Molecular and Cellular Pathogenesis of Emery-Dreifuss Muscular Dystrophy

描述（由申请人提供）：本申请的总体目标是确定与Emery-Dreifuss肌营养不良（EDMD）相关的心肌病的分子发病机制。常染色体显性EDMD由编码a型层蛋白的基因突变引起，a型层蛋白是参与维持核结构的中间纤维。此外，a型层蛋白（以及sun和nesprins）是LINC复合物的一个组成部分，它在细胞核和肌动蛋白细胞骨架之间建立了物理连接。在EDMD小鼠模型中，丝裂原活化蛋白（MAP）激酶ERK和JNK在心肌病发生前在心脏组织中被激活。由于MAP激酶激活已被证实与心肌病有关，因此确定a型纤层蛋白突变如何导致MAP激酶激活将为心肌病的分子发病机制提供关键信息。我们假设EDMD引起的突变lamin A通过两种不同的机制促进MAP激酶的激活：1)突变的lamin A可能破坏UNC复合物，损害核-细胞骨架的完整性并产生激活MAP激酶途径的应激反应；2)通过直接和间接机制破坏MAP激酶锚点的功能。Lamin A突变可能直接抑制Lamin A作为MAP激酶隔离锚点促进核易位的功能。此外，层粘连蛋白A的突变可能会破坏LINC复合物，从而破坏需要完整细胞骨架的细胞质锚点的功能。为了验证我们的假设，我们提出了两个具体目标。目的1将确定破坏原代心肌细胞中的LINC复合物是否会激活MAP激酶。我们将通过表达突变形式的层粘连蛋白A、太阳蛋白和nesprins来破坏LINC复合物，这些突变形式阻止了建立LINC复合物所需的相互作用，并通过标准生化和荧光显微镜技术评估MAP激酶的激活。目的2将通过各种方法表征完整细胞中纤层蛋白A和MAP激酶的相互作用，以及纤层蛋白A突变对这种相互作用的影响。我们将通过FRAP和FLIP分析测量MAP激酶的流动性来评估层合蛋白A突变对MAP激酶锚点的功能后果。心肌损伤是EDMD最严重和危及生命的症状。了解细胞核缺陷蛋白如何导致edmd相关的心肌损伤将为设计有效的治疗策略提供必要的知识。这一知识也可能适用于由EDMD以外的疾病引起的类似心肌损伤。

DESCRIPTION (provided by applicant): The overall goal of this application is to define the molecular pathogenesis of cardiomyopathy associated with Emery-Dreifuss Muscular Dystrophy (EDMD). Autosomal dominant EDMD arises from mutations in a gene encoding A-type lamins, which are intermediate filaments involved in the maintenance of nuclear structure. Further, A-type lamins (along with suns and nesprins) are a component of the LINC complex, which establish a physical connection between the nucleus and the actin cytoskeleton. In EDMD mouse models of EDMD, mitogen activated protein (MAP) kinases ERK and JNK are activated in the heart tissue prior to the development of cardiomyopathy. As MAP kinase activation is well established to be linked to cardiomyopathy, determining how A-type lamin mutations cause activation of MAP kinases will provide key information on the molecular pathogenesis of cardiomyopathy. We hypothesize that EDMD causing mutations lamin A facilitate MAP kinase activation via two distinct mechanisms: 1) mutant lamin A may disrupt the UNC complex, compromising the integrity of the nucleo- cytoskeleton and generating a stress response that activates the MAP kinase pathway and 2) functional disruption of MAP kinase anchors by direct and indirect mechanisms. Lamin A mutations may directly inhibit the function of lamin A as a putative MAP kinase sequestering anchor to facilitate nuclear translocation. Further, mutations in lamin A may disrupt the LINC complex and hence, the function of cytoplasmic anchors that require intact cytoskeleton. To test our hypothesis, we propose two specific aims. Aim 1 will determine whether disrupting the LINC complex in primary cardiomyocytes will activate MAP kinases. We will disrupt the LINC complex by expressing mutant forms of lamin A, suns, and nesprins that prevent interactions necessary to establish the LINC complex and assess for MAP kinase activation by standard biochemical and fluorescence microscopy techniques. Aim 2 will characterize lamin A and MAP kinase interactions in intact cells by various methods and the effect of lamin A mutations on this interaction. We will assess the functional consequence of lamin A mutation on MAP kinase anchors by measuring the mobility of MAP kinases by FRAP and FLIP analysis. Heart muscle damage is the most serious and life-threatening symptom of EDMD. Understanding how defective proteins of the cell nucleus lead to EDMD-associated heart muscle damage will provide the knowledge necessary to devise an effective treatment strategy. This knowledge may also be applicable to similar heart muscle damage resulting from disorders other than EDMD.