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

细胞内 Aβ1-42 相关神经元死亡的基因表达谱分析

• 批准号: 16500233
• 负责人: UCHIDA Yoko
• 金额: $ 2.5万
• 依托单位: Tokyo Metropolitan Institute of Gerontology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16500233/
• 关键词: β-amyloid; intracellular Aβ; neuronal death; microarray; AICD; 細胞内Ass; 細胞死関連遺伝子

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.

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。研究结果如下。(1)首先通过APP-CTFβ(C99)-Myc-IRES-hrGFP基因对培养的大脑皮层神经元的转染实验，确定了细胞内Aβ对神经元死亡的影响。C99在神经元中的表达确实诱导了细胞的死亡：然而，对表达C99的细胞进行不同的检测程序会导致不同的时程和不同程度的神经元死亡。抗myc抗体比hrGFP荧光法检测到更多表达C99的死亡神经元，提示C99在细胞内沉积可能导致神经元死亡。(2)利用基因芯片技术分析C99蓄积引起的细胞死亡相关基因表达谱。46个基因被鉴定为通过C99在细胞内的积累而改变了它们的表达。为了证实10个基因的表达变化，我们通过比较表达myc和表达126MAP1B的细胞的基因表达情况，我们进行了QRT-PCR，没有显示细胞死亡的细胞。虽然表达C99的细胞和表达myc的细胞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变化。(3)利用QRT-PCR技术检测C99表达细胞与APP-CTFα或C99-D644A的差异表达。