Ethanol exposure induces neurotoxicity that is related to neuroinflammation. Microglia serve as immune cells in the central nervous system (CNS) and have been implicated as the primary contributor to oxidative and nitrosative stress on neurons;however, little is known regarding the role of microglia in alcohol-induced neuronal dysfunction, especially in the context of neuron-microglia interplay. In this proposal, we will focus on elucidating the role of nitrosative stress in alcohol-induced microglial activation that ultimately leads to subsequent neuronal injury. We will employ novel mass spectrometric and proteomic methods to identify and quantify differential protein expression and changes in nitrosative stress-related protein modifications to test our working hypothesis that alcohol-induced neurotoxicity is mediated, at least in part, by nitrosative injury from microglial activation whichis in turn modulated by interaction with surrounding neurons. We propose two specific aims to determine the response of microglia to ethanol exposure and how that response is modified by interaction with neurons, and define the role of nitrosative protein modification in cellular dysfunction induced by ethanol exposure. In SA1, we will employ stable isotope labeling with amino acids in cell culture (SILAC) followed by quantitative mass spectrometric analysis to profile differential protein expression in these cell culture systems. In SA2, novel activity-based protein profiling (ABPP) probes will be utilized to accurately measure (through affinity purification followed by semi-quantitative mass spectrometry or fluorescence visualization) a pro-inflammatory protein marker, inducible nitric oxide synthase, in the cell culture models described in SA1. The results from our proposed studies will provide further insight into the role of microglia in ethanol-induced neurodegeneration and identify potential targets for new therapeutic strategies for the treatment or prevention of neuronal injury brought about by excessive and long-term alcohol consumption.
Details regarding the central nervous system (CNS) response to chronic consumption of alcohol and subsequent impact on neuronal function are unclear but a growing body of evidence suggests that neuroinflammation mediated by microglia (the resident immune cells in the brain) plays an important role in this process. The goal of this research is to define the role of microglia in ethanol-induced neurotoxicity using cutting-edge technologies to identify changes in protein abundance as well as changes in oxidative stress- induced modifications of CNS-related proteins on a global scale in order to survey the potential impact of alcohol on activation of microglia and subsequent factors leading to neuronal injury. We anticipate that the results of our proposed studies will lead to the identification of novel therapeutic strategies for the management of alcoholism or treatment of alcohol-induced tissue injury.
|Zhang, Ping; Culver-Cochran, Ashley E; Stevens Jr, Stanley M et al. (2016) Characterization of a SILAC method for proteomic analysis of primary rat microglia. Proteomics 16:1341-6|
|Mahajan, Shikha; Manetsch, Roman; Merkler, David J et al. (2015) Synthesis and evaluation of a novel adenosine-ribose probe for global-scale profiling of nucleoside and nucleotide-binding proteins. PLoS One 10:e0115644|
|Bell-Temin, Harris; Culver-Cochran, Ashley E; Chaput, Dale et al. (2015) Novel Molecular Insights into Classical and Alternative Activation States of Microglia as Revealed by Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-based Proteomics. Mol Cell Proteomics 14:3173-84|
|Seeley, Kent W; Fertig, Alison R; Dufresne, Craig P et al. (2014) 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. Int J Mol Sci 15:6265-85|