Blood and Brain Gene Expression Signatures of Chronic Intermittent Ethanol Consumption in Mice

Alcohol Use Disorder (AUD) is a chronic, relapsing syndrome diagnosed by a heterogeneous set of behavioral signs and symptoms. There are no laboratory tests that provide direct objective evidence for diagnosis. Microarray and RNA-Seq technologies enable genome-wide transcriptome profiling at low costs and provide an opportunity to identify biomarkers to facilitate diagnosis, prognosis, and treatment of patients. Brain gene expression patterns can discriminate alcohol-dependent and non-dependent people and predict drugs that reduce drinking in rodents. However, access to brain tissue in living patients is not possible. Blood contains cellular and extracellular RNAs that provide disease-relevant information for some brain diseases. We hypothesized that blood gene expression profiles can be used to diagnose AUD. We profiled brain (prefrontal cortex, amygdala, and hypothalamus) and blood gene expression levels in C57BL/6J mice using RNA-seq one week after chronic intermittent ethanol (CIE) exposure, a mouse model of alcohol dependence. To determine the preservation of gene expression levels between blood and brain, we calculated the Spearman correlation coefficient between blood and brain mean gene expression levels across all subjects and found a high degree of preservation (rho range: [0.50, 0.67]) with hundreds of transcripts in blood correlated with their brain transcript levels. To determine whether the transcriptional response to alcohol dependence was similar in blood and brain, we studied the overlapping differentially expressed genes (DEGs) and gene coexpression networks. Although there was small overlap between blood and brain DEGs, there was considerable overlap of gene networks perturbed after CIE related to cell-cell signaling (e.g., GABA and glutamate receptor signaling, endocannabinoid signaling, synaptogenesis), immune responses (e.g., antigen presentation, communication between innate and adaptive immune systems), and protein processing / mitochondrial functioning (e.g., ubiquitination, unfolded protein responses, oxidative phosphorylation). To determine whether blood gene expression can predict alcohol dependence status, blood gene expression data were used to train classifiers (logistic regression, random forest, and partial least squares discriminant analysis), which were highly accurate at predicting alcohol dependence status (maximum AUC for females: 90.1%; males: 80.5%). These results suggest that gene expression profiles from peripheral blood samples contain a biological signature of alcohol dependence that can discriminate between alcohol-dependent and non-dependent subjects. Overall design: Alcohol dependence was induced using chronic intermittent ethanol (CIE) treatment with voluntary alcohol drinking tests (two bottle choice drinking in the dark 2BC DID) N = 40 C57Bl/6J mice; 10 per group; male and female; alcohol-dependent (ethanol vapor) and non-dependent (air vapor). CIE was implemented as described in previous studies. Thirty minutes before the dark cycle, mice were singly housed for two hours with access to two drinking tubes, one containing 15% ethanol and the other containing water. Ethanol and water consumption during these 2-hour periods was recorded. Following this baseline period of two-bottle choice (2BC) drinking, which lasted 20 days (5 days per week for 4 weeks), mice were divided into two balanced groups with similar distributions of ethanol and water consumption. One group was exposed to intermittent ethanol vapor and the other to control air in identical chambers. The ethanol vapor group was administered 1.75 g/kg ethanol plus 68.1 mg/kg pyrazole to inhibit alcohol dehydrogenase, and then was placed in the chambers to receive intermittent vapor for 4 days (16 hours vapor on, 8 hours vapor off). Once per week, immediately following a 16-hour bout of vapor, mice were removed, and tail blood was sampled for blood alcohol determination. Target blood alcohol levels were 175-250 mg%. Following the fourth day of exposure, mice were allowed 72 hours of undisturbed time. The mice were then given 5 days of 2BC drinking. The control group was injected with 68.1 mg/kg pyrazole in saline and placed in chambers delivering air for the same periods as the ethanol vapor group and then received 2BC testing at the same time as the vapor groups. Mice were subjected to four cycles of vapor or air exposure followed by 5 days of 2BC drinking. All mice then received one final 4-day vapor or air exposure. One week later, mice were euthanized and tissue collected for RNA sequencing.