1. Prosapip1-Dependent Adaptations In The Nucleus Accumbens Drive Alcohol Intake, Seeking And Reward
Sophie Laguesse1, Nadege Morisot1, Feng Liu1, Marcelo Lopez2, Khanhky Phamluong1, William C Griffin2, Howard Becker2, Kevin Bender1 and Dorit Ron1
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
Model of excessive alcohol drinking
Introduction
RNA-seq
Hypothesis and aims
Deptor
Pras40
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).
Hypothesis
mTORC1-dependent translation of synaptic proteins contributes to neuroadaptations that underlie alcohol drinking behaviors
Intermittent access 20% alcohol two-bottle choice drinking paradigm
Water +
Vehicle
Rapamycin
Alcohol
Polysomal RNA
mRNA purification
AAAA 3’
fragmentation
Deptor
Pras40
mTORC1 M W F
Days
Weeks
1 - 7 8 P P
NAc isolation
cDNA library
Aim
Identification by RNA-seq and characterization of proteins whose translation is controlled by mTORC1 in the NAc after alcohol consumption
Polysomal RNA purification
A+W
24h
W only
Withdrawal
Rapamycin 3h
Binge
Translation Initiation
Factor Complex
AAAA
m7G
Illumina Hiseq
High-throughput sequencing
Alignment& data analysis
RNA-seq analysis
Ref gene
mRNA translation
Results
1. RNA-seq identified Prosapip1 as downstream target of mTORC1
2. Alcohol consumption increases Prosapip1 protein levels
3. Alcohol consumption promotes F-actin assembly via Prosapip1
shProsap
G F
CTL
CIE
SCR
Actin
Prosapip1
Alcohol increase Prosapip1 translation in the NAc in a mTORC1-dependent manner **
G-actin
CTL
Prosap 25 50 75
100 d a
CTL
Prosap **
F-actin
F/G-Actin/Actin
(%of the control) 50
100
150
200 a
G F
G F
CTL
Prosapip1
Actin
Water
Alcohol (B)
Prosapip1
GAPDH
Total homogenate
Prosapip1/GAPDH
(% of the control) W B
*** 50
100
150
200
Synaptic fraction **
Prosapip1/Actin
(% of the control) 50
100
150
200
RNA-seq
qRT-PCR b
Water
Alcohol (B)
Prosapip1
Actin
Prosapip1 FPKM 10 20 30 40 * **
W+V
W+R
A+V
A+R
100
200
300 * **
Prosapip1/ GAPDH
(% of control) 20 40 60 80
100
120
(%of the control)
F-Actin/Actin
F-actin
Water
CIE **
500 mm
F-actin **
F/G-Actin/Actin
(%of the control)
SCR
shProsap 50
100
150 **
G-actin
SCR
shProsap 50
100
150 b
G F
G F
SCR
shProsapip1
Actin
Prosapip1
CTL+SCR
CTL+shProsap
CIE+SCR
CIE+shProsap c
Water
Alcohol (WD)
Prosapip1
GAPDH **
Total homogenate
Prosapip1/GAPDH
(% of the control) W WD 50
100
150
200 **
Synaptic fraction
Prosapip1/Actin
(% of the control) W WD 50
100
150
200 20 40 60 80
100
120
(%of the control)
G-Actin/Actin
G-actin
Water
CIE ** W B **
F-actin
F/G-Actin/Actin
(%of the control) 50
100
150
***
G-actin W B 25 50 75
100 e c
G F
G F
Water
Alcohol (B)
Actin
Prosapip1 d
Water
Alcohol (WD)
Prosapip1
Actin
F- 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.
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
(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.
Conclusions
4. Prosapip1 contributes to alcohol-dependent structural plasticity
5. Prosapip1 drives alcohol self-administration and contributes to alcohol reward
Ribosomes
G-Actin
mTORC1
pS6K
p4EBP a a b M W F
M-F
Days
Weeks
1 - 7
8 - 11
Surgery
Active
lever
Operant chamber
Inactive
Alcohol
IA2BC SA
SCR
shProsapip1
Water
Alcohol M W F
Days
Weeks
1 - 4 5
Surgery
IA2BC
A+W
Dissection
(Binge)
6-8 A 9 4h
No Alcohol b c d
SCR
shProsapip1
Alcohol (or saline)
Saline
Pre-cond. 1 2 3 4 5 6 7 10
Conditioning
Post-cond.
Days
Surgery
-24 8 9 30 60 90
120
Lever press * 2 4 6 8 10
Frequency of active press * k 40 80
120 30 60 90
Time (min)
Cumulative
active press *
mTORC1 g d 5 10 15
Spine density
Spines/10μm ns
Water
Alcohol c
0.5
1.0
1.5
Spine length
Spine length (μm) ns
Water
Alcohol
Spines subtypes 20 40 60
*** **
Filopodia
Thin
Stubby
Mushroom *
% of spines
p4EBP
pS6K e f g P P 4 8 12 16
Frequency of port visit * 50
100
150
200
Port visit * 40 80
120 50
100
150
200
Time (min) *
Cumulative
port visit
Saline+SCR
Alcohol+SCR
Saline+shProsap
Alcohol+shProsap
Ribosomes
Prosapip1 l
100
200
300
400
500
CPP score (s) *
***
Alcohol e f h
Prosapip1 i j
Head diameter (μm)
Head diameter
0.2
0.4
0.6
***
Water
Alcohol
Spine area (μm2)
spine area
0.5
1.0
1.5
*** **
Water
Alcohol 40 80
120 9 18 27 36
alcohol delivery
Cumulative * 1 2 3 4
Frequency of alcohol delivery *
0.0
0.5
1.0
1.5
2.0
Alcohol consumed
(g/kg/2h) *
F-Actin
Behavioral Adaptations
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.
(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-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
(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.
This work was supported by NIH P50 AA (D.R.) and the Belgian American Educational Foundation (S.L.)