Roles of the SCN1A and SCN8A Voltage-Gated Sodium Channel Genes in Neurological Disease 公开
Inglis, George Andrew (Spring 2020)
Abstract
The neuronal voltage-gated sodium channel genes, SCN1A and SCN8A, play a critical role in the control of normal neuronal excitability. Mutations in both genes have been linked to several neurological disorders, most prominently epilepsy. Though both channels are detectable in a variety of neuronal subtypes, loss-of-function mutations in SCN1A particularly impact the activity of GABAergic interneurons that drive neuronal inhibition. Therefore, modulating SCN1A transcription may have broad translational relevance to neurological disorders associated with impaired neuronal inhibition, such as epilepsy, schizophrenia, or Alzheimer’s disease. In contrast, increased SCN8A activity in excitatory pyramidal neurons is sufficient to induce seizures in mice, suggesting that SCN8A mutations may drive neuronal hyperexcitability. However, patients with SCN8A mutations exhibit a diverse range of clinical phenotypes, thereby complicating efforts to develop efficacious treatments. Given the broad contributions of SCN1A and SCN8A to neurological disease, the aims of this dissertation were (1) to identify genetic elements or pathways contributing toward transcriptional regulation of SCN1A and (2) to characterize the phenotypic impacts of novel, overlapping Scn8a mutations. For the first aim, we analyzed publicly available neuronal open chromatin data to identify putative functional genomic elements in SCN1A, such as transcriptional enhancer sequences. Given that SCN1A is robustly expressed in GABAergic interneurons, we also performed ATAC-seq and mRNA-seq at three time points during interneuron development from human-derived iPSCs, to identify additional genetic elements associated with elevated SCN1A expression. At the same time, we developed a comprehensive profile of genome-wide epigenomic and transcriptomic changes concordant with GABAergic interneuron development. From this study, we identified several genes that may play an important role in interneuron function and the pathogenesis of schizophrenia. In line with our second aim, we generated three mouse lines with varying degrees reduced activity of the Scn8a channel protein, Nav1.6: Δ9, ∇3, and Δ35. Our results suggest that hypomorphic Scn8a alleles may exert effects on Nav1.6 function that are distinct from null alleles, potentially due to aberrant channel heterodimerization. Altogether, the results presented in this dissertation expand our existing knowledge of SCN1A regulation, the pathways contributing toward a GABAergic interneuron cell fate, and the phenotypic impact of hypomorphic and loss-of-function Nav1.6 mutations.
Table of Contents
CHAPTER 1: Introduction.......................................................................................................... 1
1.1 The voltage-gated sodium channel gene family.................................................................... 1
1.2 Voltage-gated sodium channels in the central nervous system............................................. 3
1.2.1 Overview of the neuronal voltage-gated sodium channels............................................ 3
1.2.2 SCN1A, SCN8A, and the excitatory-inhibitory balance................................................. 5
1.3 SCN1A and SCN8A dysfunction in neurological disease...................................................... 5
1.3.1 Familial hemiplegic migraine......................................................................................... 5
1.3.2 SCN1A-derived epilepsy................................................................................................. 6
1.3.3 SCN8A-derived epilepsy................................................................................................. 6
1.3.4 Neuropsychiatric disorders and dementia.................................................................... 10
1.4 Regulation of SCN1A and SCN8A....................................................................................... 11
1.4.1 Pharmacological modulation of Nav1.1 and Nav1.6 activity........................................ 11
1.4.2 Transcriptional regulation of SCN1A and SCN8A expression...................................... 12
1.4.3 Detecting additional putative functional genomic elements........................................ 13
1.5 Summary and goals of dissertation..................................................................................... 17
CHAPTER 2: Towards the identification of transcriptional regulatory elements for the voltage-gated sodium channel gene, SCN1A 19
2.1 Abstract............................................................................................................................... 19
2.2 Introduction......................................................................................................................... 20
2.3 Materials and Methods........................................................................................................ 21
2.3.1 Identification of putative functional genomic elements (FGEs) in the SCN1A locus.. 21
2.3.2 Generation of luciferase reporter vectors..................................................................... 22
2.3.3 SH-SY5Y cell culture and transfection........................................................................ 24
2.3.4 Quantitative real-time PCR (qRT-PCR)....................................................................... 25
2.3.5 Statistical analysis........................................................................................................ 25
2.4 Results................................................................................................................................. 27
2.4.1 Open chromatin and transcription factor ChIP-seq data predict 32 putative functional genomic elements (FGEs) within the SCN1A locus............................................................................................................................................... 27
2.4.2 FGE2 and FGE17 moderately enhance luciferase reporter activity............................. 30
2.4.3 SCN1A FGEs may jointly contribute to transcriptional regulation of reporter activity 32
2.4.4 Epilepsy-associated SNPs do not robustly affect luciferase reporter activity.............. 34
2.5 Discussion........................................................................................................................... 36
2.6 Acknowledgments............................................................................................................... 38
CHAPTER 3: Transcriptomic and epigenomic dynamics associated with development of human iPSC-derived GABAergic interneurons....................................................................................................................................................... 39
3.1 Abstract............................................................................................................................... 39
3.2 Introduction......................................................................................................................... 40
3.3 Materials and Methods........................................................................................................ 42
3.3.1 In vitro differentiation to GABAergic interneurons (GINs)........................................ 42
3.3.2 Immunostaining for neuronal markers......................................................................... 43
3.3.3 RNA-seq and data analysis........................................................................................... 44
3.3.4 Quantitative real-time PCR (qRT-PCR)....................................................................... 46
3.3.5 Assay for transposase-accessible chromatin (ATAC-seq) and data analysis............... 46
3.3.6 Transcription factor (TF) motif analysis...................................................................... 48
