Jing-Qiong (Katty) Kang, MD, PhD Department of …...1sec 1sec 100uV 100uV A B D Bilateral...
Transcript of Jing-Qiong (Katty) Kang, MD, PhD Department of …...1sec 1sec 100uV 100uV A B D Bilateral...
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SLC6A1 mutations in cell and mouse models
Jing-Qiong (Katty) Kang, MD, PhDDepartment of Neurology,Vanderbilt Brain Institute
Vanderbilt University Kennedy Center of Human Development
Vanderbilt University Medical CenterNashville, TN, 37232
The speaker has no conflicts of interest
Modeling SLC6A1mutations from stem cell
to mouse models
Jing-Qiong (Katty) Kang, MD, PhDDepartment of Neurology & Pharmacology
Vanderbilt Brain InstituteVanderbilt University Kennedy Center of Human
DevelopmentVanderbilt University Medical Center
Nashville, TN, 37232
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GABR,
GABRA1,3,5,6, GABRB1, 2, 3,
GABRG2, GABRD
STXBP1
Mutations in genes directly or indirectly affecting GABAergic pathway and the
associated epilepsy syndromes, autism and intellectual disability
CAE=Childhood absence
IAE=Idiopathic absence
epilepsy
ID=intellectual disability
LGS=Lennox-Gastautsyndrome
JME=Juvenile myoclonic
epilepsy
LGS=Lennox-Gastaut
syndrome
MAE=Myoclonic astatic
epilepsy
GRIN2A-BSCN, SCN1A,
2A, 3A, 8A, SCN1B
*
* Gene will be studied
IGE, CAE, JME, IAE, MAE,LGS, ID, autism
Figure 1SLC6A1
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astrocyte
GADGlutamic acid
GABAergic interneuron
GABA
Post-synaptic neuron
GABA-T
GABAA receptor
GABAB receptor
GABAB receptor
Antiseizure drugs
VGBTGB
Vigabatrin (VGB)
Tiagabine (TGB)
Levetiracetam (LEV)Clobazam (CBZ)
Clonazepam (CZP)
Phenobarbital(PB)
GAT-1LEV
GABAergic neurotransmission is a major pathway for epilepsy
Receptor,
neurotransmitter,
transporter
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γOur previous work has mainly focused on GABAA receptor
We identified GABA transporter shares a lot of features with an
ion channel protein
γ
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GABAA receptor binding sites
Many sedative, anticonvulsant drugs work bymodulating GABAA receptors and GABA pathway
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Previously reported phenotypesAutismIntellectual disabilityDevelopmental disorderLennox-Gastaut syndromeEpilepsy with myoclonic-atonic seizures
R44Q
NH2
COOH
L151Rfs*35
C164Y
W193X
G270SA288V
G299V
F339L
G457Hfs*10
S459R
G232V
G234S
A128V
G550R
V511M
A334P
L36Gfs*171
Q534*
Q397*
P361T
Phenotypes of P361T:Epilepsy + autism
Extracellular
S295L
Mutations identified in SLC6A1
12 transmembrane domains
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Video showing multiple mutations in GAT-1 associated
with epilepsy, autism and intellectual disability
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Fp1-AVG
Fp2-AVG
F3-AVG
F4-AVG
C3-AVG
C4-AVG
P3-AVG
P4-AVG
O1-AVG
O2-AVG
T1-AVG
T2-AVG
F7-AVG
F8-AVG
T3-AVG
T4-AVG
T5-AVG
T6-AVG
Fz-AVG
Cz-AVG
Pz-AVG
ECG
EMG-L
EMG-R
Fp1-AVG
Fp2-AVG
F3-AVG
F4-AVG
C3-AVG
C4-AVG
P3-AVG
P4-AVG
O1-AVG
O2-AVG
T1-AVG
T2-AVG
F7-AVG
F8-AVG
T3-AVG
T4-AVG
T5-AVG
T6-AVG
Fz-AVG
Cz-AVG
Pz-AVG
ECG
EMG-L
EMG-R
SLC6A1(P361T) associated with autism and epilepsy in a 6 -year-old girl, bilateral prefrontal
2.0-3.0 HZ spike and slow waves
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Fp1-AVG
Fp2-AVG
F3-AVG
F4-AVG
C3-AVG
C4-AVG
P3-AVG
P4-AVG
O1-AVG
O2-AVG
T1-AVG
T2-AVG
F7-AVG
F8-AVG
T3-AVG
T4-AVG
T5-AVG
T6-AVG
Fz-AVG
Cz-AVG
Pz-AVG
ECG
EMG-L
EMG-R
Fp1-AVG
Fp2-AVG
F3-AVG
F4-AVG
C3-AVG
C4-AVG
P3-AVG
P4-AVG
O1-AVG
O2-AVG
T1-AVG
T2-AVG
F7-AVG
F8-AVG
T3-AVG
T4-AVG
T5-AVG
T6-AVG
Fz-AVG
Cz-AVG
Pz-AVG
ECG
EMG-L
EMG-R
1sec1sec
100uV100uV
A B
D
Bilateral multifocal 2.0-3 HZ spike and slow wave discharges
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Figure 6
Machine learning and structure modeling showing loss
of a hydrogen bond, resulting in a destabilized mutant
protein
P361T361
DC
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Machine learning and structure modeling showing
form of more hydrogen bonds.
