Glutamate uptake and release by astrocytes are enhanced by Clostridium botulinum C3 protein.
Inhibition of Rho activity by Clostridium botulinum C3 transferase (C3bot) versatily changes functional properties of neural cells. Using cultivated mouse astrocytes, we show here that C3bot increases both uptake and secretion of glutamate. The enhanced glutamate uptake is initiated by an NFkappaB-dependent up-regulation of the glial glutamate transporter 1 that is efficaciously sorted to the plasma membrane. The increase in cytosolic glutamate concentration promotes vesicular glutamate storage in astrocytes treated with C3bot. Parallel to the increased storage, C3-induced impairment of Rho-dependent pathways strongly enhances Ca(2+)-dependent secretion of glutamate. This is accompanied by higher levels of the SNARE protein synaptobrevin. Synaptobrevin inactivation by botulinum neurotoxin D almost completely inhibits Ca(2+)-dependent glutamate secretion triggered by C3bot, indicating that the enhanced release of glutamate mainly originates from exocytosis. In addition, C3bot increases the exocytosis/endocytosis turnover, as analyzed by the stimulated accumulation of the fluorescent dye AM1-43. The release of glutamine, the main metabolite of glutamate, is only moderately affected by C3bot. In conclusion, inhibition of Rho-dependent pathways shifts astrocytes to a secretory active stage in which they may modulate neuronal excitability.
Höltje
Markus
M
Centrum für Anatomie, Institut für Integrative Neuroanatomie, Charité-Universitätsmedizin Berlin, Philippstrasse 12, 10115 Berlin, Germany. markus.holtje@charite.de
Hofmann
Fred
F
Lux
Romy
R
Veh
Rüdiger W
RW
Just
Ingo
I
Ahnert-Hilger
Gudrun
G
D036002:ADP Ribose Transferases / Q000494:pharmacology*; D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism*; D001905:Botulinum Toxins / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004705:Endocytosis / Q000187:drug effects; D027342:Excitatory Amino Acid Transporter 2 / Q000378:metabolism; D005089:Exocytosis / Q000187:drug effects; D018698:Glutamic Acid / Q000378:metabolism*; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D011726:Pyridinium Compounds / Q000494:pharmacology; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D050683:R-SNARE Proteins / Q000378:metabolism; D022142:Secretory Vesicles / Q000378:metabolism*; D015854:Up-Regulation / Q000187:drug effects; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
C507005:AM1-43 compound; D027342:Excitatory Amino Acid Transporter 2; D016328:NF-kappa B; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D050683:R-SNARE Proteins; C096325:botulinum toxin type D; D018698:Glutamic Acid; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D001905:Botulinum Toxins; D020741:rho GTP-Binding Proteins; D002118:Calcium