Absence Of Glutamate Research Articles

Glutamate's Effects on the N-Methyl-D-Aspartate (NMDA) Receptor Ion Channel in Alzheimer's Disease Brain: Challenges for PET Radiotracer Development for Imaging the NMDA Ion Channel.

In an effort to further understand the challenges facing in vivo imaging probe development for the N-methyl-D-aspartate (NMDA) receptor ion channel, we have evaluated the effect of glutamate on the Alzheimer's disease (AD) brain. Human post-mortem AD brain slices of the frontal cortex and anterior cingulate were incubated with [3H]MK-801 and adjacent sections were tested for Aβ and Tau. The binding of [3H]MK-801 was measured in the absence and presence of glutamate and glycine. Increased [3H]MK-801 binding in AD brains was observed at baseline and in the presence of glutamate, indicating a significant increase (>100%) in glutamate-induced NMDA ion channel activity in AD brains compared to cognitively normal brains. The glycine effect was lower, suggesting a decrease of the co-agonist effect of glutamate and glycine in the AD brain. Our preliminary findings suggest that the targeting of the NMDA ion channel as well as the glutamate site may be appropriate in the diagnosis and treatment of AD. However, the low baseline levels of [3H]MK-801 binding in the frontal cortex and anterior cingulate in the absence of glutamate and glycine indicate significant hurdles for in vivo imaging probe development and validation.

A human relevant mixture of persistent organic pollutants (POPs) and perfluorooctane sulfonic acid (PFOS) differentially affect glutamate induced excitotoxic responses in chicken cerebellum granule neurons (CGNs) in vitro

Primary cultures of cerebellar granule neurons (CGNs) derived from chicken embryos were used to explore the effects on developmental neurotoxicity by a complex defined mixture of persistent organic pollutants (POPs). Its chemical composition and concentrations were based on blood levels in the Norwegian/Scandinavian population. Perfluorooctane sulfonic acid (PFOS) alone, its most abundant compound was also evaluated. Different stages of CGNs maturation, between day in vitro (DIV) 1, 3, and 5 were exposed to the POP mixture, or PFOS alone. Their combination with glutamate, an excitatory endogenous neurotransmitter important in neurodevelopment, also known to cause excitotoxicity was evaluated. Outcomes with the mixture at 500x blood levels were compared to PFOS at its corresponding concentration of 20 μM. The POP mixture reduced tetrazolium salt (MTT) conversion at earlier stages of maturation, compared to PFOS alone. Glutamate-induced excitotoxicity was enhanced above the level of that induced by glutamate alone, especially in mature CGNs at DIV5. Glutathione (GSH) concentrations seemed to set the level of sensitivity for the toxic insults from exposures to the pollutants. The role of N-methyl-D-aspartate receptor (NMDA-R) mediated calcium influx in pollutant exposures was investigated using the non-competitive and competitive receptor antagonists MK-801 and CGP 39551. Observations indicate a calcium-independent, but still NMDA-R dependent mechanism in the absence of glutamate, and a calcium- and NMDA-R dependent one in the presence of glutamate. The outcomes for the POP mixture cannot be explained by PFOS alone, indicating that other chemicals in the mixture contribute its overall effect.

Two Ways To Convert a Low-Affinity Potassium Channel to High Affinity: Control of Bacillus subtilis KtrCD by Glutamate.

