Frédérique Masmejean

Mechanisms Contributing to the Phase-Dependent Regulation of Neurogenesis by the Novel Antidepressant, Agomelatine, in the Adult Rat Hippocampus

Agomelatine is a novel antidepressant acting as a melatonergic receptor agonist and serotonergic (5-HT2C) receptor antagonist. In adult rats, chronic agomelatine treatment enhanced cell proliferation and neurogenesis in the ventral hippocampus (VH), a region pertinent to mood disorders. This study compared the effects of agomelatine on cell proliferation, maturation, and survival and investigated the cellular mechanisms underlying these effects. Agomelatine increased the ratio of mature vs immature neurons and enhanced neurite outgrowth of granular cells, suggesting an acceleration of maturation. The influence of agomelatine on maturation and survival was accompanied by a selective increase in the levels of BDNF (brain-derived neurotrophic factor) vs those of VEGF (vascular endothelial factor) and IGF-1 (insulin-like growth factor 1), which were not affected. Agomelatine also activated several cellular signals (extracellular signal-regulated kinase1/2, protein kinase B, and glycogen synthase kinase 3β) known to be modulated by antidepressants and implicated in the control of proliferation/survival. Furthermore, as agomelatine possesses both melatonergic agonist and serotonergic (5-HT2C) antagonist properties, we determined whether melatonin and 5-HT2C receptor antagonists similarly influence cell proliferation and survival. Only the 5-HT2C receptor antagonists, SB243,213 or S32006, but not melatonin, mimicked the effects of agomelatine on cell proliferation in VH. The promoting effect of agomelatine on survival was not reproduced by the 5-HT2C receptor antagonists or melatonin alone. However, it was blocked by a melatonin antagonist, S22153. These results show that agomelatine treatment facilitates all stages of neurogenesis and suggest that a joint effect of melatonin agonism and 5HT2C antagonism may be involved in promotion by agomelatine of survival in the hippocampus.

The neuronal excitatory amino acid transporter EAAC1/EAAT3: does it represent a major actor at the brain excitatory synapse?

EAAC1/EAAT3 is a transporter of glutamate (Glu) present at the post‐synaptic neuronal element, in opposition to the two other main transporters, GLAST/EAAT1 and GLT1/EAAT2, expressed at the excitatory amino acid (EAA) synapse by surrounding astrocytes. Although, in the adult, EAAC1/EAAT3 exhibits a rather low expression level and is considered to make a minor contribution to Glu removal from the synapse, its early expression during brain development, before the astrocytes are functional, suggests that such a neuronal transporter is involved in the developmental effects of EAA and, possibly, in the biosynthesis and trophic role of GABA, which is excitatory in nature in different brain regions during the earlier stages of brain development. This neuronal Glu transporter is considered to have a dual action as it is apparently involved in the neuronal uptake of cysteine, which acts as a key substrate for the synthesis of glutathione, a major anti‐oxidant, because the neurones do not express the Xc– transport system in the mature brain. Interestingly, EAAC1/EAAT3 activity/expression was shown to be highly regulated by neuronal activity as well as by intracellular signalling pathways involving primarily α protein kinase C (αPKC) and phosphatidylinositol‐3‐kinase (PI3K). Such regulatory processes could act either at the post‐traductional level or at the transcriptional level. It is worth noting that EAAC1/EAAT3 exhibits specificity, compared with other EAA transporters, because it is present mainly in the intracellular compartment and only for about 20% at the plasma membrane. Variations in neuronal Glu uptake were shown to be associated with rapid changes in the trafficking of the transporter protein altering the membranar location of the transporter. More recent data show that astrocyte‐secreted factors such as cholesterol could also influence rapid changes in the location of EAAC1/EAAT3 between the plasma membrane and the cytoplasmic compartment. Such a highly regulated process of EAAC1/EAAT3 activity/expression may have implications in the physiopathology of major diseases affecting EAA brain signalling, which is further supported by data obtained in animal models of hypoxia–anoxia, for example.

Differential regulation by protein kinases of activity and cell surface expression of glutamate transporters in neuron-enriched cultures

This study described the involvement of short-term PKA, PKC or PI3K phosphorylation-mediated processes in the regulation of activity and trafficking of the excitatory amino acid transporters EAAC1, GLAST and GLT-1 endogenously expressed in neuron-enriched cultures. Glutamate uptake was dose-dependently decreased by inhibitors of protein kinase A (PKA), [N-[2-(p-bromocinnamylamino)-ethyl]-5-(isoquinolinesulfonamide)] (H89) or phosphatidylinositol 3-kinase (PI3K) (wortmannin), but not altered after protein kinase C (PKC) inhibition (staurosporine) or activation phorbol-12-myristate-13-acetate (PMA). Biotinylation and immunoblotting results (% of controls) showed that EAAC1 membrane expression was significantly decreased by H89 (71.9+/-4.7%) and wortmannin (63.3+/-20.0%) and increased by PMA (137.7+/-15.5%). H89 and PMA induced a significant decrease of the cell surface fraction of GLAST (54.0+/-34.1% and 73.3+/-14.3%, respectively) whereas wortmannin significantly increased this fraction (119.8+/-9.3%). After treatment with H89, the GLT-1 membrane level showed a two-fold increase (179.4+/-19.7%). Conversely, PMA and wortmannin induced a significant decrease of the cell surface expression of GLT-1 (49.0+/-15.4% and 40.7+/-33.7%, respectively). Confocal microscopy revealed a wortmannin-induced clustering of EAAC1 in the intracellular compartment. These data suggest that trafficking of glutamate transporters can be differentially regulated by PKA-, PKC- and PI3K-dependent signaling pathways and could therefore control total glutamate uptake activity. These processes may represent rapid adaptive responses to changes in the cellular environment, which significantly contribute to regulation of EAA transmission and further prevent possible excitotoxic events.

