| Epilepsy is a chronic seizure disorder and affects more than 50 million people worldwide. Advances in genome sequencing have identified >1200 mutations in voltage-gated sodium ion channel gene SCN1A that result in genetic epilepsy . Yet, we know very little about the underlying changes in channel function caused by these different mutations. The main focus of the O’Dowd lab is to evaluate cellular and behavioral effects of patient-specific SCN1A mutations resulting in two major epilepsy disorders – genetic epilepsy with febrile seizures plus (GEFS+) and Dravet Syndrome (DS). |
| Our goal – In order to develop efficient patient-specific anti-convulsant therapies, we use a comparative approach with three complementary model systems- Drosophila, transgenic mice and isogenic pairs of human induced pluripotent stem cells (iPSCs). Key effects of the mutation that are conserved across multiple species (Drosophila, mice and humans) are most likely to reflect cellular mechanisms contributing to the disease phenotype in humans. |
| Modeling epilepsy in fruit flies Cost-effective and efficient, the fruit fly (Drosophila melanogaster) has been used to make many key discoveries in the field of neuroscience and to model several neurological disorders. Using CRISPR/Cas9 gene editing, we have introduced patient-specific SCN1A mutations into the Drosophila sodium channel gene, para. Behavioral tests show that flies with epilepsy-causing mutations have seizures resembling those present in humans. To understand how specific epilepsy-causing mutations influence epilepsy-associated behaviors, we perform electrophysiological recordings of neuronal firing and sodium channel activity in mutant flies. Our lab uses an ex vivo preparation in which neurons in the adult fly brain can be recorded from in real-time in different contexts, in different environments and in the presence of potential drugs. |
| Transgenic mouse model of epilepsy Recent gene editing technology of CRISPR/Cas9 has made it possible to more efficiently generate knock-in models for a variety of genetic diseases including those associated with large complex genes like the sodium channel. We generated a transgenic mouse model carrying human GEFS+ causing mutation K1270T in SCN1A gene. Our research is directed towards understanding how specific mutations in the SCN1A sodium channel gene result in GEFS+. We study different seizure behavior paradigms and compliment it with molecular, immunohistochemical and electrophysiology-based studies to understand cellular correlates of hyperexcitability in neuronal circuits – which is the most likely causes of seizures in epilepsy. |
| Modeling human stem cell lines with epilepsy mutations We are working to generate human iPSC lines with CRISPR/Cas9 gene editing that harbor one of two mutations at a specific locus in the SCN1A gene. These single base-pair alterations result in an amino acid change from arginine (R) to cysteine (C) or histidine (H) (referred to as R-C and R-H respectively). Despite being located at the same position, R-C was identified in patients with a severe seizure disorder known as Dravet Syndrome while R-H was identified in a family with a more moderate seizure disorder known as GEFS+. We aim to better understand how these two mutations contribute to seizure disorders of varying severity by analyzing the electrophysiological properties of neurons derived from each line including the isogenic parental line from which they were generated. Patient derived human iPSC cell lines: Complementary to our mouse model for studying K1270T SCN1A mutation, we used CRISPR/Cas9 to generate two pairs of isogenic iPSC lines, one by introducing the mutation into control iPSCs derived from an unaffected patient sibling, and the other by correcting the mutation in patient iPSCs from a GEFS+ individual. Using a differentiation protocol we optimized (Xie et al., 2018), we pattern isogenic iPSCs into functionally active neurons. To determine the cellular mechanism of the mutation, we perform whole-cell recording to compare the electrophysiological properties of neurons derived from isogenic lines. |