Cell, network and mouse modelling of genetic epilepsies for mechanism, diagnosis and therapy

December 7th 2013

Steven Petrou, PhD Deputy Director, The Florey Institute

Deputy Director, The Centre for Neural Engineering The University of Melbourne, Australia

American Epilepsy Society | Annual Meeting

Disclosure

Name of Commercial Interest

NONE

American Epilepsy Society | 2013 Annual Meeting

Type of Financial Relationship

NONE

Learning Objectives

• Understand how different models of genetic epilepsy can reveal “direct” and “emergent” pathologies

• Understand how this knowledge may inform therapeutic strategy

American Epilepsy Society | 2013 Annual Meeting

• Examined 264 trios (792 exomes)

• 329 variants discovered

Opportunity for translation provided by explosion in genetics knowledge

Models for revealing disease state biomarkers and pathology

1. Mouse

2. Single Cell

a. Biophysics

b. Structure-function

3. Neuronal Network a. Primary cultures

b. Human stem cell based

1. MOUSE MODELS

Mouse model reveals emergent pathologies

Novel epileptogenic mechanism in model of Early Onset Epileptic

Encephalopathy (EOEE) suggests targeted therapeutic intervention

Defined by frequent and severe epileptic seizures with progressive developmental regression, possible psychomotor deficits as seen in Dravet syndrome

Difficult to treat

Increasing recognition of underlying genetic defects

SCN1B gene, homozygous R125C (Patino, 2009)

Patient presented with Dravet Syndrome

We previously developed the het SCN1B(C121W) mouse

Does the homozygous mouse model Dravet?

Early onset epileptic encephalopathies (EOEE)

Modified from: Patino, G.A. (2009), A functional null mutation of SCN1B …

Characteristics of the homozygous C121W mouse

• Altered gait

• Premature death

• Spontaneous seizures

• More rapid progression to tonic-

clonic seizure following heating

Petrou, unpublished data 2013

Survival

Pharmacosensitivity of the homozygous C121W mouse similar to Dravet Syndrome

• Similar pharmacosensitivity to human Dravet patients

• Suggests shared pathological mechanism

Petrou, unpublished data 2013

In stark contrast to SCN1A based Dravet models, CA1 interneurons appear normal

Petrou, unpublished data 2013

BUT, hippocampal excitatory neurons from our homozygous C121W mice are more excitable

WT (n =8), Hom (n = 8)

Petrou, unpublished data 2013

Changes in input resistance explains neuronal excitability

WT (n=15), Hom (n=10); *P<0.05

Petrou, unpublished data 2013

• Increased input resistance (Rin) may be major contributor to increased excitability

Reduced neuronal arborisation might explain the changes in cellular properties

Petrou, unpublished data 2013

• Drugs that specifically target and reduce Rin may rescue the seizure phenotype

• Retigabine

– Activates KCNQ2/3 channels

– Reduces Rin

Can we use our information on cellular dysfunction to predict drug efficacy?

Modified from: Surti TS (2005), Identification by mass spectrometry and…

In vitro: Retigabine reverses the neuronal deficit

Reduces input resistance in homozygous neurons

Shifts homozygous input-output relationship

Petrou, unpublished data 2013

In vivo: Retigabine reduces seizure susceptibility

Petrou, unpublished data 2013

• Homozygous mice as a mouse model of Dravet – Phenotype corresponds to SCN1A mouse model of Dravet

• Thermogenic seizure susceptibility

• Premature death, unstable gait, severe seizures

• Altered excitatory neuron firing distinguishes this pathology from that of SCN1A based models – Increased Rin underlies changes in firing properties

– Mutant neurons display reduced dendritic arborisation as primary pathology • Emergent properties

– Retigabine reverses cellular and behavioral deficits

• Potential example of disease mechanism based therapy

Summary

2. SINGLE CELL MODELS

Genotype-phenotype correlation and pharmacological modulation of hKCNT1 channel

mutations causing epilepsy

KCNT1 based epilepsies

• In 2011 ADNFLE (Autosomal Dominant Nocturnal Frontal Lobe Epilepsy) was thought to be a disorder of nicotinic acetylcholine receptors

• In 2012 KCNT1 was discovered (Heron et al., 2012, Nature Genetics) as a gene for more severe ADNFLE with psychiatric features expanding the genetic architecture

• At the same time Barcia et al (2012, Nature Genetics) showed that KCNT1 was also associated with a very severe epileptic encephalopathy, Epilepsy of Infancy with Migrating Focal Seizures (EIMFS) characterised by drug-resistant seizures and developmental delay

• EIMFS and ADNFLE are allelic yet clinically distinct and the mechanism by which KCNT1 mutations produce this phenotypic spectrum is intriguing

KCNT1: (Slo2.2, KCa4.1, SLACK)

