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Cited by 216Mitochondrial Function and PathologyMetabolism and Genetic DisordersAutophagy in Disease and Therapy

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Opa1 Overexpression Ameliorates the Phenotype of Two Mitochondrial Disease Mouse Models

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Civiletto, Gabriele, Varanita, Tatiana, Cerutti, Raffaele, Gorletta, Tatiana, Barbaro, Serena, Marchet, Silvia, Lamperti, Costanza, Viscomi, Carlo, Scorrano, Luca, Zeviani, Massimo·2015·Cell Metabolism
DOI 10.1016/j.cmet.2015.04.016PMC PMC4457891
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Abstract

Increased levels of the mitochondria-shaping protein Opa1 improve respiratory chain efficiency and protect from tissue damage, suggesting that it could be an attractive target to counteract mitochondrial dysfunction. Here we show that Opa1 overexpression ameliorates two mouse models of defective mitochondrial bioenergetics. The offspring from crosses of a constitutive knockout for the structural complex I component Ndufs4 (Ndufs4(-/-)), and of a muscle-specific conditional knockout for the complex IV assembly factor Cox15 (Cox15(sm/sm)), with Opa1 transgenic (Opa1(tg)) mice showed improved motor skills and respiratory chain activities compared to the naive, non-Opa1-overexpressing, models. While the amelioration was modest in Ndufs4(-/-)::Opa1(tg) mice, correction of cristae ultrastructure and mitochondrial respiration, improvement of motor performance and prolongation of lifespan were remarkable in Cox15(sm/sm)::Opa1(tg) mice. Mechanistically, respiratory chain supercomplexes were increased in Cox15(sm/sm)::Opa1(tg) mice, and residual monomeric complex IV was stabilized. In conclusion, cristae shape amelioration by controlled Opa1 overexpression improves two mouse models of mitochondrial disease.

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Graphical abstract CC BY-NC-ND · PubMed Central
Figure 1 Improved Motor Function and Extended Lifespan in Ndufs4−/−::Opa1tg Double Mutants (A) Rotarod analysis (n = 6/group). Dashed blue: WT; dashed red: Opa1tg; solid blue: Ndufs4−/−; solid red: Ndufs4−/−::Opa1tg. Error bars represent SEM. The asterisks represent the significance levels calculate by unpaired, two-tail Student’s t test, ∗p < 0.05, ∗∗∗p < 0.005, and refer to the comparison between Ndufs4−/−::Opa1tg and Ndufs4−/−. (B) Kaplan-Meier survival probability. Significance was assessed by the log rank test. Solid blue: Ndufs4−/− (n = 16); solid red: Ndufs4−/−::Opa1tg (n = 9). (C) Oxygen consumption measurements (nmol O2/min/mg of protein). Solid blue: WT (n = 5); blue outline: Opa1tg (n = 5); solid red: Ndufs4−/− (n = 5); red outline: Ndufs4−/−::Opa1tg (n = 6). The asterisks represent the significance levels calculate by unpaired, two-tail Student’s t test ∗p < 0.05. (D) MRC activities (nmol/min/mg of protein) in isolated brain mitochondria. Solid blue: WT (n = 8); blue outline: Opa1tg (n = 5); solid red: Ndufs4−/− (n = 7); red outline: Ndufs4−/−::Opa1tg (n = 8). Error bars represent SEM. The asterisks represent the significance levels calculate by unpaired, two-tail Student’s t test ∗p < 0.05. (E) Western blot analysis of subunits from CI (NDUFA9 and NDUFB8), CII (SDHA), CIII (UQCRC2), CIV (COX1), and CV (ATP5A) in brain isolated mitochondria. VDAC is taken as an internal control. (F) Electron microscopy of brain specimens from the different genotypes. Note that mitochondrial morphology is well preserved in Ndufs4−/−::Opa1tg versus Ndufs4−/−. Scale bar 1 μm.
