The role of nucleo-cytoskeletal link proteins in skeletal muscle

描述（由申请人提供）：最近的数据表明，Nesprin 1和2的突变可能与emry - dreifuss肌营养不良（EDMD）的发病机制有关，EDMD的特征是进行性骨骼肌无力伴相关肌肉挛缩和可变心脏缺陷。40%的EDMD患者有Emerin或Lamin A/C突变，这两个基因分别编码内膜（INM）及其底层层的蛋白质。INM、外核膜（ONM）和核层组成核膜，它通过SUN和Nesprin蛋白家族的成员与细胞骨架相连。大约60%的EDMD患者没有Emerin或LMNA突变。有趣的是，Nesprin 1和2的突变与EDMD有关。Nesprins属于一个新发现的哺乳动物幽灵重复蛋白家族。缺乏Lamin A/C的小鼠表现出EDMD的特征。从这些小鼠中分离的骨骼肌培养物的研究表明，Lamin a /C在骨骼肌成肌细胞分化和通过维持核-细胞骨架完整性来维持机械刚度方面发挥了关键作用。相比之下，Emerin敲除小鼠不表现出EDMD。然而，在Emerin缺失小鼠的骨骼肌培养物中，成肌细胞分化受到与Lamin A/C缺失细胞相同程度的干扰。此外，尽管Emerin无细胞不像Lamin A/C无细胞那样表现出机械刚度的降低，但观察到与机械转导相关的基因表达的扰动。nesprin1和nesprin2都是普遍表达的。为了研究Nesprin 1和Nesprin 2的功能作用，我们为Nesprin 1和Nesprin 2生成了floxed等位基因。通过使用鱼精蛋白Cre小鼠，我们产生了Nesprin 1和Nesprin 2 （Nesprin 1-/-和Nesprin 2-/-）的全局功能丧失突变体。我们的研究表明，Nesprin 2-/-小鼠是可存活的，没有明显的基础表型，而大约60%的Nesprin 1-/-小鼠在围产期死亡。剩下的幸存者体重减轻，运动能力受损。我们还发现Nesprin 1-/-的围产期死亡率为100%；Nesprin 2-/-双突变小鼠。Nesprin 1-/-小鼠与Nesprin 1-/-的组织学分析Nesprin 2-/-小鼠显示非突触核定位异常，突触核簇消失。本提案的总体目标是验证Nesprin 1和2在骨骼肌核定位、核膜完整性、骨骼肌成肌细胞分化、机械刚度、机械转导和肌肉功能中具有不同且重叠作用的假设。我们将通过对现有四种基因工程小鼠系的骨骼肌表型进行全面的分子、生化、组织学和生理学分析来实现这些目标。结果将阐明Nesprin突变导致肌病的机制。

DESCRIPTION (provided by applicant): Recent data suggest that mutations in Nesprin 1 and 2 may be involved in the pathogenesis of Emery-Dreifuss muscular dystrophy (EDMD), which is characterized by progressive skeletal muscle weakness with associated muscle contractures, and variable cardiac defects. 40% of patients with EDMD have been shown to have mutations in Emerin or Lamin A/C, two genes encoding proteins localized to the inner nuclear membrane (INM) and its underlying lamina, respectively. The INM, the outer nuclear membrane (ONM), and the nuclear lamina comprise the nuclear envelope, which is linked to the cytokeleton by members of both the SUN and Nesprin protein families. Approximately 60% of EDMD patients do not have mutations in either Emerin or LMNA. Intriguingly, mutations in Nesprin 1 and 2 have been associated with EDMD. Nesprins belong to a newly discovered family of mammalian spectrin-repeat proteins. Mice lacking Lamin A/C exhibit features of EDMD. Studies on skeletal muscle cultures isolated from these mice have demonstrated a critical role for Lamin A/C in skeletal myoblast differentiation and in mechanical stiffness by maintaining nucleo-cytoskeletal integrity. In contrast, Emerin knockout mice do not exhibit EDMD. However, in skeletal muscle cultures from Emerin null mice, myoblast differentiation is perturbed to the same extent as observed in Lamin A/C null cells. Moreover, although Emerin null cells do not exhibit decreased mechanical stiffness as found in Lamin A/C null cells, perturbations in gene expression associated with mechanotransduction are observed. Both Nesprin 1 and 2 are ubiquitously expressed. To investigate the functional roles of Nesprin 1 and 2, we have generated floxed alleles for Nesprin 1 and 2. By utilizing protamine Cre mice, we have generated global loss of function mutants for Nesprin 1 and 2 (Nesprin 1-/- and Nesprin 2-/-). Our studies reveal that Nesprin 2-/- mice are viable and have no obvious basal phenotype, whereas approximately 60% of Nesprin 1-/- mice die perinatally. Remaining survivors have reduced body weight and compromised exercise capacity. We also found 100% perinatal lethality in Nesprin 1-/-;Nesprin 2-/- double mutant mice. Histological analyses of Nesprin 1-/- mice and Nesprin 1-/-;Nesprin 2-/- mice revealed abnormal positioning of non-synaptic nuclei and disappearance of clusters of synaptic nuclei. The overall goals of this proposal are to test the hypothesis that Nesprin 1 and 2 have distinct and overlapping roles in skeletal muscle nuclear positioning, nuclear membrane integrity, skeletal myoblast differentiation, mechanical stiffness, mechano-transduction, and muscle function. We will achieve these goals by comprehensive molecular, biochemical, histological, and physiological analysis of the skeletal muscle phenotypes in our four existing genetically engineered mouse lines. Results will shed light into mechanisms by which mutations in Nesprin contribute to myopathies.