API

Event: 1488

Key Event Title

?

Glutamate dyshomeostasis

Short name

?

Glutamate dyshomeostasis

Biological Context

?

Level of Biological Organization
Cellular

Cell term

?

Cell term
neural cell


Organ term

?

Organ term
brain


Key Event Components

?

Process Object Action

Key Event Overview


AOPs Including This Key Event

?


Stressors

?


Taxonomic Applicability

?


Life Stages

?


Sex Applicability

?


Key Event Description

?


Glutamate (Glu) is the major excitatory neurotransmitter in the mammalian central nervous system (CNS), where it plays major roles in multiple aspects, such as development, learning, memory and response to injury (Featherstone, 2010). However, it is well recognized that Glu at high concentrations at the synaptic cleft acts as a toxin, inducing neuronal injury and death (Meldrum, 2000; Ozawa et al., 1998) secondary to activation of glumatergic N-methyl D-aspartate (NMDA) receptors and Ca2+ influx. Glu dyshomeostasis is a consequence of perturbation of  astrocyte/neuron interactions and the transport of this amino acid, as will be discussed below.

Astrocytes are critically involved in neuronal function and survival, as they produce neurotrophic factors, such as brain-derived neurotrophic factor (BDNF) and glia-derived neurotrophic factor (GDNF), as well as express two main glutamate transporters responsible for the removal of excessive Glu from the synaptic clefts (Chai et al., 2013; Sheldon et al., 2007). Glutamate is the major excitatory neurotransmitter in the CNS, playing a major role in memory and cognitive function (Platt, 1997), and Glu transporters as such prevent the overstimulation of post-synaptic glutamate receptors that lead to excitotoxic neuronal injury (Sattler et al., 2001; Dobble, 1999). Among the five subtypes of Glu transporters identified, glutamate aspartate transporter (GLAST) and Glu transporter-1 (GLT-1) [excitatory amino acid transporter (EAAT) 1 and 2 in humans, respectively], are predominantly expressed in astrocytes. They are responsible for the uptake of excess glutamate from the extracellular space (Furuta et al., 1997; Lehre et al., 1995; Tanaka, 2000), supported by the fact that knockdown of either GLT-1 or GLAST in mice increases extracellular glutamate levels, leading to excitotoxicity related neurodegeneration and progressive paralysis (Bristol and Rothstein, 1996). In the adult brain, EAAT2 accounts for >90% of extracellular glutamate clearance (Danbolt, 2001; Kim et al., 2011; Rothstein et al., 1995), and genetic deletion of both alleles of GLT-1 in mice leads to the development of lethal seizures (Rothstein et al., 1996). On the other hand, EAAT1-3 play a major role during human brain development, in particular in corticogenesis, where they are expressed in proliferative zones and in radial glia, and alterations of Glu transporters contributes to disorganized cortex seen in migration disorders (Furuta et al., 2005;Regan et al., 2007). Indeed, disruption of glutamate signaling is thought to be part of the etiology underlying some neurodevelopmental disorders such as autism and schizophrenia (Chiocchetti et al., 2014; Schwartz et al., 2012). Genes involved in glutamatergic pathways, affecting receptor signalling, metabolism and transport, were enriched in genetic variants associated with autism spectrum disorders (Chiocchetti et al, 2014).

Extracellular Glu released by neurons is taken up by astrocytes, which is converted into glutamine (Gln) by glutamine synthetase (GS), a thiol-containing enzyme (cf MIE, Binding to SH-/seleno containing proteins). Intercellular compartmentation of Gln and Glu, the so-called Gln/Glu-GABA cycle (GGC), is critical for optimal CNS function.13C NMR studies have demonstrated that the ratio of Gln/Glu is extremely high and increases with brain activity (Shen et al., 1999). Thus the GGC gives rise to the amino acid neurotransmitters Glu and GABA via dynamic astrocyte neuron interactions. Glu released at synaptic terminals is taken up by surrounding astrocytes via GLT-1 and GLAST (Rothstein et al., 1994; 1996). A small proportion of the astrocytic formed Gln via a reaction mediated by GS is transported into the extracellular space by Gln carriers, with a predominant role for System N/A transporter (SNAT3), which belongs to the bidirectional transporter System N (Chaudhry et al., 2002).

In addition to System N, release of Gln from astrocytes is mediated by other transport systems, including Systems L (LAT2) and ASC (ASCT2). Extracellular Gln is taken up into GABAergic and Glu-ergic neurons by the unidirectional System A transporters SNAT1 (Melone et al., 2004) and SNAT2 (Grewal et al., 2009). Once in neurons, Gln is converted to Glu by the mitochondrial enzyme phosphate-activated glutaminase (Kvamme et al., 2001). Additionally, Glu is packaged into synaptic vesicles by the vesicular VGLUT transporter (Bellocchio et al., 1998), released into the extracellular space and taken up by astrocytes where it is converted back to Gln by GS, thus completing the GGC (Fig. 1).

