A Single-Cell Transcriptomic Atlas Elucidates a Microglial Gene Signature Linking Ferroptosis to Mitochondrial Dysfunction in Epilepsy

Background: Epilepsy is a chronic neurological disorder caused by abnormal synchronous discharges of neurons, with ferroptosis and mitochondrial dysfunction implicated in its progression. However, a lack of resolution at the level of specific cell types has obscured critical roles and molecular mechanisms within the epileptic microenvironment. Methods: This study integrated single-nucleus RNA sequencing (snRNA-seq) and bulk RNA-seq data from epilepsy and control samples. We performed dimensionality reduction and clustering to identify 13 cell subpopulations, and then assessed the expression of ferroptosis-related genes (FRGs) and mitochondrial-related genes (MRGs) within 7 major cell types. Three machine learning algorithms were further applied to identify key genes in microglia. Subsequent analyses included immune infiltration, pathway enrichment, and drug–target interaction. Molecular docking and molecular dynamics simulations were used to evaluate the potential binding affinity of the predicted drug. In vivo validation included histopathology, mitochondrial electron microscopy, JC-1 staining, and immunohistochemistry (IHC). Results: We identified six key genes ( ATM, TGFBR1, ZEB1, PARP14, GJA1 , and ALOX5 ), which link ferroptosis and mitochondrial dysfunction with a central role for microglia in epilepsy. These genes were associated with immune infiltration, including increased Th1 cell levels and reduced CD8+ T cell abundance. Enrichment analyses implicated these hub genes in orchestrating key epileptogenic processes, including neuroinflammatory pathways, ferroptosis, and interferon response. PARP14 was upregulated in epileptic rats, and GJA1 exhibited stable binding with bleomycin. Histopathological and mitochondrial assays confirmed the presence of ferroptosis and mitochondrial damage in epilepsy. Conclusion: This study highlights the interplay between ferroptosis and mitochondrial dysfunction in epilepsy, identifying six key microglial genes as high-priority candidates for future mechanistic and therapeutic investigation. Notably, PARP14 is implicated in epileptogenesis for the first time. The stable binding between GJA1 and bleomycin offers a novel avenue for epilepsy treatment, warranting further experimental validation. Keywords: epilepsy, ferroptosis, mitochondrial dysfunction, microglia, single-cell transcriptome sequencing, PARP14