Profiling of gene expression related neuronal death by intracellular Aβ1-42

本研究的目的是通过基因表达谱分析阐明胞内Aβ诱导神经元死亡的分子机制。结果如下。(1)首先，我们通过将APP-CTFβ（C99）-myc-IRES-hrGFP cDNA转染到培养的皮层神经元中来确定细胞内Aβ对神经元死亡的影响。C99在神经元中的表达确实引起细胞死亡：然而，不同的检测方法导致不同的时间过程和不同程度的神经元死亡。通过抗myc抗体检测到的死亡的C99表达神经元的比例比通过hrGFP荧光检测到的比例更大，这表明C99的细胞内沉积可以诱导神经元死亡。(2)然后利用基因芯片技术分析了C99积累引起细胞死亡的相关基因表达谱。46个基因被鉴定为通过C99的细胞内积累改变其表达的基因。为了证实10个基因的表达改变，我们通过比较表达myc或表达△ 126 MAP 1B的细胞中的基因表达来进行QRT-PCR，这些细胞没有显示细胞死亡。表达C99的细胞与表达myc的细胞基因表达无显著差异，但表达C99的细胞与表达△ 126 MAP 1B的细胞相比，有几个基因的表达发生了改变。(3)现在，我们正在使用QRT-PCR检测C99表达细胞中这些基因的表达，并与APP-CTF α或C99-D 644 A进行比较。

The purpose of this study is to clarify the molecular mechanisms of neuronal death induced by intracellular Aβ by using gene expression profiling. Results are as follows. (1) First we determined the effect of intracellular Aβ on neuronal death by using transfection experiments of APP-CTFβ(C99)-myc-IRES-hrGFP cDNA into cultured cortical neurons. C99 expressed in neurons really induced cell death: however different detection procedures for C99-expressing cells resulted in different time course and different degree of neuronal death. Larger proportion of dead C99-expressing neurons were detected by anti-myc antibodies than that by hrGFP fluorescence, indicating that intracellular deposition of C99 may induce neuronal death. (2) Then we analyzed the gene expression profiles related to cell death caused by the accumulation of C99 by using microarray technology. Forty-six genes were identified as the genes altered their expressions by the intracellular accumulation of C99. To confirm the altered expressions of 10 genes, we performed QRT-PCR by comparing the gene expressions in myc-expressing or △126MAP1B-expressing cells, which did not show cell death. Although there was no significant difference in gene expressions between C99-expressing cell and myc-expressing cells, the expressions of several genes were altered in C99-expressing cell compared with △126MAP1B-expressing cells. (3) Now we are examining these gene expressions in C99-expressing cell compared with APP-CTF alpha or with C99-D644A by using QRT-PCR.