[Exogenous leptin improves cerebral ischemia-reperfusion-induced glutamate excitotoxic injury in mice by up-regulating GLT-1 and GLAST expression in astrocytes].

OBJECTIVE: To investigate the protective effect of exogenous leptin against focal cerebral ischemia-reperfusion (I/R) injury in mice and explore the underlying mechanism. METHODS: A total of 100 C57BL/6 mice were randomly divided into 5 groups, including a sham-operated group, cerebral I/R model group, and 3 leptin treatment groups with intraperitoneal injections of 0.5, 1.0 or 2.0 leptin immediately after occlusion of the internal carotid artery. At 24 h after reperfusion, neurological function scores of the mice were assessed, and TTC staining was used to determine the area of cerebral infarction. The pathological changes in the cortical brain tissue of the mice were observed using HE staining, and degenerative damage of the cortical neurons were assessed with Fluoro-Jade C staining. The expression of glial fibrillary acidic protein in cortical brain tissues was detected using immunohistochemistry and Western blotting. In another 45 C57BL/6 mice with sham operation, I/R modeling, or leptin (1 mg/kg) treatment, glutamic acid in the cortical brain tissue was detected using glutamate assay, and cortical glutamate-aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) protein expressions were detected using immunohistochemistry. RESULTS: Compared with the I/R model mice, the leptin-treated mice had significantly lower neurological deficit scores, smaller cerebral infarct area, milder pathologies in the cortical brain tissue, and lessened cortical neuronal damage with normal morphology and less excessive proliferation of the astrocytes. Leptin treatment significantly up-regulated the expressions of GLT-1 and GLAST and lowered the content of glutamic acid in the brain tissue of the I/R mice. CONCLUSION: Exogenous leptin has obvious neuroprotective effect against cerebral I/R injury in mice, mediated probably by controlling excessive astrocyte proliferation and up-regulating cortical GLT-1 and GLAST expressions to reduce glutamate-mediated excitotoxic injury of the astrocytes.

The astrocyte-enriched gene deathstar plays a crucial role in the development, locomotion, and lifespan of D. melanogaster.

The Drosophila melanogaster brain is a complex organ with various cell types, orchestrating the development, physiology, and behaviors of the fly. While each cell type in Drosophila brain is known to express a unique gene set, their complete genetic profile is still unknown. Advances in the RNA sequencing techniques at single-cell resolution facilitate identifying novel cell type markers and/or re-examining the specificity of the available ones. In this study, exploiting a single-cell RNA sequencing data of Drosophila optic lobe, we categorized the cells based on their expression pattern for known markers, then the genes with enriched expression in astrocytes were identified. CG11000 was identified as a gene with a comparable expression profile to the Eaat1 gene, an astrocyte marker, in every individual cell inside the Drosophila optic lobe and midbrain, as well as in the entire Drosophila brain throughout its development. Consistent with our bioinformatics data, immunostaining of the brains dissected from transgenic adult flies showed co-expression of CG11000 with Eaat1 in a set of single cells corresponding to the astrocytes in the Drosophila brain. Physiologically, inhibiting CG11000 through RNA interference disrupted the normal development of male D. melanogaster, while having no impact on females. Expression suppression of CG11000 in adult flies led to decreased locomotion activity and also shortened lifespan specifically in astrocytes, indicating the gene's significance in astrocytes. We designated this gene as 'deathstar' due to its crucial role in maintaining the star-like shape of glial cells, astrocytes, throughout their development into adult stage.

Identification of Polymorphisms in EAAT1 Glutamate Transporter Gene SLC1A3 Associated with Reduced Migraine Risk.

