FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2164485346> ?p ?o ?g. }

• W2164485346 endingPage "1737" @default.
• W2164485346 startingPage "1727" @default.
• W2164485346 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. 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) 2The abbreviations used are: EAAT, excitatory amino acid transporter; ALS, amyotrophic lateral sclerosis; AD, Alzheimer disease; SOD1, superoxide dismutase; RLM, RNA ligase-mediated; RACE, rapid amplification of cDNA ends; nt, nucleotide(s); UTR, untranslated region; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; RT, reverse transcription; pAb, polyacrylamide; PBS, phosphate-buffered saline; GFAP, glial fibrillary acid protein; EGF, epidermal growth factor; CMV, cytomegalovirus. 2The abbreviations used are: EAAT, excitatory amino acid transporter; ALS, amyotrophic lateral sclerosis; AD, Alzheimer disease; SOD1, superoxide dismutase; RLM, RNA ligase-mediated; RACE, rapid amplification of cDNA ends; nt, nucleotide(s); UTR, untranslated region; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; RT, reverse transcription; pAb, polyacrylamide; PBS, phosphate-buffered saline; GFAP, glial fibrillary acid protein; EGF, epidermal growth factor; CMV, cytomegalovirus. 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) (1Shashidharan P. Plaitakis A. Biochim. Biophys. Acta. 1993; 1216: 161-164Crossref PubMed Scopus (60) Google Scholar, 2Storck T. Schulte S. Hofmann K. Stoffel W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10955-10959Crossref PubMed Scopus (1088) Google Scholar), GLT-1 (EAAT2) (3Shashidharan P. Wittenberg I. Plaitakis A. Biochim. Biophys. Acta. 1994; 1191: 393-396Crossref PubMed Scopus (75) Google Scholar, 4Pines G. Danbolt N.C. Bjoras M. Zhang Y. Bendahan A. Eide L. Koepsell H. Storm-Mathisen J. Seeberg E. Kanner B.I. Nature. 1992; 360: 464-467Crossref PubMed Scopus (1127) Google Scholar), EAAC1 (EAAT3) (5Shashidharan P. Huntley G.W. Meyer T. Morrison J.H. Plaitakis A. Brain Res. 1994; 662: 245-250Crossref PubMed Scopus (55) Google Scholar, 6Kanai Y. Hediger M.A. Nature. 1992; 360: 467-471Crossref PubMed Scopus (1187) Google Scholar), EAAT4 (7Fairman W.A. Vandenberg R.J. Arriza J.L. Kavanaugh M.P. Amara S.G. Nature. 1995; 375: 599-603Crossref PubMed Scopus (1005) Google Scholar), and EAAT5 (8Arriza J.L. Eliasof S. Kavanaugh M.P. Amara S.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4155-4160Crossref PubMed Scopus (789) Google Scholar). EAAT2 is expressed mainly in glial cells throughout the brain and is responsible for up to 90% of all glutamate transport in adult tissue (9Tanaka K. Watase K. Manabe T. Yamada K. Watanabe M. Takahashi K. Iwama H. Nishikawa T. Ichihara N. Kikuchi T. Okuyama S. Kawashima N. Hori S. Takimoto M. Wada K. Science. 1997; 276: 1699-1702Crossref PubMed Scopus (1448) Google Scholar, 10Danbolt N.C. Storm-Mathisen J. Kanner B.I. Neuroscience. 1992; 51: 295-310Crossref PubMed Scopus (367) Google Scholar). 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 (11Ikonomidou C. Qin Qin Y. Labruyere J. Olney J.W. J Neuropathol. Exp. Neurol. 1996; 55: 211-224Crossref PubMed Scopus (105) Google Scholar, 12Rothstein J.D. Jin L. Dykes-Hoberg M. Kuncl R.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6591-6595Crossref PubMed Scopus (584) Google Scholar). 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 (13Rothstein J.D. Tsai G. Kuncl R.W. Clawson L. Cornblath D.R. Drachman D.B. Pestronk A. Stauch B.L. Coyle J.T. Ann. Neurol. 1990; 28: 18-25Crossref PubMed Scopus (559) Google Scholar) 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 (14Rothstein J.D. Martin L.J. Kuncl R.W. N. Engl. J. Med. 1992; 326: 1464-1468Crossref PubMed Scopus (1034) Google Scholar). Subsequent studies demonstrated a dramatic and selective loss of EAAT2 protein in the affected areas of ALS (15Rothstein J.D. Van Kammen M. Levey A.I. Martin L.J. Kuncl R.W. Ann. Neurol. 