Voltage-gated Sodium Channels as Modifiers of Scn1a-derived Epilepsy Open Access

Abstract

Mutations in the brain expressed voltage-gated sodium channels (SCN1A, SCN2A, SCN3A, and SCN8A) are responsible for an increasing number of epilepsy disorders. The most clinically important of these VGSCs is SCN1A, which is responsible for a spectrum of disorders ranging from genetic epilepsy with febrile seizures plus (GEFS+) to the severe encephalopathy Dravet syndrome (DS). One hallmark of SCN1A disorders is the clinical heterogeneity observed. Within a single GEFS+ pedigree, for example, the phenotype may range from mild febrile seizures to DS. This phenotypic heterogeneity suggests the presence of additional environmental or genetic factors that can influence the phenotype. One such factor is a genetic modifier, an independently segregating gene that can alter the expression of the disease gene. Identifying and investigating candidate modifier genes may improve our understanding of the complex etiology of epilepsy and provide additional gene targets for therapeutic intervention. The goal of this dissertation was to investigate the role of the VGSC genes SCN3A and SCN9A, as epilepsy genes and as candidate modifiers for SCN1A-derived epilepsy. In Chapter 2, we reported a novel, trafficking-deficient SCN3A mutation in a patient with partial epilepsy, providing further evidence that SCN3A deficiency results in increased seizure risk. We then demonstrated that partial loss of Scn3a expression is sufficient to increase seizure susceptibility and produce motor deficits in a hypomorphic mouse line. In Chapter 3, we determined that partial loss of Scn3a increases susceptibility to flurothyl-induced seizures but does not alter survival or the behavioral characteristics of a GEFS+ mouse model. Furthermore, in Appendix B, we demonstrated that the presence of a Scn9a mutation does not increase the seizure susceptibility observed in the GEFS+ mouse model. In summary, the results of this dissertation provide greater insight into the role of Scn3a and Scn9a in epilepsy.

Table of Contents

CONTENTS

Chapter 1: Sodium Channels as Disease Genes and Genetic Modifiers in Mendelian Epilepsy: The myth of the monogenic syndrome 1

1.1 Introduction.............................................................................................................. 2

1.2 The History of Gene Discovery in Monogenic Epilepsies.................................. 4

1.3 Voltage-gated sodium channels: Structure, Physiology, and Distribution...... 5

1.3.A. Molecular Structure and Physiology............................................................ 5

1.3.B. Genomic, Regional and Cellular Distribution of Subunits..................... 9

1.4 Role of Voltage-gated Sodium Channels in Epilepsy....................................... 14

1.4.A. SCN1A in Epilepsy.......................................................................................... 14

1.4.B SCN3A in Epilepsy........................................................................................... 16

1.4.C. SCN9A in Epilepsy.......................................................................................... 19

1.4.D. SCN2A in Epilepsy......................................................................................... 19

1.4.E. SCN8A in Epilepsy.......................................................................................... 20

1.5 Genetic Modifiers in Epilepsy............................................................................. 20

1.5.A. Human Studies.............................................................................................. 21

1.5.B. Animal Studies.............................................................................................. 24

1.6 Specific Aims......................................................................................................... 28

1.7 Conclusions............................................................................................................ 29

Chapter 2: SCN3A deficiency associated with increased seizure susceptibility..... 30

2.1 Abstract................................................................................................................... 31

2.2 Introduction............................................................................................................ 32

2.3 Material and Methods........................................................................................... 34

2.4. Results................................................................................................................... 47

2.4.A. The SCN3A-L247P variant is associated with childhood epilepsy and encodes a trafficking deficient channel47

2.4.B. Scn3a mRNA and protein expression is reduced in Scn3a^+/Hyp and Scn3a^Hyp/Hyp mice 51

2.4.C. Scn3a^+/Hyp mice exhibit increased seizure susceptibility...................... 55

2.4.D. Scn3a^+/Hyp mutants do not exhibit spontaneous seizures...................... 58

