1) NKCC1, a chloride importer implicated in autism, is upregulated in Eif4ebp2 knockout mice compared to wildtype. This may cause excitatory-inhibitory imbalances linked to autism. 2) Phosphorylated forms of eIF4E, mTOR, and ERK are also upregulated in knockouts, and may be important in translational control of NKCC1. Total eIF4E is downregulated. 3) Total levels of mTOR, ERK, and Akt, as well as phosphorylated Akt, show trends of change but are not significantly different between knockout and wildtype mice.

1. Translational Control of Autism Spectrum Disorders in Eif4ebp2 knockout Mouse Models
Rey Christian Pacis, Jelena Popic, Nahum Sonenberg
Department of Biochemistry and Goodman Cancer Research Centre, McGill University, 1160
Pine Ave. West, H3A 1A3, Montreal, QC, Canada
Abstract
Knockout of the eukaryotic initiation factor (eIF) 4E binding protein (4E-BP2) can induce
autistic-like phenotypes in mouse models. In this study, we focus on the effect of Eif4ebp2
knockout on protein expression in cortical lysates of postnatal development day 14 mice,
particularly the levels of certain proteins that have been causally linked to autism spectrum
disorders (ASD). One such protein is NKCC1, a chloride-ion importer that has been shown to be
responsible for the excitatory effects of GABA neurotransmission during early postnatal
development. Our present study finds that NKCC1 is upregulated in Eif4ebp2 knockouts
compared to wildtype mice, and that its upregulation may be responsible for autistic-like
phenotypes exhibited by the Eif4ebp2 knockouts. Phosphorylated isoforms of eIF4E, the
mammalian target of rapamycin (mTOR), and the extracellular signal-regulated kinase (ERK)
were also shown to be significantly upregulated, and may have importance in the translational
control of NKCC1. Additionally, total eIF4E levels were seen to be significantly downregulated,
and changes in protein levels of total mTOR, total ERK, and total protein kinase B (Akt), as well
as two phosphorylated isoforms of Akt (Ser437 and Thr308) were observed, but not significant.
Electrical imbalance and translational dysregulation have both been implicated as major
contributors to the development of ASD. The underlying translational control of NKCC1
regulation during this early development period must, then, be of importance in elucidating the
translational basis of autism spectrum disorder.
1. Introduction
Autism spectrum disorder describes a varying degree of neuro-developmental disorders
that can manifest as diminished social interactions, difficulty in verbal and nonverbal
communication, narrow and restricted interests, and repetitive behaviors1
. Due to its current
ambiguity, the molecular and genetic underpinnings that give rise to ASDs in individuals is of
particular interest for research. Presently, most methods and tests to diagnose autism can only be
implemented after a certain age when the baby or toddler begins to exhibit characteristic signs

2. and symptoms2
. The characterization of the molecular basis of ASD may not only explain the
conditions and mechanisms that cause ASD to develop, which might aid in early detection, but
also provide potential for design of drug treatments that may reverse the impairments that arise
from ASDs.
Translation is the final step of protein biosynthesis during which polypeptides are formed
from the mRNA transcript and fold into functional proteins3
. The initiation of translation is, thus,
a critical step in the synthesis of proteins, and, therefore, must have a highly regulated
mechanism. Among a class of proteins known as eukaryotic initiation factors, which aid in
translation initiation, eIF4E is responsible for recruitment of mRNA modified at the 5’ end with
a 7-methyl-GTP cap structure3
. It then complexes with two other eIFs to form a stable eIF4F
complex that can successfully initiate translation4
. A family of proteins known to bind to eIF4E,
known as 4E-binding proteins (4E-BPs), inhibits the function of eIF4E by preventing formation
of the eIF4F complex, and ultimately inhibiting translation4
. The major 4E-binding protein in the
brain, 4E-BP2, is responsible for regulating cap-dependent translation of mRNA transcripts that
will eventually code for proteins involved in synaptogenesis and neurogenesis.
Translational control of certain proteins is particularly important during the early
postnatal development days. One such example is the regulation of the chloride-ion importer and
exporter NKCC1 and KCC2, respectively5
. Early in postnatal development, the neurotransmitter
γ-aminobutyric acid (GABA), can function in excitatory neurotransmission, which is opposite its
regularly inhibitory role in mammalian adults5
. GABA-mediated excitation results from high
intracellular chloride concentrations caused by postnatally low levels of the exporter KCC2 and
by chloride import by NKCC1. When GABA channels open, the resulting chloride efflux may
result in sufficient depolarization for action potential generation. Normally, KCC2 is upregulated
with age, and GABA resumes its original inhibitory role as intracellular chloride concentrations
decrease. However, overexpression of NKCC1, arising most likely from a lack of translational
control, may cause excitatory-inhibitory imbalances that persist with age. Such imbalances have
been implicated in the development of ASD-like phenotypes5
, among other neurological
disorders, including anxiety disorders6
, schizophrenia, stiff-person syndrome, and convulsive
seizures7
.

