Ligands of peroxisome proliferator-activated receptor-alpha promote glutamate transporter-1 endocytosis in astrocytes

Astrocytes, a stellate-shape glial population in the central nervous system (CNS), maintain glutamate homeostasis in adult CNS by undergoing glutamate uptake at the synapse through their glutamate transporter-1 (GLT-1). Peroxisome proliferator-activated receptor-α (PPARα) can be activated by endogenous saturated fatty acids to regulate astrocytic lipid metabolism and functions. However, it is unclear if PPARα can exert the regulatory action on GLT-1 expression in astrocytes. This study showed that treatment with palmitic acid (PA) and the other two PPARα agonists (GW 7647 and WY 14,643) caused no change in the morphology of astrocytes, whereas membranous GLT-1 protein levels in astrocytes were significantly decreased by PA and PPARα agonists. Through lentivirus-mediated overexpression of GLT-1 tagged with red fluorescent protein (GLT-1-RFP), we also observed that GLT-1-RFP puncta in the processes of astrocytes were inhibited by the PPARα agonists. This reduction was prevented by the addition of the PPARα antagonist, GW6471. GLT-1-RFP was co-localized to the early endosome marker–EEA1 in astrocytes treated with the PPARα agonists. Moreover, PPARα-induced inhibition in membranous GLT-1 expression was abolished by the addition of dynamin inhibitor (dynasore). Furthermore, the co-treatment of astrocytes with PPARα agonists and dynasore, or with PPARα agonists and protein kinase C (PKC) inhibitor bis-indolylmaleimide 1 (BIS1), prevented the endocytosis of GLT-1-RFP. Based on the results, we conclude that the PPARα agonists increased GLT-1 endocytosis in astrocytes possibly through the PKC signaling pathway. In addition, our findings provide important information of PPARα involvement in the downregulation of astrocytic glutamate uptake via the promoted GLT-1 endocytosis.     

GPR30 regulates glutamate transporter GLT-1 expression in rat primary astrocytes

The G protein-coupled estrogen receptor GPR30 contributes to the neuroprotective effects of 17β-estradiol (E2); however, the mechanisms associated with this protection have yet to be elucidated. Given that E2 increases astrocytic expression of glutamate transporter-1 (GLT-1), which would prevent excitotoxic-induced neuronal death, we proposed that GPR30 mediates E2 action on GLT-1 expression. To investigate this hypothesis, we examined the effects of G1, a selective agonist of GPR30, and GPR30 siRNA on astrocytic GLT-1 expression, as well as glutamate uptake in rat primary astrocytes, and explored potential signaling pathways linking GPR30 to GLT-1. G1 increased GLT-1 protein and mRNA levels, subject to regulation by both MAPK and PI3K signaling. Inhibition of TGF-α receptor suppressed the G1-induced increase in GLT-1 expression. Silencing GPR30 reduced the expression of both GLT-1 and TGF-α and abrogated the G1-induced increase in GLT-1 expression. Moreover, the G1-induced increase in GLT-1 protein expression was abolished by a protein kinase A inhibitor and an NF-κB inhibitor. G1 also enhanced cAMP response element-binding protein (CREB), as well as both NF-κB p50 and NF-κB p65 binding to the GLT-1 promoter. Finally, to model dysfunction of glutamate transporters, manganese was used, and G1 was found to attenuate manganese-induced impairment in GLT-1 protein expression and glutamate uptake. Taken together, the present data demonstrate that activation of GPR30 increases GLT-1 expression via multiple pathways, suggesting that GPR30 is worthwhile as a potential target to be explored for developing therapeutics of excitotoxic neuronal injury.     

Modulation of Glutamate Transport and Receptor Binding by Glutamate Receptor Antagonists in EAE Rat Brain

