二甲双胍改善由C9ORF72肌萎缩侧索硬化/额颞叶痴呆相关多聚甘氨酸-精氨酸诱导的线粒体损伤

doi:10.3969/j.issn.1674-8115.2023.07.006

上海交通大学学报（医学版） ›› 2023, Vol. 43 ›› Issue (7): 839-847.doi: 10.3969/j.issn.1674-8115.2023.07.006

• 论著 · 基础研究 • 上一篇

二甲双胍改善由C9ORF72肌萎缩侧索硬化/额颞叶痴呆相关多聚甘氨酸-精氨酸诱导的线粒体损伤

冯奕源¹(), 徐忠匀¹, 尹雅芙¹, 王辉¹, 程维维¹^,²()

^1.上海交通大学医学院附属新华医院核医学科，上海 200092
^2.上海交通大学医学院附属新华医院心血管发育与再生医学研究所，上海 200092

收稿日期:2022-11-29 接受日期:2023-05-15 出版日期:2023-07-28 发布日期:2023-07-28
通讯作者: 程维维 E-mail:fengyy1019@sina.com;wcheng37@outlook.com
作者简介:冯奕源（1997—），女，硕士生；电子信箱：fengyy1019@sina.com。
基金资助:
国家自然科学基金(81901162);上海市青年科技启明星计划(20QA1406300)

Metformin ameliorates the mitochondrial damage induced by C9ORF72 amyotrophic lateral sclerosis/frontotemporal dementia-related poly-GR

FENG Yiyuan¹(), XU Zhongyun¹, YIN Yafu¹, WANG Hui¹, CHENG Weiwei¹^,²()

^1.Department of Nuclear Medicine, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China
^2.Institute for Developmental and Regenerative Cardiovascular Medicine, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China

Received:2022-11-29 Accepted:2023-05-15 Online:2023-07-28 Published:2023-07-28
Contact: CHENG Weiwei E-mail:fengyy1019@sina.com;wcheng37@outlook.com
Supported by:
National Natural Science Foundation of China(81901162);Shanghai Rising-Star Program(20QA1406300)

摘要/Abstract

摘要：

目的·探索C9ORF72肌萎缩侧索硬化/额颞叶痴呆（amyotrophic lateral sclerosis/frontotemporal dementia，ALS/FTD）相关的多聚甘氨酸-精氨酸（poly-glycine-arginine，poly-GR）对线粒体形态及功能的影响，并分析二甲双胍对由poly-GR诱导的线粒体损伤的修复作用及其可能的机制。方法·采用慢病毒感染法分别构建能够稳定表达50个重复甘氨酸-精氨酸序列［（glycine-arginine）₅₀，（GR）₅₀］和绿色荧光蛋白（green fluorescent protein，GFP）的SK-N-SH细胞，即（GR）₅₀-SK细胞株和GFP CTRL-SK细胞株。采用蛋白质印迹法（Western blotting）验证已构建细胞中（GR）₅₀蛋白的表达水平，并采用荧光显微镜观察GFP CTRL-SK细胞的GFP表达。采用碘化丙啶（propidium iodine，PI）染色分别检测（GR）₅₀-SK、GFP CTRL-SK细胞的凋亡水平。利用免疫荧光（immunofluorescence，IF）染色分析（GR）₅₀蛋白在细胞内的定位情况。采用超氧化物指示剂MitoSOX Red分别对（GR）₅₀-SK、GFP CTRL-SK细胞的氧自由基进行染色，并利用荧光显微镜观察红色荧光强度以评估线粒体活性氧水平的变化。采用透射电镜分别观察（GR）₅₀-SK、GFP CTRL-SK细胞的线粒体形态。采用Western blotting检测（GR）₅₀-SK、GFP CTRL-SK细胞的蛋白激酶B（protein kinase B，PKB，又称AKT）及其磷酸化水平。利用SC79激活（GR）₅₀-SK细胞中的AKT，并采用MitoSOX Red染色及PI染色实验分析AKT磷酸化后细胞的线粒体活性氧水平及凋亡水平。利用二甲双胍处理（GR）₅₀-SK、GFP CTRL-SK细胞，随后通过上述方法以及ATP检测试剂盒检测细胞的凋亡水平、线粒体活性氧水平、线粒体形态、AKT及其磷酸化水平、ATP浓度情况。结果·Western blotting结果提示（GR）₅₀-SK细胞构建成功，荧光显微镜的观察结果显示GFP CTRL-SK细胞构建成功。PI染色结果显示，（GR）₅₀-SK细胞较GFP CTRL-SK细胞的凋亡水平更高（P=0.016）。IF结果提示，（GR）₅₀-SK细胞中（GR）₅₀蛋白与线粒体存在部分共定位。与GFP CTRL-SK细胞相比，（GR）₅₀-SK细胞的线粒体形态及结构存在明显异常，其活性氧水平明显上升。（GR）₅₀-SK细胞中的AKT水平与GFP CTRL-SK细胞相仿，但磷酸化AKT水平显著下降。SC79处理（GR）₅₀-SK细胞后，可显著上调其AKT磷酸化水平，并下调其活性氧水平及凋亡水平。二甲双胍处理可明显上调（GR）₅₀-SK细胞中的磷酸化AKT水平，但对AKT水平无影响；可重塑该细胞中的部分线粒体形态结构、降低活性氧水平、增加ATP的生成（P=0.000），并下调细胞的凋亡水平（P=0.000）。结论·（GR）₅₀可通过下调AKT磷酸化引起线粒体形态及功能异常，并促进细胞凋亡；而二甲双胍则可抑制由（GR）₅₀蛋白诱导的上述病理事件的发生。

