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

项目摘要/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。 先天缺陷的证据。异位畸形(HTX)、左-右(LR)构型不当和 内脏器官，是冠心病的高危人群。患者-父母三人组的整个外显子组测序揭示了许多 CHD/HTX候选基因。DNAJC6就是其中之一，它编码神经元特异性的网状蛋白外壳 HSP70分子伴侣，欧西林。虽然已经在网状蛋白囊泡去涂层和 青少年帕金森氏症，其在胚胎发育中的作用和CHD/HTX并非如此。初步的功能丧失 (LOF)筛查显示，DNAJC6缺失会导致15%的胚胎出现异常的心脏循环，并且 在40%的胚胎中，Pitx2c的表达异常。这些都表明了异常的LR图案。没有以前的研究 在早期发育中将DNAJC6与LR模式或心脏形态发生联系起来。因此，这一目标是 建议阐明生长素(DNAJC6)导致LR异常的分子机制 可能导致CHD/HTX的图案。第一个目标将使用LOF实验来确定何时进入LR 图案级联DNAJC6最早的作用是在左右组织者中可视化Dand5的表达。 Will Focus早期发育过程中DNAJC6时空表达的测定 机械分析。第二个目标是分析DNAJC6 LOF是否通过破坏 内吞作用。荧光显微镜和透射电子显微镜将揭示内吞作用、笼蛋白包裹的小泡 在缺乏生长素的胚胎中，去涂层和网状蛋白池的维持受到影响。表观复制与DNAJC6 利用网状蛋白介导的内吞成分进行的LofLR图案缺陷修复实验将表明 生长素在LR构图中的作用是否依赖于笼蛋白。最终目标将调查阿司匹林是否 通过干扰Wnt或Notch信号来影响LR模式，这两个信号都受到网状蛋白介导的影响 内吞作用。下游产物的荧光素酶报告蛋白和蛋白质印迹(WNT和缺口的β-连环蛋白 Notch的胞内域)将展示哪个信令级联被激活。物候学和LOF 用下游信号抑制剂和组件进行的救援实验将表明，生长素在细胞周期中的作用 LR图案依赖于WNT或凹槽。综上所述，这些实验将阐明生长素的作用 (DNAJC6)在左右模式、心脏发育和冠心病发病机制中的作用。这将有助于未来的患者 除了CHD表型外，他们的诊断和治疗结果还可以针对基因。这 申请书还概述了申请者在高级课程、研究指导、新 技术和专业技能，包括书面和口头数据演示。这项培训计划将为 有能力从事独立研究的科学家和教授的申请者。

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.