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Potent amyloidogenicity and pathogenicity of Ab43

© 2011 Nature America, Inc. All rights reserved.

Takashi Saito1,8, Takahiro Suemoto1,8, Nathalie Brouwers2,3, Kristel Sleegers2,3, Satoru Funamoto4,
Naomi Mihira1, Yukio Matsuba1, Kazuyuki Yamada5, Per Nilsson1, Jiro Takano1, Masaki Nishimura6,
Nobuhisa Iwata1,7, Christine Van Broeckhoven2,3, Yasuo Ihara4 & Takaomi C Saido1
The amyloid-b peptide Ab42 is known to be a primary amyloidogenic and pathogenic agent in Alzheimer’s disease. However,
the role of Ab43, which is found just as frequently in the brains of affected individuals, remains unresolved. We generated
knock-in mice containing a pathogenic presenilin-1 R278I mutation that causes overproduction of Ab43. Homozygosity wasembryonic lethal, indicating that the mutation involves a loss of function. Crossing amyloid precursor protein transgenic micewith heterozygous mutant mice resulted in elevated Ab43, impairment of short-term memory and acceleration of amyloid-b
pathology, which accompanied pronounced accumulation of Ab43 in plaque cores similar in biochemical composition to thoseobserved in the brains of affected individuals. Consistently, Ab43 showed a higher propensity to aggregate and was moreneurotoxic than Ab42. Other pathogenic presenilin mutations also caused overproduction of Ab43 in a manner correlating with
Ab42 and with the age of disease onset. These findings indicate that Ab43, an overlooked species, is potently amyloidogenic,
neurotoxic and abundant in vivo.
Alzheimer’s disease, the most common form of dementia, is characterized by two pathological features in the brain, extracellular senile
plaques and intracellular neurofibrillary tangles. Senile plaquesconsist of amyloid-β peptide (Aβ) that is generated from amyloid
precursor protein (APP) through sequential proteolytic processingby β-secretase and γ-secretase. Two major forms of Aβ exist, Aβ40
and Aβ42, with Aβ42 being more neurotoxic as a result of its higherhydrophobicity, which leads to faster oligomerization and aggregation1. A number of mutations associated with early-onset familial
Alzheimer’s disease (FAD) have been identified in the APP, PSEN1and PSEN2 genes, and these mutations lead to accelerated productionof Aβ42 or an increase in the Aβ42/Aβ40 ratio. Together, these findings indicate that Aβ42 is essential for the initiation of pathogenesis.
However, the possible involvement of longer Aβ species that also exist
in the brains of individuals with Alzheimer’s disease has not yet been
fully investigated.
Thus far, various longer Aβ species, such as Aβ43, Aβ45, Aβ48,
Aβ49 and Aβ50, have been qualitatively described in the brains ofindividuals with Alzheimer’s disease2. Similar Aβ species have also
been found in transgenic mice that overexpress APP carrying FADlinked mutations3. Further quantitative studies have revealed that
Aβ43 is deposited more frequently than Aβ40 in both sporadic
Alzheimer’s disease (SAD) and FAD4–7.
How these Aβ species with different C-terminal ends are generated
from the precursor has mainly been investigated by cell biologicaland biochemical methods. A number of studies8,9 have found thatγ and ε cleavage by γ-secretase controls the fate of the C-terminal
end. Aβ43, generated from Aβ49 via Aβ46, is subsequently converted
to Aβ40 by γ-secretase, whereas Aβ42 is independently generated
from Aβ48 via Aβ45. It has also been reported that the FAD-­associatedI213T mutation in the PSEN1 gene increases the generation of longer
Aβ species, such as Aβ43, Aβ45 and those even longer than Aβ46, inaddition to Aβ42 (ref. 10). It is also noteworthy that Aβ43 appears tobe as prone to aggregate in vitro as Aβ42 (ref. 11), leading to faster
formation of oligomers than occurs in the case of Aβ40 (ref. 12).
These observations imply that Aβ43 could be produced as a physio­
logical or pathological metabolite of γ-secretase and may affect Aβamyloidosis in the brain.
To address whether Aβ43 contributes to Alzheimer’s disease patho­
logy, we decided to take advantage of the molecular phenotype of
the presenilin-1 (PS1) R278I mutation, as this mutation results inselective overt production of Aβ43 in vitro13, an effect that occurs
to a much greater extent than in the case of other mutations, suchas R278K, R278S and R278T. The R278I mutation has been independently reported in a pedigree bearing atypical Alzheimer’s disease with language impairment14. A follow-up survey revealed that
one of the affected individuals subsequently progressed to more
severe cognitive impairment, and another individual from a different branch of the family with the mutation showed Alzheimer’s
disease–associated symptoms with an early loss of episodic memory
and with a clinical onset of the disease at 59 years of age (M.N. Rossor,
University College London, personal communication).

1Laboratory for Proteolytic Neuroscience, RIKEN Brain Science Institute, Wako-shi, Saitama, Japan. 2Neurodegenerative Brain Diseases Group, Department of
Molecular Genetics, Flanders Interuniversity Institute for Biotechnology, Antwerpen, Belgium. 3Laboratory of Neurogenetics, Institute Born-Bunge, University of
Antwerp, Antwerpen, Belgium. 4Faculty of Life Sciences, Doshisha University, Kyoto, Japan. 5Research Resource Center, RIKEN Brain Science Institute, Wakoshi, Saitama, Japan. 6Molecular Neuroscience Research Center, Shiga University of Medical Science, Shiga, Japan. 7Department of Molecular Medicinal Sciences,
Division of Biotechnology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan. 8These authors contributed equally to this work.
Correspondence should be addressed T.C.S. (saido@brain.riken.jp).

Received 17 March; accepted 13 May; published online 3 July 2011; doi:10.1038/nn.2858

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8I

8I

27
27

/R

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c

R

/R
R

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8I

8I

27
R

27

+

+/

+

R278I/R278I+/+/R278I

–/–

+/

b
+/+

+/

a

Homologous recombination, germline transmission and geno­type wereconfirmed by Southern blotting and PCR (Supplementary Fig. 1b,c).
The expression levels of the mutant and wild-type PS1 in theembryonic mouse brains were identical (Supplementary Fig. 1d).
Unexpectedly, homozygous knock-in (R278I/R278I) was embryoniclethal at embryonic day 15–18 (E15–18; Fig. 1a). The mutant embryos
showed an overall size reduction, a stubby tail, limb ateliosis andhemorrhage in the CNS as compared with wild-type littermate controls (Fig. 1a).
This phenotype is similar to that of PS1-deficient mice andNotch1-related mutant mice15,16, although the adverse phenotypeof the PS1-R278I knock-in mice appeared a few days later than thatof PS1-deficient mice. In contrast, we observed no developmental deficits in heterozygous knock-in (+/R278I) mice (Fig. 1a andSupplementary Fig. 2). The lethal phenotype of the R278I mutation appears to be caused by a loss of developmental function thatmanifests only under the recessive condition. We generated two linesof double-mutant mice: R278I knock-in/PS1 knockout and M146V
knock-in/PS1 knockout. The phenotype of former was embryoniclethal and the ­latter was normal (Supplementary Fig. 3a–d). Thisobservation ­suggests that the R278 mutation is a loss-of-­function
mutation. To the best of our knowledge, this is the first case ofdevelopmental ­ abnormality being caused by a FAD-linked PS1
point mutation.

R

RESULTS
Generation of PS1-R278I knock-in mice
To generate PS1 knock-in mice, we constructed a targeting vector carrying a point mutation that results in the replacement of arginine 278
to isoleucine in exon 8 of the PSEN1 allele (Supplementary Fig. 1a).

