The proposed project addresses the following questions: 1) across different species and different models are there (from the transcriptional perspective) common neuroimmune mechanisms regulating excessive ethanol consumption; 2) do the ?common mechanisms? extend across brain regions; 3) are there differences between males and females in the transcriptional signatures; 4) are long non-coding RNAs relevant to our understanding of the neuroimmune hypothesis; 5) does gene splicing participate in the neuroimmune hypothesis; 6) from addressing 1-5, can we extract and validate specific targets for manipulating excessive consumption. To address these questions, three specific aims have been proposed.
Specific Aim 1 will examine using RNA-Seq the transcriptonal features associated with the risk for developing and the individual variation in mouse lines selected for binge ethanol consumption.
The aim will focus on the high drinking in the dark selected lines (HDID-1 & HDID-2; Crabbe et al. 2012); transcriptional data will be obtained both in ethanol nave animals (risk) and in animals three weeks following the standard 4-day DID trial (individual variation). RNA-Seq data will be obtained from the central nucleus of the amygdala (CeA), the nucleus accumbens shell (NAc-sh) and the ventral tegmental area (VTA). Data are analyzed using a network centric approach that focuses on both gene coexpression and cosplicing (Iancu et al. 2015) and emphasizes the detection of hub genes.
Specific Aim 2 will analyze in collaboration with INIA-Stress samples from rhesus macaques chronically exposed to ethanol. In ongoing studies we have examined the brain transcriptome in both rhesus and cynomolgus macaques chronically and continuously exposed to ethanol; the data strongly point to the involvement of neuroimmune genes. We now propose to extend this work to two new groups of animals. One group will have undergone chronic intermittent exposure (CIE) The second group will be formed from both male and female animals chronically (12 months) exposed to ethanol and controls.
Specific Aim 3 will prioritize and validate targets generated from specific aims 1 and 2. The analysis strategy emphasizes detecting coexpressed or cospliced gene clusters that are closely aligned with excessive ethanol consumption. Neuroimmune-related clusters, especially those that cross species and models, will have the highest priority for further analysis; the very highest priority will go to those clusters for which it appears that the repurposing of existing drugs can be used to affect cluster structure (Mayfield/Farris/Ponomarev node). For some gene clusters, drug repurposing will not be the most efficient strategy for gene manipulation and it will be necessary to rely on gene knockouts & knockins (Homanics node) or vector based approaches (Lasek node). The actual behavioral testing will occur in the Crabbe/Ozburn, Blednov/Messing and Bell nodes. Finally, a special arm of this aim is to provide the Homanics node with lncRNA targets for evaluation. The goal is to find hub lncRNAs within relevant gene modules, focusing on those modules associated with neuroimmune function.
There is now compelling evidence that the brain uses some of the same signaling molecules that are used in the immune system to regulate synaptic function. There is also evidence that some of these immune signaling elements are involved in regulating excessive ethanol consumption. The proposed studies, in collaboration with other members of the INIA consortium, will attempt to find the most relevant of these alcohol-immune signaling pathways and determine if there are drugs which can be repurposed to affect these alcohol-immune signaling pathways.
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