Division of Pharmacology, Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Samsung Biomedical Research Institute, Suwon, Gyeonggi-do 440-746, Korea.
College of Pharmacy, Chosun University, Gwangju 501-759, Korea.
Int J Mol Sci. 2017 Oct 3;18(10):2093. doi: 10.3390/ijms18102093.
Aging is considered the major risk factor for neurodegenerative diseases including Parkinson's disease (PD). Telomere shortening is associated with cellular senescence. In this regard, pharmacological or genetic inhibition of telomerase activity has been used to model cellular aging. Here, we employed CRISPR-Cas9 technology to instantly remove the telomere to induce aging in a neuroblastoma cell line. Expression of both Cas9 and guide RNA targeting telomere repeats ablated the telomere, leading to retardation of cell proliferation. Instant deletion of telomere in SH-SY5Y cells impaired mitochondrial function with diminished mitochondrial respiration and cell viability. Supporting the pathological relevance of cell aging by CRISPR-Cas9 mediated telomere removal, alterations were observed in the levels of PD-associated proteins including PTEN-induced putative kinase 1, peroxisome proliferator-activated receptor γ coactivator 1-α, nuclear respiratory factor 1, parkin, and aminoacyl tRNA synthetase complex interacting multifunctional protein 2. Significantly, α-synuclein expression in the background of telomere removal led to the enhancement of protein aggregation, suggesting positive feed-forward interaction between aging and PD pathogenesis. Collectively, our results demonstrate that CRISPR-Cas9 can be used to efficiently model cellular aging and PD.
衰老是包括帕金森病（PD）在内的神经退行性疾病的主要风险因素。端粒缩短与细胞衰老有关。在这方面，端粒酶活性的药理学或遗传学抑制已被用于模拟细胞衰老。在这里，我们使用 CRISPR-Cas9 技术即时去除端粒以诱导神经母细胞瘤细胞系衰老。表达 Cas9 和靶向端粒重复序列的指导 RNA 可消除端粒，导致细胞增殖减缓。SH-SY5Y 细胞中端粒的即时缺失损害了线粒体功能，导致线粒体呼吸和细胞活力下降。CRISPR-Cas9 介导的端粒去除导致 PD 相关蛋白水平发生变化，包括 PTEN 诱导的假定激酶 1、过氧化物酶体增殖物激活受体 γ 共激活因子 1-α、核呼吸因子 1、Parkin 和氨酰 tRNA 合成酶复合物相互作用多功能蛋白 2。值得注意的是，端粒去除背景下的 α-突触核蛋白表达导致蛋白聚集增强，表明衰老和 PD 发病机制之间存在正反馈相互作用。总之，我们的结果表明，CRISPR-Cas9 可用于有效地模拟细胞衰老和 PD。