1995; 38: 73-84Crossref PubMed Scopus (1234) Google Scholar). Defective glutamate transport and loss of EAAT2 protein were also reported in the affected areas of AD patients (16Li S. Mallory M. Alford M. Tanaka S. Masliah E. J. Neuropathol. Exp. Neurol. 1997; 56: 901-911Crossref PubMed Scopus (289) Google Scholar, 17Masliah E. Alford M. DeTeresa R. Mallory M. Hansen L. Ann. Neurol. 1996; 40: 759-766Crossref PubMed Scopus (364) Google Scholar). This phenomenon also occurs in several rodent models of the diseases, including transgenic mice or rats expressing ALS-linked mutant superoxide dismutase (SOD1) (18Dunlop J. Beal McIlvain H. She Y. Howland D.S. J. Neurosci. 2003; 23: 1688-1696Crossref PubMed Google Scholar, 19Howland D.S. Liu J. She Y. Goad B. Maragakis N.J. Kim B. Erickson J. Kulik J. DeVito L. Psaltis G. DeGennaro L.J. Cleveland D.W. Rothstein J.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1604-1609Crossref PubMed Scopus (686) Google Scholar, 20Bendotti C. Tortarolo M. Suchak S.K. Calvaresi N. Carvelli L. Bastone A. Rizzi M. Rattray M. Mennini T. J. Neurochem. 2001; 79: 737-746Crossref PubMed Scopus (161) Google Scholar, 21Bruijn L.I. Becher M.W. Lee M.K. Anderson K.L. Jenkins N.A. Copeland N.G. Sisodia S.S. Rothstein J.D. Borchelt D.R. Price D.L. Cleveland D.W. Neuron. 1997; 18: 327-338Abstract Full Text Full Text PDF PubMed Scopus (1092) Google Scholar) and transgenic mice expressing AD-linked mutant amyloid precursor protein (22Masliah E. Alford M. Mallory M. Rockenstein E. Moechars D. Van Leuven F. Exp. Neurol. 2000; 163: 381-387Crossref PubMed Scopus (125) Google Scholar). These rodent models demonstrated that down-regulation of EAAT2 protein occurs during the final stage of the pathology. We (23Guo H. Lai L. Butchbach M.E. Stockinger M.P. Shan X. Bishop G.A. Lin C.L. Hum. Mol. Genet. 2003; 12: 2519-2532Crossref PubMed Scopus (211) Google Scholar) modestly overexpressed EAAT2 in the SOD1G93A mouse to compensate for the loss of EAAT2 and demonstrated a delay in disease onset accompanied by a prolonged survival of motor neurons. In addition, Rothstein et al. (24Rothstein J.D. Patel S. Regan M.R. Haenggeli C. Huang Y.H. Bergles D.E. Jin L. Hoberg M.Dykes Vidensky S. Chung D.S. Toan S.V. Bruijn L.I. Su Z.Z. Gupta P. Fisher P.B. Nature. 2005; 433: 73-77Crossref PubMed Scopus (1222) Google Scholar) recently reported that increased expression of EAAT2 by ceftriaxone ameliorated symptoms and prolonged the survival of SOD1G93A mice. These studies imply that impairment of glutamate transport is a late contributing mechanism and not a major culprit for neuron degeneration. The mechanism underlying the loss of EAAT2 is still unclear. It appears that this occurs due to disturbance at the post-transcriptional level, because EAAT2 mRNA is not decreased (15Rothstein J.D. Van Kammen M. Levey A.I. Martin L.J. Kuncl R.W. Ann. Neurol. 1995; 38: 73-84Crossref PubMed Scopus (1234) Google Scholar, 16Li S. Mallory M. Alford M. Tanaka S. Masliah E. J. Neuropathol. Exp. Neurol. 1997; 56: 901-911Crossref PubMed Scopus (289) Google Scholar, 25Bristol L.A. Rothstein J.D. Ann. Neurol. 1996; 39: 676-679Crossref PubMed Scopus (203) Google Scholar). In this study, we demonstrate that translation of EAAT2 mRNA is regulated by many factors. It is possible that reduced EAAT2 protein levels in ALS and AD may be partially due to lack of stimulating factors or the presence of inhibiting factors that repress translation of EAAT2 transcripts. Reagents—Corticosterone, retinol, T3 (triodo-l-thyronine), biotin, vitamin B12, and cycloheximide were obtained from Sigma. B-27, B-27 without antioxidants, and N-2 were obtained from Invitrogen. Identification of Initiation Sites—The 5′ end of the EAAT2 transcript was