An Intracellular Threonine of Amyloid-b Precursor
Protein Mediates Synaptic Plasticity Deficits and Memory
Franco Lombino1, Fabrizio Biundo1, Robert Tamayev1, Ottavio Arancio2, Luciano D’Adamio1*
1Department of Microbiology & Immunology, Albert Einstein College of Medicine, Bronx, New York, United States of America, 2Department of Pathology & Cell Biology,
Taub Institute for Research on Alzheimer’s Disease and the Aging Brain, Columbia University, New York, New York, United States of America
Mutations in Amyloid-ß Precursor Protein (APP) and BRI2/ITM2b genes cause Familial Alzheimer and Danish Dementias (FAD/
FDD), respectively. APP processing by BACE1, which is inhibited by BRI2, yields sAPPß and ß-CTF. ß-CTF is cleaved by
gamma-secretase to produce Aß. A knock-in mouse model of FDD, called FDDKI, shows deficits in memory and synaptic
plasticity, which can be attributed to sAPPß/ß-CTF but not Aß. We have investigated further the pathogenic function of ß-
CTF focusing on Thr668of ß-CTF because phosphorylation of Thr668is increased in AD cases. We created a knock-in mouse
bearing a Thr668Ala mutation (APPTAmice) that prevents phosphorylation at this site. This mutation prevents the
development of memory and synaptic plasticity deficits in FDDKImice. These data are consistent with a role for the carboxyl-
terminal APP domain in the pathogenesis of dementia and suggest that averting the noxious role of Thr668is a viable
therapeutic strategy for human dementias.
Citation: Lombino F, Biundo F, Tamayev R, Arancio O, D’Adamio L (2013) An Intracellular Threonine of Amyloid-b Precursor Protein Mediates Synaptic Plasticity
Deficits and Memory Loss. PLoS ONE 8(2): e57120. doi:10.1371/journal.pone.0057120
Editor: Riqiang Yan, Cleveland Clnic Foundation, United States of America
Received November 19, 2012; Accepted January 17, 2013; Published February 22, 2013
Copyright: ? 2013 Lombino et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the Alzheimer’s Association (IIRG-09-129984 and ZEN-11-201425 to L.D.), the Edward N. & Della L. Thome
Memorial Foundation grant (to L.D.), the National Institutes of Health (NIH; R01AG033007 to L.D. and R01NS049442 to O.A.), the Training Program in Cellular and
Molecular Biology and Genetics T32 GM007491 to R.T. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of
Competing Interests: The AECOM has a patent on the commercial use of FDDKI mice. Luciano D’Adamio is a co-inventor on this patent. AECOM has licensed
the patent to Remegenix, a company of which Luciano D’Adamio is a co-founder and a Board member. As a co-founder Luciano D’Adamio owns ,35% of
Remegenix. The patent and the licensing only covers commercial use of the mice and does not pose any obstacle to distribution of the mice to academic
laboratories. There are no further patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS
ONE policies on sharing data and materials, as detailed online in the guide for authors.
* E-mail: email@example.com
Familial dementias are caused by mutations in APP  and
genes that regulate APP processing. These include the PSEN1/2
genes, which code for the catalytic component of the gamma-
secretase, and the BRI2/ITM2b gene, whose protein product
BRI2 binds APP and inhibits APP processing [1–8]. Cases
caused by APP/PSEN mutations are classified as FAD and those
caused by mutations in BRI2/ITM2b as FDD or Familial British
dementia (FBD). The prevailing pathogenic model for these
dementias posits that amyloid peptides trigger dementia. In AD,
the amyloid peptide Aß is a part of APP; in FDD and FBD, the
amyloidogenic peptides, called ADan and ABri respectively, are
generated from the mutant BRI2 proteins [2,8]. FDD patients
present mixed amyloid plaques containing both Ab and ADan.
However, recent data suggest that these dementias share
pathogenic mechanisms involving synaptic-toxic APP metabo-
lites distinct from Ab [9,10].
In FDD, a 10-nucleotide duplication in the BRI2/ITM2B
gene leads to the synthesis of a longer BRI2 protein . In
normal individuals, BRI2 is synthesized as an immature type-II
membrane protein (imBRI2) that is cleaved at the C-terminus
into mature BRI2 and a 23aa soluble C-terminal fragment .
