L 1 element as an inrinsic factor of mammalian aging

描述（由申请人提供）：本提案的目的是了解细胞对L1活性反应的机制方面，评价L1诱导的诱变及其对DNA损伤和相关衰老的贡献，确定这种贡献是否随年龄增加，并找出这种贡献在个体中的潜在变化。这些目标是重要的，因为现有的数据表明，由L1编码的DNA内切核酸酶造成的双链断裂（DSB）形式的DNA损伤可能远大于通过插入诱变由元件引起的基因组损伤。我们检测到内源性L1的表达在一些体细胞。因此，这种类型的DNA损伤对整个生物体都有影响，而不仅仅是对它的后代。我们还希望解决L1对人类干细胞中DNA损伤和相关衰老的贡献。对于这些研究，我们将操纵L1的表达水平，并通过蛋白质印迹分析，g-H2 AX焦点染色，和在正常细胞系中的COMET测定，将L1活性与所产生的DNA损伤相关联。我们将使用报告基因试验来定量L1元素的致突变潜力。我们将使用培养的原代细胞模型来确定L1加速体细胞和干细胞衰老的潜力。报道的L1元件造成DNA损伤的能力及其在许多人体组织中的内源性表达从根本上改变了我们对L1表达的意义及其对宿主健康影响的理解。该项目旨在确定体细胞和干细胞中持续的低水平L1表达是否会导致突变的稳定积累，这是由于L1内切核酸酶产生的DSB的易错修复。这一发现的直接健康意义将是L1对哺乳动物衰老和/或年龄相关疾病如癌症的贡献。由于保健不断改善，越来越多的老年人更容易患上与年龄有关的疾病。了解衰老的分子机制将有助于我们预防衰老或年龄相关疾病的发生。

DESCRIPTION (provided by applicant): The goal of this proposal is to understand the mechanistic aspects of cellular responses to L1 activity, to evaluate L1 -induced mutagenesis and its contribution to DNA damage and associated aging, to determine whether this contribution increases with age, and to find out potential variation of this contribution among individuals. These goals are important because the existing data indicate that the DNA damage in the form of double-strand breaks (DSBs) inflicted by the L1-encoded DNA endonuclease is potentially much greater than the insult to the genome caused by the element via insertional mutagenesis. We detected endogenous L1 expression in a number of somatic cells. Therefore this type of DNA damage has consequences for the whole organism, not only for its progeny. We also wish to address the L1 contribution to the DNA damage and associated aging in human stem cells. For these studies we will manipulate L1 expression levels and correlate L1 activity with the resulting DNA damage by western blot analysis, g-H2AX foci staining, and COMET assays in normal cell lines. We will use reporter assay to quantify the mutagenic potential of L1 elements. We will use cultured primary cell models to determine L1 potential to accelerate cellular senescence in somatic and stem cells. Reported ability of L1 elements to inflict DNA damage and its endogenous expression in a number of human tissues fundamentally changes our understanding of the significance of the L1 expression and its impact on the health of the host. This project is designed to determine whether ongoing low levels of L1 expression in somatic and stem cells lead to a steady accumulation of mutations due to the error-prone repair of the DSBs created by the L1 endonuclease. The direct health implication of this discovery would be L1 contribution to mammalian aging and/or age- associated diseases such as cancer. Due to the constant improvement of the health care there is a growing population of older people that is more susceptible to age-associated diseases. Understanding of the molecular mechanisms of aging would contribute to our potential to prevent the onset of aging or age- associated diseases.