Functional and Phenotypic Characterization of a New FSGS Gene

描述(由申请人提供)：局灶性节段性肾小球硬化(FSGS)是肾功能衰竭的常见原因，是改变肾小球滤过屏障(GFB)功能完整性的致病改变的结果。对家族性FSGS病例的研究指出足细胞在其发病机制中的中心作用。我们的长期目标是通过识别对维持GFB功能完整性至关重要的通路来了解FSGS的分子发病机制，并确定FSGS的新治疗靶点。这项应用的总体目标是研究F-肌动蛋白结合细胞周期基因ANLN突变导致FSGS的机制。我们的方法是可行的，因为我们最近发现ANLN的F-肌动蛋白结合域R431C突变是家族性FSGS的原因之一。我们发现，在FSGS塌陷的人和小鼠的肾活检组织中，苯胺素表达上调。表达R431C突变的足细胞株表现出与足细胞关键蛋白CD2AP结合缺陷、AKT激活和细胞运动异常。此外，敲除斑马鱼胚胎中的苯胺素会扰乱GFB。我们的主要假设是，苯丙胺的F-肌动蛋白结合域的突变影响F-肌动蛋白细胞骨架聚合，导致足细胞异常增殖、凋亡和迁移；足细胞动态平衡的破坏，进而扰乱正常的GFB功能，导致FSGS的发病。我们将通过以下具体目标来探讨这一假说：1)通过表征a)R431C突变激活的信号级联反应来确定ANLN R431C破坏足细胞动态平衡的机制；b)确定R431C突变对细胞凋亡、细胞增殖和细胞迁移的功能影响，以及c)PI3K/AKT偶联信号通路的药物抑制剂对该突变诱导的表型的影响。2)通过体内互补实验确定R431C突变对斑马鱼胚胎GFB的功能影响，以确定R431C ANLN突变的等位基因致病性，并以肾小球滤过作为生理相关读数来评估药物拯救。3)通过对FSGS患者ANLN基因外显子进行测序，并比较有无突变的患者的疾病表型，分析一组FSGS患者的ANLN基因突变。创新：这项提案代表了第一项旨在定义苯青素变异导致FSGS的机制的研究。意义：解开ANLN突变导致FSGS的机制可能会确定对维持足细胞细胞骨架功能完整性至关重要的途径。此外，通过探讨苯青素在细胞增殖、凋亡和运动中的作用，我们将 洞察足细胞更新在健康和疾病中的机制。我们的遗传和机制方法将促进我们对FSGS足细胞表型变化的分子发病机制的理解，并导致识别新的治疗靶点和毒性较低的药理学方法。

DESCRIPTION (provided by applicant): Focal segmental glomerulosclerosis (FSGS), a common cause of kidney failure, is the result of pathogenic changes that alter the functional integrity of the glomerular filtration barrier (GFB). The study of familial FSGS cases points to a central role of the podocyte in its pathogenesis. Our long term goals are to understand the molecular pathogenesis of FSGS by identifying pathways that are critical for the maintenance of the functional integrity of the GFB and identify novel therapeutic targets for FSGS. The overall objective of this application is to study the mechanisms by which mutations in an F-actin binding cell cycle gene, ANLN, cause FSGS. Our approach is feasible because we recently identified a mutation in F-actin binding domain of ANLN, R431C, as a cause of familial FSGS. We showed that anillin is upregulated in kidney biopsies of humans and mice with collapsing FSGS. Podocyte cell lines expressing the R431C mutation demonstrate defective binding to CD2AP a key podocyte protein, activation of AKT and aberrant cell motility. Also, knockdown of anillin in zebrafish embryos disrupts the GFB. Our overarching hypothesis is that mutations in the F-actin binding domain of anillin affect F-actin cytoskeleton polymerization and lead to aberrant podocyte proliferation, apoptosis and migration; disruption of podocyte homeostasis then disrupts normal GFB function and leads to the pathogenesis of FSGS. We will explore this hypothesis through the following specific aims: 1) Determine the mechanisms by which podocyte homeostasis is disrupted by ANLN R431C by characterizing a) the signaling cascades activated by the R431C mutation b) determine the functional effect of the R431C mutation on apoptosis, cell proliferation and cell migration, and c) the effect of pharmacologic inhibitors of the PI3K/AKT coupled signaling pathways on the phenotype induced by the mutation. 2) Determine the functional effect of the R431C mutation on the GFB of zebrafish embryos using an in vivo complementation assay to determine allele pathogenicity of R431C ANLN mutation and assess pharmacologic rescue using glomerular filtration as a physiologically relevant readout. 3) Analyze mutations of the ANLN gene in a cohort of patients with FSGS by sequencing the exons of ANLN in FSGS patients and comparing the disease phenotype in subjects with and without mutations. Innovation: This proposal represents the first study designed to define the mechanisms by which anillin variants cause FSGS. Significance: Unraveling the mechanisms by which mutations in ANLN cause FSGS may identify pathways that are important for maintaining the functional integrity of the podocyte cytoskeleton. Furthermore, by probing the role of anillin in cell proliferation, apoptosis, and motility, we will provide insight into the mechanisms of podocyte renewal in health and disease. Our genetic and mechanistic approaches will advance our understanding of the molecular pathogenesis of podocyte phenotype changes in FSGS and lead to identification of novel therapeutic targets and less toxic pharmacologic approaches.