Marcos Emilio dos Santos Frizzo

Riluzole enhances glutamate uptake in rat astrocyte cultures

Abstract1. Riluzole is used for the treatment of amyotrophic lateral sclerosis and reported to have neuroprotective effects in animal models of Parkinsons disease, Huntingtons disease, and brain ischemia. The neuroprotective action of riluzole has been attributed to its ability to inhibit glutamate release (A. Doble, Neurology47(4):233S–241S, 1996).2. The effect of riluzole on L-[2,3-3H] glutamate uptake was investigated in rat cortical astrocyte cultures.3. Riluzole showed a biphasic concentration-dependent effect on basal glutamate uptake. At low concentrations (1 and 10 μM) riluzole significantly increased glutamate uptake, whereas from 100 μM promoted a slight reduction.4. Considering the large range of glutamate levels in the synaptic cleft, we studied the 1 μM riluzole effect on uptake of glutamate at different concentrations (1–1000 μM). Riluzole was more effective at low glutamate concentrations (10 μM), enhancing the basal glutamate uptake up to 42%.5. The action of riluzole on astrocytic glutamate uptake could be an additional mechanism to its neuroprotective role, perhaps suggesting a modulatory action on glutamatergic system involving glutamate clearance from synaptic cleft.

Guanosine Enhances Glutamate Uptake in Brain Cortical Slices at Normal and Excitotoxic Conditions

Abstract1. The effect of guanosine on L-[2,3-3H]glutamate uptake was investigated in brain cortical slices under normal or oxygen–glucose deprivation (OGD) conditions.2. In slices exposed to physiological conditions, guanosine (1–100 μM) stimulated glutamate uptake (up to 100%) in a concentration-dependent manner when a high (100 μM) but not a low (1 μM) concentration of glutamate was used.3. In slices submitted to OGD, guanosine 1 and 100 μM also increased 100 μM glutamate uptake (38 and 70%, respectively).4. The increasing of glutamate and taurine released to the incubation medium in cortical slices submitted to OGD were significantly attenuated by the presence of guanosine in the incubation medium.5. Guanosine prevented the increase in propidium iodide incorporation into cortical slices induced by OGD, indicating a protective role against ischemic injury.6. These results support the hypothesis of a protective role for guanosine during brain ischemia, possibly by activating glutamate uptake into neural cells.

Effect of orally administered guanosine on seizures and death induced by glutamatergic agents.

Intraperitoneal guanosine has been shown to prevent quinolinic acid-induced seizures in mice. In this study, we investigated the effect of orally administered guanosine on seizures induced by the glutamate agonists quinolinic acid and kainate, and the endogenous glutamate releaser alpha-dendrotoxin. Guanosine (7.5 mg/kg, per os), administered 75 min in advance, prevented 70% of seizures induced by i.c.v. quinolinic acid, being as efficient as the NMDA channel blocker MK-801 administered intraperitoneally. Guanosine was ineffective against kainate-induced seizures, but significantly reversed the potentiation of seizures and death caused by the concomitant injection of MK-801. Guanosine also significantly prevented seizures and death induced by i.c.v. alpha-dendrotoxin, whereas MK-801 and phenobarbital only prevented death. Altogether, our findings underscore the therapeutic potential of oral administration of guanosine for treating diseases involving glutamatergic excitotoxicity, including epilepsy.

Chronically administered guanosine is anticonvulsant, amnesic and anxiolytic in mice

Acute administration of intraperitoneal and oral guanosine has been shown to prevent quinolinic acid and alpha-dendrotoxin-induced seizures in rats and mice. In this study, we investigated the effects of 2 weeks ad libitum consumption of guanosine (0.5 mg/ml) added to mice water supply on seizures and lethality induced by the alpha-dendrotoxin, hole-board behavior, inhibitory avoidance task, locomotor activity, motor coordination, rectal temperature, body weight, and water and food consumption. Guanosine prevented seizures in 40% and death in 50% on mice treated with i.c.v. alpha-dendrotoxin; it also impaired inhibitory avoidance memory and increased head-dipping behavior and locomotor activity on the hole-board test. Guanosine consumption did not alter any of the other parameters evaluated. The anticonvulsant, amnesic, and anxyolytic-like effects may be associated with the ability of guanosine in modulating the glutamatergic excitatory system. Adding to previously reported data, these findings suggest a potential role for chronic guanosine in the management of diseases associated with glutamatergic excitotoxicity, including epilepsy and anxiety.

Activation of glutamate uptake by guanosine in primary astrocyte cultures.

Guanine-based purines have been shown to modulate the effects of glutamate, which is essential for brain function and mediates excitotoxicity. In the search for a mechanism involving the interaction between purine nucleoside guanosine and glutamate, we found that guanosine dose-dependently, significantly (63%) and potently (EC50 = 2.47 μM) enhanced glutamate uptake in cultured astrocytes. This effect was not inhibited by the blocker of nucleoside transporter dipyridamole nor by the adenosine antagonist theophylline, suggesting an extracellular site of action without the involvement of adenosine receptors. These results indicate a regulatory role of guanosine on extracellular levels of glutamate, possibly contributing for protecting neural cells against glutamate-induced excitotoxicity.

Computational Challenges in miRNA Target Predictions: To Be or Not to Be a True Target?

