Abstract
Background
Leber’s hereditary optic neuropathy (LHON) is a maternally inherited eye disease due to mutations in mitochondrial DNA. However, there is no effective treatment for this disease. LHON-linked ND6 14484T > C (p.M64V) mutation caused complex I deficiency, diminished ATP production, increased production of reactive oxygen species (ROS), elevated apoptosis, and impaired mitophagy. Here, we investigated if the allotopic expression of human mitochondrial ND6 transgene corrected the mitochondrial dysfunctions due to LHON-associated m.14484T > C mutation.
Methods
Nucleus-versions of ND6 was generated by changing 6 non-universal codons with universal codons and added to mitochondrial targeting sequence of COX8. Stable transfectants were generated by transferring human ND6 cDNA expressed in a pCDH-puro vector into mutant cybrids carrying the m.14484T > C mutation and control cybrids. The effect of allotopic expression of ND6 on oxidative phosphorylation (OXPHOS) was evaluated using Blue Native gel electrophoresis and extracellular flux analyzer. Assessment of ROS production in cell lines was performed by flow cytometry with MitoSOX Red reagent. Analyses for apoptosis and mitophagy were undertaken via flow cytometry, TUNEL and immunofluorescence assays.
Results
The transfer of human ND6 into the cybrids carrying the m.14484T > C mutation raised the levels of ND6, ND1 and ND4L but did not change the levels of other mitochondrial proteins. The overexpression of ND6 led to 20~23% increases in the assembly and activity of complex I, and ~ 53% and ~ 33% increases in the levels of mitochondrial ATP and ΔΨm in the mutant cybrids bearing m.14484T > C mutation. Furthermore, mutant cybrids with overexpression of ND6 exhibited marked reductions in the levels of mitochondrial ROS. Strikingly, ND6 overexpression markedly inhibited the apoptosis process and restored impaired mitophagy in the cells carrying m.14484T > C mutation. However, overexpression of ND6 did not affect the ND6 level and mitochondrial functions in the wild-type cybrids, indicating that this ND6 level appeared to be the maximum threshold level to maintain the normal cell function.
Conclusion
We demonstrated that allotopic expression of nucleus-versions of ND6 restored complex I, apoptosis and mitophagy deficiencies caused by the m.14484T > C mutation. The restoration of m.14484T > C mutation-induced mitochondrial dysfunctions by overexpression of ND6 is a step toward therapeutic interventions for LHON and mitochondrial diseases.
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Introduction
Leber’s hereditary optic neuropathy (LHON) is the most common maternally inherited eye disease that presents with the loss of central vision in young adults, due to the degeneration of retinal ganglion cells and their axons [1,2,3,4,5,6]. The majority of LHON cases globally results from one of three mitochondrial DNA (mtDNA) mutations (ND1 3460G > A, ND4 11778G > A, and ND6 14484T > C), which affects the essential subunits of complex I (NADH: ubiquinone oxidoreductase) [7,8,9,10,11,12,13]. These mtDNA mutations resulted in the complex I deficiency, diminished ATP synthesis and an increasing generation of reactive oxygen species (ROS) [14,15,16,43]. The improvement of both OXPHOS and ΔΨm would reduce the production of ROS [36]. In this investigation, mutant cell lines with overexpression of ND6 exhibited marked reductions in the levels of mitochondrial ROS and three antioxidant enzymes, SOD2 in the mitochondrion and SOD1 and catalase in the cytosol catalase. The lower production of ROS can reduce a vicious cycle of oxidative stress in the mitochondria, thereby decreasing the damage of mitochondrial and cellular proteins, lipids and nuclear acids [50].
Mitochondrial dysfunctions caused by LHON-associated mtDNA mutations impaired the cell viability and affected the apoptotic process as well as mitophagy [19, 20, 45, 51,52,53]. In particular, mutant cybrids bearing the m.14484T > C mutation exhibited increasing ratio of Annexin V-positive cells and elevated releases of cytochrome c into cytosol than those in control cybrids [20]. In the present study, we demonstrated that the overexpression of ND6 suppressed the m.14484T > C mutation-induced the impairment of apoptosis. Lines of evidence from Annexin V/PI-based flow cytometry, TUNEL and immunocytostaining assays indicated much less apoptosis in the mutant cybrids with overexpression of ND6 than those in the parental mutant cybrids. These were further supported by decreasing levels in cytochrome c, and BAX which mediate cell death by apoptosis [54], raising expressions of Bcl-xL which has anti-apoptotic activity [55], and rescuing levels of apoptosis activated proteins: caspases 7, 9, and 3 in the mutant cybrids with overexpression of ND6, as compared with than those in the parental mutant cybrids [56, 57]. Notably, the levels of apoptosis in the mutant cybrids with overexpression of ND6 were comparable with those in the control cybrids. Furthermore, our previous study showed that the m.14484T > C mutation impaired the mitophagy [20]. In this study, we demonstrated that the overexpression of ND6 restored impaired mitophagy due to the m.14484T > C mutation by immunocytostaining and Western blot analyses. In particular, the average levels of LC3II/I + II, P62, PINK1 and Parkin in the mutant cybrids expressing nucleus-version of ND6 were significantly elevated, as compared with these in the parental mutant cybrids. These data indicated that overexpression of human ND6 reversed the abnormal cell apoptosis and mitophagy which caused by m.14484T > C mutation. Therefore, the overexpression of ND6 in mutant cells may reprogram energy metabolism and prevent the dysfunction or death of retinal ganglion cells due to the m.14484T > C mutation [58].
Conclusion
In this investigation, we demonstrated that allotopic expression of human ND6 restored complex I, apoptosis and mitophagy deficiencies caused by LHON-linked m.14484T > C mutation. The restoration of m.14484T > C mutation-induced mitochondrial dysfunctions by overexpression of ND6 is a step toward therapeutic interventions for LHON and other mitochondrial diseases.
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Acknowledgements
This paper is dedicated to the memory of Dr. Yi Tong, former Professor of Fujian Medical University, a pioneer Chinese clinician in LHON. We are grateful to all patients and family members for their participation.
Funding
This work was supported by the Grants 2021YFC2700900 from National Key Research and Development Program of China (M.X.G), 31970557 (M.X.G) and 82171847(J.Y.) from the National Natural Science Foundation of China.
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MXG designed the experiments, monitored the project progression, data analysis and interpretation. JW, YJ, CA, JRC and DG performed the biochemical analyses, JW, JZ and JQM performed the data analysis. JW and YJ prepared the initial draft of the manuscript. MXG made the final version of the manuscript. All authors reviewed the manuscript.
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Wang, J., Ji, Y., Ai, C. et al. Optimized allotopic expression of mitochondrial ND6 transgene restored complex I and apoptosis deficiencies caused by LHON-linked ND6 14484T > C mutation. J Biomed Sci 30, 63 (2023). https://doi.org/10.1186/s12929-023-00951-1
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DOI: https://doi.org/10.1186/s12929-023-00951-1