In FDD patients, cleavage of the BRI2 mutant protein leads to
the release of the longer ADan peptide . To model FDD we
generated FDDKI mice that like FDD patients , carry one
wild type Bri2/Itm2b allele and the other one has the Danish
mutation . FDDKI mice develop synaptic and memory
deficits due to loss of Bri2 protein, but do not develop
amyloidosis . BRI2 binds to APP and inhibits cleavage of
APP by secretases [4–7]. Owing to the loss of BRI2, processing
of APP is increased in FDD [14,15]. Memory and synaptic
deficits of FDDKI mice require APP , and are mediated by
sAPPß and/or ß-CTF produced during synaptic plasticity and
memory acquisition. Inhibition of c-secretase, the enzyme that
processes b-CTF to yield Aß, worsens memory deficits and is
associated with an accumulation of ß-CTF [10,16,17]. In
addition, caspase-9 in activated in FDDKImice and caspase-9
Overall, these results suggest that ß-CTF, rather than Aß, is a
major toxic species causing dementia. Here, we have investi-
gated further the pathogenic role of the carboxyl-terminal
region of APP and especially the role of residue Thr668.
PLOS ONE | www.plosone.org1February 2013 | Volume 8 | Issue 2 | e57120
Thr668of APP Mediates Object Recognition Deficits found
Recent findings suggest that products of BACE1-processing of
APP (predominantly ß-CTF) trigger several pathological features
related to human dementias both in a mouse model of FDD
[10,16] and human neurons derived from familial and sporadic
AD . Thus, we decided to probe in more details the pathogenic
function of the carboxyl-terminal region of APP, focusing on the
intracellular Thr668residue (following the numbering of the
APP695isoform). The phosphorylation status of Thr668either
creates or destroys docking sites for intracellular proteins that
interact with APP [19–22]. In addition, phosphorylation at Thr668
is increased in AD cases  suggesting potential pathogenic
implications. We generated mice expressing APP with a Thr668Ala
mutation, called APPTA. Western blot analysis of hippocampal
synaptosomes from either APPWT/WTor APPTA/TAmice shows
that the Thr668Ala mutation abolishes phosphorylation at Thr668
Thus, the APPTAmice are an ideal genetic tool to study the role
of Thr668and its phosphorylation in the pathogenesis of dementia.
To this end, we utilized FDDKImice, which develop severe aging-
dependent memory and synaptic plasticity deficits that first
become measurable at ,5 months of age . Most importantly,
these deficits are prevented when FDDKImice lack one allele of
APP, reducing the APP protein load , and require production
of APP ß-CTF [10,16]. Thus, since memory and synaptic deficits
of FDDKImice are dependent on endogenous APP, we can test
the pathogenic role of Thr668by introducing this APP mutation on
generated littermates of the following 6 genotypes: WT, FDDKI,
FDDKI/APPTA/TA, FDDKI/APPTA/WT, APPTA/TAand APPTA/
WT. To test memory, six-month-old mice were subjected to the
novel object recognition (NOR) task, which is a non-aversive
task that relies on the mouse’s natural exploratory behavior.
Open field studies showed that FDDKI, FDDKI/APPTA/TA,
FDDKI/APPTA/WT, APPTA/TAand APPTA/WTmice have no
defects in habituation and locomotor behavior, sedation, risk
assessment and anxiety-like behavior in novel environments
(Figure 1b and c). During the training session, mice of all
genotypes spent the same amount of time exploring the two
identical objects during the training phase (Figure 1d). The
following day, when a novel object was introduced, FDDKI
spent the same amount of time exploring the two objects as if
they were both novel to them, while the WT, APPTA/TA, and
APPTA/WTmice still spent more time exploring the novel object
(Figure 1e). Notably, FDDKI/APPTA/TAand FDDKI/APPTA/WT
mice behaved like the WT mice and explored preferentially the
novel object (Figure 1e), demonstrating a prevention of the
defect of the FDDKImice. We subjected the mice to the NOR
task at 9 months, and also at 12 months to confirm that this is
a true prevention of deficits and not a delay. We found similar
data to the data at 6 months with the FDDKImice showing no
preference between the two objects on the second day, while
the FDDKI/APPTA/TA, FDDKI/APPTA/WT, APPTA/TA, APPTA/WT
mice all behaved similar to the WT mice (Figure 1f and 1g).
These data confirm that memory is impaired in FDDKI mice
upon aging in an ethologically relevant, non-aversive behavioral
prevented by changing the Thr668residue on the intracellular
region of APP to an Alanine.
