Hervé MOINE Test de Dgkk comme cible thérapeutique du syndrome de l' X fragile dans les souris Fmr1- KO - Fondation maladies rares
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LA RECHERCHE DANS
LES MALADIES RARES
28 Février 2020 -
STRASBOURG
Test de Dgkk comme cible thérapeutique
du syndrome de l’X fragile dans les souris Fmr1- KO
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) X-fragile unaffected
CGGn
> 200 mutation Fragile X
Methylation
55-200 premutation FXTAS, POF
M 5-50
normal
Irwin et al. 2002
CpG
FMR1
5’UTR 3’UTR
No treatment
FMRP
Function?
FMRP: A protein involved in mRNA metabolism
soma
Dendritic spine
Postsynaptic
compartment
Polyribosomes
Nucleus
Neuronal granules
dendrite
Stress granules
Loss of FMRP: excessive mGluR signaling
mGluRI
(Huber et al. 2002,
Bear et al. 2004)
soma
Dendritic spine
Postsynaptic
compartment
Nucleus
Polyribosomes
+
Neuronal granules
Alteration of
Synapse functioning
mGluRI-LTD increase & LTP decrease
dendrite
Stress granules
FMRP function?
soma
Dendritic spine
Postsynaptic
compartment
Polyribosomes
Nucleus
Neuronal granules
dendrite
mRNA targets?
Stress granules
FMRP CLIP-microarray : DGKk mRNA is a main target of FMRP
Fmr1 KO Fmr1 Wt
soma
Dendritic spine
Postsynaptic
compartment
Log10 (pValue)
Polyribosomes
Nucleus
Neuronal granules
Log2 (fold change) n=5
dendrite
[AIWt CLIP/AIWt Input]/[AIKO CLIP/AIKO Input]
Stress granules
FMRP controls DGKk synthesis Bogi Zambo / Oktay Cakil
Human cerebellum
HeLa+DGKk
CTR FXS
1 2 3 1 2 3 4
Dgkk DGKk 250
100
FMRP
FMRP 35
Dgkk mRNA GAPDH
COS-1 cells
***
DGKk-FL
100
siC siFmr1
DGKk(HA)
FMRP
GAPDH
0
r1
C
si
Fm
si
DGKk : Diacylglycerol kinase kappa
Diacylglycerol Phosphatidic Acid
(DAG) (PA)
Dgkk
Dgkk
DAG PA
FMRP
Dgkk mRNA
DGKk : a master regulator of lipid signaling
Presynaptic
neuron
Glutamate
PiP2
DAG PA Excitatory synapse
mGluRI
Gq
PLC
Dgkk
FMRP
Dgkk mRNA
Dendritic spine
Postsynaptic
neuron
DGKk : a master regulator of lipid signaling
Presynaptic
neuron
Glutamate
PiP2
DAG PA AMPAR Excitatory synapse
mGluRI
Gq
PLC
Ras PKC
Homer GRP
PIKE-L Ras Dgkk
PI3K
RAF
Akt FMRP
Dgkk mRNA mGluR-LTD
TSC1/2 MEK1/2
S6K
Rheb ERK1/2
mTOR Mnk
4E-BP eIF4E
Local protein translation
(spine growth)DGKk : a master regulator of lipid signaling
Presynaptic
neuron
Glutamate
PiP2
DAG PA Excitatory synapse
mGluRI
Gq
PLC
Ras PKC
Homer Rac
GRP PAK
PIKE-L Ras Dgkk Actin filament stabilization
PI3K (spine maintenance)
RAF
Akt FMRP
Dgkk mRNA
TSC1/2 MEK1/2
S6K
Rheb ERK1/2
mTOR Mnk
4E-BP eIF4E
Local protein translation
(spine growth)A novel FXS pathomechanism: DAG/PA unbalance
Presynaptic
neuron
Glutamate
PiP2
mGluRI DAG PA
Excitatory synapse
Gq
PLC
Ras PKC
Homer Rac
GRP PAK
PIKE-L
PI3K
Ras
RAF
Dgkk
-
Akt FMRP
Dgkk mRNA
Fragile X
MEK1/2
TSC1/2 Actin filament stabilization
S6K
Rheb ERK1/2 (spine maintenance)
mTOR Mnk PA
4E-BP eIF4E DAG
Enhanced
+
in F X S
Decreased Local protein translation
in F X S
(spine growth)Knockdown of DGKk recapitulates FXS phenotype
AAV9-Gfp-shRNA Scramble
AAV9-Gfp-shRNA DGKK
Day 1 Day 28
Fmr1 KO Injection Analysis
Dgkk KD
hippocampal slices CA1 LTP LTD
Fmr1-Wt Fmr1-KO
SH-control SH-DGKk LFS (900 pulses at 5 Hz)
TBS ( 3x5 bursts at 5 Hz)
Tabet et al. PNAS 2016DGKk : a therapeutic target for FXS ?