Dysfunction in ion channels or processes involved in maintaining ionic homeostasis is thought to lower the threshold for cortical spreading depression (CSD), and plays a role in susceptibility to associated neurological disorders, including pathogenesis of a migraine. Rare pathogenic variants in specific ion channels have been implicated in monogenic migraine subtypes. In this study, we further examined the channelopathic nature of a migraine through the analysis of common genetic variants in three selected ion channel or transporter genes: SLC4A4, SLC1A3, and CHRNA4. Using the Agena MassARRAY platform, 28 single-nucleotide polymorphisms (SNPs) across the three candidate genes were genotyped in a case-control cohort comprised of 182 migraine cases and 179 matched controls. Initial results identified significant associations between migraine and rs3776578 (p = 0.04) and rs16903247 (p = 0.05) genotypes within the SLC1A3 gene, which encodes the EAAT1 glutamate transporter. These SNPs were subsequently genotyped in an independent cohort of 258 migraine cases and 290 controls using a high-resolution melt assay, and association testing supported the replication of initial findings-rs3776578 (p = 0.0041) and rs16903247 (p = 0.0127). The polymorphisms are in linkage disequilibrium and localise within a putative intronic enhancer region of SLC1A3. The minor alleles of both SNPs show a protective effect on migraine risk, which may be conferred via influencing the expression of SLC1A3.

ABCG2 and SLC1A5 functionally interact to rewire metabolism and confer a survival advantage to cancer cells under oxidative stress.

ABCG2, a member of the ABC transporter superfamily, is overexpressed in many human tumors and has long been studied for its ability to export a variety of chemotherapeutic agents, thereby conferring a multidrug resistance (MDR) phenotype. However, several studies have shown that ABCG2 can also confer an MDR-independent survival advantage to tumor cells exposed to stress. While investigating the mechanism by which ABCG2 enhances survival in stressful milieus, we have identified a physical and functional interaction between ABCG2 and SLC1A5, a member of the solute transporter superfamily and the primary transporter of glutamine in cancer cells. This interaction was accompanied by increased glutamine uptake, increased glutaminolysis, and rewired cellular metabolism, as evidenced by an increase in key metabolic enzymes and alteration of glutamine-dependent metabolic pathways. Specifically, we observed an increase in glutamine metabolites shuttled to the TCA cycle, and an increase in the synthesis of glutathione, accompanied by a decrease in basal levels of reactive oxygen species and a marked increase in cell survival in the face of oxidative stress. Notably, the knockdown of SLC1A5 or depletion of exogenous glutamine diminished ABCG2-enhanced autophagy flux, further implicating this solute transporter in ABCG2-mediated cell survival. This is, to our knowledge, the first report of a functionally significant physical interaction between members of the two major transporter superfamilies. Moreover, these observations may underlie the protective role of ABCG2 in cancer cells under duress and suggest a novel role for ABCG2 in the regulation of metabolism in normal and diseased states.

Thyroid hormone deficiency affects anxiety-related behaviors and expression of hippocampal glutamate transporters in male congenital hypothyroid rat offspring.

Thyroid hormones are crucial for brain development and their deficiency during fetal and postnatal periods can lead to mood and cognitive disorders. We aimed to examine the consequences of thyroid hormone deficiency on anxiety-related behaviors and protein expression of hippocampal glutamate transporters in congenital hypothyroid male offspring rats. Possible beneficial effects of treadmill exercise have also been examined. Congenital hypothyroidism was induced by adding propylthiouracil (PTU) to drinking water of pregnant Wistar rats from gestational day 6 until the end of the weaning period (postnatal day 28). Next, following 4 weeks of treadmill exercise (5 days per week), anxiety-related behaviors were examined using elevated plus maze (EPM) and light/dark box tests. Thereafter, protein expression of astrocytic (GLAST and GLT-1) and neuronal (EAAC1) glutamate transporters were measured in the hippocampus by immunoblotting. Hypothyroid rats showed decreased anxiety-like behavior, as measured by longer time spent in the open arms of the EPM and in the light area of the light/dark box, compared to control rats. Hypothyroid rats had significantly higher GLAST and GLT-1 and lower EAAC1 protein levels in the hippocampus than did the euthyroid rats. Following exercise, anxiety levels decreased in the euthyroid group while protein expression of EAAC1 increased and returned to normal levels in the hypothyroid group. Our findings indicate that thyroid hormone deficiency was associated with alterations in protein expression of glutamate transporters in the hippocampus. Up-regulation of hippocampal GLAST and GLT-1 could be at least one of the mechanisms associated with the anxiolytic effects of congenital hypothyroidism.