Thr668of APP Mediates Short-term Memory Deficits
Found in FDDKIMice
To further test memory, WT, FDDKI, FDDKI/APPTA/TA,
FDDKI/APPTA/WT, APPTA/TA, APPTA/WTmice were subjected
at 5.5 months of age to the radial arm water maze (RAWM) task, a
spatial working memory test that depends upon hippocampal
function . This task tests short-term memory, which is the
memory affected in early stages of AD. The six genotypes were
required to learn and memorize the location of a hidden platform
in one of the arms of a maze with respect to spatial cues. WT,
APPTA/TA, and APPTA/WTmice were able to acquire (A) and retain
(R) memory of the task. FDDKImice showed severe abnormalities
during both acquisition and retention of the task (Figure 2a),
confirming that FDDKImice have severe impairment in short-
term spatial memory for platform location during both acquisition
and retention of the task. This defect was due to a deficit in
memory per se and not to deficits in vision, motor coordination or
motivation because testing with the visible platform showed no
difference in the swimming speed and the time needed to find the
platform between the FDDKIand WT mice (Figure 2c and d).
Both the FDDKI/APPTA/TAand the FDDKI/APPTA/WTmice
showed no defects in the memory test (Figure 2a), showing that
mutating the intracellular APP residue Thr668to an alanine
prevented the RAWM deficit of FDDKImice, and confirming the
data seen in NOR. To ensure that this was not simply a delay of
the deficit, the mice were re-tested at 9 months in the RAWM task,
and once again the FDDKI/APPTA/TAand the FDDKI/APPTA/WT
mice did not show the deficit seen in the FDDKImice (Figure 2b).
Thr668of APP Mediates Synaptic Deficits Found in FDDKI
The FDDKImice have compromised long-term potentiation
(LTP) , a long-lasting form of synaptic plasticity that is thought
to be associated with learning and memory. Like for memory, the
LTP deficit of FDDKImice are prevented when APP protein levels
are halved , and by inhibiting processing of APP by BACE1
(also known as b-secretase) [10,16]. Thus, we tested if this one
amino acid change in APP could also prevent the synaptic
plasticity defect found in the FDDKI mice. To this end, we
investigated synaptic transmission and plasticity using the
Schaeffer collateral pathway in hippocampal slices from WT,
APPTA/TA, FDDKIand FDDKI/APPTA/TAmice. As expected, LTP
was reduced in FDDKI mice compared with WT littermates
(Figure 3). Strikingly, the APPTA/TApoint mutation prevented LTP
impairments in FDDKImice (Figure 3). Taken together, these
findings provide compelling genetic evidence that APP and BRI2
functionally interact, and that the synaptic and memory deficien-
cies due to loss of Bri2 function require the APP intracellular
In this manuscript, we have pinpointed an intracellular residue
of APP that is required for memory and synaptic plasticity deficits.
FDDKImice allow for a genetic analysis of pathogenic pathways
on a genetic background that is congruous to the human disease.
We showed that haplodeficiency in APP prevented all FDDKI
mice’s deficits at all ages. Now we take this further by showing that
mutation in just one residue of APP, the intracellular amino acid
Thr668, can also prevent the memory and synaptic deficits.
We studied the functional relevance of Thr668of APP because
APPpThr668is enriched in AD patients , suggesting a
pathogenic role for phosphorylation at this residue, and because
it has profound effects on APP protein/protein interactions and
Essential Role of APP-Thr668 in Neurodegeneration
PLOS ONE | www.plosone.org2 February 2013 | Volume 8 | Issue 2 | e57120
Figure 1. A Thr668Ala mutation on APP prevents the object recognition memory deficit of FDDKImice. (a) Western blot analysis of
hippocampal synaptosomal preparations shown that the Thr to Ala mutation abolishes phosphorylation of Thr668(APPpThr668). Interestingly, only the
mature form of APP (mAPP) and not the immature (imAPP), is found phosphorylated on this Thr in hippocampal synaptic fractions of WT mice. (b and
c) Open field is a sensorimotor test for habituation, exploratory, emotional behavior, and anxiety-like behavior, in novel environments. The percent of
time in the center (b) and the number of entries into the center (c) are indicators of anxiety levels. The more the mouse enters the center and
explores it, the lower the level of anxiety-like behavior. Since the FDDKI, FDDKI/APPTA/TA, FDDKI/APPTA/WT, APPTA/TA, APPTA/WTmice are similar to the WT
animals there is no deficit or excess of anxiety. (d) All six genotypes (WT, FDDKI, FDDKI/APPTA/TA, FDDKI/APPTA/WT, APPTA/TA, APPTA/WT) mice spent similar
amounts of time exploring the two identical objects on day 1. (e) FDDKI/APPTA/TAand FDDKI/APPTA/WTmice behaved similarly to WT mice and
prevented the deficit in the NOR tests found in FDDKImice at 6 months of age (FDDKIversus FDDKI/APPTA/TAP=0.011; FDDKIversus FDDKI/APPTA/WT
P=0.0083; FDDKIversus WT P,0.001), (f) 9 months of age (FDDKIversus FDDKI/APPTA/TAP=0.01; FDDKIversus FDDKI/APPTA/WTP=0.347; FDDKIversus
WT P=0.000995), and (g) 12 months of age (FDDKIversus FDDKI/APPTA/TAP=0.0003; FDDKIversus FDDKI/APPTA/WTP=0.0002; FDDKIversus WT
P,0.0001). Thus the APPTApoint mutation prevented the novel object recognition deficit of FDDKImice.