Figure 1: Schematic representation of Glu and Gln transport systems related to the GGC. From Sidorik-Wegrzynowicz and Aschner, 2013)


How It Is Measured or Detected

?


 L-Glu transporter activities can be quantified by direct or indirect methods:

・For direct quantification, L-Glu transporter activities are determined by the amount of 3H-labeled ligand (L-Glu or D-aspartate) taken up by the cells (Primary mixed astrocyte and neuron cultures [Perez-Dominguez et al., 2014]; primary astrocyte cultures [Matos et al., 2008; Li et al., 2006; Hazell et al., 2003]; Xenopus laevis oocyte overexpressing the L-Glu transporter subtype of interest [Sogaard et al., 2013; Trotti et al., 2001]; transfected HeLa cells [Zhang and Qu, 2012]) or tissues (ex. Hippocampal tissue [Selkirk et al., 2005])

・For indirect quantification, L-Glu transporter activities are determined by the L-Glu residue in the medium or buffer after incubation with cells expressing the different L-Glu transporters (Brison et al., 2014; Xin et al., 2019; Jin et al., 2015; Gu et al., 2014; Abe et alt.2000], .).

・The transport activity of the different L-Glu transporter subtypes should be determined in the presence of the appropriate inhibitors as shown in the table 1. Ex: The glutamate uptake activity via EAAT1 can be determined in the presence of dihydrokainic acid (DHK), a specific inhibitor for GLT-1, as described in Mutkus et al. (2005). Expression level of L-Glu transporter subtypes should be confirmed using Western blotting or immunocytochemistry.  It is interesting to note that pure astrocyte culture express only GLAST (EAAT1) (Danbolt et al., 2016); whereas  In mixed astrocyte and neuron cultures, GLAST (EAAT1) and GLT-1(EAAT2) are expressed (Danbolt et al., 2016). The expression of GLT1 (EAAT2) is suggested to be induced by soluble factors (Gegelashvili et al., 1997, 2000; Plachez et al., 2000; Martinez-Lozada et al., 2016).  

For measuring glutamate release, load 3H glutamate for several hours and then look at release over time, as descibed in (Arizza et al., 1994)

The L-Glu concentrations in medium or in incubation buffer can be quantified by commercially-available kits quantifying the final products of the redox reaction in which L-Glu is a substrate.   

The kits using colorimetric final products (OD=450 nm)

■Glutamate Assay Kit from Abcam (ab83389)

■Glutamate Colorimetric Assay Kit from BioVision (K629)

■Glutamate Assay Kit from Merck (MAK004)  

The kit using the bioluminescent metabolite

■Glutamate-Glo™ Assay from Promega (J7021)

Table 1 is a summary based on the reviews of Murphy-Royal et al., 2017 and Pajarillo et al., 2019, with some modification.  Concerning the physiological functions of each EAAT subtype, see the review by Danbolt (Danbolt, 2001).

human

rodent

distribution

Non-specific inhibitors

Specific inhibitors

EAAT1

GLAST

Astrocytes, cerebellum,

High expression in astrocytes at developmental stage

DL-threo-b-benzyloxyaspartate

(TBOA) and its variants (e.g. PMBTBOA

and TFB-TBOA) (Bridges et al.,

1999; Lebrun et al., 1997; Shimamoto et al., 1998; Shimamoto,

2008)

UCPH101 (Erichsen et al., 2010)

EAAT2

GLT-1

Astrocytes

(>90% adult CNS L-Glu uptake)

Neuronal terminals (hippocampus, cortex, still controversial)

WAY213613 (Dunlop et al., 2005)

DHK (Arriza et al.,

1994; Bridges et al., 1999

EAAT3

EAAC1

Neurons

Especially high in hippocampal neurons.

Also function as  Cys transporter (Watts et al., 2014)

 

EAAT4

EAAT4

Purkinje cells in the cerebellum

 

EAAT5

EAAT5

Retina,

Very weak in the CNS

 

 


Domain of Applicability

?


The involvement of glutamate in learning and memory processes is well conserved in all taxa, from invertebrates (ex. Drosophila) to vertebrates (Fagnou and Tuchek, 1995).


References

?


Abe K, Abe Y, Saito H. Evaluation of L-glutamate clearance capacity of cultured rat cortical astrocytes. Biol Pharm Bull. 2000 Feb;23(2):204-7.