Repeated antibiotic drug treatment negatively affects memory function and glutamatergic nervous system of the hippocampus in mice.

The gut microbiota is associated with memory; however, the relationship between dysbiosis-induced memory deficits and hippocampal glutamatergic neurons remains unclear. In our study, a mouse dysbiosis model showed impaired memory-related behavior in the passive avoidance test; decreased expression levels of glutaminase, excitatory amino acid transporter (EAAT)1, EAAT2, vesicular glutamate transporter 2, synaptophysin, brain-derived neurotrophic factor, doublecortin, neuronal nuclear protein, glial fibrillary acidic protein, and S100ß; and decreased phosphorylation of N-methyl-D-aspartate receptor subunit 1, calmodulin-dependent protein kinase II, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit 1, and cAMP response element-binding protein in the hippocampus. This suggests that dysbiosis-induced memory dysfunction is associated with the hippocampal glutamatergic nervous system.

Corpora cavernosa fibroblasts mediate penile erection.

Penile erection is mediated by the corpora cavernosa, a trabecular-like vascular bed that enlarges upon vasodilation, but its regulation is not completely understood. Here, we show that perivascular fibroblasts in the corpora cavernosa support vasodilation by reducing norepinephrine availability. The effect on penile blood flow depends on the number of fibroblasts, which is regulated by erectile activity. Erection dynamically alters the positional arrangement of fibroblasts, temporarily down-regulating Notch signaling. Inhibition of Notch increases fibroblast numbers and consequently raises penile blood flow. Continuous Notch activation lowers fibroblast numbers and reduces penile blood perfusion. Recurrent erections stimulate fibroblast proliferation and limit vasoconstriction, whereas aging reduces the number of fibroblasts and lowers penile blood flow. Our findings reveal adaptive, erectile activity-dependent modulation of penile blood flow by fibroblasts.

Fibroblasts enable penile erection.

Perivascular fibroblasts may underlie erectile dysfunction.

Mechanistic study of the anti-excitatory amino acid toxicity of Bushen Zhichan decoction for Parkinson's disease based on the transcriptional regulation of EAAT1 by YY1.

