Project Summary/Abstract Alzheimer’s disease (AD) and other neurodegenerative diseases are characterized by age-related onset and progressive neuronal loss. Neuroinflammation mediated by disease-associated microglia is increasingly implicated in AD pathogenesis. As the primary immune cells in the brain, abnormally reactive microglia have been recently found to create a neuroinflammatory environment that results in neuronal death, yet the mechanisms by which age and genetic factors interact remain largely unknown. Somatic mutations accumulate in various cell types during the development and aging process of the human body. Clonal expansion, driven by somatic mutations in genes regulating cell proliferation, is associated with an increased risk of cancer with age, but has only recently been linked to a growing list of non-cancer neurological diseases. Notably, somatic BRAF mutation in the microglial lineage has been implicated in histiocytosis-associated neurodegenerative conditions. Our preliminary results from three AD cohorts and with three sequencing technologies consistently show an excess of clonal somatic mutations in AD brains, particularly in proliferation- related genes of microglia. This new study aims to examine if the accumulation of somatic mutation contributes to an age-related increase in AD risk by driving clonal expansion of microglia, which subsequently induces neuroinflammation and neuronal loss in AD brains. The first Aim of the study is to identify somatic mutations by re-analysis of the existing bulk RNA-seq datasets from large AD cohorts, and compare the transcriptome-wide burden and distribution of somatic mutation between different brain regions of AD patients and matched controls. In the second Aim of the study, molecule-barcoded ultra-deep panel sequencing will be applied to screen for somatic mutations more sensitively among genes that regulate cell cycle and proliferation in AD and control brains; we will further use amplicon sequencing to quantify the mutant allele fraction across different cell types sorted from AD brains, to verify if these somatic mutations are specifically carried by microglia. The third Aim of the study will focus on the functional impact of proliferation-related somatic mutations in AD microglia, by using both an in silico method based on AD single-nucleus RNA-seq data and an in vitro method based on a human stem- cell differentiated microglia model. Our proposed study will shed new light on the contribution of somatic mutation to increased AD risk, and highlight the clonal expansion of microglia driven by somatic mutations in proliferation-related genes as a potential mechanism involved in AD pathogenesis.