Plasma membrane expression of the neuronal glutamate transporter EAAC1 is regulated by glial factors: evidence for different regulatory pathways associated with neuronal maturation.
At the glutamatergic synapse the neurotransmitter is removed from the synaptic cleft by high affinity amino acid transporters located on neurons (EAAC1) and astrocytes (GLAST and GLT1), and a coordinated action of these cells is necessary in order to regulate glutamate extracellular concentration. We show here that treatment of neuronal cultures with glial soluble factors (GCM) is associated with a redistribution of EAAC1 and GLAST to the cell membrane and we analysed the effect of membrane cholesterol depletion on this regulation. In enriched neuronal culture (90% neurons and 10% astrocytes), GCM treatment for 10 days increases EAAC1 and GLAST cell surface expression with no change in total expression. In opposite, GLT1 surface expression is not modified by GCM but total expression is increased. When cholesterol is acutely depleted from the membrane by 10 mM methyl-beta-cyclodextrin (beta5-MCD, 30 min), glutamate transport activity and cell surface expressions of EAAC1 and GLAST are decreased in the enriched neuronal culture treated by GCM. In pure neuronal culture addition of GCM also increases EAAC1 cell membrane expression but surprisingly acute treatment with beta5-MCD decreases glutamate uptake activity but not EAAC1 cell membrane expression. By immunocytochemistry a modification in the distribution of EAAC1 within neurons was undetectable whatever the treatment but we show that EAAC1 was no more co localized with Thy-1 in the enriched neuronal culture treated by GCM suggesting that GCM have stimulated polarity formation in neurons, an index of maturation. In conclusion we suggest that different regulatory mechanisms are involved after GCM treatment, glutamate transporter trafficking to and from the plasma membrane in enriched neuronal culture and modulation of EAAC1 intrinsic activity and/or association with regulatory proteins at the cell membrane in the pure neuronal culture. These different regulatory pathways of EAAC1 are associated with different neuronal maturation stages.
Lortet
S
S
IC2N-IBDML, Centre National de la Recherche Scientifique, UMR 6216, Parc Scientifique et Technologique de Luminy, Marseille Cedex 09, France. lortet@ibdml.univ-mrs.fr
Canolle
B
B
Masmejean
F
F
Nieoullon
A
A
D000818:Animals; D001253:Astrocytes / Q000502:physiology; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism / Q000502:physiology; D050580:Excitatory Amino Acid Transporter 3 / Q000096:biosynthesis*; D018698:Glutamic Acid / Q000378:metabolism; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics; D009457:Neuroglia / Q000502:physiology*; D009474:Neurons / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000502:physiology*; D051269:Sodium-Glucose Transporter 1 / Q000096:biosynthesis / Q000235:genetics; D047392:beta-Cyclodextrins / Q000494:pharmacology
D050580:Excitatory Amino Acid Transporter 3; D008565:Membrane Proteins; C493398:Slc1a1 protein, rat; C495676:Slc5a1 protein, rat; D051269:Sodium-Glucose Transporter 1; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D018698:Glutamic Acid; D002784:Cholesterol