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Objectives: Evidence suggests that decreased dopamine secretion in mesocorticolimbic pathways could predispose to increased susceptibility to substance addiction. It has been proposed to define such a phenomenon as the reward deficit syndrome (RDS). Dopaminergic projections of the reward system receive glutaminergic projections from cortex. Research indicates that a reduction in the stimulating glutamatergic transmission on the dopaminergic system could represent an alternative phenotype of RDS. Potential source of this type of abnormality is glutamate reuptake which depends on excitatory amino acid transport proteins (EAAT) function. The most important of them is EAAT2, polymorphisms of which have been linked to several mental disorders., Methods: We analyzed the genetic and psychometric data of 125 young adults (n = 125) for the effect of the rs4354668 polymorphism of the SLC1A2 gene for EAAT2 on the risky or harmful drug use (RHDU). After exploratory analysis we used logistic regression models to assess the probability of RHDU in individual groups., Results: In the final model T/T variant of rs4354668 was significantly associated with a lower probability of RHDU occurrence compared to G/G variant (OR: 0.021; 95% CI: 0.001 - 0.275; p = 0.009). Other significant predictors of RHDU were smoking status and risky or harmful drinking of alcohol., Conclusions: The results obtained may indicate a possible relationship of the risk of harmful drug use with variants of the rs4354668 polymorphism of the SLC1A2 gene for EAAT2. Subjects with the T/T variant of this polymorphism appear to be less at risk of developing drug use disorders.

The Chinese hamster ovary cell line CHO is widely used for biopharmaceutical production. Genome editing makes it possible to improve the growth properties of cells, their auxotrophy, and the functioning of the apoptosis and autophagy induction systems. Simultaneous editing of multiple genes makes it possible to obtain a cell line with the required genotype faster than several consecutive rounds of genomic knockout, but the probability of success is lower. Simultaneous editing of the dhfr, glul, bak1, and bax genes in the CHO S cells genome yielded 24 clones with signs of auxotrophy for thymidine and glutamine. Five of them turned out to be dhfr +/- , all five contained a knockout of one or two glul alleles. In one clone, 7 out of 8 target alleles were inactivated by a frameshift, and the second dhfr allele was partially inactivated by insertion of the GAA triplet, which reduced the enzyme activity 2.5 times. The probability of simultaneous knockout of both dhfr alleles increased to 50% when the genome was edited with a pair of guide RNAs directed to one exon of the dhfr gene., (© 2022. Pleiades Publishing, Ltd.)

Obesity is associated with inflammation and insulin resistance (IR), but the regulation of insulin sensitivity (IS) and connections between IS and inflammation remain unclear. We investigated the role of miR-467a-5p, a miRNA induced by hyperglycaemia, in regulating inflammation and blood glucose handling. We previously demonstrated that miR-467a-5p is induced by hyperglycaemia and inhibits the production of thrombospondin-1 (TSP-1), a protein implicated in regulating inflammation. To investigate the role of miR-467 in blood glucose handling and tissue inflammation, WT C57BL/6 mice were fed chow or Western diet from 5 to 32 weeks of age and injected weekly with miR-467a-5p antagonist. Inhibiting miR-467a-5p resulted in 47% increase in macrophage infiltration and increased Il6 levels in adipose tissue, higher plasma insulin levels (98 ng/mL vs 63 ng/mL), and 17% decrease in glucose clearance without increase in weight or HDL/LDL. The antagonist effect was lost in mice on Western diet. Mice lacking TSP-1 lost some but not all of the miR-467 effects, suggesting Thbs1 (and other unknown transcripts) are targeted by miR-467 to regulate inflammation. miR-467a-5p provides a physiological feedback when blood glucose is elevated to avoid inflammation and increased blood glucose and insulin levels, which may prevent IR., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Background: The carotid body (CB) plays a critical role in cyclic intermittent hypoxia (CIH)-induced chemosensitivity; however, the underlying mechanism remains uncertain. We have demonstrated the presence of multiple inotropic glutamate receptors (iGluRs) in CB, and that CIH exposure alters the level of some iGluRs in CB. This result implicates glutamatergic signaling in the CB response to hypoxia. The glutamatergic neurotransmission is not only dependent on glutamate and glutamate receptors, but is also dependent on glutamate transporters, including vesicular glutamate transporters (VGluTs) and excitatory amino acid transporters (EAATs). Here, we have further assessed the expression and distribution of VGluTs and EAATs in human and rat CB and the effect of CIH exposure on glutamate transporters expression., Methods: The mRNA of VGluTs and EAATs in the human CB were detected by RT-PCR. The protein expression of VGluTs and EAATs in the human and rat CB were detected by Western blot. The distribution of VGluT3, EAAT2 and EAAT3 were observed by immunohistochemistry staining and immunofluorescence staining. Male Sprague-Dawley (SD) rats were exposed to CIH (FIO 2 10-21%, 3 min/3 min for 8 h per day) for 2 weeks. The unpaired Student's t-test was performed., Results: Here, we report on the presence of mRNAs for VGluT1-3 and EAAT1-3 in human CB, which is consistent with our previous results in rat CB. The proteins of VGluT1 and 3, EAAT2 and 3, but not VGluT2 and EAAT1, were detected with diverse levels in human and rat CB. Immunostaining showed that VGluT3, the major type of VGluTs in CB, was co-localized with tyrosine hydroxylase (TH) in type I cells. EAAT2 and EAAT3 were distributed not only in type I cells, but also in glial fibrillary acidic protein (GFAP) positive type II cells. Moreover, we found that exposure of SD rats to CIH enhanced the protein level of EAAT3 as well as TH, but attenuated the levels of VGluT3 and EAAT2 in CB., Conclusions: Our study suggests that glutamate transporters are expressed in the CB, and that glutamate transporters may contribute to glutamatergic signaling-dependent carotid chemoreflex to CIH.

Excitatory amino acid transporter (EAAT)1 and EAAT2 mediate glutamatergic neurotransmission and prevent excitotoxicity through binding and transportation of glutamate into glia. These EAATs may be regulated by metabotropic glutamate receptor 5 (mGluR5), which is also expressed by glia. Whilst we have data from an Affymetrix™ Human Exon 1.0 ST Array showing higher levels of EAAT1 mRNA (+36%) in Brodmann's are (BA)9 of subjects with schizophrenia, there is evidence that EAAT1 and EAAT2, as well as mGluR5 levels, are altered in the cortex of subjects with the disorder. Hence, we measured mRNA levels of these genes in other cortical regions in subjects with that disorder. EAAT1, EAAT2 and mGluR5 mRNA were measured, in triplicate, using Quantitative PCR in BA10 and BA46 from subjects with schizophrenia (n = 20) and age and sex matched controls (n = 18). Levels of mRNA were normalised to the geometric mean of two reference genes, transcription factor B1, mitochondrial (TFB1M) and S-phase kinase-associated protein 1A (SKP1A), for which mRNA did not vary between diagnostic groups in either region. Normalised levels of EAAT1 and EAAT2 mRNA were significantly higher in BA10 (EAAT1: U = 58, p = 0.0002; EAAT2 U = 70, p = 0.0009), but not BA46 (EAAT1: U = 122, p = 0.09; EAAT2: U = 136, p = 0.21), from subjects with schizophrenia compared to controls. mGluR5 levels in BA10 (U = 173, p=0.85) and BA46 (U = 178, p = 0.96) did not vary by cohort. Our data suggests that region-specific increases in cortical EAAT1 and EAAT2 mRNA are involved in schizophrenia pathophysiology and that disrupted glutamate uptake in schizophrenia may be of particular significance in BA10., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Excitatory amino acid transporter 2 (EAAT 2 ) plays a pivotal role in glutamate clearance in the adult brain, thereby preventing excitotoxic effects. Considering the high efficacy of EAAT 2 for glutamate uptake, we hypothesized that the expression of this transporter in mesenchymal stem cells (MSCs) for systemic administration could yield a cell-based glutamate-grabbing therapy, combining the intrinsic properties of these cells with excitotoxic protection., Methods: To address this hypothesis, EAAT 2 -encoding cDNA was introduced into MSCs and human embryonic kidney 293 cells (HEK cells) as the control cell line. EAAT 2 expression and functionality were evaluated by in vitro assays. Blood glutamate-grabbing activity was tested in healthy and ischemic rat models treated with 3 × 10 6 and 9 × 10 6 cells/animal., Findings: The expression of EAAT 2 in both cell types conferred the expected glutamate-grabbing activity in in vitro and in vivo studies. The functional improvement observed in ischemic rats treated with EAAT 2 -HEK at low dose, confirmed that this effect was indeed mediated by the glutamate-grabbing activity associated with EAAT 2 functionality. Unexpectedly, both cell doses of non-transfected MSCs induced higher protection than transfected EAAT 2 -MSCs by another mechanism independent of the glutamate-grabbing capacity., Interpretation: Although the transfection procedure most likely interferes with some of the intrinsic protective mechanisms of mesenchymal cells, the results show that the induced expression of EAAT 2 in cells represents a novel alternative to mitigate the excitotoxic effects of glutamate and paves the way to combine this strategy with current cell therapies for cerebral ischemia., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Immune stimulation might be involved in the pathophysiology of major depressive disorder (MDD). This stimulation induces indoleamine 2,3-dioxygenase (IDO), an enzyme that reduces the tryptophan bioavailability to synthesize serotonin. IDO products, kynurenine metabolites, exert neurotoxic/neuroprotective actions through glutamate receptors. Thus, we study elements of these pathways linked to kynurenine metabolite activity examining whether antidepressants (ADs) can modulate them. Male Wistar rats were exposed to chronic mild stress (CMS), and some of them were treated with ADs. The expression of elements of the IDO pathway, including kynurenine metabolites, and their possible modulation by ADs was studied in the frontal cortex (FC). CMS increased IDO expression in FC compared to control group, and ADs restored the IDO expression levels to control values. CMS-induced IDO expression led to increased levels of the excitotoxic quinolinic acid (QUINA) compared to control, and ADs prevented the rise in such levels. Neither CMS nor ADs changed significantly the antiexcitotoxic kynurenic acid (KYNA) levels. The QUINA/KYNA ratio, calculated as excitotoxicity risk indicator, increased after CMS and ADs prevented this increase. CMS lowered excitatory amino acid transporter (EAAT)-1 and EAAT-4 expression, and some ADs restored their expression levels. Furthermore, CMS decreased N-methyl-D-aspartate receptor (NMDAR)-2A and 2B protein expression, and ADs mitigated this decrease. Our research examines the link between CMS-induced pro-inflammatory cytokines and the kynurenine pathway; it shows that CMS alters the kynurenine pathway in rat FC. Importantly, it also reveals the ability of classic ADs to prevent potentially harmful situations related to the brain scenario caused by CMS.

