7/11 Biochemical and Genetic Determinants of Alcohol Consumption

PROJECT SUMMARY
This project is a continuation and further development of a previous INIA project, which was based on studies
showing ethanol-induced changes in neuroimmune gene expression in animal models and humans. Those
data suggested that ethanol dysregulates Toll-like receptor (TLR) signaling through the myeloid differentiation
primary response gene 88 (MyD88) and thereby promotes excessive ethanol intake. Our most recent work
showed that genetic and pharmacological manipulation of another branch of TLR signaling via the TIR-domain-
containing adapter-inducing interferon-β (TRIF) protein also regulates ethanol intake. In exploring downstream
mechanisms by which these signaling disruptions act, we found that chronic alcohol consumption increased
several TRIF-dependent signaling components, including type 1 interferons (IFN1s). These findings lead us to
the central hypothesis of this renewal application, which is that chronic alcohol exposure activates pathways
leading to IFN1 production and expression of interferon-stimulated genes (ISGs), which increase alcohol
consumption. To test this hypothesis, we will study alcohol intake and alcohol-related behaviors in mice
deficient in critical components of pathways leading to IFN1 production and signaling, and in mice administered
compounds that block IFN1 signaling. The proposal has three Specific Aims. Specific Aim 1 will study
changes in alcohol consumption in mice undergoing every other day two-bottle choice (EOD-2BC) drinking for
at least 4 weeks. To reduce IFN1 production, we will examine these behaviors in mice with genetic deletion of
the transcription factors Irf3 or Irf7. To examine the role of IFN1 signaling, we will use Ifnar1 knockout mice
which carry a null mutation in the IFN1 receptor. We will also study wild-type mice treated with inhibitors of the
kinase TYK2, which mediates IFN1 receptor signaling. Specific Aim 2 will determine if EOD-2BC drinking
induces IFN1 responses in specific brain regions by detecting Fos expression in Mx1GFP mice in which the
interferon-stimulated response element of the Mx1 gene drives GFP expression. Data from these whole brain
imaging studies will be shared with the Harsan-Keiffer project to be incorporated into their multimodal
connectome analyses. To determine if IFN1 regulates alcohol responses in certain regions, we will knockout
Ifnar1 using local microinjection of Cre recombinase in floxed Ifnar mice and use Fos-Cre-ER (TRAP2) mice to
knockout or knockdown Ifnar1 in activated neurons and glia. Specific Aim 3 will identify ISGs induced by
EOD-2BC alcohol drinking in brain regions identified in Aim 2. The Mayfield project will help us analyze
transcriptomic data to identify ISGs. To investigate causality, groups of ISGs will be knocked down using
multiplex CRISPR interference in collaboration with the Farris-Homanics project. We anticipate that some other
INIA projects will identify additional proteins that require behavioral testing with pharmacological agents or
genetically modified mice, which we will carry out as part of this consortium.
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Grant Number: 5U01AA013520-25
NIH Institute/Center: NIH
Principal Investigator: YURI BLEDNOV