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

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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.