Panet H, Barzilai A, Daily D, Melamed E, Offen D
Department of Neurology, Rabin Medical Center and the Laboratory for Neurosciences, Felsenstein Medical Research Center, Petah Tikva and Sackler School of Medicine, Tel Aviv, Israel.
J Neurochem. 2001 Apr;77(2):391-8. doi: 10.1046/j.1471-4159.2001.00213.x.
The etiology of Parkinson's disease is still unknown, though current investigations support the notion of the pivotal involvement of oxidative stress in the process of neurodegeneration in the substantia nigra (SN). In the present study, we investigated the molecular mechanisms underlying cellular response to a challenge by dopamine, one of the local oxidative stressors in the SN. Based on studies showing that nuclear factor kappa B (NF-kappaB) is activated by oxidative stress, we studied the involvement of NF-kappaB in the toxicity of PC12 cells following dopamine exposure. We found that dopamine (0.1-0.5 m M) treatment increased the phosphorylation of the IkappaB protein, the inhibitory subunit of NF-kappaB in the cytoplasm. Immunoblot analysis demonstrated the presence of NF-kappaB-p65 protein in the nuclear fraction and its disappearance from the cytoplasmic fraction after 2 h of dopamine exposure. Dopamine-induced NF-kappaB activation was also evidenced by electromobility shift assay using radioactive labeled NF-kappaB consensus DNA sequence. Cell-permeable NF-kappaB inhibitor SN-50 rescued the cells from dopamine-induced apoptosis and showed the importance of NF-kappaB activation to the induction of apoptosis. Furthermore, flow cytometry assay demonstrated a higher level of translocated NF-kappaB-p65 in the apoptotic nuclei than in the unaffected nuclei. In conclusion, our findings suggest that NF-kappaB activation is essential to dopamine-induced apoptosis in PC12 cells and it may be involved in nigral neurodegeneration in patients with Parkinson's disease.
帕金森病的病因仍不清楚，尽管目前的研究支持氧化应激在黑质神经退行性变过程中起关键作用这一观点。在本研究中，我们调查了细胞对多巴胺（黑质局部氧化应激源之一）刺激作出反应的分子机制。基于显示核因子κB（NF-κB）被氧化应激激活的研究，我们研究了NF-κB在多巴胺暴露后PC12细胞毒性中的作用。我们发现多巴胺（0.1 - 0.5 mM）处理增加了细胞质中NF-κB抑制亚基IκB蛋白的磷酸化。免疫印迹分析表明，多巴胺暴露2小时后，核组分中存在NF-κB-p65蛋白，而细胞质组分中该蛋白消失。使用放射性标记的NF-κB共有DNA序列进行的电泳迁移率变动分析也证明了多巴胺诱导的NF-κB激活。细胞可渗透的NF-κB抑制剂SN-50使细胞免受多巴胺诱导的凋亡，并表明NF-κB激活对凋亡诱导的重要性。此外，流式细胞术分析表明，凋亡细胞核中易位的NF-κB-p65水平高于未受影响的细胞核。总之，我们的研究结果表明，NF-κB激活对多巴胺诱导的PC12细胞凋亡至关重要，并且它可能参与帕金森病患者的黑质神经退行性变。
