High Levels Of Extracellular Glutamate Research Articles

Effect of ACEA 1021 on Neurotoxicity Induced by Dexamethasone — Initial Behavioral Study

Considerable evidence suggests that glucocorticoids (GCs) play an important role in neurodegeneration. Chronic elevated levels of GCs can result in neuronal degeneration of the hippocampal piramidal neurons, which are paralleled by cognitive deficits. Moreover, GCs potentiate stress or ischemia-induced accumulation of excitatory amino acids (EAA) in the extracellular space of hippocampus. ACEA 1021 (licostinel), a selective antagonist of the N-methyl-D-aspartate (NMDA) receptor, has been reported to prevent the excitotoxic action of high extracellular glutamate levels. The aim of this study was to investigate the effect of ACEA 1021 on neurotoxic effect of dexamethasone (DEX - a synthetic GCs receptor agonist). The experiments were carried out on Albino Swiss mice (25-30 g). ACEA 1021, at the doses: 1.25 and 2.5 mg/kg/day, ip, was administered 15 min before DEX (16 mg/kg/day, ip). The long-term memory acquisition (passive avoidance test) and the motor performance (“chimney” test) were evaluated 14 days after the drugs administration. The prolongation of climbing time in the “chimney” test and decrease of the retention time in the memory task of mice treated with DEX for 14 days. In mice treated with DEX for 14 days, ACEA 1021 at the both doses reduced the climbing time in the “chimney” test, at the dose of 1.25 mg/kg improved memory acquisition. The above findings suggest that ACEA 1021 could prevent the neurotoxic effects induced by DEX, but further study needs to be carried out to explain this effect.

Cannabidiol Modifies the Glutamate Over-Release in Brain Tissue of Patients and Rats with Epilepsy: A Pilot Study.

Drug-resistant epilepsy (DRE) is associated with high extracellular levels of glutamate. Studies support the idea that cannabidiol (CBD) decreases glutamate over-release. This study focused on investigating whether CBD reduces the evoked glutamate release in cortical synaptic terminals obtained from patients with DRE as well as in a preclinical model of epilepsy. Synaptic terminals (synaptosomes) were obtained from the epileptic neocortex of patients with drug-resistant temporal lobe epilepsy (DR-TLE, n = 10) or drug-resistant extratemporal lobe epilepsy (DR-ETLE, n = 10) submitted to epilepsy surgery. Synaptosomes highly purified by Percoll-sucrose density gradient were characterized by confocal microscopy and Western blot. Synaptosomes were used to estimate the high KCl (33 mM)-evoked glutamate release in the presence of CBD at different concentrations. Our results revealed responsive tissue obtained from seven patients with DR-TLE and seven patients with DR-ETLE. Responsive tissue showed lower glutamate release (p < 0.05) when incubated with CBD at low concentrations (less than 100 µM) but not at higher concentrations. Tissue that was non-responsive to CBD (DR-TLE, n = 3 and DR-ELTE, n = 3) showed high glutamate release despite CBD exposure at different concentrations. Simultaneously, a block of the human epileptic neocortex was used to determine its viability through whole-cell and extracellular electrophysiological recordings. The electrophysiological evaluations supported that the responsive and non-responsive human epileptic neocortices used in the present study exhibited proper neuronal viability and stability to acquire electrophysiological responses. We also investigated whether the subchronic administration of CBD could reduce glutamate over-release in a preclinical model of temporal lobe epilepsy. Administration of CBD (200 mg/kg, p.o. every 24 h for 7 days) to rats with lithium-pilocarpine-evoked spontaneous recurrent seizures reduced glutamate over-release in the hippocampus. The present study revealed that acute exposure to low concentrations of CBD can reduce the glutamate over-release in synaptic terminals obtained from some patients with DRE. This effect is also evident when applied subchronically in rats with spontaneous recurrent seizures. An important finding was the identification of a group of patients that were non-responsive to CBD effects. Future studies are essential to identify biomarkers of responsiveness to CBD to control DRE.

The Key Role of Astrocytes in Amyotrophic Lateral Sclerosis and Their Commitment to Glutamate Excitotoxicity.