3. Dysregulation of translational signaling pathways have also been observed in ASD8
. Akt
and mTOR, two cellular kinases involved in a signaling cascade that regulate cap-dependent
translation8
, and ERK, a kinase involved in translation of genes encoding several neural adhesion
molecules and scaffolding proteins9
, have all been shown to be dysregulated in ASD.
The present study uses protein extracts obtained from the cortices of postnatal-day-14
(P14) mice that were either wildtype (WT) or Eif4ebp2 knockouts (KOs). Knockout of 4E-BP2
has been shown to induce ASD-like phenotypes (Gkogkas et. al. 2013), arising potentially from
dysregulation of cap-mediated translation. We investigated the expression levels of several
proteins that have been previously shown to be associated with ASD: (1) NKCC1, the chloride-
ion importer shown to be overexpressed and causing excitatory-inhibitory imbalances5
, (2) eIF4E
and p-eIF4E, which are the target of 4E-BP2 and responsible for cap-dependent translation
initiation3, 10
, (3) mTOR and p-mTOR, (4) ERK and p-ERK, and (5) protein kinase B (Akt) and
its serine-437 and threonine-308 phosphorylated isoforms. Specifically, upregulated activity of
mTOR and ERK by phosphorylation increases transcription, translation, and potentially other
cellular processes that may overall contribute to the development of ASD8
. Our results show that
in Eif4ebp2 knockout mice, which have been shown to develop ASD-like behaviors as adults,
exhibit upregulation of NKCC1, p-eIF4E, p-mTOR, and p-ERK and downregulation of eIF4E.
No significant change in total mTOR, total ERK, and Akt, total or phosphorylated, were
observed. The findings in this report present proteins that may be causally linked to ASD
pathogenesis and perhaps, with further research, may prove useful as potential diagnostic tools,
or even treatment targets.
2. Materials and Methods
2.1. Animals
Fourteen-day-old (P14) male WT and BP2KO were obtained from animal colony at McGill
University. Throughout the study period, pups were kept with their mothers, and were housed in a
controlled environment (temperature 22◦C, humidity 50–60%, 12 h light/12 h dark schedule) and
efforts were made to minimize discomfort and the number of animals used. The animal
experimentation protocol was approved by the McGill University Animal Care Committee and
conducted in accordance with the Guide to the Care and Use of Experimental Animals of the
Canadian Council on Animal Care. Genotypes were confirmed by western blot analyses.

4. 2.1. SDS Polyacrylamide Gel Electrophoresis
Brain tissue was collected, and homogenized in Radioimmunoprecipitation assay (RIPA)
buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 5
mM EDTA pH 8.0, 1 mM EGTA pH 8.0, 10 mM NaF, 1 mM β-glycerophosphate, 1 mM Na
orthovanadate) containing protease inhibitor cocktail (Roche). The homogenates were sonicated
and centrifuged at 12000 rpm for 10 minutes at 4°C. The supernatant were collected and stored at
-80°C until future use. Protein concentrations were measured using Bradford assay with bovine
serum albumin (BSA) as standard11
. Ten micrograms of protein extracts were heated, denatured
and resolved by 4-12% gradient gel (Novex) and transferred onto a 0.2 µm nitrocellulose
membrane.
2.3. Western Blot Analysis
The membranes were washed on a shaker ten minutes in milli-Q H2O, followed by
another ten minutes in Tris-buffered saline/0.1% Tween-20 (TBS-T). After washing, the
membranes were blocked for sixty minutes in 3% bovine serum albumin in TBS-T. The
membranes were incubated overnight with primary antibody on a shaker at +4o
C. The next day,
the membranes were washed three times for ten minutes each in TBS-T and incubated with
secondary antibody for an hour at room temperature, followed by three ten-minute washes in
TBS-T. The primary antibodies diluted for the following proteins were used: GAPDH (sc-32233,
Santa Cruz, 1:8000), NKCCI (1:1000), eIF4E (610270, BD Transduction Laboratories,1:1000),
p-eIF4E (NB-100-79938, Novus Biologicals, 1:20000), mTOR (1:1000), p-mTOR (1:1000),
ERK (sc-93, Santa Cruz, 1:2000), p-ERK (9106, Cell Signaling, 1:1000), Akt (1:1000), p-
AktSer473 (1:1000), and p-AktThr308 (1:2000). The secondary anti-mouse and anti-rabbit (GE
Healthcare, 1:10000) were diluted in TBS-T. Values were normalized to GAPDH for total
proteins, while phosphorylated isoforms were normalized to total proteins.
Immunoreactivity was detected by enhanced chemiluminescence (plus-ECL; Perkin
Elmer Inc.) after exposure of an X-ray film (Denville Scientific Inc.).
2.4. Stripping Protocol
To measure amount of phosphorylated proteins, membranes were first incubated with an
antibody against the phospho-protein, after which membranes were washed three times for 10