Potassium and glutamate are the major cation and anion, respectively, in every living cell. Due to the high concentrations of both ions, the cytoplasm of all cells can be regarded as a potassium glutamate solution. This implies that the concentrations of both ions need to be balanced. While the control of potassium uptake by glutamate is well established for eukaryotic cells, much less is known about the mechanisms that link potassium homeostasis to glutamate availability in bacteria. Here, we have discovered that the availability of glutamate strongly decreases the minimal external potassium concentration required for the highly abundant Bacillus subtilis potassium channel KtrCD to accumulate potassium. In contrast, the inducible KtrAB and KimA potassium uptake systems have high apparent affinities for potassium even in the absence of glutamate. Experiments with mutant strains revealed that the KtrD subunit responds to the presence of glutamate. For full activity, KtrD synergistically requires the presence of the regulatory subunit KtrC and of glutamate. The analysis of suppressor mutants of a strain that has KtrCD as the only potassium uptake system and that experiences severe potassium starvation identified a mutation in the ion selectivity filter of KtrD (Gly282 to Val) that similarly results in a strongly glutamate-independent increase of the apparent affinity for potassium. Thus, this work has identified two conditions that increase the apparent affinity of KtrCD for potassium, i.e., external glutamate and the acquisition of a single point mutation in KtrD.IMPORTANCE In each living cell, potassium is required for maintaining the intracellular pH and for the activity of essential enzymes. Like most other bacteria, Bacillus subtilis possesses multiple low- and high-affinity potassium uptake systems. Their activity is regulated by the second messenger cyclic di-AMP. Moreover, the pools of the most abundant ions potassium and glutamate must be balanced. We report two conditions under which the low-affinity potassium channel KtrCD is able to mediate potassium uptake at low external potassium concentrations: physiologically, the presence of glutamate results in a severely increased potassium uptake. Moreover, this is achieved by a mutation affecting the selectivity filter of the KtrD channel. These results highlight the integration between potassium and glutamate homeostasis in bacteria.

Electrical Stimulation of C6 Glia-Precursor Cells In Vitro Differentially Modulates Gene Expression Related to Chronic Pain Pathways.

Glial cells comprise the majority of cells in the central nervous system and exhibit diverse functions including the development of persistent neuropathic pain. While earlier theories have proposed that the applied electric field specifically affects neurons, it has been demonstrated that electrical stimulation (ES) of neural tissue modulates gene expression of the glial cells. This study examines the effect of ES on the expression of eight genes related to oxidative stress and neuroprotection in cultured rodent glioma cells. Concentric bipolar electrodes under seven different ES types were used to stimulate cells for 30 min in the presence and absence of extracellular glutamate. ES consisted of rectangular pulses at 50 Hz in varying proportions of anodic and cathodic phases. Real-time reverse-transcribed quantitative polymerase chain reaction was used to determine gene expression using the ∆∆Cq method. The results demonstrate that glutamate has a significant effect on gene expression in both stimulated and non-stimulated groups. Furthermore, stimulation parameters have differential effects on gene expression, both in the presence and absence of glutamate. ES has an effect on glial cell gene expression that is dependent on waveform composition. Optimization of ES therapy for chronic pain applications can be enhanced by this understanding.

PT-31, a putative α2-adrenoceptor agonist, is effective in schizophrenia cognitive symptoms in mice.

Evidence of changes in central noradrenergic activity has been reported in schizophrenic patients and studies indicate that activation of the α2-adrenoceptor improves memory and neuroprotection. In this study, a new imidazolidine derivative 3-(2-chloro-6-fluorobenzyl)-imidazolidine-2,4-dione, PT-31, a putative α2A-adrenoceptor agonist, was evaluated in mouse models predictive of efficacy in the treatment of positive and cognitive symptoms of schizophrenia, as well as its ability to promote cerebellar granule cell survival in vitro, in the presence or absence of glutamate (100 µmol/l). PT-31 prevented apomorphine-induced climbing and the ketamine-induced hyperlocomotion, without inducing catalepsy or motor impairment. PT-31 protected against the impairment of prepulse inhibition induced by apomorphine, (±)-DOI, and ketamine. The molecule did not affect mouse short nor long-term memory per se, but it protected against ketamine-induced memory impairment when administered at different stages of the memory process (acquisition, consolidation, and retrieval) in the novel object recognition task. When added to cultured cerebellar granule neurons, PT-31 was not toxic per se and protected neurons from glutamate-induced apoptosis. In conclusion, PT-31 displayed a preclinical pharmacology predictive of neuroprotective effects and efficacy in relieving schizophrenia symptoms, without inducing motor side effects, suggesting that it could represent a molecular scaffold for antipsychotic drug development.

Melanin-concentrating hormone neurons promote rapid eye movement sleep independent of glutamate release.

