BBB proteomic analysis reveals that complex febrile seizures in infancy enhance susceptibility to epilepsy in adulthood through dysregulation of ECM-receptor interaction signaling pathway

BACKGROUND: Complex febrile seizures (CFS) have been associated with an increased risk of epilepsy in adulthood. However, the specific link between blood-brain barrier (BBB) and the predisposition to epilepsy in adults who experienced CFS during infancy remains unclear. The objective of this study was to investigate the alteration of BBB in adult mice who had experienced CFS during infancy, and to explore the mechanisms of increased susceptibility to epilepsy after CFS. METHODS: The CFS pup model was induced using hot air, and the seizure susceptibility was examined using low-dose pentylenetetrazole (PTZ) after 8 W. The brain microvessels representing BBB function were isolated and their protein expression changes were analyzed using data-independent acquisition (DIA) proteomic techniques. Subsequently, the bioinformatic analyses were performed using ClusterProfiler, STRING, Gene Set Enrichment Analysis (GSEA), etc. The enriched pathways, changes in the expression of BBB-related proteins, and alterations in metabolites including certain neurotransmitters were subsequently validated by Western Blotting, quantitative real-time polymerase chain reaction (qRT-PCR), and mass spectrometric imaging (MSI). In addition, we selected the MMP inhibitor Incyclinide to verify that dysregulation of the ECM-receptor interaction signaling pathway increases epilepsy susceptibility in adult mice. RESULTS: Mice that experienced CFS in infancy show increased susceptibility to epilepsy in adulthood, and BBB proteomic profile was significantly altered in the CFS mice. The network analysis suggests that dysregulation of the extracellular matrix (ECM)-receptor interaction pathway is a key mechanism. Moreover, MSI analysis uncovered notable changes in differential metabolites, including amino acids and nucleotide-derived neurotransmitters associated with the function of BBB maintaining neuronal homeostasis. Subsequent validation experiments showed that dysregulation of the ECM-receptor interaction signaling pathway exacerbated epilepsy susceptibility in adult mice. CONCLUSION: Our research represents the pioneering demonstration of the modified BBB proteomics associated with epilepsy susceptibility in adult mice previously exposed to CFS in infancy. Notably, the increased susceptibility is attributed to the dysregulation of the ECM-receptor interaction pathway. These findings may help to elucidate the role of BBB alterations in the progression of epilepsy susceptibility, and provide new orientations for subsequent prevention and treatment of epilepsy.