Hypothesis
mTORC1-dependent translation
of synaptic proteins contributes
to neuroadaptations that underlie
alcohol drinking behaviors
Deptor
Pras40
mTORC1
Aim
Identification by RNA-seq and
characterization of proteins
whose translation is controlled by
mTORC1 in the NAc after alcohol
consumption
Hypothesis and aims
P
Prosapip1-Dependent Adaptations In The Nucleus Accumbens
Drive Alcohol Intake, Seeking And Reward
Sophie Laguesse
1
, Nadege Morisot
1
, Feng Liu
1
, Marcelo Lopez
2
, Khanhky Phamluong
1
, William C Griffin
2
,
Howard Becker
2
, Kevin Bender
1
and Dorit Ron
1
1
Department of Neurology, Alcohol Center for Translational Genetics, University of California San Francisco, CA 94143
2
Charleston Alcohol Research Center, Department of Psychiatry and Behavioral Sciences, Medical University of South Carolina, Charleston, SC 29425
This work was supported by NIH P50 AA017072 (D.R.) and the Belgian American Educational Foundation (S.L.) Email: dorit.ron@ucsf.edu
the mammalian target of rapamycin complex 1 (mTORC1) is a
macromolecular complex localized at dendrites that plays a role in
synaptic plasticity by promoting the translation of synaptic proteins
(Buffington et al, Ann Rev Neurosci, 2014). mTORC1
phosphorylates its downstream effectors the ribosomal S6 Kinase
(S6K) and the 4E-binding protein (4E-BP), both proteins being
involved in the assembly of the translation initiation complex
(Lipton and Sahin, Neuron, 2014)
We previously showed that excessive alcohol consumption
activates the H-Ras/ PI3K/AKT signaling in the nucleus accumbens
(NAc), which leads to the activation of mTORC1 (Neasta et al,
PNAS, 2010; Ben Hamida et al, J Neurosci, 2012). Rapamycin, a
specific inhibitor of mTORC1, inhibits rodents alcohol seeking and
drinking (Neasta et al, PNAS, 2010).
Deptor Pras40 P P Translation Initiation Factor Complex mRNA translation
Intermittent access 20% alcohol
two-bottle choice drinking
paradigm
M W F
M
Days
Weeks
1 - 7
8
A+W
24h
24h
W only
Withdrawal
Rapamycin
3h
Binge
5. Prosapip1 drives alcohol self-administration and contributes
to alcohol reward
4. Prosapip1 contributes to alcohol-dependent structural
plasticity
Introduction
1. RNA-seq identified Prosapip1 as downstream
target of mTORC1
(a) Prosapip1 Fragment per Kilobase of transcript per Million mapped reads (FPKM) value from RNA-seq. n=3 (b) Quantitative Real-time PCR determined prosapip1 polysomal mRNA levels. n= 5.*p<0.05, **p<0.01.
a
b
2. Alcohol consumption increases Prosapip1
protein levels
After 7-8 weeks of IA-2BC, NAc were removed after binge drinking of alcohol (B) (a,b) or after 24 hours of Withdrawal (WD) (c,d). Prosapip1 protein levels were determined by western blot in the total homogenate (a, c) or in the purified crude synaptic fraction (b,d). n=4 water, 5 alcohol. **p<0.01, ***p<0.001
e
c
d
Water Alcohol (B) Prosapip1 GAPDHTotal homogenate
P
ro
sa
p
ip
1/
G
A
P
D
H
(%
o
f
th
e
c
o
n
tr
o
l)
W B***
0 50 100 150 200 Water Alcohol (B) Prosapip1 ActinSynaptic fraction
**
P
ro
s
ap
ip
1/
A
ct
in
(%
o
f
th
e
co
n
tr
o
l)
0 50 100 150 200 Water Alcohol (WD) Prosapip1 GAPDH**
Total homogenate
P
ro
sa
p
ip
1/
G
A
P
D
H
(%
o
f
th
e
co
n
tr
o
l)
W WD 0 50 100 150 200 Water Alcohol (WD) Prosapip1 Actin**
Synaptic fraction
P
ro
s
ap
ip
1/
A
ct
in
(%
o
f
th
e
co
n
tr
o
l)
W WD 0 50 100 150 2003. Alcohol consumption promotes F-actin assembly via
Prosapip1
G F G F CTL Prosapip1 Actin Prosapip1 CTL Prosap**
F-actin
F
/G
-A
ct
in
/A
ct
in
(%
o
f
th
e
c
o
n
tr
o
l)
0 50 100 150 200**
G-actin
CTL Prosap 0 25 50 75 100 G F G F Water Alcohol (B) Actin Prosapip1 W B**
F-actin
F
/G
-A
ct
in
/A
c
ti
n
(%
o
f
th
e
co
n
tr
o
l)
0 50 100 150***
G-actin
W B 0 25 50 75 100 CTL+SCR CTL+shProsap CIE+SCR CIE+shProsap 0 20 40 60 80 100 120F
-A
ct
in
/A
ct
in
(%
o
f
th
e
c
o
n
tr
o
l)
F-actin
Water CIE**
**
0 20 40 60 80 100 120G
-A
ct
in
/A
ct
in
(
%
o
f
th
e
co
n
tr
o
l)
G-actin
Water CIE**
**
G F G F SCR shProsapip1 Actin Prosapip1**
G-actin
SCR shProsap 0 50 100 150 500 mF-actin
**
F
/G
-A
ct
in
/A
ct
in
(%
o
f
th
e
c
o
n
tr
o
l)
SCR shProsap 0 50 100 150a
b
c
d
shProsap G F G F G F G F CTL CIE SCR shProsap SCR Actin Prosapip1F- and G-actin contents were determined by western blot in the NAc after (a) lentivirus-mediated Prosapip1 overexpression, (b) lentivirus-mediated Prosapip1 knockdown and (c) alcohol binge drinking (B). (d) NAc were infused with lentivirus delivering shRNA against Prosapip1 (shProsap) or a scrambled sequence (SCR). Mice underwent 2 weeks of Chronic Intermittent Exposure (CIE) to alcohol vapors whereas controls had air exposure (CTL). F- and G-actin contents were determined 24 hours after the last CIE exposure. n=4. **p<0.01, ***p<0.001.
