM670/671NL)5and Beyreuther/Iberian (I716F) mutations6,7. The Swedish mutation
elevates the total amount of AB40 and AB42, whereas the Beyreuther/Iberian mutation increases the ratio of AB42 to AB40. We first humanized the murine AB sequence and inserted Swedish mutations(AppNL mice). We then created another line that also possessed
the Beyreuther/Iberian mutation (AppNL-F mice), although putting
the two independent mutations together may potentially exert
unexpected effects. The presence of intron 16 was indispensable forcorrect expression of APP (data not shown).
Wild-type (Appwt/wt), AppNL/NL and AppNL-F/NL-F mice generated equal amounts of APP and APP intracellular domain (AICD)(Supplementary Fig. 3a,b). Because Swedish mutations facilitate
B-cleavage of APP8, the levels of C-terminal fragment B (CTF-B)
increased similarly in AppNL/NL and AppNL-F/NL-F mice in a gene dose–
dependent manner. These observations indicate that AppNL/NL mice are
relevant negative controls for AppNL-F/NL-F mice. In contrast, APP-Tg
mice (APP23) produced much higher levels of APP, CTF-B/A andAICD than Appwt/wt and AppNL-F/NL-F mice (Supplementary Fig. 4a).

The AppNL-F/NL-F mice produced more AB42 than any of the other mice;the ratio of AB42/AB40 was also highest in these mice (Supplementary
Fig. 4b–e). The AB42 levels increased in an age-dependent manner(Fig. 1a), accompanying progressive AB pathology (Fig. 1b). We
detected initial deposition of AB at 6 months in the AppNL-F/NL-F
mice (Supplementary Fig. 5) and at 12 months in APP-Tg mice
(Supplementary Fig. 6a,b). Heterozygous AppNL-F/wt mice exhibited
cortical amyloidosis only after 24 months (Supplementary Fig. 7).
We next investigated the N- and C-terminal structures of AB in theplaques of AppNL-F/NL-F mice9. AB1–42 appeared as an initial species,
followed by AB3(pE)–42 (Fig. 1c and Supplementary Fig. 5). ABX–40 was
a minor species. This observation, consistent with human pathology(Supplementary Fig. 8a), implies that conversion of AB1–42 toAB3(pE)–42 occurs after deposition of AB1–42. In contrast, the APP-Tg
mice mainly accumulated AB1–40 (Supplementary Fig. 6c).
We observed accumulation of microglia and activated astrocytes(Fig. 1d), signs of neuroinflammation, around the AB plaques.
We also detected synaptic alterations, as indicated by losses of synaptophysin and PSD95 immunoreactivities (Fig. 1e) similar to thoseobserved in AD brains (Supplementary Fig. 8b–d). We then examined memory function in the AppNL-F/NL-F mice using the Y-maze test
(Fig. 1f). The mutant mice showed impairment at 18 months, whereas
AppNL/NL mice were normal. This suggests that the increase in CTF-B
levels in the AppNL-F/NL-F mice was not the cause of impairment, as
AppNL-F/NL-F and AppNL/NL mice produce equal amounts of CTF-B(Supplementary Fig. 3a,b). However, extremely high levels of CTF-B
may affect cognitive function in APP transgenic mice10.
Deficiency in calpastatin augments AD-associated pathology in
APP-Tg mice11. The most notable feature of the double mutant isearly lethality: half of them die by 10 weeks. We had thus argued
that the calpain-calpastatin system might have a major role in AD
pathogenesis. To validate these phenotypes, we generated AppNL-F/NL-F
and AppNL/NL mice that were deficient in calpastatin (AppNL-F/NL-F ×
Cast−/− and AppNL/NL × Cast−/−). Calpastatin deficiency had no effecton the survival of AppNL-F/NL-F and AppNL/NL mice (Supplementary
Fig. 9a). We also failed to reproduce other phenotypes such asincreased tau phosphorylation and somatodendritic atrophy, as analyzed in a previously described manner11 (data not shown). These are
all likely artifacts caused by APP overexpression.
There were, however, a few confirmable phenotypes. Calpastatin
deficiency exacerbated AB amyloidosis in the AppNL-F/NL-F mice
(Supplementary Fig. 9b), as in APP-Tg mice. We also observed concomitantly higher microgliosis and astrocytosis in AppNL-F/NL-F × Cast−/−mice than in AppNL-F/NL-F mice (Supplementary Fig. 9c). Calpastatin
deficiency increased the biochemical levels of AB in AppNL-F/NL-F mice,but not in AppNL/NL mice (Supplementary Fig. 9d), suggesting that
calpastatin deficiency affects processes associated with the deposition,

