Beard Richard S, Hoettels Brian A, McAllister Jessica M, Meegan Jamie E, Wertz Travis S, Self Desiree A, Hrkach Dylan E, Greiner Daniel, Chapman Kristina, Villalba Nuria, Yang Xiaoyuan, Cha Byeong J, Jorcyk Cheryl L, Oxford Julia T, Wu Mack H, Yuan Sarah Y
Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida, Tampa, FL, USA.
Department of Biological Sciences, Boise State University, Boise, ID, USA.
J Cereb Blood Flow Metab. 2025 Feb 7:271678X251318620. doi: 10.1177/0271678X251318620.
Blood-brain barrier (BBB) dysfunction occurs in numerous central nervous system disorders. Unfortunately, a limited understanding of the mechanisms governing barrier function hinders the identification and assessment of BBB-targeted therapies. Previously, we found that non-muscle myosin light chain kinase (nmMLCK) negatively regulates the tight junction protein claudin-5 in brain microvascular endothelial cells (BMVECs) under inflammatory conditions. Here, we used complementary animal and primary cell co-culture models to further investigate nmMLCK and claudin-5 during neuroinflammation. We found that -knockout mice resisted experimental autoimmune encephalomyelitis (EAE), including paralysis, demyelination, neutrophil infiltration, and BBB dysfunction. However, transiently silencing claudin-5 culminated in a fulminant disease course. In parallel, we found that neutrophil-secreted factors triggered a biphasic loss in the barrier quality of wild-type BMVEC monolayers, plus pronounced neutrophil migration during the second phase. Conversely, -knockout monolayers resisted barrier dysfunction and neutrophil migration. Lastly, we found an inverse relationship between claudin-5 expression in BMVECs and neutrophil migration. Overall, our findings support a pathogenic role for nmMLCK in BMVECs during EAE that includes BBB dysfunction and neutrophil infiltration, reveal that claudin-5 contributes to the immune barrier properties of BMVECs, and underscore the harmful effects of claudin-5 loss during neuroinflammation.
血脑屏障（BBB）功能障碍在众多中枢神经系统疾病中都会出现。遗憾的是，对调控屏障功能机制的了解有限，这阻碍了针对血脑屏障的治疗方法的识别和评估。此前，我们发现非肌肉肌球蛋白轻链激酶（nmMLCK）在炎症条件下对脑微血管内皮细胞（BMVECs）中的紧密连接蛋白claudin-5起负调控作用。在此，我们使用互补的动物和原代细胞共培养模型，在神经炎症过程中进一步研究nmMLCK和claudin-5。我们发现，敲除小鼠能抵抗实验性自身免疫性脑脊髓炎（EAE），包括瘫痪、脱髓鞘、中性粒细胞浸润和血脑屏障功能障碍。然而，短暂沉默claudin-5会导致病情迅速发展。同时，我们发现中性粒细胞分泌的因子会引发野生型BMVEC单层细胞屏障质量的双相性丧失，且在第二阶段会出现明显的中性粒细胞迁移。相反，敲除单层细胞能抵抗屏障功能障碍和中性粒细胞迁移。最后，我们发现BMVECs中claudin-5的表达与中性粒细胞迁移呈负相关。总体而言，我们的研究结果支持nmMLCK在EAE期间BMVECs中的致病作用，包括血脑屏障功能障碍和中性粒细胞浸润，揭示claudin-5有助于BMVECs的免疫屏障特性，并强调神经炎症期间claudin-5缺失的有害影响。