In the last two decades, there has been increasing evidence supporting non-neuronal cells as active contributors to neurodegenerative disorders. Among glial cells, astrocytes play a pivotal role in driving amyotrophic lateral sclerosis (ALS) progression, leading the scientific community to focus on the "astrocytic signature" in ALS. Here, we summarized the main pathological mechanisms characterizing astrocyte contribution to MN damage and ALS progression, such as neuroinflammation, mitochondrial dysfunction, oxidative stress, energy metabolism impairment, miRNAs and extracellular vesicles contribution, autophagy dysfunction, protein misfolding, and altered neurotrophic factor release. Since glutamate excitotoxicity is one of the most relevant ALS features, we focused on the specific contribution of ALS astrocytes in this aspect, highlighting the known or potential molecular mechanisms by which astrocytes participate in increasing the extracellular glutamate level in ALS and, conversely, undergo the toxic effect of the excessive glutamate. In this scenario, astrocytes can behave as "producers" and "targets" of the high extracellular glutamate levels, going through changes that can affect themselves and, in turn, the neuronal and non-neuronal surrounding cells, thus actively impacting the ALS course. Moreover, this review aims to point out knowledge gaps that deserve further investigation.

Cerebral Malaria and Cytotoxic Lesions of the Corpus Callosum.

To the Editors: Malaria is a common cause of morbidity and mortality worldwide. According to the WHO, 229 million cases and 409,000 deaths were reported worldwide in 2020.1 Children account for a high burden of malaria-related mortality and are at a higher risk of developing cerebral malaria (CM).1 A 12-year-old, previously healthy female from Guinea-Bissau was admitted 1 day after arriving in Portugal with a 2-day history of headache, generalized myalgia, and high-grade fever. Physical examination was unremarkable, except for pallor, icteric sclerae, and nontender hepatomegaly (4 cm). The patient was hemodynamically stable (BP, 101/52 mmHg; HR, 111 bpm), but shortly after admission, vomiting, lethargy, and impaired consciousness were observed (Glasgow coma score 11). She presented with anemia (hemoglobin, 10.0 g/dL; normal reference [NR]: 12.0–16.0), thrombocytopenia (78 × 109/L; NR: 150–450) and significant elevation of inflammatory markers (C-reactive protein, 301.0 mg/L; NR, <5; and procalcitonin, >100 ng/mL; NR, <0.1). No electrolyte disturbances, hypoglycemia, or acidosis. Plasmodium falciparum blood smear was positive, and severe malaria was diagnosed due to high parasitemia (28.5%/erythrocytes–11.872.000 parasites/µL) and central nervous system (CNS) involvement other infections were excluded. The cerebrospinal fluid revealed pleocytosis (15 cells), generalized slowing on the electroencephalogram, and a lesion in the splenium of the corpus callosum with T2 hypersignal and restricted diffusion (Fig. 1), compatible with a cytotoxic lesion of the corpus callosum (CLOCCs) on brain magnetic resonance imaging (MRI). CM was diagnosed based on severely impaired consciousness, CLOCCs, and no other identifiable causes of coma.FIGURE 1.: MRI axial images T2 weighted (A), DWI (B) and ADC map (C): oval lesion in the splenium of the corpus callosum (arrow) with T2 hypersignal and diffusion restriction with DWI hypersignal and ADC map hyposignal, corresponding to cytotoxic edema.The patient was treated with parenteral quinine and clindamycin, due to the immediate unavailability of artesunate, plus ceftriaxone. She became afebrile with progressive improvement in neurological status after 48 h and complete parasite clearance on day 3. Artemether-lumefantrine was initiated on day 5, and the patient was discharged on day 8. CM is defined as a disturbance of consciousness in a patient with Plasmodium parasitemia and no other identifiable cause.2 Despite the reported low mortality rate of pediatric imported malaria in Europe (<1%), CM entails a risk of life-threatening acute CNS effects with diffuse encephalopathy and often rapidly progressive coma, as observed in our patient.3 Distinctive brain MRI features involving both white and gray matter structures have been reported in a large cohort of Malawian children with CM. Increased volume, T2 hyperintensity, and Diffusion-weighted imaging (DWI) abnormalities affecting the cortical area (61.7%), subcortical and periventricular white matter (71.7%), basal ganglia (84.2%) and the corpus callosum (49.2%) were the most frequent features.4 CLOCCS was a common MRI finding in CM, as suggested by DWI/Apparent diffusion coefficient mismatch with DWI increased signal and Apparent diffusion coefficient low signal,4 as observed in our patient (Fig. 1). The corpus callosum, mostly the splenium, is a region vulnerable to inflammatory cytokinopathy, driven by malaria, seizures or other causes.5 The higher density of cytokines and glutamate receptors in this area leads to high extracellular glutamate levels, inducing cytotoxic edema upon infection, which is frequently reversible.5 CM in children is the most severe complication of P. falciparum infection and needs a high suspicion index in nonendemic countries. Altered MRI and CLOCCs, although unspecific, are associated with CM.

