Test de Dgkk comme cible thérapeutique

du syndrome de l’X fragile dans les souris Fmr1- KO

LA RECHERCHE DANS LES MALADIES RARES

28 Février 2020 -STRASBOURG

Hervé MOINE

Fragile X-syndrome- First cause of familial intellectual disability (1/4000 m, 1/8000 f)

- hyperactivity, autism, hypersensitivity, stereotypies…- facial abnormalities, macroorchidism…

- "Synaptopathy"- abnormal dendritic spines in patients + Fmr1 KO mice- synaptic plasticity alterations in mouse FXS (mGluR-LTD & LTP altered)

- Triplet repeat disease (Verkerk et al. 1991; Kremer et al., 1991; Fu et al., 1991)

Irwin et al. 2002

X-fragile unaffected

> 200

M

mutation Fragile X

FMRP

Methylation

premutation FXTAS, POF

CpG

CGGn

5’UTR 3’UTR

normal

FMR1

Function?

55-200

5-50

No treatment

dendrite

soma

Nucleus

Stress granules

Polyribosomes

Neuronal granules

Postsynapticcompartment

Dendritic spine

FMRP: A protein involved in mRNA metabolism

dendrite

soma

Nucleus

mGluRI

Stress granules

Polyribosomes

Neuronal granules

Alteration ofSynapse functioning

mGluRI-LTD increase & LTP decrease

+

(Huber et al. 2002, Bear et al. 2004)

Postsynapticcompartment

Dendritic spine

Loss of FMRP: excessive mGluR signaling

dendrite

soma

Nucleus

Stress granules

Polyribosomes

Neuronal granules

Postsynapticcompartment

Dendritic spine

mRNA targets?

FMRP function?

dendrite

soma

Nucleus

Stress granules

Polyribosomes

Neuronal granules

Postsynapticcompartment

Dendritic spine

FMRP CLIP-microarray : DGKk mRNA is a main target of FMRP

n=5

Log1

0 (p

Valu

e)

Log2 (fold change)

Fmr1 KO Fmr1 Wt

[AIWt CLIP/AIWt Input]/[AIKO CLIP/AIKO Input]

Dgkk

DGKk(HA)

FMRP

GAPDH

DGKk-FL100

0

siCsiF

mr1

***

siC siFmr1

DgkkmRNAFMRP

FMRP controls DGKk synthesis

COS-1 cells

FXS

HeLa+D

GKk

CTR

100

35

250DGKk

FMRP

GAPDH

1 2 3 1 2 3 4

Human cerebellum

Bogi Zambo / Oktay Cakil

DgkkmRNAFMRP

Phosphatidic Acid(PA)

Diacylglycerol(DAG)

DGKk : Diacylglycerol kinase kappa

Dgkk

Dgkk

PADAG

DgkkmRNAFMRP

mGluRI Excitatory synapse

Dendritic spine

Postsynapticneuron

PLC

Gq

PADAGPiP2

Glutamate

Presynapticneuron

DGKk : a master regulator of lipid signaling

Dgkk

DgkkmRNAFMRP

Excitatory synapse

PLC

Gq

PADAGPiP2

Glutamate

Presynapticneuron

RasGRP

PKC

Akt

TSC1/2Rheb

mTOR

4E-BP

PI3KRas

ERK1/2 S6K

HomerPIKE-L

RAF

Mnk

eIF4E

MEK1/2

Local protein translation(spine growth)

AMPAR

mGluR-LTD

mGluRI

Dgkk

DGKk : a master regulator of lipid signaling

DgkkmRNAFMRP

Excitatory synapse

PLC

Gq

PADAGPiP2

Glutamate

Presynapticneuron

Local protein translation(spine growth)

RacPAK

Actin filament stabilization(spine maintenance)

mGluRI

DgkkRasGRP

PKC

Akt

TSC1/2Rheb

mTOR

4E-BP

PI3KRas

ERK1/2 S6K

HomerPIKE-L

RAF

Mnk

eIF4E

MEK1/2

DGKk : a master regulator of lipid signaling

Dgkk mRNAFMRP

Excitatory synapse

PLC

Gq

PADAGPiP2

Glutamate

Presynapticneuron

A novel FXS pathomechanism: DAG/PA unbalance

RasGRP

PKC

Akt

TSC1/2

Rheb

mTOR

4E-BP

PI3K

Ras

ERK1/2S6K

HomerPIKE-L

RAF

Mnk

eIF4E

MEK1/2

RacPAK -

+Local protein translation(spine growth)

