The role of nucleo-cytoskeletal link proteins in skeletal muscle

核细胞骨架连接蛋白在骨骼肌中的作用

基本信息

批准号：
8003272
负责人：
Ju Chen
金额：
$ 34.76万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-07-01 至 2015-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8003272
关键词：
Ablation Address Adult Age-Months Alleles Binding Biochemical Biological Process Biology Body Weight C-terminal Cardiac Cell Nucleus Cells Congenital Heart Defects Contracture Cytoskeleton Data Defect Desmin Emery-Dreifuss Muscular Dystrophy Exercise Exhibits Family Gene Expression Genes Genetically Engineered Mouse Goals In Vitro Intermediate Filaments Kinetics Knock-out Knockout Mice Lamin Type A Light Link Mechanics Membrane Proteins Microfilaments Modeling Molecular Mus Muscle Muscle Fibers Muscle Weakness Muscle function Muscular Dystrophies Mutant Strains Mice Mutation Myoblasts Myopathy N-terminal Nuclear Nuclear Envelope Nuclear Inner Membrane Nuclear Lamina Nuclear Outer Membrane Null Lymphocytes Organism Pathogenesis Patients Perinatal Phenotype Physiological Positioning Attribute Protamines Protein Family Proteins Respiratory Failure Role Signal Transduction Skeletal Muscle Skeletal Myoblasts Spectrin Stretching Survivors Synapses Tamoxifen Testing calponin emerin improved in vivo insight link protein loss of function member mutant public health relevance research study retinal rods satellite cell

项目摘要

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. PUBLIC HEALTH RELEVANCE: Mutations in Nesprin 1 and 2 may be involved in the pathogenesis of Emery-Dreifuss muscular dystrophy (EDMD). The proposed studies will help us to understand the biological function of Nesprin 1 and 2 in skeletal muscle at molecular, cellular, and physiological levels and to gain insight into mechanisms by which mutations in Nesprin 1 and 2 are involved in the pathogenesis of EDMD, thereby improving our general understanding of EDMD and other types of muscular dystrophy. In addition, the mouse lines will be useful as test models for potential therapies.

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 Emery-Dreifuss 肌营养不良症 (EDMD) 的发病机制有关，其特征是进行性骨骼肌无力并伴有肌肉挛缩和各种心脏缺陷。 40% 的 EDMD 患者已被证明存在 Emerin 或 Lamin A/C 突变，这两个基因分别编码位于内核膜 (INM) 及其底层的蛋白质。 INM、外核膜 (ONM) 和核纤层构成核膜，核膜通过 SUN 和 Nesprin 蛋白家族的成员与细胞骨架相连。大约 60% 的 EDMD 患者在 Emerin 或 LMNA 中没有突变。有趣的是，Nesprin 1 和 2 的突变与 EDMD 相关。 Nesprin 属于新发现的哺乳动物血影蛋白重复蛋白家族。缺乏核纤层蛋白 A/C 的小鼠表现出 EDMD 的特征。对从这些小鼠中分离出的骨骼肌培养物进行的研究表明，Lamin A/C 在骨骼肌成肌细胞分化和通过维持核细胞骨骼完整性来维持机械刚度方面发挥着关键作用。相比之下，Emerin 基因敲除小鼠不表现出 EDMD。然而，在 Emerin 缺失小鼠的骨骼肌培养物中，成肌细胞分化受到干扰的程度与在 Lamin A/C 缺失细胞中观察到的程度相同。此外，尽管 Emerin 无效细胞没有像 Lamin A/C 无效细胞那样表现出机械刚度降低，但观察到与机械转导相关的基因表达的扰动。 Nesprin 1 和 2 均普遍表达。为了研究 Nesprin 1 和 2 的功能作用，我们生成了 Nesprin 1 和 2 的 floxed 等位基因。通过利用鱼精蛋白 Cre 小鼠，我们生成了 Nesprin 1 和 2 的整体功能丧失突变体（Nesprin 1-/- 和 Nesprin 2） -/-)。我们的研究表明，Nesprin 2-/- 小鼠能够存活，并且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，运动能力下降。我们还发现 Nesprin 1-/-；Nesprin 2-/- 双突变小鼠的围产期死亡率为 100%。 Nesprin 1-/- 小鼠和 Nesprin 1-/-;Nesprin 2-/- 小鼠的组织学分析显示非突触核定位异常和突触核簇消失。该提案的总体目标是检验 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、机械传导和肌肉功能中具有不同且重叠的作用的假设。我们将通过对我们现有的四个基因工程小鼠品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。结果将揭示 Nesprin 突变导致肌病的机制。公共卫生相关性：Nesprin 1 和 2 的突变可能与 Emery-Dreifuss 肌营养不良症 (EDMD) 的发病机制有关。拟议的研究将帮助我们在分子、细胞和生理水平上了解 Nesprin 1 和 2 在骨骼肌中的生物学功能，并深入了解 Nesprin 1 和 2 的突变参与 EDMD 发病的机制，从而提高我们对 EDMD 和其他类型肌营养不良症的总体了解。此外，小鼠品系可用作潜在疗法的测试模型。