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修复如何引导神经系统的发育和维护。这种ROL集中于一种特殊的DNA修复形式，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发挥关键作用。在发育中的大脑中，细胞程序性死亡是一个与神经发生和分化相协调的调控过程。缺乏NHEJ或其他DNA修复过程的基因敲除小鼠通常会增加胚胎中枢神经系统的凋亡和围产期的致死率。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对神经元存活和分化的影响。为了实现这一广泛目标，我们有四个具体目标。第一个建议比较正常和NHEJ缺陷小鼠胚胎大脑皮层中神经前体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，并检测细胞周期和其他分子标志物的表达。我们还将使用重组酶技术产生CNS特异性的NHEJ基因的条件性敲除，以评估神经前体细胞和神经元中失败的NHEJ基因是否导致了细胞凋亡的差异。第二个目的将确定NHEJ缺陷的胚胎中枢神经系统培养物是否对缺氧、自由基损伤或兴奋性毒性损伤表现出更强的凋亡反应。第三个目的将研究NHEJ缺乏和累积的DNA断裂加上缺氧或兴奋毒性损伤是否激活P53和下游的凋亡效应，如线粒体功能障碍。我们将产生缺乏NHEJ基因和P53或Bax的复合基因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，以询问外源性生长因子或自由基清除剂是否可以延缓或阻止NHEJ缺陷小鼠的下游凋亡信号。

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.