analyzed using the RNA ligase-mediated rapid amplification of cDNA ends (5′-RLM-RACE) method according to the protocol provided with the FirstChoice™ RLMRACE kit (Ambion, Austin, TX). Briefly, total RNA (10 μg) was dephosphorylated with 2 μl of calf intestinal phosphatase. The cap structure was subsequently removed with 2 μl of tobacco acid pyrophosphatase to produce a phosphorylated RNA at the 5′ end, to which the 5′-RACE adapter (5′-GCUGAUGGCGAUGAAUGAACACUGCGUUUGCUGGCUUUGAUGAAA-3′) was ligated. The resulting RNA was reverse-transcribed using EAAT2 gene-specific primers (primer A1, 5′-GCTTGGGTTCCTCTGAGCCAAGATGACTGT-3′, corresponding to position +59 downstream of ATG; primer B1, 5′-GGTGGCAGGAGCCCAGGATCTAAG-3′, corresponding to position –771 upstream of ATG) and 200 units of SuperScript II reverse transcriptase. The resulting cDNAs were amplified by PCR using the RACE 5′ outer primer (5′-GCTGATGGCGATGAATGAACACTG-3′) and EAAT2-specific primers A1 and B1. The resulting PCR products were amplified further by nested PCR using the RACE 5′ inner primer (5′-CGCGGATCCGAACACTGCGTTTGCTGGCTTTGATG-3′) and EAAT2-specific primers (A2, 5′-CCACCTGCTTGGGCATATTGTTGGCAC-3′, corresponding to position +17 downstream of ATG; B2, 5′-CTATTGTTTCCCCTGAAGCCCGC-3′, corresponding to position –800 upstream of ATG). The PCR conditions were as follows: 94 °C for 2 min, 85 °C for 2 min, followed by 10 cycles at 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min and then 25 cycles at 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min (increasing 0.5 s for each cycle). The reaction was then extended for 10 min at 72 °C. Nested PCR products were purified from 1% agarose gel and cloned using the PCR-Script™ Amp cloning kit (Stratagene, La Jolla, CA). Individual clones were sequenced for determination of the transcription start sites. Abundance of EAAT2 mRNAs—Normal human frontal cortex mRNA was isolated from postmortem frozen tissues using TRIzol (Invitrogen) followed by Oligotex Suspension kit (Qiagen, Valencia, CA). First-strand cDNA was synthesized with Thermoscript reverse transcriptase (Invitrogen) using primer A1 (described above). As a control EAAT2 RNA, an EAAT2 transcript that contains the 565-nucleotide (nt) 5′-UTR and 395 nt of coding region was generated by in vitro transcription using RiboMAX™ large scale RNA production systems (Promega, Madison, WI). The template for the generation of this control RNA was prepared from pcDNA3/EAAT2 by restriction digestion with ScaI (Invitrogen). The following primer combinations (indicated in Fig. 1C) were used for PCR: primers A1 + C (5′-CCCGGCGTCCGCTTTCTCCCT-3′, corresponding to position –72 upstream of ATG), primers A1 + D (5′-CTGGGCGCATCGCTCTCTCG-3′, –310 upstream of ATG), and primers E (5′-GGTAAGCCCTTTAGCGCCTC-3′, –405 upstream of ATG) + F(5′-AAACCTTGCAATCCCTCCCTGGCCG-3′, –525 upstream of ATG). The MasterTaq kit (Eppendorf, Westbury, NY), which is designed to increase reproducible yields from GC-rich templates, was used, and PCR conditions were as follows: 95 °C for 3 min, 85 °C for 2 min; 95 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min for 30 cycles followed by 10 min of extension at 72 °C. The PCR products were resolved in 2% agarose gel, and the intensity of each band was analyzed by Kodak one-dimensional image analysis software (Eastman Kodak Co.). Cell Cultures—Mouse primary cortical cultures were obtained as described previously (23Guo H. Lai L. Butchbach M.E. Stockinger M.P. Shan X. Bishop G.A. Lin C.L. Hum. Mol. Genet. 