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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
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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
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dendrite
soma
Nucleus
Stress granules
Polyribosomes
Neuronal granules
Postsynapticcompartment
Dendritic spine
FMRP: A protein involved in mRNA metabolism
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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
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dendrite
soma
Nucleus
Stress granules
Polyribosomes
Neuronal granules
Postsynapticcompartment
Dendritic spine
mRNA targets?
FMRP function?
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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]
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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
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DgkkmRNAFMRP
Phosphatidic Acid(PA)
Diacylglycerol(DAG)
DGKk : Diacylglycerol kinase kappa
Dgkk
Dgkk
PADAG
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DgkkmRNAFMRP
mGluRI Excitatory synapse
Dendritic spine
Postsynapticneuron
PLC
Gq
PADAGPiP2
Glutamate
Presynapticneuron
DGKk : a master regulator of lipid signaling
Dgkk
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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
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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
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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
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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
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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 ?
+
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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
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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 (%
)
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Dgkk+
PADAG
Normal
PA
DAG
Fragile X
Conclusions :
+ Pioglitazone
up-regulation of DGK activity ameliorates Fmr1-KO phenotypes
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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
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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