The etiology of multiple sclerosis (MS) is currently unknown. However, one potential mechanism involved in the disease may be excitotoxicity. The elevation of glutamate in cerebrospinal fluid, as well as changes in the expression of glutamate receptors (iGluRs and mGluRs) and excitatory amino acid transporters (EAATs), have been observed in the brains of MS patients and animals subjected to experimental autoimmune encephalomyelitis (EAE), which is the predominant animal model used to investigate the pathophysiology of MS. In the present paper, the effects of glutamatergic receptor antagonists, including amantadine, memantine, LY 367583, and MPEP, on glutamate transport, the expression of mRNA of glutamate transporters (EAATs), the kinetic parameters of ligand binding to N-methyl-D-aspartate (NMDA) receptors, and the morphology of nerve endings in EAE rat brains were investigated. The extracellular level of glutamate in the brain is primarily regulated by astrocytic glutamate transporter 1 (GLT-1) and glutamate-aspartate transporter (GLAST). Excess glutamate is taken up from the synaptic space and metabolized by astrocytes. Thus, the extracellular level of glutamate decreases, which protects neurons from excitotoxicity. Our investigations showed changes in the expression of EAAT mRNA, glutamate transport (uptake and release) by synaptosomal and glial plasmalemmal vesicle fractions, and ligand binding to NMDA receptors; these effects were partially reversed after the treatment of EAE rats with the NMDA antagonists amantadine and memantine. The antagonists of group I metabotropic glutamate receptors (mGluRs), including LY 367385 and MPEP, did not exert any effect on the examined parameters. These results suggest that disturbances in these mechanisms may play a role in the processes associated with glutamate excitotoxicity and the progressive brain damage in EAE.     

The GLT-1 and GLAST glutamate transporters are expressed on morphologically distinct astrocytes and regulated by neuronal activity in primary hippocampal cocultures

The GLT-1 and GLAST astroglial transporters are the glutamate transporters mainly involved in maintaining physiological extracellular glutamate concentrations. Defects in neurotransmitter glutamate transport may represent an important component of glutamate-induced neurodegenerative disorders (such as amyotrophic lateral sclerosis) and CNS insults (ischemia and epilepsy). We characterized the protein expression of GLT-1 and GLAST in primary astrocyte-neuron cocultures derived from rat hippocampal tissues during neuron differentiation/maturation. GLT-1 and GLAST are expressed by morphologically distinct glial fibrillary acidic protein-positive astrocytes, and their expression correlates with the status of neuron differentiation/maturation and activity. Up-regulation of the transporters paralleled the content of the synaptophysin synaptic vesicle marker p38, and down-regulation was a consequence of glutamate-induced neuronal death or the reduction of synaptic activity. Finally, soluble factors in neuronal-conditioned media prevented the down-regulation of the GLT-1 and GLAST proteins. Although other mechanisms may participate in regulating GLT-1 and GLAST in the CNS, our data indicate that soluble factors dependent on neuronal activity play a major regulating role in hippocampal cocultures.     

Primary Cultures From Cerebral Cortex and Hippocampus Enriched in Glutamatergic and GABAergic Neurons

The aim was to define a primary culture system enriched in neurons using a defined culture medium, and characterize the model system as to cellular morphology and neuronal phenotypes. We found that these primary neuron enriched cultures from either newborn rat cerebral cortex or hippocampus contain small GABAergic and large glutamatergic neurons as well as astrocytes and microglia. Astrocytes in these cultures are morphologically differentiated with long, slender processes and interact with soluble factors responsible for induction and expression of the glutamate transporter GLT-1. The cultures achieve the highest expression of the vesicular glutamate transporter 1 (VGLUT1) and GLT-1 after 20 days in vitro. Conditioned media from these neuron enriched cultures also induced GLT-1 expression in primary astrocytic cultures, which were free from neurons. The amount of glutamatergic neurons guides the morphological maturation of astrocytes and GLT-1 expression both in the neuron enriched cultures and in the conditioned media supplemented astrocytic cultures. Interestingly, these cultures were not influenced or activated by the inflammatory stimulus lipopolysaccharide. This suggests that soluble factors from neurons protect microglia and astrocytes to become inflammatory reactive. In conclusion we have developed a well characterized culture model system enriched in neurons, taken from newborn rats and cultured in defined media. The neurons express different neuronal phenotypes. Such a model system is valuable when studying interactions between neurons and glial cells.     