Cytotoxic lesion of the corpus callosum in a patient with aphasic status epilepticus

A 47-year-old man with a history of aphasic seizures presented to the emergency room with a 12-hour global aphasia. Upon admission, brain MRI did not reveal acute lesions, and EEG showed sharp waves in the left frontal-temporal region. An Aphasic Status Epilepticus was diagnosed and antiepileptic treatment was initiated with adequate response. A week after the episode, a new brain MRI showed a high-signal ovoid lesion on T2-weighted and FLAIR sequences in the central part of the splenium of the corpus callosum. On diffusion-weighted images (DWI) the lesion was hyperintense with decreased apparent diffusion coefficient (ADC) values, indicating restricted diffusion consistent with a cytotoxic lesion of the corpus callosum (CLOCC). Follow-up MRI one month later showed complete image resolution. CLOCCs are secondary lesions associated with various entities in which high levels of cytokines and extracellular glutamate cause intracellular edema and reduced diffusion, a condition called cytotoxic edema, which affects vulnerable brain regions such as the splenium of the corpus callosum. In epileptic patients, CLOCCs may be due to the effect of seizures, especially prolonged ones, as well as antiepileptic treatment itself. CLOCCs are rare radiological findings and must be recognized to avoid misdiagnosis.

Cytotoxic lesion of the corpus callosum in a patient with aphasic status epilepticus

A 47-year-old man with a history of aphasic seizures presented to the emergency room with a 12-hour global aphasia. Upon admission, brain MRI did not reveal acute lesions, and EEG showed sharp waves in the left frontal-temporal region. An Aphasic Status Epilepticus was diagnosed and antiepileptic treatment was initiated with adequate response. A week after the episode, a new brain MRI showed a high-signal ovoid lesion on T2-weighted and FLAIR sequences in the central part of the splenium of the corpus callosum. On diffusion-weighted images (DWI) the lesion was hyperintense with decreased apparent diffusion coefficient (ADC) values, indicating restricted diffusion consistent with a cytotoxic lesion of the corpus callosum (CLOCC). Follow-up MRI one month later showed complete image resolution. CLOCCs are secondary lesions associated with various entities in which high levels of cytokines and extracellular glutamate cause intracellular edema and reduced diffusion, a condition called cytotoxic edema, which affects vulnerable brain regions such as the splenium of the corpus callosum. In epileptic patients, CLOCCs may be due to the effect of seizures, especially prolonged ones, as well as antiepileptic treatment itself. CLOCCs are rare radiological findings and must be recognized to avoid misdiagnosis.

