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Review
. 2011 Oct;226(10):2484-93.
doi: 10.1002/jcp.22609.

Role of excitatory amino acid transporter-2 (EAAT2) and glutamate in neurodegeneration: opportunities for developing novel therapeutics

Keetae Kim  1 , Seok-Geun Lee, Timothy P Kegelman, Zhao-Zhong Su, Swadesh K Das, Rupesh Dash, Santanu Dasgupta, Paola M Barral, Michael Hedvat, Paul Diaz, John C Reed, John L Stebbins, Maurizio Pellecchia, Devanand Sarkar, Paul B Fisher
Affiliations
Review

Role of excitatory amino acid transporter-2 (EAAT2) and glutamate in neurodegeneration: opportunities for developing novel therapeutics

Keetae Kim et al. J Cell Physiol. 2011 Oct.

Abstract

Glutamate is an essential excitatory neurotransmitter regulating brain functions. Excitatory amino acid transporter (EAAT)-2 is one of the major glutamate transporters expressed predominantly in astroglial cells and is responsible for 90% of total glutamate uptake. Glutamate transporters tightly regulate glutamate concentration in the synaptic cleft. Dysfunction of EAAT2 and accumulation of excessive extracellular glutamate has been implicated in the development of several neurodegenerative diseases including Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis. Analysis of the 2.5 kb human EAAT2 promoter showed that NF-κB is an important regulator of EAAT2 expression in astrocytes. Screening of approximately 1,040 FDA-approved compounds and nutritionals led to the discovery that many β-lactam antibiotics are transcriptional activators of EAAT2 resulting in increased EAAT2 protein levels. Treatment of animals with ceftriaxone (CEF), a β-lactam antibiotic, led to an increase of EAAT2 expression and glutamate transport activity in the brain. CEF has neuroprotective effects in both in vitro and in vivo models based on its ability to inhibit neuronal cell death by preventing glutamate excitotoxicity. CEF increases EAAT2 transcription in primary human fetal astrocytes through the NF-κB signaling pathway. The NF-κB binding site at -272 position was critical in CEF-mediated EAAT2 protein induction. These studies emphasize the importance of transcriptional regulation in controlling glutamate levels in the brain. They also emphasize the potential utility of the EAAT2 promoter for developing both low and high throughput screening assays to identify novel small molecule regulators of glutamate transport with potential to ameliorate pathological changes occurring during and causing neurodegeneration.

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Figures

Figure 1
Figure 1
Schematic representation of pathways and inhibitors affecting EAAT2-Prom activity. EGF-R, EGF receptor; TNFR, TNF-α receptor; TRADD, TNF receptor-1-associated death domain protein; TRAF2, TNF receptor-associated factor 2; NIK, NF-κB-induced kinase; IκK, I-κB kinase; IκB, inhibitor of NF-κB; ERK, extracellular signal-regulated kinase.
Figure 2
Figure 2
Activation of NF-κB by CEF. A, PHFA were treated with 10 μM for 4 days. Nuclear extracts (NE) and cytoplasmic extracts (CE) were prepared and immunoblotted with the indicated antibodies. B, PHFA were infected with Ad.vec or Ad.IκBα-m32. The infected cells were transfected with 3κB-Luc, containing three tandem NF-κB binding sites. C, PHFA were transfected with the EAAT2Pro-954 and pcDNA or p65 expression vector together with pSV-β-galactosidase plasmid as an internal control. One day after transfection, cells were treated with 10 μM ceftriaxone for 2 days. D, PHFA were transfected with the EAAT2Pro-954 and control siRNA or p65 siRNA together with a pSV-β-galactosidase plasmid as an internal control. P65 siRNA treatment abolished ceftriaxone-mediated induction of EAAT2 promoter activity. (Data from Lee et al., J Biol Chem, 2008)
Figure 3
Figure 3
Schematic representation of screening paradigm to identify regulators of EAAT2 expression affecting glutamate transport in the brain. Using the EAAT2 promoter (EAAT2-Prom) linked to a luciferase reporter gene (EAAT2-Luc) we have established Telomerase-immortalized primary human fetal astrocyte (IM-PHFA) clones, expressing EAAT2-Luc (IM-PHFA-EAAT2-Luc). These clones can be used to screen various small molecule libraries, including Combi-Chem, ASINEX and Natural Product Libraries, to identify novel agonist and antagonists of the EAAT2-Prom. Once putative regulators of EAAT2 have been identified they can be tested for activity in in vitro neuronal/astrocyte co-culture systems and animal models of neurodegeneration. Positive compounds can be modified chemically to produce potentially active/safe compounds that pass the blood brain barrier and can be used for clinical trials to define active agents capable of preventing or treating various neurodegenerative conditions.
Figure 4
Figure 4
In vitro and in vivo neuroprotection by CEF. A, Oxygen glucose deprivation (OGD) preconditioning or ceftriaxone pre-treatment (1 μM) was protective compared to no treatment. B, CEF treatment of spinal cord cultures prevented threo-hydroxyaspartate (THA)-induced loss of motor neuron but not in GLT1-null mouse tissue. C, CEF treatment delayed loss of muscle strength in G93A SOD1 ALS mice. (Data from Rothstein et al., Nature, 2005)
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