Critical assessment of DNA adenine methylation in brain cells from healthy aging and Alzheimer's disease

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PROJECT SUMMARY/ABSTRACT DNA methylation contributes to epigenetic regulation of many important biological processes. The prevailing dogma is that DNA methylation almost exclusively occurs at the fifth position of cytosine (5mC) in eukaryotes. This dogma has been revised in the past five years as multiple studies reported the existence of N6- methyladenine (6mA) across eukaryotes including the mouse and human genome. A few studies have found evidence that suggests the diverse functions 6mA plays in mammals. Some studies have also reported increased 6mA level in certain neuronal cells and upon social stress, suggesting 6mA may play important roles in health and diseases. However, several studies have challenged the presence of 6mA in mammalian genomes, highlighting multiple sources of confounding factors. The active debate has created unusual confusions in the epigenetic community. Yet, tens of studies continued to report new discoveries about 6mA in the human genome including some papers at high-profile journals. A few ongoing research studies have set out to examine the functional roles of 6mA in brain cells and brain disorders, especially in Alzheimer’s disease. To unambiguously assess the abundance and prevalence of 6mA in the human genome, it is imperative to employ a reliable and sensitive 6mA mapping method. However, past and ongoing studies mostly employ antibody-based methods, which are associated with non-specificity. Although Single Molecule Real-Time sequencing (SMRT-seq) has been widely used to map 6mA at base resolution in bacteria, recent work by us and others have found that existing methods are not sensitive enough for eukaryotic genomes with low 6mA abundance. To address this fundamental technological gap, we will build on our >10-yr experience in SMRT- seq to develop a new method for sensitive 6mA detection, and take a neutral perspective to critically examine 6mA in the human genome and in brain tissues from patients with Alzheimer’s disease (AD), Primary age- related tauopathy (PART, another type of dementia) and controls. This project is significant and very timely because (1) if the novel method supports the existence of 6mA in the human genome, it will provide ongoing and future studies with a much-needed tool to detect 6mA in certain cell types and Alzheimer’s diseases; (2) if the novel method does not support a significant level of 6mA as reported, it will serve as a much-needed direct evidence to clarify the current debate.