ETHNOPHARMACOLOGICAL RELEVANCE: Bushen Zhichan decoction (BSZCF) is derived from Liuwei Dihuang Pill, a famous Chinese herbal formula recorded in the book Key to Therapeutics of Children's Diseases. It has been widely used as a basic prescription for nourishing and tonifying the liver and kidneys to treat Parkinson's disease (PD), but its mechanism remains to be explored. AIM OF THE STUDY: BSZCF, a Chinese herbal formula comprising five herbs: Rehmannia glutinosa (Gaertn.) DC., Dioscorea oppositifolia L., Cornus officinalis Siebold & Zucc., Fallopia multiflora (Thunb.) Haraldson and Cistanche tubulosa (Schenk) Wight, is used clinically to treat PD. In vivo and in vitro experiments were designed to elucidate the mechanism of BSZCF in the protection of dopamine (DA) neurons and the treatment of PD. The toxicity of excitatory amino acids (EAA) may be attenuated by inhibiting the transcription factor Yin Yang 1 (YY1) and up-regulating the expression of excitatory amino acid transporter 1 (EAAT1). MATERIALS AND METHODS: IN VIVO: After 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was intraperitoneally injected into specific pathogen free (SPF) C57BL/6J mice, model mice were intragastrically given adamantane hydrochloride tablets (AHT) or different doses of BSZCF for 14 days. Both open field and pole-climbing tests were conducted to assess behavioral changes. In vitro: 1-Methyl-4-phe-nylpyridiniumiodide (MPP+)-injured human neuroblastoma cells (SH-SY5Y) were utilized to construct PD cell models. Primary astrocytes were transfected with EAAT1 and YY1 lentiviruses for EAAT1 gene knockout and YY1 gene knockout astrocytes, respectively. The high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of BSZCF was performed to control the quality of blood drugs. The optimal concentration and time of PD cell models treated by BSZCF were determined by the use of Cell Counting Kit-8 (CCK8). Enzyme-linked immunosorbent assay (ELISA) was used for measuring glutamate (Glu) in the peripheral blood and cells of each group. Western blotting (WB) and real-time quantitative polymerase chain reaction (qPCR) were used to detect tyrosine hydroxylase (TH), dopamine transporters (DAT), EAAT1 and YY1 protein and mRNA. After the blockade of EAAT1, immunofluorescence (IF) assay was used to detect the TH protein in each group. RESULTS: In vivo research showed that BSZCF improved the behavioral symptoms of PD mice, and reduced the death of DA neurons and the level of Glu. The mechanism may be related to the decrease of YY1 expression and the increase of EAAT1 levels. In vitro experiments showed that the anti-excitatory amino acid toxicity of BSZCF was achieved by inhibiting YY1 expression and regulating EAAT1. CONCLUSIONS: By inhibiting YY1 to increase the expression of EAAT1 and attenuating the toxicity of Glu, BSZCF exerts the effect of protecting DA neurons and treating PD-like symptoms in mice.

Chemically Functionalized Single-Walled Carbon Nanotubes Prevent the Reduction in Plasmalemmal Glutamate Transporter EAAT1 Expression in, and Increase the Release of Selected Cytokines from, Stretch-Injured Astrocytes in Vitro.

We tested the effects of water-soluble single-walled carbon nanotubes, chemically functionalized with polyethylene glycol (SWCNT-PEG), on primary mouse astrocytes exposed to a severe in vitro simulated traumatic brain injury (TBI). The application of SWCNT-PEG in the culture media of injured astrocytes did not affect cell damage levels, when compared to those obtained from injured, functionalization agent (PEG)-treated cells. Furthermore, SWCNT-PEG did not change the levels of oxidatively damaged proteins in astrocytes. However, this nanomaterial prevented the reduction in plasmalemmal glutamate transporter EAAT1 expression caused by the injury, rendering the level of EAAT1 on par with that of control, uninjured PEG-treated astrocytes; in parallel, there was no significant change in the levels of GFAP. Additionally, SWCNT-PEG increased the release of selected cytokines that are generally considered to be involved in recovery processes following injuries. As a loss of EAATs has been implicated as a culprit in the suffering of human patients from TBI, the application of SWCNT-PEG could have valuable effects at the injury site, by preventing the loss of astrocytic EAAT1 and consequently allowing for a much-needed uptake of glutamate from the extracellular space, the accumulation of which leads to unwanted excitotoxicity. Additional potential therapeutic benefits could be reaped from the fact that SWCNT-PEG stimulated the release of selected cytokines from injured astrocytes, which would promote recovery after injury and thus counteract the excess of proinflammatory cytokines present in TBI.

Changes in excitatory amino acid transporters in response to remote ischaemic preconditioning and glutamate excitotoxicity.

