Ni B, Wu X, Du Y, Su Y, Hamilton-Byrd E, Rockey P K, Rosteck P, Poirier G G, Paul S M
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, USA.
J Neurosci. 1997 Mar 1;17(5):1561-9. doi: 10.1523/JNEUROSCI.17-05-01561.1997.
Several members of the IL-1beta-converting enzyme (ICE) family of proteases recently have been implicated in the intracellular cascade mediating the apoptotic death of various cell types. It is unclear, however, whether ICE-related proteases are involved in apoptosis of mammalian neurons and, if so, how they are activated. Here we report the cloning of an ICE-related protease (IRP) from rat brain, which displays strong sequence identity to human CPP32. In situ hybridization histochemistry reveals that this IRP mRNA is expressed in neuron-enriched regions of the developing and adult rat brain but is profoundly downregulated in the adult (compared with developing) brain. To investigate whether this IRP is involved in the death of neurons in the developing brain, we studied IRP expression in cultured cerebellar granule neurons. In cultured cerebellar granule neurons, reduction of extracellular K+ reliably induces apoptosis and stimulates overexpression of IRP mRNA. The latter is especially prominent 4 hr after switching from high K+ to low K+ medium. The expression of IRP mRNA was maintained at this level for at least 8 hr and was followed by apoptotic death of these neurons. Induction of IRP mRNA and cell death are blocked completely by adding depolarizing concentrations of K+ </=90 min after switching to low K+ medium (i.e., before the commitment point for apoptosis) and partially blocked by brain-derived neurotrophic factor (BDNF), which also partially rescues granule neurons from low K+-induced apoptosis. In addition, overexpression of IRP cDNA in HeLa cells results in cell death accompanied by strong internucleosomal cleavage of DNA, a typical feature of apoptosis. Finally, we detected cleavage of the putative death substrate poly (ADP-ribose) polymerase (PARP), beginning 8 hr after changing from high K+ to low K+ medium, coinciding with the time course of induced expression of the IRP gene. Our data suggest that transcriptional activation of IRP could be one of the mechanisms involved in the apoptotic death of cerebellar granule neurons.
白细胞介素-1β转化酶（ICE）蛋白酶家族的几个成员最近被认为参与了介导各种细胞类型凋亡死亡的细胞内级联反应。然而，尚不清楚ICE相关蛋白酶是否参与哺乳动物神经元的凋亡，如果参与，它们是如何被激活的。在此，我们报道了从大鼠脑中克隆出一种ICE相关蛋白酶（IRP），它与人类CPP32具有很强的序列同源性。原位杂交组织化学显示，这种IRP mRNA在发育中和成年大鼠脑富含神经元的区域表达，但在成年（与发育中相比）脑中显著下调。为了研究这种IRP是否参与发育中脑神经元的死亡，我们研究了培养的小脑颗粒神经元中IRP的表达。在培养的小脑颗粒神经元中，细胞外钾离子的减少可靠地诱导凋亡并刺激IRP mRNA的过度表达。从高钾培养基转换到低钾培养基后4小时，后者尤为明显。IRP mRNA的表达在这个水平维持至少8小时，随后这些神经元发生凋亡死亡。在转换到低钾培养基后（即凋亡的决定点之前）≤90分钟添加去极化浓度的钾离子可完全阻断IRP mRNA的诱导和细胞死亡，脑源性神经营养因子（BDNF）可部分阻断，BDNF也可部分挽救颗粒神经元免受低钾诱导的凋亡。此外，IRP cDNA在HeLa细胞中的过度表达导致细胞死亡，并伴有DNA的强烈核小体间切割，这是凋亡的典型特征。最后，我们检测到从高钾培养基转换到低钾培养基8小时后开始出现假定的死亡底物聚（ADP-核糖）聚合酶（PARP）的切割，这与IRP基因诱导表达的时间进程一致。我们的数据表明，IRP的转录激活可能是参与小脑颗粒神经元凋亡死亡的机制之一。