Essential Role of APP-Thr668 in Neurodegeneration
PLOS ONE | www.plosone.org3 February 2013 | Volume 8 | Issue 2 | e57120
APP biology. For example, Thr668phosphorylation impairs APP/
Fe65 interaction [20,21] but promotes Pin1 binding . In
addition, this phosphorylation regulates trafficking of APP and
APP derived metabolites . Previous studies in mice suggested a
protective role for phosphorylation of Thr668in the pathogenesis
of AD by showing that Pin1 decreases APP processing and Aß
production by binding APP phosphorylated on Thr668.
However, analysis of the APPTAmice has shown that preventing
phosphorylation by mutating Thr668into an Ala does not change
Aß levels in vivo [28,29].
If Aß were a major neuro-toxic peptide in dementia, FDDKI/
APPTA/TAmice should either have deficits comparable to FDDKI
mice based on the evidence that the Thr668Ala mutation does not
change Aß levels [28,29], or should present with a worsened
phenotype based on the hypothesis that binding of Pin1 to
APPpThr668reduces Aß levels . Instead, we have found that
the Thr668Ala mutation on one or both alleles of APP prevents all
the memory and synaptic deficits found in the FDDKImice. This is
seen in a short-term memory test, such as the RAWM task, and
also in an ethologically relevant, non-aversive behavioral context,
such as the NOR task. The memory deficits were prevented at
their start and no deficits could be found even as late as 9–12
months of age. The same was true for the synaptic plasticity at 12
months old. The FDDKImice show strong synaptic defects in the
Schaffer collateral pathway, however, FDDKI/APPTA/TAthe mice
showed no such deficits.
In this context, it is worth noting that mutation of another
phosphorylated amino acid present in the APP intracellular
region, namely Tyr682, results in a different (almost opposite)
phenotype. This tyrosine is comprised in the intracellular
682YENPTY687sequence of APP, a docking region for numerous
APP-binding proteins that regulate processing and functions of
APP [19,30–35]. Phosphorylation of Tyr682is consequential.
Some proteins, such as Grb2 , Shc [37,38], Grb7 and Crk 
interact with APP only when Tyr682is phosphorylated; others, like
Fe65, Fe65L1 and Fe65L2 only when this tyrosine is not
Figure 2. A Thr668Ala mutation on APP prevents the short-term memory deficit of FDDKImice. (a and b) In RAWM testing, FDDKI/APPTA/
TA, FDDKI/APPTA/WT, APPTA/TA, APPTA/WTmice made the same number of errors as WT mice at both 5.5 months and 9 months of age. At 5.5 months of
age, FDDKImice made significantly more errors at A4 (versus FDDKI/APPTA/TAP=0.0007; versus FDDKI/APPTA/WTP=0.0028; versus WT P=0.0005) and
R (versus FDDKI/APPTA/TAP=0.0017; versus FDDKI/APPTA/WTP=0.0005; versus WT P=0.0005) (a). Similar results are found at 9 months of age; FDDKI
mice made significantly more errors at A4 (versus FDDKI/APPTA/TAP=0.0004; versus FDDKI/APPTA/WTP=0.019; versus WT P=0.0003) and R (versus
FDDKI/APPTA/TAP=0.0006; versus FDDKI/APPTA/WTP=0.004; versus WT P,0.0001). Thus, the APPTA/TAand APPTA/WTpoint mutations prevent the
development of working memory deficits in FDDKImice (b). (c and d) WT, FDDKI, FDDKI/APPTA/TA, FDDKI/APPTA/WT, APPTA/TAand APPTA/WTmice have
similar speed (c) and need similar time (d) to reach a visible platform.
