Abstract
Mutations in genes affecting mitochondrial proteins are increasingly recognised in patients with epilepsy, but the factors determining cell fate during seizure activity in these mutations remain unknown. Fluorescent dye imaging techniques were applied to fibroblast cell lines from patients suffering from common mitochondrial mutations and to age-matched controls. Using live cell imaging techniques in fibroblasts, we show that fibroblasts with mutations in the mitochondrial genome had reduced mitochondrial membrane potential and NADH pools and higher redox indices, indicative of respiratory chain dysfunction. Increasing concentrations of ferutinin, a Ca2+ ionophore, led to oscillatory Ca2+ signals in fibroblasts resembling dynamic Ca2+ changes that occur during seizure-like activity. Co-monitoring of mitochondrial membrane potential (ΔΨm) changes induced by ferutinin showed accelerated membrane depolarisation and cell collapse in fibroblasts with mutations in the mitochondrial genome when compared to controls. Ca2+ flash photolysis using caged Ca2+ confirmed impaired Ca2+ handling in fibroblasts with mitochondrial mutations. Findings indicate that intracellular Ca2+ levels cannot be compensated during periods of hyperexcitability, leading to Ca2+ overload and subsequent cell death in mitochondrial diseases.
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19 May 2018
The original version of this article unfortunately contained mistake. The author’s family name “Kov ac” was written with space thus this should be corrected to “Kovac”.
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Acknowledgements
The authors would like to thank the MRC Centre for Neuromuscular Diseases BioBank London for providing the additional fibroblast lines for this study.
Funding
This work was supported by the Medical Faculty of the University of Münster (17-003 fellowship to SK) and the NIHR Queen Square Dementia Biomedical Research Unit. This work was undertaken at UCLH/UCL which receives a proportion of funding from the Department of Health’s NIHR Biomedical Research Centres funding scheme.
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SK and AYA conceived the study. SK performed and designed the experiments. EP and HH contributed toward the experiments. SK and AYA analysed the data. SK, AYA and MCW wrote the manuscript. All authors critically discussed the data and approved the final version of the manuscript.
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The original version of this article was revised to correct the author's family name “Kov ac” to “Kovac”.
Electronic supplementary material
Supplementary Figure 1
Mitochondrial mass. Bar chart summarizing mitochondrial mass in different fibroblast cell lines. Mitochondrial mass is expressed as mitochondrial volume / total cell volume (%). (GIF 59 kb)
Supplementary Figure 2
Method used to determine MPTP opening in fibroblasts treated with ferutinin. Traces represent mean (± SEM) TMRM fluorescence changes measured over mitochondria (solid triangles) and the nucleus (open triangle). Fluorescence measured over mitochondria show depolarization of ΔΨm in a stepwise fashion. Once a cumulative concentration of 13.5 μM ferutinin is reached, depolarization ΔΨm is accelerated as suggested by the change in the slope of TMRM fluorescence. This change indicates mitochondrial permeability transition pore opening. Due to redistribution of the dye, this appears to coincide with peak TMRM fluorescence in the nucleus. Nuclear peak fluorescence was therefore used to determine MPTP opening. (GIF 216 kb)
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Kovac, S., Preza, E., Houlden, H. et al. Impaired Bioenergetics in Mutant Mitochondrial DNA Determines Cell Fate During Seizure-Like Activity. Mol Neurobiol 56, 321–334 (2019). https://doi.org/10.1007/s12035-018-1078-9
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DOI: https://doi.org/10.1007/s12035-018-1078-9