Single-Nucleus RNA Sequencing Reveals Ferroptosis as a Potential Contributor to the Pathogenesis of Focal Cortical Dysplasia

Objective: Focal cortical dysplasia (FCD) is a leading cause of drug-resistant epilepsy, while its molecular and cellular mechanisms remain poorly understood. This study aimed to characterize the cellular heterogeneity of FCD and investigate the function of ferroptosis in FCD pathogenesis.Methods: Single-nucleus RNA sequencing (snRNA-seq) was carried out on epileptogenic cortical tissues from 18 patients with FCD and 6 perilesional control samples with normal histology. Data were analyzed using Uniform Manifold Approximation and Projection (UMAP) for dimensionality reduction and visualization. Differentially expressed genes (DEGs) were identified and subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. UCell scoring and gene set enrichment analysis (GSEA) were applied to assess pathway activity. Expression levels of ferroptosis-related genes (FRGs) were validated by immunofluorescence, and biochemical assays quantified the levels of superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA), and lipid peroxides (LPO) in frozen cortical tissues.Results: A total of 170,747 nuclei were profiled, resolving 5 major cell types, including inhibitory neurons, excitatory neurons, astrocytes, oligodendrocytes, and microglia. DEGs across these populations were significantly enriched in ferroptosis and oxidative stress-associated pathways. UCell and GSEA highlighted remarkable alterations in ferroptosis, apoptosis, and oxidative stress, particularly in inhibitory neurons and astrocytes. Immunofluorescence confirmed upregulation of key FRGs, including FTL, FTH1, PCBP1, MAP1LC3B, and PRNP, in FCD tissues. Concordantly, biochemical assays demonstrated reduced SOD and GSH levels, alongside elevated MDA and LPO levels, confirming the transcriptional and histological findings.Conclusions: These results indicate that ferroptosis may be an important participant or a concurrent mechanism in the pathogenesis of FCD, potentially contributing to cellular damage and epileptogenesis. Our comprehensive analysis suggests that targeting ferroptosis-related pathways may hold promise as a potential therapeutic strategy for FCD, offering new insights into the molecular basis of this condition.