The Role of Auxilin, a Clathrin Uncoating Chaperone, in Congenital Heart Disease and Left-Right Patterning

网格蛋白脱衣伴侣辅助素在先天性心脏病和左右模式中的作用

• 批准号: 10450641
• 负责人: Delfina Pearledith Gonzalez
• 金额: $ 3.16万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10450641
• 关键词: Adolescent; Affect; Auxilins; Bilateral; Biology; Cardiac; Cardiac development; Cells; Childhood; Cilia; Clathrin; Clathrin Heavy Chains; Clathrin-Coated Vesicles; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Communities; Congenital Abnormality; Curiosities; Data; Defect; Development; Diagnosis; Disease; Drosophila genus; Embryo; Embryonic Development; Endocytosis; Epidermis; Extracellular Fluid; Fluorescence; Future; Gene Expression; Genes; Genetic; Genotype; Goals; Health; Heart; Heart Abnormalities; Heat-Shock Proteins 70; Human; Immunofluorescence Microscopy; In Situ Hybridization; Lateral; Lead; Left; Life; Link; Live Birth; Luciferases; Maintenance; Measures; Mediating; Mediator of activation protein; Mentorship; Mesoderm; Microscopy; Molecular; Molecular Chaperones; Morphogenesis; Mutant Strains Mice; Neurons; Nodal; Oral; Organ; Outcome; Output; Parents; Parkinson Disease; Pathogenesis; Pathogenicity; Pathway interactions; Patient-Focused Outcomes; Patients; Pattern; Phenocopy; Phenotype; Photoreceptors; Prevalence; Reporter; Reporting; Research; Role; Scientist; Side; Signal Pathway; Signal Transduction; Situs Inversus; Students; Tadpoles; Techniques; Testing; Training; Translating; Transmission Electron Microscopy; United States; Vesicle; Visceral; WNT Signaling Pathway; Western Blotting; Wing; Xenopus; beta catenin; cardiogenesis; career; congenital heart disorder; disease phenotype; exome sequencing; experimental study; fluid flow; genome wide association study; high risk; improved; infant death; inhibitor; loss of function; mutant; notch protein; overexpression; professor; skills; spatiotemporal; stem

Project Summary/Abstract Congenital heart disease (CHD) afflicts approximately 1% of live births and is among the most life-threatening of the birth defects. Patients with heterotaxy (HTX), improper left-right (LR) patterning and orientation of the visceral organs, are at high risk of CHD. Whole exome sequencing of patient-parent trios revealed many candidate CHD/HTX genes. Among these is DNAJC6, which codes for the neuronal-specific clathrin-coat HSP70 chaperone, Auxilin. Although auxilin has been studied in the context of clathrin vesicle uncoating and Juvenile Parkinson's, its role in embryonic development and CHD/HTX has not. A preliminary loss of function (LOF) screen revealed that DNAJC6 depletion causes abnormal cardiac looping in 15% of embryos, and abnormal pitx2c expression in 40% of embryos. These indicate abnormal LR patterning. No previous studies have linked DNAJC6 with LR patterning or heart morphogenesis in early development. Thus, the goal of this proposal is to elucidate the molecular mechanism by which auxilin (DNAJC6) leads to abnormal LR patterning that can lead to CHD/HTX. The first aim will use LOF experiments to determine when in the LR patterning cascade DNAJC6 has its earliest effects by visualizing dand5 expression in the left-right organizer. Determination of spatiotemporal DNAJC6 expression in early development in will focus subsequent mechanistic analysis. The second aim will analyze whether DNAJC6 LOF affects LR patterning by disrupting endocytosis. Fluorescence and TEM microscopy will reveal whether endocytosis, clathrin-coated vesicle uncoating, and clathrin pool maintenance are affected in auxilin-depleted embryos. Phenocopy and DNAJC6 LOF LR patterning defect rescue experiments with clathrin-mediated endocytosis components will indicate whether auxilin's role in LR patterning is clathrin-dependent. The final aim will investigate whether auxilin impacts LR patterning by disturbing Wnt or Notch signaling, both of which are affected by clathrin-mediated endocytosis. Luciferase reporters and western blots for downstream outputs (β-catenin for Wnt and the notch intracellular domain for Notch) will demonstrate which signaling cascade is activated. Phenocopy and LOF rescue experiments with downstream signaling inhibitors and components will indicate whether auxilin's role in LR patterning is Wnt or Notch dependent. Together, these experiments will elucidate the role of auxilin (DNAJC6) in left-right patterning, cardiac development, and CHD pathogenesis. This will aid future patient diagnosis and outcomes as their treatment can be targeted to genotype in addition to CHD phenotype. This application also outlines the applicant's training plan in advanced coursework, research mentorship, new techniques, and professional skills including written and oral data presentation. This training plan will equip the applicant with the skills to pursue a career as an independent research scientist and professor.

项目总结/摘要 先天性心脏病（CHD）约占活产婴儿的1%，是最危及生命的疾病之一 出生缺陷。异位（HTX）、左右（LR）模式和方向不正确的患者 内脏器官，是冠心病的高危人群。患者-父母三人组的全外显子组测序揭示了许多 候选CHD/HTX基因。其中之一是DNAJC 6，它编码神经元特异性的网格蛋白外套 HSP 70分子伴侣蛋白，HSP 70蛋白。尽管已经在网格蛋白囊泡脱壳和细胞分裂的背景下研究了生长素， 青少年帕金森氏症，其在胚胎发育中的作用与CHD/HTX无关。初步丧失功能 (LOF)筛查显示，DNAJC 6缺失导致15%的胚胎出现异常心脏循环， 40%的胚胎中pitx 2c表达异常。这些表明LR模式异常。无既往研究 已经将DNAJC 6与LR模式或早期发育中的心脏形态发生联系起来。因此， 本研究旨在阐明生长素（DNAJC 6）导致LR异常的分子机制 可能导致CHD/HTX的模式。第一个目标将使用LOF实验来确定何时处于LR 图案化级联DNAJC 6通过可视化左右组织者中的dand 5表达而具有其最早的效应。 在早期发育中时空DNAJC 6表达的测定将关注随后的 机理分析第二个目的是分析DNAJC 6 LOF是否通过破坏LR的表达来影响LR的模式。 内吞作用荧光和TEM显微镜将揭示是否内吞，网格蛋白包被的囊泡 脱壳和网格蛋白库的维持在生长素耗尽的胚中受到影响。表型和DNAJC 6 用网格蛋白介导的内吞作用成分进行的LOF LR图案化缺陷拯救实验表明， 生长素在LR模式中的作用是否依赖网格蛋白。最终的目标是研究生长素是否 通过干扰Wnt或Notch信号影响LR模式，这两种信号都受到网格蛋白介导的 内吞作用荧光素酶报告基因和蛋白质印迹用于下游输出（β-连环蛋白用于Wnt，缺口蛋白用于Wnt）。 Notch的胞内结构域）将证明哪个信号级联被激活。表型和LOF 下游信号抑制剂和组分的拯救实验将表明生长素在 LR模式化是Wnt或Notch依赖性的。总之，这些实验将阐明生长素的作用， （DNAJC 6）在左右模式，心脏发育和CHD发病机制中的作用。这将有助于未来的患者 诊断和结果，因为他们的治疗可以针对基因型除了CHD表型。这 申请还概述了申请人在高级课程，研究导师，新的 技术和专业技能，包括书面和口头数据介绍。该培训计划将使 申请人具有从事独立研究科学家和教授职业的技能。