Functional and Phenotypic Characterization of a New FSGS Gene

新 FSGS 基因的功能和表型特征

• 批准号: 8932678
• 负责人: Rasheed Adebayo Gbadegesin
• 金额: $ 35.66万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-24 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8932678
• 关键词: Actins; Address; Affect; Age; Age of Onset; Alleles; Animal Model; Apoptosis; Binding; Biological Assay; Biopsy; Cell Line; Cell Proliferation; Cells; Confocal Microscopy; Coupled; Critical Pathways; Cytokinesis; Cytoskeletal Proteins; Cytoskeleton; Data; Disease; Dominant-Negative Mutation; Embryo; End stage renal failure; Exons; F-Actin; Focal Segmental Glomerulosclerosis; Genes; Genetic; Goals; Health; Homeostasis; Human; Hypertrophy; Immunofluorescence Microscopy; Induced Mutation; Kidney; Kidney Diseases; Kidney Failure; Lead; Maintenance; Mind; Mitotic; Modeling; Molecular; Monitor; Mus; Mutate; Mutation; Outcome; PI3K/AKT; Pathogenesis; Pathogenicity; Pathway interactions; Patients; Pattern; Phenotype; Play; Proteins; Proto-Oncogene Proteins c-akt; Public Health; Publishing; Renal glomerular disease; Reporting; Research Design; Role; Signal Pathway; Signal Transduction; Staining method; Stains; Subfamily lentivirinae; Transfection; Transmission Electron Microscopy; Variant; WT1 gene; Zebrafish; anillin; base; cdc Genes; cell motility; cohort; disease phenotype; gene delivery system; glomerular filtration; in vivo; inhibitor/antagonist; innovation; insight; loss of function; migration; mutant; nephrin; new therapeutic target; novel; overexpression; podocyte; polymerization; protein activation; research study; response; slit diaphragm; targeted treatment; therapeutic target

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.

描述（由申请人提供）：局灶节段性肾小球硬化症（FSGS）是肾衰竭的常见原因，是改变肾小球滤过屏障（GFB）功能完整性的致病变化的结果。对家族性 FSGS 病例的研究表明足细胞在其发病机制中发挥着核心作用。我们的长期目标是通过确定对维持 GFB 功能完整性至关重要的途径来了解 FSGS 的分子发病机制，并确定 FSGS 的新治疗靶点。本申请的总体目标是研究 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 的突变是家族性 FSGS 的原因。我们发现，在 FSGS 崩溃的人类和小鼠的肾活检中，苯尼林的表达上调。表达 R431C 突变的足细胞细胞系表现出与关键足细胞蛋白 CD2AP 的结合缺陷、AKT 激活和异常细胞运动。此外，斑马鱼胚胎中苯胺的敲低也会破坏 GFB。我们的总体假设是，anillin 的 F-肌动蛋白结合域的突变会影响 F-肌动蛋白细胞骨架的聚合，并导致异常的足细胞增殖、凋亡和迁移。足细胞稳态的破坏会破坏正常的 GFB 功能并导致 FSGS 的发病机制。我们将通过以下具体目标探索这一假设：1) 通过表征 a) R431C 突变激活的信号级联 b) 确定 R431C 突变对细胞凋亡的功能影响，确定 ANLN R431C 破坏足细胞稳态的机制，细胞增殖和细胞迁移，c) PI3K/AKT 偶联信号通路的药理学抑制剂对突变诱导的表型的影响。 2) 使用体内互补测定确定 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，并使用肾小球滤过作为生理相关读数来评估药理救援。 3) 通过对 FSGS 患者的 ANLN 外显子进行测序并比较有突变和无突变的受试者的疾病表型，分析 FSGS 患者队列中 ANLN 基因的突变。创新：该提案代表了第一项旨在定义阿尼林变体导致 FSGS 机制的研究。意义：揭示 ANLN 突变导致 FSGS 的机制可能会确定对于维持足细胞细胞骨架功能完整性非常重要的途径。此外，通过探讨苯胺在细胞增殖、凋亡和运动中的作用，我们将 提供对健康和疾病中足细胞更新机制的深入了解。我们的遗传和机制方法将增进我们对 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小的药理学方法。