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GLT-1 is the major glutamate transporter in the brain, and is expressed in astrocytes and in axon terminals in the hippocampus, cortex, and striatum. Neuronal GLT-1 accounts for only 5-10% of total brain GLT-1 protein, and its function is uncertain. In HD, synaptic dysfunction of the corticostriate synapse is well-established. Transcriptional dysregulation is a key feature of HD. We hypothesized that deletion of neuronal GLT-1, because it is expressed in axon terminals in the striatum, might produce a synaptopathy similar to that present in HD. If true, then some of the gene expression changes observed in HD might also be observed in the neuronal GLT-1 knockout. In situ hybridization using 33 P labeled oligonucleotide probes was carried out to assess localization and expression of a panel of genes known to be altered in expression in HD. We found changes in the expression of cannabinoid receptors 1 and 2, preproenkaphalin, and PDE10A in the striatum of mice in which the GLT-1 gene was inactivated in neurons by expression of synapsin-Cre, compared to wild-type littermates. These changes in expression were observed at 12 weeks of age but not at 6 weeks of age. No changes in DARPP-32, PDE1B, NGFIA, or β-actin expression were observed. In addition, we found widespread alteration in expression of the dynamin 1 gene. The changes in expression in the neuronal GLT-1 knockout of genes thought to exemplify HD transcriptional dysregulation suggest an overlap in the synaptopathy caused by neuronal GLT-1 deletion and HD. These data further suggest that specific changes in expression of cannabinoid receptors, preproenkephalin, and PDE10A, considered to be the hallmark of HD transcriptional dysregulation, may be produced by an abnormality of glutamate homeostasis under the regulation of neuronal GLT-1, or a synaptic disturbance caused by that abnormality, independently of mutation in huntingtin., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

GLT-1 (EAAT2; slc1a2) is the major glutamate transporter in the brain, and is predominantly expressed in astrocytes, but at lower levels also in excitatory terminals. We generated a conditional GLT-1 knock-out mouse to uncover cell-type-specific functional roles of GLT-1. Inactivation of the GLT-1 gene was achieved in either neurons or astrocytes by expression of synapsin-Cre or inducible human GFAP-CreERT2. Elimination of GLT-1 from astrocytes resulted in loss of ∼80% of GLT-1 protein and of glutamate uptake activity that could be solubilized and reconstituted in liposomes. This loss was accompanied by excess mortality, lower body weight, and seizures suggesting that astrocytic GLT-1 is of major importance. However, there was only a small (15%) reduction that did not reach significance of glutamate uptake into crude forebrain synaptosomes. In contrast, when GLT-1 was deleted in neurons, both the GLT-1 protein and glutamate uptake activity that could be solubilized and reconstituted in liposomes were virtually unaffected. These mice showed normal survival, weight gain, and no seizures. However, the synaptosomal glutamate uptake capacity (Vmax) was reduced significantly (40%). In conclusion, astrocytic GLT-1 performs critical functions required for normal weight gain, resistance to epilepsy, and survival. However, the contribution of astrocytic GLT-1 to glutamate uptake into synaptosomes is less than expected, and the contribution of neuronal GLT-1 to synaptosomal glutamate uptake is greater than expected based on their relative protein expression. These results have important implications for the interpretation of the many previous studies assessing glutamate uptake capacity by measuring synaptosomal uptake., (Copyright © 2015 the authors 0270-6474/15/355187-15$15.00/0.)

Oxidative stress has been implicated in the pathogenesis of Huntington's disease (HD), however, the origin of the oxidative stress is unknown. System xc(-) plays a role in the import of cystine to synthesize the antioxidant glutathione. We found in the STHdh(Q7/Q7) and STHdh(Q111/Q111) striatal cell lines, derived from neuronal precursor cells isolated from knock-in mice containing 7 or 111 CAG repeats in the huntingtin gene, that there is a decrease in system xc(-) function. System xc(-) is composed of two proteins, the substrate specific transporter, xCT, and an anchoring protein, CD98. The decrease in function in system xc(-) that we observed is caused by a decrease in xCT mRNA and protein expression in the STHdh(Q111/Q111) cells. In addition, we found a decrease in protein and mRNA expression in the transgenic R6/2 HD mouse model at 6weeks of age. STHdh(Q111/Q111) cells have lower basal levels of GSH and higher basal levels of ROS. Acute inhibition of system xc(-) causes greater increase in oxidative stress in the STHdh(Q111/Q111) cells than in the STHdh(Q7/Q7) cells. These results suggest that a defect in the regulation of xCT may be involved in the pathogenesis of HD by compromising xCT expression and increasing susceptibility to oxidative stress., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Excitotoxicity is thought to be important in the pathogenesis of Huntington's disease (HD). Glutamate is the predominant excitatory neurotransmitter in the brain, and excess activation of glutamate receptors can cause neuronal dysfunction and death. Glutamate transporters regulate the extracellular concentration of glutamate. GLT-1 is the most abundant glutamate transporter, and accounts for most of the glutamate transport in the brain. Administration of ceftriaxone, an antibiotic that increases the functional expression of GLT-1, can improve the behavioral phenotype of the R6/2 mouse model of HD. To test the hypothesis that GLT-1 expression critically affects the HD disease process, we generated a novel mouse model that is heterozygous for the null allele of GLT-1 and carries the R6/2 transgene (double mutation). We demonstrated that the protein expression of total GLT-1, as well as two of its isoforms, is decreased within the cortex and striatum of 12-week-old R6/2 mice, and that the expression of EAAC1 was decreased in the striatum. Protein expression of GLT-1 was further decreased in the cortex and striatum of the double mutation mice compared with the R6/2 mice at 11 weeks. However, the effects of the R6/2 transgene on weight loss, accelerating rotarod, climbing and paw-clasping were not exacerbated in these double mutants. Na(+) -dependent glutamate uptake into synapatosomes isolated from the striatum and cortex of 11-week-old R6/2 mice was unchanged compared with controls. These results suggest that changes in GLT-1 expression or function per se are unlikely to potentiate or ameliorate the progression of HD., (© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Excitotoxicity may contribute to the pathogenesis of Huntington's disease. High affinity Na+ dependent glutamate transporters, residing in the plasma membrane, clear glutamate from the extracellular space and are the primary means of protection against excitotoxicity. Many reports suggest that Huntington's disease is associated with a decrease in the expression and function of glutamate transporters. We studied the expression and function of these transporters in a cellular model of Huntington's disease, STHdh(Q111/Q111) and STHdh(Q7/Q7) cells. We found that only GLT-1b and EAAC1 were expressed in these cell lines and only EAAC1 significantly contributed to the glutamate uptake. Surprisingly, there was an increase in Na+-dependent glutamate uptake in STHdh(Q111/Q111) cells accompanied by an increase in surface expression of EAAC1. We studied the influence of the Akt pathway on EAAC1 mediated uptake, since EAAC1 surface expression is influenced by Akt and previous studies have shown increased Akt expression in STHdh(Q111/Q111) cells. Glutamate uptake was inhibited by Akt pathway inhibitors in both the STHdh(Q7/Q7) and the STHdh(Q111/Q111) cell lines. We found no difference in Akt activation between the two cell lines under our conditions of culture. Therefore a difference in Akt activation does not seem to explain the increase in EAAC1 mediated uptake in the STHdh(Q111/Q111) cells., (Copyright © 2013 Elsevier Ltd. All rights reserved.)