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The Paternal Age Effect - Enhanced Germ Cell Mutagenesis Modulated by the TRP53/APE1/MDM2 Tumor Suppressor Axis

父亲年龄效应 - TRP53/APE1/MDM2 肿瘤抑制轴调节的增强生殖细胞诱变

基本信息

批准号：
10646448
负责人：
Christi A Walter
金额：
$ 35.58万
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：
2020
资助国家：
美国
起止时间：
2020-09-30 至 2025-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10646448
关键词：
Address Age Aging Animals Automobile Driving Base Excision Repairs Biological Birth Birth Rate Cell division Cells Child Child Health Chromatin Chromosomes Chronic DNA Damage DNA Repair DNA sequencing Data Diagnosis Embryonic Development Enzymes Epidemic Fathers Fertility Frequencies Generations Genes Genetic Genetic Diseases Genotype Germ Cells Germ-Line Mutation Goals Heterozygote Immunofluorescence Microscopy Immunoprecipitation Infertility Intervention Knock-out Knockout Mice Label Life Link MDM2 gene Mass Spectrum Analysis Mediating Meiosis Methodology Methods Modeling Mus Mutagenesis Mutation National Institute of Child Health and Human Development Paint Paternal Age Phosphorylation Physiology Play Population Proteins Recurrence Reproduction Research Risk Risk Factors Risk Reduction Role Signal Transduction Site Spermatids Spermatocytes Spermatogenic Cell TP53 gene Testing Transcription Coactivator Transgenic Mice Transgenic Organisms Tumor Suppressor Proteins Ubiquitination age effect autism spectrum disorder cell type chromatin protein cytotoxic driving force endonuclease genome integrity human old age (65+)in vivo inhibitor innovation insight male men middle age multicatalytic endopeptidase complex mutant novel older men overexpression postmitotic preservation repaired reproductive reproductive senescence senescence sperm cell stem cell aging transcription factor young adult young man

项目摘要

PROJECT SUMMARY Birth rates to older fathers increased approximately 60% since 1980, while births to younger fathers declined. Of the nearly 4 million registered births in 2015, 11% (~440,000) were born to older fathers. Sperm from older men have more mutations compared with young men, increasing the risk that their children will be afflicted with a genetic disease. This increase in mutations, the paternal age effect, is increasingly significant and is directly relevant to male reproductive aging and to child health. DNA repair declines in spermatogenic cells as mice age, promoting an increase in mutations. Reduced levels of a base excision repair protein is causal to reduced repair activity and increased mutagenesis in spermatogenic cells at older age. Our preliminary data indicate the mechanisms driving increased germline mutagenesis with aging occur in meiotic pachytene spermatocytes and involve APEX1, TRP53 and MDM2, tumor suppressors that normally function to protect against mutagenesis. We hypothesize that chronic activation of TRP53 activates MDM2 ubiquitination of APEX1, resulting in reduced APEX1. In Aim 1 we will test if phosphorylation of TRP53 at Ser18/23 triggers degradation of APEX1 in pachytene spermatocytes, but not post-meiotic round spermatids, from older mice. In Aim 2. we will test if MDM2 ubiquitinates APEX1, leading to greater amounts of highly ubiquitinated APEX1 in pachytene spermatocytes, not round spermatids, of older mice, proteasomal degradation of APEX1, and a resulting greater spontaneous mutation frequency. In Aim 3, based upon preliminary data showing a unique distribution of APEX1, we will test if APEX1 uniquely interacts with chromatin in meiotic cells. Methods: Defined populations of enriched specific spermatogenic cell types will be prepared from male mice carrying targeted genetic changes in Mdm2 and Trp53 to test whether 1) these tumor suppressors dysregulate APEX1 abundance in old mice, and 2) become chronically activated during aging resulting in decreased APEX1 abundance, decreased base excision repair, and increased mutagenesis. The importance of proteasome degradation and MDM2 activity will be tested using proteasome and MDM2 inhibitors. Proximity labeling will be used in vivo to identify APEX1 interacting proteins in meiotic cells. Age-associated mutation frequency and spectra will be determined for mouse genes using duplex sequencing. The proposed studies test a novel model in which a tumor suppressor axis (i.e. TRP53, MDM2 and APEX1) that normally functions to safeguard genome integrity, instead compromises gamete genetic quality by reducing APEX1 abundance and resulting in elevated mutagenesis in the unique biological context of aging meiotic germ cells. If confirmed, this is a paradigm shift from the widely held view that the major driving force of increased mutations in male gametes is a passive accumulation. The proposed studies will address a fundamental biological mechanism driving increased mutagenesis that leads to the paternal age effect and may ultimately reveal potential treatment targets to protect against genetic diseases mediated through mutagenesis in spermatogenic cells.