Evidence demonstrated that the glutamatergic system is implicated in mediating relapse to several drugs of abuse, including methamphetamine (METH). Glutamate homeostasis is maintained by a number of glutamate transporters, such as glutamate transporter type 1 (GLT-1), cystine/glutamate transporter (xCT), and glutamate aspartate transporter (GLAST). In addition, group II metabotropic glutamate receptors (mGluR2/3) were found to be implicated in relapse-seeking behavior. Ample evidence showed that β-lactam antibiotics are effective in upregulating GLT-1 and xCT expression, thus improving glutamate homeostasis and attenuating relapse to drugs of abuse. In this study, we investigated the reinstatement of METH using conditioned place preference (CPP) in male alcohol-preferring (P) rats exposed to home-cage free choice ethanol drinking. Here, we tested the effect of clavulanic acid (CA), a β-lactam, on the reinstatement of METH-seeking and ethanol drinking. In addition, we examined the expression of GLT-1, xCT, and GLAST as well as metabotropic glutamate receptor (mGluR2/3) in the nucleus accumbens (NAc) shell, NAc core, and dorsomedial prefrontal cortex (dmPFC). A priming i.p. injection of METH reinstated preference in METH-paired chamber following extinction. Chronic exposure to ethanol decreased the expression of GLT-1 and xCT in the NAc shell, but not in the NAc core or dmPFC. CA treatment blocked the reinstatement of METH-seeking, decreased ethanol intake, and restored the expression of GLT-1 and xCT in the NAc shell. In addition, the expression of mGluR2/3 was increased by CA treatment in the NAc shell and dmPFC. These findings suggest that these glutamate transporters and mGluR2/3 might be potential therapeutic targets for the attenuation of reinstatement to METH-seeking.

Cognitive deficits represent core features of schizophrenia, affecting quality of life and functioning. The excitatory amino acid transporter 2 (EAAT2) is responsible for the majority of glutamate reuptake and its activity is crucial for glutamatergic neurotransmission, prevention of excitotoxic damage and cerebral metabolism. Different studies reported that EAAT2 rs4354668 (-181 T/G) influences cognitive functions and brain structures in patients with schizophrenia. Specifically, the G allele, linked to lower EAAT2 expression, was associated with impaired prefrontal cognitive performance and reduced grey matter volumes. Cognitive remediation therapy (CRT) is one of the best available tool to treat cognitive deficits in schizophrenia, able to induce a neuroplastic modulation of cognitive functions. The present study aims to investigate the effects of rs4354668 on CRT outcome, also considering possible genotype interaction with antipsychotic (AP) treatment, since EAAT2 expression is negatively influenced by clozapine. We examined rs4354668 in 88 clinically stabilized patients with schizophrenia, treated with CRT and assessed at enrolment, at the end of CRT and after 3 months. We observed greater working memory improvements among patients carrying the T/T genotype, regardless of AP treatment. Moreover, we reported a significant interaction between pharmacological treatment and rs4354668 on executive functions, with greater improvements among T/T patients treated with APs other than clozapine. These observations suggest that impaired EAAT2 expression may attenuate CRT outcome. Moreover, our results indicate the possibility that rs4354668 could also differentially influence the response to CRT depending on the AP treatment., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Neuronal hyperactivity is one of the earliest events observed in Alzheimer's disease (AD). Moreover, alterations in the expression of glutamate transporters have been reported to exacerbate amyloid pathology and cognitive deficits in transgenic AD mouse models. However, the molecular links between these pathophysiological changes remain largely unknown. Here, we report novel interaction between presenilin 1 (PS1), the catalytic component of the amyloid precursor protein-processing enzyme, γ-secretase, and a major glutamate transporter-1 (GLT-1). Our data demonstrate that the interaction occurs between PS1 and GLT-1 expressed at their endogenous levels in vivo and in vitro, takes place in both neurons and astrocytes, and is independent of the PS1 autoproteolysis and γ-secretase activity. This intriguing discovery may shed light on the molecular crosstalk between the proteins linked to the maintenance of glutamate homeostasis and Aβ pathology.

Preterm delivery is associated with neurodevelopmental impairment caused by environmental and genetic factors. Dysfunction of the excitatory amino acid transporter 2 (EAAT2) and the resultant impaired glutamate uptake can lead to neurological disorders. In this study, we investigated the role of single nucleotide polymorphisms (SNPs; g.-200C>A and g.-181A>C) in the EAAT2 promoter in susceptibility to brain injury and neurodisability in very preterm infants born at or before 32-week gestation. DNA isolated from newborns' dried blood spots were used for pyrosequencing to detect both SNPs. Association between EAAT2 genotypes and cerebral palsy, cystic periventricular leukomalacia and a low developmental score was then assessed. The two SNPs were concordant in 89.4% of infants resulting in three common genotypes all carrying two C and two A alleles in different combinations. However, in 10.6% of cases, non-concordance was found, generating six additional rare genotypes. The A alleles at both loci appeared to be detrimental and consequently, the risk of developing cerebral palsy increased four- and sixfold for each additional detrimental allele at -200 and -181 bp, respectively. The two SNPs altered the regulation of the EAAT2 promoter activity and glutamate homeostasis. This study highlights the significance of glutamate in the pathogenesis of preterm brain injury and subsequent development of cerebral palsy and neurodevelopmental disabilities. Furthermore, the described EAAT2 SNPs may be an early biomarker of vulnerability to neurodisability and may aid the development of targeted treatment strategies.

The Fibronectin Leucine-Rich Transmembrane protein 2 (FLRT2) has been implicated in several hormone -and sex-dependent physiological and pathological processes (including chondrogenesis, menarche and breast cancer); is known to regulate developmental synapses formation, and is expressed in the hippocampus, a brain structure central for learning and memory. However, the role of FLRT2 in the adult hippocampus and its relevance in sex-dependent brain functions remains unknown. We here used adult single-allele FLRT2 knockout (FLRT2 +/- ) mice and behavioral, electrophysiological, and molecular/biological assays to examine the effects of FLRT2 haplodeficiency on synaptic plasticity and hippocampus-dependent learning and memory. Female and male FLRT2 +/- mice presented morphological features (including body masses, brain shapes/weights, and brain macroscopic cytoarchitectonic organization), indistinguishable from their wild type counterparts. However, in vivo examinations unveiled enhanced hippocampus-dependent spatial memory recall in female FLRT2 +/- animals, concomitant with augmented hippocampal synaptic plasticity and decreased levels of the glutamate transporter EAAT2 and beta estrogen receptors. In contrast, male FLRT2 +/- animals exhibited deficient memory recall and decreased alpha estrogen receptor levels. These observations propose that FLRT2 can regulate memory functions in the adulthood in a sex-specific manner and might thus contribute to further research on the mechanisms linking sexual dimorphism and cognition.

