A Cannabinoid link between mitochondria and memory 2

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A Cannabinoid link between mitochondria and memory 2 1942 1964 O OH Δ 9 -Tetrahydrocannabinol 1990 CB1 1992 O NH Anandamide OH

Skin GI tract Bone HPA axis Neuronal plasticity Giovanni Pain perception Muscle Marsicano EndoCannabinoids Tumor and

2 A Cannabinoid link between mitochondria and memory O OH Δ 9 -Tetrahydrocannabinol 1990 CB O NH Anandamide OH FEAR 3000 a.c. Skin GI tract Bone HPA axis Neuronal plasticity Giovanni Pain perception Muscle Marsicano EndoCannabinoids Tumor and NeuroAdaptation Food Intake INSERM U1215 NeuroCentre Magendie, Rewarding Development Circulation Bordeaux, France Inflammation NeuroFrance Reproduction 2017 Neurogenesis Sleep Mitochondria and Brain Functions, 19/05/17 Locomotion Heart Liver Sexual Behaviour

3 A Cannabinoid link between mitochondria and memory 3 How do CB1 receptors do all these beautiful things?

4 Text Book: Mechanisms of action of CB1 4 THC/AEA CB1 + MAPK G i/o - adenylyl cyclase + K + ir - Ca 2+ camp + - P ATP K + - Neuronal hyperpolarization A - Decrease of neuronal excitability

Is that all? Does CB1 signal also INSIDE the cells?

The hippy scientists at work Effect of 1-tetrahydrocannabinol on ATPase activity

1971 Feb;20(2):473-5 Biochemical effects and morphological changes in rat liver

5 Is that all? Does CB1 signal also INSIDE the cells? 5 Cannabinoids are lipids, that can travel through membranes, and The Seventies: The hippy scientists at work Effect of 1-tetrahydrocannabinol on ATPase activity of rat liver mitochondria Chari-Bitron A, Bino T. Biochem Pharmacol Feb;20(2):473-5 Biochemical effects and morphological changes in rat liver mitochondria exposed to 1-tetrahydrocannabinol Bino T, Chari-Bitron A, Shahar A. Biochim Biophys Acta Oct 23;288(1):

Interesting stuff, isn t it? Yes Pedro, I have a question for you Tell me In your EM studies of immunogold staining of CB1 in the brain, did you ever observe CB1 in the mitochondria?

6 A serendipitous discovery 6 Movie Script: Time: Interior day Location: SfN Meeting Poster session, Washington DC, November 2008 Characters: G. Marsicano and Pedro Grandes (Neuroanatomist, Univ. of Basque Country, Spain, Expert in electron microscopy) G.M.: P.G.: G.M.: P.G.: G.M.: Hi, Pedro, how are you? Good! Interesting stuff, isn t it? Yes Pedro, I have a question for you Tell me In your EM studies of immunogold staining of CB1 in the brain, did you ever observe CB1 in the mitochondria? P.G.: Sometimes, but we consider it as background, like everybody Why you ask? G.M.: Just like that a crazy thought What about if I send you some CB1-KO mice as negative controls and you check if this staining is really unspecific? P.G.: OK

7 A serendipitous Wild-Type discovery 7 CB1-KO % CB1 mitocondrial particles on total P. Grandes N. Puente Purified brain mitochondria CB1 receptors are present on mitochondrial membranes of neurons (mtcb1) Benard et al Nat. Neurosci 2012

CB1 Intermembrane space INNER MEMBRANE Matrix Adenylyl G Proteins H + H + H+ Cyt.

8 What are Mitochondria? 8 Energy ROS production Calcium signalling Apoptosis Metabolism Cytosol OUTER MEMBRANE CB1 Intermembrane space INNER MEMBRANE Matrix Adenylyl G Proteins H + H + H+ Cyt.c e Cyclase - e - Q QH 2 e e - e - - H+ H + H + Succ. Fum. NADH NAD + H + O 2 Complex I Complex II Complex III Complex IV H 2 O + - ADP+Pi H + H + ATP Complex V

9 Does mtcb1 influence brain mitochondrial functions? 9 PURIFICATION of mitochondria from mouse brains (WT and CB1-KO) Mitochondrial respiration and biochemistry in the presence of an exogenous CB1 agonist (WIN) ng Oxygen/min/mg prot Mitochondrial Respiration WT CB1-KO Giovanni Benard mtcb1 signalling DIRECTLY regulates mitochondrial functions

10 Does mtcb1 influence brain mitochondrial functions? 10 mtcb1 signalling DIRECTLY regulates mitochondrial functions What is mtcb1 good for? Cover Charlie Padgett

11 Need of tools: What is the signaling of mtcb1? 11 mtcb 1 Gα i Etienne Hebert-Chatelain Soluble Adenylyl cyclase (sac) I II III IV V O 2 ATP

Hebert-Chatelain Soluble Adenylyl cyclase (sac) sac is

present in mitochondria sac is specifically inhibited by

12 Need of tools: What is the signaling of mtcb1? 12 mtcb 1 Gα i Soluble Adenylyl Cyclase (sac) Etienne Hebert-Chatelain Soluble Adenylyl cyclase (sac) sac is enriched in mitochondria sac is the only adenylyl cyclase present in mitochondria sac is specifically inhibited by O 2 I II III IV V KH7 KH7 can be reasonably used ATP to block mtcb1 signaling

13 mtcb 1 A role of mtcb1 in amnesic effects of cannabinoids? Gα i Object recognition Memory (ORM) Intra-hippocampal KH7 13 Tifany Desprez Soluble Adenylyl cyclase (sac) KH7 I II III IV V KH7 blocks the effect of cannabinoids on O 2 ATP long-term ORM