Stearoylethanolamide interferes with retrograde endocannabinoid signalling and supports the blood-brain barrier integrity under acute systemic inflammation

Neuroinflammation plays a prominent role in the onset of demyelinating diseases, major depressive disorder and delayed neurodegeneration. An open question remains whether pharmacological suppression of inflammation can effectively reduce the progression of these states. Bioactive lipid mediators such as N-acylethanolamines (NAEs) have an anti-inflammatory activity and are of pharmacological interest due to their endogenous on-demand production and the existence of distinct biological targets in humans and animals.Here we demonstrate for the first time, that treatment with stearoylethanolamide (SEA), a prevailing endogenously formed NAE, is neuroprotective against LPS-induced neuroinflammation in C57BL/6 male mice. SEA restricted the spreading of peripheral inflammation to the brain, and averted the activation of resident microglia and leukocyte trafficking to the brain parenchyma. Treatment with SEA per se increased the neuronal expression of cannabinoid receptors CB1/2 and brain levels of the most potent endogenous CB1/2 agonist 2-arachidonoylglycerol in vivo. SEA enhanced the amplitude of synaptic vesicle release, supported the balanced signal-to-noise ratio in glutamate- and GABAergic neurotransmission and decreased the excitotoxic risk associated with higher extracellular glutamate levels under neuroinflammation.The interference of SEA with the endocannabinoid system and presynaptic neurotransmitter release may represent an intrinsic neuroprotective mechanism that is triggered by inflammation and glutamate excitotoxicity. Thus, our data allows to consider SEA for the preventive therapy of acute and late-onset neuroinflammation-associated synaptic dysfunction and neurodegeneration.

Ex vivo electrochemical measurement of glutamate release during spinal cord injury

Excessive glutamate release following traumatic spinal cord injury (SCI) has been associated with exacerbating the extent of SCI. However, the mechanism behind sustained high levels of extracellular glutamate is unclear. Spinal cord segments mounted in a sucrose double gap recording chamber are an established model for traumatic spinal cord injury. We have developed a method to record, with micro-scale printed glutamate biosensors, glutamate release from ex vivo rat spinal cord segments following injury. This protocol would work equally well for similar glutamate biosensors.

Neonatal exposure to monosodium glutamate results in dysmorphology of orofacial lower motor neurons

Glutamate is the most abundant excitatory neurotransmitter in the central nervous system, and is stored and released by both neurons and astrocytes. Despite the important role of glutamate as a neurotransmitter, high levels of extracellular glutamate can result in excitotoxicity and apoptosis. Monosodium glutamate (MSG) is a naturally occurring sodium salt of glutamic acid that is used as a flavour enhancer in many processed foods. Neonatal exposure to MSG has been shown to result in neurodegeneration in several forebrain regions, characterised by neuronal loss and neuroendocrine abnormalities. However, the brainstem effects of neonatal MSG exposure have not been investigated. It is therefore hypothesized that MSG exposure during the early postnatal period would impact brainstem lower motor neurons involved in feeding behaviour. The effect of neonatal MSG exposure on brainstem lower motor neurons was investigated by exposing rat pups to either 4 mg/g MSG or saline from postnatal day (P) 4 through 10. On P28, brains were preserved by vascular perfusion with fixative, frozen sectioned and stained for Nïssl substance. The number, size and shape of brainstem motor neurons were compared between MSG and saline-exposed animals. MSG exposure had no impact on the total number of neurons in the nuclei examined. However, MSG exposure was associated with a significant increase in the number of round somata in both the trigeminal and facial nuclei. Furthermore, MSG exposure resulted in significantly smaller neurons in all motor nuclei examined. These results suggest that neonatal exposure to MSG impacts the development of brainstem lower motor neurons which may impact feeding and swallowing behaviours in young animals.

Metabotropic Glutamate Receptor 2 Positive Allosteric Modulators: Closing the Gate on Drug Abuse?

Abnormally high levels of extracellular glutamate, the principal excitatory neurotransmitter in the central nervous system, have been implicated in elevated drug seeking and taking as well as drug addiction (1). Presynaptic metabotropic glutamate receptors (mGluRs) can limit glutamate release by acting as autoreceptors on glutamatergic terminals (2). Among the eight subtypes of mGluRs, group II (mGluR2 and mGluR3) and group III (mGluR4, mGluR7, and mGluR8) are known to act as autoreceptors at excitatory synapses in the mammalian brain. Group II mGluRs have received considerable attention in recent years as contributing factors and therapeutic targets for drug abuse disorders (2). For example, several studies demonstrated that agonists of group II mGluRs decrease operant self-administration of drugs, including cocaine, alcohol, nicotine, and methamphetamine. In addition, activation of group II mGluRs attenuates cue-induced reinstatement of cocaine, heroin, alcohol, and methamphetamine self-administration as well as drug priming–induced reinstatement of nicotine, methamphetamine, and cocaine seeking. Conversely, an antagonist of group II mGluRs increases alcohol drinking (3). There is an evolving idea that presynaptic group II mGluRs may provide a “gatekeeper” function, limiting glutamate release at key synapses in addiction circuitry.