Results
Ribosomes
G-Actin
mTORC1
pS6K
p4EBP
Behavioral Adaptations
F-Actin
pS6K
p4EBP
P
P
Ribosomes
Prosapip1
Prosapip1
H
ea
d
d
ia
m
e
te
r
(μ
m
)
Head diameter
0 0.2 0.4 0.6***
***
***
Water AlcoholS
p
in
e
a
re
a
(μ
m
2)
spine area
0 0.5 1.0 1.5***
**
Water Alcohol 0 0.5 1.0 1.5Spine length
S
p
in
e
le
n
g
th
(
μ
m
)
ns Water Alcohol 0 5 10 15Spine density
S
p
in
es
/1
0μ
m
ns Water Alcohol SCR shProsapip1 Water Alcohol(a-b) After 4 weeks of IA20%-2BC, NAc were infused with Ltv-shProsapip1 or Ltv-SCR at low titer (1X10*5pg/mL). Mice underwent 3 more weeks of IA-2BC. After the last binge drinking, the dendritic spine morphology of shell GFP-positive MSNs was analyzed : spine density (c), spine length (d), spine head diameter (e) and spine area (f). (g) Percentage of Filopodia, Thin, Stubby and Mushroom-type spines in MSNs of the NAc shell.
Spines subtypes
0 20 40 60***
***
***
***
***
**
Filopodia Thin Stubby Mushroom
*
%
o
f
sp
in
e
s
M W F Days Weeks 1 - 4 5 Surgery IA2BC A+W Dissection (Binge) W F M 6-8 A W M 9 4ha
b
c
d
e
f
g
0 40 80 120 0 30 60 90 120 Time (min)C
u
m
u
la
ti
ve
a
ct
iv
e
p
re
s
s
*
0 2 4 6 8 10F
re
q
u
en
cy
o
f
ac
ti
ve
p
re
ss
*
0 4 8 12 16F
re
q
u
en
cy
o
f
p
o
rt
vi
si
t
*
0 40 80 120 0 50 100 150 200 Time (min)*
C
u
m
u
la
ti
ve
p
o
rt
v
is
it
0 50 100 150 200 P o rt v is it*
0 40 80 120 0 9 18 27 36C
u
m
u
la
ti
v
e
al
co
h
o
l
d
el
iv
er
y
*
0 1 2 3 4F
re
q
u
en
cy
o
f
al
co
h
o
l
d
el
iv
er
y
*
0.0 0.5 1.0 1.5 2.0 A lc o h o l c o n s u m e d (g /k g /2 h )*
0 100 200 300 400 500C
P
P
s
co
re
(
s)
*
***
Saline+SCR Alcohol+SCR Saline+shProsap Alcohol+shProsap Alcohol (or saline)Saline Pre -co nd . 1 2 3 4 5 6 7 10 Conditioning Po st-c on d. Days Su rger y -24 8 9
(a) Mice underwent IA20%-2BC for 7-8 weeks and were trained to self-administer alcohol in operant chambers. NAc were infused with lentivirus delivering shRNA against Prosapip1 (shProsap) or a scrambled sequence (SCR), and, self-administration was resumed. (b) number of active and inactive lever presses on the last self-self-administration session. (c, f, i) Cumulative number, (d,g,j) Frequency of (b-d) active lever press, (e-g) port visits and (h-j) alcohol delivery. (k) 4 weeks after Ltv infusion, mice underwent CPP experiment. (l) CPP score is expressed as the time spent in the drug-paired compartment during the post-conditioning test minus the time spent on the pre-conditioning test. n=10. *p<0.05
b
c
d
e
f
g
h
i
j
k
l
a
No Alcohol
Alcohol
In absence of alcohol, mTORC1 shows a basal activity in the NAc. Excessive alcohol drinking activates mTORC1 in the NAc, resulting in an increased translation of Prosapip1 mRNA. Prosapip1 accumulation promotes F-actin assembly that results in the enlargement of dendritic spines, which in turn promotes alcohol seeking, drinking, and reinforces alcohol rewarding effects.
