Line M. Levy

Brain Glutamate Transporter Proteins Form Homomultimers

Removal of excitatory amino acids from the extracellular fluid is essential for synaptic transmission and for avoiding excitotoxicity. The removal is accomplished by glutamate transporters located in the plasma membranes of both neurons and astroglia. The uptake system consists of several different transporter proteins that are carefully regulated, indicating more refined functions than simple transmitter inactivation. Here we show by chemical cross-linking, followed by electrophoresis and immunoblotting, that three rat brain glutamate transporter proteins (GLAST, GLT and EAAC) form homomultimers. The multimers exist not only in intact brain membranes but also after solubilization and after reconstitution in liposomes. Increasing the cross-linker concentration increased the immunoreactivity of the bands corresponding to trimers at the expense of the dimer and monomer bands. However, the immunoreactivities of the dimer bands did not disappear, indicating a mixture of dimers and trimers. GLT and GLAST do not complex with each other, but as demonstrated by double labeling post-embedding electron microscopic immunocytochemistry, they co-exist side by side in the same astrocytic cell membranes. The oligomers are held together noncovalently in vivo. In vitro, oxidation induces formation of covalent bonds (presumably -S-S-) between the subunits of the oligomers leading to the appearance of oligomer bands on SDS-polyacrylamide gel electrophoresis. Immunoprecipitation experiments suggest that GLT is the quantitatively dominant glutamate transporter in the brain. Radiation inactivation analysis gives a molecular target size of the functional complex corresponding to oligomeric structure. We postulate that the glutamate transporters operate as homomultimeric complexes.

Down-regulation of Glial Glutamate Transporters after Glutamatergic Denervation in the Rat Brain

Membrane‐localized transporter proteins, expressed in both neurons and glial cells, are responsible for removal of extracellular glutamate in the mammalian CNS. The amounts and activities of these transporters may be under regulatory control. We demonstrate here that cortical lesions, which decrease striatal glutamate uptake in synaptosome‐containing homogenates by ∼50%, also decrease the striatal concentrations of the astrocytic glutamate transporter proteins, GLT‐1 and GLAST by ∼20–30%. Since GABA uptake activity was not decreased and glial fibrillary acidic protein was increased in the same samples, the lesion‐induced losses of GLT‐1 and GLAST were not caused by a general impairment of neuronal or glial function. The observed reduction in the two astrocytic glutamate transporters after corticostriatal nerve terminal degeneration indicates that their levels of expression are dependent on glutamatergic innervation.

Cell-specific expression of the glutamine transporter SN1 suggests differences in dependence on the glutamine cycle.

Glutamine is involved in a variety of metabolic processes, including recycling of the neurotransmitters glutamate and γ‐aminobutyric acid (GABA). The system N transporter SN1 mediates efflux as well as influx of glutamine in glial cells [Chaudhry et al. (1999), Cell, 99, 769–780]. We here report qualitative and quantitative data on SN1 protein expression in rat. The total tissue concentrations of SN1 in brain and in kidney are half and one‐quarter, respectively, of that in liver, but the average concentration of SN1 could be higher in astrocytes than in hepatocytes. Light and electron microscopic immunocytochemistry shows that glutamatergic, GABAergic and, surprisingly, purely glycinergic boutons are ensheathed by astrocytic SN1 laden processes, indicating a role of glutamine in the production of all three rapid transmitters. A dedication of SN1 to neurotransmitter recycling is further supported by the lack of SN1 immunoreactivity in oligodendrocytes (cells rich in glutamine but without perisynaptic processes). All neuronal structures appear unlabelled implying that a different protein mediates glutamine uptake into nerve endings. In several regions, SN1 immunoreactivity is higher in association with GABAergic than glutamatergic synapses, in agreement with observations that exogenous glutamine increases output of transmitter glutamate but not GABA. Nerve terminals with low transmitter reuptake or high prevailing firing frequency are associated with high SN1 immunoreactivity in adjacent glia. Bergmann glia and certain other astroglia contain very low levels of SN1 immunoreactivity compared to most astroglia, including retinal Müller cells, indicating the possible existence of SN isoforms and alternative mechanisms for transmitter recycling.

