Role of m6A RNA Modifications in Microglial Dysfunction and Alzheimer's Disease Pathogenesis

ABSTRACT Alzheimer’s Disease (AD) and AD-related dementias (ADRD) are devastating neurodegenerative disorders characterized by progressive cognitive decline and neuroinflammation. While epigenetic and protein-coding gene alterations have been widely studied in relation to brain aging, the role of RNA modifications, particularly N6-methyladenosine (m6A), remains less understood. m6A modifications influence gene expression, RNA stability, and cellular responses, but their specific contribution to AD/ADRD pathogenesis is largely unexplored. Microglia, the brain’s resident immune cells, play a central role in AD, contributing to processes such as amyloid-beta clearance and synaptic remodeling. Many AD-associated genetic risk loci, including TREM2 and APOE, are highly expressed in microglia, and microglia-specific regulatory regions capture a significant proportion of AD heritability. However, the post-transcriptional mechanisms driving microglial responses in AD, particularly through m6A RNA modifications, are poorly understood. Given microglia's critical role in neuroinflammation and AD pathogenesis, understanding how RNA modifications in these cells contribute to disease could reveal novel therapeutic targets. We hypothesize that dysregulation of m6A RNA modifications in microglia plays a key role in AD/ADRD pathogenesis by altering gene expression, disrupting cellular homeostasis, and exacerbating neuroinflammation. To investigate this, we will systematically map m6A RNA modification landscapes in microglia isolated from human brain tissue and human induced pluripotent stem cell (hiPSC)-derived microglia models using advanced long-read RNA sequencing. We will also use CRISPR-based perturbation of m6A writers, erasers, and readers to assess the functional consequences of these RNA modifications on microglial activities such as phagocytosis, cytokine production, and inflammatory responses. Aim 1 will profile m6A RNA modifications in microglia from AD and control brain tissues using long-read RNA sequencing, identifying differential m6A sites and investigating their interactions with AD risk loci to understand how these modifications influence microglial function. Aim 2 will utilize CRISPR-based approaches to perturb key m6A regulatory proteins in hiPSC-derived microglia and assess the impact on functions relevant to AD pathology. By integrating cutting-edge genomic technologies with functional assays, this study will provide crucial insights into how m6A RNA modifications contribute to AD-related neuroinflammation and microglial dysfunction. Ultimately, this research has the potential to uncover novel therapeutic targets, advancing efforts to develop RNA-based interventions for AD/ADRD.