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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.

项目总结自1980年以来，年长父亲的出生率上升了约60%，而年轻父亲的出生率下降了。的在2015年登记的近400万新生儿中，11%(约44万)是年龄较大的父亲所生。老年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遗传病。这种突变的增加，即父亲的年龄效应，越来越显著，而且直接与男性生殖老化和儿童健康有关。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会导致修复减少老年生精细胞的活性和诱变性增加。我们的初步数据显示随着年龄增长的生殖系突变增加的机制发生在减数分裂粗线期精母细胞和涉及APEX1、TrP53和MDM2，这三种肿瘤抑制因子通常具有防止突变的功能。我们假设TrP53的慢性激活激活了APEX1的MDM2泛素化，导致减少 APEX1.在目标1中，我们将测试Ser18/23处Trp53的磷酸化是否触发APEX1的降解来自老年小鼠的粗线期精母细胞，而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 泛素化APEX1，导致粗线期精母细胞中大量高度泛素化的APEX1，老年小鼠的非圆形精子细胞，APEX1的蛋白酶体降解，以及由此导致的更大的自发性突变频率。在目标3中，基于显示APEX1独特分布的初步数据，我们将测试如果APEX1在减数分裂细胞中与染色质唯一地相互作用。方法：确定特定的富集型人群将从携带MDM2和Trp53靶向基因变化的雄性小鼠中制备生精细胞类型为了测试1)这些肿瘤抑制因子是否会失调老年小鼠的APEX1丰度，以及2)是否会在衰老过程中被长期激活，导致APEX1丰度降低，碱基切除修复减少，和更多的诱变。蛋白酶体降解和MDM2活性的重要性将通过蛋白酶体和MDM2抑制剂。邻近标记技术将在体内用于鉴定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链测序。所提出的研究测试了一种新的模型，在该模型中肿瘤抑制器轴(即TRP53， MDM2和APEX1)通常起到保护基因组完整性的作用，而不是损害配子遗传通过减少APEX1丰度和在独特的生物学背景下导致突变增加来提高质量成熟的减数分裂生殖细胞。如果得到证实，这是一种范式转变，不再是人们普遍认为的主要驱动因素雄配子中突变增加的力量是一种被动积累。拟议的研究将针对驱动突变增加的基本生物学机制，导致父代年龄效应，并可能最终揭示潜在的治疗目标，以防止通过突变介导的遗传病在生精细胞中。