T H E J O U R N A L O F C E L L B I O L O G Y
The Journal of Cell Biology, Vol. 171, No. 2, October 24, 2005 327–335
The Rockefeller University Press$8.00
Activation of GSK-3 and phosphorylation of
CRMP2 in transgenic mice expressing APP
Kathleen A. Ryan and Sanjay W. Pimplikar
Department of Pathology and Cell Biology Program, Case Western Reserve University, Cleveland, OH 44106
myloid precursor protein (APP), implicated in
Alzheimer’s disease, is a trans-membrane pro-
tein of undetermined function. APP is cleaved by
-secretase that releases the APP intracellular domain
(AICD) in the cytoplasm. In vitro studies have implicated
AICD in cell signaling and transcriptional regulation, but
its biologic relevance has been uncertain and its in vivo
function has not been examined. To investigate its func-
tional role, we generated AICD transgenic mice, and
found that AICD causes significant biologic changes in
vivo. AICD transgenic mice show activation of glycogen
CRMP2 protein, a GSK-3
substrate that plays a crucial
role in Semaphorin3a-mediated axonal guidance. Our
data suggest that AICD is biologically relevant, causes
significant alterations in cell signaling, and may play a
role in axonal elongation or pathfinding.
) and phosphorylation of
Amyloid precursor protein (APP), a cell surface protein of un-
known function, is implicated in the pathogenesis of Alzhei-
mer’s disease (AD) (Price et al., 1998; Annaert and De Strooper,
2002; Selkoe, 2005). APP topology resembles that of a mem-
brane receptor protein; it has a large extracellular portion, a sin-
gle transmembrane segment, and a cytoplasmic tail domain that
interacts with several proteins, including Fe65. Although the
function of APP is not understood completely, it has been impli-
cated in a variety of processes, including signal transduction, cell
migration, and axonal elongation (see De Strooper and Annaert,
2000). APP is cleaved initially by
the extracellular portion and generates membrane-associated
COOH-terminal fragments (APP-CTFs) that are cleaved further
-secretase within the plane of the membrane. The
age results in the extracellular secretion of P3 or 40/42 residue–
peptides (which accumulate in amyloid plaques in AD
brains), and simultaneous release of the APP intracellular do-
main (AICD) within the cell. The function of AICD or the rele-
-secretase cleavage in APP biology is unknown.
The generation of AICD peptide follows the general
steps of “regulated intramembrane proteolysis” which re-
-secretase, which sheds
sults in the release of a membrane-tethered transcriptional
regulator in response to an external signal (Brown et al.,
2000). We and other investigators have shown that cleaved
AICD enters the nucleus and regulates gene expression in
vitro (Cao and Sudhof, 2001; Gao and Pimplikar, 2001;
Baek et al., 2002). The AICD target genes are not firmly
known, and a majority of support for its transcriptional role
comes from the use of an artificial reporter gene. Although
additional in vitro studies showed that AICD also alters cell
signaling (Leissring et al., 2002) and induces apoptosis
(Passer et al., 2000; Kinoshita et al., 2002), the physiologic
relevance of AICD has been uncertain because its steady-
state levels are reported to be low (Cupers et al., 2001; Kim-
berly et al., 2001). To examine the in vivo role of AICD, we
generated transgenic mice that express AICD and Fe65 in
the forebrain and hippocampal regions of the postnatal brain.
We report that the transgenic mice show two- to threefold
higher levels of AICD than control mice, and display robust
activation of glycogen synthase kinase-3
creased phosphorylation of a downstream substrate, CRMP2, a
key component of the axonal guidance signaling pathway.
We also demonstrate the presence of endogenous AICD in
the membrane fractions from control mice, which suggests
that the steady-state levels of AICD are higher than previ-
ously believed. Together, our in vivo findings support a bio-
logic role for AICD in regulating gene expression and cell
) and in-
Correspondence to Sanjay W. Pimplikar: email@example.com
Abbreviations used in this paper: AD, Alzheimer’s disease; AICD, APP intracel-
lular domain; APP, amyloid precursor protein; CRMP2, collapsin responsive
mediator protein–2; CTF, COOH-terminal fragment; ERK, extracellular signal-
regulated kinase; FAD, familial Alzheimer’s disease; GSK, glycogen synthase
kinase; Sema3a, Semaphorin3a.
JCB • VOLUME 171 • NUMBER 2 • 2005328
AICD transgenic mice
To examine the in vivo effects of AICD, we generated double
transgenic mice expressing AICD and Fe65. The steady-state
levels of AICD are reported to be exceedingly low. Ectopically
expressed AICD is turned over rapidly in tissue culture cells,
but can be stabilized when coexpressed with Fe65 (Cupers et
al., 2001; Kimberly et al., 2001). Fe65 is a cytoplasmic protein
that binds the “Y
ENPTY” motif in APP cytoplasmic domain
through its PTB2 domain (Borg et al., 1996). The binding of
Fe65 to holo-APP at the plasma membrane was proposed to reg-
ulate cell migration and control the growth cone movement in
the neurons (Sabo et al., 2001, 2003). Fe65 also is required for
the transcriptional activity of AICD (Cao and Sudhof, 2001;
Baek et al., 2002). Therefore, we reasoned that coexpression
of Fe65 might be required to stabilize AICD and to observe its
full effects in transgenic animals. The specificity of AICD ef-
fects can be determined by comparing the AICD
transgenic mice with Fe65 single transgenic animals. We used
promoter to drive the transgene expression, be-
cause its activity is restricted to forebrain and hippocampal re-
gions of the brain (Abel et al., 1997), the areas that are affected
widely in AD. Moreover, CaMKII
only at the 2-wk postnatal stage, thus avoiding possible lethal
side effects during embryonic development. We expressed the
59-residue long AICD peptide, which is a product of “
age” of APP. APP-CTF also undergoes “
generates a 50-residue long AICD (Gu et al., 2001; Yu et al.,
2001). The present study focused on characterizing the in vivo
activity of AICD59 (referred to here as AICD).
