Project Summary/Abstract. Neuroimmune signaling through TLR (Toll-like-receptor) pathways modulate ethanol behaviors in mammals and flies. An output of these pathways are NFκB transcription factors. The fly NFκB gene called Dif is an output of the Toll signaling pathway in adult flies. Dif modulates ethanol sensitivity— increased Dif activity reduces sensitivity, while decreased Dif increases sensitivity. Because NFκB is a well- known transcription factor, it has been axiomatic that its effects are produced by an action in the nucleus. In addition to the well-known nuclear and cytoplasmic locations, NFκB has also been observed at the postsynap- tic density. In mammals, synaptic activity has been described as causing the translocation of NFκB from the synapse to the nucleus where it modifies gene expression in ways that alter synaptic plasticity. However, from Drosophila there is strong evidence that NFκB proteins can directly modulate the synaptic machinery. The Dif gene produces two NFκB protein isoforms by alternative mRNA splicing. These are DifA and DifB. The focus of this proposal is DifB. The DifB isoform is only expressed in Drosophila CNS neurons, where it is specific to the mushroom bodies (well known to influence ethanol behaviors), antennal lobes, and the subesophageal gan- glion of the brain. The DifB isoform lacks a nuclear localization signal and a transcription activation domain. DifB biochemically co-fractionates with a synaptic protein and away from a nuclear protein. Immunohistochemi- cal staining confirms that DifB localizes to synaptic regions but does not enter the nucleus even following im- mune activation. Splice variant–specific mutations show that the DifB isoform modulates the sensitivity to seda- tion with ethanol but does not contribute to immunity. Whereas the nuclear DifA isoform is essential in the immune response but does not contribute to the ethanol response.The objective of this proposal is to identify DifB interacting proteins in order to better predict how DifB modulates ethanol sensitivity. We have used CRISPR to build flies that express a variety of epitope-tagged DifB (& DifA) isoforms. These will be used in coimmunoprecipitation/mass spectrometry experiments to identify proteins that physically interact with DifB. Based on biochemical fractionation, imaging, and evolutionary comparison with a related protein, we ex- pect the interacting proteins to be synaptic proteins. The extragenomic action of an NFκB is an unexplored and rapid way in which Toll pathway signaling can modulate ethanol responses. The description of the protein:pro- tein interactome of the extragenomic NFκB encoded by DifB will be a significant contribution because it will provide us with the keys for understanding how this protein modulates neuronal signaling and behavior. Fur- thermore, understanding how an NF-kB can directly modulate the synapse may also contribute to our under- standing of the origins of the unusual consequences of neu...