Recently, a genome-wide association study of a Caucasian population identified variant rs3794087 in intron 4 of the SLC1A2 gene, which may increase the risk of essential tremor (ET). Considering the overlap in the pathological features and clinical manifestations of ET and Parkinson's disease (PD), several studies on the association between rs3794087 and PD were later performed in other populations. However, results about the role of SLC1A2 rs3794087 in PD were inconsistent. We thus performed a case-control study in a Chinese Han population to investigate the role of SLC1A2 rs3794087 in Chinese patients with PD. Overall, 1096 subjects comprising 546 patients with PD and 550 control subjects were genotyped. A meta-analysis of the data obtained from the current sample-set and those available from prior studies was performed. Taking all patients and controls into consideration, rs3794087 was found to have no significant effect on PD susceptibility in analyses using allelic (p = .486), genotype (p = .736), additive (p = .764), dominant (p = .438), and recessive (p = .878) genetic models. The results of the meta-analysis were in agreement with our findings (the pooled OR was 0.97 and 95% CI = 0.85, 1.10). Our study suggested that rs3794087 does not lead to an increased risk of PD in the Chinese Han population. The role of single nucleotide polymorphism rs3794087 in the development of PD remains to be further studied., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Recently, heterozygous de novo mutations in SCL1A2 have been reported to underlie severe early-onset epileptic encephalopathy. In one male presenting with epileptic seizures and visual impairment, we identified a novel homozygous splicing variant in SCL1A2 (c.1421 + 1G > C) by using exome sequencing. Functional studies on cDNA level confirmed a consecutive loss of function. Our findings suggest that not only de novo mutations but also biallelic variants in SLC1A2 can cause epilepsy and that there is an additional autosomal recessive mode of inheritance. These findings also contribute to the understanding of the genetic mechanism of autosomal dominant SLC1A2 -related epileptic encephalopathy as they exclude haploinsufficiency as exclusive genetic mechanism., Competing Interests: Disclosure The authors report no conflicts of interest in this work., (Georg Thieme Verlag KG Stuttgart · New York.)

Targeted expression of genes coding for proteins specific to astrocytes, oligodendrocytes and myelin was performed in frontal cortex area 8 of Creutzfeldt-Jakob disease methionine/methionine and valine/valine (CJD MM1 and VV2, respectively) compared with controls. GFAP (glial fibrillary acidic protein) mRNA was up-regulated whereas SLC1A2 (solute carrier family 1 member 2, coding for glutamate transporter 1: GLT1), AQ4 (aquaporin 4), MPC1 (mitochondrial pyruvate carrier 1) and UCP5 (mitochondrial uncoupled protein 5) mRNAs were significantly down-regulated in CJD MM1 and CJD VV2, and GJA1 (connexin 43) in CJD VV2. OLIG1 and OLIG2 (oligodendocyte transcription factor 1 and 2, respectively), SOX10 (SRY-Box10) and oligodendroglial precursor cell (OPC) marker NG2 (neuronal/glial antigen) 2 were preserved, but GALC (coding for galactosylceramidase), SLC2A1 (solute carrier family 2 member 1: glucose transporter member 1: GLUT1) and MCT1 (monocarboxylic acid transporter 1) mRNA expression levels were significantly reduced in CJD MM1 and CJD VV2. Expression levels of most genes linked to myelin were not altered in the cerebral cortex in CJD. Immunohistochemistry to selected proteins disclosed individual variations but GFAP, Olig-2, AQ4 and GLUT1 correlated with mRNA levels, whereas GLT1 was subjected to individual variations. However, MPC1, UCP5 and MCT1 decrease was more closely related to the respective reduced neuronal immunostaining. These observations support the idea that molecular deficits linked to energy metabolism and solute transport in astrocytes and oligodendrocytes, in addition to neurons, are relevant in the pathogenesis of cortical lesions in CJD.

Termination of Saccharomyces cerevisiae RNA polymerase II (Pol II) transcripts occurs through two alternative pathways. Termination of mRNAs is coupled to cleavage and polyadenylation while noncoding transcripts are terminated through the Nrd1-Nab3-Sen1 (NNS) pathway in a process that is linked to RNA degradation by the nuclear exosome. Some mRNA transcripts are also attenuated through premature termination directed by the NNS complex. In this paper we present the results of nuclear depletion of the NNS component Nab3. As expected, many noncoding RNAs fail to terminate properly. In addition, we observe that nitrogen catabolite-repressed genes are upregulated by Nab3 depletion., (Copyright © 2017 American Society for Microbiology.)

Massively parallel sequencing has revealed many de novo mutations in the etiology of developmental and epileptic encephalopathies (EEs), highlighting their genetic heterogeneity. Additional candidate genes have been prioritized in silico by their co-expression in the brain. Here, we evaluate rare coding variability in 20 candidates nominated with the use of a reference gene set of 51 established EE-associated genes. Variants within the 20 candidate genes were extracted from exome-sequencing data of 42 subjects with EE and no previous genetic diagnosis. We identified 7 rare non-synonymous variants in 7 of 20 genes and performed Sanger sequence validation in affected probands and parental samples. De novo variants were found only in SLC1A2 (aka EAAT2 or GLT1) (c.244G>A [p.Gly82Arg]) and YWHAG (aka 14-3-3γ) (c.394C>T [p.Arg132Cys]), highlighting the potential cause of EE in 5% (2/42) of subjects. Seven additional subjects with de novo variants in SLC1A2 (n = 1) and YWHAG (n = 6) were subsequently identified through online tools. We identified a highly significant enrichment of de novo variants in YWHAG, establishing their role in early-onset epilepsy, and we provide additional support for the prior assignment of SLC1A2. Hence, in silico modeling of brain co-expression is an efficient method for nominating EE-associated genes to further elucidate the disorder's etiology and genotype-phenotype correlations., (Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

The role of glutamate in the regulation of neurogenesis is well-established, but the role of vesicular glutamate transporters (VGLUTs) and excitatory amino acid transporters (EAATs) in controlling adult neurogenesis is unknown. Here we investigated the implication of VGLUTs in the differentiation of subventricular zone (SVZ)-derived neural precursor cells (NPCs). Our results show that NPCs express VGLUT1-3 and EAAT1-3 both at the mRNA and protein level. Their expression increases during differentiation closely associated with the expression of marker genes. In expression analyses we show that VGLUT1 and VGLUT2 are preferentially expressed by cultured SVZ-derived doublecortin+ neuroblasts, while VGLUT3 is found on GFAP+ glial cells. In cultured NPCs, inhibition of VGLUT by Evans Blue increased the mRNA level of neuronal markers doublecortin, B3T and MAP2, elevated the number of NPCs expressing doublecortin protein and promoted the number of cells with morphological appearance of branched neurons, suggesting that VGLUT function prevents neuronal differentiation of NPCs. This survival- and differentiation-promoting effect of Evans blue was corroborated by increased AKT phosphorylation and reduced MAPK phosphorylation. Thus, under physiological conditions, VGLUT1-3 inhibition, and thus decreased glutamate exocytosis, may promote neuronal differentiation of NPCs.

