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Ca2+ and reactive oxygen species in staurosporine-induced neuronal apoptosis
J H Prehn1, J Jordán, G D Ghadge
1Department of Pharmacology and Toxicology, Philipps-University, Marburg, Germany.
Abstract:
Staurosporine (0.03-0.5 microM) induced a dose-dependent, apoptotic degeneration in cultured rat hippocampal neurons that was sensitive to 24-h pretreatments with the protein synthesis inhibitor cycloheximide (1 microM) or the cell cycle inhibitor mimosine (100 microM). To investigate the role of Ca2+ and reactive oxygen species in staurosporine-induced neuronal apoptosis, we overexpressed calbindin D28K, a Ca2+ binding protein, and Cu/ Zn superoxide dismutase, an antioxidative enzyme, in the hippocampal neurons using adenovirus-mediated gene transfer. Infection of the cultures with the recombinant adenoviruses (100 multiplicity of infection) resulted in a stable expression of the respective proteins assessed 48 h later. Overexpression of both calbindin D28K and Cu/Zn superoxide dismutase significantly reduced staurosporine neurotoxicity compared with control cultures infected with a beta-galactosidase overexpressing adenovirus. Staurosporine-induced neuronal apoptosis was also significantly reduced when the culture medium was supplemented with 10 or 30 mM K+, suggesting that Ca2+ influx via voltage-sensitive Ca2+ channels reduces this apoptotic cell death. In contrast, neither the glutamate receptor agonist NMDA (1-10 microM) nor the NMDA receptor antagonist dizocilpine (MK-801; 1 microM) was able to reduce staurosporine neurotoxicity. Cultures treated with the antioxidants U-74500A (1-10 microM) and N-acetylcysteine (100 microM) also demonstrated reduced staurosporine neurotoxicity. These results suggest a fundamental role for both Ca2+ and reactive oxygen species in staurosprine-induced neuronal apoptosis.
Insights
Staurosporine triggers neuronal apoptosis, but this process is mitigated by blocking protein synthesis or cell cycle progression. Overexpressing protective proteins or increasing extracellular potassium ions significantly reduced this cell death, highlighting roles for calcium and reactive oxygen species.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- Staurosporine is a potent inducer of apoptosis in various cell types, including neurons.
- Understanding the mechanisms underlying staurosporine-induced neuronal apoptosis is crucial for developing neuroprotective strategies.
Purpose of the Study:
- To investigate the roles of calcium (Ca2+) and reactive oxygen species (ROS) in staurosporine-induced apoptosis of rat hippocampal neurons.
- To evaluate the neuroprotective effects of overexpressing calbindin D28K and Cu/Zn superoxide dismutase against staurosporine toxicity.
Main Methods:
- Cultured rat hippocampal neurons were treated with staurosporine to induce apoptosis.
- Adenovirus-mediated gene transfer was used to overexpress calbindin D28K and Cu/Zn superoxide dismutase.
- Neuroprotection was assessed by measuring neuronal survival and apoptosis following staurosporine treatment under various conditions, including altered extracellular potassium levels and antioxidant administration.
Main Results:
- Staurosporine induced dose-dependent apoptotic degeneration in hippocampal neurons.
- Pretreatment with cycloheximide or mimosine conferred significant protection against staurosporine-induced apoptosis.
- Overexpression of calbindin D28K and Cu/Zn superoxide dismutase significantly reduced staurosporine neurotoxicity.
- Increased extracellular potassium (10-30 mM) reduced staurosporine-induced apoptosis, suggesting a role for Ca2+ influx.
- Antioxidants U-74500A and N-acetylcysteine also reduced staurosporine neurotoxicity.
Conclusions:
- Both Ca2+ influx and reactive oxygen species play fundamental roles in staurosporine-induced neuronal apoptosis.
- Targeting Ca2+ signaling and reducing oxidative stress may represent viable therapeutic approaches for preventing staurosporine-induced neurodegeneration.