Graduation Year

2024

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Molecular Biosciences

Major Professor

Stanley M. Stevens Jr., Ph.D.

Committee Member

Paula Bickford, Ph.D.

Committee Member

Brant Burkhardt, Ph.D.

Committee Member

Bin Liu, Ph.D.

Committee Member

Meera Nanjundan, Ph.D.

Keywords

DDA library generation, DIA-PASEF, Ethanol, Microglia, PBIT, Proteomics

Abstract

Through their involvement in various processes such as phagocytosis of foreign materials and damaged cellular debris, regulatory factor release, and surveillance, the macrophages of the brain, microglia, maintain homeostasis within the central nervous system. These diverse microglial functions therefore require transition to a range of phenotypic states. The epigenetic regulation of this activity, especially through trimethylation of lysine 4 on histone H3, and its regulator, lysine demethylase (KDM)5B, is an emerging research area. KDM5B has been implicated in several diseases including cancer, inflammatory disorders, and alcohol use disorder (AUD), the latter being the focus of this dissertation project. Deep proteomic characterization of microglia and related neuroimmune response to alcohol, provided through mass spectrometry advancements, could allow for crucial developments in AUD research. Here we sought to utilize deep proteomics to characterize the role of microglia and the epigenetic regulator KDM5B to further elucidate key mechanisms of ethanol-induced microglial reactivity. Within this context, enhancement of proteomic methodology included the use of ion mobility fractionation combined with streamlined liquid-phase fractionation to improve peptide separation, which generated a deep protein library from adult-derived mouse microglia. This improved methodology was used for proteomic characterization of KDM5B deficiency and inhibition using in vitro and in vivo microglia, respectively. Microglial proteome coverage was increased to approximately 8,000 proteins in a single shot. Furthermore, genetic reduction in KDM5B blunted in vitro inflammatory pathways overall, while in vitro pharmacological inhibition increased migration and oxidative stress control. In vivo, within the male population of mice, an increase in inflammation-related pathways was observed with both KDM5B inhibition and ethanol treatment while the female population displayed a decreased immune response. By using improved methodology to comprehensively characterize the microglial proteome, we generated key insights into mechanisms involved in KDM5B regulation of microglial reactivity, which could lead to better understanding of epigenetic control of neuroimmune responses in AUD.

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