The use of antipsychotics carries the risk of metabolic side effects, such as weight gain and new onset type-2 diabetes mellitus. The mechanisms of the observed metabolic alterations are not fully understood. We compared the effects of two atypical antipsychotics, one known to favor weight gain (olanzapine), the other not (aripiprazole), on glucose metabolism. Primary human peripheral blood mononuclear cells (PBMC) were isolated and stimulated with olanzapine or aripiprazole for 72 h. Cellular glucose uptake was analyzed in vitro by 18F-FDG uptake. Further measurements comprised mRNA expression of glucose transporter (GLUT) 1 and 3, GLUT1 protein expression, DNA methylation of GLUT1 promoter region, and proteins involved in downstream glucometabolic processes. We observed a 2-fold increase in glucose uptake after stimulation with aripiprazole. In contrast, olanzapine stimulation decreased glucose uptake by 40%, accompanied by downregulation of the cellular energy sensor AMP activated protein kinase (AMPK). GLUT1 protein expression increased, GLUT1 mRNA expression decreased, and GLUT1 promoter was hypermethylated with both antipsychotics. Pyruvat-dehydrogenase (PDH) complex activity decreased with olanzapine only. Our findings suggest that the atypical antipsychotics olanzapine and aripiprazole differentially affect energy metabolism in PBMC. The observed decrease in glucose uptake in olanzapine stimulated PBMC, accompanied by decreased PDH point to a worsening in cellular energy metabolism not compensated by AMKP upregulation. In contrast, aripiprazole stimulation lead to increased glucose uptake, while not affecting PDH complex expression. The observed differences may be involved in the different metabolic profiles observed in aripiprazole and olanzapine treated patients., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Exposure to ethanol (EtOH) or methamphetamine (MA) can lead to increase in extracellular glutamate concentration in the brain. Although studies from ours showed the effects of EtOH exposure on key glial glutamate transporters, little is known about the effects of sequential exposure to EtOH and MA or MA alone on certain glial glutamate transporters. In this study, we investigated the effects of sequential exposure to EtOH and MA on the expression of the major glutamate transporters, glutamate transporter 1 (GLT-1), as well as cystine/glutamate antiporter (xCT) and glutamate aspartate transporter (GLAST) in striatum and hippocampus. We also tested the effects of ceftriaxone (CEF), known to upregulate GLT-1, in animals administered EtOH and MA. Wistar rats were orally gavaged with EtOH (6 g/kg) or water for 7 days. On the following day (day 8), the rats received four intraperitoneal (i.p.) injections of MA (10 mg/kg) or saline (vehicle) occurring every 2 h. The rats were then treated with CEF (200 mg/kg/day, i.p.) or saline on days 8, 9, and 10. EtOH or MA exposure caused a significant downregulation of GLT-1 expression as compared to control groups in striatum and hippocampus. Furthermore, sequential exposure of EtOH and MA caused a significant downregulation of GLT-1 expression as compared to either drug administered alone in both brain regions. Importantly, GLT-1 expression was restored following CEF treatment. There were no significant differences on xCT and GLAST expression in striatum and hippocampus between all groups. These findings demonstrated that sequential exposure to EtOH and MA has additive effect in downregulation of GLT-1 and this effect can be attenuated by CEF treatment.

Lactose plays a crucial role in controlling milk volume by inducing water toward into the mammary secretory vesicles from the mammary epithelial cell cytoplasm, thereby maintaining osmolality. In current study, we determined the expression of several lactose synthesis related genes, including glucose transporters (glucose transporter 1, glucose transporter 8, sodium-glucose cotransporter 1, sodium-glucose cotransporter 3, and sodium-glucose cotransporter 5), lactose synthases (α-lactalbumin and β1,4-galactosyltransferase), and hexokinases (hexokinase-1 and hexokinase-2) in sow mammary gland tissue at day 17 before delivery, on the 1st day of lactation and at peak lactation. The data showed that glucose transporter 1 was the dominant glucose transporter within sow mammary gland and that expression of each glucose transporter 1, sodium-glucose cotransporter 1, hexokinase-1, hexokinase-2, α-lactalbumin, and β1,4-galactosyltransferase were increased (p < 0.05) when the sows transited from late pregnancy to peak lactation. AKT1 over-expressed mammary epithelial cells were then constructed, and the results indicated that AKT1 increases (p < 0.01) the expression of hexokinase-1 and glucose transporter 1. In summary, lactose synthesis was significantly elevated with the increase of milk production and AKT1 could positively regulate lactose synthesis.

Astrocytes play a fundamental role in maintaining the health and function of the central nervous system. Increasing evidence indicates that astrocytes undergo both cellular and molecular changes at an early stage in neurological diseases, including Alzheimer's disease (AD). These changes may reflect a change from a neuroprotective to a neurotoxic phenotype. Given the lack of current disease-modifying therapies for AD, astrocytes have become an interesting and viable target for therapeutic intervention. The astrocyte transport system covers a diverse array of proteins involved in metabolic support, neurotransmission and synaptic architecture. Therefore, specific targeting of individual transporter families has the potential to suppress neurodegeneration, a characteristic hallmark of AD. A small number of the 400 transporter superfamilies are expressed in astrocytes, with evidence highlighting a fraction of these are implicated in AD. Here, we review the current evidence for six astrocytic transporter subfamilies involved in AD, as reported in both animal and human studies. This review confirms that astrocytes are indeed a viable target, highlights the complexities of studying astrocytes and provides future directives to exploit the potential of astrocytes in tackling AD., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

Background: Genome-wide association studies have revealed a link between essential tremor (ET) and the gene SLC1A2, which encodes excitatory amino acid transporter type 2 (EAAT2). We explored EAAT biology in ET by quantifying EAAT2 and EAAT1 levels in the cerebellar dentate nucleus, and expanded our prior analysis of EAAT2 levels in the cerebellar cortex., Objective: To quantify EAAT2 and EAAT1 levels in the cerebellar dentate nucleus and cerebellar cortex of ET cases vs., Methods: We used immunohistochemistry to quantify EAAT2 and EAAT1 levels in the dentate nucleus of a discovery cohort of 16 ET cases and 16 controls. Furthermore, we quantified EAAT2 levels in the dentate nucleus in a replicate cohort (61 ET cases, 25 controls). Cortical EAAT2 levels in all 77 ET cases and 41 controls were quantified., Results: In the discovery cohort, dentate EAAT2 levels were 1.5-fold higher in 16 ET cases vs. 16 controls (p = 0.007), but EAAT1 levels did not differ significantly (p = 0.279). Dentate EAAT2 levels were 1.3-fold higher in 61 ET cases vs. 25 controls in the replicate cohort (p = 0.022). Cerebellar cortical EAAT2 levels were 20% and 40% lower in ET cases vs. controls in the discovery and the replicate cohorts (respective p values = 0.045 and < 0.001)., Conclusion: EAAT2 expression is enhanced in the ET dentate nucleus, in contrast to differentially reduced EAAT2 levels in the ET cerebellar cortex, which might reflect a compensatory mechanism to maintain excitation-inhibition balance in cerebellar nuclei., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Transmembrane transporter proteins allow the passage of essentially all biologically important molecules across the lipid membranes of cells and organelles and are therefore of central importance to all forms of life. Current methods of transporter measurement, however, are lacking in several dimensions. Herein, a method is presented in which oscillating stimuli are presented to transporter-expressing cells, and activity is measured through imaging the corresponding oscillating responses of intracellular fluorescent sensors. This approach yields continuous temporal readouts of transporter activity and can therefore be used to measure time-dependent responses to drugs and other stimuli. Because of the periodic nature of the response, temporal Fourier transforms can be used to identify and quantify regions of interest in the xy plane and to overcome noise. This technique, called the Oscillating Stimulus Transporter Assay (OSTA), should greatly facilitate both functional characterization of transporters as well as high-throughput screening of drugs for transporters of particular pathophysiological interest., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Excitatory amino acid transporter 2 (EAAT2), also known as glial glutamate transporter type 1 (GLT-1), plays an important role in maintaining the extracellular glutamate concentrations below neurotoxic levels. The highly conserved TM2 transmembrane domain of GLT-1 maintains a stable position during the transport cycle; however, the effect of the transport cycle on the topology of TM4 in not well established. To further reveal the function of TM4, two cysteine pairs between TM2 and TM4 were introduced using site-directed mutagenesis. A significant decrease of transport activity was observed in the I93C/V241C and I97C/V241C mutants upon application of the oxidative cross-linking reagent, copper (II) (1,10-phenanthroline) 3 (CuPh), which suggests that a conformational shift is essential for transporter activity. Furthermore, the decrease in activity by CuPh crosslinking was enhanced in external media with glutamate or potassium, which suggests that TM2 and TM4 assume closer proximity in the inward-facing conformation of the transporter. Our results suggest that the TM4 domain of GLT-1, and potentially other glutamate transporters, undergoes a complex conformational shift during substrate translocation, which involves an increase in the proximity of the TM2 and TM4 domains in the inward-facing conformation.

