Benjamins Joyce A, Nedelkoska Liljana, George Edwin B
Department of Neurology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Neurochem Res. 2003 Jan;28(1):143-52. doi: 10.1023/a:1021612615554.
Mature mouse oligodendrocytes (OLs) are susceptible to death in demyelinating diseases such as multiple sclerosis and in brain injury following neurotrauma, ischemia, or stroke. To understand mechanisms leading to death of mature OLs and develop strategies for protection, we utilized cultures of mature mouse OLs to investigate the role of caspases and calpains in OL cell death mediated by different mechanisms. The agents used were (i) staurosporine, which induces apoptotic death via inhibition of protein kinases; (ii) kainate, which activates non-NMDA glutamate receptors; (iii) thapsigargin, which releases intracellular calcium stores; and (iv) SNAP, which releases active NO species and causes necrotic cell death. Inhibitors blocking primary effector caspases (including caspase 3), the FAS (death receptor)-mediated initiator caspases (including caspase 8), and stress-induced caspases (including caspase 9), were tested for their protective effects. Inhibition of caspases 3, 8, and 9 each robustly protected OLs following insult with staurosporine, thapsigargin, or kainate when added at optimal times. The time of addition of the inhibitors for maximal protection varied with the agent, from 1 h of preincubation before addition of staurosporine to 6 h after addition of kainate. Much less protection was seen for the NO generator SNAP under any condition. The role of calcium in OL death in each model was investigated by chelating extracellular Ca++ with EGTA, and by inhibiting the Ca++-activated calpain proteases. Calcium chelation did not protect against staurosporine, but decreased OL death initiated by kainate, thapsigargin, or NO. The calpain inhibitors PD150606 and calpain inhibitor I protected from cell death initiated by staurosporine, kainate, and thapsigargin, but not from cell death initiated by the NO donor SNAP.
成熟的小鼠少突胶质细胞（OLs）在诸如多发性硬化症等脱髓鞘疾病以及神经创伤、缺血或中风后的脑损伤中易发生死亡。为了了解导致成熟OLs死亡的机制并制定保护策略，我们利用成熟小鼠OLs培养物来研究半胱天冬酶和钙蛋白酶在不同机制介导的OL细胞死亡中的作用。所使用的试剂有：（i）星形孢菌素，其通过抑制蛋白激酶诱导凋亡性死亡；（ii）海人藻酸，其激活非NMDA谷氨酸受体；（iii）毒胡萝卜素，其释放细胞内钙储存；（iv）硝普钠（SNAP），其释放活性NO物质并导致坏死性细胞死亡。测试了阻断主要效应半胱天冬酶（包括半胱天冬酶3）、FAS（死亡受体）介导的起始半胱天冬酶（包括半胱天冬酶8）和应激诱导的半胱天冬酶（包括半胱天冬酶9）的抑制剂的保护作用。在最佳时间添加时，抑制半胱天冬酶3、8和9在星形孢菌素、毒胡萝卜素或海人藻酸损伤后均能有力地保护OLs。抑制剂添加以实现最大保护的时间因试剂而异，从添加星形孢菌素前预孵育1小时到添加海人藻酸后6小时不等。在任何条件下，对于NO生成剂SNAP的保护作用都要小得多。通过用乙二醇双四乙酸（EGTA）螯合细胞外Ca++以及抑制Ca++激活的钙蛋白酶来研究钙在每种模型中OL死亡中的作用。钙螯合不能保护细胞免受星形孢菌素的影响，但可减少由海人藻酸、毒胡萝卜素或NO引发的OL死亡。钙蛋白酶抑制剂PD150606和钙蛋白酶抑制剂I可保护细胞免受由星形孢菌素、海人藻酸和毒胡萝卜素引发的细胞死亡，但不能保护细胞免受NO供体SNAP引发的细胞死亡。