We cloned myc-tagged Fe65 or AICD in plasmid NN265
that contained intron and SV40 polyadenylation sequences
(Abel et al., 1997). A fragment that contained the intron, the
promoter becomes active
transgene open reading frame, and polyA signal was excised
and cloned into MM403, downstream of the 8-kb CaMKII
promoter (Fig. 1 A). We mixed AICD and Fe65 expressing
plasmids in 1:1 proportion, and co-injected the linearized plas-
mids into oocytes of C57BL/6 mice. Injected oocytes were im-
planted in pseudopregnant C57BL/6 mice; by PCR on tail
DNA, 9 out of 49 pups obtained were found to have incorpo-
rated both transgenes. All 9 founder mice were mated with
C57BL/6 mice. Germline transmission was observed in five
lines, of which four of the founder lines transmitted both trans-
genes to F1 pups (unpublished data). In the current study, we
present data from two of these four independent lines (named
.12 and FeC
.25). The fifth line, called Fe.27, did not
transmit the AICD transgene to pups (Fig. 1 B), and thereby,
fortuitously created a Fe65 single transgenic line. The expres-
sion levels of Fe65 transgene were determined by Western blot
analysis. Total brain homogenates (40
two animals from each transgenic line or two nontransgenic lit-
termates was separated by SDS-PAGE on a 10% gel, trans-
ferred to a nitrocellulose membrane, and probed using anti-myc
antibodies to detect the transgene or anti-Fe65 3H6 antibody to
detect total Fe65 (endogenous
signal was apparent in mice from all three transgenic lines, but
was absent in nontransgenic littermates (Fig. 1 C, top panel).
The total levels of Fe65 in the three transgenic lines (Fig. 1 D;
top panel) were comparable, and were approximately twice as
high as the nontransgenic control animals, when normalized for
the levels of tubulin (Fig. 1 E). The Fe65 levels in Fe.27 mice
were not significantly different from those in FeC
.25 mice (P
0.04 by Bonferroni/Dunn test).
g protein each) from
transgene). The myc-Fe65
AICD transgene levels in transgenic mice
We next determined the AICD levels by Western blot follow-
ing a protocol (see “Materials and Methods”) described by
The horizontal lines with arrows shows the location of transgene specific primers. (B) A PCR reaction on tail DNA isolated from three pups from Fe.27 line
from three different litters (lanes 1–3) using Fe65 (left) or AICD primers (right) was performed together with primers for mouse Xist gene. Lanes denoted
“?” contained DNA from the founder mouse (Fe.27). Note that none of the pups carries the transgene for AICD. (C and D) Western blot analysis of brain
homogenates from two animals from double transgenic lines (FeC?.12 and FeC?.25), single Fe65 transgenic line (Fe.27), and nontransgenic littermate
controls. Blots were probed with anti-myc 9E10 (C; top panel) or anti Fe65 antibody 3H6 (D; top panel), and visualized by ECL. The blots were stripped
and reprobed with anti-tubulin DM1A antibody as an internal control (bottom panels). (E) Quantitative analysis of total Fe65 levels as detected by 3H6 an-
tibody. Protein levels were normalized to tubulin by reprobing the same blots after stripping. Quantification from three independent experiments. Values
are the mean ? SEM; n ? 6. Fe65 levels in FeC?.12 and FeC?.25 mice were significantly different from nontransgenic (nTg) animals (P ? 0.0001), but
not from Fe.27 mice (P ? 0.04) by Bonferroni/Dunn test.
Generation and characterization of double transgenic FeC? and single Fe65 transgenic mice. (A) Construction of AICD and Fe65 transgenes.
CELL SIGNALING CHANGES IN AICD TRANSGENIC MICE • RYAN AND PIMPLIKAR329
Pinnix et al. (2001), using antibody 0443, which is highly
sensitive in detecting AICD. We fractionated total brain ho-
mogenates into cytosol (Fig. 2 A), and membrane or nuclear
fractions (Fig. 2 B); 20
g protein was separated on a
NuPAGE Bis-Tris 4–12% gel and probed with antibody 0443
that was raised against the COOH-terminal 20 residues of
APP. As a positive control, we loaded cell extract from COS-1
cells that were cotransfected with AICD and Fe65 (lane 1).
We readily detected AICD59 in the cytosolic fraction from
.25 mice (Fig. 2 A, top panel: lanes 6 and 7). A longer ex-
posure of the same blot also revealed the presence of AICD in
.12 mice (bottom panel: lanes 4, 5), but not in the cyto-
solic fractions from Fe.27 (lanes 8 and 9) or nontransgenic con-
trol animals (lanes 2 and 3). The expression levels of AICD
transgene parallel those of Fe65 transgene because FeC
mice express lower levels of both transgenes compared with
.25 animals (Fig. 1 C).
We next determined the presence of AICD in the mem-
brane and nuclear fractions (Fig. 2 B) from the brains of these
animals. Whereas AICD is detected in the cytosolic fraction of
only AICD transgenic mice (Fig. 2 B, upper panel), an AICD
co-migrating band was observed in the membrane fractions of
all animals (middle panel), and seemed to be present in slightly
higher levels in the transgenic mice (lanes 4–7) compared with
the control or Fe.27 mice. This finding is unexpected, because it
is believed that the endogenously present AICD in brain only
can be detected with a combined approach of immunoprecipita-
tion and immunoblot. To rule out the possibility that this band
was recognized nonspecifically by antibody 0443, we probed
brain membrane fractions from APP knock-out (KO) mice
and R1.40 transgenic mice that overexpress human APP with
“Swedish mutation” by two- to threefold (Lamb et al., 1997).