+/++/R278I R278IR278I (kDa)

FL

720

PS1 NTF
NTF
E15

PS1

480

PS1 CTF

242

Nct
720

Pen-2
PS2

Aph-1

480

PS2 CTF

242/R
R
27 278I
8I+/ /R2
R
27 78
8I I

8I

8I

27

R

R

+/

+/
+

+/
+

27

27
8
27 I
8I
–/ /R2
–
7

+/
+

e

R

/R

–/

–

8I

27

R

+/
+

27

8I

27

d

+/
R

8I

8I

Figure 1 Phenotypic and biochemical
characterization of PS1-R278I knockin mice. (a) Embryonic lethality inhomozygous PS1-R278I knock-in
NctFL
mice. An overall size reduction,
Nct
PS1 NTF
FL APP
stubby tail (arrowhead), limb ateliosis
NTF
IgG (H)(yellow arrows) and hemorrhage
IgG (L)CTF-β
IgG (L)in the CNS (white arrow) were
CTF-αFL
APP CTF
Pen-2
observed. Scale bars represent 2 mm.
*
Pen-2(b–f) Embryonic brains (b–e) andPS1 CTF
AICD
IgG (L)IgG (L) Aph-1
MEFs (f) were analyzed by western blot
Aph-1
Long exposure(see Supplementary Fig. 4). Antibodies
CTF
AICD
APP CTF
are listed to the left of each panel.
Immunoprecipitation: αPS1-NTF
N-Cad(b) Expression of γ-secretase
FL N-cad+/+
R278I/R278I+/R278I–/–
components. FL, full-length PS1.
N-Cad
CTF N-cad(c) BN-PAGE analysis of native
Myc-∆Notch
Cleaved
Myc-NICD
NICD
γ-secretase complexes. −/− indicates
Notch1
homozygous PS1 knockout mice.
β-actinArrows indicate the position of thenative wild-type, 360-kDa PS1 andPS2 γ-secretase complexes, whereas arrowhead points to the atypical high molecular weight (750 kDa) γ-secretase complex. (d) Immunoprecipitation by
antibodies to PS1-NTF. IgG(H) and IgG(L) indicate immunoglobulin heavy and light chains, respectively. (e) γ-secretase activity in PS1-R278I knock-in
brains. Brain extracts were analyzed by western blotting to detect endogenous APP CTF-β, APP CTF-α, APP intracellular domain (AICD), full-lengthN-cadherin, N-cadherin CTF and NICD products. * indicates an additional signal, smaller than that of CTF-α, which appeared in the knock-in mice.
(f) Notch1 processing in PS1-R278I knock-in MEFs. Myc-tagged ∆Notch was transiently expressed in the MEFs, and cell lysates were subjected to western
blot analysis using antibody to Myc. β-actin levels are shown as internal controls.
+/R© 2011 Nature America, Inc. All rights reserved.

We generated PS1-R278I knock-in mice to assess the biologicalimportance of the mutation and the pathological effect of Aβ43 on
Aβ amyloidosis. Homozygosity in knock-in mice was embryoniclethal, presumably because of a partial loss of γ-secretase activity
that resulted in a failure in Notch1 processing. Consistent with this,mouse embryonic fibroblasts (MEFs) derived from the homozygous
knock-in mice exhibited a failure in PS1 endoproteolysis and Notch1
processing, implying that the particular selectivity of the PS1-R278I
mutation for Aβ43 production is closely associated with the partial loss of γ-secretase activity, that is, suppression of the Aβ43-toAβ40 conversion, which could also be caused by some of the otherPS1 mutations. We crossed heterozygous knock-in mice with APPtransgenic mice (APP23 mice carrying the human APP isoform 751
transgene ­ harboring the Swedish mutation (K651N M652L)) andfound that the progeny exhibited short-term memory loss before
plaque formation and developed considerably accelerated amyloid
pathology, indicating that Aβ43 is potently amyloidogenic and pathogenic in vivo.

f

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7
4

Aβ43

Aβ40

h 0.4

GuHCl

0.9

+/+
+/R278I

0.6

Aβ40

75

0

Aβ43

Aβ42

Aβ43

**

50

**
**

**

25

8I
/–
27

8I

8I

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R
27

27
R

+/
+

ND

/R
27

ND

+/

l 1.2

Aβ43/Aβ42

**

0.75

*

0.6

TS

m4

Aβ42/Aβ40

**

0.50

0

GuHCl
Aβ43/Aβ40

**

2

TS

n4

3

**

Aβ43

0.25

0

GuHCl

0.9

**

0

TS

Aβ42

j 1.000.1

GuHCl

Aβ43/Aβ42

**

**

3

**

2

0.3

1

1

0

0

0

+ +/
R /R2 +
27
8I 78I
/R
27
R 8I
27
8I
/–

k 100

Aβ42

Aβ40

Aβ43/Aβ40

0.2

0.2

Ratio

Aβ40

0

Aβ43

i 0.3

*

0.1

0.3
0

Aβ42

Aβ42/Aβ40

0.3
Ratio

**

0.05

Ratio

g 1.2

0.10

Ratio

Aβ42

Ratio

Aβ40

+/+
+/R278I

0.15

0.5 1 2 5 0.5 1 2 5 0.5 1 2 5 (ng)

0.5 1 2 5 0.5 1 2 5 0.5 1 2 5 (ng)TS+
R +/R /+
27 2
8I 78
/R I
27
R 8I
27
8I
/–

7
4

0.20

Ratio

16

f

C43

–1

(kDa)
45
34

16

Aβ (pmol g–1)

Figure 2 Aβ levels in adult PS1-R278I knock-in
mouse brains and MEFs. (a,b) Establishment of
ELISA system to specifically quantify Aβ42 andAβ43 (see Supplementary Figs. 6a,b and 7).
(c–e) Specificity of the antibodies to Aβ40, Aβ42
and Aβ43 used in this ELISA system. Synthetic
Aβ1–40, Aβ1–42 and Aβ1–43 were separated
by tris/tricine PAGE (15% polyacrylamide gel)and subjected to western blotting. Antibodies toAβ C40, C42 and C43 specifically recognizedAβ1–40, Aβ1–42 and Aβ1–43, respectively (seeSupplementary Fig. 6c–e). (f–n) Quantificationof Aβ40, Aβ42 and Aβ43 by ELISA in adultmouse brains (f–j) and MEFs (k–n). (f–j) Cortical
hemispheres from 24-month-old wild-type andheterozygous knock-in mice were homogenized
and fractionated into Tris-HCl–buffered saline–
soluble (TS) and GuHCl-soluble fractions. Data
represent mean ± s.e.m. (n = 9). *P < 0.05 and**P < 0.01 between wild-type and heterozygous
knock-in mice, Student’s t test. (k–n) Aβ
concentrations in conditioned medium from MEFs.
We inoculated 8 × 105 cells in a 1-ml culture.
The conditioned medium was collected after
24 h and subjected to ELISA. R278I/– indicatesa double heterozygote PS1-R278I knock-in × PS1
knockout mice. Data represent mean ± s.d.
from two independent experiments (n = 16).
**P < 0.01 compared with wild type, one-way
ANOVA with Scheffe’s F test. ND, not detected.

e(kDa) C4245
34

Aβ (pmol g )d+
R +/R /+
27 2
7
8I 8
/R I
2
R 78I
27
8I
/–

O.D.

in the PSEN1 gene (PS1-∆E9)17 and from SH-SY5Y cells treated with
Abnormal PS1 endoproteolysis and Notch1 processing
To assess the functional importance of the R278I mutation in the PS1 a γ-secretase inhibitor, L-685,458 (ref. 18). PS1-∆E9 and PS1-∆T440
knock-in line, we analyzed the biochemical properties of PS1-R278I also cause PS1 endoproteolytic abnormality in a similar manner toγ-secretase in the embryonic brains before degeneration (Fig. 1a). the PS1-R278I mutation19–21. The presence of this high molecularWestern blotting revealed a marked decrease in the levels of the weight γ-secretase may reflect a conformational change in the multiN-terminal fragment (NTF) and C-terminal fragment (CTF) of PS1, component complex or binding of additional factor(s) to the complex,
indicating a failure of PS1 endoproteolysis, in homozygous knock- although its mechanistic involvement remains unclear.
in brains, whereas the γ-secretase components, including Nicastrin
We then examined the effect of the mutation on the metabolism(Nct), presenilin enhancer-2 (Pen-2) and anterior pharynx defective-1 of the γ-secretase substrates. Both the CTF-α and CTF-β of APP
protein (Aph-1), were expressed at normal levels (Fig. 1b). The NTF and the CTF of N-cadherin accumulated at substantial levels in theand CTF in the homozygous knock-in mice were, however, clearly homozygous PS1-R278I knock-in mouse brain, but not in the wilddetectable, indicating that a fraction of the endoproteolytic activ- type or heterozygous brains (Fig. 1e). Conversely, the APP intracelity of PS1 still remained (Fig. 1b and Supplementary Fig. 3e). It is lular domain and Notch1 intracellular domain (NICD) could not bealso noteworthy that the endoproteolysis was partially blocked in the detected in the homozygous knock-in. An additional signal smaller
heterozygous PS1-R278I brain, suggesting that the process is at least than that of CTF-α appeared in the knock-in mice (Fig. 1e), prepartly autolytic.
sumably representing an aberrant proteolytic product of CTF-α andWe next investigated whether the PS1-R278I mutation affects CTF-β. These data indicate that the PS1-R278I mutation leads to a
the assembly of the γ-secretase complex by Blue Native PAGE (BN- substantial loss of γ-secretase activity in a recessive manner.
PAGE)17. A major signal corresponding to a molecular weight of
To further analyze the mutant γ-secretase, we established MEFs
360 kDa, the normal molecular weight of the native PS1 γ-secretase from knock-in mice and littermate controls. Western blotting
complex, was detected in both wild-type and knock-in brains in (Supplementary Fig. 4a), BN-PAGE (Supplementary Fig. 4b) anda manner similar to that of the PS2 γ-secretase complex (Fig. 1c). immunoprecipitation experiments (Supplementary Fig. 4c) revealed
Immunoprecipitation experiments further demonstrated that the that the biochemical properties of mutant presenilin in MEFs weremutant PS1 formed a complex with Nct, Pen-2
and Aph-1 (Fig. 1d). These data indicate
a 0.6 Aβ42
b 0.7 Aβ43cthat the PS1-R278I mutation does not affect(kDa) C40
0.6
y = 0.003x + 0.1694
y = 0.0047x + 0.0997
0.5
the formation of the γ-secretase complex.
2245
R = 0.9866
=
0.958
R
0.5
34
0.4
Notably, BN-PAGE detected a minor signal
Aβ40
Aβ40
0.4
Aβ42
0.3
Aβ42
corresponding to a higher molecular weight of
16
Aβ43
0.3
Aβ43
0.2
750 kDa in the homozygous knock-in brains
0.2
7
0.1
0.1
(Fig. 1c). A similar higher molecular weight400
signal has been described in preparations
0 20 40 60 80 100 120
0 20 40 60 80 100 120
0.5 1 2 5 0.5 1 2 5 0.5 1 2 5 (ng)from an individual with a deletion of exon 9
Aβ concentration (pM)Aβ concentration (pM)
Aβ42
Aβ43
Aβ40