2003; 12: 2519-2532Crossref PubMed Scopus (211) Google Scholar). Briefly, cortices were dissected out of the newborn (P0–P1) brains and incubated in activated papain for 30 min at 37 °C, triturated by repeated pipetting with a small bore pipette and plated onto poly-d-lysine (0.1 mg/ml)-coated plastic culture dishes or glass slides. These cultures were maintained in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) containing 25 mm glucose, 1 mm sodium pyruvate, 19.4 μm pyridoxine hydrochloride, 2 mm glutamine, and 1% B-27 supplement (Invitrogen). Rat primary astrocyte cultures were obtained from Dr. Richard W. Burry (Department of Neuroscience, Ohio State University). The cells were maintained in DMEM with 10% fetal bovine serum (FBS) (Invitrogen). Human embryonic kidney cells (HEK293 cells) were cultured in DMEM with 10% FBS. Generation of Plasmid DNA Constructs—To generate pcDNA3/EAAT2 with 76-nt 5′-UTR, DNA containing EAAT2 coding region and 76 bp of 5′-UTR was amplified by PCR using the primer set 5′-CCCGGCGTCCGCTTTCTCCCT-3′ and 5′-GGATCCAGACTCATATCCTTATTTCTCACG-3′ and pcDNA3/EAAT2 as template; this PCR product was inserted into pPCR-ScriptAmpSK(+) (Stratagene, La Jolla, CA) and then subcloned into pcDNA3 with EcoRI and NotI. pcDNA3/EAAT2 with a 310-nt 5′-UTR was generated in the same way as above except that the following PCR primers were used: 5′-CTGGGCGCATCGCTCTCTCG-3′ and 5′-GGATCCAGACTCATATCCTTATTTCTCACG-3′. To generate pcDNA3/EAAT2 with a 1091-nt 5′-UTR, the 5′-UTR sequence from –1117 to –239 was subcloned from TOPO/EAAT2 promoter into pcDNA3/EAAT2 using SspI and SacII. Generation of Stably Transfected Primary Astrocyte Cell Lines—Low passage (four passages) rat primary astrocyte cells were plated onto cell culture dishes and transfected with plasmid DNA (pcDNA3/EAAT2) using Lipofectamine Plus (Invitrogen) according to standard protocol. The medium was replaced with fresh medium containing Geneticin (0.9 mg/ml) 48 h post-transfection to select for EAAT2-expressing cells. Selection medium was replaced every 3 days until colonies formed (18–21 days later). Colonies were examined for EAAT2 expression by RT-PCR using EAAT2-specific primers and also by immunoblotting using a rabbit anti-EAAT2 pAb. RT-PCR—Total RNA from primary astrocytes or primary cortical cultures was isolated with TRIzol (Invitrogen), and first strand cDNA was synthesized with Moloney murine leukemia virus reverse transcriptase (Invitrogen) using an EAAT2-specific primer (5′-ACGCTGGGGAGTTTATTCAAGAAT-3′). β-Actin was used as an internal control (primer, 5′-TGTCAAAGAAAGGGTGTAAAACGCAGC-3′). PCR primers 5′-GGCAACTGGGGATGTACA-3′ and 5′-ACGCTGGGGAGTTTATTCAAGAAT-3′ were used for EAAT2 cDNA, and primers 5′-CGGGACCTGACAGACTACCTCAT-3′ and 5′-TGTCAAAGAAAGGGTGTAAAACGCAGC-3′ were used for actin cDNA. PCR conditions were as follows: 95 °C for 3 min, 85 °C for 2 min; 95 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min for 30 cycles followed by 10 min of extension at 72 °C. Immunoblotting—Immunoblotting was performed as described previously (26Guo H. Lai L. Butchbach M.E. Lin C.L. Mol. Cell Neurosci. 2002; 21: 546-560Crossref PubMed Scopus (38) Google Scholar). Briefly, the harvested samples were sonicated in PBS containing Complete protease inhibitor mixture (Roche Applied Science), assayed for protein concentration, resolved by SDS-PAGE (8% polyacrylamide), and transferred onto polyvinylidene difluoride membranes. The following primary antibodies were used: rabbit anti-EAAT2 pAb (1:4000), rabbit anti-EAAT1 pAb (1:200), rabbit anti-EAAT3 pAb (1:200), rabbit anti-glial fibrillary acid protein (GFAP) pAb (1:1000; Promega, Madison, WI), and goat anti-actin (1:2000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The immunoreactive bands were detected using the SuperSignal West Pico Chemiluminescent Substrate (Pierce) according to the manufacturer's directions. Band intensities were analyzed with Scion Image Release Beta 4.0.2 (Scion Corp.). [3H]Glutamate Uptake Assay—Uptake of radiolabeled glutamate was monitored in cultured cells as described previously (27Butchbach M.E. Tian G. Guo H. Lin C.L. J. Biol. Chem. 2004; 279: 