关键词: C9ORF72肌萎缩侧索硬化症/额颞叶痴呆, 多聚甘氨酸-精氨酸, 线粒体, 磷酸化蛋白激酶B, 二甲双胍

Abstract:

Objective ·To investigate the effect of C9ORF72 amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD)-related poly-glycine-arginine (poly-GR) on mitochondrial morphology and function, and analyze the rescue effect of metformin on mitochondrial damage induced by poly-GR and its underlying mechanism. Methods ·SK-N-SH cells stably overexpressing 50 repeated glycine-arginine sequences [(GR)₅₀] or green fluorescent protein (GFP) were constructed by lentivirus infection, which were respectively named as (GR)₅₀-SK cell line and GFP CTRL-SK cell line. (GR)₅₀ expression in (GR)₅₀-SK cells was verified by Western blotting. GFP expression in GFP GTRL-SK cells was observed by fluorescence microscope. Propidium iodide (PI) staining was used to detect the apoptosis levels of (GR)₅₀-SK and GFP CTRL-SK cells. Immunofluorescence (IF) staining was performed to determine the subcellular location of (GR)₅₀. Reactive oxygen species (ROS) level of mitochondria was evaluated by staining cells with MitoSOX Red followed by observing the intensity of red fluorescence under fluorescence microscope. The mitochondrial morphology of (GR)₅₀-SK and GFP CTRL-SK cells was observed by transmission electron microscopy. Western blotting was used to detect protein kinase B (PKB, also known as AKT) and its phosphorylation levels in (GR)₅₀-SK and GFP CTRL-SK cells. SC79 was used to activate AKT in (GR)₅₀-SK cells, and MitoSOX Red staining and PI staining were used to analyze mitochondrial ROS and apoptosis levels after phosphorylated AKT increased. Metformin was used to treat (GR)₅₀-SK and GFP CTRL-SK cells, respectively, and the apoptosis levels, mitochondrial ROS levels, mitochondrial morphology, AKT and its phosphorylation levels, and ATP concentrations of the two cells were detected by the above methods and ATP detection kit, respectively. Results ·Western blotting showed that the construction of (GR)₅₀-SK cells was successful, and fluorescence microscopy showed that the construction of GFP CTRL-SK cells was also successful. PI staining results showed that the apoptosis level of (GR)₅₀-SK cells was higher than that of the GFP CTRL-SK cells (P=0.016). IF staining results showed that there was partial co-localization of (GR)₅₀ in the mitochondria of (GR)₅₀-SK cells. Compared with GFP CTRL-SK cells, the mitochondrial morphology and structure of (GR)₅₀-SK cells were significant abnormalities, with a significantly increased ROS levels. The AKT levels in (GR)₅₀-SK cells were similar to those in the GFP CTRL-SK cells, but there was a significant decrease in phosphorylated AKT levels. After (GR)₅₀-SK cells were treated with SC79, the AKT phosphorylation level was significantly upregulated, and ROS level and apoptosis level were significantly downregulated. Metformin could significantly up-regulate the phosphorylated AKT levels in (GR)₅₀-SK cells, but had no effect on AKT levels; it could reshape the morphology and structure of some mitochondria, reduce ROS levels, increase ATP production (P=0.000), and down-regulate the level of cell apoptosis (P=0.000). Conclusion ·(GR)₅₀ can cause mitochondrial morphology and function abnormalities by down-regulating AKT phosphorylation, and promote cell apoptosis. Metformin can effectively reduce the occurrence of the above pathological events induced by (GR)₅₀.

