FMRP Mediates mGluR5-Dependent Translation
of Amyloid Precursor Protein
Cara J. Westmark*, James S. Malter
Department of Pathology and Laboratory Medicine, Waisman Center for Developmental Disabilities, University of Wisconsin, Madison, Wisconsin, United States of America
Amyloid precursor protein (APP) facilitates synapse formation in the developing brain, while beta-amyloid (Ab)
accumulation, which is associated with Alzheimer disease, results in synaptic loss and impaired neurotransmission.
Fragile X mental retardation protein (FMRP) is a cytoplasmic mRNA binding protein whose expression is lost in fragile X
syndrome. Here we show that FMRP binds to the coding region of APP mRNA at a guanine-rich, G-quartet–like
sequence. Stimulation of cortical synaptoneurosomes or primary neuronal cells with the metabotropic glutamate
receptor agonist DHPG increased APP translation in wild-type but not fmr-1 knockout samples. APP mRNA
coimmunoprecipitated with FMRP in resting synaptoneurosomes, but the interaction was lost shortly after DHPG
treatment. Soluble Ab40or Ab42levels were significantly higher in multiple strains of fmr-1 knockout mice compared to
wild-type controls. Our data indicate that postsynaptic FMRP binds to and regulates the translation of APP mRNA
through metabotropic glutamate receptor activation and suggests a possible link between Alzheimer disease and
fragile X syndrome.
Citation: Westmark CJ, Malter JS (2007) FMRP mediates mGluR5-dependent translation of amyloid precursor protein. PLoS Biol 5(3): e52. doi:10.1371/journal.pbio.0050052
Alzheimer disease (AD) is a neurodegenerative disorder
characterized by senile plaques and neurofibrillary tangles.
The plaques are predominantly composed of beta-amyloid
(Ab), a 39–42 amino acid peptide cleaved from the amyloid
precursor protein (APP). APP is likely important for synapse
formation in the developing brain , while excess Ab causes
impaired synaptic function . Disordered synaptic trans-
mission is also a hallmark of other neuronal disorders, such as
epilepsy and fragile X mental retardation syndrome (FXS).
FXS is the most prevalent form of inherited mental
retardation, affecting one in 4,000 men and one in 8,000
women. This X chromosome–linked disorder is characterized
by moderate to severe mental retardation (overall IQ ,70),
autistic-like behavior, seizures, facial abnormalities (large,
prominent ears and long, narrow face) and macroorchidisim
. At the neuroanatomic level, FXS is distinguished by an
overabundance of long, thin, tortuous dendritic spines with
prominent heads and irregular dilations [4,5]. The increased
length, density, and immature morphology of dendritic
spines in FXS suggest an impairment of synaptic pruning
In the majority of cases, FXS results from a trinucleotide
(CGG) repeat expansion to .200 copies in the 59-UTR of the
fmr-1 gene (located at Xq27.3) . The CGG expansion is
associated with hypermethylation of the surrounding DNA,
chromatin condensation, and subsequent transcriptional
silencing of the fmr-1 gene, resulting in the loss of expression
of fragile X mental retardation protein (FMRP) .
FMRP is an mRNA-binding protein that is ubiquitously
expressed throughout the body, with significantly higher
levels in young animals . The protein has two heteroge-
neous nuclear ribonucleoprotein (hnRNP) K homology
domains and one RGG box as well as nuclear localization
and export signals. FMRP interacts with BC1 RNA as well as a
number of RNA-binding proteins, including nucleolin and
YB1 and the FMRP homologs FXR1 and FXR2 . FMRP has
been implicated in translational repression [10–15], and in
the brain, cosediments with both translating polyribosomes
 and with mRNPs . The RGG box of FMRP binds to
intramolecular G quartet sequences in target mRNAs ,
while the KH2 domain has been proposed to bind to so-called
kissing complex RNAs based on in vitro selection assays .
In addition, FMRP binds to uridine-rich mRNAs [19,20]. In
aggregate, more than 500 mRNA ligands for FMRP have been
identified, many with the potential to influence synaptic
formation and plasticity [10,17].
FMRP is required for type 1 metabotropic glutamate
receptor (mGluR)–dependent translation of synaptic pro-
teins, including FMRP and postsynaptic density 95 (PSD-95)
[21,22]. Both PSD-95 and FMRP mRNAs contain putative G-
quartets in their 39-UTR and coding sequence, respectively
[22,23]. Database searches revealed that APP mRNA possesses
a G-quartet–like motif in the coding region (position 825–846
of the mouse sequence) embedded within a guanine-rich
domain (694–846) containing several DWGG repeats. APP
mRNAs (70% of APP695 and 50% of APP751/770) are
associated with polyribosomes in rat brain , suggesting
that translational regulation could play an important role in
Academic Editor: Mark F. Bear, Massachusetts Institute of Technology, United
States of America
Received March 8, 2006; Accepted December 18, 2006; Published February 13,
Copyright: ? 2007 Westmark and Malter. 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.
Abbreviations: AD, Alzheimer disease; APP, amyloid precursor protein; Ab, beta-
amyloid; ELISA, enzyme-linked immunosorbent assay; FMRP, fragile X mental
retardation protein; FXS, fragile X syndrome; IP, immunoprecipitate; KO, knockout;
mGluR, metabotropic glutamate receptor; PSD-95, postsynaptic density 95 protein;
RNP, ribonucleoprotein; RTqPCR, real-time quantitative PCR; SN, synpatoneuro-
* To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
PLoS Biology | www.plosbiology.org March 2007 | Volume 5 | Issue 3 | e520629
P PL Lo oS S BIOLOGY
APP production. Indeed, APP contains a 59-UTR iron
response element previously implicated in translation control
. Therefore, we hypothesized that APP mRNA translation
would be regulated by FMRP.
We now show that after stimulation with the mGluR
agonist DHPG, APP levels increased significantly in wild-type
(WT) but not synaptoneurosomes (SNs) or cultured neurons
from knockout (KO) animals. In KO SNs or neurons, APP was
constitutively elevated. APP mRNA coimmunoprecipitated
with FMRP in WT, resting SNs, but this interaction was lost
with DHPG treatment. FMRP monomer bound to the 59 end
of the G-rich sequence in the coding region of APP mRNA.
Our data indicate that FMRP represses the translation of APP
through mGluR-dependent interactions with APP mRNA.
Consistent with constitutively elevated APP levels, the
proteolytic products Ab40and Ab42are elevated in the brains
of fmr-1 KO mice compared to WT.
APP mRNA Coimmunoprecipitates with Anti-FMRP
Our laboratory has shown that FMRP and PSD-95 mRNAs
are rapidly translated in mouse primary cortical neurons in
response to the type 1 mGluR agonist DHPG . Normal
regulation was lost in fmr-1 KO-derived neurons, implicating
FMRP in this process. These and other FMRP-regulated
mRNAs contain G-quartets, which have been proposed as at
least one site of mRNA/FMRP interaction . Database
searches of brain mRNAs revealed that the coding region of
human, mouse, and rat APP mRNAs contained a G-quartet–
like sequence (Figure 1A) within a G-rich domain containing
several DWGG repeats (Figure 1B). The putative G-quartet
motif in APP mRNA has the potential to form a stable
structure containing three guanine planes (Figure S1). FMRP
binds to G-rich sequences (so-called G-quartets; consensus
site: DWGG-N(0–2)-DWGG-N(0–1)-DWGG-N(0–1)-DWGG, where
D is any nucleotide except C and W is A or U)  arranged in
a planar conformation and stabilized by Hoogsteen-type
hydrogen bonds. In the human APP mRNAs, the G-rich
region containing the putative G-quartet motif is found in all
three splice variants (APP695, APP751, and APP770; 87
nucleotides upstream of the sequence coding for the
Kunitz-type protease inhibitor domain, which is missing in
APP695). FMRP also binds to kissing complex sites , but
APP mRNA lacks such a site. Therefore, we prepared cortical
lysates as well as SNs from WT mice and immunoprecipitated
FMRP. Contrary to a previous report utilizing a different
protocol for the preparation of SNs , APP mRNA is
present in SNs, and reverse transcription (RT)–PCR revealed
that the message was brought down with specific, but not
control, antisera in cortical lysates as well as SNs (unpub-
lished data). Thus, APP mRNA is a potential target of FMRP,
presumably via the coding region putative G-quartet.
