Cell, network and mouse modelling of genetic epilepsies for mechanism, diagnosis and therapy December 7 th 213 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 Type of Financial Relationship NONE American Epilepsy Society 213 Annual Meeting
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 213 Annual Meeting
Opportunity for translation provided by explosion in genetics knowledge Examined 264 trios (792 exomes) 329 variants discovered
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
Early onset epileptic encephalopathies (EOEE) 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, 29) Patient presented with Dravet Syndrome We previously developed the het SCN1B(C121W) mouse Does the homozygous mouse model Dravet? Modified from: Patino, G.A. (29), A functional null mutation of SCN1B
Characteristics of the homozygous C121W mouse Survival Altered gait Premature death Spontaneous seizures More rapid progression to tonicclonic seizure following heating Petrou, unpublished data 213
Pharmacosensitivity of the homozygous C121W mouse similar to Dravet Syndrome Similar pharmacosensitivity to human Dravet patients Suggests shared pathological mechanism Petrou, unpublished data 213
In stark contrast to SCN1A based Dravet models, CA1 interneurons appear normal Petrou, unpublished data 213
BUT, hippocampal excitatory neurons from our homozygous C121W mice are more excitable WT (n =8), Hom (n = 8) Petrou, unpublished data 213
Changes in input resistance explains neuronal excitability Increased input resistance (R in ) may be major contributor to increased excitability WT (n=15), Hom (n=1); *P<.5 Petrou, unpublished data 213
Reduced neuronal arborisation might explain the changes in cellular properties Petrou, unpublished data 213
Can we use our information on cellular dysfunction to predict drug efficacy? Drugs that specifically target and reduce R in may rescue the seizure phenotype Retigabine Activates KCNQ2/3 channels Reduces R in Modified from: Surti TS (25), 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 213
In vivo: Retigabine reduces seizure susceptibility Petrou, unpublished data 213
Summary 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 R in 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
2. SINGLE CELL MODELS
Genotype-phenotype correlation and pharmacological modulation of hkcnt1 channel mutations causing epilepsy
KCNT1 based epilepsies In 211 ADNFLE (Autosomal Dominant Nocturnal Frontal Lobe Epilepsy) was thought to be a disorder of nicotinic acetylcholine receptors In 212 KCNT1 was discovered (Heron et al., 212, Nature Genetics) as a gene for more severe ADNFLE with psychiatric features expanding the genetic architecture At the same time Barcia et al (212, Nature Genetics) showed that KCNT1 was also associated with a very severe epileptic encephalopathy, Epilepsy of Infancy with Migrating Focal Seizures (EIMFS) characterised by drugresistant 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 mutations in ADNFLE and EIMFS KCNT1: (Slo2.2, K Ca 4.1, SLACK) Na+ activated potassium channel Thought to contribute to RMP and to slow hyperpolarisation following repetitive firing NH 2 ADNFLE mutants M896I R398Q Y796H R928C Least severe Disease severity Y796H NAD + binding [Na + domain ] R428Q A934T P924L M896I P924L R928C (R97C) Most severe (R899C) A934T EIMFS mutants I76M R398Q R428Q R474H RCK domains [Na + ] COOH
KCNT1 epilepsy mutations show increased R 3 9 8 Q R 3 9 8 Q 7 9 6 H current magnitude and altered kinetics 1 Y 1 1 1 1 Y 1 1 7 9 6 H 1 1 1 1 M W T R 1 WT W T M8961 M 8 9 6 I 8 9 6 I 1 R 9 2 8 CR928C R 9 2 8 C 1 R428Q R 4 2 8 Q 1 1 1 4 2 8 Q 1 1 Y 1 1 1 1 1 1 R 3 9 8 Q R 3 9 8 Q Y 7 9 6 H 7 9 6 H A 9 3 4 T A 9 3 4 T 1 1 1 1 R398Q Y796H A934T 1 P924L 1 P 9 2 4 L P 9 2 4 L 2 A 2 1 m s 1 1 1 1 1 1 1 1 R R 9 2 8 C R 9 2 8 C 1 1 R 4 2 8 Q 4 2 8 Q Petrou, unpublished data 213 1 1
current (μa) c u r re n t a t + 1 m V ( A ) current (μa) c u r re n t a t + 1 m V ( A ) KCNT1 gain of function correlates with disease severity 8 6 **** **** 6 **** **** **** **** 4 **** 4 **** ** **** 2 2 W T M 8 9 6 I R 3 9 8 Q Y 7 9 6 H R 9 2 8 C R 4 2 8 Q A 9 3 4 T P 9 2 4 L W T A D N F L E E IM F S Petrou, unpublished data 213
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? Cinchona Tree Bark Yang et al., 26 quinine quinidine
Quinidine blocks human WT and mutant channels Petrou, unpublished data 213
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 MEA dish Active Area Contact Pads Spikes and Bursts
Units Spikes Number of Units Bursts POC from mouse genetic model neurons: Sensitivity to genetic variation Wild Type Spiking C121W 25 Bursting 15 2 1 15 1 5 5-5 2 4 6 Week -5 2 4 6 Week N=6 WT C121W Petrou, unpublished data 213
POC: Sensitivity to drugs Spiking Week 1 2 3 4 Bath Retigabine (1uM) Wash Petrou, unpublished data 213
POC: Sensitivity to drugs Petrou, unpublished data 213
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