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. 2024 Nov 30;15(11):870.
doi: 10.1038/s41419-024-07165-9.

OPA1 and disease-causing mutants perturb mitochondrial nucleoid distribution

J Macuada  1 , I Molina-Riquelme  1 , G Vidal  2   3 , N Pérez-Bravo  1 , C Vásquez-Trincado  1 , G Aedo  1   2 , D Lagos  1   4 , P Yu-Wai-Man  4   5   6   7 , R Horvath  4 , T J Rudge  2   3   8 , B Cartes-Saavedra  1   9 , V Eisner  10
Affiliations

OPA1 and disease-causing mutants perturb mitochondrial nucleoid distribution

J Macuada et al. Cell Death Dis. .

Abstract

Optic atrophy protein 1 (OPA1) mediates inner mitochondrial membrane (IMM) fusion and cristae organization. Mutations in OPA1 cause autosomal dominant optic atrophy (ADOA), a leading cause of blindness. Cells from ADOA patients show impaired mitochondrial fusion, cristae structure, bioenergetic function, and mitochondrial DNA (mtDNA) integrity. The mtDNA encodes electron transport chain subunits and is packaged into nucleoids spread within the mitochondrial population. Nucleoids interact with the IMM, and their distribution is tightly linked to mitochondrial fusion and cristae shaping. Yet, little is known about the physio-pathological relevance of nucleoid distribution. We studied the effect of OPA1 and ADOA-associated mutants on nucleoid distribution using high-resolution confocal microscopy. We applied a novel model incorporating the mitochondrial context, separating nucleoid distribution into the array in the mitochondrial population and intramitochondrial longitudinal distribution. Opa1-null cells showed decreased mtDNA levels and nucleoid abundance. Also, loss of Opa1 led to an altered distribution of nucleoids in the mitochondrial population, loss of cristae periodicity, and altered nucleoids to cristae proximity partly rescued by OPA1 isoform 1. Overexpression of WT OPA1 or ADOA-causing mutants c.870+5 G > A or c.2713 C > T in WT cells, showed perturbed nucleoid array in the mitochondria population associated with cristae disorganization, which was partly reproduced in Skeletal muscle-derived fibroblasts from ADOA patients harboring the same mutants. Opa1-null and cells overexpressing ADOA mutants accumulated mitochondria without nucleoids. Interestingly, intramitochondrial nucleoid distribution was only altered in Opa1-null cells. Altogether, our results highlight the relevance of OPA1 in nucleoid distribution in the mitochondrial landscape and at a single-organelle level and shed light on new components of ADOA etiology.

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Conflict of interest statement

Competing interests: PYWM is a consultant for GenSight Biologics, Neurophth Therapeutics, Stoke Therapeutics, and PYC Therapeutics, and has received research support from GenSight Biologics and Santhera Pharmaceuticals. All other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Ethics approval: All methods were performed based on Biosafety Protocol #220621005 following the guidelines and regulations from the Research Biosecurity Committee, at Pontificia Universidad Católica de Chile (PUC). Fibroblasts derived from ADOA patients and control individuals were provided by Newcastle Research Biobank for Rare and Neuromuscular Diseases, based on a Material Transference Agreement (MTA) with PUC. The patient´s informed consent was considered as part of the MTA.

