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肌营养不良症(EDMD)相关的心肌病的分子发病机制。常染色体显性EDMD源于编码A型板层蛋白的基因突变，A型板层蛋白是参与维持核结构的中间丝。此外，A-型层粘连蛋白(连同SUN和NESprint)是LINC复合体的组成部分，它建立了细胞核和肌动蛋白细胞骨架之间的物理连接。在EDMD小鼠模型中，有丝分裂原激活蛋白(MAP)激酶ERK和JNK在心肌病发生之前在心脏组织中被激活。由于MAP激酶的激活与心肌病密切相关，因此确定A型lamin突变如何导致MAP激酶的激活将为心肌病的分子发病机制提供关键信息。我们假设，引起突变的EDMD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复合体，破坏核-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激酶锚定的功能。Lamin A突变可能直接抑制了Lamin A作为MAPK隔离锚的功能，从而促进了核转位。此外，层蛋白A的突变可能会破坏LINC复合体，从而破坏需要完整细胞骨架的细胞质锚的功能。为了检验我们的假设，我们提出了两个具体目标。目标1将确定破坏原代心肌细胞中的LINC复合体是否会激活MAP激酶。我们将通过表达突变形式的层蛋白A、SUN和nesiprs来破坏LINC复合体，这些突变形式阻止了建立LINC复合体所必需的相互作用，并通过标准的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层蛋白A和MAPK的相互作用，以及层蛋白A突变对这种相互作用的影响。我们将通过FRAP和翻转分析来测量MAP激酶的迁移率，以评估Lamin A突变对MAP激酶锚定的功能后果。心肌损伤是EDMD最严重、最危及生命的症状。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的心肌损伤。

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.