Yvette Dehnes

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.

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.

A QUANTITATIVE ASSESSMENT OF GLUTAMATE UPTAKE INTO HIPPOCAMPAL SYNAPTIC TERMINALS AND ASTROCYTES : NEW INSIGHTS INTO A NEURONAL ROLE FOR EXCITATORY AMINO ACID TRANSPORTER 2 (EAAT2)

The relative distribution of the excitatory amino acid transporter 2 (EAAT2) between synaptic terminals and astroglia, and the importance of EAAT2 for the uptake into terminals is still unresolved. Here we have used antibodies to glutaraldehyde-fixed d-aspartate to identify electron microscopically the sites of d-aspartate accumulation in hippocampal slices. About 3/4 of all terminals in the stratum radiatum CA1 accumulated d-aspartate-immunoreactivity by an active dihydrokainate-sensitive mechanism which was absent in EAAT2 glutamate transporter knockout mice. These terminals were responsible for more than half of all d-aspartate uptake of external substrate in the slices. This is unexpected as EAAT2-immunoreactivity observed in intact brain tissue is mainly associated with astroglia. However, when examining synaptosomes and slice preparations where the extracellular space is larger than in perfusion fixed tissue, it was confirmed that most EAAT2 is in astroglia (about 80%). Neither d-aspartate uptake nor EAAT2 protein was detected in dendritic spines. About 6% of the EAAT2-immunoreactivity was detected in the plasma membrane of synaptic terminals (both within and outside of the synaptic cleft). Most of the remaining immunoreactivity (8%) was found in axons where it was distributed in a plasma membrane surface area several times larger than that of astroglia. This explains why the densities of neuronal EAAT2 are low despite high levels of mRNA in CA3 pyramidal cell bodies, but not why EAAT2 in terminals account for more than half of the uptake of exogenous substrate by hippocampal slice preparations. This and the relative amount of terminal versus glial uptake in the intact brain remain to be discovered.

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.

The density of EAAC1 (EAAT3) glutamate transporters expressed by neurons in the mammalian CNS.

The extracellular levels of excitatory amino acids are kept low by the action of the glutamate transporters. Glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) are the most abundant subtypes and are essential for the functioning of the mammalian CNS, but the contribution of the EAAC1 subtype in the clearance of synaptic glutamate has remained controversial, because the density of this transporter in different tissues has not been determined. We used purified EAAC1 protein as a standard during immunoblotting to measure the concentration of EAAC1 in different CNS regions. The highest EAAC1 levels were found in the young adult rat hippocampus. Here, the concentration of EAAC1 was ∼0.013 mg/g tissue (∼130 molecules μm−3), 100 times lower than that of GLT-1. Unlike GLT-1 expression, which increases in parallel with circuit formation, only minor changes in the concentration of EAAC1 were observed from E18 to adulthood. In hippocampal slices, photolysis of MNI-d-aspartate (4-methoxy-7-nitroindolinyl-d-aspartate) failed to elicit EAAC1-mediated transporter currents in CA1 pyramidal neurons, and d-aspartate uptake was not detected electron microscopically in spines. Using EAAC1 knock-out mice as negative controls to establish antibody specificity, we show that these relatively small amounts of EAAC1 protein are widely distributed in somata and dendrites of all hippocampal neurons. These findings raise new questions about how so few transporters can influence the activation of NMDA receptors at excitatory synapses.

Rapid affinity purification of erythropoietin from biological samples using disposable monoliths

Identification of post-translational modifications of proteins in biological samples often requires access to preanalytical purification and concentration methods. In the purification step high or low molecular weight substances can be removed by size exclusion filters, and high abundant proteins can be removed, or low abundant proteins can be enriched, by specific capturing tools. In this paper is described the experience and results obtained with a recently emerged and easy-to-use affinity purification kit for enrichment of the low amounts of EPO found in urine and plasma specimens. The kit can be used as a pre-step in the EPO doping control procedure, as an alternative to the commonly used ultrafiltration, for detecting aberrantly glycosylated isoforms. The commercially available affinity purification kit contains small disposable anti-EPO monolith columns (6 μL volume, Ø7 mm, length 0.15 mm) together with all required buffers. A 24-channel vacuum manifold was used for simultaneous processing of samples. The column concentrated EPO from 20 mL urine down to 55 μL eluate with a concentration factor of 240 times, while roughly 99.7% of non-relevant urine proteins were removed. The recoveries of Neorecormon (epoetin beta), and the EPO analogues Aranesp and Mircera applied to buffer were high, 76%, 67% and 57%, respectively. The recovery of endogenous EPO from human urine was 65%. High recoveries were also obtained when purifying human, mouse and equine EPO from serum, and human EPO from cerebrospinal fluid. Evaluation with the accredited EPO doping control method based on isoelectric focusing (IEF) showed that the affinity purification procedure did not change the isoform distribution for rhEPO, Aranesp, Mircera or endogenous EPO. The kit should be particularly useful for applications in which it is essential to avoid carry-over effects, a problem commonly encountered with conventional particle-based affinity columns. The encouraging results with EPO propose that similar affinity monoliths, with the appropriate antibodies, should constitute useful tools for general applications in sample preparation, not only for doping control of EPO and other hormones such as growth hormone and insulin but also for the study of post-translational modifications of other low abundance proteins in biological and clinical research, and for sample preparation prior to in vitro diagnostics.