Figure 1 Improved Motor Function and Extended Lifespan in Ndufs4−/−::Opa1tg Double Mutants (A) Rotarod analysis (n = 6/group). Dashed blue: WT; dashed red: Opa1tg; solid blue: Ndufs4−/−; solid red: Ndufs4−/−::Opa1tg. Error bars represent SEM. The asterisks represent the significance levels calculate by unpaired, two-tail Student’s t test, ∗p < 0.05, ∗∗∗p < 0.005, and refer to the comparison between Ndufs4−/−::Opa1tg and Ndufs4−/−. (B) Kaplan-Meier survival probability. Significance was assessed by the log rank test. Solid blue: Ndufs4−/− (n = 16); solid red: Ndufs4−/−::Opa1tg (n = 9). (C) Oxygen consumption measurements (nmol O2/min/mg of protein). Solid blue: WT (n = 5); blue outline: Opa1tg (n = 5); solid red: Ndufs4−/− (n = 5); red outline: Ndufs4−/−::Opa1tg (n = 6). The asterisks represent the significance levels calculate by unpaired, two-tail Student’s t test ∗p < 0.05. (D) MRC activities (nmol/min/mg of protein) in isolated brain mitochondria. Solid blue: WT (n = 8); blue outline: Opa1tg (n = 5); solid red: Ndufs4−/− (n = 7); red outline: Ndufs4−/−::Opa1tg (n = 8). Error bars represent SEM. The asterisks represent the significance levels calculate by unpaired, two-tail Student’s t test ∗p < 0.05. (E) Western blot analysis of subunits from CI (NDUFA9 and NDUFB8), CII (SDHA), CIII (UQCRC2), CIV (COX1), and CV (ATP5A) in brain isolated mitochondria. VDAC is taken as an internal control. (F) Electron microscopy of brain specimens from the different genotypes. Note that mitochondrial morphology is well preserved in Ndufs4−/−::Opa1tg versus Ndufs4−/−. Scale bar 1 μm.CC BY-NC-ND · PubMed Central
Figure 2 Improved Motor Function and Extended Lifespan in Cox15sm/sm−::Opa1Tg Animals (A) Treadmill analysis of motor performance. Dashed blue: WT (n = 8); dashed red: Opa1tg (n = 9); solid blue: Cox15sm/sm (n = 10); solid red Cox15sm/sm::Opa1tg (n = 8). Error bars represent SEM. The asterisks represent the significance levels calculate by unpaired, two-tail Student’s t test, ∗∗∗p < 0.005, and refer to the comparison between Cox15sm/sm::Opa1tg and Cox15sm/sm. (B) Kaplan–Meier survival probability. Significance was assessed by the log rank test. Solid blue: Cox15sm/sm (n = 23); solid red: Cox15sm/sm::Opa1tg (n = 17).
Figure 2 Improved Motor Function and Extended Lifespan in Cox15sm/sm−::Opa1Tg Animals (A) Treadmill analysis of motor performance. Dashed blue: WT (n = 8); dashed red: Opa1tg (n = 9); solid blue: Cox15sm/sm (n = 10); solid red Cox15sm/sm::Opa1tg (n = 8). Error bars represent SEM. The asterisks represent the significance levels calculate by unpaired, two-tail Student’s t test, ∗∗∗p < 0.005, and refer to the comparison between Cox15sm/sm::Opa1tg and Cox15sm/sm. (B) Kaplan–Meier survival probability. Significance was assessed by the log rank test. Solid blue: Cox15sm/sm (n = 23); solid red: Cox15sm/sm::Opa1tg (n = 17).CC BY-NC-ND · PubMed Central
Figure 3 Improved Morphological Features of Cox15sm/sm::Opa1Tg Skeletal Muscle (A) H&E staining of skeletal muscles from mice of the different genotypes. Scale bar 100 μm. (B) Quantification of the number of centralized nuclei in the different genotypes. 600 fibers/sample were analyzed (n = 4/genotype). Solid blue: WT; blue outline: Opa1tg; solid red: Cox15sm/sm; red outline: Cox15sm/sm::Opa1tg. Statistical significance was calculated by unpaired, two-tail Student’s t test: ∗∗∗p < 0.005. Error bars represent SEM. (C) Cross-sectional area of muscle fibers of gastrocnemius. 600 fibers/sample were analyzed (n = 4/genotype). Solid blue: WT; blue outline: Opa1tg; solid red: Cox15sm/sm; red outline: Cox15sm/sm::Opa1Tg. Statistical significance was calculated by unpaired, two-tail Student’s t test: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.005. Error bars represent SEM.