Aβ (pM)© 2011 Nature America, Inc. All rights reserved.

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9 months

GuHCl-Aβ40
15
4,000
12

3,000

9

e
**

2,000

6

1,000

3
0

g

3 months

3 months0Aβ42/Aβ40

450

1.2

300

0.6

150

h

0.36

0.24
0.18
0.12

0.09

0.06

0

0

TS GuHCl TS GuHCl
3 months 9 months

APP

1026

c 4 TS-Aβ43

0

**

2

1

**

180
120

0.2
0

**

60
0

3 months

2.0
1.5
1.0

*

Aβ43/Aβ42

*

2.4
1.6
0.8
0

**

5

6
9
Age (month)j 100
Percentage alternation

10

3

0

12

*

80
60
40
20

P

8I
R
27

ty
pe

1-

ild
W

PS

P
PS AP
1- P
R ×
27
8I

0

established a specific and highly sensitive ELISA system that could
distinguish between Aβ40, Aβ42 and Aβ43 over a broad concentration range (Fig. 2a,b and Supplementary Figs. 6a,b and 7). The
antibodies that we used were highly specific to each Aβ species(Fig. 2c–e and Supplementary Fig. 6c–e). Brain tissue fractions thatwere ­soluble in Tris-HCl–buffered saline and those that were solublein 6 M ­guanidine-HCl (GuHCl) were subjected to quantification.
There was a significant decrease in the steady-state levels of Aβ40
in the GuHCl-soluble fraction in the brains of aged (24 months old)hetero­zygous PS1-R278I knock-in mice as compared with wild-type
animals (P < 0.01), although no differences were recorded in the levelsof Aβs in the Tris-HCl–buffered saline fraction, or in the levels of Aβ42
and Aβ43 in the GuHCl-soluble fraction (Fig. 2f,g). This reduction ofAβ40 in the GuHCl-soluble fraction resulted in a significant elevationof the Aβ42/Aβ40 (P < 0.05) and Aβ43/Aβ40 (P < 0.01) ratios in the
GuHCl-soluble fraction (Fig. 2h,i). Notably, the Aβ43/Aβ42 ratio
was also significantly increased in the GuHCl-soluble fraction of theheterozygous PS1-R278I knock-in mouse brain (P < 0.05; Fig. 2j). In
younger PS1-R278I knock-in mice (3 months old), Aβ43 levels were
too low to detect, although the GuHCl-Aβ40 levels were again significantly reduced in the knock-in mice (P < 0.05; Supplementary Fig. 8).
These results indicate that Aβ43 levels in the mouse brain increaseon aging, and that the increase in the Aβ42/Aβ40 and Aβ43/Aβ40
ratios observed in the older heterozygous mice appears to be primarily
caused by a decrease in Aβ40. Furthermore, the R278I mutation led
to an elevation in the Aβ43/Aβ42 ratio in aged mice. Taken together,
these findings indicate that the PS1-R278I mutation gives rise to amodest in vivo effect in terms of the levels of endogenous Aβ species
under heterozygous conditions.
We next quantified the Aβ variants in conditioned medium fromknock-in MEFs (Fig. 2k–n). The steady-state levels of Aβ40 were

9 months

4.0
3.2

APP × PS1-R278I

9 months

240

0.4

**

TS GuHCl TS GuHCl
3 months 9 months0

3 months

0.6

i

**

2

**

1

0.8

9 months

**

12

15

8I

6
9
Age (month)3

GuHCl-Aβ43
1.0
300

**

*

4

3

0

f

Aβ43/Aβ40
0.30

0.27
0.18

3 months

**

AP

3

*

20

R
27

0

*

25

ty
pe

**

1.0

*

1-

2.0
1.5

i 30

W
ild

Plaque area (%)

2.5

0.5

9 months

1.8

**

PS

APP × PS1-R278I

APP
Hippocampus

3.0*

GuHCl-Aβ42
2.4
600

0

9 months

0.45

3 monthsf

Number of entries

h 3.5

Aβ (pmol g–1)

0

**

2.5

0

Aβ (pmol g–1)

0

7
6
5
4
3
2
1
0

PS1-R278I

AP

e

3.0

0.5

Ratio

4

–1

8

4

Aβ (pmol g )

12

8

–1

12

6
5
4
3
2
1
0

Aβ (pmol g )

16

Ratio

–1

Aβ (pmol g )–1

Aβ (pmol g )d16

b 7 TS-Aβ42*

20g 4.0 CortexWild type
Plaque area (%)

d

Ab40, Ab42 and Ab43 in PS1-R278I knock-in brains and MEFs
Because homozygous R278I knock-in mice are embryonic lethal,
we went on to analyze adult heterozygous knock-in mice. The adult­heterozygous mice were normal in terms of development and anatomy at both 3 and 24 months of age (Supplementary Fig. 2), whereas
various biochemical properties of PS1, such as partial abnormality of endoproteolysis, the molecular weight of γ-secretase and theidentity of the complex components, were identical to those of theheterozygous embryonic brain (Supplementary Fig. 5). We thena 20 TS-Aβ40c3.5

identical to those in the embryonic brains. We then expressed Myctagged ∆Notch1 in the mutant and wild-type MEFs. Western blot
analysis revealed that conversion of Myc-∆Notch1 to Myc-NICD bylimited proteolysis occurred in wild-type and heterozygous knock-in
MEFs, but not in the homozygous knock-in or PS1 knockout MEFs
(Fig. 1f). These results indicate that the R287I mutation induces
developmental deficits by abolishing of γ-secretase–dependent
Notch1 processing.

Ratio© 2011 Nature America, Inc. All rights reserved.

b

PS AP
1- P
R ×
27
8I

a

Figure 3 Acceleration of Aβ pathology and shortterm memory impairment by the R278I knock-in
mutation in APP mice. (a–f) Brain sections from
APP × PS1-R278I mice (3 (a), 6 (b) and9 months (c,d) old) and 9-month-old single
APP mice (d,f) were immunostained with the
4G8 antibody to Aβ (a–d) and antibody to GFAP
(green) with 4G8 counterstaining (red) (e,f).
Aβ-immunostained brain sections from cortex (g)and hippocampus (h) of 3-, 6-, 9- and 12-monthold wild-type, APP and heterozygous PS1-R278I
knock-in mice, as well as APP × PS1-R278I mice
were analyzed (n = 5–6 each genotype). *P < 0.05
and **P < 0.01 compared with APP mice, two-way
ANOVA with Scheffe’s F test. Scale bars represent
500 µm (a–d) and 50 µm (e,f). (i,j) Y-maze test
was performed before plaque formation using
3–4-month-old male wild-type, PS1-R278I knock-in,
APP and APP × PS1-R278I mice. Data represent
mean ± s.e.m. (n = 10 each genotype). *P < 0.05
compared with wild-type or PS1-R278I knock-in
mice, one-way ANOVA with Scheffe’s F test.

** **TS GuHCl TS GuHCl
3 months 9 months

Figure 4 Aβ40, Aβ42 and Aβ43 in APP × PS1-R278I mice. (a–i) The
levels of Aβ40 (a,d), Aβ42 (b,e) and Aβ43 (c,f) were quantified by ELISA
and the ratios of the Aβ species (g–i) were subsequently determined.
Cortical hemispheres from single APP and APP × PS1-R278I mousebrain (3 and 9 months old) were homogenized and fractionated into
Tris-HCl–buffered saline–soluble fractions (a–c) and GuHCl-extractablefractions (d–f). Data represent mean ± s.e.m. (n = 7, 3 months old;n = 5, 9 months old). *P < 0.05 and **P < 0.01 between APP miceand APP × PS1-R278I mice, Student’s t test.

