Thomas Rauen

Differential expression of three glutamate transporter subtypes in the rat retina

Abstract.The immunocytochemical distribution of the three excitatory amino acid transporter subtypes GLT-1, GLAST-1 and EAAC-1 was studied in the rat retina using antibodies raised against synthetic peptides corresponding to the C-terminus of each transporter subtype (Rothstein et al. 1994). A comparative immunoblot analysis of rat cortex, cerebellum and retina membrane proteins suggested the following rank order of glutamate transporter subtype expression in retina: GLAST-1≥EAAC-1>GLT-1. GLAST-1 immunoreactivity was seen in Müller cells and astrocytes. EAAC-1 was found in horizontal cells, in amacrine and displaced amacrine cells, and in ganglion cells. A minority of bipolar cells also expressed EAAC-1. GLT-1 was preferentially expressed by different types of bipolar cells; however, it was also found in some amacrine cells. The functional role of this differential distribution of glutamate transporters in the retina is discussed.

High-affinity glutamate transporters in the rat retina: a major role of the glial glutamate transporter GLAST-1 in transmitter clearance

Abstract Glutamate is the major excitatory neurotransmitter of the mammalian retina and glutamate uptake is essential for normal transmission at glutamatergic synapses. The reverse transcriptase-polymerase chain reaction (RT-PCR) has revealed the presence of three different high-affinity glutamate transporters in the rat retina, viz. GLAST-1, GLT-1 and EAAC-1. No message has been found in the retina for EAAT-4, a transporter recently cloned from human brain. By using membrane vesicle preparations of total rat retina, we show that glutamate uptake in the retina is a high-affinity electrogenic sodium-dependent transport process driven by the transmembrane sodium ion gradient. Autoradiography of intact and dissociated rat retinae indicates that glutamate uptake by Müller glial cells dominates total retinal glutamate transport and that this uptake is strongly influenced by the activity of glutamine synthetase. RT-PCR, immunoblotting and immunohistochemistry have revealed that Müller cells express only GLAST-1. The Km for glutamate of GLAST-1 is 2.1±0.4 µM. This study suggests a major role for the Müller cell glutamate transporter GLAST-1 in retinal transmitter clearance. By regulating the extracellular glutamate concentration, the action of GLAST-1 in Müller cells may extend beyond the protection of neurons from excitotoxicity; we suggest a mechanism by which Müller cell glutamate transport might play an active role in shaping the time course of excitatory transmission in the retina.

Discovery of new risk loci for IgA nephropathy implicates genes involved in immunity against intestinal pathogens

We performed a genome-wide association study (GWAS) of IgA nephropathy (IgAN), the most common form of glomerulonephritis, with discovery and follow-up in 20,612 individuals of European and East Asian ancestry. We identified six new genome-wide significant associations, four in ITGAM-ITGAX, VAV3 and CARD9 and two new independent signals at HLA-DQB1 and DEFA. We replicated the nine previously reported signals, including known SNPs in the HLA-DQB1 and DEFA loci. The cumulative burden of risk alleles is strongly associated with age at disease onset. Most loci are either directly associated with risk of inflammatory bowel disease (IBD) or maintenance of the intestinal epithelial barrier and response to mucosal pathogens. The geospatial distribution of risk alleles is highly suggestive of multi-locus adaptation, and genetic risk correlates strongly with variation in local pathogens, particularly helminth diversity, suggesting a possible role for host–intestinal pathogen interactions in shaping the genetic landscape of IgAN.

Localization of the glutamate transporter GLT-1 in rat and macaque monkey retinae.

Antibodies directed against a glutamate transporter (GLT-1) purified from rat brain were applied to cryostat sections of rat and macaque monkey retinae. In the brain, GLT-1 expression is found mainly in astrocytes, and therefore it has been suggested that GLT-1 may be a glutamate transporter specific to glial cells. However, in the rat retina, cones and two distinct cone bipolar cell types were strongly immunoreactive. In the monkey retina, flat midget bipolars and one diffuse bipolar cell type (DB2)), were found to be labelled. Müller cells or astrocytes, the neuroglial cells of rat and monkey retinae, were not GLT-1-immunoreactive.