FMRP Regulates APP Translation
APP is highly expressed in neurons and dendrites and may
promote synaptic maturation . Conversely, overexpression
of APP and its proteolytic product, Ab, have been implicated
in the synaptic losses seen early in the development of AD
. Therefore, we asked if APP translation was regulated by
dendritic FMRP. We utilized fmr-1 KO mice, a rodent model
for FXS, that display dendritic spine anomalies similar to that
in the human disorder [28–30]. Cortical SNs were prepared
from both WT and fmr-1 KO mice, and overall protein
synthesis was analyzed in response to DHPG (100 lM)–
induced mGluR activation. SNs from either animal were
metabolically active with equivalent total
ration (Figure 2). Therefore, FMRP was not required for basal
protein synthesis, which is in agreement with a prior report
. However, we did not observe an increase in overall
protein synthesis in response to DHPG, whereas Weiler and
colleagues  observed a 1.3-fold increase in
incorporation after 5 min of stimulation.
To assess de novo synthesis,
were immunoprecipitated with anti-APP. After 15 min of
incubation, untreated WT SNs translated modest amounts of
APP, which rapidly increased by 2.7-fold with DHPG treat-
ment. After 1 hr, APP remained elevated in stimulated SNs
over the control, but the difference was less (1.6-fold) than at
15 min, suggesting more persistent translation in the
unstimulated controls, slowing of new synthesis after stim-
ulation, and/or compensatory protein turnover in the DHPG-
treated samples (Figure 3A and 3B). In KO SNs, APP synthesis
was less than in WT SNs and showed a minimal response to
DHPG. The translational inhibitor anisomycin blocked
DHPG-mediated synthesis of APP, as did the specific mGluR5
inhibitor MPEP (Figure 3C and 3D).
In order to assess changes in steady-state levels, rather than
new protein synthesis, APP was measured in WT and KO SNs
in response to DHPG by Western blot analysis (Figure 4). In
WT SNs, there was a rapid increase in total APP levels within
5 min of DHPG treatment (1.6-fold, n ¼ 3), which was
completely absent in KO SNs. Regardless of treatment, APP
levels remained nearly constant over time in KO SNs, as did
b-actin. In the absence of DHPG, steady-state levels of APP
were substantially higher in KO SNs compared to WT SNs.
Within 20 min of DHPG treatment, APP levels in WT SNs
approached those seen in unstimulated KO SNs (Figure 4).
Protease inhibitors increased steady-state levels of APP in WT
SNs to those seen in KO SNs (unpublished data). These data
35S-labeled WT or KO SNs
Alzheimer disease (AD) and fragile X syndrome (FXS) are devastating
neurological disorders associated with synaptic dysfunction result-
ing in cognitive impairment and behavioral deficits. Despite these
similar endpoints, the pathobiology of AD and FXS have not
previously been linked. We have established that translation of
amyloid precursor protein (APP), which is cleaved to generate
neurotoxic bamyloid, is normally repressed by the fragile X mental
retardation protein (FMRP) in the dendritic processes of neurons.
Activation of a particular subtype of glutamate receptor (mGluR5)
rapidly increases translation of APP in neurons by displacing FMRP
from a guanidine-rich sequence in the coding region of APP mRNA.
In the absence of FMRP, APP synthesis is constitutively increased
and nonresponsive to mGluR-mediated signaling. Excess APP is
proteolytically cleaved to generate significantly elevated bamyloid
in multiple mutant mouse strains lacking FMRP compared to wild
type. Our data support a growing consensus that FMRP binds to
guanine-rich domains of some dendritic mRNAs, suppressing their
translation and suggest that AD (neurodegenerative disorder) and
FXS (neurodevelopmental disorder) may share a common molecular
pathway leading to the overproduction of APP and its protein-
PLoS Biology | www.plosbiology.orgMarch 2007 | Volume 5 | Issue 3 | e520630
FMRP Regulates APP Translation
suggest that APP mRNA is translationally repressed by FMRP
in unstimulated WT SNs. mGluR activation rapidly dere-
presses APP synthesis as shown for FMRP and PSD-95 [21,22].
APP levels during maximal derepression approach those seen
constitutively in fmr-1 KO cells. After the cessation of mGluR
signaling, APP levels presumably drop due to degradation,
which appears more robust in WT than KO cells.
FMRP Regulates Dendritic APP Levels in Cultured Neurons
SNs are a relatively crude preparation of pre- and
postsynaptic densities that are contaminated with other cell
types, such as astrocytes, which form synapses with neurons.
Thus, we prepared primary embryonic day–18 cortical
neuron cultures from WT and fmr-1 KO brains and assessed
dendritic APP levels by immunofluorescence. APP was found
in the cell body as well as dendritic puncta of both WT and
fmr-1 KO neurons (Figure 5A). There was a 21% increase in
the basal level of APP in untreated fmr-1 KO neurons
compared to WT (Figure 5B). Neurons stimulated with DHPG
for 10 and 20 min prior to cell fixation showed a 18%–25%
increase in dendritic APP levels in WT but no increase in fmr-
1 KO cultures (Figure 5B). These data confirm our findings in
SNs that (1) fmr-1 KO mice have higher basal synaptic levels of
APP, and (2) DHPG increases APP levels selectively in WT
samples. These data also demonstrate that FMRP and mGluR
activation regulate APP synthesis in both FVB and C57BL/6
mice, as the SNs were prepared from the former strain, and
the primary cortical neurons from the latter strain.
mGluR Activation Does Not Affect APP mRNA Stability
FMRP and homologs have been implicated in the control of
mRNA decay. There are increased APP mRNA levels in the
cerebral cortex, hippocampus, and cerebellum in a FXS
mouse model , and FXR1P, an FMRP homolog, is an AU-
rich element–binding protein that binds to and regulates
TNFa mRNA stability and translation . APP mRNA
contains two 39-UTR cis-elements within 200 bases of the
stop codon that mediate message stability. Hence, we
analyzed APP mRNA and 18S rRNA decay in SNs by real-
time PCR. APP mRNA did not decay over 120 min regardless
of mGluR activation in WT and KO SNs (Figure S2). These
data indicate that mGluR-dependent APP translation was
independent of mRNA stabilization. APP mRNA has a half-
life of approximately 5 h in resting immune cells, which is
prolonged in activated cells [34,35] or rat PC12 (Westmark
and Malter, unpublished data). Thus, APP mRNA decays with
comparable kinetics in SNs and mammalian cells.
mGluR Activation Dislodges APP mRNA from an mRNP
Complex Containing FMRP
The mechanism underlying FMRP-mediated translational
repression is controversial . Alterations in the association
of FMRP with polyribosomes, small nontranslated RNAs, or
Figure 1. The Coding Region of APP mRNA Contains a Putative G-Quartet Sequence within a G-Rich Region Containing Several DWGG Repeats
(A) Alignment of the canonical G-quartet motif with the putative G-quartet sequence in human, mouse, and rat APP mRNAs.
(B) Alignment of the G-rich region of the human, mouse, and rat APP genes. The predicted G-quartet sequence is located at position 825–846 of the
mouse gene and is highlighted.
Figure 2. SNs Prepared from WT and fmr-1 KO Cortices Are Translation-
SDS-PAGE analysis of35S-Met–labeled SNs without (C lanes) or with (D
lanes) DHPG stimulation for times shown in minutes. The data are
representative of multiple experiments: n ¼ 6 (WT); n ¼ 5 (KO).
PLoS Biology | www.plosbiology.org March 2007 | Volume 5 | Issue 3 | e520631
FMRP Regulates APP Translation
other proteins have all been proposed [9,12,37,38]. We asked
if the APP mRNA/FMRP interaction changed after DHPG.
