The Nucleocytoskeleton in Progeria and Aging

描述（申请人提供）：哈钦森-吉尔福德早衰综合征（HGPS）是一种罕见的遗传疾病，类似于生理衰老，但出现在儿童早期。了解HGPS触发衰老的机制可能会揭示正常衰老的新方面。HGPS是由LMNA基因突变引起的，LMNA基因编码a型核层蛋白，与核膜相关的中间丝蛋白。这些突变增加了一个隐剪接位点的使用和一个蛋白水解加工位点的缺失，导致法酰化的前层蛋白（一种被称为progerin的变体）在核层中异常积累。编码处理前纤层蛋白A的蛋白酶的ZMPSTE24基因的突变导致类似的法酰化前纤层蛋白A的积累，并导致限制性皮肤病（RD），这是一种相关但更严重的早衰疾病。在正常细胞中也检测到低水平的progerin，它们可能导致衰老表型。尽管在HGPS、RD和正常衰老中描述了核膜的形态改变，但progerin导致细胞功能异常和疾病的机制尚不清楚。这很可能是由于progerin的表达改变了核膜，从而影响了细胞的其他结构。越来越多的证据表明，细胞核通过一种称为LINC复合物的相互作用蛋白质复合物与细胞骨架相连。这些蛋白质包括层粘连蛋白、太阳蛋白和nesprin蛋白。太阳是核膜内的跨膜蛋白，直接与层粘连蛋白和内层粘连蛋白相互作用。Nesprins位于外核膜中，与sun和细胞骨架元件（如肌动蛋白）相关。我们假设，progerin的表达导致LINC复合物的破坏，导致核细胞骨架连接缺陷，从而改变核运动和中心体定位。通过一个极化成纤维细胞的模型系统，我们获得了初步的数据，表明progerin的表达改变了细胞核中nesprin2G的定位，干扰了细胞核的运动和中心体的定位。在本实验的目的1中，我们将测试法酰化的progerin和prelamin A是否会干扰LINC复合物的形成和功能，并确定progerin在核运动和中心体定位的途径中起作用。在Aim 2中，我们将通过检查LINC复合物组分在核膜中的迁移率及其相互作用来确定progerin如何影响LINC复合物组分的生物物理行为。在Aim 3中，我们将通过检查正常衰老细胞是否在LINC复合体中表现出缺陷，以及这些缺陷是否可以通过减少progerin表达或法尼化的干预来改善，来测试progerin是否是正常衰老的重要组成部分。这些研究将确定核胞质相互作用的缺陷是否会导致早衰症和正常衰老的细胞功能障碍。

DESCRIPTION (provided by applicant): Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder that resembles physiological aging but presents in early childhood. Understanding the mechanism by which HGPS triggers aging may reveal novel aspects of normal aging. HGPS is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins associated with the inner nuclear membrane. The mutations increase use of a cryptic splice site and the deletion of a proteolytic processing site resulting in the abnormal accumulation of farnesylated prelamin A variant called progerin in the nuclear lamina. Mutations in the ZMPSTE24 gene encoding the protease that processes prelamin A result in a similar accumulation of farnesylated prelamin A and cause restrictive dermopathy (RD), a related but more severe progeriod disease. Low levels of progerin have also been detected in normal cells where they may contribute to aged phenotypes. Although morphological alterations in the nuclear envelope have been described in HGPS, RD and normal aging, the mechanism by which progerin leads to abnormal cellular function and disease is unknown. It is likely that progerin expression alters the nuclear lamina and that this affects other structures in the cell. An emerging body of evidence has suggested that the nucleus is connected to the cytoskeleton via a complex of interacting proteins known as the LINC complex. These proteins include the lamins, Suns and nesprin proteins. Suns are transmembrane proteins in the inner nuclear membrane that interact directly with lamins and nesprins. Nesprins are in the outer nuclear membrane and associate with Suns and cytoskeletal elements such as actin. We hypothesize that progerin expression leads to a disruption in the LINC complex, resulting in defective nucleocytoskeletal connections and consequently alterations in nuclear movement and centrosome positioning. Using a model system of fibroblasts undergoing polarization, we have obtained preliminary data showing that expression of progerin alters localization of nesprin2G in the nucleus and interferes with nuclear movement and centrosome positioning. In Aim1 of the proposed experiments, we will test whether farnesylated progerin and prelamin A interfere with LINC complex formation and function and identify where in the pathways for nuclear movement and centrosome positioning progerin acts. In Aim 2, we will determine how progerin affects the biophysical behavior of LINC complex components by examining their mobility in the nuclear membranes and their interactions with each other. In Aim 3, we will test whether progerin is an important component of normal aging by examining whether normal aged cells exhibit defects in the LINC complex and whether the defects can be ameliorated by interventions that reduce progerin expression or farnesylation. These studies will establish whether defects in nucleocytoplasmic interactions contribute to cellular dysfunction in progeria and in normal aging.