Glutamate transporters around the tripartite synapse

Glutamate is the major excitatory neurotransmitter in the mammalian nervous system. It is inactivated by cellular uptake catalyzed by a family of glutamate transporter proteins (GLT1, GLAST, EAAC1, EAAT4 and EAAT5). The main aim of the present thesis was to determine the contributions of the individual glutamate transporter subtypes to the total glutamate uptake around hippocampal synapses focusing on the EAAC1 subtype and on the mysterious transporter responsible for nerve terminal uptake of glutamate. The first step on this endeavor was to make antibodies to EAAC1. As outlined in Paper I, it turned out to be more difficult to make good antibodies to EAAC1 than to the other glutamate transporters. Specificity testing using tissue from EAAC1 knockout mice as negative controls revealed highly specific interactions with unrelated proteins. Paper II summarizes of the lessons learnt about immunocytochemical specificity testing, and Paper III illustrates how the antigen pre-adsorption test can be misleading. After having overcome methodological problems, we were in position to address the original question. In Paper IV a new procedure for immunoisolation of EAAC1 was developed, and known amounts of pure EAAC1 protein was used as standard to quantify EAAC1 concentrations in brain tissue extracts. EAAC1 was found to be present at 13 μg per gram hippocampal protein. This is 100 times less than GLT1 and argues against a significant contribution of EAAC1 to rapid transmitter activation. EAAC1is selectively expressed in neuronal somata and dendrites throughout the brain, and thereby in a total surface area similar to that of astrocytes. In Paper V we show that nerve terminal glutamate uptake fully depends on GLT1, and that about 10% of hippocampal GLT1 protein is expressed terminals. This also explains why high levels of GLT1 mRNA is present in CA3 pyramidal cells. In Paper VI we describe antibodies to GLT1 splice variants and show that GLT1a represents about 90 % of total hippocampal GLT1, while GLT1b and GLT1c represented 6 and 1 %, respectively. We also provide the first data on the distribution of the GLT1b and show that this variant does not contribute to nerve terminal uptake

Expression of glutamate transporters in mouse liver, kidney, and intestine

Glutamate transport activities have been identified not only in the brain, but also in the liver, kidney, and intestine. Although glutamate transporter distributions in the central nervous system are fairly well known, there are still uncertainties with respect to the distribution of these transporters in peripheral organs. Quantitative information is mostly lacking, and few of the studies have included genetically modified animals as specificity controls. The present study provides validated qualitative and semi-quantitative data on the excitatory amino acid transporter (EAAT)1-3 subtypes in the mouse liver, kidney, and intestine. In agreement with the current view, we found high EAAT3 protein levels in the brush borders of both the distal small intestine and the renal proximal tubules. Neither EAAT1 nor EAAT2 was detected at significant levels in murine kidney or intestine. In contrast, the liver only expressed EAAT2 (but 2 C-terminal splice variants). EAAT2 was detected in the plasma membranes of perivenous hepatocytes. These cells also expressed glutamine synthetase. Conditional deletion of hepatic EAAT2 did neither lead to overt neurological disturbances nor development of fatty liver

Effects of 3 weeks GMP oral administration on glutamatergic parameters in mice neocortex

Overstimulation of the glutamatergic system (excitotoxicity) is involved in various acute and chronic brain diseases. Several studies support the hypothesis that guanosine-5′-monophosphate (GMP) can modulate glutamatergic neurotransmission. The aim of this study was to evaluate the effects of chronically administered GMP on brain cortical glutamatergic parameters in mice. Additionally, we investigated the neuroprotective potential of the GMP treatment submitting cortical brain slices to oxygen and glucose deprivation (OGD). Moreover, measurements of the cerebrospinal fluid (CSF) purine levels were performed after the treatment. Mice received an oral administration of saline or GMP during 3 weeks. GMP significantly decreases the cortical brain glutamate binding and uptake. Accordingly, GMP reduced the immunocontent of the glutamate receptors subunits, NR2A/B and GluR1 (NMDA and AMPA receptors, respectively) and glutamate transporters EAAC1 and GLT1. GMP treatment significantly reduced the immunocontent of PSD-95 while did not affect the content of Snap 25, GLAST and GFAP. Moreover, GMP treatment increased the resistance of neocortex to OGD insult. The chronic GMP administration increased the CSF levels of GMP and its metabolites. Altogether, these findings suggest a potential modulatory role of GMP on neocortex glutamatergic system by promoting functional and plastic changes associated to more resistance of mice neocortex against an in vitro excitotoxicity event

Comparative analysis of antibodies to xCT (Slc7a11): Forewarned is forearmed

The cystine/glutamate antiporter or system Xc- exchanges cystine for glutamate, thereby supporting intracellular glutathione synthesis and nonvesicular glutamate release. The role of system Xc- in neurological disorders can be dual and remains a matter of debate. One important reason for the contradictory findings that have been reported to date is the use of nonspecific anti-xCT (the specific subunit of system Xc-) antibodies. Often studies rely on the predicted molecular weight of 55.5 kDa to identify xCT on Western blots. However, using brain extracts from xCT knockout (xCT(-/-) ) mice as negative controls, we show that xCT migrates as a 35-kDa protein. Misinterpretation of immunoblots leads to incorrect assessment of antibody specificity and thereby to erroneous data interpretation. Here we have verified the specificity of most commonly used commercial and some in-house-developed anti-xCT antibodies by comparing their immunoreactivity in brain tissue of xCT(+/+) and xCT(-/-) mice by Western blotting and immunohistochemistry. The Western blot screening results demonstrate that antibody specificity not only differs between batches produced by immunizing different rabbits with the same antigen but also between bleedings of the same rabbit. Moreover, distinct immunohistochemical protocols have been tested for all the anti-xCT antibodies that were specific on Western blots in order to obtain a specific immunolabeling. Only one of our in-house-developed antibodies could reveal specific xCT labeling and exclusively on acetone-postfixed cryosections. Using this approach, we observed xCT protein expression throughout the mouse forebrain, including cortex, striatum, hippocampus, midbrain, thalamus, and amygdala, with greatest expression in regions facing the cerebrospinal fluid and meninges. J. Comp. Neurol., 2015. © 2015 Wiley Periodicals, Inc.status: publishe