The Journal of Neuroscience, September 21, 2016 • 36(38):9933–9936 • 9933

Neurobiology of Disease

Calpain Activation in Alzheimer’s Model Mice Is an Artifactof APP and Presenilin Overexpression
Takashi Saito, Yukio Matsuba, Naomi Yamazaki, Shoko Hashimoto, and XTakaomi C. Saido
Laboratory for Proteolytic Neuroscience, RIKEN Brain Science Institute, Wako 351-0198, Japan

Intraneuronal calcium stimulates the calpain-dependent conversion of p35 to p25, a CDK5 activator. It is widely believed thatamyloid ␤ peptide (A␤) induces this conversion that, in turn, has an essential role in Alzheimer’s disease pathogenesis. However,in vivo studies on p25 generation used transgenic mice overexpressing mutant amyloid precursor protein (APP) and presenilin(PS). Here, using single App knock-in mice, we show that p25 generation is an artifact caused by membrane protein overexpression.
We show that massive A␤42 accumulation without overexpression of APP or presenilin does not produce p25, whereas p25
generation occurred with APP/PS overexpression and in postmortem mouse brain. We further support this finding using micedeficient for calpastatin, the sole calpain-specific inhibitor protein. Thus, the intracerebral environment of the APP/PS mousebrain and postmortem brain is an unphysiological state.
Key words: amyloid; calpain; Nav1.1; p25

Significance Statement
We recently estimated using single App knock-in mice that accumulate amyloid ␤ peptide without transgene overexpression that
60% of the phenotypes observed in Alzheimer’s model mice overexpressing mutant amyloid precursor protein (APP) or APP andpresenilin are artifacts (Saito et al., 2014). The current study further supports this estimate by invalidating key results from papers
that were published in Cell. These findings suggest that more than 3000 publications based on APP and APP/PS overexpression
must be reevaluated.

Introduction
Since 1995 (Games et al., 1995), experimental AD research incell and animal models has suffered from artifactual findings
caused by protein overexpression paradigms (Nilsson et al.,2014; Saito et al., 2014). Membrane protein overexpression
results in chronic ER stress that elevates cytoplasmic calcium
concentrations (Chaudhari et al., 2014; Borkham-Kamphorst
Received June 14, 2016; revised July 8, 2016; accepted Aug. 5, 2016.
Author contributions: T.S., S.H., and T.C.S. designed research; T.S., Y.M., N.Y., and T.C.S. performed research; T.S.,
Y.M., N.Y., and T.C.S. contributed unpublished reagents/analytic tools; T.S., Y.M., N.Y., and T.C.S. analyzed data;
T.C.S. wrote the paper.
This work was supported by a Grant-in-Aid for Scientific Research (B) from the Ministry of Education, Culture,Sports, Science and Technology of Japan (T.S.); the Japan Science and Technology Agency Precursory Research for
Embryonic Science and Technology (T.S.); and the Brain Mapping by Integrated Neurotechnologies for Disease
Studies project of the Japan Agency for Medical Research and Development (T.C.S.). We thank Kazuhiro Yamakawa
for providing antibodies to Nav1.1 and Charles Yokoyama for editing this manuscript.
T.S., Y.M., and T.C.S. serve as advisor, director, and CEO, respectively, for RIKEN BIO Co. Ltd., a RIKEN venture basedat the Brain Science Institute, Central Building, Wako, Japan.
This article is freely available online through the J Neurosci Author Open Choice option.
Correspondence should be addressed to Takaomi C. Saido at the above address. E-mail: saido@brain.riken.jp.
DOI:10.1523/JNEUROSCI.1907-16.2016
Copyright © 2016 Saito et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License
Creative Commons Attribution 4.0 International, whichpermitsunrestricteduse,distributionandreproductioninany
medium provided that the original work is properly attributed.

et al., 2016). For instance, Seo et al. (2014) used transgenic
mice that overexpressed APP and presenilin carrying 5 familial
AD-associated mutations (5XFAD mice; Oakley et al., 2006).
Although the two transgenes inserted impair at least twogene loci of the host animals, these and other transgenic mice
have never been sequenced, to our knowledge. In this case, theasserted disease mechanisms were evidently studied without
sufficient mouse genetic controls.