Antibody 0443 recognized the full-length APP in FeC
(Fig. 2 C, top panel), which was present in increased amounts in
R1.40 mice (lanes 6 and 7) and absent in APP KO mice (lanes
4 and 5). Similarly, APP-CTF fragments were detected in
.12 and R1.40 mice, but not in the APP-KO mice (middle
panel). The AICD59 (lane 1) co-migrating band was present
only in FeC
.12 and R1.40 mice, but not in APP-KO mice
(arrow, middle panel). These data show that the co-migrating
band is recognized specifically by antibody 0443, and must be
an APP product because it is absent in APP-KO mice.
To understand why the membrane-associated AICD was
not detected in previous studies, we separated membrane pro-
teins from R1.40 brains in duplicate lanes on the same gel, and
electrophoretically transferred them onto a nitrocellulose mem-
brane. The membrane was cut lengthwise in two. One part re-
ceived the antigen retrieval treatment (see “Materials and
Methods”), whereas the other part was kept in PBS at room
temperature. Both blots were blocked in 10% calf serum and
processed identically. Fig. 2 D shows that although the AICD
band is detected clearly upon antigen retrieval (arrow, lanes 4
and 5), it is not detected in the absence of the treatment, even
though APP-CTFs are clearly visible (lanes 2 and 3). We veri-
fied these results further by comparing brain membranes from
R1.40 mice with control C57BL/6 mice. R1.40 mice, which
produce increased amounts of A
peptides (Lamb et al., 1997),
the membrane fraction. (A) Cytosolic proteins from control (con) or two
transgenic mice from indicated lines were separated on a 4–12% Bis-Tris
NuPAGE gel, and the blots were probed with antibody 0443 after antigen
retrieval as described in “Methods and materials.” Cell lysate from COS-1
cells cotransfected with AICD and Fe65 was run as a positive control (lane
?). AICD band is clearly visible in FeC?.25 mice (top panel, lanes 6 and
7), whereas a longer exposure shows the AICD protein present in FeC?.12
mice (bottom panel, lanes 4 and 5), but not in control (lanes 2 and 3) or
Fe.27 mice (lanes 8 and 9). (B) Brain homogenates from two control (con)
transgenic mice from indicated lines were separated into cytosolic (Cyto.),
membrane (Memb.), or nuclear (Nucl.) fractions, and the blots were probed
with antibody 0443 as indicated above. Cell lysate from COS-1 cells
cotransfected with AICD and Fe65 was run as a positive control (lane ?).
Although AICD was detected in the cytosol of only FeC? transgenic mice
(arrow), the membrane and nuclear fractions of all animals showed the
AICD co-migrating band (arrows). (C) The AICD co-migrating band is ab-
sent in APP-KO mice. Brain membranes from two FeC?.12 mice (lanes 2
and 3), APP-KO mice (lanes 4 and 5), and R1.40 transgenic mice express-
ing human APP with “Swedish mutation” (lanes 6 and 7) were probed with
antibody 0443, as described above, on a NuPAGE gel. Top panel shows
that the APP band is present in FeC?.12 and in higher amounts in R1.40
mice, but is absent in APP-KO mice. Similarly, the middle panel shows the
absence of APP-CTFs in the APP-KO mice. Note that the AICD co-migrating
band (arrow) also is absent in APP-KO mice. (D) The AICD peptide becomes
detectable only after antigen retrieval treatment (ART). Equal amounts of
brain membrane proteins from two R1.40 mice were separated in duplicate
on a NuPAGE gel. After electrophoretic transfer, the membrane was cut
lengthwise to give two identical gels. One was exposed to boiling PBS for
5 min (ART), whereas the other was kept in PBS at room temperature (con).
Arrow shows the AICD band, which is detectable only in the ART samples.
(E) AICD band (arrow) is present in increased amounts in R1.40 transgenic
mice compared with the controls (con) and is absent in APP-KO mice. Mem-
brane fractions from indicated mice were probed with antibody 0443.
AICD transgenic mice show barely detectable levels of AICD in
JCB • VOLUME 171 • NUMBER 2 • 2005 330
also should give rise to increased amounts of AICD. AICD was
present in greater amounts in R1.40 mice (Fig. 2 E, lanes 4 and
5) compared with controls (lanes 2 and 3), and was absent in
APP-KO mice (lanes 6 and 7).
Together, these results show that the endogenous AICD
can be detected in the membrane, but not the cytosolic fractions,
of wild-type C57BL/6 animals (Fig. 2 B, also compare Fig. 2 E
with Fig. 2 A). More importantly, the AICD transgenic mice ex-
press AICD at greater levels than do nontransgenic control mice,
and similar to those levels observed in APP transgenic mice with
familial Alzheimer’s disease (FAD) mutation (Fig. 2 C). This
lends strong support to the validity of our transgenic model.
