Skin fibroblasts derived from Leber′s hereditary optic neuropathy patients present mitochondrial dysfunction_Chinese Journal of Ocular Fundus Diseases

Authors：

Yao Shun , Yang Mingzhu , Li Ya ,  Lei Bo

Henan Provincial People's Hospital, Henan Provincial Eye Hospital, Zhengzhou 450003, China;

Corresponding author：

Lei Bo, Email: bolei99@126.com

Keywords：

Optic atrophy, hereditary, Leber; Fibroblasts; Skin; Mitochondria; Cell survival

DOI：

10.3760/cma.j.cn511434-20220627-00385

Video：

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Objective To investigate the feasibility of immoribund skin fibroblast cell line derived from Leber′s hereditary optic neuropathy (LHON) patients as a cell model. Methods A basic research. Two LHON patients and 2 healthy volunteers were recruited from Department of Ophthalmology of Genetic Clinic of Henan Provincial Eye Hospital. The skin tissue of participants was obtained, and the 4 immortalized skin fibroblasts were constructed by SV40 virus infection, including 2 LHON patient cells (LHON-1 and LHON-2 cells) and 2 healthy volunteers cells (NC-1 and NC-2 cells). Mitochondrial morphology in cells was observed by electron microscope. The levels of reactive oxygen species (ROS), nicotinamide adenine dinucleotide-oxidation state (NAD⁺), nicotinamide adenine dinucleotide-reduction state (NADH) and adenosine triphosphate (ATP) in fibroblasts were detected. Cellular oxygen consumption was measured by seahorse mitochondrial pressure assay. Cell viability was detected using cell counting kit-8 (CCK8). One-way ANOVA was performed to compare the levels of ROS, NAD⁺, NADH and ATP in LHON and NC cells, as well as basal oxygen consumption, maximal oxygen consumption, ATP-coupled oxygen consumption, and cell viability. Results Compared with NC-1 and NC-2, the number of mitochondrial crest in LHON fibroblasts was significantly reduced, indicating abnormal mitochondrial morphology. Biochemical analysis showed that ROS levels in LHON cells increased, but NAD⁺/NADH and ATP levels decreased, and the oxygen consumption was significantly inhibited, indicating the presence of mitochondrial damage and respiratory dysfunction. The results of CCK-8 detection showed that the survival ability of LHON-1 and LHON-2 cells was worse under stress conditions. Conclusion Immortalized skin fibroblast cell lines from LHON patients presented mitochondrial dysfunction.

Citation： Yao Shun, Yang Mingzhu, Li Ya, Lei Bo. Skin fibroblasts derived from Leber′s hereditary optic neuropathy patients present mitochondrial dysfunction. Chinese Journal of Ocular Fundus Diseases, 2022, 38(8): 675-680. doi: 10.3760/cma.j.cn511434-20220627-00385 Copy

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10.	Yu-Wai-Man P, Griffiths PG, Chinnery PF. Mitochondrial optic neuropathies-disease mechanisms and therapeutic strategies[J]. Prog Retin Eye Res, 2011, 30(2): 81-114. DOI: 10.1016/j.preteyeres.2010.11.002.
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20.	Ji Y, Zhang J, Yu J, et al. Contribution of mitochondrial ND1 3394T>C mutation to the phenotypic manifestation of Leber's hereditary optic neuropathy[J]. Hum Mol Genet, 2019, 28: 1515-1529. DOI: 10.1093/hmg/ddy450.
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24.	Civiletto G, Varanita T, Cerutti R, et al. Opa1 overexpression ameliorates the phenotype of two mitochondrial disease mouse models[J]. Cell Metab, 2015, 21(6): 845-854. DOI: 10.1016/j.cmet.2015.04.016.
25.	Carelli V, Ross-Cisneros FN, Sadun AA. Mitochondrial dysfunction as a cause of optic neuropathies[J]. Prog Retin Eye Res, 2004, 23(1): 53-89. DOI: 10.1016/j.preteyeres.2003.10.003.

