Advanced paternal age exacerbates neuroinflammation in offspring via m6A modification-mediated intergenerational inheritance

The trend of postponing childbearing age is prevalent worldwide. Advanced paternal age (APA) is associated with adverse pregnancy outcomes and offspring health. However, the underlying mechanism by which paternal aging affects the risk of offspring neuropsychiatric disorders is unclear. Our study aims to explore the behavioral phenotypes and the pathologic epigenetic alterations of APA offspring inherited from aging sperm. Behavioral tests, ELISA assay, immunofluorescence and western blotting were performed on offspring mice. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA immunoprecipitation sequencing (RIP-seq) were used to investigate the modified N6-methyladenosine (m6A) profiles of paternal sperm and offspring hippocampus. Intervention of gene expression by lentivirus and adeno-associated virus in both vivo and vitro examined the potential therapeutic targets of intergenerational inherited neuroinflammation. In our study, APA offspring exhibit cognitive impairment and autism-like behavior. An increase in neuroinflammation in APA offspring is associated with microglial overactivation, which manifests as abnormal morphology and augmented engulfment. MeRIP-seq of F0 sperm and F1 hippocampus reveal that Nr4a2 is hypermethylated with decreased expression in APA offspring involving in synaptic plasticity and microglial function. In addition, Ythdc1, an m6A reader protein, is markedly elevated in aging sperm and remains elevated in adult hippocampus of APA group. Enhanced Ythdc1 recognizes and suppresses the hypermethylated Nr4a2, thereby contributing to the abnormal phenotype in offspring. The overexpression of Ythdc1 triggers microglial activation in vitro and its suppression in the hippocampus of APA progeny alleviates behavioral aberrations and attenuates neuroinflammation. Our study provides additional evidence of the abnormal behavioral phenotypes of APA offspring and reveals potential epigenetic inheritance signatures and targeted genes for future research.