Intensive Supportive Care plus Immunosuppression in IgA Nephropathy

BACKGROUND The outcomes of immunosuppressive therapy, when added to supportive care, in patients with IgA nephropathy are uncertain. METHODS We conducted a multicenter, open-label, randomized, controlled trial with a two-group, parallel, group-sequential design. During a 6-month run-in phase, supportive care (in particular, blockade of the renin-angiotensin system) was adjusted on the basis of proteinuria. Patients who had persistent proteinuria with urinary protein excretion of at least 0.75 g per day were randomly assigned to receive supportive care alone (supportive-care group) or supportive care plus immunosuppressive therapy (immunosuppression group) for 3 years. The primary end points in hierarchical order were full clinical remission at the end of the trial (protein-to-creatinine ratio <0.2 [with both protein and creatinine measured in grams] and a decrease in the estimated glomerular filtration rate [eGFR] of <5 ml per minute per 1.73 m(2) of body-surface area from baseline) and a decrease in the eGFR of at least 15 ml per minute per 1.73 m(2) at the end of the trial. The primary end points were analyzed with the use of logistic-regression models. RESULTS The run-in phase was completed by 309 of 337 patients. The proteinuria level decreased to less than 0.75 g of urinary protein excretion per day in 94 patients. Of the remaining 162 patients who consented to undergo randomization, 80 were assigned to the supportive-care group, and 82 to the immunosuppression group. After 3 years, 4 patients (5%) in the supportive-care group, as compared with 14 (17%) in the immunosuppression group, had a full clinical remission (P=0.01). A total of 22 patients (28%) in the supportive-care group and 21 (26%) in the immunosuppression group had a decrease in the eGFR of at least 15 ml per minute per 1.73 m(2) (P=0.75). There was no significant difference in the annual decline in eGFR between the two groups. More patients in the immunosuppression group than in the supportive-care group had severe infections, impaired glucose tolerance, and weight gain of more than 5 kg in the first year of treatment. One patient in the immunosuppression group died of sepsis. CONCLUSIONS The addition of immunosuppressive therapy to intensive supportive care in patients with high-risk IgA nephropathy did not significantly improve the outcome, and during the 3-year study phase, more adverse effects were observed among the patients who received immunosuppressive therapy, with no change in the rate of decrease in the eGFR. (Funded by the German Federal Ministry of Education and Research; STOP-IgAN ClinicalTrials.gov number, NCT00554502.).

Reduced synaptic clustering of GABA and glycine receptors in the retina of the gephyrin null mutant mouse

Clustering of neurotransmitter receptors in postsynaptic densities involves proteins that aggregate the receptors and link them to the cytoskeleton. In the case of glycine and GABAA receptors, gephyrin has been shown to serve this function. However, it is unknown whether gephyrin is involved in the clustering of all glycine and GABAA receptors or whether it interacts only with specific isoforms. This was studied in the retinae of mice, whose gephyrin gene was disrupted, with immunocytochemistry and antibodies that recognize specific subunits of glycine and GABAA receptors. Because homozygous (geph −/−) mutants die around birth, an organotypic culture system of the mouse retina was established to study the clustering of gephyrin and the receptors in vitro. We found that all gephyrin and all glycine receptor clusters (hot spots) were abolished in the geph (−/−) mouse retina. In the case of GABAA receptors, there was a significant reduction of clusters incorporating the γ2, α2, and α3 subunits; however, a substantial number of hot spots was still present in geph (−/−) mutant retinae. This shows that gephyrin interacts with all glycine receptor isoforms but with only certain forms of GABAA receptors. In heterozygous geph (+/−) mutants, no reduction of hot spots was observed in the retina in vivo, but a significant reduction was found in the organotypic cultures. This suggests that mechanisms may exist in vivo that allow for the compensation of a partial gephyrin deficit. J. Comp. Neurol. 427:634–648, 2000.

Glutamate forward and reverse transport: From molecular mechanism to transporter-mediated release after ischemia

Glutamate transporters remove the excitatory neurotransmitter glutamate from the extracellular space after neurotransmission is complete, by taking glutamate up into neurons and glia cells. As thermodynamic machines, these transporters can also run in reverse, releasing glutamate into the extracellular space. Because glutamate is excitotoxic, this transporter‐mediated release is detrimental to the health of neurons and axons, and it, thus, contributes to the brain damage that typically follows a stroke. This review highlights current ideas about the molecular mechanisms underlying glutamate uptake and glutamate reverse transport. It also discusses the implications of transporter‐mediated glutamate release for cellular function under physiological and patho‐physiological conditions.