Thus, FMRP was immunoprecipitated from WT SNs (60 min
after DHPG), and the pellet was reverse transcribed and
analyzed by real-time quantitative PCR (qPCR). APP mRNA
was readily detected in anti-FMRP pellets in untreated WT
SNs (Figure 6A). However, APP mRNA associated with FMRP
could not be detected in DHPG-stimulated WT SN immuno-
preciptates (IPs) or in the KO with or without DHPG within
40 cycles of real-time PCR. The negative controls for this
experiment were duplicate IPs in the absence of 7G1–1 FMRP
antibody, which also did not produce any real-time PCR Ct
values for APP mRNA within 40 cycles (data not shown). The
.60-fold difference in FMRP-associated APP mRNA was
highly significant. Evaluation at earlier times revealed that the
APP mRNA–FMRP complex was lost within 5 min of DHPG
treatment (unpublished data).
Immunoprecipitation of FMRP from WT SNs followed by
Western blotting (Figure 6B) or
analysis (unpublished data) demonstrated that DHPG treat-
ment does not interfere with the ability of anti–7G1–1
antibody to bind to FMRP. In fact, in both assays there was
more FMRP immunoprecipitated from the DHPG-treated
WT SNs, which is in agreement with previous reports that
DHPG stimulates the dendritic translation of FMRP [22,39].
Our data suggest that physical interactions between FMRP
and APP mRNA underlie translational repression, with
mGluR activation rapidly moderating these events. Presum-
ably, the loss of FMRP/APP mRNA interaction results in
rapid, pulsatile protein expression in dendrites.
FMRP RNP Binds to G-Rich Sequences
FMRP is a component of large RNP complexes . The
data presented here demonstrate that APP mRNA is also
associated with this RNP. To determine the likely interaction
site, in vitro RNase protection assays were performed on
FMRP IPs from whole-cortex lysates. Residual APP mRNA was
mapped by RTqPCR with primers immediately surrounding
the predicted G-quartet (Figure 7A). Surprisingly, the G-rich
area immediately preceding the G-quartet (nt 699–796) was
approximately 4-fold more protected from nuclease digestion
than fragments containing the predicted G-quartet (825–846).
Although this protected area does not contain a canonical G-
Figure 3. mGluR Activation Increases APP Translation in SNs
(A) Immunoprecipitated,35S-labeled APP (120-kDa band) from WT (15 min) and KO and WT (60 min) SNs analyzed by SDS-PAGE and (B) plotted as a
percentage of APP synthesis; n ¼ 3 repetitions. Asterisk indicates significant differences, with p ¼ 0.008 between 6DHPG samples at 15 min and p ¼
0.016 between control at 15 min and DHPG at 60 min. For the control samples at 15 and 60 min, p¼0.056, and for the samples with or without DHPG at
60 min, p ¼ 0.05. (C) Immunoprecipitated,35S-labeled APP (120-kDa band) from WT SNs treated with DHPG, anisomycin, and MPEP, analyzed by SDS-
PAGE, and (D) plotted as a percentage of APP synthesis; n¼3 repetitions (DHPG), n¼4 (anisomycinþDHPG and anisomycin), and n¼5 (MPEPþDHPG
Figure 4. Differential Regulation of APP Levels in WT and KO SNs
Western blots of WT (top panel) and KO (bottom panel) SN treated with
or without DHPG (5, 10, and 20 min) and hybridized with anti-APP and
anti–b-actin antibodies. The data are representative of three experi-
ments, and quantitation with ImageQuant software demonstrates a 1.6–
1.8-fold increase in APP between untreated and DHPG-stimulated WT
SNs at all of the times tested.
PLoS Biology | www.plosbiology.org March 2007 | Volume 5 | Issue 3 | e520632
FMRP Regulates APP Translation
quartet motif, the sequence is very G-rich and contains
several closely spaced DWGG repeats. The smallest amplicon
(nt 774–871) containing the predicted G-quartet motif
amplified a 98-base fragment, of which 46 nucleotides were
guanines (47% G-rich; Table S1). Although this is the most G-
rich amplicon of those tested, and T1 ribonuclease cuts 39 of
single-stranded G-residues, the 98-nt protected fragment
(amplicon 699–796) was 40% G-rich, providing nearly
equivalent numbers of targets for digestion. Thus, nucleotides
699–796 in the coding region of APP mRNA possess a G-rich
sequence that is protected from nuclease digestion by an RNP
complex containing FMRP.
The FMRP-containing RNP complex likely protects other
cis-elements in APP mRNA as well. Our laboratory has
defined a 29-base element located 200 nucleotides down-
stream of the stop codon in APP mRNA that regulates
message decay . We have also identified two proteins,
nucleolin and hnRNP C, that bind to this 29-base element
. Since nucleolin interacts with FMRP to form multi-
protein complexes , we would expect the 29-base element
to be protected from T1 ribonuclease digestion of anti-FMRP
IPs, as shown in Figure 7A (nt 2318–2416). Our data suggest
that multiple cis-regulatory elements of APP mRNA interact
with the FMRP-containing RNP complex.
FMRP Monomer Binds to APP mRNA
Despite the presence of an RNase-protected, G-rich
sequence, APP mRNA might not associate directly with
FMRP. To determine if this was the case and to further
characterize the interaction, we performed a modified CLIP
assay . SNs were cross-linked with ultraviolet light,
immunoprecipitated with anti-FMRP, digested with T1
ribonuclease, and analyzed by SDS-PAGE. FMRP immuno-
reactive material (80 kDa) was excised and analyzed by
RTqPCR. The amplicon encompassing the G-rich sequence
(nucleotides 699–796) of APP mRNA gave a positive signal
that was approximately 5-fold greater than that of the
predicted G-quartet motif–containing sequence (nt 774–
871) immediately downstream (Figure 7B). Thus, our data
define the G-rich region immediately preceding the predicted
G-quartet as the binding site between FMRP and APP mRNA.
The loss of FMRP binding at the G-rich region presumably
derepresses APP translation, as it was contemporaneous.
Soluble Ab40and Ab42Are Increased in fmr-1 KO
Increased translation of APP provides more targets for
cleavage by b- and c-secretases. Therefore, we would expect
fmr-1 KO mice to have exacerbated Ab production with aging.
Right-brain hemispheres from middle-aged FVB mice (11–13
mo old) were homogenized in protein extraction buffer
containing 1% Triton X-100 and protease inhibitors and the
soluble material was analyzed by enzyme-linked immuno-
sorbent assay (ELISA) for Ab40and Ab42. The fmr-1 KO mouse
brain contained 1.6 times more Ab40and 2.5 times more Ab42
than WT controls (Figure 8A). We also tested Ab40/42levels in
C57BL/6 mice (12–14 mo old) to ensure that this was not a
strain-specific event. We did not observe an increase in
soluble Ab40or Ab42levels in fmr-1 KO C57BL/6 brain samples
(unpublished data), but guanidine-soluble fractions showed a
2.8-fold increase in Ab40 and a 1.2-fold increase in Ab42
(Figure 8B). Therefore, the brains of two distinct murine
Figure 5. DHPG Enhances APP Translation in WT but Not fmr-1 KO Neurons
(A) Immunofluorescent confocal images of WT (top) and KO (bottom) neuronal cells treated with or without DHPG (0, 10, and 20 min) and hybridized
with anti-22C11 APP primary and anti-mouse rhodamine-conjugated secondary antibodies. The dashed yellow rectangles encompass segments of
dendrites, which are enlarged and displayed below the photos.
(B) Dendritic APP levels were quantitated with ImageJ software and plotted as a percentage of untreated WT samples. Asterisks indicate significant
differences, with p , 0.001 between the pairs.
PLoS Biology | www.plosbiology.org March 2007 | Volume 5 | Issue 3 | e520633
FMRP Regulates APP Translation
strains lacking fmr-1 both showed increased APP and
accumulated pathogenic Ab species over time.
Synaptic plasticity is required for normal learning and
memory and is impaired in FXS. High dendritic spine density
is normal for young mice, but synapse pruning during
postnatal development is absent in the KO, resulting in
increased spine density in adulthood . The molecular
basis for defective pruning in fmr-1 KO mice is unknown, but
likely reflects the loss of FMRP-regulated translation of
synaptic mRNA. FMRP regulates group 1 mGluR-dependent
translation of mRNA targets important in diverse neuronal
functions . For example, FMRP normally represses the
translation of microtubule-associated protein 1B (MAP1B)
mRNA during synaptogenesis. In FXS, MAP1B expression is
constitutively elevated, leading to abnormally increased
microtubule stability . Therefore, it is of great interest
to identify FMRP-dependent synaptic mRNAs that contribute
to dendritic structure and function.
