Department of Anatomy, Physiology and Genetics, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA
Mol Cell Neurosci. 2011 Sep;48(1):62-71. doi: 10.1016/j.mcn.2011.06.006. Epub 2011 Jun 25.
Peroxynitrite has been suggested to be the potent oxidant causing toxicity to neurons and oligodendrocytes (OLs). Our previous studies have illustrated that intracellular zinc liberation contributes to peroxynitrite toxicity to mature OLs. In this study, we further investigated the signaling pathways involved in this event and identified protein kinase C (PKC) as an important early signaling molecule. We found that a non-selective PKC inhibitor bisindolylmaleimide-1 blocked OL toxicity induced by a peroxynitrite generator SIN-1 and exogenous zinc. The protective effects were due to its inhibition on ERK1/2 phosphorylation and ROS generation. The same phenomenon was also observed in OLs following prolonged treatment with phorbol 12 myristate 13 acetate (PMA), which downregulates the conventional and the novel PKC isoforms (cPKCs and nPKCs). To determine the role of specific PKC isoforms, we found that a specific nPKC inhibitor rottlerin significantly reduced SIN-1- or zinc-induced toxicity, whereas Go6976, a cPKC inhibitor, reduced OL toxicity triggered by zinc, but not by SIN-1 at high concentrations. Rottlerin was more potent than Go6976 to attenuate ERK1/2 phosphorylation and ROS generation induced by SIN-1 or zinc. Surprisingly, zinc only induced phosphorylation of PKCθ, but not PKCδ. Knockdown of PKCθ using lentiviral shRNA attenuated SIN-1- or zinc-induced toxicity. These results suggest that PKCθ might be the major PKC isoform involved in peroxynitrite and zinc toxicity to mature OLs, and provide a rationale for development of specific inhibitors of PKCθ in the treatment of multiple sclerosis and other neurodegenerative diseases, in which peroxynitrite formation plays a pathogenic role.
过氧亚硝酸盐被认为是导致神经元和少突胶质细胞（OL）毒性的有效氧化剂。我们之前的研究表明，细胞内锌的释放有助于过氧亚硝酸盐对成熟 OL 的毒性。在这项研究中，我们进一步研究了涉及这一事件的信号通路，并确定蛋白激酶 C（PKC）是一个重要的早期信号分子。我们发现，一种非选择性 PKC 抑制剂双吲哚马来酰亚胺-1（bisindolylmaleimide-1）阻断了过氧亚硝酸盐生成剂 SIN-1 和外源性锌诱导的 OL 毒性。这种保护作用是由于其对 ERK1/2 磷酸化和 ROS 生成的抑制作用。在长时间用佛波醇 12-肉豆蔻酸 13-醋酸酯（phorbol 12-myristate 13-acetate，PMA）处理 OL 后，也观察到了同样的现象，PMA 下调了传统和新型 PKC 同工型（cPKC 和 nPKC）。为了确定特定 PKC 同工型的作用，我们发现，一种特定的 nPKC 抑制剂rottlerin 显著降低了 SIN-1 或锌诱导的毒性，而 cPKC 抑制剂 Go6976 降低了锌但不是高浓度 SIN-1 触发的 OL 毒性。与 Go6976 相比，rottlerin 更能减弱 SIN-1 或锌诱导的 ERK1/2 磷酸化和 ROS 生成。令人惊讶的是，锌只诱导了 PKCθ的磷酸化，而不是 PKCδ。使用慢病毒 shRNA 敲低 PKCθ 可减弱 SIN-1 或锌诱导的毒性。这些结果表明，PKCθ 可能是参与成熟 OL 中过氧亚硝酸盐和锌毒性的主要 PKC 同工型，并为开发针对 PKCθ 的特异性抑制剂治疗多发性硬化症和其他神经退行性疾病提供了依据，其中过氧亚硝酸盐的形成起着致病作用。
