Table_1_Endothelial β-Catenin Deficiency Causes Blood-Brain Barrier Breakdown via Enhancing the Paracellular and Transcellular Permeability.DOCX

Disruption of the blood-brain barrier (BBB) causes or contributes to neuronal dysfunction and several central nervous system (CNS) disorders. Wnt/β-catenin signaling is essential for maintaining the integrity of the adult BBB in physiological and pathological conditions, including stroke. However, how the impairment of the endothelial Wnt/β-catenin signaling results in BBB breakdown remains unclear. Furthermore, the individual contributions of different BBB permeability-inducing mechanisms, including intercellular junction damage, endothelial transcytosis, and fenestration, remains unexplored. Here, we induced β-catenin endothelial-specific conditional knockout (ECKO) in adult mice and determined its impact on BBB permeability and the underlying mechanism. β-catenin ECKO reduced the levels of active β-catenin and the mRNA levels of Wnt target genes in mice, indicating downregulation of endothelial Wnt/β-catenin signaling. β-catenin ECKO mice displayed severe and widespread leakage of plasma IgG and albumin into the cerebral cortex, which was absent in wild-type controls. Mechanistically, both the paracellular and transcellular transport routes were disrupted in β-catenin ECKO mice. First, β-catenin ECKO reduced the tight junction protein levels and disrupted the intercellular junction ultrastructure in the brain endothelium. Second, β-catenin ECKO substantially increased the number of endothelial vesicles and caveolae-mediated transcytosis through downregulating Mfsd2a and upregulating caveolin-1 expression. Interestingly, fenestration and upregulated expression of the fenestration marker Plvap were not observed in β-catenin ECKO mice. Overall, our study reveals that endothelial Wnt/β-catenin signaling maintains adult BBB integrity via regulating the paracellular as well as transcellular permeability. These findings may have broad applications in understanding and treatment of CNS disorders involving BBB disruption.

血脑屏障（blood-brain barrier, BBB）的破坏可引发或加重神经元功能障碍与多种中枢神经系统（central nervous system, CNS）疾病。Wnt/β-连环蛋白信号通路（Wnt/β-catenin signaling）在生理及病理状态下（包括脑卒中）对维持成人BBB的完整性至关重要，但目前内皮Wnt/β-连环蛋白信号通路受损如何导致BBB破坏的具体机制仍不明确。此外，包括细胞间连接损伤、内皮转胞吞作用及窗孔形成在内的各类BBB通透性诱导机制，其各自的独立贡献尚未得到系统探究。本研究通过在成年小鼠中构建内皮特异性β-连环蛋白条件性敲除（endothelial-specific conditional knockout, ECKO）模型，探究其对BBB通透性的影响及潜在分子机制。结果显示，β-连环蛋白ECKO小鼠的活性β-连环蛋白水平及Wnt靶基因的mRNA表达量均显著降低，证实内皮Wnt/β-连环蛋白信号通路受到有效抑制。β-连环蛋白ECKO小鼠表现出严重且广泛的血浆IgG与白蛋白渗漏进入大脑皮层的表型，而野生型对照组未观察到此类渗漏现象。机制层面，β-连环蛋白ECKO小鼠的细胞旁转运与转细胞转运通路均遭到破坏：其一，β-连环蛋白ECKO可降低脑内皮细胞的紧密连接蛋白水平，并破坏其细胞间连接的超微结构；其二，β-连环蛋白ECKO通过下调Mfsd2a的表达、上调小窝蛋白-1（caveolin-1）的表达，显著增加了内皮囊泡数量及小窝介导的转胞吞作用水平。值得注意的是，β-连环蛋白ECKO小鼠未观察到窗孔形成及窗孔标记物Plvap表达上调的情况。综上，本研究揭示内皮Wnt/β-连环蛋白信号通路可通过调控细胞旁及转细胞通透性来维持成人BBB的完整性，上述发现可为理解及治疗涉及BBB破坏的中枢神经系统疾病提供重要的理论依据与应用前景。