Figures

Fig. 1
Fig. 1. Opa1 determines nucleoid abundance and array in the mitochondrial population and intramitochondrial longitudinal distribution.
A Mitochondrial DNA abundance in WT MEF, Opa1−/− and Opa1−/− cells stably expressing a lentiviral plasmid carrying OPA1 cDNA, quantified by qPCR and expressed as mt-Nd4 to Gapdh ratio. Data are mean ± SEM of ≥ 3 independent experiments. B Schematic representation of the components of nucleoid distribution. C Representative images of WT MEF, Opa1−/− and Opa1−/− cells with WT OPA1 acute expression, co-transfected with mCherry-Omp25 (OMM) and Tfam-GFP (nucleoids) cDNA. Bottom panel, insets of Tfam-GFP (top) and merge (bottom). D Percentage of mitochondria with different number of nucleoid clusters. E Mean nucleoid cluster number per mitochondrion. F Mean nucleoid cluster number per mitochondrion area, manually quantified. G Number of nucleoid clusters per total mitochondrial area, semi-automatically quantified by the MiNuD algorithm. For Opa1−/− cells, DNA was labeled using PicoGreen, and mitochondria were labeled with mCherry-Omp25 (OMM) or MitoTracker Deep Red. H Mean nucleoid cluster number per mitochondrion length. I Simple regression of the relationship between nucleoid cluster number and mitochondrion length in individual organelles. Data are mean ± SEM of ≥ 281 objects from ≥ 15 cells of ≥ 3 independent experiments, and for G data are mean ± SEM of ≥ 10 cells of ≥ 2 independent experiments (****p < 0.0001; ***p < 0.0005; *p < 0.05).
Fig. 2
Fig. 2. The stable expression of WT OPA1 rescues mitochondrial cristae periodicity and nucleoid-cristae proximity in Opa1−/− cells.
A Representative TEM images of WT MEF, Opa1−/− and Opa1−/− cells stably expressing a lentiviral plasmid carrying OPA1 cDNA. B Mitochondrial cristae shape classification and C their quantification. D Mitochondrial cristae periodicity classification and E their quantification. Data are from ≥ 98 objects from ≥ 2 independent experiments. F Representative immunofluorescence images of WT MEF, Opa1−/− and Opa1−/− cells stably expressing a lentiviral plasmid carrying OPA1 cDNA. Cells were labeled with MitoTracker Deep Red and antibodies against dsDNA and Mic60. The white boxes correspond to 5 x 5 μm insets. (Right) Intensity profile of dsDNA and Mic60 signals along the yellow line from left to right (2 µm). G, H Colocalization coefficients for dsDNA and Mic60 in the cells shown in F. Each point represents one cell. Data are mean ± SEM from ≥ 70 cells from 4 independent experiments (****p < 0.0001; *p < 0.05). In A, B and D the scale bar is 1 μm.
Fig. 3
Fig. 3. Acute expression of WT and pathogenic OPA1 variants perturb mitochondrial nucleoid cluster abundance and distribution.
A Schematic representation of OPA1 isoform 1 domains and ADOA-causing mutants: OPA1 c.870+5 G > A (GTPase domain) and OPA1 c.2713 C > T (GTPase effector domain, GED). MTS: mitochondrial targeting sequence. TM: transmembrane domain. S1: Processing site 1. CC1: coiled-coil domain 1. B Mitochondrial DNA abundance of WT MEF stably expressing a lentiviral plasmid carrying control cDNA (CTL), OPA1 WT, or pathogenic OPA1 variants; quantified by qPCR and expressed as mt-Nd4 to Gadph ratio. Data are mean ± SEM from ≥ 5 independent experiments. C Representative images of WT MEF, WT MEF overexpressing OPA1 WT, or pathogenic OPA1 variants, co-transfected with mCherry-Omp25 (OMM) and Tfam-GFP (nucleoids) cDNA. Bottom panel, insets of Tfam-GFP (top), and merge (bottom). D Percentage of mitochondria with a different number of nucleoid clusters. E Mean nucleoid cluster number per mitochondrion. F Mean nucleoid cluster number per mitochondrion area, manually quantified. G Mean nucleoid cluster number per total mitochondrial area, quantified semi-automatically by the MiNuD algorithm. H Mean nucleoid cluster number per mitochondrion length. I A simple regression of the relation between mitochondrial length and the number of nucleoids in single organelles. Data are mean ± SEM of ≥ 228 objects from ≥ 18 cells of ≥ 4 independent experiments. For G, data are mean ± SEM of ≥ 11 cells of ≥ 4 independent experiments (****p < 0.0001; **p < 0.005; *p < 0.05).
Fig. 4
Fig. 4. Cristae ultrastructure and nucleoid-cristae proximity in cells expressing OPA1 ADOA-causing mutants.
A Representative TEM images of WT MEF stably expressing a lentiviral plasmid containing control cDNA (CTL), OPA1 WT, or pathogenic OPA1 variants. Scale bar is 1 μm. B Quantification of mitochondrial cristae shape descriptors. C Quantification of mitochondrial cristae periodicity. Data are from ≥ 112 objects from ≥ 3 independent experiments. D Representative immunofluorescence images of Skeletal muscle-derived fibroblasts from ADOA patients carrying OPA1 variants. Cells were labeled with antibodies against dsDNA and Mic60. The white boxes correspond to the 5 x 5 μm insets. (Right) Intensity profile of dsDNA and Mic60 signals along the yellow line from left to right (2 µm). E, F Colocalization coefficients for dsDNA and Mic60 in the cells shown in D. Each point represents one cell. Data are mean ± SEM from ≥ 70 cells from 4 independent experiments (****p < 0.0001).
Fig. 5
Fig. 5. Summary of main findings.
The cartoon and table show mitochondrial morphology and cristae ultrastructure changes, as well as nucleoid (green circles) abundance and nucleoid distribution alterations in the studied models. Mitochondrial fusion data were obtained from Cartes-Saavedra, et al. [48].
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