Familial hemiplegic migraine type III (FHM3) is an autosomal dominant disease that results from gain-of-function mutations in SCN1A, which encodes the voltage-gated sodium channel Nav1.1 [1]. This causes migraines with aura, which can include visual, sensory, motor, and auditory symptoms followed by a headache. The aura symptoms must include fully reversible motor impairment (hemiplegia) along with at least one other type of aura [2]. Migraines with aura are thought to come from cortical spreading depression (CSD), which is a wave of rapid depolarization across the cortex [1]. Due to its rarity, there has not been extensive research into SCN1A and FHM3, so its role in causing hemiplegia is currently unknown.
My primary goal is to investigate how mutations in SCN1A cause reversible motor impairment. I will use mice (Mus musculus) as my model organism due to the gene being highly conserved along with ease of behavioral assays and CSD induction through KCl application. Mutations at serine sites can disrupt phosphorylation, potentially altering cell signaling and protein function. I hypothesize that conserved serines between species are important for CSD induction, and in turn, hemiplegia. My long-term goal is to understand how gain-of-function mutations in this gene cause hemiplegia as an aura symptom.
Aim 1: Identify conserved amino acids in the SCN1A that are essential for Nav1.1 inactivation.
Rationale: CSD, and in turn, hemiplegia, is induced when the Nav1.1 channel does not properly close
Approach: I will obtain FASTA protein sequences of orthologs from Ensembl and will conduct Multiple Sequence Alignment using MEGA. I will use Pfam to identify domains that act on Nav1.1 inactivation and select a target mutation site that is conserved among species. Mutations will be induced using CRISPR/Cas9, and the open field test will be used in the mutated mice to assess motor impairment.
Hypothesis: Mice with knock-ins in a conserved serine will have dysfunctional fast inactivation in Nav1.1 channels, causing reversible motor impairment.
Aim 2: Perform single-cell RNA-sequencing to look at differentially expressed genes in confirmed mutant mice.
Rationale: Mutations in SCN1A may affect gene expression of other genes that may aid in propagation of reversible motor impairment.
Approach: I will perform single-cell RNA-seq in confirmed mutant and WT mice in which CSD has been induced. This single-cell RNA-seq will be analyzed for genetic impact of CSD on SCN1A-expressing neurons between WT mice and mutant mice. Differentially expressed genes will be analyzed and sorted according to GO terms.
Hypothesis: Altering SCN1A regulation will produce changes in the expression of other genes that are involved in action potential propagation and voltage-gated sodium channels in the brain.
Aim 3: Use BioID to identify novel proteins that function with SCN1A sodium channels in the brain.
Rationale: It is unknown if SCN1A-regulating proteins contribute to propagation of CSD through modulation of SCN1A domain activity.
Approach: SCN1A proteins will be biotin-labeled in vivo, purified, and analyzed in both WT and confirmed mutant mice. Proteins will be identified using mass spectrometry. These proteins will be analyzed for GO terms associated with SCN1A. Proteins found in WT mice but not in mutants will be knocked out through the use of CRISPR/Cas9. I will then run behavioral assays to confirm that these proteins have differential expression in WT vs mutant mice.
Hypothesis: Proteins that interact with SCN1A and are involved in action potential propagation will have lower expression levels in mutant mice compared to the control mice.
My primary goal is to investigate how mutations in SCN1A cause reversible motor impairment. I will use mice (Mus musculus) as my model organism due to the gene being highly conserved along with ease of behavioral assays and CSD induction through KCl application. Mutations at serine sites can disrupt phosphorylation, potentially altering cell signaling and protein function. I hypothesize that conserved serines between species are important for CSD induction, and in turn, hemiplegia. My long-term goal is to understand how gain-of-function mutations in this gene cause hemiplegia as an aura symptom.
Aim 1: Identify conserved amino acids in the SCN1A that are essential for Nav1.1 inactivation.
Rationale: CSD, and in turn, hemiplegia, is induced when the Nav1.1 channel does not properly close
Approach: I will obtain FASTA protein sequences of orthologs from Ensembl and will conduct Multiple Sequence Alignment using MEGA. I will use Pfam to identify domains that act on Nav1.1 inactivation and select a target mutation site that is conserved among species. Mutations will be induced using CRISPR/Cas9, and the open field test will be used in the mutated mice to assess motor impairment.
Hypothesis: Mice with knock-ins in a conserved serine will have dysfunctional fast inactivation in Nav1.1 channels, causing reversible motor impairment.
Aim 2: Perform single-cell RNA-sequencing to look at differentially expressed genes in confirmed mutant mice.
Rationale: Mutations in SCN1A may affect gene expression of other genes that may aid in propagation of reversible motor impairment.
Approach: I will perform single-cell RNA-seq in confirmed mutant and WT mice in which CSD has been induced. This single-cell RNA-seq will be analyzed for genetic impact of CSD on SCN1A-expressing neurons between WT mice and mutant mice. Differentially expressed genes will be analyzed and sorted according to GO terms.
Hypothesis: Altering SCN1A regulation will produce changes in the expression of other genes that are involved in action potential propagation and voltage-gated sodium channels in the brain.
Aim 3: Use BioID to identify novel proteins that function with SCN1A sodium channels in the brain.
Rationale: It is unknown if SCN1A-regulating proteins contribute to propagation of CSD through modulation of SCN1A domain activity.
Approach: SCN1A proteins will be biotin-labeled in vivo, purified, and analyzed in both WT and confirmed mutant mice. Proteins will be identified using mass spectrometry. These proteins will be analyzed for GO terms associated with SCN1A. Proteins found in WT mice but not in mutants will be knocked out through the use of CRISPR/Cas9. I will then run behavioral assays to confirm that these proteins have differential expression in WT vs mutant mice.
Hypothesis: Proteins that interact with SCN1A and are involved in action potential propagation will have lower expression levels in mutant mice compared to the control mice.
swansonspecificaims041624draft2.docx | |
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References
[1] Chever, O., et al. (2021). Initiation of migraine-related cortical spreading depolarization by hyperactivity of GABAergic neurons and nav1.1 channels. Journal of Clinical Investigation, 131(21). https://doi.org/10.1172/jci142203
[2] Kazemi, H., et al. (2014). Familial hemiplegic migraine and spreading depression. Iranian Journal of Child Neurology, 8(3).
[2] Kazemi, H., et al. (2014). Familial hemiplegic migraine and spreading depression. Iranian Journal of Child Neurology, 8(3).
About the website
This web page was produced as an assignment for Genetics 564, an undergraduate capstone course at UW-Madison
Joely Swanson, [email protected]
Last updated April 29th, 2024
Genetics 564 website
Joely Swanson, [email protected]
Last updated April 29th, 2024
Genetics 564 website