Actin filament stabilization(spine maintenance)

Enhancedin FXSDecreasedin FXS

PADAG

Fragile X

mGluRI

Dgkk

Fmr1 KODgkk KD

Fmr1-KOFmr1-Wt

hippocampal slices CA1

SH-DGKkSH-control

LTP LTD

TBS ( 3x5 bursts at 5 Hz) LFS (900 pulses at 5 Hz)

Injection Analysis

Day 1 Day 28

AAV9-Gfp-shRNA Scramble

AAV9-Gfp-shRNA DGKK

Knockdown of DGKk recapitulates FXS phenotype

Tabet et al. PNAS 2016

Could up-regulation of DGK activity rescue Fmr1-KO phenotypes?

Gene therapy : AAV-DGKk

1)

2)

Pharmacological rescue : Pioglitazone

Fmr1 KO Fmr1 KO

- Dgkk Dgkk

DGKk : a therapeutic target for FXS ?

+

hippocampal organotypic cultures (12 DIV)

SH-scramble SH-DGKk

Fmr1-KOFmr1-Wt SH-scrambleSH-DGKKFmr1-KO 100

80

60

40

20

0 5 24 48 72Time (h)

Spin

est

abili

ty(%

)

*SH-scrambleSH-DGKKFmr1-KO

hippocampal slices CA1

+∆N-DGKkFmr1-KO+∆N-DGKk

AAV-∆N-DGKk rescue of Fmr1-KO mouse∆N-DGKk transgene rescues dendritic spines of Fmr1-KO neurons

AAV9-∆N-DGKk transduces efficiently neurons in vitro and in vivo

AAV9-GFP

AAV9-∆N-DGKk

Cortical neurons (14 days in vitro)

Fmr1 KO

GETEX maturation project In progress

4 weeks transduction

Fmr1-KO +∆N-DGKk

+ DgkkHA DAPI

AAV9-∆N-DGKk

NT

Molecular analyses

n=24

4 weeks

W1 W2 W4Behavioral tests

W5W3

Rh10, intracranial injection, 4 sites : striatum + hippocampe (10^11 VG/mouse)PHPeB retroorbital injection (5.10^11 VG/mouse)

AAV-Dgkk gene therapy approach in Fmr1-KO mouse

W6 W7 W8 W10W9 W12W11 W14W13

8 weeksBehavioral tests Electrophysiological analyses

W16W15 W18W17

AAV injection

Actimétrie (32h)Elevated plus mazeOpen field test Novel object recognition testStereotypies (Head shakes)GroomingNestingSocial preference / recognitionKO

KO+AAV

Wt

Dis

tanc

e tr

avel

ed

KO KO+AAV

Wt KO KO+AAV

Wt KO KO+AAV

Wt KO KO+AAV

Wt KO KO+AAV

Wt KO KO+AAV

Wt

Rh10-∆N-DGKK (+4 weeks)

Molecular analyses

Tota

l arm

ent

ries

% ti

me

in o

pen

arm

s

open

arm

sen

trie

s

% b

urie

dm

arbl

es

Soci

al m

emor

y-sn

iffin

gdu

ratio

n (%

)

Dgkk+

PADAG

Normal

PA

DAG

Fragile X

Conclusions :

+ Pioglitazone

up-regulation of DGK activity ameliorates Fmr1-KO phenotypes

4APABP

40S

AAAAAA

eIF4E FMRP

? - Binding site ? Clip-seq- FMRP/ribosome interaction

- Mechanism of Dgkk control by FMRP ?

- Functions of DGKk in higher brain functions?

DGKk mRNA

DGKk-KO Fmr1-KO?=

eIF4G

Perspectives :

- Preclinical validation of Dgkk targeting

Present membersKarima Habbas

Boggi Zambo

Oktay Cakil

Eric Flatter

Jean-Louis Mandel

Former membersRicardos TabetAndréa Geoffroy Fumi HoshinoZaiane Schmitt

AcknowledgmentsCollaboratorsHélène Puccio

Arnaud Duchon

Yann Herault

Françoise Piguet ICM Paris

Nicolas Vitale INCI Strasbourg

Dimitri Heintz IBMP Strasbourg

Olivier Manzoni INMED Marseille

Flora Tassone UC Davis

Antonina Fedorova SATT Conectus

Barbara Bardoni IPMC Nice

IGBMC

Fonds Paul Mandel