The peroxisome proliferator activated receptor gamma agonist pioglitazone increases functional expression of the glutamate transporter excitatory amino acid transporter 2 (EAAT2) in human glioblastoma cells.

Glioma cells release glutamate through expression of system xc-, which exchanges intracellular glutamate for extracellular cysteine. Lack of the excitatory amino acid transporter 2 (EAAT2) expression maintains high extracellular glutamate levels in the glioma microenvironment, causing excitotoxicity to surrounding parenchyma. Not only does this contribute to the survival and proliferation of glioma cells, but is involved in the pathophysiology of tumour-associated epilepsy (TAE). We investigated the role of the peroxisome proliferator activated receptor gamma (PPARγ) agonist pioglitazone in modulating EAAT2 expression in glioma cells. We found that EAAT2 expression was increased in a dose dependent manner in both U87MG and U251MG glioma cells. Extracellular glutamate levels were reduced with the addition of pioglitazone, where statistical significance was reached in both U87MG and U251MG cells at a concentration of ≥ 30 μM pioglitazone (p < 0.05). The PPARγ antagonist GW9662 inhibited the effect of pioglitazone on extracellular glutamate levels, indicating PPARγ dependence. In addition, pioglitazone significantly reduced cell viability of U87MG and U251MG cells at ≥ 30 μM and 100 μM (p < 0.05) respectively. GW9662 also significantly reduced viability of U87MG and U251MG cells with 10 μM and 30 μM (p < 0.05) respectively. The effect on viability was partially dependent on PPARγ activation in U87MG cells but not U251MG cells, whereby PPARγ blockade with GW9662 had a synergistic effect. We conclude that PPARγ agonists may be therapeutically beneficial in the treatment of gliomas and furthermore suggest a novel role for these agents in the treatment of tumour associated seizures through the reduction in extracellular glutamate.

OP35 * THE PPAR AGONIST PIOGLITAZONE UPREGULATES EAAT2 EXPRESSION IN HUMAN GLIOBLASTOMA CELLS

INTRODUCTION: Glioblastoma multiforme (GBM) cells release glutamate through expression of system xc−, which exchanges intracellular glutamate for extracellular cysteine. Lack of glutamate uptake through low excitatory amino acid transporter 2 (EAAT2) expression permits high extracellular glutamate levels, causing damaging excitotoxicty to surrounding parenchyma and epileptogenesis at the peritumoural edge. PPARγ agonists upregulate EAAT2 expression and prevent glutamate mediated excitotoxicity in stroke models. We investigated PPARγ agonists in GBM glutamate regulation. METHOD: Glioma cell lines U87MG and U251, and Glioma stem cells (GSCs) isolated from freshly resected tumour specimens were exposed to increasing concentrations of Pioglitazone. Effects on cell viability, protein expression and extracellular glutamate levels were evaluated with an ATP assay, Western Blotting and HPLC respectively. RESULTS: EAAT2 expression was upregulated in a dose and time dependent manner in both U87MG and U251 cells. Glutamate levels were reduced with the addition of pioglitazone, where statistical significance was reached in U251 cells at a concentration of 30 µM pioglitazone (n = 3, p < 0.05). Pioglitazone significantly reduced the cell viability of U87MG and U251 cells at concentrations of ≥ 30 µM (n = 4, p < 0.05) and 100 µM (n = 6, p < 0.05) respectively. Photomicrographs revealed morphological changes in the glioma cell lines and inability of the GSCs to form neurospheres with increasing pioglitazone concentrations. CONCLUSION: This study provides preliminary evidence of PPARγ-mediated regulation of EAAT2 expression in human glioblastoma cells and suggests a novel treatment strategy for glioma associated seizures.

