神経変性疾患の細胞モデルを用いた、発症機序と予防法についての研究

利用细胞模型研究神经退行性疾病发病机制及预防方法

• 批准号: 16790687
• 负责人: 田中 有史
• 金额: $ 2.18万
• 依托单位: Okayama University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Young Scientists (B)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16790687/
• 关键词: Parkinson's disease; inducible cell model; alpha-synuclein; ubiquitin; NF-kappaB; IkappaB; cell death; apoptosis; パーキンソン病; 細胞死; α-シヌクレイン; パーキン; ユビキチン; 細胞内シグナル伝達; アポトーシス

We have been studying the mechanisms underlying cell death caused by alpha-synuclein using our inducible PC12 cell model of Parkinson's disease. We confirmed induction and suppression of alpha-synuclein in the absence and presence of doxycycline, respectively, with immunocytochemistry and western blotting experiments. We observed cytoplasmic inclusions stained for alpha-synuclein and ubiquitin in A53T mutant cell lines. In cell toxicity evaluation, we employed the trypan-blue exclusion assay and DAPI nuclear staining, finding increased apoptotic cell death after neuron-like differentiation and induction of A53T alpha-synuclein expression. Cell death we observed here was in an alpha-synuclein expression dependent manner.We have proposed to elucidate the role for the NF-kappaB pathway in cell death caused by mutant alpha-synuclein. We found that cytoplasmic inclusions in A53T cell lines were positive for IkappaB immunostaining. We tried three NF-kappaB inhibitors including PDTC,DDTC, and SN5O, finding attenuation of A53T alpha-synuclein-caused cell death in the presence of the inhibitors.Our findings suggest that the NF-kappaB pathway may play a role in mutant alpha-synuclein-caused cell death. In addition, the NF-kappaB pathway may be a potential target to treat neural cell death caused by alpha-synuclein.

我们一直在使用我们的帕金森氏病可诱导PC12细胞模型来研究α-突触核蛋白导致细胞死亡的机制。通过免疫细胞化学和免疫印迹实验，我们分别证实了在没有和存在多西环素的情况下α-突触核蛋白的诱导和抑制。我们在A53T突变细胞系中观察到α-突触核蛋白和泛素染色的细胞质包涵体。在细胞毒性评价中，我们采用台盼蓝排斥实验和DAPI核染色，发现诱导A53Tα-突触核蛋白表达和神经元样分化后的细胞凋亡率增加。我们在这里观察到的细胞死亡是α-突触核蛋白表达依赖的方式。我们建议阐明核因子-kappaB通路在突变型α-突触核蛋白引起的细胞死亡中的作用。我们发现A53T细胞系胞质内包涵体呈IkappaB免疫染色阳性。我们尝试了三种核因子-kappaB抑制剂，包括PDTC、DDTC和SN5O，发现在这些抑制剂的存在下，A53Tα-突触核蛋白引起的细胞死亡被减弱。我们的发现表明，核因子-kappaB通路可能在突变型α-突触核蛋白引起的细胞死亡中发挥作用。此外，核因子-kappaB通路可能是治疗α-突触核蛋白引起的神经细胞死亡的潜在靶点。