Glutamate enhances the surface distribution and release of Munc18 in cerebral cortical neurons.
OBJECTIVE
Munc18 is considered as an intracellular protein that plays an important role in exocytosis of neurotransmitters. Previous studies have demonstrated the presence of autoantibodies against Munc18 in a subgroup of Rasmussen's encephalitis patients. However, the machinery of Munc18 autoimmunity is still elusive. The present study was aimed to investigate Munc18 release from primary cultured neurons, Munc18 distribution on the outer plasma membrane of neurons, and the neurotoxicity of Munc18 antibody.


METHODS
The cerebral cortical neurons from embryonic day 17 Sprague-Dawley rats were prepared and cultured in neurobasal medium. The proteins in culture medium were precipitated with 10 % trichloroacetic acid, and analyzed by immunoblotting. The proteins on neuronal surface were biotinylated with EZ-Link-sulfo-NHS-LC-Biotin, and collected with avidin-conjugated agarose beads followed by immunoblotting analysis. For cell surface immunofluorescent staining, the living neurons were labeled with anti-Munc18 antibody at 4 degrees C. Neuronal injury was assessed by lactate dehydrogenase(LDH) release.


RESULTS
Munc18 was detected in culture medium by immunoblotting analysis. After treatment with 50 micromol/L glutamate for 1 h, Munc18 content in medium was increased. Meanwhile, beta-actin and syntaxin1 were not detected in culture medium, and LDH release was not significantly increased. Moreover, glutamate enhanced Munc18 distribution on outer plasma membrane. Living neuron staining also demonstrated the localization of Munc18 on neuronal surface after glutamate treatment, especially at contacting regions between neurons. Glutamate-induced increase of surface Munc18 distribution was suppressed by NMDA receptor antagonist MK801, but not by AMPA receptor antagonist NBQX. Moreover, compared with c-Fos antibody, Munc18 antibody could induce neuronal injury, when culture medium contained the components of serum.


CONCLUSIONS
A portion of Munc18 can be released from neurons or distributed on neuronal surface, which can be enhanced by glutamate treatment via activation of NMDA receptors. Besides, Munc18 antibody-induced neuronal injury depends on the serum components.
Wan
Ping
P
State Key Laboratory of Medical Neurobiology, Shanghai Medical College, Fudan University, Shanghai, China.
Zhang
Yan-Ping
YP
Yan
Jie
J
Xu
Yu-Xia
YX
Wang
Hong-Quan
HQ
Yang
Ru
R
Zhu
Cui-Qing
CQ
D000818:Animals; D019921:Biotinylation / Q000379:methods; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology*; D016291:Dizocilpine Maleate / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004622:Embryo, Mammalian; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D018698:Glutamic Acid / Q000494:pharmacology*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D051938:Munc18 Proteins / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects*; D021381:Protein Transport / Q000187:drug effects; D011810:Quinoxalines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors
D018691:Excitatory Amino Acid Antagonists; D051938:Munc18 Proteins; D011810:Quinoxalines; C062865:2,3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline; D018698:Glutamic Acid; D016291:Dizocilpine Maleate; D007770:L-Lactate Dehydrogenase