Bioinformatics analyses reveal age-specific neuroimmune modulation as a target for treatment of high ethanol drinking.

BACKGROUND: Use of in silico bioinformatics analyses has led to important leads in the complex nature of alcoholism at the genomic, epigenomic, and proteomic level, but has not previously been successfully translated to the development of effective pharmacotherapies. In this study, a bioinformatics approach led to the discovery of neuroimmune pathways as an age-specific druggable target. Minocycline, a neuroimmune modulator, reduced high ethanol (EtOH) drinking in adult, but not adolescent, mice as predicted a priori. METHODS: Age and sex-divergent effects in alcohol consumption were quantified in FVB/NJ × C57BL/6J F1 mice given access to 20% alcohol using a 4 h/d, 4-day drinking-in-dark (DID) paradigm. In silico bioinformatics pathway overrepresentation analysis for age-specific effects of alcohol in brain was performed using gene expression data collected in control and DID-treated, adolescent and adult, male mice. Minocycline (50 mg/kg i.p., once daily) or saline alone was tested for an effect on EtOH intake in the F1 and C57BL/6J (B6) mice across both age and gender groups. Effects of minocycline on the pharmacokinetic properties of alcohol were evaluated by comparing the rates of EtOH elimination between the saline- and minocycline-treated F1 and B6 mice. RESULTS: Age and gender differences in DID consumption were identified. Only males showed a clear developmental increase difference in drinking over time. In silico analyses revealed neuroimmune-related pathways as significantly overrepresented in adult, but not in adolescent, male mice. As predicted, minocycline treatment reduced drinking in adult, but not adolescent, mice. The age effect was present for both genders, and in both the F1 and B6 mice. Minocycline had no effect on the pharmacokinetic elimination of EtOH. CONCLUSIONS: Our results are a proof of concept that bioinformatics analysis of brain gene expression can lead to the generation of new hypotheses and a positive translational outcome for individualized pharmacotherapeutic treatment of high alcohol consumption.

Alcohol-induced one-carbon metabolism impairment promotes dysfunction of DNA base excision repair in adult brain.

The brain is one of the major targets of chronic alcohol abuse. Yet the fundamental mechanisms underlying alcohol-mediated brain damage remain unclear. The products of alcohol metabolism cause DNA damage, which in conditions of DNA repair dysfunction leads to genomic instability and neural death. We propose that one-carbon metabolism (OCM) impairment associated with long term chronic ethanol intake is a key factor in ethanol-induced neurotoxicity, because OCM provides cells with DNA precursors for DNA repair and methyl groups for DNA methylation, both critical for genomic stability. Using histological (immunohistochemistry and stereological counting) and biochemical assays, we show that 3-week chronic exposure of adult mice to 5% ethanol (Lieber-Decarli diet) results in increased DNA damage, reduced DNA repair, and neuronal death in the brain. These were concomitant with compromised OCM, as evidenced by elevated homocysteine, a marker of OCM dysfunction. We conclude that OCM dysfunction plays a causal role in alcohol-induced genomic instability in the brain because OCM status determines the alcohol effect on DNA damage/repair and genomic stability. Short ethanol exposure, which did not disturb OCM, also did not affect the response to DNA damage, whereas additional OCM disturbance induced by deficiency in a key OCM enzyme, methylenetetrahydrofolate reductase (MTHFR) in Mthfr(+/-) mice, exaggerated the ethanol effect on DNA repair. Thus, the impact of long term ethanol exposure on DNA repair and genomic stability in the brain results from OCM dysfunction, and MTHFR mutations such as Mthfr 677C→T, common in human population, may exaggerate the adverse effects of ethanol on the brain.

An in vitro model for studying the effects of continuous ethanol exposure on N-methyl-D-aspartate receptor function.

Long-term ethanol exposure has deleterious effects on both glial and neuronal function. We assessed alterations in both astrocytic and neuronal viability, and alterations in N-methyl-d-aspartate receptor (NMDAR) function, in cocultures of rat cerebellar granule cells (CGCs) and astrocytes after continuous ethanol exposure (CEE). Treatment of cells with 100 mM EtOH once every 24 h for 4 days resulted in a mean ethanol concentration of 57.3 ± 2.1 mM. Comparisons between control and post-ethanol-treated cells were made 4 days after the last ethanol treatment. CEE did not alter glial cell viability, as indicated by the absence of either changes in astrocytic morphology, actin depolymerization, or disruption of astrocytic intracellular mitochondrial distribution at any day postethanol treatment. The CGCs were healthy and viable after CEE, as indicated by phase-contrast microscopy and the trypan-blue exclusion method. Whole-cell patch-clamp experiments indicated that NMDA-induced currents (I(NMDA)) were altered by CEE treatment. Similar to previous results obtained during the withdrawal phase from chronic ethanol exposure, I(NMDA) from CEE-treated cells were significantly larger than I(NMDA) from NMDARs in control CGCs, but returned to control values by the fourth day post-CEE. However, after the last ethanol dosing and during a time when ethanol concentrations remained high, I(NMDA) were significantly smaller than control values. Identical results were observed in CGCs expressing the NR2A or NR2B subunit. In summary, both neurons and astrocytes remained healthy following exposure to CEE with no signs of neurotoxicity at the cellular level, and modulation of NMDAR function is consistent with findings from prior experiments. Thus, we conclude that the CEE paradigm in glial-neuronal cocultures readily lends itself to long-term in vitro studies of ethanol effects that include glial-neuronal interactions and the ability to study ethanol withdrawal-induced neurotoxicity.