Very low levels of methylmercury induce cell death of cultured rat cerebellar neurons via calpain activation.
Methylmercury, an environmental neurotoxicant, induces the apoptotic death of cerebellar granule cells in vitro at a low concentration. To further understand the mechanism of cell death, we used a rat cerebellar granule cell culture system to investigate whether the calpain/cyclin-dependent kinase 5 (cdk5)/p35 cascade, an important cascade for neuronal apoptosis, is involved in the methylmercury-induced death. A noteworthy finding was that the cerebellar granular cell death was increased at a very low concentration of methylmercury, 30 nM, which is lower than that previously reported. The high sensitivity to methylmercury indicates that this culture system is useful for studying methylmercury toxicity at very low concentrations. Using this system, we here found that the methylmercury-induced death was inhibited by the calpain inhibitor II. Furthermore, it was shown that, in methylmercury-exposed cells, alpha-fodrin and tau, calpain substrates, were cleaved to the fragments that disappeared by treatment with the calpain inhibitor II. We next assayed and showed that the intracellular Ca(2+) concentration in cerebellar granule cells increased after methylmercury exposure in a time- and dose-dependent manner, significantly even at 30 nM. These results indicated that a very low concentration of methylmercury causes the intracellular Ca(2+) concentration to increase, activates calpain in the cells, and then induces cell death. We further found that the p35 protein was also processed to p25 that forms the cdk5-p25 complex, a hyperactive kinase for tau. However, an immunoblot using the anti-phosphorylated tau antibody showed that there was no increase of phosphorylated tau in methylmercury-exposed cells. These results suggested that methylmercury-induced cell death via calpain activation should not involve the stimulation of tau phosphorylation activity.
Department of Public Health, School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan. sakauem@pharm.kitasato-u.ac.jp
Motoharu
M
Sakaue
Maiko
M
Okazaki
Shuntaro
S
Hara
D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D002470:Cell Survival / Q000502:physiology; D002525:Cerebellar Cortex / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D015151:Immunoblotting; D008767:Methylmercury Compounds / Q000633:toxicity*; D008840:Microfilament Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D009842:Oligopeptides / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013050:Spectrometry, Fluorescence; D016875:tau Proteins / Q000378:metabolism; D013049:Spectrin
D002118:Calcium; D002154:Calpain; D002352:Carrier Proteins; D015853:Cysteine Proteinase Inhibitors; D008767:Methylmercury Compounds; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D009842:Oligopeptides; D016875:tau Proteins; D013049:Spectrin; C089495:neuronal Cdk5 activator (p25-p35); C069861:calpain inhibitor 2; C004925:methylmercuric chloride