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FL APP

500

500

400

400

300

300

200

200

100

100

0

0

M
oc
k
W
A7 T
9
V9 V
4M
I1
4
A2 3T
31
L2 V
62
L2 F
82
G V
38
4A

W
T
A7
9V
V9
4M
I1
43
A2 T
31
V
L2
62
L2 F
82
V
G
38
4A

M
oc
k

β-actin600

e 120 Aβ42
100

G384A

d 120 Aβ42

A231V
V94M

40

A79V

50
60
Age of onset

70

60

*

**

40
20
0

80

*

*

25

**

80

y = 0.38x + 483.8
2
R = 0.024

30

**

100

WT
L262F

I143T

f 30 Aβ43

y = –1.05x + 103.9
R 2 = 0.646

L282V

M
oc
k
W
T
A7
9
V9 V
4M
I1
4
A2 3T
31
L2 V
6
L2 2F
82
G V
38
4A

pM

NTF

c 700 Aβ40

600

pM

b 700 Aβ40

FL

PS1 NTF

pM

ag 30 Aβ43
25

y = – 0.31x + 28.7
2

significantly reduced in a gene dose–dependent manner in the PS1R278I MEFs as compared with wild-type MEFs (P < 0.01). In contrast, Aβ43 markedly increased in the homozygous knock-in MEFs,
whereas Aβ42 levels remained unchanged in all genotypes (Fig. 2k).
Thus, the ratios of longer Aβ species significantly increased inhomozygous PS1-R278I knock-in MEFs (P < 0.01; Fig. 2l–n).
Notably, there was no increase in Aβ43 levels in conditioned
medium from heterozygous knock-in MEFs (Fig. 2k). To unravel
the underlying mechanism, we crossed heterozygous R278I knockin mice with PS1 knockout mice (R278I/–) and measured the ­levelsof Aβs present in conditioned medium from cultured MEFs. Aβ43
levels were increased, implying that wild-type PS1 processes Aβ43
to Aβ40 in heterozygous PS1-R278I knock-in MEFs (Fig. 2k).

b

APP × PS1-M146V

pM

Aβ

Aβ40

Aβ42

Aβ43

Aβ42

Aβ43

d 0.6 Aβ42**

0.4

Area (%)

0.8

0e 0.6 Aβ43
0.5

0.5

1.0

0.2

f

APP × PS1-R278I

Aβ40

c 1.2 Aβ40
0.6Furthermore, no Aβ43 was detected in heterozygous PS1 knockout MEFs (data not shown). Together, the data suggests that theγ-secretase substrate can be transferred between separate PS1 molecules or between dimers of PS1, as previously suggested 22, or evenbetween PS1 molecules in the PS1 complexes, for further processing. The fact that total Aβ (Aβ40 + Aβ42 + Aβ43) was decreasedin heterozygous knock-in MEFs, as compared with homozygous
knock-in MEFs, is of particular interest. This might be a result ofa dysfunctional PS1 heterodimer, with wild-type PS1 being either
directly affected by PS1-R278I or overloaded with Aβ43 generated
by PS1-R278I. Further experiments are required to resolve the reason behind the decreased total Aβ level (Fig. 2k and Supplementary
Fig. 9b). Taken together, our data indicate that the R278I ­mutation

Aβ

0.4
0.3
0.1

ND

0
APP

**

0.2
ND

APP × PS1-M146V

Area (%)

aArea (%)© 2011 Nature America, Inc. All rights reserved.

M
oc
k
W
T
A7
9V
V9
4M
I1
43
A2 T
31
L2 V
62
L2 F
82
G V
38
4A

pM

pM

R = 0.698
G384A
Figure 5 Effect of various pathogenic
G384A
* *
20
80
20
L262F
L282V
PS1 mutations on Aβ43 production.
15
60
15
A231V
The constructs containing PSEN1
L282V
A231V
I143T
L262F
10
40
10
WT
with FAD-associated mutations were
I143T
V94M
WT
A79V
5
20
5
V94M A79V
transfected into HEK293 cells stably
expressing APP with the Swedish
0
0
0
30
40
50
60
70
80
30
40
50
60
70
80
mutation. (a) Expression levels of
Age of onset
Age of onset
PS1-FAD mutants. (b–g) Quantificationof the steady-state levels of Aβ40, Aβ42 and Aβ43 and the correlation between Aβ levels and the age of disease onset. Age of onset is shown as follows: wild type
(WT, 75 years old), A79V (59.3), V94M (53), I143T (32.5), A231V (58), L262F (50.3), L282V (44) and G384A (34.9)24,44. Data represent mean ± s.d. fromfive independent series each consisting of duplicate measurements. *P < 0.05 and **P < 0.01 compared with wild type, one-way ANOVA with Dunnett test.

0.4

**

Aβ

Aβ40

Aβ/Aβ40

Aβ

Aβ42

Aβ/Aβ42Aβ

Aβ43

Aβ/Aβ43

g

h

0.3
0.2
0.1
0

ND

APP × PS1-R278I

Figure 6 Localization of Aβ species in amyloid plaques of APP × PS1-R278I mice. (a,b) A set of serial brain sections from 9-month-old APP ×
PS1-M146V mice (a) and APP × PS1-R278I mice (b) were immunostained with the following antibodies to Aβ: 4G8 (total Aβ), C40 (Aβ1–40), C42(Aβ1–42) and C43 (Aβ1–43). (c–e) The immunoreactive areas in single APP (left), APP × PS1-M146V (middle) and APP × PS1-R278I (right) mice
were quantified as indicated (n = 6). **P < 0.01 between APP × PS1-M146V mice and APP × PS1-R278I mice, one-way ANOVA with Scheffe’s F test.
ND, not detected. (f–h) Double-staining with 4G8 (green) and Aβ40 (f), Aβ42 (g) or Aβ43 (h) (red). The images in the left (green) and middle (red) are
merged (yellow) on the right. Scale bars represent 500 µm (a,b) and 50 µm (f–h).

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a r t ic l e s
APP × PS1-M146V
APP × PS1-R278I
Figure 7 Mature amyloid plaques in APP × PS14G8
Thioflavin S
Thioflavin S
R278I mice and in vitro aggregation property
Thioflavin S**
and neural cell toxicity of Aβ43. (a–f) A set of
60
800
**
700
serial brain sections from 9-month-old APP ×
50600
PS1-M146V mice (a,c) and APP × PS1-R278I
40
500
mice (b,d) were stained with thioflavin S (a,b)
400
30
and immunostained with 4G8 (c,d). Thioflavin
4G8
4G8
300
20
S–positive plaque are marked with arrows (a,b)
200
and the corresponding plaques in the serial
10100
brain sections are also marked (c,d). Scale00bars represent 500 µm. (e,f) The intensity ofcortical and hippocampal Aβ immunoreactivity
and thioflavin S signals were quantified (e),and the ratio of thioflavin S/total Aβ signal of*amyloid plaques was determined (f) (n = 12).
40,000**60,000140**
Data represent mean ± s.e.m. **P < 0.01
35,000120
50,000**30,000
between APP × PS1-M146V mice and APP ×
100
40,00025,000
PS1-R278I mice, Student’s t test. (g,h) In vitro
80**
20,00030,000Aβ aggregation experiments. Incorporation of60
15,000
thioflavin T into Aβ aggregates was measured
20,00040
10,000by fluorescence spectroscopy. The aggregation20
10,0005,000
properties of 20 µM Aβ40, Aβ42 and Aβ43000
10 µM 3 µM 1 µM
at 20 µM were measured individually in g.
Aβ40 Aβ42 Aβ43
Aβ40 (20 µM)+ + +SH-SY5Y cells
The effect of Aβ40, Aβ42 and Aβ43 at aAβ42 (2 µM)+ + +Aβ concentration****
Aβ40 (0.2 µM)(20 µM each)+ – –concentration of 0.2 µM on the mixture of120
Aβ42 (0.2 µM)– + –20 µM Aβ40 and 2 µM Aβ42 was then assessed100**
Aβ43 (0.2 µM)– – +in h. Data represent mean ± s.d. from three80**
independent series each consisting of 6–8
80
35
**
**
60
individual measurements. **P < 0.01 between
30 **
*
**
40
60
Aβ40 and Aβ42 or between Aβ42 and Aβ43,
25
20*P < 0.05 between Aβ40 and Aβ43, one-way
20
40
ANOVA with Scheffe’s F test. (i,j) Neural cell**** *
150**
10 µM 3 µM 1 µM
toxicity of Aβ43. Cell viability (i) and lactate
1020Primary neuron
dehydrogenase (LDH) release as a measure
5of cell toxicity (j) were assayed. Aβs were00
10 µM 3 µM 1 µM Veh
10 µM 3 µM 1 µM Vehadministrated at 1, 3 and 10 µM, respectively.
Primary neuron
SH-SY5Y cells
The results obtained after treatment with
Aβ40 (white), Aβ42 (gray) and Aβ43 (black)are indicated, and vehicle (veh) treatment was also indicated by open column in (j). Data represent mean ± s.d. from three independent series eachconsisting of six individual measurements. **P < 0.01 between Aβ40 and Aβ42 or between Aβ42 and Aβ43, and *P < 0.05 between Aβ40 and Aβ43,
two-way ANOVA with Scheffe’s F test or Dunnett test.
Thioflavin S / total plaque (%)