Neuroprotective Effects of Propofol in a Model of Ischemic Cortical Cell Cultures Role of Glutamate and Its Transporters

Background During cerebral ischemia, excess of glutamate release and dysfunction of its high affinity transport induce an accumulation of extracellular glutamate, which plays an important role in neuronal death. The authors studied the relationship among propofol neuroprotection, glutamate extracellular concentrations, and glutamate transporter activity in a model of ischemic cortical cell cultures. Methods Thirteen-day-old primary cortical neuronal-glial cultures were exposed to a 90-min combined oxygen–glucose deprivation (OGD) in an anaerobic chamber, followed by reoxygenation. Propofol was added only during the OGD period, and its effect was compared to that of the N-methyl-d-aspartate receptor antagonist dizocilpine (MK-801). Twenty-four hours after the injury, cell death was quantified by lactate dehydrogenase release and cell viability by reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). Extracellular concentrations of glutamate in culture supernatants and glutamate uptake were performed at the end of OGD period by high-performance liquid chromatography and incorporation of l-[3H]glutamate into cells, respectively. Results At clinically relevant concentrations (0.05–10 &mgr;m), propofol offered protection equivalent to that of MK-801. It significantly reduced lactate dehydrogenase release and increased the reduction of MTT. At the end of the ischemic injury, propofol was able to reverse the OGD-induced increase in glutamate extracellular concentrations and decrease of glutamate uptake. The inhibition of the glial GLT1 transporter by 3-methyl-glutamate did not further modify the effect of propofol on glutamate uptake, suggesting that GLT1 was not the major target of propofol. Conclusion Propofol showed a neuroprotective effect in this in vitro model of OGD, which was apparently mediated by a GLT1-independent restoration of the glutamate uptake impaired during the injury.

Sevoflurane Protects Rat Mixed Cerebrocortical Neuronal–glial Cell Cultures against Transient Oxygen–glucose Deprivation: Involvement of Glutamate Uptake and Reactive Oxygen Species

Background:The purpose of this study was to clarify the role of glutamate and reactive oxygen species in sevoflurane-mediated neuroprotection on an in vitro model of ischemia–reoxygenation. Methods:Mature mixed cerebrocortical neuronal–glial cell cultures, treated or not with increasing concentrations of sevoflurane, were exposed to 90 min combined oxygen–glucose deprivation (OGD) in an anaerobic chamber followed by reoxygenation. Cell death was quantified by lactate dehydrogenase release into the media and cell viability by reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium by mitochondrial succinate dehydrogenase. Extracellular concentrations of glutamate and glutamate uptake were assessed at the end of the ischemic injury by high-performance liquid chromatography and incorporation of L-[3H]glutamate into cells, respectively. Free radical generation in cells was assessed 6 h after OGD during the reoxygenation period using 2′,7′-dichlorofluorescin diacetate, which reacts with intracellular radicals to be converted to its fluorescent product, 2′,7′-dichlorofluorescin, in cell cytosol. Results:Twenty-four hours after OGD, sevoflurane, in a concentration-dependent manner, significantly reduced lactate dehydrogenase release and increased cell viability. At the end of OGD, sevoflurane was able to reduce the OGD-induced decrease in glutamate uptake. This effect was impaired in the presence of threo-3-methyl glutamate, a specific inhibitor of the glial transporter GLT1. Sevoflurane counteracted the increase in extracellular level of glutamate during OGD and the generation of reactive oxygen species during reoxygenation. Conclusion:Sevoflurane had a neuroprotective effect in this in vitro model of ischemia–reoxygenation. This beneficial effect may be explained, at least in part, by sevoflurane-induced antiexcitotoxic properties during OGD, probably depending on GLT1, and by sevoflurane-induced decrease of reactive oxygen species generation during reoxygenation.