The successful implementation of remote ischaemic conditioning as a clinical neuroprotective strategy requires a thorough understanding of its basic principles, which can be modified for each patient. The mechanisms of glutamate homeostasis appear to be a key component. In the current study, we focused on the brain-to-blood glutamate shift mediated by glutamate transporters (excitatory amino acid transports [EAATs]) and the effect of remote ischaemic preconditioning (RIPC) as a mediator of ischaemic tolerance. We used model mimicking ischaemia-mediated excitotoxicity (intracerebroventricular administration of glutamate) to avoid the indirect effect of ischaemia-triggered mechanisms. We found quantitative changes in EAAT2 and EAAT3 and altered membrane trafficking of EAAT1 on the cells of the choroid plexus. These changes could underlie the beneficial effects of ischaemic tolerance. There was reduced oxidative stress and increased glutathione level after RIPC treatment. Moreover, we determined the stimulus-specific response on EAATs. While glutamate overdose stimulated EAAT2 and EAAT3 overexpression, RIPC induced membrane trafficking of EAAT1 and EAAT2 rather than a change in their expression. Taken together, mechanisms related to glutamate homeostasis, especially EAAT-mediated transport, represents a powerful tool of ischaemic tolerance and allow a certain amount of flexibility based on the stimulus used.

Neuroprotective Potential of L-Glutamate Transporters in Human Induced Pluripotent Stem Cell-Derived Neural Cells against Excitotoxicity.

Human induced pluripotent stem cell (hiPSC)-derived neural cells have started to be used in safety/toxicity tests at the preclinical stage of drug development. As previously reported, hiPSC-derived neurons exhibit greater tolerance to excitotoxicity than those of primary cultures of rodent neurons; however, the underlying mechanisms remain unknown. We here investigated the functions of L-glutamate (L-Glu) transporters, the most important machinery to maintain low extracellular L-Glu concentrations, in hiPSC-derived neural cells. We also clarified the contribution of respective L-Glu transporter subtypes. At 63 days in vitro (DIV), we detected neuronal circuit functions in hiPSC-derived neural cells by a microelectrode array system (MEA). At 63 DIV, exposure to 100 µM L-Glu for 24 h did not affect the viability of neural cells. 100 µM L-Glu in the medium decreased to almost 0 µM in 60 min. Pharmacological inhibition of excitatory amino acid transporter 1 (EAAT1) and EAAT2 suppressed almost 100% of L-Glu decrease. In the presence of this inhibitor, 100 µM L-Glu dramatically decreased cell viability. These results suggest that in hiPSC-derived neural cells, EAAT1 and EAAT2 are the predominant L-Glu transporters, and their uptake potentials are the reasons for the tolerance of hiPSC-derived neurons to excitotoxicity.

Conserved allosteric inhibition mechanism in SLC1 transporters.

Excitatory amino acid transporter 1 (EAAT1) is a glutamate transporter belonging to the SLC1 family of solute carriers. It plays a key role in the regulation of the extracellular glutamate concentration in the mammalian brain. The structure of EAAT1 was determined in complex with UCPH-101, apotent, non-competitive inhibitor of EAAT1. Alanine serine cysteine transporter 2 (ASCT2) is a neutral amino acid transporter, which regulates pools of amino acids such as glutamine between intracellular and extracellular compartments . ASCT2 also belongs to the SLC1 family and shares 58% sequence similarity with EAAT1. However, allosteric modulation of ASCT2 via non-competitive inhibitors is unknown. Here, we explore the UCPH-101 inhibitory mechanisms of EAAT1 and ASCT2 by using rapid kinetic experiments. Our results show that UCPH-101 slows substrate translocation rather than substrate or Na+ binding, confirming a non-competitive inhibitory mechanism, but only partially inhibits wild-type ASCT2. Guided by computational modeling using ligand docking and molecular dynamics simulations, we selected two residues involved in UCPH-101/EAAT1 interaction, which were mutated in ASCT2 (F136Y, I237M, F136Y/I237M) in the corresponding positions. We show that in the F136Y/I237M double-mutant transporter, 100% of the inhibitory effect of UCPH-101 could be restored, and the apparent affinity was increased (Ki = 4.3 µM), much closer to the EAAT1 value of 0.6 µM. Finally, we identify a novel non-competitive ASCT2 inhibitor, through virtual screening and experimental testing against the allosteric site, further supporting its localization. Together, these data indicate that the mechanism of allosteric modulation is conserved between EAAT1 and ASCT2. Due to the difference in binding site residues between ASCT2 and EAAT1, these results raise the possibility that more potent, and potentially selective ASCT2 allosteric inhibitors can be designed .