Wildtype GAT-1(S295) mutant GAT-1(L295)
Mutation from Serine to Leucine may bring huge impact.The side chain polarity is mutated from polar to nonpolar, and Serine is hydrophilic but Leucine is hydrophobic. According to the images, more hydrogen bonds formed.
mutant GAT-1(L295)
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Overview and experimental approaches
Patient iPSCs
Figure 6We have studied the traffick and function of mutant
GAT-1 in HEK293T, iPSCs, mouse neurons and
astrocytes
How to study the impact of a mutation?
iPSCs
astrocytes
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wt
wt+
LC-9
66
wt+
No-7
66
untran
sfec
ted
G75
R
Y14
0C
Y14
5F
W19
3X
A28
8V
S29
5L
A33
4P
P36
1T
G36
2R
A35
7V
F385
L
V51
1M
0
25
50
75
100
uptake in neuron multi mutations% 3H GABA uptake
un 966 711
un=untreated
966 =Cl-966 (100µM)
711=NNC711 (70µM)
wt
mock GR YC YF WX AV SL AP PT GR2 AV FL VM
AV=A288V
SL= S295L
AP= A334P
PT= P361T
RQ=R44Q
GR=G75R
YC= Y140C
YF= Y145F
WX= W193X
GR2=G362R
AV2= A357V
FL= F385L
VM= V511M75
A
Neuron
%
What is the function of the mutant transporters?
RQ GR YC YF WX AV SL AP PT GR2 AV2 FL VM
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wt
mock
R44QL73F
G75R
Y140C
Y145F
W193X
A288V
S295L
A305T
A334P
P361T
G362R
A357V
F385L
G457Hfs
V511M
0
25
50
75
100
surface multi mutations
A B
GAT-1YFP
C
U wt mutant
Anti-GAT-1
surface
GAPDH
wt mutant0.5
0.6
0.7
0.8
0.9
1.0
1.1
wt mutant0.5
0.6
0.7
0.8
0.9
1.0
1.1
Normalized FI
GAT-1YFP
Dsurface
surface
wt
mutant
controlKDa
108
37
EWt=100
%
Normalized IDVs
GAT-1YFP
Phenotypes
LGS, MAE, CAE, Autism,
ID
flow
Surface
biotinylation
Impaired trafficking is a major defect
GAT-1YFP
Can mutant GAT-1 traffick to cell surface?
Using a high-throughput assay
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wt
P361T
+Tuni
overlay GAT-1YFP ERCFP
10µm
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Wt S295LP361T
Live
Astrocytes
Live
neurons
Astrocytes could be a good tool for GAT-1 trafficking study
GAT-1YFP
10µm
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GAT-1 immunoreactivity in cortex (red) and hippocampus
(green)
3 months old mouse in C57/BL/6J mouse background
Mouse tissue
Cortex Hippocampus
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Wt S295LP361T
Live
Astrocytes
Live
neurons
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overlay oct4 GAT-1 To-pro-3
10µm
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wt
mutant
overlay GAT-1YFP GAT-1 To-pro-3
mutant
10µm
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2. Immnuoprecipitation with anti-GABA
transporter anitserum
1. Immnuoprecipitation with the affinity-purified
antibody against GABA transporter
Dimer ?35S
HeLa cells
Biochemistryl Will GAT-1 form dimers?l
monomer
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corrected patient10X
C Pat 1 2
iPSCs adult mice
C=corrected
Pat=patient
GAT-1
KDa
250
75
iPSCs
37LC
Day 1
Day 4
A
B
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iPSC iPSC iPSC iPSC FibroblastPatientPatient CorrectCorrectnormal lympho
cytes neuronmouse
cortex
ATCC
FibroblastKDa
75
50
GAT-1
1. GAT-1 is expressed in iPSCs, fibroblasts and lymphocytes
2. GAT-1 in stem cells and mature neurons may have
different posttranslational modifications
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3H GABA uptake
Normalized to HEK293T baseline
activity
HEK
iPSC con
corre Pat
LC-966
0.0
0.5
1.0
1.5
2.0
2.5
Data 5
Human iPSCs only had minimal GABA uptake
activity
LC-966=100µm 30min
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con WT S295L A288V P361T WT S295L A288V P361T
GAT-1
LC
B KDa
100
75
Astrocytes
mutant mixed
Wtcopy
Mutant GAT-1 interfered the expression of the endogenous GAT-1
GAT-1 GABA uptake activity was measured at day 5 (P0) in dish.
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100
17 days old mouse cortical neurons
day 5th transfectiontotal
wt S295L A305T A334P A357V P361T G362R F385L V511M
LC
LC
GAT-1YFP
GAT-1YFP
KDaMarker
A
Mutant GAT-1 had diverse expression pattern in mouse neurons
Neurons
KDa
100
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Slc6a1+/A288V mutant pups have better survival
rate than Gabrb3+/N328D pups (LGS)
SLC6A1+/A288V
Gabrb3+/N328D
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Righting reflex
The mutant Slc6a1+/A288V mice had
seizures
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Anxiety in the mutant Slc6a1+/A288V mice
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Pretraining
Orientation
Training
Learning
Probe
Memory
Barnes Maze Overview
Barnes Maze is less stressful than Water Maze and was used for
cognition test .
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Barnes Maze
Mutant mouse Wildtype mouse
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Probe Trial
Hole
Tim
e S
pen
t (s
ec)
25
30
35
40
45Wild-TypeSLC6A1
+/-SLC6A1+/A288VLC
Ongoing work with Barnes maze test in
the mutant Slc6a1+/A288V mice
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Musical city convention center in Nashville
Thanks for the invitation!
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mutant
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mutations
mouse
model
patient
iPSC line
models
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End
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Wt R44Q S295L G234S
GAT-1
GAT-1