Figure 3 Improved Morphological Features of Cox15sm/sm::Opa1Tg Skeletal Muscle (A) H&E staining of skeletal muscles from mice of the different genotypes. Scale bar 100 μm. (B) Quantification of the number of centralized nuclei in the different genotypes. 600 fibers/sample were analyzed (n = 4/genotype). Solid blue: WT; blue outline: Opa1tg; solid red: Cox15sm/sm; red outline: Cox15sm/sm::Opa1tg. Statistical significance was calculated by unpaired, two-tail Student’s t test: ∗∗∗p < 0.005. Error bars represent SEM. (C) Cross-sectional area of muscle fibers of gastrocnemius. 600 fibers/sample were analyzed (n = 4/genotype). Solid blue: WT; blue outline: Opa1tg; solid red: Cox15sm/sm; red outline: Cox15sm/sm::Opa1Tg. Statistical significance was calculated by unpaired, two-tail Student’s t test: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.005. Error bars represent SEM.CC BY-NC-ND · PubMed Central
Figure 4 Amelioration of Mitochondrial Ultrastructure in Cox15sm/sm::Opa1Tg Skeletal Muscle (A) EM microphotographs of specimens from the different genotypes. Note that while there is accumulation of inter-myofibrillar amorphous material and disorganization of the mitochondrial cristae (inset) in the Cox15sm/sm, the myofibrillary array and cristae structure (inset) are both well preserved in the Cox15sm/sm::Opa1Tg. Mitochondria appear larger and with denser cristae in Opa1Tg than in WT. Scale bar 2 μm; scale bar of the inset: 200 nm. (B) Morphometric quantification of the area, perimeter, length and width of mitochondria from the different genotypes. A total of 200 mitochondria from WT and Opa1Tg mice (n = 2) were analyzed, and 500 from Cox15sm/sm and Cox15sm/sm::Opa1Tg (n = 2).
Figure 4 Amelioration of Mitochondrial Ultrastructure in Cox15sm/sm::Opa1Tg Skeletal Muscle (A) EM microphotographs of specimens from the different genotypes. Note that while there is accumulation of inter-myofibrillar amorphous material and disorganization of the mitochondrial cristae (inset) in the Cox15sm/sm, the myofibrillary array and cristae structure (inset) are both well preserved in the Cox15sm/sm::Opa1Tg. Mitochondria appear larger and with denser cristae in Opa1Tg than in WT. Scale bar 2 μm; scale bar of the inset: 200 nm. (B) Morphometric quantification of the area, perimeter, length and width of mitochondria from the different genotypes. A total of 200 mitochondria from WT and Opa1Tg mice (n = 2) were analyzed, and 500 from Cox15sm/sm and Cox15sm/sm::Opa1Tg (n = 2).CC BY-NC-ND · PubMed Central
Figure 5 Increased Respiration and Mrc activities in Cox15sm/sm::Opa1Tg Skeletal Muscle (A) Oxygen consumption rate (nmol O2/min/mg of protein). Solid blue: WT (n = 5); blue outline: Opa1tg (n = 5); solid red: Cox15sm/sm (n = 5); red outline: Cox15sm/sm::Opa1tg (n = 6). Error bars represent SEM. The asterisks represent the significance levels calculate by unpaired, two-tail Student’s t test, ∗p < 0.05. (B) MRC complex activities (nmol/min/mg of protein) in skeletal muscle mitochondria shown as a percentage of CS activity. Solid blue: WT (n = 5); blue outline: Opa1tg (n = 5); solid red: Cox15sm/sm (n = 5); red outline: Cox15sm/sm::Opa1tg (n = 6). Error bars represent SD. The asterisks represent the significance levels calculate by unpaired, two-tail Student’s t test, ∗p < 0.05. (C) COX/SDH histochemical double staining. Scale bar: 50 μm.
