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来阐明细胞内Aβ诱导神经元死亡的分子机制。结果如下：(1)首先通过应用APP-CTFβ(C99)-myc-IRES-hrGFP cDNA转染培养的皮质神经元实验，确定细胞内Aβ对神经元死亡的影响。C99在神经元中表达确实会导致细胞死亡，但不同的检测方法导致表达C99的细胞死亡的时间进程和程度不同。与hrGFP荧光相比，抗myc抗体检测到表达C99的神经元死亡比例更大，表明细胞内C99沉积可能导致神经元死亡。(2)利用微阵列技术分析C99积累导致细胞死亡的相关基因表达谱。46个基因被鉴定为细胞内C99积累改变了表达的基因。为了证实10个基因的表达改变，我们通过QRT-PCR比较表达myc和表达△126map1b的细胞中的基因表达，没有显示细胞死亡。虽然表达c99的细胞与表达myc的细胞之间的基因表达没有显著差异，但与表达△126map1b的细胞相比，表达c99的细胞中有几个基因的表达发生了改变。(3)现在我们用QRT-PCR的方法对比APP-CTF α和C99-D644A在c99表达细胞中的表达情况。

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.