Essential Role of APP-Thr668 in Neurodegeneration
PLOS ONE | www.plosone.org4 February 2013 | Volume 8 | Issue 2 | e57120
phosphorylated , suggesting that phosphorylation–dephos-
phorylation on Tyr682modulates APP functions. To test the in vivo
function of Tyr682we have created mice with Tyr682replaced by a
Gly. This knock-in mutation alters the function of APP in memory
formation, development/aging [41,42] and changes APP process-
ing, leading to a significant decrease in Aß levels . Thus, while
the Thr668Ala mutation on APP, which does not reduce Aß
production, prevents memory deficits of FDDKI mice, the
Tyr682Gly mutation, which reduces Aß production, causes
cognitive defects on its own . These data show that the
intracellular region of APP has a fundamental role in memory
formation, a role that is not linked to Aß.
New evidence points to b-derived metabolites of APP, especially
ß-CTF, as the synaptic-toxic APP fragments mediating synaptic
and memory impairments. The data presented here suggest that
the synaptic-toxic activity of ß-CTF requires Thr668(Figure 4a–c).
It is possible that this synaptic-toxic activity necessitates or is
enhanced by phosphorylation of Thr668(Figure 4d–f), which is
abolished by the Thr668Ala mutation. It is interesting to note that
this mutation does not alter essential biological functions of APP
during development , suggesting that targeting the role of
Thr668, and perhaps its phosphorylation, in dementia may be an
effective and safe therapeutic approach to dementias. Since the
APPTAmutation prevents memory and synaptic deficits in
heterozygosis, a partial reduction of the noxious pathogenic
functions mediated by Thr668will be therapeutically efficient.
The animals used for these studies were backcrossed to C57Bl6/
J mice for at least 14 generations. Mice were handled according to
the Ethical Guidelines for Treatment of Laboratory Animals of
Albert Einstein College of Medicine. The procedures were
described and approved in animal protocol number 200404.
The Institutional Animal Care and Use Committee (IACUC)
approved this protocol. IACUC is a federally mandated committee
that oversees all aspects of the institution’s animal care and use
program, facilities and procedures. The regulations of the USDA
and PHS require institutions using animals to appoint an IACUC.
The members of the IACUC are appointed by the Dean of Albert
Einstein College of Medicine of Yeshiva University (Einstein).
Hippocampi were homogenized in H buffer [5 mM Hepes/
NaOH pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.32 M sucrose,
plus phosphatase/protease inhibitors at 10% (w/v) and centri-
fuged at 800 g for 10 min. The supernatant (S1) was separated to
supernatant (S2) and pellet (P2) by spinning at 9,200 g for 15 min.
P2 represents crude synaptosomal fraction.
The following antibodies were used: anti-APP (Chemicon), anti-
APP-CTF (Invitrogen), anti-APPpThr668and : anti-Akt (Cell
Signaling). Secondary antibodies conjugated with horse-radish-
peroxidase are from Southern Biotechnology.
Figure 3. A Thr668Ala mutation on APP prevents the synaptic deficits of FDDKImice. Normal LTP in FDDKI/APPTA/TAand APPTA/TAcompared
with WT mice by two-way ANOVA (FDDKI/APPTA/TAversus WT mice: F(1,12)=1.936; P=0.187; APPTA/TAversus WT F(1,12)=0.989; P=0.338). Two-way
ANOVA shows impaired LTP in FDDKImice when compared with WT (F(1,13)=15.125; P=0.002), to FDDKI/APPTA/TA(F(1,13)=12.759; P=0.004) or to
APPTA/TAmice littermates (F(1,13)=22.396; P,0.0001).
Essential Role of APP-Thr668 in Neurodegeneration
PLOS ONE | www.plosone.org5 February 2013 | Volume 8 | Issue 2 | e57120
Electrophysiology and Behavior
Only male mice were used to avoid variations due to hormonal
fluctuations during the estrous female cycle, which influence
severely behavioral and electrophysiological tests.
Spatial Working Memory
A six-armed maze was placed into white tank filled with water
(24–25uC) and made opaque by the addition of nontoxic white
paint. Spatial cues were presented on the walls of the testing room.
At the end of one of the arms was positioned a clear 10 cm
submerged platform that remained in the same location for every
trial in 1 d but was moved approximately randomly from day to
day. On each trial, the mouse started the task from a different
randomly chosen arm. Each trial lasted 1 min, and errors were
counted each time the mouse entered the wrong arm or needed
more than 10 s to reach the platform. After each error, the mouse
was pulled back to its starting position. After four consecutive
acquisition trials, the mouse was placed in its home cage for
30 min, then returned to the maze and administered a fifth
retention trial. The scores for each mouse on the last 3 days of
testing were averaged and used for statistical analysis.