34388-34396Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Cultured cells grown on 6-well plates were washed with uptake sample buffer (320 mm sucrose in 50 mm Tris-HCl, pH 7.4) and then incubated for 10 min at 37 °C with l-[3H]glutamate (0.5 μCi; Amersham Biosciences) in either Na+-containing or Na+-free Kreb's buffer supplemented with 40 μm unlabeled glutamate. The cells were washed with ice-cold PBS and lysed in 1 mm NaOH. The amount of radiolabeled glutamate was measured using a Beckman Coulter LS6500 multipurpose scintillation counter (Beckman Coulter, Fullerton, CA). The amount of l-[3H]glutamate transported into the cells was calculated as previously described (27Butchbach M.E. Tian G. Guo H. Lin C.L. J. Biol. Chem. 2004; 279: 34388-34396Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Na+-dependent l-[3H]glutamate uptake was calculated by subtracting Na+-independent l-[3H]glutamate uptake (in Na+-free Kreb's buffer) from the total l-[3H]glutamate uptake (in Na+-containing Kreb's buffer). Protein concentrations were determined with the Coomassie Plus protein assay (Pierce). Na+-dependent l-[3H]glutamate uptake was expressed as nmol of l-[3H]glutamate/mg of protein/min. Immunofluorescence—Fixation of cultured cells and immunofluorescent staining were accomplished as described previously (27Butchbach M.E. Tian G. Guo H. Lin C.L. J. Biol. Chem. 2004; 279: 34388-34396Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). The following primary antibodies were used in this study: purified rabbit anti-EAAT2 pAb (1:200), purified rabbit anti-EAAT3 pAb (1:200), rabbit anti-GFAP pAb (1:1000; Promega, Madison, WI), and mouse anti-MAP2 monoclonal antibody (1:1000; Neomarker, Fremont, CA). Images were obtained using a Zeiss Axioskop 2 inverted microscope and AxioVision software. Cell Surface Biotinylation—Labeling of proteins on the plasma membrane was accomplished by cell surface biotinylation as described previously (23Guo H. Lai L. Butchbach M.E. Stockinger M.P. Shan X. Bishop G.A. Lin C.L. Hum. Mol. Genet. 2003; 12: 2519-2532Crossref PubMed Scopus (211) Google Scholar). Cells were washed twice with PBS/CaMg (100 μm CaCl2 and 1 mm MgCl2 in PBS, pH 7.4) at room temperature and then incubated with biotinylation buffer (1 mg/ml EZ-Link® Sulfo-NHS-SS-Biotin (Pierce) in PBS/CaMg) for 20 min at 4 °C with constant shaking. Cells were then incubated with quenching buffer (100 mm glycine in PBS/CaMg) for 30 min at 4 °C with constant shaking and scraped in quenching buffer. The samples were sonicated in PBS containing Complete protease inhibitor mixture (Roche Applied Science) and lysed in 1% Triton X-100 (Roche Applied Science) for 1 h at 4 °C. Biotinylated proteins were recovered by incubation with 100 μl of immobilized NeutrAvidin (50% slurry; Pierce) at 4 °C overnight with end-over-end rotation. The avidin beads were recovered by centrifugation (12,000 × g for 5 min at 4 °C) and then washed four times with washing buffer (1% Triton X-100 in PBS containing Complete protease inhibitor mixture). After washing, the beads were resuspended in 1× SDS-PAGE loading dye. Pulse-Chase Experiments—PA-EAAT2 cells were first cultured in 10% FBS for 24 h, washed twice with sterile PBS/CaMg (100 μm CaCl2 and 1 mm MgCl2 in PBS, pH 7.4) at room temperature, and then incubated with sterile biotinylation buffer (1 mg/ml EZ-Link® Sulfo-NHS-SS-Biotin (Pierce) in PBS/CaMg) for 20 min at 37 °C with gentle shaking every 5 min. Cells were then incubated with sterile quenching buffer (100 mm glycine in PBS/CaMg) for 30 min at 37 °C with gentle shaking every 5 min. After biotinylation, cells were cultured in DMEM with corticosterone (0.2 μg/ml) for 8 or 24 h and then harvested for biotin-labeled EAAT2 protein level analysis as described above. In Vitro Translation—Each form of EAAT2 cDNA in pcDNA3 vector was used as a