dinhibits Aβ43 to Aβ40 conversion, leading to increased Aβ43
levels and concomitant decrease of Aβ40 without altering Aβ42
levels. A similar Aβ-processing pathway has been described
previously9 (Fig. 2k and Supplementary Fig. 10).
Ab pathology and memory impairment of APP mice
Overexpression of wild-type human APP in the above-stated
MEFs using a semliki-forest virus vector23 resulted in a significant
increase in Aβ43 in the heterozygous R278I knock-in cells (P < 0.05;
Supplementary Fig. 9). The presence of the excessive γ-secretase substrates, that is, APP CTF-α and CTF-β, appeared to force the mutant
PS1 to participate in APP processing. These observations prompted us
to crossbreed heterozygous PS1-R278I knock-in mice with APP mice
to assess the effect of Aβ43 in vivo. APP × PS1-R278I mice started toaccumulate pathological Aβ deposits at around 6 months of age,whereas it took about 12 months for APP transgenic mice to begin toshow signs of such deposition (Fig. 3a–h). Massive astrocytosis was
also detected around the amyloid plaques by 9 months age in the APP ×
PS1-R278I mice, but not in single transgenic mice (Fig. 3e,f). Behaviorally,
3–4-month-old APP × PS1-R278I mice exhibited short-term ­memory
impairment as compared with single transgenic mice when theirperformance was evaluated in a Y-maze test (Fig. 3i,j). A similartendency was also observed in the Morris water-maze test, although
the difference in this case did not reach statistical significance1028

LDH release forcytotoxiciy (%)

PS AP
1- P
R ×
27
8I

Cell viability(% of vehicle)

PS A
1- PP
M ×
14
6V

i

Cell viability(% of vehicle)

Thioflavin T fluorescence(a.u.)

Thioflavin T fluorescence (a.u.)hj

LDH release forcytotoxicity (%)© 2011 Nature America, Inc. All rights reserved.

g

PS A
1- PP
M ×
14
6V
PS AP
1- P
R ×
27
8I

c

f

e

3

b

Intensity (× 10 )a

(P = 0.051; data not shown). Taken together, these findings indicatethat the PS1-R278I mutation leads to accelerated Aβ pathology withan accompanying inflammatory response and that the cognitive
impairment occurs even before plaque formation.
We next quantified the steady-state levels of Aβ40, Aβ42 and Aβ43
in the brains of APP and APP × PS1-R278I mice at 3 and 9 months.
Notably, only the double-mutant mice exhibited selective elevation ofAβ43 in both Tris-HCl–buffered saline–soluble and GuHCl-soluble
brain fractions at 3 months, which is a time before the ­pathologicaldeposition of Aβ (Fig. 4a–f), but by which the double-mutant micealready showed short-term memory impairment (Fig. 3i,j). In contrast, Aβ40 and Aβ42 levels started to increase at around 9 months.
Consequently, both the Aβ43/Aβ40 and Aβ43/Aβ42 ratios werehigher in the double mutant mice than in the single APP transgenic mice at both 3 and 9 months, whereas the Aβ42/Aβ40 ratio
remained unaltered (Fig. 4g–i). It is worth noting that the elevationof biochemically detectable Aβ43 levels preceded plaque formation,
implying that Aβ43 may be the initial seeding species and the trigger
of memory impairment in this mouse model. The steady-state levelof Aβ43 also increased in an age-dependent manner in the single
APP transgenic mouse brains, beginning before plaque formation(Fig. 3g,h and Supplementary Fig. 11).
In addition, we observed that a variety of FAD-associated PS1 mutations resulted in overproduction of Aβ43 in a manner ­correlating withVOLUME 14 | NUMBER 8 | AUGUST 2011

nature NEUROSCIENCE

a r t ic l e s
a

Aβ

b

Aβ40

f

Aβ42

g

Aβ43

c

Aβ42

d

Aβ43

h

Aβ42

i

Aβ43

j

Aβ

k

Aβ43

l

Aβ/Aβ43m

Thioflavin S© 2011 Nature America, Inc. All rights reserved.

the quantity of Aβ42 as well as with the age of onset of the affectedindividuals24 (Fig. 5 and Supplementary Fig. 12). These observations
suggest that there is an intrinsic mechanism by which Aβ43 is physio­
logically generated and that both Aβ42 and Aβ43 may be involvedin Alzheimer’s disease pathogenesis. The reason for the correlation
between Aβ42 and Aβ43 remains elusive.
APP × PS1-R278I versus APP × PS1-M146V mice
We generated another line of double-mutant mice by crossbreeding
the APP transgenic mice with PS1-M146V knock-in mice, which
served as a positive control with which to compare the APP × PS1R278I mice, as the former mutation results in overproduction of Aβ42
rather than Aβ43 (ref. 25). As expected, the PS1-M146V mutation,
unlike the PS1-R278I mutation, resulted in selective accumulation ofAβ42 (Supplementary Fig. 13). Although the steady-state levels ofAβ42 in the APP × PS1-M146V mice was about tenfold greater thanthat of Aβ43 in APP × PS1-R287I mice at 9 months, the total plaqueareas, as determined by immunohistochemistry, were similar (Fig. 6).
Both double-mutant mice accumulated Aβ40 and Aβ42, whereas Aβ43
was much more abundant in the APP × PS1-R278I mice (Fig. 6a,b).
Quantitative image analyses yielded consistent results (Fig. 6c–e).
Aβ43 immunoreactivity colocalized with the plaque cores in a manner
similar to that of Aβ42, but not to that of Aβ40 (Fig. 6f–h). Notably,Aβ species with the third N-terminal residue converted to pyroglutamate (N3pE-Aβ), a potently pathogenic Aβ subspecies26–29, also colocalized with plaque cores and deposits were more abundant in APP ×
PS1-R278I than in APP × PS1-M146V mice (Supplementary Fig. 14).
Although the underlying mechanism that accounts for the elevated
N3pE-Aβ generation in the APP × PS1-R278I mice remains unclear,
the observation is consistent with a previous finding that some presenilin mutations increase the quantity of N-terminally truncated Aβin the brains of individuals with FAD30.
Although APP × PS1-M146V mice accumulated greater numbersof Aβ plaques in the cortical and hippocampal areas than APP ×
PS1-R278I mice, the density of thioflavin S–positive plaques per
total plaques was significantly greater in the APP × PS1-R278I mice
(P < 0.01; Fig. 7a–f). This observation indicates that Aβ43 is evenmore prone to seed cores in plaque formation than Aβ42. To test thishypothesis in vitro, we carried out thioflavin T–binding experiments
using an equal amount of Aβ40, Aβ42 and Aβ43 (20 µM each). Aβ43
induced the highest incorporation of thioflavin T into Aβ aggregates (Fig. 7g). In addition, stoichiometric experiments, in which we
added a relatively small quantity of Aβ40, Aβ42 or Aβ43 (0.2 µM) tonature NEUROSCIENCE

VOLUME 14 | NUMBER 8 | AUGUST 2011

Percentage of area

e100
80

**

60
40
20
0
A
β4
A 0
β4
A 2
β4
3

Figure 8 Aβ43 in amyloid plaques in
Alzheimer’s disease brains. (a–m) Serial
sections of the hippocampal region (a–d,h,i,j–m)
and the frontal cortical region of brains fromindividuals with Alzheimer’s disease (f,g) were
stained with 4G8 (total Aβ), C40 (Aβ1–40),
C42 (Aβ1–42) and C43 (Aβ1–43), as wellas thioflavin S, as indicated. The single
staining (a–d,f–i) was developed using
3,3′-diaminobenzidine, whereas the double
staining (j–m) used the fluorescent dyes
fluorescein (green, Aβ) and rhodamine(red, Aβ43). The images in j and k are merged(yellow) in l. Scale bars represent 250 µm (a–d)
and 25 µm (f–m). The ratio of Aβ40, Aβ42 and Aβ43 of theplaque areas in the hippocampal region of brain sections from fourindividuals with Alzheimer’s disease were quantified in e. **P < 0.01
between Aβ40 and Aβ43, one-way ANOVA with Scheffe’s F test(see Supplementary Fig. 15).