Neurons containing melanin-concentrating hormone (MCH) in the posterior lateral hypothalamus play an integral role in rapid eye movement sleep (REMs) regulation. As MCH neurons also contain a variety of other neuropeptides [e.g., cocaine- and amphetamine-regulated transcript (CART) and nesfatin-1] and neurotransmitters (e.g., glutamate), the specific neurotransmitter responsible for REMs regulation is not known. We hypothesized that glutamate, the primary fast-acting neurotransmitter in MCH neurons, is necessary for REMs regulation. To test this hypothesis, we deleted vesicular glutamate transporter (Vglut2; necessary for synaptic release of glutamate) specifically from MCH neurons by crossing MCH-Cre mice (expressing Cre recombinase in MCH neurons) with Vglut2flox/flox mice (expressing LoxP-modified alleles of Vglut2), and studied the amounts, architecture and diurnal variation of sleep-wake states during baseline conditions. We then activated the MCH neurons lacking glutamate neurotransmission using chemogenetic methods and tested whether these MCH neurons still promoted REMs. Our results indicate that glutamate in MCH neurons contributes to normal diurnal variability of REMs by regulating the levels of REMs during the dark period, but MCH neurons can promote REMs even in the absence of glutamate.

Anterior Cingulate Glutamate and GABA Associations on Functional Connectivity in Schizophrenia.

The underlying neurobiological mechanism for abnormal functional connectivity in schizophrenia (SCZ) remains unknown. This project investigated whether glutamate and GABA, 2 metabolites that contribute to excitatory and inhibitory functions, may influence functional connectivity in SCZ. Resting-state functional magnetic resonance imaging and proton magnetic resonance spectroscopy were acquired from 58 SCZ patients and 61 healthy controls (HC). Seed-based connectivity maps were extracted between the anterior cingulate cortex (ACC) spectroscopic voxel and all other brain voxels. Magnetic resonance spectroscopy (MRS) spectra were processed to quantify glutamate and GABA levels. Regression analysis was performed to describe relationships between functional connectivity and glutamate and GABA levels. Reduced ACC functional connectivity in SCZ was found in regions associated with several neural networks including the default mode network (DMN) compared to HC. In the HC, positive correlations were found between glutamate and both ACC-right inferior frontal gyrus functional connectivity and ACC-bilateral superior temporal gyrus functional connectivity. A negative correlation between GABA and ACC-left posterior cingulate functional connectivity was also observed in HC. These same relationships were not statistically significant in SCZ. The present investigation is one of the first studies to examine links between functional connectivity and glutamate and GABA levels in SCZ. Results indicate that glutamate and GABA play an important role in the functional connectivity modulation in the healthy brain. The absence of glutamate and GABA correlations in areas where SCZ showed significantly reduced functional connectivity may suggest that this chemical-functional relationship is disrupted in SCZ.

The Role of Glutamate in Promoting Prostate Cancer Cell Proliferation, Motility, Invasiveness and Survival

Prostate cancer (PC) is one of the most common cancers affecting men in the western world. Androgen deprivation was the most common approach for treatment of metastatic PC. Unfortunately, this strategy is accompanied with a several side effects such as diabetes and cardiovascular diseases. Moreover, a tumor relapse was indicated in 80-90% of cases, after 1-3 years of treatment, with a life span 1-2 year due to the development of androgen resistant tumors. This study aimed to identify metabotropic glutamate receptor 1 (mGR1) as a possible target for PC therapy. Androgen sensitive PC cell line LNcap were cultured in presence and absence of glutamate (Glu) release inhibitor Riluzole and mGR1 antagonist BAY36-7620. Functional validation of the effect of Glu deprivation on LNcap cell proliferation, motility, invasiveness was conducted using MTT, wound healing and invasion assays, respectively. Molecular validation of Glu signaling and cell apoptosis was conducted using western blot approach. The data revealed a significant effect of glutamate downmodulation on inhibiting LNcap cell proliferation, motility and invasiveness at concentration of 25μM of Riluzole and BAY36-7620. Molecular analysis showed a marked effect of Glu inhibitors on induction of pro-apoptotic markers c-PARP, c-caspases 3, 7 and 9 and inhibition of Akt dependent cell proliferation. In conclusion, in addition to Androgens, Glu plays an essential role in PC cell proliferation, motility, invasiveness and survival.

