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持。该R01集中于称为非同源末端连接（NHEJ）的DNA修复的特定形式，其是CNS的适当发育所需的。参与NHEJ和其他DNA修复的酶在神经退行性疾病如阿尔茨海默病和ALS中的基因组稳定性和神经元存活中起关键作用。在发育中的大脑中，程序性细胞死亡是与神经发生和分化一致的调节过程。敲除小鼠缺乏NHEJ或其他DNA修复过程通常具有增加的胚胎CNS细胞凋亡和围产期致死性。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。为了实现这一目标，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志物的表达。我们还将使用cre重组酶技术产生CNS特异性条件性敲除NHEJ基因，以评估神经祖细胞和神经元中失败的NHEJ是否是导致细胞凋亡差异的原因。第二个目标将确定是否NHEJ缺陷的胚胎中枢神经系统培养物表现出增加的细胞凋亡反应缺氧，自由基损伤，或兴奋性毒性损伤。第三个目标将检查NHEJ缺乏和累积的DNA断裂与缺氧或兴奋性毒性损伤是否激活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的复合敲除小鼠来测试这一假设。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.