Ranmal A. Samarasinghe, Ph.D. - US grants

Project Summary/Abstract Epilepsy is a severe and debilitating disease and a significant public health concern. Epilepsy is also a disease without a medical cure, and a disease where about 1 in 3 patients fails to respond to anti-seizure medications. In the most severe epilepsy syndromes of childhood, medical control of seizures can be even more challenging. Novel experimental platforms have the potential to play a critical role in advancing our understanding and treatment of epilepsy. Brain organoids derived from human embryonic or induced pluripotent stem cells are one such novel technology that has enormous potential. This is particularly true for severe childhood epilepsies, as organoids are ideally suited to model early neural development. Organoids are 3D structures that recapitulate complex elements of human brain such as its laminar organization and cell types seen in all six layers of human cortex. Since they can be human induced-pluripotent stem cell (hiPSC) derived, an organoid can be produced directly from patient tissue. Recent advances in organoid technology have resulted in the ability to generate distinct brain region-like organoids such as forebrain cortex and hippocampus and to make ?fusion? structures with integration of inhibitory and excitatory cell types. In the following proposal I will leverage these advances and build on an organoid platform that I have recently developed to model brain circuit formation and dysfunction in epilepsy. Previously, l was able to recapitulate hyperexcitable electrographic features in organoids derived from a patient with Rett syndrome, a neurological disorder highly associated with seizures and epilepsy. I have now generated cortical and hippocampal organoids from hiPSCs harboring mutations in the SCN8A gene. This mutation results in a severe childhood epilepsy. I have found that the SCN8A mutant cortex organoids have a highly hyperexcitable pattern of physiological activity compared to controls, whereas the SCN8A mutant hippocampus lacks a particular type of neural oscillation that is important for memory consolidation called a sharp wave ripple. This finding suggests that the SCN8A mutation results in different physiological activity patterns in distinct brain regions. Based on published studies, I hypothesize that this difference is primarily due to dysfunction of excitatory neurons in the cortex versus inhibitory interneurons in the hippocampus. I will now use an array of techniques such as calcium indicator imaging, extracellular recordings, immunohistochemistry, and manipulation of the genetic background of excitatory and inhibitory neurons within the organoid to test this hypothesis. To increase the rigor and generalizability of my data, I will use hiPSC from three different patients with pathogenic SCN8A mutations. Finally, I will perform drug testing to further isolate the role of specific cell types to the observed phenotypes and for consideration as therapeutic agents in patients. I expect that this will both provide a blueprint for a novel methodology for epilepsy research and enhance our treatment and understanding of epilepsy and neural circuit dysfunction resulting from SCN8A mutations.