1. Prosapip1 – A New Target For Alcohol
Sophie Laguesse
Dorit Ron
From genes
To behavior
Ron Lab

2. Synaptic Plasticity and Alcohol Addiction?
Reward circuit
Synaptic Proteins
Neuroadaptations
Caroni et al., Nat. Rev. Neurosci., 2012.

3. mTORC1 and mRNA Local Dendritic Translation
mRNA translation
Synaptic plasticity
Learning and memory
Deptor
Pras40
4E-BP P
S6Kinase P
Translation Initiation complex

4. Phospho Protein / Total Protein(% of control)
Alcohol Activates mTORC1 and mRNA Translation in the Nac
mRNA translation
Synaptic plasticity
Learning and memory
Deptor
Pras40 50
100
150
200
250
Saline
Alcohol
P-S6K
P-4E-BP *
Phospho Protein / Total Protein(% of control)
S6K
4E-BP
Mouse NAc
4E-BP P
S6Kinase P
pS6 5X
20X
Alcohol
Water
NAc core
NAc Shell
Translation Initiation complex
Neasta et al., PNAS, 2010
Neasta et al, Biol. Psy, 2011
Barak et al, Nat Neurosci, 2013
Beckley et al, J Neurosci, 2016

5. Rapamycin – A Selective mTORC1 Inhibitor
Inhibition of mTORC1 reduces alcohol drinking
Rapamycin – A Selective mTORC1 Inhibitor
0.1
0.2
0.3
0.4
0.5
0.6
Alcohol consumed
(g/kg/30 min) *
Veh
Rapamycin
(10 mg/kg)
Benjamin, et al. (2011) Nat Rev Drug Discov
Neasta et al., PNAS, 2010
Neasta et al, Biol. Psy, 2011
Barak et al, Nat Neurosci, 2013
Beckley et al, J Neurosci, 2016

6. Polysomes purification and RNA-seq
Hypothesis and Aims
Hypothesis: mTORC1-dependent translation of synaptic proteins contributes to neuroadaptations that underlie alcohol drinking behaviors
Aim: Identification and characterization of proteins whose translation is controlled by mTORC1 in the nucleus accumbens after alcohol consumption ?
Polysomes purification and RNA-seq ?

7. Identification of Synaptic Proteins whose Translation Is Controlled by mTORC1 in the Nac after Alcohol Consumption
Experimental design: Intermittent access two bottle choice 20% alcohol
24 hours 2BC
24 hours withdrawal
3 hours Rapamycin
Sanchis-Segura and Spanagel, Addiction Biol., 2006.
24h
Alcohol (20%)
Water
48h
Water +
Vehicle
Rapamycin
Alcohol
Polysomal RNA purification
RNA-sequencing
AAAA 3’
mRNA
Ref gene
45%
15%
Sucrose gradient centrifugation
AAAA
m7G
Non-translating mRNA
Actively translating mRNA

8. mRNA expression/GAPDH
Polysomal RNA-seq Identified Prosapip1 as a Target of mTORC1
421
575 12
Translation increased by alcohol
Translation decreased by rapamycin
RNA-seq
Prosapip1: ProSAP2 (Shank3)-interacting protein 1
SPAR
Shank3
qRT-PCR
100
200
300
mRNA expression/GAPDH
(%of the control) **
Vehicle
Rapamycin
Water
Alcohol
Brain specific, enriched in the striatum
Highly enriched in the post-synaptic density
Water
Alcohol 10 20 30 40 50 *
***
FPKM
Vehicle
Rapamycin
Prosapip1
Interacts with Shank3: morphology of medium spiny neurons
Interacts with SPAR (spine-associated Rap-specific GTPase-activating protein): spine morphology and actin cytoskeleton regulation

9. Translation Transcription
Does Alcohol Consumption Increases Prosapip1 Translation?
Polysomal mRNA
total mRNA
water
Alcohol (binge) 50
100
150
200
***
Prosapip1 mRNA/GAPDH level (% of the control) ns
water
Alcohol (binge) 20 40 60 80
100
Prosapip1 mRNA/GAPDH level (% of the control)
Binge
Translation
Transcription
Withdrawal
water
Alcohol (WD) 50
100
150
200
250
Prosapip1 mRNA/GAPDH level (% of the control) *
water
Alcohol (WD) 20 40 60 80
100 ns
Prosapip1 mRNA/GAPDH level (% of the control)

10. Conclusion 1
Excessive alcohol intake increases Prosapip1 translation in the Nac in a mTORC1- dependent manner
Are Prosapip1 protein levels increased in the nucleus accumbens after alcohol consumption?