Herein, we show that APP mRNA is a previously unappre-
ciated target for FMRP-mediated translational repression at
the synapse. The normal physiologic role of APP remains ill
defined, but increasing evidence suggests an important role
in synapse formation [44,45] and maturation . APP local-
izes to postsynaptic densities, axons, dendrites, and neuro-
muscular junctions [1,46]. APP/APP-like protein 2 double-KO
mice exhibit defective neuromuscular junctions, excessive
nerve terminal sprouting, and defective synaptic transmission
. APP is developmentally regulated with maximal ex-
pression during synaptogenesis and subsequently declines
when mature connections are completed [48,49]. Therefore,
synaptic overexpression of APP during early development
may contribute to the immature dendritic spines and
inadequate synaptic pruning characteristic of FXS.
We have identified a G-rich region located within
Figure 6. mGluR Activation Dislodges FMRP from APP mRNA
(A) APP mRNA was coimmunoprecipitated with FMRP from WT and KO
SNs with or without DHPG treatment for 60 min, analyzed by RTqPCR,
and plotted as the fold increase in APP mRNA. The data are the average
of three experiments.
(B) FMRP was immunoprecipitated from WT SNs with or without DHPG
for 60 min and analyzed by Western blotting. The data are representative
of two experiments.
Figure 7. FMRP Binds to a G-Rich Sequence in APP mRNA
(A) Relative positions of the G-rich, predicted G-quartet and 29 base
elements in nucleotides 446-2500 of APP (top). FMRP IPs digested with
ribonuclease T1, analyzed by RTqPCR, and plotted as a percentage of
(B) FMRP IPs analyzed by the modified CLIP method and plotted as a
percentage of APP699–796mRNA.
PLoS Biology | www.plosbiology.orgMarch 2007 | Volume 5 | Issue 3 | e520634
FMRP Regulates APP Translation
nucleotides 699–796 in the coding region of APP mRNA as an
FMRP-binding site. The G-quartet–like sequence immediately
downstream of this G-rich region was not protected from
nuclease digestions of FMRP IPs. This result was surprising
because the intramolecular G-quartet motif has been
identified by in vitro RNA selection assays as the site of
interaction with FMRP . As expected from the FMRP
interaction site mapping results, alignment of the G-rich
region (nt 699–796) and DWGG repeats of mouse APP mRNA
is highly conserved with both the human (86%) and rat (93%)
sequences. Our data suggest that there may be flexibility in
the spacing of the DWGG repeats for G-quartet formation
and agrees with previous findings that the presence of a G-
quartet does not ensure binding by FMRP .
We have determined that FMRP associates with APP mRNA
in SN preparations, and that translation of APP mRNA
increases in response to DHPG. DHPG-upregulated trans-
lation of APP can be blocked by the translational inhibitor
anisomycin or the mGluR5-specific inhibitor MPEP. mGluR-
mediated translation is concurrent with FMRP dissociation
from APP mRNA and is independent of mRNA decay. The
rapid dissociation of FMRP from APP mRNA, in response to
mGluR activation, suggests that post-translational modifica-
tions, such as phosphorylation, may regulate FMRP binding
activity. Ceman and colleagues have shown that FMRP is
phosphorylated N-terminal to the RGG box and that
phosphorylation/dephosphorylation status of the protein is
correlated with binding to stalled versus active polyribosomes
. Our data support a model developing in the literature
whereby FMRP acts as an immediate early-response protein
that regulates translation at the synapse. When FMRP is
bound to APP or other synaptic mRNAs, translation is
repressed. Upon mGluR activation, FMRP is released from
the nontranslating RNP, resulting in prompt protein syn-
thesis. In FXS, high levels of protein are constitutively
produced that are normally translationally repressed by
We would predict that constitutively upregulated APP
would lead to increased processing to Ab. Indeed, increased
Ab40and Ab42are present in two mouse models for FXS. To
date, the only abnormal neurpathologic observations in the
human FXS brain have involved impaired synaptic pruning
and maturation ; however, a very limited number of aged
FXS brains have been studied [4,29,52], so other neuro-
pathologies, such as increased amyloid burden and synaptic
degeneration normally associated with AD, cannot be
excluded. It is difficult to measure cognitive decline in
mentally retarded individuals; however, in support of our
prediction, fragile X–associated tremor/ataxia syndrome in
males is associated with dementia .
The normal physiologic function(s) of APP are not well
understood, albeit the protein is likely important for synapse
formation in the developing brain . A recent report
demonstrates that children with severe autism and aggression
express .2-fold more secreted bAPP (1,200 pg/ml) than
children without autism (500 pg/ml) . Many people with
FXS (67% of men and 23% of women) are also autistic .
Interestingly, the highest levels of secreted bAPP were found
in two children with FXS . Thus, overproduction of
secreted bAPP may contribute to the developmental dis-
abilities observed in patients with FXS and autism. In
addition, FMRP mRNA and protein expression are down-
regulated as a function of aging in the mouse brain ,
suggesting that repressed transcripts, such as APP, would be
upregulated with aging, a well-known phenomenon in
animals and humans.
In conclusion, FMRP represses translation of APP mRNA in
dendrites, suggesting a link between two neurodevelopmental
disorders, FXS and autism, and a neurodegenerative disease,
Materials and Methods
Materials. The anti-FMRP antibody (mAb7G1–1)  was obtained
from the Developmental Studies Hybridoma Bank, University of Iowa
(http://www.uiowa.edu/;dshbwww). The anti-APP polyclonal antibody
(catalog number 51–2700) was purchased from Zymed Laboratories
(http://www.invitrogen.com), and the anti-mouse b-actin antibody
(catalog number A5441), protease inhibitor cocktail (catalog number
P2714), ribonuclease T1 (catalog number R1003), and poly(D)-lysine
Figure 8. Increased Ab40and Ab42Levels in fmr-1 KO Mice
(A) Soluble brain lysates from 1-y-old WT and fmr-1 KO mice (FVB strain) analyzed by ELISA and plotted as a percentage of soluble Ab compared to WT
controls. Student t-tests: p ¼ 0.06 (Ab40) and p ¼ 0.001 (Ab42).
(B) GnHCl-soluble brain lysates from 1-y-old WT and fmr-1 KO mice (C57BL/6 strain) analyzed by ELISA and plotted as a percentage of GnHCl-soluble Ab
compared to WT controls. Student t-tests: p , 0.001 (Ab40) and p ¼ 0.39 (Ab42).
PLoS Biology | www.plosbiology.org March 2007 | Volume 5 | Issue 3 | e520635
FMRP Regulates APP Translation
(catalog number P6407) were purchased from Sigma Chemical
Company (http://www.sigmaaldrich.com). The anti-rabbit and anti-
mouse HRP-conjugated secondary antibodies, percoll, Redivue Pro-
Mix-L [35S] (catalog number AGQ0080) and enhanced chemilumi-
nescence detection reagents were obtained from Amersham Pharma-
cia (http://www5.amershambiosciences.com). Anti-22C11 APP
antibody (mAB348) was acquired from Chemicon (http://www.
chemicon.com). The rabbit polyclonal Ab40(catalog number 9131),
Ab42(catalog number 9134), and rodent Ab (catalog number 9154)
antibodies were purchased from Signet Laboratories (http://www.
signetlabs.com). DHPG (catalog number 0805) was obtained from
Tocris Cookson (http://www.tocris.com). Omniscript RT was acquired
from Qiagen (http://www.qiagen.com). The MagnaBind Protein A
beads, PAGEprep advance kit, and micro BCA protein assay reagent
kit were obtained from Pierce Biotechnology (http://www.piercenet.
com). DNA oligonucleotides were synthesized by Integrated DNA
Technologies (http://www.idtdna.com), and SYBR Green PCR master
mix was obtained from Applied Biosystems (http://www.
appliedbiosystems.com). NeuroBasal medium, B27 supplement, goat
anti-mouse rhodamine-conjugated antibody, and ProLong Gold
Antifade with DAPI were from Invitrogen (http://www.invitrogen.
com). TRI-Reagent was purchased from Molecular Research Center
(http://www.mrcgene.com). MPEP was purchased from Tocris Cook-
son or synthesized by Technically (http://www.technically.com) and
provided by FRAXA Research Foundation (http://www.fraxa.org).
