Abstracts

Rationale:
Mammalian Target of Rapamycin (mTOR) mediated cortical malformations like Focal Cortical Dysplasia (FCD), Hemimegalencephaly (HME) and Tuberous Sclerosis (TSC) cause medication-resistant pediatric epilepsy. While all three conditions arise due to somatic mutations in the mTOR signaling pathway, they each also have distinct neuroanatomical and etiologic features, that make it hard to examine the relationship between them. In order to understand the developmental origin and shared molecular features of these mTOR-linked epilepsies, we used single nucleus sequencing strategies in donated patient surgical specimens to characterize the transcriptomic identity, and molecular features of all cell types present within the malformations.
Method:
De-identified tissue from surgical resections are collected with previous patient consent for research in strict observance of legal & institutional ethical regulations. Brain regions with cortical lesions & epileptogenic foci are resected en-bloc, flash-frozen & sectioned to preserve cellular architecture. For single-nucleus sequencing, individual nuclei are isolated & barcoded according to standard protocols, followed by reverse-transcription, amplification & sequencing to generate single-nucleus expression profiles. The sequencing data is clustered using an unbiased clustering algorithm to group nuclei with similar transcriptomic profiles into a single cluster. Further bio-informatic analyses are used to compare identified cell types with cell clusters in published databases of human cortex, and to investigate their molecular signatures. Results30000 cells from patients with cortical malformations (n = 8 FCD, 3 TSC and 2 HME patients) were isolated and characterized. Using unbiased clustering approaches, generated molecularly distinct transcriptomic clusters of cells, corresponding to identifiable cell types. (1) By comparing with published transcriptomic databases, we identified transcriptomically unique cell types in FCDIIB, HME and TSC lesions that correspond to dysplastic balloon cells. These cells have a shared molecular identity irrespective of malformation type, co-expressing markers associated with progenitor cells and neurons, indicating a shared developmental origin. Using immunofluorescence staining, we validated our findings with marker co-expression in tissue sections. (2) We also identified transcriptomic changes in pyramidal neurons, interneurons and glial cells within the epileptic region, that may highlight the molecular and cellular responses to epilepsy in these cell types. With further validation, these datasets can be used to comprehensively explore disease phenotype & identify unique molecular pathways to investigate cellular responses, and as bio-markers for diagnosis and therapy.
Conclusion:
We identified key developmental signatures, normally present in progenitor cells within populations of balloon neurons present in FCD, HME and TSC lesions, highlighting shared molecular & developmental origins of this disease-associated cell type. We have also identified transcriptomic changes in pyramidal neurons, interneurons and glial cells, reflecting disease-relevant changes in the epileptic regions. The outcome of this study is a thorough transcriptomic characterization of all cell types in mTOR-linked epileptic cortical malformations, that will be an important community resource.
Funding:
:NIH/NINDS Re-entry Supplement, CURE Taking Flight Award & NARSAD Young Investigator Award to L.S.; NIH/NINDS K08 award to M.P. ; NIH/NINDS R35 grant NS097305 to A.R.K.