Identification of a novel apoptotic pathway in the developing mammalian embryo

鉴定发育中的哺乳动物胚胎中新的细胞凋亡途径

• 批准号: 9180055
• 负责人: Ayumi Nakamura
• 金额: $ 4.09万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-11-16 至 2018-11-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9180055
• 关键词: Animals; Apoptosis; Apoptotic; BCL2 gene; BCL2L1 gene; Bax protein; Binding; Brain; Brain region; Bypass; CASP3 gene; CASP9 gene; Caenorhabditis elegans; Caspase; Cell Death; Cell Line; Cells; Cellular biology; Cessation of life; Complex; Congenital Abnormality; Cytoplasm; Data; Defect; Development; Dorsal; Embryo; Embryonic Development; Embryonic and Fetal Development; Enterobacteria phage P1 Cre recombinase; Event; Exencephalies; Exhibits; Failure; Homeostasis; Homologous Gene; Hyperplasia; Invertebrates; Knockout Mice; Knowledge; Lead; Maintenance; Mammalian Cell; Mass Spectrum Analysis; Maternal and Child Health; Mediating; Mitochondria; Modeling; Molecular Conformation; Mus; Nervous system structure; Neural Tube Closure; Neuraxis; Neurodevelopmental Disorder; Neurons; Organ; Pathogenesis; Pathway interactions; Phenotype; Protein Isoforms; Proteins; Research Proposals; Reverse Transcriptase Polymerase Chain Reaction; Small Interfering RNA; Telencephalon; Testing; Tissues; WD Repeat; apoptotic protease-activating factor 1; bcl-xlong protein; cell type; cranium; cytochrome c; developmental disease; embryonic stem cell; in vivo; knock-down; nerve stem cell; nervous system disorder; neural precursor cell; novel; postnatal; pro-apoptotic protein; programs; public health relevance; small hairpin RNA; small molecule inhibitor; virtual

DESCRIPTION (provided by applicant): Apoptosis is critical for the maintenance of tissue homeostasis and for the maturation of various organs in the developing embryo. Failure to activate the cell death program results in embryonic lethality and severe central nervous system (CNS) defects, including hyperplasia of cells in the CNS, exencephaly (protrusion of brain matter from the skull), and incomplete neural tube closure. Although the apoptotic pathway has been well characterized in certain mammalian cell lines, exactly how cell death is regulated in primary embryonic stem cells (ESCs) and in the neural precursor cells (NPCs) of the CNS remains largely unknown. In this proposal, I will explore a novel apoptotic pathway engaged by ESCs and NPCs that primes them for rapid death in the developing embryo. This proposal will test the hypothesis that these cells utilize a novel C. elegans-like apoptotic pathway for activating caspases and cell death independently of the mitochondria. In particular, I hypothesize that, similar to what is observed in cells undergoing apoptosis in the developing C. elegans embryo, ESCs and NPCs express a previously uncharacterized short form of the pro-apoptotic protein, sApaf-1, that exists in complex with, and is inhibited by, the anti-apoptotic Bc-xL protein. In Aim 1, I will investigate how the sApaf-1/Bcl-xL interaction regulates apoptosis in ESCs and NPCs. Specifically, I will examine the physical interaction between sApaf-1 and Bcl-xL and determine whether Bcl-xL inhibition or knockdown is sufficient to allow sApaf-1 to activate caspases and induce rapid apoptosis in both cell types. Importantly, I will test whether this novel pathway in ESCs and NPCs induces apoptosis by bypassing the mitochondria as observed in the C. elegans embryo. In Aim 2, I will investigate the sApaf-1/Bcl-xL apoptotic pathway in the developing brain in vivo. This aim will explore the increased cell death phenotype of conditional knockout mice that are deficient in Bcl-xL in the developing brain, and whether the cell death observed in these animals can be rescued by simultaneous deletion of Apaf-1. The results from this proposal will be important for uncovering key features of ESC and NPC biology that determine how cell death is regulated in early mammalian embryonic development.

描述（由申请人提供）：凋亡对于维持组织稳态和发育中各种器官的成熟至关重要。未能激活细胞死亡程序会导致胚胎致死性和严重的中枢神经系统（CNS）缺陷，包括CNS中细胞的增生，脑外部（头骨上的脑物质突出）和不完全的神经管闭合。尽管凋亡途径在某些哺乳动物细胞系中的特征很好，但准确地在原代胚胎干细胞（ESC）和中枢神经系统的神经前体细胞（NPC）中调节细胞死亡仍在很大程度上未知。在此提案中，我将探索ESC和NPC所涉及的一种新颖的凋亡途径，该途径在发育中的胚胎中快速死亡。该提案将检验以下假设：这些细胞利用新型的秀丽隐杆线虫样凋亡途径来独立于线粒体激活胱天蛋白酶和细胞死亡。特别是，我假设，类似于发育中的秀丽隐杆线虫胚胎，ESCS和NPC在经历凋亡中观察到的类似，表达了一种先前未经表征的促凋亡蛋白SAPAF-1的简短形式，它与抗蛋白质BC-XL蛋白相似，并抑制了抗蛋白质BC-XL蛋白。在AIM 1中，我将研究SAPAF-1/BCL-XL相互作用如何调节ESC和NPC中的凋亡。具体而言，我将检查SAPAF-1和BCL-XL之间的物理相互作用，并确定BCl-XL抑制或敲低是否足以使SAPAF-1激活胱天蛋白酶并诱导两种细胞类型的快速凋亡。重要的是，我将测试这种新型ESC和NPC中的新途径是否通过在秀丽隐杆线虫胚胎中观察到的线粒体来诱导凋亡。在AIM 2中，我将研究体内发育中的大脑中的SAPAF-1/BCL-XL凋亡途径。该目标将探索发育中大脑中BCL-XL缺乏的条件敲除小鼠的细胞死亡表型，以及在这些动物中观察到的细胞死亡是否可以通过同时删除APAF-1来挽救。该提案的结果对于发现ESC和NPC生物学的关键特征将很重要，这些特征决定了如何在早期哺乳动物胚胎发育中调节细胞死亡。