Materials and Methods
Mutant mice. AppNL-F/NL-F and calpastatin (Cast) KO mice were maintained as described previously (Saito et al., 2014). All the miceused in the experiments were male and established on a C57BL/6J
background. All animal experiments were performed according
to the RIKEN Brain Science Institute’s guidelines for animal
experimentation.
Western blot analysis. We prepared brain extracts from postmortem
mouse brain excised at 1 or 2 h after demise as a positive control for
calpain activation (Taniguchi et al., 2001). AD model mouse brains
were prepared as described previously (Higuchi et al., 2005). We
subjected the samples to Western blot analysis using anti-p35/25 antibody (C64B10, Cell Signaling Technology) and anti-␤-tubulin antibody (SAP.4G5, Abcam).
We also analyzed the expression levels of Nav1.1. Brain extracts
prepared as described previously (Ogiwara et al., 2013) were sub-Saito et al. • Artifacts in Alzheimer Model Mice9934 • J. Neurosci., September 21, 2016 • 36(38):9933–9936

(kDa)
35p35

p25

2050β-tubulin

lane

234567AppNL-F

1 hr 2 hr Wild Type
Postmortem

Results

89AppNL-F

10APP23x Cast KOBrainp25/β-tubulin

P2

350ildWxO
as
C10ApApp NL

p NL-F-FxW

APtKp NL

ildTy

pe0-F20*15-F40**C
astKOAP
P2360pe80

20p NL100**

25Ap120Ty% of postmortem brain (2 hr)

p35/β-tubulin% of postmortem brain (2 hr)

Figure 1. Generation of p25 in App knock-in and APP-overexpressing mice. We performed a Western blot analysis of wild-type
postmortem brain, wild-type control brain, 24 month old AppNL-F/NL-F brain, AppNL-F/NL-F X Cast KO brain, and APP-overexpressing(APP23) brain using antibodies to p35/p25 and to ␤-tubulin. The band intensities were quantified as shown in the graph (n ⫽ 4).
*p ⬍ 0.05; **p ⬍ 0.01 (one-way ANOVA, Scheffe’s F test). Data represent mean ⫾ SEM.
(kDa)
250

Nav1.150β-tubulin

lane1234561 hr 2 hr Wild Type AppNL-F
Postmortem

78AppNL-F

910APP23x Cast KOBrain

Nav1.1/β-tubulin
100806040

3KOP2AP-Fp NL-Fx

Castp NL

TyApW
ildstomortem

0pe20

Ap% of Wild Type120

PoWe crossed APP-overexpressing micewith Cast KO mice and observed peculiar
phenotypes (Higuchi et al., 2012), including early lethality, where half of the micedied in 10 weeks. This result contradictsthe chronic, progressive nature of AD.
Conversely, when we crossed mutant humanized App knock-in mice, overproducing A␤42 without overexpressing APP,
with Cast KO mice, the double mutant
mice lived as long as wild-type mice, or⬎2 years (Saito et al., 2014), indicatingthat the phenotype of early lethality wasan overexpression artifact. More than3000 papers have been published using
these old generation APP mouse models,
and we conclude that the results described in these papers require reevaluation
using new generation models. As a firststep, we examined A␤-induced calpaindependent p25 generation (Oakley et al.,2006; Seo et al., 2014), based on studies of
the calpain– calpastatin system (Saido etal., 1994).
We confirmed conversion of p35 to
p25 by calpain in postmortem mousebrain as a positive control (Taniguchi etal., 2001; Fig. 1, first through fourthlanes). This conversion is caused by intraneuronal ATP depletion resulting in anelevation of cellular calcium concentration (Lipton, 1999). We then examined
aged 24-month-old AppNL-F/L-F mice(Saito et al., 2014; Fig. 1, fifth and sixthlanes) and observed no conversion of p35
to p25 despite massive A␤ deposition(Saito et al., 2014). This finding disagrees
with the results of Oakley et al. (2006) andSeo et al. (2014).
We crossed AppNL-F/NL-F mice with
Cast KO mice, in which calpain is hyperactivatable (Higuchi et al., 2005; Takanoet al., 2005). Even in the absence of calpastatin, we saw no conversion of p35 top25 (Fig. 1, seventh and eighth lanes) despite the observation that calpastatin deficiency increases A␤ amyloidosis (Saito etal., 2014). Thus, we conclude that p25
generation in 5XFAD mice is an artifact.

