Translational Control of Glial Glutamate Transporter EAAT2 Expression

Abstract

Glutamate is the major excitatory neurotransmitter in the central nervous system. Its activity is carefully modulated in the synaptic cleft by glutamate transporters. The glial glutamate transporter EAAT2 is the main mediator of glutamate clearance. Reduced EAAT2 function could lead to accumulation of extracellular glutamate, resulting in a form of cell death known as excitotoxicity. In amyotrophic lateral sclerosis and Alzheimer disease, EAAT2 protein levels are significantly decreased in affected areas. EAAT2 mRNA levels, however, remain constant, indicating that alterations in EAAT2 expression are due to disturbances at the post-transcriptional level. In the present study, we found that some EAAT2 transcripts contained 5-untranslated regions (5-UTRs) greater than 300 nucleotides. The mRNAs that bear long 5-UTRs are often regulated at the translational level. We tested this possibility initially in a primary astrocyte line that constantly expressed an EAAT2 transcript containing the 565-nt 5-UTR and found that translation of this transcript was regulated by many extracellular factors, including corticosterone and retinol. Moreover, many disease-associated insults affected the efficiency of translation of this transcript. Importantly, this translational regulation of EAAT2 occurred in vivo (i.e. both in primary cortical neurons-astrocytes mixed cultures and in mice). These results indicate that expression of EAAT2 protein is highly regulated at the translational level and also suggest that translational regulation may play an important role in the differential EAAT2 protein expression under normal and disease conditions. Excitatory amino acid transporters (EAATs)2 in the central nervous system maintain extracellular glutamate concentrations below excitotoxic levels and contribute to the clearance of glutamate released during neurotransmission. Five sodium-dependent glutamate transporter subtypes have been identified and characterized: GLAST-1 (EAAT1) (1, 2), GLT-1 (EAAT2) (3, 4), EAAC1 (EAAT3) (5, 6), EAAT4 (7), and EAAT5 (8). EAAT2 is expressed mainly in glial cells throughout the brain and is responsible for up to 90% of all glutamate transport in adult tissue (9, 10).A malfunction in the glutamate transport system can lead to accumulation of excessive glutamate in the synapse, which is harmful to neurons and could result in neurodegeneration (11,12). Over the past 15 years, it has been demonstrated that glutamate-mediated toxicity may play an important role in the pathogenesis of amyotrophic lateral sclerosis (ALS) and Alzheimer disease (AD). Rothstein and colleagues (13) reported a significant increase in levels of glutamate in the cerebrospinal fluid of ALS patients and defective glutamate transport in the affected areas of ALS (14). Subsequent studies demonstrated a dramatic and selective loss of EAAT2 protein in the affected areas of ALS (15). Defective glutamate transport and loss of EAAT2 protein were also reported in the affected areas of AD patients (16,17). This phenomenon also occurs in several rod...

EAAT2 regulation and splicing: relevance to psychiatric and neurological disorders

“…Recent work has demonstrated that there are variants with long 5 0 -UTRs that are regulated at the translational level. 38 In this study, the transcripts with the longer 5 0 -UTRs were regulated by corticosterone, retinol and several Upstream open reading frames (ORFs), which may be translated or inhibit translation of the main protein product, are present in some of these 5 0 -UTRs. 34,36 Variants that encode upstream ORFs (such as the molecules annotated as HBGTIIB and HBGTIIC) appear to be less abundant than those that do not encode upstream ORFs.…”

“…GLT1 expression is also regulated by glucocorticoids including corticosterone and the synthetic molecule dexamethasone. 38,84,85 Corticosterone was found to regulate translation of GLT1 isoforms with longer 5 0 -UTRs, but not to affect transcription or protein stability. 38 This molecule led to a dosedependent increase in GLT1 protein levels in cultured astrocytes, further supporting the notion that isoforms of GLT1 are differentially regulated in response to stress.…”

“…In addition, treating cultured astrocytes with a low dose of b-amyloid, which makes up the plaques found in AD brains, led to enhanced translation of EAAT2 mRNA. 38 Another group found that low concentrations of b-amyloid increased EAAT2 expression through a pathway involving the transcription factor NF-kB. 95 Thus, b-amyloid appears to influence EAAT2 expression at both the transcriptional and translational levels.…”

Clarin-1, Encoded by the Usher Syndrome III Causative Gene, Forms a Membranous Microdomain

“…Cell Surface Biotinylation-Proteins on the plasma membrane were labeled by biotinylation as described previously (19). Briefly, cells were incubated with 1 mg/ml EZ-Link Sulfo-NHS-SS-Biotin in PBS buffer (137 mM NaCl, 2.7 mM KCl, 10.1 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.4, 100 M CaCl 2 , and 1 mM MgCl 2 ) for 30 min at 4°C.…”

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“…A small 11-amino acid fragment of the fulllength peptide, Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35), is a convenient alternative in Alzheimer's disease investigations as the peptide mimics several toxicological and oxidative stress properties of the native full-length peptide 4,18,19 . Recently, our group demonstrated that treatment of mixed cell cultures with 10 µM Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) for 12 h and 24 h reduces SAT1 protein level as demonstrated by immunocytochemical and quantitative immunoreactivity analysis 20 .…”