Background/objective: Despite many data suggesting a role of genetic factors in the risk for essential tremor (ET), the responsible genes have not been identified. We analyzed in ET Spanish families three single nucleotide polymorphisms (SNPs): DRD3 rs6280, SLC1A2 rs3794087, and MAPT rs1052553) previously related to an increased risk for developing the disease., Methods: We recruited 45 subjects with ET and 13 subjects without tremor belonging to 11 families who were evaluated because of familial tremor. Diagnosis of probable or definite ET was done according to TRIG criteria. Genotyping of the 3 SNPs was done using TaqMan-based qPCR assays. Data were compared with those of healthy controls of our laboratory. Family-based association testing for disease traits was performed as well., Results: rs6280 and rs3794087 genotype and allelic frequencies did not differ significantly between subjects with ET and healthy controls. However, rs1052553AA genotype and the allele rs1052553A allele were significantly more frequent among ET patients. rs1052553A allele was non-significantly overrepresented in ET patients compared with controls when considering only the more severely affected member of each ET family. Family-based association test for disease traits showed lack of association between ET and the three SNPs studied., Conclusions: Our results showed a lack of association between rs6280 and rs3794087 with the risk for ET, though a marginal increased risk for ET was observed among the rs1052553A allele carriers, which was not confirmed with a family-based association study.

Epileptic encephalopathies (EEs) are the most clinically important group of severe early-onset epilepsies. Next-generation sequencing has highlighted the crucial contribution of de novo mutations to the genetic architecture of EEs as well as to their underlying genetic heterogeneity. Our previous whole-exome sequencing study of 264 parent-child trios revealed more than 290 candidate genes in which only a single individual had a de novo variant. We sought to identify additional pathogenic variants in a subset (n = 27) of these genes via targeted sequencing in an unsolved cohort of 531 individuals with a diverse range of EEs. We report 17 individuals with pathogenic variants in seven of the 27 genes, defining a genetic etiology in 3.2% of this unsolved cohort. Our results provide definitive evidence that de novo mutations in SLC1A2 and CACNA1A cause specific EEs and expand the compendium of clinically relevant genotypes for GABRB3. We also identified EEs caused by genetic variants in ALG13, DNM1, and GNAO1 and report a mutation in IQSEC2. Notably, recurrent mutations accounted for 7/17 of the pathogenic variants identified. As a result of high-depth coverage, parental mosaicism was identified in two out of 14 cases tested with mutant allelic fractions of 5%-6% in the unaffected parents, carrying significant reproductive counseling implications. These results confirm that dysregulation in diverse cellular neuronal pathways causes EEs, and they will inform the diagnosis and management of individuals with these devastating disorders., (Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Background: The association between the polymorphism rs3794087 in the solute carrier family 1, member 2 (SLC1A2) and the risk of essential tremor (ET) among different studies is controversial. Considering the overlap of the clinical manifestations and pathological characteristics of ET, Parkinson's disease (PD), multiple system atrophy (MSA), as well as amyotrophic lateral sclerosis (ALS), we explored the possible genetic association of rs3794087 with ET, PD, MSA and ALS in a Chinese cohort., Methods: A total of 112 ET, 621 PD, 356 MSA, 513 sporadic ALS (SALS) patients and 437 healthy controls (HCs) were genotyped for rs3794087 using the Sequenom iPLEX Assay technology., Results: Significant association was found between SLC1A2 rs3794087 and PD in the additive model (p=0.006), which was more obvious in early onset PD. The minor allele of rs3794087 decreased the risk for early onset PD (p=0.011, OR: 0.73, 95% CI: 0.56-0.94). However, no significant differences in the genotype distributions and allele frequency were observed in the allelic, additive, dominant or recessive genetic models of SLC1A2 rs3794087 between ET patients and HCs, between SALS patients and HCs, and between MSA and HCs., Conclusions: Our results suggested SLC1A2 rs3794087 may decrease the risk for PD in a Chinese cohort, but do not support a role in the susceptibility to SALS or MSA., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Genomic profiling of immortalized human mammary epithelial (hTERT-HME1) cells identified several metabolic genes, including the membrane glutamate transporter, SLC1A1, as 1,25-dihydroxyvitamin D3 (1,25D) regulated. In these studies we have surveyed the effects of 1,25D on known glutamate transporters and evaluated its impact on cellular glutamate handling. We confirm that expression of SLC1A1 and all of its known transcript variants are significantly upregulated in hTERT-HME1 cells following 1,25D treatment. Expression of the full-length cognate protein, EAAT3, is correspondingly increased in 1,25D treated hTERT-HME1 cells. Under the same conditions, the expression of two other glutamate transporters--SLC1A6 (EAAT4) and SLC1A2 (EAAT2 or GLT-1)--is enhanced by 1,25D while that of SLC1A3 (EAAT1 or GLAST) and SLC7A11 (xCT) is decreased. Glutamate is not essential for growth of hTERT-HME1 cells, and supplemental glutamate (up to 0.5 mM) does not abrogate the growth inhibitory effects of 1,25D. These data suggest that extracellular glutamate is not a major contributor to cellular energy metabolism in hTERT-HME1 cells under basal conditions and that the growth inhibitory effects of 1,25D are not secondary to its effects on glutamate handling. Instead, the effects of 1,25D on glutamate transporters translated to a decrease in cellular glutamate concentration and an increase in media glutamate concentration, suggesting that one or more of these transporters functions to export glutamate in response to 1,25D exposure. The reduced cellular glutamate concentration may also reflect its incorporation into the cellular glutathione (GSH) pool, which is increased upon 1,25D treatment. In support of this concept, the expression of GCLC (which codes for the rate-limiting enzyme in GSH synthesis) and genes which generate reducing equivalents in the form of NADPH (ie, G6PD, PGD, IDH2) are elevated in 1,25D-treated cells. Taken together, these data identify 1,25D as a physiological regulator of multiple membrane glutamate transporters that impacts on overall cellular glutamate handling., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. After release from presynaptic nerve terminals, glutamate is quickly removed from the synaptic cleft by a family of five glutamate transporters, the so-called excitatory amino acid transporters (EAAT1-5). EAATs are prototypic members of the growing number of dual-function transport proteins: they are not only glutamate transporters, but also anion channels. Whereas the mechanisms underlying secondary active glutamate transport are well understood at the functional and at the structural level, mechanisms and cellular roles of EAAT anion conduction have remained elusive for many years. Recently, molecular dynamics simulations combined with simulation-guided mutagenesis and experimental analysis identified a novel anion-conducting conformation, which accounts for all experimental data on EAAT anion currents reported so far. We here review recent findings on how EAATs accommodate a transporter and a channel in one single protein.

The 150-year-long search for treatments of amyotrophic lateral sclerosis (ALS) is still fueled by frustration over the shortcomings of available therapeutics. Contributing to the therapeutic limitations might be the targeting of a single aspect of this multifactorial-multisystemic disease. In an attempt to overcome this, we devised a novel multifactorial-cocktail treatment, using lentiviruses encoding: EAAT2, GDH2, and NRF2, that act synergistically to address the band and width of the effected excito-oxidative axis, reducing extracellular-glutamate and glutamate availability while improving the metabolic state and the anti-oxidant response. This strategy yielded particularly impressive results, as all three genes together but not separately prolonged survival in ALS mice by an average of 19-22 days. This was accompanied by improvement in every parameter evaluated, including body-weight loss, reflex score, neurologic score, and motor performance. We hope to provide a novel strategy to slow down disease progression and alleviate symptoms of patients suffering from ALS.

Glutamate transporters maintain synaptic concentration of the excitatory neurotransmitter below neurotoxic levels. Their transport cycle consists of cotransport of glutamate with three sodium ions and one proton, followed by countertransport of potassium. Structural studies proposed that a highly conserved serine located in the binding pocket of the homologous GltPh coordinates L-aspartate as well as the sodium ion Na1. To experimentally validate these findings, we generated and characterized several mutants of the corresponding serine residue, Ser-364, of human glutamate transporter SLC1A2 (solute carrier family 1 member 2), also known as glutamate transporter GLT-1 and excitatory amino acid transporter EAAT2. S364T, S364A, S364C, S364N, and S364D were expressed in HEK cells and Xenopus laevis oocytes to measure radioactive substrate transport and transport currents, respectively. All mutants exhibited similar plasma membrane expression when compared with WT SLC1A2, but substitutions of serine by aspartate or asparagine completely abolished substrate transport. On the other hand, the threonine mutant, which is a more conservative mutation, exhibited similar substrate selectivity, substrate and sodium affinities as WT but a lower selectivity for Na(+) over Li(+). S364A and S364C exhibited drastically reduced affinities for each substrate and enhanced selectivity for L-aspartate over D-aspartate and L-glutamate, and lost their selectivity for Na(+) over Li(+). Furthermore, we extended the analysis of our experimental observations using molecular dynamics simulations. Altogether, our findings confirm a pivotal role of the serine 364, and more precisely its hydroxyl group, in coupling sodium and substrate fluxes., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