1Ap

jected to immunoblotting using rabbit antiC-terminal Nav1.1 (IO1; Ogiwara et al.,2007) and anti-␤-tubulin antibody. Each setof experiments was repeated at least threetimes. The band intensity was determinedwith a densitometer (LAS4000, Fujifilm).
Statistical analysis. All data are shown asmeans ⫾ SEM. For comparisons among fourgroups, one-way ANOVA followed by post hoctest (Scheffe’s F test) was used, using Statcel 3(add-in software on Microsoft Excel).

Figure2. ExpressionlevelsofNav1.1inAppknock-inandAPP-overexpressingmice.WeperformedaWesternblotanalysisofwild-typecontrol brain, 24 month old AppNL-F/NL-F brain, AppNL-F/NL-F X Cast KO brain, and APP-overexpressing (APP23) brain using antibodies toNav1.1 and to ␤-tubulin. The band intensities were quantified as shown in the graph (n ⫽ 4). Data represent mean ⫾ SEM.

Saito et al. • Artifacts in Alzheimer Model Mice

Consistently, we observed p25 generation in APP23 mice thatoverexpress APP (Fig. 1, ninth and tenth lanes, bottom).
Protein overexpression paradigms are risky in biologicalscience particularly with membrane proteins. In this regard,
APP and PS possess one and nine transmembrane domains,respectively (Chen, 2015). In contrast, overexpression of mutant tau, a cytosolic protein, exhibited no effect on p25 levels(data not shown). Membrane protein overexpression resultsin chronic ER stress that elevates cytoplasmic calcium concentration (Chaudhari et al., 2014; Borkham-Kamphorst et al.,2016). This nonspecific calcium rise probably activates calpain, resulting in p25 generation in 5XFAD mice (Oakley etal., 2006; Seo et al., 2014). Likewise, 5XFAD mice exhibit
intraneuronal A␤ that is supposed to cause neurodegeneration (Oakley et al., 2006). Indeed, when we crossed APPoverexpressing mice with Atg-7 KO mice lacking autophagy,
we observed intraneuronal A␤ and enhanced neurodegeneration (Nilsson et al., 2013). However, autophagy deficiency,
which induced intraneuronal A␤ accumulation in single App
knock-in mice, caused no neurodegeneration (unpublisheddata).
The overexpression paradigm may also explain the epileptic
phenotypes of APP-overexpressing mice, which show a downregulation of Nav1.1, a sodium channel expressed in PV-positive
interneurons (Verret et al., 2012), because Nav1.1 may also be
another substrate of calpain (Ebensperger et al., 2005). Conversely, we observed no changes in Nav1.1 levels in App knock-in,
APP-overexpressing, or postmortem brains (Fig. 2), suggestingthat Nav1.1 downregulation is a phenomenon unique to the J-20
model mice.

Discussion
Of the five phenotypes that we observed in APP-overexpressing
mice crossbred with Cast KO mice (Higuchi et al., 2012), only
two were reproduced using single App knock-in mice crossbred
with Cast KO mice (Saito et al., 2014). This result allows an estimation that ⬃60% of the phenotypes observed using APPoverexpressing mice may be artifacts. We emphasize that bothbasic and clinical research communities must accept and remit
this reality for the reasons outlined below.
All APP-overexpressing mice overproduce an APP fragment generated by ␤-secretase [C-terminal fragment ␤ (CTF␤); Saito et al., 2014, their Supplemental Fig. 4a], which does
not accumulate in the AD brain (Nilsson et al., 2014). CTF-␤
is more neurotoxic than A␤ (Mitani et al., 2012). To ourknowledge, almost all therapeutic anti-A␤ antibodies bind toCTF-␤ because A␤ and CTF-␤ share common epitopes near
the amino terminus or midregion of the A␤ sequence (Lannfelt et al., 2014). Therefore, the experimental passive immunization of APP-overexpressing mice may have improved
cognitive function by removing CTF-␤ rather than A␤. If thisproves true, the immunotherapeutic prevention trials conducted on thousands of human volunteers may not provideclear information about its beneficial effect. Alternatively,
therapeutic antibodies may have bound to vascular amyloid
in humans because immunotherapy marginally reduced A␤burdens only in apolipoprotein E4 genotype carriers (Salloway et al., 2014), where apolipoprotein E4 genotype isan independent risk factor for cerebral amyloid angiopathy(Shinohara et al., 2016).
In addition, essentially all candidate medications that failed inclinical trials (Mangialasche et al., 2010) have depended on oldgeneration models for preclinical studies. Therefore, we suggestJ. Neurosci., September 21, 2016 • 36(38):9933–9936 • 9935

