G. Hoogland

Increased glutamine synthetase but normal EAAT2 expression in platelets of ALS patients.

Amyotrophic lateral sclerosis is a fatal neurodegenerative disease and glutamate excitotoxicity has been implicated in its pathogenesis. Platelets contain a glutamate uptake system and express components of the glutamate-glutamine cycle, such as the predominant glial excitatory amino acid transporter 2 (EAAT2). In several neurological diseases platelets have proven to be systemic markers for the disease. We compared properties of key components of the glutamate-glutamine cycle in blood platelets of ALS patients and healthy controls. Platelets were analyzed for (3)H-glutamate uptake in the presence or absence of thrombin and for EAAT2 and glutamine synthetase protein expression by Western blotting. Platelets of ALS patients showed a 37% increase in expression of glutamine synthetase, but normal expression of glutamate transporter EAAT2. Glutamate uptake in resting or thrombin-stimulated platelets did not differ significantly between platelets from ALS patients and controls. Thrombin-stimulation resulted in about a seven-fold increase in glutamate uptake. Our data suggest that glutamine synthetase may be a peripheral marker of ALS and encourage further investigation into the role of this enzyme in ALS.

Alternative splicing of glutamate transporter EAAT2 RNA in neocortex and hippocampus of temporal lobe epilepsy patients

RATIONALE Altered expression of glutamate transporter EAAT2 protein has been reported in the hippocampus of patients with temporal lobe epilepsy (TLE). Two alternative EAAT2 mRNA splice forms, one resulting from a partial retention of intron 7 (I7R), the other from a deletion of exon 9 (E9S), were previously implicated in the loss of EAAT2 protein in patients with amyotrophic lateral sclerosis. METHODS By RT-PCR we studied the occurrence of I7R and E9S in neocortical and hippocampal specimens from TLE patients and non-neurological controls. RESULTS Both splice forms were found in all neocortical specimens from TLE patients (100% I7R, 100% E9S). This was significantly more than in controls (67% I7R, 60% E9S; P < 0.05). We also detected I7R and E9S in all seven motor cortex post-mortem samples from patients with amyotrophic lateral sclerosis. Within the TLE patient group, both splice variants appeared significantly more in non-sclerotic (100%), than in sclerotic hippocampi (69%, P < 0.05). CONCLUSION These data indicate that the epileptic brain, especially that of TLE patients without hippocampal sclerosis, is highly prone to alternative EAAT2 mRNA splicing. Our data confirm that the presence of alternative EAAT2 splice forms is not disease specific.

Thrombin-stimulated glutamate uptake in human platelets is predominantly mediated by the glial glutamate transporter EAAT2

Glutamate toxicity has been implicated in the pathogenesis of various neurological diseases. Glial glutamate transporters play a key role in the regulation of extracellular glutamate levels in the brain by removing glutamate from the extracellular fluid. Since human blood platelets possess an active glutamate uptake system, they have been used as a peripheral model of glutamate transport in the central nervous system (CNS). The present study is aimed at identifying the glutamate transporter on blood platelets, and to asses the influence of platelet activation on glutamate uptake. Platelets from healthy donors showed Na+-dependent glutamate uptake (Km, 3.5+/-0.9 microM; Vmax, 2.8+/-0.2 pmol glutamate/75 x 10(6)platelets/30 min), which could be blocked dose-dependently by the EAAT specific inhibitors DL-threo-E-benzyloxyaspartate (TBOA), L-trans-pyrrolidine-2,4-dicarboxylic acid (tPDC) and high concentrations of the EAAT2 inhibitor dihydrokainate (DHK). Analysis of platelet homogenates on Western blots showed EAAT2 as the predominant glutamate transporter. Platelet activation by thrombin caused an increase in glutamate uptake, which could be inhibited by TBOA and the EAAT2 inhibitor DHK. Kinetic analysis showed recruitment of new transporters to the membrane. Indeed, Western blot analysis of subcellular fractions revealed that alpha-granules, which fuse with the membrane upon thrombin stimulation, contained significant EAAT2 immunoreactivity. Inhibition of the second messengers involved in alpha-granule secretion (protein kinase C, phosphatidylinositol-3-kinase) inhibited thrombin-stimulated uptake, but not basal uptake. These data show that the glial EAAT2 is the predominant glutamate transporter on blood platelets and suggest, that thrombin increases glutamate uptake capacity by recruiting new transporters (EAAT2) from alpha-granules.

