Analyses of molecular mechanisms of caspases during neural development

Caspase-3、caspase-9、APAF-1等与细胞凋亡相关的基因在细胞死亡诱导中起着重要作用。然而，一些证据表明，caspase的微小激活不会导致细胞死亡，但需要神经发育和分化，因为caspase-3和caspase-9基因敲除的小鼠表现出混乱的神经发育。结果：1.鉴定了caspase在神经发育过程中的表达，RT-PCR检测到caspase-7在原代神经元培养早期表达增加，而caspase-3在神经分化过程中表达增加；2.在大肠杆菌中产生活性caspase。将人caspase-3和caspase-7基因克隆到pET表达载体中，在大肠杆菌中诱导表达活性caspase，并进行纯化。3.caspase-7底物的筛选总共有17000个基因。使用体外转录和翻译产生的355标记蛋白，在caspase活性存在的情况下，通过体外切割试验筛选底物。4.人脑cdna中caspase-7底物的鉴定。从96个文库中鉴定出18个基因为caspase-7底物。其中5个为未知基因，10个为全长基因，2个不含编码区。我们将分析底物对神经发育的作用。

Apoptotic related genes, such as caspase-3, caspase-9, and Apaf-1, have a critical roles as cell death inducer. However, several evidences suggest that trivial activation of caspases, which do not induce cell death, needs to neural development and differentiation because knockout mice of caspase-3 and caspase-9 show disorganized neural development. In this project, we planned to identify caspases to be activated during neural development and screen its substrates to analyze the molecular roles of caspases.Results1.Identification of caspases expression during neural development.The expression of caspase-7 was increased at early stage of primary neuronal culture by RT-PCR, whereas caspase-3 was increased during neural differentiation.2.Generation of active caspase in E. coli.Human caspase-3 and caspase-7 genes were cloned into pET expression vectors and active caspases were induced in E. coli., and purified.3.Screening of substrates for caspase-7.Small pool cDNA library contained about 100 genes from human brain per pool, which comes to 17,000 genes in total. Substrates were screened by in vitro cleavage assay in the presence of active caspase using 355-labeled proteins, which were generated by in vitro transcription and translation. Autoradiograph showed the cleaved product by caspase.4.Identification of caspase-7 substrates from human brain cDNA.Eighteen genes were identified as caspase-7 substrates from 96 pools cDNA library. Five of eighteen were unknown genes, ten genes included full-length, and two plasmids had no coding region. We are going to analyze the function of substrates for neural development.