Chapter 3 Properties and localization of glutamate transporters

Publisher Summary The glutamate transporters in the plasma membranes of astrocytes and neurons are essential for the normal functioning of the nervous system. They represent the only mechanism capable of quickly removing glutamate from the extracellular fluid. It is important to maintain a low concentration of glutamate extracellularly for two reasons. First, glutamate is the major excitatory neurotransmitter and a high signal-to-noise ratio requires the removal of extracellular glutamate so that the concentration fluctuates with synaptic release. Second, glutamate is highly toxic to neurons expressing glutamate receptors and glutamate receptors are found on most neurons and even on many glial cells. There is experimental evidence for the idea that the transporters may be actively involved in the regulation of synaptic transmission because they can modify the time course of synaptic events. The sodium-dependent glutamate transporters use the transmembrane gradients of sodium, potassium, and pH as driving forces.

A monoclonal antibody raised against an [Na+K+]coupled l‐glutamate transporter purified from rat brain confirms glial cell localization

A monoclonal antibody (9C4) shows that an [Na+K+]coupled glutamate transporter protein purified from rat brain runs electrophoretically as a wide band and is localized in neuroglial cell bodies and processes, but not in neurons. This confirms the findings with polyclonal antibodies [Neuroscience 51 (1992) 295‐310], and shows that the apparent heterogeneity in relative molecular mass is accounted for by a single antigenic epitope. By testing several synthetic peptides derived from the deduced amino acid sequences of two cloned rat brain glutamate transporters, the antigenic epitope was identified as residing within the peptide TQSVYDDTKNHRESNSNQC (residues 518–536) of one of these [Nature 360 (1992) 464‐467].

Localization of transporters using transporter-specific antibodies.

Publisher Summary This chapter discusses the localization of transporters using transporter-specific antibodies. Neurotransmitter transporters in the plasma membranes of neurons and glial cells play essential roles in the nervous system by removing the transmitters from the extracellular fluid surrounding the receptors. This is required for securing a high signal-to-noise ratio and for avoiding the overstimulation of receptors. Two different families of plasma membrane neurotransmitter transporters are identified by molecular cloning. One family includes the transporters for γ-aminobutyric acid (GABA), noradrenaline, dopamine, serotonin, proline, choline, and glycine. The other family includes the four different glutamate transporters and a transporter for neutral amino acids. Some neurotransmitter transporters are regulated and highly differentially localized. For determining their physiological and pathophysiological roles, it is essential to know their exact localizations, not only qualitatively, but also quantitatively. Thus, the chapter describes methods used by laboratory for the immunocytochemical investigation of glutamate and glycine transporters. These techniques depend on specific antibodies, optimal tissue and antigen preservation, and proper controls.

Expression of glial glutamate transporters GLT-1 and GLAST is unchanged in the hippocampus in fully kindled rats

In situ hybridization techniques and quantitative western blotting were used to study the expression of the glial glutamate transporter GLT-1 and GLAST in the brains of normal (implanted, non-kindled) and fully kindled rats. Wistar rats were implanted with stimulating electrodes in the basolateral amygdala, and killed 28 days after the stimulated group had shown stage 5 seizures on five occasions. The brains were processed for in situ hybridization of messenger RNA for GLT-1 using 35S-labelled oligonucleotide probes or digoxigenin-labelled riboprobes. Paired (kindled and non-kindled) sections were used for qualitative and quantitative analyses. Image analysis of autoradiograms showed no change in expression of GLT-1 messenger RNA in any region of the hippocampus or in the cortex. An increase in expression of GLT-1 messenger RNA (expressed as percentage difference of control) was observed bilaterally in the striatum in kindled animals (16-21%, P<0.05). Nuclear emulsion-dipped sections showed predominant glial cell labelling in the hippocampus. Particle density analysis revealed reduced cell labelling in some kindled vs control pairs but overall there was no significant reduction in labelling in CA1. Equivalent results were found in CA1 using digoxigenin-labelled riboprobes. Quantitative immunoblotting also revealed no change in GLT-1 or GLAST transporter protein in the hippocampus of kindled animals. From these data we conclude that the enduring seizure susceptibility associated with the fully kindled state is unlikely to involve alterations in hippocampal GLT-1 messenger RNA or GLT-1 and GLAST transporter protein expression.

