Mechanism of Mn(II)-mediated dysregulation of glutamine-glutamate cycle: focus on glutamate turnover.
Manganese (Mn) has been implicated in the impairment of the glutamate-glutamine cycling (GGC) by deregulation of Glu and glutamine (Gln) turnover in astrocytes. Here, we have examined possible mechanisms involved in the Mn(II)-mediated disruption of Glu turnover, including those related to protein degradation, such as the proteasomal and lysosomal machinery. Our study revealed that lysosome but not proteasomal inhibition is responsible for down-regulation of the Glu transporter after Mn(II) treatment. Because protein kinase C (PKC) activation leads to the down-regulation of Glu carriers, and Mn(II) increases PKC activity, we hypothesized that the PKC signaling contributes to the Mn(II)-mediated disruption of Glu turnover. Our results show that PKC activation causes a decrease in Glu uptake and that inhibition of PKC reverses Mn(II)-dependent down-regulation of Glu influx as well as glutamate transporter 1 (GLT1) and glutamate-aspartate transporter (GLAST) protein level. Co-immunoprecipitation studies show association of GLT1 with the PKCδ and PKCα isoforms and Mn(II)-induced specific increase in PKCδ-GLT1 interaction. In addition, astrocytes transfected with shRNA against PKCδ show decreased sensitivity to Mn(II) compared with those transfected with control shRNA or shRNA targeted against PKCα. Taken together, these findings demonstrate that PKCδ signaling is involved in the Mn(II)-induced deregulation of Glu turnover in astrocytes.
Department of Pediatrics Vanderbilt University Medical Center, Nashville, TN 37232, USA. michael.aschner@vanderbilt.edu
Marta
M
Sidoryk-Wegrzynowicz
Eunsook
E
Lee
Michael
M
Aschner
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism; D015153:Blotting, Western; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D002712:Chlorides / Q000633:toxicity*; D003600:Cytosol / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects; D018698:Glutamic Acid / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism*; D047468:Immunoprecipitation; D007202:Indicators and Reagents; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D017895:Manganese Compounds; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011493:Protein Kinase C / Q000502:physiology; D051571:Protein Kinase C-alpha / Q000378:metabolism; D051745:Protein Kinase C-delta / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects; D014178:Translocation, Genetic / Q000187:drug effects; D054875:Ubiquitination / Q000187:drug effects
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D002712:Chlorides; D007202:Indicators and Reagents; D017895:Manganese Compounds; D034741:RNA, Small Interfering; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D005973:Glutamine; D018698:Glutamic Acid; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D020169:Caspases; D046988:Proteasome Endopeptidase Complex; C025340:manganese chloride