Role of the Voltage Gated Sodium Channel Scn8a in Epilepsy 公开

Abstract

Voltage gated sodium channels (VGSCs) are critical regulators of neuronal excitability and synchrony. Alterations in the function of the CNS VGSCs are associated with susceptibility to epilepsy, cognitive impairment, and emotional disturbances. Mice with mutations in the VGSC Scn8a exhibit increased resistance to induced seizures; however, interestingly, Scn8a mutant mice also experience frequent spontaneous absence seizures. Epilepsy-causing mutations in the human SCN8A gene were recently identified but little is currently known about the mechanisms by which altered SCN8A function leads to epilepsy, or perhaps can confer seizure protection.

Using genetic approaches, we probed the developmental and brain-region dependence of Scn8a-mediated seizure protection in order to establish the feasibility of achieving seizure protection by selectively targeting Scn8a during adulthood. We established that increased resistance to induced seizures is achieved when Scn8a is inactivated in the adult mouse.

In order to provide a more complete understanding of the consequences of altering Scn8a function, we investigated the seizure, behavioral, and electrophysiological effects of a novel mutation in the Scn8a gene, R1627H. Interestingly, heterozygous knock-in mice (Scn8a^RH/+) exhibited increased resistance to induced seizures, but this was not observed in homozygous Scn8a^RH/RH mutants. Scn8a^RH/RH mice were also susceptible to audiogenic seizures and displayed a recessive movement disorder.

Lastly, we propose a cellular mechanism to explain the paradoxical seizure outcomes that arise from loss of Scn8a activity. We found that selective reduction of Scn8a expression in excitatory neurons results in increased seizure resistance without the generation of absence seizures. In contrast, the deletion of Scn8a in interneurons leads to spontaneous absence seizures but does not influence seizure thresholds. Furthermore, Scn8a immunoreactivity and electrophysiological recordings raise the possibility that Scn8a expression in the thalamic reticular nucleus is important for absence seizure generation.

In conclusion this work 1) provides basic information about the development of Scn8a-related seizure phenotypes 2) expands the phenotypes associated with altered Scn8a function, and 3) identifies critical cell-types mediating increased seizure resistance and absence seizure generation in Scn8a mutant animals.

Table of Contents

CHAPTER 1 : Introduction and Background

1.1. Voltage Gated Sodium Channel Structure and Function.............................................. 2-5

1.1.A. Overview............................................................................................................... 2

1.1.B. Molecular and Genomic Organization of VGSCs............................................... 2-5

1.2. Genomic Organization of VGSCs.................................................................................. 6

1.3. Distribution of VGSCs.............................................................................................. 7-11

1.3.A. Overview............................................................................................................... 7

1.3.B. Scn1a Distribution.............................................................................................. 7-8

1.3.C. Scn8a Distribution.............................................................................................. 8-9

1.3.D. Scn2a Distribution................................................................................................. 9

1.3.E. Scn3a Distribution................................................................................................. 9

1.3.F. Glial Expression of VGSCs............................................................................... 9-10

1.4. Overview of Epilepsy............................................................................................. 12-12

1.4.A. What is Epilepsy................................................................................................ 12

1.4.B. Idiopathic Epilepsies.......................................................................................... 13

1.5. VGSC Epilepsy Mutations...................................................................................... 14-16

1.5.A. SCN1A Epilepsy Mutations............................................................................ 14-15

1.5.B. SCN2A Epilepsy Mutations.................................................................................. 15

1.5.C. SCN3A, SCN8A, and SCN9A Epilepsy Mutations........................................... 15-16

1.5.D. Epilepsy Mutations in VGSC Beta Subunits........................................................ 16

1.6. VGSC and Epilepsy: Mechanisms.......................................................................... 16-21

1.6.A. Mechanisms of SCN1A Epilepsies.................................................................. 17-18

1.6.B. Mechanisms of SCN8A Epilepsies.................................................................. 19-20

1.6.C. Mechanisms of SCN2A and SCN3A Epilepsies............................................... 20-21

