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Abstract

Microglia are the resident immune cells in the brain and play a critical role in maintaining homeostasis and regulating neuroinflammation, a hallmark of neurodegenerative diseases like Alzheimer’s disease (AD). These cells constantly monitor their environment and respond to injury or inflammatory stimuli, influencing disease progression. Microglial function is regulated by genetic mechanisms, including enhancers, which are non-coding DNA elements that control gene expression. Recent studies have identified AD-associated single nucleotide polymorphisms (SNPs) in enhancers active in microglia, suggesting that these variants may alter gene expression and contribute to AD pathogenesis. Specifically, the rs1377416 SNP has been linked to increased SPI1 expression and late-onset AD, but the underlying mechanisms remain unclear. The SPI1 gene encodes the transcription factor PU.1, which is essential for microglial differentiation and function. This project explores how the AD-associated SPI1 enhancer variant influences PU.1 levels and microglial-driven inflammation in response to lipopolysaccharide (LPS) stimulation. Using a mouse model carrying the AD-associated SPI1 enhancer variant, this study examines changes in PU.1 protein levels, microglial morphology, and gene expression. Additionally, the impact of the enhancer variant on histone modification markers such as H3K27ac is assessed in microglia. By evaluating the regulation of PU.1 and enhancer activity, this research aims to infer molecular alterations associated with the enhancer variant. RT-qPCR analysis showed a significant effect of LPS treatment on Spi1 and Madd expression, particularly in females. Flow cytometry analyses of PU.1 and H3K27ac expression in microglia and other immune cell types demonstrated no significant main effects or interactions. Morphological analysis of microglia identified a significant increase in hypertrophic microglia following LPS treatment in males and females that was significantly blunted in the male SPI1 mutants. Overall, the findings of this study suggest the enhancer mutation may not have a primary driving role in altering microglial function but instead contributes to a more complex involvement in AD pathology potentially by modulating impacts of inflammatory stimuli on microglial responses.