Validation of AICD transgenic mice
Although no bona fide target genes of AICD have been identi-
fied genetically, a chromatin immunoprecipitation assay was
used recently to show that AICD is recruited to
moter and stimulates its transcription (Baek et al., 2002; Von
Rotz et al., 2004). We sought to validate our animal model by
examining the expression of
mice. We probed the membrane fractions from nontransgenic lit-
termate controls, two FeC
transgenic lines, and Fe.27 animals
with anti-KAI1 antibody (Fig. 3 A, top panel). A doublet of
KAI1 protein band was visible in FeC
(lanes 3–6), but not in nontransgenic controls (lanes 1 and 2) nor
in Fe65 transgenic Fe.27 mice (lanes 7 and 8). When normalized
for protein loading (Fig. 3 A, bottom panel), FeC
express higher levels of KAI1 compared with FeC
data confirm and extend in vivo the previous observations that
gene expression (Baek et al., 2002; Von
Rotz et al., 2004). These findings also validate the supposition
-secretase cleaved, 59-residue long AICD exhibits bio-
logic effects that are observed when APP is cleaved in vivo to
generate AICD (Cao and Sudhof, 2001; Baek et al., 2002).
Although no gene, other than KAI1, has been shown con-
clusively to be an AICD target, a study implicated AICD in
regulating genes that are involved in Ca
et al., 2002). Therefore, we examined the levels of SERCA 2b,
a ubiquitously expressed Ca-ATPase that maintains the ER
levels, in AICD transgenic mice. Membrane fractions
from animals were analyzed by Western blotting using an anti-
body that specifically detects mouse SERCA 2b in nonmus-
cle tissue. We detected no consistent, significant changes in
SERCA 2b levels in FeC
.12 or FeC
control animals (Fig. 3 B), when normalized for protein load-
ing. SERCA 2b levels in Fe.27 mice were variable. Although it
is possible that a small increase in SERCA 2b levels was not
detected in the whole brain membrane fraction, a recent study
(Mueller et al., 2004) also found no differences in SERCA 2b
levels in AICD expressing cells by DNA microarray (Muller,
T., and R. Egensperger, personal communication).
gene in the AICD transgenic
.12 and FeC
.25 seem to
.25 mice compared with
Elevated levels of active glycogen
synthase kinase–3 in transgenic mice
Glycogen synthase kinase (GSK)–3
Thr kinase that is implicated in AD pathogenesis (Jope and
is a proline-directed Ser/
Johnson, 2004). In vitro observations suggest that ectopic ex-
pression of AICD results in a significant increase in the mRNA
and protein levels of GSK-3
(Kim et al., 2003; Von Rotz et
al., 2004). To examine whether AICD activates GSK-3
vivo, we determined the status of GSK-3 by using antibodies
that recognize the activated or inhibited forms of the enzyme.
Phosphorylation of GSK-3
at Y216 stimulates its kinase ac-
tivity (active form), whereas phosphorylation at S9 potently re-
presses (inactive form) the kinase activity (Jope and Johnson,
2004). Brain cytosolic fractions were immunoblotted with anti-
body anti-pY216/279 that recognizes the phospho-Y216 on
and the equivalent Y279 residue on GSK-3
shows that activated forms of GSK-3
.12 and FeC
.25 mice (top panel, lanes 3–6) when
compared with nontransgenic controls (lanes 1 and 2) or Fe.27
mice (lanes 7 and 8). Densitometric analysis revealed (Fig. 4
1.7-fold increase in pGSK-3
showed similar changes). We also analyzed the total
levels by stripping the blots and reprobing with anti-
bodies that recognize GSK-3
changes in the protein levels in transgenic mice compared with
the control animals (Fig. 4 A, bottom panel).
We corroborated these findings further by determining
the levels of the inactive form of GSK-3
body that specifically detects phospho-Ser9–GSK-3
double transgenic mice showed a dramatic reduction in the
levels as compared with Fe.27 mice or nontrans-
genic littermates (Fig. 4 C, lanes 3–6, top panel), whereas the
levels of total GSK-3
remained unaltered (bottom panel). A
reduction in pS9–GSK-3
levels also was observed in a differ-
ent double transgenic line (FeC
tification of these data shows that the levels of pS9–GSK-3
were reduced by 70% as compared with controls (Fig. 4 D).
Together, these data show that low levels of AICD are able to
. Fig. 4 A
levels in FeC
and observed no significant
by using an anti-
.22; unpublished data). Quan-
transgenic mice, but not the control or Fe.27 mice, show expression of KAI1
gene. Membrane fractions from indicated mice were probed with anti-KAI1
antibody. FeC?.25 mice showed higher expression of KAI1 protein com-
pared with FeC?.12 mice. (B) Membrane fractions from indicated mice
were probed with anti-SERCA 2b antibody. No significant changes in
SERCA 2b levels were reproducibly observed in FeC? transgenic mice.
Increased KAI1 levels in AICD transgenic mice. (A) The AICD
CELL SIGNALING CHANGES IN AICD TRANSGENIC MICE • RYAN AND PIMPLIKAR331
In addition to the GSK-3 pathway, the extracellular signal-
regulated kinase (ERK) pathway also is activated in AD brains
(Zhu et al., 2003). To determine the selectivity of the AICD ef-
fect, we examined the levels of phosho-ERK1 and -ERK2 in
the transgenic animals. Brain homogenates from FeC
and nontransgenic littermate animals were immunoblotted
using pERK antibodies that recognize activated ERK1 and
ERK2. We observed no significant differences in the levels of
activated ERK1 or ERK2 (Fig. 4 E) in control or transgenic
mice. Thus, these findings show that the ERK is not activated
in 8–12-wk old FeC
mice, and that AICD does not cause ERK
activation. The lack of AICD on ERK activation is consistent
with the reports the ERK activation occurs by way of A
tides (Bell et al., 2004). However, the possibility cannot be
ruled out that AICD modulates some other signaling pathways.
