Programmed Cell Death and DNA Damage Signals

描述（由申请人提供）：我们的长期目标是了解程序性细胞死亡和DNA修复如何指导神经系统的发育和维护。本研究聚焦于一种特殊形式的DNA修复，称为非同源末端连接（NHEJ），这是中枢神经系统正常发育所必需的。参与NHEJ和其他DNA修复的酶在阿尔茨海默氏症和肌萎缩侧索硬化症等神经退行性疾病的基因组稳定性和神经元存活中起着关键作用。在发育中的大脑中，程序性细胞死亡是与神经发生和分化相协调的调控过程。缺失NHEJ或其他DNA修复过程的敲除小鼠通常会增加胚胎中枢神经系统的凋亡和围产期死亡率。我们将利用NHEJ缺陷小鼠结合细胞和分子方法来研究基因组不稳定性对神经元存活和分化的影响。要实现这个大目标，我们有四个具体目标。首先，比较正常和nhej缺陷小鼠胚胎大脑皮层神经祖细胞和有丝分裂后神经元的分子表型。我们将监测细胞死亡并检查细胞周期和其他分子标记的表达。我们还将使用cree -recombinase技术产生cns特异性的NHEJ基因条件敲除，以评估神经祖细胞和神经元中NHEJ的失败是否导致细胞凋亡的差异。第二个目的是确定缺乏NHEJ的胚胎CNS培养是否对缺氧、自由基损伤或兴奋性毒性损伤表现出增加的凋亡反应。第三个目的是研究NHEJ缺乏和积累的DNA断裂是否与缺氧或兴奋性损伤一起激活p53和下游凋亡效应，如线粒体功能障碍。我们将产生缺乏NHEJ基因和p53或Bax的复合敲除小鼠来验证这一假设。我们的第四个目标将扩展这些研究，以了解外源性生长因子或自由基清除剂是否可以延迟或阻止NHEJ缺陷小鼠的下游凋亡信号传导。

DESCRIPTION (provided by applicant): Our long-term objective is to understand how programmed cell death and DNA repair guide development and maintenance of the nervous system. This ROl focuses a specific form of DNA repair called Non-Homologous End Joining (NHEJ), which is required for proper development of the CNS. Enzymes involved in NHEJ and other DNA repairs serve critical roles in genomic stability and neuronal survival in neurodegenerative disorders such as Alzheimer's and ALS. In the developing brain, programmed cell death is regulated process acting in concert with neurogenesis and differentiation. Knockout mice deficient NHEJ or other DNA repair processes typically have increased apoptosis in the embryonic CNS and perinatal lethality. We will utilize NHEJ deficient mice in combination with cellular and molecular approaches to study the impact of genomic instability on neuronal survival and differentiation. To accomplish this broad goal, we have four Specific aims. The first proposes to compare the molecular phenotypes of neural progenitors and postmitotic neurons in the cerebral cortex of normal and NHEJ-deficient mouse embryos. We will monitor cell death and examine the expression of cell cycle and other molecular markers. We will also generate CNS-specific conditional knockouts of NHEJ genes using cre-recombinase technology to evaluate whether failed NHEJ in neural progenitors and neurons is responsible for the differences in apoptosis. The second aim will determine if NHEJ deficient embryonic CNS cultures exhibit increased apoptotic responses to hypoxia, free radical damage, or excitotoxic injury. The third aim will examine whether NHEJ deficiency and accumulated DNA breaks coupled with hypoxia or excitotoxic injury activate p53 and downstream apoptotic effects such as mitochondrial dysfunction. We will generate compound knockout mice lacking NHEJ genes and either p53 or Bax to test this hypothesis. Our fourth aim will extend these studies to ask whether exogenous growth factors or free radical scavengers can delay or prevents downstream apoptotic signaling in NHEJ deficient mice.