1. Yu-Wai-Man P, Griffiths PG, Brown DT, et al. The epidemiology of Leber hereditary optic neuropathy in the North East of England[J]. Am J Hum Genet, 2003, 72(2): 333-339. DOI: 10.1086/346066.
2. Wallace DC, Singh G, Lott MT, et al. Mitochondrial DNA mutation associated with Leber's hereditary optic neuropathy[J]. Science, 1988, 242(4884): 1427-1430. DOI: 10.1126/science.3201231.
3. Johns DR, Neufeld MJ, Park RD. An ND-6 mitochondrial DNA mutation associated with Leber hereditary optic neuropathy[J]. Biochem Biophys Res Commun, 1992, 187(3): 1551-1557. DOI: 10.1016/0006-291x(92)90479-5.
4. Huoponen K, Vilkki J, Aula P, et al. A new mtDNA mutation associated with Leber hereditary optic neuroretinopathy[J]. Am J Hum Genet, 1991, 48(6): 1147-1153.
5. 杨雪莹, 陈长征. Leber遗传性视神经病变的细胞及动物模型应用研究进展[J]. 中华眼底病杂志, 2021, 37(10): 825-830. DOI: 10.3760/cma.j.cn511434-20210207-00073.Yang XY, Chen CZ. Research progress in cell and animal models of Leber hereditary optic neuropathy[J]. Chin J Ocul Fundus Dis, 2021, 37(10): 825-830. DOI: 10.3760/cma.j.cn511434-20210207-00073.
6. Kishita Y, Ishikawa K, Nakada K, et al. A high mutation load of m.14597A>G in MT-ND6 causes Leigh syndrome[J/OL]. Sci Rep, 2021, 11(1): 11123[2021-05-27]. https://pubmed.ncbi.nlm.nih.gov/34045482/. DOI: 10.1038/s41598-021-90196-5.
7. Morvan D, Demidem A. NMR metabolomics of fibroblasts with inherited mitochondrial complex Ⅰ mutation reveals treatment-reversible lipid and amino acid metabolism alterations[J]. Metabolomics, 2018, 14(5): 55. DOI: 10.1007/s11306-018-1345-9.
8. Uittenbogaard M, Brantner CA, Fang Z, et al. The m. 11778A>G variant associated with the coexistence of Leber's hereditary optic neuropathy and multiple sclerosis-like illness dysregulates the metabolic interplay between mitochondrial oxidative phosphorylation and glycolysis[J]. Mitochondrion, 2019, 46: 187-194. DOI: 10.1016/j.mito.2018.06.001.
9. Yao S, Zhou QR, Yang MZ, et al. Multi-mtDNA variants may be a factor contributing to mitochondrial function variety in the skin-derived fibroblasts of Leber's hereditary optic neuropathy patients[J/OL]. Front Mol Neurosci, 2022, 15: 920221[2022-07-13]. https://pubmed.ncbi.nlm.nih.gov/35909448/. DOI: 10.3389/fnmol.2022.920221.
10. Yu-Wai-Man P, Griffiths PG, Chinnery PF. Mitochondrial optic neuropathies-disease mechanisms and therapeutic strategies[J]. Prog Retin Eye Res, 2011, 30(2): 81-114. DOI: 10.1016/j.preteyeres.2010.11.002.
11. Lin CS, Sharpley MS, Fan W, et al. Mouse mtDNA mutant model of Leber hereditary optic neuropathy[J]. Proc Natl Acad Sci USA, 2012, 109(49): 20065-20070. DOI: 10.1073/pnas.1217113109.
12. Yang X, Jiang J, Li Z, et al. Strategies for mitochondrial gene editing[J]. Comput Struct Biotechnol J, 2021, 19: 3319-3329. DOI: 10.1016/j.csbj.2021.06.003.
13. Gammage PA, Rorbach J, Vincent AI, et al. Mitochondrially targeted ZFNs for selective degradation of pathogenic mitochondrial genomes bearing large-scale deletions or point mutations[J]. EMBO Mol Med, 2014, 6(4): 458-466. DOI: 10.1002/emmm.201303672.