Mutation in glutamate transporter homologue GltTk provides insights into pathologic mechanism of episodic ataxia 6.

Episodic ataxias (EAs) are rare neurological conditions affecting the nervous system and typically leading to motor impairment. EA6 is linked to the mutation of a highly conserved proline into an arginine in the glutamate transporter EAAT1. In vitro studies showed that this mutation leads to a reduction in the substrates transport and an increase in the anion conductance. It was hypothesised that the structural basis of these opposed functional effects might be the straightening of transmembrane helix 5, which is kinked in the wild-type protein. In this study, we present the functional and structural implications of the mutation P208R in the archaeal homologue of glutamate transporters GltTk. We show that also in GltTk the P208R mutation leads to reduced aspartate transport activity and increased anion conductance, however a cryo-EM structure reveals that the kink is preserved. The arginine side chain of the mutant points towards the lipidic environment, where it may engage in interactions with the phospholipids, thereby potentially interfering with the transport cycle and contributing to stabilisation of an anion conducting state.

Yes-associated protein regulates glutamate homeostasis through promoting the expression of excitatory amino acid transporter-2 in astrocytes via ß-catenin signaling.

The homeostasis of glutamate is mainly regulated by the excitatory amino acid transporters (EAATs), especially by EAAT2 in astrocytes. Excessive glutamate in the synaptic cleft caused by dysfunction or dysregulation of EAAT2 can lead to excitotoxicity, neuronal death and cognitive dysfunction. However, it remains unclear about the detailed regulation mechanism of expression and function of astrocytic EAAT2. In this study, first, we found increased neuronal death and impairment of cognitive function in YAPGFAP -CKO mice (conditionally knock out Yes-associated protein [YAP] in astrocytes), and identified EAAT2 as a downstream target of YAP through RNA sequencing. Second, the expression of EAAT2 was decreased in cultured YAP-/- astrocytes and the hippocampus of YAPGFAP -CKO mice, and glutamate uptake was reduced in YAP-/- astrocytes, but increased in YAP-upregulated astrocytes. Third, further investigation of the mechanism showed that the mRNA and protein levels of ß-catenin were decreased in YAP-/- astrocytes and increased in YAP-upregulated astrocytes. Wnt3a activated YAP signaling and up-regulated EAAT2 through ß-catenin. Furthermore, over-expression or activation of ß-catenin partially restored the downregulation of EAAT2, the impairment of glutamate uptake, neuronal death and cognitive decline that caused by YAP deletion. Finally, activation of EAAT2 also rescued neuronal death and cognitive decline in YAPGFAP -CKO mice. Taken together, our study identifies an unrecognized role of YAP signaling in the regulation of glutamate homeostasis through the ß-catenin/EAAT2 pathway in astrocytes, which may provide novel insights into the pathogenesis of brain diseases that closely related to the dysfunction or dysregulation of EAAT2, and promote the development of clinical strategy.

Selective Clearance of Senescent Chondrocytes in Osteoarthritis by Targeting Excitatory Amino Acid Transporter Protein 1 to Induce Ferroptosis.