Visible Platform Testing
Visible platform training to test visual and motor deficits was
performed in the same pool as in the RAWM; however, the arms
of the maze were removed. The platform was marked with a black
flag and positioned randomly from trial to trial. Time to reach the
platform and speed were recorded with a video tracking system
(HVS 2020; HVS Image).
Open Field and Novel Object Recognition
After 30 min to acclimate to the testing room, each mouse was
placed into a 40 cm640 cm open field chamber with 2 ft high
opaque walls. Each mouse was allowed to habituate to the normal
open field box for 10 min, and repeated again 24 hours later, in
which the video tracking system (HVS 2020; HVS Image)
quantifies the number of entries into and time spent in the center
of the locomotor arena. Novel object recognition was performed as
previously described . Results were recorded as an object
discrimination ratio (ODR), which is calculated by dividing the
time the mice spent exploring the novel object, divided by the total
amount of time exploring the two objects.
Transverse hippocampal slices (400 mm) were transferred to a
recording chamber where they were maintained at 29uC and
perfused with artificial cerebrospinal fluid (ACSF) continuously
bubbled with 95% O2and 5% CO2. The ACSF composition in
mMwas: 124 NaCl,4.4 KCl,
2 CaCl2, 2 MgSO4, and 10 glucose. CA1 field-excitatory-post-
synaptic potentials (fEPSPs) were recorded by placing both the
stimulating and the recording electrodes in CA1 stratum radiatum.
For LTP experiments, a 30 min baseline was recorded every
minute at an intensity that evoked a response approximately 35%
of the maximum evoked response. LTP was induced using a teta-
burst stimulation (four pulses at 100 Hz, with bursts repeated at
5 Hz and each tetanus including one ten-burst train). Responses
were recorded for 90 min after tetanization and plotted as
percentage of baseline fEPSP slope.
1 Na2HPO4,25 NaHCO3,
Figure 4. Model depicting the mechanisms by which Thr668may lead to memory and synaptic plasticity deficits. (a and b), Due to loss
of BRI2 protein, APP processing is increased during synaptic transmission and memory acquisition in FDD leading to increased production of ß-CTF.
This event compromises synaptic plasticity and memory acquisition leading to memory deficits. (c), Thr668is essential for the pathogenic role of ß-
CTF, as shown by the evidence that mutating this residue into an Ala prevents development of memory/synaptic deficits. (d–f), Phosphorylation of
Thr668may be required or facilitate the synaptic-toxic role of ß-CTF, since the Thr668Ala mutation prevents phosphorylation.
Essential Role of APP-Thr668 in Neurodegeneration
PLOS ONE | www.plosone.org6 February 2013 | Volume 8 | Issue 2 | e57120
Statistical Analysis Download full-text
All data are shown as mean 6 s.e.m. Experiments were
performed in blind. Statistical tests included two-way ANOVA for
repeated measures and t-test when appropriate.
We thank Alessia P.M. Barbagallo for technical help.
Conceived and designed the experiments: LD. Performed the experiments:
LD FL FB RT. Analyzed the data: LD FL FB RT. Contributed reagents/
materials/analysis tools: OA. Wrote the paper: LD.
1. Bertram L, Lill CM, Tanzi RE (2010) The genetics of Alzheimer disease: back to
the future. Neuron 68: 270–281.
2. Vidal R, Frangione B, Rostagno A, Mead S, Revesz T, et al. (1999) A stop-
codon mutation in the BRI gene associated with familial British dementia.
Nature 399: 776–781.
3. St George-Hyslop PH, Petit A (2005) Molecular biology and genetics of
Alzheimer’s disease. C R Biol 328: 119–130.
4. Matsuda S, Giliberto L, Matsuda Y, Davies P, McGowan E, et al. (2005) The
familial dementia BRI2 gene binds the Alzheimer gene amyloid-beta precursor
protein and inhibits amyloid-beta production. J Biol Chem 280: 28912–28916.
5. Fotinopoulou A, Tsachaki M, Vlavaki M, Poulopoulos A, Rostagno A, et al.
(2005) BRI2 interacts with amyloid precursor protein (APP) and regulates
amyloid beta (Abeta) production. J Biol Chem 280: 30768–30772.
6. Matsuda S, Matsuda Y, Snapp EL, D’Adamio L (2011) Maturation of BRI2
generates a specific inhibitor that reduces APP processing at the plasma
membrane and in endocytic vesicles. Neurobiol Aging 32: 1400–1408.
7. Matsuda S, Giliberto L, Matsuda Y, McGowan EM, D’Adamio L (2008) BRI2
inhibits amyloid beta-peptide precursor protein processing by interfering with
the docking of secretases to the substrate. J Neurosci 28: 8668–8676.
