Tumor Suppressors Mediate a Reduction in Male Gamete Quality with Aging

肿瘤抑制剂介导雄性配子质量随着衰老而降低

• 批准号: 9564362
• 负责人: Christi A Walter
• 金额: $ 53.69万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-30 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9564362
• 关键词: APEX1 gene; Address; Affect; Age; Aging; Animals; Automobile Driving; Base Excision Repairs; Biological; Birth Rate; Cell Aging; Cell division; Cells; Child; Child health care; Chronic; Congenital Abnormality; DNA Damage; Data; Death Rate; Embryonic Development; Enzymes; Fathers; Female; Frequencies; Genetic; Genetic Risk; Genetically Engineered Mouse; Genome; Genotype; Germ Cells; Goals; Hereditary Disease; Hospitalized Child; Human; Infant; Infant Mortality; Knock-out; Knockout Mice; Knowledge; Lead; MDM2 gene; Measures; Mediating; Methods; Modeling; Molecular; Mus; Mutagenesis; Mutation; Parents; Paternal Age; Pathway interactions; Phosphorylation; Play; Population; Prevalence; Proteasome Inhibitor; Proteins; Publishing; Reproduction; Reproductive Health; Role; Source; Spermatocytes; Spermatogenic Cell; Stem cells; TP53 gene; Testing; Tissues; Transgenic Mice; Transgenic Organisms; Tumor Suppressor Proteins; Ubiquitination; Wild Type Mouse; age effect; age related; biological adaptation to stress; cellular engineering; design; disorder risk; driving force; genetically modified cells; genome integrity; inhibitor/antagonist; innovation; insight; male; middle age; multicatalytic endopeptidase complex; mutant; next generation sequencing; novel; statistics; stem cell population; stressor; transcription factor

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.

项目摘要 患有遗传性疾病或出生缺陷的儿童住院年龄较小，住院时间较长，并且有 死亡率高于因其他原因住院的儿童。在美国出生的每33个婴儿中就有一个出生 缺陷;婴儿死亡的头号原因。我们的长期目标是改变这些可怕的统计数据， 描述了通过增加雄性配子的突变来降低游戏质量的机制， 年龄增长，即，父亲的年龄效应随着年龄的增长，父亲年龄效应越来越显著 出生率与年龄较大的父亲有关，并且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，减少碱基切除修复蛋白APE 1，导致减少碱基切除 随着年龄的增长，生精细胞的修复和突变增加。初步数据显示 验证MDM 2泛素化APE 1是由TRP 53 Ser 18/23磷酸化触发的假设 导致APE 1降低。目的1：测试MDM 2是否泛素化APE 1，导致更大量的 老年小鼠生殖细胞中高度泛素化的APE 1，APE 1的蛋白酶体降解， 自发突变频率目的2：测试TRP 53在Ser 18/23处的磷酸化是否触发 老年小鼠生精细胞中APE 1的降解。目的3：测试APE 1的变化是否 丰度和驱动这些变化的机制起源于精原干细胞群。 方法：从携带Mdm 2靶向变化的雄性小鼠中制备确定的生精细胞 和Trp 53来测试这些肿瘤抑制因子是否调节年轻小鼠中APE 1的丰度， 在老年野生型小鼠中长期激活，导致APE 1丰度降低，碱基切除修复减少 和增加的诱变。将检测蛋白酶体降解和MDM 2活性的重要性 使用抑制剂。彗星芯片阵列将分析单细胞中的DNA损伤，以确定是否增加了DNA 损伤可触发TRP 53激活。双链体标签下一代测序将确定 突变频率起始于精原干细胞群体。我们提出了一个新的模型， 肿瘤抑制因子正常发挥保护基因组完整性的作用， 在生殖细胞的独特生物学背景下，通过减少APE 1丰度来提高质量和诱变 和衰老。这是一个范式的转变，从广泛持有的观点突变积累被动干 细胞作为雄性配子遗传质量下降的主要驱动力与老化和TRP 53的作用 和MDM 2作为基因组的保护者。