Golgi-associated NO-cGMP Signaling Defect in Muscular Dystrophy

描述(由申请人提供)：Duchenne肌营养不良症的机制通常归因于由于细胞外基质和皮质肌动蛋白细胞骨架之间的连接中断而导致的细胞膜脆性。Dystrophin及其相关蛋白的这种结构作用得到了大量证据的支持。此外，肌营养不良蛋白复合体是肌膜信号蛋白的支架，包括神经元型一氧化氮合酶(nNOS5，肌肉特异性异构体)、几种激酶、水和离子通道以及其他蛋白质。在MDX小鼠肌肉中转基因表达nNOS可以改善营养不良的表型，这一证明引起了人们对该信号蛋白的关注。最近，我们发现这种酶的第二个亚型nNOS2定位在高尔基体上，甚至在营养不良的第一个表型迹象明显之前，它在这个位置的表达就在MDX肌肉中减少了。此外，我们还发现NO激活的可溶性鸟苷环化酶(SGC)和cGMP激活的蛋白激酶G(PKG)与nNOS2在高尔基体上共定位。同时缺乏nNOS5和nNOS2(KN2)的小鼠有许多缺陷，包括严重的肌病缺陷。基于这些初步结果，我们将从结构、生化和功能水平上对KN2小鼠的肌肉进行表征。此外，我们还将验证这样一种假设，即在骨骼肌中转基因表达nNOS2将挽救/改善KN2和MDX表型。NNOS2在钝化收缩肌肉中的1-肾上腺素能血管收缩中的作用将为该酶在缓解肌营养不良的一个组成部分功能性缺血中的重要性提供信息。最后，我们将确定NO/cGMP/PKG途径和直接蛋白质亚硝化的高尔基底物。最后，我们将确定调节(S)NO/cGMP/PKG通路的磷酸二酯酶(PDE)。对这一信号通路的全面了解将使我们能够更合理地选择治疗靶点，包括PDE抑制剂，它可以减缓肌肉退化的进展，改善营养不良的表型。 公共卫生相关性：这里提出的研究将提供有关肌肉营养不良症导致肌肉退化的机制的新信息。高尔基体是一种调节蛋白质修饰和运输到表面膜的细胞器，对新型信号机制的关注可能会揭示减缓肌肉退化进程的新的治疗靶点。总的来说，这个项目产生的知识将与许多类型的肌肉疾病相关。

DESCRIPTION (provided by applicant): The mechanism by which the loss of dystrophin leads to Duchenne Muscular Dystrophy is generally attributed to membrane fragility due to disruption of the linkage between the extracellular matrix and the cortical actin cytoskeleton. This structural role for dystrophin and its associated proteins is supported by substantial evidence. In addition, the dystrophin complex serves as a sarcolemmal scaffold for signaling proteins, including neuronal nitric oxide synthase (nNOS5, the muscle-specific isoform), several kinases, water and ion channels and other proteins. The demonstration that transgenic expression of nNOS in mdx mouse muscle ameliorates the dystrophic phenotype has focused attention on this signaling protein. Recently, we have found that a second isoform of this enzyme, nNOS2, is localized on the Golgi, and that its expression at this site is reduced in mdx muscle, even before the first phenotypic signs of dystrophy are evident. Furthermore, we find that soluble guanylate cyclase (sGC), which is activated by NO, and cGMP-activated protein kinase G (PKG) are colocalized with nNOS2 on the Golgi. Mice lacking both nNOS5 and nNOS2 (KN2) have numerous deficiencies, including a severe myopathic defect. Based on these preliminary results, we will characterize muscle from KN2 mice at the structural, biochemical and functional levels. In addition, we will test the hypothesis that transgenic expression of nNOS2 in skeletal muscle will rescue/ameliorate the KN2 and mdx phenotypes. The role of nNOS2 in blunting 1-adrenergic vasoconstriction in contracting muscle will provide information on the importance of this enzyme in alleviating functional ischemia, a component of muscular dystrophy. Finally, we will determine the Golgi substrates of the NO/cGMP/PKG pathway and of direct protein nitrosylation. Finally, we will identify the phosphodiesterase(s) (PDEs) that regulate(s) the NO/cGMP/PKG pathway. A complete understanding of this signaling pathway will enable a more rationale approach to selecting therapeutic targets, including PDE inhibitors, which slow the progression of muscle degeneration and ameliorate the dystrophic phenotype. PUBLIC HEALTH RELEVANCE: The research proposed here will provide new information about the mechanisms that lead to muscle degeneration in muscular dystrophies. The focus on a novel signaling mechanism at the Golgi, a cellular organelle that regulates protein modification and trafficking to the surface membrane, may reveal new therapeutic targets for slowing the progress of muscle degeneration. In general, the knowledge generated by this project will be relevant to many types of muscle diseases.