Ann D. Mitrovic

Glutamate transporter variants reduce glutamate uptake in Alzheimer's disease

A characteristic of Alzheimers disease (AD) is that neuron populations in the temporal, frontal, and parietal cortices are selectively vulnerable. Several neurotransmitters have been proposed to play roles in neural destruction as AD progresses, including glutamate. Failure to clear the synaptic cleft of glutamate can overstimulate postsynaptic glutamate receptors, promoting neuronal death. Excitatory amino acid transporter 2 (EAAT2), which is concentrated in perisynaptic astrocytes, performs 90% of glutamate uptake in mammalian central nervous system. Alternative splicing of EAAT2 mRNA could regulate glutamate transport in normal and disease states. We report disease- and pathology-specific variations in EAAT2 splice variant expression in AD brain obtained at autopsy. While wild type EAAT2 showed a global reduction in expression, brain regions susceptible to neuronal loss demonstrated greater expression of transcripts that reduced glutamate transport in an in vitro assay. Functional splice variant EAAT2b showed no significant variation with disease state. These results have implications for the treatment of AD as modulators of EAAT2 splicing and/or glutamate uptake would augment current therapies aimed at blocking glutamate receptors.

Identification of Functional Domains of the Human Glutamate Transporters EAAT1 and EAAT2

Glutamate transporters serve the important function of mediating removal of glutamate released at excitatory synapses and maintaining extracellular concentrations below excitotoxic levels. Excitatory amino acid transporter subtypes EAAT1 and EAAT2 have a high degree of sequence homology and similar predicted topology and yet display a number of functional differences. Several recombinant chimeric transporters were generated to identify domains that contribute to functional differences between EAAT1 and EAAT2. Wild-type transporters and chimeric transporters were expressed in Xenopus laevis oocytes, and electrogenic transport was studied under voltage clamp conditions. The differential sensitivity of EAAT1 and EAAT2 to transport blockers, kainate,threo-3-methylglutamate, and (2S,4R)-4-methylglutamate as well asl-serine-O-sulfate transport and chloride permeability were employed to characterize chimeric transporters. One particular region, transmembrane domains 9 and 10, plays an important role in defining these functional differences. The intracellular carboxyl-terminal region may also play a minor role in conferring an effect on chloride permeability. This study provides important insight into the identification of functional domains that determine differences among glutamate transporter subtypes.

Serine-O-sulphate transport by the human glutamate transporter, EAAT2

Expression of the recombinant human excitatory amino aid transporters, EAAT1 and EAAT2, in Xenopus laevis oocytes allows electrogenic transport to be studied under voltage clamp conditions. We have investigated the transport of the pharmacological substrate, L‐serine‐O‐sulphate transport by EAAT1 and EAAT2. The EC50 values for L‐serine‐O‐sulphate transport by EAAT2 showed a steep voltage‐dependence, increasing from 152±11 μM at −100 mV to 1930±160 μM at 0 mV. In contrast to EAAT2, EC50 values for L‐serine‐O‐sulphate transport by EAAT1 were relatively constant over the membrane potential range of −100 mV to 0 mV. The EC50 values for L‐glutamate and D‐aspartate transport, by EAAT2, were also relatively constant over this membrane potential range. Chloride ions modulated the voltage‐dependent changes in EC50 values for transport by EAAT2. This effect was most apparent for L‐serine‐O‐sulphate transport, and to a lesser extent for L‐glutamate and not at all for D‐aspartate transport by EAAT2. Extracellular sodium and proton concentrations also modulated the voltage‐dependence of L‐serine‐O‐sulphate EC50 values for EAAT2. We speculate that these different properties of L‐serine‐O‐sulphate transport by EAAT2 compared to other substrates may be due to the much stronger acidity of the sulphate group of L‐serine‐O‐sulphate compared to carboxyl groups of L‐glutamate or D‐aspartate. These results highlight some of the differences in the way different glutamate transporter subtypes transport substrates. This may be used to understand further the transport process and develop subtype selective inhibitors of glutamate transport.

Influence of the oestrous cycle on L-glutamate and L-aspartate transport in rat brain synaptosomes

Oestrous cycle and sex differences in sodium-dependent transport of L-[3H]glutamate and L-[3H]aspartate were investigated employing well washed synaptosomes prepared from rat brain cortex. Transport was best analysed on the basis of two components, a high and low affinity transport site. Oestrous cycle and sex differences were observed for both substrates. The high affinity transporter displayed highest affinity for glutamate transport in synaptosomes from female rats during proestrous and oestrous. This differed significantly from glutamate transport during dioestrous and in male rats. High affinity aspartate transport displayed highest affinity during oestrous and differed significantly from transport during dioestrous. Maximal velocity of high affinity glutamate transport was higher in synaptosomes from females during dioestrous compared with oestrous and lower in synaptosomes from male rats when compared with female rats in dioestrous and metoestrous. The low affinity sodium-dependent glutamate transporter displayed a 10-fold higher affinity for glutamate during proestrous than during the other three phases of oestrous and in male rats. Exogenously applied oestradiol and progesterone to synaptosomes from male rats showed no effect on glutamate or aspartate transport. No acute effect of oestradiol or progesterone on glutamate currents in oocytes expressing EAAT1 or EAAT2 subtype of glutamate transporter was observed. These results suggest hormonal regulation of high and low affinity sodium-dependent excitatory amino acid transporters over the four day oestrous cycle in synaptosomes from rat cortex. This regulation is unlikely to be due to a direct effect of oestradiol or progesterone on glutamate transporters.

