TUMOR SUPPRESSORS MEDIATE A REDUCTION IN MALE GAMETE QUALITY WITH AGING

Children with a genetic disease or birth defect are hospitalized at a younger age, stay longer, and have ahigher death rate than children hospitalized for other reasons. One in 33 infants born in the US has a birthdefect; the number one cause of infant mortality. Our long-term goal is to change these dire statistics bydelineating the mechanisms that reduce game quality by increasing mutagenesis in male gametes withincreasing age, i.e., the paternal age effect. The paternal age effect is increasingly significant with increasingbirth rates to older fathers, and is directly relevant to male reproductive health and child health. Previouslypublished studies revealed that reduced base excision repair protein APE1, results in reduced base excisionrepair and increased mutagenesis in spermatogenic cells with increasing age. Preliminary data leads us totest the hypothesis that MDM2 ubiquitination of APE1 is triggered by TRP53 Ser18/23 phosphorylationresulting in reduced APE1. Aim 1: Test whether MDM2 ubiquitinates APE1, leading to greater amounts ofhighly ubiquitinated APE1 in germ cells of older mice, proteasomal degradation of APE1, and a greaterspontaneous mutation frequency. Aim 2: Test whether phosphorylation of TRP53 at Ser18/23 triggersdegradation of APE1 in spermatogenic cells from older mice. Aim 3: Test whether changes in APE1abundance, 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 Mdm2and Trp53 to test whether these tumor suppressors regulate APE1 abundance in young mice and becomechronically activated in old wild type mice resulting in reduced APE1 abundance, reduced base excision repairand increased mutagenesis. The importance of proteasome degradation and MDM2 activity will be testedusing inhibitors. CometChip arrays will analyze DNA damage in single cells to determine if increased DNAdamage may trigger TRP53 activation. Duplex tag next generation sequencing will determine if increases inmutation frequency initiate in the spermatogonial stem cell population. We propose a novel model in whichtumor suppressors that function normally to safeguard genome integrity, instead cause decreased gametequality and increased mutagenesis by reducing APE1 abundance in the unique biological context of germ cellsand aging. This is a paradigm shift from the widely-held view of mutations accumulating passively in stemcells as the major driving force of reduced genetic quality in male gametes with aging and for the role of TRP53and MDM2 as protectors of the genome.