DNA template for TNT quick coupled transcription/translation systems in the presence of T7 RNA polymerase and [35S]methionine according to the manufacturer's protocols (Promega, Madison, WI). Equivalent copy numbers of plasmid DNAs were used in each assay. The reactions were carried out at 30 °C for 90 min. The synthesized protein products were electrophoretically resolved through an 8% SDS-polyacrylamide gel, and the gel was then dried and exposed to autoradiography film for 24 h. Drug Administration—Intrathecal administration of drugs was performed as described previously by Hylden and Wilcox (28Hylden J.L.K. Wilcox G.L. Eur. J. Phamacol. 1980; 67: 313-316Crossref PubMed Scopus (1725) Google Scholar). Normal FVB mice (20–25 g) were used throughout these experiments. Briefly, a disposable 30-gauge one-half-inch needle (BD Biosciences) attached to a 50-μl Hamilton syringe (Hamilton, Reno, NV) was inserted into the intervertebral space between L5 and L6 level of the spinal cord. The accuracy of each injection was indicated by a characteristic reflexive flick of the tail. 1 μg/μl of corticosterone in 10% ethanol was injected in a volume of 5 μl/mouse, and 5 μl of 10% ethanol was injected into the control mice. Statistical Analysis—The quantitative data in this study were expressed as the mean ± S.E. Statistical analysis was performed using the unpaired Student's t test. There Are Multiple Transcriptional Initiation Sites of the EAAT2 Gene—Eukaryotic translation can be subdivided into three sequential phases of initiation, elongation, and termination. Frequently, it is the initiation phase that is targeted in translational regulation (29Gingras A.C. Gygi S.P. Raught B. Polakiewicz R.D. Abraham R.T. Hoekstra M.F. Aebersold R. Sonenberg N. Genes Dev. 1999; 13: 1422-1437Crossref PubMed Scopus (985) Google Scholar). Such regulation is mainly determined by structural properties of the mRNA primarily within the 5′-UTR. Su et al. (30Su Z.Z. Leszczyniecka M. Kang D.C. Sarkar D. Chao W. Volsky D.J. Fisher P.B. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1955-1960Crossref PubMed Scopus (165) Google Scholar) have reported that the major transcript of EAAT2 in primary human fetal astrocytes is initiated from an adenosine residue located 283 nt upstream of the ATG start codon. However, we found in a previous study (31Lin C.L. Bristol L.A. Jin L. Dykes-Hoberg M. Crawford T. Clawson L. Rothstein J.D. Neuron. 1998; 20: 589-602Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar) that some human EAAT2 transcripts contain at least 428 nt of 5′-UTR. In addition, a human EAAT2 cDNA that we obtained from another laboratory contained the 565-nt 5′-UTR. Furthermore, mouse EAAT2 transcripts contain at least 660 nt of 5′-UTR (32Utsunomiya-Tate N. Endou H. Kanai Y. FEBS Lett. 1997; 416: 312-316Crossref PubMed Scopus (112) Google Scholar), and rat EAAT2 transcripts contain at least 621 nt of 5′-UTR (33Rozyczka J. Engele J. Brain Res. Mol. Brain Res. 2005; 133: 157-161Crossref PubMed Scopus (20) Google Scholar). Su et al. (30Su Z.Z. Leszczyniecka M. Kang D.C. Sarkar D. Chao W. Volsky D.J. Fisher P.B. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1955-1960Crossref PubMed Scopus (165) Google Scholar) used primer extension analysis to determine the transcriptional initiation site. Shorter primer extension products can be generated if the primer target sequence is not close to the 5′ terminus of the mRNA. This is due to the tendency of reverse transcriptase to stop or pause in a region of high secondary structure in the template RNA. We used the computer program MFOLD (available on the World Wide Web at bioweb.pasteur.fr/seqanal/interfaces/mfold-simple.html) (34Zuker M. Nucleic Acids Res. 2003; 31: 3406-3415Crossref PubMed Scopus (9968) Google Scholar) to predict secondary structure of EAAT2 5′-UTR. There is a predicted region of strong secondary structure (ΔG = –99.3 kcal/mol) located from position –136 to –340 upstream of ATG. Moreover, we performed many RT-PCR experiments to amplify EAAT2 5′-UTR and learned that it is difficult to pass through the region around 250 nt upstream of ATG when using standard RT conditions. 