Niels C. Danbolt

GLUTAMATE TRANSPORTERS IN GLIAL PLASMA MEMBRANES : HIGHLY DIFFERENTIATED LOCALIZATIONS REVEALED BY QUANTITATIVE ULTRASTRUCTURAL IMMUNOCYTOCHEMISTRY

The glutamate transporters GLT-1 and GLAST were studied by immunogold labeling on ultrathin sections of rat brain tissue embedded in acrylic resins at low temperature after freeze substitution. Both proteins were selective markers of astrocytic plasma membranes. GLT-1 was much higher in hippocampal astrocytes than in cerebellar astrocytes. Astroglial membrane GLAST densities ranked as follows: Bergmann > cerebellar granular layer approximately hippocampus > cerebellar white matter. No astrocyte appeared unlabeled. Astrocytic membranes facing capillaries, pia, or stem dendrites were lower in glutamate transporters than those facing nerve terminals, axons, and spines. Parallel fiber boutons (glutamatergic) synapsin on interneuron dendritic shafts were surrounded by lower transporter densities than those synapsing on Purkinje cell spines. Our findings suggest the localizations of glutamate transporters are carefully regulated.

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.

An [Na+ + K+]coupledl-glutamate transporter purified from rat brain is located in glial cell processes

Polyclonal antibodies were generated against the major polypeptide (73,000 mol. wt) present in a highly purified preparation of the [Na+ + K+]coupled L-glutamate transporter from rat brain. These antibodies were able to selectively immunoprecipitate the 73,000 mol. wt polypeptide as well as most of the L-glutamate transport activity--as assayed upon reconstitution--from crude detergent extracts of rat brain membranes. The immunoreactivity in the various fractions obtained during the purification procedure [Danbolt et al. (1990) Biochemistry 29, 6734-6740] closely correlated with the L-glutamate transport activity. Immunoblotting of a crude sodium dodecyl sulphate brain extract, separated by two-dimensional isoelectric focusing-sodium dodecyl sulphate-polyacrylamide gel electrophoresis, showed that the antibodies recognized one 73,000 mol. wt protein species only. Deglycosylation of the protein gave a 10,000 reduction in molecular mass, but no reduction in immunoreactivity. These findings establish that the 73,000 mol. wt polypeptide represents the L-glutamate transporter or a subunit thereof. The antibodies also recognize a 73,000 mol. wt polypeptide and immunoprecipitate L-glutamate transport activity in extracts of brain plasma membranes from rabbit, pig, cow, cat and man. Using the antibodies, the immunocytochemical localization of the transporter was studied at the light and electron microscopic levels in rat central nervous system. In all regions examined (including cerebral cortex, caudatoputamen, corpus callosum, hippocampus, cerebellum, spinal cord) it was found to be located in glial cells rather than in neurons. In particular, fine astrocytic processes were strongly stained. Putative glutamatergic axon terminals appeared non-immunoreactive. The uptake of glutamate by such terminals (for which there is strong previous evidence) therefore may be due to a subtype of glutamate transporter different from the glial transporter demonstrated by us.

SOD1 mutants linked to amyotrophic lateral sclerosis selectively inactivatea glial glutamate transporter

The mechanism by which Cu2+/Zn2+ superoxide dismutase (SOD1) mutants lead to motor neuron degeneration in familial amyotrophic lateral sclerosis (FALS) is unknown. We show that oxidative reactions triggered by hydrogen peroxide and catalyzed by A4V and I113T mutant but not wild-type SOD1 inactivated the glutamate transporter human GLT1. Chelation of the copper ion of the prosthetic group of A4V prevented GLT1 inhibition. GLT1 was a selective target of oxidation mediated by SOD1 mutants, and its reactivity was confined to the intracellular carboxyl-terminal domain. The antioxidant Mn(III)TBAP rescued GLT1 from inhibition. Because inactivation of GLT1 results in neuronal degeneration, we propose that toxic properties of SOD1 mutants lead to neuronal death via an excitotoxic mechanism in SOD1-linked FALS.

The Perivascular Astroglial Sheath Provides a Complete Covering of the Brain Microvessels: An Electron Microscopic 3D Reconstruction

The unravelling of the polarized distribution of AQP4 in perivascular astrocytic endfeet has revitalized the interest in the role of astrocytes in controlling water and ion exchange at the brain–blood interface. The importance of the endfeet is based on the premise that they constitute a complete coverage of the vessel wall. Despite a number of studies based on different microscopic techniques this question has yet to be resolved. We have made an electron microscopic 3D reconstruction of perivascular endfeet in CA1 (stratum moleculare) of rat hippocampus. The endfeet interdigitate and overlap, leaving no slits between them. Only in a few sites do processes—tentatively classified as processes of microglia—extend through the perivascular glial sheath to establish direct contact with the endothelial basal lamina. In contrast to the endfoot covering of the endothelial tube, the endfoot covering of the pericyte is incomplete, allowing neuropil elements to touch the basal lamina that enwraps this type of cell. The 3D reconstruction also revealed large bundles of mitochondria in the endfoot processes that came in close apposition to the perivascular endfoot membrane. Our data support the idea that in pathophysiological conditions, the perivascular astrocytic covering may control the exchange of water and solutes between blood and brain and that free diffusion is limited to narrow clefts between overlapping endfeet.

