Biological mechanism of familial focal segmental glomerulosclerosis-1

描述(由申请人提供)：α-肌动蛋白-4基因ACTN4突变导致一种常染色体显性形式的局灶性和节段性肾小球硬化(FSGS)。ACTN4介导的FSGS是显性的，成体起病，缓慢进展，提示足细胞功能随时间和压力的微妙变化。指导我们研究的假设是，α-肌动蛋白-4肌动蛋白结合域的显性功能获得突变导致肌动蛋白细胞骨架改变，机械功能改变，对压力的反应能力降低。我们将继续努力，利用我们开发的新的生化、细胞和动物工具来了解这种肾脏疾病。我们的目标是：(1)确定突变的α-肌动蛋白-4对肌动蛋白细胞骨架的生物力学性质和肌动蛋白动力学的影响：我们将确定α-肌动蛋白-4突变是否以功能获得的方式改变肌动蛋白细丝网络结构和肌动蛋白动力学。我们将确定α-肌动蛋白-4突变对肌动蛋白动力学和体内聚合的影响，以及对肌动蛋白细胞骨架超微结构的影响。我们还将研究肌动蛋白和α-肌动蛋白-4相关分子突触素是否改变突变的α-肌动蛋白-4的生化特性。(2)确定突变的α-肌动蛋白-4生物对细胞过程的下游影响：我们将确定ACTN4介导的细胞骨架变化是否会对细胞产生有害的后果。我们将检测突变细胞的生物力学特性。我们将检查由疾病相关突变引起的α-肌动蛋白和α-肌动蛋白的聚集是否对蛋白酶体功能有毒性影响。我们还将确定细胞骨架的变化是否会导致线粒体功能改变、ROS产生增加和细胞凋亡。(3)明确环境和遗传因素在Actn4突变疾病中对肾小球功能的影响：我们将对小鼠进行应激，以确定K255E杂合子是否增加了小鼠对肾小球损伤的易感性。我们将用血管紧张素转换酶抑制剂治疗小鼠，以确定减少突变足细胞上的机械力是否会改变疾病的进展。由于突触素似乎调节α-肌动蛋白-4/F-肌动蛋白的相互作用，我们将通过产生突触素缺陷/α-肌动蛋白-4突变小鼠并对其进行表型分析来研究这种相互作用。与公共卫生相关：肾衰竭是发病率和死亡率不断增加的原因。FSGS是一种频率越来越高的肾脏疾病。我们的研究将有助于理解特定人类基因ACTN4的缺陷如何导致肾脏疾病，并最终可能为FSGS的治疗提供新的见解。

DESCRIPTION (provided by applicant): Mutations in the alpha-actinin-4 gene ACTN4 cause an autosomal dominant form of focal and segmental glomerulosclerosis (FSGS). ACTN4-mediated FSGS is dominant, adult onset, and slowly progressive, suggesting subtle time and stress dependent alterations in podocyte function. The hypothesis guiding our studies is that dominant, gain-of-function mutations in the actin-binding domain of alpha-actinin-4 leads to an altered actin cytoskeleton, altered mechanical function, and a decreased ability to respond to stress. We will continue our efforts to understand this kidney disease, utilizing new biochemical, cellular, and animal tools that we have developed. We aim to: (1) Define the effect of mutant alpha-actinin-4 on the biomechanical properties of the actin cytoskeleton and on actin dynamics: We will determine if alpha-actinin-4 mutations act in a gain-of-function manner to alter the structure of the actin filament network and actin dynamics. We will determine the effect of alpha-actinin-4 mutations on actin dynamics and polymerization in vitro and in vivo, as well as the effect on the ultrastructure of the actin cytoskeleton. We will also examine if the actin and alpha-actinin-4-associated molecule synaptopodin alters the biochemical properties of mutant alpha-actinin-4. (2) Define the downstream effects of mutant alpha-actinin-4 biology on cellular processes: We will determine if ACTN4-mediated alterations in the cytoskeleton have detrimental cellular consequences. We will examine the biomechanical properties of mutant cells. We will examine if aggregation of alpha-actinin and actin caused by disease-associated mutations has toxic effects on proteasome function. We will also determine if the cytoskeletal changes lead to altered mitochondrial function, increased ROS production, and apoptosis. (3) Define the role of envirionmental and genetic factors on glomerular function in Actn4-mutant disease in vivo: We will stress mice to determine if heterozygosity for K255E in mice increases susceptibility to glomurular injury. We will treat mice with angiotensin converting enzyme inhibitors, to determine if reducing the mechanical forces on mutant podocytes alters disease progression. Because synaptopodin appears to modulate the alpha-actinin-4/F-actin interaction, we will examine this interaction genetically by generating and phenotyping synaptopodin-deficient/alpha-actinin-4 mutant mice. Public health relatedness: Kidney failure is an increasing cause of morbidity and mortality. FSGS is a form of kidney disease of increasing frequency. Our studies will help understand how defects in a particular human gene ACTN4 lead to kidney disease and, ultimately, may give new insights into treatment of FSGS.