SLC1A2 is reported to be responsible for the majority of glutamate uptake, which has a crucial role in neural development and synaptic plasticity, and a disturbance in glutamatergic transmission has been suggested to be involved in the pathophysiology of schizophrenia (SCZ) and cognition. To evaluate the relationship of common variants within SLC1A2 with SCZ and cognition in Han Chinese, 28 tag SNPs were genotyped in the discovery stage, which included 1117 cases and 2289 controls; significantly associated markers were genotyped in the replication stage with 2128 cases and 3865 controls. The rs4354668 SNP was identified to be significantly associated with SCZ in both datasets, and a similar pattern was also observed in the two-stage study on conducting imputation and haplotype association analyses. In addition, significant associations between the rs4354668 SNP and cognition were observed when processing the perseverative error of the Wisconsin Card Sorting Test in patients and controls. Our results provide supportive evidence for an effect of SLC1A2 on the etiology of SCZ, suggesting that genetic variation (rs4354668 and its haplotypes) in SLC1A2 may be involved in impaired executive function, which adds to the current body of knowledge regarding the risk of SCZ and the impairment of cognitive performance., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Crystal structures of the archaeal homologue GltPh have provided important insights into the molecular mechanism of transport of the excitatory neurotransmitter glutamate. Whereas mammalian glutamate transporters can translocate both glutamate and aspartate, GltPh is only one capable of aspartate transport. Most of the amino acid residues that surround the aspartate substrate in the binding pocket of GltPh are highly conserved. However, in the brain transporters, Thr-352 and Met-362 of the reentrant hairpin loop 2 are replaced by the smaller Ala and Thr, respectively. Therefore, we have studied the effects of T352A and M362T on binding and transport of aspartate and glutamate by GltPh. Substrate-dependent intrinsic fluorescence changes were monitored in transporter constructs containing the L130W mutation. GltPh-L130W/T352A exhibited an ~15-fold higher apparent affinity for l-glutamate than the wild type transporter, and the M362T mutation resulted in an increased affinity of ~40-fold. An even larger increase of the apparent affinity for l-glutamate, around 130-fold higher than that of wild type, was observed with the T352A/M362T double mutant. Radioactive uptake experiments show that GltPh-T352A not only transports aspartate but also l-glutamate. Remarkably, GltPh-M362T exhibited l-aspartate but not l-glutamate transport. The double mutant retained the ability to transport l-glutamate, but its kinetic parameters were very similar to those of GltPh-T352A alone. The differential impact of mutation on binding and transport of glutamate suggests that hairpin loop 2 not only plays a role in the selection of the substrate but also in its translocation., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background/objective: Recently, a genome-wide association study showed a statistically significant association between the rs3794087 single nucleotide polymorphism (SNP) in the solute carrier family 1--glial affinity glutamate transporter, member 2 (SLC1A2) and the risk for essential tremor (ET). However, four further association studies showed controversial results.We carried out a systematic review and a meta-analysis including all the studies published on the risk of ET related to this SNP., Materials and Methods: The systematic review was performed using several databases, the meta-analysis was carried out using the software Meta-DiSc 1.1.1, and heterogeneity between studies was tested using the Q statistic., Results: The meta-analysis included five association studies for the SLC1A2 rs3794087 SNP (1925 ET patients, 4914 controls) and the risk for ET. The global diagnostic odds ratio (95% confidence intervals) was 1.08 (0.79-1.48) for the total group. After excluding data from the discovery series (which was responsible for a high degree of heterogeneity), the global diagnostic odds ratio (95% confidence intervals) was 0.96 (0.74-1.23). The separate analysis in White and Asiatic individuals on the frequency of the minor allele of rs3794087 did not show significant differences between ET patients and controls in both subgroups after excluding the discovery series., Conclusion: The results of the meta-analysis suggest that rs3794087 is not associated with the risk for ET.

Understanding the storage and release of the excitatory neurotransmitter, L-glutamate by synaptic vesicles has lagged behind receptor characterizations due to a lack of pharmacological agents. We report that the glutamate analog, 3-aminoglutarate (3-AG) is a "silent" false transmitter for glutamate neurons that may be a useful tool to study storage and release mechanisms. Like L-glutamate itself, 3-AG is a high-affinity substrate for both the plasma membrane (EAATs) and vesicular (vGLUT) glutamate transporters. As such, EAATs facilitate 3-AG entry into neuronal cytoplasm allowing 3-AG to compete with L-glutamate for transport into vesicles thus reducing glutamate content. In a synaptosomal preparation, 3-AG inhibited calcium-dependent endogenous L-glutamate release. Unlike L-glutamate, 3-AG had low affinity for both ionotropic (NMDA and AMPA) and G-protein coupled (mGlu1-8) receptors. Consequently, 3-AG behaves as a "silent" false transmitter that may be used in physiological experiments to probe synaptic vesicle storage and release mechanisms for L-glutamate. The companion paper by Wu et al. (2015) describes initial experiments that explore the effects of 3-AG on glutamate synaptic transmission under physiological and pathophysiological conditions., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The SLC1A2 gene encodes the excitatory amino acid transporter 2 (EAAT2). Glutamate is the major mediator of excitatory neurotransmission and EAAT2 is responsible for clearing the neurotransmitter from the synaptic cleft. Genetic variation in SLC1A2 has been implicated in a range of neurological and neuropsychiatric conditions including schizophrenia (SZ), autism and in core phenotypes of bipolar disorder (BD). The coding and putative regulatory regions of SLC1A2 gene were screened for variants using high resolution melting or sequenced in 1099 or in 32 BD subjects. Thirty-two variants were detected in the SLC1A2 gene. Fifteen potentially etiological variants were selected for genotyping in 1099 BD and 1095 control samples. Five amino acid changing variants were also genotyped in 630 participants suffering from SZ. None of the variants were found to be associated with BD or SZ or with the two diseases combined. However, two recurrent missense variants (rs145827578:G>A, p.(G6S); rs199599866:G>A, p.(R31Q)) and one recurrent 5'-untranslated region (UTR) variant (ss825678885:G>T) were detected in cases only. Combined analysis of the recurrent-case-only missense variants and of the case-only missense and 5'-UTR variants showed nominal evidence for association with the combined diseases (Fisher's P=0.019 and 0.0076). These findings are exploratory in nature and await replication in larger cohorts, however, they provide intriguing evidence that potentially functional rare variants in the SLC1A2 gene may confer susceptibility to psychotic disorders.

Transplantation-based replacement of lost and/or dysfunctional astrocytes is a promising therapy for spinal cord injury (SCI) that has not been extensively explored, despite the integral roles played by astrocytes in the central nervous system (CNS). Induced pluripotent stem (iPS) cells are a clinically-relevant source of pluripotent cells that both avoid ethical issues of embryonic stem cells and allow for homogeneous derivation of mature cell types in large quantities, potentially in an autologous fashion. Despite their promise, the iPS cell field is in its infancy with respect to evaluating in vivo graft integration and therapeutic efficacy in SCI models. Astrocytes express the major glutamate transporter, GLT1, which is responsible for the vast majority of glutamate uptake in spinal cord. Following SCI, compromised GLT1 expression/function can increase susceptibility to excitotoxicity. We therefore evaluated intraspinal transplantation of human iPS cell-derived astrocytes (hIPSAs) following cervical contusion SCI as a novel strategy for reconstituting GLT1 expression and for protecting diaphragmatic respiratory neural circuitry. Transplant-derived cells showed robust long-term survival post-injection and efficiently differentiated into astrocytes in injured spinal cord of both immunesuppressed mice and rats. However, the majority of transplant-derived astrocytes did not express high levels of GLT1, particularly at early times post-injection. To enhance their ability to modulate extracellular glutamate levels, we engineered hIPSAs with lentivirus to constitutively express GLT1. Overexpression significantly increased GLT1 protein and functional GLT1-mediated glutamate uptake levels in hIPSAs both in vitro and in vivo post-transplantation. Compared to human fibroblast control and unmodified hIPSA transplantation, GLT1-overexpressing hIPSAs reduced (1) lesion size within the injured cervical spinal cord, (2) morphological denervation by respiratory phrenic motor neurons at the diaphragm neuromuscular junction, and (3) functional diaphragm denervation as measured by recording of spontaneous EMGs and evoked compound muscle action potentials. Our findings demonstrate that hiPSA transplantation is a therapeutically-powerful approach for SCI., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A CD44-SLC1A2 fusion has recently been discovered in a subset of primary gastric cancers, and an APIP-SLC1A2 fusion has been described in a colon cancer cell line (SNU-C1); however, whether such SLC1A2 fusions occur in primary colorectal cancer (CRC) and whether such fusions are specific for gastrointestinal cancers remain uncertain. In the present study, we examined 90 primary CRCs and 112 primary non-small cell lung cancers (NSCLCs) for CD44-SLC1A2 and APIP-SLC1A2 fusion transcripts using RT-PCR and subsequent sequencing analyses. Although the expression of both types of SLC1A2 fusion transcripts was not detected in any of the NSCLCs, the expression of CD44-SLC1A2, but not the APIP-SLC1A2 fusion transcript, was detected in one (1.1 %) CRC. The CD44-SLC1A2 fusion transcript was expressed in cancerous tissue but not in corresponding non-cancerous tissue, and the fusion occurred between exon 1 of CD44 and exon 2 of SLC1A2; it was expected that a slightly truncated but functional SLC1A2 protein would be produced under the CD44 promoter. A quantitative RT-PCR analysis revealed that SLC1A2 mRNA expression was upregulated in CRC containing SLC1A2 fusion transcripts, while it was downregulated in most other CRCs. The SLC1A2 fusion-positive carcinoma was located on the right-side of colon, was a mucinous adenocarcinoma, was immunohistochemically negative for MSH2 mismatch repair protein, and contained no APC or KRAS mutations. Together, these results suggest that the expression of SLC1A2 fusion transcripts is related to a subset of primary CRCs and may contribute to the elucidation of the characteristics of SLC1A2 fusion-positive CRCs in the future.

