Prosapip1-a novel target of alcohol drinking behaviors

Prosapip1 – A New Target For Alcohol

Sophie Laguesse

Dorit Ron

Synaptic Proteins

Neuroadaptations

Synaptic Plasticity and Alcohol Addiction

Caroni et al., Nat. Rev. Neurosci., 2012.

Reward circuit

mTORC1 and mRNA Local Dendritic Translation

mRNA translation

Synaptic plasticity

Learning and memory

Deptor Pras40

S6Kinase

P

4E-BP

P

Translation

Initiation complex

Alcohol Activates mTORC1 and mRNA Translation in the Nac

mRNA translation

Synaptic plasticity

Learning and memory

Deptor Pras40

S6Kinase

P

4E-BP

P

Neasta et al., PNAS, 2010 Neasta et al, Biol. Psy, 2011 Barak et al, Nat Neurosci, 2013 Beckley et al, J Neurosci, 2016

Translation

Initiation complex

0 50 100 150 200 250 Saline_Alcohol P-S6K P-4E-BP * * P h o sp h o P ro te in / T o ta l P ro te in (% o f co n tr o l) Saline Alcohol P-S6K S6K P-4E-BP 4E-BP Mouse NAc

pS6

5X 20X 5X 20X Alcohol Water NAc core NAc Shell

Neasta et al., PNAS, 2010 Neasta et al, Biol. Psy, 2011 Barak et al, Nat Neurosci, 2013 Beckley et al, J Neurosci, 2016 Benjamin, et al. (2011) Nat Rev Drug Discov

Rapamycin – A Selective mTORC1 Inhibitor

Inhibition of mTORC1 reduces alcohol drinking

0 0.1 0.2 0.3 0.4 0.5 0.6 A lc o h o l co n su m ed (g /k g /3 0 m in )

*

Veh Rapamycin (10 mg/kg)

Aim: Identification and characterization of proteins

whose translation is controlled by mTORC1 in the

nucleus accumbens after alcohol consumption

Polysomes purification and RNA-seq

Hypothesis and Aims

?

?

Hypothesis: mTORC1-dependent translation of

synaptic proteins contributes to neuroadaptations

that underlie alcohol drinking behaviors

Water + Vehicle Water + Rapamycin Alcohol + Vehicle Alcohol + Rapamycin

Polysomal RNA purification

45% 15% Sucrose gradient centrifugation AAAA m7G Non-translating mRNA AAAA m7G

Actively translating mRNA

24 hours 2BC 24 hours withdrawal

3 hours Rapamycin

24h

Alcohol (20%) Water

24h 24h 24h 24h 48h

Experimental design: Intermittent access two bottle choice 20% alcohol

Sanchis-Segura and Spanagel, Addiction Biol., 2006.

Identification of Synaptic Proteins whose Translation Is Controlled by

mTORC1 in the Nac after Alcohol Consumption

RNA-sequencing

AAAA 3’ mRNA

Polysomal RNA-seq Identified Prosapip1 as a Target of mTORC1

Water Alcohol 0 10 20 30 40 50

*

***

F P K M Vehicle Rapamycin

Prosapip1

qRT-PCR

0 100 200 300 m R N A e xp re ss io n /G A P D H ( % o f th e c o n tr o l)

**

_**

Vehicle Rapamycin Water Alcohol

Prosapip1: ProSAP2 (Shank3)-interacting protein 1

• Brain specific, enriched in the striatum

• Highly enriched in the post-synaptic density

Shank3

• Interacts with Shank3: morphology of

medium spiny neurons

SPAR

• Interacts with SPAR (spine-associated

Rap-specific GTPase-activating protein): spine

morphology and actin cytoskeleton

regulation

421 12 575

Translation increased by alcohol Translation decreased by rapamycin

Binge

Withdrawal

Polysomal mRNA

total mRNA

water Alcohol (binge) 0 50 100 150 200

***

P ro s a p ip 1 m R N A /G A P D H l e v e l (% o f th e c o n tr o l)

ns

water Alcohol (binge)

0 20 40 60 80 100 P ro s a p ip 1 m R N A /G A P D H l e v e l (% o f th e c o n tr o l)

Translation

Transcription

Does Alcohol Consumption Increases Prosapip1 Translation?

water Alcohol (WD) 0 50 100 150 200 250 P ro s a p ip 1 m R N A /G A P D H l e v e l (% o f th e c o n tr o l)

*

water Alcohol (WD) 0 20 40 60 80 100

ns

P ro s a p ip 1 m R N A /G A P D H l e v e l (% o f th e c o n tr o l)

Excessive alcohol intake increases Prosapip1

translation in the Nac in a mTORC1- dependent manner

Conclusion 1

Are Prosapip1 protein levels increased in the

nucleus accumbens after alcohol consumption?

Are Prosapip1 Protein Levels increased after Alcohol Consumption?

0 50 100 150

***

p ro te in /G A P D H l e v e l (% o f th e c o n tr o l)

Water Alcohol (binge)

Prosapip1 GAPDH water Alcohol (WD) Water Alcohol (WD) 0 50 100 150 200

**

P ro s a p ip 1 /G A P D H (% o f th e c o n tr o l)

Water Alcohol (binge)

0 50 100 150 200

_**

P ro s a p ip 1 /A c ti n (% o f th e c o n tr o l)

Synaptic fraction

Nucleus accumbens homogenates

Water Alcohol (WD) 0 50 100 150 200 P ro s a p ip 1 /A c ti n (% o f th e c o n tr o l)

**

Prosapip1 GAPDH

water Alcohol (binge)

Prosapip1 Actin Alcohol (binge) water Prosapip1 Actin water Alcohol (WD) H S1 P1 S2 P2

Conclusion 2

Prosapip1 translation

What are the functional consequences

of an enhanced Prosapip1 translation?