Regulation of glutamate transporter GLT-1 by MAGI-1

The sodium-dependent glutamate transporter, glutamate transporter subtype 1 (GLT-1) is one of the main glutamate transporters in the brain. GLT-1 contains a COOH-terminal sequence similar to one in an isoform of Slo1 K(+) channel protein previously shown to bind MAGI-1 (membrane-associated guanylate kinase with inverted orientation protein-1). MAGI-1 is a scaffold protein which allows the formation of complexes between certain transmembrane proteins, actin-binding proteins, and other regulatory proteins. The glutathione S-transferase pull-down assay demonstrated that MAGI-1 was a binding partner of GLT-1. The interaction between MAGI-1 and GLT-1 was confirmed by co-immunoprecipitation. Immunofluorescence of MAGI-1 and GLT-1 demonstrated that the distribution of MAGI-1 and GLT-1 overlapped in astrocytes. Co-expression of MAGI-1 with GLT-1 in C6 Glioma cells resulted in a significant reduction in the surface expression of GLT-1, as assessed by cell-surface biotinylation. On the other hand, partial knockdown of endogenous MAGI-1 expression by small interfering RNA in differentiated cultured astrocytes increased glutamate uptake and the surface expression of endogenous GLT-1. Knockdown of MAGI-1 increased dihydrokainate-sensitive, Na(+) -dependent glutamate uptake, indicating that MAGI-1 regulates GLT-1 mediated glutamate uptake. These data suggest that MAGI-1 regulates surface expression of GLT-1 and the level of glutamate in the hippocampus.     

Reversed Actrocytic GLT-1 during Ischemia is Crucial to Excitotoxic Death of Neurons, but Contributes to the Survival of Astrocytes themselves

During ischemia, the operation of astrocytic/neuronal glutamate transporters is reversed and glutamate and Na⁺ are co-transported to the extracellular space. This study aims to investigate whether this reversed operation of glutamate transporters has any functional meanings for astrocytes themselves. Oxygen/glucose deprivation (OGD) of neuron/astrocyte co-cultures resulted in the massive death of neurons, and the cell death was significantly reduced by treatment with either AP5 or DHK. In cultured astrocytes with little GLT-1 expression, OGD produced Na⁺ overload, resulting in the reversal of astrocytic Na⁺/Ca²⁺-exchanger (NCX). The reversed NCX then caused Ca²⁺ overload leading to the damage of astrocytes. In contrast, the OGD-induced Na⁺ overload and astrocytic damage were significantly attenuated in PACAP-treated astrocytes with increased GLT-1 expression, and the attenuation was antagonized by treatment with DHK. These results suggested that the OGD-induced reversal of GLT-1 contributed to the survival of astrocytes themselves by releasing Na⁺ with glutamate via reversed GLT-1.     

Characterisation of the expression of NMDA receptors in human astrocytes

Astrocytes have long been perceived only as structural and supporting cells within the central nervous system (CNS). However, the discovery that these glial cells may potentially express receptors capable of responding to endogenous neurotransmitters has resulted in the need to reassess astrocytic physiology. The aim of the current study was to characterise the expression of NMDA receptors (NMDARs) in primary human astrocytes, and investigate their response to physiological and excitotoxic concentrations of the known endogenous NMDAR agonists, glutamate and quinolinic acid (QUIN). Primary cultures of human astrocytes were used to examine expression of these receptors at the mRNA level using RT-PCR and qPCR, and at the protein level using immunocytochemistry. The functionality role of the receptors was assessed using intracellular calcium influx experiments and measuring extracellular lactate dehydrogenase (LDH) activity in primary cultures of human astrocytes treated with glutamate and QUIN. We found that all seven currently known NMDAR subunits (NR1, NR2A, NR2B, NR2C, NR2D, NR3A and NR3B) are expressed in astrocytes, but at different levels. Calcium influx studies revealed that both glutamate and QUIN could activate astrocytic NMDARs, which stimulates Ca2+ influx into the cell and can result in dysfunction and death of astrocytes. Our data also show that the NMDAR ion channel blockers, MK801, and memantine can attenuate glutamate and QUIN mediated cell excitotoxicity. This suggests that the mechanism of glutamate and QUIN gliotoxicity is at least partially mediated by excessive stimulation of NMDARs. The present study is the first to provide definitive evidence for the existence of functional NMDAR expression in human primary astrocytes. This discovery has significant implications for redefining the cellular interaction between glia and neurons in both physiological processes and pathological conditions.     

Thiamine deficiency results in release of soluble factors that disrupt mitochondrial membrane potential and downregulate the glutamate transporter splice-variant GLT-1b in cultured astrocytes