5′ RLM-RACE is a reliable way to determine the transcriptional initiation site of the mRNA. Only authentic capped 5′ ends of mRNA are detected by RLM-RACE. We first used the computer program Promoter 2.0 (www.cbs.dtu.dk/services/Promoter/) (35Knudsen S. Bioinformatics. 1999; 15: 356-361Crossref PubMed Scopus (264) Google Scholar) to predict the potential transcriptional initiation sites. The result showed that there are three potential initiation sites within 1.2 kb upstream of ATG, each located at around –74, –345, and –1153 upstream of ATG. Primer A, which corresponds to position +59 downstream of ATG, was used to test the putative –74 and –345 initiation sites, whereas primer B, which corresponds to position –771 upstream of ATG, was used to test the putative –1153 initiation site. Tobacco acid pyrophosphatase-treated human frontal cortex mRNA was used as a template, since tobacco acid pyrophosphatase removes the cap structure from full-length mRNA, thereby leaving a free 5′-monophosphate to be ligated to an adapter for amplification. As shown in Fig. 1A, two PCR fragments were obtained when using primer A, and one PCR fragment was obtained when using primer B. No product was obtained when RNA was not treated with tobacco acid pyrophosphatase, indicating that these PCR fragments were derived from full-length capped mRNA. These PCR fragments were cloned and sequenced. They all contained adapter sequence and mapped the 5′ end of the mRNA to 76, 310, and 1091 nt upstream of the ATG start codon (Fig. 1B). These results indicate that there are multiple transcriptional initiation sites of the EAAT2 gene. What are the relative abundances of these three forms of EAAT2 mRNA in vivo? We initially attempted to answer this question by a ribonuclease protection assay but were not able to obtain reliable results, probably due to the strong secondary structure of the 5′-UTR. We then approached this question by using quantitative RT-PCR. mRNA samples from normal human front cortices (n = 3) and the control EAAT2 RNA, generated by in vitro transcription using EAAT2 cDNA as template, were subjected to RT. Primer A, which corresponds to position +59 downstream of ATG, was used for RT, which was performed at 60 °C using Thermoscript so as to easily pass through regions of high secondary structure. Serial dilutions of RT product were then subjected to PCR using primer sets indicated in Fig. 1C. The PCR product amplified by primer A and primer C represents all three forms of EAAT2 mRNA, whereas the PCR product amplified by primer A and primer D represents those mRNAs containing the 310- and 1091-nt 5′ UTRs, and the PCR product amplified by primer E and primer F represents those mRNAs containing a 1091-nt 5′-UTR. PCR products were resolved by agarose gel electrophoresis (Fig. 1D). The intensity of each band was meas" @default.
• W2164485346 created "2016-06-24" @default.
• W2164485346 creator A5016687250 @default.
• W2164485346 creator A5028163347 @default.
• W2164485346 creator A5036284331 @default.
• W2164485346 creator A5052372774 @default.
• W2164485346 creator A5066169695 @default.
• W2164485346 creator A5079377537 @default.
• W2164485346 creator A5084306990 @default.
• W2164485346 date "2007-01-01" @default.
• W2164485346 modified "2023-10-15" @default.
• W2164485346 title "Translational Control of Glial Glutamate Transporter EAAT2 Expression" @default.
• W2164485346 cites W1480533650 @default.
• W2164485346 cites W1574724477 @default.
• W2164485346 cites W1585174469 @default.