Mouse husbandry. The WT and fmr-1 KO mice in the FVB and
C57BL/6 backgrounds were a generous gift from Aaron Grossman
and Dr. Bill Greenough (University of Illinois at Urbana-Champaign).
The fmr-1 KO mice were originally developed by Frank Kooy and
backcrossed .11 times to FVB mice, albeit these FVB mice have the
genes for pigmentation and normal vision . Mice were housed two
to four per microisolator cage on a 6am–6pm light cycle with ad
libitum food (Purina 5015 mouse diet; http://www.purina.com) and
water. The cages contained seeds and a neslet as the only sources of
environmental enrichment. All animal husbandry and euthanasia
procedures were performed in accordance with the National
Institutes of Health and an approved University of Wisconsin–
Madison animal care protocol through the Research Animal
Resources Center. fmr-1 genotypes were determined by PCR analysis
of DNA extracted from tail biopsies. The FVB strain was used for all
experiments described herein except for preparing the cultured
neuronal cells (Figure 5) and the Ab ELISAs (Figure 8B).
SN preparation and stimulation. SNs were prepared from WT and
fmr-1 KO mouse cortical tissue [57,58]. Briefly, mouse pups aged 14–
17 d were killed by carbon dioxide asphyxiation followed by removal
of the brain cortices. The cortices were washed in ice-cold gradient
medium (GM buffer: 0.25 M sucrose, 5 mM Tris [pH 7.5], and 0.1 mM
EDTA), transferred to a glass dounce homogenizer containing ice-
cold GM buffer, and gently homogenized with five strokes of the loose
pestle followed by five strokes of the tight pestle. The homogenate
was spun at 1000g for 10 min at 4 8C in round-bottom tubes to pellet
cellular debris and nuclei. The supernatant (2 ml aliquots) was
applied to percoll gradients (layers ¼ 2 ml each of 23%, 15%, 10%,
and 3% isomotic percoll) and spun at speed (32,500g) for 5 min at
48C. The third band from the top of the gradient (the 23%/15%
interface) containing intact SNs was removed and pooled for the
experiments. The two higher-molecular-weight bands at the 15%/
10% and 10%/3% interfaces contain broken membranes. The salt
concentration of the SNs was adjusted by adding one-tenth volume of
103stimulation buffer (100 mM Tris [pH 7.5], 5 mM Na2HPO4, 4 mM
KH2PO4, 40 mM NaHCO3, 800 mM NaCl). To suppress nonspecific
excitation, 1 lM tetrodotoxin was added. The protein concentration
of the SNs was determined by Bradford assay and ranged from 200–
SNs were equilibrated to room temperature by rotation on a
nutator mixer for a minimum of 10 min. DHPG was dissolved in 13
stimulation buffer immediately prior to use and added to the SNs
(100 lM final concentration). Samples were mixed at room temper-
ature in 1.5 ml Eppendorf tubes for the indicated times.
Radiolabeling SNs with35S-Met and immunoprecipitation of APP.
WT and KO SNs (450 ll) were mixed with 25 ll Redivue Pro-Mix-L
[35S] for 5 min prior to stimulation with 25 ll 2 mM DHPG. Samples
were flash frozen at the indicated times. To analyze new protein
synthesis, SN lysates were cleared of free isotope, percoll, and sucrose
by purification with the PAGEprep Advance kit per the manufac-
turer’s directions. Protein concentrations were determined by the
BCA assay, and 15 lg protein was denatured and loaded per lane on
12% SDS gels. The gels were dried and exposed to a phosphorimager
To specifically analyze APP synthesis, WT and KO SN lysates (500
ll) were immunoprecipitated with APP antibody. Briefly, SN lysates
were precleared with protein A magnetic beads in 1 ml volumes
containing 500 ll SNs, 500 ll 23 IP buffer (20 mM HEPES [pH 7.4],
400 mM NaCl, 60 mM EDTA [pH 8], and 2% Triton X-100), protease
inhibitor cocktail, and 100 ll packed fresh protein A magnetic beads.
For the immunoprecipitations, 10 lg anti-APP antibody (Zymed
catalog number 51–2700) and fresh protein A magnetic beads were
added and mixed overnight at 4 8C. The beads were washed three
times with IP buffer, and the final, washed pellets were suspended in
40 ll 23 SDS sample buffer and boiled for 5 min; the proteins were
then separated on 12% SDS gels. The gels were transferred to
nitrocelluose membrane, dried, exposed to a phosphorimager screen,
and scanned on a STORM 860 phosphorimager (Molecular Dynamics,
http://www6.amershambiosciences.com). The 120-kDa APP bands
were quantitated with ImageQuant software (GE Healthcare Life
For the inhibitor studies, SNs (425 ll) were preincubated with 25 ll
anisomycin (40 lM final concentration) or MPEP (10 lM final
concentration) for 10 min prior to the addition of 25 ll35S-Met for 5
min and stimulation with DHPG (100 lM final concentration) for 15
min. Samples were processed as described in the preceding para-
Western blot analysis. Aliquots of SNs were collected at 5, 10, and
20 min after DHPG treatment, quenched with an equal volume of 23
SDS sample buffer (8% SDS, 24% glycerol, 100 mM Tris [pH 6.8], 4%
b-mercaptoethanol, 0.02% bromophenol blue, 2% Triton X-100, 2%
deoxycholate, 2% NP-40 alternative, and 2% sarkosyl) and boiled for
5 min prior to analysis by 12% SDS-PAGE. The separated proteins
were transferred to 0.45 lm nitrocellulose membrane in Towbin
buffer containing 20% MeOH with a Criterion Blotter (Bio-Rad,
http://www.bio-rad.com; 100 V at 4 8C for 75 min). The membranes
were blocked in 5% nonfat dry milk and hybridized with anti-rabbit
APP antibody (dilution, 1 lg/ml) and anti-mouse b-actin antibody
(dilution, 1:20,000) followed by hybridization with anti-rabbit or anti-
mouse HRP-conjugated secondary antibodies (dilution, 1:2000).
Proteins were visualized by enhanced chemiluminescence on a
STORM 860 phosphorimager.
Neuronal cell culture, confocal microscopy, and image analysis.
Pregnant females (embryonic day 18) were anesthetized with
halothane prior to decapitation and transfer of the uterine sac to
ice-cold HBSS. Cortices were removed, washed with ice-cold HBSS,
lysed with 0.5 mg/ml trypsin for 25 min at 37 8C, washed with HBSS,
suspended in NeuroBasal medium (supplemented with 2% B27
supplement, penicillin/streptomycin, and 0.5 mM glutamine), tritu-
rated 703 with a 10-ml pipet, and passed through a 70-lm cell
strainer. Cells were counted by trypan blue dye exclusion and plated
at 1.25 3 105cells/ml on poly(D)-lysine–coated glass coverslips in 12-
well tissue-culture dishes and cultured for 11 d at 37 8C/5% CO2. Half
of the culture medium was removed and replaced with fresh, warm
medium on day 4.
Neuronal cells were treated with 100 lM DHPG, washed with PBS
containing 2% FBS, fixed in 4% PHA for 10 min at room
temperature, and permeabilized with MeOH (?20 8C) for 15 min.
Fixed, permeabilized cells were stained with anti-22C11 against the
amino-terminus of APP (Chemicon number mAB348; 1:2000 for 1 h)
and visualized with goat anti-mouse rhodamine-conjugated secon-
dary antibody (Invitrogen; 1:500 for 30 min in the dark). All washes
and antibody dilutions were in PBS containing 2% FBS. Coverslips
were fixed to slides with 12 ll ProLong Gold Antifade with DAPI
(Invitrogen) and dried overnight.