Loss of astrocytic glutamate transporters is a major feature of both thiamine deficiency (TD) and Wernicke’s encephalopathy. However, the underlying basis of this process is not well understood. In the present study we have investigated the possibility of release of astrocytic soluble factors that might be involved in the regulation of the glutamate transporter GLT-1b in these cells. Treatment of naïve astrocytes with conditioned media from astrocytes exposed to TD conditions resulted in a progressive decrease in glutamate uptake over 24 h. Immunoblotting and flow cytometry measurements indicated this was accompanied by a 20–40% loss of GLT-1b. Astrocytes exposed to either TD or TD conditioned media showed increased disruption of mitochondrial membrane potential compared to control cells, and treatment of astrocytes with TD resulted in an increase in the pro-inflammatory cytokine TNF-α and elevated levels of phospho-IκB fragment, indicative of increased activation of NF-κB. Inhibition of TNF-α activity with the use of a neutralizing antibody blocked the increased NF-κB activation, while inhibition of NF-κB ameliorated the decrease in GLT-1b and reversed the decrease in glutamate uptake occurring with TD treatment. Together, these findings indicate that astrocytes exposed to TD conditions show responses suggesting that soluble factors released by these cells under conditions of TD play a regulatory role in terms of glutamate transport function and mitochondrial integrity.     

Down-expressed GLT-1 in PSD astrocytes inhibits synaptic formation of NSC-derived neurons in vitro

Little is known about the effect of astroglial GLT-1 of post-stroke depression (PSD) rat model on the function of neural stem cells (NSCs). This study aimed to investigate whether astroglial GLT-1 of PSD rats affect differentiation of NSCs from neonatal rat hippocampus and synaptic formation of NSC-derived neurons. Astrocytes were isolated from the left hippocampus of normal adult SD rats and PSD rats. A lentiviral vector was used to silence the expression of GLT-1 in astrocytes of PSD rats. NSCs were respectively co-cultured with normal (control), PSD, and GLT-1 silenced astrocytes for 7 days. GLT-1, GFAP, MAP2, Synaptophysin (SYN), glutamate (Glu) and glutamine (Gln) were respectively measured by qRT-PCR, western blot, immunofluorescence and efficient mass spectrometry (MS). PSD astrocytes increased the number of NSC-derived astrocytes, but inhibited the expression of GLT-1 of NSC-derived astrocytes and synapses of NSC-derived neurons. On the basis of the low expression of GLT-1 in PSD astrocytes, we further silenced GLT-1 in PSD astrocytes. Interestingly, GLT-1 silenced PSD astrocytes more obviously inhibited synapses of NSC-derived neurons, but increased the number of NSC-derived neurons and reversed the expression of GLT-1 in NSC-derived astrocytes. At the same time, concentration of glutamate in the medium elevated, and glutamine in the medium gradually reduced. In NSC-derived neurons and astrocytes, glutamate metabolism was also affected by changed GLT-1. Down-expressed GLT-1 in PSD astrocytes stimulated NSCs differentiating into astrocytes, but inhibiting the formation of functional synapses by influencing glutamate metabolism in vitro.     

Methylmercury alters the in vitro uptake of glutamate in GLAST- and GLT-1-transfected mutant CHO-K1 cells

In order to maintain normal functioning of the brain, glutamate homeostasis and extracellular levels of excitotoxic amino acids (EAA) must be tightly controlled. This is accomplished, in large measure, by the astroglial high-affinity Na⁺-dependent EAA transporters glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1). Methylmercury (MeHg) is a potent neurotoxicant. Astrocytes are known targets for MeHg toxicity, representing a site for mercury localization. Mehg is known to cause astrocytic swelling, EAA release, and uptake inhibition in astrocytes, leading to increased extracellular glutamate levels and ensuing neuronal excitotoxicity and degeneration. However, the mechanisms and contribution of specific glutamate transporters to MeHg-induced glutamate dyshomeostasis remain unknown. Accordingly, the present study was carried out to investigate the effects of MeHg on the transport of [d-2, 3-³H]-d-aspartate, a nonmetabolizable glutamate analog in Chinese hamster ovary cells (CHO) transfected with the glutamate transporter subtypes GLAST or GLT-1. Additional studies examined the effects of MeHg on mRNA and protein levels of these transporters. Our results indicate the following (1) MeHg selectively affects glutamate transporter mRNA expression. MeHg treatment (6 h) led to no discernible changes in GLAST mRNA expression; however, GLT-1 mRNA expression significantly (p<0.001) increased following treatments with 5 or 10 μM MeHg. (2) Selective changes in the expression of glutamate transporter protein levels were also noted. GLAST transporter protein levels significantly (p<0.001, both at 5 and 10 μM MeHg) increased and GLT-1 transporter protein levels significantly (p<0.001) decreased followign MeHg exposure (5 μM). (3) MeHg exposure led to significant inhibition (p<0.05) of glutamate uptake by GLAST (both 5 and 10 μM MeHg), whereas GLT-1 transporter activity was significantly (p<0.01) increased following exposure to 5 and 10 μM MeHg. These studies suggest that MeHg contributes to the dysregulation of glutamate homeostasis and that its effects are distinct for GLAST and GLT-1.     