• W2164485346 cites W1629378997 @default.
• W2164485346 cites W1722754210 @default.
• W2164485346 cites W1882321051 @default.
• W2164485346 cites W1969400861 @default.
• W2164485346 cites W1969786907 @default.
• W2164485346 cites W1971639674 @default.
• W2164485346 cites W1976387924 @default.
• W2164485346 cites W1977620415 @default.
• W2164485346 cites W1983197885 @default.
• W2164485346 cites W1988718039 @default.
• W2164485346 cites W1988931872 @default.
• W2164485346 cites W1991701465 @default.
• W2164485346 cites W1996589674 @default.
• W2164485346 cites W1998140931 @default.
• W2164485346 cites W1999613366 @default.
• W2164485346 cites W2000724016 @default.
• W2164485346 cites W2002782460 @default.
• W2164485346 cites W2007872436 @default.
• W2164485346 cites W2012851706 @default.
• W2164485346 cites W2018879809 @default.
• W2164485346 cites W2019689561 @default.
• W2164485346 cites W2022367303 @default.
• W2164485346 cites W2032653084 @default.
• W2164485346 cites W2034102455 @default.
• W2164485346 cites W2037337146 @default.
• W2164485346 cites W2038293737 @default.
• W2164485346 cites W2039849406 @default.
• W2164485346 cites W2048032676 @default.
• W2164485346 cites W2050523782 @default.
• W2164485346 cites W2056033864 @default.
• W2164485346 cites W2057745494 @default.
• W2164485346 cites W2062660352 @default.
• W2164485346 cites W2068732348 @default.
• W2164485346 cites W2070243481 @default.
• W2164485346 cites W2075770826 @default.
• W2164485346 cites W2083204774 @default.
• W2164485346 cites W2086367273 @default.
• W2164485346 cites W2086689324 @default.
• W2164485346 cites W2092034907 @default.
• W2164485346 cites W2092358916 @default.
• W2164485346 cites W2093015515 @default.
• W2164485346 cites W2094673798 @default.
• W2164485346 cites W2094996078 @default.
• W2164485346 cites W2101823052 @default.
• W2164485346 cites W2107768418 @default.
• W2164485346 cites W2110089243 @default.
• W2164485346 cites W2113197426 @default.
• W2164485346 cites W2121726351 @default.
• W2164485346 cites W2123321988 @default.
• W2164485346 cites W2126925362 @default.
• W2164485346 cites W2135718342 @default.
• W2164485346 cites W2141157874 @default.
• W2164485346 cites W2149497936 @default.
• W2164485346 cites W2151927746 @default.
• W2164485346 cites W2154549928 @default.
• W2164485346 cites W2160079255 @default.
• W2164485346 cites W2160644313 @default.
• W2164485346 cites W2403978899 @default.
• W2164485346 cites W2429572096 @default.
• W2164485346 doi "https://doi.org/10.1074/jbc.m609822200" @default.
• W2164485346 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17138558" @default.
• W2164485346 hasPublicationYear "2007" @default.
• W2164485346 type Work @default.
• W2164485346 sameAs 2164485346 @default.
• W2164485346 citedByCount "77" @default.
• W2164485346 countsByYear W21644853462012 @default.
• W2164485346 countsByYear W21644853462013 @default.
• W2164485346 countsByYear W21644853462014 @default.
• W2164485346 countsByYear W21644853462015 @default.
• W2164485346 countsByYear W21644853462016 @default.
• W2164485346 countsByYear W21644853462017 @default.
• W2164485346 countsByYear W21644853462018 @default.
• W2164485346 countsByYear W21644853462019 @default.
• W2164485346 countsByYear W21644853462020 @default.
• W2164485346 countsByYear W21644853462021 @default.
• W2164485346 countsByYear W21644853462022 @default.
• W2164485346 crossrefType "journal-article" @default.
• W2164485346 hasAuthorship W2164485346A5016687250 @default.
• W2164485346 hasAuthorship W2164485346A5028163347 @default.
• W2164485346 hasAuthorship W2164485346A5036284331 @default.
• W2164485346 hasAuthorship W2164485346A5052372774 @default.
• W2164485346 hasAuthorship W2164485346A5066169695 @default.
• W2164485346 hasAuthorship W2164485346A5079377537 @default.

• next