Cerebral Compression Disrupts the Blood–Brain Barrier via Claudin-5 Ubiquitination in the Rat Somatosensory Cortex

studies implicating phosphorylation, ubiquitination, and redistribution of endothelial tight junction proteins in BBB disruption, we hypothesized that similar mechanisms contribute to BBB breakdown in our animal model of epidural compression. Immunoblotting showed that the expression levels of claudin-5, occludin, and JAM-1 were unchanged at 3 days post-compression, although we previously reported that BBB disruption was evident at this time point. Antioxidant treatment selectively increased claudin-5 expression, whereas occludin and JAM-1 levels remained unchanged. Immunoprecipitation and subcellular fractionation analyses demonstrated that 3-day compression markedly increased the phosphorylation and ubiquitination of claudin-5 in both cytosolic and membrane fractions of endothelial cells. Ascorbate not only prevented claudin-5 phosphorylation and ubiquitination but also increased its abundance in the membrane fraction. Epsin1, Eps15, and Hrs are adaptor proteins involved in endocytosis and intracellular trafficking of membrane proteins. Accordingly, we examined their interactions with claudin-5 and found that these interactions were significantly enhanced following compression. Ascorbate attenuated the interactions of claudin-5 with Epsin1 and Eps15, but not with Hrs. Collectively, these findings indicate that epidural compression disrupts BBB primarily by inducing phosphorylation, ubiquitination, and redistribution of claudin-5. This study highlights the therapeutic potential of targeting superoxide during the early phase of cortical compression, particularly under conditions associated with BBB compromise.