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

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.

Keywords

• c9orf72 amyotrophic lateral sclerosis/ frontotemporal dementia (c9orf72 als/ftd)
• poly-glycine-arginine (poly-gr)
• mitochondrion
• phosphorylated protein kinase b
• metformin