3.3.7 Statistical analysis........................................................................................................ 48
3.3.8 Data availability........................................................................................................... 49
3.4 Results................................................................................................................................. 50
3.4.1 Human induced pluripotent stem cells (iPSCs) are differentiated to GABAergic interneurons (GINs) with high efficiency 50
3.4.2 RNA-seq confirms enrichment of genes and pathways associated with GIN function 52
3.4.3 HC1- and HC2-derived GINs exhibit similar changes in chromatin accessibility across differentiation 56
3.4.4 Changes in chromatin accessibility are correlated with GIN-specific gene expression 58
3.4.5 Motif enrichment analysis implicates distinct groups of transcription factors in GIN differentiation 60
3.4.6 A subset of DEGs may represent novel risk factors for schizophrenia........................ 62
3.5 Discussion........................................................................................................................... 67
3.6 Acknowledgments............................................................................................................... 72
CHAPTER 4: Mutations in the Scn8a DIIS4 voltage sensor reveal new distinctions among hypomorphic and null Nav1.6 sodium channels....................................................................................................................................................... 73
4.1 Abstract............................................................................................................................... 73
4.2 Introduction......................................................................................................................... 74
4.3 Materials and Methods........................................................................................................ 77
4.3.1 Generation of founder mice.......................................................................................... 77
4.3.2 Genotyping................................................................................................................... 77
4.3.3 Animal maintenance..................................................................................................... 78
4.3.4 Protein modeling.......................................................................................................... 79
4.3.5 Survival and weight analysis........................................................................................ 79
4.3.6 Western blot analysis.................................................................................................... 79
4.3.7 6 Hz psychomotor seizure induction............................................................................ 80
4.3.8 Flurothyl seizure induction........................................................................................... 80
4.3.9 EEG surgery and analysis............................................................................................. 81
4.3.10 Behavioral assessment................................................................................................ 81
4.3.11 Acoustic startle response............................................................................................ 82
4.3.12 Auditory brainstem response...................................................................................... 82
4.3.13 Experimental conditions for motor assessment.......................................................... 83
4.3.14 Rotarod....................................................................................................................... 83
4.3.15 Grip strength............................................................................................................... 83
4.3.16 Nerve conduction analysis.......................................................................................... 84
4.3.17 Statistical analysis...................................................................................................... 84
4.4 Results................................................................................................................................. 86
4.4.1 Identification of new Scn8a mutants with severe motor impairments......................... 86
4.4.2 Δ9 mutants express normal levels of Nav1.6................................................................ 90
4.4.3 Scn8aΔ9/+, Scn8a∇3/+, and Scn8aΔ35/+ heterozygous mutants are resistant to induced seizures 92
4.4.4 Δ9 and ∇3 are predicted hypomorphic alleles of Scn8a............................................... 94
4.4.5 Scn8aΔ9/+ and Scn8a∇3/+ mutants do not exhibit increased anxiety or deficits in learning and sociability 94
4.4.6 Scn8aΔ9/+ and Scn8a∇3/+ mutants exhibit a decreased acoustic startle response........... 94
4.4.7 Scn8aΔ9/+ and Scn8a∇3/+ heterozygous mutants exhibit motor impairments................ 98
4.4.8 Δ9 and ∇3 homozygous mutants exhibit reduced nerve conduction velocity............ 100
4.5 Discussion......................................................................................................................... 102
4.6 Acknowledgments............................................................................................................. 108
CHAPTER 5: Conclusions and future directions.................................................................. 109
5.1 Summary of dissertation research..................................................................................... 109
5.2 The state of SCN1A transcriptional regulation.................................................................. 110
5.2.1 Endogenous regulation of SCN1A mRNA levels....................................................... 110
5.2.2 dCas9-based strategies to elevate SCN1A expression................................................ 114
5.2.3 The shift towards single-cell analyses........................................................................ 115
5.3 Assessment of novel targets for GABAergic interneuron function.................................. 116
5.3.1 Putative genetic regulatory elements in genes relevant to GABAergic function....... 116
5.3.2 Examining the contribution of CPLX2 and TRPC4 to neurological disease.............. 117
5.4 The phenotypic spectrum of loss-of-function mutations in SCN8A.................................. 118
5.4.1 Examining the phenotypic impact of hypomorphic and null SCN8A alleles............. 118
5.4.2 Future characterization of the Scn8a Δ9 and∇3 alleles.............................................. 120
5.4.3 The contribution of Nav1.6 to the acoustic startle response and hearing................... 120
5.5 Conclusions....................................................................................................................... 121
APPENDIX A: Supporting data for Chapter 2...................................................................... 147
APPENDIX B: Supporting data for Chapter 3...................................................................... 152
APPENDIX C: Supporting data for Chapter 4...................................................................... 160
C.1 Supporting materials and methods................................................................................... 160
C.1.1 Open field test and novel object recognition............................................................. 160
C.1.2 Three-chamber social interaction.............................................................................. 160
C.1.3 Reciprocal social interaction...................................................................................... 161
C.2 Supporting figures and tables........................................................................................... 162
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