ABSTRACT
Alcohol use disorder (AUD) is a significant health crisis in the U.S., which can result in numerous damaging
effects on multiple organ systems including the brain. Although various cell-type-specific and associated
molecular mechanisms underlie these negative effects of alcohol in the brain, the neuroimmune response,
modulated in part by microglia, has been considered a key pathological driver during alcohol misuse. Microglia,
the resident immune cells of the brain, exhibit a broad range of reactivity from alcohol exposure that is context-
dependent; however, the full molecular landscape of this phenotypic spectrum has yet to be fully characterized.
Additionally, most studies that have investigated alcohol-induced microglial reactivity utilize rodent animal
models, which have limited translational relevance to neuroimmune-specific outcomes related to AUD in
humans. To address these limitations, we propose a novel in vivo model called Chimera-BioOrthogonal Non-
Canonical Amino acid Tagging (BONCAT), which will allow comprehensive, unbiased, and cell-type-specific
characterization of the human microglial response to alcohol in an in vivo environment. In order to rigorously test
the utility of this innovative model to study human microglial reactivity to alcohol at the proteome level, we have
developed the following Specific Aims: 1) Characterization of human microglia derived from induced pluripotent
stem cells (iPSCs) bearing mutant MetRS (an enzyme necessary to carry out the BONCAT approach) and
determine in vitro reactivity to alcohol and 2) Characterization of the human microglial response to alcohol in vivo
using Chimera-BONCAT, which will utilize a chimeric mouse model with human microglia that are engineered
for BONCAT labeling. This project will be the first of its kind to investigate alcohol-induced reactivity of human
microglia using an in vivo chimeric model as well as a novel approach to selectively enrich human microglia from
the chimeric mouse brain for downstream proteomic analysis. The results from this study will provide key insights
into alcohol-induced phenotypic changes that occur in human microglia with potentially higher translational
values compared to conventional models.
