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：
10264033
负责人：
Christi A Walter
金额：
$ 36.07万
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/10264033
关键词：
APEX1 gene 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 Genetic Risk Genotype Germ Cells Germ-Line Mutation Goals Immunofluorescence Microscopy Immunoprecipitation Infertility Intervention Knock-out Knockout Mice Label Life Link MDM2 gene Mass Spectrum Analysis Mediating Meiosis Methodology Methods Mitotic Modeling Mus Mutagenesis Mutation National Institute of Child Health and Human Development Paint Paternal Age Phosphorylation Physiology Play Population Proteins Reproduction Research Risk Risk Factors 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 base cell type chromatin protein cytotoxic driving force endonuclease genome integrity human old age (65+)in vivo inhibitor/antagonist innovation insight male men middle age multicatalytic endopeptidase complex mutant novel older men overexpression 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.

项目摘要自1980年以来，年龄较大的父亲的出生率增长了约60％，而对年轻父亲的出生下降。的 2015年的近400万注册出生，较老的父亲出生了11％（〜440,000）。老年男子的精子与年轻人相比，有更多的突变，增加了孩子的风险遗传疾病。突变的增加，父亲年龄的效应，越来越重要，直接与男性生殖衰老和儿童健康有关。随着小鼠的年龄，DNA修复在生精细胞中的下降，促进突变的增加。碱基切除修复蛋白的降低是为了减少修复的因果年龄较大的精子生成细胞中的活性和诱变增加。我们的初步数据表明在减数分裂的精子细胞中，驱动的机制随着衰老而增加了种系诱变，并且涉及APEX1，TRP53和MDM2，通常功能可防止诱变的肿瘤抑制剂。我们假设TRP53的慢性激活激活了Apex1的MDM2泛素化，从而减少了 Apex1。在AIM 1中，我们将测试在Ser18/23处Trp53的磷酸化是否在Apex1中的触发下降 pachytene的精子细胞，但不是来自老鼠的脂肪生成后圆形精子。在AIM 2中。我们将测试MDM2是否泛素化Apex1，导致pachytene精子细胞中的高度泛素化APEX1，不是圆形的小鼠的圆形精子，Apex1的蛋白酶体降解和自发性更大的自发性突变频率。在AIM 3中，根据显示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相互作用。方法：定义的富集特定种群将从携带MDM2和TRP53中靶向遗传变化的雄性小鼠制备精子细胞类型测试1）这些肿瘤抑制剂是否使老鼠的Apex1丰度失调，而2）长期激活在衰老过程中，导致Apex1丰度降低，碱性切除修复减少，并增加诱变。蛋白酶体降解和MDM2活性的重要性将使用蛋白酶体和MDM2抑制剂。接近标记将在体内使用来识别APEX1相互作用蛋白在减数分裂细胞中。使用年龄相关的突变频率和光谱将用于小鼠基因双工测序。提出的研究测试了一个新型模型，其中肿瘤抑制轴（即TRP53， MDM2和APEX1）通常起作用以保护基因组完整性，而是损害了配子遗传通过减少Apex1丰度，并在独特的生物学环境中升高诱变来质量减素生殖细胞衰老。如果得到确认，这是从主要驾驶的广泛观点的范式转变男配子中突变增加的力是被动积累。拟议的研究将解决驱动的基本生物学机制增加了诱变，从而导致父亲年龄的影响，并且可能最终揭示了潜在的治疗靶标，以防止通过诱变介导的遗传疾病在生精细胞中。