Epilepsy, characterized by sudden, unprovoked seizures resulting from abnormal brain activity, affects individuals of all ages (Fisher et al., 2014). At the heart of this neurological puzzle lies the SCN2A gene. The SCN2A gene controls Nav1.2 sodium channels predominantly expressed in the central nervous system, where they play a crucial role in neuronal excitability and synaptic transmission regulation (Ogiwara et al., 2012). Dysregulation of Nav1.2 channel function, due to mutations in SCN2A, can lead to hyperexcitability and seizure generation (Catterall et al., 2010). It's noteworthy that epilepsy is observed when the SCN2A gene is upregulated (Ogiwara et al., 2012). Gap in Knowledge The role of the SCN2A gene its regulation of Nav1.2 channels is still unclear.In order to address this gap I will be using mouse (Mus musculus) models due to similarities in their domains, and sodium channel pathways as the ones in humans,, as well as their genomic surroundings are the same as humans. (Sanders et al., 2018).The primary purpose is to determine the role of SCN2A in sodium channel regulation in the brain. I hypothesize that the knockout of R853q and R1882q, within the SCN2A gene will both restore normal sodium channel activity within mutant types.
Aim 1: Identify conserved amino acids in the SCN2A that are essential for sodium channel function.: Hypothesis- I hypothesize that mice with mutations at particular glutamine sites in Nav1.2 channels will experience abnormal brain activity and seizures. These phenotypes will be detectable through EEG recordings and visibly observed in the animals. Rationale:. This gene is mainly expressed in the central nervous system (CNS), particularly in excitatory and glutamatergic neurons. According to literature the highly upregulated R1882q which encodes for Nav1.2 channel, is located on a glutamatergic site. In addition, a close by location should be suspected of doing the same. Approach: I will conduct a multiple sequence alignment using FASTA data obtained from Ensembl, MEGA, and Clustal Omega and literature. Subsequently, I will identify one additional point conserved point within this region for mutation. Specifically, I will focus on two regions: R1882q, which is suspected to be associated with a mutation in the gene, and R853q, which is nearby and also highly suspected. Mutations will be induced using CRISPR/Cas9, and electroencephalogram (EEG) tests will be attached to the mice to assess seizure activity. As a control, I will utilize a wild-type mouse
Aim2: Determine genes important for sodium channel regulation in the brain. Hypothesis Inducing neuronal activity will reveal differential gene expression between control and mutant R853q, Rationale: Since the upregulation of R1882q changes gene expression of Nav 1.2 sodium channels similar expression should be seen in R853q due to them being close in location Approach: RNAseq will be used to compare gene expression in control and mutants from tissue samples from the cortex after inducing neuronal activity with Cu-Znb. Aim 3: Quantify protein levels in wild-type and SCN2A mutants to identify proteins necessary for sodium channel regulation. Hypothesis Most proteins will have no affect to the action potentials with the exception of the Nav 1.2. Rationale: litature explains that Nav 1.2 is vital for the SCN2A which means that its protein will be the most affected in mutants. Approach: BioID will be used to tag and quantify the proteins in mutant models to find those driving increased glutamatergic neuronal activity. Using GO term analysis, relevant proteins are identified. CRISPR/Cas9 then targets and knocks out these proteins to alleviate the phenotype.
Expected Outcomes. In these investigations, we anticipate gaining a deeper understanding of how SCN2A and associated protein networks cooperate to prevent seizures. This knowledge not only has implications for elucidating the pathophysiology of epilepsy but also provides new avenues for preventative and therapeutic interventions. future research endeavors, aim to manipulate both dietary composition and age parameters in order to further elucidate their respective roles in the observed phenomena.
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