5. minutes in stripping buffer (2.5 mM glycine-HCl, 1% SDS, pH of 2.0), followed by three washes
in TBS-T for 10 minutes and later incubated with total protein.
2.5. Statistics
Densitometric analysis of the developed films for evaluation of protein levels was
achieved by the image analysis software ImageQuant 5.2. The data are presented as percentages
(mean ± standard error of mean) relative to the wildtype control samples, which were normalized
to 100%. Differences in the experimental group were tested using the paired, two-tailed student’s
t-test, and significance was accepted at P<0.05. Statistical analyses was performed using
Statistica software.
3. Results
3.1. The chloride-ion transporter NKCCI is upregulated in Eif4ebp2 knockout mice
To test the hypothesis that chloride transport is dysregulated in ASD, we have used western
blot to determine the expression of main chloride importer, NKCC1 in the cortex of P14 WT and
Eif4ebp2 KO mice pups. We have shown that NKCC1 is significantly increased (about 50%) in
the cortex of KO compared to WT P14 mice (p=0.016) (Figure 1).
Fig. 1. NKCC1 is upregulated in Eif4ebp2 knockout mice. Western blot analysis of protein extracts from the cortex
of P14 mice was used to determine NKCC1 expression, p = 0157. The data is expressed as a percent to the control,
as mean ± SEM (n = 4). An asterisk is used to indicate a significant difference, as per the student’s t-test (P<0.05).
3.2 Significant upregulation of phosphorylated of eIF4E, mTOR, and ERK in Eif4ebp2 knockout
mice.
Our results show that p-eIF4E (Fig. 2B), p-mTOR (Fig. 3B), and p-ERK (Fig.4B) were
all upregulated in the knockout mice. Additionally, downregulation of total eIF4E was also
observed (Fig. 2A). However, this may simply be an artifact of the conversion to its
0
50
100
150
200
wt bp2ko
NKCC1/GAPDH(%)
*
Eif4ebp2KO

6. phosphorylated isoform. Both total mTOR (Fig. 3A) and total ERK (Fig. 4A) did not
significantly change in concentration between wildtype and knockout samples.
Fig. 2. eIF4E is downregulated and p-eIF4E is upregulated in Eif4ebp2 knockout mice. Western blot analysis of
protein extracts from the cortex of P14 was used to determine expression of (A) eIF4E, p = 0.00433, and (B) p-
eIF4E, p = 0.000045. Each bar graph is accompanied by its respective immunoblot. The data is expressed as a
percent to the control, as mean ± SEM (n = 4). An asterisk is used to indicate a significant difference, as per the
student’s t-test (P<0.05).
Fig. 3. Eif4ebp2 knockout in mice does not affect mTOR, but upregulates phospho-mTOR in P14 mice. Western
blot analysis of protein extracts from the cortex of P14 was used to determine expression of (A) mTOR, p = 0.932,
and (B) p-mTOR, p = 0.0264. Each bar graph is accompanied by its respective immunoblot. The data is expressed as
a percent to the control, as mean ± SEM (n = 4). An asterisk is used to indicate a significant difference, as per the
student’s t-test (P<0.05).
90
95
100
105
wt bp2ko
mTOR/GAPDH(%)
80
90
100
110
120
wt bp2ko
p-mTOR/mTOR(%)
A
B *
0
50
100
150
wt bp2ko
eIF4E/GAPDH(%)
0
100
200
wt bp2ko
p-eIF4E/eIF4E(%)
A
B
*
*
eIF4E (25kDa)
GAPDH (37kDa)
p-eIF4E (28kDa)
GAPDH (37kDa)
Eif4ebp2KO
Eif4ebp2KO
Eif4ebp2KO
Eif4ebp2KO