Hierarchical mutational events compensate for glutamate auxotrophy of a Bacillus subtilis gltC mutant.

Glutamate is the major donor of nitrogen for anabolic reactions. The Gram-positive soil bacterium Bacillus subtilis either utilizes exogenously provided glutamate or synthesizes it using the gltAB-encoded glutamate synthase (GOGAT). In the absence of glutamate, the transcription factor GltC activates expression of the GOGAT genes for glutamate production. Consequently, a gltC mutant strain is auxotrophic for glutamate. Using a genetic selection and screening system, we could isolate and differentiate between gltC suppressor mutants in one step. All mutants had acquired the ability to synthesize glutamate, independent of GltC. We identified (i) gain-of-function mutations in the gltR gene, encoding the transcription factor GltR, (ii) mutations in the promoter of the gltAB operon and (iii) massive amplification of the genomic locus containing the gltAB operon. The mutants belonging to the first two classes constitutively expressed the gltAB genes and produced sufficient glutamate for growth. By contrast, mutants that belong to the third class appeared most frequently and solved glutamate limitation by increasing the copy number of the poorly expressed gltAB genes. Thus, glutamate auxotrophy of a B. subtilis gltC mutant can be relieved in multiple ways. Moreover, recombination-dependent amplification of the gltAB genes is the predominant mutational event indicating a hierarchy of mutations.

Glycine Transporter 1 Inhibitors and the Promise of a New Schizophrenia Therapy.

Patent Application Title: 4,5Dihydropyrazole Derivatives, Pharmaceutical Compositions Containing Them, and Their Use in Therapy Patent Application Number: US 2016/0075691 A1 Publication date: March 17, 2016 Priority Application: US 62/049,231 Priority date: September 11, 2014 Inventors: Amberg, W.; Brewer, J.; Hutchins, C.; Lange, U.; Lao, Y.; Li, H.-Q.; Pohlki, F.; Vasudevan, A.; Wang, Y.; Zhao, H. Applicants: AbbVie Deutschland GMBH & Co. KG, Wiesbaden (DE) AbbVie Inc., North Chicago, IL, USA Disease Area: Schizophrenia Biological Target: Glycine transporter-1 (GlyT1) Summary: The present invention relates to 4,5-dihydropyrazole derivatives represented generally by formula (I). These compounds are GlyT1 inhibitors and may be beneficial in the treatment of symptoms of schizophrenia patients. Glutamate is the conjugate base of glutamic acid (2-aminopentanedioic acid). Glutamate is abundant in the human nervous system and in the brain. It is a very important excitatory neurotransmitter that plays a major role in neural activation. It acts as the brain’s main excitatory neurotransmitter, and it is also used as the precursor for the biosynthesis of γ-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the brain. Glutamate receptors are synaptic receptors located primarily on the membranes of neuronal cells. They are responsible for the glutamate-mediated postsynaptic excitation of neural cells and are important for neural communication, memory formation, learning, and regulation. One of the glutamate receptors is the N-methyl-D-aspartate (NMDA) receptor. It was named because the agonist moleculeN-methyl-D-aspartate can bind to it selectively but not to any other glutamate receptors. NMDA receptor is activated by glutamate in addition to glycine as a coagonist. Its activation allows positively charged ions to flow through the cell membrane. The NMDA receptor is very important for controlling synaptic plasticity and memory function. Many of the central nervous system (CNS) disorders are caused by dysfunction of the glutamatergic pathways. These include schizophrenia, cognitive deficits, dementia, Parkinson disease, Alzheimer disease, bipolar disorder, and other disorders. Schizophrenia is a complex disease that affects patients’ thinking, feelings, and actions. It affects about 1% of the world population and requires lifelong therapy. A large number of animal models studies support what is known as the NMDA hypofunction hypothesis of schizophrenia. The hypothesis focuses on the hypofunction of the NMDA glutamate transmitter system as a cause of schizophrenia, and therefore, the modulation of that systemmay potentially provide a promising treatment for schizophrenia. Researchers have identified two specific glycine transporters, GlyTl and GlyT2. They belong to the Na/Cl-dependent family of neurotransmitter transporters, which also includes taurine, GABA, proline, monoamines, and orphan transporters. GlyT1 and GlyT2 have only about 50% similarities at the amino acid level. GlyT2 is expressed in spinal cord, brainstem, and cerebellum, while GlyT1 is expressed in these regions as well as in the forebrain areas such as cortex, hippocampus, septum, and thalamus. Rat studies have shown that GlyT2 is expressed by glycinergic nerve endings in rat spinal cord, whereas GlyT1 appears to be preferentially expressed by glial cells. These studies suggest that GlyT2 is predominantly responsible for glycine uptake at glycinergic synapses, whereasGlyT1 is involved inmonitoring glycine concentration in the vicinity ofNMDA receptor expressing synapses.Other recent rat studies have demonstrated that inhibition ofGlyT1 potentiatesNMDA receptor activity and affects NMDA receptor-dependent long-term potentiation. Molecular cloning has identified three variants of GlyTl, named GlyT-1a, GlyT-1b, and GlyT-1c. They differ in their N-terminal regions and display unique distributions in the brain and peripheral tissues. The function of theNMDA receptor can bemodulated by varying the levels of the glycine coagonist. The inhibition of GlyT1 slows the process of removal of glycine from the synapse, which elevates the level of synaptic glycine. The increase in the synaptic concentration of glycine does not produce an activation of NMDA receptors in the absence of glutamate and that maintains the activity-dependent activation of the NMDA receptor. Since high affinity transport mechanismsmaintain tight levels of the synaptic glutamate, an increased activation of the glycine site will only enhance the NMDA component of activated synapses.