Electrogenic Glutamate Transporters in the CNS: Molecular Mechanism, Pre-steady-state Kinetics, and their Impact on Synaptic Signaling

Glutamate is the major excitatory neurotransmitter in the mammalian CNS. The spatiotemporal profile of the glutamate concentration in the synapse is critical for excitatory synaptic signalling. The control of this spatiotemporal concentration profile requires the presence of large numbers of synaptically localized glutamate transporters that remove pre-synaptically released glutamate by uptake into neurons and adjacent glia cells. These glutamate transporters are electrogenic and utilize energy stored in the transmembrane potential and the Na+/K+-ion concentration gradients to accumulate glutamate in the cell. This review focuses on the kinetic and electrogenic properties of glutamate transporters, as well as on the molecular mechanism of transport. Recent results are discussed that demonstrate the multistep nature of the transporter reaction cycle. Results from pre-steady-state kinetic experiments suggest that at least four of the individual transporter reaction steps are electrogenic, including reactions associated with the glutamate-dependent transporter halfcycle. Furthermore, the kinetic similarities and differences between some of the glutamate transporter subtypes and splice variants are discussed. A molecular mechanism of glutamate transport is presented that accounts for most of the available kinetic data. Finally, we discuss how synaptic glutamate transporters impact on glutamate receptor activity and how transporters may shape excitatory synaptic transmission.

Cloning, Transport Properties, and Differential Localization of Two Splice Variants of GLT-1 in the Rat CNS: Implications for CNS Glutamate Homeostasis

At least two splice variants of GLT‐1 are expressed by rat brain astrocytes, albeit in different membrane domains. There is at present only limited data available as to the spatial relationship of such variants relative to the location of synapses and their functional properties. We have characterized the transport properties of GLT‐1v in a heterologous expression system and conclude that its transport properties are similar to those of the originally described form of GLT‐1, namely GLT‐1α. We demonstrate that GLT‐1α is localized to glial processes, some of which are interposed between multiple synapse types, including GABAergic synapses, whereas GLT‐1v is expressed by astrocytic processes, at sites not interposed between synapses. Both splice variants can be expressed by a single astrocyte, but such expression is not uniform over the surface of the astrocytes. Neither splice variant of GLT‐1 is evident in brain neurons, but both are abundantly expressed in some retinal neurons. We conclude that GLT‐1v may not be involved in shaping the kinetics of synaptic signaling in the brain, but may be critical in preventing spillover of glutamate between adjacent synapses, thereby regulating intersynaptic glutamatergic and GABAergic transmission. Furthermore, GLT‐1v may be crucial in ensuring that low levels of glutamate are maintained at extrasynaptic locations, especially in pathological conditions such as ischemia, motor neurone disease, and epilepsy.

A new GLT1 splice variant: cloning and immunolocalization of GLT1c in the mammalian retina and brain

We have identified a novel carboxyl-terminal splice-variant of the glutamate transporter GLT1, which we denote as GLT1c. Within the rat brain only low levels of protein and message were detected, protein expression being restricted to end feet of astrocytes apposed to blood vessels or some astrocytes adjacent to the ventricles. Conversely, within the retina, this variant was selectively and heavily expressed in the synaptic terminals of both rod- and cone-photoreceptors in both humans and rats. Double-immunolabelling with antibodies to the carboxyl region of GLT1b/GLT1v, which is strongly expressed in apical dendrites of bipolar cells and in cone photoreceptors revealed that in the rat GLT1c was co-localised with GLT1b/GLT1v in cone photoreceptors but not with GLT1b/GLT1v in bipolar cells. GLT1c expression was developmentally regulated, only appearing at around postnatal day 7 in the rat retina, when photoreceptors first exhibit a dark current. Since the glutamate transporter EAAT5 is also expressed in terminals of rod photoreceptor terminals these data indicate that rod photoreceptors express two glutamate transporters with distinct properties. Similarly, cone photoreceptors express two glutamate transporters. We suggest that differential usage of these transporters by rod and cone photoreceptors may influence the kinetics of glutamate transmission by these neurons.