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: firstname.lastname@example.org
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 PIMPLIKAR335
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