Jean-François Cloix

Epilepsy, Regulation of Brain Energy Metabolism and Neurotransmission

Seizures are the result of a sudden and temporary synchronization of neuronal activity, the reason for which is not clearly understood. Astrocytes participate in the control of neurotransmitter storage and neurotransmission efficacy. They provide fuel to neurons, which need a high level of energy to sustain normal and pathological neuronal activities, such as during epilepsy. Various genetic or induced animal models have been developed and used to study epileptogenic mechanisms. Methionine sulfoximine induces both seizures and the accumulation of brain glycogen, which might be considered as a putative energy store to neurons in various animals. Animals subjected to methionine sulfoximine develop seizures similar to the most striking form of human epilepsy, with a long pre-convulsive period of several hours, a long convulsive period during up to 48 hours and a post convulsive period during which they recover normal behavior. The accumulation of brain glycogen has been demonstrated in both the cortex and cerebellum as early as the pre-convulsive period, indicating that this accumulation is not a consequence of seizures. The accumulation results from an activation of gluconeogenesis specifically localized to astrocytes, both in vivo and in vitro. Both seizures and brain glycogen accumulation vary when using different inbred strains of mice. C57BL/6J is the most “resistant” strain to methionine sulfoximine, while CBA/J is the most “sensitive” one. The present review describes the data obtained on methionine sulfoximine dependent seizures and brain glycogen in the light of neurotransmission, highlighting the relevance of brain glycogen content in epilepsies.

Chronic inhibition of glutamine synthetase is not associated with impairment of learning and memory in mice

The convulsant methionine sulfoximine (MSO) is a byproduct of the agenized flour commonly used for feeding domestic animals decades ago. MSO is a powerful glycogenic and epileptogenic agent, and it is an irreversible inhibitor of glutamine synthetase. This latter effect was hypothesized to be responsible for the increase in the incidence of some neuropathologies in humans, such as Alzheimers disease or Parkinsons disease. In order to test this hypothesis, we chronically administered MSO to two inbred strains of mice, C57BL/6J and BALB/cJ, and analyzed possible alterations in learning and memory features of these mice. Mice were given 20 mg/kg of MSO three times a week for 10 weeks. Spatial learning capabilities assessed with a radial maze were not affected by the long-term MSO treatment, although activity was significantly decreased in BALB/cJ mice. Thus, our data suggest that long-term administration of non-convulsive and non-glycogenic doses of MSO do not alter the spatial memory of mice. Our results do not support the hypothesis that chronic treatment with MSO influences hippocampus-dependent learning abilities in mice.

In vivo and in vitro glycogenic effects of methionine sulfoximine are different in two inbred strains of mice.

We investigated the relationship between brain glycogen anabolism and methionine sulfoximine (MSO)-induced seizures in two inbred mouse strains that presented differential susceptibility to the convulsant. CBA/J was considered a MSO-high-reactive strain and C57BL/6J a MSO-low-reactive strain. Accordingly, the dose of MSO needed to induce seizures in CBA/J mice is lower than that in C57BL/6J mice, and CBA/J mice which had seizures, died during the first convulsion. In addition, the time--course of the MSO effect is faster in CBA/J mice than that in C57BL/6J mice. Analyses were performed in C57BL/6J and CBA/J mice after administration of 75 (subconvulsive dose) and 40 mg/kg of MSO (subconvulsive dose, not lethal dose), respectively. In the preconvulsive period, MSO induced an increase in the brain glycogen content of C57BL/6J mice only. Twenty-four hours after MSO administration, the brain glycogen content increased in both strains. The activity and expression of fructose-1,6-bisphosphatase, the last key enzyme of the gluconeogenic pathway, were increased in MSO-treated C57BL/6J mice as compared to control mice, at all experimental time points, whereas they were increased in CBA/J mice only 24 h after MSO administration. These latter results correspond to CBA/J mice that did not have seizures. Interestingly, the differences observed in vivo were consistent with results in primary cultured astrocytes from the two strains. This data suggests that the metabolism impairment, which was not a consequence of seizures, could be related to the difference in seizure susceptibility between the two strains, depending on their genetic background.

Stable transfection of cDNAs targeting specific steps of glycogen metabolism supports the existence of active gluconeogenesis in mouse cultured astrocytes

In order to assess the participation of astrocytic gluconeogenesis in the synthesis of glycogen, mouse astrocytes were stably transfected with antisense cDNA of fructose‐1,6‐bisphosphatase (FBPase) and with sense and antisense cDNAs of glycogen synthase (GS). The antisenses of FBPase and GS have similar significant effect in decreasing astrocyte glycogen content by 60%, while sense GS significantly increased glycogen content by 100%. The FBPase activity was decreased by all three cDNAs used, while glycogen phosphorylase was not altered. The activity of GS was decreased by the antisense GS and increased by the sense GS. These data demonstrate that the gluconeogenesis in astrocytes is involved in the glycogenesis modulation. GLIA 37:379–382, 2002.

Various fructose-1,6-bisphosphatase mRNAs in mouse brain, liver, kidney and heart.

THE mouse fructose-1,6-bisphosphatase (FBPase) cDNA was previously cloned from testicular teratocarcinoma cultured cells (F9 cells). Using this published nucleotide sequence four primer sets were defined and used to amplify FBPase transcript from cerebral cortex, heart, kidney, liver and testis of male C57B1/6 mice. Only one primer set was efficient in all total RNA prepared from the various tissues. The restriction maps of these RNA amplification products suggested the existence of three different FBPase transcripts; this was confirmed by the nucleotide sequences of the FBPase transcripts and by the deduced amino acid sequences. These data are consistent with the existence of three different FBPase genes. This may be relevant in neurological diseases in which abnormalities of brain glucose metabolism are involved.