1.7. VGSC Dysfunction in Cognition and Behavior...................................................... 21-23

1.7.A. SCN1A in Cognition and Behavior................................................................. 21-22

1.7.B. SCN8A in Cognition and Behavior................................................................. 22-23

1.7.C. SCN2A and SCN3A in Cognition and Behavior.................................................... 23

1.8. VGSCs as Antiepileptic Drug Targets...................................................................... 23-24

CHAPTER 2 : Partial Knockdown of Nav1.6 (Scn8a) Sodium Channels in the hippocampus Confers Seizure Protection

2.1. Summary..................................................................................................................... 26

2.2. Introduction............................................................................................................ 27-28

2.3. Results.................................................................................................................... 28-50

2.4. Discussion.............................................................................................................. 51-55

2.5. Materials and Methods........................................................................................... 55-64

2.6. Acknowledgments....................................................................................................... 65

CHAPTER 3 : Effects of an Epilepsy Mutation in the Voltage Sensor of Scn8a on Seizure Susceptibility and Behavior

3.1. Summary..................................................................................................................... 67

3.2. Introduction............................................................................................................ 68-69

3.3. Results.................................................................................................................... 69-90

3.4. Discussion.............................................................................................................. 91-93

3.5. Materials and Methods......................................................................................... 93-103

3.6. Acknowledgments..................................................................................................... 104

CHAPTER 4: Contrasting Effects on Seizure Susceptibility of Conditional Cell-Type Specific Inactivation of the Scn8a Gene

4.1. Summary................................................................................................................... 106

4.2. Introduction........................................................................................................ 107-108

4.3. Results................................................................................................................ 108-128

4.4. Discussion.......................................................................................................... 129-131

4.5. Materials and Methods....................................................................................... 132-135

4.6. Acknowledgments..................................................................................................... 135

CHAPTER 5 : Overall Discussion and Conclusions

5.1. Overview............................................................................................................ 137-138

5.2. Reconciliation of Seizure Outcomes Associated with Scn8a Mutations.............. 138-141

5.3. A Circuit Model of SCN1A-Epilepsy and Genetic Modification by SCN8A Mutations 142-143

5.4. Persistent Sodium Current: Unifying VGSC Epilepsy Mechanisms?.......................... 144

5.5. A Circuit Model of Scn8a-Absence Epilepsy...................................................... 144-147

5.6. Therapies for VGSC-Derived Epilepsies............................................................ 149-150

5.7. Concluding Remarks.......................................................................................... 150-151

REFERENCES ......................................................................................................... 152-181

Figure Index

Figure 1.1. Schematic representation of α and β subunits of a voltage gated sodium channel 5

Figure 1.2. Schematic representation of VGSC subcellular Localization............................. 11

Figure 1.3. Loss of inhibition model of Scn1a dysfunction leading to epilepsy.................. 18

Figure 2.1. Spontaneous seizure-like burst discharges in CA3 pyramidal cell layer induced by elevated potassium in hippocampal slices from Scn8a^med/+ mutant and WT mice....................................... 31-32

Figure 2.2. Analysis of burst characteristics between Scn8a^med/+ and WT mice.................. 33

Figure 2.3. Developmental expression of VGSCs in the hippocampus of Scn8a^med/+ mutant and WT mice 36-37

Figure 2.4. Tamoxifen-induced global deletion of the Scn8a gene................................ 39-40

Figure 2.5. Inducible deletion of the Scn8a gene in the adult mouse is sufficient to increase the latency to flurothyl- and KA-induced seizures and to protect against electrically induced 6-Hz psychomotor seizures 42-43

Figure 2.6. Lentiviral Cre-mediated knockdown of Scn8a in the hippocampus reduces seizure activity following PT administration.......................................................................................................... 46-47

Figure 2.7. Efficient adeno-assoicated viral shRNA-mediated knockdown of the Scn8a gene in the hippocampus of Scn1a^+/- and WT mice............................................................................................. 48-49