1Laboratory for Proteolytic Neuroscience, RIKEN Brain Science Institute, Wako-shi, Saitama, Japan. 2Japan Science and Technology Agency, Saitama, Japan.
3Laboratory for Behavioral Genetics, RIKEN Brain Science Institute, Wako-shi, Saitama, Japan. 4Department of Molecular Medicinal Sciences, Division of
Biotechnology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan. Correspondence should be addressed to T.C.S. (saido@brain.riken.jp).

Received 5 January; accepted 12 March; published online 13 April 2014; doi:10.1038/nn.3697

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Figure 1 Neuropathology and memory impairment of AppNL-F/NL-F mice. (a) Biochemical quantities of AB40 and AB42 in AppNL-F/NL-F brains. AB levels in
the Tris-HCl–buffered saline (TS) and GuHCl fractions of cortex from 2–24-month-old mice were quantified by sandwich ELISA. Data represent mean os.e.m. (n = 4, 4, 5, 6, 6, 6, 4 and 6 mice per indicated time point, respectively). (b) AB pathology in AppNL-F/NL-F brains. Brain sections from 9–24month-old APPNL-F/NL-F mice were immunostained with antibody to AB (4G8). Plaque areas were quantified as indicated in the graph on the right (n = 4,
5, 6, 6, 6, 4 and 6 mice per indicated time point, respectively). Scale bars represent 500 Mm. (c) N- and C-terminal structures of AB in the AppNL-F/NL-F
brain. Brain sections from 24-month-old AppNL-F/NL-F mice (n = 4) were immunostained with endo-specific antibodies as indicated. Scale bars represent
100 Mm. (d) Neuroinflammation in AppNL-F/NL-F mouse brains. Inflammatory responses were detected by triple staining using fluorostyryl benzene (FSB),
antibody to GFAP and antibody to Iba1 as markers of cored AB plaque, astrocytosis and microgliosis, respectively. Cortical immunoreactivities were
quantified as shown in Supplementary Figure 3c. Scale bars represent 25 Mm. (e) Synaptic alterations in AppNL-F/NL-F mouse brains. Double staining
was performed using 4G8 antibody with a presynaptic marker (antibody to synaptophysin) and with a postsynaptic marker (antibody to PSD95). Scalebars represent 10 Mm. (f) Memory impairment in AppNL-F/NL-F knock-in mice. The Y-maze test was performed using 18-month-old mice. Data represent
mean o s.e.m. (n = 10, one-way ANOVA with Sheffe’s F test, *P < 0.05).