Na+ activated potassium channel

Thought to contribute to RMP and to slow

hyperpolarisation following repetitive firing

NH2

COOH

R398Q

Y796H

R928C

(R907C)

(R899C)

RCK domains NAD+ binding

domain [Na+

]

M896I

A934T

I760M R428Q

R474H

P924L

[Na+]

KCNT1 mutations in ADNFLE and EIMFS

M896I R398Q Y796H R928C R428Q A934T P924L

ADNFLE mutants EIMFS mutants

Most severe Least severe Disease severity

KCNT1 epilepsy mutations show increased current magnitude and altered kinetics

Petrou, unpublished data 2013

W T

0 1

0

1 0

M 8 9 6 I

0 1

0

1 0

R 3 9 8 Q

0 1

0

1 0

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R 9 2 8 C

0 1

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1 0 0 0

0

1 0

A 9 3 4 T

0 1 0 0 0

0

1 0

P 9 2 4 L

1 0 0 0

0

1 0

2 A

1 0 0 m s

W T

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0

1 0

M 8 9 6 I

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R 3 9 8 Q

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0

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Y 7 9 6 H

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R 4 2 8 Q

1 0 0 0

0

1 0

A 9 3 4 T

0 1 0 0 0

0

1 0

P 9 2 4 L

1 0 0 0

0

1 0

2 A

1 0 0 m s

W T

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0

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1 0 0 0

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1 0 0 0

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2 A

1 0 0 m s

W T

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1 0 0 0

0

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A 9 3 4 T

0 1 0 0 0

0

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P 9 2 4 L

1 0 0 0

0

1 0

2 A

1 0 0 m s

WT M8961 R928C R428Q

R398Q Y796H A934T P924L

KCNT1 gain of function correlates with disease severity

Petrou, unpublished data 2013

WT

M896I

R398Q

Y796H

R928C

R428Q

A934T

P924L

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curr

ent (μA

)

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ent (μA

)

Cinchona Tree Bark

quinine

quinidine

Yang et al., 2006

Quinidine as a potential therapy?

• The clinical severity of KCNT1 epilepsies

creates an urgent need for intervention

• Quinidine is an FDA approved antiarrhythmic

drug shown to inhibit rodent SLACK channels

• Does it act on hKCNT1 and how does it

interact with mutant channels?

Quinidine blocks human WT and mutant channels

Petrou, unpublished data 2013

3. NEURONAL NETWORK MODELS

The perfect storm for rapidly understanding the effect of mutations and drugs

INPUTS a. Genetics discoveries in epilepsy

b. Stem cell technology improvements

c. Multi-electrode array readiness

OUTPUTS a. Diagnostic markers

b. Efficacy markers

Convergence of technologies to enable breakthroughs in seizure neurobiology

and drug discovery toward the promise of precision medicine

Modeling complex genetic interactions

• Role of genetic background in determining clinical heterogeneity

– Compare multiple mutations in isogenic cell lines to simplify mutant analysis

– Compare patient mutation in cell line versus patient iPS neurons

– “Rescue” patient mutant to understand residual phenotype

• Create “disease state” models to explore drug action

CRISPR/Cas (Clustered Regularly Interspaced Palindromic Repeats/CRISPR-associated)

• Genome editing technologies

– Zinc Finger Nucleases

– TALENs

– CRISPR/Cas

CRISPR/Cas Homology Directed Repair

• Addressing off target effects:

• Nickase version of Cas

• WES of cell lines with established bioinformatics filtering

• Scalable

Stem Cell Differentiation

• Goal is to create a functioning network of neurons that can display sufficiently complex behavior to respond to genetic changes and drug exposure

• Differentiate human stem cells into

– Inhibitory neurons

– Excitatory neurons

– Glia

Multi Electrode Array: “Epilepsy in a dish”

Spikes and Bursts

MEA dish

Active Area

Contact Pads

POC from mouse genetic model neurons: Sensitivity to genetic variation

2 4 6

-50

0

50

100

150

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Week

Nu

mb

er

of U

nits

2 4 6

-5000

0

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10000

15000

Week

Un

its

Wild Type C121W

N=6

Sp

ike

s

Bu

rsts

Spiking

WT

C12

1W

0

20

40

60

80

ms

WT

C121W

Petrou, unpublished data 2013

Bursting

POC: Sensitivity to drugs

Petrou, unpublished data 2013

Retigabine (10uM)

Wash

Spiking

Bath

Week 1 2 3 4

Petrou, unpublished data 2013

POC: Sensitivity to drugs

MEA profiling

Acknowledgements

• Chris Reid • Carol Milligan • Melody Li • Emma Morrisroe • Sophie Crux • Nuttawat “Pop”

Suphanatarida • Kay Richards • Elena Gazina • Verena Wimmer • Bryan Leaw

• Sam Berkovic • David Goldstein • Ingrid Scheffer • Leanne Dibbens • Sarah Heron