Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Human Anatomy and Histology, University of Bari Medical School, Bari, Italy.
Lab Invest. 2013 May;93(5):592-610. doi: 10.1038/labinvest.2013.46. Epub 2013 Mar 25.
The mdx mouse, the most widely used animal model of Duchenne muscular dystrophy (DMD), develops a seriously impaired blood-brain barrier (BBB). As glucocorticoids are used clinically to delay the progression of DMD, we evaluated the effects of chronic treatment with α-methyl-prednisolone (PDN) on the expression of structural proteins and markers in the brain and skeletal muscle of the mdx mouse. We analyzed the immunocytochemical and biochemical expression of four BBB markers, including endothelial ZO-1 and occludin, desmin in pericytes, and glial fibrillary acidic protein (GFAP) in glial cells, and the expression of the short dystrophin isoform Dp 71, the dystrophin-associated proteins (DAPs), and aquaporin-4 (AQP4) and α-β dystroglycan (DG) in the brain. We evaluated the BBB integrity of mdx and PDN-treated mdx mice by means of intravascular injection of horseradish peroxidase (HRP). The expression of DAPs was also assessed in gastrocnemius muscles and correlated with utrophin expression, and laminin content was measured in the muscle and brain. PDN treatment induced a significant increase in the mRNA and protein content of the BBB markers; a reduction in the phosphorylation of occludin in the brain and of AQP4/β DG in both tissues; an increase of Dp71 protein content; and an increase of both mRNA and protein levels of the AQP4/α-β DG complex. The latter was associated with enhanced laminin and utrophin in the muscle. The HRP assay demonstrated functional restoration of the BBB in the PDN-treated mdx mice. Specifically, mdx mice showed extensive perivascular labeling due to escape of the marker, while HRP was exclusively intravascular in the PDN-treated mice and the controls. These data illustrate for the first time that PDN reverses the BBB alterations in the mdx mouse and re-establishes the proper expression and phosphorylation of β-DG in both the BBB and skeletal muscle. Further, PDN partially protects against muscle damage. The reduction in AQP4 and occludin phosphorylation, coupled with their anchoring to glial and endothelial membranes in PDN-treated mice, suggests that the drug may target the glial and endothelial cells. Our results suggest a novel mechanism for PDN action on cerebral and muscular function, restoring the link between DAPs and the extracellular matrix, most likely through protein kinase inactivation.
mdx 小鼠是杜氏肌营养不良症（DMD）最广泛使用的动物模型，其血脑屏障（BBB）严重受损。由于糖皮质激素临床上用于延缓 DMD 的进展，我们评估了慢性使用α-甲基泼尼松龙（PDN）对 mdx 小鼠脑和骨骼肌中结构蛋白和标志物表达的影响。我们分析了四种 BBB 标志物的免疫细胞化学和生化表达，包括内皮细胞紧密连接蛋白 ZO-1 和 occludin、周细胞中的结蛋白和神经胶质细胞中的胶质纤维酸性蛋白（GFAP），以及短肌营养不良蛋白同工型 Dp71、肌营养不良蛋白相关蛋白（DAPs）和水通道蛋白-4（AQP4）和α-β 二聚糖（DG）在大脑中的表达。我们通过静脉内注射辣根过氧化物酶（HRP）评估了 mdx 和 PDN 处理的 mdx 小鼠的 BBB 完整性。还评估了 DAPs 在比目鱼肌中的表达，并与 utrophin 表达相关，同时测量了肌肉和大脑中的层粘连蛋白含量。PDN 治疗诱导 BBB 标志物的 mRNA 和蛋白含量显著增加；大脑中 occludin 和两种组织中 AQP4/βDG 的磷酸化减少；Dp71 蛋白含量增加；以及 AQP4/α-βDG 复合物的 mRNA 和蛋白水平增加。后者与肌肉中层粘连蛋白和 utrophin 的增加有关。HRP 测定表明 PDN 处理的 mdx 小鼠 BBB 功能得到恢复。具体而言，mdx 小鼠由于标记物逃逸而出现广泛的血管周围标记，而 PDN 处理的小鼠和对照组中 HRP 仅为血管内。这些数据首次表明，PDN 可逆转 mdx 小鼠的 BBB 改变，并在 BBB 和骨骼肌中重新建立β-DG 的适当表达和磷酸化。此外，PDN 部分保护肌肉免受损伤。AQP4 和 occludin 磷酸化减少，以及它们在 PDN 处理的小鼠中与神经胶质和内皮膜结合，表明该药物可能靶向神经胶质和内皮细胞。我们的结果表明了 PDN 对大脑和肌肉功能的作用的新机制，通过蛋白激酶失活恢复 DAPs 与细胞外基质之间的联系。