SPECIFICITY OF ANTIBODIES: UNEXPECTED CROSS-REACTIVITY OF ANTIBODIES DIRECTED AGAINST THE EXCITATORY AMINO ACID TRANSPORTER 3 (EAAT3)

UNLABELLED Specific antibodies are essential tools for identifying individual proteins in biological samples. While generation of antibodies is often straightforward, determination of the antibody specificity is not. Here we illustrate this by describing the production and characterization of antibodies to excitatory amino acid transporter 3 (EAAT3). We synthesized 13 peptides corresponding to parts of the EAAT3 sequence and immunized 6 sheep and 30 rabbits. All sera were affinity purified against the relevant immobilized peptide. Antibodies to the peptides were obtained in almost all cases. Immunoblotting with tissue extracts from wild type and EAAT3 knockout animals revealed that most of the antibodies did not recognize the native EAAT3 protein, and that some recognized other proteins. Several immunization protocols were tried, but strong reactions with EAAT3 were only seen with antibodies to the C-terminal peptides. In contrast, good antibodies were obtained to several parts of EAAT2. EAAT3 was only detected in neurons. However, rabbits immunized with an EAAT3-peptide corresponding to residues 479-498 produced antibodies that labeled axoplasm and microtubules therein particularly strongly. On blots, these antibodies recognized both EAAT3 and a slightly smaller, but far more abundant protein that turned out to be tubulin. The antibodies were fractionated on columns with immobilized tubulin. One fraction contained antibodies apparently specific for EAAT3 while another fraction contained antibodies recognizing both EAAT3 and tubulin despite the lack of primary sequence identity between the two proteins. Addition of free peptide to the incubation solution blocked immunostaining of both EAAT3 and tubulin. CONCLUSIONS Not all antibodies to synthetic peptides recognize the native protein. The peptide sequence is more important than immunization protocol. The specificity of an antibody is hard to predict because cross-reactivity can be specific and to unrelated molecules. The antigen preabsorption test is of little value in testing the specificity of affinity purified antibodies.

Evaluation of Drugs Acting at Glutamate Transporters in Organotypic Hippocampal Cultures: New Evidence on Substrates and Blockers in Excitotoxicity*

Removal of L-glutamate (Glu) from the synapse is critical to maintain normal transmission and to prevent excitotoxicity, and is performed exclusively by excitatory amino acid transporters (EAATs). We investigated the effects of substrates and blockers of EAATs on extracellular Glu and cellular viability in organotypic cultures of rat hippocampus. Seven-day treatment with a range of drugs (L-trans-pyrrolidine-2,4-dicarboxylate, (2S,4R)-4-methyl-glutamate, (±)-threo-3-methylglutamate and DL-threo-β-benzyloxyaspartate), in the presence of 300 μM added Glu, resulted in increased extracellular Glu and a significant correlation between Glu concentration and cellular injury (as indicated by lactate dehydrogenase release). In contrast, (2S,3S,4R)-2-(carboxycyclopropyl)glycine (L-CCG-III) exerted a novel neuroprotection against this toxicity, and elevations in extracellular Glu were not toxic in the presence of this compound. Similar results were obtained following two-week treatment of cultures without added Glu. Whilst blockade of GLT-1 alone was relatively ineffective in producing excitotoxic injury, heteroexchange of Glu by EAAT substrates may exacerbate excitotoxicity.

Effect of single doses of methoxypolyethylene glycol‐epoetin beta (CERA, Mircera™) and epoetin delta (Dynepo™) on isoelectric erythropoietin profiles and haematological parameters

Erythropoietin (EPO) has been misused in sports for many years due to its performance-enhancing effect. In the last decade, detection of abuse has been possible with isoelectric focusing (IEF) based on the different isoform profiles of endogenous and recombinant EPO. The release of new EPOs on the market, such as the recombinant erythropoietin epoetin delta (Dynepo™) and the chemically modified EPO, CERA (Mircera™) potentially represents analytical challenges to the fight against doping. This study set out to investigate the possibility of and the time window for detecting the administration of a single dose of Dynepo™ and CERA. Our results are in agreement with earlier findings that detection of Dynepo™ is best achieved by combining IEF with SDS-PAGE. Haematological parameters were monitored for possible effects due to the long half-life (130 hours) of CERA in blood. Interestingly, although several haematological parameters were significantly changed after the injection of CERA, the endogenous EPO signal was still present in all collected samples. Due to the long half-life and the large size of the CERA molecule (about 60 kDa), it was uncertain whether CERA would be excreted into urine in detectable amounts unless urine collection was preceded by strenuous physical exercise. We find that CERA can be detected in urine without prior exercise in several, but not all, subjects. CERA is nevertheless best detected in serum with regard to both probability and length of detection, in addition to stability in matrix over time.

Black market products confiscated in Norway 2011–2014 compared to analytical findings in urine samples

Doping agents are widely and illicitly distributed through the Internet. Analysis of these preparations is useful in order to monitor the availability of prohibited substances on the market, and more importantly to predict which substances are expected to be found in urine samples collected from athletes and to aid clinical and forensic investigations. Based on a close collaboration with the Norwegian police and the Norwegian custom authorities, the Norwegian Doping Control Laboratory has performed analyses of confiscated material suspected of containing doping agents. The analyses were performed using gas chromatography (GC) and liquid chromatography (LC) combined with mass spectrometry (MS). The majority (67%) of the analyzed black market products contained anabolic- androgenic steroids (AAS) as expected, whereas peptide- and protein-based doping substances were identified in 28% of the preparations. The Norwegian Doping Control Laboratory receives samples collected from recreational and elite athletes in addition to samples collected in clinical and forensic investigations. The findings in the seized material reflected the findings in the urine samples analyzed regarding the anabolic steroids. Thus, analyzing material seized in Norway may give a good indication of doping agents available on the local market.