Samples fromfour individualsa ­mixture of Aβ40 (20 µM) and Aβ42 (2 µM), revealed that, of thethree, Aβ43 most potently accelerated the incorporation of thioflavin T(Fig. 7h). These data indicate that Aβ43 contributes to the formationof the thioflavin T–positive β-sheeted structure to a greater extent thaneither Aβ40 or Aβ42, a finding that may account for the observationthat a relatively small amount of Aβ43 is sufficient to accelerate Aβamyloidosis and induce plaque core formation in vivo.
Neural toxicity and amyloid pathology of Ab43
Consistent with Aβ42 having higher hydrophobicity and higher
toxicity than Aβ40 in vitro and in vivo, a large number of studies
have found that Aβ42 contributes to synaptic dysfunctions 31–34.
We therefore compared the toxicity of Aβ40, Aβ42 and Aβ43.
Aβ43 showed a higher potent neural toxicity in a dose-dependent
manner as compared with Aβ40 and Aβ42 (Fig. 7i,j). These results
indicate that Aβ43 directly affects neural toxicity and induces
synaptic dysfunction, which would contribute to short-term memory
impairments before the amyloidogenesis (Fig. 3i,j).
Finally, we performed immunohistochemical experiments on brain
sections from individuals with SAD to explore the possible involvement of Aβ43 in human neuropathology. Aβ43 accumulated in thebrains more frequently than Aβ40 (Fig. 8a–e and Supplementary
Fig. 15), and was present in both diffuse (Fig. 8f,g) and dense-cored(Fig. 8h,i) plaques, similar to Aβ42 and N3pE-Aβ (Supplementary
Fig. 16a–d). Furthermore, thioflavin S fluorescence signals colocalized well with Aβ43 immunoreactivity (Fig. 8j–m), as well as with
N3pE-Aβ (Supplementary Fig. 16e–g). These observations are consistent with those of previous studies, which found that a substantial
amount of Aβ43 accumulates in SAD and FAD brains4–7.
DISCUSSION
Previous studies using Bri-Aβ fusion proteins have shown that Aβ42
is essential for amyloid deposition in vivo31 and that Aβ40 inhibits
this deposition32. The difference between Aβ40 and Aβ42 lies in the
C-terminal amino-acid sequence, that is, the additional presence ofisoleucine and alanine residues in Aβ42. Because both isoleucine
and alanine are hydrophobic amino acids, it is reasonable to assume1029

© 2011 Nature America, Inc. All rights reserved.

a r t ic l e sthat Aβ42 is more prone to form a β-sheet structure than Aβ40. In
contrast, the carboxyl-terminal amino acid of Aβ43 is threonine,
which carries a hydrophilic alcohol group (together with a hydrophobic methyl group) and could therefore reverse the hydrophobicityof Aβ42. Thus, the amyloidogenicity of Aβ43, a natural product ofγ-secretase activity8,9, has remained elusive.
We focused on Aβ43, an overlooked species in Alzheimer’s disease research, and investigated its role in the amyloidogenesis and­pathogenesis of Alzheimer’s disease. To date, the major focus ofresearch into Alzheimer’s disease has been placed on the amyloido­
genecity of Aβ42 and, in numerous studies, BC05, an antibody to Aβ42that has been used to demonstrate that Aβ42 is the major pathogenic
species in Alzheimer’s disease. As partial crossreactivity of BC05 to
Aβ43 had already been reported35, Aβ42(43) was noted in some ofthe studies that used BC05. However, many studies have overestimatedAβ42 levels and ignored the possible changes in Aβ43 levels. Almost
all FAD-associated PS1 mutations result in an increased Aβ42/Aβ40
ratio that is caused by an increase of Aβ42. However, some of the PS1mutations lead to a decrease of Aβ40 with or without alteration ofAβ42 levels, which also leads to an increased Aβ42/Aβ40 ratio. One
explanation of the association between decreased Aβ40 and FAD could
be that Aβ40 is involved in protection from plaque fromation32. We
found that decreased Aβ40 levels accompanied increased Aβ43 levels
in PS1-R278I knock-in mice. Furthermore, the increased Aβ43 levels accelerated Aβ pathology, contributing to the early onset of thedisease. Thus, we propose that Aβ43 should be separately analyzed
from Aβ42.
In an effort to explore the role of Aβ43 in Aβ amyloidosis, we generated PS1-R278I knock-in mice, as this mutation causes overproduction of Aβ43 in vitro13. We chose to use this presenilin mutation
knock-in procedure rather than the overexpression strategy for thefollowing reasons. First, the R278I mutation is known to be clinicallypathogenic. Second, the knock-in procedure is less artificial thantransgenic overexpression approaches in general, and the knock-in
mice could potentially be used to generate a relevant Alzheimer’s
disease model by crossbreeding with other mice, such as mutant
APP transgenic or knock-in mice. Unexpectedly, the phenotype ofthe homozygous knock-in mice proved to be embryonic lethal in
association with abnormal PS1 endoproteolysis. Limited proteolysisof APP CTF-α and CTF-β, N-cadherin, and Notch1 was also hampered in the homozygous knock-in embryos, although the γ-secretase
components appeared to have been properly assembled as a 360-kDa
complex. On the basis of previous studies, it appears that the disturbance in Notch1 processing represents the primary cause of thepremature death that we observed16,36. Compared with PS1 knockout, the embryonic lethality of PS1-R278I knock-in mice occurs at aslightly later stage. Taking into account the fact that a 50% reductionof γ-secretase activity in heterozygous PS1-R278I or in heterozygous
PS1 knockout mice does not lead to embryonic lethality and thata 90% reduction in homozygous PS1-R278I mice is lethal, it seems
that the γ-secretase activity threshold for survival is somewhere
between 10–50% of wild type. The remaining 10% γ-secretase activity in homozygous PS1-R278I knock-in mice could account for thedelayed lethality compared with PS1 knockout mice (Supplementary
Fig. 10c). Taken together, these results strongly suggested that the
primary phenotype of the R278I mutation was a partial loss of function of γ-secretase activity.
Despite this, MEFs prepared from homozygous embryos produced
extremely high steady-state levels of Aβ43 (approximately 20-fold greaterthan that of wild-type MEFs); this accompanied a substantial decreasein Aβ40 production and no changes in Aβ42 levels. Previous in vitro
1030