rather than the generation, of AB. Furthermore, calpastatin deficiency
increased the memory impairment of AppNL-F/NL-F mice by 3 months(Supplementary Fig. 9e), consistent with accelerated AB pathology.
Some of the phenotypes that we observed in APP-Tg mice therefore
turned out to be artifacts, presumably resulting from high levels of
APP and non-AB fragments, whereas other phenotypes did not. This
makes AppNL-F/NL-F mice convenient tools for distinguishing facts
from artifacts in the phenotypes thus far reported using APP-Tg micecrossbred with other genetically modified mice. In particular, the rolesof tau12 and apolipoprotein E13 are important. It is also possible thatsome of the clinical trials of candidate AD therapeutics failed because
the researchers immediately progressed from APP-Tg mice to humans
without any additional validation.
We also generated another line of mice that carry the Arctic mutation14,15 in addition to the Swedish and Beyreuther/Iberian mutations(AppNL-G-F) (Supplementary Fig. 1b). AppNL-G-F/NL-G-F mice exhibited
APP expression and processing identical to that of AppNL-F/NL-F mice
(Supplementary Fig. 10a,b). However, this mutation, located in the
middle of the AB sequence, altered the binding properties of various
antibodies to AB, as analyzed by sandwich ELISA and immunohistochemistry (Supplementary Fig. 11). Antibodies to the N and C termini appeared to bind to both AB species in a similar manner. Using
an appropriate combination of the antibodies, we observed aggressive
AB amyloidosis in AppNL-G-F/NL-G-F mice in an age-dependent manner(Fig. 2a,b). Notably, the cortical deposition began by 2 months andwas almost saturated by 7 months. The heterozygous mice began AB
deposition at 4 months (Supplementary Fig. 12).
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Unlike AppNL-F/NL-F mice, AppNL-G-F/NL-G-F mice also showed
subcortical amyloidosis after 4 months (Fig. 3b), consistent withthe pathology of human Arctic mutation carriers16. This subcorticalamyloidosis was not detected in mice overexpressing human APP
with the Arctic mutation, presumably because an artificial promoter
was used to drive the transgene expression17. We observed greater
microgliosis and astrocytosis in 9-month-old AppNL-G-F/NL-G-F micethan in 18-month-old AppNL-F/NL-F mice (Fig. 2c), implying that the
Arctic AB may be more proinflammatory than wild-type AB. The
reduction in synaptophysin and PSD95 immunoreactivities near AB
plaques was similar in these mutant mice (Figs. 1e and 2d). Consistent
with the pronounced AB pathology, AppNL-G-F/NL-G-F mice showedmemory impairment by 6 months (Fig. 2e), approximately three times
faster than AppNL-F/NL-F mice.
The memory impairment that we observed in our mutant mice islikely to reflect preclinical cognitive decline in humans18. We therefore
consider these mice to be useful tools for identifying and validating pathways by which AB amyloidosis induces subsequent pathological changes.
A number of approaches can now be adopted, including pathway analyses based on various ‘omics’ data. We can also analyze the tau interactome
to identify protein(s) that may link AB amyloidosis and tauopathy. In any
case, the advantages lie in the existence of relevant negative controls andthe lack of concern about overexpression artifact(s).
AppNL-F/NL-F mice will also provide a better tool for identifying
upstream factors that affect AB amyloidosis than AppNL-G-F/NL-G-F
mice, given that the sequence of AB is identical to that of wild-type
AB, which accumulates in most familial and sporadic AD patients.

