Abstracts

Rationale: Focal Cortical Dysplasia (FCD) is a major cause of medication-resistant pediatric epilepsy. FCD originates in the embryonic cerebral cortex as errors in neuronal differentiation and migration. These errors lead to focal cortical disorganization, characterized by disrupted lamination, misplaced neurons, dysplastic neurons and focal seizures. Recent studies have linked FCD with somatic mutations in genes regulating the mammalian Target of Rapamycin (mTOR) signaling pathway. We used immunofluorescence assays and advanced single-nuclear genomic approaches in donated patient surgical specimens to identify unique progenitor-cell signatures in the patient tissue. We then developed an ex-vivo organotypic slice culture system to explore the effect of modulating mTOR signaling in progenitor cells. Using this approach, we have been able to explore the cell biology behind the cortical disorganization in patients and develop a model of pediatric epilepsy. Methods: 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 genomic screens and histological studies. We also developed an ex-vivo organotypic slice culture system that allows us to label and modulate progenitor cells during cortical development. We then used pharmacological treatments to block as well as activate mTOR signaling in this model.  Results: 1) Using immuno-fluorescence assays, we show that cytomegalic neurons and balloon cells in patient tissues retain expression of markers expressed by outer radial glial (oRG) cells, a distinct group of progenitor cells that are thought to contribute to the cortical expansion in gyrencephalic mammals (n = 5). 2) Using advanced single-nuclear genomics approach in surgically resected tissue from 4 patients, we show that abnormal cytomegalic neurons and balloon cells in FCD patients have a unique transcriptomic identity that is distinct from the normal neuronal and glial cells in the cortex. This approach has allowed us to identify unique markers for these cells that can be used to further understand the identity of these neurons and their contribution to the disease phenotype.3) Using an ex-vivo organotypic slice culture system of human cortical development, we show that modulation of mTOR signaling directly affects the generation of the basal process in the radial and outer radial glial cells of the developing human cortex. Inhibition of mTOR signaling with rapamycin, results in a 33% reduction in the length of the basal process. These observations have been validated with multiple markers in over 15 different slices in 4 independent experiments (p < 0.005). We find that activation of mTOR signaling within these same progenitor cells also This provides a framework to understand the role of mTOR signaling in the developmental programs that underlie the generation of the abnormal cells in FCD patients. Conclusions: We established that abnormal neurons from FCD patients retain expression of oRG cell markers suggesting a maturation defect in these neurons. Using our novel slice culture system, we show that changes in mTOR signaling affect the structure and function of oRG cells during development, suggesting a likely mechanism for the etiology of FCD. Funding: NIH/NINDS Research Re-entry Supplement; CURE Epilepsy Taking Flight Award