br Materials and methods br Results

Materials and methods
Results Analysis of the post-mortem human prefrontal cortex by western blotting indicated that GLAST expression was increased 1.6-fold in the extracts of alcoholic brains relative to controls (Fig. 1A and B). To resolve the distinct sub-pools of GLAST within our samples we have extracted protein separately as cytosolic and microsomal fractions (which would include plasma membranes; PM) and studied GLAST expression in both extracts. Alcohol appeared to cause a moderate if any increase (not statistically significant at P < 0.05) in GLAST expression in cytosolic fraction but the increase was more pronounced (2.2 fold) in the plasma membrane fraction (Fig. 1A and B). GLAST (EAAT1) is normally expressed in astrocytes (review: Šerý et al., 2015) but our data come from whole tissue, including both astrocytes and neurons (as well as from other structures present in cgrp such as oligodendrocytes and blood vessels). The data in Fig. 1 are, therefore, best interpreted as virtually all of the alcohol-induced GLAST being located in astrocytic plasma membranes. The GLAST protein complex was immunoisolated by anti-GLAST antibody and the isolated protein was further analysed through shotgun proteomics. The analysis detected 48 proteins in anti-rabbit GLAST samples and 10 proteins were in rabbit IgG (negative control); these 10 proteins were eliminated as not uniquely related to GLAST. The remaining 38 proteins were considered specifically bound by GLAST (Table 2). These GLAST-associated proteins can be broadly classified as being involved in cell structure (6 proteins; 16%), energy and general metabolism (18 proteins; 47%), neurotransmitter (glutamate and GABA) metabolism (three proteins; 8%), signalling (6 proteins: 16%), neurotransmitter storage/release at synapses (three proteins; 8%) and calcium buffering (two proteins; 5%).
Discussion
Conclusion Overall, our findings suggest that the complexity of the GLAST interactome that we identify in this study may mean that any alcohol-related changes in GLAST expression and cytoplasmic versus plasmalemmal distribution that we have found are likely to have widely ramified effects on astrocyte biology and function. It is unlikely that the changes in glutamate transport will be the only parameter that is impacted since the co-associated proteins are also likely to change their cellular distributions and abundance if the molecular interactions with GLAST that we identify are indicative of functional biologically relevant interactions at the level of molecular pathways. Accordingly, the data presented here may serve to expose further molecular pathways – such as energy metabolism – that may be significant when studying the effects of alcohol on brain. Conversely, some of the interactions may be indicative of a convergence in mechanisms with other types of brain injury where profound changes in proteins such as glutamine synthetase (an enzyme in glial cells to which GLAST supplies glutamate; Table 2) that have also been described both in alcoholism and neurodegenerative disease (Matsuda-Matsumoto et al., 2007; Lee et al., 2010), observations which may highlight pathological changes in astrocytes in multiple disease states.
Introduction The amino acid l-glutamate is considered a major mediator of excitatory signaling in the central nervous system (CNS), including the retina from different species (Martins and Pearson, 2008, de Souza et al., 2012). Glutamatergic activity is mediated by a variety of ionotropic and metabotropic receptors in the CNS. Ionotropic N-methyl-d-aspartate (NMDA) receptors are involved in many events during development, including dendritic spine formation, maintenance and remodeling (McKinney, 2010). Prolonged activation of ionotropic glutamate receptors can lead to excitotoxicity (Ferreira et al., 1996). Therefore its extracellular levels must be highly regulated in order to avoid neuronal injury (Ishikawa, 2013).