Brain infection with Staphylococcus aureus leads to high extracellular levels of glutamate, aspartate, γ‐aminobutyric acid, and zinc

Staphylococcal brain infections may cause mental deterioration and epileptic seizures, suggesting interference with normal neurotransmission in the brain. We injected Staphylococcus aureus into rat striatum and found an initial 76% reduction in the extracellular level of glutamate as detected by microdialysis at 2 hr after staphylococcal infection. At 8 hr after staphylococcal infection, however, the extracellular level of glutamate had increased 12-fold, and at 20 hr it had increased >30-fold. The extracellular level of aspartate and γ-aminobutyric acid (GABA) also increased greatly. Extracellular Zn(2+) , which was estimated at ∼2.6 µmol/liter in the control situation, was increased by 330% 1-2.5 hr after staphylococcal infection and by 100% at 8 and 20 hr. The increase in extracellular glutamate, aspartate, and GABA appeared to reflect the degree of tissue damage. The area of tissue damage greatly exceeded the area of staphylococcal infiltration, pointing to soluble factors being responsible for cell death. However, the N-methyl-D-aspartate receptor antagonist MK-801 ameliorated neither tissue damage nor the increase in extracellular neuroactive amino acids, suggesting the presence of neurotoxic factors other than glutamate and aspartate. In vitro staphylococci incubated with glutamine and glucose formed glutamate, so bacteria could be an additional source of infection-related glutamate. We conclude that the dramatic increase in the extracellular concentration of neuroactive amino acids and zinc could interfere with neurotransmission in the surrounding brain tissue, contributing to mental deterioration and a predisposition to epileptic seizures, which are often seen in brain abscess patients.

Glaucoma: role of neuroprotective agents

Glaucoma is an optic neuropathy, considered as the second leading cause of blindness worldwide. Glaucoma is characterized by selective death of retinal ganglion cells (RGC) and a progressive loss of vision. Elevated intraocular pressure (IOP) is one of the most important risk factors for developing glaucoma and hence we mainly focus on lowering IOP to arrest the progression of glaucoma. However, many patients continue to demonstrate a clinically downhill course despite the control of initially raised IOP. In fact, some patients develop what is called normal tension glaucoma, not associated to an increased IOP. This emphasizes that several pressure-independent mechanisms are responsible for the development and progression of glaucomatous neuropathy and that high IOP and vascular insufficiency in the optic nerve head are only risk factors for the development of glaucoma, and are not the only target for the treatment of glaucoma. The reason is that the process of RGC death is thought to be biphasic, and the primary injury is followed by a slower secondary degeneration related to a noxious environment surrounding the apoptotic cells. This environment is characterized by changes in the extra-cellular ionic concentrations, increased amounts of free radicals, neurotrophins (NT) depletion and increased glutamate-induced excitotoxicity due to high extra-cellular glutamate levels, which binds to N-methyl-D-aspartate (NMDA) receptors leading to an abnormally high intracellular Ca2+ concentration. Neuroprotection is a process that attempts to preserve the remaining cells that are still vulnerable to damage, and the main aim of neuroprotective therapy is to employ pharmacologic or other means to attenuate the hostility of the environment surrounding the degenerating cells, or to supply the cells with the tools to deal with this aggression, providing resilience to the insult. Several agents have been reported neuroprotective in glaucoma, both in clinical assays, such as Ca2+ channel blockers, and in experimental studies, such as betaxolol, brimonidine, NMDA antagonists, nitric oxide synthase inhibitors, NT and Ginkgo biloba extract. Most neuroprotective agents for glaucoma have proved beneficial effects over RGC, not showing effects over IOP. However, when analyzing classically used medications for glaucoma, it becomes difficult to understand if its effect over the progression of glaucoma is due to neuroprotective pathways or by means of lowering IOP. The ideal anti-glaucoma drug would be one that when applied topically, reduces IOP, but also probes to reach the retina in appropriate amounts, and activates specific receptors in the retina to attenuate RGC death. In this review, we will examine currently advocated neuroprotective drug-based strategies in the potential management of glaucoma.