Could up-regulation of DGK activity rescue Fmr1-KO phenotypes?
- Dgkk + Dgkk
Fmr1 KO Fmr1 KO
1)
Pharmacological rescue : Pioglitazone
2) Gene therapy : AAV-DGKkAAV-∆N-DGKk rescue of Fmr1-KO mouse
∆N-DGKk transgene rescues dendritic spines of Fmr1-KO neurons
Fmr1-KO SH-scramble
Fmr1-Wt
SH-DGKK
SH-scramble SH-DGKk +∆N-DGKk Fmr1-KO 100
Spine stability (%)
Fmr1-KO
80 SH-scramble
+∆N-DGKk
60 * SH-DGKK
40 Fmr1-KO
20 Fmr1-KO
+∆N-DGKk
hippocampal organotypic cultures (12 DIV) 0 5 24 48 72
hippocampal slices CA1 Time (h)
AAV9-∆N-DGKk transduces efficiently neurons in vitro and in vivo
HA DAPI
NT
AAV9-GFP
+ Dgkk
AAV9-∆N-DGKk Fmr1 KO
AAV9-∆N-DGKk
GETEX maturation
4 weeks transduction
project In progress
Cortical neurons (14 days in vitro)AAV-Dgkk gene therapy approach in Fmr1-KO mouse
Rh10, intracranial injection, 4 sites : striatum + hippocampe (10^11 VG/mouse) Actimétrie (32h)
PHPeB retroorbital injection (5.10^11 VG/mouse) Elevated plus maze
Open field test
Wt Novel object recognition test
AAV injection Stereotypies (Head shakes)
Grooming
Nesting
KO Social preference / recognition
8 weeks
Behavioral tests Electrophysiological analyses Molecular analyses
4 weeks
Behavioral tests Molecular analyses
KO+AAV
W1 W2 W3 W4 W5 W6 W7 W8 W9 W10 W11 W12 W13 W14 W15 W16 W17 W18
Rh10-∆N-DGKK (+4 weeks)
Social memory-sniffing duration (%)
Distance traveled
% time in open arms
open arms entries
Total arm entries
% buried marbles
Wt KO KO Wt KO KO Wt KO KO Wt KO KO Wt KO KO Wt KO KO
n=24 +AAV +AAV +AAV +AAV +AAV +AAVConclusions :
+ Pioglitazone
+ Dgkk
Fragile X Normal
PA
DAG DAG PA
up-regulation of DGK activity ameliorates Fmr1-KO phenotypesPerspectives :
- Preclinical validation of Dgkk targeting
- Mechanism of Dgkk control by FMRP ?
G
4eIF4E
40S ? -- Binding site ? Clip-seq
FMRP/ribosome interaction
e IF FMRP
4A
P
AAAABP
AAA DGKk mRNA
- Functions of DGKk in higher brain functions?
DGKk-KO Fmr1-KO
?
=Acknowledgments
Present members
Collaborators
Karima Habbas
Hélène Puccio
Boggi Zambo
Arnaud Duchon IGBMC
Oktay Cakil
Yann Herault
Eric Flatter
Françoise Piguet ICM Paris
Jean-Louis Mandel
Nicolas Vitale INCI Strasbourg
Dimitri Heintz IBMP Strasbourg
Former members
Olivier Manzoni INMED Marseille
Ricardos Tabet
Andréa Geoffroy Flora Tassone UC Davis
Fumi Hoshino Antonina Fedorova SATT Conectus
Zaiane Schmitt Barbara Bardoni IPMC Nice
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