studies have found that Aβ43 is processed to Aβ40, whereas Aβ42 is
independently produced from Aβ45 in the presence of γ-­secretase8,9.
Consistent with these findings, our results from crossbreeding heterozygous PS1-R278I mice with PS1 knockout mice, which showed substantial levels of both Aβ40 and Aβ43, indicate that Aβ43 was indeed
converted to Aβ40 independently of Aβ42 production (Fig. 2k).
Furthermore, we carried out in vitro γ-secretase assays and found thatthe ratio of production of Aβ46 in homozygous PS1-R278I MEFs wasincreased with a concomitant increase of Aβ43 and decrease of Aβ40
(Supplementary Fig. 10), suggesting that production of Aβ40 andAβ43 also depends on Aβ46 production, as previously postulated8–10.
Thus, inhibition of this Aβ43-to-Aβ40 conversion could account for
the increase in Aβ43 and the concomitant decrease in Aβ40 in theknock-in MEFs. Notably, treatment of PS1-∆E9–expressing cells with
L-685,458 results in elevated Aβ43 production37, consistent with thenotion that multiple processes are involved in the generation of various Aβ species and that a partial loss of γ-secretase activity might giverise to a particular Aβ species. However, in vitro γ-secretase activityof heterozygous and homozygous PS1-R278I was markedly reduced
in a gene dose–dependent ­manner, whereas there were no substantial differences in the steady-state levels of total MEF-produced Aβcompared with wild-type MEFs. To elucidate the reason behind thiscontradiction, it will be necessary to investigate other mechanisms,
such as intracellular trafficking and secretion of Aβ, in depth.
The molecular mechanism that allows Aβ43 production, but
not other proteolytic processes, remains to be clarified, but it likely
involves specific conformational changes of the γ-secretase complex38.
Because Aβ42 is produced independently of Aβ43 in the presence ofγ-secretase, some of the FAD-associated PS1 mutations that cause adecrease in Aβ40 without an increase in Aβ42, such as A79V, A231V,
C263F, L282V, L166P and G384A24,39, might actually result in theelevation of Aβ43 in a manner similar to the R278I mutation. In addition, PS1-∆E10, an artificial PS1 mutation located to the loop domainof PS1 where R278I is present, leads to a substantial reduction of thesteady-state levels of Aβ40 without any alteration of Aβ42 levels, similar to our results; however, Aβ43 levels were not measured40. It will
therefore be important to investigate whether these FAD-associatedmutations give rise to increased Aβ43 levels and to scrutinize theiramyloidogenicity. In fact, the I143T, L262F, L282V and G384A mutations did lead to substantial production of Aβ43 in our transfection
assays. Notably, Aβ43 levels and the ratio of Aβ43/Aβ40 substantiallycorrelated well with the age of disease onset in a manner similar toAβ42 levels and the ratio of Aβ42/Aβ40. In addition, a PS1-I143T
carrier in a Swedish family with FAD gave rise to high levels of Aβ43
(ref. 7). These observations highlight the possibility that compounds
that facilitate the Aβ43-to-Aβ40 and Aβ42-to-Aβ38 conversions
might be beneficial for prevention and treatment of Alzheimer’s­disease by decreasing both Aβ42 and Aβ43. In support of this notion,an oral vaccination with an adeno-associated virus vector carrying
Aβ1–43 cDNA was reported to result in a marked reduction of Aβburdens and improvement of behavioral performances in Tg2576 APP
transgenic mice41,42.
Although we originally thought to generate APP × homozygous
PS1-R278I mice, we also explored the possible utility of heterozygous
PS1-R278I knock-in mice, given that overexpression of APP in hetero­
zygous PS1-R278I knock-in MEFs resulted in selective elevationof Aβ43. Consistent with this, APP × heterozygous PS1-R278I mice
exhibited short-term memory impairment, selective biochemical
accumulation of Aβ43 at an early stage before plaque formation andsubstantial acceleration of Aβ pathology thereafter as compared with
APP mice. It should also be noted that the APP × PS1-R278I mice
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a r t ic l e s
exhibited a greater density of the thioflavin S–positive signal per plaquethan APP × PS1-M146V mice, which overproduced Aβ42 instead of
Aβ43. Consistent with previous reports6,7, we observed Aβ43-positiveplaques more often than Aβ40-positive ones in the brains of individuals with Alzheimer’s disease. Aβ43 has previously been found in amyloid plaques in individuals with Alzheimer’s disease4,6,7, as well as inaged gorillas43 and in some Alzheimer’s disease model mice harboring
PS1 or APP FAD mutations3,10. In addition, it has been suggested thatthe amount of Aβ43 in plaques correlates with cognitive decline5. We
also found that Aβ43 exhibited potent neural toxicity, comparable toor even greater than that of Aβ42. These findings establish that Aβ43
is indeed amyloidogenic in vivo and likely to be pathogenic. Thus, theC-terminal amino acid residue of Aβ43, threonine, appears tostrengthen the hydrophobicity of the peptide rather than reversing it.
Notably, biochemical accumulation of Aβ43 preceded pathologicaldeposition in the APP × PS1-R278I mice and in the single APP mice.
In addition, the basal Aβ43 levels substantially increased with aging inwild-type mice up to at least 18 months of age (data not shown). Theseobservations suggest that Aβ43 is potentially valuable as a biomarker for presymptomatic diagnosis of Alzheimer’s disease. We believethat it would be worth trying to quantify Aβ43 levels in cerebrospinal
fluid from individuals with Alzheimer’s disease and controls. We also
detected the presence of N3pE-Aβ in APP × PS1-R278I mouse brains,
a finding that is supported by a previous report quantitatively describing N3pE-Aβ42 and N3pE-Aβ43 in the brains of individuals with FAD
or SAD2. It is of particular interest that Pittsburgh Compound B, aprobe for amyloid imaging by positron emission tomography, selectively binds to N3pE-Aβ26, implying that N3pE-Aβ42/43 could be particularly prone to seed deposition of other Aβ species, consistent withprevious findings28. It is also possible that the mutation might affectthe interaction of PS1 with other substrates or alter its property of nonγ-secretase activity, such as regulation of neurotransmitter release29.
In summary, our results indicate that Aβ43, which has largely been
overlooked, is potently amyloidogenic and toxic, and highlight thepotential value of Aβ43, that is, cerebrospinal fluid Aβ43 levels, as
an early marker for some of the detrimental effects of aging in theadult brain. We propose that inhibition of Aβ43 generation, suchas by facilitating the conversion of Aβ43 to Aβ40 in the γ-secretase
complex, should be beneficial for prevention of Aβ amyloidosis.
Methods
Methods and any associated references are available in the online ­versionof the paper at http://www.nature.com/natureneuroscience/.
Note: Supplementary information is available on the Nature Neuroscience website.
Acknowledgments
We thank M.N. Rossor (University College London) for sharing clinicalinformation about the R278I mutation carriers, J.Q. Trojanowski and V.M.-Y. Lee(University of Pennsylvania) for providing postmortem brain tissues, R. Kopan(Washington University) for providing Myc-tagged ∆Notch1 plasmid,
A. Takashima (RIKEN Brain Science Institute) for providing antibody to Aph-1,
and J. Hardy (University College London) for valuable discussions. This work was
supported by research grants from RIKEN Brain Science Institute, the Ministryof Education, Culture, Sports, Science and Technology, the Ministry of Health,
Labor and Welfare of Japan, the TAKEDA Science Foundation, the Fund for
Scientific Research – Flanders (FWO-V), the Interuniversity Attraction Poles
program P6/43 of the Belgian Federal Science Policy Office and a Methusalem
Excellence Grant of the Flemish Government to C.V.B. N.B receives a FWO-V
postdoctoral fellowship.
AUTHOR CONTRIBUTIONSThis study was jointly designed by T. Saito, T. Suemoto and T.C.S. Experiments
were performed by T. Saito, T. Suemoto, N.M., Y.M., K.Y. and S.F. T. Saito,
T. Suemoto, S.F., K.Y., P.N., J.T., M.N., N.I., C.V.B., Y.I. and T.C.S. jointly analyzed

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and interpreted data. N.B., K.S. and C.V.B. identified pathogenic PS1 mutations inpatients and families and generated PSEN1 vector constructs for expression studies.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Published online at http://www.nature.com/natureneuroscience/.
Reprints and permissions information is available online at http://www.nature.com/
reprints/index.html.
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ONLINE METHODS

Generation of PS1-R278I knock-in mice. The genomic DNA of mouse PSEN1
was isolated from the bacterial artificial chromosome (BAC) library from the
129/Sv mouse genome, and one BAC clone that included introns 5–11 of thePSEN1 gene was obtained (Supplementary Fig. 1). The fragment from the ApaIsite of intron 5 to the HindIII site of intron 11 provided the basis for constructionof the targeting vector. To introduce the PS1-R278I mutation, we subcloned the
SmaI/BamHI fragment containing introns 7 and 8 of the PSEN1 gene into pBluescript vector. To introduce the R278I mutation, we used 5′-GGT TGA AAC AGC
TCA GGA AAT AAA TGA GAC TCT CTT TCC AGC-3′ (underlined, original G
to T mutation) as our primer, using GeneEditor Mutagenesis System (Promega)according to the manufacturer’s protocol. This fragment was used to replace theoriginal sequence of the PSEN1 gene. Finally, a pgk-neo gene cassette was inserted
for positive selection at the EcoRI/SmaI sites located in intron 7, and a diphtheria
toxin A fragment cassette was inserted for negative selection at the HindIII site in
intron 11. We used the ApaI/EcoRI fragment spanning from intron 5 to intron 7(4.3 kb) as the long arm and the BamHI/HindIII fragment spanning from intron 8
to intron 11 (3.8 kb) as the short arm of the targeting vector.
Embryonic stem cell cultures and gene-targeting experiments were carriedout as described previously. Targeted embryonic stem cells were microinjected
into 129/Sv blastocysts. DNA was extracted from the biopsied tail of mouse pups,
and the F1 generation of the mutant animals was identified by Southern blot
analysis with a 3′ external probe that was produced by PCR using 5′-AAT GGA
TAA TCA GAG CCT GCC-3′ and 5′-TCC TCA CAA CTA ACT ACC CAA
GG-3′ as primers.
The heterozygous mice were crossbred with EIIa-Cre transgenic mice toremove the pgk-neo gene, after which the generated PS1-R278I knock-in mice
were backcrossed to the C57BL6/J strain. When the pgk-neo gene was removed
by Cre excision, a short sequence ranging from the EcoRI to the SmaI sites of
intron 7 was also removed. Deletion of this short sequence in intron 7 enables
detection of the genotype of mutant mice. To genotype the PS1-R278I knock-in
mouse, tail DNA was isolated and subjected to PCR analysis using 5′-AGT TTC
AGA CCA GCC TAG GCC AC-3′ and 5′-AGG AAG GGA GAC TTG ACA
GC-3′ as primers.
Other mutant mice. PS1 knockout mice and PS1-M146V knock-in mice werepurchased from the Jackson Laboratory. APP23 mice carrying the human APP
isoform 751 transgene harboring the Swedish mutation (K651N M652L)45 havebeen described previously46. All animal experiments were carried out according
to the RIKEN Brain Science Institute’s guidelines for animal experimentation.
MEFs. MEFs were prepared from E13–14 embryos of wild-type, PS1-R278I
knock-in and PS1 knockout mice, and inoculated in Dulbecco’s modified Eagle
medium supplemented with 10% fetal bovine serum (FBS, vol/vol). The conditioned medium and cell lysates from MEFs (passage <8) were subjected tobiochemical analyses, including ELISA, native PAGE and western blotting.
Transfection of the MEFs with the Myc-tagged ∆Notch construct47 was performed using FuGENE 6 Transfection Reagent (Roche) according to the manufacturer’s instructions.
Blue native-PAGE (BN-PAGE). Non-denaturing native PAGE was performed
to confirm the integrity of the γ-secretase complexes17 using the Novex Bis-Tris
gel system (Invitrogen) according to the manufacturer’s instructions. Samples
were extracted from embryonic brains and MEFs using the sample buffer fromthe Novex Bis-Tris gel system that contains 1% digitonin. Equal amounts ofproteins as determined using the BCA Protein Assay Kit (Pierce) were loaded
on a 3–12% gradient Bis-Tris acrylamide gel. Immunoblotting was performed
using the antibodies H70 (to the PS1 N terminus, Santa Cruz) and Ab-2 (toPS2, Calbiochem).
Immunoprecipitation assay and western blot analysis. Brain homogenates fromembryonic brains (E14–16) and cell lysates of MEFs were immunoprecipitatedwith H70, and then captured by Dynabead-conjugating protein G (Invitrogen).
Immunoprecipitants were subjected to western blot analysis using antibodies H70,
MAB5232 (to the PS1 loop, Chemicon), Ab-2, PA1-758 (to Nicastrin, Affinity
Bioreagents) and ACS-01 (to Aph1)10, and antibody to Pen-2 (Zymed). In addition, we used antibody to Aβ1-12 (6E10, Covance), antibody to the N terminus