GSK-3? mRNA levels are not increased
in transgenic mice
In contrast to the in vitro observations that AICD causes an in-
crease in the GSK-3? protein levels in tissue culture cells, we
did not observe an increase in the GSK-3? protein levels in the
transgenic mice (Fig. 4, A and C). To examine this discrepancy
in detail, we measured the mRNA levels in brain extracts by
real-time PCR. Total RNA was extracted from brain tissue,
subjected to reverse transcriptase reaction, and real-time PCR
was performed. We used primers for GSK-3? and KAI1, and
normalized the values by using ?-actin. FeC?.12 mice showed
a 1.8-fold increase in KAI1 message when compared with non-
transgenic or Fe.27 mice (Fig. 5 A). By contrast, we detected
no change in GSK-3? transcripts when compared with non-
transgenic or Fe.27 mice (Fig. 5 B). These observations suggest
that GSK-3? activation in FeC? transgenic mice is not due to
increased transcription or translation of GSK-3? gene. Because
GSK-3? kinase activity is regulated largely at the posttransla-
tional level by upstream kinases and phosphatases (Jope and
Johnson, 2004), it is possible that AICD activates GSK-3? by
modulating upstream regulators. However, because the trans-
genes are expressed only in certain neuronal cells, we cannot
rule out the possibility that a slight increase in GSK-3? mRNA
or protein was undetected in whole brain extracts.
Phosphorylation of collapsin responsive
mediator protein–2 in AICD transgenic
Many proteins are phosphorylated by GSK-3? on multiple Ser/
Thr residues; the microtubule binding proteins, tau, microtu-
bule-associated protein 1B, and collapsin responsive mediator
protein–2 (CRMP2), are among the known GSK-3 substrates.
These proteins bind and stimulate microtubule stability, but
they fail to bind microtubules upon phosphorylation and cause
microtubule depolymerization (Fukata et al., 2002; Jope and
Johnson, 2004). We examined the status of CRMP2, a neu-
blot analysis of brain cytosol from animals from indicated lines was probed with anti-GSK antibody (pY279/216) that specifically recognizes the activated
forms of GSK-3? and -3? enzymes (top panel). Note that mice from both AICD transgenic lines show higher levels of activated GSK-3? and -3? (lanes 3–6)
as compared with control (con; lanes 1 and 2) or Fe.27 mice (lanes 7 and 8). Total GSK-3? protein levels are not changed (bottom panel). (B) Quantitative
analysis of phospho–GSK-3? levels in transgenic and control mice. Quantification of GSK-3? levels gave similar results (not depicted). Protein levels were
normalized to tubulin by reprobing the same blots after stripping. This experiment was repeated twice, and was performed on animals from an independent
FeC?.22 line. Values are the mean ? SEM; n ? 6. *, P ? 0.05 against nontransgenic (nTg) or Fe.27 mice by Fisher’s PLSD test. (C and D) AICD transgenic
mice show a dramatic reduction in the levels of inhibited form of GSK-3?, as detected by pS9–GSK-3? antibody (top panel). The total GSK-3? levels were
not changed (bottom panel). Quantitative analysis of data in shown in (D). The protein levels were normalized to tubulin. This experiment was repeated
twice and performed on animals from an independent FeC?.22 line. Values are the mean ? SEM; n ? 6. **, P ? 0.001 against nontransgenic or Fe.27
mice. (E) Activated ERK1 and ERK2 levels are not altered significantly in transgenic mice as detected by pERK1/2 antibodies.
The AICD transgenic mice show activated GSK-3 levels. (A) Higher levels of activated form of GSK-3? and -3? in AICD transgenic mice. Immuno-
JCB • VOLUME 171 • NUMBER 2 • 2005332
ronal-specific protein that is involved in axonal repulsion and
neuronal polarity, in our transgenic mice. CRMP2 mediates the
repulsive action of Semaphorin3a (Sema3a), which binds the
Plexin receptors in growth cones, activates GSK-3?, and phos-
phorylates CRMP2 on T509 and S522 (Uchida et al., 2005). An
antibody, 3F4, which originally was raised against the neu-
rofibrillary tangles (NFTs) from AD brains (Yoshida et al.,
1998; Gu et al., 2000), specifically recognizes CRMP2 phosphor-
ylated at T509 and S522 (Uchida et al., 2005). We examined
the phosphorylation status of CRMP2 in our FeC? transgenic
mice by using the following antibodies: antibody 3F4 to detect
CRMP2 phosphorylated at T509 and S522 (Uchida et al.,
2005); antibody pT514 to recognize CRMP2 phosphorylated at
T514 (Yoshimura et al., 2005); and antibody C4G that recog-
nizes total CRMP2 (Gu et al., 2000). We immunoblotted brain
cytosolic fractions from the indicated mice with antibody 3F4,
and observed that the levels of phosphoCRMP2 were signifi-
cantly higher in FeC?.12 and FeC?.25 animals compared with
nontransgenic control or Fe.27 mice (Fig. 6 A). We stripped the
blot and reprobed with antibody C4G that recognizes total
CRMP2, and found that the total levels of CRMP2 were unal-
tered (Fig. 6 B). Quantification of the protein levels showed a
two- to threefold increase in phosho-CRMP2 in the AICD trans-
genic mice compared with Fe.27 or normal mice (Fig. 6 C)
CRMP2 also is phosphorylated at T514 by GSK-3? when
primed by Cdk5; this phosphorylation event plays a crucial role
in determining axonal versus dendritic fate of hippocampal neu-
rites (Yoshimura et al., 2005). We used a pT514-specific anti-
body to test whether phosphorylation of CRMP2 at T514 also
was stimulated in FeC? mice. Fig. 6 D (top panel) shows that the
levels of pT514-CRMP2 were not increased in our FeC? double
transgenic animals. We stripped the blot and reprobed with anti-
tubulin antibody to show that equal amounts of proteins were
loaded (Fig. 6 D, bottom panel). Together, these findings show
that the phosphorylation of T509/S522, but not of T514, residue
in CRMP2 protein is induced in FeC? double transgenic mice.
