glutamate transporters
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2022 ◽  
Vol 15 ◽  
Author(s):  
Pauline Beckers ◽  
Olaya Lara ◽  
Ines Belo do Nascimento ◽  
Nathalie Desmet ◽  
Ann Massie ◽  
...  

Disruption of the glutamatergic homeostasis is commonly observed in neurological diseases and has been frequently correlated with the altered expression and/or function of astrocytic high-affinity glutamate transporters. There is, however, a growing interest for the role of the cystine-glutamate exchanger system xc– in controlling glutamate transmission. This exchanger is predominantly expressed in glial cells, especially in microglia and astrocytes, and its dysregulation has been documented in diverse neurological conditions. While most studies have focused on measuring the expression of its specific subunit xCT by RT-qPCR or by Western blotting, the activity of this exchanger in tissue samples remains poorly examined. Indeed, the reported use of sulfur- and carbon-radiolabeled cystine in uptake assays shows several drawbacks related to its short radioactive half-life and its relatively high cost. We here report on the elaborate validation of a method using tritiated glutamate as a substrate for the reversed transport mediated by system xc–. The uptake assay was validated in primary cultured astrocytes, in transfected cells as well as in crude synaptosomes obtained from fresh nervous tissue samples. Working in buffers containing defined concentrations of Na+, allowed us to differentiate the glutamate uptake supported by system xc– or by high-affinity glutamate transporters, as confirmed by using selective pharmacological inhibitors. The specificity was further demonstrated in primary astrocyte cultures from transgenic mice lacking xCT or in cell lines where xCT expression was genetically induced or reduced. As such, this assay appears to be a robust and cost-efficient solution to investigate the activity of this exchanger in physiological and pathological conditions. It also provides a reliable tool for the screening and characterization of new system xc– inhibitors which have been frequently cited as valuable drugs for nervous disorders and cancer.


2021 ◽  
Author(s):  
Francis Valiyaveetil ◽  
Erika Riederer ◽  
Pierre Moenne-Loccoz

Glutamate transporters carry out the concentrative uptake of glutamate by harnessing the ionic gradients present across cellular membranes. A central step in the transport mechanism is the coupled binding of Na+ and substrate. The sodium coupled Asp transporter, GltPh is an archaeal homolog of glutamate transporters that has been extensively used to probe the transport mechanism. Previous studies have shown that hairpin-2 (HP2) functions as the extracellular gate for the aspartate binding site and plays a key role in the coupled binding of sodium and aspartate to GltPh. The binding sites for three Na+ ions (Na1-3) have been identified in GltPh but the specific roles of the individual Na+ sites in the binding process has not been elucidated. In this study, we developed assays to probe Na+ binding to the Na1 and Na3 sites and to monitor the conformational switch in the NMDGT motif. We used these assays along with a fluorescence assay to monitor HP2 movement and EPR spectroscopy to show that Na+ binding to the Na3 site is required for the NMDGT conformational switch while Na+ binding to the Na1 site is responsible for the partial opening of HP2. Complete opening of HP2 requires the conformational switch of the NMDGT motif and therefore Na+ binding to both the Na1 and the Na3 sites. Based on our studies we also propose an alternate pathway for the coupled binding of Na+ and Asp.


2021 ◽  
pp. 1-19
Author(s):  
Dicson Sheeja Malar ◽  
Mani Iyer Prasanth ◽  
James Michael Brimson ◽  
Kanika Verma ◽  
Anchalee Prasansuklab ◽  
...  

BACKGROUND: Glutamate toxicity is involved in several neurodegenerative conditions, including Alzheimer’s disease. OBJECTIVE: The study aims to investigate the neuroprotective efficacy of ethanol extract of Hibiscus sabdariffa calyces (HS) against glutamate-induced toxicity in HT-22 cells and induce anti-aging property in Caenorhabditis elegans. METHODS: HT-22 cells were pre-treated with HS followed by glutamate and evaluated for the neuroprotective effect using cell viability assay, confocal microscopic analysis, qPCR, Western blot, and docking analysis. Induction of anti-aging property in C. elegans with HS extract was analyzed through physiological assays and qPCR analysis. RESULTS: GC-MS analysis of the HS extract showed the presence of 19 compounds with antioxidant properties including oleamide,2-(diethoxymethyl)furan and 5-methylfurfural. In vitro studies reveal that glutamate exerted toxicity in HT-22 cells by inducing oxidative stress, depleting glutathione, downregulating glutamate transporters, antioxidant genes, inducing autophagy (Beclin-1, Atg-5, Atg-7, LC3-II) by the activation of MAPK (p38, JNK) pathway, and causing apoptosis. However, pre-treatment with HS extract (5, 10μg/ml) reversed the effect and offered neuroprotection. In silico studies showed that the compounds of HS extract can bind effectively and inhibit the activity of NMDAR, calpain-1 and GSK-3β. In C. elegans, HS extended lifespan, reduced the accumulation of lipofuscin, modulated healthspan-related genes and downregulated the expression of daf-2. CONCLUSION: Our results indicate that HS with its bioactive components exhibits neuroprotective activity by upregulating glutamate transporters, inhibiting autophagy and exerts anti-aging property through DAF-16 dependent mechanism.


