Watabe Masahiko, Nakaki Toshio
Department of Pharmacology, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi Ward, Tokyo 173-8605, Japan.
J Pharmacol Exp Ther. 2004 Dec;311(3):948-53. doi: 10.1124/jpet.104.071381. Epub 2004 Jul 27.
Chronic complex I inhibition caused by rotenone induces features of Parkinson's disease in rats, including selective nigrostriatal dopaminergic degeneration and Lewy bodies with alpha-synuclein-positive inclusions. To determine the mechanisms underlying rotenone-induced neuronal death, we used an in vitro model of human dopaminergic SH-SY5Y cells. In rotenone-induced cell death, rotenone induced Bad dephosphorylation without changing the amount of Bad proteins. Rotenone also increased the amount of alpha-synuclein in cells showing morphological changes in response to rotenone. Because Bad and alpha-synuclein are known to bind to 14-3-3 proteins, we examined the effects of rotenone on these complexes. Whereas a decreased Bad amount bound to 14-3-3 proteins, rotenone increased alpha-synuclein binding to these proteins. Because dephosphorylation by calcineurin activates Bad, we examined the possible involvement of Bad activation in rotenone-induced apoptosis by using the calcineurin inhibitor tacrolimus (FK506). Tacrolimus suppressed two rotenone-induced actions: Bad dephosphorylation and apoptosis. Furthermore, the inhibition of caspase-9, which functions downstream from Bad, completely suppressed rotenone-induced apoptosis. Our findings demonstrate that Bad activation plays a role in rotenone-induced apoptosis of SH-SY5Y cells.
鱼藤酮引起的慢性复合体I抑制可诱导大鼠出现帕金森病特征，包括选择性黑质纹状体多巴胺能神经元变性以及含有α-突触核蛋白阳性包涵体的路易小体。为了确定鱼藤酮诱导神经元死亡的机制，我们使用了人多巴胺能SH-SY5Y细胞的体外模型。在鱼藤酮诱导的细胞死亡中，鱼藤酮诱导了Bad的去磷酸化，而Bad蛋白的量没有改变。鱼藤酮还增加了细胞中α-突触核蛋白的量，这些细胞在对鱼藤酮的反应中表现出形态变化。由于已知Bad和α-突触核蛋白与14-3-3蛋白结合，我们研究了鱼藤酮对这些复合物的影响。与14-3-3蛋白结合的Bad量减少，而鱼藤酮增加了α-突触核蛋白与这些蛋白的结合。由于钙调神经磷酸酶的去磷酸化激活了Bad，我们通过使用钙调神经磷酸酶抑制剂他克莫司（FK506）来研究Bad激活在鱼藤酮诱导的细胞凋亡中的可能作用。他克莫司抑制了鱼藤酮诱导的两种作用：Bad去磷酸化和细胞凋亡。此外，对在Bad下游起作用的半胱天冬酶-9的抑制完全抑制了鱼藤酮诱导的细胞凋亡。我们的研究结果表明，Bad激活在鱼藤酮诱导的SH-SY5Y细胞凋亡中起作用。