that a careful revalidation of all the results of scientific and clinical importance obtained using APP-overexpressing mice areurgently required. Depending on expression levels, not all AD
model mice may produce artificial phenotypes. In this context,
p25 generation can be one criterion in the reevaluation.

References
Borkham-Kamphorst E, Steffen BT, Van de Leur E, Haas U, Tihaa L, Friedman SL, Weiskirchen R (2016) CCN1/CYR61 overexpression in hepatic
stellate cells induces ER stress-related apoptosis. Cell Signal 28:34 – 42.
CrossRef Medline
Chaudhari N, Talwar P, Parimisetty A, Lefebvre d’Hellencourt C, Ravanan P(2014) A molecular web: endoplasmic reticulum stress, inflammation,and oxidative stress. Front Cell Neurosci 8:213. Medline
Chen M (2015) The maze of APP processing in Alzheimer’s disease: where
did we go wrong in reasoning? Front Cell Neurosci: 9:186. CrossRef
Ebensperger G, Ebensperger R, Herrera EA, Riquelme RA, Sanhueza EM,Lesage F, Marengo JJ, Tejo RI, Llanos AJ, Reyes RV (2005) Fetal brain
hypometabolism during prolonged hypoxaemia in the llama. J Physiol
567:963–975. CrossRef
Games D, Adams D, Alessandrini R, Barbour R, Berthelette P, Blackwell C,Carr T, Clemens J, Donaldson T, Gillespie F, et al. (1995) Alzheimertype neuropathology in transgenic mice overexpressing V717F ␤-amyloid
precursor protein. Nature 373:523–527. CrossRef Medline
Higuchi M, Tomioka M, Takano J, Shirotani K, Iwata N, Masumoto H, MakiM, Itohara S, Saido TC (2005) Distinct mechanistic roles of calpain andcaspase activation in neurodegeneration as revealed in mice overexpressing their specific inhibitors. J Biol Chem 280:15229 –15237. CrossRef
MedlineHiguchi M, Iwata N, Matsuba Y, Takano J, Suemoto T, Maeda J, Ji B, Ono
M, Staufenbiel M, Suhara T, Saido TC (2012) Mechanistic involvement of the calpain-calpastatin system in Alzheimer neuropathology.
FASEB J 26:1204 –1217. CrossRef Medline
Lannfelt L, Relkin NR, Siemers ER (2014) Amyloid-␤-directed immunotherapy for Alzheimer’s disease. J Intern Med 275:284 –295. CrossRef
Medline
Lipton P (1999) Ischemic cell death in brain neurons. Physiol Rev 79:
1431–1568. Medline
Mangialasche F, Solomon A, Winblad B, Mecocci P, Kivipelto M (2010)
Alzheimer’s disease: clinical trials and drug development. Lancet Neurol
9:702–716. CrossRef Medline
Mitani Y, Yarimizu J, Saita K, Uchino H, Akashiba H, Shitaka Y, Ni K, Matsuoka N (2012) Differential effects between ␥-secretase inhibitors andmodulators on cognitive function in amyloid precursor proteintransgenic and nontransgenic mice. J Neurosci 32:2037–2050. CrossRefMedline
Nilsson P, Loganathan K, Sekiguchi M, Matsuba Y, Hui K, Tsubuki S, Tanaka
M, Iwata N, Saito T, Saido TC (2013) A␤ secretion and plaque formation depend on autophagy. Cell Rep 5:61– 69. CrossRef Medline
Nilsson P, Saito T, Saido TC (2014) New mouse model of Alzheimer’s. ACS
Chem Neurosci 5:499 –502. CrossRef
Oakley H, Cole SL, Logan S, Maus E, Shao P, Craft J, Guillozet-Bongaarts A,Ohno M, Disterhoft J, Van Eldik L, Berry R, Vassar R (2006) Intraneuronal ␤-amyloid aggregates, neurodegeneration, and neuron loss intransgenic mice with five familial Alzheimer’s disease mutations: potential factors in amyloid plaque formation. J Neurosci 26:10129 –10140.
CrossRef Medline
Ogiwara I, Miyamoto H, Morita N, Atapour N, Mazaki E, Inoue I, Takeuchi
T, Itohara S, Yanagawa Y, Obata K, Furuichi T, Hensch TK, Yamakawa K(2007) Nav1.1 localizes to axon of parvalbumin-positive inhibitory interneurons: a circuit basis for epileptic seizures in mice carrying an Scn1agene mutation. J Neurosci 27:5903–5914. CrossRef Medline
Ogiwara I, Iwasato T, Miyamoto H, Iwata R, Yamagata T, Mazaki E, Yanagawa Y, Tamamaki N, Hensch TK, Itohara S, Yamakawa K (2013)
Nav1.1 haploinsufficiency in excitatory neurons ameliorates seizureassociated sudden death in a mouse model of Dravet syndrome. Hum Mol
Genet 22:4784 – 4804. CrossRef Medline
Saido TC, Sorimachi H, Suzuki K (1994) Calpain: new perspectives inmolecular diversity and physiological-pathological involvement.
FASEB J 8:814 – 822. Medline