Glutamate Limitation, BvgAS Activation, and (p)ppGpp Regulate the Expression of the Bordetella pertussis Type 3 Secretion System.

Bordetella pertussis is a bacterium that is considered to be highly adapted to humans, and it has not been isolated from the environment. As this bacterium does not utilize sugars, the abundant supply of glutamate in Stainer Scholte (SS) medium enables B. pertussis to grow efficiently in liquid culture in vitro, and as such, SS medium is a popular choice for laboratory experiments. However, the concentration of glutamate in the in vivo niche of B. pertussis is quite low. We investigated the bacterial response to low concentrations of glutamate to elucidate bacterial physiology via the expression of the type 3 secretion system (T3SS), and we discuss its relationship to the Bvg mode in which the two-component regulator of pathogenesis (BvgAS) is activated. Glutamate limitation induced the expression of both the T3SS apparatus and effector genes at the transcriptional level. (p)ppGpp, a modulator of the stringent response, was necessary for maximum expression of the T3SS genes. These observations indicate that the expression of the T3SS is managed by nutrient starvation. In addition, the autoaggregation ability was high in the absence of glutamate and no autoaggregation was observed in glutamate-replete medium. Taken together, glutamate-limited conditions in Bvg(+) mode elicit the high expression of T3SS genes in B. pertussis and promotes its sessile form. Bordetella pertussis is a highly contagious pathogen that causes respiratory infectious disease. In spite of the increasing use of vaccination, the number of patients with pertussis is increasing. The proteins produced in vivo often are different from the protein profile under laboratory conditions; therefore, the development of conditions reflecting the host environment is important to understand native bacterial behavior. In the present study, we examined the effect of glutamate limitation, as its concentration in vivo is much lower than that in the culture medium currently used for B. pertussis experiments. As predicted, the T3SS was induced by glutamate limitation. These results are suggestive of the importance of regulation by nutrient conditions and in the pathogenicity of B. pertussis.

Effects of Agonist and Regulator Binding on the Structure and Conformational Flexibility of the Ligand Binding Domains of Ionotropic Glutamate Receptors

Ionotropic Glutamate receptors (iGluRs) are tetrameric proteins with 4 domains, an amino terminal domain, the ligand binding domain, the transmembrane ion channel and a largely disordered intracellular C terminal region. The ligand binding domain binds glutamate, a major fast excitatory neurotransmitter, which activates the receptor channels. There are three main families of iGluRs, AMPA, NMDA, and kainite which among other things differ in their responses to two endogenous neurosteroids, pregnenalone sulfate (PS) and pregnanalone sulfate (PREGAS). To investigate the role that conformational flexibility of the ligand binding domain plays in response to either glutamate or the neurosteroids (in the presence or absence of glutamate) we have used Multiwavelength Collisional Quenching (MWCQ) which reports on the exposure and charge environment of multiple tyrosines and tryptophans in the protein. While Glutamate has different effects on flexibility depending upon the family of receptor, the use of differently charged quenching molecules shows glutamate induced changes in the charge environment that can be correlated with steroid binding ability and that the presence of glutamate augments the effects of the steroid leading to enhanced global rigidity of the domains. NSF Grant MCB-104995 to EB