8. Vidal R, Revesz T, Rostagno A, Kim E, Holton JL, et al. (2000) A decamer
duplication in the 39 region of the BRI gene originates an amyloid peptide that is
associated with dementia in a Danish kindred. Proc Natl Acad Sci U S A 97:
9. Israel MA, Yuan SH, Bardy C, Reyna SM, Mu Y, et al. (2012) Probing sporadic
and familial Alzheimer’s disease using induced pluripotent stem cells. Nature
10. Tamayev R, Matsuda S, Arancio O, D’Adamio L (2012) beta- but not gamma-
secretase proteolysis of APP causes synaptic and memory deficits in a mouse
model of dementia. EMBO Mol Med 4: 171–179.
11. Garringer HJ, Murrell J, D’Adamio L, Ghetti B, Vidal R (2009) Modeling
familial British and Danish dementia. Brain Struct Funct.
12. Giliberto L, Matsuda S, Vidal R, D’Adamio L (2009) Generation and Initial
Characterization of FDD Knock In Mice. PLoS One 4: e7900.
13. Tamayev R, Matsuda S, Fa M, Arancio O, D’Adamio L (2010) Danish
dementia mice suggest that loss of function and not the amyloid cascade causes
synaptic plasticity and memory deficits. Proc Natl Acad Sci U S A 107: 20822–
14. Tamayev R, Matsuda S, Giliberto L, Arancio O, D’Adamio L (2011) APP
heterozygosity averts memory deficit in knockin mice expressing the Danish
dementia BRI2 mutant. EMBO J 30: 2501–2509.
15. Matsuda S, Tamayev R, D’Adamio L (2011) Increased AbetaPP processing in
familial Danish dementia patients. J Alzheimers Dis 27: 385–391.
16. Tamayev R, Matsuda S, D’Adamio L (2012) beta - but not gamma-secretase
proteolysis of APP causes synaptic and memory deficits in a mouse model of
dementia. Mol Neurodegener 7 Suppl 1: L9.
17. Tamayev R, D’Adamio L (2012) Inhibition of gamma-secretase worsens
memory deficits in a genetically congruous mouse model of Danish dementia.
Mol Neurodegener 7: 19.
18. Tamayev R, Akpan N, Arancio O, Troy CM, L DA (2012) Caspase-9 mediates
synaptic plasticity and memory deficits of Danish dementia knock-in mice:
caspase-9 inhibition provides therapeutic protection. Mol Neurodegener 7: 60.
19. Scheinfeld MH, Ghersi E, Davies P, D’Adamio L (2003) Amyloid beta protein
precursor is phosphorylated by JNK-1 independent of, yet facilitated by, JNK-
interacting protein (JIP)-1. J Biol Chem 278: 42058–42063.
20. Ando K, Iijima KI, Elliott JI, Kirino Y, Suzuki T (2001) Phosphorylation-
dependent regulation of the interaction of amyloid precursor protein with Fe65
affects the production of beta-amyloid. J Biol Chem 276: 40353–40361.
21. Tamayev R, Zhou D, D’Adamio L (2009) The interactome of the Amyloid
betaeta Precursor Protein family members is shaped by phosphorylation of their
intracellular domains. Mol Neurodegener 4: 28.
22. Balastik M, Lim J, Pastorino L, Lu KP (2007) Pin1 in Alzheimer’s disease:
multiple substrates, one regulatory mechanism? Biochim Biophys Acta 1772:
23. Shin RW, Ogino K, Shimabuku A, Taki T, Nakashima H, et al. (2007) Amyloid
precursor protein cytoplasmic domain with phospho-Thr668 accumulates in
Alzheimer’s disease and its transgenic models: a role to mediate interaction of
Abeta and tau. Acta Neuropathol 113: 627–636.
24. Barbagallo AP, Wang Z, Zheng H, D’Adamio L (2011) The intracellular
threonine of amyloid precursor protein that is essential for docking of Pin1 is
dispensable for developmental function. PLoS One 6: e18006.
25. Diamond DM, Park CR, Heman KL, Rose GM (1999) Exposing rats to a
predator impairs spatial working memory in the radial arm water maze.
Hippocampus 9: 542–552.
26. Matsushima T, Saito Y, Elliott JI, Iijima-Ando K, Nishimura M, et al. (2012)
Membrane-microdomain localization of amyloid beta-precursor protein (APP)
C-terminal fragments is regulated by phosphorylation of the cytoplasmic Thr668
residue. J Biol Chem 287: 19715–19724.
