Paired Regional Electrophysiologic Mapping and Single-nucleus RNA Sequencing in Focal Epilepsies Reveal Distinct Cellular and Molecular Characteristics in the Seizure Focus and Penumbra
Ashwin Viswanathan1, Molly Murch2, Abby Brand3, Julia Furnari4, Archana Yadav2, Nathaniel Rolfe1, Clara Stucke4, Aayushi Mahajan4, Juncheng Li3, Osama Al-Dalahmah3, Jeffrey Bruce4, Brian Gill4, Brett Youngerman4, Neil Feldstein4, Guy McKhann4, Peter Canoll3, Catherine Schevon2, Vilas Menon2, Melodie Winawer2
1Columbia University Vagelos College of Physicians and Surgeons, 2Department of Neurology, 3Department of Pathology and Cell Biology, 4Department of Neurological Surgery, Columbia University Irving Medical Center
Objective:
To determine cellular and transcriptional features of the seizure focus and penumbra in patients undergoing surgical resection for intractable focal epilepsy.
Background:
While the electrophysiologic features of focal epilepsy are actively used clinically to guide surgical resections, studies have yet to probe underlying tissue features. The seizure focus is defined by homogenous synchronous neuronal bursting, whereas the penumbra exhibits excitatory synaptic spread without local neuronal bursting due to intact inhibition. Directly probing tissue-based alterations in these distinct regions may provide advances in understanding ictogenesis and refining therapeutic approaches for focal epilepsies.
Design/Methods:
We developed a novel electrographically-guided, MRI-localized approach to sample paired biopsies from 12 patients with diverse focal epilepsy etiologies undergoing resective surgery as epilepsy treatment. Regions were defined using pre-operative invasive stereo-EEG, and tissue was analyzed with single-nucleus RNA sequencing (snRNASeq) and immunohistochemistry (IHC). Differential gene expression, gene set enrichment analysis (GSEA), and single-cell hierarchical Poisson factorization (scHPF) analyses were used to identify region-specific cellular and transcriptional features.
Results:
snRNAseq revealed differentially increased microglial abundance and reduced proportion of PV+ interneurons within the seizure focus without a corresponding change in total neuronal abundance, findings which were validated via IHC. We also identified a relative decrease in RORB+ pyramidal neurons and VIP+ interneurons in the seizure focus. GSEA and scHPF analyses revealed elevated expression of gene signatures associated with neuronal plasticity, synapse formation, and axonogenesis in both excitatory and inhibitory neurons within the penumbra, relative to the focus.
Conclusions:
Our findings demonstrate distinct features in cellular composition and transcriptional programs present in the seizure focus and ictal penumbra across focal epilepsy etiologies. The enrichment of plasticity-associated gene signatures in the penumbra suggests region-specific neuronal alterations that play a role in ictogenesis and seizure spread. These findings provide grounds for future work targeting alterations to synaptic plasticity as a strategy to treat focal epilepsies.
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