doi:10.1038/nn.2858

of APP (22C11, Chemicon), antibody to APP CTF (Sigma), antibody to Myc(9B11, Cell Signaling), antibody to Notch1 (mN1A, BD Bioscience) and antibody
to β-actin (AC-15, Sigma).
ELISA. Soluble materials from mouse cortical hemispheres were dissolved in
Tris-HCl–buffered saline and the insoluble materials were dissolved in guanidineHCl solution as described previously48. Samples from the brains and from theconditioned medium of MEFs were analyzed using an Aβ-ELISA kit (Wako) toquantify Aβ40. To specifically quantify the levels of Aβ42 and Aβ43, we established an Aβ42- and Aβ43-specific sandwich ELISA system using the Aβ-ELISA
kit (Wako). Given that BC05, a detection antibody of this kit, cross-reacts with
Aβ42 and Aβ43 (ref. 35), we used the Aβ42- and Aβ43-specific antibodies C42(Aβ42 specific, IBL) and C43 (Aβ43 specific, IBL). The specificities of these antibodies are shown in Figure 2c–e and Supplementary Figure 6c–e. Samples wereincubated overnight at 4 °C in a 96-well plate coated with the capture antibody,
BNT77 (antibody to Aβ11–28)49. Aβ from samples captured in the ELISA wereincubated with C42 or C43 (1:100, 3 h at 20–25 °C), after which horseradish
peroxidase–conjugated antibody to rabbit IgG (1:500, 2 h at 20–25 °C) was added
as a detection antibody. Synthesized Aβ42 or Aβ43 peptide (Peptide Institute)
was used for the preparation of a standard curve, and diluted with the diluents
solutions provided in the kit. For consistency, when we quantified the amountof Aβ40, a synthesized Aβ40 peptide (Peptide Institute) was also used for the
preparation of a standard curve. This system also worked in broader concentration range of Aβ42 and Aβ43 (Supplementary Fig. 6a,b). Furthermore, ahighly sensitive Aβ43 system, based on modified protocols, was established forthe measurement of samples containing small amounts of Aβ43, such as samples
derived from non–APP transgenic mice and cells that are not overexpressing APP(Supplementary Fig. 7).
Immunohistochemical and histochemical studies. Paraffin-embedded
mouse brain sections were immunostained with 4G8 (antibody to Aβ17-24,
Covance), C40 (specific antibody to Aβ40, IBL), C42, C43 and MAB3402(antibody to GFAP, Chemicon), with or without tyramide signal amplification(PerkinElmer Life Sciences) as described previously48. Quantification of immuno­
reactivity from brain sections were carried out using MetaMorph imaging
software (Universal Imaging) as previously described48.
Y-maze test. Mice were housed individually before transferring to the behavioral
laboratory. They were kept during the behavioral analysis. The light condition was
12-h:12-h (lights on 8:00). The laboratory was air-conditioned and maintained
at a temperature of approximately 22–23 °C and a humidity of approximately
50–55%. Food and water were freely available except during experimentation.
Large tweezers were used to handle mice to avoid individual differences in thehandling procedure. All of the experiments were conducted in the light phase(9:00–18:00), and the starting time of the experiments was kept constant.
The Y-maze apparatus (O’Hara) was made of gray plastic and consisted of threecompartments (3-cm (width) bottom and 10-cm (width) top, 40 cm (length) and12 cm (height)) radiating out from the center platform (3 × 3 × 3 cm triangle).
The maze was positioned 80 cm above the floor, surrounded by a number ofdesks and test apparatuses around the maze to act as spatial cues. In this test,
each mouse was placed in the center of the maze facing toward one of the arms
and was then allowed to explore freely for 5 min. Experiments were performed
at a light intensity of 150 lx at the platform. An arm entry was defined as four legs
entering one of the arms, and the experimenter counted the sequence of entries
by watching a TV monitor behind a partition. An alternation was defined as entry
into all three arms on consecutive choices (the maximum number of alternations
was the total number of entries minus 2). The percent alternation was calculated
as (actual alternations divided by maximum alternations) × 100. The percent
alternation was designated as the spontaneous alternation behavior of the mouse,
was taken as a measure of memory performance.
Thioflavin T–binding assay. The thioflavin T–binding assay was performed bymixing aliquots of Aβ. Human Aβ1–40, Aβ 1–42 and Aβ1–43 were purchasedfrom the Peptide Institute. We first examined the aggregation properties of Aβ40,
Aβ42 and Aβ43 individually by incubating the peptides separately at 20 µM in
50 mM potassium phosphate buffer (pH 7.4) at 37 °C for 24 h with agitation. The
stoichiometric effect of different Aβ species on aggregation was investigated innature NEUROSCIENCE© 2011 Nature America, Inc. All rights reserved.

the mixture of Aβ40 and Aβ42 by adding and mixing Aβs in 50 mM potassium
phosphate buffer (pH 7.4) at molar concentrations of 20:2:0.2 µM (Aβ40:Aβ42:
Aβ40, Aβ42 or Aβ43 = 100:10:1) and incubating them at 37 °C for 24 h withagitation. After incubation, thioflavin T was added to a final concentration of
5 µM and thioflavin T fluorescence was measured at excitation and emission
wavelengths of 442 nm and 485 nm, respectively.
Neural cell toxicity assay. Primary cortical neurons were isolated as previously
described23 and plated at a density of 5 × 104 cells per well in 96-well plate (n = 6
wells in each experimental conditions). We treated 10–14 d in vitro cultures withsynthesized Aβ40, Aβ42 and Aβ43 peptide (Peptide Institute) at 0.1 to 10 µM
of Aβs for 72 h. These Aβ peptides were dissolved in 10 mM phosphate buffer(pH 7.4, 90%) and 60 mM NaOH (10%), which was used as the vehicle33. SH-SY5Y
cells were plated at a density of 2 × 104 cells per well with 10% FBS supplemented
medium in 96-well plate (n = 6 wells in each experimental conditions), and incubated for 24 h. Then the medium was replaced with medium containing 1% FBS(vol/vol), and treated with each Aβ peptides for 48 h. Cell viability was determined using MTS assay (CellTiter 96 Aqueous One Solution Cell Proliferation
Assay Kit, Promega)50, and lactate dehydrogenase release as cell toxicity was
performed using CytoTox-ONE Homogeneous Membrane Integrity Assay Kit(Promega)33, according to the manufacturer’s instructions and compared tovehicle treated cells.

nature NEUROSCIENCE

Alzheimer’s disease brain sections. Post-mortem Alzheimer’s disease brain
tissues were kindly provided by J.Q. Trojanowski and V.M.-Y. Lee (University ofPennsylvania). The tissues had been fixed with ethanol or formalin and embedded in paraffin. This study was approved by the Institutional Review Board of
the RIKEN Brain Science Institute.

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