Targeting Translation Control with p70 S6 Kinase 1 Inhibitors
to Reverse Phenotypes in Fragile X Syndrome Mice
Aditi Bhattacharya , Maggie Mamcarz, Caitlin Mullins, Ayesha Choudhury,
Robert G. Boyle, Daniel G. Smith , David W. Walker , Eric Klann
Supplementary Information
4 Supplementary Figures and Legends
2 Supplementary Tables and Legends
Supplementary Behavioral Methods
1
Supplementary Figure 1
Figure S1: A) Dose response of PF-4708671 and FS-115 to inhibit S6 phosphorylation in wild-type
hippocampal slices. Phosphorylation of S6 was measured in response to stimulation with 1 µM insulin in
presence of increasing concentrations of either PF-4708671 or FS-115. n=4 slices from two mice per
condition per inhibitor. B) PF-4708671 and FS-115 do not impact protein synthesis in S6K1 KO mice.
BONCAT experiments showing incorporation of AHA into WT and S6K1 KO vehicle (veh)-, 10uM PF4708671 (PF)- and 10uM FS-115 (FS)-treated hippocampal slices. 40µM anisomycin (aniso) was used as
a negative control. Histogram shows quantification of protein synthesis rates in each condition. n=3 mice,
3 slices per treatment per genotype. Blots were counterstained to monitor phospho-S6 levels and S6K1
levels. C) Representative western blots for BONCAT in cortical lysates isolated from WT mice and Fmr1
KO littermates. 5 minutes post incubation with AHA, 10µM PF-4708671, FS-115 or vehicle were added to
individual aliquots and lysates were incubated for an additional 10 mins. Equal volumes were loaded on
gels and probed for the anti-biotin signal. n=4 per genotype. D) Representative western blots for
BONCAT in cortical lysates isolated from chronically dosed WT mice and Fmr1 KO littermates. Cortical
lysates were incubated for 15 mins with AHA and again equal volumes were loaded on gels and probed
for the anti-biotin signal. n=4 per genotype per treatment. Please see Fig. 1D and Fig. 2. I-J, for
corresponding quantification. E) Dose response of inhibiting S6 and eEF2 phosphorylation by increasing
doses of FS-115 delivered via acute gavage. n=3 WT mice per treatment. A dose response curve for PF4708671 can be found in Huynh et al., 2014.
2
Supplementary Figure 2
Figure S2: A-B) Acute injection of PF-4708671 decreases S6 240/44 phosphorylation in both wild-type
(WT) and Fmr1 knockout (Fmr1 KO, Fmr1 in graphs) mice. n=5 mice per genotype per treatment;
*p<0.05 with a two-way ANOVA (genotype x treatment) followed by Tukey post hoc tests. Significant main
effects were found in vehicle vs drug-treated for WT and Fmr1 KO mice F(1,16)=15.5808, Fcrit=4.49. C-D)
Acute gavage of FS-115 (FS) decreases S6 240/44 phosphorylation, but not eEF2 phosphorylation, in
both WT and Fmr1 KO mice. n=4 mice per genotype per condition. *p<0.05 and **p<0.01 respectively
with a two-way ANOVA (genotype x treatment) followed by Tukey post hoc tests. Significant main effects
were found in vehicle vs drug treated for WT and Fmr1 KO mice F(1,15)=15.1035, Fcrit=4.74. E-F) The
phosphorylation of p-eIF4B did not show appreciable changes in response to acute exposure of wild-type
and Fmr1 knockout (KO) mice to either PF-4708671 or FS-115. n=3 per genotype per condition. Error
bars denote S.E.M.
3
Supplementary Figure 3
Figure S3: A) Chronic treatment of wild-type and Fmr1 knockout (KO) mice with PF-4708671 does not
alter social preference in a 3-chambered social arena. All groups showed significantly more interaction
with a mouse as compared to an object. **p<0.01, ***p<0.001 with a pair-wise Student's t-test for each
group. B) Graph showing performance of all genotype and treatment groups across the training, longterm memory, and reversal stages of the Y-maze choice reversal test. *** p<0.001 for comparison
between wild-type+vehicle and Fmr1 KO+vehicle, ** p<0.01 for Fmr1 KO+vehicle and Fmr1 KO+PF4708671, statistical analyses by repeated measures ANOVA (treatment x time), followed by BonferroniDunn post-hoc comparisons. C) Chronic treatment of wild-type and Fmr1 knockout (KO) mice with FS115 does not alter social preference in a 3-chambered social arena. All experimental groups showed
significantly more interaction with a mouse as compared to an object. *p<0.05, **p<0.01, ***p<0.001
statistical analyses with a pair-wise Student's t-test for each group. D) Graph showing performance of all
genotype and treatment groups across the training, long-term memory, and reversal stages of the Ymaze choice arm reversal test. *p<0.05 for comparison between wild-type + vehicle vs. Fmr1 KO +
vehicle and Fmr1 KO + vehicle vs. Fmr1 KO + FS-115 by non-parametric ANOVA by Friedman Test,
followed by Dunn’s multiple comparison test. Error bars denote S.E.M. Please also refer to
Supplementary Behavioral Protocols.