27. Pastorino L, Sun A, Lu PJ, Zhou XZ, Balastik M, et al. (2006) The prolyl
isomerase Pin1 regulates amyloid precursor protein processing and amyloid-beta
production. Nature 440: 528–534.
28. Sano Y, Nakaya T, Pedrini S, Takeda S, Iijima-Ando K, et al. (2006)
Physiological mouse brain Abeta levels are not related to the phosphorylation
state of threonine-668 of Alzheimer’s APP. PLoS ONE 1: e51.
29. Barbagallo AP, Weldon R, Tamayev R, Zhou D, Giliberto L, et al. (2010)
Tyr682 in the Intracellular Domain of APP Regulates Amyloidogenic APP
Processing In Vivo. PLoS One 5: e15503.
30. Vitale M, Renzone G, Matsuda S, Scaloni A, D’Adamio L, et al. (2012)
Proteomic characterization of a mouse model of familial Danish dementia.
J Biomed Biotechnol 2012: 728178.
31. Scheinfeld MH, Matsuda S, D’Adamio L (2003) JNK-interacting protein-1
promotes transcription of A beta protein precursor but not A beta precursor-like
proteins, mechanistically different than Fe65. Proc Natl Acad Sci U S A 100:
32. Scheinfeld MH, Roncarati R, Vito P, Lopez PA, Abdallah M, et al. (2002) Jun
NH2-terminal kinase (JNK) interacting protein 1 (JIP1) binds the cytoplasmic
domain of the Alzheimer’s beta-amyloid precursor protein (APP). J Biol Chem
33. Roncarati R, Sestan N, Scheinfeld MH, Berechid BE, Lopez PA, et al. (2002)
The gamma-secretase-generated intracellular domain of beta-amyloid precursor
protein binds Numb and inhibits Notch signaling. Proc Natl Acad Sci U S A 99:
34. Matsuda S, Matsuda Y, D’Adamio L (2003) Amyloid beta protein precursor
(AbetaPP), but not AbetaPP-like protein 2, is bridged to the kinesin light chain
by the scaffold protein JNK-interacting protein 1. J Biol Chem 278: 38601–
35. D’Ambrosio C, Arena S, Fulcoli G, Scheinfeld MH, Zhou D, et al. (2006)
Hyperphosphorylation of JNK-interacting protein 1, a protein associated with
Alzheimer disease. Mol Cell Proteomics 5: 97–113.
36. Zhou D, Noviello C, D’Ambrosio C, Scaloni A, D’Adamio L (2004) Growth
factor receptor-bound protein 2 interaction with the tyrosine-phosphorylated tail
of amyloid beta precursor protein is mediated by its Src homology 2 domain.
J Biol Chem 279: 25374–25380.
37. Tarr PE, Roncarati R, Pelicci G, Pelicci PG, D’Adamio L (2002) Tyrosine
phosphorylation of the beta-amyloid precursor protein cytoplasmic tail promotes
interaction with Shc. J Biol Chem 277: 16798–16804.
38. Russo C, Dolcini V, Salis S, Venezia V, Zambrano N, et al. (2002) Signal
transduction through tyrosine-phosphorylated C-terminal fragments of amyloid
precursor protein via an enhanced interaction with Shc/Grb2 adaptor proteins
in reactive astrocytes of Alzheimer’s disease brain. J Biol Chem 277: 35282–
39. Tamayev R, Zhou D, D’Adamio L (2009) The interactome of the amyloid beta
precursor protein family members is shaped by phosphorylation of their
intracellular domains. Mol Neurodegener 4: 28.
40. Zhou D, Zambrano N, Russo T, D’Adamio L (2009) Phosphorylation of a
tyrosine in the amyloid-beta protein precursor intracellular domain inhibits Fe65
binding and signaling. J Alzheimers Dis 16: 301–307.
41. Matrone C, Luvisetto S, La Rosa LR, Tamayev R, Pignataro A, et al. (2012)
Tyr682 in the Abeta-precursor protein intracellular domain regulates synaptic
connectivity, cholinergic function, and cognitive performance. Aging Cell 11:
42. Barbagallo AP, Weldon R, Tamayev R, Zhou D, Giliberto L, et al. (2010)
Tyr(682) in the intracellular domain of APP regulates amyloidogenic APP
processing in vivo. PLoS One 5: e15503.
43. Bevins RA, Besheer J (2006) Object recognition in rats and mice: a one-trial non-
matching-to-sample learning task to study ‘recognition memory’. Nat Protoc 1:
Essential Role of APP-Thr668 in Neurodegeneration
PLOS ONE | www.plosone.org7February 2013 | Volume 8 | Issue 2 | e57120