9936 • J. Neurosci., September 21, 2016 • 36(38):9933–9936
Saito T, Matsuba Y, Mihira N, Takano J, Nilsson P, Itohara S, Iwata N, Saido
TC (2014) Single App knock-in mouse models of Alzheimer’s disease.
Nat Neurosci 217:661– 663. CrossRef
Salloway S, Sperling R, Fox NC, Blennow K, Klunk W, Raskind M, Sabbagh
M, Honig LS, Porsteinsson AP, Ferris S, Reichert M, Ketter N, Nejadnik B,
Guenzler V, Miloslavsky M, Wang D, Lu Y, Lull J, Tudor IC, Liu E, et al.
(2014) Two phase 3 trials of bapineuzumab in mild-to-moderate Alzheimer’s disease. N Engl J Med 370:322–333. CrossRef Medline
Seo J, Giusti-Rodríguez P, Zhou Y, Rudenko A, Cho S, Ota KT, Park C, Patzke H,Madabhushi R, Pan L, Mungenast AE, Guan JS, Delalle I, Tsai LH (2014)
Activity-dependent p25 generation regulates synaptic plasticity and A␤induced cognitive impairment. Cell 157:486 – 498. CrossRef Medline
Shinohara M, Murray ME, Frank RD, Shinohara M, DeTure M, Yamazaki Y,Tachibana M, Atagi Y, Davis MD, Liu CC, Zhao N, Painter MM, Petersen
RC, Fryer JD, Crook JE, Dickson DW, Bu G, Kanekiyo T (2016) Impact

Saito et al. • Artifacts in Alzheimer Model Miceof sex and APOE4 on cerebral amyloid angiopathy in Alzheimer’s disease.
Acta Neuropathol 132:225–234. CrossRef Medline
Takano J, Tomioka M, Tsubuki S, Higuchi M, Iwata N, Itohara S, Maki M,
Saido TC (2005) Calpain mediates excitotoxic DNA fragmentation
via mitochondrial pathways in adult brains: evidence from calpastatin mutant mice. J Biol Chem 280:16175–16184. CrossRefMedline
Taniguchi S, Fujita Y, Hayashi S, Kakita A, Takahashi H, Murayama S, SaidoTC, Hisanaga S, Iwatsubo T, Hasegawa M (2001) Calpain-mediateddegradation of p35 to p25 in postmortem human and rat brains. FEBSLett 489:46 –50. CrossRef Medline
Verret L, Mann EO, Hang GB, Barth AM, Cobos I, Ho K, Devidze N, Masliah
E, Kreitzer AC, Mody I, Mucke L, Palop JJ (2012) Inhibitory interneuron deficit links altered network activity and cognitive dysfunction inAlzheimer model. Cell 149:708 –721. CrossRef Medline