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Figure 2 Neuropathology and memory impairment of AppNL-G-F/NL-G-F mice. (a) Biochemical quantities of AB in AppNL-G-F/NL-G-F brains.
We quantified AB40 and AB42 in the TS and GuHCl fractions of cortex from 2–9-month-old mice by sandwich ELISA. We generated standard
curves for calibration using synthetic AB1–40 and AB1–42 with E22G amino acid replacement (Supplementary Fig. 11). Data represent mean o s.e.m.
(n = 4, 4, 6 and 6 mice per indicated time point, respectively). (b) AB deposition in AppNL-G-F/NL-G-F brains. Brain sections from 2–9-month-old
AppNL-G-F/NL-G-F mice were immunostained using antibody to AB42. Cortical, hippocampal and subcortical immunoreactive plaque areas were
quantified as shown in the graph on the right (n = 3, 3, 4 and 4 mice per indicated time point, respectively). Scale bars represent 500 Mm.
(c) Neuroinflammation in AppNL-G-F/NL-G-F mouse brains. Inflammatory responses were detected by triple staining of frozen sections from 18-month-old
AppNL-F/NL-F mice and from 9-month-old AppNL-G-F/NL-G-F mice. Bottom, superimposed images of the boxed area in the upper right panel (AppNL-G-F/NL-G-F
mice) at a higher magnification. Cortical immunoreactivities were quantified as shown in Supplementary Figure 10a. Scale bars represent 100 Mm(top) and 25 Mm (bottom). (d) Synaptic alterations in AppNL-G-F/NL-G-F mouse brains. Double staining was performed using antibody to AB1–x with
antibodies to synaptophysin and PSD95. Scale bars represent 10 Mm. (e) Memory impairment in AppNL-G-F/NL-G-F mice. The Y-maze test was performed
using 6-month-old mice. Data represent mean o s.e.m. (n = 10 Appwt/wt, 9 AppNL/NL, 9 AppNL-F/NL-F and 10 AppNL-G-F/NL-G-F, one-way ANOVA with
Sheffe’s F test, *P < 0.05 and **P < 0.01).

It will be interesting to examine the phenotypes of AppNL-F/NL-F
mice lacking autophagosomal function, as Atg7 deficiency induces
impairment of AB secretion, accumulation of intracellular AB andneurodegeneration with no sign of tauopathy19, implying the presenceof pathway(s) that lead from intracellular AB to neurodegeneration
independent of tauopathy. On the basis of these facts and assumptions, we propose that AppNL-F/NL-F and AppNL-G-F/NL-G-F mice be usedas standard mouse models for identifying mechanisms and pathways
upstream and downstream of AB amyloidosis.

AUTHOR CONTRIBUTIONSThis study was designed by T.S. and T.C.S. Generation of the knock-in mice was
supported by S.I. Experiments were performed by T.S., Y.M., N.M. and J.T. T.S.,
J.T., P.N., N.I. and T.C.S. jointly analyzed and interpreted data.

METHODS
Methods and any associated references are available in the online
version of the paper.

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14. Hashimoto, T. et al. J. Biol. Chem. 286, 27081–27091 (2011).
15. Tsubuki, S., Takaki, Y. & Saido, T.C. Lancet 361, 1957–1958 (2003).
16. Kalimo, H. et al. Acta Neuropathol. Commun. 1, 60 (2013).
17. Cheng, I.H. et al. Nat. Med. 10, 1190–1192 (2004).
18. Cash, D.M. et al. Neurology 81, 1425–1433 (2013).
19. Nilsson, P. et al. Cell Reports 5, 61–69 (2013).

Note: Any Supplementary Information and Source Data files are available in the
online version of the paper.
ACKNOWLEDGMENTS
We thank C.A. Lemere (Brigham and Woman’s Hospital) and M. Higuchi(National Institute of Radiological Science) for valuable discussions. We also
thank E. Takano, R. Fujioka and Y. Tomita for technical assistance. This work was
supported by research grants from RIKEN Brain Science Institute, the Ministryof Education, Culture, Sports, Science and Technology, the Ministry of Healthand Welfare, grants-in-aid for the Molecular Imaging Program, Japan Science andTechnology Agency Precursory Research for Embryonic Science and Technology,
the Cell Science Research Foundation and the Takeda Science Foundation.

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COMPETING FINANCIAL INTERESTS
The authors declare competing financial interests: details are available in the online
version of the paper.
Reprints and permissions information is available online at http://www.nature.com/
reprints/index.html.

