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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要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，并且住院时间较长。死亡率高于因其他原因住院的儿童。美国每 33 名婴儿中就有 1 名出生缺点;婴儿死亡的第一大原因。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：描述了通过增加雄性配子的诱变来降低游戏质量的机制年龄增加，即父亲年龄效应。随着父亲年龄的增长，父亲年龄的影响越来越显着。高龄父亲的出生率，与男性生殖健康和儿童健康直接相关。之前已发表的研究表明，碱基切除修复蛋白 APE1 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。初步数据引导我们检验 APE1 的 MDM2 泛素化是由 TRP53 Ser18/23 磷酸化触发的假设导致 APE1 减少。目标 1：测试 MDM2 是否泛素化 APE1，从而导致更多的老年小鼠生殖细胞中 APE1 高度泛素化、APE1 的蛋白酶体降解以及更大的影响自发突变频率。目标 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是否发生变化丰度以及驱动这些变化的机制起源于精原干细胞群。方法：从携带 Mdm2 靶向变化的雄性小鼠中制备确定的生精细胞和 Trp53 来测试这些肿瘤抑制因子是否调节年轻小鼠中的 APE1 丰度并成为在年老的野生型小鼠中长期激活，导致 APE1 丰度减少，碱基切除修复减少并增加诱变。将测试蛋白酶体降解和 MDM2 活性的重要性使用抑制剂。 CometChip 阵列将分析单个细胞中的 DNA 损伤，以确定 DNA 是否增加损伤可能会触发 TRP53 激活。双工标签下一代测序将确定是否增加突变频率在精原干细胞群中起始。我们提出了一种新颖的模型，其中肿瘤抑制因子正常发挥作用以保护基因组完整性，但却导致配子减少通过降低生殖细胞独特生物背景中的 APE1 丰度来提高质量并增加诱变和老化。这是对突变在干细胞中被动积累的广泛持有观点的范式转变。细胞是雄性配子遗传质量随着衰老而降低的主要驱动力以及 TRP53 的作用和 MDM2 作为基因组的保护者。