Identification and characterization of estrogen receptor-related receptor alpha and gamma in human glioma and astrocytoma cells

The purpose of this study was to examine expression and function of estrogen receptor-related receptors (ERRs) in human glioma and astrocytoma cell lines. These estrogen receptor-negative cell lines expressed ERRalpha and ERRgamma proteins to varying degree in a cell context dependent manner, with U87MG glioma cells expressing both orphan nuclear receptors. Cell proliferation assays were performed in the presence of ERR isoform-specific agonists and antagonists, and the calculated EC(50) and IC(50) values were consistent with previous reported values determined in other types of cancer cell lines. Induction of luciferase expression under the control of ERR isoform-specific promoters was also observed in these cells. These results indicate that ERRalpha and ERRgamma are differentially expressed in these tumor cell lines and likely contribute to agonist-dependent ERR transcriptional activity.

Selection of two lines of mice based on latency to onset of methionine sulfoximine seizures

Purpose:  In various animals methionine sulfoximine (MSO) induces tonic–clonic seizures resembling the most striking form of human epilepsies. The aim of the present study was to select two lines of mice based upon differences in their latency to MSO‐dependent seizures, in order to characterize them.

Glycogen Metabolism and Brain Pathologies

Glucose is the main fuel for cell life, and supports a number of different processes in providing cells with energy. Excess glucose is polymerized into glycogen, which is an energy-glucose store. Alterations in glycogen content and/or synthesis have been reported in human neuropathologies, such as Alzheimers disease, epilepsies and cancer. Epileptic foci are hypometabolic during the interictal period, and probably hypermetabolic during crisis. Animal models of epilepsies are used for studying the reasons why neurons suddenly and temporally synchronize their activity. One model associates seizures of the “grand mal” type with cortical glycogen accumulation: induction of epileptiform crisis by methionine sulfoximine (MSO). The glycogen accumulation, observed in astrocytes only, occurs as soon as the preconvulsive period. High glycogen has also been demonstrated in primary cultures of astrocytes. Abnormal glycogen content has been characterized in various types of cancers, including gliomas. High invasion properties, spontaneous resistance to chemotherapeutic drugs, and a mean prognosis of 12 months characterize glioblastomas, the highest grade of gliomas that inevitably leads to death. The various therapeutic means, including surgery, chemicaland radio-therapies, and gene therapy have thus far been inefficient in significantly improving patient survival. Glycogen synthesis was targeted in cell lines from murine and human glioblastomas by an antisense glycogen synthase cDNA strategy; and the inhibition of glycogen synthesis in these cell lines decreases both in vitro and in vivo invasiveness. Glycogen can therefore be considered as putatively involved in at least two different pathologies of the brain, such as epilepsies and cancer. This abnormal glycogen content and synthesis can be proposed as putative diagnostic and therapeutic targets in brain pathologies.

A new putative target for antisense gene therapy of glioma: Glycogen synthase

The treatment of malignant brain gliomas remains a challenge, despite the availability of the classical triad of surgery, radiotherapy, and chemotherapy. There is thus the need for investigations into other forms of treatment strategies, such as gene therapy. Using antisense technology we have targeted glycogen metabolism, since malignant astrocytes present a high content of glycogen. In vitro rat C6‑glioma cells, transfected with antisense glycogen synthase (C6‑AS cells) exhibited a decreased expression of glycogen synthase and reduced activity of glycogen synthesis, along with attenuated invasiveness. In vivo tumors induced by C6‑AS cells in nude mice exhibited a significant reduction in tumor growth compared with controls. This reduction could be mediated by the induction of MCH‑I expression. The inhibition of glycogen synthesis by antisense glycogen synthase validates a putative target and a new approach for further study to advance the much‑needed efficacy of intervention strategies for malignant gliomas.

Monoamines and glycogen levels in cerebral cortices of fast and slow methionine sulfoximine-inbred mice

The experimental model of seizures which depends upon methionine sulfoximine (MSO) simulates the most striking form of human epilepsy. MSO generates epileptiform seizures in a large variety of animals, increases brain glycogen content and induces brain monoamines modifications. We selected two inbred lines of mice based upon their latency toward MSO-dependent seizures, named as MSO-Fast (sensitive), having short latency toward MSO, and MSO-Slow (resistant) with a long latency. We determined 13 monoamines and glycogen contents in brain cortices of the MSO-Fast and slow lines in order to determine the relationships with MSO-dependent seizures. The present data show that using these MSO-Fast and MSO-Slow inbred lines it could be demonstrated that: (1) in basal conditions the neurotransmitter 5-HT is significantly higher in MSO-Fast mice than in MSO-Slow ones; (2) MSO in both lines induced a significant increase in brain content of DOPAC (3,4-dihydroxyphenylacetic acid), HVA (homovanillic acid), MHPG (3-methoxy-4-hydroxyphenylglycol), and 5-HT (serotonin); a significant decrease in MSO-Slow mice in brain content of NME (normetepinephrine), and 5-HIAA (5-hydroxyindoleacetic acid) and the variation of other monoamines were not significant; (3) the brain glycogen content is significantly higher in MSO-Fast mice than in MSO-Slow ones, both in basal conditions and after MSO administration. From our data, we propose that brain glycogen content may constitute a defense against epileptic attack, as glycogen may be degraded down to glucose-6-phosphate that can be used to either postpone the epileptic attack or to provide neurons with energy when they needed it. Brain glycogen might therefore be considered as a molecule that can contribute to struggle seizures, at least in MSO-dependent seizure. The 5-HT content may constitute a defense against MSO-dependent epilepsy.