Images were acquired with a Nikon C1 laser-scanning confocal
microscope with EZ-C1 v2.20 software (Nikon, http://www.nikon.com)
at 603 magnification. APP levels in the puncta of four to seven
dendrites per sample were quantitated with IMAGE J software using
the Analyze Particles function (minimum of 205 puncta analyzed per
treatment) (Rasband, W.S., Image J, U.S. National Institutes of Health,
http://rsb.info.nih.gov/ij; 1997–2006). Figures were assembled with
Adobe Photoshop 8.0 (Adobe Systems, http://www.adobe.com). All
DHPG-treated and fmr-1 KO samples were highly statistically differ-
ent from untreated WT samples by t-test analyses (p ,0.001) and
expressed as SEM.
APP mRNA measurements. Aliquots of SNs were collected at the
indicated timepoints and flash frozen at ?80 8C. The samples were
thawed and vortexed to prepare SN lysates. To directly reverse-
transcribe RNA from SN lysates without an RNA purification step, a
modified method for the detection of mRNA in single neurons was
utilized . Briefly, 2 ll SN lysate was added per standard RT
reaction containing RNase-free DNase I and random nonamer
primer. The reactions were incubated at 37 8C for 15 min to destroy
any contaminating genomic DNA, 65 8C for 5 min to inactivate the
PLoS Biology | www.plosbiology.org March 2007 | Volume 5 | Issue 3 | e520636
FMRP Regulates APP Translation
DNase I, and 20 8C for 10 min to anneal the random primer.
Omniscript RT was added and reverse transcription proceeded at 37
8C for 60 min before inactivation at 93 8C for 5 min. The RT reactions
were diluted 5-fold with DEPC water prior to real-time PCR analysis.
For the statistical analysis, APP mRNA levels from triplicate experi-
ments were determined, normalized to 18S rRNA, and plotted as a
percentage of total APP mRNA. Error bars depict SEM.
Real-time PCR controls, standard curves, and analyses. The PCR
primers were designed with Primer Express software from Applied
Biosystems, and BLAST homology searches of the amplicons revealed
that the primers were gene specific. PCR reactions were optimized for
primer and template concentrations and contained 500 nM APP
primers (forward: 1701-ccgtggcacccttttgg-1717; and reverse: 1774-
gggcgggcgtcaaca-1760) or 300 nM 18S primers (forward: 98-cattaaat-
cagttatggttcctttgg-123; and reverse: 181-tcggcatgtattagctctagaattacc-
155), 10.5 ll 1:5 diluted RT reaction and 12.5 ll SYBR green PCR mix
in a 25 ll reaction volume. The cycle conditions were as follows: 2
min at 50 8C and 10 min at 95 8C (40 cycles: 15 s at 95 8C, 1 min at 60
8C), followed by a dissociation stage for 15 s at 95 8C, 1 min at 60 8C,
and 15 s at 95 8C. The average PCR efficiencies for the APP and 18S
primers over a 200-fold concentration range were 100% (APP) and
101% (18S) (n ¼ 9 experiments each), with a delta slope of 0.079. As
the difference in slopes between the sample PCR (APP) and the
normalization control (18S) was less than 0.1, the comparative CT
method was utilized to calculate the relative concentration of APP
mRNA normalized to 18S rRNA. SNs are void of nuclei; however to
ensure there was no genomic DNA contamination, control RT
reactions on SN templates in the absence of reverse transcriptase
were analyzed by real-time PCR and found void of APP PCR product.
The final APP and 18S PCR products were analyzed by agarose gel
electrophoresis and were single bands of the correct molecular
weight by EtBr staining (74 bp for APP; 84 bp for 18S).
FMRP IPs and real-time PCR analysis. SN lysates were precleared
with protein A magnetic beads and immunoprecipitated with 10 ll
RNasin, 10 lg 7G1–1 FMRP antibody (or no antibody controls), and
100 ll packed fresh protein A magnetic beads for 3 h at 4 8C. The IPs
were washed with IP buffer (10 mM HEPES [pH 7.4], 200 mM NaCl, 30
mM EDTA [pH 8], and 0.5% Triton X-100) and suspended in 1 ml
TRI-Reagent. Total RNA was isolated and precipitated in the
presence of 2 lg tRNA. The final pellet was suspended in DEPC
water, solubilized 10 min at 60 8C, and reverse transcribed with
Qiagen Omniscript and random nonamer primer (60 min at 37 8C, 5
min at 93 8C). The cDNA was diluted 5-fold and analyzed for APP by
qPCR as described immediately above.
Preparation of whole-cortex lysate. The cortices from six WT mice
(13 d old) were torn into pieces and homogenized in cold
immunoprecipitation buffer (10 mM HEPES [pH 7.4], 200 mM NaCl,
30 mM EDTA [pH 8], and 0.5% Triton X-100) containing 23protease
inhibitor cocktail and 0.4 U/ll RNasin. The homogenate was spun at
1,000g for 10 min at 4 8C to remove nuclei and unlysed cells, and the
pellet was discarded. The cleared lysate was flash frozen in aliquots at
Ribonuclease TI digestions and modified CLIP assay. Pellets from
anti-FMRP immunoprecipitations of whole-cortex lysate were washed
once with immunoprecipitation buffer and once with DPBS before
digestion with ribonuclease TI (0.8–4.0 U) in a 100-ll reaction volume
for 30 min at 37 8C with occasional mixing to disperse the magnetic
protein A beads. The digested samples were washed twice with DPBS
to remove RNA fragments. Protected RNA was isolated with TRI-
Reagent and analyzed by RTqPCR. The primer sequences for the real
time PCR are listed in Table S2. The delta Ctbetween undigested and
T1-digested samples was calculated and plotted as a percentage of
For the modified CLIP assay , cleared cortical lysate was cross-
linked with 400 mJ/cm2ultraviolet light in an UV Stratalinker 2400
(Stratagene, http://www.stratagene.com), immunoprecipitated with
anti-FMRP, and digested with ribonuclease TI. The washed pellets
were suspended in 40 ll SDS loading buffer containing no reducing
agents, heated for 10 min at 70 8C, applied to 12% SDS/PA gels, and
transferred to 0.45 lm nitrocellulose membrane in Towbin buffer.
Western blotting of a duplicate membrane indicated that FMRP
migrates at 80 kDa. A band encompassing approximately the 75–85
kDa molecular weight range was excised, transferred to TRI-Reagent,
and vortexed vigorously for 15 min at 37 8C. RNA was isolated and
analyzed by RTqPCR.
Ab40and Ab42ELISAs. For soluble brain lysates, right hemispheres
from four WT (aged 11, 13, 13, and 13 mo) and three KO mice (aged
11, 12, and 12 mo; FVB strain) and four WT (aged 13.5, 13.5, 12, and
12 mo) and four KO mice (aged 14, 14, 12, and 12 mo; C57BL/6 strain)
were homogenized in 500 ll protein extraction buffer (10 mM Tris
[pH 7.6], 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.25% NP-40,
and 13 protease inhibitor cocktail). Insoluble material was removed
by centrifugation at 12,000 rpm for 10 min, and aliquots of the
soluble fraction were flash frozen. For total brain lysates, left
hemispheres were homogenized in cold, 5 M GnHCl, mixed for 3–4
h at room temperature, and frozen at ?80 8C. Sandwich ELISAs with
the Signet Ab40/9131 and Ab42/9134 capture antibodies and the
rodent Ab/9154 reporter antibody were performed as previously
described . Ab levels were quantified based upon standard curves
run on the same ELISA plate and then expressed as a percentage of
Ab compared to WT controls.
Figure S1. G-Quartet Model
A model for the putative G-quartet sequence located in the coding
region of APP mRNA. Canonical G-quartets form two guanine planes,
while the putative APP G-quartet has the potential to form three
Found at doi:10.1371/journal.pbio.0050052.sg001 (46 KB TIF).
Figure S2. mGluR-Dependent APP Translation Is Independent of
WT and KO SNs with or without DHPG (5, 10, 15, and 30 min) were
analyzed by RTqPCR. APP mRNA levels were normalized to 18S
rRNA and plotted as a percentage of total APP mRNA.
Found at doi:10.1371/journal.pbio.0050052.sg002 (166 KB TIF).
Table S1. G-Richness of APP Amplicons
Found at doi:10.1371/journal.pbio.0050052.st001 (15 KB XLS).
Table S2. Real-Time PCR Sequencing Primers
Found at doi:10.1371/journal.pbio.0050052.st002 (16 KB XLS).
