Activation of NMDA receptor partly involved in beta-bungarotoxin-induced neurotoxicity in cultured primary neurons.
In this study, we demonstrated that a snake presynaptic toxin, beta-bungarotoxin (beta-BuTX), was capable of binding to NMDA receptors of the cultured primary neurons (cerebellar granule neurons, CGNs). We labeled beta-BuTX with fluorescent FITC (FITC-beta-BuTX) and showed that the binding of FITC-beta-BuTX was inhibited by unlabeled beta-BuTX and MK801 (an NMDA receptor antagonist). Meanwhile, the binding of [3H]-MK801 was also reduced by unlabeled MK801 and beta-BuTX. In addition, beta-BuTX produced a very potent neurotoxic effect on mature CGNs with the EC(50) of 3ng/ml (equivalent to 144pM), but was less effective in immature CGNs. We explored the signaling pathway of neuronal death and found that it was apparently due to the excessive production of reactive oxygen species (ROS) induced by beta-BuTX. MK801 and antioxidants (Vitamin C, N-acetylcysteine (NAC), melatonin, epigallocatechin gallate (EGCG), superoxide dismutase (SOD) and catalase) attenuated not only ROS production but also beta-BuTX-neurotoxicity. The downstream signaling of ROS was identified as the activation of caspase-3. Caspase inhibitor (z-DEVD-fmk) and antioxidants depressed both caspase-3 activation and neurotoxicity. Based on these findings and our previous reports, we conclude that the binding and activation of NMDA receptors by beta-BuTX was crucial step to produce the potent neurotoxic effect. The binding of NMDA receptors resulted in excessive Ca(2+) influx, followed by ROS production and activation of caspase-3. This snake toxin is considered not only to be a useful tool for exploring the death-signaling pathway of neurotoxicity, but also provides a model for searching neuroprotective agents.
Tseng
Wen-Pei
WP
Institute of Pharmacology, College of Medicine, National Taiwan University, No. 1, Section 1, Jen-Ai Road, Taipei 10043, Taiwan.
Lin-Shiau
Shoei-Yn
SY
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D002038:Bungarotoxins / Q000633:toxicity*; D020169:Caspases / Q000502:physiology; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002470:Cell Survival; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology; D016291:Dizocilpine Maleate / Q000378:metabolism; D018691:Excitatory Amino Acid Antagonists / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D016501:Neurites / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000187:drug effects* / Q000648:ultrastructure; D009498:Neurotoxins / Q000633:toxicity*; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D016194:Receptors, N-Methyl-D-Aspartate / Q000819:agonists* / Q000037:antagonists & inhibitors; D015398:Signal Transduction / Q000187:drug effects; D013778:Tetrazolium Salts; D013844:Thiazoles
D000975:Antioxidants; D002038:Bungarotoxins; D018691:Excitatory Amino Acid Antagonists; D009498:Neurotoxins; D017382:Reactive Oxygen Species; D016194:Receptors, N-Methyl-D-Aspartate; D013778:Tetrazolium Salts; D013844:Thiazoles; D016291:Dizocilpine Maleate; D020169:Caspases; C022616:thiazolyl blue