Kim In-Su, Ganesan Palanivel, Choi Dong-Kug
Department of Biotechnology, Konkuk University, Chungju 380-701, Korea.
Nanotechnology Research Center and Department of Applied Life Science, College of Biomedical and Health Science, Konkuk University, Chungju 380-701, Korea.
Int J Mol Sci. 2016 Nov 1;17(11):1819. doi: 10.3390/ijms17111819.
Neuronal apoptosis in the substantia nigra par compacta (SNpc) appears to play an essential role in the pathogenesis of Parkinson's disease. However, the mechanisms responsible for the death of dopaminergic neurons are not fully understood yet. To explore the apoptotic mechanisms, we used a well-known parkinsonian toxin, 1-methyl-4-phenylpyridine (MPP⁺), to induce neuronal apoptosis in the human dopaminergic SH-SY5Y cell line. The most common method of interaction between cells is gap junctional intercellular communication (GJIC) mediated by gap junctions (GJs) formed by transmembrane proteins called connexins (Cx). Modulation of GJIC affects cell viability or growth, implying that GJIC may have an important role in maintaining homeostasis in various organs. Here, we hypothesized that increasing the level of the gap junction protein Cx43 in SH-SY5Y neuroblastoma cells could provide neuroprotection. First, our experiments demonstrated that knocking down Cx43 protein by using Cx43-specific shRNA in SH-SY5Y neuroblastoma cells potentiated MPP⁺-induced neuronal apoptosis evident from decreased cell viability. In another experiment, we demonstrated that over-expression of Cx43 in the SH-SY5Y cell system decreased MPP⁺-induced apoptosis based on the MTT assay and reduced the Bax/Bcl-2 ratio and the release of cytochrome C based on Western blot analysis. Taken together, our results suggest that Cx43 could mediate resistance against MPP⁺-induced apoptosis in SH-SY5Y neuroblastoma cells via modulating the mitochondrial apoptosis pathway.
黑质致密部（SNpc）中的神经元凋亡似乎在帕金森病的发病机制中起着至关重要的作用。然而，多巴胺能神经元死亡的机制尚未完全明确。为了探究凋亡机制，我们使用一种著名的帕金森毒素1-甲基-4-苯基吡啶（MPP⁺）在人多巴胺能SH-SY5Y细胞系中诱导神经元凋亡。细胞间最常见的相互作用方式是由连接蛋白（Cx）形成的间隙连接（GJ）介导的间隙连接细胞间通讯（GJIC）。GJIC的调节会影响细胞活力或生长，这意味着GJIC可能在维持各器官的内环境稳定中发挥重要作用。在此，我们假设提高SH-SY5Y神经母细胞瘤细胞中间隙连接蛋白Cx43的水平可以提供神经保护作用。首先，我们的实验表明，在SH-SY5Y神经母细胞瘤细胞中使用Cx43特异性短发夹RNA敲低Cx43蛋白会增强MPP⁺诱导的神经元凋亡，这从细胞活力下降中明显可见。在另一项实验中，我们证明基于MTT分析，SH-SY5Y细胞系统中Cx43的过表达降低了MPP⁺诱导的凋亡，并且基于蛋白质印迹分析降低了Bax/Bcl-2比值以及细胞色素C的释放。综上所述，我们的结果表明，Cx43可能通过调节线粒体凋亡途径介导SH-SY5Y神经母细胞瘤细胞对MPP⁺诱导凋亡的抗性。