The GenBank (http://www.ncbi.nlm.nih.gov/Genbank) accession num-
bers for the gene products mentioned in this paper are human APP
mRNA (NM_000484), mouse APP mRNA (X59379), rat APP mRNA
(X07648), and 18S mRNA (M27358).
The mAb7G1–1 antibody developed by Dr. Stephen T. Warren was
obtained from the Developmental Studies Hybridoma Bank under
the auspices of the National Institute of Child Health and Human
Development and maintained by The University of Iowa, Department
of Biological Sciences (Iowa City, Iowa, United States). We are
indebted to Aaron Grossman and Dr. Bill Greenough (University of
Illinois at Champaign-Urbana) for generously providing WT and fmr-
1 KO mice in the FVB and C57BL/6 backgrounds as well as breeding
and genotyping protocols. We thank Denice Springman, Dr. Albee
Messing, and Dr. Tracy Hagemann for training in animal handling
and mouse tail biopsies, and for the DNA extraction protocol; Dr.
Ste ´phane Esnault for training in real-time PCR; Dr. Dandan Sun
(Department of Neurosurgery, University of Wisconsin at Madison)
for advice in culturing cortical neuronal cells; Dr. Pamela Westmark
for fluorescent staining protocols and training and assistance in
confocal microscopy and image analysis; and Dr. Luigi Puglielli
(Department of Medicine, University of Wisconsin at Madison) for
detailed Ab ELISA protocols and troubleshooting advice. We thank
FRAXA Research Foundation for providing MPEP. We acknowledge
the expert technical assistance provided by the University of
Wisconsin at Madison animal care staffs at the Waisman Center
and Rennebohm Pharmacy, in particular Sharon Hildebrandt.
Author contributions. CJW conceived and designed the experi-
ments and performed the experiments. CJW and JSM analyzed the
data, contributed reagents/materials/analysis tools, and wrote the
Funding. This work was supported by National Institutes of Health
Grants R01 AG10675 (to J.S.M.), P30 HD03352 (to the Waisman
Center), State of WI grant money (to the Wisconsin Comprehensive
Memory Program), and by a private donation by Bill and Doris Willis
(to the Waisman Center)
Competing interests. The authors have declared that no competing
PLoS Biology | www.plosbiology.orgMarch 2007 | Volume 5 | Issue 3 | e52 0637
FMRP Regulates APP Translation
1. Akaaboune M, Allinquant B, Farza H, Roy K, Magoul R, et al. (2000)
Developmental regulation of amyloid precursor protein at the neuro-
muscular junction in mouse skeletal muscle. Mol Cell Neurosci 15: 355–367.
2. Kamenetz F, Tomita T, Hsieh H, Seabrook G, Borchelt D, et al. (2003) APP
processing and synaptic function. Neuron 37: 925–937.
3. Hagerman RJ, Hagerman PJ (2002) Physical and behavioral phenotype.
Hagerman RJ, Cronister A, editors. Baltimore: John Hopkins University
Press. pp. 3–109.
4. Rudelli RD, Brown WT, Wisniewski K, Jenkins EC, Laure-Kamionowska M,
et al. (1985) Adult fragile X syndrome. Clinico-neuropathologic findings.
Acta Neuropathol (Berl) 67: 289–295.
5. Wisniewski KE, Segan SM, Miezejeski CM, Sersen EA, Rudelli RD (1991) The
Fra(X) syndrome: Neurological, electrophysiological, and neuropatholog-
ical abnormalities. Am J Med Genet 38: 476–480.
6. Verkerk AJ, Pieretti M, Sutcliffe JS, Fu YH, Kuhl DP, et al. (1991)
Identification of a gene (FMR-1) containing a CGG repeat coincident with a
breakpoint cluster region exhibiting length variation in fragile X
syndrome. Cell 65: 905–914.
7. Oberle I, Rousseau F, Heitz D, Kretz C, Devys D, et al. (1991) Instability of a
550-base pair DNA segment and abnormal methylation in fragile X
syndrome. Science 252: 1097–1102.
8. Khandjian EW, Fortin A, Thibodeau A, Tremblay S, Cote F, et al. (1995) A
heterogeneous set of FMR1 proteins is widely distributed in mouse tissues
and is modulated in cell culture. Hum Mol Genet 4: 783–789.
9. Bagni C, Greenough WT (2005) From mRNP trafficking to spine
dysmorphogenesis: The roots of fragile X syndrome. Nat Rev Neurosci 6:
10. Brown V, Jin P, Ceman S, Darnell JC, O’Donnell WT, et al. (2001)
Microarray identification of FMRP-associated brain mRNAs and altered
mRNA translational profiles in fragile X syndrome. Cell 107: 477–487.
11. Miyashiro KY, Beckel-Mitchener A, Purk TP, Becker KG, Barret T, et al.
(2003) RNA cargoes associating with FMRP reveal deficits in cellular
functioning in Fmr1 null mice. Neuron 37: 417–431.
12. Zalfa F, Giorgi M, Primerano B, Moro A, Di Penta A, et al. (2003) The fragile
X syndrome protein FMRP associates with BC1 RNA and regulates the
translation of specific mRNAs at synapses. Cell 112: 317–327.
13. Laggerbauer B, Ostareck D, Keidel EM, Ostareck-Lederer A, Fischer U
(2001) Evidence that fragile X mental retardation protein is a negative
regulator of translation. Hum Mol Genet 10: 329–338.
14. Li Z, Zhang Y, Ku L, Wilkinson KD, Warren ST, et al. (2001) The fragile X
mental retardation protein inhibits translation via interacting with mRNA.
Nucleic Acids Res 29: 2276–2283.
15. Mazroui R, Huot ME, Tremblay S, Filion C, Labelle Y, et al. (2002) Trapping
of messenger RNA by Fragile X Mental Retardation protein into
cytoplasmic granules induces translation repression. Hum Mol Genet 11:
16. Stefani G, Fraser CE, Darnell JC, Darnell RB (2004) Fragile X mental
retardation protein is associated with translating polyribosomes in neuro-
nal cells. J Neurosci 24: 7272–7276.
17. Darnell JC, Jensen KB, Jin P, Brown V, Warren ST, et al. (2001) Fragile X
mental retardation protein targets G quartet mRNAs important for
neuronal function. Cell 107: 489–499.
18. Darnell JC, Fraser CE, Mostovetsky O, Stefani G, Jones TA, et al. (2005)
Kissing complex RNAs mediate interaction between the Fragile-X mental
retardation protein KH2 domain and brain polyribosomes. Genes Dev 19:
19. Chen L, Yun SW, Seto J, Liu W, Toth M (2003) The fragile X mental
retardation protein binds and regulates a novel class of mRNAs containing
U rich target sequences. Neuroscience 120: 1005–1017.
20. Dolzhanskaya N, Sung YJ, Conti J, Currie JR, Denman RB (2003) The fragile
X mental retardation protein interacts with U-rich RNAs in a yeast three-
hybrid system. Biochem Biophys Res Commun 305: 434–441.
21. Greenough WT, Klintsova AY, Irwin SA, Galvez R, Bates KE, et al. (2001)
Synaptic regulation of protein synthesis and the fragile X protein. Proc
Natl Acad Sci U S A 98: 7101–7106.
22. Todd PK, Mack KJ, Malter JS (2003) The fragile X mental retardation
protein is required for type-I metabotropic glutamate receptor-dependent
translation of PSD-95. Proc Natl Acad Sci U S A 100: 14374–14378.
23. Schaeffer C, Bardoni B, Mandel JL, Ehresmann B, Ehresmann C, et al.
(2001) The fragile X mental retardation protein binds specifically to its
mRNA via a purine quartet motif. EMBO J 20: 4803–4813.
24. Denman R, Potempska A, Wolfe G, Ramakrishna N, Miller DL (1991)
Distribution and activity of alternatively spliced Alzheimer amyloid
peptide precursor and scrapie PrP mRNAs on rat brain polysomes. Arch
Biochem Biophys 288: 29–38.
25. Rogers JT, Randall JD, Cahill CM, Eder PS, Huang X, et al. (2002) An iron-
responsive element type II in the 59-untranslated region of the Alzheimer’s
amyloid precursor protein transcript. J Biol Chem 277: 45518–45528.