Loss of glutamine synthetase in the human epileptogenic hippocampus: possible mechanism for raised extracellular glutamate in mesial temporal lobe epilepsy.

BACKGROUND High extracellular glutamate concentrations have been identified as a likely trigger of epileptic seizures in mesial temporal lobe epilepsy (MTLE), but the underlying mechanism remains unclear. We investigated whether a deficiency in glutamine synthetase, a key enzyme in catabolism of extracellular glutamate in the brain, could explain the perturbed glutamate homoeostasis in MTLE. METHODS The anteromedial temporal lobe is the focus of the seizures in MTLE, and surgical resection of this structure, including the hippocampus, leads to resolution of seizures in many cases. By means of immunohistochemistry, western blotting, and functional enzyme assays, we assessed the distribution, quantity, and activity of glutamine synthetase in the MTLE hippocampus. FINDINGS In western blots, the expression of glutamine synthetase in the hippocampus was 40% lower in MTLE than in non-MTLE samples (median 44 [IQR 30-58] vs 69 [56-87]% of maximum concentration in standard curve; p=0.043; n=8 and n=6, respectively). The enzyme activity was lower by 38% in MTLE vs non-MTLE (mean 0.0060 [SD 0.0031] vs 0.0097 [0.0042] U/mg protein; p=0.045; n=6 and n=9, respectively). Loss of glutamine synthetase was particularly pronounced in areas of the MTLE hippocampus with astroglial proliferation, even though astrocytes normally have high content of the enzyme. Quantitative immunoblotting showed no significant change in the amount of EAAT2, the predominant glial glutamate transporter in the hippocampus. INTERPRETATION A deficiency in glutamine synthetase in astrocytes is a possible molecular basis for extracellular glutamate accumulation and seizure generation in MTLE. Further studies are needed to define the cause, but the loss of glutamine synthetase may provide a new focus for therapeutic interventions in MTLE.

Hippocampal GABA and glutamate transporter immunoreactivity in patients with temporal lobe epilepsy

Objective: Sodium-coupled transporters remove extracellular neurotransmitters and alterations in their function could enhance or suppress synaptic transmission and seizures. This study determined hippocampal gamma-aminobutyric acid (GABA) and glutamate transporter immunoreactivity (IR) in temporal lobe epilepsy (TLE) patients. Methods: Hippocampal sclerosis (HS) patients (n = 25) and non-HS cases (mass lesion and cryptogenic; n = 20) were compared with nonseizure autopsies (n = 8). Hippocampal sections were studied for neuron densities along with IR for glutamate decarboxylase (GAD; presynaptic GABA terminals), GABA transporter-1 (GAT-1; presynaptic GABA transporter), GAT-3 (astrocytic GABA transporter), excitatory amino acid transporter 3 (EAAT3; postsynaptic glutamate transporter), and EAAT2-1 (glial glutamate transporters). Results: Compared with autopsies, non-HS cases with similar neuron counts showed: 1) increased GAD IR gray values (GV) in the fascia dentata outer molecular layer (OML), hilus, and stratum radiatum; 2) increased GAT-1 OML GVs; 3) increased astrocytic GAT-3 GVs in the hilus and Ammon’s horn; and 4) no IR differences for EAAT3-1. HS patients with decreased neuron densities demonstrated: 1) increased OML and inner molecular layer GAD puncta; 2) decreased GAT-1 puncta relative to GAD in the stratum granulosum and pyramidale; 3) increased GAT-1 OML GVs; 4) decreased GAT-3 GVs; 5) increased EAAT3 IR on remaining granule cells and pyramids; 6) decreased glial EAAT2 GVs in the hilus and CA1 stratum radiatum associated with neuron loss; and 7) increased glial EAAT1 GVs in CA2/3 stratum radiatum. Conclusions: Hippocampal GABA and glutamate transporter IR differ in TLE patients compared with autopsies. These data support the hypothesis that excitatory and inhibitory neurotransmission and seizure susceptibility could be altered by neuronal and glial transporters in TLE patients.

Neuronal soluble factors differentially regulate the expression of the GLT1 and GLAST glutamate transporters in cultured astroglia.

