Rahim, A.; Zhan, X.; Han, Q.; O'Donnell, A.; Jeong, A.; Madugundu, G.-S.; Pujari, S.; Kruk, M.; Luo, X.; Li, L.; Wu, T. P.; Tretyakova, N. Y.

N6-methyldeoxyadenosine (N6medA) is a recently identified endogenous DNA modification widely found in bacteria, plants, and eukaryotes. In mammals, N6medA has been implicated in brain function, immunity, and response to environmental stress, but its relevance to gene regulation and mammalian aging remains controversial due to its extremely low abundance (< 1 per 10 million adenines) and an uncertainty regarding its genomic origin. We have developed and validated an ultrasensitive isotope dilution nano liquid chromatography-nanospray ionization Orbitrap mass spectrometry methodology to quantify N6medA in genomic DNA. Applying this approach to human prefrontal cortex tissues, we found that genomic N6medA levels increase linearly with chronological age (Pearson correlation coefficient, 0.95). Individuals with mild cognitive impairment (MCI) and Alzheimer's disease (AD) exhibited a trend toward elevated cortical N6medA levels relative to age-matched controls. Genome-wide profiling of N6medA in human prefrontal cortex was conducted using two independent methods: NAME-Seq and MeDIP-Seq, which revealed age associated adenine methylation changes reminiscent of established epigenetic aging signatures such as the 5-methylcytosine clocks. N6medA mapping experiments identified a subset of genomic loci that were altered in MCI and AD. Pathway analysis of cross-validated adenine methylation sites revealed an enrichment of genes involved in neuronal function and age-related neurological processes, including glutamatergic synapse, axon guidance, and long-term depression. Finally, mass-spectrometry-based photoaffinity proteomics with synthetic DNA representing a region of the APP gene identified N6medA reader proteins with known roles in DNA repair, replication and transcription. Together, these findings identify N6medA as an age-associated DNA modification in the human brain and suggest that its accumulation and recognition by specific protein readers may contribute to molecular processes underlying brain aging and age-related neurodegeneration.