Semi-quantitative distribution of excitatory amino acid (glutamate) transporters 1-3 (EAAT1-3) and the cystine-glutamate exchanger (xCT) in the adult murine spinal cord.

Proper glutamatergic neurotransmission requires a balance between glutamate release and removal. The removal is mainly catalyzed by the glutamate transporters EAAT1-3, while the glutamate-cystine exchanger (system xc- with specific subunit xCT) represents one of the release mechanisms. Previous studies of the spinal cord have focused on the cellular distribution of EAAT1-3 with special reference to the dorsal horn, but have not provided quantitative data and have not systematically compared multiple segments. Here we have studied the distribution of EAAT1-3 and xCT in sections of multiple spinal cord segments using knockout tissue as negative controls. EAAT2 and EAAT3 were evenly expressed in all gray matter areas at all segmental levels, albeit with slightly higher levels in laminae 1-4 (dorsal horn). Somewhat higher levels of EAAT2 were also seen in lamina 9 (ventral horn), while EAAT3 was also detected in the lateral spinal nucleus. EAAT1 was concentrated in laminae 1-3, lamina 10, the intermediolateral nucleus and the sacral parasympathetic nucleus, while xCT was concentrated in laminae 1-3, lamina 10 and the leptomeninges. The levels of these four transporters were low in white matter, which represents 42% of the spinal cord volume. Quantitative immunoblotting revealed that the average level of EAAT1 in the whole spinal cord was 0.6 ± 0.1% of that in the cerebellum, while the levels of EAAT2, EAAT3 and xCT were, respectively, 41.6 ± 12%, 39.8 ± 7.6%, and 30.8 ± 4.3% of the levels in the hippocampus (mean values ± SEM). Conclusions: Because the hippocampal tissue content of EAAT2 protein is two orders of magnitude higher than the content of the EAAT3, it follows that most of the gray matter in the spinal cord depends almost exclusively on EAAT2 for glutamate removal, while the lamina involved in the processing of autonomic and nociceptive information rely on a complex system of transporters.

SLC1A1, SLC16A9, and CNTN3 Are Potential Biomarkers for the Occurrence of Colorectal Cancer.

BACKGROUND: This study is aimed at identifying unknown clinically relevant genes involved in colorectal cancer using bioinformatics analysis. METHODS: Original microarray datasets GSE107499 (ulcerative colitis), GSE8671 (colorectal adenoma), and GSE32323 (colorectal cancer) were downloaded from the Gene Expression Omnibus. Common differentially expressed genes were filtered from the three datasets above. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed, followed by construction of a protein-protein interaction network to identify hub genes. Kaplan-Meier survival analysis and TIMER database analysis were used to screen the genes related to the prognosis and tumour-infiltrating immune cells of colorectal cancer. Receiver operating characteristic curves were used to assess whether the genes could be used as markers for the diagnosis of ulcerative colitis, colorectal adenoma, and colorectal cancer. RESULTS: A total of 237 differentially expressed genes common to the three datasets were identified, of which 60 were upregulated, 125 were downregulated, and 52 genes that were inconsistently up- and downregulated. Common differentially expressed genes were mainly enriched in the cellular component of extracellular exosome and integral component of membrane categories. Eight hub genes, i.e., CXCL3, CXCL8, CEACAM7, CNTN3, SLC1A1, SLC16A9, SLC4A4, and TIMP1, were related to the prognosis and tumour-infiltrating immune cells of colorectal cancer, and these genes have diagnostic value for ulcerative colitis, colorectal adenoma, and colorectal cancer. CONCLUSION: Three novel genes, CNTN3, SLC1A1, and SLC16A9 were shown to have diagnostic value with respect to the occurrence of colorectal cancer and should be verified in future studies.