Synaptosomal glutamate and GABA transport in patients with temporal lobe epilepsy

High‐affinity glutamate and GABA transporters found in the plasma membrane of neurons and glial cells terminate neurotransmission by rapidly removing extracellular transmitter. Impairment of transporter function has been implicated in the pathophysiologic mechanisms underlying epileptogenesis. We characterized glutamate and γ‐aminobutyric acid (GABA) transport in synaptosomes, isolated from neocortical and hippocampal biopsies of patients with temporal lobe epilepsy (TLE). We analyzed K+‐evoked release in the presence and absence of Ca2+ to determine vesicular and transporter‐mediated release, respectively. We also analyzed 3H‐glutamate and 3H‐GABA uptake, the effect of glutamate uptake inhibitors L‐trans‐pyrrolidine‐2,4‐dicarboxylic acid (tPDC) and DL‐threo‐β‐benzyloxyaspartate (TBOA), and GABA uptake inhibitor N‐(4,4‐diphenyl‐3‐butenyl)‐3‐piperidinecarboxylic acid (SK&F 89976‐A). Neocortical synaptosomes from TLE patients did not show vesicular glutamate release, strongly reduced transporter‐mediated release, and an increased basal release compared to that in rat synaptosomes. Furthermore, basal release was less sensitive to tPDC, and 3H‐glutamate uptake was reduced compared to that in rat synaptosomes. Vesicular GABA release from neocortical synaptosomes of TLE patients was reduced compared to that in rat synaptosomes, whereas transporter‐mediated release was hardly affected. Furthermore, basal GABA release was more than doubled, but neither basal nor stimulated release were increased by SK&F 89976‐A, which did significantly increase both types of GABA release in rat synaptosomes. Finally, 3H‐GABA uptake by synaptosomes from TLE patients was reduced significantly in hippocampus (0.19 ± 0.04%), compared to that in neocortex (0.32 ± 0.04%). Control experiments with human peritumoral cortical tissue suggest that impaired uptake of glutamate, but not of GABA, was caused in part by the hypoxic state of the biopsy. Our findings provide evidence for impaired function of glutamate and GABA transporters in human TLE.

Characterization of neocortical and hippocampal synaptosomes from temporal lobe epilepsy patients.

To investigate epilepsy-associated changes in the presynaptic terminal, we isolated and characterized synaptosomes from biopsies resected during surgical treatment of drug-resistant temporal lobe epilepsy (TLE) patients. Our main findings are: (1) The yield of synaptosomal protein from biopsies of epilepsy patients was about 25% of that from rat brain. Synaptosomal preparations were essentially free of glial contaminations. (2) Synaptosomes from TLE patients and naive rat brain, quickly responded to K(+)-depolarization with a 70% increase in intrasynaptosomal Ca(2+) ([Ca(2+)](i)), and a 40% increase in B-50/GAP-43 phosphorylation. (3) Neocortical and hippocampal synaptosomes from TLE patients contained 20-50% of the glutamate and gamma-aminobutyric acid (GABA) contents of rat cortical synaptosomes. (4) Although the absolute amount of glutamate and GABA released under basal conditions from neocortical synaptosomes of TLE patients was lower than from rat synaptosomes, basal release expressed as percentage of total content was higher (16.4% and 17.3%, respectively) than in rat (11.5% and 9. 9%, respectively). (5) Depolarization-induced glutamate and GABA release from neocortical synaptosomes from TLE patients was smaller than from rat synaptosomes (3.9% and 13.0% vs. 21.9% and 25.0%, respectively). (6) Analysis of breakdown of glial fibrillary acid protein (GFAP) indicates that resection time (anoxic period during the operation) is a critical parameter for the quality of the synaptosomes. We conclude that highly pure and viable synaptosomes can be isolated even from highly sclerotic human epileptic tissue. Our data show that in studies on human synaptosomes it is of critical importance to distinguish methodological (i.e., resection time) from pathology-related abnormalities.

