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）：细胞凋亡对于组织稳态的维持以及发育中胚胎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死亡和严重的中枢神经系统（CNS）缺陷，包括中枢神经系统细胞增生、脑外畸形（脑物质从头骨中突出）和神经管闭合不完全。尽管细胞凋亡途径在某些哺乳动物细胞系中已得到很好的表征，但在原代胚胎干细胞（ESC）和中枢神经系统的神经前体细胞（NPC）中细胞死亡的确切调控方式仍然很大程度上未知。在本提案中，我将探索 ESC 和 NPC 参与的一种新的细胞凋亡途径，使它们在发育中的胚胎中快速死亡。该提案将检验以下假设：这些细胞利用一种新的线虫样凋亡途径来独立于线粒体激活半胱天冬酶和细胞死亡。特别是，我假设，与在发育中的线虫胚胎中经历凋亡的细胞中观察到的情况类似，ESC 和 NPC 表达一种先前未表征的促凋亡蛋白 sApaf-1 的短形式，该蛋白与抗凋亡 Bc-xL 蛋白复合存在，并被抗凋亡 Bc-xL 蛋白抑制。在目标 1 中，我将研究 sApaf-1/Bcl-xL 相互作用如何调节 ESC 和 NPC 中的细胞凋亡。具体来说，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让 sApaf-1 激活半胱天冬酶并诱导两种细胞类型快速凋亡。重要的是，我将测试 ESC 和 NPC 中的这一新途径是否如线虫胚胎中观察到的那样通过绕过线粒体来诱导细胞凋亡。在目标 2 中，我将研究体内发育中的大脑中的 sApaf-1/Bcl-xL 凋亡途径。该目标将探讨发育中大脑中缺乏 Bcl-xL 的条件性敲除小鼠的细胞死亡表型增加，以及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。该提案的结果对于揭示 ESC 和 NPC 生物学的关键特征非常重要，这些特征决定了早期哺乳动物胚胎发育中细胞死亡的调节方式。