Group I metabotropic glutamate receptors: a potential target for regulation of proliferation and differentiation of an immortalized human neural stem cell line.

Human neural stem cells (NSCs) from the developing embryo or the subventricular zone of the adult brain can potentially elicit brain repair after injury or disease, either via endogenous cell proliferation or by cell transplantation. Profound knowledge of the diverse signals affecting these cells is, however, needed to realize their therapeutic potential. Glutamate and group I metabotropic glutamate receptors (mGluRs) affect proliferation and survival of rodent NSCs both during embryonic and post-natal development. To investigate the role of group I mGluRs (mGluR1 and mGluR5) on human NSCs, we differentiated an immortalized, forebrain-derived stem cell line in the presence or absence of glutamate and with addition of either the group I mGluR agonist DHPG or the selective antagonists, MPEP (mGluR5) and LY367385 (mGluR1). Characterization of differentiated cells revealed that both mGluR1 and mGluR5 were present on the cells. Addition of glutamate to the growth medium significantly increased cell proliferation and reduced cell death, resulting in increased cell numbers. In the presence of glutamate, selective activation of group I mGluRs reduced gliogenesis, whereas selective inhibition of group I mGluRs reduced neurogenesis. Our results substantiate the importance of glutamate signalling in the regulation of human NSCs and may as such be applied to promote proliferation and neuronal differentiation.

Neuroprotective effect of N-acyl 5-hydroxytryptamines on glutamate-induced cytotoxicity in HT-22 cells.

Some endocannabinoids have been known to express anti-inflammatory and antioxidant actions independent of cannabinoid receptors. In this respect, we investigated whether N-acyl 5-hydroxytryptamines (5-HTs) might prevent against glutamate-induced oxidative cytotoxicity in HT-22 cells, and attempted to elucidate the mechanism for their cytoprotective action. N-acyl 5-HTs with palmitoyl, stearoyl, arachidonoyl or docosahexaenoyl chain expressed a remarkable protective effect on glutamate-induced cytotoxicity. Additionally, glutamate-induced oxidative stress, represented by the increase of reactive oxygen species level and the reduction of glutathione level, was prevented markedly by N-acyl 5-HTs at submicromolar levels. Further, N-palmitoyl 5-HT, the most cytoprotective, enhanced antioxidant defense by up-regulating the expression of antioxidant enzymes such as heme oxygenase-1, glutamate-cysteine ligase catalytic subunit or NAD(P)H quinine oxidoreductase-1 in the presence or absence of glutamate. Consistent with this, N-palmitoyl 5-HT stimulated nuclear translocation of Nrf2 in early phase (2h), and this effect was remarkably suppressed by inhibitors of PI3K, PDK-1, Akt or p38 MAPK. Additionally, N-palmitoyl 5-HT suppressed glutamate-induced activation of ERK in late phase (12h), but not in early phase (2h), presumably supporting the implication of MEK/ERK pathway in glutamate-induced cytotoxicity. Collectively, it is suggested that N-acyl 5-HTs may attenuate glutamate-induced cytotoxicity via the activation of PI3K/PDK-1/Akt- and p38 MAPK-dependent Nrf2 signaling in early phase as well as the suppression of MEK/ERK pathway in late phase.

Identification and functional expression of a glutamate- and avermectin-gated chloride channel from Caligus rogercresseyi, a southern Hemisphere sea louse affecting farmed fish.

