A tetracycline-regulated adenovirus encoding dominant-negative caspase-9 is regulated in rat brain and protects against neurotoxin-induced cell death in vitro, but not in vivo
A tetracycline-regulated adenovirus encoding dominant-negative caspase-9 is regulated in rat brain and protects against neurotoxin-induced cell death in vitro, but not in vivo
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DOI：
10.1016/j.expneurol.2004.08.024
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发表时间：
2005-02-01
影响因子：
5.3
通讯作者：
Bohn, MC
中科院分区：
文献类型：
--
作者：
Ebert, AD;Chen, F;Bohn, MC
Caspase-9是参与细胞凋亡的关键下游效应分子，细胞凋亡是一种细胞死亡过程，被认为参与受帕金森病（PD）影响的黑质（SN）中多巴胺（DA）神经元的死亡。在这项研究中，我们确定了四环素调节腺病毒携带的显性负性形式的caspase-9（Casp 9DN）和标记基因，增强型绿色荧光蛋白（EGFP），在一个双向启动子的控制下，可以分别在体外和体内调节多西环素。我们接下来观察到，Casp 9DN基因递送显著保护HeLa细胞中TNF α和环己酰亚胺诱导的染色质凝聚，并防止染色质凝聚和6-羟基多巴胺（6-OHDA）处理的MN 9D细胞（多巴胺能细胞系）中早期凋亡标记物膜联蛋白V的出现。还评估了Casp 9DN对体内DA神经元的影响。用荧光金（FG）逆行标记DA神经元，并用Casp 9DN和EGFP或单独的EGFP转导。纹状体注射6-OHDA 1周后，DA能神经元出现进行性损害。损伤后2周，黑质FG+神经元的形态计量学分析显示，Casp 9DN组FG+神经元的平均细胞直径分别比EGFP组和PBS组大8%和21%（P < 0.05）。然而，治疗组之间在受损SN中剩余的神经元数量方面没有差异。这些结果表明，虽然在胱天蛋白酶-9水平抑制凋亡在体外具有保护作用，但在体内对6-OHDA诱导的细胞死亡没有保护作用。(C)2004年爱思唯尔公司All rights reserved.
Caspase-9 is a critical downstream effector molecule involved in apoptosis, a cell death process thought to be involved in the demise of dopamine (DA) neurons in the substantia nigra (SN) affected by Parkinson's disease (PD). In this study, we determined that a tetracycline-regulated adenovirus harboring a dominant-negative form of caspase-9 (Casp9DN) and the marker gene, enhanced green fluorescent protein (EGFP), under the control of a bidirectional promoter could each be regulated in vitro and in vivo by doxycycline. We next observed that Casp9DN gene delivery significantly protected against TNFalpha and cycloheximide-induced chromatin condensation in HeLa cells and prevented chromatin condensation and the appearance of the early apoptotic marker annexin V in 6-hydroxydopamine (6-OHDA) treated MN9D cells, a dopaminergic cell line. Effects of Casp9DN on DA neurons in vivo were also assessed. DA neurons were retrogradely labeled with fluorogold (FG) and transduced with Casp9DN and EGFP or EGFP alone. A progressive lesion of DA neurons was induced by striatal injection of 6-OHDA 1 week later. At 2 weeks post-lesion, a morphometric analysis of FG+ neurons in the SN revealed that the mean cell diameter of FG labeled neurons in the Casp9DN group was 8% and 21% larger than the EGFP and PBS groups, respectively (P < 0.05). However, there was no difference among the treatment groups in the number of neurons remaining in the lesioned SN. These results suggest that while inhibiting apoptosis at the level of caspase-9 is protective in vitro, it is not protective against 6-OHDA-induced cell death in vivo. (C) 2004 Elsevier Inc. All rights reserved.