The biologic relevance of AICD to APP physiology or AD pa-
thology has been proposed, but in vivo evidence that supports
the hypothesis has been lacking. The current study was aimed at
examining this hypothesis by expressing AICD postnatally and
selectively in the forebrain and hippocampal regions of mouse
brain. Here, we first show that AICD is present in brain mem-
branes from normal control mice and can be detected by West-
ern blot alone. We further demonstrate that the transgenic mice
that were generated in this study express AICD at levels that are
similar to those found in APP transgenic mice with FAD muta-
tion, which are two- to threefold higher than in nontransgenic
mice. These data strongly support the validity of our mouse
model, which was verified further by observing increased ex-
pression of KAI1 gene in transgenic mice. Finally, we present
evidence that the AICD transgenic mice display robust changes
in GSK-3 signaling, which supports an in vivo role for AICD.
The finding that an AICD59–co-migrating peptide is pres-
ent in the membrane fraction of the normal, wild-type C57BL/6
animals is novel and unanticipated because it was not detected
in previous studies. We provide compelling data, by using
various controls, that the membrane-associated, AICD59–
co-migrating band is a product of APP. These include APP KO
mice, which do not show the presence of AICD, and APP trans-
genic mice with FAD mutation, which show elevated AICD
levels compared with control animals. The AICD peptide is not
detected by Western blotting in the absence of antigen-retrieval
treatment; this may explain why previous studies failed to detect
AICD. Antigen-retrieval techniques have been used routinely in
immunohistochemical detection of antigens in tissue sections,
and our present findings indicate that such techniques also can be
useful in Western blotting. The absence of AICD in the cytosol
and its association with the membrane suggest that the AICD
peptide is hydrophobic in nature. Studies that reported the
“?-cleavage” of APP-CTF found the resultant AICD peptide
(AICD50) to be soluble and to fractionate in the cytosolic, but
not in the membrane, fraction (Gu et al., 2001; Yu et al., 2001).
These considerations make the AICD–co-migrating band that
was identified in the present study more likely to be AICD59/57
than AICD50. The possibility that the endogenous band repre-
sents AICD57, rather than AICD59, cannot be ruled out. Future
studies are required to establish unequivocally the length of the
endogenous, membrane-associated AICD. In any case, our find-
ings show that the steady-state levels of endogenously produced
AICD peptides are higher than previously believed.
The FeC? double transgenic mice that were generated in
the current study showed robust stimulation of GSK-3? activ-
ity, without any significant changes in the mRNA or GSK-3?
protein levels. These changes are caused primarily by AICD,
from at least three animals was isolated, subjected to reverse tran-
scriptase, and real-time PCR was performed using BioRad iCycler and
primers for KAI1 gene (A) or GSK-3? gene (B). Mouse ?-actin primers
were used as an internal control. Data are expressed as levels of KAI1 or
GSK-3? transcripts relative to ?-actin. Values are the mean ? SEM; n ? 6.
P ? 0.08 for KAI1 transcript levels in FeC?.12 against nontransgenic or
Fe.27 mice by Fisher’s PLSD test.
Analysis of mRNA transcript levels by real-time PCR. Total RNA
CELL SIGNALING CHANGES IN AICD TRANSGENIC MICE • RYAN AND PIMPLIKAR333
because the Fe.27 mice express similar levels of Fe65 (Fig. 1 E)
but do not exhibit altered GSK-3 signaling. However, it is pos-
sible that AICD may not be able to activate GSK-3 without
Fe65. We do not know how AICD stimulates the GSK-3? ac-
tivity in mice. Because the levels of mRNA transcripts and to-
tal protein of GSK-3? are not changed in the transgenic mice,
our findings suggest a posttranscriptional signaling role for
AICD in GSK-3 activation. Recently, Cao and Sudhof (2004)
suggested that AICD acts catalytically in the context of mem-
brane to “activate” Fe65, and bring about its biologic effects
without necessarily being present in the nucleus. We detected
AICD in the membrane and nuclear fractions in transgenic and
control mice (Fig. 2 B). The presence of AICD in the membrane
fraction is consistent with the above hypothesis. Nonetheless,
the presence of AICD in the nuclear, but not in the cytosolic
fraction of control mice shows that AICD accumulates in the
nucleus and likely performs its function. Incidentally, our
transgenic mice demonstrate the biologic relevance of the
“?-cleaved” AICD because they show that AICD59 peptide in
vivo replicates the known activities of AICD (expression of
KAI1 gene and activation of GSK-3?). Future studies on
AICD50 transgenic mice will be required to determine the bio-
logic effects of the “?-cleaved” AICD peptide, which is not
likely to remain associated with membranes.
The finding that AICD stimulates GSK-3 activity in vivo
could be relevant to AD pathology. GSK-3? activity is elevated
in AD brains (Pei et al., 1997; Kaytor and Orr, 2002; Bhat et al.,
2004) and in the “Swedish mutation” mouse model, Tg2576, of
AD (Tomidokoro et al., 2001). Higher GSK-3 activity in AD
brains is responsible for phosphorylating the microtubule-binding
protein, tau (Augustinack et al., 2002). Hyperphosphorylated tau
aggregates into paired helical filaments and forms NFTs, a hall-
mark of AD pathology (Pei et al., 1999; Ishizawa et al., 2003).
