The Nucleocytoskeleton in Progeria and Aging

描述（由申请人提供）：Hutchinson-Gilford早衰综合征（HGPS）是一种罕见的遗传性疾病，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型核纤层蛋白，与内核膜相关的中间丝蛋白。这些突变增加了隐蔽剪接位点的使用和蛋白水解加工位点的缺失，导致称为早老蛋白的法尼基化前核层蛋白A变体在核层中的异常积累。ZMPSTE 24基因编码的蛋白酶，加工前层蛋白A的突变导致类似的积累法尼基化前层蛋白A，并导致限制性皮肤病（RD），相关的，但更严重的早衰症。在正常细胞中也检测到低水平的早老蛋白，它们可能有助于老化表型。尽管在HGPS、RD和正常衰老中已经描述了核膜中的形态学改变，但早老蛋白导致异常细胞功能和疾病的机制尚不清楚。很可能早老蛋白的表达改变了核纤层，这影响了细胞中的其他结构。一个新兴的证据表明，细胞核是通过一个复杂的相互作用的蛋白质称为LINC复合体连接到细胞骨架。这些蛋白质包括核纤层蛋白、Suns和nesprin蛋白。SUNS是位于内核膜中的跨膜蛋白，与核纤层蛋白和nesprins直接相互作用。巢蛋白位于外核膜中，与太阳和细胞骨架元件如肌动蛋白结合。我们假设早老蛋白的表达导致LINC复合体的破坏，导致核细胞骨架连接缺陷，从而改变核运动和中心体定位。使用模型系统的成纤维细胞进行极化，我们已经获得了初步的数据显示，早老蛋白的表达改变了nesprin 2G在细胞核中的定位，并干扰核运动和中心体定位。在拟议实验的目标1中，我们将测试法尼基化的早老蛋白和前层蛋白A是否干扰LINC复合物的形成和功能，并确定核运动和中心体定位早老蛋白作用的途径。在目标2中，我们将通过检查它们在核膜中的迁移率及其相互作用来确定早老蛋白如何影响LINC复合物组分的生物物理行为。在目标3中，我们将通过检查正常老化细胞是否表现出LINC复合物的缺陷以及这些缺陷是否可以通过减少早老蛋白表达或法尼基化的干预来改善来测试早老蛋白是否是正常老化的重要组分。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中的细胞功能障碍。 公共卫生相关性：核蛋白核纤层蛋白A的一种异常形式在Hutchinson-Gilford早衰综合征患者的细胞中以高水平表达，而在正常衰老细胞中以低水平表达。异常核纤层蛋白A如何影响细胞功能和衰老尚不清楚。我们将探讨异常核纤层蛋白A是否改变了核膜中介导细胞核与细胞质中细胞骨架丝附着的蛋白质，从而导致细胞迁移和极性缺陷，这可能是衰老的基础。

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. PUBLIC HEALTH RELEVANCE: An abnormal form of the nuclear protein lamin A is expressed at high levels in cells from patients with the Hutchinson-Gilford progeria syndrome and at low levels in normal aged cells. How the abnormal lamin A affects cell function and aging is unknown. We will explore whether the abnormal lamin A alters proteins in the nuclear membrane that mediate the attachment of the nucleus to cytoskeletal filaments in the cell's cytoplasm, thereby causing defects in cell migration and polarity that could underlie aging.