Cuproptosis and Immune Microenvironment Interplay in Temporal Lobe Epilepsy: Identification of Key Molecular Signatures and Therapeutic Targets

Background: Temporal lobe epilepsy (TLE), the predominant drug-resistant focal epilepsy, involves neuroinflammation and neuronal hyperexcitability. Cuproptosis-a copper-dependent cell death pathway triggered by mitochondrial copper overload and lipoylated protein aggregation-remains unexplored in epilepsy. This study investigates its molecular basis, neuroinflammatory crosstalk, and therapeutic implications in TLE. Methods: Bulk RNA-seq and single-cell RNA-seq datasets from GEO were analyzed using weighted gene co-expression network analysis (WGCNA) and consensus clustering to stratify cuproptosis-associated TLE subtypes. Ten machine learning algorithms identified hub genes linked to cuproptosis-immune crosstalk. Experimental validation in a pilocarpine-induced TLE mouse model confirmed gene expression changes via Western blot and immunohistochemistry. Results: Two TLE subtypes were stratified: cuproptosis-related gene (CRG) -high with upregulated cuproptosis drivers, heightened macrophage/T-cell infiltration, and NF-κB-mediated neuroinflammation, and CRG-low exhibiting disrupted copper homeostasis. Hub genes (CD44, PDE5A, TUBA1A) linked cuproptosis to astrocyte-driven immune interactions, endothelial dysfunction, and neuronal stress. Single-cell analysis localized CD44 to astrocytes interacting with microglia, while PDE5A and TUBA1A correlated with blood-brain barrier leakage and neuronal hyperexcitability. Experimental validation confirmed decreased CD44 and elevated PDE5A/TUBA1A in TLE mice, aligning with seizure severity. Conclusion: This study firstly establishes cuproptosis as a mechanistic bridge between copper dysregulation and TLE pathology, driving neuroinflammation via NF-κB and neuronal-glial dysfunction. The CRG-based subtyping offers novel disease classification, while CD44, PDE5A, and TUBA1A emerge as therapeutic targets to mitigate copper-mediated neurotoxicity. These findings reposition cuproptosis as a key pathway in epilepsy, providing a roadmap for precision therapy in drug-resistant TLE.