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ONLINE METHODS
Generation of AppNL/NL, AppNL-F/NL-F and AppNL-G-F/NL-G-F mice. As describedin Supplementary Figure 1, we isolated mouse App genomic DNA from thebacterial artificial chromosome (BAC) library carrying the C57BL/6 mouse
genome, which included introns 15–17. We humanized the AB sequence andintroduced Swedish mutations into exon 16, and Beyreuther/Iberian and Arctic
mutations into exon 17. Finally, we inserted a pgk-neo gene cassette with alox/FRT sequence in intron 16 for positive selection. We used the fragment
spanning intron 15 to exon 16 (genomic mouse App from 206,484 to 207,984;
approximately 1.5 kbp) and the fragment spanning exon 17 to intron 17 (genomic
mouse App from 210,922 to 216,617; approximately 5.7 kbp) as the short-arm
and long-arm targeting vectors, respectively.
Embryonic stem (ES) cell culture and gene targeting were carriedout as previously described20. Targeted ES cells were microinjected into
C57BL/6 blastocysts. We extracted DNA from the biopsied tails of mouse
pups and identified the F1 generation by Southern blotting using thefollowing 5` and 3` external probes. The former was generated by PCR
using a primer pair of 5`-ACCTGTTCCAATAACTCTACAGCC-3` and
5`-ATGTGGCAGTGACATGAATGCTC-3`, and the latter by 5`-CACTCACAGTCATTCACAGTGC-3` and 5`-GGCATCTACTTGTGTTACAGCAC-3`.
We then crossbred heterozygous mutant mice with EIIa-Cre Tg mice toremove the pgk-neo gene. Finally, we removed the EIIa-Cre transgene by crossing the mice with wild-type C57BL/6 mice. We genotyped the mice by PCR
using the following cocktail of primers: 5`-ATCTCGGAAGTGAAGATG-3`,
5`-ATCTCGGAAGTGAATCTA-3`, 5`-TGTAGATGAGAACTTAAC-3` and5`-CGTATAATGTATGCTATACGAAG-3`.
Other mutant mice. APP23 mice, which overexpress Swedish mutation–
containing human APP751 under the control of the neuron-specific Thy-1
promoter, were maintained as previously described4. Calpastatin knockout(Cast−/−) mice have also been described previously20. All the mice used in theexperiments were on the C57BL/6J background. All animal experiments werecarried out according to the RIKEN Brain Science Institute’s guidelines foranimal experimentation.
Northern blot analysis. Total RNA from brain tissues were subjected to northern blotting to quantify APP expression levels using a North2South labeling anddetection kit (Thermo Scientific), according to the manufacturer’s instructions.
Specific probes to identify the RNA of mouse App and Actb were produced by
PCR using the following primer pairs: 5`-ATGTGCAGAATGGAAAGT-3` and5`-CAGCATACAAACTCTACC-3` for the former, and 5`-TCATGAAGTGTG
ACGTTGACATCCGT-3` and 5`-CTTAGAAGCATTTGCGGTGCACGATG-3`
for the latter. Full-length blots for this experiment are shown in Supplementary
Figure 13.

