Functional epitranscriptomic profiling of m6A RNA modifications in Alzheimer's Disease - PROJECT SUMMARY/ABSTRACT N6-methyladenosine (m6A) is a post-transcriptional RNA modification. Understanding of its role in synaptic regulation of neurons is rapidly evolving. We discovered that m6A modifications are increased in Alzheimer’s Disease (AD), the most predominant neurodegenerative disorder marked by amyloid beta (Aβ) accumulation, tau aggregation, and synaptic deterioration. We determined that m6A-modified RNA and tau aggregates directly interact in AD. These data led me to hypothesize that pathological tau alters the m6A epitranscriptome in AD, which I will begin to investigate by (aim 1) determining which m6A-modified sites and transcripts are changed in human post-mortem AD brains. My preliminary m6A-RIP-seq studies using human and mouse AD brains suggest that differentially methylated transcripts in AD are associated with synaptic dysregulation. I will dissect this finding by (aim 2) determining the relative contributions of pathological Aβ and tau in driving disease-linked changed in m6A-modified transcripts, particularly between synaptosomal and non-synaptosomal RNA preps. To achieve these goals, I will perform a novel and robust m6A sequencing technique that I have already optimized, termed DART-seq, on both human post-mortem AD brains and brains from mouse models expressing pathological Aβ and tau. Next, I will apply computational pipelines to analyze the identity and infer function of m6A-modified transcripts, cross-reference these results to public AD databases, and investigate these results in a synaptic context. The fellowship training plan will extend my training to include translational application by processing human post-mortem brain tissue, computational transcriptomics by generating RNA-seq datasets, bioinformatic approaches by analyzing and interpreting RNA-seq datasets, data integration by cross-referencing DART-seq results to publicly available AD datasets, subcellular fractionation by isolating synaptosomes, and scientific communication by disseminating my results through conference presentations, seminars, and publications. The environment of the research training will occur at Boston University, Massachusetts, an extraordinary institution located in one of the top global scientific hubs rich with faculty, facilities, and technologies pertaining to the advancement of neurodegenerative breakthroughs. The research training will take place in the laboratory of the sponsor and advisor, Dr. Benjamin Wolozin, an internationally acclaimed neuroscientist with decades of highly impactful work investigating the molecular and cellular underpinnings of neurodegeneration. Broadly stated, the long-term objectives of this proposal are two-fold: 1) elucidate the key sites and transcripts that become differentially m6A-modified in AD and in response to tau aggregation, 2) determine the functional significance of these differential m6A-modified transcripts in synaptic expression, localization, and translation. These objectives align with the mission of NIA to provide fundamental knowledge about the nature of aging and how the m6A epitranscriptome changes in AD.
