Transcriptome-wide mapping of brain m6A methylome in Alzheimer’s disease - Alzheimer’s disease (AD) affects over 55 million people worldwide, and this number is expected to nearly triple by 2050. AD is characterized clinically by progressive cognitive decline, and pathologically by amyloid plaques, neurofibrillary tangles, and loss of neurons in the brain. A thorough understanding of its mechanism is a prerequisite for discovering effective therapeutic interventions. N6-methyladenosine (m6A) is the most prevalent post-transcriptional RNA modification that plays important roles in gene regulation and many other biological processes including neurodevelopment, learning and memory. m6A is particularly abundant in the brain and its dysregulation has been associated with neurological disorders. Yet, we still do not have a complete map of m6A in the brain of older individuals and the mechanism by which m6A dysregulation may causally contribute to AD also remains an enigma. We have recently developed several novel technologies for transcriptome-wide quantitative m6A profiling at base-resolution. We have also validated the methods in a pilot study consisting of 60 postmortem prefrontal cortex and identified multiple m6A alterations associated with cognitive phenotypes and AD neuropathology (e.g., amyloid-β, tau tangles). Using these novel technologies, the current study will test the hypothesis that brain m6A dysregulation is causally implicated in AD pathology. Our objectives here are to 1) create the first high-resolution reference map of brain m6A methylome (i.e., all m6A sites in brain) in the brain of older adults; 2) identify specific m6A alterations associated with AD and its clinical and pathological endophenotypes; and 3) elucidate the mechanism by which m6A dysregulation may causally contribute to AD pathogenesis. To achieve these goals, we leverage a large collection of human postmortem brain tissue samples (dorsolateral prefrontal cortex) in two community-based cohorts of aging and dementia: Religious Orders Study (ROS) and Rush Memory and Aging Project (MAP). Deep clinicopathological phenotypes and rich brain omics data sets (e.g., GWAS, epigenomics, transcriptomics, proteomics) are available in both cohorts. In Aim 1, we will perform quantitative profiling of brain m6A methylome and identify specific m6A alterations associated with AD neuropathology. Aim 2 will integrate m6A data with other brain omics data, including genomics (GWAS), epigenomics (DNA methylation, histone acetylation, and miRNA), transcriptomics (RNA-seq) and proteomics, in the same brain cortex of same individuals, to decipher the mechanism by which altered m6A methylation may trigger AD pathology. In Aim 3, we will use a dCas13b-FTO fusion for m6A editing in human induced pluripotent stem cells (iPSC)-derived neurons to functionally validate the top-ranked genes and determine the causal role of m6A dysregulation in AD pathogenesis. Such results will provide novel mechanistic insight into the role of m6A dysregulation in AD pathology and inform the development of therapeutic interventions targeting m6A and its regulatory pathways for AD treatment.
