Klettner A, Baumgrass R, Zhang Y, Fischer G, Bürger E, Herdegen T, Mielke K
Institute of Pharmacology, Christian-Albrechts-University, Hospitalstrasse 4, 24105, Kiel, Germany.
Brain Res Mol Brain Res. 2001 Dec 16;97(1):21-31. doi: 10.1016/s0169-328x(01)00286-8.
The immunosuppressant FK506 displays substantial neuroprotective and neuroregenerative effects. It is not fully understood to which extent these effects depend on the inhibition of the calcineurin phosphatase (PP2B). The present study has re-addressed this issue using Lie120, a novel highly specific inhibitor of calcineurin, which does not block the enzymatic activity of FKBPs or cyclophilins, respectively. We have determined the effect of FK506 (10-500 nM), V-10,367 (a FK506 derivative which does not block calcineurin; 1-5 microM) and Lie120 (a novel specific inhibitor of calcineurin, 0.1-5 microM) on the cellular survival and the pro-degenerative JNK activity of PC12 and Neuro2A cells following application of 200 microM H(2)O(2). FK506 and V-10,367, but not Lie120, protected both cell lines against H(2)O(2)-mediated death, whereas an increase in JNK1 activity was blocked by FK506 and Lie120, but not by V-10,367. Co-incubation of FK506 and V-10,367 with the mRNA synthesis inhibitor actinomycin D abolished the protective effect of FK506 and V-10,367. This antagonization was effective when actinomycin D was applied 30 min or 1 h, but not 2 or 4 h, after H(2)O(2) suggesting that FKBP-ligands confer their neuroprotection by rapid de novo synthesis of (functionally) anti-apoptotic proteins. The search for the corresponding effector genes revealed that the expression of FKBP25, FKBP38 and FKBP52 (analysis by reverse transcription-polymerase chain reaction (RT-PCR) did not change following H(2)O(2) or FK506, and this was also true for the expression of apoptosis-related genes caspase 3, bax, bcl-2 and bcl-xL (analysis by Multiplex-PCR). Summarizing, neuronal protection by FKBP-ligands is not mediated either by calcineurin or by JNK1 in this experimental set-up, whereas the FK506 mediated inhibition of JNK1 is realized by the inhibition of calcineurin, an effective activator of JNK1 in neurons.
免疫抑制剂FK506具有显著的神经保护和神经再生作用。目前尚不完全清楚这些作用在多大程度上依赖于钙调神经磷酸酶（PP2B）的抑制。本研究使用Lie120重新探讨了这个问题，Lie120是一种新型的高度特异性钙调神经磷酸酶抑制剂，它不会分别阻断FK506结合蛋白（FKBPs）或亲环蛋白的酶活性。我们测定了FK506（10 - 500 nM）、V - 10,367（一种不阻断钙调神经磷酸酶的FK506衍生物；1 - 5 μM）和Lie120（一种新型的钙调神经磷酸酶特异性抑制剂，0.1 - 5 μM）对200 μM H₂O₂处理后的PC12和Neuro2A细胞的细胞存活及促变性JNK活性的影响。FK506和V - 10,367可保护两种细胞系免受H₂O₂介导的死亡，而Lie120则不能，然而FK506和Lie120可阻断JNK1活性的增加，V - 10,367则不能。FK506和V - 10,367与mRNA合成抑制剂放线菌素D共同孵育可消除FK506和V - 10,367的保护作用。当在H₂O₂处理后30分钟或1小时应用放线菌素D时，这种拮抗作用有效，但在2或4小时应用时则无效，这表明FKBP配体通过快速从头合成（功能性）抗凋亡蛋白来赋予其神经保护作用。对相应效应基因的搜索显示，FKBP25、FKBP38和FKBP52的表达（通过逆转录 - 聚合酶链反应（RT - PCR）分析）在H₂O₂或FK506处理后没有变化，凋亡相关基因caspase 3、bax、bcl - 2和bcl - xL的表达（通过多重PCR分析）也是如此。总之，在本实验设置中，FKBP配体的神经元保护作用既不是由钙调神经磷酸酶介导，也不是由JNK1介导，而FK506介导的JNK1抑制是通过抑制钙调神经磷酸酶实现的，钙调神经磷酸酶是神经元中JNK1的有效激活剂。
