Vanderluit J L, McPhail L T, Fernandes K J, McBride C B, Huguenot C, Roy S, Robertson G S, Nicholson D W, Tetzlaff W
CORD (Collaboration on Repair Discoveries), University of British Columbia, Vancouver, British Columbia, Canada.
Eur J Neurosci. 2000 Oct;12(10):3469-80. doi: 10.1046/j.1460-9568.2000.00241.x.
In this report, we examined the possible functions of the cell death protease, caspase-3, in the axotomy-induced apoptosis of facial motoneurons in newborn rodents. Using in situ hybridization and Western blot, we found higher levels of caspase-3 mRNA and pro-caspase-3 protein expression in motoneurons of neonatal and 2-week-old rats than adult rats. Following facial motoneuron axotomy, caspase-3 mRNA and protein expression increased in motoneurons of both neonatal and adult rats. However, using an antibody directed to the activated form of the caspase-3 protease, we found that catalytically active caspase-3 was present only in axotomized neonatal motoneurons. As motoneurons in neonatal but not adult rodents are susceptible to axotomy-induced apoptosis, we hypothesized that caspase-3 may play a role in their demise. To determine the necessity of caspase-3 activation in axotomy-induced apoptosis, we counted the number of surviving motoneurons at 4 and 7 days following axotomy in wild type mice and caspase-3 gene-deleted mice. There were nearly three times more surviving motoneurons in caspase-3 gene-deleted mice than in wild type mice at both 4 days (mean 1074 vs. 464, P<0.005) and 7 days (mean 469 vs. 190, P<0.005) following injury, indicating a slower rate of death. Examination of the dying motoneurons using TUNEL staining (for fragmented DNA) and bisbenzimide staining (for nuclear morphology) revealed incomplete nuclear condensation in caspase-3-deficient motoneurons. These results demonstrate that caspase-3 activation plays important roles in the rapid demise of axotomized neonatal motoneurons.
在本报告中，我们研究了细胞死亡蛋白酶caspase-3在新生啮齿动物面神经运动神经元轴突切断诱导的凋亡中可能发挥的作用。通过原位杂交和蛋白质免疫印迹法，我们发现新生大鼠和2周龄大鼠运动神经元中caspase-3 mRNA和前体caspase-3蛋白的表达水平高于成年大鼠。面神经运动神经元轴突切断后，新生大鼠和成年大鼠运动神经元中caspase-3 mRNA和蛋白表达均增加。然而，使用针对caspase-3蛋白酶活化形式的抗体，我们发现具有催化活性的caspase-3仅存在于轴突切断的新生运动神经元中。由于新生而非成年啮齿动物的运动神经元易受轴突切断诱导的凋亡影响，我们推测caspase-3可能在其死亡过程中发挥作用。为了确定caspase-3激活在轴突切断诱导的凋亡中的必要性，我们对野生型小鼠和caspase-3基因缺失小鼠轴突切断后4天和7天存活的运动神经元数量进行了计数。在损伤后4天（平均1074个对464个，P<0.005）和7天（平均469个对190个，P<0.005），caspase-3基因缺失小鼠存活的运动神经元数量几乎是野生型小鼠的三倍，表明死亡速度较慢。使用TUNEL染色（用于检测DNA片段化）和双苯甲酰胺染色（用于检测核形态）对濒死运动神经元进行检查，发现caspase-3缺陷的运动神经元中核浓缩不完全。这些结果表明，caspase-3激活在轴突切断的新生运动神经元的快速死亡中起重要作用。