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Supplementary Figure 4
Figure S4: Top) Representation of dendritic spine types as used previously (16). A) Bar graph showing
the breakdown of specific spine categories as a fraction of the total number of spines counted per neuron
per condition from the PF-4708671-treated cohort of mice. n=3 mice (3-5 neurons per mouse, n=9-15
neurons per condition). B) Bar graph showing the breakdown of specific spine categories as a fraction of
the total number of spines counted per neuron per condition from the FS-115-treated cohort of mice. n=3
mice (4-6 neurons per mouse, n=12-18 neurons per condition). Statistical analyses for both graphs was
done with Kruskal-Wallis non-parametric ANOVA followed by Dunn’s multiple comparison; *p<0.05,
**p<0.01, ***p<0.001. Error bars denote S.E.M. C) Representative dendrites from each treatment group.
Please note that the graph in Figure 4 shows pooled data of stubby and mushroom spines with the
corresponding statistics.
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Supplementary Table 1
Table S1: Kinase profiles of PF-4708671 and FS-115
Characteristic
PF-4708671
FS-115
S6K1[IC50]
0.16μM
0.021μM
S6K2 [IC50]
65μM
2.06μM
Percentage Inhibition of other kinases
PF-4708671 (1μM)
FS-115 (0.5μM)
Akt 1 and 2
31
1
AMPK (α1 and α2)
12
11
eEF2K
27
0
mTOR
n.d
11
NEK 6
25
7
PDK1
14
3
PI3K (α and β)
-7
0
PKA
17
67
PKCα
+6
1
PKG
n.d
68
ROCKII
9
96
Rsk1
n.d
34
Rsk2
13
0
S6K1
77
91
S6K2
35
38% (at 1.1 µM)
Sgk1
35
18
Table S1: Comparative kinase specificity profiles and pharmacokinetics of PF-4708671 and FS115. A) Table shows the impact of 1 µM PF-4708671 and 0.5 µM FS-115 on the activity of common
kinases. The PF-4708671 inhibitory profile is adapted from supplementary data in Pearce et al. 2010,
whereas the FS-115 data was generated in experiments described in the Supplementary Information.
Abbreviations: S6K1 (p70 ribosomal S6Kinase 1); Akt (protein kinase B); AMPK (AMP-activated protein
kinase); eEF2K- eEF2 Kinase, mTOR (mechanistic target of rapamycin); NEK6 (NIMA related Kinase 6);
PDK1(protein linase D); PI3K (phosphatidylinositol 3-kinase); PKCα (Protein Kinase C alpha); PKA
(Protein Kinase A); PKG (Protein Kinase G); PRK (protein kinase C-related kinase); ROCKII (Rhodependent protein kinase); Rsk (p90 ribosomal S6 kinase 1); Sgk1 (Serum Glucocorticoid Related Kinase
1). n.d- not determined. + sign indicates increased kinase activity.
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Table S2: Pharmacokinetic profile and blood-brain distribution of PF-4708671 and FS-115
Supplementary
Table 2
in mice
PF-4708671
Time (h)
Plasma (ng/ml)
Brain (ng/g)
Brain: Plasma ratio
0.5
834
423
0.51
1
3680
2080
0.56
2
15100
8164
0.54
Time (h)
Plasma (ng/ml)
Brain (ng/g)
Brain: Plasma ratio
0.5
1890
6427
3.4
1.1
3013
15233
5.1
2.6
4740
19167
4.0
FS-115
Table S2: Pharmacokinetics and blood-brain distribution profiles of PF-4708671 and FS-115. PF4708671 pharmacokinetics were obtained from male C57/Bl6 mice given an IP injection of 32 mg/kg
using in-house protocol by Pfizer. n=3 mice per time point. FS-115 pharmacokinetics were obtained from
male CD-1 given a single oral gavage of 25 mg/kg. n=3 mice per time point.