Arriza JL, Fairman WA, Wadiche JI, Murdoch GH, Kavanaugh MP, Amara SG (1994) Functional comparisons of three glutamate transporter subtypes cloned from human motor cortex. J Neurosci 14(9):5559–5569.

Bellocchio EE, Hu H, Pohorille A, Chan J, Pickel VM, Edwards RH. The localization of the brain-specific inorganic phosphate transporter suggests a specific presynaptic role in glutamatergic transmission. J Neurosci. 1998;18:8648–59.

Bridges RJ, Kavanaugh MP, Chamberlin AR. A pharmacological review of competitive inhibitors and substrates of high-affinity, sodium-dependent glutamate transport in the central nervous system. 1999; Curr. Pharm. Des. 5, 363e379.

Brison E, Jacomy H, Desforges M, Talbot PJ. Novel Treatment with Neuroprotective and Antiviral Properties against a Neuroinvasive Human Respiratory Virus. J Virol. 2014 Feb; 88(3): 1548–1563.

Bristol LA, Rothstein JD. Glutamate transporter gene expression in amyotrophic lateral sclerosis motor cortex. Annals of neurology. 1996;39:676–679.

Chai L, Guo H, Li H, Wang S, Wang YL, Shi F, Hu LM, Liu Y, Adah D. Scutellarin and caffeic acid ester fraction, active components of Dengzhanxixin injection, upregulate neurotrophins synthesis and release in hypoxia/reoxygenation rat astrocytes. Journal of ethnopharmacology. 2013;150:100–107.

Chaudhry FA, Reimer RJ, Edwards RH. The glutamine commute: take the N line and transfer to the A. J Cell Biol. 2002;157:349–55.

Chiocchetti, A. G., H. S. Bour and C. M. Freitag (2014). "Glutamatergic candidate genes in autism spectrum disorder: an overview." J Neural Transm (Vienna) 121(9): 1081-1106.

Danbolt NC. Glutamate uptake. Progress in neurobiology. 2001;65:1–105.

Danbolt NC, Furness DN, Zhou Y. Neuronal vs glial glutamate uptake: Resolving the conundrum. Neurochem Int. 2016 Sep;98:29-45.

Doble A. The role of excitotoxicity in neurodegenerative disease: implications for therapy. Pharmacology & therapeutics. 1999;81:163–221.

Dunlop J, McIlvain HB, Carrick TA, Jow B, Lu Q, Kowal D, Lin S, Greenfield A, Grosanu C, Fan K, Petroski R, Williams J, Foster A, Butera J. Characterization of novel aryl-ether, biaryl, and fluorene aspartic acid and diaminopropionic acid analogs as potent inhibitors of the high-affinity glutamate transporter EAAT2. Mol Pharmacol. 2005 Oct;68(4):974-82.

Erichsen MN, Huynh TH, Abrahamsen B, Bastlund JF, Bundgaard C, Monrad O, Bekker-Jensen A, Nielsen CW, Frydenvang K, Jensen AA, Bunch L. Structure-activity relationship study of first selective inhibitor of excitatory amino acid transporter subtype 1: 2-Amino-4-(4-methoxyphenyl)-7-(naphthalen-1-yl)-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (UCPH-101). J Med Chem. 2010 Oct 14;53(19):7180-91.

Fagnou DD, Tuchek JM (1995) The biochemistry of learning and memory. Mol Cell Biochem 149-150:279-86

Featherstone, D. E. (2010). "Intercellular glutamate signaling in the nervous system and beyond." ACS Chem Neurosci 1(1): 4-12.

Furuta A, Rothstein JD, Martin LJ. Glutamate transporter protein subtypes are expressed differentially during rat CNS development. The Journal of neuroscience : the official journal of the Society for Neuroscience. 1997;17:8363–8375.

Furuta, A., S. Takashima, H. Yokoo, J. D. Rothstein, K. Wada and T. Iwaki (2005). "Expression of glutamate transporter subtypes during normal human corticogenesis and type II lissencephaly." Brain Res Dev Brain Res 155(2): 155-164.

Gegelashvili G, Danbolt NC, Schousboe A, Neuronal soluble factors differentially regulate the expression of the GLT1 and GLAST glutamate transporters in cultured astroglia. J. Neurochem. 1997; 69, 2612e2615.

Gegelashvili G, Dehnes Y, Danbolt NC, Schousboe A, The high-affinity glutamate transporters GLT1, GLAST and EAAT4 are regulated via different signalling mechanisms. Neurochem. Int. 2000; 37, 163e170.