Abstract: The glutamate transporters in the plasma membranes of neural cells secure termination of the glutamatergic synaptic transmission and keep the glutamate levels below toxic concentrations. Astrocytes express two types of glutamate transporters, GLAST (EAAT1) and GLT1 (EAAT2). GLT1 predominates quantitatively and is responsible for most of the glutamate uptake activity in the juvenile and adult brain. However, GLT1 is severely down‐regulated in amyotrophic lateral sclerosis, a progressive neurodegenerative disease. Furthermore, selective loss of this transporter occurs in cultured astroglia. Expression of GLAST, but not of GLT1, seems to be regulated via the glutamate receptor signalling. The present study was undertaken to examine whether neuronal factors, other than glutamate, influence the expression of astroglial glutamate transporters. The expression of GLT1 and GLAST was examined in primary cultures of cerebellar granule neurons, cortical neurons, and astrocytes under different experimental conditions, including those that mimic neuron‐astrocyte interactions. Pure astroglial cultures expressed only GLAST, whereas astrocytes grown in the presence of neurons expressed both GLAST (at increased levels) and GLT1. The induction of GLT1 protein and its mRNA was reproduced in pure cortical astroglial cultures supplemented with conditioned media from cortical neuronal cultures or from mixed neuron‐glia cultures. This treatment did not change the levels of GLAST. These results suggest that soluble neuronal factors differentially regulate the expression of GLT1 and GLAST in cultured astroglia. Further elucidation of the molecular nature of the secreted neuronal factors and corresponding signalling pathways regulating the expression of the astroglial glutamate transporters in vitro may reveal mechanisms important for the understanding and treatment of neurological diseases.

The high-affinity glutamate transporters GLT1, GLAST, and EAAT4 are regulated via different signalling mechanisms.

High-affinity glutamate transporters ensure termination of glutamatergic neurotransmission and keep the synaptic concentration of this amino acid below excitotoxic levels. However, neuronal glutamate transporters, EAAC1 and EAAT4, are located outside the synaptic cleft and contribute less significantly to the glutamate uptake in the brain than two astroglial transporters, GLAST and GLT1. Aberrant functioning of the glutamate uptake system seems to be linked to some neurodegenerative disorders (eg amyotrophic lateral sclerosis, ALS). Expression of glutamate transporters is differentially regulated via distinct cellular mechanisms. GLT1, which is expressed at very low levels in cultured astrocytes, is strongly induced in the presence of neurons. The present immunocytochemical data provide further evidence that neuronal soluble factors, rather than physical contact between neurons and glia, determine the induction of GLT1 in astrocytes. This effect is apparently mediated by yet undefined growth factor(s) via the tyrphostin-sensitive receptor tyrosine kinase (RTK) signalling, that in turn, supports the downstream activation of p42/44 MAP kinases and the CREM and ATF-1 transcription factors. RTK-independent simultaneous activation of the CREB transcription factor suggests a possible involvement of complementary pathway(s). Neuronal soluble factors do not affect expression of GLAST, but induce supporting machinery for differential regulation of GLAST via the astroglial metabotropic glutamate receptors, mGluR3 and mGluR5. Thus, long-term treatment with the group I mGluR agonist, DHPG, causes down-regulation of GLAST, whereas the group II agonist, DCG-IV, has an opposite effect on the expression of GLAST in astrocytes. However, in BT4C glioma cells glutamate or other transportable substrates (D-aspartate and L-2,4-trans-PDC) induced cell-surface expression of EAAT4 in a receptor-independent manner. The activity-dependent trafficking of this transporter which also exhibits properties of a glutamate-gated chloride channel may play functional roles not only in neuronal excitability, but in glioma cell biology as well.

Differential Developmental Expression of the Two Rat Brain Glutamate Transporter Proteins GLAST and GLT

The extracellular concentration of the excitatory neurotransmitter glutamate is kept low by the action of glutamate transporters in the plasma membranes of both neurons and glial cells. These transporters may play important roles, not only in the adult brain, but also in the developing brain, as glutamate is thought to modulate the formation and elimination of synapses as well as neuronal migration, proliferation and apoptosis. Here we demonstrate the developmental changes in the expression of two glutamate transporters, GLAST and GLT, by quantitative immunoblotting and by light and electron microscopic immunocytochemistry. At birth, GLT is not detectable, but GLAST is present at significant concentrations both in the forebrain and in the cerebellum. GLT is first detected in the forebrain and cerebellum in the second and third week, respectively. Both transporters reach adult levels by postnatal week 5. The development of the total glutamate uptake activity in the forebrain, as determined by solubilization and reconstitution of the transporters in liposomes, parallels that of GLT, in agreement with the observation that GLT is the predominant transporter in the adult brain. The regional distributions of both GLAST and GLT in the tissue are similar in young and adult rats. Only GLAST is detectable in the external germinal layer of the cerebellar cortex. Electron microscopical investigation demonstrated GLAST and GLT exclusively in glial cells in young as well as in adult animals.