Figure 2.8. Adeno-associated viral shRNA-mediated knockdown of the Scn8a gene in the hippocampus increases flurothyl-induced seizure thresholds in Scn1a^+/- and WT mice..................................... 50

Figure 3.1. Characterization of the biophysical properties of Scn8a-R1627H channels...... 70

Figure 3.2. Generation of the R1627H mutant mouse line............................................. 72-73

Figure 3.3. Homozygous Scn8a-R1627H mutants have reduced weight............................. 75

Figure 3.4. Recessive motor impairment in R1627H mice.................................................. 76

Figure 3.5. Effects of the R1627H mutation on flurothyl- and 6 Hz-induced seizure susceptibility and hippocampal bursting................................................................................................................... 81-82

Figure 3.6. The Scn8a-R1627H mutation increases lifespan and seizure resistance in a mouse model of GEFS+ (Scn1a-R1648H)....................................................................................................................... 83

Figure 3.7. Scn8a^RH/RH mice are susceptiable to audiogenic seizures............................. 86-87

Figure 3.8. Scn8a^RH/RH mice have reduced auditory brainstem nuclei responses (ABRs).... 88

Figure 3.9. Flurothyl seizure thresholds and response to acoustic stimuli in mice carrying the Scn8a-medjo mutation..................................................................................................................................... 90

Figure 4.1. The floxed Scn8a allele does not alter Nav1.6 (Scn8a) protein levels or flurothyl-induced seizure thresholds in the absence of Cre but is efficiently recombined in expected brain regions by the Emx1- and Dlx5/6-Cre drivers................................................................................................................................... 110

Figure 4.2. Seizure thresholds are increased by loss of Scn8a from excitatory but not inhibitory neurons 115

Figure 4.3. Inactivation of Scn8a in excitatory forebrain neurons increases resistance to induced epileptiform hippocampal bursting activity.............................................................................. 116-117

Figure 4.4. Resistance to flurothyl-induced seizures is influenced by the level of inactivation of Scn8a in excitatory forebrain neurons....................................................................................................... 118

Figure 4.5. Selective inactivation of Scn8a in excitatory forebrain neurons in Scn1a mutant mice improves survival and increases resistance to flurothyl-induced seizures................................................ 119-120

Figure 4.6. Inhibitory neuron-specific but not forebrain excitatory neuron-specific inactivation of the Scn8a gene is sufficient to produce SWDs................................................................................ 122-123

Figure 4.7. Evaluation of Scn8a expression in interneuronal populations of thalamocortical circuitry 125

Figure 4.8. nRT neurons of Scn8a-deficient mice have impaired tonic firing................... 126

Figure 4.9. Developmental increases in SWDs in Scn8a^med/+ mice.................................... 128

Figure 5.1. Homozygous Scn8a-R1627H mice exhibit deficits in interneuron firing in response to large depolarization................................................................................................................................... 141

Figure 5.2. Loss of inhibition model of Scn1a dysfunction leading to generalized tonic-clonic epilepsy and reversal by loss of Scn8a.............................................................................................................. 143

Figure 5.3. Thalamocortical circuit models of conditional cell-type specific inactivation of Scn8a leading to SWD generation............................................................................................................ 146-147

Tables

Table 2.1. decreased Scn8a expression reduced the occurrence of bursting in hippocampal slices from Scn1a^RH/+ mutant mice .................................................................................................................. 34

Table 3.1. Summary of behavioral tasks performed on the R1627H line ............................ 78

Table 4.1. Summary of colocalization of Dlx5/6-Cre and Emx1-Cre expression in the CNS 111

About this Dissertation

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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
关键词	Epilepsy Seizure Nav1.1 Animal Models Scn1a Nav1.6
Committee Chair / Thesis Advisor	Escayg, Andrew, Emory University
Committee Members	Weinshenker, David, Emory University Traynelis, Stephen, Emory University Dingledine, Raymond J, Emory University Hess, Ellen, Emory University

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