Significantly, the spatiotemporal distribution of active GSK-3?
in AD brains coincides with the progression of NFTs and neuro-
degeneration, but not with the amyloid deposition (Pei et al.,
1997; Pei et al., 1999). Thus, activated GSK-3? found in AD
brains is one of the critical factors in the formation of NFTs. It is
not clear what induces GSK-3? activation in AD brains, and our
transgenic mice suggest that AICD could be a contributing factor.
We observed elevated AICD levels in R1.40 transgenic mice that
carry the “Swedish mutation” (Fig. 2), and our preliminary find-
ings show that GSK-3 also is activated in these animals (unpub-
lished data); however, R1.40 mice also show increased A? load.
Because some in vitro studies suggest that A? can activate GSK-
3?, it will be crucial to determine the contribution of these two
APP-derived fragments toward GSK-3 activation in AD. We
have initiated analysis of phospho-tau, a known substrate of
CRMP2 phosphorylated at T509 and S522. Note the elevated levels of phospho-CRMP2 in FeC? transgenic mice compared with Fe.27 or nontransgenic
control mice. (B). The blot used above was stripped and reprobed with antibody C4G, which recognizes total CRMP2. The levels of total CRMP2 are not
changed. (C) Quantitative analysis of phospho-CRMP2 levels as detected by 3F4 antibody. Protein levels were normalized to tubulin by reprobing the
same blots after stripping. Quantification from three independent experiments. Values are the mean ? SEM; n ? 6. *, P ? 0.05 against nontransgenic
(nTg) or Fe.27 mice by Fisher’s PLSD test. (D) Levels of CRMP2 phosphorylated at T514 were not changed in transgenic mice (top panel). The blot was
stripped and reprobed with antitubulin antibodies as a loading control (bottom panel). (E) A hypothetical cascade of signaling events, similar to Sema3a
signaling pathway, suggests a role for APP in axonal guidance. The cleavage of APP to release AICD activates GSK-3? and results in phosphorylation of
CRMP-2 at S522 and T509–the same residues that mediate the repulsive action of Sema3a upon binding to Plexin/Neuropilin receptors. F-spondin, an
extracellular signaling protein of floor plate and hippocampus which is involved in axonal pathfinding and neurite outgrowth, could be a candidate
signaling protein (shown by “?”) because it binds APP and inhibits AICD production.
Phosphorylation of CRMP2, a GSK-3? substrate, in transgenic mice. (A) Brain cytosol fractions were probed with antibody 3F4, which recognizes
JCB • VOLUME 171 • NUMBER 2 • 2005334
GSK-3 in FeC? transgenic mice. In the young animals that we
examined so far (8–12 weeks old), we did not detect any signifi-
cant changes in tau phosphorylation as detected by antibodies
AT-8, AT-100, and TG-3 (not shown). It is not an uncommon ob-
servation that abnormal tau phosphorylation is detected in older
animals, but not in young animals (Andorfer et al., 2003).
The FeC? transgenic mice described here recapitulated the
known in vitro effects of AICD, and revealed a new observation
that AICD induces phosphorylation of CRMP2 on the residues
that are crucially involved in growth cone collapse and axonal
guidance. The phosphorylation of T509/S522 in CRMP2 plays a
crucial role in mediating the repulsive action of Sema3a (Uchida
et al., 2005). Sema3a is an extracellular matrix protein that binds
Plexin/Neuropilin receptors in growth cones, activates GSK-3?,
and phosphorylates CRMP2 on T509 and S522. These two resi-
dues are crucial in axonal guidance, because mutation of either
residue abolishes the action of Sema3a and eliminates 3F4-reac-
tivity (incidentally, 3F4-reactive phospho-CRMP2 is known to
accumulate in the NFTs in AD brains [Yoshida et al., 1998; Gu
et al., 2000; Cole et al., 2004]). Thus, increased phosphorylation
of CRMP2 in the transgenic mice suggests an AICD-mediated
signaling role for APP in axonal guidance or elongation (Fig.
6 E). The identity of an upstream ligand that binds APP and ini-
tiates the signaling cascade is unknown. F-spondin, a contact-
repellent protein that is present in floor plate and hippocampus,
may be a candidate because it binds APP and inhibits AICD pro-
duction (Ho and Sudhof, 2004). The proposed role of AICD in
mediating APP signaling and axonal elongation is consistent
with the cortical dysplasia that is observed in mice lacking all
three members of the APP family (Herms et al., 2004), or Fe65
and Fe65L1 proteins (Guenette et al., 2003).
Materials and methods
A cDNA encoding the 59 residue–long amyloid precursor protein–
COOH-terminal fragment (APP-CTF) cleaved at the ?-secretase site was
cloned into the vector NN265. A fragment containing an upstream intron,
the APP intracellular domain (AICD)59 ORF, and a downstream SV40
polyadenylation signal was excised from this plasmid and cloned into
MM403 plasmid containing the 5? regulatory region of the CaMKII? gene
(both plasmids were the gift of T. Abel, University of Pennsylvania, Phila-
delphia, PA). Full-length cDNA encoding the entire ORF of rat Fe65 (gift of
B. Margolis, University of Michigan, Ann Arbor, MI) was cloned as
above. The transgenic constructs were linearized by digesting with BssHII
(mixed in 1:1 proportion), microinjected into the male pronucleus of
C57Bl/6, and implanted into pseudopregnant C57Bl/6 females. Founder
mice were identified by PCR on tail DNA, and by using a forward primer
(F1) in the intron region (GCGCTAAGATTGTCAGTTTCC) and reverse
primer for AICD-R1 (TCTGCTGCATCTTGGACAGG) or Fe65-R1 (ACATT-
TCGGTTCTGGTCTCG). The sequence of primers that was used for the
mouse endogenous Xist gene was as follows: forward: GGGACCTAACT-
GTTGGCTTTATCAG, reverse: GAAGTGAATTGAAGTTTTGGTCTAG. We
also performed PCR using primers in the SV40 polyadenylation signal to
ensure that the complete transgene was integrated.