Expression and distribution of 'high affinity' glutamate transporters GLT1, GLAST, EAAC1 and of GCPII in the rat peripheral nervous system.

l-Glutamate is one of the major excitatory neurotransmitters in the mammalian central nervous system, but recently it has been shown to have a role also in the transduction of sensory input at the periphery, and in particular in the nociceptive pathway. An excess of glutamate is implicated in cases of peripheral neuropathies as well. Conventional therapeutic approaches for treating these diseases have focused on blocking glutamate receptors with small molecules or on reducing its synthesis of the receptors through the inhibition of glutamate carboxypeptidase II (GCPII), the enzyme that generates glutamate. In vivo studies have demonstrated that the pharmacological inhibition of GCPII can either prevent or treat the peripheral nerve changes in both BB/Wor and chemically induced diabetes in rats. In this study, we characterized the expression and distribution of glutamate transporters GLT1, GLAST, EAAC1 and of the enzyme GCPII in the peripheral nervous system of female Wistar rats. Immunoblotting results demonstrated that all glutamate transporters and GCPII are present in dorsal root ganglia (DRG) and the sciatic nerve. Immunofluorescence localization studies revealed that both DRG and sciatic nerves were immunopositive for all glutamate transporters and for GCPII. In DRG, satellite cells were positive for GLT1 and GCPII, whereas sensory neurons were positive for EAAC1. GLAST was localized in both neurons and satellite cells. In the sciatic nerve, GLT1 and GCPII were expressed in the cytoplasm of Schwann cells, whereas GLAST and EAAC1 stained the myelin layer. Our results give for the first time a complete characterization of the glutamate transporter system in the peripheral nervous system. Therefore, they are important both for understanding glutamatergic signalling in the PNS and for establishing new strategies to treat peripheral neuropathies.

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.

Deficits of neuronal glutamatergic markers in the caudate nucleus in schizophrenia.

Abnormal glutamate neurotransmission has been implicated in the pathophysiology of schizophrenia. In the present study we investigated two potential neuronal glutamatergic markers, the Excitatory Amino Acid Transporter 3 (EAAT3) and the Vesicular Glutamate Transporter 1 (VGluT1), in post-mortem striatal tissue from control subjects and from subjects with schizophrenia (n = 15 per group). We also investigated the possible influence of chronic antipsychotic administration (typical and atypical) on striatal VGluT1 expression in the rat brain. We found deficits in EAAT3 in all striatal regions examined in schizophrenia when compared to controls. Following correction for confounding factors (post-mortem interval), these deficits only remained significant in the caudate nucleus (p = 0.019). We also found significant deficits in VGluT1 in the caudate nucleus (p = 0.009) in schizophrenia. There were no significant differences in VGluT1 in the striatum of antipsychotic treated rats when compared to their vehicle treated controls. The data provides additional evidence for a glutamatergic synaptic pathology in the caudate nucleus in schizophrenia and may reflect a loss of glutamatergic cortico-striatal pathways. The absence of an effect of antipsychotic administration on VGluT1 indicates that the deficits in schizophrenia are unlikely to be a consequence of pharmacotherapy and thus likely to be a correlate of the disease process.

Specificity controls for immunocytochemistry: the antigen preadsorption test can lead to inaccurate assessment of antibody specificity.

The biomedical research community relies directly or indirectly on immunocytochemical data. Unfortunately, validation of labeling specificity is difficult. A common specificity test is the preadsorption test. This test was intended for testing crude antisera but is now frequently used to validate monoclonal and affinity purified polyclonal antibodies. Here, the authors assess the power of this test. Nine affinity purified antibodies to different epitopes on 3 proteins (EAAT3, slc1a1; EAAT2, slc1a2; BGT1, slc6a12) were tested on samples (tissue sections and Western blots with or without fixation). The selected antibodies displayed some degree of cross-reactivity as defined by labeling of samples from knockout mice. The authors show that antigen preadsorption blocked all labeling of both wild-type and knockout samples, implying that preadsorption also blocked binding to cross-reactive epitopes. They show how this can give an illusion of specificity and illustrate sensitivity-specificity relationships, the importance of good negative controls, that fixation can create new epitopes, and that cross-reacting epitopes present in sections may not be present on Western blots and vice versa. In conclusion, they argue against uncritical use of the preadsorption test and, in doing so, address a number of other issues related to immunocytochemistry specificity testing.

