Chronic Voluntary Binge Ethanol Consumption Causes Sex-Specific Differences in Microglial Signaling Pathways and Withdrawal-associated Behaviors in Mice.

BACKGROUND: Microglia are the resident immune cells in the brain where they play essential roles in the development and maintenance of physiological functions of this organ. Aberrant activation of microglia is speculated to be involved in the pathogenesis of a variety of neurological disorders, including alcohol use disorders. Repeated binge ethanol (EtOH) consumption can have a profound impact on the function and integrity of the brain resulting in changes in behaviors such as withdrawal and reward. However, the microglial molecular and cellular pathways associated with EtOH binge consumption remain poorly understood. METHOD: In this study, adult C57BL/6J male and female mice were subjected daily to a gelatin-based drinking-in-the-dark voluntary EtOH consumption paradigm (3 h/d for 4 months) to characterize EtOH consumption and withdrawal-associated and anxiety-like behaviors. Brain microglia were isolated at the end and analyzed for protein expression profile changes using unbiased mass spectrometry-based proteomic analysis. RESULTS: Both male and female mice consistently consumed binge quantities of EtOH daily, resulting in blood EtOH levels > 80 mg/dl measured at the end of the 3-hour daily consumption period. Although female mice consumed a significantly greater amount of EtOH than male mice, EtOH withdrawal-associated anxiety-like behaviors measured by marble-burying, light-dark box, and elevated plus maze tests were predominantly observed in male mice. Proteomic analysis of microglia isolated from the brains of animals at the end of the 4-month binge EtOH consumption identified 117 and 37 proteins that were significantly up- or downregulated in EtOH-exposed male and female mice, respectively, compared to their pair-fed controls. Protein expression profile-based pathway analysis identified several cellular pathways that may underlie the sex-specific and EtOH withdrawal-associated behavioral abnormalities. CONCLUSION: Taken together, our findings revealed sex-specific changes in EtOH withdrawal-associated behaviors and signaling pathways in the mouse brain microglia and may help advance our understanding of the molecular, cellular, and behavioral changes related to human binge EtOH consumption.

Evaluation of a method for nitrotyrosine site identification and relative quantitation using a stable isotope-labeled nitrated spike-in standard and high resolution fourier transform MS and MS/MS analysis.

The overproduction of reactive oxygen and nitrogen species (ROS and RNS) can have deleterious effects in the cell, including structural and possible activity-altering modifications to proteins. Peroxynitrite is one such RNS that can result in a specific protein modification, nitration of tyrosine residues to form nitrotyrosine, and to date, the identification of nitrotyrosine sites in proteins continues to be a major analytical challenge. We have developed a method by which 15N-labeled nitrotyrosine groups are generated on peptide or protein standards using stable isotope-labeled peroxynitrite (O15NOO-), and the resulting standard is mixed with representative samples in which nitrotyrosine formation is to be measured by mass spectrometry (MS). Nitropeptide MS/MS spectra are filtered using high mass accuracy Fourier transform MS (FTMS) detection of the nitrotyrosine immonium ion. Given that the nitropeptide pair is co-isolated for MS/MS fragmentation, the nitrotyrosine immonium ions (at m/z=181 or 182) can be used for relative quantitation with negligible isotopic interference at a mass resolution of greater than 50,000 (FWHM, full width at half-maximum). Furthermore, the standard potentially allows for the increased signal of nitrotyrosine-containing peptides, thus facilitating selection for MS/MS in a data-dependent mode of acquisition. We have evaluated the methodology in terms of nitrotyrosine site identification and relative quantitation using nitrated peptide and protein standards.