Self‐induced accumulation of glutamate in striatal astrocytes and basal ganglia excitotoxicity

Excitotoxicity induced by high levels of extracellular glutamate (GLU) has been proposed as a cause of cell degeneration in basal ganglia disorders. This phenomenon is normally prevented by the astrocytic GLU-uptake and the GLU-catabolization to less dangerous molecules. However, high-GLU can induce reactive gliosis which could change the neuroprotective role of astrocytes. The striatal astrocyte response to high GLU was studied here in an in vivo rat preparation. The transient striatal perfusion of GLU (1 h) by reverse microdialysis induced complex reactive gliosis which persisted for weeks and which was different for radial-like glia, protoplasmic astrocytes and fibrous astrocytes. This gliosis was accompanied by a persistent cytosolic accumulation of GLU (immunofluorescence quantified by confocal microscope), which persisted for weeks (self-induced glutamate accumulation), and which was associated to a selective decrease of glutamine synthetase activity. This massive and persistent self-induced glutamate accumulation in striatal astrocytes could be an additional factor for the GLU-induced excitotoxicity, which has been implicated in the progression of different basal ganglia disorders.

Mislocalization of the exitatory amino-acid transporters (EAATs) in human astrocytoma and non-astrocytoma cancer cells: effect of the cell confluence

BackgroundAstrocytomas are cancers of the brain in which high levels of extracellular glutamate plays a critical role in tumor growth and resistance to conventional treatments. This is due for part to a decrease in the activity of the glutamate transporters, i.e. the Excitatory Amino Acid Transporters or EAATs, in relation to their nuclear mislocalization in astrocytoma cells. Although non-astrocytoma cancers express EAATs, the localization of EAATs and the handling of L-glutamate in that case have not been investigated.MethodsWe looked at the cellular localization and activity of EAATs in human astrocytoma and non-astrocytoma cancer cells by immunofluorescence, cell fractionation and L-glutamate transport studies.ResultsWe demonstrated that the nuclear mislocalization of EAATs was not restricted to astrocytoma and happened in all sub-confluent non-astrocytoma cancer cells we tested. In addition, we found that cell-cell contact caused the relocalization of EAATs from the nuclei to the plasma membrane in all human cancer cells tested, except astrocytoma.ConclusionsTaken together, our results demonstrated that the mislocalization of the EAATs and its associated altered handling of glutamate are not restricted to astrocytomas but were also found in human non-astrocytoma cancers. Importantly, we found that a cell contact-dependent signal caused the relocalization of EAATs at the plasma membrane at least in human non-astrocytoma cancer cells, resulting in the correction of the altered transport of glutamate in such cancer cells but not in astrocytoma.

The potential role of glutamate in the current diabetes epidemic

In the present article, we propose the perspective that abnormal glutamate homeostasis might contribute to diabetes pathogenesis. Previous reports and our recent data indicate that chronically high extracellular glutamate levels exert direct and indirect effects that might participate in the progressive loss of β-cells occurring in both T1D and T2D. In addition, abnormal glutamate homeostasis may impact all the three accelerators of the "accelerator hypothesis" and could partially explain the rising frequency of T1D and T2D.

Effects of hippocampal high‐frequency electrical stimulation in memory formation and their association with amino acid tissue content and release in normal rats

Hippocampal high frequency electrical stimulation (HFS) at 130 Hz has been proposed as a therapeutical strategy to control neurological disorders such as intractable temporal lobe epilepsy (TLE). This study was carried out to determine the effects of hippocampal HFS on the memory process and the probable involvement of amino acids. Using the autoshaping task, we found that animals receiving hippocampal HFS showed augmented short-term, but not long-term memory formation, an effect blocked by bicuculline pretreatment and associated with enhanced tissue levels of amino acids in hippocampus. In addition, microdialysis experiments revealed high extracellular levels of glutamate, aspartate, glycine, taurine, and alanine during the application of hippocampal HFS. In contrast, GABA release augmented during HFS and remained elevated for more than 1 h after the stimulation was ended. HFS had minimal effects on glutamine release. The present results suggest that HFS has an activating effect on specific amino acids in normal hippocampus that may be involved in the enhanced short-term memory formation. These data further provide experimental support for the concept that hippocampus may be a promising target for focal stimulation to treat intractable seizures in humans.

