Doctor of Philosophy (Ph.D.)
Degree Granting Department
Biology (Cell Biology, Microbiology, Molecular Biology)
Brant Burkhardt, Ph.D.
Stanley Stevens, Ph.D.
Paula Bickford, Ph.D.
Bin Liu, Ph.D.
Epigenetics, Ethanol, LPS, Microglia, Proteomics
Microglia, the resident immune cells of the brain, can exhibit a broad range of activation phenotypes and have been implicated in several diseases and disorders of the central nervous system. Here, we described a method optimized for sensitive and rapid quantitative proteomic analysis of microglia that involves suspension trapping (S-Trap) for efficient and reproducible protein extraction from a microglial cell count expected from an individual mouse brain (~300K) while also simultaneously providing the first comprehensive proteomic characterization of a novel adult-derived mouse microglial cell line. This enhanced method was used throughout all subsequent works and was especially necessary when we attempted to fully characterize ethanol-exposed primary microglia and microglia exposed to the pro-inflammatory stimulus, LPS.
This global study yielded the deepest proteome coverage to date of microglia after acute ethanol exposure and showed clear and distinct differences between ethanol induced alternative activation of microglia and the pro-inflammatory activation of microglia induced by LPS. The proteomic dataset generated from this study helped to further support that acute ethanol exposure produces an alternative activation phenotype in microglia that does not fully align with the full activation that can be detrimental to neuronal function. Several predicted pathways and downstream functions could be considered targets for mechanistic understanding of ethanol-induced microglial activation including inhibition of pathways such as cell migration and phagocytosis.
Finally, we applied both the methods and novel pathways to investigate a more mechanistic hypothesis behind ethanol-induced microglial activation. Novel predicted downstream functional outcomes such as inhibition of phagocytosis and cell migration were identified. Additionally, previous data from our lab, which suggests the possible role of histone methylation, in particular H3K4me3 through the activity of the histone demethylase KDM5B, was hypothesized as an upstream regulator associated with microglial activation. Taken together, these pathways could be responsible for the phenotypic changes found in ethanol-induced microglial activation. Therefore, we aimed to determine a possible mechanism for ethanol-induced microglial activation and how ethanol affects microglial response to a pro-inflammatory stimulus. Specifically, we investigated the role of H3K4me3 in LPS- and ethanol-treated mouse primary microglia and determined genes associated with this mark through ChIP-seq analysis and how enrichment of this mark is altered based on these treatments. Overlap of gene/gene promoters identified by ChIP-seq analysis with differentially expressed proteins identified by proteomics provided insight into the transcriptional regulation associated with H3K4me3 in the context of LPS- and ethanol-induced microglial activation phenotype. Lastly, in terms of downstream functional outcomes associated with ethanol-treated microglia, we focused on ethanol effects on phagocytosis and inflammatory response predicted from our proteomic data and the possible role of TREM2 (an important regulator of microglial phagocytosis and immune response).
Scholar Commons Citation
Guergues, Jennifer Guergues, "Advanced Proteomic and Epigenetic Characterization of Ethanol-Induced Microglial Activation" (2019). Graduate Theses and Dissertations.