Preferential vulnerability of mesencephalic dopamine neurons to glutamate transporter dysfunction

Nigral depletion of the main brain antioxidant GSH is the earliest biochemical event involved in Parkinson’s disease pathogenesis. Its causes are completely unknown but increasing number of evidence suggests that glutamate transporters [excitatory amino acid transporters (EAATs)] are the main route by which GSH precursors may enter the cell. In this study, we report that dopamine (DA) neurons, which express the excitatory amino acid carrier 1, are preferentially affected by EAAT dysfunction when compared with non‐DA neurons. In rat embryonic mesencephalic cultures, l‐trans‐pyrrolidine‐2,4‐dicarboxylate, a substrate inhibitor of EAATs, is directly and preferentially toxic for DA neurons by decreasing the availability of GSH precursors and lowering their resistance threshold to glutamate excitotoxicity through NMDA‐receptors. In adult rat, acute intranigral injection of l‐trans‐pyrrolidine‐2,4‐dicarboxylate induces a large regionally selective and dose‐dependent loss of DA neurons and α‐synuclein aggregate formation. These data highlight for the first time the importance of excitatory amino acid carrier 1 function for the maintenance of antioxidant defense in DA neurons and suggest its dysfunction as a candidate mechanism for the selective death of DA neurons such as occurring in Parkinson’s disease.

A new class of scorpion toxin binding sites related to an A-type K+ channel: pharmacological characterization and localization in rat brain.

A new scorpion toxin (3751.8 Da) was isolated from the Buthus martensi venom, sequenced and chemically synthesized (sBmTX3). The A‐type current of striatum neurons in culture completely disappeared when 1 μM sBmTX3 was applied (K d=54 nM), whereas the sustained K+ current was unaffected. 125I‐sBmTX3 specifically bound to rat brain synaptosomes (maximum binding=14 fmol mg−1 of protein, K d=0.21 nM). A panel of toxins yet described as specific ligands for K+ channels were unable to compete with 125I‐sBmTX3. A high density of 125I‐sBmTX3 binding sites was found in the striatum, hippocampus, superior colliculus, and cerebellum in the adult rat brain.

Effects of PKA and PKC modulators on high affinity glutamate uptake in primary neuronal cell cultures from rat cerebral cortex

In this study, the effects of various agents known to alter protein phosphorylation, via protein kinase C or A, on high affinity glutamate uptake were investigated in primary neuronal cell cultures of rat cerebral cortex. Incubating the culture dishes with chelerythrine or H89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide), which inhibit PKC and PKA, respectively, dramatically decreased the glutamate uptake in a dose-dependent manner. Saturation kinetic analysis showed that chelerythrine and H89 decreased the Vmax (chelerythrine: -61%, P < 0.06; -59%, P < 0.05) without affecting the Km of the transport process as compared to the control values. These inhibitory effects were counteracted by the corresponding protein kinase activators, i.e. PMA (phorbol-12-myristate 13-acetate) in the case of PKC and forskolin in the case of PKA, although these protein kinase activators alone did not significantly affect the glutamate uptake. These results provide evidence that, in primary cultures of neuronal cells, the high affinity glutamate uptake may be regulated by both PKA and PKC-mediated phosphorylation processes.

ABCA2 is a marker of neural progenitors and neuronal subsets in the adult rodent brain.

The notion that the ATP‐binding cassette transporter‐A2 (ABCA2) may be involved in brain sterol homeostasis and is associated with early onset Alzheimers disease led us to explore its neural expression. Our data support and extend the previous reports on ABCA2 expression by oligodendrocytes. They evidence that ABCA2 (i) is located in intracellular vesicles, identified in transfected cells as lysosome‐related organelles only partially overlapping with classical endolysosomes; (ii) is a marker of neural progenitors as it is expressed in the subventricular zone of the lateral ventricle and the dentate gyrus of the hippocampal formation, sites of continual neurogenesis in the adult brain, and in nestin+ cells differentiated in vitro from embryonic stem cells; (iii) persists, in the adult rodent brain, in a subset of GABAergic and glutamatergic neurons. Considering that the latter are targets of Alzheimers lesions, these data provide a new rationale to explore the neuropathological consequences of ABCA2 functional dysregulations.

Glial soluble factors regulate the activity and expression of the neuronal glutamate transporter EAAC1: implication of cholesterol

A co‐ordinated regulation between neurons and astrocytes is essential for the control of extracellular glutamate concentration. Here, we have investigated the influence of astrocytes and glia‐derived cholesterol on the regulation of glutamate transport in primary neuronal cultures from rat embryonic cortices. Glutamate uptake rate and expression of the neuronal glutamate transporter EAAC1 were low when neurons were grown without astrocytes and neurons were unable to clear extracellular glutamate. Treatment of the neuronal cultures with glial conditioned medium (GCM) increased glutamate uptake Vmax, EAAC1 expression and restored the capacity of neurons to eliminate extracellular glutamate. Thus, astrocytes up‐regulate the activity and expression of EAAC1 in neurons. We further showed that cholesterol, present in GCM, increased glutamate uptake activity when added directly to neurons and had no effect on glutamate transporter expression. Furthermore, part of the GCM‐induced effect on glutamate transport activity was lost when cholesterol was removed from GCM (low cholesterol‐GCM) and was restored when cholesterol was added to low cholesterol‐GCM. This demonstrates that glia‐derived cholesterol regulates glutamate transport activity. With these experiments, we provide new evidences for neuronal glutamate transport regulation by astrocytes and identified cholesterol as one of the factors implicated in this regulation.