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Supplementary Behavioral Methods
Overall design and rationale
Performance on the behavioral tests was key to our overall evaluation of the treatment outcomes of the
two S6K1 inhibitors. In preliminary studies we chose to use the open field test to determine whether
particular doses of the drugs were making the mice hypoactive. . Following this initial scree, two major
cohorts were used for each drug (separately) comprising of 6-7, 3-4 month-old male mice. The behavioral
tests were conducted in increasing order of difficulty and stress ranging from marble burying, social
interaction, and Y-maze choice arm reversal. PF-4708671-treated cohorts of mice were injected for 10
days before starting behavioral tests. On day 1 OFA was done, followed marble burying on day 2, day 3
to 6 was devoted to social testing where a maximum of 6 mice were tested in a day (this limitation was so
that stimulus mice were not used more than once), day 7-9 were Y-maze. The FS-115-treated cohorts
were gavaged for 3 days followed by the same sequence of behavioural tests. Because gavage can be
stressful, we acclimatized all animals to saline-gavage three days before delivering either vehicle or FS115. Mice were either injected or gavaged at the same time every day between 9 and 10 am throughout
the pre-test phase and during the test phase. During days of behavioral testing, mice were
injected/gavaged and then taken to experimental rooms for habituation. All tests began within 90
minutess of either drug or vehicle being administered and were performed as described previously
(Bhattacharya et al., 2012); these descriptions are given below. Mice were coded before the start of
dosing, weighed 2-3 throughout the entire pre-test and test periods, and the experimenter was blind to
genotype and treatment. Analyses and scoring were done by a separate individual who also was blind to
genotype and treatment.
Open Field Analysis (OFA)
Mice were habituated to experimental room for 30 minutes before start of the test. Mice were placed into
the center of a clear Plexiglas (40x40x30) cm open-field arena and allowed to explore for 15 minutes.
Illumination was provided by overhead lights (~800 lux) inside the arenas and experiments were done in
the presence of white noise at 55 dB . Data were collected in 2 minute intervals controlled by Digiscan
optical animal activity system (RXYZCM, Accuscan Electronics). Data was pooled for computerdesignated peripheral and central sectors and expressed as an average per genotype.
Marble Burying
20 small black marbles arranged in 5x4 row arrangement in clean Plexiglas cages containing fresh 5 cm
deep bedding. Mice were individually introduced into this test arena and allowed to remain for 30 minutes
under dim light and white-noise conditions. At the end of 30 minutes, the mouse was removed and the
number of unburied marbles were counted by a tester who was blind to genotype. A threshold of 50%
coverage was used to determine if marbles were buried. A one-way ANOVA was used for statistical
analysis with p < 0.05 as significance criteria.
Social Interaction Test
Social anxiety was examined using the three-chambered social box (Crawley, 2004). The test is
comprised of 3 phases. Initially, mice was habituated to the empty arena for 5 minutes after which empty
wire cages were introduced and the animal was allowed to inspect these for another 10 minutes. The
second phase of the test measured social preference involving a 15-minute session where the
experimental mouse was exposed to an inanimate object (wooden geometric shape) under one of the
wire cages and a stimulus mouse (different genotype male mouse aged 2 months, housed in different
cages and location). The placement of both social and non-social targets was counterbalanced between
experimental subjects. Parameters measured were a) time spent in each chamber, b) time spent sniffing
each target and c) time spent 1cm in the vicinity of the targets. Measurements were done with either
tracking software (Ethovision XT, Noldus) or manually by an observer blind to genotypes. We found good
correlation in most cases. The third phase examined social novelty, during which the inanimate object
was replaced by a novel stimulus mouse. Similar parameters were monitored as in the social preference
test. Time spent interacting with the target mice was scored by an observer blind to genotype. A Student’s
t-test was used for statistical analysis with p < 0.05 as significance criteria. All experiments were carried
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out under dim lights and white noise.
Arm Reversal in Y-Maze
Mice were habituated to the maze for 15 minutes on day 1. The mice were trained to locate a submerged
platform in one of the two arms of the Y-maze for 20 trials on day 2. Training threshold was set at 50%
(10 consecutive correct choices in 20 trials). On day 3, the mice were tested to see if they were able to
find the platform in the Y-maze from the training done the previous day. Mice that were successfully able
to locate the platform in four trials out of five then were placed in the reversal phase of the experiment.
Here, the platform was moved into the opposite arm. The mice were tested to determine how long and
whether they could find the platform. Mice were allowed only 20 seconds to make an arm choice. Mice
that made the incorrect choice were trapped the incorrect arm for 20 seconds and were returned to their
home cage. No direction or guidance was given to the mice at this stage. Mice that were able to correctly
locate the new position of the platform 9 out of 10 trials were deemed to have succeeded and were not
tested further. Mice were assigned randomly to either left or right arms at the beginning of training and the
researcher was blind to genotype during training/testing. Repeated measures ANOVA tests were used for
statistical analysis with p < 0.05 as significance criteria followed by post-hoc analyses.
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