Dysregulation of the glutamate transporters EAAT1 and EAAT2 and their isoforms have been implicated in schizophrenia. EAAT1 and EAAT2 expression has been studied in different brain regions but the prevalence of astrocytic glutamate transporter expression masks the more subtle changes in excitatory amino acid transporters (EAATs) isoforms in neurons in the cortex. Using laser capture microdissection, pyramidal neurons were cut from the anterior cingulate cortex of postmortem schizophrenia (n = 20) and control (n = 20) subjects. The messenger RNA (mRNA) levels of EAAT1, EAAT2 and the splice variants EAAT1 exon9skipping, EAAT2 exon9skipping and EAAT2b were analyzed by real time PCR (RT-PCR) in an enriched population of neurons. Region-level expression of these transcripts was measured in postmortem schizophrenia (n = 25) and controls (n = 25). The relationship between selected EAAT polymorphisms and EAAT splice variant expression was also explored. Anterior cingulate cortex pyramidal cell expression of EAAT2b mRNA was increased (P < 0.001; 67%) in schizophrenia subjects compared with controls. There was no significant change in other EAAT variants. EAAT2 exon9skipping mRNA was increased (P < 0.05; 38%) at region level in the anterior cingulate cortex with no significant change in other EAAT variants at region level. EAAT2 single-nucleotide polymorphisms were significantly associated with changes in EAAT2 isoform expression. Haloperidol decanoate-treated animals, acting as controls for possible antipsychotic effects, did not have significantly altered neuronal EAAT2b mRNA levels. The novel finding that EAAT2b levels are increased in populations of anterior cingulate cortex pyramidal cells further demonstrates a role for neuronal glutamate transporter splice variant expression in schizophrenia.

Excitatory amino acid transporters (EAATs) are essential for terminating glutamatergic synaptic transmission. They are not only coupled glutamate/Na(+)/H(+)/K(+) transporters but also function as anion-selective channels. EAAT anion channels regulate neuronal excitability, and gain-of-function mutations in these proteins result in ataxia and epilepsy. We have combined molecular dynamics simulations with fluorescence spectroscopy of the prokaryotic homolog GltPh and patch-clamp recordings of mammalian EAATs to determine how these transporters conduct anions. Whereas outward- and inward-facing GltPh conformations are nonconductive, lateral movement of the glutamate transport domain from intermediate transporter conformations results in formation of an anion-selective conduction pathway. Fluorescence quenching of inserted tryptophan residues indicated the entry of anions into this pathway, and mutations of homologous pore-forming residues had analogous effects on GltPh simulations and EAAT2/EAAT4 measurements of single-channel currents and anion/cation selectivities. These findings provide a mechanistic framework of how neurotransmitter transporters can operate as anion-selective and ligand-gated ion channels., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background/aims: Klotho, a transmembrane protein expressed in chorioid plexus of the brain, kidney, and several other tissues, is required for inhibition of 1,25(OH)2D3 formation by FGF23. The extracellular domain of Klotho protein could be cleaved off, thus being released into blood or cerebrospinal fluid. At least in part by exerting β-glucuronidase activity, soluble klotho regulates several ion channels and carriers. Klotho protein deficiency accelerates the appearance of age related disorders including neurodegeneration and muscle wasting and eventually leads to premature death. The present study explored the effect of Klotho protein on the excitatory glutamate transporters EAAT1 (SLC1A3) and EAAT2 (SLC1A2), Na+ coupled carriers clearing excitatory amino acids from the synaptic cleft and thus participating in the regulation of neuronal excitability., Methods: cRNA encoding EAAT1 or EAAT2 was injected into Xenopus laevis oocytes and glutamate (2 mM)-induced inward current (IGlu) taken as measure of glutamate transport. Measurements were made without or with prior 24 h treatment with soluble ß-Klotho protein (30 ng/ml) in the absence and presence of β-glucuronidase inhibitor D-saccharic acid 1,4-lactone monohydrate (DSAL,10 µM)., Results: IGlu was observed in EAAT1 and in EAAT2 expressing oocytes but not in water injected oocytes. In both, EAAT1 and EAAT2 expressing oocytes IGlu was significantly increased by treatment with soluble ß-Klotho protein, an effect reversed by DSAL. Treatment with ß-klotho protein increased significantly the maximal transport rate without significantly modifying the affinity of the carriers., Conclusion: ß-Klotho up-regulates the excitatory glutamate transporters EAAT1 and EAAT2 and thus participates in the regulation of neuronal excitation., (© 2015 The Author(s) Published by S. Karger AG, Basel.)

Objective: Mesial temporal lobe epilepsy (MTLE) is one of the most common types of the intractable epilepsies and is most often associated with hippocampal sclerosis (HS), which is characterized by pronounced loss of hippocampal pyramidal neurons. microRNAs (miRNAs) have been shown to be dysregulated in epilepsy and neurodegenerative diseases, and we hypothesized that miRNAs could be involved in the pathogenesis of MTLE and HS., Methods: miRNA expression was quantified in hippocampal specimens from human patients using miRNA microarray and quantitative real-time polymerase chain reaction RT-PCR, and by RNA-seq on fetal brain specimens from domestic pigs. In situ hybridization was used to show the spatial distribution of miRNAs in the human hippocampus. The potential effect of miRNAs on targets genes was investigated using the dual luciferase reporter gene assay., Results: miRNA expression profiling showed that 25 miRNAs were up-regulated and 5 were down-regulated in hippocampus biopsies of MTLE/HS patients compared to controls. We showed that miR-204 and miR-218 were significantly down-regulated in MTLE and HS, and both were expressed in neurons in all subfields of normal hippocampus. Moreover, miR-204 and miR-218 showed strong changes in expression during fetal development of the hippocampus in pigs, and we identified four target genes, involved in axonal guidance and synaptic plasticity, ROBO1, GRM1, SLC1A2, and GNAI2, as bona fide targets of miR-218. GRM1 was also shown to be a direct target of miR-204., Significance: miR-204 and miR-218 are developmentally regulated in the hippocampus and may contribute to the molecular mechanisms underlying the pathogenesis of MTLE and HS., (Wiley Periodicals, Inc. © 2014 International League Against Epilepsy.)

MicroRNAs (miRNAs) are potential therapeutic targets in a variety of pathological conditions in the brain; however, their clinical application is hampered by lack of efficient delivery modes. Mesenchymal stromal stem cells (MSCs) migrate to sites of injury and inflammation and exert therapeutic effects in various neurological disorders. Here, we examined the ability of MSCs to deliver exogenous miRNA mimics and pre-miRNAs to human neural progenitor cells (NPCs) and astrocytes and characterized the functional impact of this delivery. We found that MSCs efficiently delivered fluorescent-labeled miR-124 and miR-145 mimics to cocultured NPCs and astrocytes. We further demonstrated the delivery of the miRNAs using novel reporter plasmids that contain a sequence complementary to miR-124 or miR-145 downstream of luciferase or mCherry. Binding of the specific miRNAs to these sequences results in decreased luciferase activity or mCherry fluorescence and therefore enable analysis of miRNA delivery in living cells. The delivered exogenous miR-124 significantly decreased the expression of the target gene Sox9 by targeting its 3'-UTR, and increased the neuronal differentiation of the NPCs. In addition, the delivered miR-124 increased the expression of the glutamate transporters, EAAT1 in NPCs and EAAT2 in both NPCs and astrocytes. Similar results were obtained with MSCs transfected with pre-miR-124. The miRNA delivery was mediated by MSC-derived exosomes and was cell contact independent. These results suggest that MSCs can functionally deliver exogenous miRNAs to neural cells and provide an efficient route of therapeutic miRNA delivery to the brain in pathological conditions with clinical implications for regenerative medicine.