Aims: This study aimed at exploring the mechanism of ferroptosis (an iron-dependent form of nonapoptotic cell death) resistance in senescent chondrocytes (SenChos). Results: In this study, by utilizing metabolomics and single-cell RNA sequencing, we found that hyperactivation of ferroptosis metabolism was one of the most prominent metabolic features in SenChos. Interestingly, however, SenChos were able to survive in this state and were resistant to ferroptosis-induced cell death. Next, we elucidated that this survival mechanism of SenChos could be primarily attributed to overexpression of the membrane protein excitatory amino acid transporter protein 1 (EAAT1), which can increase intracellular glutamate (Glu) levels and activate the glutathione system to counteract ferroptosis. In addition, 2-amino-5,6,7,8-tetrahydro-4-(4-methoxyphenyl)-7-(naphthalen-1-yl)-5-oxo-4H-chromene-3-carbonitrile (UCPH-101) (a specific inhibitor of EAAT1) and siRNA-EAAT1 were able to substantially increase the sensitivity of SenChos to ferroptosis and to induce cell death, with no apparent effects on the normal cells. Administration of an intraarticular injection of UCPH-101 caused inhibition of EAAT1 selectively, cleared SenChos from cartilage, improved the cartilage homeostasis, and significantly delayed the progression of osteoarthritis (OA). Innovation: This work supports a relevant role for EAAT1 in ferroptosis resistance mechanism for SenChos, revealing a potential therapeutic target of OA. Conclusions: EAAT1-Glu-glutathione peroxidase 4 anti-ferroptosis axis is a key survival mechanism for SenChos, and EAAT1 is an effective and specific target for anti-senescence therapy in OA. Antioxid. Redox Signal. 39, 262-277.

EphA4/ephrinA3 reverse signaling mediated downregulation of glutamate transporter GLAST in Müller cells in an experimental glaucoma model.

Deficiency of glutamate transporter GLAST in Müller cells may be culpable for excessive extracellular glutamate, which involves in retinal ganglion cell (RGC) damage in glaucoma. We elucidated how GLAST was regulated in rat chronic ocular hypertension (COH) model. Western blot and whole-cell patch-clamp recordings showed that GLAST proteins and GLAST-mediated current densities in Müller cells were downregulated at the early stages of COH. In normal rats, intravitreal injection of the ephrinA3 activator EphA4-Fc mimicked the changes of GLAST in COH retinas. In purified cultured Müller cells, EphA4-Fc treatment reduced GLAST expression at mRNA and protein levels, which was reversed by the tyrosine kinase inhibitor PP2 or transfection with ephrinA3-siRNA (Si-EFNA3), suggesting that EphA4/ephrinA3 reverse signaling mediated GLAST downregulation. EphA4/ephrinA3 reverse signaling-induced GLAST downregulation was mediated by inhibiting PI3K/Akt/NF-κB pathways since EphA4-Fc treatment of cultured Müller cells reduced the levels of p-Akt/Akt and NF-κB p65, which were reversed by transfecting Si-EFNA3. In Müller cells with ephrinA3 knockdown, the PI3K inhibitor LY294002 still decreased the protein levels of NF-κB p65 in the presence of EphA4-Fc, and the mRNA levels of GLAST were reduced by LY294002 and the NF-κB inhibitor SN50, respectively. Pre-injection of the PI3K/Akt pathway activator 740 Y-P reversed the GLAST downregulation in COH retinas. Western blot and TUNEL staining showed that transfecting of Si-EFNA3 reduced Müller cell gliosis and RGC apoptosis in COH retinas. Our results suggest that activated EphA4/ephrinA3 reverse signaling induces GLAST downregulation in Müller cells via inhibiting PI3K/Akt/NF-κB pathways, thus contributing to RGC damage in glaucoma.

Comparing the mitochondrial signatures in ESCs and iPSCs and their neural derivations.

