The role of ubiquitin in neuronal apoptosis

测定沙鼠短暂脑缺血后海马中泛素含量。用放射免疫法或ELISA定量测定CA1区和CA3/树突区的游离和共轭泛素。结果与我们之前的免疫染色结果一致。短暂性缺血后，游离泛素在CA1区下降，且从未恢复，而游离泛素在CA3/齿状区短暂下降，48小时内恢复到对照水平。共轭泛素在这两个区域的含量显著增加。CA3/齿状区降至正常水平，CA1缺血48小时后仍维持较高水平。泛素mRNA原位杂交显示海马全区信号明显增加。由于游离的泛素在CA1中没有恢复，泛素mRNA的翻译在该区域被阻断。另一方面，在耐受诱导的海马中，泛素mRNA被翻译，游离泛素迅速恢复。游离泛素的减少导致泛素-蛋白酶体蛋白水解系统的紊乱。我们研究了泛素-蛋白酶体系统的紊乱是细胞死亡过程的简单结果，还是一个可能导致细胞降解的活跃过程。蛋白酶体抑制剂抑制大鼠皮层神经元蛋白酶体活性。该过程导致caspase-3激活和随后的神经元凋亡。这些结果表明，泛素和蛋白酶体在海马延迟性神经元死亡过程中有着深刻而积极的关系。

Ubiquitin content was measured in the gerbil hippocampus following brief cerebral ischemia. Free and conjugated ubiquitin of the CA1 sector and CA3/dendate region are quantitated by radio-immunoassay or ELISA.The result was parallel with our former result by immunostaining. Following transient ischemia, free ubiquitin decreased in CA1 and it never recovered, whereas free ubiquitin transiently decreased in CA3/dentate region and then recovered to the control level within 48 hours. The content of conjugated ubiquitin remarkably increased in both of the regions. It decreased down to the normal level in CA3/dentate region but it maintained a high level at 48 hours following ischemia in CA1. In situ hybridization of ubiquitin mRNA demonstrated a marked increase of signal in the whole area of the hippocampus. Since free ubiquitin never recovered in CA1, translation of ubiquitin mRNA was blocked in this region. On the other hand, ubiquitin mRNA is translated and free ubiquitin rapidly recovered in tolerance-induced hippocampus. Decrease in free ubiquitin results in disturbance of ubiquitin-proteasome proteolytic system. We examined whether the disturbance of ubiquitin-proteasome system is a simple result of cell death process or it is an active process that could contribute to cell degradation. Proteasome activity was inhibited by proteasome inhibitor in cultured rat cortical neurons. The procedure resulted in caspase-3 activation and subsequent neuronal apoptosis. These results indicated that ubiquitin and proteasome are deeply and actively related in the process of delayed neuronal death in the hippocampus.