Alu RNA激活溶酶体TRPML1通道清除过剩活性氧从而延缓细胞衰老

The abilities of fighting stress and disease gradually decline and a variety of age-related diseases occur during human aging process. It has been proved that Alu RNA participated in the formation of stress granules. Our previous works have showed that Alu RNA regulated gene expression and prolonged the culture passage numbers of human fibroblasts. Reactive oxygen species (ROS) are produced mainly as byproducts of mitochondrial respiration, and their cytosolic levels are tightly controlled by multiple anti-oxidant mechanisms. High levels of ROS may additionally cause severe oxidative damage of proteins and lipids, the aging rate has been associated with the production of high levels of ROS. Lysosomes are organelles that ‘host’ important nutrient-sensitive molecules. Lysosomes are required for quality-control regulation of mitochondria and recent studies suggest that mitochondria, the major source of endogenous ROS, are localized in close physical proximity to lysosomes. Lipoprotein transient receptor potential channel 1(TRPML1) is a ROS sensor localized on the lysosomal membrane that orchestrates an autophagy-dependent programme to alleviate oxidative stress in the cell. Lysosomal Ca2+ release is believed to be mediated by TRPML1, a key Ca2+-conducting channel on the lysosomal membrane that releases Ca2+ from the lumen into the cytosol. Lysosomal Ca2+ release leads to a decrease in the phosphorylation of transcription factor (TF)EB, a master transcriptional regulator of both autophagy and lysosomal biogenesis. Dephosphorylated TFEB proteins translocate rapidly to the nucleus from the cytosol and lysosomes, inducing the expression of a unique set of genes that are related specifically to autophagosome and lysosome biogenesis. In this project, we will prove whether Alu RNA can remove excess ROS and alleviate damage of mitochondrial DNA in cultured senescent human fibroblasts; we will use patch-clamp methods to investigate whether Alu RNA activate whole-endolysosome TRPML1-mediated currents (ITRPML1) and then lead to lysosomal Ca2+ release and improve lysosomal autophagy functions; we will prove whether Alu RNA induce calcineurin-dependent TFEB-nuclear translocation and increases of LC3-II expression that participate in autophagy formation by using western blotting method.

衰老过程使人体对抗压力和疾病的能力下降，出现多种年龄相关疾病。已经证实Alu RNA参与压力颗粒的形成，调节基因表达，延长人成纤维细胞培养代数。活性氧（ROS）是线粒体呼吸作用的副产品，高水平ROS导致细胞衰老。溶酶体参与线粒体质量控制的调节，通过自我吞噬抑制ROS产生。TRPML1是位于溶酶体膜上的ROS传感器，协调自我吞噬依赖的机制减缓细胞的氧化应激。TRPML1通道介导钙离子释放，溶酶体钙释放刺激转录因子TFEB核转位，激活一系列基因表达，引起自噬性溶酶体形成。本项目预证明Alu RNA是否能清除衰老的人成纤维细胞中过剩的ROS缓解线粒体DNA损伤；用膜片钳技术证实Alu RNA是否激活衰老细胞的溶酶体TRPML1通道引起钙释放，提升溶酶体自噬功能；用Western杂交证明Alu RNA是否通过激活TRPML1通道介导TFEB转录因子核转位，提升参与自噬形成的LC3-Ⅱ的表达。

1、.项目的背景. 人口老龄化是全球性问题。已经证明高水平活性氧（ROS）的产生在细胞衰老的起始和维持中起关键作用。干预细胞中过剩的ROS，清除自由基，增加抗氧化防御能力，是目前广泛采用的抗衰老策略。Alu重复序列是人基因组中最重要的重复序列，这些Alu序列被RNA聚合酶Ⅲ转录为Alu RNA。我们的前期研究证明转染Alu RNA能延长培养的人成纤维细胞培养代数（即延缓人成纤维细胞衰老）。.2、.主要研究内容.（1）证明Alu RNA是否降低衰老的人成纤维细胞（高培养代数细胞）中ROS的水平。（2）观察Alu RNA是否改善衰老的人成纤维细胞线粒体DNA（mtDNA）的损伤。（3）观察Alu RNA是否改善衰老的人成纤维细胞的线粒体膜电位。（4）证实Alu RNA是否激活衰老的人成纤维细胞溶酶体的自我吞噬。（5）观察Alu RNA是否改变衰老的人成纤维细胞LC3-II / I的比值。（6）证实Alu RNA是否激活衰老的人成纤维细胞溶酶体的TRPML1通道。（7）证实Alu RNA是否通过TRPML1触发衰老的人成纤维细胞溶酶体的钙释放。（8）证明Alu RNA的处理是否改变衰老的人成纤维细胞的溶酶体Ph值。（9）证实Alu RNA延缓成纤维细胞衰老是通过TRPML1通道介导TFEB转录因子活化。.3、.重要结果和关键数据.（1）已经制备出了基因工程人源Alu RNA，人源Alu反义RNA （Aluas RNA），鼠源B1 RNA和鼠源B1反义RNA（B1as RNA）。（2）证明了B1as RNA通过降低氧化损伤以及调节衰老相关基因的表达从而发挥延缓小鼠衰老的作用。（3）证明了Aluas RNA改善丙酮醛引起的人晶状体上皮细胞的凋亡是通过激活了 Nrf2/ARE信号途径。（4）证明了B1as RNA可以改善老年小鼠的年龄相关性白内障，是通过调节p53信号途径。这些研究结果已经总结在巴基斯坦博士生的毕业论文中。（5）正式发表SCI论文3篇，英文论文2篇，核心期刊论文1篇，参加学术会议2次。另2篇SCI论文正在审稿中。.4、.科学意义. 本项目探讨了Aluas RNA以及B1as RNA延缓衰老及治疗年龄相关性白内障的分子机理。这些研究将为Aluas RNA提高人类寿命和改善人体衰老症状，成为有效的核酸制剂提供理论依据。