14 What is mtcb1 good for? More precise tools 14 oluble Adenylyl cyclase (sac) Gα i Gigett Bellocchio Peggy Vincent Tifany Despréz Edgar Soria Giovanni Benard Etienne Hebert- Chatelain IN SILICO calculation of mitochondrial targeting probability (MitoProt and PSort ) High CB1 40% Anna Delamarre I II III IV V DN22-CB1 1% O 2 ATP 22 a.a. Low

15 What is mtcb1 good for? More precise tools 15 Gα i Gigett Bellocchio Peggy Vincent Tifany Despréz Edgar Soria Giovanni Benard Etienne Hebert- Chatelain Anna Delamarre oluble Adenylyl cyclase (sac) Prediction of mitochondrial targeting probability DN22-CB1 I II III IV V O 2 ATP mtcb1 pmcb1

of DN22-CB1 expression Viral expression of CB1 and DN22-CB1

16 What is mtcb1 good for? More precise tools 16 oluble Adenylyl cyclase (sac) Gα i Gigett Bellocchio Peggy Vincent Tifany Despréz Edgar Soria Giovanni Benard Etienne Hebert- Chatelain IN VIVO analysis of DN22-CB1 expression Viral expression of CB1 and DN22-CB1 in the hippocampus of CB1-KO mice Anna Delamarre I II III IV V CB1-KO AAV-CB1 or AAV-DN22/GFP O 2 ATP

Bellocchio Peggy Vincent Tifany Despréz Edgar Soria Giovanni Benard IN

DN22-CB1 in the hippocampus of CB1-KO mice CB1-KO WT AAV-CB1 Etienne

17 What is mtcb1 good for? More precise tools 17 oluble Adenylyl cyclase (sac) Gα i Gigett Bellocchio Peggy Vincent Tifany Despréz Edgar Soria Giovanni Benard IN VIVO analysis of DN22-CB1 expression Viral expression of CB1 and DN22-CB1 in the hippocampus of CB1-KO mice CB1-KO WT AAV-CB1 Etienne Hebert- Chatelain Anna Delamarre ANATOMY CB1-KO AAV-DN22-CB1 O 2 I II III IV V ATP M M M M Pedro Grandes, L. Reguero, N. Puente, U. Elezgarai

18 What is mtcb1 good for? More precise tools 18 oluble Adenylyl cyclase (sac) O 2 Gα i Hippocampal mtcb1 is necessary for ATP Gigett Bellocchio I III IV V cannabinoid- II induced amnesia (Hebert-Chatelain et al. Nature 2016) Peggy Vincent Mt-CB1 in object recognition memory Memory Performance Tifany Despréz Edgar Soria Giovanni Benard Etienne Hebert- Chatelain Anna Delamarre

19 CONCLUSIONS 19 CB1 receptors are functionally present on brain mitochondria (mtcb1) MtCB1 signaling depends on intramitochondrial Gi proteins, sac and Soluble AC and intramitochondrial mediate amnesic effects of cannabinoids DN22-CB1 is a tool to exclude CB1 from mitochondrial membranes Hippocampal mtcb1 signaling is necessary for synaptic and amnesic effects of cannabinoids via regulation of intramitochondrial sac and For those who believe, no proof is necessary. For those who don t believe, no proof is possible (Stuart Chase, American engineer and economist, )

The Lab Francis Grumpy Chaouloff Federico Sick Massa Astrid Mama Cannich Virginie British Morales Gigett Pitbull Bellocchio Roman François Serrat Marjorie Black Swann Varilh Edgar Guest Soria Arnau

20 The Lab Francis Grumpy Chaouloff Federico Sick Massa Astrid Mama Cannich Virginie British Morales Gigett Pitbull Bellocchio Roman François Serrat Marjorie Black Swann Varilh Edgar Guest Soria Arnau Blade Runner Busquets Su Elegans Melser Antonio Braces Pagano Laurie Tamoxifen Robin Jose Swimmer Cruz Bastien Naughty Boy Redon Geoffrey Jesus Terral Zhe Xiao Long Zhao Christofer Robertson, Christina Ioannnidou, Dave Saraswat, Yohan Wards, Paula Sotres, Sarra Zaghbouni, Simone Corinti, Thanks to Old members Etienne Passport Chatelain Giovanni Petit Benard Tifany Mito Desprez Mathilde Peace & Love Metna, Joana It s OK Lourenço, Pauline Prof Lafenêtre, Barbara Smile Bosier, Michelangelo Buonarroti Colavita, Sarah Mona Lisa Dubreucq, Hu Chinagirl Bo, Isabel Air-conditioning Matias, Ilaria Pizza Belluomo, Dany Blondie Verrier, Peggy Betty Boop Vincent Collaborations Pedro GRANDES, Nagore PUENTE, Leire REGUERO, Uzaskun ELZGARAI, Gabriel BARREDA (Bilbao), Rodrigue ROSSIGNOL (Bordeaux), Beat LUTZ (Germany), Daniela COTA, Stephane OLIET, Aude PANATIER, Pier-Vi PIAZZA (Bordeaux)

Cannabinoid Type 1 Receptors in Astrocytes. Giovanni Marsicano, PhD

Cannabinoid Type 1 Receptors in Astrocytes EndoCannabinoids and NeuroAdaptation INSERM U862 NeuroCentre Magendie Bordeaux, France 1 Cannabinoid type 1 receptors in astrocytes 1964 OH O 9 -Tetrahydrocannabinol