2.4.E. Scn3a^+/Hyp mutants are hypoactive and display deficits in motor learning 59

2.5. Discussion............................................................................................................. 64

2.5.A. A novel SCN3A variant (L247P) is associated with childhood epilepsy and displays reduced trafficking to the cell surface64

2.5.B. In vivo characterization of Scn3a haploinsufficiency.............................. 65

2.5.C. A putative role for Scn3a in motor function and coordination............. 68

2.6. Conclusions........................................................................................................... 68

Chapter 3: Exploring the possibility that Scn3a can function as a genetic modifier in Scn1a mutant mice 70

3.1 Abstract................................................................................................................... 71

3.3 Materials and Methods......................................................................................... 75

3.4 Results.................................................................................................................... 77

3.4.A. Differences in lifespan and pre-weaning weight among Scn3a^+/Hyp/Scn1a^+/RH mice and littermates are sex-dependent. 77

3.4.B. Scn3a^+/Hyp/Scn1a^+/RH mice are more susceptible to flurothyl-induced seizures than either single heterozygote.80

3.4.C. Scn3a^+/Hyp/Scn1a ^+/RH and Scn1a^+/RH mice exhibit similar thresholds to hyperthermia-induced seizures and 6 Hz-induced seizures..................................................................................................................................... 82

3.4.D Behavioral responses of the Scn3a^+/Hyp/Scn1a ^+/RH mice were similar to Scn3a^+/Hyp mutants. 87

3.5 Discussion.............................................................................................................. 90

Chapter 4: Conclusions and Future Directions.......................................................... 93

4.1 Overview................................................................................................................. 94

4.2 The Role of SCN3A in Partial Epilepsy: Different Mechanisms, Common Outcome 95

4.3 Potential Mechanisms of Scn3a Epilepsy........................................................... 98

4.3.A. Susceptibility to Flurothyl-Induced Seizures........................................... 98

4.3.B. Susceptibility to 6 Hz Corneal Stimulation.............................................. 99

4.3.C. Susceptibility to Seizures Induced by Kainic Acid................................. 100

4.4 The role of Scn3a in motor behavior................................................................. 100

4.5 Scn3a as a Modifier of Scn1a............................................................................. 102

4.5.A. Effects on Seizure Susceptibility and Behavior..................................... 102

4.5.B. Role of Scn3a Dosage on Modifier Effects.............................................. 102

4.6 Scn9a as a Modifier of Scn1a............................................................................. 103

4.7. Conclusions......................................................................................................... 104

Appendix A: Additional Data and Figures from Chapter 2...................................... 105

A.1 Material and Methods........................................................................................ 106

A.2 Results.................................................................................................................. 110

A.2.A. No significant changes in Scn1a, Scn2a, and Scn8a expression were observed in Scn3a^+/Hyp mutants110

A.2.B. Scn3a^+/Hyp mutants performed normally in behavioral testing.......... 111

Appendix B: Scn9a does not modify the seizure phenotype of a GEFS+ mouse model. 117

B.1 Abstract................................................................................................................ 118

B.2 Introduction......................................................................................................... 119

B.3 Materials and Methods...................................................................................... 120

B.4 Results.................................................................................................................. 122

B.4.A. The susceptibility of Scn9a^NY mutants to hyperthermia-induced and flurothyl-induced seizures is comparable to WT littermates................................................................................................................................... 122

B.4.B. Scn1a^+/RH/Scn9a^+/NY mutants are not more susceptible to hyperthermia-induced or flurothyl-induced seizures than Scn1a^+/RH mice................................................................................................................................... 123

B.5 Discussion............................................................................................................ 126

REFERENCES............................................................................................................... 128

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Neuroscience
Keyword	epilepsy sodium channels
Committee Chair / Thesis Advisor	Escayg, Andrew, Emory University
Committee Members	Caspary, Tamara, Emory University Rainnie, Donald, Emory University Weinshenker, David, Emory University Hess, Ellen, Emory University

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