DNA Adenine Methylation Clock in Brain Aging and Alzheimer's Disease Progression

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.