Genetic variation influences glutamate concentrations in brains of patients with multiple sclerosis

Glutamate is the main excitatory neurotransmitter in the mammalian brain. Appropriate transmission of nerve impulses through glutamatergic synapses is required throughout the brain and forms the basis of many processes including learning and memory. However, abnormally high levels of extracellular brain glutamate can lead to neuroaxonal cell death. We have previously reported elevated glutamate levels in the brains of patients suffering from multiple sclerosis. Here two complementary analyses to assess the extent of genomic control over glutamate levels were used. First, a genome-wide association analysis in 382 patients with multiple sclerosis using brain glutamate concentration as a quantitative trait was conducted. In a second approach, a protein interaction network was used to find associated genes within the same pathway. The top associated marker was rs794185 (P < 6.44 x 10(-7)), a non-coding single nucleotide polymorphism within the gene sulphatase modifying factor 1. Our pathway approach identified a module composed of 70 genes with high relevance to glutamate biology. Individuals carrying a higher number of associated alleles from genes in this module showed the highest levels of glutamate. These individuals also showed greater decreases in N-acetylaspartate and in brain volume over 1 year of follow-up. Patients were then stratified by the amount of annual brain volume loss and the same approach was performed in the 'high' (n = 250) and 'low' (n = 132) neurodegeneration groups. The association with rs794185 was highly significant in the group with high neurodegeneration. Further, results from the network-based pathway analysis remained largely unchanged even after stratification. Results from these analyses indicated that variance in the activity of neurochemical pathways implicated in neurodegeneration is explained, at least in part, by the inheritance of common genetic polymorphisms. Spectroscopy-based imaging provides a novel quantitative endophenotype for genetic association studies directed towards identifying new factors that contribute to the heterogeneity of clinical expression of multiple sclerosis.

Molecular Signalling Mediating the Protective Effect of A1Adenosine and mGlu3 Metabotropic Glutamate Receptor Activation against Apoptosis by Oxygen/Glucose Deprivation in Cultured Astrocytes

Astrocyte death may occur in neurodegenerative disorders and complicates the outcome of brain ischemia, a condition associated with high extracellular levels of adenosine and glutamate. We show that pharmacological activation of A(1) adenosine and mGlu3 metabotropic glutamate receptors with N(6)-chlorocyclopentyladenosine (CCPA) and (-)2-oxa-4-aminocyclo-[3.1.0]hexane-4,6-dicarboxylic acid (LY379268), respectively, protects cultured astrocytes against apoptosis induced by a 3-h exposure to oxygen/glucose deprivation (OGD). Protection by CCPA and LY379268 was less than additive and was abrogated by receptor blockade with selective competitive antagonists or pertussis toxin. Both in control astrocytes and in astrocytes exposed to OGD, CCPA and LY379268 induced a rapid activation of the phosphatidylinositol-3-kinase (PI3K) and extracellular signal-regulated kinases 1 and 2 (ERK1/2)/mitogen-activated protein kinase (MAPK) pathways, which are known to support cell survival. In cultures exposed to OGD, CCPA and LY379268 reduced the activation of c-Jun N-terminal kinase and p38/MAPK, reduced the levels of the proapoptotic protein Bad, increased the levels of the antiapoptotic protein Bcl-X(L), and were highly protective against apoptotic death, as shown by nuclear 4'-6-diamidino-2-phenylindole staining and measurements of caspase-3 activity. All of these effects were attenuated by treatment with 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126) and 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002), which inhibit the MAPK and the PI3K pathways, respectively. These data suggest that pharmacological activation of A(1) and mGlu3 receptors protects astrocytes against hypoxic/ischemic damage by stimulating the PI3K and ERK1/2 MAPK pathways.