Cavß3 Contributes to the Maintenance of the Blood-Brain Barrier and Alleviates Symptoms of Experimental Autoimmune Encephalitis.

ABSTRACT

BACKGROUND: Tight control of cytoplasmic Ca2+ in endothelial cells is essential for the regulation of endothelial barrier function. Here, we investigated the role of Cavß3, a subunit of voltage-gated Ca2+ (Cav) channels, in modulating Ca2+ signaling in brain microvascular endothelial cells (BMECs) and how this contributes to the integrity of the blood-brain barrier. METHODS: We investigated the function of Cavß3 in BMECs by Ca2+ imaging and Western blot, examined the endothelial barrier function in vitro and the integrity of the blood-brain barrier in vivo, and evaluated disease course after induction of experimental autoimmune encephalomyelitis in mice using Cavß3-/- (Cav ß3-deficient) mice as controls. RESULTS: We identified Cavß3 protein in BMECs, but electrophysiological recordings did not reveal significant Cav channel activity. In vivo, blood-brain barrier integrity was reduced in the absence of Cavß3. After induction of experimental autoimmune encephalomyelitis, Cavß3-/- mice showed earlier disease onset with exacerbated clinical disability and increased T-cell infiltration. In vitro, the transendothelial resistance of Cavß3-/- BMEC monolayers was lower than that of wild-type BMEC monolayers, and the organization of the junctional protein ZO-1 (zona occludens-1) was impaired. Thrombin stimulates inositol 1,4,5-trisphosphate-dependent Ca2+ release, which facilitates cell contraction and enhances endothelial barrier permeability via Ca2+-dependent phosphorylation of MLC (myosin light chain). These effects were more pronounced in Cavß3-/- than in wild-type BMECs, whereas the differences were abolished in the presence of the MLCK (MLC kinase) inhibitor ML-7. Expression of Cacnb3 cDNA in Cavß3-/- BMECs restored the wild-type phenotype. Coimmunoprecipitation and mass spectrometry demonstrated the association of Cavß3 with inositol 1,4,5-trisphosphate receptor proteins. CONCLUSIONS: Independent of its function as a subunit of Cav channels, Cavß3 interacts with the inositol 1,4,5-trisphosphate receptor and is involved in the tight control of cytoplasmic Ca2+ and Ca2+-dependent MLC phosphorylation in BMECs, and this role of Cavß3 in BMECs contributes to blood-brain barrier integrity and attenuates the severity of experimental autoimmune encephalomyelitis disease.