Inhibitors of interleukin-1 beta-converting enzyme-family proteases (caspases) prevent apoptosis without affecting decreased cellular ability to reduce 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide in cerebellar granule neurons.
We assessed the possible role of interleukin-1beta-converting enzyme-family proteases (caspases) in apoptosis in cultured rat cerebellar granule neurons. CPP32 (caspase-3)-like protease activity was augmented by low KCl treatment, preceding neuronal cell death. Agents such as brain-derived neurotrophic factor (BDNF), dibutylyl cAMP, NMDA, actinomycin D, S-adenosyl-L-methionine, and spermine prevented apoptosis. For various neuroprotective agents, the degree of apoptosis prevention correlated with the prevention of the activation of CPP32-like protease. Furthermore, Z-Asp-2, 6-dichlorobenzoyloxy-methylketone (Z-Asp-CH2-DCB), Boc-Asp-fluoromethylketone (Boc-Asp-FMK), and Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK), which are inhibitors of caspases, also prevented apoptosis. In contrast to many other neuroprotective agents, these inhibitors of caspases showed little effect on the decrease of cellular 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) reduction activity after low KCl treatment. The neurons rescued by these inhibitors of caspases during low KCl treatment were in a hypoenergic state in their ATP levels and vulnerable to subsequent treatment with medium containing high KCl or glutamate which induce an influx of Ca2+, but which are less toxic to normal neurons. These results suggest that caspase(s) are involved in the apoptosis of cerebellar granule neurons and that several agents protect neurons from death by blocking the activation of the protease(s). Although several caspase inhibitors examined in this study protect neurons from apoptosis, rescued neurons are vulnerable to subsequent stimuli that induce necrotic cell death.
Harada
J
J
Neuroscience Research Laboratories, Sankyo, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan. jyunha@shina.sankyo.co.jp
Sugimoto
M
M
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism / Q000502:physiology; D020170:Caspase 1 / Q000378:metabolism; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D002531:Cerebellum / Q000187:drug effects / Q000201:enzymology*; D004396:Coloring Agents / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000201:enzymology*; D004734:Energy Metabolism / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D009474:Neurons / Q000187:drug effects / Q000201:enzymology; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013778:Tetrazolium Salts / Q000378:metabolism*; D013844:Thiazoles / Q000378:metabolism*
D061945:Caspase Inhibitors; D004396:Coloring Agents; D015853:Cysteine Proteinase Inhibitors; D013778:Tetrazolium Salts; D013844:Thiazoles; D011189:Potassium Chloride; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D020170:Caspase 1; C022616:thiazolyl blue; D002118:Calcium