Glutamate and γ-aminobutyric acid content and release of synaptosomes from temporal lobe epilepsy patients

During surgical intervention in medically refractory temporal lobe epilepsy (TLE) patients, diagnosed with either mesial temporal lobe sclerosis (MTS)‐ or tumor (T)‐associated TLE, biopsies were taken from the anterior temporal neocortex and the hippocampal region. Synaptosomes, isolated from these biopsies were used to study intrasynaptosomal Ca2+ levels ([Ca2+]i), and glutamate and γ‐aminobutyric acid (GABA) contents and release. All synaptosomal preparations demonstrated a basal [Ca2+]i of about 200 nM, except neocortical synaptosomes from MTS‐associated TLE patients (420 nM). K+‐induced depolarization resulted in a robust increase of the basal [Ca2+]i in all preparations. Neocortical synaptosomes from TLE patients contained 22.9 ± 3.0 nmol glutamate and 4.6 ± 0.5 nmol GABA per milligram synaptosomal protein, whereas rat cortical synaptosomes contained twice as much glutamate and four times as much GABA. Hippocampal synaptosomes from MTS‐associated TLE patients, unlike those from T‐associated TLE patients, contained about 70% less glutamate and 55% less GABA than neocortical synaptosomes. Expressed as percentage of total synaptosomal content, synaptosomes from MTS‐associated TLE patients exhibited an increased basal and a reduced K+‐induced glutamate and GABA release compared to rat cortical synaptosomes. In MTS‐associated TLE patients, only GABA release from neocortical synaptosomes was partially Ca2+‐dependent. Control experiments in rat synaptosomes demonstrated that at least part of the reduction in K+‐induced release can be ascribed to resection‐induced hypoxia in biopsies. Thus, synaptosomes from MTS‐associated TLE patients exhibit a significant K+‐induced increase in [Ca2+]i, but the consequent release of glutamate and GABA is severely impaired. Our data show that at least part of the differences in glutamate and GABA content and release between human biopsy material and fresh rat tissue is due to the resection time. J. Neurosci. Res. 60:686–695, 2000

Phosphoproteins and the Regulation of Vesicular Neurotransmitter Release

Neurotransmission requires rapid docking, fusion and recycling of neurotransmitter vesicles in the nerve terminal. Many of the proteins involved in this complex mechanism have now been identified (reviewed by Scheller, 1995; Sudhof, 1995) and are present in presynaptic nerve terminals, where they constitute the molecular apparatus for the storage and secretion of neurotransmitters and termination of their effects. Presynaptic nerve terminals store a variety of different neurotransmitters in vesicles which are secreted into the extracellular space by means of regulated exocytosis i.e. upon a specific signal, usually a depolarization-induced Ca2+ influx. Neurotransmitter-containing vesicles firmly dock at the presynaptic plasma membrane prior to exocytosis and are rapidly reclaimed and recycled after fusion and secretion. In mammalian brain nerve terminals two classes of secretory neurotransmitter vesicles can be distinguished on an ultrastructural basis, namely small clear-cored vesicles and large dense-cored vesicles (reviewed by Kelly, 1993). Small clear-cored vesicles, which store neurotransmitters such as glutamate, GABA, glycine, acetylcholine and possibly catecholamines, are released swiftly at the active synaptic zone and undergo multiple cycles of exo- and endocytosis within the nerve terminal. Large dense-cored vesicles, which contain catecholamines or a variety of neuropeptides, on the other hand, cannot refill after exocytosis and therefore need to be recycled via the trans Golgi network.