Parasitic sea lice represent a major sanitary threat to marine salmonid aquaculture, an industry accounting for 7% of world fish production. Caligus rogercresseyi is the principal sea louse species infesting farmed salmon and trout in the southern hemisphere. Most effective control of Caligus has been obtained with macrocyclic lactones (MLs) ivermectin and emamectin. These drugs target glutamate-gated chloride channels (GluCl) and act as irreversible non-competitive agonists causing neuronal inhibition, paralysis and death of the parasite. Here we report the cloning of a full-length CrGluClα receptor from Caligus rogercresseyi. Expression in Xenopus oocytes and electrophysiological assays show that CrGluClα is activated by glutamate and mediates chloride currents blocked by the ligand-gated anion channel inhibitor picrotoxin. Both ivermectin and emamectin activate CrGluClα in the absence of glutamate. The effects are irreversible and occur with an EC50 value of around 200 nM, being cooperative (nH = 2) for ivermectin but not for emamectin. Using the three-dimensional structure of a GluClα from Caenorabditis elegans, the only available for any eukaryotic ligand-gated anion channel, we have constructed a homology model for CrGluClα. Docking and molecular dynamics calculations reveal the way in which ivermectin and emamectin interact with CrGluClα. Both drugs intercalate between transmembrane domains M1 and M3 of neighbouring subunits of a pentameric structure. The structure displays three H-bonds involved in this interaction, but despite similarity in structure only of two these are conserved from the C. elegans crystal binding site. Our data strongly suggest that CrGluClα is an important target for avermectins used in the treatment of sea louse infestation in farmed salmonids and open the way for ascertaining a possible mechanism of increasing resistance to MLs in aquaculture industry. Molecular modeling could help in the design of new, more efficient drugs whilst functional expression of the receptor allows a first stage of testing of their efficacy.

How do Glutamate Transporters Function as Transporters and Ion Channels?

Plasma-membrane glutamate transporters move the main excitatory neurotransmitter in the mammalian brain, glutamate, across the membrane against its concentration gradient. This uphill transport is achieved by coupling transmembrane glutamate movement to the cotransport of three Na+ ions down their own concentration gradient. In addition, one potassium ion is countertransported.

Abstract 78: Fractionated whole brain irradiation modulates Homer1a expression in a brain region specific manner.

Abstract About 500,000 new cancer patients will develop brain metastases in 2012. The primary treatment modality for ∼250,000 of these patients is partial or whole brain irradiation. While effective for controlling metastatic brain tumors, there are significant late effects (> 6 months) following radiotherapy which include a progressive, irreversible cognitive impairment. Although the exact mechanism(s) behind this radiation-induced brain injury are unknown, preclinical studies suggest that radiation alters neuronal plasticity. Indeed irradiating the rodent brain decreases long term potentiation, alters N-methyl-D-aspartate (NMDA) and gamma-aminobutyric acid (GABA) receptor expression and ablates neurogenesis. In this study, we used microarray analysis to show that Homer1a, a transcriptionally-regulated early response gene, was transiently upregulated in the hippocampus and downregulated in the cortex at 48 hours after fractionated (total dose of 40 Gy delivered as 2 Gy/fraction, 2X/week) whole brain irradiation (fWBI) in young adult male Fischer 344 X Brown Norway rats. Two months after fWBI Homer1a gene expression was downregulated in both the hippocampus and cortex. Homer1a binds the consensus sequence on the C-terminus of the group I metabotropic glutamate receptors (mGluR); thereby, disrupting the cross-linking of constitutively expressed forms of Homer1 and activating mGluR in the absence of glutamate. Radiation did not alter phosphorylation of mGluR1, total GluR2 or phosphorylation of GluR2/3 in the hippocampus or cortex at 2 months after fWBI. However, the radiation-induced changes in Homer1a expression appeared to produce a significant downregulation in mGluR1 in the hippocampus, and a significant upregulation in the cortex. Memory/learning paradigms suggest that trafficking of 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid (AMPA) receptors containing mGluR1 from intracellular compartments to the plasma membrane is an “early” event during neural plasticity; thus decreased mGluR1 may result in alterations in hippocampal neural plasticity following fWBI. These data suggest a novel mechanism for the development of radiation-induced cognitive impairment. (Supported by CA122318) Citation Format: Elizabeth Moore, Mitra Kooshki, Lance Miller, Mike Robbins. Fractionated whole brain irradiation modulates Homer1a expression in a brain region specific manner. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 78. doi:10.1158/1538-7445.AM2013-78