The data presented here are from multiple 8–12-wk old, mixed gender an-
imals from two double transgenic lines (FeC?.12 and FeC?.25) and a sin-
gle Fe65 transgenic line (Fe.27). We used nontransgenic littermates as
controls. Mice were killed by cervical dislocation and their brains were re-
moved, divided sagittally (after removing cerebellum), and quick frozen.
Tissues were homogenized in 10 volumes of Tris-buffered saline (50 mM
Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA) with freshly added 1 mM
PMSF and protease and phosphatase inhibitor cocktail (Sigma-Aldrich).
Total homogenates were centrifuged briefly to remove nuclei and unbro-
ken tissue, and centrifuged at 150,000 g for 60 min to obtain cytosol (su-
pernatant) and crude membrane (pellet) fractions. The nuclear extracts
were prepared using the NE-PER kit (Pierce Chemical Co.). Protein deter-
mination was performed using a Bio-Rad Laboratories kit. Equal amounts
of proteins (usually 10–25 ?g per lane) were loaded on the gel.
Immunoblotting was performed as described before (Zheng et al., 1998;
Gao and Pimplikar, 2001) using a 10% SDS-PAGE gel, except for AICD
detection, which was performed as described by Pinnix et al. (2001), with
some modifications. In brief, the proteins were separated using NuPAGE
Novex Bris-Tris 4–12% gel (Invitrogen) and transferred on to nitrocellulose
membrane. The membranes were incubated with boiling PBS for 5 min (an-
tigen-retrieval treatment) before blocking in TBS containing 10% newborn
calf serum for 2 h at room temperature. The blots were incubated with up to
1:10,000 dilution of antibody 0443 (gift of K. Sambamurti, Medical Uni-
versity of South Carolina, Charleston, SC) overnight at 4?C, and the blots
were visualized using ECL. Other antibodies used are as follows: antibody
369 (gift of S. Gandy) to probe AICD; anti-myc antibody 9E10 (CLON-
TECH Laboratories, Inc.) to detect myc-Fe65 transgene; and antibody 3H6
(Upstate Biotechnology) to detect total Fe65 (this antibody detects mouse
and rat Fe65). Anti-pS9–GSK-3? antibody to detect inhibited kinase was
from Cell Signaling; anti-pY279/216 to detect activated GSK-3 (Cell Sig-
naling) was a gift of M. Smith (Case Western Reserve University, Cleveland
OH); and SERCA2 antibody, MA3-919, was from Affinity BioReagents, Inc.
Antibody 3F4 recognizes CRMP2 phosphorylated at S522 and T509, and
antibody C4G binds unphosphorylated CRMP2 (both antibodies were the
gifts of Y. Ihara and Y. Morishima, University of Tokyo, Tokyo, Japan). Anti-
body that recognizes pT514 CRMP2 was a gift of K. Kaibuchi. Anti-
pERK1/2 antibody was from Cell Signaling. Protein bands were visualized
by ECL using Pierce SuperSignal system. Protein levels were quantified by
scanning the films in Photoshop (Adobe), and measuring the band pixel
densities in NIH Image J software (National Institutes of Health). The results
were analyzed by ANOVA with Fisher’s protected least significance differ-
ence (PLSD) test using StatView software (Abacus Concepts). The data pre-
sented here are from multiple animals from FeC?.12 and FeC?.25 lines and
were repeated in the FeC?.22 transgenic line.
Quantitative real-time PCR
Animals were killed by cervical dislocation, brains were dissected, cere-
bellum was discarded, and total RNA was isolated from the brain tissue
with RNeasy kit (QIAGEN). First-strand cDNA synthesis was performed us-
ing 4 ?g RNA with the Superscript First-Strand Synthesis system for RT-PCR
(Invitrogen) according to the manufacturer’s protocol. The cDNA mixture
was diluted 1:20; 5 ?l of the cDNA product was used for real-time PCR
performed on the iCycler (Bio-Rad Laboratories), and detected by using iQ
SYBR Green Supermix. The amplification cycle was 95?C for 10 s, 65?C
for 15 s, and 72?C for 15 s. Primers were designed by using Primer 3 soft-
ware to amplify the 3? region of the ORF. Primer sequences were: ?-actin
(forward: TACAGCTTCACCACCACAGC; reverse: ATGCCACAGGATTC-
CATACC), KAI1 (forward: TCTGTGGGAGACAGGGTAGG; reverse: CTG-
CCAAGAAACACCAGTCC), and GSK-3? (forward: TCCATTCCTTTG-
GAATCTGC; reverse: CAATTCAGCCAACACACACAGC). Melting curve
analysis confirmed that only one product was amplified. Specificity was
confirmed by electrophoresis of PCR products through 1.5% agarose gels
(stained with ethidium bromide). Only one product was observed with
each primer set, and the product size matched that predicted from pub-
lished cDNA sequences. Expression was normalized to ?-actin.
We thank the two anonymous reviewers whose comments resulted in the vast
improvement in this manuscript. We thank B. Lamb for R1.40 mice, his advice
in generating the AICD transgenic mice, and for his comments on the manu-
This work was supported by a grant from the Alzheimer’s Association
to S.W. Pimplikar (IIRG-3894) and by a grant from the National Institute on
Aging to UAC/CWRU ADRC (P50-AG08012).
Submitted: 13 May 2005
Accepted: 16 September 2005
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