Figure 5 Increased Respiration and Mrc activities in Cox15sm/sm::Opa1Tg Skeletal Muscle (A) Oxygen consumption rate (nmol O2/min/mg of protein). Solid blue: WT (n = 5); blue outline: Opa1tg (n = 5); solid red: Cox15sm/sm (n = 5); red outline: Cox15sm/sm::Opa1tg (n = 6). Error bars represent SEM. The asterisks represent the significance levels calculate by unpaired, two-tail Student’s t test, ∗p < 0.05. (B) MRC complex activities (nmol/min/mg of protein) in skeletal muscle mitochondria shown as a percentage of CS activity. Solid blue: WT (n = 5); blue outline: Opa1tg (n = 5); solid red: Cox15sm/sm (n = 5); red outline: Cox15sm/sm::Opa1tg (n = 6). Error bars represent SD. The asterisks represent the significance levels calculate by unpaired, two-tail Student’s t test, ∗p < 0.05. (C) COX/SDH histochemical double staining. Scale bar: 50 μm.CC BY-NC-ND · PubMed Central
Figure 6 Molecular Analysis of Cox15sm/sm::Opa1tg Skeletal Muscle (A) Western blot analysis of OPA1, SDHA (CII), ATP5A (CV), UQCRC2 (CIII), NDUFA9 (CI), COX1, and COX4 (CIV); GAPDH is taken as a loading reference. (B) Densitometric analysis of MRC subunits. Solid blue: WT (n = 4); blue outline: Opa1tg (n = 4); solid red: Cox15sm/sm (n = 4); red outline: Cox15sm/sm::Opa1tg (n = 4). Error bars represent SEM. The asterisks represent the significance levels calculate by unpaired, two-tail Student’s t test. ∗∗p < 0.01; ∗∗∗p < 0.005. (C) mRNA expression analysis of CIV- and mitochondrial-biogenesis-related genes. Gene transcripts, retrotranscribed into cDNA, were quantified by RT-PCR; normalized to the amount of the β-Actin gene transcript; taken as a standard; and expressed as time-fold variation relative to the WT. Error bars represent SEM. The asterisks represent the significance levels calculate by unpaired, two-tail Student’s t test. ∗∗p < 0.01; ∗∗∗p < 0.005.
Figure 6 Molecular Analysis of Cox15sm/sm::Opa1tg Skeletal Muscle (A) Western blot analysis of OPA1, SDHA (CII), ATP5A (CV), UQCRC2 (CIII), NDUFA9 (CI), COX1, and COX4 (CIV); GAPDH is taken as a loading reference. (B) Densitometric analysis of MRC subunits. Solid blue: WT (n = 4); blue outline: Opa1tg (n = 4); solid red: Cox15sm/sm (n = 4); red outline: Cox15sm/sm::Opa1tg (n = 4). Error bars represent SEM. The asterisks represent the significance levels calculate by unpaired, two-tail Student’s t test. ∗∗p < 0.01; ∗∗∗p < 0.005. (C) mRNA expression analysis of CIV- and mitochondrial-biogenesis-related genes. Gene transcripts, retrotranscribed into cDNA, were quantified by RT-PCR; normalized to the amount of the β-Actin gene transcript; taken as a standard; and expressed as time-fold variation relative to the WT. Error bars represent SEM. The asterisks represent the significance levels calculate by unpaired, two-tail Student’s t test. ∗∗p < 0.01; ∗∗∗p < 0.005.CC BY-NC-ND · PubMed Central
Figure 7 Stabilization of Respiratory Complexes and Supercomplexes in Cox15sm/sm::Opa1Tg Skeletal Muscle (A) CIV-specific BNGE in-gel activity. Note the increased staining of the bands corresponding to CIV-holoenzyme and CIV-containing sc in Cox15sm/sm::Opa1tg versus Cox15sm/sm. (B) CI-specific BNGE in-gel activity. Note the increased staining of the bands corresponding to the sc in Cox15sm/sm::Opa1tg versus Cox15sm/sm. (C) Western blot analysis of MRC CIV from BNGE of digitonin-treated isolated mitochondria. The blot was immunodecorated with an anti-COX1 antibody. (D) Western blot analysis of MRC CI- and CIV-containing sc’s from BNGE of digitonin-treated isolated mitochondria. The blot was immunodecorated with an anti-NDUFB8 antibody.
