How do CB1 receptors do all these beautiful

things?

A Cannabinoid link between mitochondria and memory 3

Text Book: Mechanisms of action of CB1

Ca 2+ - K

+ ir

+

G i/o

adenylyl cyclase

-

ATP cAMP

+

PKA

K + A

- P

CB1

+ MAPK

THC/AEA

-  Neuronal hyperpolarization -  Decrease of neuronal excitability

(retrograde inhibition of neurotransmission)

4

The Seventies: The “hippy scientists” at work

Is that all? Does CB1 signal also INSIDE the cells? Cannabinoids are lipids, that can travel through membranes, and…

Biochemical effects and morphological changes in rat liver mitochondria exposed to 1-tetrahydrocannabinol Bino T, Chari-Bitron A, Shahar A. Biochim Biophys Acta. 1972 Oct 23;288(1):195-202.

Effect of 1-tetrahydrocannabinol on ATPase activity of rat liver mitochondria Chari-Bitron A, Bino T. Biochem Pharmacol. 1971 Feb;20(2):473-5

1976

5

A serendipitous discovery

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.: Hi, Pedro, how are you? P.G.: Good! Interesting stuff, isn’t it? G.M.: Yes… Pedro, I have a question for you… P.G.: Tell me G.M.: 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…

6

Wild-Type CB1-KO

0.0

10

20

% C

B1

mito

cond

rial

part

icle

s on

tota

l

Purified brain mitochondria

CB1 receptors are present on mitochondrial membranes of neurons (mtCB1) Benard et al Nat. Neurosci 2012

P. Grandes N. Puente

A serendipitous discovery 7

INNER MEMBRANE

Intermembrane space

Matrix

OUTER MEMBRANE

Cytosol

ADP+Pi

H+

ATP Complex V

-

+

• Energy

• ROS production

• Calcium signalling

• Apoptosis

• Metabolism

What are Mitochondria?

H+ H+ H+ H+

H+ H+ H+

QH2 Q

NADH NAD + H+

Cyt.c

H2O Succ. Fum.

e- e- e-

Complex I Complex II Complex III

Complex IV

e- e-

O2

CB1

Adenylyl Cyclase

G Proteins

PKA

8

Does mtCB1 influence brain mitochondrial functions?

PURIFICATION of mitochondria from mouse brains (WT and CB1-KO) Mitochondrial respiration and biochemistry in the presence of an exogenous CB1 agonist (WIN)

0

50

100

ng

Oxy

gen/

min

/mg

prot

.

0 10 20 50 100

Mitochondrial Respiration

WT CB1-KO

mtCB1 signalling DIRECTLY regulates mitochondrial functions

Giovanni Benard

9

Does mtCB1 influence brain mitochondrial functions?

Cover Charlie Padgett

mtCB1 signalling DIRECTLY regulates mitochondrial functions

What is mtCB1 good for?

10

O2 ATP

Soluble Adenylyl cyclase (sAC)

PKA

mtCB1

I II III IV V

Gαi Etienne Hebert-Chatelain

Need of tools: What is the signaling of mtCB1? 11

sAC is enriched in mitochondria

sAC is the only adenylyl cyclase present in mitochondria

KH7

sAC is specifically inhibited by…

O2 ATP


PKA

mtCB1

I II III IV V

Gαi Etienne Hebert-Chatelain

Soluble Adenylyl Cyclase (sAC)

KH7 can be reasonably used to block mtCB1 signaling

Need of tools: What is the signaling of mtCB1? 12

A role of mtCB1 in amnesic effects of cannabinoids?

O2 ATP


PKA

mtCB1

I II III IV V

Gαi

Object recognition Memory (ORM)

Intra-hippocampal KH7

KH7

KH7 blocks the effect of cannabinoids on long-term ORM

Tifany Desprez

13

PKA


O2 ATP

I II III IV V

Gαi

IN SILICO calculation of mitochondrial targeting probability

(MitoProt and PSort ) High

CB1 40%

1%

Low 22 a.a.

DN22-CB1

What is mtCB1 good for? More precise tools

Giovanni Benard

Etienne Hebert- Chatelain

Tifany Despréz

Edgar Soria

Peggy Vincent

Anna Delamarre

Gigett Bellocchio

14

pmCB1

PKA


O2 ATP

I II III IV V

Gαi

Prediction of mitochondrial targeting probability

mtCB1

CB1DN22-CB1


Giovanni Benard


Tifany Despréz

Edgar Soria

Peggy Vincent

Anna Delamarre

Gigett Bellocchio

15

PKA


O2 ATP

I II III IV V

Gαi

IN VIVO analysis of DN22-CB1 expression

CB1-KO

Viral expression of CB1 and DN22-CB1 in the hippocampus of CB1-KO mice

AAV-CB1 or AAV-DN22/GFP


Giovanni Benard


Tifany Despréz

Edgar Soria

Peggy Vincent

Anna Delamarre

Gigett Bellocchio

16

WT CB1-KO AAV-CB1 CB1-KO

AAV-DN22-CB1

PKA


O2 ATP

I II III IV V

Gαi

IN VIVO analysis of DN22-CB1 expression

Viral expression of CB1 and DN22-CB1 in the hippocampus of CB1-KO mice ANATOMY

M M

M

M


Pedro Grandes, L. Reguero, N. Puente, U. Elezgarai

Giovanni Benard


Tifany Despréz

Edgar Soria

Peggy Vincent

Anna Delamarre

Gigett Bellocchio

17

PKA


O2 ATP

I II III IV V

Gαi

Mt-CB1 in object recognition memory


Mem

ory

Perf

orm

ance

Hippocampal mtCB1 is

necessary for cannabinoid-

induced amnesia (Hebert-Chatelain et al. Nature 2016)

Giovanni Benard


Tifany Despréz

Edgar Soria

Peggy Vincent

Anna Delamarre

Gigett Bellocchio

18

CONCLUSIONS

•  CB1 receptors are functionally present on brain mitochondria (mtCB1)

•  MtCB1 signaling depends on intramitochondrial Gi proteins, sAC and PKA

•  Soluble AC and intramitochondrial PKA 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 PKA

“For those who believe, no proof is necessary. For those who don’t believe, no proof is possible”

(Stuart Chase, American engineer and economist, 1888-1985)

19

Thanks to 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)

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,

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

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