Key words: C9ORF72 amyotrophic lateral sclerosis/ frontotemporal dementia (C9ORF72 ALS/FTD), poly-glycine-arginine (poly-GR), mitochondrion, phosphorylated protein kinase B, metformin

中图分类号:

R744.8

冯奕源, 徐忠匀, 尹雅芙, 王辉, 程维维. 二甲双胍改善由C9ORF72肌萎缩侧索硬化/额颞叶痴呆相关多聚甘氨酸-精氨酸诱导的线粒体损伤[J]. 上海交通大学学报（医学版）, 2023, 43(7): 839-847.

FENG Yiyuan, XU Zhongyun, YIN Yafu, WANG Hui, CHENG Weiwei. Metformin ameliorates the mitochondrial damage induced by C9ORF72 amyotrophic lateral sclerosis/frontotemporal dementia-related poly-GR[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(7): 839-847.

图/表 3

图1 稳转细胞株的构建及(GR)50 蛋白对细胞凋亡水平、线粒体功能、形态的影响Note： A. Detection of FLAG-tag protein expression in cells by Western blotting. B. Observation of GFP signal in GFP CTRL-SK cells by fluorescence microscope. Scale bar=50 μm. C. Detection of cell apoptosis level by PI staining. Scale bar=50 μm. D. Detection of subcellular location of (GR)50 and TOM20 or HSP90 by IF. Scale bar=1 μm. E. Detection of mitochondrial ROS level of (GR)50-SK and GFP CTRL-SK cells by MitoSOX Red superoxide indicator staining. Red fluorescence represents intracellular ROS, and blue fluorescence represents DAPI-stained nucleus. Scale bar=100 μm. F. Observation of mitochondrial morphology and structure of cells by transmission electron microscope. Scale bar=2 μm. G. Quantification analysis of the proportion of normal mitochondria and length of mitochondrial short diameter.

Fig 1 Construction of stable transfected cell lines and effect of (GR)50 on cell apoptosis levels, mitochondrial function and morphology

图2 (GR)50 对AKT磷酸化水平的影响及调控AKT磷酸化对线粒体ROS水平、细胞凋亡水平的影响Note： A. Detection of the expression levels of AKT and phosphorylated AKT in cells by Western blotting. B. Detection of the effect of SC79 on AKT and phosphorylated AKT levels in (GR)50-SK cells by Western blotting. C. Detection of the effect of SC79 on mitochondrial ROS level of (GR)50-SK cells by MitoSOX Red superoxide indicator staining. Red fluorescence represents intracellular ROS, and blue fluorescence represents DAPI-stained nucleus. Scale bar=10 μm. D. Detection of the effect of SC79 on cell apoptosis level by PI staining. Scale bar=100 μm.

Fig 2 Effect of (GR)50 on AKT phosphorylation level and the effect of regulating AKT phosphorylation on mitochondrial ROS level and cell apoptosis level

图3 二甲双胍对由(GR)50 诱导的AKT磷酸化水平异常、线粒体形态及功能损伤、细胞凋亡水平的影响Note： A. Detection of the expression level of AKT and phosphorylated AKT in cells of different groups by Western blotting. B. Detection of the effect of metformin on mitochondrial ROS level of cells by MitoSOX Red superoxide indicator staining. Red fluorescence represents intracellular ROS, and blue fluorescence represents DAPI-stained nucleus. Scale bar=100 μm. C. Observation of mitochondrial morphology of (GR)50-SK cells treated with metformin by transmission electron microscope. Scale bar=100 μm. D. Quantification analysis of the proportion of normal mitochondria and length of mitochondrial short diameter of (GR)50-SK cells. E. Detection of the effect of metformin on ATP level in cells of different groups by ATP detection assay. F. Detection of the effect of metformin on apoptosis levels in cells of different groups by PI staining. Met—metformin.

Fig 3 Effects of metformin on (GR)50-induced abnormal AKT phosphorylation level, mitochondrial morphology and function impairment and cell apoptosis levels

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二甲双胍改善由C9ORF72肌萎缩侧索硬化/额颞叶痴呆相关多聚甘氨酸-精氨酸诱导的线粒体损伤

Metformin ameliorates the mitochondrial damage induced by C9ORF72 amyotrophic lateral sclerosis/frontotemporal dementia-related poly-GR

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