项目概要自 1980 年以来，高龄父亲的出生率增加了约 60%，而年轻父亲的出生率则下降。的 2015 年登记的近 400 万新生儿中，11%（约 44 万）是高龄父亲所生。年长男性的精子与年轻男性相比，他们有更多的突变，增加了他们的孩子患上这种疾病的风险遗传病。这种突变的增加，即父亲年龄效应，越来越显着，并且直接影响与男性生殖衰老和儿童健康有关。随着小鼠年龄的增长，生精细胞的 DNA 修复能力下降，促进突变的增加。碱基切除修复蛋白水平降低是修复减少的原因老年生精细胞的活性和诱变增加。我们的初步数据表明随着年龄的增长，驱动种系突变增加的机制发生在减数分裂粗线期精母细胞中，并且涉及 APEX1、TRP53 和 MDM2，这些肿瘤抑制因子通常具有防止突变的作用。我们假设 TRP53 的慢性激活会激活 APEX1 的 MDM2 泛素化，从而导致顶点1。在目标 1 中，我们将测试 Ser18/23 位点的 TRP53 磷酸化是否会触发 APEX1 的降解来自老年小鼠的粗线期精母细胞，但不是减数分裂后的圆形精子细胞。在目标 2 中，我们将测试 MDM2 是否泛素化 APEX1，导致粗线期精母细胞中高度泛素化的 APEX1 数量增加，老年小鼠的精子细胞不呈圆形，APEX1 的蛋白酶体降解，以及由此产生的更大的自发性突变频率。在目标 3 中，根据显示 APEX1 独特分布的初步数据，我们将测试 APEX1 是否与减数分裂细胞中的染色质发生独特的相互作用。方法：定义富集特定群体生精细胞类型将从携带 Mdm2 和 Trp53 目标基因变化的雄性小鼠中制备测试 1) 这些肿瘤抑制因子是否会失调老年小鼠中的 APEX1 丰度，以及 2) 在衰老过程中长期激活，导致 APEX1 丰度下降、碱基切除修复减少，并增加诱变。将使用以下方法测试蛋白酶体降解和 MDM2 活性的重要性蛋白酶体和 MDM2 抑制剂。邻近标记将用于体内鉴定 APEX1 相互作用蛋白在减数分裂细胞中。将使用以下方法确定小鼠基因与年龄相关的突变频率和谱双工测序。拟议的研究测试了一种新模型，其中肿瘤抑制轴（即 TRP53， MDM2 和 APEX1）通常起到保护基因组完整性的作用，但却损害了配子遗传通过降低 APEX1 丰度并导致在独特的生物背景下诱变增加来提高质量衰老的减数分裂生殖细胞。如果得到证实，这将是一种范式转变，与广泛持有的观点相比，即主要驱动力雄配子突变增加的力量是一种被动积累。拟议的研究将解决驱动诱变增加的基本生物学机制导致父亲年龄效应，并可能最终揭示潜在的治疗靶点，以预防通过诱变介导的遗传性疾病在生精细胞中。