CX3CL1/CX3CR1 axis dysregulation contributes to epileptogenic mechanisms in focal cortical dysplasia

Abstract Focal cortical dysplasia (FCD) is a predominant cause of drug-resistant epilepsy in children, yet the neuro-immune mechanisms underlying this condition are not well understood. Through transcriptomic screening combined with machine learning approaches (LASSO, SVM-RFE, and RF) and weighted gene co-expression network analysis (WGCNA), we implicated CX3CR1 in FCD-associated epileptogenesis, identifying it as a candidate diagnostic marker. Single-cell RNA sequencing revealed a distinct cellular distribution, with CX3CR1 enriched in microglia and its ligand, CX3CL1, enriched in interneurons within FCD lesions. Consistent with this, RT-qPCR, western blot, and immunohistochemical analyses confirmed significant upregulation of the CX3CL1/CX3CR1 axis in human FCD subtypes (Ia, IIa, and IIb) compared to non-epileptic controls. To establish causality, we investigated this axis in a neonatal rat model of FCD. We found that abnormal CX3CL1 and CX3CR1 expression was associated with core pathological features: cortical malformation, microglial polarization, and increased seizure susceptibility. Critically, direct overexpression of secretory CX3CL1 (sCX3CL1) in rats was sufficient to recapitulate spontaneous epileptiform discharges, reduce seizure latency, and prolong seizure duration. Taken together, our findings reveal sCX3CL1 as a pivotal epileptogenic factor and underscore the CX3CL1/CX3CR1 axis as a promising target for therapeutic intervention in FCD-related epilepsy, offering potential for novel treatment strategies aimed at mitigating this challenging condition.