Regional differences in the inhibition of L-glutamate and L-aspartate sodium-dependent high affinity uptake systems in rat CNS synaptosomes by L-trans-pyrrolidine-2,4-dicarboxylate, threo-3-hydroxy-D-aspartate and D-aspartate.

The sodium-dependent high affinity transport of L-[3H]glutamate and L-[3H]aspartate into synaptosomal fractions prepared from three different regions was employed to investigate the inhibitors L-trans-pyrrolidine-2,4-dicarboxylate, threo-3-hydroxy-D-aspartate and D-aspartate. These substances showed regional heterogeneity as inhibitors of sodium-dependent high affinity uptake of L-glutamate and L-aspartate. L-trans-Pyrrolidine-2,4-dicarboxylate was a more potent inhibitor of the uptake of L-glutamate than of L-aspartate in the cortex (IC50 8 microM vs L-glutamate and 13 microM vs L-aspartate) and cerebellum (IC50 4 microM v L-glutamate and 8 microM vs L-aspartate). threo-3-Hydroxy-D-aspartate was a more potent inhibitor of the uptake of L-glutamate than of L-aspartate in the cortex (IC50 9 microM vs L-glutamate and 13 microM vs L-aspartate) and hippocampus (IC50 6 microM v L-glutamate and 11 microM v L-aspartate). D-Aspartate was a more potent inhibitor of the uptake of L-glutamate than of L-aspartate only in the cortex (IC50 8 microM vs L-glutamate and 15 microM vs L-aspartate). These results thus support other evidence that there is regional heterogeneity in sodium-dependent high affinity acidic amino acid uptake sites in the brain.

Exon-skipping Splice Variants of Excitatory Amino Acid Transporter-2 (EAAT2) Form Heteromeric Complexes with Full-length EAAT2

The glial transporter excitatory amino acid transporter-2 (EAAT2) is the main mediator of glutamate clearance in brain. The wild-type transporter (EAAT2wt) forms trimeric membrane complexes in which each protomer functions autonomously. Several EAAT2 variants are found in control and Alzheimer-diseased human brains; their expression increases with pathological severity. These variants might alter EAAT2wt-mediated transport by abrogating membrane trafficking, or by changing the configuration or functionality of the assembled transporter complex. HEK293 cells were transfected with EAAT2wt; EAAT2b, a C-terminal variant; or either of two exon-skipping variants: alone or in combination. Surface biotinylation studies showed that only the exon-7 deletion variant was not trafficked to the membrane when transfected alone, and that all variants could reach the membrane when co-transfected with EAAT2wt. Fluorescence resonance energy transfer (FRET) studies showed that co-transfected EAAT2wt and EAAT2 splice variants were expressed in close proximity. Glutamate transporter function was measured using a whole cell patch clamp technique, or by changes in membrane potential indexed by a voltage-sensitive fluorescent dye (FMP assay): the two methods gave comparable results. Cells transfected with EAAT2wt or EAAT2b showed glutamate-dependent membrane potential changes consistent with functional expression. Cells transfected with EAAT2 exon-skipping variants alone gave no response to glutamate. Co-transfection of EAAT2wt (or EAAT2b) and splice variants in various ratios significantly raised glutamate EC50 and decreased Hill coefficients. We conclude that exon-skipping variants form heteromeric complexes with EAAT2wt or EAAT2b that traffic to the membrane but show reduced glutamate-dependent activity. This could allow glutamate to accumulate extracellularly and promote excitotoxicity.

Glycine transporter 1 associates with cholesterol-rich membrane raft microdomains

Membrane rafts, the highly-ordered, cholesterol-rich microdomains of the plasma membrane play important roles in cellular functions. In this study, GLYT1-CFP and GLYT2-CFP were constructed, followed by investigation of whether the tagged transporters associate with a fluorescence probe that labels membrane rafts (DilC16) by using Fluorescence Resonance Energy Transfer. A close association was observed between DiIC16 and GLYT1-CFP, but not for GLYT2-CFP. The glycine transport ability of GLYT1 is also highly dependent on the integrity of this area. Together, the results suggest that GLYT1 and membrane rafts are co-localized in the membrane, and that this influences the rate of glycine transport.

Site-directed mutagenesis in the study of membrane transporters.

One of the major goals in membrane transporter research is to understand how transporter proteins work at the molecular level. Ideally, this research would be carried out with a detailed knowledge of the three-dimensional structure of the protein. However, in the absence of atomic resolution structures for many membrane transporters other molecular tools need to be employed. In vitro site-directed mutagenesis is one method that has the capacity to provide both structural information and identification of the role of individual residues and/or regions of a protein that are involved in function.

Medicinal chemistry and molecular pharmacology of GABA receptors and glutamate transporters : Complementary structure-activity relationships

GABA and glutamate are the major inhibitory and excitatory neurotransmitters, respectively, in the human brain. Many GABA and glutamate receptors and transporters are key protein targets for drug development, and many known CNS drugs act on these targets. There have been a substantial number of traditional studies of structure–activity relationships in this area. The advent of modern molecular biology using recombinant DNA technology enables studies of structure–activity relationships to be carried out on these protein targets, thus complementing structure–activity relationships for the ligands interacting with these targets. This is illustrated with examples from our investigations of subtypes of GABAC receptors and glutamate transporters using both native and chimeric proteins of known amino acid sequence expressed in Xenopus oocytes. Studies of such complementary structure–activity relationships involving structural variations of both the ligands and their targets will play important roles in drug development. Such studies are vital to the development of drugs that interact selectively with particular native and mutant protein receptor/transporter subtypes. Drug Dev. Res. 46:255–260, 1999.