Many RNA regulatory proteins controlling pre-messenger RNA splicing contain serine:arginine (SR) repeats. Here, we found that these SR domains bound hydrogel droplets composed of fibrous polymers of the low-complexity domain of heterogeneous ribonucleoprotein A2 (hnRNPA2). Hydrogel binding was reversed upon phosphorylation of the SR domain by CDC2-like kinases 1 and 2 (CLK1/2). Mutated variants of the SR domains changing serine to glycine (SR-to-GR variants) also bound to hnRNPA2 hydrogels but were not affected by CLK1/2. When expressed in mammalian cells, these variants bound nucleoli. The translation products of the sense and antisense transcripts of the expansion repeats associated with the C9orf72 gene altered in neurodegenerative disease encode GRn and PRn repeat polypeptides. Both peptides bound to hnRNPA2 hydrogels independent of CLK1/2 activity. When applied to cultured cells, both peptides entered cells, migrated to the nucleus, bound nucleoli, and poisoned RNA biogenesis, which caused cell death., (Copyright © 2014, American Association for the Advancement of Science.)

EAAT2 is a predominantly astroglial glutamate transporter responsible for the majority of synaptic glutamate clearance in the mammalian central nervous system (CNS). Its dysfunction has been linked with many neurological disorders, including amyotrophic lateral sclerosis (ALS). Decreases in EAAT2 expression and function have been implicated in causing motor neuron excitotoxic death in ALS. Nevertheless, increasing EAAT2 expression does not significantly improve ALS phenotype in mouse models or in clinical trials. In the SOD1-G93A mouse model of inherited ALS, the cytosolic carboxy-terminal domain is cleaved from EAAT2, conjugated to SUMO1, and accumulated in astrocytes where it triggers astrocyte-mediated neurotoxic effects as disease progresses. However, it is not known whether this fragment is sumoylated after cleavage or if full-length EAAT2 is already sumoylated prior to cleavage as part of physiological regulation. In this study, we show that a fraction of full-length EAAT2 is constitutively sumoylated in primary cultures of astrocytes in vitro and in the CNS in vivo. Furthermore, the extent of sumoylation of EAAT2 does not change during the course of ALS in the SOD1-G93A mouse and is not affected by the expression of ALS-causative mutant SOD1 proteins in astrocytes in vitro, indicating that EAAT2 sumoylation is not driven by pathogenic mechanisms. Most interestingly, sumoylated EAAT2 localizes to intracellular compartments, whereas non-sumoylated EAAT2 resides on the plasma membrane. In agreement, promoting desumoylation in primary astrocytes causes increased EAAT2-mediated glutamate uptake. These findings could have implications for optimizing therapeutic approaches aimed at increasing EAAT2 activity in the dysfunctional or diseased CNS., (© 2014 Wiley Periodicals, Inc.)

Astrocyte elevated gene-1 (AEG-1), a novel human immunodeficiency virus (HIV)-1 and tumor necrosis factor (TNF)-α-inducible oncogene, has generated significant interest in the field of cancer research as a therapeutic target for many metastatic aggressive tumors. However, little is known about its role in astrocyte responses during HIV-1 central nervous system (CNS) infection and whether it contributes toward the development of HIV-associated neurocognitive disorders (HAND). Therefore, in this study, we investigated changes in AEG-1 CNS expression in HIV-1-infected brain tissues and elucidated a potential mechanism of AEG-1-mediated regulation of HAND. Immunoblotting and immunohistochemical analyses of HIV-1 seropositive and HIV-1 encephalitic human brain tissues revealed significantly elevated levels of AEG-1 protein. Immunohistochemical analyses of HIV-1 Tat transgenic mouse brain tissues also showed a marked increase in AEG-1 staining. Similar to in vivo observations, cultured astrocytes expressing HIV-1 Tat also revealed AEG-1 and cytokine up-regulation. Astrocytes treated with HAND-relevant stimuli, TNF-α, interleukin (IL)-1β, and HIV-1, also significantly induced AEG-1 expression and nuclear translocation via activation of the nuclear factor (NF)-κB pathway. Co-immunoprecipitation studies demonstrated IL-1β- or TNF-α-induced AEG-1 interaction with NF-κB p65 subunit. AEG-1 knockdown decreased NF-κB activation, nuclear translocation, and transcriptional output in TNF-α-treated astrocytes. Moreover, IL-1β treatment of AEG-1-overexpressing astrocytes significantly lowered expression of excitatory amino acid transporter 2, increased expression of excitatory amino acid transporter 2 repressor ying yang 1, and reduced glutamate clearance, a major transducer of excitotoxic neuronal damage. Findings from this study identify a novel transcriptional co-factor function of AEG-1 and further implicate AEG-1 in HAND-associated neuroinflammation., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Single-strand conformational polymorphism (SSCP) is still a frequently used genotyping method across different fields for the detection of single nucleotide polymorphisms (SNPs) due to its simplicity, requirement for basic equipment accessible in most laboratories and low cost. This technique was previously used to detect rs4354668:A > C (g.-181A > C) SNP in the promoter of astroglial glutamate transporter (EAAT2) and the same approach was initially used here to investigate this promoter region in a cohort of newborns., Results: Unexpectedly, four distinct DNA migration patterns were identified by SSCP. Sanger sequencing revealed two additional SNPs: g.-200C > A and g.-168C > T giving a rise to a total of ten EAAT2 promoter variants. SSCP failed to distinguish these variants reliably and thus pyrosequencing assays were developed. g.-168C > T was found in heterozygous form in one infant only with minor allele frequency (MAF) of 0.0023. In contrast, g.-200C > A and -181A > C were more common (with MAF of 0.46 and 0.49, respectively) and showed string evidence of linkage disequilibrium (LD). In a systematic comparison, 16% of samples were miss-classified by SSCP with 25-31% errors in the identification of the wild-type and homozygote mutant genotypes compared to pyrosequencing or Sanger sequencing. In contrast, SSCP and pyrosequencing of an unrelated single SNP (rs1835740:C > T), showed 94% concordance., Conclusion: Our data suggest that SSCP cannot always detect reliably several closely located SNPs. Furthermore, caution is needed in the interpretation of the association studies linking only one of the co-inherited SNPs in the EAAT2 promoter to human diseases.

Previous human postmortem studies have shown that expression of glutamate transporters (SLC1A2 and SLC1A3) and gamma-aminobutyric acid-synthesizing enzyme [glutamic acid decarboxylase 1 (GAD1)] are reduced in the dorsolateral prefrontal cortex (dlPFC) in subjects with major depressive disorder (MDD). However, no studies have explored the association between these two molecules and its related biological processes in MDD because of limited postmortem sample availability. Data sharing using the Stanley neuropathology consortium integrative database (SNCID), a web-based tool that integrates datasets from the same postmortem brain samples, allowed us to reanalyze existing postmortem data efficiently. We found two datasets where the mRNA levels of GAD1 and SLC1A2 in subregions of the dlPFC were significantly and marginally lower in subjects with MDD (n = 15) than in controls (n = 15) (p = 0.045 and 0.057, respectively). In addition, there was a positive correlation between these two molecules (n = 30, p < 0.05). Spearman's rank correlation analysis using all available datasets revealed that the expression levels of both GAD1 and SLC1A2 mRNAs were commonly correlated with the expression levels of several neuropathological markers in the dlPFC in all of the SNCID subjects (n = 60, p < 0.001). Most of these markers are known to be involved in the RAF/MEK/ERK signal transduction pathway. This exploratory study provides an initial step for future studies to investigate an association between the reductions in SLC1A2 and GAD1 mRNA expression and their relation to the attenuation of the RAF/MEK/ERK signaling pathway in the dlPFC in MDD. The integration of the existing archival data may shed light on one important aspect of the pathophysiology of MDD.