Journal of Shanghai Jiao Tong University (Medical Science) >

2023 , Vol. 43 >Issue 7: 839 - 847

DOI: https://doi.org/10.3969/j.issn.1674-8115.2023.07.006

Basic research

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

  • Yiyuan FENG ,
  • Zhongyun XU ,
  • Yafu YIN ,
  • Hui WANG ,
  • Weiwei CHENG
Expand
  • 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
CHENG Weiwei, E-mail: wcheng37@outlook.com.

Received date: 2022-11-29

  Accepted date: 2023-05-15

  Online published: 2023-07-28

Supported by

National Natural Science Foundation of China(81901162);Shanghai Rising-Star Program(20QA1406300)

Fold

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

Cite this article

Yiyuan FENG , Zhongyun XU , Yafu YIN , Hui WANG , Weiwei CHENG . 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 . DOI: 10.3969/j.issn.1674-8115.2023.07.006

References

1 GIJSELINCK I, VAN LANGENHOVE T, VAN DER ZEE J, et al. A C9orf72 promoter repeat expansion in a Flanders-Belgian cohort with disorders of the frontotemporal lobar degeneration-amyotrophic lateral sclerosis spectrum: a gene identification study[J]. Lancet Neurol, 2012, 11(1): 54-65.
2 KWON I, XIANG S H, KATO M, et al. Poly-dipeptides encoded by the C9orf72 repeats bind nucleoli, impede RNA biogenesis, and kill cells[J]. Science, 2014, 345(6201): 1139-1145.
3 TAO Z T, WANG H F, XIA Q, et al. Nucleolar stress and impaired stress granule formation contribute to C9orf72 RAN translation-induced cytotoxicity[J]. Hum Mol Genet, 2015, 24(9): 2426-2441.
4 SABERI S, STAUFFER J E, JIANG J, et al. Sense-encoded poly-GR dipeptide repeat proteins correlate to neurodegeneration and uniquely co-localize with TDP-43 in dendrites of repeat-expanded C9orf72 amyotrophic lateral sclerosis[J]. Acta Neuropathol, 2018, 135(3): 459-474.
5 LOPEZ-GONZALEZ R, LU Y B, GENDRON T F, et al. Poly(GR) in C9ORF72-related ALS/FTD compromises mitochondrial function and increases oxidative stress and DNA damage in iPSC-derived motor neurons[J]. Neuron, 2016, 92(2): 383-391.
6 ZHANG Y J, GENDRON T F, EBBERT M T W, et al. Poly(GR) impairs protein translation and stress granule dynamics in C9orf72-associated frontotemporal dementia and amyotrophic lateral sclerosis[J]. Nat Med, 2018, 24(8): 1136-1142.
7 LI S, WU Z, TANTRAY I, et al. Quality-control mechanisms targeting translationally stalled and C-terminally extended poly(GR) associated with ALS/FTD[J]. Proc Natl Acad Sci USA, 2020, 117(40): 25104-25115.
8 GAYATRI M B, GAJULA N N, CHAVA S, et al. High glutamine suppresses osteogenesis through mTORC1-mediated inhibition of the mTORC2/AKT-473/RUNX2 axis[J]. Cell Death Discov, 2022, 8(1): 277.
9 LESNIK C, GOLANI-ARMON A, ARAVA Y. Localized translation near the mitochondrial outer membrane: an update[J]. RNA Biol, 2015, 12(8): 801-809.
10 CHOI S Y, LOPEZ-GONZALEZ R, KRISHNAN G, et al. C9ORF72-ALS/FTD-associated poly(GR) binds Atp5a1 and compromises mitochondrial function in vivo[J]. Nat Neurosci, 2019, 22(6): 851-862.
11 LI S, WU Z, LI Y, et al. Altered MICOS morphology and mitochondrial ion homeostasis contribute to poly(GR) toxicity associated with C9-ALS/FTD[J]. Cell Rep, 2020, 32(5): 107989.
12 XIE X X, SHU R N, YU C N, et al. Mammalian AKT, the emerging roles on mitochondrial function in diseases[J]. Aging Dis, 2022, 13(1): 157-174.
13 WANG H, CHEN S, ZHANG Y, et al. Electroacupuncture ameliorates neuronal injury by Pink1/Parkin-mediated mitophagy clearance in cerebral ischemia-reperfusion[J]. Nitric Oxide, 2019, 91: 23-34.
14 WANG S, KANDADI M R, REN J. Double knockout of Akt2 and AMPK predisposes cardiac aging without affecting lifespan: role of autophagy and mitophagy[J]. Biochim Biophys Acta Mol Basis Dis, 2019, 1865(7): 1865-1875.
15 MOR D E, SOHRABI S, KALETSKY R, et al. Metformin rescues Parkinson's disease phenotypes caused by hyperactive mitochondria[J]. Proc Natl Acad Sci USA, 2020, 117(42): 26438-26447.
16 ZU T, GUO S, BARDHI O, et al. Metformin inhibits RAN translation through PKR pathway and mitigates disease in C9orf72 ALS/FTD mice[J]. Proc Natl Acad Sci USA, 2020, 117(31): 18591-18599.
Options
Outlines

/

〈 〉