11. Are Prosapip1 Protein Levels increased after Alcohol Consumption?
Nucleus accumbens homogenates
water
Alcohol (binge) 50
100
150
***
protein/GAPDH level
(% of the control)
Water
Alcohol (binge)
Water
Alcohol (WD) 50
100
150
200 **
Prosapip1/GAPDH
(% of the control)
Prosapip1
GAPDH
Prosapip1
GAPDH
water
Alcohol (WD)
Synaptic fraction H S1 P1 S2 P2
Prosapip1
Actin
Alcohol (binge)
water
Water
Alcohol (WD) 50
100
150
200
Prosapip1/Actin
(% of the control) **
Water
Alcohol (binge) 50
100
150
200 **
Prosapip1/Actin
(% of the control)
Prosapip1
Actin
water
Alcohol (WD)

12. Conclusion 2
What are the functional consequences of an enhanced Prosapip1 translation?
Prosapip1 translation

13. Does Prosapip1 promote F-actin assembly?
G-actin

14. Does Prosapip1 promotes F-actin assembly?
N2A cells
F-actin
GFP
Prosapip1-GFP 25 50 75
100
125
150
175 **
F-Actin/total Actin
(%of the control)
G-actin
GFP
Prosapip1-GFP 25 50 75
100 **
G-Actin/total Actin
(%of the control)
G F
G F
Actin
GFP
Prosapip1-GFP
Prosapip1
In vivo: nucleus accumbens
Ltv-GFP
Ltv-Prosapip1-GFP
GFP
NeuN
Merge
100 um
500 um
GFP
Prosapip1-GFP 25 50 75
100
125
150
175
F-Actin/total Actin
(%of the control)
F-actin **
GFP
Prosapip1-GFP 20 40 60 80
100
G-Actin/total Actin
(%of the control)
G-actin **
G F
G F
Ltv-GFP
Ltv-Prosapip1-GFP
Actin
Prosapip1

15. Excessive Alcohol Drinking Promotes F-actin Assembly in the Nucleus Accumbens
water
Alcohol (binge) 25 50 75
100
125
150
F-Actin/total Actin
(%of the control)
F-actin
*** 25 50 75
100
G-Actin/total Actin
(%of the control)
water
Alcohol (binge)
***
G-actin
G F
G F
Water
Alcohol (Binge)
Actin
Prosapip1

16. Does Prosapip1 contribute to alcohol drinking behaviors?
Conclusion 3
Alcohol consumption increases mTORC1-dependent mRNA translation of Prosapip1 in the nucleus accumbens
Prosapip1 promotes F-actin assembly
Does Prosapip1 contribute to alcohol drinking behaviors?
Prosapip1 translation

17. Alcohol Self-Administration in Mice
Experimental time line:
Self-administration
Surgery
IA-20% 2BC 1 7 12 14 18
weeks
Alcohol 20%
R (-)
L (+)
Self-administration
Nucleus accumbens
Ltv-GFP
Ltv-Prosapip1-GFP

18. Prosapip1 Exacerbates Self-Administration of Alcohol
100
200
300
400
500
600 1 7 14 21 28
Active lever press
Sessions
Fixed Ratio 2 3
Virus
infusion
Ltv-ProSAPIP1
Ltv-GFP 50
100
150
200
250
300
Reward port entries 1 7 14 21 28
Sessions
Virus
infusion
Fixed Ratio 2 3
100
200
300
400
500
600
Inactive lever press 1 7 14 21 28
Sessions
Virus
infusion
Fixed Ratio 2 3 20 40 60 80
100
120
Time (min)
Representative patterns of active lever press

19. Prosapip1 exacerbates self-administration of alcohol in mice
Conclusion 4
Prosapip1 exacerbates self-administration of alcohol in mice
Does Prosapip1 knock-down reduce alcohol self-administration ?

20. Prosapip1 Knock-down Reduces Self-Administration of Alcohol
Ltv-shSCR-GFP
Ltv-shProsapip1- GFP
Prosapip1
GAPDH
GFP
Ltv-shSCR-GFP
Ltv-shProsapip1-GFP
Nucleus accumbens
GFP
NeuN
Merge
100 mm

21. Prosapip1 Knock-Down Reduces Self-Administration of Alcohol
Ltv-shProSAPIP1
Ltv-shSCR
Active lever press
Fixed Ratio 2
Virus
infusion
Fixed Ratio 2
Reward port entries
Inactive lever press
Fixed Ratio 2

22. General Conclusions
Alcohol consumption activates mTORC1 and increases the translation of Prosapip1 in the nucleus accumbens
Prosapip1 promotes F-actin assembly
Prosapip1 regulates alcohol self-administration in mice
Prosapip1
F-actin
G-actin

23. To behavior From genes Thank you for attention Prof. Dorit Ron
Ron Lab
Thank you for attention
Prof. Dorit Ron
Nadege Morisot
Khanky Phamluong
Ron lab members
Feng Liu
Howard Becker
Marcelo Lopez

24. Is Prosapip1 expression increased after Alcohol Vaporization?
H. Becker
M. Lopez
chronic intermittent ethanol vaporization [CIE]
CIE
CTL
4 days
3 days
24h
pS6K
control
alcohol (CIE)
100
200
300
(% of the control)
pS6K/S6K **
p4E-BP 25 50 75
100
125
150
175
(% of the control)
p4E-BP/4E-BP *
control
alcohol (CIE)
pS6K
S6K
control
ethanol
p4E-BP
4E-BP
control
ethanol
Chronic Intermittent Ethanol Vapor
Exposure (16 hr/day x 4 days)
Control Air Inhalation
72 hr Abstinence Period
prosapip1 50
100
150
200
250
Prosapip1/GAPDH
(% of the control) *
control
alcohol (CIE)
GAPDH
Prosapip1
control
ethanol