The Protective Signaling of Metabotropic Glutamate Receptor 1 Is Mediated by Sustained, β-Arrestin-1-dependent ERK Phosphorylation

Metabotropic glutamate receptor 1 (mGlu1) is a G protein-coupled receptor that enhances the hydrolysis of membrane phosphoinositides. In addition to its role in synaptic transmission and plasticity, mGlu1 has been shown to be involved in neuroprotection and neurodegeneration. In this capacity, we have reported previously that in neuronal cells, mGlu1a exhibits the properties of a dependence receptor, inducing apoptosis in the absence of glutamate, while promoting neuronal survival in its presence (Pshenichkin, S., Dolińska, M., Klauzińska, M., Luchenko, V., Grajkowska, E., and Wroblewski, J. T. (2008) Neuropharmacology 55, 500-508). Here, using CHO cells expressing mGlu1a receptors, we show that the protective effect of glutamate does not rely on the classical mGlu1 signal transduction. Instead, mGlu1a protective signaling is mediated by a novel, G protein-independent, pathway which involves the activation of the MAPK pathway and a sustained phosphorylation of ERK, which is distinct from the G protein-mediated transient ERK phosphorylation. Moreover, the sustained phosphorylation of ERK and protective signaling through mGlu1a receptors require expression of beta-arrestin-1, suggesting a possible role for receptor internalization in this process. Our data reveal the existence of a novel, noncanonical signaling pathway associated with mGlu1a receptors, which mediates glutamate-induced protective signaling.

A Conserved Aspartate Determines Pore Properties of Anion Channels Associated with Excitatory Amino Acid Transporter 4 (EAAT4)

Excitatory amino acid transporter (EAAT) glutamate transporters function not only as secondary active glutamate transporters but also as anion channels. Recently, a conserved aspartic acid (Asp(112)) within the intracellular loop near to the end of transmembrane domain 2 was proposed as a major determinant of substrate-dependent gating of the anion channel associated with the glial glutamate transporter EAAT1. We studied the corresponding mutation (D117A) in another EAAT isoform, EAAT4, using heterologous expression in mammalian cells, whole cell patch clamp, and noise analysis. In EAAT4, D117A modifies unitary conductances, relative anion permeabilities, as well as gating of associated anion channels. EAAT4 anion channel gating is characterized by two voltage-dependent gating processes with inverse voltage dependence. In wild type EAAT4, external l-glutamate modifies the voltage dependence as well as the minimum open probabilities of both gates, resulting in concentration-dependent changes of the number of open channels. Not only transport substrates but also anions affect wild type EAAT4 channel gating. External anions increase the open probability and slow down relaxation constants of one gating process that is activated by depolarization. D117A abolishes the anion and glutamate dependence of EAAT4 anion currents and shifts the voltage dependence of EAAT4 anion channel activation by more than 200 mV to more positive potentials. D117A is the first reported mutation that changes the unitary conductance of an EAAT anion channel. The finding that mutating a pore-forming residue modifies gating illustrates the close linkage between pore conformation and voltage- and substrate-dependent gating in EAAT4 anion channels.

Glutamine synthetase down-regulation reduces astrocyte protection against glutamate excitotoxicity to neurons

Although the role of astrocyte glutamate transporters in glutamate clearance is well illustrated, the role of glutamine synthetase (GS) that influences this process remains to be elucidated. We examined whether GS affected the uptake of glutamate in astrocytes in vitro. The glutamate uptake was assessed by measuring the concentration of glutamate and glutamine in culture medium in the presence or absence of glutamate. We demonstrated that inhibition of GS in astrocytes by MSO significantly impaired glutamate uptake and glutamine release. Conversely, induction of GS expression in astrocytes by gene transfer significantly enhanced the glutamate uptake and glutamine release. When an inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) was applied to the cultures, it significantly reduced GS expression and inhibited glutamate-induced GS activation resulting in increased excitotoxicity to neurons. These results suggest that GS in astrocytes may represent a novel target for neuroprotection against neuronal dysfunction and death that occur in many neurological disorders.