26. Sung YJ, Weiler IJ, Greenough WT, Denman RB (2004) Selectively enriched
mRNAs in rat synaptoneurosomes. Brain Res Mol Brain Res 126: 81–87.
27. Lacor PN, Buniel MC, Chang L, Fernandez SJ, Gong Y, et al. (2004) Synaptic
targeting by Alzheimer’s-related amyloid beta oligomers. J Neurosci 24:
28. (1994) Fmr1 knockout mice: A model to study fragile X mental retardation.
The Dutch-Belgian Fragile X Consortium. Cell 78: 23–33.
29. Irwin SA, Patel B, Idupulapati M, Harris JB, Crisostomo RA, et al. (2001)
Abnormal dendritic spine characteristics in the temporal and visual
cortices of patients with fragile-X syndrome: A quantitative examination.
Am J Med Genet 98: 161–167.
30. McKinney BC, Grossman AW, Elisseou NM, Greenough WT (2005)
Dendritic spine abnormalities in the occipital cortex of C57BL/6 Fmr1
knockout mice. Am J Med Genet B Neuropsychiatr Genet. 136: 98–102.
31. Weiler IJ, Spangler CC, Klintsova AY, Grossman AW, Kim SH, et al. (2004)
Fragile X mental retardation protein is necessary for neurotransmitter-
activated protein translation at synapses. Proc Natl Acad Sci U S A 101:
32. D’Agata V, Warren ST, Zhao W, Torre ER, Alkon DL, et al. (2002) Gene
expression profiles in a transgenic animal model of fragile X syndrome.
Neurobiol Dis 10: 211–218.
33. Garnon J, Lachance C, Di Marco S, Hel Z, Marion D, et al. (2005) Fragile X-
related protein FXR1P regulates proinflammatory cytokine tumor necrosis
factor expression at the post-transcriptional level. J Biol Chem 280: 5750–
34. Westmark CJ, Malter JS (2001) Extracellular-regulated kinase controls beta-
amyloid precursor protein mRNA decay. Brain Res Mol Brain Res 90: 193–
35. Zaidi SH, Malter JS (1994) Amyloid precursor protein mRNA stability is
controlled by a 29-base element in the 39-untranslated region. J Biol Chem
36. Bear MF, Huber KM, Warren ST (2004) The mGluR theory of fragile X
mental retardation. Trends Neurosci 27: 370–377.
37. Feng Y, Absher D, Eberhart DE, Brown V, Malter HE, et al. (1997) FMRP
associates with polyribosomes as an mRNP, and the I304N mutation of
severe fragile X syndrome abolishes this association. Mol Cell 1: 109–118.
38. Ceman S, Brown V, Warren ST (1999) Isolation of an FMRP-associated
messenger ribonucleoprotein particle and identification of nucleolin and
the fragile X-related proteins as components of the complex. Mol Cell Biol
39. Weiler IJ, Irwin SA, Klintsova AY, Spencer CM, Brazelton AD, et al. (1997)
Fragile X mental retardation protein is translated near synapses in
response to neurotransmitter activation. Proc Natl Acad Sci U S A 94:
40. Zaidi SH, Malter JS (1995) Nucleolin and heterogeneous nuclear ribonu-
cleoprotein C proteins specifically interact with the 39-untranslated region
of amyloid protein precursor mRNA. J Biol Chem 270: 17292–17298.
41. Ule J, Jensen KB, Ruggiu M, Mele A, Ule A, et al. (2003) CLIP identifies
Nova-regulated RNA networks in the brain. Science 302: 1212–1215.
42. Galvez R, Greenough WT (2005) Sequence of abnormal dendritic spine
development in primary somatosensory cortex of a mouse model of the
fragile X mental retardation syndrome. Am J Med Genet A 135: 155–160.
43. Lu R, Wang H, Liang Z, Ku L, O’Donnell WT, et al. (2004) The fragile X
protein controls microtubule-associated protein 1B translation and
microtubule stability in brain neuron development. Proc Natl Acad Sci U
S A 101: 15201–15206.
44. Torroja L, Packard M, Gorczyca M, White K, Budnik V (1999) The Drosophila
beta-amyloid precursor protein homolog promotes synapse differentiation
at the neuromuscular junction. J Neurosci 19: 7793–7803.
45. Yang G, Gong YD, Gong K, Jiang WL, Kwon E, et al. (2005) Reduced
synaptic vesicle density and active zone size in mice lacking amyloid
precursor protein (APP) and APP-like protein 2. Neurosci Lett. 384: 66–71.
46. Shigematsu K, McGeer PL, McGeer EG (1992) Localization of amyloid
precursor protein in selective postsynaptic densities of rat cortical
neurons. Brain Res 592: 353–357.
47. Wang P, Yang G, Mosier DR, Chang P, Zaidi T, et al. (2005) Defective
neuromuscular synapses in mice lacking amyloid precursor protein (APP)
and APP-Like protein 2. J Neurosci 25: 1219–1225.
48. Loffler J, Huber G (1992) Beta-amyloid precursor protein isoforms in
various rat brain regions and during brain development. J Neurochem 59:
49. Moya KL, Benowitz LI, Schneider GE, Allinquant B (1994) The amyloid
precursor protein is developmentally regulated and correlated with
synaptogenesis. Dev Biol 161: 597–603.
50. Ceman S, O’Donnell WT, Reed M, Patton S, Pohl J, et al. (2003)
Phosphorylation influences the translation state of FMRP-associated
polyribosomes. Hum Mol Genet 12: 3295–3305.
51. Comery TA, Harris JB, Willems PJ, Oostra BA, Irwin SA, et al. (1997)
Abnormal dendritic spines in fragile X knockout mice: Maturation and
pruning deficits. Proc Natl Acad Sci U S A 94: 5401–5404.
52. Hinton VJ, Brown WT, Wisniewski K, Rudelli RD (1991) Analysis of
neocortex in three males with the fragile X syndrome. Am J Med Genet 41:
53. Hagerman PJ, Hagerman RJ (2004) Fragile X-associated tremor/ataxia
syndrome (FXTAS). Ment Retard Dev Disabil Res Rev 10: 25–30.
54. Sokol DK, Chen D, Farlow MR, Dunn DW, Maloney B, et al. (2006) High
levels of Alzheimer beta-amyloid precursor protein (APP) in children with
severely autistic behavior and aggression. J Child Neurol 21: 444–449.
55. Clifford S, Dissanayake C, Bui QM, Huggins R, Taylor AK, et al. (2006)
PLoS Biology | www.plosbiology.orgMarch 2007 | Volume 5 | Issue 3 | e52 0638
FMRP Regulates APP Translation
Autism spectrum phenotype in males and females with fragile X full Download full-text
mutation and premutation. J Autism Dev Disord. In press.
56. Singh K, Gaur P, Prasad S (2006) Fragile X mental retardation (Fmr-1) gene
expression is down regulated in brain of mice during aging. Mol Biol Rep,
Epub ahead of print.
57. Dunkley PR, Heath JW, Harrison SM, Jarvie PE, Glenfield PJ, et al. (1988) A
rapid Percoll gradient procedure for isolation of synaptosomes directly
from an S1 fraction: Homogeneity and morphology of subcellular
fractions. Brain Res 441: 59–71.
58. Bagni C, Mannucci L, Dotti CG, Amaldi F (2000) Chemical stimulation of
synaptosomes modulates alpha-Ca2þ/calmodulin-dependent protein kinase
II mRNA association to polysomes. J Neurosci 20: RC76.
59. Comer AM, Gibbons HM, Qi J, Kawai Y, Win J, et al. (1999) Detection of
mRNA species in bulbospinal neurons isolated from the rostral ventro-
lateral medulla using single-cell RT-PCR. Brain Res Brain Res Protoc 4:
60. Costantini C, Weindruch R, Della Valle G, Puglielli L (2005) A TrkA-to-
p75NTR molecular switch activates amyloid beta-peptide generation
during aging. Biochem J 391: 59–67.
PLoS Biology | www.plosbiology.org March 2007 | Volume 5 | Issue 3 | e520639
FMRP Regulates APP Translation