title: L-glutamate released from activated microglia downregulates astrocytic L-glutamate transporter expression in neuroinflammation: the 'collusion' hypothesis for increased extracellular L-glutamate concentration in neuroinflammation.
abstract: BACKGROUND
  In the central nervous system, astrocytic L-glutamate (L-Glu) transporters maintain extracellular L-Glu below neurotoxic levels, but their function is impaired with neuroinflammation. Microglia become activated with inflammation; however, the correlation between activated microglia and the impairment of L-Glu transporters is unknown.
  
  
  METHODS
  We used a mixed culture composed of astrocytes, microglia, and neurons. To quantify L-Glu transporter function, we measured the extracellular L-Glu that remained 30 min after an application of L-Glu to the medium (the starting concentration was 100 μM). We determined the optimal conditions of lipopolysaccharide (LPS) treatment to establish an inflammation model without cell death. We examined the predominant subtypes of L-Glu transporters and the changes in the expression levels of these transporters in this inflammation model. We then investigated the role of activated microglia in the changes in L-Glu transporter expression and the underlying mechanisms in this inflammation model.
  
  
  RESULTS
  Because LPS (10 ng/mL, 72 h) caused a significant increase in the levels of L-Glu remaining but did not affect cell viability, we adopted this condition for our inflammation model without cell death. GLAST was the predominant L-Glu transporter subtype, and its expression decreased in this inflammation model. As a result of their release of L-Glu, activated microglia were shown to be essential for the significant decrease in L-Glu uptake. The serial application of L-Glu caused a significant decrease in L-Glu uptake and GLAST expression in the astrocyte culture. The hemichannel inhibitor carbenoxolone (CBX) inhibited L-Glu release from activated microglia and ameliorated the decrease in GLAST expression in the inflammation model. In addition, the elevation of the astrocytic intracellular L-Glu itself caused the downregulation of GLAST.
  
  
  CONCLUSIONS
  Our findings suggest that activated microglia trigger the elevation of extracellular L-Glu through their own release of L-Glu, and astrocyte L-Glu transporters are downregulated as a result of the elevation of astrocytic intracellular L-Glu levels, causing a further increase of extracellular L-Glu. Our data suggest the new hypothesis that activated microglia collude with astrocytes to cause the elevation of extracellular L-Glu in the early stages of neuroinflammation.
authors.affiliation: Laboratory of Neuropharmacology, Division of Pharmacology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Tokyo 158-8501, Japan.
authors.forename: Junpei
authors.initials: J
authors.lastname: Takaki
authors.forename: Koki
authors.initials: K
authors.lastname: Fujimori
authors.forename: Marie
authors.initials: M
authors.lastname: Miura
authors.forename: Takeshi
authors.initials: T
authors.lastname: Suzuki
authors.forename: Yuko
authors.initials: Y
authors.lastname: Sekino
authors.forename: Kaoru
authors.initials: K
authors.lastname: Sato
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001224:Aspartic Acid / Q000494:pharmacology; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D001578:Benzopyrans / Q000494:pharmacology; D002229:Carbenoxolone / Q000494:pharmacology; D016923:Cell Death; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D018920:Coculture Techniques; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology*; D027341:Excitatory Amino Acid Transporter 1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D045604:Extracellular Fluid / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D018698:Glutamic Acid / Q000378:metabolism* / Q000494:pharmacology; D007770:L-Lactate Dehydrogenase; D008070:Lipopolysaccharides / Q000494:pharmacology; D017628:Microglia / Q000187:drug effects / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D018709:Statistics, Nonparametric; D013778:Tetrazolium Salts; D013844:Thiazoles
chemical_list: C553295:2-amino-4-(4-methoxyphenyl)-7-(naphthalen-1-yl)-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile; D001578:Benzopyrans; D027341:Excitatory Amino Acid Transporter 1; D008070:Lipopolysaccharides; C493392:Slc1a3 protein, rat; D013778:Tetrazolium Salts; D013844:Thiazoles; C120673:benzyloxyaspartate; D001224:Aspartic Acid; D018698:Glutamic Acid; D007770:L-Lactate Dehydrogenase; C022616:thiazolyl blue; D002229:Carbenoxolone