Figure 7 Stabilization of Respiratory Complexes and Supercomplexes in Cox15sm/sm::Opa1Tg Skeletal Muscle (A) CIV-specific BNGE in-gel activity. Note the increased staining of the bands corresponding to CIV-holoenzyme and CIV-containing sc in Cox15sm/sm::Opa1tg versus Cox15sm/sm. (B) CI-specific BNGE in-gel activity. Note the increased staining of the bands corresponding to the sc in Cox15sm/sm::Opa1tg versus Cox15sm/sm. (C) Western blot analysis of MRC CIV from BNGE of digitonin-treated isolated mitochondria. The blot was immunodecorated with an anti-COX1 antibody. (D) Western blot analysis of MRC CI- and CIV-containing sc’s from BNGE of digitonin-treated isolated mitochondria. The blot was immunodecorated with an anti-NDUFB8 antibody.CC BY-NC-ND · PubMed Central

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Authors
Civiletto, G., Varanita, T., Cerutti, R., Gorletta, T., Barbaro, S., Marchet, S., Lamperti, C., Viscomi, C., Scorrano, L., & Zeviani, M.
Affiliations
  • Fondazione IRCCS Istituto Neurologico Carlo Besta (IT)
  • Veneto Institute of Molecular Medicine (IT)
  • MRC Mitochondrial Biology Unit (GB)
  • University of Padua (IT)
Corresponding author
  • Scorrano, Luca
  • Zeviani, Massimo
ORCID
Venue
Cell Metabolism
Publisher
Elsevier BV
Published
2015-06
Volume
21
Issue
6
Pages
845-854
ISSN
1550-4131 (ISSN-L)
DOI
10.1016/j.cmet.2015.04.016
PMC
PMC4457891
OpenAlex
W2169501919
Cited by
216
References
50
Citations by year
2026: 4 · 2025: 12 · 2024: 12 · 2023: 15 · 2022: 19 · 2021: 26 · 2020: 26 · 2019: 28 · 2018: 22 · 2017: 18 · 2016: 24 · 2015: 10
FWCI
10.65
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell Metabolism (publishedVersion, cc-by-nc-nd)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • Archivio Istituzionale della Ricerca (Universita Degli Studi Di Milano) (submittedVersion, other-oa)
  • Padua Research Archive (University of Padova) (submittedVersion, cc-by-nc-nd)
  • Padua Research Archive (University of Padova) (submittedVersion, other-oa)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mitochondrial Function and Pathology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Clinical Biochemistry › Metabolism and Genetic Disorders
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Autophagy in Disease and Therapy
Keywords
OPA1, mitochondrial disease, mitochondrial cristae, mitochondrial fusion, NDUFS4, complex I, cytochrome c oxidase, respiratory chain supercomplexes, mitochondrial bioenergetics, motor function, lifespan extension, transgenic mice, mitochondrial dysfunction
MeSH terms
Animals, Electron Transport Complex IV, Mitochondria, Oxygen Consumption, Gene Expression Regulation, Enzymologic, Mice, Knockout, GTP Phosphohydrolases, Mitochondrial Diseases, Electron Transport Complex I, Mice

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