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Mesiotemporal sclerosis (MTS), the most frequent form of drug-resistant temporal lobe epilepsy, often develops after an initial precipitating injury affecting the immature brain. To analyse early processes in epileptogenesis we used the juvenile pilocarpine model to study status epilepticus (SE)-induced changes in expression of key components in the glutamate-glutamine cycle, known to be affected in MTS patients. SE was induced by Li(+) /pilocarpine injection in 21-day-old rats. At 2-19 weeks after SE hippocampal protein expression was analysed by immunohistochemistry and neuron damage by FluoroJade staining. Spontaneous seizures occurred in at least 44% of animals 15-18 weeks after SE. As expected in this model, we did not observe loss of principal hippocampal neurons. Neuron damage was most pronounced in the hilus, where we also detected progressive loss of parvalbumin-positive GABAergic interneurons. Hilar neuron loss (or end-folium sclerosis), a common feature in patients with MTS, was accompanied by a progressively decreased glutamine synthetase (GS)-immunoreactivity from 2 (-15%) to 19 weeks (-33.5%) after SE. Immunoreactivity for excitatory amino-acid transporters, vesicular glutamate transporter 1 and glial fibrillary acidic protein was unaffected. Our data show that SE elicited in 21-day-old rats induces a progressive reduction in hilar GS expression without affecting other key components of the glutamate-glutamine cycle. Reduced expression of glial enzyme GS was first detected 2 weeks after SE, and thus clearly before spontaneous recurrent seizures occurred. These results support the hypothesis that reduced GS expression is an early event in the development of hippocampal sclerosis in MTS patients and emphasize the importance of astrocytes in early epileptogenesis., (© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Childhood status epilepticus (SE) initiates an epileptogenic process that leads to spontaneous seizures and hippocampal pathology characterized by neuronal loss, gliosis and an imbalance between excitatory and inhibitory neurotransmission. It remains unclear whether these changes are a cause or consequence of chronic epilepsy. In this study, in vivo MRS was used in a post-SE juvenile rat model of temporal lobe epilepsy (TLE) to establish the temporal evolution of hippocampal injury and neurotransmitter imbalance. SE was induced in P21 rats by injection of lithium and pilocarpine. Four and eight weeks after SE, in vivo (1) H and γ-aminobutyric acid (GABA)-edited MRS of the hippocampus was performed in combination with dedicated ex vivo immunohistochemistry for the interpretation and validation of MRS findings. MRS showed a 12% decrease (p<0.0001) in N-acetylaspartate and a 15% increase (p=0.0226) in choline-containing compound concentrations, indicating neuronal death and gliosis, respectively. These results were confirmed by FluoroJade and vimentin staining. Furthermore, severe and progressive decreases in GABA (-41%, p<0.001) and glutamate (Glu) (-17%, p<0.001) were found. The specific severity of GABAergic cell death was confirmed by parvalbumin immunoreactivity (-68%, p<0.001). Unexpectedly, we found changes in glutamine (Gln), the metabolic precursor of both GABA and Glu. Gln increased at 4 weeks (+36%, p<0.001), but returned to control levels at 8 weeks. This decrease was consistent with the simultaneous decrease in glutamine synthase immunoreactivity (-32%, p=0.037). In vivo MRS showed gliosis and (predominantly GABAergic) neuronal loss. In addition, an increase in Gln was detected, accompanied by a decrease in glutamine synthase immunoreactivity. This may reflect glutamine synthase downregulation in order to normalize Gln levels. These changes occurred before spontaneous recurrent seizures were present but, by creating a pre-epileptic state, may play a role in epileptogenesis. MRS can be applied in a clinical setting and may be used as a noninvasive tool to monitor the development of TLE., (Copyright © 2012 John Wiley & Sons, Ltd.)

Temporal lobe epilepsy (TLE) is one of the most common focal epilepsy syndromes. In a genome-wide expression study of the human TLE hippocampus we previously showed up-regulation of genes involved in chemokine signalling. Here we investigate in the rat pilocarpine model for TLE, whether changes in chemokine signalling occur during epileptogenesis and are persistent. Therefore we analysed hippocampal protein expression and cellular localisation of CCL2, CCL4, CCR1 and CCR5 after status epilepticus. We found increased CCL4 (but not CCL2) expression in specific populations of hilar astrocytes at 2 and 19 weeks after SE concomitant with a persistent up-regulation of its receptor CCR5. Our results show an early and persistent up-regulation of CCL4/CCR5 signalling during epileptogenesis and suggest that CCL4 signalling, rather than CCL2 signalling, could have a role in the epileptogenic process., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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.

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.

Background: Increased levels of glutamate have been reported in the epileptogenic hippocampus of patients with temporal lobe epilepsy (TLE). This sustained increase, which may contribute to the initiation and propagation of seizure activity, indicates impaired clearance of glutamate released by neurons. Glutamate is predominantly cleared by glial cells through the excitatory amino acid transporter 2 (EAAT2) and its subsequent conversion to glutamine by the glial enzyme glutamine synthetase (GS)., Methods: The authors examined the hippocampal distribution of GS, EAAT2, and glial fibrillary acidic protein (GFAP) by immunohistochemistry in TLE patients with (HS group) and without hippocampal sclerosis (non-HS group), and in autopsy controls. In hippocampal homogenates the authors measured relative protein amounts by immunoblotting and GS enzyme activity., Results: In the autopsy control and non-HS group GS immunoreactivity (IR) was predominantly found in glia in the neuropil of the subiculum, of the pyramidal cell layer of all CA fields, and in the supragranular layer of the dentate gyrus. In the HS group, GS and EAAT2 IR were markedly reduced in subfields showing neuron loss (CA1 and CA4), whereas GFAP IR was increased. The reduction in GS IR in the HS group was confirmed by immunoblotting and paralleled by decreased GS enzyme activity., Conclusions: Glial glutamine synthetase is downregulated in the hippocampal sclerosis (HS) hippocampus of temporal lobe epilepsy (TLE) patients in areas with severe neuron loss. This downregulation appears to be pathology-related, rather than seizure-related, and may be part of the mechanism underlying impaired glutamate clearance found in the hippocampus of TLE patients with HS.