2021 ◽  
Author(s):  
Ada G Rodríguez-Campuzano ◽  
Luisa C. Hernández-Kelly ◽  
Arturo Ortega

Abstract Exposure to xenobiotics has a significant impact in brain physiology, that could be liked to an excitotoxic processes induced by a massive release of the main excitatory neurotransmitter, L-glutamate. Overstimulation of post-synaptic and extra-synaptic glutamate receptors leads to a disturbance of intracellular calcium homeostasis that is critically involved in neuronal death. Hence, glutamate extracellular levels are tightly regulated through its uptake by glial glutamate transporters. It has been observed that glutamate regulates its own removal, both in the short-time frame via a transporter-mediated decrease in the uptake, and in the long-term through the transcriptional control of its gene expression, a process mediated by glutamate receptors that involves the Ca2+/diacylglycerol-dependent protein kinase and the transcription factor Ying Yang 1. Taking into consideration that this transcription factor is as a member of the Polycomb complex and thus, part of repressive and activating chromatin remodeling factors, it might direct the interaction of DNA methyltransferases or dioxygenases of methylated cytosines to their target sequences. Since glial glutamate transporters promoters are targets of Ying-Yang 1, in this contribution, we explored the role of dynamic DNA methylation in the expression and function of glial glutamate transporters. To this end, we used the well-characterized models of primary cultures of chick cerebellar Bergmann glia cells and a human retina-derived Müller glia cell line. A time and dose-dependent increase in global DNA methylation was found upon glutamate exposure. Under hypomethylation conditions, both glial glutamate transporters expression and function were increased. These results, favor the notion that a dynamic methylation program triggered by glutamate in glia cells modulates one of its major functions: glutamate removal.


Author(s):  
Numan Taspinar ◽  
Ahmet Hacimuftuoglu ◽  
Selcuk Butuner ◽  
Basak Togar ◽  
Gokhan Arslan ◽  
...  

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Maria Vrettou ◽  
Liying Yan ◽  
Kent W. Nilsson ◽  
Åsa Wallén-Mackenzie ◽  
Ingrid Nylander ◽  
...  

AbstractDNA methylation and gene expression can be altered by early life stress (ELS) and/or ethanol consumption. The present study aimed to investigate whether DNA methylation of the Vesicular Glutamate Transporters (Vglut)1-3 is related to previously observed Vglut1-3 transcriptional differences in the ventral tegmental area (VTA), nucleus accumbens (Acb), dorsal striatum (dStr) and medial prefrontal cortex (mPFC) of adult rats exposed to ELS, modelled by maternal separation, and voluntary ethanol consumption. Targeted next-generation bisulfite sequencing was performed to identify the methylation levels on 61 5′-cytosine-phosphate-guanosine-3′ sites (CpGs) in potential regulatory regions of Vglut1, 53 for Vglut2, and 51 for Vglut3. In the VTA, ELS in ethanol-drinking rats was associated with Vglut1-2 CpG-specific hypomethylation, whereas bidirectional Vglut2 methylation differences at single CpGs were associated with ELS alone. Exposure to both ELS and ethanol, in the Acb, was associated with lower promoter and higher intronic Vglut3 methylation; and in the dStr, with higher and lower methylation in 26% and 43% of the analyzed Vglut1 CpGs, respectively. In the mPFC, lower Vglut2 methylation was observed upon exposure to ELS or ethanol. The present findings suggest Vglut1-3 CpG-specific methylation signatures of ELS and ethanol drinking, underlying previously reported Vglut1-3 transcriptional differences in the mesocorticolimbic brain.


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