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

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

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

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

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

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 ?

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

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

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)

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?

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)

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

Does Prosapip1 promote F-actin assembly? G-actin

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

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

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

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

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

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 ?

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

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

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

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

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