Chimera-BONCAT: A novel in vivo model for in-depth characterization of the human microglial response to alcohol

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.