Tumor Suppressors Mediate a Reduction in Male Gamete Quality with Aging

项目总结 患有遗传病或出生缺陷的儿童住院年龄较小，住院时间较长，并有 死亡率高于因其他原因住院的儿童。在美国出生的婴儿中，每33人中就有一人出生 缺陷；婴儿死亡的头号原因。我们的长期目标是通过以下方式改变这些可怕的统计数据 通过增加雄配子的诱变来描述降低游戏质量的机制 年龄增长，即父亲的年龄效应。父系年龄效应随着年龄的增加而日益显著 年龄较大的父亲的出生率，与男性生殖健康和儿童健康直接相关。先前 已发表的研究表明，碱基切除修复蛋白APE1的减少会导致碱基切除减少 随着年龄的增长，生精细胞的修复和突变增加。初步数据引导我们 验证由Trp53 Ser18/23磷酸化触发APE1的MDM2泛素化的假设 导致APE1值降低。目标1：测试MDM2是否使APE1无处不在，导致更多的 老年小鼠生殖细胞中高度泛素化的APE1，APE1的蛋白酶体降解，以及更大的 自发突变频率。目的2：测试Ser18/23处TrP53的磷酸化是否触发 老年小鼠生精细胞中APE1的降解。目标3：测试APE1是否发生变化 丰度和驱动这些变化的机制起源于精原干细胞群体。 方法：从携带MDM2靶向改变的雄性小鼠中制备明确的生精细胞 和TrP53来测试这些肿瘤抑制因子是否调节年轻小鼠的APE1丰度，并成为 在老年野生型小鼠中慢性激活导致APE1丰度降低，碱基切除修复减少 和更多的诱变。将测试蛋白酶体降解和mdm2活性的重要性。 使用抑制剂。CometChip阵列将分析单细胞中的DNA损伤，以确定DNA是否增加 损伤可能触发TrP53的激活。双工标签下一代测序将决定是否增加 突变频率始于精原干细胞群体。我们提出了一个新的模型，在这个模型中 正常发挥作用以保护基因组完整性的肿瘤抑制因子反而会导致配子减少 在生殖细胞独特的生物学背景下，通过减少APE1丰度来提高质量和增加突变 和衰老。这是一种范式的转变，不同于普遍认为的突变在茎中被动积累的观点 细胞是雄配子随年龄增长遗传质量下降的主要驱动力和TrP53的作用 和MDM2作为基因组的保护者。

PROJECT SUMMARY Children with a genetic disease or birth defect are hospitalized at a younger age, stay longer, and have a higher death rate than children hospitalized for other reasons. One in 33 infants born in the US has a birth defect; the number one cause of infant mortality. Our long-term goal is to change these dire statistics by delineating the mechanisms that reduce game quality by increasing mutagenesis in male gametes with increasing age, i.e., the paternal age effect. The paternal age effect is increasingly significant with increasing birth rates to older fathers, and is directly relevant to male reproductive health and child health. Previously published studies revealed that reduced base excision repair protein APE1, results in reduced base excision repair and increased mutagenesis in spermatogenic cells with increasing age. Preliminary data leads us to test the hypothesis that MDM2 ubiquitination of APE1 is triggered by TRP53 Ser18/23 phosphorylation resulting in reduced APE1. Aim 1: Test whether MDM2 ubiquitinates APE1, leading to greater amounts of highly ubiquitinated APE1 in germ cells of older mice, proteasomal degradation of APE1, and a greater spontaneous mutation frequency. Aim 2: Test whether phosphorylation of TRP53 at Ser18/23 triggers degradation of APE1 in spermatogenic cells from older mice. Aim 3: Test whether changes in APE1 abundance, and mechanisms driving those changes, originate in the spermatogonial stem cell population. Methods: Defined spermatogenic cells will be prepared from male mice carrying targeted changes in Mdm2 and Trp53 to test whether these tumor suppressors regulate APE1 abundance in young mice and become chronically activated in old wild type mice resulting in reduced APE1 abundance, reduced base excision repair and increased mutagenesis. The importance of proteasome degradation and MDM2 activity will be tested using inhibitors. CometChip arrays will analyze DNA damage in single cells to determine if increased DNA damage may trigger TRP53 activation. Duplex tag next generation sequencing will determine if increases in mutation frequency initiate in the spermatogonial stem cell population. We propose a novel model in which tumor suppressors that function normally to safeguard genome integrity, instead cause decreased gamete quality and increased mutagenesis by reducing APE1 abundance in the unique biological context of germ cells and aging. This is a paradigm shift from the widely-held view of mutations accumulating passively in stem cells as the major driving force of reduced genetic quality in male gametes with aging and for the role of TRP53 and MDM2 as protectors of the genome.