Regulation of glutathione synthesis via interaction between glutamate transport-associated protein 3-18 (GTRAP3-18) and excitatory amino acid carrier-1 (EAAC1) at plasma membrane.

Regulation of the cysteine transporter known as excitatory amino acid carrier-1 (EAAC1) for intracellular glutathione (GSH) content was investigated using human embryonic kidney (HEK) 293 cells as a model system. GSH content was significantly reduced by l-aspartate-beta-hydroxamate (50-250 microM), an inhibitor of both EAAC1 and GLT1, both of which are transporters to take up cysteine, whereas dihydrokainate (1-100 microM), a specific inhibitor of GLT1, failed to do so. This indicates that EAAC1 is involved in GSH content in HEK293 cells. We examined the effect of glutamate transport-associated protein 3-18 (GTRAP3-18), which is capable of interacting with EAAC1. The GSH content decreased when the GTRAP3-18 protein level at the plasma membrane was increased by methyl-beta-cyclodextrin (250 microM), rendering the cells more vulnerable to oxidative stress. Intracellular GSH increased when the GTRAP3-18 protein level at the plasma membrane was decreased by antisense oligonucleotides, rendering the cells more resistant to oxidative stress. Furthermore, we found that the increase in GSH content produced by stimulating protein kinase C, a translocator and activator of EAAC1, was inhibited by an increase in cell surface GTRAP3-18 protein. These results show GTRAP3-18 to negatively and dominantly regulate cellular GSH content via interaction with EAAC1 at the plasma membrane.

Electroconvulsive seizure-induced gene expression profile of the hippocampus dentate gyrus granule cell layer.

Electroconvulsive shock (ECS) is the most effective treatment for depression, but the mechanism underlying the therapeutic action of this treatment is still unknown. To better understand the molecular changes that may be necessary for the clinical effectiveness of ECS we have combined the technologies of gene expression profiling using cDNA microarrays with T7-based RNA amplification and laser microdissection to identify regulated genes in the dentate gyrus granule cell layer of the hippocampus. We have identified genes previously reported to be up-regulated following ECS, including brain-derived neurotrophic factor, neuropeptide Y, and thyrotrophin releasing hormone, as well as several novel genes. Notably, we have identified additional genes that are known to be involved in neuroprotection, such as growth arrest DNA damage inducible beta (Gadd45beta), and the excitatory amino acid transporter-1 (EAAC1/Slc1A1). In addition, via in situ hybridization we show that EAAC1 is specifically up-regulated in the dentate gyrus, but not in other hippocampal subfields. This study demonstrates the utility of microarray analysis of microdissected subregions of limbic brain regions and identifies novel ECS-regulated genes.

Feedback loops link odor and pheromone signaling with reproduction.

Pheromones can have profound effects on reproductive physiology and behavior in mammals. To investigate the neural circuits underlying these effects, we used a genetic transneuronal tracer to identify neurons that synapse with GnRH (LHRH) neurons, the key regulators of reproduction. We then asked whether the connected neurons are presynaptic or postsynaptic to GnRH neurons and analyzed their responses to chemosensory cues. Surprisingly, these experiments indicate that GnRH neurons receive pheromone signals from both odor and pheromone relays in the brain and may also receive common odor signals. Moreover, feedback loops are evident whereby GnRH neurons could influence both odor and pheromone processing. Remarkably, approximately 800 GnRH neurons communicate with approximately 50,000 neurons in 53 functionally diverse brain areas, with some connections exhibiting sexual dimorphism. These studies reveal a complex interplay between reproduction and other functions in which GnRH neurons appear to integrate information from multiple sources and modulate a variety of brain functions.