Prosapip1

F-actin G-actin

Does Prosapip1 promote F-actin assembly?

Does Prosapip1 promotes F-actin assembly?

G F G F Actin GFP Prosapip1-GFP Prosapip1

• N2A cells

F-actin

GFP Prosapip1-GFP 0 25 50 75 100 125 150 175

_**

F -A ct in /t o ta l A ct in (% o f th e c o n tr o l)

G-actin

GFP Prosapip1-GFP 0 25 50 75 100

**

G -A ct in /t o ta l A c ti n (% o f th e co n tr o l)

• In vivo: nucleus accumbens

G F G F Ltv-GFP Ltv-Prosapip1-GFP Actin Prosapip1 GFP Prosapip1-GFP 0 25 50 75 100 125 150 175 F -A ct in /t o ta l A ct in (% o f th e c o n tr o l)

F-actin

**

GFP Prosapip1-GFP 0 20 40 60 80 100 G -A ct in /t o ta l A ct in (% o f th e co n tr o l)

G-actin

**

GFP NeuN Merge 100 um 500 um

Ltv-GFP

Ltv-Prosapip1-GFP

water Alcohol (binge) 0 25 50 75 100 125 150 F -A c ti n /t o ta l A ct in (% o f th e co n tr o l)

F-actin

***

0 25 50 75 100 G -A ct in /t o ta l A ct in ( % o f th e co n tr o l)

water Alcohol (binge)

***

G-actin

Excessive Alcohol Drinking Promotes F-actin Assembly in the

Nucleus Accumbens

G F G F

Water Alcohol (Binge)

Actin

Conclusion 3

Prosapip1 translation

Does Prosapip1 contribute to alcohol

drinking behaviors?

Prosapip1 promotes F-actin assembly

Alcohol consumption increases

mTORC1-dependent mRNA translation of Prosapip1 in

Alcohol Self-Administration in Mice

Experimental time line:

Alcohol 20% R (-) L (+) Self-administration Self-administration Self-administration

Surgery

IA-20% 2BC 1 7 12 14 18 weeks Ltv-GFP Ltv-Prosapip1-GFP Nucleus accumbens

0 20 40 60 80 100 120

Time (min)

Representative patterns of active lever press

Prosapip1 Exacerbates Self-Administration of Alcohol

Ltv-ProSAPIP1 Ltv-GFP 0 100 200 300 400 500 600 1 7 14 21 28 A ct iv e le ve r p re ss Sessions Fixed Ratio 2 Fixed Ratio 3 Virus infusion

Active lever press

0 50 100 150 200 250 300 R ew ar d p o rt e n tr ie s 1 7 14 21 28 Sessions Virus infusion Fixed Ratio 2 Fixed Ratio 3

Reward port entries

0 100 200 300 400 500 600 In ac ti ve l ev er p re ss 1 7 14 21 28 Sessions Virus infusion Fixed Ratio 2 Fixed Ratio 3

Prosapip1 exacerbates self-administration of

alcohol in mice

Conclusion 4

Does Prosapip1 knock-down reduce alcohol

self-administration ?

Prosapip1 Knock-down Reduces Self-Administration of Alcohol

Ltv-shSCR-GFP Ltv-shProsapip1-GFP Lt v-shSC R-G FP Lt v-shPr osap ip GFP Prosapip1 GAPDH GFP GFP NeuN Merge 100 mm Nucleus accumbens

Prosapip1 Knock-Down Reduces Self-Administration of Alcohol

Inactive lever press

Fixed Ratio 2 Fixed Ratio 2

Reward port entries

Ltv-shProSAPIP1 Ltv-shSCR

Active lever press

Fixed Ratio 2 Virus

General Conclusions

• Alcohol consumption activates mTORC1 and increases the

translation of Prosapip1 in the nucleus accumbens

Prosapip1

• Prosapip1 promotes F-actin assembly

• Prosapip1 regulates alcohol self-administration in mice

F-actin G-actin

Thank you for attention

Prof. Dorit Ron

Nadege Morisot

Khanky Phamluong

Ron lab members

Feng Liu

Howard Becker

Marcelo Lopez

Is Prosapip1 expression increased after Alcohol Vaporization?

• chronic intermittent ethanol vaporization [CIE]

pS6K S6K control ethanol p4E-BP 4E-BP control ethanol GAPDH Prosapip1 control ethanol

H. Becker

M. Lopez

CIE CTL CIE CTL

4 days _{3 days} 4 days 24h

pS6K

control alcohol (CIE)

0 100 200 300 p S 6 K /S 6 K (% o f th e c o n tr o l)

_**

p4E-BP 0 25 50 75 100 125 150 175 p 4 E -B P /4 E -B P (% o f th e c o n tr o l)

*

control alcohol (CIE)

prosapip1 0 50 100 150 200 250 P ro s a p ip 1 /G A P D H (% o f th e c o n tr o l)

*