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，内斯普林1和2中的突变可能参与了emery-dreifuss肌肉营养不良（EDMD）的发病机理，该发病机理的特征是渐进的骨骼肌无力与相关的肌肉相关的肌肉缩减和可变心脏缺陷。 EDMD患者中有40％的患者在Emerin或lamin A/C中具有突变，这是编码局部核膜（INM）及其基础层状蛋白的两个基因。 INM，外核膜（ONM）和核薄片构成核包膜，核包膜与太阳和内斯普林蛋白家族的成员都与细胞骨架有关。大约60％的EDMD患者在Emerin或LMNA中没有突变。有趣的是，Nesprin 1和2中的突变与EDMD有关。 Nesprins属于新发现的哺乳动物谱蛋白重复蛋白的家族。缺乏层lamin A/C的小鼠EDMD展示了特征。从这些小鼠中分离出的骨骼肌培养物的研究表明，通过保持核细胞骨骼完整性，通过维持骨骼肌细胞分化和机械刚度在机械刚度中起着关键作用。相比之下，Emerin敲除小鼠不表现出EDMD。然而，在来自烯烃无效小鼠的骨骼肌培养物中，成肌细胞分化的扰动程度与在层粘连蛋白A/C无效细胞中观察到的程度相同。此外，尽管在层粘连蛋白A/C零细胞中发现的烯烃无效细胞没有表现出降低的机械刚度，但观察到与机械传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，我们为Nesprin 1和2产生了flox的等位基因。通过利用精蛋白CRE小鼠，我们为Nesprin 1和2（Nesprin 1 - / - 和Nesprin 2-和Nesprin 2 - / - ）产生了全局功能突变体损失。我们的研究表明，Nesprin 2 - / - 小鼠是可行的，没有明显的基础表型，而Nesprin 1 - / - 小鼠约有60％的围产期死亡。其余的幸存者减轻了体重和锻炼能力。我们还发现Nesprin 1 - / - ; Nesprin 2 - / - 双重突变小鼠中的100％围产期致死性。 Nesprin 1 - / - 小鼠和Nesprin 1 - / - ; Nesprin 2 - / - 小鼠的组织学分析显示非突触核的异常定位以及突触核的簇消失。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，骨骼成肌细胞分化，机械刚度，机械转换和肌肉功能中具有独特且重叠的作用的假设。我们将通过在我们现有的四种基因工程小鼠系中对骨骼肌表型的全面分子，生化，组织学和生理分析实现这些目标。结果将阐明内斯普林突变会导致肌病的机制。公共卫生相关性：Nesprin 1和2中的突变可能参与Emery-Dreifuss肌肉营养不良（EDMD）的发病机理。拟议的研究将帮助我们了解Nesprin 1和2在分子，细胞和生理水平下骨骼肌中的生物学功能，并深入了解Nesprin 1和2中的Nesprin 1和2中的突变参与EDMD的发病机理，从而改善了我们对EDMD和其他类型的肌肉性肌肉的一般理解。此外，小鼠系将作为潜在疗法的测试模型有用。