All microRNA (miRNA) target—finder algorithms return lists of candidate target genes. How valid is that output in a biological setting? Transcriptome analysis has proven to be a useful approach to determine mRNA targets. Time course mRNA microarray experiments may reliably identify downregulated genes in response to overexpression of specific miRNA. The approach may miss some miRNA targets that are principally downregulated at the protein level. However, the high-throughput capacity of the assay makes it an effective tool to rapidly identify a large number of promising miRNA targets. Finally, loss and gain of function miRNA genetics have the clear potential of being critical in evaluating the biological relevance of thousands of target genes predicted by bioinformatic studies and to test the degree to which miRNA-mediated regulation of any “validated” target functionally matters to the animal or plant.

Ebselen protects against methylmercury-induced inhibition of glutamate uptake by cortical slices from adult mice

Methylmercury (MeHg) is a highly neurotoxic compound and the inhibition of glutamate uptake by astrocytes has been pointed as an important mechanism involved in MeHg-induced glutamate excitotoxicity. We examined the effect of oral exposure to MeHg (10 and 40 mg/l in drinking water) on glutamate uptake by brain cortical slices of adult mice. Moreover, the possible protective role of ebselen (20 mg/kg, subcutaneously) against MeHg effect was also examined. In addition, it was measured the glutathione peroxidase and catalase activities in mice brain. Our results demonstrated, for the first time, that in vivo exposure to MeHg causes a dose-dependent decrease in glutamate uptake and that ebselen, which did not affect the uptake per se, reverted this effect. MeHg decreased glutathione peroxidase activity and increased catalase activity, effects which were also prevented by ebselen. These results may indirectly indicate that: (i) the in vivo inhibitory effect of MeHg on glutamate uptake could be probably related to overproduction of H(2)O(2); (ii) the protective effect of ebselen on MeHg-induced inhibition of glutamate uptake could be related to its ability to detoxify H(2)O(2).

Ontogenetic profile of glutamate uptake in brain structures slices from rats: sensitivity to guanosine

The excitotoxicity of the neurotransmitter glutamate has been shown to be connected with many acute and chronic diseases of the CNS. High affinity sodium-dependent glutamate transporters play a key role in maintaining adequate levels of extracellular glutamate. In the present study, we used slices of striatum, hippocampus and cortex from rat brain to describe the in vitro profile of glutamate uptake during development and ageing, and its sensitivity to guanosine. In all structures, glutamate uptake was higher in immature animals. There was a maximum decrease in glutamate uptake in striatum and hippocampus in 15-month-old rats, which later increased, while in cortex there was a significant decrease in rats aged 60 days old. The effect of guanosine seems to be age and structure dependent since the increase in basal glutamate uptake was only seen in slices of cortex from 10-day-old animals.

Quinolinic acid promotes seizures and decreases glutamate uptake in young rats: reversal by orally administered guanosine

Quinolinic acid (QA) has been used as a model for experimental overstimulation of the glutamatergic system. Glutamate uptake is the main mechanism involved in the maintenance of extracellular glutamate below toxic levels. Guanosine systemically administered prevents quinolinic acid-induced seizures in adult mice and increases basal glutamate uptake by cortical astrocyte culture and slices from young rats. The immature brain differs from the adult brain in its susceptibility to seizures, seizure characteristics, and responses to antiepileptic drugs (AED). Here we investigated the effect of guanosine p.o. on QA-induced seizures in young rats (P12-14) and upon ex vivo glutamate uptake by cortical slices from these animals. I.c.v. infusion of 250 nmol QA induced seizures in all animals and decreased glutamate uptake. I.p. injection of MK-801 and phenobarbital 30 min before QA administration prevented seizures in all animals. Guanosine (7.5 mg/kg) 75 min before QA prevented seizures in 50% of animals as well as prevented the decrease of glutamate uptake in the protected animals. To investigate if the anticonvulsive effect of guanosine was specific for QA-induced seizures, the picrotoxin-induced seizures model was also performed. Pretreatment with phenobarbital i.p. (60 mg/kg-30 min) prevented picrotoxin-induced seizures in all animals, whereas guanosine p.o. (7.5 mg/kg-75 min) and MK-801 i.p. (0.5 mg/kg-30 min) had no effect. Thus, guanosine protection on the QA-induced seizures in young rats and on the decrease of glutamate uptake showed some specificity degree towards the QA-induced toxicity. This points that guanosine could be considered for treatments of epilepsy, and possibly other neurological disorders in children.

Effects of chronic administered guanosine on behavioral parameters and brain glutamate uptake in rats

Oral and intraperitoneal administration of the nucleoside guanosine have been shown to prevent quinolinic acid‐ (QA) and α‐dendrotoxin‐induced seizures, impair memory, and impair anxiety in rats and mice. We investigated the effect of 2‐weeks ad lib orally administered guanosine (0.5 mg/ml) on seizures induced by QA, inhibitory avoidance memory, and locomotor performance in rats. We also studied the mechanism of action of guanosine through the measurement of its concentration in the cerebrospinal fluid (CSF) and its effect on glutamate uptake in cortical slices of rats. QA produced seizures in 85% of rats, an effect partially prevented by guanosine (53% of seizures; P = 0.0208). Guanosine also impaired retention on the inhibitory avoidance task (P = 0.0278) and decreased locomotor activity on the open field test (P = 0.0101). The CSF guanosine concentration increased twofold in the treated group compared to that in the vehicle group (P = 0.0178). Additionally, QA promoted a 30% decrease in glutamate uptake as compared to that with intracerebroventricular saline administration, an effect prevented by guanosine in animals protected against QA‐induced seizures. Altogether, these findings suggest a potential role of guanosine for treating diseases involving glutamatergic excitotoxicity such as epilepsy. These effects seem to be related to modulation of glutamate uptake.