The Nucleocytoskeleton in Progeria and Aging

描述(申请人提供)：Hutchinson-Gilford早衰症(HGPS)是一种罕见的遗传性疾病，类似生理性衰老，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，即与内核膜相关的中间丝蛋白。这些突变增加了对隐蔽剪接位点的使用和蛋白质降解加工位点的缺失，导致法尼化的前层蛋白A变异体在核层中异常积累，称为孕激素。ZMPSTE24基因编码处理Prelamin A的蛋白酶的突变会导致类似的法尼化Prelamin A积聚，并导致限制性皮肤病(RD)，这是一种相关但更严重的程序性疾病。在正常细胞中也检测到低水平的孕激素，它们可能与衰老表型有关。虽然在HGPS、RD和正常衰老中已经描述了核膜的形态变化，但孕激素导致细胞功能异常和疾病的机制尚不清楚。孕激素的表达很可能改变了核层，从而影响了细胞中的其他结构。一组新出现的证据表明，细胞核通过一种称为LINC复合体的相互作用蛋白质复合体与细胞骨架相连。这些蛋白质包括Lamins、Suns和Nesprin蛋白质。SUN是内核膜中的跨膜蛋白，直接与Lamins和Nesprint相互作用。Nesprint位于外核膜，与太阳和细胞骨架元素(如肌动蛋白)相关。我们假设孕激素的表达导致LINC复合体的破坏，导致核细胞骨架连接缺陷，从而改变核移动和中心体的位置。利用极化的成纤维细胞模型系统，我们获得了初步的数据，表明孕激素的表达改变了Nesprint 2G在核中的定位，并干扰了核的运动和中心体的定位。在拟议的实验中，我们将测试法尼化的孕激素和前层蛋白A是否干扰LINC复合体的形成和功能，并确定孕激素在核运动和中心体定位的通路中的作用。在目标2中，我们将通过检测LINC复杂成分在核膜中的迁移率以及它们之间的相互作用来确定孕激素如何影响LINC复杂成分的生物物理行为。在目标3中，我们将通过检测正常衰老的细胞是否存在LINC复合体中的缺陷，以及是否可以通过减少孕激素表达或法尼化的干预来改善这些缺陷，来测试孕激素是否是正常衰老的重要组成部分。这些研究将确定核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.