Immunohistochemical and histochemical studies. We immunostained paraffinembedded and frozen mouse brain sections using antibodies specific to the
N termini of AB (AB1–X and AB3(pE)–X9), the N-terminal region of AB (82E1,
IBL), AB17–24 (4G8, Covance), ABX–40 (IBL), ABX–42 (IBL), phosphorylatedtau (AT8, Invitrogen), synaptophysin (conjugated with FITC; clone SY38,
PROGEN), the PSD95 PDZ domain (Synaptic Systems), Iba1 (Wako) and GFAP
(MAB3402, Millipore). We used tyramide signal amplification (PerkinElmer Life
Sciences), when necessary, as previously described23. 1-fluoro-2,5-bis(3-carboxy4-hydroxystyryl)benzene (FSB) was used for detection of amyloidosis11.
We performed antigen retrieval by autoclave (121 °C for 5 min) for 82E1 and 6E10
staining or by formic acid treatment (90% formic acid for 5 min at 20–25 °C) for
immunohistochemistry of 4G8, ABX–40 and ABX–42 antibodies. We quantified theimmunoreactive areas using MetaMorph imaging software (Universal Imaging)as previously described23. To reduce the variance among tissue sections, we used
the average of data from at least four sections per mouse as an individual value.
Y-maze and Morris water maze tests. The Y-maze test was performed with aslight modification as previously described24. Mice were caged in a group of
three to five individuals before transferring to a behavioral laboratory. The light
condition was 12-h:12-h (lights on 8:00). All of the experiments were conductedin the light phase (9:00–18:00), and the starting time of the experiments was
kept constant.
The Y-maze apparatus (O’Hara & Co), made of gray plastic, consisted of threecompartments (3 cm (W) bottom and 10 cm (W) top, 40 cm (L) and 12 cm (H))
radiating out from the center platform (3 × 3 × 3 cm triangle), and positioned
60 cm above the floor. In this test, each mouse was placed in the center of themaze facing toward one of the arms and then allowed to explore freely for
5 min. The light intensity of the platform was kept at 80 lx. We recorded andanalyzed the activity and spontaneous behavioral alternations of the mice using
Time YM2 for Y-maze (Two Maze System, O’Hara & Co). A reduction in the
behavioral alternations corresponds to memory impairment25.
The Morris water maze test was performed as previously described19, with minor
modifications using Time MWM for the Morris water maze (O’Hara & Co). Althougha tendency toward memory impairment was detected in 18-month-old AppNL-F/NL-F
mice, it did not reach statistical significance (P = 0.893; data not shown).
AD brain sections. Postmortem AD brain tissues were kindly provided by
J.Q. Trojanowski and V.M.-Y. Lee (University of Pennsylvania). The tissues
had been fixed with ethanol or formalin and embedded in paraffin. Paraffinembedded brain (neocortical and hippocampal region) sections from normal
humans and AD patients were also purchased from Wako (Japan). The researchplans to use human materials were approved by the Institutional Review Boardof the RIKEN Brain Science Institute.

Western blot analysis of APP and APP-derived fragments. Brain homogenates
prepared as previously described21 were subjected to western blotting using antibody to human AB1–12 (6E10, Covance), antibody to APP N terminus (22C11,
Millipore) and antibody to APP-CTF (A8717, Sigma). Each set of experiments
was repeated at least three times to confirm the results. The band intensity wasdetermined with a densitometer, LAS4000 (Fujifilm). Full-length blots for theseexperiments are shown in Supplementary Figure 14.

Statistical analysis. All data are shown as mean o s.e.m. For comparison between
two groups, statistical analysis was performed by Student t test. For comparisonsamong three or more groups, we used one-way analysis of variance (ANOVA) or
repeated-measures ANOVA followed by post hoc test (Scheffe F test). Normality
was tested using Statcel 3 (add-in software for Excel, Microsoft). The data werecollected and processed in a randomized and blinded manner. No statisticalmethods were used to predetermine sample size, but our sample sizes were similar
to those generally employed in the field.

Enzyme-linked immunosorbent assay (ELISA). Soluble materials from mousecortical hemispheres were dissolved in TS fraction and insoluble materials in
guanidine-HCl solution (GuHCl fraction) as previously described22. We
quantified ABX–40 and ABX–42 in these fractions using an AB ELISA kit (Wako)
according to the manufacturer’s instructions. We quantified ABX–40 and ABX–42
carrying the Arctic mutation based on standard curves using synthetic humanArctic AB peptides (Peptide Institute).

20. Takano, J. et al. J. Biol. Chem. 280, 16175–16184 (2005).
21. Saito, T. et al. Nat. Med. 11, 434–439 (2005).
22. Iwata, N. et al. J. Neurosci. 24, 991–998 (2004).
23. Enya, M. et al. Am. J. Pathol. 154, 271–279 (1999).
24. Saito, T. et al. Nat. Neurosci. 14, 1023–1032 (2011).
25. Sarnyai, Z. et al. Proc. Natl. Acad. Sci. USA 97, 14731–14736 (2000).

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