14. Komor AC, Kim YB, Packer MS, et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage[J]. Nature, 2016, 533(7603): 420-424. DOI: 10.1038/nature17946.
15. Yahata N, Matsumoto Y, Omi M, et al. TALEN-mediated shift of mitochondrial DNA heteroplasmy in MELAS-iPSCs with m.13513G>A mutation[J/OL]. Sci Rep, 2017, 7(1): 15557[2017-11-14]. https://pubmed.ncbi.nlm.nih.gov/29138463/. DOI: 10.1038/s41598-017-15871-y.
16. Giordano L, Deceglie S, d'Adamo P, et al. Cigarette toxicity triggers Leber's hereditary optic neuropathy by affecting mtDNA copy number, oxidative phosphorylation and ROS detoxification pathways[J/OL]. Cell Death Dis, 2015, 6(12): e2021[2015-12-17]. https://pubmed.ncbi.nlm.nih.gov/26673666/. DOI: 10.1038/cddis.2015.364.
17. Catarino CB, Ahting U, Gusic M, et al. Characterization of a Leber's hereditary optic neuropathy (LHON) family harboring two primary LHON mutations m. 11778G>A and m. 14484T>C of the mitochondrial DNA[J]. Mitochondrion, 2017, 36: 15-20. DOI: 10.1016/j.mito.2016.10.002.
18. Falabella M, Forte E, Magnifico MC, et al. Evidence for detrimental cross interactions between reactive oxygen and nitrogen species in Leber's hereditary optic neuropathy cells[J/OL]. Oxid Med Cell Longev, 2016, 2016: 3187560[2015-12-31]. https://pubmed.ncbi.nlm.nih.gov/26881022/. DOI: 10.1155/2016/3187560.
19. Brown MD, Trounce IA, Jun AS, et al. Functional analysis of lymphoblast and cybrid mitochondria containing the 3460, 11778, or 14484 Leber's hereditary optic neuropathy mitochondrial DNA mutation[J]. J Biol Chem, 2000, 275(51): 39831-39836. DOI: 10.1074/jbc.M006476200.
20. Ji Y, Zhang J, Yu J, et al. Contribution of mitochondrial ND1 3394T>C mutation to the phenotypic manifestation of Leber's hereditary optic neuropathy[J]. Hum Mol Genet, 2019, 28: 1515-1529. DOI: 10.1093/hmg/ddy450.
21. Zhang J, Jiang P, Jin X, et al. Leber's hereditary optic neuropathy caused by the homoplasmic ND1 m. 3635G>A mutation in nine Han Chinese families[J]. Mitochondrion, 2014, 18: 18-26. DOI: 10.1016/j.mito.2014.08.008.
22. Hung SS, Van Bergen NJ, Jackson S, et al. Study of mitochondrial respiratory defects on reprogramming to human induced pluripotent stem cells[J]. Aging, 2016, 8(5): 945-957. DOI: 10.18632/aging.100950.
23. Yang TC, Yarmishyn AA, Yang YP, et al. Mitochondrial transport mediates survival of retinal ganglion cells in affected LHON patients[J]. Hum Mol Genet, 2020, 29(9): 1454-1464. DOI: 10.1093/hmg/ddaa063.
24. Civiletto G, Varanita T, Cerutti R, et al. Opa1 overexpression ameliorates the phenotype of two mitochondrial disease mouse models[J]. Cell Metab, 2015, 21(6): 845-854. DOI: 10.1016/j.cmet.2015.04.016.
25. Carelli V, Ross-Cisneros FN, Sadun AA. Mitochondrial dysfunction as a cause of optic neuropathies[J]. Prog Retin Eye Res, 2004, 23(1): 53-89. DOI: 10.1016/j.preteyeres.2003.10.003.

Chinese Journal of Ocular Fundus Diseases

Skin fibroblasts derived from Leber′s hereditary optic neuropathy patients present mitochondrial dysfunction

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