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

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

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

γOur previous work has mainly focused on GABAA receptor

We identified GABA transporter shares a lot of features with an

ion channel protein

γ

GABAA receptor binding sites

Many sedative, anticonvulsant drugs work bymodulating GABAA receptors and GABA pathway

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

Video showing multiple mutations in GAT-1 associated

with epilepsy, autism and intellectual disability

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

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

Figure 6

Machine learning and structure modeling showing loss

of a hydrogen bond, resulting in a destabilized mutant

protein

P361T361

DC

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)

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

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

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

wt

P361T

+Tuni

overlay GAT-1YFP ERCFP

10µm

Wt S295LP361T

Live

Astrocytes

Live

neurons

Astrocytes could be a good tool for GAT-1 trafficking study

GAT-1YFP

10µm

GAT-1 immunoreactivity in cortex (red) and hippocampus

(green)

3 months old mouse in C57/BL/6J mouse background

Mouse tissue

Cortex Hippocampus

Wt S295LP361T

Live

Astrocytes

Live

neurons

overlay oct4 GAT-1 To-pro-3

10µm

wt

mutant

overlay GAT-1YFP GAT-1 To-pro-3

mutant

10µm

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

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

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

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

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.

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

Slc6a1+/A288V mutant pups have better survival

rate than Gabrb3+/N328D pups (LGS)

SLC6A1+/A288V

Gabrb3+/N328D

Righting reflex

The mutant Slc6a1+/A288V mice had

seizures

Anxiety in the mutant Slc6a1+/A288V mice

Pretraining

Orientation

Training

Learning

Probe

Memory

Barnes Maze Overview

Barnes Maze is less stressful than Water Maze and was used for

cognition test .

Barnes Maze

Mutant mouse Wildtype mouse

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

Musical city convention center in Nashville

Thanks for the invitation!

mutant

mutations

mouse

model

patient

iPSC line

models

End

Wt R44Q S295L G234S

GAT-1

GAT-1