Expression of SOD1 G93A or wild-type SOD1 in primary cultures of astrocytes down-regulates the glutamate transporter GLT-1: lack of involvement of oxidative stress

Glutamate excitotoxicity is implicated in the aetiology of amyotrophic lateral sclerosis (ALS) with impairment of glutamate transport into astrocytes a possible cause of glutamate-induced injury to motor neurons. It is possible that mutations of Cu/Zn superoxide dismutase (SOD1), responsible for about 20% of familial ALS, down-regulates glutamate transporters via oxidative stress. We transfected primary mouse astrocytes to investigate the effect of the FALS-linked mutant hSOD1(G93A) and wild-type SOD1 (hSOD1wt) on the glutamate uptake system. Using western blotting, immunocytochemistry and RT-PCR it was shown that expression of either hSOD1(G93A) or hSOD1wt in astrocytes produced down-regulation of the levels of a glutamate transporter GLT-1, without alterations in its mRNA level. hSOD1(G93A) or hSOD1wt expression caused a decrease of the monomeric form of GLT-1 without increasing oxidative multimers of GLT-1. The effects were selective to GLT-1, since another glutamate transporter GLAST protein and mRNA levels were not altered. Reflecting the decrease in GLT-1 protein, [3H]d-aspartate uptake was reduced in cultures expressing hSOD1(G93A) or hSOD1wt. The hSOD1-induced decline in GLT-1 protein and [3H]d-aspartate uptake was not blocked by the antioxidant Trolox nor potentiated by antioxidant depletion using catalase and glutathione peroxidase inhibitors. Measurement of 2',7'-dichlorofluorescein (DCF)-induced fluorescence revealed that expression of hSOD1(G93A) or hSOD1wt in astrocytes does not lead to detectable increase of intracellular reactive oxygen species. This study suggests that levels of GLT-1 protein in astrocytes are reduced rapidly by overexpression of hSOD1, and is due to a property shared between the wild-type and G93A mutant form, but does not involve the production of intracellular oxidative stress.     

Loss of the glutamate transporter splice-variant GLT-1b in inferior colliculus and its prevention by ceftriaxone in thiamine deficiency

Downregulation of astrocytic glutamate transporters is a feature of thiamine deficiency (TD), the underlying cause of Wernicke's encephalopathy, and plays a major role in its pathophysiology. Recent investigations suggest that ceftriaxone, a β-lactam antibiotic, stimulates GLT-1 expression and confers neuroprotection against ischemic and motor neuron degeneration. Thus, ceftriaxone treatment may be a protective strategy against excitotoxic conditions. In the present study, we examined the effects of ceftriaxone on the glutamate transporter splice-variant GLT-1b in rats with TD and in cultured astrocytes under TD conditions. Our results indicate that ceftriaxone protects against loss of GLT-1b levels in the inferior colliculus during TD, but with no significant effect in the thalamus and frontal cortex by immunoblotting and immunohistochemistry. Ceftriaxone also normalized the loss of GLT-1b in astrocyte cultures under conditions of TD. These results suggest that ceftriaxone has the ability to increase GLT-1b levels in astrocytes during TD, and may be an important pharmacological strategy for the treatment of excitotoxicity in this disorder.     

Insulin increases glutamate transporter GLT1 in cultured astrocytes

The astroglial cell-specific glutamate transporter subtype 2 (excitatory amino acid transporter 2, GLT1) plays an important role in excitotoxicity that develops after damage to the central nervous system (CNS) is incurred. Both the protein kinase C signaling pathway and the epidermal growth factor (EGF) pathway have been suggested to participate in the modulation of GLT1, but the modulatory mechanisms of GLT1 expression are not fully understood. In the present study, we aimed to evaluate the effects of insulin on GLT1 expression. We found that short-term stimulation of insulin led to the upregulation of both total and surface expressions of GLT1. Akt phosphorylation increased after insulin treatment, and triciribine, the inhibitor of Akt phosphorylation, significantly inhibited the effects of insulin. We also found that the upregulation of GLT1 expression correlated with increased kappa B motif-binding phosphoprotein (KBBP) and GLT1 mRNA levels. Our results suggest that insulin may modulate the expression of astrocytic GLT1, which might play a role in reactive astrocytes after CNS injuries.