Abstracts

Rationale: Focal Cortical Dysplasia (FCD) is a cortical malformation that causes medication resistant pediatric epilepsy. Being a neurodevelopmental condition, FCD originates in the embryo and has been linked to defects in progenitors, neuronal differentiation or neuronal migration. Such errors lead to focal cortical disorganization, characterized by disrupted lamination, misplaced neurons, dysplastic neurons and focal seizures. FCD has been linked to somatic mutations in the mammalian Target of Rapamycin (mTOR) signaling pathway. In order to understand the developmental origin of FCD and other mTOR-linked epilepsies, we used ex vivo and in vitro models to examine the effects of modulating mTOR signaling during human brain development. Using single nucleus sequencing strategies in donated patient surgical specimens, we identified key signatures that link developmental errors with patient malformations. These signatures link the cell biology of abnormal cortical development with the cortical phenotypes in patients.  We have also identified transcriptomic signatures that are unique to the epileptic region, that can be validated further to generate potential diagnostic markers or therapeutic targets.   Methods: 1) We have developed an ex-vivo organotypic slice culture system and human-specific organoid culture models to examine the role of mTOR signaling in cortical development. We then used genetic manipulations and pharmacological treatments to modulate mTOR signaling in these models.  2) De-identified tissue samples from surgical resections are collected with previous patient consent for research in strict observance of legal & institutional ethical regulations. The brain regions with cortical lesions and epileptogenic foci are resected en-bloc and brought to the lab, where they are flash-frozen and sectioned to preserve cellular architecture. Dysplastic areas are separated from adjacent normal cortex, which serves as a control. This very valuable tissue pipeline is used for both advanced transcriptomic screens and histological studies. Results: 1) Using pharmacological and genetic modulations in organotypic slice cultures and cortical organoids, we found that both inactivation and hyper-activation of mTOR signaling resulted in profound changes to the morphology, migration and positioning of outer radial glial (oRG) cells. oRG cells are a distinct group of progenitor cells that contribute to the radial scaffold and cortical expansion in gyrencephalic mammals. The basal process of oRG cells was significantly shortened by changing the level of mTOR signaling (p < 0.005; >15 slices each condition from >8 independent experiments). These effects are mediated by the activity of the low molecular weight GTPase, CDC42, that regulates the cytoskeleton. We propose that by controlling oRG morphology and migration of oRG cells, mTOR signaling plays a vital role in the organization of the radial scaffold, providing a model for the cortical malformations seen in patients. 2) Single nuclear transcriptomic studies in surgically resected tissue (n = 4 patients) have identified changes in cell diversity within epileptic tissue. Using immuno-fluorescence assays, we show that cytomegalic neurons and balloon cells in patient tissues retain gene expression associated with oRG cells (n = 6). Both gene ontology studies and immunofluorescence assays show a change in in the cytoskeletal organization and polarity of the abnormal neurons consistent with developmental errors in oRG cells (n = 3). 3) In addition to the abnormal pyramidal neurons, we have identified transcriptomic changes associated with interneurons, glial cells and the vasculature within the epileptic region. With further validation, these datasets can be used to comprehensively explore the disease phenotype, helping develop potential diagnostic markers or therapeutic targets. Conclusions: We­ show that changes in mTOR signaling regulate the cytoskeleton and affect the morphology and migration of oRG cells during development, suggesting a likely mechanism for the developmental origin of FCD. We observe similar changes to the cytoskeleton in patient samples, identified oRG signatures within the lesions and identified epilepsy-associated transcriptomic changes within all cell types in the epileptic cortex. With further validation, these datasets can be used to comprehensively explore the disease phenotype, helping develop potential diagnostic markers or therapeutic targets. Funding: CURE Taking Flight Award 2018; NARSAD Young Investigator Award 2018