Grewal S, Defamie N, Zhang X, et al. SNAT2 amino acid transporter is regulated by amino acids of the SLC6 gamma-aminobutyric acid transporter subfamily in neocortical neurons and may play no role in delivering glutamine for glutamatergic transmission. J Biol Chem. 2009;284:11224–36.

Gu L, Xu H, Wang F, Xu G, Sinha D, Wang J, Xu JY, Tian H, Gao F, Li W, Lu L, Zhang J, Xu GT, Erythropoietin Exerts a Neuroprotective Function Against Glutamate Neurotoxicity in Experimental Diabetic Retina. Invest. Ophthalmol. Vis. Sci., Dec 2014; 55: 8208 - 8222.

Hazell AS, Pannunzio P, Rama Rao KV, Pow DV, Rambaldi A. Thiamine deficiency results in downregulation of the GLAST glutamate transporter in cultured astrocytes. Glia. 2003 Aug;43(2):175-84.

Jin LW, Horiuchi M, Wulff H, Liu XB, Cortopassi GA, Erickson JD, Maezawa I., Rett Syndrome Microglia: A Mechanism for Mitochondrial Dysfunction and Neurotoxicity. J. Neurosci., Feb 2015; 35: 2516 – 2529

Kim K, Lee SG, Kegelman TP, Su ZZ, Das SK, Dash R, Dasgupta S, Barral PM, Hedvat M, Diaz P, Reed JC, Stebbins JL, Pellecchia M, Sarkar D, Fisher PB. Role of excitatory amino acid transporter-2 (EAAT2) and glutamate in neurodegeneration: opportunities for developing novel therapeutics. Journal of cellular physiology. 2011;226:2484–2493.

Kvamme E, Torgner IA, Roberg B. Kinetics and localization of brain phosphate activated glutaminase. J Neurosci Res. 2001;66:951–8.

Lebrun B, Sakaitani M, Shimamoto K, Yasudakamatani Y, Nakajima T, New beta-hydroxyaspartate derivatives are competitive blockers for the bovine glutamate/aspartate transporter. 1997; J. Biol. Chem. 272, 20336e20339.

Lehre KP, Levy LM, Ottersen OP, Storm-Mathisen J, Danbolt NC. Differential expression of two glial glutamate transporters in the rat brain: quantitative and immunocytochemical observations. The Journal of neuroscience : the official journal of the Society for Neuroscience. 1995;15:1835–1853.

Li LB, Toan SV, Zelenaia O, Watson DJ, Wolfe JH, Rothstein JD, Robinson MB. Regulation of astrocytic glutamate transporter expression by Akt: evidence for a selective transcriptional effect on the GLT-1/EAAT2 subtype. J Neurochem. 2006 May;97(3):759-71.

Martinez-Lozada Z, Guillem AM, Robinson MB. Transcriptional Regulation of Glutamate Transporters: From Extracellular Signals to Transcription Factors. Adv Pharmacol. 2016;76:103-45.

Meldrum, B. S. (2000). "Glutamate as a neurotransmitter in the brain: review of physiology and pathology." J Nutr 130(4S Suppl): 1007S-1015S.

Melone M, Quagliano F, Barbaresi P, Varoqui H, Erickson JD, Conti F. Localization of the glutamine transporter SNAT1 in rat cerebral cortex and neighboring structures, with a note on its localization in human cortex. Cereb Cortex. 2004;14:562–74.

Matos M, Augusto E, Oliveira CR, Agostinho P. Amyloid-beta peptide decreases glutamate uptake in cultured astrocytes: involvement of oxidative stress and mitogen-activated protein kinase cascades. Neuroscience. 2008 Oct 28;156(4):898-910.

Murphy-Royal C, Dupuis J, Groc L, Oliet SHR. Astroglial glutamate transporters in the brain: Regulating neurotransmitter homeostasis and synaptic transmission. J Neurosci Res. 2017 Nov;95(11):2140-2151.

Mutkus, L., J. L. Aschner, T. Syversen and M. Aschner (2005). "Methylmercury alters the in vitro uptake of glutamate in GLAST- and GLT-1-transfected mutant CHO-K1 cells." Biol Trace Elem Res 107(3): 231-245.

Ozawa, S., H. Kamiya and K. Tsuzuki (1998). "Glutamate receptors in the mammalian central nervous system." Prog Neurobiol 54(5): 581-618.

Pajarillo E, Rizor A, Lee J, Aschner M, Lee E. The role of astrocytic glutamate transporters GLT-1 and GLAST in neurological disorders: Potential targets for neurotherapeutics. Neuropharmacology. 2019 Mar 6:107559.