Highly differential expression of SN1, a bidirectional glutamine transporter, in astroglia and endothelium in the developing rat brain

The transmitters glutamate and GABA also subserve trophic action and are required for normal development of the brain. They are formed from glutamine, which may be synthesized in glia or extracted from the blood. In the adult, the glutamine transporter SN1 is expressed in the astroglia. SN1 works in both directions, depending on the concentration gradients of its substrates and cotransported ions, and is thought to regulate extracellular glutamine and to supply the neurons with the transmitter precursor. In this article, we have quantified the expression and studied the localization of SN1 at different developmental stages. SN1 is expressed in astroglia throughout the CNS from embryonic stages through adulthood. No indication of SN1 staining in neuronal elements has been obtained at any stage. Quantitative immunoblotting of whole brain extracts demonstrates increasing expression of SN1 from P0, reaching a peak at P14, twice the adult level. A moderate and slower rise and fall of the expression levels of SN1 occurs in the cerebellum. Strong transient SN1‐like staining is also found in Bergmann glia and vascular endothelium in the first postnatal weeks. Strong intracellular staining in the same time period suggests a high rate of SN1 synthesis in the early postnatal period. This coincides with the increasing levels of glutamate and GABA in the CNS and with the time course of synaptogenesis. This study suggests that the expression of SN1 is highly regulated, correlating with the demand for glutamine during the critical period of development. GLIA 41:260–275, 2003.

Ischemia induced changes in expression of the astrocyte glutamate transporter GLT1 in hippocampus of the rat.

Changes in cellular uptake of glutamate following transient cerebral ischemia is of possible importance to ischemia induced cell death. In the present study, we employed in situ hybridization and immunohistochemistry to investigate the influence of cerebral ischemia on expression of mRNA and protein of the astrocyte glutamate transporter GLT1, and of glial fibrillary acidic protein. Different subfields of CA1 and CA3 of the rat hippocampus were studied at various time-points after ischemia (days 1, 2, 4, and 21). In CA1, GLT1-mRNA was decreased at all time-points after ischemia except from day 2, whereas in CA3, decreases were seen only on day 1. Expression of GLT1-protein in CA1 was unchanged during the initial days after ischemia, but decreased markedly from day 2 to 4. In CA3, GLT1-protein increased progressively throughout the observation period after ischemia. Following the degeneration of CA1 pyramidal cells, a positive correlation between the number of CA1 pyramidal cells and expression of either GLT1-mRNA or -protein was evident selectively in CA1. Increases in expression of mRNA and protein of glial fibrillary acidic protein were present from day 2, most notable in CA1. The present data provide evidence that expression of GLT1 in CA1 of the hippocampus is not decreased persistently before the degeneration of CA1 pyramidal cells, but is downregulated in response to loss of these neurons. Since the reduction in GLT1 expression evolved concomitantly with the degeneration of CA1 pyramidal cells, it may contribute to the severity of CA1 pyramidal cell loss. A progressive postischemic increase in GLT1 expression in CA3 may be linked to the resistance of CA3 neurons to ischemic cell damage.

Inducible expression of the GLT-1 glutamate transporter in a CHO cell line selected for low endogenous glutamate uptake

Inducible expression of the mammalian glial cell glutamate transporter GLT‐1 has been established in a CHO cell line selected for low endogenous Na+‐dependent glutamate uptake by [3H]aspartate suicide selection. Culturing the cells in doxycycline‐containing medium, to activate GLT‐1 expression via the Tet‐On system, increased uptake of the GLT‐1 substrate d‐aspartate 280‐fold, and increased cell size. Applying glutamate to whole‐cell clamped, doxycycline‐treated cells evoked a transporter‐mediated current with characteristics appropriate for GLT‐1. This cell line provides a useful tool for further examination of the electrical, biochemical and pharmacological properties of GLT‐1, the most abundant glutamate transporter in the brain.