Abstract
24(S)-Hydroxycholesterol (24S-OHC) produced by cholesterol 24-hydroxylase expressed mainly in neurons plays an important physiological role in the brain. Conversely, it has been reported that 24S-OHC possesses potent cytotoxicity. The molecular mechanisms of 24S-OHC-induced cell death have not yet been fully elucidated. In this study, using human neuroblastoma SH-SY5Y cells and primary cortical neuronal cells derived from rat embryo, we characterized the form of cell death induced by 24S-OHC. SH-SY5Y cells treated with 24S-OHC exhibited neither fragmentation of the nucleus nor caspase activation, which are the typical characteristics of apoptosis. 24S-OHC-treated cells showed necrosis-like morphological changes but did not induce ATP depletion, one of the features of necrosis. When cells were treated with necrostatin-1, an inhibitor of receptor-interacting serine/threonine kinase 1 (RIPK1) required for necroptosis, 24S-OHC-induced cell death was significantly suppressed. The knockdown of RIPK1 by transfection of small interfering RNA of RIPK1 effectively attenuated 24S-OHC-induced cell death. It was found that neither SH-SY5Y cells nor primary cortical neuronal cells expressed caspase-8, which was regulated for RIPK1-dependent apoptosis. Collectively, these results suggest that 24S-OHC induces neuronal cell death by necroptosis, a form of programmed necrosis.
Highlights
Hydroxycholesterol (24S-OHC),3 which can be transported across the blood-brain barrier (4)
Several lines of evidence have revealed that the inhibitor of RIPK1, methylthiohydantoin-DL-tryptophan, can inhibit certain types of caspase-independent cell death with the features of necrosis in experimental paradigms such as cell cultures treated with a combination of an apoptosis inducer and a caspase inhibitor, cells treated with an activator of the receptor-interacting protein, and neurons in vivo after ischemic brain injury (21–24)
This study clearly shows that neuronal cell death induced by 24S-OHC, at least partly, involves the necroptosis pathway
Summary
24S-OHC, 24(S)-hydroxycholesterol; AMC, aminomethylcoumarin; LDH, lactate dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PI, propidium iodide; PS, phosphatidylserine; ROS, reactive oxygen species; STS, staurosporine. 24S-OHC Induces Neuronal Cell Death through Necroptosis unknown. The term “necroptosis” has been used to designate one particular type of programmed necrosis that depends on the receptor-interacting serine/threonine kinase 1 (RIPK1) (21, 22). Several lines of evidence have revealed that the inhibitor of RIPK1, methylthiohydantoin-DL-tryptophan (necrostatin-1; Nec-1), can inhibit certain types of caspase-independent cell death with the features of necrosis in experimental paradigms such as cell cultures treated with a combination of an apoptosis inducer and a caspase inhibitor, cells treated with an activator of the receptor-interacting protein, and neurons in vivo after ischemic brain injury (21–24). The death-inhibiting effect of Nec-1 has been reported to be highly specific for this form of cell death, which has been called necroptosis. We found that 24S-OHC induced necroptosis of neuronal cells in a cell type-dependent manner
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