Pérez-Domínguez M, Hernández-Benítez R, Peña Segura C, Pasantes-Morales H. Thrombin-facilitated efflux of D-[3H]-aspartate from cultured astrocytes and neurons under hyponatremia and chemical ischemia. Neurochem Res. 2014 Jul;39(7):1219-31.

Plachez C, Danbolt NC, Recasens M. Transient expression of the glial glutamate transporters GLAST and GLT in hippocampal neurons in primary culture. 2000; J. Neurosci. Res. 59, 587e593.

Platt SR. The role of glutamate in central nervous system health and disease--a review. Veterinary journal (London, England : 1997) 2007;173:278–286.

Regan MR, Huang YH, Kim YS, Dykes-Hoberg MI, Jin L, Watkins AM, Bergles DE, Rothstein JD. Variations in promoter activity reveal a differential expression and physiology of glutamate transporters by glia in the developing and mature CNS. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2007;27:6607–6619.

Rothstein JD, Martin L, Levey AI, et al. Localization of neuronal and glial glutamate transporters. Neuron. 1994;13:713–25.

Rothstein JD, Van Kammen M, Levey AI, Martin LJ, Kuncl RW. Selective loss of glial glutamate transporter GLT-1 in amyotrophic lateral sclerosis. Annals of neurology. 1995;38:73–84.

Rothstein JD, Dykes-Hoberg M, Pardo CA, Bristol LA, Jin L, Kuncl RW, Kanai Y, Hediger MA, Wang Y, Schielke JP, Welty DF. Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate. Neuron. 1996;16:675–686.

Sattler R, Tymianski M. Molecular mechanisms of glutamate receptor-mediated excitotoxic neuronal cell death. Molecular neurobiology. 2001;24:107–129.

Schwartz, C. E. and G. Neri (2012). "Autism and intellectual disability: two sides of the same coin." Am J Med Genet C Semin Med Genet 160C(2): 89-90.

Selkirk JV, Nottebaum LM, Vana AM, Verge GM, Mackay KB, Stiefel TH, Naeve GS, Pomeroy JE, Petroski RE, Moyer J, Dunlop J, Foster AC. Role of the GLT-1 subtype of glutamate transporter in glutamate homeostasis: the GLT-1-preferring inhibitor WAY-855 produces marginal neurotoxicity in the rat hippocampus. Eur J Neurosci. 2005 Jun;21(12):3217-28.

Sheldon AL, Robinson MB. The role of glutamate transporters in neurodegenerative diseases and potential opportunities for intervention. Neurochemistry international. 2007;51:333–355.

Shen J, Petersen KF, Behar KL, et al. Determination of the rate of the glutamate/glutamine cycle in the human brain by in vivo 13C NMR. Proc Natl Acad Sci U S A. 1999;96:8235–40.

Shimamoto K, Lebrun B, Yasudakamatani Y., Sakaitani M, Shigeri Y, Yumoto N, Nakajima T. DL-threo-beta-benzyloxyaspartate, a potent blocker of excitatory amino acid transporters. 1998; Mol. Pharmacol. 53, 195e201.

Shimamoto K. Glutamate transporter blockers for elucidation of the function of excitatory neurotransmission systems, 2008; Chem Rec. 8, 182e199.

Sidoryk-Wegrzynowicz M1Aschner M. Manganese toxicity in the central nervous system: the glutamine/glutamate-γ-aminobutyric acid cycle. J Intern Med. 2013 May;273(5):466-77. doi: 10.1111/joim.12040.

Sogaard R, Borre L, Braunstein TH, Madsen KL, MacAulay N. Functional modulation of the glutamate transporter variant GLT1b by the PDZ domain protein PICK1. J Biol Chem. 2013 Jul 12;288(28):20195-207.

Tanaka K. Functions of glutamate transporters in the brain. Neuroscience research. 2000;37:15–19.

Trotti D, Peng JB, Dunlop J, Hediger MA. Inhibition of the glutamate transporter EAAC1 expressed in Xenopus oocytes by phorbol esters. Brain Res. 2001 Sep 28;914(1-2):196-203.

Watts SD, Torres-Salazar D, Divito CB, Amara SG, Cysteine transport

through excitatory amino acid transporter 3 (EAAT3). PLoS One 2014; 9 e109245.

Xin W, Oligodendrocytes Support Neuronal Glutamatergic Transmission via Expression of Glutamine Synthetase. Cell Rep., May 2019; 31116973.

Zhang X, Qu S. The Accessibility in the External Part of the TM5 of the Glutamate Transporter EAAT1 Is Conformationally Sensitive during the Transport Cycle. PLoS One. 2012; 7(1): e30961.