7. Fig. 4. Eif4ebp2 knockout in mice does not affect ERK, but upregulates phospho-ERK in P14 mice. Western blot
analysis of protein extracts from the cortex of P14 was used to determine expression of (A) ERK, p = 0.0731, and
(B) p-ERK, p = 0.0276. Each bar graph is accompanied by its respective immunoblot. The data is expressed as a
percent to the control, as mean ± SEM (n = 4). An asterisk is used to indicate a significant difference, as per the
student’s t-test (P<0.05).
3.3. No significant Change in Total Akt or its Ser473- and Thr308-phosphorylated isoforms.
Total Akt, as well as two phosphorylated isoforms, p-AktSer473 and p-AktThr308, was
also studied by western blot analysis. Although protein levels were observed to decrease in
Eif4ebp2 knockout mice, downregulation was not observed to the desired significance of P<0.05.
Fig. 5. Eif4ebp2 knockout in mice may cause downregulation of total, as well as Thr308 and Ser437 isoforms of,
Akt. Western blot analysis of protein extracts from the cortex of P14 was used to determine expression of (A) total
Akt, p = 0.137; (B) p-AktThr308, p = 0.232; and (C) p-AktSer437, p = 0.0879. Each bar graph is accompanied by its
respective immunoblot. The data is expressed as a percent to the control, as mean ± SEM (n = 4). An asterisk is used
to indicate a significant difference, as per the student’s t-test (P<0.05).
80
85
90
95
100
105
wt bp2ko
Akt/GAPDH(%)
0
50
100
150
wt bp2ko
p-AktSer473/GAPDH
(%)
0
50
100
150
wt bp2ko
p-AktThr308/GAPDH
(%)
A B C
A
B
0
50
100
150
wt bp2ko
ERK/GAPDH(%)
0
100
200
300
wt bp2ko
p-ERK/ERK(%)
*
ERK (42/44 kDa)
GAPDH (37kDa)
p-ERK (42/44 kDa)
GAPDH (37kDa)
WT KO WT KO WT KO WT KO WT KO WT KO WT KO WT KO WT KO WT KO WT KO WT KO
Akt (60kDa)
GAPDH (37kdA)
p-Akt Thr308 (60kDa)
GAPDH (37kdA)
p-AktSer437 (60kDa)
GAPDH (37kdA)
Eif4ebp2KO
Eif4ebp2KO
Eif4ebp2KO Eif4ebp2KOEif4ebp2KO

8. 4. Discussion
The focus of this study was to investigate the protein levels in the cortex of postnatal-day
14 wildtype and Eif4ebp2 knockout mice in order to better understand the roles that various
proteins play in the development of ASD. Excitatory-inhibitory (E/I) imbalances and
translational dysregulation that occur in in early postnatal development have been implicated in
the development of ASD in animal models5
. Upregulation of the chloride-ion importer NKCC1
has been shown to play a role in GABAergic imbalances5
. Our results show that in Eif4ebp2
knockout mice, NKCC1 is upregulated, most likely arising from the lack of cap-mediate
translation regulation by 4E-BP2. Overexpression of NKCC1 can cause neurotransmission
imbalances by generating and perpetuating abnormally high levels of intracellular chloride, even
as KCC2, the main chloride-ion exporter, is upregulated with age5
. Chloride-ion efflux from the
cell when GABA anion channels open may occasionally be depolarizing enough to induce an
action potential. This may deter, or even prevent, GABA from functioning in its naturally
inhibitory manner5
.
Further, our results show that phosphorylated isoforms of the proteins eIF4E, mTOR, and
ERK are all significantly upregulated in Eif4ebp2 knockout mice during early postnatal
development. Phosphorylated eIF4E has been shown to cause increased cap-dependent
translation by making it easier for the eIF4F cap complex to dissociate from the transcript, which
may facilitate initiation and the loading of more ribosomes10
. Activation of ERK by
phosphorylation may lead to behavioral abnormalities characteristic of ASD by increasing the
translation of neurogenic adhesion molecules and scaffolding proteins which may cause
excitatory-inhibitory imbalances that have been implicated in ASD9
.
The main issue in ASD research, as with many neurodegenerative disorders, is the
ambiguity surrounding the molecular and genetic mechanisms that underlie its development.
Current methods of diagnosing autism in humans rely mostly on interactive tests conducted a
few years after birth when autistic phenotypes begin to show. Treatment for autism involves
therapies and reducing the characteristic symptoms; few approved medical treatments are
available. The significant upregulation of NKCC1, p-eIF4E, p-mTOR, and p-ERK observed in
our study suggests that these proteins do, indeed, may play a key role in the development of

9. ASD. Perhaps future research may aim to further elucidate the mechanistic underpinnings of
these proteins so as to be able to use them as early diagnostic criteria for ASD, and potentially
even in the design of drug treatment.

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