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have distinct origins: ESCs are derived from pre-implanted embryos while iPSCs are reprogrammed somatic cells. Both have their own characteristics and lineage specificity, and both are valuable tools for studying human neurological development and disease. Thus far, few studies have analyzed how differences between stem cell types influence mitochondrial function and mitochondrial DNA (mtDNA) homeostasis during differentiation into neural and glial lineages. In this study, we compared mitochondrial function and mtDNA replication in human ESCs and iPSCs at three different stages - pluripotent, neural progenitor and astrocyte. We found that while ESCs and iPSCs have a similar mitochondrial signature, neural and astrocyte derivations manifested differences. At the neural stem cell (NSC) stage, iPSC-NSCs displayed decreased ATP production and a reduction in mitochondrial respiratory chain (MRC) complex IV expression compared to ESC-NSCs. IPSC-astrocytes showed increased mitochondrial activity including elevated ATP production, MRC complex IV expression, mtDNA copy number and mitochondrial biogenesis relative to those derived from ESCs. These findings show that while ESCs and iPSCs are similar at the pluripotent stage, differences in mitochondrial function may develop during differentiation and must be taken into account when extrapolating results from different cell types.Abbreviation: BSA: Bovine serum albumin; DCFDA: 2',7'-dichlorodihydrofluorescein diacetate; DCX: Doublecortin; EAAT-1: Excitatory amino acid transporter 1; ESCs: Embryonic stem cells; GFAP: Glial fibrillary acidic protein; GS: Glutamine synthetase; iPSCs: Induced pluripotent stem cells; LC3B: Microtubule-associated protein 1 light chain 3ß; LC-MS: Liquid chromatography-mass spectrometry; mito-ROS: Mitochondrial ROS; MMP: Mitochondrial membrane potential; MRC: Mitochondrial respiratory chain; mtDNA: Mitochondrial DNA; MTDR: MitoTracker Deep Red; MTG: MitoTracker Green; NSCs: Neural stem cells; PDL: Poly-D-lysine; PFA: Paraformaldehyde; PGC-1α: PPAR-γ coactivator-1 alpha; PPAR-γ: Peroxisome proliferator-activated receptor-gamma; p-SIRT1: Phosphorylated sirtuin 1; p-ULK1: Phosphorylated unc-51 like autophagy activating kinase 1; qPCR: Quantitative PCR; RT: Room temperature; RT-qPCR: Quantitative reverse transcription PCR; SEM: Standard error of the mean; TFAM: Mitochondrial transcription factor A; TMRE: Tetramethylrhodamine ethyl ester; TOMM20: Translocase of outer mitochondrial membrane 20.

EAAT1-dependent slc1a3 Transcriptional Control depends on the Substrate Translocation Process.

SUMMARY STATEMENT: EAAT1/GLAST down-regulates its expression and function at the transcriptional level by activating a signaling pathway that includes PI3K, PKC and NF-κB, favoring the notion of an activity-dependent fine-tuning of glutamate recycling and its synaptic transactions through glial cells.

Glutamate-aspartate transporter 1 attenuates oxygen-glucose deprivation-induced injury by promoting glutamate metabolism in primary cortical neurons.

Ischemic stroke is a common cerebral disease. However, the treatment for the disease is limited. Daurian ground squirrel (GS; Spermophilus dauricus), a hibernating mammalian species, is highly tolerant to ischemia. In the present study, GS neurons in a non-hibernating state were found to be more resistant to oxygen-glucose deprivation (OGD), an ischemic model in vitro. We leveraged the differences in the endurance capacity of GS and rats to investigate the mechanisms of resistance to ischemia in GS neurons. We first identified glutamate-aspartate transporter 1 (GLAST) as a cytoprotective factor that contributed to tolerance against OGD injury of GS neurons. The expression of GLAST in GS neurons was much higher than that in rat neurons. Overexpression of GLAST rescued viability in rat neurons, and GS neurons exhibited decreased viability following GLAST knockdown under OGD conditions. Mechanistically, more glutamate was transported into neurons after GLAST overexpression and served as substrates for ATP production. Furthermore, eukaryotic transcription initiation factor 4E binding protein 1 was downregulated by GLAST to rescue neuronal viability. Our findings not only revealed an important molecular mechanism underlying the survival of hibernating mammals but also suggested that neuronal GLAST may be a potential target for ischemic stroke therapy.