Tobias Hévor

Mesoporous silica nanoparticles enhance MTT formazan exocytosis in HeLa cells and astrocytes.

We report on the observation that mesoporous silica nanoparticles (MSNs), after being endocytosed, interfere with the MTT test in HeLa cells and astrocytes by accelerating the exocytosis of formazan crystals. The stimulation of MTT formazan exocytosis is probably related to perturbation of intracellular vesicle trafficking by MSN uptake as revealed by experiments in presence of chloroquine and genistein. Similar effect has been previously observed with a number of chemicals, especially with neurotoxic beta amyloid peptides, but not with nanoparticles. We showed also that MTT reduction test gives an overestimation of the cytotoxicity of mesoporous silica nanoparticles compared to other tests such as LDH activity, WST-1 test and flow cytometry. These findings show that MTT assay should not be used for the study of MSN toxicity, and that perturbation of intracellular trafficking has to be taken into account in evaluating biocompatibility of MSNs.

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.

Necrotic cell death induced by δ‐aminolevulinic acid in mouse astrocytes. Protective role of melatonin and other antioxidants

Abstract: Accumulation of δ‐aminolevulinic acid (ALA), as it occurs in acute intermittent porphyria (AIP), is the origin of an endogenous source of reactive oxygen species (ROS), which can exert oxidative damage to cell structures. In the present work we examined the ability of different antioxidants to revert ALA‐promoted damage, by incubating mouse astrocytes with 1.0 mm ALA for different times (1–4 hr) in the presence of melatonin (2.5 mm), superoxide dismutase (25 units/mL), catalase (200 units/mL) or glutathione (0.5 mm). The defined relative index [(malondialdehyde levels/accumulated ALA) × 100], decreases with incubation time, reaching values of 76% for melatonin and showing that the different antioxidants tested can protect astrocytes against ALA‐promoted lipid peroxidation. Concerning porphyrin biosynthesis, no effect was observed with catalase and superoxide dismutase whereas increases of 57 and 87% were obtained with glutathione and melatonin, respectively, indicating that these antioxidants may prevent the oxidation of porphobilinogen deaminase, reactivating so that the AIP genetically reduced enzyme. Here we showed that ALA induces cell death displaying a pattern of necrosis. This pattern was revealed by loss of cell membrane integrity, marked nuclear swelling and double labeling with annexin V and propidium iodide. In addition, no caspase 3‐like activity was detected. These findings provide the first experimental evidence of the involvement of ALA‐promoted ROS in the damage of proteins related to porphyrin biosynthesis and the induction of necrotic cell death in astrocytes. Interestingly, melatonin decreases the number of enlarged nuclei and shows a protective effect on cellular morphology.

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.

Early, middle, and late stages of neural cells from ovine embryo in primary cultures.

The utilization of neural cells in culture has importantly increased the knowledge of the nervous system biology. In most studies, the investigations are performed on biological materials coming from common laboratory animals and the extrapolation of the results to other animals is not easy. For some studies, such as developmental biology of the nervous system, prion disease investigations, or agronomical production, the utilization of ovine neural cell cultures presents many advantages. Unfortunately, there are few data on the conditions of culture of such cells. In the present work, we investigated simple ways to obtain neurons and astrocytes from sheep brain. Viable neuronal cell cultures were obtained from 40 to 50 day old fetuses. Their morphologies were quite similar to that of neurons from rodent or chick brain and they were labeled by antineurofilament antibodies. Stages older than 50 days of pregnancy were unable to give viable culture of neurons. The stages of 40 day old fetus to newborn lamb were able to give viable astrocyte cultures. The common protoplasmic astrocytes were obtained and they were labeled by antiglial fibrillary acidic protein antibodies. The astrocytes contained glycogen, thus looking like the common astrocytes from rodents. Neuronal or astroglial cultures can be derived from 26 day old embryos, but the cultures contained contaminating cells. Among the latter cells, there were undifferentiated cells which were flat and epitheloid and which were grouped as islets. These cells could be maintained in culture for a time duration over 7 months, even after two passages. They differentiated principally in astrocytes with a radial configuration. This work shows how some neural cells can be simply and easily cultured from sheep brain. For the first time, neurons were cultured from the sheep embryonic brain. Moreover, stem cells were cultured for more than 7 months and, finally, glycogen accumulation in sheep astrocytes was shown to be the same as that in rodent astrocytes. The oligodendrocyte culture was already documented. Thus, sheep can easily be used as well as other models for neural cell studies.

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.

Isolation of carbohydrate metabolic clones from cultured astrocytes.

Astrocytes are the principal sites of glycogen synthesis in the nervous tissue. Growing evidence shows that there are many types of astrocytes. The aim of the present investigation was to isolate different types of astrocytes that display different carbohydrate anabolism. Astrocytes from newborn rat brain were directly cloned from primary cultures without a previous transformation. Many clones were obtained, and they were termed CP clones. Another series of clones, termed SV clones, were obtained after the transfection of the primary cultures by the SV40 T antigen. The effectiveness of the transfection was verified by the rate of DNA synthesis using flow cytometry and by the presence of plasmid DNA in the genomic DNA of the astrocytes using the Southern blot method. After the transfection, the growth velocity increased greatly. The size and shape of the astrocytes were the same for each cell in a given clone, regardless of the cloning method utilized. However, these sizes and shapes could be different from one clone to another in CP clones, whereas all the astrocytes of all the SV clones looked like each other. All the clones obtained stained positively with anti‐glial fibrillary acidic protein antibodies. Glycogen stained in the clones using concanavalin A‐horseradish peroxidase. The glycogen content was also measured using biochemical analysis. Concordant results obtained using these two methods showed that some clones contained an important quantity of glycogen while other clones contained a small amount, in the CP series as well as in the SV series. This property was the same for the intracellular glucose concentrations. The activity of the gluconeogenic enzyme fructose‐1,6‐bisphosphatase was measured in each clone using spectrophotometry. This activity was also significantly different from one clone to another. The clones containing large amounts of glycogen had important fructose‐1,6‐bisphosphatase activity. The present results show that it is possible to clone astrocytes either directly from primary cultures without immortalization or after their transformation. When analyzing these clones, it appears that carbohydrate anabolism can be significantly different from one astrocyte to another. This difference may also exist in vivo.